**Meet the editors**

Dr Patricia Hernández-Rodríguez is the Director of Research Group BIOMIGEN (Molecular Biology and Immunogenetics) in the Biology Program, Department of Basic Science; she is also a member of Animal Medicine and Reproduction Research Center (CIMRA) of the Faculty of Agricultural Sciences, Universidad de La Salle (Colombia). She holds a Biology degree, a Specialization in Epidemiology, a Master of Science in Molecular Biology and she is a PhD student. She has served as a professor to undergraduate and graduate students. He has lectured at scientific events in Colombia and other countries such as Argentina, Cuba, Spain, Mexico and as guest lecturer at the National University of Asuncion in Paraguay. Fields of Research: Infectious Diseases, Genetics and Molecular Biology.

Dr Patricia Gómez is a Veterinarian and holds a PhD from the Universidad Nacional de Colombia. She is an associate lecturer in the Faculty of Agricultural Sciences, Universidad de La Salle and a member of Animal Medicine and Reproduction Research Center (CIMRA) of Universidad de La Salle. Her professional focus is on the Avian Medicine and Molecular Biology. She has a teaching experience on Histology, Physiology and Avian Medicine as well as wide training in research and laboratory.

Contents

**Preface IX** 

Chapter 1 **Application of PCR in Diagnosis** 

Chapter 3 **Role of Polymerase Chain Reaction in Forensic Entomology 51**  Tock Hing Chua and Y. V. Chong

Chapter 4 **PCR for Screening Potential** 

Duška Delić

**of Peste des Petits Ruminants Virus (PPRV) 1** 

Chapter 2 **Application of PCR-Based Methods to Dairy Products and to** 

Maurilia Rojas-Contreras, María Esther Macías-Rodríguez

**Perspectives from the Microbiology Standpoint 119**  Saúl Flores-Medina, Diana Mercedes Soriano-Becerril

Chapter 5 **Polymerase Chain Reaction for Phytoplasmas Detection 91** 

**High Resolution Probe Melting Analysis 143**  Jennifer E. Hardingham, Ann Chua, Joseph W. Wrin,

Patricia Hernández-Rodríguez and Arlen Gomez Ramirez

Christophe Monnet and Bojana Bogovič Matijašić

Muhammad Abubakar, Farida Mehmood, Aeman Jeelani and Muhammad Javed Arshed

**Non-Dairy Probiotic Products 11** 

**Probiotic Lactobacilli for Piglets 65** 

and José Alfredo Guevara Franco

Chapter 6 **Molecular Diagnostics of Mycoplasmas:** 

and Francisco Javier Díaz-García

Chapter 7 **BRAF V600E Mutation Detection Using** 

Aravind Shivasami, Irene Kanter, Niall C. Tebbutt and Timothy J. Price

**Types, Utilities and Limitations 157** 

Chapter 8 **Polymerase Chain Reaction:** 

## Contents

## **Preface XIII**


X Contents


Contents VII

Chapter 20 **PCR Advances Towards the Identification of Individual and Mixed Populations of Biotechnology Microbes 443** 

Chapter 21 **Lack of Evidence for Contribution of eNOS, ACE and AT1R** 

Chapter 22 **Analysis of Genomic Instability and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 469** Nikola Tanic, Jasna Bankovic and Nasta Tanic

**in Turkish Subjects in Trakya Region 455**

Chapter 23 **Analysis of Alternatively Spliced Domains** 

**Glycoprotein Tenascin C 487**  Ursula Theocharidis and Andreas Faissner

P. Veeramuthumari and W. Isabel

Chapter 27 **PCR-RFLP and Real-Time PCR Techniques** 

Fousseyni S. Touré Ndouo

Chapter 26 **Detection of Bacterial Pathogens** 

Chapter 24 **Submicroscopic Human Parasitic Infections 501** 

Chapter 25 **Identification of Genetic Markers Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 517**

> **in River Water Using Multiplex-PCR 531**  C. N. Wose Kinge, M. Mbewe and N. P. Sithebe

**in Molecular Cancer Investigations 555**  Uzay Gormus, Nur Selvi and Ilhan Yaylim-Eraltan

**Gene Polymorphisms with Development of Ischemic Stroke** 

**in Multimodular Gene Products - The Extracellular Matrix** 

P. S. Shwed

Tammam Sipahi

Chapter 10 **PCR in Food Analysis 195**  Anja Klančnik, Minka Kovač, Nataša Toplak, Saša Piskernik and Barbara Jeršek


Chapter 20 **PCR Advances Towards the Identification of Individual and Mixed Populations of Biotechnology Microbes 443**  P. S. Shwed

VI Contents

Chapter 9 **The Application of PCR-Based Methods** 

Saša Piskernik and Barbara Jeršek

Chapter 11 **PCR in Disease Diagnosis of WND 221**  Asifa Majeed, Abdul Khaliq Naveed, Natasha Rehman and Suhail Razak

Chapter 10 **PCR in Food Analysis 195** 

Chapter 13 **Recent Advances and** 

Xiangyang Miao

Ayse Gul Mutlu

Chapter 16 **Application of PCR Technologies** 

**from Central Africa 309**

Chapter 17 **Study of Mycobacterium Tuberculosis** 

Chapter 18 **Development of a Molecular Platform** 

Sylvia Broeders, Nina Papazova,

Chapter 19 **Overview of Real-Time PCR Principles 405** 

**in Food Control Agencies – A Review 173**  Azuka Iwobi, Ingrid Huber and Ulrich Busch

Anja Klančnik, Minka Kovač, Nataša Toplak,

Chapter 12 **Real-Time PCR for Gene Expression Analysis 229**

Chapter 14 **Measuring of DNA Damage by Quantitative PCR 283**

**in Tissues of Pear Using** *In Situ* **RT-PCR and Primed** *In Situ* **Labeling 295**  Na Liu, Jianxin Niu and Ying Zhao

**to Humans, Animals, Plants and Pathogens**

Ouwe Missi Oukem-Boyer Odile, Migot-Nabias Florence, Born Céline, Aubouy Agnès and Nkenfou Céline

**by Molecular Methods in Northeast Mexico 349**

**for GMO Detection in Food and Feed on the Basis of** 

Morteza Seifi, Asghar Ghasemi, Siamak Heidarzadeh,

Mahmood Khosravi, Atefeh Namipashaki, Vahid Mehri Soofiany,

H. W. Araujo-Torres, J. A. Narváez-Zapata, M. G. Castillo-Álvarez, MS. Puga-Hernández, J. Flores-Gracia and M. A. Reyes-López

**"Combinatory qPCR" Technology 363** 

Marc Van den Bulcke and Nancy Roosens

Ali Alizadeh Khosroshahi and Nasim Danaei

Chapter 15 **Detection of** *Apple Chlorotic Leaf Spot Virus*

Akin Yilmaz, Hacer Ilke Onen, Ebru Alp and Sevda Menevse

**Applications of Transgenic Animal Technology 255**


Preface

beings.

life.

diseases that affect living beings.

This book is intended to present current concepts in molecular biology with the emphasis on the application to animal, plant and human pathology, in various aspects such as etiology, diagnosis, prognosis, treatment and prevention of diseases as well as the use of these methodologies in understanding the pathophysiology of various

It is known today that molecular biology has revolutionized the study and understanding of health and disease. Significant developments occurred after 1953, based on the impact generated in many disciplines, especially those life-related such as medicine. Furthermore, the advances in molecular biology have revolutionized industry, agriculture, pharmacology, and animal and plant production, among others. Technology based on Molecular Chain reaction Polymerase (PCR) is advancing rapidly since it is fundamental for improving the health of all living

Importantly, the most of the research in biology and medicine requires a series of molecular strategies that allow the generation of new knowledge, in order to enable better understanding of the mechanisms of life and the cellular changes that affect all living things. Molecular biology has transformed the way we see and understand the physiological and pathological changes of cells, organs and systems. In this sense, this book presents the fundamentals, applications, advantages and disadvantages of various molecular techniques from the research process in biology, medicine, agriculture and environment in basic and applied science. Each chapter explains molecular techniques through various experiments offering new knowledge in different disciplines with applications trying to ultimately improve the conditions of

The book includes the participation of different authors and co-authors of various nationalities, all of them experts in the field. The book will be useful to professionals, students, teachers and researchers interested in expanding their knowledge in

molecular biology, one of the most exciting areas of work today.

## Preface

This book is intended to present current concepts in molecular biology with the emphasis on the application to animal, plant and human pathology, in various aspects such as etiology, diagnosis, prognosis, treatment and prevention of diseases as well as the use of these methodologies in understanding the pathophysiology of various diseases that affect living beings.

It is known today that molecular biology has revolutionized the study and understanding of health and disease. Significant developments occurred after 1953, based on the impact generated in many disciplines, especially those life-related such as medicine. Furthermore, the advances in molecular biology have revolutionized industry, agriculture, pharmacology, and animal and plant production, among others. Technology based on Molecular Chain reaction Polymerase (PCR) is advancing rapidly since it is fundamental for improving the health of all living beings.

Importantly, the most of the research in biology and medicine requires a series of molecular strategies that allow the generation of new knowledge, in order to enable better understanding of the mechanisms of life and the cellular changes that affect all living things. Molecular biology has transformed the way we see and understand the physiological and pathological changes of cells, organs and systems. In this sense, this book presents the fundamentals, applications, advantages and disadvantages of various molecular techniques from the research process in biology, medicine, agriculture and environment in basic and applied science. Each chapter explains molecular techniques through various experiments offering new knowledge in different disciplines with applications trying to ultimately improve the conditions of life.

The book includes the participation of different authors and co-authors of various nationalities, all of them experts in the field. The book will be useful to professionals, students, teachers and researchers interested in expanding their knowledge in molecular biology, one of the most exciting areas of work today.

#### XIV Preface

I am grateful for the possibility of editing this book and sending a message to all readers: perform with passion, responsibility and dedication your projects in life; in my case – it is the research.

> **Patricia Hernandez-Rodriguez** Universidad De La Salle, Bogota, Colombia

## **Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV)**

Muhammad Abubakar\*, Farida Mehmood, Aeman Jeelani and Muhammad Javed Arshed *National Veterinary Laboratory (NVL), Park Road, Islamabad Pakistan* 

## **1. Introduction**

a. Global perspective of PPRV

A Peste des petits ruminant (PPR) is a viral disease of sheep, goats and wild ruminants. It is acute disease which is endemic in many countries of Africa, Arabian Peninsula, Middle east and India. 7, 12, 13

It was first reported in Côte d'Ivoire in West Africa 14 and was named as Kata, psuedorinderpest, pneumoenterititis complex and stomatitis-pneumenteritis syndrome 15 . Then in 1972 a sort of disease in goats in Sudan was identified to be PPR 16 . In recent years either the presence of antibodies to the virus or viral nucleic acid has been confirmed from the countries like Burkina Faso (2008), Ghana (2010), Nigeria (2007) and Senegal (2010) 17 .

Recently detection of PPRV in East Africa countries is shown by the detection of Antibodies in Kenya (1999 and 2009) and Uganda (2005 and 2007) 18. It has also been detected in North Africa (Egypt) in 1987 and 1990.

In Saudi Arabia, an outbreak of PPRV has been reported in April, 2002 in Sheeps and Goats 1. In Pakistan PPRV has been reported since 1991 which was confirmed by PCR in 1994. 19 In India the was first reported in 1987 11. In Iran the disease was reported in 1995 20 while in Iraq it was first detected in 2000 21 .

b. Disease picture of PPRV

Peste des petits ruminants (PPR) represents one of the most economically important animal diseases in areas that rely on small ruminants. Outbreaks tend to be associated with contact of immuno-naïve animals with animals from endemic areas. In addition to occurring in extensive-migratory populations, PPR can occur in village and urban settings though the number of animals is usually too small to maintain the virus in these situations.


 \* Corresponding Author

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV) 3

A close contact between the infected animals which is in the febrile stage and susceptible animals is a source of transmission of the disease 15. During sneezing and coughing the virus spread from animal to animal 22. Indirect transmission seems to be unlikely in view of the

Epidemiology pattern vary from area to area, for example in the humid Guinean zone where PPR occurs in an epizootic form can cause mortality between 50-80% while in arid and semiarid regions, PPR is seldomly fatal but usually occurs as a subclinical or inapparent infection opening the door for other infections such as Pasteurellosis 4 . In Saudi Arabia a high morbidity of 90% was reported, 2 3-8 months animal are more susceptible to disease than

PPRV belong to *Morbillivirus* genus. For a long time it was thought to be a variant of RP that was adapted to sheeps and goats and had lost its virulence for cattles. 3 The causative agent of PPR is RNA virus which is single strand and non-segmented. It belongs to the family *Paramyxoviridae* and *genus Morbillivirus* which also includes measles virus, rinderpest virus (RPV), canine-distemper virus, phocinedistemper virus, and dolphin and porpoise morbilliviruses24. All the viruses belonging to the genus morbilli are serologically related.

The genome contains six tandemly arranged transcription units which encodes six structural proteins i.e the surface glycoproteins F and H, the nucleocapsid (N), the matrix (M), the polymerase or large (L) and the polymerase-associated (P) proteins. The cistron directing the synthesis of this later protein is encoding the virus non-structural proteins C and V by the use of two other open reading frames (ORF) of the messengers. The gene order is 3'N-P-M-F-H-L5', as determined by transcriptional mapping. 25 The genome is flanked by extragenic

For viruses of the family *Paramyxoviridae*, the genome promoter (GP) contains 107 nucleotides comprising the leader sequence and the adjacent non-coding region of the N gene at the 3' end of the negative-strand. While antigenome promoter (AGP) contain 109 nucleotides that encompass the trailer sequence and the proximal untranslated region of the L gene. Both GP and the AGP contains the polymerase binding sites and the RNA encapsidation signals for the replication of the full genome while the production of messengers (m-RNA) is a function of the GP 26. So GP and AGP have an impact on the

Genes and promoters of *Morbillivirus*; the protein coding regions (N, P, V, C, M, F, H, and L), noncoding intergenic regions and the leader and trailer regions along with the specialized sequence motifs are shown. The genome promoter includes the leader sequence and the non coding regions N at the 3' end of the genomic RNA. The antigenome promoter includes the trailer sequence and the untranslated regions of the L gene at 5' end. Gene start (GS) and

• Cattle and pigs develop in-apparent infections and do not transmit disease

low resistance of the virus in the environment and its sensitivity to lipid solvent. 4

Phylogenetic analysis also shows that there is high degree of homology.

sequences at the 3' ((52 nucleotides, leader) and 5' ends (37 nucleotides, trailer).

gene end (GE), enclosing the intergenic trinucleotide motifs are also shown.

• May be associated with limited disease events in camels

**2. Molecular epidemiology of PPRV** 

either of adults or unweaned animals 23.

a. Genome Organization of PPRV:

virulence of virus.

• Both morbidity and mortality rates are lower in endemic areas and in adult animals when compared to young ones.

Fig. 1. Geographic distribution of PPRV lineages (Dhar *et al*., 2002)

	- Breed-linked predisposition in goats

Fig. 2. Clinical Picture and Severity of the Disease

Wildlife host range not fully understood


## **2. Molecular epidemiology of PPRV**

2 Polymerase Chain Reaction

• Both morbidity and mortality rates are lower in endemic areas and in adult animals

**Lineage 1**

**Lineage 2** 

**Lineage 3** 

**Lineage 4** 

**Uncharacterised** 

• documented disease in captive wild ungulates: Dorcas gazelle (Gazelle dorcas), Thomson's gazelles (Gazella thomsoni), Nubian ibex (Capra ibex nubiana), Laristan

• Experimentally the American white-tailed deer (Odocoileus virginianus) is fully

sheep (Ovis gmelini laristanica) and gemsbok (Oryx gazella)

when compared to young ones.

c. Hosts Range

• Goats (predominantly) and sheep

• Breed-linked predisposition in goats

Fig. 2. Clinical Picture and Severity of the Disease

Wildlife host range not fully understood

susceptible

Fig. 1. Geographic distribution of PPRV lineages (Dhar *et al*., 2002)

A close contact between the infected animals which is in the febrile stage and susceptible animals is a source of transmission of the disease 15. During sneezing and coughing the virus spread from animal to animal 22. Indirect transmission seems to be unlikely in view of the low resistance of the virus in the environment and its sensitivity to lipid solvent. 4

Epidemiology pattern vary from area to area, for example in the humid Guinean zone where PPR occurs in an epizootic form can cause mortality between 50-80% while in arid and semiarid regions, PPR is seldomly fatal but usually occurs as a subclinical or inapparent infection opening the door for other infections such as Pasteurellosis 4 . In Saudi Arabia a high morbidity of 90% was reported, 2 3-8 months animal are more susceptible to disease than either of adults or unweaned animals 23.

a. Genome Organization of PPRV:

PPRV belong to *Morbillivirus* genus. For a long time it was thought to be a variant of RP that was adapted to sheeps and goats and had lost its virulence for cattles. 3 The causative agent of PPR is RNA virus which is single strand and non-segmented. It belongs to the family *Paramyxoviridae* and *genus Morbillivirus* which also includes measles virus, rinderpest virus (RPV), canine-distemper virus, phocinedistemper virus, and dolphin and porpoise morbilliviruses24. All the viruses belonging to the genus morbilli are serologically related. Phylogenetic analysis also shows that there is high degree of homology.

The genome contains six tandemly arranged transcription units which encodes six structural proteins i.e the surface glycoproteins F and H, the nucleocapsid (N), the matrix (M), the polymerase or large (L) and the polymerase-associated (P) proteins. The cistron directing the synthesis of this later protein is encoding the virus non-structural proteins C and V by the use of two other open reading frames (ORF) of the messengers. The gene order is 3'N-P-M-F-H-L5', as determined by transcriptional mapping. 25 The genome is flanked by extragenic sequences at the 3' ((52 nucleotides, leader) and 5' ends (37 nucleotides, trailer).

For viruses of the family *Paramyxoviridae*, the genome promoter (GP) contains 107 nucleotides comprising the leader sequence and the adjacent non-coding region of the N gene at the 3' end of the negative-strand. While antigenome promoter (AGP) contain 109 nucleotides that encompass the trailer sequence and the proximal untranslated region of the L gene. Both GP and the AGP contains the polymerase binding sites and the RNA encapsidation signals for the replication of the full genome while the production of messengers (m-RNA) is a function of the GP 26. So GP and AGP have an impact on the virulence of virus.

Genes and promoters of *Morbillivirus*; the protein coding regions (N, P, V, C, M, F, H, and L), noncoding intergenic regions and the leader and trailer regions along with the specialized sequence motifs are shown. The genome promoter includes the leader sequence and the non coding regions N at the 3' end of the genomic RNA. The antigenome promoter includes the trailer sequence and the untranslated regions of the L gene at 5' end. Gene start (GS) and gene end (GE), enclosing the intergenic trinucleotide motifs are also shown.

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV) 5

mice28 .Identification of B- and T-cell epitopes on the protective antigens of PPRV would

Sheep and goats are unlikely to be infected more than once in their economic life12. Lambs or kids receiving colostrum from previously exposed or vaccinated with RP tissue culture vaccine were found to acquire a high level of maternal antibodies that persist for 3-4 months. The maternal antibodies were detectable up to 4 months using virus neutralization test compared to 3 month with competitive ELISA29. Measles vaccine did not protect against

Before collecting or sending any samples from animals with a suspected foreign animal disease, the proper authorities should be contacted. Samples should only be sent under secure conditions and to authorized laboratories to prevent the spread of the disease. In live animals, swabs of ocular and nasal discharges, and debris from oral lesions should be collected; a spatula can be rubbed across the gum and inside the lips to collect samples from oral lesions. Whole, unclotted blood (in heparin or EDTA) should be taken for virus isolation and PCR. Biopsy samples of lymph nodes or spleen may also be useful. Samples for virus isolation should be collected during the acute stage of the disease, when clinical signs are present; whenever possible, these samples should be taken from animals with high fever and before the onset of diarrhea. At necropsy, samples can be collected from lymph nodes (particularly the mesenteric and mediastinal nodes), lungs, spleen, tonsils and affected sections of the intestinal tract (e.g. ileum and large intestine). These samples should be taken from euthanized or freshly dead animals. Samples for virus isolation should be transported chilled on ice. Similar samples should be collected in formalin for histopathology. Whenever possible, paired sera should be taken rather than single samples. However, in countries that are PPR-free, a single serum sample (taken at least a week after

Conventional techniques such as the Agar Gel Immuno Diffusion (AGID) test are not routinely used for standard diagnosis as they lack sensitivity when compared to other assays. However, Haemagglutination tests (HA) and Haemagglutination Inhibition tests (HI) tests can be used for routine screening purposes in control programmes as they display

Virus isolation in cell culture can be attempted with several different cell lines where samples permit. Although Vero cells have been the choice for isolation and propagation of PPRV, it is reported that B95a, an adherent cell line derived from Epstein-Barr virustransformed marmoset B-lymphoblastoid cells, is more sensitive and support better growth of PPRV lineage IV as compared to Vero cells. More recently, Vero cells expressing the SLAM receptor have been used as an effective alternative for isolation in cell culture. The fragility of morbillivirus virions generally renders techniques such as virus isolation redundant for routine diagnostic use, especially where sample quality is poor. Such

comparative sensitivity alongside being simple to perform and cheap to produce.

PPR, but a degree of cross protection existed between PPR and canine distemper. 30

open up avenues to design novel epitope based vaccines against PPR.

**3. Specimen collection, processing and shipment** 

the onset of clinical signs) may be diagnostic.

a. Conventional Methods of PPRV Diagnosis

**4. Laboratory diagnosis of PPR** 

Fig. 3. Genome of PPR virus

b. Antigenic and Immunogenic Epitopes:

Surface glycoproteins hemagglutinin (H) and fusion protein (F) of morbilliviruses are highly immmunogenic and helps in providing the immunity. PPRV is closely related to rinderpest virus (RPV). Antibodies against PPRV are both cross neutralizing and Cross protective. A vaccinia virus double recombinant expressing H and F glycoproteins of RPV has been shown to protect goats against PPR disease though the animals developed virusneutralizing antibodies only against the RPV and not against PPRV. Capripox recombinants expressing the H protein or the F protein of RPV or the F protein of PPRV conferred protection against PPR disease in goats, but without production of PPRV-neutralizing antibodies27 or PPRV antibodies detectable by ELISA (Berhe *et al*, 2003). These results suggested that cell-mediated immune responses could play a crucial role in protection. Goats immunized with a recombinant baculovirus expressing the H glycoprotein generated both humoral and cell-mediated immune responses.28 The responses generated against PPRV-H protein in the experimental goats are also RPV crossreactive suggesting that the H protein presented by the baculovirus recombinant 'resembles' the native protein present on PPRV.28

Lymphoproliferative responses were demonstrated in these animals against PPRV-H and RPV-H antigens 28. N-terminal T cell determinant and a C-terminal domain harboring potential T cell determinant(s) in goats were mapped. Though the sub-set of T cells (CD4+ and CD8+ T cells) in PBMC that responded to the recombinant protein fragments and the synthetic peptide could not be determined, this could potentially be a CD4+ helper T cell epitope, which has been shown to harbor an immunodominant H restricted epitope in

Surface glycoproteins hemagglutinin (H) and fusion protein (F) of morbilliviruses are highly immmunogenic and helps in providing the immunity. PPRV is closely related to rinderpest virus (RPV). Antibodies against PPRV are both cross neutralizing and Cross protective. A vaccinia virus double recombinant expressing H and F glycoproteins of RPV has been shown to protect goats against PPR disease though the animals developed virusneutralizing antibodies only against the RPV and not against PPRV. Capripox recombinants expressing the H protein or the F protein of RPV or the F protein of PPRV conferred protection against PPR disease in goats, but without production of PPRV-neutralizing antibodies27 or PPRV antibodies detectable by ELISA (Berhe *et al*, 2003). These results suggested that cell-mediated immune responses could play a crucial role in protection. Goats immunized with a recombinant baculovirus expressing the H glycoprotein generated both humoral and cell-mediated immune responses.28 The responses generated against PPRV-H protein in the experimental goats are also RPV crossreactive suggesting that the H protein presented by the baculovirus recombinant 'resembles' the native protein present on PPRV.28 Lymphoproliferative responses were demonstrated in these animals against PPRV-H and RPV-H antigens 28. N-terminal T cell determinant and a C-terminal domain harboring potential T cell determinant(s) in goats were mapped. Though the sub-set of T cells (CD4+ and CD8+ T cells) in PBMC that responded to the recombinant protein fragments and the synthetic peptide could not be determined, this could potentially be a CD4+ helper T cell epitope, which has been shown to harbor an immunodominant H restricted epitope in

Fig. 3. Genome of PPR virus

b. Antigenic and Immunogenic Epitopes:

mice28 .Identification of B- and T-cell epitopes on the protective antigens of PPRV would open up avenues to design novel epitope based vaccines against PPR.

Sheep and goats are unlikely to be infected more than once in their economic life12. Lambs or kids receiving colostrum from previously exposed or vaccinated with RP tissue culture vaccine were found to acquire a high level of maternal antibodies that persist for 3-4 months. The maternal antibodies were detectable up to 4 months using virus neutralization test compared to 3 month with competitive ELISA29. Measles vaccine did not protect against PPR, but a degree of cross protection existed between PPR and canine distemper. 30

## **3. Specimen collection, processing and shipment**

Before collecting or sending any samples from animals with a suspected foreign animal disease, the proper authorities should be contacted. Samples should only be sent under secure conditions and to authorized laboratories to prevent the spread of the disease. In live animals, swabs of ocular and nasal discharges, and debris from oral lesions should be collected; a spatula can be rubbed across the gum and inside the lips to collect samples from oral lesions. Whole, unclotted blood (in heparin or EDTA) should be taken for virus isolation and PCR. Biopsy samples of lymph nodes or spleen may also be useful. Samples for virus isolation should be collected during the acute stage of the disease, when clinical signs are present; whenever possible, these samples should be taken from animals with high fever and before the onset of diarrhea. At necropsy, samples can be collected from lymph nodes (particularly the mesenteric and mediastinal nodes), lungs, spleen, tonsils and affected sections of the intestinal tract (e.g. ileum and large intestine). These samples should be taken from euthanized or freshly dead animals. Samples for virus isolation should be transported chilled on ice. Similar samples should be collected in formalin for histopathology. Whenever possible, paired sera should be taken rather than single samples. However, in countries that are PPR-free, a single serum sample (taken at least a week after the onset of clinical signs) may be diagnostic.

## **4. Laboratory diagnosis of PPR**

a. Conventional Methods of PPRV Diagnosis

Conventional techniques such as the Agar Gel Immuno Diffusion (AGID) test are not routinely used for standard diagnosis as they lack sensitivity when compared to other assays. However, Haemagglutination tests (HA) and Haemagglutination Inhibition tests (HI) tests can be used for routine screening purposes in control programmes as they display comparative sensitivity alongside being simple to perform and cheap to produce.

Virus isolation in cell culture can be attempted with several different cell lines where samples permit. Although Vero cells have been the choice for isolation and propagation of PPRV, it is reported that B95a, an adherent cell line derived from Epstein-Barr virustransformed marmoset B-lymphoblastoid cells, is more sensitive and support better growth of PPRV lineage IV as compared to Vero cells. More recently, Vero cells expressing the SLAM receptor have been used as an effective alternative for isolation in cell culture. The fragility of morbillivirus virions generally renders techniques such as virus isolation redundant for routine diagnostic use, especially where sample quality is poor. Such

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV) 7

Molecular techniques such as reverse transcription polymerase chain reaction (RT- PCR) and nucleic acid hybridization are generally used. These genome based techniques are largely used because of their high specificity and sensitivity. However, modern one step real-time RT-PCR assays specific for PPRV and loop-mediated isothermal amplification techniques are more sensitive techniques for PPRV detection but do not allow genetic typing of positive samples. RT-PCR coupled with ELISA have also been used to increase the analytical sensitivity of visualization of RT-PCR products and to overcome the drawbacks of electrophoresis-based detection such as use of ethidium bromide, exposure to UV light etc. The assay is reported to detect viral RNA in infected tissue culture fluid with a virus titre as low as 0.01 TCID50/100 µL and has been reported as being 100 and 10,000 times more

b. Molecular Methods for PPRV Diagnosis

**diagnosis of PPR** 

percent, respectively 31 .

sensitive than the sandwich ELISA and RT-PCR, respectively. 31

**5. Potential and application of PCR technique for future advances in** 

Among the various techniques developed for the detection of PPRV, PCR technique has been the most popular and highly sensitive tool so far for diagnosis of PPR. The routine serological techniques and virus isolation are normally used to diagnose morbillivirus infection in samples submitted for laboratory diagnosis. However, such techniques are not suitable for use on decomposed tissue samples, the polymerase chain reaction (PCR), has proved invaluable for analysis of such poorly preserved field samples. The PCR test consists of repetitive cycles of DNA denaturation, primer annealing and extension by a DNA polymerase effectively doubling the target with each cycle leading, theoretically, to an exponential rise in DNA product. There placement of the polymerase now fragment by thermo-stable polymerase derived from Thermus aquaticus (Taq) has greatly improved the usefulness of PCR. These qualities have made the PCR one of the essential techniques in molecular biology today and it is starting to have a wide use in laboratory disease diagnosis. Since the genome of all Morbilliviruses consists of a single strand of RNA, it must be first copied into DNA, using reverse transcriptase, in a two-step reaction known as reverse transcription polymerase chain reaction (RT-PCR).Among the various techniques developed for the detection of PPRV, however, polymerase chain reaction (PCR) technique developed using F-gene primers has been the most popular tool so far, for diagnosis as well as molecular epidemiological studies. RT-PCR using phosphoprotein (P) universal primer and fusion (F) protein gene specific primer sets to detect and differentiate between PPR and RP are described by 8, 24, 32 developed a RT-PCR test, using phosphoprotein (P) gene and fusion protein(F) gene specific primer sets to detect and differentiate RPV and PPRV. They observed that RT-PCR was able to detect virus secretion in ocular swabs at four days post infection (PI) in experimentally infected goats, as compared to eight days PI by IcELISA. RT-PCR assay preclude the need for virus isolation and, because of the rapidity with which completely specific results could be obtained, the assay appeared to be the test of choice for PPRV detection. Relative specificity and sensitivity of F-gene based RT-PCR with sandwich-ELISA was 100 and 12.5

techniques are also considered to be time-consuming and cumbersome. Virus isolation does, however, play an important role from a research perspective.

ELISA tests using monoclonal antibodies are often used for serological diagnosis and antigen detection for diagnostic and screening purposes. For PPR antibodies detection, the competitive ELISA is the most suitable choice as it is sensitive, specific, reliable, and has a high diagnostic specificity (99.8%) and sensitivity (90.5%). Immunocapture ELISA (ICE) is a rapid, sensitive and virus specific test for PPRV antigen detection and it can differentiate between RPV and PPRV and has been reported to be more sensitive than the AGID test.

For rapid diagnosis to enable a swift implementation of control measures, further development and validation of pen-side tests such as the chromatographic strip test and the dot ELISA that can be performed without the need for equipments or technical expertise are highly desirable.


Table 1. Detail of conventional methods for the detection and confirmation of PPR

#### b. Molecular Methods for PPRV Diagnosis

6 Polymerase Chain Reaction

techniques are also considered to be time-consuming and cumbersome. Virus isolation does,

ELISA tests using monoclonal antibodies are often used for serological diagnosis and antigen detection for diagnostic and screening purposes. For PPR antibodies detection, the competitive ELISA is the most suitable choice as it is sensitive, specific, reliable, and has a high diagnostic specificity (99.8%) and sensitivity (90.5%). Immunocapture ELISA (ICE) is a rapid, sensitive and virus specific test for PPRV antigen detection and it can differentiate between RPV and PPRV and has been reported to be more sensitive than the AGID test.

For rapid diagnosis to enable a swift implementation of control measures, further development and validation of pen-side tests such as the chromatographic strip test and the dot ELISA that can be performed without the need for equipments or technical expertise are

**(Lab or Field)** 

CIEP Both Both

Lab Antigen

Both Both

cELISA Lab Antibody

Ic-ELISA Lab Antigen

**Feature Detected (Antigen or Antibody)** 

however, play an important role from a research perspective.

**Test Name Acronym Application** 

1 Agar gel immuno-diffusion AGID Both Both

3 Dot enzyme immunoassay -- Lab Antigen

IH staining

6 Immuno-filtration IF Lab Antigen 7 Latex agglutination tests LA Field Antigen 8 Virus isolation VI Lab Antigen

10 Novel sandwich ELISA sELISA Lab Antigen

Table 1. Detail of conventional methods for the detection and confirmation of PPR

HA and HI

highly desirable.

2 Counter Immunoelectrophoresis

sections

inhibition tests

(c-ELISA)

assay

4 Differential immuno-histochemical staining of tissue

5 Haemagglutination and Haemagglutination

9 Competitive enzyme-linked Immuno-sorbent assay

11 Immuno-capture enzymelinked immunosorbent

**Sr #** 

Molecular techniques such as reverse transcription polymerase chain reaction (RT- PCR) and nucleic acid hybridization are generally used. These genome based techniques are largely used because of their high specificity and sensitivity. However, modern one step real-time RT-PCR assays specific for PPRV and loop-mediated isothermal amplification techniques are more sensitive techniques for PPRV detection but do not allow genetic typing of positive samples. RT-PCR coupled with ELISA have also been used to increase the analytical sensitivity of visualization of RT-PCR products and to overcome the drawbacks of electrophoresis-based detection such as use of ethidium bromide, exposure to UV light etc. The assay is reported to detect viral RNA in infected tissue culture fluid with a virus titre as low as 0.01 TCID50/100 µL and has been reported as being 100 and 10,000 times more sensitive than the sandwich ELISA and RT-PCR, respectively. 31

#### **5. Potential and application of PCR technique for future advances in diagnosis of PPR**

Among the various techniques developed for the detection of PPRV, PCR technique has been the most popular and highly sensitive tool so far for diagnosis of PPR. The routine serological techniques and virus isolation are normally used to diagnose morbillivirus infection in samples submitted for laboratory diagnosis. However, such techniques are not suitable for use on decomposed tissue samples, the polymerase chain reaction (PCR), has proved invaluable for analysis of such poorly preserved field samples. The PCR test consists of repetitive cycles of DNA denaturation, primer annealing and extension by a DNA polymerase effectively doubling the target with each cycle leading, theoretically, to an exponential rise in DNA product. There placement of the polymerase now fragment by thermo-stable polymerase derived from Thermus aquaticus (Taq) has greatly improved the usefulness of PCR. These qualities have made the PCR one of the essential techniques in molecular biology today and it is starting to have a wide use in laboratory disease diagnosis. Since the genome of all Morbilliviruses consists of a single strand of RNA, it must be first copied into DNA, using reverse transcriptase, in a two-step reaction known as reverse transcription polymerase chain reaction (RT-PCR).Among the various techniques developed for the detection of PPRV, however, polymerase chain reaction (PCR) technique developed using F-gene primers has been the most popular tool so far, for diagnosis as well as molecular epidemiological studies. RT-PCR using phosphoprotein (P) universal primer and fusion (F) protein gene specific primer sets to detect and differentiate between PPR and RP are described by 8, 24, 32 developed a RT-PCR test, using phosphoprotein (P) gene and fusion protein(F) gene specific primer sets to detect and differentiate RPV and PPRV. They observed that RT-PCR was able to detect virus secretion in ocular swabs at four days post infection (PI) in experimentally infected goats, as compared to eight days PI by IcELISA. RT-PCR assay preclude the need for virus isolation and, because of the rapidity with which completely specific results could be obtained, the assay appeared to be the test of choice for PPRV detection. Relative specificity and sensitivity of F-gene based RT-PCR with sandwich-ELISA was 100 and 12.5 percent, respectively 31 .

Application of PCR in Diagnosis of Peste des Petits Ruminants Virus (PPRV) 9

[16] Diallo, A., Barrett, T., Barbron, M., Subbarao, S.M., Taylor, W.P., 1988. Differentiation of

[17] El-Yuguda, A., Chabiri, L., Adamu, F. & Baba, S. (2010). Peste des petits ruminants

[18] Saeed, I. K., Ali, Y. H., Khalafalla, A. I. & Rahman-Mahasin, E. A. (2010). Current

[19] Amjad, H., Qamar ul, I., Forsyth, M., Barrett, T. & Rossiter, P. B. (1996). Peste des petits [20] Bazarghani, T. T., Charkhkar, S., Doroudi, J. & Bani Hassan, E. (2006). A review on

[21] Barhoom, S., Hassan, W. & Mohammed, T. (2000). Peste des petits ruminants in sheep

[22] Housawi, F., Abu Elzein, E., Mohamed, G., Gameel, A., Al-Afaleq, A., Hagazi, A. & Al-

[23] Taylor, W. P. Abusaidy, S., Barret, T. (1990) The epidemiology of PPR in the sultanate of

[24] Barrett, T., C. Amarel-Doel, R.P. Kitching and A. Gusev, 1993a. Use of the polymerase

[25] Dowling, P.C., Blumberg, B.M., Menonna, J., Adamus, J.E., Cook, P., Crowley, J.C.,

[26] Walpita, P. (2004). An internal element of the measles virus antigenome promoter

[27] Romero, C.H., Barrett, T., Kitching, R.P., Bostock, C., Black, D.N. (1995) Protection of

[29] Libeau, G., A. Diallo, F. Colas and L. Gaerre, 1994. Rapid differential diagnosis of

[30] Gibbs, P.J.E., Taylor, W.P. Lawman, M.P. and Bryant, J. (1979) Classification of the peste

Vaccine 13 : 36-40 ruminants in goats in Pakistan. *Vet Rec* 139, 118–119. [28] Sinnathamby, G., G.J. Renukaradhya, M. Rajasekhar, R. Nayak, M.S. Shaila (2001)

modulates replication efficiency. Virus Research 100: 199-211.

*Infect Dis Vet Public Health* 53 (Suppl. 1), 17–18. Medline

Eastern Saudi Arabia. *Rev Elev Med Vet Pays Trop* 57, 31–34. http://osp.mans.edu.eg/elsawalhy/Inf-Dis/PPR.htm

with attenuated RP virus. Res. Vet. Sci. 27: 321-324.

animals. Rev. Sci. Tech. Off. Int. Epiz., 12: 865–72

rinderpest and peste des petits ruminants viruses using specic cDNA clones. J.

virus Experimental PPR (goat plague) in Goats and sheep. Canadian J. Vet. Res. 52,

situation of peste des petits ruminants (PPR) in the Sudan. *Trop Anim Health Prod* 

peste des petits ruminants (PPR) with special reference to PPR in Iran. *J Vet Med B* 

Bishr,B. (2004). Emergence of peste des petits ruminants virus in sheep and goats in

Oman. Vet. Micro. 22: 341-352.Taylor, W.P. (1979a) Protection of goats against PPR

chain reaction in differentiating rinderpest field virus and vaccine virus in the same

1986. (PPRV) infection among small ruminants slaughtered at the central abattoir,

goats against peste des petits ruminants with a recmbinant capripox viruses expressing the fusion and haemagglutinin protein genes of rinderpest virus.

Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of peste des petits ruminants virus: identification of a T cell

rinderpest and peste des petits ruminants using an immunocapture ELISA. Vet.

des petits ruminants virus as the fourth member of the genus Morbillivirus.

[15] Braide, V.B. (1981) Peste des petits ruminantss. World anim. Review.39: 25-28.

Virol. Methods 23, 127–136.

in Iraq. *Iraqi J Vet Sci* 13, 381–385.

Maiduguri, Nigeria.

Rec., 134: 300–4

determinant. Vaccine 19: 4816-4823.

Intervirology. 11: 268 – 274.

46-52.

42, 89–93.

## **6. Conclusion**

The conventional techniques are largely replaced by genome-based detection techniques for the diagnosis and confirmation of PPR virus. Molecular-biological techniques such as RT-PCR and nucleic acid hybridization are now in use. These genome based techniques are largely used because of their high specificity and sensitivity. However one step real-time RT-PCR assays specific for PPRV and loop-mediated isothermal amplification techniques are more sensitive techniques for PPRV detection.

## **7. References**


The conventional techniques are largely replaced by genome-based detection techniques for the diagnosis and confirmation of PPR virus. Molecular-biological techniques such as RT-PCR and nucleic acid hybridization are now in use. These genome based techniques are largely used because of their high specificity and sensitivity. However one step real-time RT-PCR assays specific for PPRV and loop-mediated isothermal amplification techniques

[1] Housawi, F., Abu Elzein, E., Mohamed, G., Gameel, A., Al-Afaleq, A., Hagazi, A. & Al-

[2] Abu Elzein, E.M.E., Hassanien, M.M., Alfaleg, A.I.A, Abd Elhadi, M.A., Housawi, F.M.T. (1990) Isolation of PPR virus from goats in Saudi Arabia. Vet. Rec., 127: 309-310. [3] Laurent, A. (1968) Aspects biologiques de la multiplication du virus de la peste des petits ruminants sur les cultures cellulaire. Rev. Elev. Méd. Vét. Pays trop. 21: 297-308. [4] Lefèvre, P.C. and Diallo, A. (1990) Peste des petites ruminants. Revue Scientifique Office

[5] Radostits OM, CC Gay, DC Blood and KW Hinchcliff, 2000. Veterinary Medicine. 9th Ed,

[6] Dhar P, BP Sreenivasa, T Barrett, M Corteyn, RP Singh and SK Bandyopadhyay, 2002.

[7] Shaila, M.S., Shamaki, D., Morag, A.F., Diallo, A., Goatley, L., Kitching, R.P. and Barrett,

[8] Forsyth, M.A. and T. Barrett, 1995. Evaluation of polymerase chain reaction for the

[9] Farooq, U., Q.M. khan and T. Barrett. (2008). Molecular Based Diagnosis of Rinderpest

[10] Albayrak, H and F.Alkan. (2009). PPR virus infection on sheep in blacksea region of

[11] Shaila, M.S., V. Purushothaman, D. Bhavasar, K. Venugopal and R.A. Venkatesan, 1989.

[12] Taylor, W.P. (1984). The distribution and epidemiology of peste des petits ruminants.

[13] Wamwayi, H.M, M. Fleming, T. Barrett. (1995). Characterisation of African isolates of

[14] Gargadennec, L. and A. Lalanne, 1942. La peste des petits ruminants. Bulletin des

Services Zoo Techniques et des Epizooties de I'Afrique Occidentale Francaise, 5:

Peste des petits ruminants of sheep in India. Vet. Rec., 125: 602

rinderpest virus. Veterinary Microbiology 44 (2–4): 151–163.

Recent epidemiology of peste des petits ruminants virus (PPRV). Vet Microbiol, 88:

T. (1996). Geographic distribution and epidemiology of peste des petits ruminants

detection and characterization of rinderpest and peste des petits ruminants viruses

and Peste Des Petits Ruminants Virus in Pakistan.international journal of

Turkey: Epidemiology and diagnosis by RT-PCR and virus isolation. Veterinary

Eastern Saudi Arabia. *Rev Elev Med Vet Pays Trop* 57, 31–34. http://osp.mans.edu.eg/elsawalhy/Inf-Dis/PPR.htm

of rinderpest virus. Vet. Microbiol. 41: 151-163.

for epidemiological studies. Virus Res. 39: 151–63

viruses Virus Res. 43: 149-153.

agriculture & biology. 10 (1): 93-96

Prey. Vet. Med., 2: 157-166.

research communications. 33 (3) 241-249.

WB Saunders Company Ltd, London, UK, pp: 563-565.

Bishr,B. (2004). Emergence of peste des petits ruminants virus in sheep and goats in

**6. Conclusion** 

**7. References** 

153-15

16–21

are more sensitive techniques for PPRV detection.


**2** 

*Thiverval-Grignon* 

*1France 2Slovenia* 

**Application of PCR-Based Methods to Dairy** 

*1UMR782 Génie et Microbiol. des Procédés Alimentaires INRA, AgroParisTech,* 

Christophe Monnet1 and Bojana Bogovič Matijašić2

**Products and to Non-Dairy Probiotic Products** 

*2Institute of Dairy Science and Probiotics, Biotechnical Faculty, University of Ljubljana* 

Many types of cheeses and fermented dairy products are produced throughout the world. They contain various types of bacteria and fungi. In many cases, their exact microbiological composition is not well known because the deliberately added microorganisms are only part of the final microbiota. These microorganisms contribute to the manufacturing of the product (aroma compound production, acidification, impact on texture, colour etc.). Occasionally, dairy products may also be contaminated by spoilage microorganisms and pathogens. PCR-based methods have many interesting applications for dairy products. They can be used to detect, identify and quantify either unwanted or beneficial microorganisms. They can also provide culture-independent microbial fingerprints. Another application is the detection or the quantification of specific genes or groups of genes, such as those involved in the generation of the functional properties. In addition, the abundance of specific mRNA transcripts can be quantified by reverse transcription real-time PCR, which is very useful for a better understanding of the physiology and activity of the

Probiotics have been defined as ''live microorganisms that, when administered in adequate amounts, confer a health benefit on the host'' (FAO/WHO, 2002). The deficiencies of the quality of probiotic products in terms of too-low numbers or the absence of labelled species are commonly observed. The facts that probiotic functionality is a strain specific trait and that several probiotic strains have very similar phenotypic properties dictate the need for more powerful and rapid methods than conventional cultivation-based methods which have several disadvantages and very limited selectivity. The use of PCR based methods especially

Conventional PCR, combined with gel electrophoresis, has been successfully used for the genus-, species- or strain-specific determination of the presence of probiotic organisms in the products or in the biological samples (faeces). An important feature of probiotics, however, is the viability which is a prerequisite for the probiotic functionality. In this regard, a common DNA-based quantification by real-time PCR is not very useful for quantification purposes since the DNA released from dead or damaged cells also

**1. Introduction** 

microorganisms present in dairy products.

has greatly expanded during recent years.


## **Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products**

Christophe Monnet1 and Bojana Bogovič Matijašić2 *1UMR782 Génie et Microbiol. des Procédés Alimentaires INRA, AgroParisTech, Thiverval-Grignon 2Institute of Dairy Science and Probiotics, Biotechnical Faculty, University of Ljubljana 1France 2Slovenia* 

## **1. Introduction**

10 Polymerase Chain Reaction

[31] Abubakar M, HA Khan, MJ Arshed, M Hussain M and Ali Q, 2011. Peste des petits

[32] Couacy-Hymann, E., Roger, F., Hurard, C., Guillou, J.P., Libeau, G., Diallo,A., 2002.

chain reaction assay. J. Virol.Methods 100, 17–25.

Rum Res, 96: 1–10.

ruminants (PPR): Disease appraisal with global and Pakistan perspective. Small

Rapid andsensitive detection of peste despetitsruminants virus by a polymerase

Many types of cheeses and fermented dairy products are produced throughout the world. They contain various types of bacteria and fungi. In many cases, their exact microbiological composition is not well known because the deliberately added microorganisms are only part of the final microbiota. These microorganisms contribute to the manufacturing of the product (aroma compound production, acidification, impact on texture, colour etc.). Occasionally, dairy products may also be contaminated by spoilage microorganisms and pathogens. PCR-based methods have many interesting applications for dairy products. They can be used to detect, identify and quantify either unwanted or beneficial microorganisms. They can also provide culture-independent microbial fingerprints. Another application is the detection or the quantification of specific genes or groups of genes, such as those involved in the generation of the functional properties. In addition, the abundance of specific mRNA transcripts can be quantified by reverse transcription real-time PCR, which is very useful for a better understanding of the physiology and activity of the microorganisms present in dairy products.

Probiotics have been defined as ''live microorganisms that, when administered in adequate amounts, confer a health benefit on the host'' (FAO/WHO, 2002). The deficiencies of the quality of probiotic products in terms of too-low numbers or the absence of labelled species are commonly observed. The facts that probiotic functionality is a strain specific trait and that several probiotic strains have very similar phenotypic properties dictate the need for more powerful and rapid methods than conventional cultivation-based methods which have several disadvantages and very limited selectivity. The use of PCR based methods especially has greatly expanded during recent years.

Conventional PCR, combined with gel electrophoresis, has been successfully used for the genus-, species- or strain-specific determination of the presence of probiotic organisms in the products or in the biological samples (faeces). An important feature of probiotics, however, is the viability which is a prerequisite for the probiotic functionality. In this regard, a common DNA-based quantification by real-time PCR is not very useful for quantification purposes since the DNA released from dead or damaged cells also

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 13

components from the cheese matrix (Rademaker et al., 2005). After their recovery, the cells are disrupted and DNA is purified from the lysed cells. Cell disruption may involve beadbeating, addition of lytic enzymes such as lysozyme, lyticase, mutanolysin or lysostaphine, addition chemical compounds, or a combination of these treatments. After cell lysis, purification of DNA may be performed by classical phenol/chloroform extraction. Phenol is a strong denaturant of proteins that leads to the partition of the proteins into the organic phase and at the interface of the organic and aqueous phases. Procedures avoiding the use of phenol, which is a toxic chemical, have been described. For example, Coppola et al. (2001), Rademaker et al. (2006), and Moschetti et al. (2001) used a commercial kit containing a synthetic resin which removes the cell lysis products that interfere with the PCR amplification. Baruzzi et al. (2005), Trmcic et al. (2008), and Furet et al. (2004) used a commercial kit in which proteins are eliminated by the use of a protein precipitation solution. Column-based or DNA-binding matrix purification methods have also been used (Rudi et al., 2005; Parayre et al., 2007; Zago et al., 2009; Le Dréan et al., 2010), sometimes as a final purification step after phenol/chloroform extraction (Stevens and Jaykus, 2004; Lopez-Enriquez et al., 2007). Separation of cells from the food matrix simplifies the subsequent steps of DNA extraction because most undesirable compounds such as matrix-associated reaction inhibitors are eliminated at the first step of extraction. In addition, large amounts of cheeses (for example more than 10 grams) can be processed in each extraction, which yields a large final amount of DNA. This is important in dairy products containing a low concentration of cells, for example at the initial steps of cheese-manufacturing, where direct DNA extraction is in most cases not possible. Furthermore, the separation of cells from the dairy food matrix eliminates in some cases the need for cultural enrichment prior to detection of pathogens. In contrast to RNA, it is unlikely that there is a large quantitative or qualitative change of the DNA present inside of the cells during the separation of the cells from the dairy food matrix. One of the drawbacks of the DNA extraction methods based on cell separation is that some DNA may be lost during the separation, due to cell lysis,

In direct DNA extraction procedures (McKillip et al., 2000; Duthoit et al., 2003; Feurer et al., 2004a; Feurer et al., 2004b; Callon et al., 2006; Monnet et al., 2006; Delbes et al., 2007; Masoud et al., 2011), the cheese samples are first homogenised in a liquid solution by a method involving bead-beating, a mortar and pestle or other mechanical treatments. Efficient treatments of casein degradation and cell lysis, followed by phenol/chloroform extractions, are then needed to remove most contaminating compounds. Contaminating RNA can be removed by a treatment with RNase. Subsequent alcohol precipitation or column-based purification is then used to further purify and/to concentrate the DNA. Carraro et al. (2011) used a column-based purification method for direct extraction of DNA from cheese samples.

Reverse transcription PCR analyses of RNA may be used in microbial diversity evaluation or for the detection or quantification of mRNA transcripts. Like for DNA, there are two types of extraction methods for RNA from dairy products, either direct extractions, or extractions after prior separation of the cells from the food matrix. The amount of RNA that can be recovered from dairy products is in general higher than for DNA. Indeed, the RNA content of microbial cells is higher than DNA. For example, in *Escherichia (E.) coli*, Bremer and Dennis (1996) reported a concentration varying from 7.6 to 18.3 µg of DNA per 109 cells,

especially for yeasts and Gram-negative strains.

**2.1.2 RNA extraction** 

contributes to the results of analysis. One of the alternative approaches for selective detection of viable bacteria is the treatment of the samples with DNA-intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) that they can penetrate only into membrane-compromised bacterial cells or dead cells where they are by photo-activation covalently linked to DNA and prevent it from PCR amplification.

## **2. Application of PCR-based methods to dairy products**

#### **2.1 Nucleic acid extraction from dairy products**

## **2.1.1 DNA extraction**

Most of the DNA present in cheeses and other fermented dairy products is from the microorganisms that are present. This DNA has to be purified before performing PCR analyses. Dairy products are compositionally complex and there are several reports of dairy matrix-associated PCR inhibition (Niederhauser et al., 1992; Rossen et al., 1992; Herman and Deridder, 1993). One can distinguish two types of DNA extraction methods from dairy products: either direct extractions, or extractions after prior separation of the cells from the food matrix. In all cases, the DNA extraction protocols have to be adapted to the cheese under investigation.

Most methods described in the literature involve prior separation of the cells (Allmann et al., 1995; Herman et al., 1997; Serpe et al., 1999; Torriani et al., 1999; McKillip et al., 2000; Coppola et al., 2001; Ogier et al., 2002; Randazzo et al., 2002; Ercolini et al., 2003; Furet et al., 2004; Ogier et al., 2004; Baruzzi et al., 2005; Rudi et al., 2005; Rademaker et al., 2006; El-Baradei et al., 2007; Lopez-Enriquez et al., 2007; Parayre et al., 2007; Rossmanith et al., 2007; Trmcic et al., 2008; Van Hoorde et al., 2008; Alegría et al., 2009; Dolci et al., 2009; Zago et al., 2009; Le Dréan et al., 2010; Mounier et al., 2010). The recovery of cells from milks or fermented milks is easier to perform than from cheeses. In most cases, homogenisation of the samples and casein solubilisation is done in a sodium citrate solution, using a mechanical blender or glass beads, and the cells are recovered subsequently by centrifugation. Part of the fat is eliminated at this step because it forms a layer at the surface after centrifugation. Serpe et al. (1999) homogenised cheese samples in a Tris-HCl buffer containing the non-anionic detergent Tween 20 to emulsify the fat fraction of the sample. Depending on the type of cheese and the ripening stage, the cell pellet obtained after centrifugation may contain a large amount of caseins. These may be removed by washing the cell pellet with a buffer once or several times, and compounds such as Triton X-100 may be added for a better removal (Baruzzi et al., 2005). Caseins may also be eliminated by pronase digestion before recovery of the cells by centrifugation (Allmann et al., 1995; Furet et al., 2004; Ogier et al., 2004; Flórez and Mayo, 2006). It has been reported that the recovery of the bacterial cells may be improved by addition of polyethylene glycol during the homogenisation step (Stevens and Jaykus, 2004). A matrix lysis buffer containing urea and SDS combined with an homogenisation in a Stomacher laboratory blender has been used by Rossmanith et al. (2007) to recover Gram-positive cells from various food samples, including cheeses. In the procedure described by Herman et al. (1997) and Bonetta et al. (2008), bacterial cells are recovered from homogenised cheese by centrifugation after chemical extraction of fat and proteins. At the surface of some cheeses, for example smear-ripened cheeses, there is a high microbial density, and therefore, a simple surface scraping is sometimes sufficient to recover the microbial cells without need to eliminate the components from the cheese matrix (Rademaker et al., 2005). After their recovery, the cells are disrupted and DNA is purified from the lysed cells. Cell disruption may involve beadbeating, addition of lytic enzymes such as lysozyme, lyticase, mutanolysin or lysostaphine, addition chemical compounds, or a combination of these treatments. After cell lysis, purification of DNA may be performed by classical phenol/chloroform extraction. Phenol is a strong denaturant of proteins that leads to the partition of the proteins into the organic phase and at the interface of the organic and aqueous phases. Procedures avoiding the use of phenol, which is a toxic chemical, have been described. For example, Coppola et al. (2001), Rademaker et al. (2006), and Moschetti et al. (2001) used a commercial kit containing a synthetic resin which removes the cell lysis products that interfere with the PCR amplification. Baruzzi et al. (2005), Trmcic et al. (2008), and Furet et al. (2004) used a commercial kit in which proteins are eliminated by the use of a protein precipitation solution. Column-based or DNA-binding matrix purification methods have also been used (Rudi et al., 2005; Parayre et al., 2007; Zago et al., 2009; Le Dréan et al., 2010), sometimes as a final purification step after phenol/chloroform extraction (Stevens and Jaykus, 2004; Lopez-Enriquez et al., 2007). Separation of cells from the food matrix simplifies the subsequent steps of DNA extraction because most undesirable compounds such as matrix-associated reaction inhibitors are eliminated at the first step of extraction. In addition, large amounts of cheeses (for example more than 10 grams) can be processed in each extraction, which yields a large final amount of DNA. This is important in dairy products containing a low concentration of cells, for example at the initial steps of cheese-manufacturing, where direct DNA extraction is in most cases not possible. Furthermore, the separation of cells from the dairy food matrix eliminates in some cases the need for cultural enrichment prior to detection of pathogens. In contrast to RNA, it is unlikely that there is a large quantitative or qualitative change of the DNA present inside of the cells during the separation of the cells from the dairy food matrix. One of the drawbacks of the DNA extraction methods based on cell separation is that some DNA may be lost during the separation, due to cell lysis, especially for yeasts and Gram-negative strains.

In direct DNA extraction procedures (McKillip et al., 2000; Duthoit et al., 2003; Feurer et al., 2004a; Feurer et al., 2004b; Callon et al., 2006; Monnet et al., 2006; Delbes et al., 2007; Masoud et al., 2011), the cheese samples are first homogenised in a liquid solution by a method involving bead-beating, a mortar and pestle or other mechanical treatments. Efficient treatments of casein degradation and cell lysis, followed by phenol/chloroform extractions, are then needed to remove most contaminating compounds. Contaminating RNA can be removed by a treatment with RNase. Subsequent alcohol precipitation or column-based purification is then used to further purify and/to concentrate the DNA. Carraro et al. (2011) used a column-based purification method for direct extraction of DNA from cheese samples.

#### **2.1.2 RNA extraction**

12 Polymerase Chain Reaction

contributes to the results of analysis. One of the alternative approaches for selective detection of viable bacteria is the treatment of the samples with DNA-intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) that they can penetrate only into membrane-compromised bacterial cells or dead cells where they are by

Most of the DNA present in cheeses and other fermented dairy products is from the microorganisms that are present. This DNA has to be purified before performing PCR analyses. Dairy products are compositionally complex and there are several reports of dairy matrix-associated PCR inhibition (Niederhauser et al., 1992; Rossen et al., 1992; Herman and Deridder, 1993). One can distinguish two types of DNA extraction methods from dairy products: either direct extractions, or extractions after prior separation of the cells from the food matrix. In all cases, the DNA extraction protocols have to be adapted to the cheese

Most methods described in the literature involve prior separation of the cells (Allmann et al., 1995; Herman et al., 1997; Serpe et al., 1999; Torriani et al., 1999; McKillip et al., 2000; Coppola et al., 2001; Ogier et al., 2002; Randazzo et al., 2002; Ercolini et al., 2003; Furet et al., 2004; Ogier et al., 2004; Baruzzi et al., 2005; Rudi et al., 2005; Rademaker et al., 2006; El-Baradei et al., 2007; Lopez-Enriquez et al., 2007; Parayre et al., 2007; Rossmanith et al., 2007; Trmcic et al., 2008; Van Hoorde et al., 2008; Alegría et al., 2009; Dolci et al., 2009; Zago et al., 2009; Le Dréan et al., 2010; Mounier et al., 2010). The recovery of cells from milks or fermented milks is easier to perform than from cheeses. In most cases, homogenisation of the samples and casein solubilisation is done in a sodium citrate solution, using a mechanical blender or glass beads, and the cells are recovered subsequently by centrifugation. Part of the fat is eliminated at this step because it forms a layer at the surface after centrifugation. Serpe et al. (1999) homogenised cheese samples in a Tris-HCl buffer containing the non-anionic detergent Tween 20 to emulsify the fat fraction of the sample. Depending on the type of cheese and the ripening stage, the cell pellet obtained after centrifugation may contain a large amount of caseins. These may be removed by washing the cell pellet with a buffer once or several times, and compounds such as Triton X-100 may be added for a better removal (Baruzzi et al., 2005). Caseins may also be eliminated by pronase digestion before recovery of the cells by centrifugation (Allmann et al., 1995; Furet et al., 2004; Ogier et al., 2004; Flórez and Mayo, 2006). It has been reported that the recovery of the bacterial cells may be improved by addition of polyethylene glycol during the homogenisation step (Stevens and Jaykus, 2004). A matrix lysis buffer containing urea and SDS combined with an homogenisation in a Stomacher laboratory blender has been used by Rossmanith et al. (2007) to recover Gram-positive cells from various food samples, including cheeses. In the procedure described by Herman et al. (1997) and Bonetta et al. (2008), bacterial cells are recovered from homogenised cheese by centrifugation after chemical extraction of fat and proteins. At the surface of some cheeses, for example smear-ripened cheeses, there is a high microbial density, and therefore, a simple surface scraping is sometimes sufficient to recover the microbial cells without need to eliminate the

photo-activation covalently linked to DNA and prevent it from PCR amplification.

**2. Application of PCR-based methods to dairy products** 

**2.1 Nucleic acid extraction from dairy products** 

**2.1.1 DNA extraction** 

under investigation.

Reverse transcription PCR analyses of RNA may be used in microbial diversity evaluation or for the detection or quantification of mRNA transcripts. Like for DNA, there are two types of extraction methods for RNA from dairy products, either direct extractions, or extractions after prior separation of the cells from the food matrix. The amount of RNA that can be recovered from dairy products is in general higher than for DNA. Indeed, the RNA content of microbial cells is higher than DNA. For example, in *Escherichia (E.) coli*, Bremer and Dennis (1996) reported a concentration varying from 7.6 to 18.3 µg of DNA per 109 cells,

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 15

Trmcic et al., 2011). In the method described by Monnet et al. (2008), the cellular processes are stopped at the very beginning of the procedure, by addition of a guanidinium thiocyanate-phenol-chloroform solution to the cheese sample, and bead-beating is immediately performed. The reagent also inactivates RNases that may be present. At this step, the samples can be kept several weeks at -80 °C without any decrease of RNA integrity, which is not possible when the cheese samples are frozen before the RNA extraction. It was found that the amount of cheese sample should not exceed 100 mg per ml of reagent, as a higher ratio affects the quality and quantity of the purified RNA. The fat, caseins and DNA are removed after recovery of the aqueous phase which is formed after addition of chloroform. Subsequent acidic phenol-chloroform extraction and column-based purification is then performed to get RNA extracts suitable for reverse transcription PCR analyses and which can be stored several months at -80 °C. Use of 7-ml bead-beating tubes allows the processing of 500 mg samples of cheese (Trmcic et al., 2011). In addition, several samples may be pooled and concentrated during the column-based purification step, which allows higher amounts of RNA to be recovered. With this procedure, sufficient amounts of RNA could be obtained for analysing gene expression of a *Lactococcus (L.) lactis* strain whose concentration was about 108 CFU per gram of cheese, with a corresponding RNA extraction

Fig. 1. RNA quality assessment with the Agilent Bioanalyzer: electrophoregrams of RNA preparations from various commercial smear-ripened cheeses using the method described by Monnet et al. (2008). 16S and 23S rRNA are from bacterial origin, and 18S and 26S rRNA are from fungi. Cheese B contains more RNA from fungi than cheeses A and C, and shows a

16S

A

B

C

16S

18S

Time (s)

23S 16S

18S

23S

18S

23S

26S

26S

26S

Fluorescence

The quality of the RNA samples has to be assessed. Absence of contaminating DNA can be checked by performing PCR amplifications with controls in which reverse transcription has not been performed. RNA concentration can be measured with a spectrophotometer at 260 nm or with a fluorometer after addition of fluorescent dyes. The RNA integrity is evaluated by gel electrophoresis or by automated capillary-based electrophoresis (e.g. 2100 Bioanalyzer equipment, Agilent). RNA is mostly constituted of ribosomal RNA (rRNA), and the sharpness of the small (16S or 18S) and large (23S or 26S) rRNA subunit bands is

yield of 4.9 x 10-6 ng RNA per CFU.

higher overall RNA integrity.

and from 20 to 211 µg of RNA per 109 cells, depending on the growth rates. Messenger RNA (mRNA) accounts for only 1-5% of the total cellular RNA. Compared to DNA, RNA is relatively unstable. This is largely due to the presence of ribonucleases (RNases), which break down RNA molecules. RNases are very stable enzymes and are difficult to inactivate. They can be present in the sample or introduced by contamination during RNA handling.

RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al., 2002; Bleve et al., 2003; Sanchez et al., 2006; Bogovic Matijasic et al., 2007; Smeianov et al., 2007; Makhzami et al., 2008; Rantsiou et al., 2008a; Rantsiou et al., 2008b; Ulvé et al., 2008; Duquenne et al., 2010; Falentin et al., 2010; Cretenet et al., 2011; La Gioia et al., 2011; Masoud et al., 2011; Rossi et al., 2011; Taïbi et al., 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above). It is unlikely that the abundance of ribosomal RNA is modified during the cell separation procedure, but changes may occur with mRNA transcripts. Indeed, steady-state transcript levels are a result of both RNA synthesis and degradation. The mean half-life of *E. coli* mRNA measured by Selinger et al. (2003) was 6.8 min. It is likely that mRNA synthesis and degradation occurs also during the separation of the cells from the food matrix. This is why all treatments before the complete inactivation of cellular processes should be as short as possible. Ulvé et al. (2008) separated bacterial cells from cheeses by homogenisation in a citrate solution at a temperature of +4 °C, and extracted RNA using a column-based purification method after disruption of the cells by bead-beating. This method was compared to a direct RNA extraction, by measurement of the transcript abundance of 29 genes (Monnet et al., 2008). For most genes, there was no difference, but a higher level was measured for genes which expression is known to be modified by heat, acid, or osmotic stresses. Different methods of bacterial cell disruption were tested by Ablain et al. (2009) for the extraction of *Staphylococcus (S.) aureus* DNA and RNA. The best results were obtained with a combination of lysostaphin treatment and bead-beating. The cell pellets recovered from Camembert cheeses were treated with Chelex beads to remove contaminating compounds that may interfere in subsequent PCR analyses. *Propionibacterium (P.) freundereichii*, a species involved in Emmental cheese ripening, has a thick cell wall surrounded with capsular exopolysaccharides. For an efficient lysis of *P. freundereichii* cells recovered from cheeses, Falentin et al. (2010) used a combination of lysozyme treatment, bead-beating and phenol-chloroform extraction. Sanchez et al. (2006) recovered lactic acid bacteria cells from milk cultures after dispersion of caseins with EDTA, and extracted RNA using guanidinium thiocyanate-phenol-chloroform (commercial TRIzol reagent), a reagent that inactivates cellular processes and allows separation of RNA from DNA and proteins (Chomczynski and Sacchi, 1987). Duquenne et al. (2010) also used this type of extraction, after disruption of the cells by bead-beating. Bacterial cells may also be separated from cheese matrices using a Nycodenz gradient (Makhzami et al., 2008). In order to limit the changes in mRNA transcript composition inside of the cells during their separation from the dairy food matrix, Taïbi et al. (2011) added to the samples a stopping solution consisting of a mixture of phenol and ethanol. Smeianov et al. (2007) added the commercial reagent RNAprotect and rifampin, an antibiotic that suppresses the initiation of RNA synthesis, during the recovery of *Lactobacillus (Lb.) helveticus* cells from milk cultures.

So far, only a few studies have involved direct RNA extraction procedures from dairy products (Duthoit et al., 2005; Bonaiti et al., 2006; Monnet et al., 2008; Carraro et al., 2011;

and from 20 to 211 µg of RNA per 109 cells, depending on the growth rates. Messenger RNA (mRNA) accounts for only 1-5% of the total cellular RNA. Compared to DNA, RNA is relatively unstable. This is largely due to the presence of ribonucleases (RNases), which break down RNA molecules. RNases are very stable enzymes and are difficult to inactivate. They can be present in the sample or introduced by contamination during RNA handling. RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al., 2002; Bleve et al., 2003; Sanchez et al., 2006; Bogovic Matijasic et al., 2007; Smeianov et al., 2007; Makhzami et al., 2008; Rantsiou et al., 2008a; Rantsiou et al., 2008b; Ulvé et al., 2008; Duquenne et al., 2010; Falentin et al., 2010; Cretenet et al., 2011; La Gioia et al., 2011; Masoud et al., 2011; Rossi et al., 2011; Taïbi et al., 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above). It is unlikely that the abundance of ribosomal RNA is modified during the cell separation procedure, but changes may occur with mRNA transcripts. Indeed, steady-state transcript levels are a result of both RNA synthesis and degradation. The mean half-life of *E. coli* mRNA measured by Selinger et al. (2003) was 6.8 min. It is likely that mRNA synthesis and degradation occurs also during the separation of the cells from the food matrix. This is why all treatments before the complete inactivation of cellular processes should be as short as possible. Ulvé et al. (2008) separated bacterial cells from cheeses by homogenisation in a citrate solution at a temperature of +4 °C, and extracted RNA using a column-based purification method after disruption of the cells by bead-beating. This method was compared to a direct RNA extraction, by measurement of the transcript abundance of 29 genes (Monnet et al., 2008). For most genes, there was no difference, but a higher level was measured for genes which expression is known to be modified by heat, acid, or osmotic stresses. Different methods of bacterial cell disruption were tested by Ablain et al. (2009) for the extraction of *Staphylococcus (S.) aureus* DNA and RNA. The best results were obtained with a combination of lysostaphin treatment and bead-beating. The cell pellets recovered from Camembert cheeses were treated with Chelex beads to remove contaminating compounds that may interfere in subsequent PCR analyses. *Propionibacterium (P.) freundereichii*, a species involved in Emmental cheese ripening, has a thick cell wall surrounded with capsular exopolysaccharides. For an efficient lysis of *P. freundereichii* cells recovered from cheeses, Falentin et al. (2010) used a combination of lysozyme treatment, bead-beating and phenol-chloroform extraction. Sanchez et al. (2006) recovered lactic acid bacteria cells from milk cultures after dispersion of caseins with EDTA, and extracted RNA using guanidinium thiocyanate-phenol-chloroform (commercial TRIzol reagent), a reagent that inactivates cellular processes and allows separation of RNA from DNA and proteins (Chomczynski and Sacchi, 1987). Duquenne et al. (2010) also used this type of extraction, after disruption of the cells by bead-beating. Bacterial cells may also be separated from cheese matrices using a Nycodenz gradient (Makhzami et al., 2008). In order to limit the changes in mRNA transcript composition inside of the cells during their separation from the dairy food matrix, Taïbi et al. (2011) added to the samples a stopping solution consisting of a mixture of phenol and ethanol. Smeianov et al. (2007) added the commercial reagent RNAprotect and rifampin, an antibiotic that suppresses the initiation of RNA synthesis, during the recovery of

*Lactobacillus (Lb.) helveticus* cells from milk cultures.

So far, only a few studies have involved direct RNA extraction procedures from dairy products (Duthoit et al., 2005; Bonaiti et al., 2006; Monnet et al., 2008; Carraro et al., 2011; Trmcic et al., 2011). In the method described by Monnet et al. (2008), the cellular processes are stopped at the very beginning of the procedure, by addition of a guanidinium thiocyanate-phenol-chloroform solution to the cheese sample, and bead-beating is immediately performed. The reagent also inactivates RNases that may be present. At this step, the samples can be kept several weeks at -80 °C without any decrease of RNA integrity, which is not possible when the cheese samples are frozen before the RNA extraction. It was found that the amount of cheese sample should not exceed 100 mg per ml of reagent, as a higher ratio affects the quality and quantity of the purified RNA. The fat, caseins and DNA are removed after recovery of the aqueous phase which is formed after addition of chloroform. Subsequent acidic phenol-chloroform extraction and column-based purification is then performed to get RNA extracts suitable for reverse transcription PCR analyses and which can be stored several months at -80 °C. Use of 7-ml bead-beating tubes allows the processing of 500 mg samples of cheese (Trmcic et al., 2011). In addition, several samples may be pooled and concentrated during the column-based purification step, which allows higher amounts of RNA to be recovered. With this procedure, sufficient amounts of RNA could be obtained for analysing gene expression of a *Lactococcus (L.) lactis* strain whose concentration was about 108 CFU per gram of cheese, with a corresponding RNA extraction yield of 4.9 x 10-6 ng RNA per CFU.

Fig. 1. RNA quality assessment with the Agilent Bioanalyzer: electrophoregrams of RNA preparations from various commercial smear-ripened cheeses using the method described by Monnet et al. (2008). 16S and 23S rRNA are from bacterial origin, and 18S and 26S rRNA are from fungi. Cheese B contains more RNA from fungi than cheeses A and C, and shows a higher overall RNA integrity.

The quality of the RNA samples has to be assessed. Absence of contaminating DNA can be checked by performing PCR amplifications with controls in which reverse transcription has not been performed. RNA concentration can be measured with a spectrophotometer at 260 nm or with a fluorometer after addition of fluorescent dyes. The RNA integrity is evaluated by gel electrophoresis or by automated capillary-based electrophoresis (e.g. 2100 Bioanalyzer equipment, Agilent). RNA is mostly constituted of ribosomal RNA (rRNA), and the sharpness of the small (16S or 18S) and large (23S or 26S) rRNA subunit bands is

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 17

widely used in studies of the bacterial diversity of dairy products. Several distinct amplicons may be produced with some strains, due to differences in sequences of the rRNA

In fungi, the internal transcribed spacer (ITS) is a region located between the 18S rRNA and 26S rRNA genes. It includes the 5.8S rRNA gene that splits the ITS into two parts: ITS1 and ITS2. The 18S, 5.8S, 26S and 5S rRNA sequences form up to hundreds of tandem repeats. The ITS region undergoes a faster rate of evolution than rRNA but its sequence remains homogenous within a species. The ITS2 region has been chosen as target for the study of the fungal biodiversity of smear-ripened cheeses (Mounier et al., 2010), and the ITS1 region for the study of the fungal diversity in cow, goat and ewe milk (Delavenne et al., 2011). Primers targeting regions of the 26S rRNA (Feurer et al., 2004b; Flórez and Mayo, 2006; Bonetta et al., 2008; Alegría et al., 2009; Dolci et al., 2009; Mounier et al., 2009) and the 18S rRNA (Callon et al., 2006; Arteau et al., 2010) were chosen to investigate the dominant yeast microflora of

Housekeeping genes are less used than rRNA in molecular studies of microbial diversity of dairy products. This is due to a lower availability in sequence databases. However, this may change in the near future, due to the rapid increase of the number of sequenced genomes. The *rpoB* gene, encoding the RNA polymerase beta subunit has been used as a target for PCR-DGGE analysis to follow lactic acid bacterial population dynamics in cheeses (Rantsiou

Defined groups of microorganisms may be studied by amplification of specific targets, either by PCR or by real-time PCR. In the latter case, quantitative data can be obtained. The primers have to be designed so that amplification occurs only from DNA of the group of interest. As for PCR-based methods of microbial diversity evaluation, rRNA sequences are frequently used as target and the specificity may be evaluated *in silico* by comparing the rRNA sequences of the group of interest to that of other microorganisms that are present in the same habitat. A high level of specificity is achieved when there is a large sequence difference with non-target microorganisms for one or both of the PCR primers. Presence of mismatches near the 3' of the primers ensures a better specificity than at the 5' end. In addition, absence, or presence of only one or two G or C residues in the last five nucleotides at the 3' end of primers, makes them less likely to hybridise transiently and to be available for non-specific extension by the DNA polymerase (Bustin, 2000). *Corynebacterium casei* cells could be quantified in cheeses by real-time PCR using a couple of primers targeting the V6 region of the 16S rRNA gene (Monnet et al., 2006). The assay was specific, as no amplification occurred with DNA from other *Corynebacterium* species present in cheeses. Primers targeting 16S rRNA genes were also used for the quantification of *Carnobacterium* cells in cheeses (Cailliez-Grimal et al., 2005), of *L. lactis* subsp. *cremoris* in fermented milks (Grattepanche et al., 2005), of *Streptococcus (Str.) thermophilus* and lactobacilli in fermented milks (Furet et al., 2004), of thermophilic bacilli in milk powder (Rueckert et al., 2005) and of bacterial species that can develop during the cold storage of milk (Rasolofo et al., 2010). Primers targeting the 16S-23S-spacer region were used for the specific detection of *Clostridium tyrobutyricum* in semi-soft and hard cheeses (Herman et al., 1997) and for the quantification of *Listeria (List.) monocytogenes* in foods, including fresh and ripened cheeses

**2.2.2 Amplification targets for specific microbial groups** 

copies.

several types of cheeses.

et al., 2004).

indicative of the global degree of RNA integrity. From the 2100 Bioanalyzer electrophoresis profile, a value, named RIN (RNA Integrity Number), is calculated. A RIN value of 10 corresponds to apparently intact material. RIN calculations can be done with either eukaryotic or prokaryotic RNA, but not when both types of RNA are present in the same sample, which would be the case for RNA samples from numerous types of cheeses. Examples of RNA electrophoregrams of RNA preparations from cheese samples are shown in Figure 1. During the ripening or storage of cheeses, some microbial populations may decline, for example by autolysis. This has a detrimental effect on RNA integrity and, in consequence, a poor RNA integrity level is not necessarily due to an inadequate sampling or RNA extraction procedure.

## **2.2 Amplification targets**

All PCR analyses rely on amplification of DNA target sequences. Concerning PCR applications to dairy products, one can distinguish targets used for PCR-based microbial diversity evaluation, and targets for PCR analysis of specific microbial groups.

## **2.2.1 Amplification targets for microbial diversity evaluation methods**

In methods of microbial diversity evaluation involving PCR, the amplification target is a sequence which has to be present in a large part of the bacterial or fungal population. The sequence variations allow the subsequent differentiation of the generated amplicons. In most cases, these techniques involve amplification of ribosomal RNA or housekeeping genes. In both prokaryotes and eukaryotes, rRNA genes usually show a high sequence homogeneity within a species (Liao, 1999), which explains why they are widely used in species identification and makes them a good target in molecular microbial diversity evaluation methods.

Bacterial 16S, 23S and 5S rRNA genes are organised into a co-transcribed operon. The typical length of theses genes is ~2900 bp (23S), ~1500 bp (16S) and ~120 bp (5S). There are multiple copies (generally <10) of the rRNA genes in most bacteria, and the rRNA operons are generally dispersed throughout the chromosome. 16S rRNA sequences are frequently used as amplification target. All 16S rRNA genes share nine hypervariable (polymorphic) regions (Neefs et al., 1993) and the sequences are easily available from public databases. The hypervariable regions are flanked by conserved sequences, which can serve for amplification with "universal" primers (Baker et al., 2003). The variable V1 (Cocolin et al., 2004; Bonetta et al., 2008), V3 (Coppola et al., 2001; Ercolini et al., 2001; Ogier et al., 2002; Duthoit et al., 2003; Ercolini et al., 2003; Mauriello et al., 2003; Andrighetto et al., 2004; Ercolini et al., 2004; Feurer et al., 2004a; Feurer et al., 2004b; Lafarge et al., 2004; Ogier et al., 2004; Duthoit et al., 2005; Flórez and Mayo, 2006; Delbes et al., 2007; El-Baradei et al., 2007; Parayre et al., 2007; Abriouel et al., 2008; Ercolini et al., 2008; Gala et al., 2008; Van Hoorde et al., 2008; Alegría et al., 2009; Casalta et al., 2009; Dolci et al., 2009; Giannino et al., 2009; Mounier et al., 2009; Serhan et al., 2009; Dolci et al., 2010; Fontana et al., 2010; Van Hoorde et al., 2010; Masoud et al., 2011), V2 (Duthoit et al., 2003; Delbes and Montel, 2005; Saubusse et al., 2007), V4-V5 (Ercolini et al., 2003), V1-V3 (Randazzo et al., 2002), V4-V8 (Randazzo et al., 2006), V5-V6 (Le Bourhis et al., 2005; Le Bourhis et al., 2007) and V6-V8 (Randazzo et al., 2002; Ercolini et al., 2008; Nikolic et al., 2008; Randazzo et al., 2010) regions of the 16S rRNA genes and the 16S-23S-spacer region (Coppola et al., 2001; Henri-Dubernet et al., 2004) are

indicative of the global degree of RNA integrity. From the 2100 Bioanalyzer electrophoresis profile, a value, named RIN (RNA Integrity Number), is calculated. A RIN value of 10 corresponds to apparently intact material. RIN calculations can be done with either eukaryotic or prokaryotic RNA, but not when both types of RNA are present in the same sample, which would be the case for RNA samples from numerous types of cheeses. Examples of RNA electrophoregrams of RNA preparations from cheese samples are shown in Figure 1. During the ripening or storage of cheeses, some microbial populations may decline, for example by autolysis. This has a detrimental effect on RNA integrity and, in consequence, a poor RNA integrity level is not necessarily due to an inadequate sampling or

All PCR analyses rely on amplification of DNA target sequences. Concerning PCR applications to dairy products, one can distinguish targets used for PCR-based microbial

In methods of microbial diversity evaluation involving PCR, the amplification target is a sequence which has to be present in a large part of the bacterial or fungal population. The sequence variations allow the subsequent differentiation of the generated amplicons. In most cases, these techniques involve amplification of ribosomal RNA or housekeeping genes. In both prokaryotes and eukaryotes, rRNA genes usually show a high sequence homogeneity within a species (Liao, 1999), which explains why they are widely used in species identification and makes them a good target in molecular microbial diversity

Bacterial 16S, 23S and 5S rRNA genes are organised into a co-transcribed operon. The typical length of theses genes is ~2900 bp (23S), ~1500 bp (16S) and ~120 bp (5S). There are multiple copies (generally <10) of the rRNA genes in most bacteria, and the rRNA operons are generally dispersed throughout the chromosome. 16S rRNA sequences are frequently used as amplification target. All 16S rRNA genes share nine hypervariable (polymorphic) regions (Neefs et al., 1993) and the sequences are easily available from public databases. The hypervariable regions are flanked by conserved sequences, which can serve for amplification with "universal" primers (Baker et al., 2003). The variable V1 (Cocolin et al., 2004; Bonetta et al., 2008), V3 (Coppola et al., 2001; Ercolini et al., 2001; Ogier et al., 2002; Duthoit et al., 2003; Ercolini et al., 2003; Mauriello et al., 2003; Andrighetto et al., 2004; Ercolini et al., 2004; Feurer et al., 2004a; Feurer et al., 2004b; Lafarge et al., 2004; Ogier et al., 2004; Duthoit et al., 2005; Flórez and Mayo, 2006; Delbes et al., 2007; El-Baradei et al., 2007; Parayre et al., 2007; Abriouel et al., 2008; Ercolini et al., 2008; Gala et al., 2008; Van Hoorde et al., 2008; Alegría et al., 2009; Casalta et al., 2009; Dolci et al., 2009; Giannino et al., 2009; Mounier et al., 2009; Serhan et al., 2009; Dolci et al., 2010; Fontana et al., 2010; Van Hoorde et al., 2010; Masoud et al., 2011), V2 (Duthoit et al., 2003; Delbes and Montel, 2005; Saubusse et al., 2007), V4-V5 (Ercolini et al., 2003), V1-V3 (Randazzo et al., 2002), V4-V8 (Randazzo et al., 2006), V5-V6 (Le Bourhis et al., 2005; Le Bourhis et al., 2007) and V6-V8 (Randazzo et al., 2002; Ercolini et al., 2008; Nikolic et al., 2008; Randazzo et al., 2010) regions of the 16S rRNA genes and the 16S-23S-spacer region (Coppola et al., 2001; Henri-Dubernet et al., 2004) are

diversity evaluation, and targets for PCR analysis of specific microbial groups.

**2.2.1 Amplification targets for microbial diversity evaluation methods** 

RNA extraction procedure.

**2.2 Amplification targets** 

evaluation methods.

widely used in studies of the bacterial diversity of dairy products. Several distinct amplicons may be produced with some strains, due to differences in sequences of the rRNA copies.

In fungi, the internal transcribed spacer (ITS) is a region located between the 18S rRNA and 26S rRNA genes. It includes the 5.8S rRNA gene that splits the ITS into two parts: ITS1 and ITS2. The 18S, 5.8S, 26S and 5S rRNA sequences form up to hundreds of tandem repeats. The ITS region undergoes a faster rate of evolution than rRNA but its sequence remains homogenous within a species. The ITS2 region has been chosen as target for the study of the fungal biodiversity of smear-ripened cheeses (Mounier et al., 2010), and the ITS1 region for the study of the fungal diversity in cow, goat and ewe milk (Delavenne et al., 2011). Primers targeting regions of the 26S rRNA (Feurer et al., 2004b; Flórez and Mayo, 2006; Bonetta et al., 2008; Alegría et al., 2009; Dolci et al., 2009; Mounier et al., 2009) and the 18S rRNA (Callon et al., 2006; Arteau et al., 2010) were chosen to investigate the dominant yeast microflora of several types of cheeses.

Housekeeping genes are less used than rRNA in molecular studies of microbial diversity of dairy products. This is due to a lower availability in sequence databases. However, this may change in the near future, due to the rapid increase of the number of sequenced genomes. The *rpoB* gene, encoding the RNA polymerase beta subunit has been used as a target for PCR-DGGE analysis to follow lactic acid bacterial population dynamics in cheeses (Rantsiou et al., 2004).

#### **2.2.2 Amplification targets for specific microbial groups**

Defined groups of microorganisms may be studied by amplification of specific targets, either by PCR or by real-time PCR. In the latter case, quantitative data can be obtained. The primers have to be designed so that amplification occurs only from DNA of the group of interest. As for PCR-based methods of microbial diversity evaluation, rRNA sequences are frequently used as target and the specificity may be evaluated *in silico* by comparing the rRNA sequences of the group of interest to that of other microorganisms that are present in the same habitat. A high level of specificity is achieved when there is a large sequence difference with non-target microorganisms for one or both of the PCR primers. Presence of mismatches near the 3' of the primers ensures a better specificity than at the 5' end. In addition, absence, or presence of only one or two G or C residues in the last five nucleotides at the 3' end of primers, makes them less likely to hybridise transiently and to be available for non-specific extension by the DNA polymerase (Bustin, 2000). *Corynebacterium casei* cells could be quantified in cheeses by real-time PCR using a couple of primers targeting the V6 region of the 16S rRNA gene (Monnet et al., 2006). The assay was specific, as no amplification occurred with DNA from other *Corynebacterium* species present in cheeses. Primers targeting 16S rRNA genes were also used for the quantification of *Carnobacterium* cells in cheeses (Cailliez-Grimal et al., 2005), of *L. lactis* subsp. *cremoris* in fermented milks (Grattepanche et al., 2005), of *Streptococcus (Str.) thermophilus* and lactobacilli in fermented milks (Furet et al., 2004), of thermophilic bacilli in milk powder (Rueckert et al., 2005) and of bacterial species that can develop during the cold storage of milk (Rasolofo et al., 2010). Primers targeting the 16S-23S-spacer region were used for the specific detection of *Clostridium tyrobutyricum* in semi-soft and hard cheeses (Herman et al., 1997) and for the quantification of *Listeria (List.) monocytogenes* in foods, including fresh and ripened cheeses

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 19

methods involving PCR amplification are based on the analysis of DNA or RNA extracted from the food product. Even if they have several potential biases, they are faster and

Denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and temporal temperature gradient gel electrophoresis (TTGE) are widely used to study cheese microbial communities (Coppola et al., 2001; Ercolini et al., 2001; Ogier et al., 2002; Randazzo et al., 2002; Ercolini et al., 2003; Mauriello et al., 2003; Andrighetto et al., 2004; Cocolin et al., 2004; Ercolini et al., 2004; Henri-Dubernet et al., 2004; Lafarge et al., 2004; Ogier et al., 2004; Rantsiou et al., 2004; Le Bourhis et al., 2005; Flórez and Mayo, 2006; Randazzo et al., 2006; Cocolin et al., 2007; El-Baradei et al., 2007; Le Bourhis et al., 2007; Parayre et al., 2007; Abriouel et al., 2008; Bonetta et al., 2008; Ercolini et al., 2008; Gala et al., 2008; Henri-Dubernet et al., 2008; Nikolic et al., 2008; Rantsiou et al., 2008b; Van Hoorde et al., 2008; Alegría et al., 2009; Casalta et al., 2009; Dolci et al., 2009; Giannino et al., 2009; Serhan et al., 2009; Dolci et al., 2010; Fontana et al., 2010; Fuka et al., 2010; Randazzo et al., 2010; Van Hoorde et al., 2010; Masoud et al., 2011). Target sequences from rRNA or housekeeping genes are amplified and separated by electrophoresis. Separation is based on decreased electrophoretic mobility of partially melted double-stranded DNA molecules in polyacrylamide gels with a thermal gradient (TGGE) or which contain a gradient of DNA denaturants (DGGE). In TTGE, the separation is based on a temporal temperature gradient that increases in a linear fashion over the length of the electrophoresis time. Even if the DNA molecules have the same size, they may be separated because of their melting temperature behaviour, which depends on the sequence. A GC-rich clamp of about 40 bases is added at the 5' end of one of the primers to stabilize the melting behaviour and to prevent the complete dissociation of the DNA fragments during electrophoresis. Assignment of the migration bands is done by comparison to a database containing the migration profiles of reference strains. DNA bands can be recovered from the gel and sequenced in order to confirm the assignments, or to find an assignment for bands which are not present in the database. DGGE, TGGE and TTGE profiles reveal a picture of the microbial diversity and can be used to compare different dairy products or to follow a given product at different

potentially more exhaustive than culture-dependent methods.

fabrication stages. However, these methods are only semi-quantitative.

confirm the assignations.

Single-strand conformation polymorphism-PCR (SSCP-PCR) is another PCR-based method for microbial diversity investigation that has been applied to dairy products (Duthoit et al., 2003; Feurer et al., 2004a; Feurer et al., 2004b; Delbes and Montel, 2005; Duthoit et al., 2005; Callon et al., 2006; Delbes et al., 2007; Saubusse et al., 2007; Mounier et al., 2009). This technique is based on the sequence-dependent differential intra-molecular folding of single strand DNA, which alters the migration speed of the molecules under non-denaturing conditions. Single strand DNA fragments having the same size may thus be separated, if their sequences generate different intramolecular interactions. After denaturation, the fluorescently labelled PCR products are separated using a capillary-based automated sequencer. In some cases, several stable conformations can be formed from one single strand DNA fragment, resulting in multiple bands. As for DGGE, TGGE and TTGE, SSCP provides community fingerprints that cannot be phylogenetically assigned directly. A database containing the migration profile of reference strains has to be created. One disadvantage of this technique is that the labelled single strand DNA fragments cannot be sequenced to

(Rantsiou et al., 2008a). rRNA sequence primers were also advised for the quantification of fungi in cheeses by real-time PCR. The variable D1/D2 domain of the 26S rRNA and the ITS1 region of the rRNA genes were targeted for the study of yeasts (Larpin et al., 2006; Makino et al., 2010) and *Penicillium roqueforti* (Le Dréan et al., 2010).

Primers of specific protein-encoding genes have been designed for the detection or the quantification of various groups of cheese microorganisms. Proteolytic lactobacilli can be detected in stretched cheeses by amplification of cell envelope proteinase genes (Baruzzi et al., 2005). Successful detection of specific bacteriocin biosynthesis genes could be achieved in microbial DNA extracted directly from several types of cheeses (Moschetti et al., 2001; Bogovic Matijasic et al., 2007; Trmcic et al., 2008). Allman et al. (1995) used specific PCR amplifications for the detection of pathogenic bacteria in dairy products. The targets were the *List. monocytogenes* listeriolysin O (*hlyA*), the *E. coli* heat-labile enterotoxin type 1 (*elt*) and heat-stable toxin 1 (*est*), and the *Campylobacter jejuni* and *Campylobacter coli* flagellin proteins (*flaA*/*flaB*). *List. monocytogenes* has also been quantified in gouda-like cheeses by real-time PCR, through *hlyA* gene amplification (Rudi et al., 2005). Another pathogen, *Brucella* spp., can be detected in soft cheeses by amplification of a fragment from a characteristic membrane antigen, protein BCSP-31 (Serpe et al., 1999). Thermonuclease (*nuc*) gene amplification has been applied for the quantification of *S. aureus* cells in cheese and milk samples (Hein et al., 2001; Hein et al., 2005; Alarcon et al., 2006; Studer et al., 2008; Aprodu et al., 2011). Manuzon et al. (2007) monitored the pool of tetracyclin resistance genes in retail cheeses in order to estimate the amount of tetracyclin resistant bacteria, which may pose a potential risk to consumers. Coliforms are a broad class of bacteria, whose presence can be used to assess the hygienic quality of foods. A real-time PCR detection method of all coliform species in a single assay has been set up (Martin et al., 2010). It is based on the amplification of a fragment of the beta-galactosidase gene (*lacZ*). *Enterococcus (E.) gilvus*, which is found in some types of cheeses, was quantified by real-time PCR using the phenylalanyl-tRNA synthase gene (*pheS*) as target (Zago et al., 2009). The procedure was selective against the highly phylogenetically related species *E. malodoratus* and *E. raffinosus*, and the *pheS* gene seems able to differentiate enterococcal species better than 16S rRNA sequences. Histamine is a toxic biogenic amine that is sometimes involved in food poisoning. In order to quantify histamine-producing bacteria in cheeses by real-time PCR, Fernandez et al. (2006) designed consensual primers targeting the histidine decarboxylase (*hdcA*) gene of Gram-positive species. Another type of undesired bacteria, *Clostridium tyrobutyricum*, responsible for late-blowing in hard and semi-hard cheeses, can be quantified in milk samples by real-time PCR amplification of the flagellin (*fla*) gene (Lopez-Enriquez et al., 2007).

It is likely that in the future, the increased availability of genome sequences will facilitate the selection of amplification targets for specific microbial groups. A good example is the study of Chen el al. (2010), in which real-time PCR primers were designed for the detection of *Salmonella enterica* strains. In this study, specific targets were generated by using a genomic analysis workflow, which compared 17 *Salmonella enterica* genome sequences to 827 non-*Salmonella* bacterial genomes.

#### **2.3 PCR-based methods for microbial diversity investigation**

Dairy products, especially cheeses, have diverse microbial compositions, which may be analysed by culture-dependent or culture-independent methods. Culture-independent

(Rantsiou et al., 2008a). rRNA sequence primers were also advised for the quantification of fungi in cheeses by real-time PCR. The variable D1/D2 domain of the 26S rRNA and the ITS1 region of the rRNA genes were targeted for the study of yeasts (Larpin et al., 2006;

Primers of specific protein-encoding genes have been designed for the detection or the quantification of various groups of cheese microorganisms. Proteolytic lactobacilli can be detected in stretched cheeses by amplification of cell envelope proteinase genes (Baruzzi et al., 2005). Successful detection of specific bacteriocin biosynthesis genes could be achieved in microbial DNA extracted directly from several types of cheeses (Moschetti et al., 2001; Bogovic Matijasic et al., 2007; Trmcic et al., 2008). Allman et al. (1995) used specific PCR amplifications for the detection of pathogenic bacteria in dairy products. The targets were the *List. monocytogenes* listeriolysin O (*hlyA*), the *E. coli* heat-labile enterotoxin type 1 (*elt*) and heat-stable toxin 1 (*est*), and the *Campylobacter jejuni* and *Campylobacter coli* flagellin proteins (*flaA*/*flaB*). *List. monocytogenes* has also been quantified in gouda-like cheeses by real-time PCR, through *hlyA* gene amplification (Rudi et al., 2005). Another pathogen, *Brucella* spp., can be detected in soft cheeses by amplification of a fragment from a characteristic membrane antigen, protein BCSP-31 (Serpe et al., 1999). Thermonuclease (*nuc*) gene amplification has been applied for the quantification of *S. aureus* cells in cheese and milk samples (Hein et al., 2001; Hein et al., 2005; Alarcon et al., 2006; Studer et al., 2008; Aprodu et al., 2011). Manuzon et al. (2007) monitored the pool of tetracyclin resistance genes in retail cheeses in order to estimate the amount of tetracyclin resistant bacteria, which may pose a potential risk to consumers. Coliforms are a broad class of bacteria, whose presence can be used to assess the hygienic quality of foods. A real-time PCR detection method of all coliform species in a single assay has been set up (Martin et al., 2010). It is based on the amplification of a fragment of the beta-galactosidase gene (*lacZ*). *Enterococcus (E.) gilvus*, which is found in some types of cheeses, was quantified by real-time PCR using the phenylalanyl-tRNA synthase gene (*pheS*) as target (Zago et al., 2009). The procedure was selective against the highly phylogenetically related species *E. malodoratus* and *E. raffinosus*, and the *pheS* gene seems able to differentiate enterococcal species better than 16S rRNA sequences. Histamine is a toxic biogenic amine that is sometimes involved in food poisoning. In order to quantify histamine-producing bacteria in cheeses by real-time PCR, Fernandez et al. (2006) designed consensual primers targeting the histidine decarboxylase (*hdcA*) gene of Gram-positive species. Another type of undesired bacteria, *Clostridium tyrobutyricum*, responsible for late-blowing in hard and semi-hard cheeses, can be quantified in milk samples

by real-time PCR amplification of the flagellin (*fla*) gene (Lopez-Enriquez et al., 2007).

**2.3 PCR-based methods for microbial diversity investigation** 

*Salmonella* bacterial genomes.

It is likely that in the future, the increased availability of genome sequences will facilitate the selection of amplification targets for specific microbial groups. A good example is the study of Chen el al. (2010), in which real-time PCR primers were designed for the detection of *Salmonella enterica* strains. In this study, specific targets were generated by using a genomic analysis workflow, which compared 17 *Salmonella enterica* genome sequences to 827 non-

Dairy products, especially cheeses, have diverse microbial compositions, which may be analysed by culture-dependent or culture-independent methods. Culture-independent

Makino et al., 2010) and *Penicillium roqueforti* (Le Dréan et al., 2010).

methods involving PCR amplification are based on the analysis of DNA or RNA extracted from the food product. Even if they have several potential biases, they are faster and potentially more exhaustive than culture-dependent methods.

Denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and temporal temperature gradient gel electrophoresis (TTGE) are widely used to study cheese microbial communities (Coppola et al., 2001; Ercolini et al., 2001; Ogier et al., 2002; Randazzo et al., 2002; Ercolini et al., 2003; Mauriello et al., 2003; Andrighetto et al., 2004; Cocolin et al., 2004; Ercolini et al., 2004; Henri-Dubernet et al., 2004; Lafarge et al., 2004; Ogier et al., 2004; Rantsiou et al., 2004; Le Bourhis et al., 2005; Flórez and Mayo, 2006; Randazzo et al., 2006; Cocolin et al., 2007; El-Baradei et al., 2007; Le Bourhis et al., 2007; Parayre et al., 2007; Abriouel et al., 2008; Bonetta et al., 2008; Ercolini et al., 2008; Gala et al., 2008; Henri-Dubernet et al., 2008; Nikolic et al., 2008; Rantsiou et al., 2008b; Van Hoorde et al., 2008; Alegría et al., 2009; Casalta et al., 2009; Dolci et al., 2009; Giannino et al., 2009; Serhan et al., 2009; Dolci et al., 2010; Fontana et al., 2010; Fuka et al., 2010; Randazzo et al., 2010; Van Hoorde et al., 2010; Masoud et al., 2011). Target sequences from rRNA or housekeeping genes are amplified and separated by electrophoresis. Separation is based on decreased electrophoretic mobility of partially melted double-stranded DNA molecules in polyacrylamide gels with a thermal gradient (TGGE) or which contain a gradient of DNA denaturants (DGGE). In TTGE, the separation is based on a temporal temperature gradient that increases in a linear fashion over the length of the electrophoresis time. Even if the DNA molecules have the same size, they may be separated because of their melting temperature behaviour, which depends on the sequence. A GC-rich clamp of about 40 bases is added at the 5' end of one of the primers to stabilize the melting behaviour and to prevent the complete dissociation of the DNA fragments during electrophoresis. Assignment of the migration bands is done by comparison to a database containing the migration profiles of reference strains. DNA bands can be recovered from the gel and sequenced in order to confirm the assignments, or to find an assignment for bands which are not present in the database. DGGE, TGGE and TTGE profiles reveal a picture of the microbial diversity and can be used to compare different dairy products or to follow a given product at different fabrication stages. However, these methods are only semi-quantitative.

Single-strand conformation polymorphism-PCR (SSCP-PCR) is another PCR-based method for microbial diversity investigation that has been applied to dairy products (Duthoit et al., 2003; Feurer et al., 2004a; Feurer et al., 2004b; Delbes and Montel, 2005; Duthoit et al., 2005; Callon et al., 2006; Delbes et al., 2007; Saubusse et al., 2007; Mounier et al., 2009). This technique is based on the sequence-dependent differential intra-molecular folding of single strand DNA, which alters the migration speed of the molecules under non-denaturing conditions. Single strand DNA fragments having the same size may thus be separated, if their sequences generate different intramolecular interactions. After denaturation, the fluorescently labelled PCR products are separated using a capillary-based automated sequencer. In some cases, several stable conformations can be formed from one single strand DNA fragment, resulting in multiple bands. As for DGGE, TGGE and TTGE, SSCP provides community fingerprints that cannot be phylogenetically assigned directly. A database containing the migration profile of reference strains has to be created. One disadvantage of this technique is that the labelled single strand DNA fragments cannot be sequenced to confirm the assignations.

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 21

community from an artisanal Sicilian cheese, Randazzo et al. (2002) compared the intensity of bands from DNA and RNA-derived DGGE profiles and concluded that some species of the samples were not very metabolically active. Other studies of RNA profiles involving either DGGE (Rantsiou et al., 2008b; Dolci et al., 2010; Masoud et al., 2011), TTGE (Le Bourhis et al., 2007), SSCP (Le Bourhis et al., 2005), T-RFLP (Sanchez et al., 2006), clone library sequencing (Carraro et al., 2011) or pyrosequencing (Masoud et al., 2011) have been

Real-time PCR (qPCR) uses fluorescent reporter dyes to combine the amplification and detection steps of the PCR reaction in a single tube format. The assay relies on measuring the increase in fluorescent signal, which is proportional to the amount of DNA produced during each PCR cycle. A quantification cycle (Cq) value is determined from the plot relating fluorescence against the cycle number. Cq corresponds to the number of cycles for which the fluorescence is higher than the background fluorescence. qPCR offers the possibility to quantify microbial populations through measurements of the abundance of a target sequence in DNA samples extracted from food products (Postollec et al., 2011). Combined with reverse transcription (RT), qPCR can also be used to estimate the amount of

Several applications of qPCR for the quantification of microbial populations in dairy products have been described (Table 1). In general, the experimental approach is the following: after extraction of DNA from the sample, qPCR is performed together with a standard curve, and the results are expressed as colony-forming-units (CFU), cell, or DNA target number per amount of dairy product. For an accurate quantification, several technical considerations have to be taken into account. First, the efficiency of recovery of the DNA from the dairy products should be constant and as high as possible. This may be verified in experiments where target cells are added to a control dairy matrix. Larpin et al. (2006) observed significant DNA losses during the extraction of DNA from cheese samples containing yeast species, and it appeared that cheese composition affected the extraction yields. DNA losses may occur during alcohol precipitation steps, especially in samples containing low amounts of DNA. A better recovery can be obtained by addition of coprecipitants such as exogenous DNA and glycogen. When column-based purification methods are used, it should be made sure that the amount of DNA loaded onto the columns does not exceed the column capacity. Another important technical consideration is that the amount of qPCR inhibitors in the DNA sample should be as limited as possible. One convenient way to evaluate the presence of inhibitors is to analyse by qPCR several dilutions of the DNA samples. The samples that need high dilution factors to reach the maximum PCR efficiency contain more inhibitors than those that need a lower dilution factor. The amount of PCR inhibitors has an impact on the detection level, as it determines the dilution factor that has to be applied in the qPCR assays. Absence of inhibitors can also be verified by inclusion of an internal amplification control (IAC). An IAC is a non-target DNA fragment that is co-amplified with the target sequence, ideally with the same primers used for the target. The forward and reverse target sequences are fused to both ends of a non-target fragment, to which a second fluorescent probe (the IAC probe) hybridises. The simultaneous use in a single reaction of two differently labelled fluorescent probes makes it

published.

RNA transcripts.

**2.4 Real-time PCR methods** 

Another PCR-based technique that has been applied to dairy products is terminal restriction fragment length polymorphism (TRFLP) (Rademaker et al., 2005; Rademaker et al., 2006; Arteau et al., 2010; Cogan and John, 2011). In TRFLP analyses, marker genes are amplified using one or two fluorescently labelled primers. The amplicons are then cut with one or several restriction enzymes and separated using a capillary-based automated sequencer. Only the end-labelled fragments are detected by the laser detector and their size can be determined by comparison with DNA size standards. One advantage of this technique is that the size of the fragments of any known DNA sequence can be determined *in silico*. This is why 16S rRNA genes, whose sequences are easily available from public databases, are frequently used in TRFLP studies. As for SSCP, a drawback of capillary electrophoresisbased TRFLP is that bands remaining unknown cannot be extracted from the gel to be identified by DNA sequencing.

In denaturing high-performance liquid chromatography (DHPLC), PCR amplicons are partially denatured and separated on a liquid chromatography column which contains chemical agents that bind more strongly to double-stranded DNA molecules. Amplicons of the same size but with sequence differences resulting in modified melting behaviours will thus have different retention times. DHPLC analyses are rapid and the elution fraction corresponding to the different amplicons can be sequenced for confirmation or identification purposes. There are not many papers concerning DHPLC analyses of dairy products (Ercolini et al., 2008; Mounier et al., 2010; Delavenne et al., 2011), but this technique will probably be increasingly used in the future.

Bacterial diversity may also be assessed by sequencing clones libraries generated from 16S rRNA gene amplification of DNA extracted from dairy products (Feurer et al., 2004a; Feurer et al., 2004b; Delbes et al., 2007; Rasolofo et al., 2010; Carraro et al., 2011). The main advantage of this technique is that no dedicated database is needed, as the sequences are already available in public genomic databases. In addition, in most cases, the 16S rRNA gene sequences permit assignments at the species level. But this technique is expensive and time-consuming, which is why it is not widely used. Second-generation DNA sequencing is a promising alternative to clone library sequencing (Cardenas and Tiedje, 2008). Masoud et al. (2011) studied the bacterial populations in Danish raw milk cheeses by pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rRNA gene. After amplification of the 16S rRNA targets, a second PCR is done by using, for each sample, a different bar-coded primer. The amplified fragments of the different samples are then mixed and sequenced together, and the sequences are assigned to bacterial taxa. A very good agreement was found with the results of PCR-DGGE analysis. In addition, minor bacterial populations that were not detected by PCR-DGGE, were found by pyrosequencing. Furthermore, pyrosequencing provides a more reliable estimate of the relative abundance of the individual bacteria. Second-generation DNA sequencing appears thus to be a powerful and promising method, which will allow a deeper investigation of the bacterial populations in dairy products.

PCR-based methods for microbial diversity investigation can also be applied to RNA samples, after reverse transcription. As the ribosomal RNA content inside of the cells increases with the growth rate (Bremer and Dennis, 1996), one can assume that higher amounts of rRNA targets will be detected in active growing cells. In addition, since RNA is less stable than DNA, it will degrade more quickly in dead cells. In a study of the bacterial community from an artisanal Sicilian cheese, Randazzo et al. (2002) compared the intensity of bands from DNA and RNA-derived DGGE profiles and concluded that some species of the samples were not very metabolically active. Other studies of RNA profiles involving either DGGE (Rantsiou et al., 2008b; Dolci et al., 2010; Masoud et al., 2011), TTGE (Le Bourhis et al., 2007), SSCP (Le Bourhis et al., 2005), T-RFLP (Sanchez et al., 2006), clone library sequencing (Carraro et al., 2011) or pyrosequencing (Masoud et al., 2011) have been published.

## **2.4 Real-time PCR methods**

20 Polymerase Chain Reaction

Another PCR-based technique that has been applied to dairy products is terminal restriction fragment length polymorphism (TRFLP) (Rademaker et al., 2005; Rademaker et al., 2006; Arteau et al., 2010; Cogan and John, 2011). In TRFLP analyses, marker genes are amplified using one or two fluorescently labelled primers. The amplicons are then cut with one or several restriction enzymes and separated using a capillary-based automated sequencer. Only the end-labelled fragments are detected by the laser detector and their size can be determined by comparison with DNA size standards. One advantage of this technique is that the size of the fragments of any known DNA sequence can be determined *in silico*. This is why 16S rRNA genes, whose sequences are easily available from public databases, are frequently used in TRFLP studies. As for SSCP, a drawback of capillary electrophoresisbased TRFLP is that bands remaining unknown cannot be extracted from the gel to be

In denaturing high-performance liquid chromatography (DHPLC), PCR amplicons are partially denatured and separated on a liquid chromatography column which contains chemical agents that bind more strongly to double-stranded DNA molecules. Amplicons of the same size but with sequence differences resulting in modified melting behaviours will thus have different retention times. DHPLC analyses are rapid and the elution fraction corresponding to the different amplicons can be sequenced for confirmation or identification purposes. There are not many papers concerning DHPLC analyses of dairy products (Ercolini et al., 2008; Mounier et al., 2010; Delavenne et al., 2011), but this technique will

Bacterial diversity may also be assessed by sequencing clones libraries generated from 16S rRNA gene amplification of DNA extracted from dairy products (Feurer et al., 2004a; Feurer et al., 2004b; Delbes et al., 2007; Rasolofo et al., 2010; Carraro et al., 2011). The main advantage of this technique is that no dedicated database is needed, as the sequences are already available in public genomic databases. In addition, in most cases, the 16S rRNA gene sequences permit assignments at the species level. But this technique is expensive and time-consuming, which is why it is not widely used. Second-generation DNA sequencing is a promising alternative to clone library sequencing (Cardenas and Tiedje, 2008). Masoud et al. (2011) studied the bacterial populations in Danish raw milk cheeses by pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rRNA gene. After amplification of the 16S rRNA targets, a second PCR is done by using, for each sample, a different bar-coded primer. The amplified fragments of the different samples are then mixed and sequenced together, and the sequences are assigned to bacterial taxa. A very good agreement was found with the results of PCR-DGGE analysis. In addition, minor bacterial populations that were not detected by PCR-DGGE, were found by pyrosequencing. Furthermore, pyrosequencing provides a more reliable estimate of the relative abundance of the individual bacteria. Second-generation DNA sequencing appears thus to be a powerful and promising method, which will allow a deeper investigation of the bacterial populations in

PCR-based methods for microbial diversity investigation can also be applied to RNA samples, after reverse transcription. As the ribosomal RNA content inside of the cells increases with the growth rate (Bremer and Dennis, 1996), one can assume that higher amounts of rRNA targets will be detected in active growing cells. In addition, since RNA is less stable than DNA, it will degrade more quickly in dead cells. In a study of the bacterial

identified by DNA sequencing.

dairy products.

probably be increasingly used in the future.

Real-time PCR (qPCR) uses fluorescent reporter dyes to combine the amplification and detection steps of the PCR reaction in a single tube format. The assay relies on measuring the increase in fluorescent signal, which is proportional to the amount of DNA produced during each PCR cycle. A quantification cycle (Cq) value is determined from the plot relating fluorescence against the cycle number. Cq corresponds to the number of cycles for which the fluorescence is higher than the background fluorescence. qPCR offers the possibility to quantify microbial populations through measurements of the abundance of a target sequence in DNA samples extracted from food products (Postollec et al., 2011). Combined with reverse transcription (RT), qPCR can also be used to estimate the amount of RNA transcripts.

Several applications of qPCR for the quantification of microbial populations in dairy products have been described (Table 1). In general, the experimental approach is the following: after extraction of DNA from the sample, qPCR is performed together with a standard curve, and the results are expressed as colony-forming-units (CFU), cell, or DNA target number per amount of dairy product. For an accurate quantification, several technical considerations have to be taken into account. First, the efficiency of recovery of the DNA from the dairy products should be constant and as high as possible. This may be verified in experiments where target cells are added to a control dairy matrix. Larpin et al. (2006) observed significant DNA losses during the extraction of DNA from cheese samples containing yeast species, and it appeared that cheese composition affected the extraction yields. DNA losses may occur during alcohol precipitation steps, especially in samples containing low amounts of DNA. A better recovery can be obtained by addition of coprecipitants such as exogenous DNA and glycogen. When column-based purification methods are used, it should be made sure that the amount of DNA loaded onto the columns does not exceed the column capacity. Another important technical consideration is that the amount of qPCR inhibitors in the DNA sample should be as limited as possible. One convenient way to evaluate the presence of inhibitors is to analyse by qPCR several dilutions of the DNA samples. The samples that need high dilution factors to reach the maximum PCR efficiency contain more inhibitors than those that need a lower dilution factor. The amount of PCR inhibitors has an impact on the detection level, as it determines the dilution factor that has to be applied in the qPCR assays. Absence of inhibitors can also be verified by inclusion of an internal amplification control (IAC). An IAC is a non-target DNA fragment that is co-amplified with the target sequence, ideally with the same primers used for the target. The forward and reverse target sequences are fused to both ends of a non-target fragment, to which a second fluorescent probe (the IAC probe) hybridises. The simultaneous use in a single reaction of two differently labelled fluorescent probes makes it

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 23

enrichment of the food samples before qPCR (Rossmanith et al., 2006; Chiang et al., 2007; Karns et al., 2007; O'Grady et al., 2009; Omiccioli et al., 2009). However, in that case, the

16S rRNA Commercial fermented

Commercial yoghurt

milks, mixed culture with *Lb. rhamnosus* and *L. lactis* subsp. *lactis* biovar. *diacetylactis*

contaminated cheeses and commercial cheeses

Experimental Emmental

Experimental Emmental

Artisanal raw milk

Lebanese raw goat's milk cheeses

contaminated milks

Experimental cheeses and commercial cheeses

contaminated cheeses and commercial cheeses

contaminated milk

contaminated cheeses

ripened cheese

cheese

cheese

cheeses

Artificially

powder

(Ongol et al., 2009)

(Grattepanche et al.,

(Furet et al., 2004)

(Cailliez-Grimal et al.,

(Monnet et al., 2006)

(Falentin et al., 2010)

(Falentin et al., 2012)

(Zago et al., 2009)

(Serhan et al., 2009)

2007)

(Lopez-Enriquez et al.,

(Fernandez et al., 2006; Ladero et al., 2008; Ladero et al., 2009)

(Manuzon et al., 2007)

(Rueckert et al., 2005)

(Martin et al., 2010)

2005)

2005)

samples

milks

Target population Target sequence Food matrix References

results can only be used for detection, and not quantification.

processing protein)

*Carnobacterium* sp. 16S rRNA Artificially

*Corynebacterium casei* 16S rRNA Commercial smear-

factor TU), *Gro*L (chaperonin GroEL)

factor TU), *Gro*L (chaperonin GroEL)

*E. gilvus pheS* (phenylalanyl-tRNA synthase

sequence

*hdc*A (histidine decarboxylase)

*tet*S (tetracycline resistance protein)

Thermophilic bacilli 16S rRNA Artificially

Coliform species *lac*Z (beta-galactosidase) Artificially

*E. faecium* Conserved *E. faecium*

16S rRNA, *tuf* (elongation

16S rRNA, *tuf* (elongation

*fla* (flagellin) Artificially

*L. lactis* subsp. *cremoris* 16S rRNA Experimental fermented

*Str. thermophilus rim*M (16S rRNA

*Str. thermophilus*, *Lb. delbrueckii*, *Lb. casei*, *Lb. paracasei*, *Lb. rhamnosus*, *Lb. acidophilus*, *Lb.* 

*P. freudenreichii* and *Lb.* 

*Str. thermophilus* and *Lb. helveticus* 

Histamine-producing

Tetracyclin resistant

*johnsonii*

*paracasei*

*Clostridium tyrobutyricum* 

bacteria

bacteria

possible to quantify the target and to assess PCR efficiency at the same time. If negative results are obtained for the target PCR, the absence of a positive IAC signal indicates that amplification has failed. Phenol extraction and repeated washing of alcohol-precipitated DNA pellets are efficient in reducing the impact of PCR inhibitors. In phenol-based purifications, the amount of PCR inhibitors may also be reduced by using a gel (Phase Lock Gel tubes) improving separation between the liquid and organic phases. For accurate qPCR quantification of microbial populations in dairy products, the level of cross-contaminations of DNA during DNA extraction and subsequent steps should be as limited as possible. This can be checked by adding several controls during the qPCR, such as water or DNA extracted from a dairy matrix that does not contain the target population. If complete absence of cross-contamination cannot be achieved, one may define a maximum Cq (quantification cycle) value, which is lower than the value obtained with the controls (e.g. five cycles lower), and over which the assay will not be considered. After qPCR amplification, melting curve analysis is carried out to confirm the absence of secondary amplification products. It is also possible to confirm amplification specificity by sequencing the resulting amplicon. Several types of standards may be used for calculating the concentration of targets in the dairy product. In the method used by Monnet et al. (2006), a standard curve is generated from different dilutions of a genomic DNA sample extracted from a pure culture of the target microorganism in liquid broth. The amount of target genomic DNA present in cheeses is then calculated and converted to colony-forming-units values, using a conversion factor determined from the pure culture DNA extract. Such calculation is valid only if the DNA recovery yield from cheeses is similar to that from cells grown in the liquid broth. Le Dréan et al. (2010) quantified *Penicillium camemberti* and *Penicillium roqueforti* mycelium in cheeses. To imitate cheese matrix effects, DNA was extracted from curd mixed with known amounts of fresh mycelium and was used as standard for further qPCR analyses. The mycelium concentration was then expressed as weight of mycelium per weight of cheese. Microbial cells may also be quantified using standard curves obtained with PCR-amplified targets. For example, Furet et al. (2004) determined the number of 16S rRNA gene targets in DNA samples prepared from dairy products and converted this value to cell numbers, taking into account the number of 16S rRNA gene copies in the chromosome of each species (http://rrndb.mmg.msu.edu, (Lee et al., 2009). Rasolofo et al. (2010) used a similar procedure for the quantification of *Staphyloccous aureus*, *Aerococcus viridans*, *Acinetobacter calcoaceticus*, *Corynebacterium variabile*, *Pseudomonas fluorescens* and *Str. uberis* in milk samples, except that standard curves were obtained from plasmids in which 16S rRNA gene sequences of the target species were inserted.

The quantification limit values for microbial cells in dairy products reported for qPCR methods are heterogeneous. They depend on factors such as the type of dairy product (cheese or fermented milk), the efficiency of DNA extraction, the target microbial population and the target DNA sequence. A value of 105 CFU/g has been reported for *Corynebacterium casei* (Monnet et al., 2006) and *Carnobacterium* species (Cailliez-Grimal et al., 2005), of 103-104 CFU/g for *List. monocytogenes* (Rantsiou et al., 2008a), of 104 CFU/g for *E. gilvus* (Zago et al., 2009), and of 103 cells/ml for lactic acid bacteria (Furet et al., 2004). In some cases, higher amounts of microorganisms are measured with qPCR analyses than with classical agar counts, which may be explained by the fact that DNA from dead cells can also be amplified. In order to lower the detection levels of pathogens, it is possible to perform culture

possible to quantify the target and to assess PCR efficiency at the same time. If negative results are obtained for the target PCR, the absence of a positive IAC signal indicates that amplification has failed. Phenol extraction and repeated washing of alcohol-precipitated DNA pellets are efficient in reducing the impact of PCR inhibitors. In phenol-based purifications, the amount of PCR inhibitors may also be reduced by using a gel (Phase Lock Gel tubes) improving separation between the liquid and organic phases. For accurate qPCR quantification of microbial populations in dairy products, the level of cross-contaminations of DNA during DNA extraction and subsequent steps should be as limited as possible. This can be checked by adding several controls during the qPCR, such as water or DNA extracted from a dairy matrix that does not contain the target population. If complete absence of cross-contamination cannot be achieved, one may define a maximum Cq (quantification cycle) value, which is lower than the value obtained with the controls (e.g. five cycles lower), and over which the assay will not be considered. After qPCR amplification, melting curve analysis is carried out to confirm the absence of secondary amplification products. It is also possible to confirm amplification specificity by sequencing the resulting amplicon. Several types of standards may be used for calculating the concentration of targets in the dairy product. In the method used by Monnet et al. (2006), a standard curve is generated from different dilutions of a genomic DNA sample extracted from a pure culture of the target microorganism in liquid broth. The amount of target genomic DNA present in cheeses is then calculated and converted to colony-forming-units values, using a conversion factor determined from the pure culture DNA extract. Such calculation is valid only if the DNA recovery yield from cheeses is similar to that from cells grown in the liquid broth. Le Dréan et al. (2010) quantified *Penicillium camemberti* and *Penicillium roqueforti* mycelium in cheeses. To imitate cheese matrix effects, DNA was extracted from curd mixed with known amounts of fresh mycelium and was used as standard for further qPCR analyses. The mycelium concentration was then expressed as weight of mycelium per weight of cheese. Microbial cells may also be quantified using standard curves obtained with PCR-amplified targets. For example, Furet et al. (2004) determined the number of 16S rRNA gene targets in DNA samples prepared from dairy products and converted this value to cell numbers, taking into account the number of 16S rRNA gene copies in the chromosome of each species (http://rrndb.mmg.msu.edu, (Lee et al., 2009). Rasolofo et al. (2010) used a similar procedure for the quantification of *Staphyloccous aureus*, *Aerococcus viridans*, *Acinetobacter calcoaceticus*, *Corynebacterium variabile*, *Pseudomonas fluorescens* and *Str. uberis* in milk samples, except that standard curves were obtained from plasmids in which 16S rRNA gene sequences of the target species were

The quantification limit values for microbial cells in dairy products reported for qPCR methods are heterogeneous. They depend on factors such as the type of dairy product (cheese or fermented milk), the efficiency of DNA extraction, the target microbial population and the target DNA sequence. A value of 105 CFU/g has been reported for *Corynebacterium casei* (Monnet et al., 2006) and *Carnobacterium* species (Cailliez-Grimal et al., 2005), of 103-104 CFU/g for *List. monocytogenes* (Rantsiou et al., 2008a), of 104 CFU/g for *E. gilvus* (Zago et al., 2009), and of 103 cells/ml for lactic acid bacteria (Furet et al., 2004). In some cases, higher amounts of microorganisms are measured with qPCR analyses than with classical agar counts, which may be explained by the fact that DNA from dead cells can also be amplified. In order to lower the detection levels of pathogens, it is possible to perform culture

inserted.

enrichment of the food samples before qPCR (Rossmanith et al., 2006; Chiang et al., 2007; Karns et al., 2007; O'Grady et al., 2009; Omiccioli et al., 2009). However, in that case, the results can only be used for detection, and not quantification.


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 25

Bovine milk samples (Rossetti et al., 2010)

(Rasolofo et al., 2010)

(Omiccioli et al., 2009)

(Larpin et al., 2006)

(Le Dréan et al., 2010)

(Makino et al., 2010)

(Rossetti et al., 2010)

Target population Target sequence Food matrix References

16S rRNA Milk during cold

storage

Artificially contaminated milk

Commercial Livarot

Model cheeses and commercial Camembert-type

contaminated milk

samples

cheeses

cheeses

Artificially contaminated fermented milk

(deoxyribodipyrimidine

*Salmonella* spp: *ttr* cluster (tetrathionate reductase

*List. monocytogenes*: *hly*A (listeriolysin O) *E. coli* O157: *rfb*E (perosamine synthetase

photolyase)

genes)

homolog)

lyase),

*Y. lipolytica*: topoisomerase II

of rRNA

gene

rRNA

*G. candidum*: *cgl* (cystathionine-gamma-

*Kluyveromyces* sp.: *lac4* 

*P. roqueforti*: ITS1 region

D1/D2 domain of 26S

*P. camemberti*: beta-tubulin

bacteriophage lysin genes Artificially

Table 1. Examples of applications of qPCR for the quantification or detection of microbial

The study of gene expression within natural environments such as dairy products is an emerging field in microbial ecology that is especially promising in the study of bacterial function even though only a few applications of reverse-transcription qPCR to dairy

*Mycoplasma bovis uvr*C

*S. aureus*, *Aerococcus viridans*, *Acinetobacter calcoaceticus*, *Corynebacterium variabile*, *Pseudomonas fluorescens* and *Str.* 

*Salmonella* spp., *List. monocytogenes* and *E.* 

*Debaryomyces hansenii*, *Geotrichum candidum*, *Kluyveromyces* sp., *Yarrowia lipolytica*

*Penicillium roqueforti*  and *Penicillium camemberti*

*Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Filobasidiella* 

*neoformans, Issatchenkia orientalis, Trichosporon asahii, and Trichosporon* 

populations in dairy products.

*jirovecii* 

*Lb. delbrueckii*  bacteriophages

*uberis*

*coli* O157


Market dairy food

samples, including

contaminated and naturally contaminated

contaminated cheeses, bovine and caprine milk

milk samples

samples

Artificially contaminated and naturally contaminated

milk samples

Commercial food samples, including

including milk and soft

contaminated cheeses and commercial gouda-

commercial cheeses

cheeses

cheese

like cheeses

products

cheeses

MAP F57 sequence Commercial raw milk

Insertion element IS*900* Milk samples and

(Singh et al., 2009)

(Omiccioli et al., 2009)

(Studer et al., 2008; Aprodu et al., 2011)

(Hein et al., 2001; Hein

(Fusco et al., 2011)

Amoroso et al., 2011)

(Omiccioli et al., 2009)

(Rantsiou et al., 2008a)

(Rudi et al., 2005)

(O'Grady et al., 2009)

(Stephan et al., 2007)

(Rodríguez-Lázaro et al., 2005; Donaghy et al., 2008; Herthnek et al., 2008; Slana et al., 2008; Botsaris et al.,

2010)

et al., 2005)

Milk samples (Boss et al., 2011)

Buffalo milk samples (Marianelli et al., 2008;

samples

cheeses

Target population Target sequence Food matrix References

*E. coli* O157:H7 virulence genes Milk samples (Karns et al., 2007)

*E. coli* O157:H7 *eae* (intimin adherence

*S. aureus egc* (enterotoxin gene

*S. aureus* genotype B *sea* (enterotoxin A), *sed*

*Brucella* spp. *rnp*B (RNA component of

*List. monocytogenes prfA* (transcriptional

*Mycobacterium avium* subsp. *paratuberculosis*

*Mycobacterium avium* subsp. *paratuberculosis* protein)

activator)

*List. monocytogenes* 16S-23S-spacer region Various foods,

*List. monocytogenes hly*A (listeriolysin O) Artificially

*List. monocytogenes ssr*A (tmRNA) Commercial dairy

protein)

*S. aureus nuc* (thermonuclease) Commercial food

*S. aureus nuc* (thermonuclease) Artificially

*S. aureus nuc* (thermonuclease) Artificially

(enterotoxin D), *luk*E (leucotoxin E)

ribonuclease P), bcsp31 (311 kDa cell surface

cluster)


Table 1. Examples of applications of qPCR for the quantification or detection of microbial populations in dairy products.

The study of gene expression within natural environments such as dairy products is an emerging field in microbial ecology that is especially promising in the study of bacterial function even though only a few applications of reverse-transcription qPCR to dairy

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 27

One disadvantage of all absolute quantification analyses is the significant reduction in the number of experimental samples that can be run on a single plate because a standard curve has to be included in each reaction run. In relative quantification methods, the amount of RNA targets in samples is expressed relative to the amount of the same target present in another sample, which is designated as the calibrator. This calibrator is chosen among the samples being compared (Cikos and Koppel, 2009). The advantage of this method is that standard curves don't have to be included in each run. However, this does not compensate for variations in reverse transcription efficiency and in RNA extraction efficiency from one sample to another. To compensate for this sample-to-sample variation, the quantity of RNA target is usually normalised to the quantity of one or several internal reference genes. These reference genes must be shown to be stable under the experimental conditions being examined, and are evaluated using software programmes such as geNorm or Bestkeeper. Two ideal reference genes are expected to have an identical expression ratio in all samples, whatever the experimental conditions. In the geNorm procedure (Vandesompele et al., 2002), the Cq values of each sample are transformed into relative quantities (Q) with a calibrator (cal) sample and using the gene-specific PCR efficiency (E), calculated as follows: Q = E (calCq – sampleCq). Normalisation is then applied by dividing the relative quantities of genes of interest by the geometric mean of the relative quantities of selected reference genes (normalisation factor). The 16S rRNA gene was used as reference gene to follow the expression of *L. lactis* nisin genes in a model cheese (Trmcic et al., 2011) . Several groups of genes could be distinguished based on expression profiles as a function of time, which contributed to a better knowledge of the regulation of nisin biosynthesis. For normalisation of gene transcripts from *Pseudomonas* spp., *Enterococcus* spp., *Pediococcus (P.) pentosaceus* and *Lb. casei* during the manufacturing of an experimental Montasio cheese, Carraro et al. (2011) used one couple of primers targeting the 16S rRNA of all bacteria present. The calculated fold-change does not reflect the specific gene expression of each population, but rather an expression taking into account the total amount of 16S rRNA. Cretenet et al. (2011) quantified the expression of several genes from *L. lactis* in model cheeses made from ultrafiltered milk, using *gyrB* (DNA gyrase subunit B) as reference gene. The histidine decarboxylase gene (*hdcA*) present in certain *Str. thermophilus* strains is involved in the synthesis histamine, a biogenic amine which may be accumulated in cheeses. The expression of *hdcA* was studied under conditions common to cheese-making, using the gene encoding the alpha subunit of the RNA polymerase (*rpoA*) as reference gene (Rossi et al., 2011). In this case, the stability of reference gene expression was assessed by absolute quantification of the transcripts obtained from fixed amounts of RNA. Up-regulation of *hdcA* occurred in the presence of free histidine and salt, and repression after thermisation. In bacteria, the gene encoding the elongation factor TU (*tuf*) is frequently used as reference gene in reverse transcription qPCR analyses. The expression of this gene by *L. lactis* was investigated in model cheeses by relative quantification using the total amount of RNA for normalisation, i.e. with reverse transcriptions performed with a fixed amount of RNA (Monnet et al., 2008). In this case, one has to check that potential biases, such as differences of reverse transcription efficiencies among the samples being studied, do not interfere. With this method, the calculated gene expression does not represent the expression relative to other mRNA transcripts, but rather the expression relative to the ribosomal RNA, which form most RNA. A large decrease of *tuf* expression, up to 100-fold, was observed after a few days. This decrease probably reflected the global decrease of mRNA transcription in the cheese matrix, after the end of growth of *L. lactis*. Duquenne et al. (2010) were able to quantify the

products have been described so far (Table 2). Reverse-transcription qPCR experiments involve the following steps: RNA extraction, evaluation of RNA integrity, DNase treatment, reverse-transcription and qPCR (Nolan et al., 2006; Bustin et al., 2009). Reverse transcriptions can be done with random hexamers, specific primers or oligo-dT primers (only for eukaryotic mRNA). Two types of quantification methods may be used: absolute quantification and relative quantification (Wong and Medrano, 2005; Nolan et al., 2006; Bustin et al., 2009; Cikos and Koppel, 2009). Absolute quantification is based on comparison of Cq values with a standard curve generated from the target sequence. The determination of a concentration of target RNA in the samples requires generating a standard curve with known amounts of RNA targets (and not DNA) that have been transcribed *in vitro*. This is necessary because the efficiencies of reverse transcription reactions are not known and vary from target to target. In addition, the reverse transcription step has been proposed as the source of most of the variability in reverse-transcription qPCR (Freeman et al., 1999), owing to the sensitivity of reverse transcriptase enzymes to inhibitors that may be present in the samples. As the production of *in vitro-*transcribed RNA standards is fastidious and timeconsuming, and there is no guarantee that the reverse transcription efficiency with these standards will be similar to that with the biological RNA samples, there are not many reports of absolute quantification in reverse transcription qPCR involving RNA standards. Absolute quantification of RNA transcripts with DNA standards (e.g. with standards that have not been reverse transcribed) is sometimes used. In that case, the exact number of RNA targets in the biological samples cannot be determined and results are expressed as "DNA gene equivalent" (Nicolaisen et al., 2008) or "cDNA". If it is assumed that the reverse transcription efficiencies for a given target are constant whatever the sample, these results can be used to compare the abundance of the same RNA target in several samples. Smeianov et al. (2007) used absolute quantification to compare the expression of *Lb. helveticus* genes during growth in milk and in MRS medium. In these experiments, the amount of cDNA before qPCR was standardised. Ulvé et al. (2008) standardised the amount of RNA before reverse transcription and compared the Cq values obtained for genes of *L. lactis* in cheeses at different ripening times. Even if it is not possible by this method to quantitatively compare the abundance of different RNA targets in the same sample (which would need *in vitro-*transcribed RNA standards), large differences in abundance may be shown. Direct comparisons of Cq values with a standardised amount of RNA have also been used to investigate the effect of cell separation from the cheese matrix before RNA extraction (Monnet et al., 2008). Bleve et al. (2003) observed a correlation between standard plate counts of yeasts and moulds present in spoiled commercial food products and the Cq values obtained by reverse transcription qPCR analysis with primers targeting the fungal actin gene. To follow gene expression of *P. freudenreichii* and *Lb. paracasei* during cheese-making, Falentin et al. (2010) measured the amount of cDNA copies of the target sequence after reverse transcription, and divided this value by the corresponding number of cells, which was measured by qPCR analysis of DNA extracted from the cheese samples. From these analyses, it could be concluded that the metabolic activity of *Lb. paracasei* cells reached a maximum during the first part of ripening, whereas the maximum activity of *P. freudenreichii* was reached later. A similar approach was used for the study of the metabolic activity of *Lb. helveticus* and *Str. thermophilus* cells during the ripening of Emmental cheese (Falentin et al., 2012).

products have been described so far (Table 2). Reverse-transcription qPCR experiments involve the following steps: RNA extraction, evaluation of RNA integrity, DNase treatment, reverse-transcription and qPCR (Nolan et al., 2006; Bustin et al., 2009). Reverse transcriptions can be done with random hexamers, specific primers or oligo-dT primers (only for eukaryotic mRNA). Two types of quantification methods may be used: absolute quantification and relative quantification (Wong and Medrano, 2005; Nolan et al., 2006; Bustin et al., 2009; Cikos and Koppel, 2009). Absolute quantification is based on comparison of Cq values with a standard curve generated from the target sequence. The determination of a concentration of target RNA in the samples requires generating a standard curve with known amounts of RNA targets (and not DNA) that have been transcribed *in vitro*. This is necessary because the efficiencies of reverse transcription reactions are not known and vary from target to target. In addition, the reverse transcription step has been proposed as the source of most of the variability in reverse-transcription qPCR (Freeman et al., 1999), owing to the sensitivity of reverse transcriptase enzymes to inhibitors that may be present in the samples. As the production of *in vitro-*transcribed RNA standards is fastidious and timeconsuming, and there is no guarantee that the reverse transcription efficiency with these standards will be similar to that with the biological RNA samples, there are not many reports of absolute quantification in reverse transcription qPCR involving RNA standards. Absolute quantification of RNA transcripts with DNA standards (e.g. with standards that have not been reverse transcribed) is sometimes used. In that case, the exact number of RNA targets in the biological samples cannot be determined and results are expressed as "DNA gene equivalent" (Nicolaisen et al., 2008) or "cDNA". If it is assumed that the reverse transcription efficiencies for a given target are constant whatever the sample, these results can be used to compare the abundance of the same RNA target in several samples. Smeianov et al. (2007) used absolute quantification to compare the expression of *Lb. helveticus* genes during growth in milk and in MRS medium. In these experiments, the amount of cDNA before qPCR was standardised. Ulvé et al. (2008) standardised the amount of RNA before reverse transcription and compared the Cq values obtained for genes of *L. lactis* in cheeses at different ripening times. Even if it is not possible by this method to quantitatively compare the abundance of different RNA targets in the same sample (which would need *in vitro-*transcribed RNA standards), large differences in abundance may be shown. Direct comparisons of Cq values with a standardised amount of RNA have also been used to investigate the effect of cell separation from the cheese matrix before RNA extraction (Monnet et al., 2008). Bleve et al. (2003) observed a correlation between standard plate counts of yeasts and moulds present in spoiled commercial food products and the Cq values obtained by reverse transcription qPCR analysis with primers targeting the fungal actin gene. To follow gene expression of *P. freudenreichii* and *Lb. paracasei* during cheese-making, Falentin et al. (2010) measured the amount of cDNA copies of the target sequence after reverse transcription, and divided this value by the corresponding number of cells, which was measured by qPCR analysis of DNA extracted from the cheese samples. From these analyses, it could be concluded that the metabolic activity of *Lb. paracasei* cells reached a maximum during the first part of ripening, whereas the maximum activity of *P. freudenreichii* was reached later. A similar approach was used for the study of the metabolic activity of *Lb. helveticus* and *Str. thermophilus* cells during the ripening of Emmental cheese

(Falentin et al., 2012).

One disadvantage of all absolute quantification analyses is the significant reduction in the number of experimental samples that can be run on a single plate because a standard curve has to be included in each reaction run. In relative quantification methods, the amount of RNA targets in samples is expressed relative to the amount of the same target present in another sample, which is designated as the calibrator. This calibrator is chosen among the samples being compared (Cikos and Koppel, 2009). The advantage of this method is that standard curves don't have to be included in each run. However, this does not compensate for variations in reverse transcription efficiency and in RNA extraction efficiency from one sample to another. To compensate for this sample-to-sample variation, the quantity of RNA target is usually normalised to the quantity of one or several internal reference genes. These reference genes must be shown to be stable under the experimental conditions being examined, and are evaluated using software programmes such as geNorm or Bestkeeper. Two ideal reference genes are expected to have an identical expression ratio in all samples, whatever the experimental conditions. In the geNorm procedure (Vandesompele et al., 2002), the Cq values of each sample are transformed into relative quantities (Q) with a calibrator (cal) sample and using the gene-specific PCR efficiency (E), calculated as follows: Q = E (calCq – sampleCq). Normalisation is then applied by dividing the relative quantities of genes of interest by the geometric mean of the relative quantities of selected reference genes (normalisation factor). The 16S rRNA gene was used as reference gene to follow the expression of *L. lactis* nisin genes in a model cheese (Trmcic et al., 2011) . Several groups of genes could be distinguished based on expression profiles as a function of time, which contributed to a better knowledge of the regulation of nisin biosynthesis. For normalisation of gene transcripts from *Pseudomonas* spp., *Enterococcus* spp., *Pediococcus (P.) pentosaceus* and *Lb. casei* during the manufacturing of an experimental Montasio cheese, Carraro et al. (2011) used one couple of primers targeting the 16S rRNA of all bacteria present. The calculated fold-change does not reflect the specific gene expression of each population, but rather an expression taking into account the total amount of 16S rRNA. Cretenet et al. (2011) quantified the expression of several genes from *L. lactis* in model cheeses made from ultrafiltered milk, using *gyrB* (DNA gyrase subunit B) as reference gene. The histidine decarboxylase gene (*hdcA*) present in certain *Str. thermophilus* strains is involved in the synthesis histamine, a biogenic amine which may be accumulated in cheeses. The expression of *hdcA* was studied under conditions common to cheese-making, using the gene encoding the alpha subunit of the RNA polymerase (*rpoA*) as reference gene (Rossi et al., 2011). In this case, the stability of reference gene expression was assessed by absolute quantification of the transcripts obtained from fixed amounts of RNA. Up-regulation of *hdcA* occurred in the presence of free histidine and salt, and repression after thermisation. In bacteria, the gene encoding the elongation factor TU (*tuf*) is frequently used as reference gene in reverse transcription qPCR analyses. The expression of this gene by *L. lactis* was investigated in model cheeses by relative quantification using the total amount of RNA for normalisation, i.e. with reverse transcriptions performed with a fixed amount of RNA (Monnet et al., 2008). In this case, one has to check that potential biases, such as differences of reverse transcription efficiencies among the samples being studied, do not interfere. With this method, the calculated gene expression does not represent the expression relative to other mRNA transcripts, but rather the expression relative to the ribosomal RNA, which form most RNA. A large decrease of *tuf* expression, up to 100-fold, was observed after a few days. This decrease probably reflected the global decrease of mRNA transcription in the cheese matrix, after the end of growth of *L. lactis*. Duquenne et al. (2010) were able to quantify the

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 29

Experimental cheeses (Falentin et al., 2012)

Experimental cheeses (Monnet et al., 2012)

Milk cultures (Thevenard et al.,

Experimental cheeses (Duquenne et al.,

2012)

2010)

(Carraro et al., 2011)

(Bleve et al., 2003)

(Ablain et al., 2009) (Fumian et al., 2009)

(Fumian et al., 2009)

Target population Target sequence Food matrix References

16S rRNA Montasio cheese

manufacturing

products, including milk and yoghurt

Artificially contaminated Camembert cheeses

Artificially

contaminated cheeses

16S rRNA, *tuf*

*Arthrobacter arilaitensis* 16S rRNA, *gyrB* (DNA

*Str. thermophilus* two-component system genes

*S. aureus gyrB* (DNA gyrase

*S. aureus* 16S rRNA, *nuc*

Noroviruses ORF1-ORF2 junction

region

(elongation factor Tu), *groL* (chaperonin GroEL)

gyrase subunit B), *ftsZ* (cell division protein), *recA* (recombinase A), *rpoB* (RNA polymerase beta chain), *rpoA* (RNA polymerase alpha chain), *tuf* (elongation factor Tu), *dnaG* (DNA primase), and genes involved in iron acquisition

Yeasts and moulds actin gene Commercial food

subunit B), *ftsZ* (cell division protein), *hu* (DNA-binding protein), *rplD* (50S ribosomal protein L4), *recA* (recombinase A), *sodA* (superoxide dismutase), *gap* (glyceraldehyde-3-

dehydrogenase), *rpoB* (RNA polymerase beta chain), *pta* (phosphate acetyltransferase), *tpi* (triose phosphate isomerase), *sea* (enterotoxin A), *sed* (enterotoxin D)

(thermonuclease)

Table 2. Examples of applications of reverse-transcription qPCR to dairy products.

phosphate

*Lb. helveticus* and *Str.* 

*Lb. casei*, *P. pentosaceus*, *Str. thermophilus*, *Enterococcus* spp., *Pseudomonas* spp.

*thermophilus*

expression of *Staphyloccus aureus* enterotoxins genes in model cheeses using a set of three stably expressed reference genes. A similar approach was applied for the study of the growth of *L. lactis* subsp. *cremoris* strains under conditions simulating cheddar cheese manufacture (Taïbi et al., 2011) and for the study of iron acquisition by *Arthrobacter arilaitensis* in experimental cheeses (Monnet et al., 2012).


expression of *Staphyloccus aureus* enterotoxins genes in model cheeses using a set of three stably expressed reference genes. A similar approach was applied for the study of the growth of *L. lactis* subsp. *cremoris* strains under conditions simulating cheddar cheese manufacture (Taïbi et al., 2011) and for the study of iron acquisition by *Arthrobacter* 

Experimental cheeses (Monnet et al., 2008)

Experimental cheeses (Trmcic et al., 2011)

Experimental cheeses (Ulvé et al., 2008)

Experimental cheeses (Cretenet et al., 2011)

Experimental cheeses (Taïbi et al., 2011)

Milk cultures (Rossi et al., 2011)

Experimental Emmental cheese

Milk cultures (La Gioia et al., 2011)

(Falentin et al., 2010)

Milk cultures (Smeianov et al., 2007)

Target population Target sequence Food matrix References

and 27 protein-encoding

nisin biosynthesis

(glyceraldehyde 3 phosphate

dehydrogenase), *purM*  (phosphoribosylaminoimidazole synthetase), *cysK* (cysteine synthase), *ldh*

dehydrogenase), *citD* (citrate lyase acyl-carrier protein), *gyrA* (DNA gyrase subunit A)

*gltD*, *lacC, gapA, gapB, pdhB, aldB, butA, noxE, murF, dnaK, chiA, pepN, gyrB, pi139, pi302*

*glyA*, *groEL*, *oppA*, *pepQ*, *purD*, *ldh*, *holin1*, *holin2*

*ldh*, *clpP*, *oppA*, *oppC*, *pepO2*, *pepT2*, *prtH*, *prtH2*, *purA*, *pyrR*

decarboxylase)

decarboxylase)

(elongation factor TU), *Gro*L (chaperonin

16S rRNA, *tuf*

GroEL)

*arilaitensis* in experimental cheeses (Monnet et al., 2012).

genes

Tu), *gapB*

(L-lactate

 *L. lactis* subsp. *lactis bcaT*, *codY*, *serA*, *cysK*,

*L. lactis* subsp*. cremoris bcaT*, *clpE*, *dnaG*, *gapA*,

*Lb. helveticus asnA*, *cysE*, *dapA*, *serA*, *L-*

*Str. thermophilus hdcA* (histidine

*Str. thermophilus tdcA* (tyrosine

*P. freudenreichii* and *Lb.* 

*paracasei*

*L. lactis* subsp. *lactis* 16S rRNA, 23S rRNA

*L. lactis* 11 genes involved in

*L. lactis* subsp. *lactis tuf* (elongation factor


Table 2. Examples of applications of reverse-transcription qPCR to dairy products.

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 31

with maltodextrin at pH 4 (Muller et al., 2010). The published data on the optimisation of DNA isolation from microencapsulated bacteria are scarce however since the first step of bacterial DNA isolation from the product is separation of the bacterial cells from the matrix, the conditions and procedures found suitable for viable count (CFU) determination in samples containing microencapsulated bacteria may be a good starting point also for DNA

Due to the specificities described above, there are no universal standard procedures and media/buffers for the rehydration of probiotic products and quantification of probiotics in such products, either by the assessment of viable counts or by PCR-based methods. Often the authors do not explain in detail the preparation of the samples of probiotic products but refer to the standards such as ISO 6887-1:2000 on the general rules for the preparation of the initial suspension and decimal dilutions of food and animal feeding stuffs, or ISO 6887- 5:2010 including specific rules for the preparation of milk and milk products which are applicable also to dried milk products and milk-based infant foods. ISO 20838:2006 provides the overall framework for qualitative methods for the detection of food-borne pathogens in or isolated from food and feed matrices using the polymerase chain reaction (PCR), but can also be applied to other matrices, for example environmental samples, or to the detection of other microorganisms under investigation. However, the standards do not contain detailed protocols which have to be developed specifically considering the properties of the

Champagne et al. (2011) recently published recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices by plate counting, but the

Microbial analysis of probiotic food supplements and pharmaceutical preparations require standardised and accurate procedures for the reactivation of dehydrated cultures. Among the resuspension buffers, ¼ Ringer solution with or without cysteine (0,05 %), peptone physiological solution (0.1% wt/vol peptone, 0.85% wt/vol NaCl) or water are used most often (Temmerman et al., 2003; Masco et al., 2005; Masco et al., 2007; Kramer et al., 2009; Bogovic Matijasic et al., 2010). For the preparation of mesophilic cultures for qPCR analysis, which present similar medium as probiotic formulations, Friedrich and Lenke (2006) used

Usually the probiotic cells are removed by centrifugation from the product matrix before being exposed to the cell lysis. Drisko et al. (2005) exceptionally resuspended the products directly in TE buffer (10 mM Tris–HCl with pH 8.0, 1 mM EDTA) and proceeded with SDS and proteinase K treatment. After the lysis of bacterial cells, phenol/chloroform extraction or different kits such as the QIAamp®DNA stool mini kit (Qiagen), the NucleoSpin® food kit (Macherey–Nagel), Wizard Genomic DNA Purification kit (Promega), Maxwell 16 Cell

Lyophilised probiotic products can also be resuspended in water and the suspension added directly in PCR mixture, without previous isolation of bacterial DNA. This way Vitali et al. (2003) for instance carried out the real-time PCR quantification of three *Bifidobacterium* strains in a pharmaceutical product VSL-3 containing lyophilised bacteria

recommendations relevant for the DNA isolation are not available.

DNA Purification Kit (Promega) are most commonly used.

PBS and sodium citrate (1% wt/vol).

isolation.

products.

and excipients.

## **3. Application of PCR-based methods to non-dairy probiotic products**

## **3.1 Nucleic acid extraction from non-dairy probiotic products**

Probiotic products comprise probiotic dairy products and probiotic food supplements which appear in several forms, like powders, capsules, tablets, suspensions etc. containing the lyophilised, dried or microencapsulated bacterial cells. Since an overview of the nucleic acid extraction and PCR application in dairy products in general have already been addressed in this chapter, we focus here on the non-dairy probiotic products such as food supplements or pharmaceutical preparations. The protocols of DNA or RNA extraction from different probiotic products have to be properly adapted to the matrix in order to achieve satisfactory yield and efficient PCR amplification. It is important to evaluate whether the components of the product other than microbial cells influence the extraction and amplification steps. Probiotic formulations may contain polysaccharides, salts, oils (microencapsulated) or proteins (milk-based) which have been demonstrated to affect the extraction or inhibit amplification by direct interaction with DNA or by interference with the polymerases used in PCR. DNA isolation from the samples containing milk which is among the common ingredients of probiotic formulations, requires multiple steps such as centrifugation, heating or cation exchange to remove proteins, calcium ions and fats (Cressier and Bissonnette, 2011).

An increasing amount of non-dairy probiotic products contain microencapsulated probiotic cells. Depending on the microencapsulation technique (spray-drying, coacervation, cocrystallisation, molecular inclusion) and the matrix and coating materials used, the physicochemical properties of microcapsules differ much. Microcapsules containing probiotic bacteria are often insoluble in water, in order to allow their controlled release in the intestine. In order to enable the release of bacterial cells and DNA to the medium, particular treatment and diluents different from the commonly used (Ringer solution, peptone saline solution, water) are needed, for example addition of emulsifiers (anionic, cationic) or nonionic detergents such as Tween 80 (Champagne et al., 2010; Burgain et al., 2011).

When probiotics are microencapsulated in alginate beads, a calcium-binding solution such as phosphates or citrates is most often used to dissolve the particles. Another problem presents dried, fat-based spray-coated probiotic bacteria which can be found in different products in a form of powders, capsules, tablets, suspension in oil or for example in chocolate. One of the concerns could be that fat coating on the particles would prevent hydration, resulting in unsatisfactory recovery of viable bacteria and under-estimation of CFU counts.

The selection of rehydration method and solutions significantly influenced the results of CFU determination by plate counting in microencapsulated *Lb. rhamnosus* R0011 or *Bifidobacterium (B.) longum* ATCC 15708 cultures spray-coated with fat. Tween 80 did not result in direct improvement of the recovery of CFU, while the addition of fat improved it. The authors concluded that the methods appropriate for the analysis of free cells in dried probiotics may not be optimal for the analysis of spray-coated ME cultures (Champagne et al., 2010). The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. Maximum recovery of *B.standardised longum* NCC3001 was achieved at 30-min reconstitution at pH 8, in the presence of 2% l-arabinose and with a ratio of 1:100 of powder to diluent, while *Lb. johnsonii* La1 showed highest recovery after reconstitution, when mixed

Probiotic products comprise probiotic dairy products and probiotic food supplements which appear in several forms, like powders, capsules, tablets, suspensions etc. containing the lyophilised, dried or microencapsulated bacterial cells. Since an overview of the nucleic acid extraction and PCR application in dairy products in general have already been addressed in this chapter, we focus here on the non-dairy probiotic products such as food supplements or pharmaceutical preparations. The protocols of DNA or RNA extraction from different probiotic products have to be properly adapted to the matrix in order to achieve satisfactory yield and efficient PCR amplification. It is important to evaluate whether the components of the product other than microbial cells influence the extraction and amplification steps. Probiotic formulations may contain polysaccharides, salts, oils (microencapsulated) or proteins (milk-based) which have been demonstrated to affect the extraction or inhibit amplification by direct interaction with DNA or by interference with the polymerases used in PCR. DNA isolation from the samples containing milk which is among the common ingredients of probiotic formulations, requires multiple steps such as centrifugation, heating or cation exchange to remove proteins, calcium ions and fats

An increasing amount of non-dairy probiotic products contain microencapsulated probiotic cells. Depending on the microencapsulation technique (spray-drying, coacervation, cocrystallisation, molecular inclusion) and the matrix and coating materials used, the physicochemical properties of microcapsules differ much. Microcapsules containing probiotic bacteria are often insoluble in water, in order to allow their controlled release in the intestine. In order to enable the release of bacterial cells and DNA to the medium, particular treatment and diluents different from the commonly used (Ringer solution, peptone saline solution, water) are needed, for example addition of emulsifiers (anionic, cationic) or non-

When probiotics are microencapsulated in alginate beads, a calcium-binding solution such as phosphates or citrates is most often used to dissolve the particles. Another problem presents dried, fat-based spray-coated probiotic bacteria which can be found in different products in a form of powders, capsules, tablets, suspension in oil or for example in chocolate. One of the concerns could be that fat coating on the particles would prevent hydration, resulting in unsatisfactory recovery of viable bacteria and under-estimation of

The selection of rehydration method and solutions significantly influenced the results of CFU determination by plate counting in microencapsulated *Lb. rhamnosus* R0011 or *Bifidobacterium (B.) longum* ATCC 15708 cultures spray-coated with fat. Tween 80 did not result in direct improvement of the recovery of CFU, while the addition of fat improved it. The authors concluded that the methods appropriate for the analysis of free cells in dried probiotics may not be optimal for the analysis of spray-coated ME cultures (Champagne et al., 2010). The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. Maximum recovery of *B.standardised longum* NCC3001 was achieved at 30-min reconstitution at pH 8, in the presence of 2% l-arabinose and with a ratio of 1:100 of powder to diluent, while *Lb. johnsonii* La1 showed highest recovery after reconstitution, when mixed

ionic detergents such as Tween 80 (Champagne et al., 2010; Burgain et al., 2011).

**3. Application of PCR-based methods to non-dairy probiotic products** 

**3.1 Nucleic acid extraction from non-dairy probiotic products** 

(Cressier and Bissonnette, 2011).

CFU counts.

with maltodextrin at pH 4 (Muller et al., 2010). The published data on the optimisation of DNA isolation from microencapsulated bacteria are scarce however since the first step of bacterial DNA isolation from the product is separation of the bacterial cells from the matrix, the conditions and procedures found suitable for viable count (CFU) determination in samples containing microencapsulated bacteria may be a good starting point also for DNA isolation.

Due to the specificities described above, there are no universal standard procedures and media/buffers for the rehydration of probiotic products and quantification of probiotics in such products, either by the assessment of viable counts or by PCR-based methods. Often the authors do not explain in detail the preparation of the samples of probiotic products but refer to the standards such as ISO 6887-1:2000 on the general rules for the preparation of the initial suspension and decimal dilutions of food and animal feeding stuffs, or ISO 6887- 5:2010 including specific rules for the preparation of milk and milk products which are applicable also to dried milk products and milk-based infant foods. ISO 20838:2006 provides the overall framework for qualitative methods for the detection of food-borne pathogens in or isolated from food and feed matrices using the polymerase chain reaction (PCR), but can also be applied to other matrices, for example environmental samples, or to the detection of other microorganisms under investigation. However, the standards do not contain detailed protocols which have to be developed specifically considering the properties of the products.

Champagne et al. (2011) recently published recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices by plate counting, but the recommendations relevant for the DNA isolation are not available.

Microbial analysis of probiotic food supplements and pharmaceutical preparations require standardised and accurate procedures for the reactivation of dehydrated cultures. Among the resuspension buffers, ¼ Ringer solution with or without cysteine (0,05 %), peptone physiological solution (0.1% wt/vol peptone, 0.85% wt/vol NaCl) or water are used most often (Temmerman et al., 2003; Masco et al., 2005; Masco et al., 2007; Kramer et al., 2009; Bogovic Matijasic et al., 2010). For the preparation of mesophilic cultures for qPCR analysis, which present similar medium as probiotic formulations, Friedrich and Lenke (2006) used PBS and sodium citrate (1% wt/vol).

Usually the probiotic cells are removed by centrifugation from the product matrix before being exposed to the cell lysis. Drisko et al. (2005) exceptionally resuspended the products directly in TE buffer (10 mM Tris–HCl with pH 8.0, 1 mM EDTA) and proceeded with SDS and proteinase K treatment. After the lysis of bacterial cells, phenol/chloroform extraction or different kits such as the QIAamp®DNA stool mini kit (Qiagen), the NucleoSpin® food kit (Macherey–Nagel), Wizard Genomic DNA Purification kit (Promega), Maxwell 16 Cell DNA Purification Kit (Promega) are most commonly used.

Lyophilised probiotic products can also be resuspended in water and the suspension added directly in PCR mixture, without previous isolation of bacterial DNA. This way Vitali et al. (2003) for instance carried out the real-time PCR quantification of three *Bifidobacterium* strains in a pharmaceutical product VSL-3 containing lyophilised bacteria and excipients.

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 33

Probiotic food supplements and pharmaceutical preparations are widespread and commercially important. The most important parameters of their quality are appropriate labelling of probiotic bacteria and adequate number of them in the products. This is still not such an easy task since standardised methods are available for only avery limited number of probiotic bacteria in dairy products such as Lb*. acidophilus* (ISO 20128/IDF 192:2006) and *Bifidobacterium* (ISO 29981/IDF 220:2010). This speaks in favour of using molecular techniques which are rapid, sensitive and specific. Several PCR tests for detection of pathogens in foods have been validated, harmonised, and commercialised to make PCR a standard tool used by food microbiology laboratories (Maurer, 2011; Postollec et al., 2011). In the probiotic field there is still much to do in terms of the application of PCR-based methods for the control of probiotic products. Conventional PCR is very useful for the detection of labelled species or genera in the probiotic products. While several applications of this technique in food, including probiotic fermented dairy products, can be found in the literature (Table 1), the reports dealing with probiotic food supplements or pharmaceutical preparations are still few (Table 3). Among the targets which have been used in PCR analysis of probiotic products in the form of capsules, tablets or powders there are most often 16S rDNA or 16S-23S intergenic spacer (IS) regions which appear in the cells in multiple copies, contain several species or genus-specific regions and enable higher sensitivity than single copy genes. In addition to the ribosomal genes, several monocopy genes have also already been used for PCR or real-time PCR of probiotics such as *htrA, pepIP, rpoA,* β-galactosidase gene, or *recA* gene (Table 3). Primers for *htrA*-trypsin-like serine protease gene were used originally by Fortina et al. (2001), for *pepIP*-immunopeptidase proline gene pepIP by Torriani et al. (2007) and for *rpoA*-RNA polymerase alpha subunit gene by Naser et al. (2007). The main advantage of the application of genes that usually appear in one copy is that they enable accurate quantification by real-time PCR also in the mixed populations of bacteria belonging to different species, while the number of rRNA

**3.2.2 Real-time PCR quantification of probiotic bacteria in non-dairy products** 

the examination of possible inhibition of PCR reaction is always required.

It is well known that many food ingredients, including fats, proteins, divalent cations, and phenolic compounds, can act as PCR inhibitors. Some of the ingredients may also hinder the adequate microbial cell separations from the sample matrix. Another common problem is non-heterogeneous distribution of target cells in the samples, the presence of microbial aggregates which are difficult to disrupt or high amounts of non-target microbiota (Brehm-Stecher et al., 2009). In the analysis of probiotic products in general the usual approach is to separate first the bacterial target cells from the matrix, which in the case of lyophilised or dried products is usually not such a difficult task and may be successfully performed by rehydration of the samples followed by centrifugation. This way most of the potential inhibitory compounds are removed. Inhibitors are further removed also during the nucleic acids purification steps which have been described above. However, as some of the inhibitors may still be present in the samples intended for quantitative PCR (qPCR) analysis,

**3.2 Detection or quantification of probiotics in non-dairy probiotic products by PCR 3.2.1 PCR detection of labelled probiotic bacteria in probiotic food supplements or** 

**pharmaceutical preparations** 

genes copies differs among the species.


Table 3. Examples of applications of PCR, qPCR or PCR-DGGE to probiotic food supplements or pharmaceutical products.

sequence

16S-23S IS, *htrA*, *pepIP*, *rpoA*

16S rDNA, 16S-23S IS

16S-23S IS, β-galactosidase

16S rDNA, *recA* genes

16S rDNA, 16S-23S IS

16S-23S IS

PCR 16S rDNA,

Table 3. Examples of applications of PCR, qPCR or PCR-DGGE to probiotic food

gene

16S rDNA capsules,

tablets

16S rDNA capsules (Kramer et al.,

capsules, powders, tablets

powder sachets

Capsules, powder, pastilles

16S rDNA not stated (Masco et al., 2005)

PCR 16S rDNA,

PCR 16S rDNA,

Form of product

capsules, tablets, powder sachets, chewable tablets, bottled products

References

capsules (Bogovic Matijasic

2003)

not stated (Drisko et al., 2005)

2009)

(Masco et al., 2007)

(Vitali et al., 2003)

(Bogovic Matijasic and Rogelj, 2006)

et al., 2010)

(Temmerman et al.,

(Aureli et al., 2010)

Target population Method Target

*B. bifidum, Bacillus coagulans, Lb. acidophilus, Lb. casei, Lb. delbrueckii* subsp. *bulgaricus, Lb. delbrueckii* subsp. *lactis, Lb. helveticus, Lb. kefiri, Lb. paracasei, Lb. plantarum, Lb. reuteri, Lb. rhamnosus, Lb. salivarius, Lc. Lactis, P. freudenreichii subsp. freudenreichii, P. freudenreichii subsp. shermanii, Str. thermophilus*

*Lb. acidophilus, Lc. lactis, E. faecium, B. bifidum, B. lactis, Lb. rhamnosus, Lb. helveticus, Bacillus cereus, Lb. delbrueckii subsp. bulgaricus, Str. thermophilus* 

*Lb. delbrueckii* subsp. *bulgaricus, Lb. salivarius, Lb. plantarum, Lb. rhamnosus, Lb. acidophilus, B. infantis, Lb. casei, Lb. brevis, B. lactis, Str. thermophilus, B. bifidum* 

*Lb. acidophilus, B. animalis subsp.* 

*B. animalis subsp. lactis, B. longum*  biotype *longum, B. bifidum, B. animalis* subsp. *lactis, B. bifidum, B. breve, B. longum* biotype *longum, B. longum* biotype

*B. animalis* subsp. *lactis, B. breve, B. bifidum, B. longum* biotype

*B.standardised infantis Y1, B.standardised breve Y8, B.standardised longum Y10* 

*Lb. acidophilus, B.standardised infantis v. liberorum, Ent. faecium, B. bifidum, Lb. delbrueckii* subsp. b*ulgaricus, Str. thermophilus, B. longum, B. breve, Lb. rhamnosus, L.* 

supplements or pharmaceutical products.

*lactis* 

*infantis*

*longum* 

*lactis* 

*Lb. gasseri, E. faecium, B. infantis* real-time

PCR

PCR-DGGE

real-time PCR

nested PCR-DGGE

real-time PCR

PCR, real-time PCR

#### **3.2.1 PCR detection of labelled probiotic bacteria in probiotic food supplements or pharmaceutical preparations**

Probiotic food supplements and pharmaceutical preparations are widespread and commercially important. The most important parameters of their quality are appropriate labelling of probiotic bacteria and adequate number of them in the products. This is still not such an easy task since standardised methods are available for only avery limited number of probiotic bacteria in dairy products such as Lb*. acidophilus* (ISO 20128/IDF 192:2006) and *Bifidobacterium* (ISO 29981/IDF 220:2010). This speaks in favour of using molecular techniques which are rapid, sensitive and specific. Several PCR tests for detection of pathogens in foods have been validated, harmonised, and commercialised to make PCR a standard tool used by food microbiology laboratories (Maurer, 2011; Postollec et al., 2011). In the probiotic field there is still much to do in terms of the application of PCR-based methods for the control of probiotic products. Conventional PCR is very useful for the detection of labelled species or genera in the probiotic products. While several applications of this technique in food, including probiotic fermented dairy products, can be found in the literature (Table 1), the reports dealing with probiotic food supplements or pharmaceutical preparations are still few (Table 3). Among the targets which have been used in PCR analysis of probiotic products in the form of capsules, tablets or powders there are most often 16S rDNA or 16S-23S intergenic spacer (IS) regions which appear in the cells in multiple copies, contain several species or genus-specific regions and enable higher sensitivity than single copy genes. In addition to the ribosomal genes, several monocopy genes have also already been used for PCR or real-time PCR of probiotics such as *htrA, pepIP, rpoA,* β-galactosidase gene, or *recA* gene (Table 3). Primers for *htrA*-trypsin-like serine protease gene were used originally by Fortina et al. (2001), for *pepIP*-immunopeptidase proline gene pepIP by Torriani et al. (2007) and for *rpoA*-RNA polymerase alpha subunit gene by Naser et al. (2007). The main advantage of the application of genes that usually appear in one copy is that they enable accurate quantification by real-time PCR also in the mixed populations of bacteria belonging to different species, while the number of rRNA genes copies differs among the species.

#### **3.2.2 Real-time PCR quantification of probiotic bacteria in non-dairy products**

It is well known that many food ingredients, including fats, proteins, divalent cations, and phenolic compounds, can act as PCR inhibitors. Some of the ingredients may also hinder the adequate microbial cell separations from the sample matrix. Another common problem is non-heterogeneous distribution of target cells in the samples, the presence of microbial aggregates which are difficult to disrupt or high amounts of non-target microbiota (Brehm-Stecher et al., 2009). In the analysis of probiotic products in general the usual approach is to separate first the bacterial target cells from the matrix, which in the case of lyophilised or dried products is usually not such a difficult task and may be successfully performed by rehydration of the samples followed by centrifugation. This way most of the potential inhibitory compounds are removed. Inhibitors are further removed also during the nucleic acids purification steps which have been described above. However, as some of the inhibitors may still be present in the samples intended for quantitative PCR (qPCR) analysis, the examination of possible inhibition of PCR reaction is always required.

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 35

2000). Real-time PCR has a potential to replace conventional enumeration of probiotic bacteria, used for routine monitoring of quality of a probiotic product and for stability studies. However, since probiotic bacteria have to be viable to exert their activity the contribution of DNA arising from non-viable cells to the result of quantification has to be

An approach using PMA or EMA treatment of the samples before the DNA isolation seems promising in this regard. Such DNA-intercalating dyes are able to bind upon exposure to bright visible light to DNA and, consequently, to inhibit PCR amplification of the DNA which is free or inside the bacterial cells with the damaged membrane. Although probiotic bacteria in the products are represented in different stages not only as viable or dead (Bunthof and Abee, 2002), the most important criterion for distinguishing between viable and irreversibly damaged cells is membrane integrity. The treatment of bacteria with EMA as a promising tool of DNA-based differentiation between viable and dead pathogenic bacteria was first proposed by Nogva et al. in 2003 (Nogva et al., 2003). In the following years several applications of this approach have been reported, where the method was optimised for different complex media such as faeces, fermented milk and environmental samples (Garcia-Cayuela et al., 2009; Fittipaldi et al., 2011; Fujimoto et al., 2011). Since ethidium monoazide has been suggested as being toxic to some viable cells, PMA has been proposed as a more appropriate alternative to EMA (Nocker et al., 2006; Fujimoto et al.,

The PMA treatment in combination with real-time PCR was applied for determination of probiotic strains *Lb. acidophilus* LA-5 and *B. animalis* ssp. *lactis* BB-12 bacteria in a pharmaceutical formulation in the form of capsules (Kramer et al., 2009). The possible effects of the ingredients of the product on PMA treatment of the samples including the photoactivation step, as well as on the PCR reaction were evaluated in the study. The ability of PMA to inhibit amplification of DNA derived from damaged bacterial cells was confirmed on bacteria from pure cultures of *Lb. acidophilus* or *B. animalis* ssp. *lactis* in a 1% (w/v) suspension of ingredients which are otherwise present in the product and on probiotic product (1% w/w). Other examples of direct application of PMA-real time PCR on the lyophilised probiotic products have not been found in the literature. The efficient PMA treatment of fermented dairy products containing the same two strains, *Lb. acidophilus* LA-5 and *B. animalis* ssp. *lactis* BB-12, have also been described (Garcia-Cayuela et al., 2009). In order to eliminate the milk ingredients prior to the PMA treatment, the samples were adjusted to pH 6.5 with 1 M NaOH, then casein micelles were dispersed by theaddition of 1 M trisodium citrate, and bacterial cells were harvested by centrifugation. The obtained cells were resuspended in water, treated with PMA and used for DNA isolation. Fujimoto et al. (2010) evaluated strain-specific qPCR with PMA treatment for quantification of viable *B. breve* strain Yakult (BbrY) in human faeces. The quantification was carried out on faecal samples spiked with BbrY strain, on the BbrY culture and on the faecal samples collected from the healthy volunteers who ingested a commercially available fermented milk product containing BbrY, once daily for 10 days. They confirmed the use of a combination of qPCR with PMA treatment and BbrY-specific primers as a quick and accurate method for

quantification of viable BbrY in faecal samples (Fujimoto et al., 2011).

Viable probiotics may be enumerated also by a qPCR-based method targeting mRNA of different housekeeping genes. The advantage of using mRNA targets over the use of DNA

excluded.

2011).

In order to exclude possible inhibition, Masco et al. (2007) prepared bacteria-free sample matrices of the food supplement, spiked them with known quantities of reference bifidobacteria and compared the standard curve slopes and efficiencies obtained during PCR amplification of pure cultures and spiked samples. The finding that amplification of pure cultures and spiked samples was equally efficient indicated that the product's matrix did not have a significant impact on DNA extraction and subsequent real-time PCR performance.

Similarly Kramer et al. (2009) prepared the standard curves from the mixture of bacterial cells of *Lb. acidophilus* or *B. animalis* ssp. *lactis* with a suspension of filler ingredients of probiotic capsules. The concentrations of Beneo synergy (0,73%), saccharose (0,11%), dextrose anhydrous (0,10%), microcrystalline cellulose (0,026 %), potato starch (0,026 %) and Mg-stearate (0,019 %) in the standard samples were the same as in the 1:100 diluted product. In addition, the negligible effect of the product ingredients on the PCR amplification efficiency was demonstrated also by the comparison of the standard curves prepared from the DNA derived from pure cultures of from the suspensions of cultures in the simulated filler.

In a further study of the same probiotic pharmaceutical preparation (Bogovič Matijašić, not published) the authors treated 1% (w/v) suspension of the product with heat (two times 120 °C/15 min). The total DNA in the suspension was mostly degraded as was demonstrated by real-time PCR amplification using *Lactobacillus* (LactoR'F/LBFR, (Songjinda et al., 2007) ) or *Bifidobacterium* (Bif-F/Bif-R, (Rinttila et al., 2004). The two-times autoclaved suspension was spiked with either of the two strains isolated from the product, and after that DNA isolated from the spiked suspension was used for the generation of standard curves.

Bogovič Matijašić et al. (2010) prepared the simulated matrix with Mg stearate (0.22%), lactose (0.39%) and starch (0.39%) corresponding to the concentrations of these ingredients in a 100-fold sample of the product in capsules. DNA was isolated by different procedures from the standard samples containing simulated matrix with a known amount of added probiotic bacteria of *Lb. gasseri, B. infantis* or *Ec. faecium*. When DNA was isolated by heat treatment (100 °C/5 min) of the standard bacterial suspensions in 1% Triton X-100, the ingredients of the prepared suspension affected the real-time PCR result. Since the filler ingredients themselves did not show any fluorescence interaction when included directly in PCR reactions, the lower concentration of probiotic determined in real-time PCR was attributed to the less effective DNA extraction by heat-triton treatment due to the presence of Mg stearate, lactose and starch. Any effect was however observed when DNA was isolated by the Maxwell system (Promega) based on the use of MagneSil paramagnetic particles (Bogovic Matijasic et al., 2010).

In all studies presented in Table 3, the real- time PCR analyses were performed by SYBR® Green I chemistry. The species specificity of the PCR was ensured by using species-specific oligonucleotide primers and additionally validated by melting point analysis.

#### **3.2.3 Viability determination of probiotics by PCR-based methods**

The viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, such as unsatisfactory selectivity, too-low a recovery, long incubation time, underestimation of cells in aggregates or chains morphology etc. (Breeuwer and Abee,

In order to exclude possible inhibition, Masco et al. (2007) prepared bacteria-free sample matrices of the food supplement, spiked them with known quantities of reference bifidobacteria and compared the standard curve slopes and efficiencies obtained during PCR amplification of pure cultures and spiked samples. The finding that amplification of pure cultures and spiked samples was equally efficient indicated that the product's matrix did not have a significant impact on DNA extraction and subsequent real-time PCR

Similarly Kramer et al. (2009) prepared the standard curves from the mixture of bacterial cells of *Lb. acidophilus* or *B. animalis* ssp. *lactis* with a suspension of filler ingredients of probiotic capsules. The concentrations of Beneo synergy (0,73%), saccharose (0,11%), dextrose anhydrous (0,10%), microcrystalline cellulose (0,026 %), potato starch (0,026 %) and Mg-stearate (0,019 %) in the standard samples were the same as in the 1:100 diluted product. In addition, the negligible effect of the product ingredients on the PCR amplification efficiency was demonstrated also by the comparison of the standard curves prepared from the DNA derived from pure cultures of from the suspensions of cultures in the simulated

In a further study of the same probiotic pharmaceutical preparation (Bogovič Matijašić, not published) the authors treated 1% (w/v) suspension of the product with heat (two times 120 °C/15 min). The total DNA in the suspension was mostly degraded as was demonstrated by real-time PCR amplification using *Lactobacillus* (LactoR'F/LBFR, (Songjinda et al., 2007) ) or *Bifidobacterium* (Bif-F/Bif-R, (Rinttila et al., 2004). The two-times autoclaved suspension was spiked with either of the two strains isolated from the product, and after that DNA isolated

Bogovič Matijašić et al. (2010) prepared the simulated matrix with Mg stearate (0.22%), lactose (0.39%) and starch (0.39%) corresponding to the concentrations of these ingredients in a 100-fold sample of the product in capsules. DNA was isolated by different procedures from the standard samples containing simulated matrix with a known amount of added probiotic bacteria of *Lb. gasseri, B. infantis* or *Ec. faecium*. When DNA was isolated by heat treatment (100 °C/5 min) of the standard bacterial suspensions in 1% Triton X-100, the ingredients of the prepared suspension affected the real-time PCR result. Since the filler ingredients themselves did not show any fluorescence interaction when included directly in PCR reactions, the lower concentration of probiotic determined in real-time PCR was attributed to the less effective DNA extraction by heat-triton treatment due to the presence of Mg stearate, lactose and starch. Any effect was however observed when DNA was isolated by the Maxwell system (Promega) based on the use of MagneSil paramagnetic

In all studies presented in Table 3, the real- time PCR analyses were performed by SYBR® Green I chemistry. The species specificity of the PCR was ensured by using species-specific

The viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, such as unsatisfactory selectivity, too-low a recovery, long incubation time, underestimation of cells in aggregates or chains morphology etc. (Breeuwer and Abee,

oligonucleotide primers and additionally validated by melting point analysis.

**3.2.3 Viability determination of probiotics by PCR-based methods** 

from the spiked suspension was used for the generation of standard curves.

particles (Bogovic Matijasic et al., 2010).

performance.

filler.

2000). Real-time PCR has a potential to replace conventional enumeration of probiotic bacteria, used for routine monitoring of quality of a probiotic product and for stability studies. However, since probiotic bacteria have to be viable to exert their activity the contribution of DNA arising from non-viable cells to the result of quantification has to be excluded.

An approach using PMA or EMA treatment of the samples before the DNA isolation seems promising in this regard. Such DNA-intercalating dyes are able to bind upon exposure to bright visible light to DNA and, consequently, to inhibit PCR amplification of the DNA which is free or inside the bacterial cells with the damaged membrane. Although probiotic bacteria in the products are represented in different stages not only as viable or dead (Bunthof and Abee, 2002), the most important criterion for distinguishing between viable and irreversibly damaged cells is membrane integrity. The treatment of bacteria with EMA as a promising tool of DNA-based differentiation between viable and dead pathogenic bacteria was first proposed by Nogva et al. in 2003 (Nogva et al., 2003). In the following years several applications of this approach have been reported, where the method was optimised for different complex media such as faeces, fermented milk and environmental samples (Garcia-Cayuela et al., 2009; Fittipaldi et al., 2011; Fujimoto et al., 2011). Since ethidium monoazide has been suggested as being toxic to some viable cells, PMA has been proposed as a more appropriate alternative to EMA (Nocker et al., 2006; Fujimoto et al., 2011).

The PMA treatment in combination with real-time PCR was applied for determination of probiotic strains *Lb. acidophilus* LA-5 and *B. animalis* ssp. *lactis* BB-12 bacteria in a pharmaceutical formulation in the form of capsules (Kramer et al., 2009). The possible effects of the ingredients of the product on PMA treatment of the samples including the photoactivation step, as well as on the PCR reaction were evaluated in the study. The ability of PMA to inhibit amplification of DNA derived from damaged bacterial cells was confirmed on bacteria from pure cultures of *Lb. acidophilus* or *B. animalis* ssp. *lactis* in a 1% (w/v) suspension of ingredients which are otherwise present in the product and on probiotic product (1% w/w). Other examples of direct application of PMA-real time PCR on the lyophilised probiotic products have not been found in the literature. The efficient PMA treatment of fermented dairy products containing the same two strains, *Lb. acidophilus* LA-5 and *B. animalis* ssp. *lactis* BB-12, have also been described (Garcia-Cayuela et al., 2009). In order to eliminate the milk ingredients prior to the PMA treatment, the samples were adjusted to pH 6.5 with 1 M NaOH, then casein micelles were dispersed by theaddition of 1 M trisodium citrate, and bacterial cells were harvested by centrifugation. The obtained cells were resuspended in water, treated with PMA and used for DNA isolation. Fujimoto et al. (2010) evaluated strain-specific qPCR with PMA treatment for quantification of viable *B. breve* strain Yakult (BbrY) in human faeces. The quantification was carried out on faecal samples spiked with BbrY strain, on the BbrY culture and on the faecal samples collected from the healthy volunteers who ingested a commercially available fermented milk product containing BbrY, once daily for 10 days. They confirmed the use of a combination of qPCR with PMA treatment and BbrY-specific primers as a quick and accurate method for quantification of viable BbrY in faecal samples (Fujimoto et al., 2011).

Viable probiotics may be enumerated also by a qPCR-based method targeting mRNA of different housekeeping genes. The advantage of using mRNA targets over the use of DNA

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 37

set was confirmed on the human faecal samples by LightCycler (Roche Diagnostics) real-

A similar approach was applied to *B. breve* strain Yakult (BbrY) by Fujimoto et al. (2011). The specificity of the BbrY-specific primer set was confirmed by PCR using DNA from 112 bacterial strains belonging to *B. breve* species, of other *Bifidobacterium* species and representatives of 11 other genera. The BbrY-specific primers were used in a real-time PCR with PMA treatment to measure the number of BbrY in the faeces of subjects who drank a

The qPCR method based on the amplification of a strain-specific DNA fragment identified by suppressive subtractive hybridisation was developed recently for specific and sensitive monitoring of *P. acidipropionici* P169 in animal feed and rumen fluid by Peng et al. (2011). The specificity and amplification efficiency was assessed on 44 *Propionibacterium* strains and also in complex microbial communities containing *P. acidipropionici* P169 (Peng et al., 2011). Certain strains have specific features that distinguish them from the other related strains, such as for example bacteriocin production. Treven et al. (submitted) evaluated the possibility of using bacteriocin-specific primers for the detection and quantification of *Lb. gasseri* K7 probiotic strain, a producer of at least two two-component bacteriocins (Zoric Peternel et al., 2010). Two pairs of primers, namely GasA\_401/610F/R and GasB\_2610- 2807F/R showed specificity for total gene cluster of gassericin K7 A (Genbank EF392861) or gassericin K7 B (Genbank AY307382) respectively as established by PCR assays using DNA of 18 reference strains belonging to *Lb. acidophilus* group and 45 faecal samples of adult volunteers who have never consumed K7 strain. GasA\_401/610F/R primers were also found to be especially useful also for real-time PCR quantification of gassericin K7 A gene cluster in faecal samples and also for *Lb. gasseri* K7-specific detection or quantification in the

Microorganisms are very important components of fermented dairy products, including probiotic food, as well as of probiotic food supplements and pharmaceutical preparations. PCR-based methods have become indispensable in the microbiological analysis of these groups of products. In the field of fermented dairy products, several applications based on PCR have been developed with the aim to detect, identify and quantify either unwanted bacteria, which may negatively influence the sensory properties of food or may be pathogenic, or beneficial microorganisms which are added as starter cultures or probiotic cultures. Beside PCR analysis of DNA, reverse transcription real-time PCR analysis of mRNA transcript is particularly useful, especially in studies of the physiology and functionality of bacteria in the food environment. In the probiotic field, PCR is expected to be increasingly applied in quality control in terms of detection and quantification of labelled probiotic bacteria in probiotic food supplements or pharmaceutical preparations, and in viability analysis of probiotics in the products. In addition to the already well-established methods described in this chapter, ever easier access to the next generation sequencing may replace some PCR approaches as molecular fingerprint, metagenomic and metatranscriptomic analyses. The access to increasing number of complete bacterial genomes may also facilitate the strain-specific analysis of probiotics or other bacteria

fermented milk product containing BbrY (Fujimoto et al., 2011).

biological samples (Treven et al. submitted).

through identification of strain-specific sequences.

**4. Conclusions** 

time PCR.

or rRNA is mainly in the instability of mRNA molecules which is degraded soon after the cell death. Reimann et al. (2010) demonstrated in *B. longum* NCC2705 a good correlation between measured mRNA levels of cysB and purB, two constitutively expressed housekeeping genes and plate counts. The 400-bp fragment of purB was degraded more quickly than the 57-bp fragments of cysB and purB, and is therefore a better marker of cell viability (Reimann et al., 2010).

With the availability of new highthroughput molecular technologies such as microarray technology and next-generation sequencing, new possibilities are now open to further development of the viability PCR approach also in the probiotic field, as has already been similarly demonstrated for selected pathogenic bacteria in environmental samples (Nocker et al., 2009; Nocker et al., 2010).

## **3.3 Strain-specific detection or quantification of probiotics**

While species- or genus-specific primers are not so difficult to construct, the problem arises when we intend to confirm different strains of the same species in the product. A variety of PCR-based genotyping techniques such as random amplified polymorphic DNA analysis (RAPD), repetitive sequence-based PCR (rep-PCR), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) ribotyping etc., are successfully used everywhere to distinguish different strains also closely related among each other (Li et al., 2009). The genotyping methods, however, require the cultivation of pure cultures of examined strains and do not enable quantification. For PCR quantification of individual probiotic strains in the probiotic products or different environments (faeces, mucosa...) strain specific primers or probes are needed. So far it has been very difficult to find strainspecific genome sequences as a target for the construction of strain-specific primers or probes.

In the study of Vitali et al. (2003), the 16S rDNA and 16S-23S rDNA-targeted strain-specific primers were designed for the quantitative detection of *B. infantis* Y1, *B. breve* Y8 and *B. longum* Y10 used in a pharmaceutical probiotic product VSL-3. These were applied in PCR, and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures (Vitali et al., 2003).

Maruo et al. (2006) generated a *L. lactis* subsp. *cremoris* FC-specific primer pair by using a specific 1164-bp long RAPD band sequence. The specificity of this primer pair has been proven with 23 *L. lactis* subsp. *cremoris* strains and 20 intestinal bacterial species, and realtime PCR determination of FC strain in the faeces was demonstrated to be successful. Marzotto et al. (2006) selected specific primers for the putative probiotic strain *Lb. paracasei*  A LcA-Fw and LcA-Rv from the terminal regions of the 250-bp RAPD fragment sequence tested the selectivity with 20 different *Lactobacillus* species and 39 *Lb. paracasei* strains. The primers were successfully applied in PCR analysis of faecal samples (Marzotto et al., 2006).

Strain-specific PCR primers and probes for real-time PCR and for conventional PCR were designed based on the sequence of RAPD products, also for *Lb. rhamnosus* GG which is one of the most studied probiotic strains (Ahlroos and Tynkkynen, 2009). The strain specificity of the primers was verified in conventional PCR using a set of strains – six *Lb. rhamnosus*, one *Lb. casei* and one *Lb. zeae*, while the applicability of the GG strain-specific primer probe set was confirmed on the human faecal samples by LightCycler (Roche Diagnostics) realtime PCR.

A similar approach was applied to *B. breve* strain Yakult (BbrY) by Fujimoto et al. (2011). The specificity of the BbrY-specific primer set was confirmed by PCR using DNA from 112 bacterial strains belonging to *B. breve* species, of other *Bifidobacterium* species and representatives of 11 other genera. The BbrY-specific primers were used in a real-time PCR with PMA treatment to measure the number of BbrY in the faeces of subjects who drank a fermented milk product containing BbrY (Fujimoto et al., 2011).

The qPCR method based on the amplification of a strain-specific DNA fragment identified by suppressive subtractive hybridisation was developed recently for specific and sensitive monitoring of *P. acidipropionici* P169 in animal feed and rumen fluid by Peng et al. (2011). The specificity and amplification efficiency was assessed on 44 *Propionibacterium* strains and also in complex microbial communities containing *P. acidipropionici* P169 (Peng et al., 2011).

Certain strains have specific features that distinguish them from the other related strains, such as for example bacteriocin production. Treven et al. (submitted) evaluated the possibility of using bacteriocin-specific primers for the detection and quantification of *Lb. gasseri* K7 probiotic strain, a producer of at least two two-component bacteriocins (Zoric Peternel et al., 2010). Two pairs of primers, namely GasA\_401/610F/R and GasB\_2610- 2807F/R showed specificity for total gene cluster of gassericin K7 A (Genbank EF392861) or gassericin K7 B (Genbank AY307382) respectively as established by PCR assays using DNA of 18 reference strains belonging to *Lb. acidophilus* group and 45 faecal samples of adult volunteers who have never consumed K7 strain. GasA\_401/610F/R primers were also found to be especially useful also for real-time PCR quantification of gassericin K7 A gene cluster in faecal samples and also for *Lb. gasseri* K7-specific detection or quantification in the biological samples (Treven et al. submitted).

## **4. Conclusions**

36 Polymerase Chain Reaction

or rRNA is mainly in the instability of mRNA molecules which is degraded soon after the cell death. Reimann et al. (2010) demonstrated in *B. longum* NCC2705 a good correlation between measured mRNA levels of cysB and purB, two constitutively expressed housekeeping genes and plate counts. The 400-bp fragment of purB was degraded more quickly than the 57-bp fragments of cysB and purB, and is therefore a better marker of cell

With the availability of new highthroughput molecular technologies such as microarray technology and next-generation sequencing, new possibilities are now open to further development of the viability PCR approach also in the probiotic field, as has already been similarly demonstrated for selected pathogenic bacteria in environmental samples (Nocker

While species- or genus-specific primers are not so difficult to construct, the problem arises when we intend to confirm different strains of the same species in the product. A variety of PCR-based genotyping techniques such as random amplified polymorphic DNA analysis (RAPD), repetitive sequence-based PCR (rep-PCR), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) ribotyping etc., are successfully used everywhere to distinguish different strains also closely related among each other (Li et al., 2009). The genotyping methods, however, require the cultivation of pure cultures of examined strains and do not enable quantification. For PCR quantification of individual probiotic strains in the probiotic products or different environments (faeces, mucosa...) strain specific primers or probes are needed. So far it has been very difficult to find strainspecific genome sequences as a target for the construction of strain-specific primers or

In the study of Vitali et al. (2003), the 16S rDNA and 16S-23S rDNA-targeted strain-specific primers were designed for the quantitative detection of *B. infantis* Y1, *B. breve* Y8 and *B. longum* Y10 used in a pharmaceutical probiotic product VSL-3. These were applied in PCR, and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic

Maruo et al. (2006) generated a *L. lactis* subsp. *cremoris* FC-specific primer pair by using a specific 1164-bp long RAPD band sequence. The specificity of this primer pair has been proven with 23 *L. lactis* subsp. *cremoris* strains and 20 intestinal bacterial species, and realtime PCR determination of FC strain in the faeces was demonstrated to be successful. Marzotto et al. (2006) selected specific primers for the putative probiotic strain *Lb. paracasei*  A LcA-Fw and LcA-Rv from the terminal regions of the 250-bp RAPD fragment sequence tested the selectivity with 20 different *Lactobacillus* species and 39 *Lb. paracasei* strains. The primers were successfully applied in PCR analysis of faecal samples (Marzotto et al., 2006). Strain-specific PCR primers and probes for real-time PCR and for conventional PCR were designed based on the sequence of RAPD products, also for *Lb. rhamnosus* GG which is one of the most studied probiotic strains (Ahlroos and Tynkkynen, 2009). The strain specificity of the primers was verified in conventional PCR using a set of strains – six *Lb. rhamnosus*, one *Lb. casei* and one *Lb. zeae*, while the applicability of the GG strain-specific primer probe

viability (Reimann et al., 2010).

et al., 2009; Nocker et al., 2010).

probes.

**3.3 Strain-specific detection or quantification of probiotics** 

characteristics in batch cultures (Vitali et al., 2003).

Microorganisms are very important components of fermented dairy products, including probiotic food, as well as of probiotic food supplements and pharmaceutical preparations. PCR-based methods have become indispensable in the microbiological analysis of these groups of products. In the field of fermented dairy products, several applications based on PCR have been developed with the aim to detect, identify and quantify either unwanted bacteria, which may negatively influence the sensory properties of food or may be pathogenic, or beneficial microorganisms which are added as starter cultures or probiotic cultures. Beside PCR analysis of DNA, reverse transcription real-time PCR analysis of mRNA transcript is particularly useful, especially in studies of the physiology and functionality of bacteria in the food environment. In the probiotic field, PCR is expected to be increasingly applied in quality control in terms of detection and quantification of labelled probiotic bacteria in probiotic food supplements or pharmaceutical preparations, and in viability analysis of probiotics in the products. In addition to the already well-established methods described in this chapter, ever easier access to the next generation sequencing may replace some PCR approaches as molecular fingerprint, metagenomic and metatranscriptomic analyses. The access to increasing number of complete bacterial genomes may also facilitate the strain-specific analysis of probiotics or other bacteria through identification of strain-specific sequences.

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 39

Baruzzi, F., Matarante, A., Caputo, L., Morea, M., 2005. Development of a culture-

Bleve, G., Rizzotti, L., Dellaglio, F., Torriani, S., 2003. Development of reverse transcription

Bogovic Matijasic, B., Koman Rajsp, M., Perko, B., Rogelj, I., 2007. Inhibition of *Clostridium tyrobutyricum* in cheese by *Lactobacillus gasseri*. Int. Dairy J. 17, 157-166. Bogovic Matijasic, B.B., Obermajer, T., Rogelj, I., 2010. Quantification of *Lactobacillus gasseri,* 

Bogovic Matijasic, B.B., Rogelj, I., 2006. Demonstration of suitability of probiotic products -

Bonaiti, C., Parayre, S., Irlinger, F., 2006. Novel extraction strategy of ribosomal RNA and

Bonetta, S., Bonetta, S., Carraro, E., Rantsiou, K., Cocolin, L., 2008. Microbiological

Boss, R., Naskova, J., Steiner, A., Graber, H.U., 2011. Mastitis diagnostics: Quantitative PCR for *Staphylococcus aureus* genotype B in bulk tank milk. J. Dairy Sci. 94, 128-137. Botsaris, G., Slana, I., Liapi, M., Dodd, C., Economides, C., Rees, C., Pavlik, I., 2010. Rapid

Breeuwer, P., Abee, T., 2000. Assessment of viability of microorganisms employing

Brehm-Stecher, B., Young, C., Jaykus, L.A., Tortorello, M.L., 2009. Sample Preparation: The

Bremer, H., Dennis, P.P., 1996. Modulation of chemical composition and other parameters of

Cellular and Molecular Biology, ASM Press, Washington DC, pp. 1553-1569. Bunthof, C.J., Abee, T., 2002. Development of a flow cytometric method to analyze

Burgain, J., Gaiani, C., Linder, M., Scher, J., 2011. Encapsulation of probiotic living cells: From laboratory scale to industrial applications. J. Food Eng. 104, 467-483. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R.,

Cailliez-Grimal, C., Miguindou-Mabiala, R., Leseine, M., Revol-Junelles, A.M., Milliere, J.B.,

polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169-193.

milk and cheese. Int. J. Food Microbiol. 141, S87-S90.

Forgotten Beginning. J. Food Prot. 72, 1774-1789.

PCR Experiments. Clin. Chem. 55, 611-622.

fluorescence techniques. Int. J. Food Microbiol. 55, 193-200.

in stretched cheese. J. Rapid Meth. Aut. Mic. 13, 177-192.

Appl. Environ. Microbiol. 69, 4116-4122.

time PCR. Food Control 21, 419-425.

Agro Food Ind. Hi-Tech 17, 38-40.

107, 171-179.

Microbiol. 25, 786-792.

Microbiol. 68, 2934-2942.

independent polymerase chain reaction-based assay for the detection of lactobacilli

(RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products.

*Enterococcus faecium* and *Bifidobacterium infantis* in a probiotic OTC drug by real-

An emphasis on survey of commercial products obtained on Slovenian market.

genomic DNA from cheese for PCR-based investigations. Int. J. Food Microbiol.

characterisation of Robiola di Roccaverano cheese using PCR-DGGE. Food

detection methods for viable *Mycobacterium avium* subspecies *paratuberculosis* in

the cell by growth rate, in: Neidhart, F.C. (Ed.), Escherichia coli and Salmonella:

subpopulations of bacteria in probiotic products and dairy starters. Appl. Environ.

Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time

2005. Quantitative polymerase chain reaction used for the rapid detection of

#### **5. References**


Ablain, W., Hallier Soulier, S., Causeur, D., Gautier, M., Baron, F., 2009. A simple and rapid

Ablain, W., Hallier Soulier, S., Causeur, D., Gautier, M., Baron, F., 2009. A simple and rapid

Abriouel, H., Martin-Platero, A., Maqueda, M., Valdivia, E., Martinez-Bueno, M., 2008.

Ahlroos, T., Tynkkynen, S., 2009. Quantitative strain-specific detection of *Lactobacillus* 

Alarcon, B., Vicedo, B., Aznar, R., 2006. PCR-based procedures for detection and

Alegría, Á., Álvarez-Martín, P., Sacristán, N., Fernández, E., Delgado, S., Mayo, B., 2009.

Allmann, M., Hofelein, C., Koppel, E., Luthy, J., Meyer, R., Niederhauser, C., Wegmuller, B.,

Amoroso, M.G., Salzano, C., Cioffi, B., Napoletano, M., Garofalo, F., Guarino, A., Fusco, G.,

Andrighetto, C., Marcazzan, G., Lombardi, A., 2004. Use of RAPD-PCR and TTGE for the

Aprodu, I., Walcher, G., Schelin, J., Hein, I., Norling, B., Rådström, P., Nicolau, A., Wagner,

Arteau, M., Labrie, S., Roy, D., 2010. Terminal-restriction fragment length polymorphism

Aureli, P., Fiore, A., Scalfaro, C., Casale, M., Franciosa, G., 2010. National survey outcomes

Baker, G.C., Smith, J.J., Cowan, D.A., 2003. Review and re-analysis of domain-specific 16S

*Brucella* spp. in water buffalo milk. Food Control 22, 1466-1470.

communities in Camembert cheese. Int. Dairy J. 20, 545-554.

cheese. Dairy Sci. Technol. 89, 69-81.

cheese. Dairy Sci. Technol. 89, 69-81.

127, 200-208.

Microbiol. 100, 352-364.

Microbiol. 38, 400-405.

Microbiol. 145, S61-S65.

primers. J. Microbiol. Meth. 55, 541-555.

Int. J. Food Microbiol. 136, 44-51.

506-514.

146, 85-97.

273.

method for the disruption of *Staphylococcus aureus*, optimized for quantitative reverse transcriptase applications: Application for the examination of Camembert

method for the disruption of *Staphylococcus aureus,* optimized for quantitative reverse transcriptase applications: Application for the examination of Camembert

Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods. Int. J. Food Microbiol.

*rhamnosus* GG in human faecal samples by real-time PCR. J. Appl. Microbiol. 106,

quantification of *Staphylococcus aureus* and their application in food. J. Appl.

Diversity and evolution of the microbial populations during manufacture and ripening of Casín, a traditional Spanish, starter-free cheese made from cow's milk.

Candrian, U., 1995. Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products. Res. Microbiol.

2011. Validation of a Real-time PCR assay for fast and sensitive quantification of

evaluation of biodiversity of whey cultures for Grana Padano cheese. Lett. Appl.

M., 2011. Advanced sample preparation for the molecular quantification of *Staphylococcus aureus* in artificially and naturally contaminated milk. Int. J. Food

and automated ribosomal intergenic spacer analysis profiling of fungal

on commercial probiotic food supplements in Italy. Int. J. Food Microbiol. 137, 265-

**5. References** 


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 41

Delavenne, E., Mounier, J., Asmani, K., Jany, J.-L., Barbier, G., Le Blay, G., 2011. Fungal diversity in cow, goat and ewe milk. Int. J. Food Microbiol. 151, 247-251. Delbes, C., Ali-Mandjee, L., Montel, M.-C., 2007. Monitoring Bacterial Communities in Raw

Delbes, C., Montel, M.C., 2005. Design and application of a *Staphylococcus*-specific single

Dolci, Barmaz, Zenato, Pramotton, Alessandria, Cocolin, Rantsiou, Ambrosoli, 2009.

Donaghy, J.A., Rowe, M.T., Rademaker, J.L.W., Hammer, P., Herman, L., De Jonghe, V.,

Drisko, J., Bischoff, B., Giles, C., Adelson, M., Rao, R.V.S., McCallum, R., 2005. Evaluation of

Duquenne, M., Fleurot, I., Aigle, M., Darrigo, C., Borezee-Durant, E., Derzelle, S., Bouix, M.,

Duthoit, F., Godon, J.-J., Montel, M.-C., 2003. Bacterial community dynamics during

Duthoit, F., Tessier, L., Montel, M.C., 2005. Diversity, dynamics and activity of bacterial

El-Baradei, G., Delacroix-Buchet, A., Ogier, J.-C., 2007. Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese. Appl. Environ. Microbiol. 73, 1248-1255. Ercolini, D., Frisso, G., Mauriello, G., Salvatore, F., Coppola, S., 2008. Microbial diversity in

Ercolini, D., Hill, P.J., Dodd, C.E.R., 2003. Bacterial community structure and location in

Stilton cheese. Appl. Environ. Microbiol. 69, 3540-3548.

Analyses. Appl. Environ. Microbiol. 73, 1882-1891.

PDO cheese. Int. J. Food Microbiol. 143, 71-75.

reaction analysis. Dig. Dis. Sci. 50, 1113-1117.

Microbiol. 77, 247-257.

Appl. Microbiol. 41, 169-174.

128-135.

76, 1367-1374.

1198-1208.

Microbiol. 69, 3840-3848.

Int. J. Food Microbiol. 124, 164-170.

the *Lactococcus lactis* Transcriptome in Cheeses Made from Milk Concentrated by Ultrafiltration Reveals Multiple Strategies of Adaptation to Stresses. Appl. Environ.

Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based

strand conformation polymorphism-PCR analysis to monitor *Staphylococcus* populations diversity and dynamics during production of raw milk cheese. Lett.

Maturing dynamics of surface microflora in Fontina PDO cheese studied by culture-dependent and -independent methods. J. Appl. Microbiol. 106, 278-287. Dolci, P., Alessandria, V., Rantsiou, K., Bertolino, M., Cocolin, L., 2010. Microbial diversity,

dynamics and activity throughout manufacturing and ripening of Castelmagno

Blanchard, B., Duhem, K., Vindel, E., 2008. An inter-laboratory ring trial for the detection and isolation of *Mycobacterium avium* subsp. *paratuberculosis* from raw milk artificially contaminated with naturally infected faeces. Food Microbiol. 25,

five probiotic products for label claims by DNA extraction and polymerase chain

Deperrois-Lafarge, V., Delacroix-Buchet, A., 2010. Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese. Appl. Environ. Microbiol.

production of registered designation of origin salers cheese as evaluated by 16S rRNA gene single-strand conformation polymorphism analysis. Appl. Environ.

populations in 'Registered Designation of Origin' Salers cheese by single-strand conformation polymorphism analysis of 16S rRNA genes. J. Appl. Microbiol. 98,

natural whey cultures used for the production of Caciocavallo Silano PDO cheese.

*Carnobacterium* species from French soft cheeses. FEMS Microbiol. Lett. 250, 163- 169.


Callon, C., Delbes, C., Duthoit, F., Montel, M.C., 2006. Application of SSCP-PCR

Cardenas, E., Tiedje, J.M., 2008. New tools for discovering and characterizing microbial

Carraro, L., Maifreni, M., Bartolomeoli, I., Martino, M.E., Novelli, E., Frigo, F., Marino, M.,

Casalta, E., Sorba, J.-M., Aigle, M., Ogier, J.-C., 2009. Diversity and dynamics of the

Champagn, C.P., Ross, R.P., Saarela, M., Hansen, K.F., Charalampopoulos, D., 2011.

Champagne, C.P., Raymond, Y., Tompkins, T.A., 2010. The determination of viable counts in

Chen, J., Zhang, L., Paoli, G.C., Shi, C., Tu, S.-I., Shi, X., 2010. A real-time PCR method for

Chiang, Y.-C., Fan, C.-M., Liao, W.-W., Lin, C.-K., Tsen, H.-Y., 2007. Real-Time PCR

Cikos, S., Koppel, J., 2009. Transformation of real-time PCR fluorescence data to target gene

Cocolin, L., Diez, A., Urso, R., Rantsiou, K., Comi, G., Bergmaier, I., Beimfohr, C., 2007.

Cocolin, L., Innocente, N., Biasutti, M., Comi, G., 2004. The late blowing in cheese: a new

Cogan, T.M., John, W.F., 2011. Cheese - Microbiology of Cheese, Encyclopedia of Dairy

Coppola, S., Blaiotta, G., Ercolini, D., Moschetti, G., 2001. Molecular evaluation of microbial

Cressier, B., Bissonnette, N., 2011. Assessment of an extraction protocol to detect the major mastitis-causing pathogens in bovine milk. Journal of Dairy Science 94, 2171-2184. Cretenet, M., Laroute, V., Ulve, V., Jeanson, S., Nouaille, S., Even, S., Piot, M., Girbal, L., Le

independent methods. Int. J. Food Microbiol. 120, 100-109.

alteration process. Int. J. Food Microbiol. 90, 83-91.

Sciences, Academic Press, San Diego, pp. 625-631.

cultures and in food matrices. Int. J. Food Microbiol. 149, 185-193.

comparative genomic analysis. Int. J. Food Microbiol. 137, 168-174.

169.

1111.

159.

414-420.

Appl. Microbiol. 29, 172-180.

Microbiol. 162, 231-239.

diversity. Curr. Opin. Biotechnol. 19, 544-549.

cheese. Int. J. Food Microbiol. 133, 243-251.

quantity. Anal. Biochem. 384, 1-10.

*Carnobacterium* species from French soft cheeses. FEMS Microbiol. Lett. 250, 163-

fingerprinting to profile the yeast community in raw milk Salers cheeses. Syst.

Cardazzo, B., 2011. Comparison of culture-dependent and -independent methods for bacterial community monitoring during Montasio cheese manufacturing. Res.

microbial community during the manufacture of Calenzana, an artisanal Corsican

Recommendations for the viability assessment of probiotics as concentrated

probiotic cultures microencapsulated by spray-coating. Food Microbiol. 27, 1104-

the detection of *Salmonella enterica* from food using a target sequence identified by

Detection of *Staphylococcus aureus* in Milk and Meat Using New Primers Designed from the Heat Shock Protein Gene htrA Sequence. J. Food Prot. 174; 70, 2855-2859. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid

guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-

Optimization of conditions for profiling bacterial populations in food by culture-

molecular approach based on PCR and DGGE to study the microbial ecology of the

diversity occurring in different types of Mozzarella cheese. J. Appl. Microbiol. 90,

Loir, Y., Loubiere, P., Lortal, S., Cocaign-Bousquet, M., 2011. Dynamic Analysis of

the *Lactococcus lactis* Transcriptome in Cheeses Made from Milk Concentrated by Ultrafiltration Reveals Multiple Strategies of Adaptation to Stresses. Appl. Environ. Microbiol. 77, 247-257.


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 43

Fujimoto, J., Tanigawa, K., Kudo, Y., Makino, H., Watanabe, K., 2011. Identification and

Fuka, M.M., Engel, M., Skelin, A., Redzepovic, S., Schloter, M., 2010. Bacterial communities

Fumian, T.M., Leite, J.P.G., Marin, V.A., Miagostovich, M.P., 2009. A rapid procedure for detecting noroviruses from cheese and fresh lettuce. J. Virol. Methods 155, 39-43. Furet, J.-P., Quenée, P., Tailliez, P., 2004. Molecular quantification of lactic acid bacteria in

Fusco, V., Quero, G.M., Morea, M., Blaiotta, G., Visconti, A., 2011. Rapid and reliable

Gala, E., Landi, S., Solieri, L., Nocetti, M., Pulvirenti, A., Giudici, P., 2008. Diversity of lactic

Garcia-Cayuela, T., Tabasco, R., Pelaez, C., Requena, T., 2009. Simultaneous detection and

using propidium monoazide and real-time PCR. Int. Dairy J. 19, 405-409. Giannino, M.L., Marzotto, M., Dellaglio, F., Feligini, M., 2009. Study of microbial diversity in

Grattepanche, F., Lacroix, C., Audet, P., Lapointe, G., 2005. Quantification by real-time PCR

Hein, I., Jorgensen, H.J., Loncarevic, S., Wagner, M., 2005. Quantification of *Staphylococcus* 

Hein, I., Lehner, A., Rieck, P., Klein, K., Brandl, E., Wagner, M., 2001. Comparison of

Henri-Dubernet, S., Desmasures, N., Guéguen, M., 2004. Culture-dependent and culture-

Henri-Dubernet, S., Desmasures, N., Gueguen, M., 2008. Diversity and dynamics of

Herman, L., Block, J.d., Renterghem, R.v., 1997. Isolation and detection of *Clostridium* 

Herman, L., Deridder, H., 1993. Cheese Components Reduce the Sensitivity of Detection of

independent methods. Int. J. Food Microbiol. 130, 188-195.

"Camembert de Normandie" cheese. Lait 84, 179-189.

142, 19-24.

97, 197-207.

528-537.

156, 554-563.

Microbiol. 125, 347-351.

Microbiol. Biotechnol. 66, 414-421.

Environ. Microbiol. 67, 3122-3126.

reaction. J. Dairy Res. 64, 311-314.

Microbiol. 54, 218-228.

29.

quantification of viable *Bifidobacterium breve* strain Yakult in human faeces by using strain-specific primers and propidium monoazide. J. Appl. Microbiol. 110, 209-217.

associated with the production of artisanal Istrian cheese. Int. J. Food Microbiol.

fermented milk products using real-time quantitative PCR. Int. J. Food Microbiol.

identification of *Staphylococcus aureus* harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR. Int. J. Food Microbiol. 144,

acid bacteria population in ripened Parmigiano Reggiano cheese. Int. J. Food

enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by

raw milk and fresh curd used for Fontina cheese production by culture-

of *Lactococcus lactis* subsp. *cremoris* in milk fermented by a mixed culture. Appl.

*aureus* in unpasteurised bovine and caprine milk by real-time PCR. Res. Microbiol.

different approaches to quantify *Staphylococcus aureus* cells by real-time quantitative PCR and application of this technique for examination of cheese. Appl.

independent methods for molecular analysis of the diversity of lactobacilli in

lactobacilli populations during ripening of RDO Camembert cheese. Can. J.

*tyrobutyricum* cells in semi-soft and hard cheeses using the polymerase chain

*Listeria monocytogenes* by the Polymerase Chain-Reaction. Neth. Milk Dairy J. 47, 23-


Ercolini, D., Mauriello, G., Blaiotta, G., Moschetti, G., Coppola, S., 2004. PCR-DGGE

Ercolini, D., Moschetti, G., Blaiotta, G., Coppola, S., 2001. The potential of a polyphasic PCR-

Falentin, H., Henaff, N., Le Bivic, P., Deutsch, S.-M., Parayre, S., Richoux, R., Sohier, D.,

FAO/WHO, 2002. Guidelines for the Evaluation of Probiotics in Food. London, Ontario,

Fernandez, M., del Rio, B., Linares, D.M., Martin, M.C., Alvarez, M.A., 2006. Real-time

Feurer, C., Irlinger, F., Spinnler, H.E., Glaser, P., Vallaeys, T., 2004a. Assessment of the rind

Feurer, C., Vallaeys, T., Corrieu, G., Irlinger, F., 2004b. Does smearing inoculum reflect the

Fittipaldi, M., Codony, F., Adrados, B., Camper, A.K., Morato, J., 2011. Viable Real-Time

Flórez, A.B., Mayo, B., 2006. Microbial diversity and succession during the manufacture and

Fontana, C., Cappa, F., Rebecchi, A., Cocconcelli, P.S., 2010. Surface microbiota analysis of

Fortina, M.G., Ricci, G., Mora, D., Parini, C., Manachini, P.L., 2001. Specific identification of

Freeman, W.M., Walker, S.J., Vrana, K.E., 1999. Quantitative RT-PCR: pitfalls and potential.

Friedrich, U., Lenke, J., 2006. Improved enumeration of lactic acid bacteria in mesophilic

bacteria: use in cheese production. J. Dairy Sci. 89, 3763-3769.

buffalo mozzarella cheese. J. Appl. Microbiol. 96, 263-270.

Food Microbiol. 144, 10-19.

Canada. April 30 and May 1, 2002.

J. Appl. Microbiol. 97, 546-556.

61, 7-12.

4171.

smear cheese? J. Dairy Sci. 87, 3189-3197.

Int. J. Food Microbiol. 138, 205-211.

BioTechniques 26, 112-122, 124-125.

Microb. Lett. 198, 85-89.

PCR-DGGE. Int. J. Food Microbiol. 110, 165-171.

culture-independent analyses. Syst. Appl. Microbiol. 24, 610-617.

end of the ripening of Emmental cheese. Food Microbiol. 29, 132-140. Falentin, H., Postollec, F., Parayre, S., Henaff, N., Le Bivic, P., Richoux, R., Thierry, A.,

fingerprints of microbial succession during a manufacture of traditional water

dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and

Thierry, A., Lortal, S., Postollec, F., 2012. Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the

Sohier, D., 2010. Specific metabolic activity of ripening bacteria quantified by realtime reverse transcription PCR throughout Emmental cheese manufacture. Int. J.

polymerase chain reaction for quantitative detection of histamine-producing

microbial diversity in a farm house-produced *vs* a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methods.

bacterial composition of the smear at the end of the ripening of a french soft, red-

PCR in Environmental Samples: Can All Data Be Interpreted Directly? Micr. Ecol.

ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by

Taleggio, Gorgonzola, Casera, Scimudin and Formaggio di Fossa Italian cheeses.

*Lactobacillus helveticus* by PCR with pepC, pepN and htrA targeted primers. FEMS

dairy starter cultures by using multiplex quantitative real-time PCR and flow cytometry-fluorescence in situ hybridization. Appl. Environ. Microbiol. 72, 4163-


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 45

Makhzami, S., Quenee, P., Akary, E., Bach, C., Aigle, M., Delacroix-Buchet, A., Ogier, J.C.,

Makino, H., Fujimoto, J., Watanabe, K., 2010. Development and evaluation of a real-time

Manuzon, M.Y., Hanna, S.E., Luo, H., Yu, Z., Harper, W.J., Wang, H.H., 2007. Quantitative

Marianelli, C., Martucciello, A., Tarantino, M., Vecchio, R., Iovane, G., Galiero, G., 2008.

Martin, M.C., Martinez, N., Rio, B.d., Ladero, V., Fernandez, M., Alvarez, M.A., 2010. A

Marzotto, M., Maffeis, C., Paternoster, T., Ferrario, R., Rizzotti, L., Pellegrino, M., Dellaglio,

affects the fecal microbiota of healthy infants. Res. Microbiol. 157, 857-866. Masco, L., Huys, G., De Brandt, E., Temmerman, R., Swings, J., 2005. Culture-dependent and

Masco, L., Vanhoutte, T., Temmerman, R., Swings, J., Huys, G., 2007. Evaluation of real-time

Masoud, W., Takamiya, M., Vogensen, F.K., Lillevang, S., Al-Soud, W.A., Sørensen, S.J.,

Mauriello, G., Moio, L., Genovese, A., Ercolini, D., 2003. Relationships between flavoring

McKillip, J.L., Jaykus, L.A., Drake, M.A., 2000. A comparison of methods for the detection of

Monnet, C., Back, A., Irlinger, F., 2012 (in press). Growth of Aerobic Ripening bacteria at the Cheese Surface is Limited by the Availability of Iron. Appl. Environ. Microbiol. Monnet, C., Correia, K., Sarthou, A.-S., Irlinger, F., 2006. Quantitative detection of

Monnet, C., Ulvé, V., Sarthou, A.-S., Irlinger, F., 2008. Extraction of RNA from cheese

electrophoresis (DGGE) and pyrosequencing. Int. Dairy J. 21, 142-148. Maurer, J.J., 2011. Rapid Detection and Limitations of Molecular Techniques. Ann. Rev.

enterococcal genes. J. Microbiol. Meth. 75, 485-490.

Buffalo Milk. J. Dairy Sci. 91, 3779-3786.

bifidobacteria. Int. J. Food Microbiol. 102, 221-230.

in probiotic products. Int. J. Food Microbiol. 113, 351-357.

industries. J. Dairy Sci. 93, 860-867.

Food Sci. Techn., Vol 2 2, 259-279.

Sci. 86, 486-497.

6972-6979.

Appl. Microbiol. 89, 49-55.

interest in dairy products. Int. J. Food Microbiol. 140, 76-83.

Time TaqMan PCR. Appl. Environ. Microbiol. 73, 1676-1677.

Serror, P., 2008. In situ gene expression in cheese matrices: application to a set of

quantitative PCR assay for detection and enumeration of yeasts of public health

Assessment of the Tetracycline Resistance Gene Pool in Cheese Samples by Real-

Evaluation of Molecular Methods for the Detection of *Brucella* Species in Water

novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting *Enterobacteriaceae* in the dairy and other food

F., Torriani, S., 2006. *Lactobacillus paracasei* A survives gastrointestinal passage and

culture-independent qualitative analysis of probiotic products claimed to contain

PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria

Jakobsen, M., 2011. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel

capabilities, bacterial composition, and geographical origin of natural whey cultures used for traditional water-buffalo mozzarella cheese manufacture. J. Dairy

*Escherichia coli* O157:H7 from artificially-contaminated dairy products using PCR. J.

*Corynebacterium casei* in cheese by real-time PCR. Appl. Environ. Microbiol. 72,

without prior separation of microbial cells. Appl. Environ. Microbiol. 74, 5724-5730.


Herthnek, D., Nielsen, S.S., Lindberg, A., Bölske, G., 2008. A robust method for bacterial

Karns, J.S., Van Kessel, J.S., McClusky, B.J., Perdue, M.L., 2007. Incidence of *Escherichia coli*

Kramer, M., Obermajer, N., Bogovic Matijasic, B., Rogelj, I., Kmetec, V., 2009. Quantification

La Gioia, F., Rizzotti, L., Rossi, F., Gardini, F., Tabanelli, G., Torriani, S., 2011. Identification

Ladero, V., Fernández, M., Alvarez, M.A., 2009. Effect of post-ripening processing on the

Ladero, V., Martínez, N., Martín, M.C., Fernández, M., Alvarez, M.A., 2008. qPCR for

Lafarge, V., Ogier, J.C., Girarda, V., Maladena, V., Leveau, J.Y., Delacroix-Buchet, A., 2004.

Larpin, S., Mondoloni, C., Goerges, S., Vernoux, J.-P., Gueguen, M., Desmasures, N., 2006.

Le Bourhis, A.G., Dore, J., Carlier, J.P., Chamba, J.F., Popoff, M.R., Tholozan, J.L., 2007.

Le Bourhis, A.G., Saunier, K., Dore, J., Carlier, J.P., Chamba, J.F., Popoff, M.R., Tholozan,

Le Dréan, G., Mounier, J., Vasseur, V., Arzur, D., Habrylo, O., Barbier, G., 2010.

Lee, Z.M.-P., Bussema, C., Schmidt, T.M., 2009. rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea. Nucleic Acids Res. 37, D489-D493. Li, W.J., Raoult, D., Fournier, P.E., 2009. Bacterial strain typing in the genomic era. FEMS

Liao, D., 1999. Concerted evolution: molecular mechanism and biological implications. Am.

Lopez-Enriquez, L., Rodriguez-Lazaro, D., Hernandez, M., 2007. Quantitative Detection of

*Clostridium tyrobutyricum* in Milk by Real-Time PCR. Appl. Environ. Microbiol. 73,

Polymerase Chain Reaction. J. Dairy Sci. 90, 3212-3219.

red-smear cheese. FEMS Yeast Res. 6, 1243-1253.

Appl. Environ. Microbiol. 71, 29-38.

Microbiol. Rev. 33, 892-916.

J. Hum. Genet. 64, 24-30.

Microbiol. 77, 1140-1144.

Dairy J. 19, 759-762.

Int. 43, 289-295.

169-178.

154-163.

138, 100-107.

3747-3751.

flow cytometry. Appl. Microbiol. Biotechnol. 84, 1137-1147.

lysis and DNA purification to be used with real-time PCR for detection of *Mycobacterium avium* subsp. *paratuberculosis* in milk. J. Microbiol. Meth. 75, 335-340.

O157:H7 and *E. coli* Virulence Factors in US Bulk Tank Milk as Determined by

of live and dead probiotic bacteria in lyophilised product by real-time PCR and by

of a Tyrosine Decarboxylase Gene (tdcA) in *Streptococcus thermophilus* 1TT45 and Analysis of Its Expression and Tyramine Production in Milk. Appl. Environ.

histamine and histamine-producing bacteria contents of different cheeses. Int.

quantitative detection of tyramine-producing bacteria in dairy products. Food Res.

Le potentiel de la TTGE pour l'étude bactérienne de quelques laits crus. Lait 84,

*Geotrichum candidum* dominates in yeast population dynamics in Livarot, a French

Contribution of *C. beijerinckii* and *C. sporogenes* in association with *C. tyrobutyricum* to the butyric fermentation in Emmental type cheese. Int. J. Food Microbiol. 113,

J.L., 2005. Development and validation of PCR primers to assess the diversity of *Clostridium* spp. in cheese by temporal temperature gradient gel electrophoresis.

Quantification of *Penicillium camemberti* and *P. roqueforti* mycelium by real-time PCR to assess their growth dynamics during ripening cheese. Int. J. Food Microbiol.


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 47

O'Grady, J., Ruttledge, M., Sedano-Balbas, S., Smith, T.J., Barry, T., Maher, M., 2009. Rapid

Omiccioli, E., Amagliani, G., Brandi, G., Magnani, M., 2009. A new platform for Real-Time

Ongol, M.P., Tanaka, M., Sone, T., Asano, K., 2009. A real-time PCR method targeting a gene

Parayre, S., Falentin, H., Madec, M.N., Sivieri, K., Le Dizes, A.S., Sohier, D., Lortal, S., 2007.

Peng, M., Smith, A.H., Rehberger, T.G., 2011. Quantification of *Propionibacterium* 

Postollec, F., Falentin, H., Pavan, S., Combrisson, J., Sohier, D., 2011. Recent advances in

Rademaker, J.L.W., Hoolwerf, J.D., Wagendorp, A.A., te Giffel, M.C., 2006. Assessment of

ripening by DNA population fingerprinting. Int. Dairy J. 16, 457-466. Rademaker, J.L.W., Peinhopf, M., Rijnen, L., Bockelmann, W., Noordman, W.H., 2005. The

T-RFLP DNA population fingerprint analysis. Int. Dairy J. 15, 785-794. Randazzo, C.L., Pitino, I., Ribbera, A., Caggia, C., 2010. Pecorino Crotonese cheese: Study of bacterial population and flavour compounds. Food Microbiol. 27, 363-374. Randazzo, C.L., Torriani, S., Akkermans, A.D.L., de Vos, W.M., Vaughan, E.E., 2002.

real-time PCR. Food Microbiol. 26, 4-7.

dairy products. J. Microbiol. Meth. 69, 431-441.

milk. Food Microbiol. 26, 615-622.

Microbiol. 77, 3898-3902.

Microbiol. 68, 1882-1785.

Int. J. Food Microbiol. 121, 99-105.

Food Microbiol. 21, 481-487.

898.

848-861.

detection of *Listeria monocytogenes* in food using culture enrichment combined with

PCR detection of *Salmonella* spp.*, Listeria monocytogenes* and *Escherichia coli* O157 in

sequence encoding 16S rRNA processing protein, rimM, for detection and enumeration of *Streptococcus thermophilus* in dairy products. Food Res. Int. 42, 893-

Easy DNA extraction method and optimisation of PCR-Temporal Temperature Gel Electrophoresis to identify the predominant high and low GC-content bacteria from

*acidipropionici* P169 Bacteria in Environmental Samples by Use of Strain-Specific Primers Derived by Suppressive Subtractive Hybridization. Appl. Environ.

quantitative PCR (qPCR) applications in food microbiology. Food Microbiol. 28,

microbial population dynamics during yoghurt and hard cheese fermentation and

surface microflora dynamics of bacterial smear-ripened Tilsit cheese determined by

Diversity, dynamics, and activity of bacterial communities during production of an artisanal sicilian cheese as evaluated by 16S rRNA analysis. Appl. Environ.

Siciliano cheese: microbial dynamics during manufacture assessed by culturing and

and vitality of *Listeria monocytogenes* in food as determined by quantitative PCR.

to follow lactic acid bacterial population dynamics during food fermentations.

Randazzo, C.L., Vaughan, E.E., Caggia, C., 2006. Artisanal and experimental Pecorino

Rantsiou, K., Alessandria, V., Urso, R., Dolci, P., Cocolin, L., 2008a. Detection, quantification

Rantsiou, K., Comi, G., Cocolin, L., 2004. The rpoB gene as a target for PCR-DGGE analysis

Rantsiou, K., Urso, R., Dolci, P., Comi, G., Cocolin, L., 2008b. Microflora of Feta cheese from

four Greek manufacturers. Int. J. Food Microbiol. 126, 36-42.

PCR-DGGE analyses. Int. J. Food Microbiol. 109, 1-8.


Moschetti, G., Blaiotta, G., Villani, F., Coppola, S., 2001. Nisin-producing organisms during

Mounier, J., Blay, G.L., Vasseur, V., Floch, G.L., Jany, J.L., Barbier, G., 2010. Application of

identification in red smear cheese surfaces. Lett. Appl. Microbiol. 51, 18-23. Mounier, J., Monnet, C., Jacques, N., Antoinette, A., Irlinger, F., 2009. Assessment of the

Muller, J.A., Stanton, C., Sybesma, W., Fitzgerald, G.F., Ross, R.P., 2010. Reconstitution

Neefs, J., Van de Peer, Y., De Rijk, P., Chapelle, S., De Wachter, R., 1993. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res. 21, 3025-3049. Nicolaisen, M.H., Baelum, J., Jacobsen, C.S., Sorensen, J., 2008. Transcription dynamics of the

Nikolic, M., Terzic-Vidojevic, A., Jovcic, B., Begovic, J., Golic, N., Topisirovic, L., 2008.

Nocker, A., Cheung, C.Y., Camper, A.K., 2006. Comparison of propidium monoazide with

Nocker, A., Mazza, A., Masson, L., Camper, A.K., Brousseau, R., 2009. Selective detection of

Nocker, A., Richter-Heitmann, T., Montijn, R., Schuren, F., Kort, R., 2010. Discrimination

Nogva, H.K., Dromtorp, S.M., Nissen, H., Rudi, K., 2003. Ethidium monoazide for DNA-

Nolan, T., Hands, R.E., Bustin, S.A., 2006. Quantification of mRNA using real-time RT-PCR.

Ogier, J.-C., Lafarge, V., Girard, V., Rault, A., Maladen, V., Gruss, A., Leveau, J.-Y.,

Ogier, J.-C., Son, O., Gruss, A., Tailliez, P., Delacroix-Buchet, A., 2002. Identification of the

removal of DNA from dead cells. J. Microbiol. Meth. 67, 310-320.

samples analyzed by 454 pyrosequencing. Int. Microbiol. 13, 59-65.

AEO106 (pRO101) in agricultural soil. Environ. Microbiol. 10, 571-579. Niederhauser, C., Candrian, U., Hofelein, C., Jermini, M., Buhler, H.P., Luthy, J., 1992. Use of

independent approaches. Int. J. Food Microbiol. 133, 31-37.

analyses. Int. J. Food Microbiol. 63, 109-116.

viability. J. Appl. Microbiol. 108, 1369-1379.

milk cheese. Int. J. Food Microbiol. 122, 162-170.

technology. J. Microbiol. Meth. 76, 253-261.

Biotechniques 34, 804-813.

Nature Protocols 1, 1559-1582.

Appl. Environ. Microbiol. 70, 5628-5643.

electrophoresis. Appl. Environ. Microbiol. 68, 3691-3701.

Environ. Microbiol. 58, 1564-1568.

traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE

denaturing high-performance liquid chromatography (DHPLC) for yeasts

microbial diversity at the surface of Livarot cheese using culture-dependent and

conditions for dried probiotic powders represent a critical step in determining cell

functional tfdA gene during MCPA herbicide degradation by *Cupriavidus necator*

Polymerase Chain-Reaction for Detection of *Listeria monocytogenes* in Food. Appl.

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's

ethidium monoazide for differentiation of live vs. dead bacteria by selective

live bacteria combining propidium monoazide sample treatment with microarray

between live and dead cells in bacterial communities from environmental water

based differentiation of viable and dead bacteria by 5 '-nuclease PCR.

Delacroix-Buchet, A., 2004. Molecular fingerprinting of dairy microbial ecosystems by use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis.

bacterial microflora in dairy products by temporal temperature gradient gel


Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products 49

Serhan, M., Cailliez-Grimal, C., Borges, F., Revol-Junelles, A.M., Hosri, C., Fanni, J., 2009.

Serpe, L., Gallo, P., Fidanza, N., Scaramuzzo, A., Fenizia, D., 1999. Single-step method for

Singh, J., Batish, V.K., Grover, S., 2009. A scorpion probe-based real-time PCR assay for detection of *E. coli* O157:H7 in dairy products. Foodborne Pathog. Dis. 6, 395-400. Slana, I., Kralik, P., Kralova, A., Pavlik, I., 2008. On-farm spread of *Mycobacterium avium*

Songjinda, P., Nakayama, J., Tateyama, A., Tanaka, S., Tsubouchi, M., Kiyohara, C.,

Stephan, R., Schumacher, S., Tasara, T., Grant, I.R., 2007. Prevalence of *Mycobacterium avium*

Stevens, K.A., Jaykus, L.A., 2004. Direct detection of bacterial pathogens in representative

Studer, E., Schaeren, W., Naskova, J., Pfaeffli, H., Kaufmann, T., Kirchhofer, M., Steiner, A.,

Taïbi, A., Dabour, N., Lamoureux, M., Roy, D., LaPointe, G., 2011. Comparative

Thevenard, B., Rasoava, N., Fourcassié, P., Monnet, V., Boyaval, P., Rul, F., 2011.

Torriani, S., Zapparoli, G., Dellaglio, F., 1999. Use of PCR-based methods for rapid

Treven, P., Trmcic, A., Obermajer, T., Rogelj, I., Bogovic Matijasic, B. Prevalence of

Detect *Staphylococcus aureus* in Milk. J. Dairy Sci. 91, 1893-1902.

Culture Medium. Appl. Environ. Microbiol. 73, 2661-2672.

Microbiol. 26, 645-652.

Biochem. 71, 2338-2342.

J. Dairy Sci. 90, 3590-3595.

Microbiol. 97, 1115-1122.

Microbiol. 69, 220-226.

J. Food Microbiol. 151, 171-181.

*lactis*. Appl. Environ. Microbiol. 65, 4351-4356.

and in adult faecal microbiota, submitted.

reaction. J. Dairy Res. 66, 313-317.

Bacterial diversity of Darfiyeh, a Lebanese artisanal raw goat's milk cheese. Food

rapid detection of *Brucella* spp. in soft cheese by gene-specific polymerase chain

subsp. *paratuberculosis* in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int. J. Food Microbiol. 128, 250-257. Smeianov, V.V., Wechter, P., Broadbent, J.R., Hughes, J.E., Rodriguez, B.T., Christensen,

T.K., Ardo, Y., Steele, J.L., 2007. Comparative High-Density Microarray Analysis of Gene Expression during Growth of *Lactobacillus helveticus* in Milk versus Rich

Shirakawa, T., Sonomoto, K., 2007. Differences in developing intestinal microbiota between allergic and non-allergic infants: A pilot study in apan. Biosci. Biotechn.

Subspecies *paratuberculosis* in Swiss Raw Milk Cheeses Collected at the Retail Level.

dairy products using a combined bacterial concentration-PCR approach. J. Appl.

Graber, H.U., 2008. A Longitudinal Field Study to Evaluate the Diagnostic Properties of a Quantitative Real-Time Polymerase Chain Reaction-Based Assay to

transcriptome analysis of *Lactococcus lactis* subsp. *cremoris* strains under conditions simulating Cheddar cheese manufacture. Int. J. Food Microbiol. 146, 263-275. Temmerman, R., Scheirlinck, I., Huys, G., Swings, J., 2003. Culture-independent analysis of

probiotic products by denaturing gradient gel electrophoresis. Appl. Environ.

Characterization of Streptococcus thermophilus two-component systems: In silico analysis, functional analysis and expression of response regulator genes in pure or mixed culture with its yogurt partner, *Lactobacillus delbrueckii* subsp. *bulgaricus*. Int.

differentiation of *Lactobacillus delbrueckii* subsp. *bulgaricus* and *L. delbrueckii* subsp.

gassericin K7 A and K7 B gene determinants in the *Lactobacillus acidophilus* group


Rasolofo, E.A., St-Gelais, D., LaPointe, G., Roy, D., 2010. Molecular analysis of bacterial

Reimann, S., Grattepanche, F., Rezzonico, E., Lacroix, C., 2010. Development of a real-time

Rinttila, T., Kassinen, A., Malinen, E., Krogius, L., Palva, A., 2004. Development of an

Rodríguez-Lázaro, D., D'Agostino, M., Herrewegh, A., Pla, M., Cook, N., Ikonomopoulos, J.,

Rossetti, B.C., Frey, J., Pilo, P., 2010. Direct detection of Mycoplasma bovis in milk and tissue

Rossi, F., Gardini, F., Rizzotti, L., La Gioia, F., Tabanelli, G., Torriani, S., 2011. Quantitative

Rossmanith, P., Krassnig, M., Wagner, M., Hein, I., 2006. Detection of *Listeria monocytogenes*

Rossmanith, P., Su, B., Wagner, M., Hein, I., 2007. Development of matrix lysis for

Rudi, K., Naterstad, K., Dromtorp, S.M., Holo, H., 2005. Detection of viable and dead *Listeria* 

Rueckert, A., Ronimus, R.S., Morgan, H.W., 2005. Development of a rapid detection and

Sanchez, J.I., Rossetti, L., Martinez, B., Rodriguez, A., Giraffa, G., 2006. Application of

Saubusse, M., Millet, L., Delbes, C., Callon, C., Montel, M.C., 2007. Application of Single

Selinger, D.W., Saxena, R.M., Cheung, K.J., Church, G.M., Rosenow, C., 2003. Global RNA

*paratuberculosis* in water and milk. Int. J. Food Microbiol. 101, 93-104. Rossen, L., Norskov, P., Holmstrom, K., Rasmussen, O.F., 1992. Inhibition of Pcr by

milk. Int. J. Food Microbiol. 138, 108-118.

morphologies. Food Microbiol. 27, 236-242.

Solutions. Int. J. Food Microbiol. 17, 37-45.

gene. Res. Microbiol. 157, 763-771.

Degradation. Genome Res. 13, 216-223.

samples by real-time PCR. Mol. Cell. Probes 24, 321-323.

Cheese Making. Appl. Environ. Microbiol. 77, 2817-2822.

1166-1177.

504-511.

301-306.

60, 155-167.

126-135.

population structure and dynamics during cold storage of untreated and treated

RT-PCR method for enumeration of viable *Bifidobacterium longum* cells in different

extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J. Appl. Microbiol. 97,

2005. Real-time PCR-based methods for detection of *Mycobacterium avium* subsp.

Components of Food Samples, Microbial Diagnostic Assays and DNA-Extraction

Analysis of Histidine Decarboxylase Gene (hdcA) Transcription and Histamine Production by *Streptococcus thermophilus* PRI60 under Conditions Relevant to

in food using a combined enrichment/real-time PCR method targeting the prfA

concentration of gram positive bacteria from food and blood. J. Microbiol. Meth. 69,

*monocytogenes* on gouda-like cheeses by real-time PCR. Lett. Appl. Microbiol. 40,

enumeration method for thermophilic bacilli in milk powders. J. Microbiol. Meth.

reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations. J. Microbiol. Meth. 65, 268-277.

Strand Conformation Polymorphism --PCR method for distinguishing cheese bacterial communities that inhibit *Listeria monocytogenes*. Int. J. Food Microbiol. 116,

Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript


**3** 

*Malaysia* 

**Role of Polymerase Chain Reaction** 

*1Department of Parasitology and Medical Diagnostics, Universiti Malaysia Sabah,* 

*2Monash University, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan* 

The history of forensic entomology dates back to the 13th century when Song Ci (or Sung Tz'u) (1186–1249), an outstanding forensic scientist in the Southern Song Dynasty, documented the first forensic entomology case in his book "Collected Cases of Injustice Rectified" (*Xi Yuan Ji Lu* which means "Washing Away of the Wrongs"). In his investigation, Song Ci identified the murderer through forensic flies which flew to the sickle used in committing the crime. This sickle had bits of soft tissue, blood, bone and hair attached to it and thus attracted the flies. The owner of the sickle then admitted to his crime

Insects (mainly flies and beetles) are the main resources in forensic entomology. They could be found in every part of the world making them a useful forensic indicator by providing

With the advancement of biotechnology, forensic entomology has become a technically well developed field. Molecular biology tools are incorporated into this field where DNA based techniques are used to help solving complicated criminal or death cases. Often, police seek scientist's help to perform these genetic techniques mainly involving Polymerase Chain

Today, the use of PCR-based methods in forensic entomology to help solve criminal and death investigations is continually increasing. In fact, it is a standard tool in most forensic

In this chapter, we will look into the various PCR-based methods that have been developed elsewhere and adopted in forensic entomology, focusing on medicocriminal entomology. We will also look into how each method contributes to the field, as well as discussing its

Forensic entomologists estimate the post-mortem interval (PMI) or the minimum time of death by analyzing the correlation between the developmental stages of the collected insect

useful clues and evidence in death and criminal cases in forensic investigations.

laboratories, and police officers are trained in molecular technology.

**2. Estimation of Post Mortem Interval (PMI)** 

**1. Introduction** 

(translated by McKnight [1]).

Reaction (PCR) analysis.

strengths and weaknesses.

**in Forensic Entomology** 

Tock Hing Chua1 and Y. V. Chong2

*Jalan UMS, Kota Kinabalu, Sabah,* 


## **Role of Polymerase Chain Reaction in Forensic Entomology**

Tock Hing Chua1 and Y. V. Chong2 *1Department of Parasitology and Medical Diagnostics, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu, Sabah, 2Monash University, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan Malaysia* 

## **1. Introduction**

50 Polymerase Chain Reaction

Trmcic, A., Monnet, C., Rogelj, I., Bogovic Matijasic, B., 2011. Expression of nisin genes in

Trmcic, A., Obermajer, T., Rogelj, I., Bogovic Matijasic, B., 2008. Short Communication:

Ulvé, V.M., Monnet, C., Valence, F., Fauquant, J., Falentin, H., Lortal, S., 2008. RNA

Van Hoorde, K., Heyndrickx, M., Vandamme, P., Huys, G., 2010. Influence of

Van Hoorde, K., Verstraete, T., Vandamme, P., Huys, G., 2008. Diversity of lactic acid

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman,

Vitali, B., Candela, M., Matteuzzi, D., Brigidi, P., 2003. Quantitative detection of probiotic

Wong, M.L., Medrano, J.F., 2005. Real-time PCR for mRNA quantitation. BioTechniques 39,

Zago, M., Bonvini, B., Carminati, D., Giraffa, G., 2009. Detection and quantification of *Enterococcus gilvus* in cheese by real-time PCR. Syst. Appl. Microbiol. 32, 514-521. Zoric Peternel, M., Canzek Majhenic, Andreja, Holo, Helge, Nes, Ingolf, Salehian, Zhian,

*Lactobacillus gasseri* K7, Probiotics & Antimicro. Prot. 2, 233-240.

of artisan Gouda-type cheeses. Food Microbiol. 27, 425-433.

94, 77-85.

929-935.

75-85.

Sci. 91, 4535-4541.

Appl. Microbiol. 105, 1327-1333.

research0034.0031 - research0034.0011.

Microbiol. 26, 269-276.

cheese-A quantitative real-time polymerase chain reaction approach. J. Dairy Sci.

Culture-Independent Detection of Lactic Acid Bacteria Bacteriocin Genes in Two Traditional Slovenian Raw Milk Cheeses and Their Microbial Consortia. J. Dairy

extraction from cheese for analysis of *in situ* gene expression of *Lactococcus lactis*. J.

pasteurization, brining conditions and production environment on the microbiota

bacteria in two Flemish artisan raw milk Gouda-type cheeses. Food Microbiol. 25,

F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3,

*Bifidobacterium* strains in bacterial mixtures by using real-time PCR. Syst. Appl.

Berlec, Ales, Rogelj, Irena., 2010. Wide-inhibitory spectra bacteriocins produced by

The history of forensic entomology dates back to the 13th century when Song Ci (or Sung Tz'u) (1186–1249), an outstanding forensic scientist in the Southern Song Dynasty, documented the first forensic entomology case in his book "Collected Cases of Injustice Rectified" (*Xi Yuan Ji Lu* which means "Washing Away of the Wrongs"). In his investigation, Song Ci identified the murderer through forensic flies which flew to the sickle used in committing the crime. This sickle had bits of soft tissue, blood, bone and hair attached to it and thus attracted the flies. The owner of the sickle then admitted to his crime (translated by McKnight [1]).

Insects (mainly flies and beetles) are the main resources in forensic entomology. They could be found in every part of the world making them a useful forensic indicator by providing useful clues and evidence in death and criminal cases in forensic investigations.

With the advancement of biotechnology, forensic entomology has become a technically well developed field. Molecular biology tools are incorporated into this field where DNA based techniques are used to help solving complicated criminal or death cases. Often, police seek scientist's help to perform these genetic techniques mainly involving Polymerase Chain Reaction (PCR) analysis.

Today, the use of PCR-based methods in forensic entomology to help solve criminal and death investigations is continually increasing. In fact, it is a standard tool in most forensic laboratories, and police officers are trained in molecular technology.

In this chapter, we will look into the various PCR-based methods that have been developed elsewhere and adopted in forensic entomology, focusing on medicocriminal entomology. We will also look into how each method contributes to the field, as well as discussing its strengths and weaknesses.

## **2. Estimation of Post Mortem Interval (PMI)**

Forensic entomologists estimate the post-mortem interval (PMI) or the minimum time of death by analyzing the correlation between the developmental stages of the collected insect

Role of Polymerase Chain Reaction in Forensic Entomology 53

(mtDNA), mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and tRNA leucine genes of blowflies (*Phormia regina* (Meigen), *Phaenicia sericata* (Meigen) and *Lucilia illustris* (Meigen)) were amplified using PCR and followed by direct sequencing. He found that there were nucleotide differences in the DNA sequences between these three

Subsequent to this research, mtDNA has been widely used for DNA analysis in forensic entomology, using COI and COII gene sequences analysis for distinguishing forensically important blow flies and flesh flies [4-14]. In purpose, such molecular work is similar to other non-entomological forensic methods in that it provides supplementary evidence in the

In China, a 278 bp region of COI was used for the identification of nine forensic flies, namely *Ophyra capensis* (Wiedemann), *Chrysomya megacephala*, *Phaenicia sericata*, *Lucilia curpina*, and *Boettcherisca peregrina* (Robineau-desvoidy) [15]. They found the species could be easily separated by molecular means except for *Phaenicia sericata* and *Lucilia curpina* because of low

In 2010, Mazzanti et al. [16] demonstrated that PCR could successfully amplify the mtDNA from empty puparial case and also fragments of the case. They could also correctly determine the eight Dipteran species (*Calliphora vicina* (Robineau-Desvoidy), *Sarcophagidae crassipalpis* (Macquart), *Phormia regina*, *Phaenicia sericata, Sarcophaga argyrostoma* (Robineau-Desvoidy), *Calliphora vomitoria* (Linnaeus), *Chrysomya megacephala, Synthesiomyia nudiseta* (Van Der Wulp)) through the amplified DNA from pupal case. This finding is particularly important as empty pupa cases left after adult emergence or fragments of it are commonly found on the corpse or in the area surrounding the corpse. The mtDNA has also been used for identification of beetle species found on corpses. In addition, COI and COII sequences have been used to study the phylogenetic relationships of carrion beetles species (Silphidae) (*Nicrophorus investigator* Zetterstedt, *Oiceoptoma novaboracense* (Fӧrster), *Necrophilia americana*

However molecular identification of species may not be accurate if it uses only the mtDNA gene [18]. More recent works also make use of internal transcribed spacer (ITS) which is a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. This transcript contains the 5' external transcribed sequence (5' ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA and finally the 3'ETS. The ITS region is widely used in taxonomy and molecular phylogeny because it is easy to amplify even from small quantities of DNA (due to the high copy number of rRNA genes) and has a high

For example, Song et al. [19] analyzed the nuclear ribosomal DNA especially internal transcribed spacer-II (ITS2) for species identification of some common necrophagous flies in southern China by phenetic approach. ITS2 gene was amplified from each individual specimen and sequences obtained were analyzed using ClustalX to construct a neighbour joining (NJ) tree. The results showed that species could be differentiated, and the identification was not affected by intra and interspecific variations. However, because of the high sequence homology between some congeneric species, more sequencing of specimens

species which could be used to differentiate their immature larval stages.

form of PMI estimate to support the charge of a suspect to the crime.

sequence divergence between these two species.

degree of variation even between closely related species.

is required before such method can be used for forensic investigations.

(Linnaeus) [17].

specimens with the approximate weather data at the time when the crime or death occurs. Within minutes of the death of a person, forensic insects are able to locate the body through the sense of smell. The female fly deposits eggs (in the case of Calliphorid flies) or larvae (for Sarcophagid flies) on open wounds or natural openings of the corpse. These larvae hatch from eggs or born alive would then feed on the corpse. The larva undergoes three developmental stages and moults into a pupa. Metamorphosis occurs within the pupa, and an adult fly emerges in about a week.

Flies are usually the insects that arrive first at the decomposing corpse, starting with Calliphorids such as *Chrysomya megacephala* (Fabricius) and *Chrysomya rufacies* (Macquart), and Sarcophagids. Following the flies, a succession of other arthropods and species of other phyla such as beetles, ants, moth, butterflies, earthworms and snails would arrive and join in decomposing the corpse. These include the beetles (Family Dermestidae and Silphidae), wasps (Family Vespidae), ants (Order Hymenoptera) and mites (gamasid and oribatid mites).

The arrival time of each individual species differ; some species such as blowfly and flesh fly arrive within five minutes prior to death while other species such as soldier fly and beetles arrive when the corpse is at the advanced decay stage [2]. The developmental rate of each species and each immature stage differs, and variation exists even among closely related species. Thus, correct species identification of the collected specimens and realistic values of the development rate of immature stages are very crucial for accurate PMI estimation.

## **3. Species identification using molecular methods**

Insect species identification has been traditionally carried out using morphological characteristics. However, morphological characteristic keys for the immature stages of many forensically important species are either not constructed yet or not easily available or appear confusing to the non experts. To overcome this problem, forensic workers have started using Polymerase Chain Reaction (PCR) in insect species identification for forensic entomology since 1994.

The DNA of the insect specimen collected from the corpse or criminal scene is extracted usually using a commercial extraction kit and the extracted DNA is amplified using a specific primer designed for a certain gene. Then, the desired amplified fragment is purified for sequencing, and the sequence obtained is analysed further using Bioinformatics tools.

For species identification in forensic entomology, further investigation subsequent to the simple PCR analysis is commonly carried out by random fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and sequence-characterized amplied region (SCAR) marker methods and real time PCR analysis.

#### **3.1 Simple PCR**

The DNA-based method as an alternative to using morphological keys for species identification was first proposed by Sperling et al. [3]. In his research, mitochondrial DNA

specimens with the approximate weather data at the time when the crime or death occurs. Within minutes of the death of a person, forensic insects are able to locate the body through the sense of smell. The female fly deposits eggs (in the case of Calliphorid flies) or larvae (for Sarcophagid flies) on open wounds or natural openings of the corpse. These larvae hatch from eggs or born alive would then feed on the corpse. The larva undergoes three developmental stages and moults into a pupa. Metamorphosis occurs within the pupa, and

Flies are usually the insects that arrive first at the decomposing corpse, starting with Calliphorids such as *Chrysomya megacephala* (Fabricius) and *Chrysomya rufacies* (Macquart), and Sarcophagids. Following the flies, a succession of other arthropods and species of other phyla such as beetles, ants, moth, butterflies, earthworms and snails would arrive and join in decomposing the corpse. These include the beetles (Family Dermestidae and Silphidae), wasps (Family Vespidae), ants (Order Hymenoptera) and mites (gamasid and oribatid

The arrival time of each individual species differ; some species such as blowfly and flesh fly arrive within five minutes prior to death while other species such as soldier fly and beetles arrive when the corpse is at the advanced decay stage [2]. The developmental rate of each species and each immature stage differs, and variation exists even among closely related species. Thus, correct species identification of the collected specimens and realistic values of the development rate of immature stages are very crucial for accurate PMI estimation.

Insect species identification has been traditionally carried out using morphological characteristics. However, morphological characteristic keys for the immature stages of many forensically important species are either not constructed yet or not easily available or appear confusing to the non experts. To overcome this problem, forensic workers have started using Polymerase Chain Reaction (PCR) in insect species identification for forensic entomology

The DNA of the insect specimen collected from the corpse or criminal scene is extracted usually using a commercial extraction kit and the extracted DNA is amplified using a specific primer designed for a certain gene. Then, the desired amplified fragment is purified for sequencing, and the sequence obtained is analysed further using

For species identification in forensic entomology, further investigation subsequent to the simple PCR analysis is commonly carried out by random fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and sequence-characterized amplied region (SCAR) marker methods and real time PCR

The DNA-based method as an alternative to using morphological keys for species identification was first proposed by Sperling et al. [3]. In his research, mitochondrial DNA

**3. Species identification using molecular methods** 

an adult fly emerges in about a week.

mites).

since 1994.

analysis.

**3.1 Simple PCR** 

Bioinformatics tools.

(mtDNA), mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and tRNA leucine genes of blowflies (*Phormia regina* (Meigen), *Phaenicia sericata* (Meigen) and *Lucilia illustris* (Meigen)) were amplified using PCR and followed by direct sequencing. He found that there were nucleotide differences in the DNA sequences between these three species which could be used to differentiate their immature larval stages.

Subsequent to this research, mtDNA has been widely used for DNA analysis in forensic entomology, using COI and COII gene sequences analysis for distinguishing forensically important blow flies and flesh flies [4-14]. In purpose, such molecular work is similar to other non-entomological forensic methods in that it provides supplementary evidence in the form of PMI estimate to support the charge of a suspect to the crime.

In China, a 278 bp region of COI was used for the identification of nine forensic flies, namely *Ophyra capensis* (Wiedemann), *Chrysomya megacephala*, *Phaenicia sericata*, *Lucilia curpina*, and *Boettcherisca peregrina* (Robineau-desvoidy) [15]. They found the species could be easily separated by molecular means except for *Phaenicia sericata* and *Lucilia curpina* because of low sequence divergence between these two species.

In 2010, Mazzanti et al. [16] demonstrated that PCR could successfully amplify the mtDNA from empty puparial case and also fragments of the case. They could also correctly determine the eight Dipteran species (*Calliphora vicina* (Robineau-Desvoidy), *Sarcophagidae crassipalpis* (Macquart), *Phormia regina*, *Phaenicia sericata, Sarcophaga argyrostoma* (Robineau-Desvoidy), *Calliphora vomitoria* (Linnaeus), *Chrysomya megacephala, Synthesiomyia nudiseta* (Van Der Wulp)) through the amplified DNA from pupal case. This finding is particularly important as empty pupa cases left after adult emergence or fragments of it are commonly found on the corpse or in the area surrounding the corpse. The mtDNA has also been used for identification of beetle species found on corpses. In addition, COI and COII sequences have been used to study the phylogenetic relationships of carrion beetles species (Silphidae) (*Nicrophorus investigator* Zetterstedt, *Oiceoptoma novaboracense* (Fӧrster), *Necrophilia americana* (Linnaeus) [17].

However molecular identification of species may not be accurate if it uses only the mtDNA gene [18]. More recent works also make use of internal transcribed spacer (ITS) which is a piece of non-functional RNA situated between structural ribosomal RNAs (rRNA) on a common precursor transcript. This transcript contains the 5' external transcribed sequence (5' ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA and finally the 3'ETS. The ITS region is widely used in taxonomy and molecular phylogeny because it is easy to amplify even from small quantities of DNA (due to the high copy number of rRNA genes) and has a high degree of variation even between closely related species.

For example, Song et al. [19] analyzed the nuclear ribosomal DNA especially internal transcribed spacer-II (ITS2) for species identification of some common necrophagous flies in southern China by phenetic approach. ITS2 gene was amplified from each individual specimen and sequences obtained were analyzed using ClustalX to construct a neighbour joining (NJ) tree. The results showed that species could be differentiated, and the identification was not affected by intra and interspecific variations. However, because of the high sequence homology between some congeneric species, more sequencing of specimens is required before such method can be used for forensic investigations.

Role of Polymerase Chain Reaction in Forensic Entomology 55

The utility of COI gene for identification of important forensic blow fly species found in Taiwan (*Chrysomya megacephala*, *Chrysomya rufifacies*, *Chrysomya pinguis*, *Hemipyrellia ligurriens* (Wiedemann), *Lucillia bazini* Seguy, *Lucilia cuprina*, *Lucillia hainanesis* Fan and *Lucilia prophyrina*) using different stages and different parts of the fly individual was also

The PCR-RFLP techniques have also been employed elsewhere, using the internal transcribed spacer (ITS) in addition to COI. For example, three major blow fly species in Taiwan (*Chrysomya megacephala*, *Chrysomya pinguis* and *Chrysomya rufacies*) could be successfully differentiated using COI and internal transcribed spacer I (ITS1) [24]. In Australia, the potential use of internal transcribed spacer II (ITS2) was investigated using PCR-RFLP analysis on all known *Chrysomya* species known from Australia [22]. All the species produced distinct restriction profiles except for the closely related species pairs, viz. between *Chrysomya latifrons* Malloch and *Chrysomya semimetallica* Malloch, and between

Recently, we carried out research on the PCR-RFLP assay for twelve Malaysian forensically important fly species. Our results (unpublished) indicate that the twelve species (*Chrysomya megacephala*, *Chrysomya rufifacies*, *Chrysomya pinguis*, *Chrysomya bezziana* (Villeneuve), *Chrysomya villenuevi, Chrysomya nigripes* (Aubertin), *Lucillia cuprina* (Wiedemann), *Ophyra spinigera* (Stein), *Sarcophaga ruficornis* (Liopygia), *Sarcophaga dux* (Thomson), *Sarcophaga peregrina* (Robineau-Desvoidy) and *Hermetia illucens* (Linnaeus)) in the study could be differentiated through COI gene digestion with three restriction enzymes (HpaII, SspI and HpyCH4V). We found that this method could be applied to immature stages and also

RAPD is another commonly used method for species identification. This method uses nonspecific primers for PCR amplification by which different regions of the DNA sample are amplified. The first RAPD typing of forensic insects was reported 1998 [4]. Eleven RAPD primers were tested to differentiate closely related species of flies and beetles found on corpse such as 'green bottle' blow flies, 'blue bottle' blow flies (Diptera: Calliphoridae) and beetles (Coleopyera: Silphidae). He found one particular primer (REP1R XIIIACGTCGICATCAGGC) was sufficient in resolving a practical forensic situation, but suggested for forensic purposes a set of at least six primers should be used to establish similarity coefcients. Nevertheless, he cautioned that in medico-legal matters, RAPD results may only be reported for so-called exclusions (where two specimens are denitely proven to be different) since an inclusion (where two specimens are shown to be similar or directly related) might induce the question of the likelihood of nding the same RAPD

**3.4 Inter simple sequence repeat (ISSR) and sequence-characterized amplied region** 

ISSRs are DNA fragments of about 100-3000 bp located between adjacent, oppositely oriented microsatellite regions, and the variation in the regions between these

tested [11], and high support for congeneric grouping of species were obtained.

*Chrysomya incisuralis* Macquart and *Chrysomya rufacies*.

incomplete specimens collected from the criminal scene.

**3.3 Randomly amplified polymorphic DNA (RAPD)** 

pattern by chance in any other animal.

**(SCAR) markers methods** 

A different section of the COI, a 250 base pair region of the gene for 16S rDNA has also being sequenced and tested [20]. They examined eight forensically important species from ten sites distributed at nine provinces in China. These were *Chrysomya megacephala*, *Chrysomya rufifacies, Calliphora vicina, Lucilia caesar* (Linnaeus), *Lucilia porphyrina*, *Phaenicia sericata* (Meigen), *Lucilia bazini* (Seguy), *Lucilia illustris* (Meigen). Their analysis of 16S rDNA sequences indicated abundant phylogenetically informative nucleotide substitutions which could identify most of the species tested except for specimens of *Lucilia caesar* and *Lucilia porphyrina.*

In a more recent study [21], species diagnosis of blowflies (*Chrysomya megacephala*, *Chrysomya pinguis* (Walker), *Phaenicia sericata*, *Lucillia porphyrina* (Walker), *Lucillia illustris*  (Meigen), *Hemipyrellia ligurriens* (Wiedemann), *Aldrichina grahami* (Aldrich) and *Musca domestica* L.) from China and Pakistan was explored using phylogenetic analysis with five gene segments. They found that more accurate results were achieved through multi gene trees compared to single gene especially in resolving evolutionary relationship between species.

Although the mitochondrial cytochrome c oxidase gene is a favourite amongst forensic entomologists resulting in vast amount of DNA data being generated, there is little agreement as to which portion of the gene to be sequenced in forensic work, as different workers used different primers and obtained different sequence lengths from different regions. This can be seen from the above works quoted in this paper, and thus sequence analysis across species may be difficult. If agreement can be reached between various workers, a COI barcode identification system can be developed for use internationally. For example, such a system, using a 658-bp fragment of the COI, was found to be suitable for the identification of *Chrysomya* species from Australia [22]. This COI barcode region can facilitate the rapid generation of a barcode database and subsequent identification of specimens.

## **3.2 Restriction fragment length polymorphism (RFLP)**

PCR-RFLP is the next method developed for species identification and separation. It is robust, easy and inexpensive. It detects the difference in homologous DNA sequences in the form of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. Thus this technique is a combination of PCR amplification and RFLP analysis, in which the desired amplified product is digested with one or more restriction enzymes. Banding patterns that are specific for each species produced from the restriction digestion can be used for identification.

Schroeder et al. [23], for example, analyzed three forensically important species in Germany using the PCR-RFLP technique. They amplied specic fragments of the COI and COII region of the mitochondrial DNA (mtDNA) which were then digested with different restriction enzymes (either DraI or HinfI). The results revealed that a short sequence of 1.3 kb of COI and COII regions could differentiate the three species (*Phaenicia sericata*, *Calliphora vicina* and *Calliphora vomitoria*). Similarly the restriction enzyme SfcI was utilised on cytochrome oxidase I gene region to distinguish between *Calliphora vicina* and *Calliphora vomitoria*, two of the main UK blowy species [12].

A different section of the COI, a 250 base pair region of the gene for 16S rDNA has also being sequenced and tested [20]. They examined eight forensically important species from ten sites distributed at nine provinces in China. These were *Chrysomya megacephala*, *Chrysomya rufifacies, Calliphora vicina, Lucilia caesar* (Linnaeus), *Lucilia porphyrina*, *Phaenicia sericata* (Meigen), *Lucilia bazini* (Seguy), *Lucilia illustris* (Meigen). Their analysis of 16S rDNA sequences indicated abundant phylogenetically informative nucleotide substitutions which could identify most of the species tested except for specimens of *Lucilia caesar* and *Lucilia* 

In a more recent study [21], species diagnosis of blowflies (*Chrysomya megacephala*, *Chrysomya pinguis* (Walker), *Phaenicia sericata*, *Lucillia porphyrina* (Walker), *Lucillia illustris*  (Meigen), *Hemipyrellia ligurriens* (Wiedemann), *Aldrichina grahami* (Aldrich) and *Musca domestica* L.) from China and Pakistan was explored using phylogenetic analysis with five gene segments. They found that more accurate results were achieved through multi gene trees compared to single gene especially in resolving evolutionary relationship between

Although the mitochondrial cytochrome c oxidase gene is a favourite amongst forensic entomologists resulting in vast amount of DNA data being generated, there is little agreement as to which portion of the gene to be sequenced in forensic work, as different workers used different primers and obtained different sequence lengths from different regions. This can be seen from the above works quoted in this paper, and thus sequence analysis across species may be difficult. If agreement can be reached between various workers, a COI barcode identification system can be developed for use internationally. For example, such a system, using a 658-bp fragment of the COI, was found to be suitable for the identification of *Chrysomya* species from Australia [22]. This COI barcode region can facilitate the rapid generation of a barcode database and subsequent identification of

PCR-RFLP is the next method developed for species identification and separation. It is robust, easy and inexpensive. It detects the difference in homologous DNA sequences in the form of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. Thus this technique is a combination of PCR amplification and RFLP analysis, in which the desired amplified product is digested with one or more restriction enzymes. Banding patterns that are specific for each species produced from the

Schroeder et al. [23], for example, analyzed three forensically important species in Germany using the PCR-RFLP technique. They amplied specic fragments of the COI and COII region of the mitochondrial DNA (mtDNA) which were then digested with different restriction enzymes (either DraI or HinfI). The results revealed that a short sequence of 1.3 kb of COI and COII regions could differentiate the three species (*Phaenicia sericata*, *Calliphora vicina* and *Calliphora vomitoria*). Similarly the restriction enzyme SfcI was utilised on cytochrome oxidase I gene region to distinguish between *Calliphora vicina* and *Calliphora* 

*porphyrina.*

species.

specimens.

**3.2 Restriction fragment length polymorphism (RFLP)** 

restriction digestion can be used for identification.

*vomitoria*, two of the main UK blowy species [12].

The utility of COI gene for identification of important forensic blow fly species found in Taiwan (*Chrysomya megacephala*, *Chrysomya rufifacies*, *Chrysomya pinguis*, *Hemipyrellia ligurriens* (Wiedemann), *Lucillia bazini* Seguy, *Lucilia cuprina*, *Lucillia hainanesis* Fan and *Lucilia prophyrina*) using different stages and different parts of the fly individual was also tested [11], and high support for congeneric grouping of species were obtained.

The PCR-RFLP techniques have also been employed elsewhere, using the internal transcribed spacer (ITS) in addition to COI. For example, three major blow fly species in Taiwan (*Chrysomya megacephala*, *Chrysomya pinguis* and *Chrysomya rufacies*) could be successfully differentiated using COI and internal transcribed spacer I (ITS1) [24]. In Australia, the potential use of internal transcribed spacer II (ITS2) was investigated using PCR-RFLP analysis on all known *Chrysomya* species known from Australia [22]. All the species produced distinct restriction profiles except for the closely related species pairs, viz. between *Chrysomya latifrons* Malloch and *Chrysomya semimetallica* Malloch, and between *Chrysomya incisuralis* Macquart and *Chrysomya rufacies*.

Recently, we carried out research on the PCR-RFLP assay for twelve Malaysian forensically important fly species. Our results (unpublished) indicate that the twelve species (*Chrysomya megacephala*, *Chrysomya rufifacies*, *Chrysomya pinguis*, *Chrysomya bezziana* (Villeneuve), *Chrysomya villenuevi, Chrysomya nigripes* (Aubertin), *Lucillia cuprina* (Wiedemann), *Ophyra spinigera* (Stein), *Sarcophaga ruficornis* (Liopygia), *Sarcophaga dux* (Thomson), *Sarcophaga peregrina* (Robineau-Desvoidy) and *Hermetia illucens* (Linnaeus)) in the study could be differentiated through COI gene digestion with three restriction enzymes (HpaII, SspI and HpyCH4V). We found that this method could be applied to immature stages and also incomplete specimens collected from the criminal scene.

## **3.3 Randomly amplified polymorphic DNA (RAPD)**

RAPD is another commonly used method for species identification. This method uses nonspecific primers for PCR amplification by which different regions of the DNA sample are amplified. The first RAPD typing of forensic insects was reported 1998 [4]. Eleven RAPD primers were tested to differentiate closely related species of flies and beetles found on corpse such as 'green bottle' blow flies, 'blue bottle' blow flies (Diptera: Calliphoridae) and beetles (Coleopyera: Silphidae). He found one particular primer (REP1R XIIIACGTCGICATCAGGC) was sufficient in resolving a practical forensic situation, but suggested for forensic purposes a set of at least six primers should be used to establish similarity coefcients. Nevertheless, he cautioned that in medico-legal matters, RAPD results may only be reported for so-called exclusions (where two specimens are denitely proven to be different) since an inclusion (where two specimens are shown to be similar or directly related) might induce the question of the likelihood of nding the same RAPD pattern by chance in any other animal.

#### **3.4 Inter simple sequence repeat (ISSR) and sequence-characterized amplied region (SCAR) markers methods**

ISSRs are DNA fragments of about 100-3000 bp located between adjacent, oppositely oriented microsatellite regions, and the variation in the regions between these

Role of Polymerase Chain Reaction in Forensic Entomology 57

profiled the expression of three genes (bcd, sll, cs) throughout the maturation of blow fly eggs, and found the expression data could predict more precisely the blow fly age (within 2 h of true age). Later, they continued to work on the larvae and pupae [32]*.* Samples were collected from the carcass at different time intervals for gene expression evaluation. The RNA of the sample was extracted and the complementary DNA (cDNA) was synthesized from the RNA using specific gene primers. The desired developmentally regulated gene expression levels were assessed by quantitative PCR, and these levels were incorporated into traditional stage and size data. They tested on 86 immature *Phaenicia sericata*, and obtained a better precision in ageing blow flies, especially for postfeeding third instars and

Real time PCR and differentially expressed genes have also been used in the determination of pupal age in *Calliphora vicina* [33]. This research indicated that expression of Arylphorin and Gene G genes is possible to determine the age of the immature stages. Arylphorin gene is highly expressed at the early stage of pupae development (at 4500 accumulated degree 14 hours or ADH) whereas Gene G is highly expressed at the end of pupae stage (at 8640ADH). On the other hand, the changes in gene expression using differential display PCR has also been investigated [34]. The data showed that different genes are expressed at different levels during pupal development of *Phaenicia sericata*. However, they admitted that their method

Further research was carried out to improve estimation of the age of blow fly (*Phaenicia sericata*) with the aim of achieving a more accurate and precise PMI approximate, through gene expression where 20 genes were analyzed using RT-PCR [32]. Nine of these genes viz. resistance to organophosphate 1 (rop-1), acetylcholine esterase (ace), chitin synthase (cs), ecdysone receptor (ecr), heat shock protein 60 and 90 (hsp60, hsp90), slalom (sll), ultraspiracle (usp), and white (w) evaluated in this study were found to be useful in

increasing the accuracy of PMI estimation for post feeding third instars and pupae.

**5. Genetic variation of forensic species population for detecting postmortem** 

Research on genetic variation of common forensically important species between populations is important for forensic studies. The genetic data of these species is likely to be different among different populations. If specimens from only one location were used in the research, the data collected might only be accurate for that particular location and it could not be applied to death investigations which occur at other locations. It might also be possible to detect post-mortem relocation of a corpse through the study of genetic variation among populations within a species. For study in a particular geographical area, PCR analysis is usually coupled with RAPD, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR). In these methods, non-specific primers are used for PCR amplification. RAPD fingerprinting could be a valuable tool for separating various

The intraspecific genetic variation of *Phaenicia sericata* between two populations in southern England has been investigated using RAPD analysis [**36**]. The genetic homogeneity of *Phaenicia sericata* was determined, basing on the RAPD data which was analysed using a

pupae.

**relocation** 

populations [35].

was not able to determine a pupa's age as yet.

microsatellites is used in ISSR PCR genotyping. The primers used are microsatellite core sequences with a few selective nucleotides as anchors into the non-repeat adjacent regions (16-18 bp). The advantage of ISSRs is that no sequence data for primer construction are needed.

The inter simple sequence repeat (ISSR) method was used to analyze the DNA polymorphism among the ve forensic y species in China, namely, *Phaenicia sericata*, *Aldrichina grahami*, *Chrysomya megacephala*, *Parasarcophaga crassipalpis* and *Musca domestica* using [25]. They found that nine ISSR primers could amplify 95 polymorphic bands which can be used to identify these species. They further converted these species-specic ISSR fragments into the sequence-characterized amplied region (SCAR) markers that can be used for the molecular diagnosis of these species.

Determination of specimens using ISSR is based on the similarity and difference in the electrophoresis result when compared with other individuals. For a high reliability of identification, such method requires a reference sample from the same species in a large database containing all species likely to be attracted to corpses in the same geographic region. On the other hand, SCAR is a genomic fragment localized in a single genetically dened locus that can be amplied by PCR using a pair of specic primers. SCARs are less sensitive to reaction conditions when compared to ISSR markers, thus allowing for a higher reliability and reproducibility among different laboratories which may use different brands of reagents and equipment. Therefore, SCARs are more appropriate diagnostic tool for practical applications.

## **3.5 Real time PCR assay**

Further development in molecular identification of species was achieved in 2010 [26]. The investigators designed a species-specic real-time polymerase chain reaction (PCR) assay to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of *Chrysomya bezziana*. It was very specic and can exclude other morphologically similar and related *Chrysomya*  and *Cochliomyia* species. With this they were able to detect one *Chrysomya bezziana* in a sample of 1000 non-target species. Similar specific system can be developed to confirm the identity of other *Chrysomya* spp.

## **4. Determination of insect developmental rate**

Immature stages of flies particularly the larvae and pupae, are often recovered from the death scene. Obtaining a better estimate of the time needed for the immature insect to develop to a certain stage will help to give a more accurate PMI. The larva and pupa stages occupy more than half of the immature development time. The developmental rate of larvae is determined by specific morphological changes and measurement of the specimen length [27-29]. For the egg and pupa which do not change size, physical measurement is not very useful. However, the changes of the pupal case and measurement of hormone level have been used for determining the pupa developmental rate of *Protophormia terraenovae* (Robineau-Desvoidy) [30].

Recently, molecular techniques involving PCR analysis have been developed for the developmental time of immature stages of the blowfly, *Phaenicia sericata.* Tarone et al. [31]

microsatellites is used in ISSR PCR genotyping. The primers used are microsatellite core sequences with a few selective nucleotides as anchors into the non-repeat adjacent regions (16-18 bp). The advantage of ISSRs is that no sequence data for primer construction are

The inter simple sequence repeat (ISSR) method was used to analyze the DNA polymorphism among the ve forensic y species in China, namely, *Phaenicia sericata*, *Aldrichina grahami*, *Chrysomya megacephala*, *Parasarcophaga crassipalpis* and *Musca domestica* using [25]. They found that nine ISSR primers could amplify 95 polymorphic bands which can be used to identify these species. They further converted these species-specic ISSR fragments into the sequence-characterized amplied region (SCAR) markers that can be

Determination of specimens using ISSR is based on the similarity and difference in the electrophoresis result when compared with other individuals. For a high reliability of identification, such method requires a reference sample from the same species in a large database containing all species likely to be attracted to corpses in the same geographic region. On the other hand, SCAR is a genomic fragment localized in a single genetically dened locus that can be amplied by PCR using a pair of specic primers. SCARs are less sensitive to reaction conditions when compared to ISSR markers, thus allowing for a higher reliability and reproducibility among different laboratories which may use different brands of reagents and equipment. Therefore, SCARs are more appropriate diagnostic tool for

Further development in molecular identification of species was achieved in 2010 [26]. The investigators designed a species-specic real-time polymerase chain reaction (PCR) assay to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of *Chrysomya bezziana*. It was very specic and can exclude other morphologically similar and related *Chrysomya*  and *Cochliomyia* species. With this they were able to detect one *Chrysomya bezziana* in a sample of 1000 non-target species. Similar specific system can be developed to confirm the

Immature stages of flies particularly the larvae and pupae, are often recovered from the death scene. Obtaining a better estimate of the time needed for the immature insect to develop to a certain stage will help to give a more accurate PMI. The larva and pupa stages occupy more than half of the immature development time. The developmental rate of larvae is determined by specific morphological changes and measurement of the specimen length [27-29]. For the egg and pupa which do not change size, physical measurement is not very useful. However, the changes of the pupal case and measurement of hormone level have been used for determining the pupa developmental rate of *Protophormia terraenovae*

Recently, molecular techniques involving PCR analysis have been developed for the developmental time of immature stages of the blowfly, *Phaenicia sericata.* Tarone et al. [31]

needed.

practical applications.

**3.5 Real time PCR assay** 

identity of other *Chrysomya* spp.

(Robineau-Desvoidy) [30].

**4. Determination of insect developmental rate** 

used for the molecular diagnosis of these species.

profiled the expression of three genes (bcd, sll, cs) throughout the maturation of blow fly eggs, and found the expression data could predict more precisely the blow fly age (within 2 h of true age). Later, they continued to work on the larvae and pupae [32]*.* Samples were collected from the carcass at different time intervals for gene expression evaluation. The RNA of the sample was extracted and the complementary DNA (cDNA) was synthesized from the RNA using specific gene primers. The desired developmentally regulated gene expression levels were assessed by quantitative PCR, and these levels were incorporated into traditional stage and size data. They tested on 86 immature *Phaenicia sericata*, and obtained a better precision in ageing blow flies, especially for postfeeding third instars and pupae.

Real time PCR and differentially expressed genes have also been used in the determination of pupal age in *Calliphora vicina* [33]. This research indicated that expression of Arylphorin and Gene G genes is possible to determine the age of the immature stages. Arylphorin gene is highly expressed at the early stage of pupae development (at 4500 accumulated degree 14 hours or ADH) whereas Gene G is highly expressed at the end of pupae stage (at 8640ADH). On the other hand, the changes in gene expression using differential display PCR has also been investigated [34]. The data showed that different genes are expressed at different levels during pupal development of *Phaenicia sericata*. However, they admitted that their method was not able to determine a pupa's age as yet.

Further research was carried out to improve estimation of the age of blow fly (*Phaenicia sericata*) with the aim of achieving a more accurate and precise PMI approximate, through gene expression where 20 genes were analyzed using RT-PCR [32]. Nine of these genes viz. resistance to organophosphate 1 (rop-1), acetylcholine esterase (ace), chitin synthase (cs), ecdysone receptor (ecr), heat shock protein 60 and 90 (hsp60, hsp90), slalom (sll), ultraspiracle (usp), and white (w) evaluated in this study were found to be useful in increasing the accuracy of PMI estimation for post feeding third instars and pupae.

## **5. Genetic variation of forensic species population for detecting postmortem relocation**

Research on genetic variation of common forensically important species between populations is important for forensic studies. The genetic data of these species is likely to be different among different populations. If specimens from only one location were used in the research, the data collected might only be accurate for that particular location and it could not be applied to death investigations which occur at other locations. It might also be possible to detect post-mortem relocation of a corpse through the study of genetic variation among populations within a species. For study in a particular geographical area, PCR analysis is usually coupled with RAPD, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR). In these methods, non-specific primers are used for PCR amplification. RAPD fingerprinting could be a valuable tool for separating various populations [35].

The intraspecific genetic variation of *Phaenicia sericata* between two populations in southern England has been investigated using RAPD analysis [**36**]. The genetic homogeneity of *Phaenicia sericata* was determined, basing on the RAPD data which was analysed using a

Role of Polymerase Chain Reaction in Forensic Entomology 59

A number of researches were conducted on the DNA extraction from the digestive tract of necrophagous larvae or the 'last meal' of these maggots. This is a useful study as the DNA profile of the host could be obtained from the extracted DNA and to determine whether the maggots used in the investigation are associated with the crime or death [40, 45]. Kondakci et al. [46] found that a complete human profile could be obtained using STR and SNP profiling of *Phaenicia sericata* third instar larvae. The STR and SNP profiles matched the identity of the host which showed that this analysis could be used to relate the maggots

With the advent of molecular techniques, forensic entomology has certainly come a long way since the days of Song Ci. Molecular technology has changed the manner by which forensic entomological investigations are being carried out, making it a sophisticated a science. This has resulted in quicker, more accurate determination of the species, as well as the age of specimens recovered from the corpse, and consequently a more accurate of PMI. From the initial use simple PCR in forensic entomology, it has progressed to RFLP, then

Initially the molecular techniques were used mainly for species identification [3, 23, 24]. However, later works extended to ageing the pupa [33], which is very useful as the size does not change during metamorphosis, and physical ageing is not possible. The accuracy of PMI

It would appear that future research is in the direction of RT-PCR assay, as this is a faster and more accurate method. Similarly studies on the intraspecific genetic variation on forensic insect populations will result in more accurate methods of population identification, and aid in deciding if a victim's body has been relocated by the criminals for

Another area which has great potential use is identification of suspect from the gut content of a fed mosquito [44] or body louse [47] at the crime scene. In the case where the body has been moved, the carrion fly larvae or pupae may help identify the deceased indicating the relocation of the corpse. Although the blood meal may be partially digestion and makes DNA extraction difficult, future research will likely to yield better technology for

Although molecular methods have advanced forensic entomology, the validity and reliability of the methods, and have also questioned the statistical basis of the sampling size have been questioned [18], and suggestions to improve have been offered. Among many things, it was suggested (a) the DNA extraction procedure should include a negative control, and the genotyping procedures should include both positive and negative controls, (b) a portion of the original tissue should be saved so that it is available for independent testing, (c) there must be an extensive record of reproducibility under specied working conditions, both when performed by the same analyst and by different analysts, (e) the analyst should have considerable experience with the particular genotyping method, and publications based on the same kind of analysis, (f) the analyst should provide a description

estimates increases if ageing of the immature stages becomes more precise.

burial to avoid suspicion, or to mislead criminal investigation.

genotyping profile with degraded or low-copy DNA template.

studied to the corpse in the investigation.

RAPD, ISSR, SCAR and finally to RT- PCR Assay.

**7. Conclusion** 

similarity coefficient method and a randomization test. They found that banding profiles (which were defined with ten random primers) from RAPD could differentiate among closely related individuals of the species. Such investigation can be used to elucidate relationships between even closely related populations of *Phaenicia sericata* and differentiate between populations, if more than a population is found on the corpse, thus helping to make a conclusion if a body had been relocated prior to its discovery.

Similarly, AFLP analysis has also been used for genetic population study of *Phormia regina* from sites spanning the contiguous United States [37]. They found there was only a very weak correlation between individual genetic and geographic distances. More interestingly, they found that adult *Phormia regina* that arrived together to the baits were closely related individuals compared to a random sample. They later applied the same method for investigating the population genetic structure of *Phaenicia sericata* from North America based on AFLP genotypes with 249 loci [38]. Although the study could not find any regional genetic variation, they nevertheless detected high local relatedness among the females in the samples. This led them to suggest that a pattern of local relatedness might support a genetic test for inferring the post-mortem relocation of a corpse.

We have conducted using similar methods a preliminary study of the population genetic variation among *Chrysomya megacephala* individuals in Malaysia. We tested the usefulness of COI gene for differentiating Malaysian *Chrysomya megacephala* individuals from four locations. Our results showed that the individuals could be put into two geographical groups based on a single nucleotide polymorphism (SNP) observed (unpublished results). It would appear possible then to infer if a corpse has been relocated from one location to another by comparing the SNP of larvae or pupae left behind at one place and those on the corpse which has been moved postmortem.

## **6. Recovery of human DNA from insects**

Many studies found that human DNA can be recovered from insects found at the scene. The recovery of DNA provides useful information for forensic cases. For example, the identity of the suspect or the deceased could be identified from a fed mosquito, fly larvae or bed bugs [39-43]. The detection of insect gut content by PCR amplification is useful for forensic entomology. DNA is extracted from the collected insect, often from the insect gut contents. Then PCR amplification usually is conducted using either short tandem repeat (STR), human mtDNA hypervariable region (HVR) or insect mtDNA for profiling. STR and HVR typing are commonly used for human profiling.

Coulson et al. [44] demonstrated the possibility of human DNA extraction, amplification and fingerprinting from *Anopheles gambiae* mosquitoes stored at different storing conditions. The results showed that it is possible to use PCR for the amplification of human DNA extracted from mosquitoes. A very interesting casework has been demonstrated in 2006 [41], where only a fresh mosquito blood stain from a smashed mosquito was found in a room of the death scene. DNA was successfully extracted from the blood stain, and PCR amplification and STRs profiling at 15 human genetic loci was then performed on the extracted DNA, using AmpFLSTR Identifiler. This produced a complete genetic profile which aided the identification of the suspect.

A number of researches were conducted on the DNA extraction from the digestive tract of necrophagous larvae or the 'last meal' of these maggots. This is a useful study as the DNA profile of the host could be obtained from the extracted DNA and to determine whether the maggots used in the investigation are associated with the crime or death [40, 45]. Kondakci et al. [46] found that a complete human profile could be obtained using STR and SNP profiling of *Phaenicia sericata* third instar larvae. The STR and SNP profiles matched the identity of the host which showed that this analysis could be used to relate the maggots studied to the corpse in the investigation.

## **7. Conclusion**

58 Polymerase Chain Reaction

similarity coefficient method and a randomization test. They found that banding profiles (which were defined with ten random primers) from RAPD could differentiate among closely related individuals of the species. Such investigation can be used to elucidate relationships between even closely related populations of *Phaenicia sericata* and differentiate between populations, if more than a population is found on the corpse, thus helping to

Similarly, AFLP analysis has also been used for genetic population study of *Phormia regina* from sites spanning the contiguous United States [37]. They found there was only a very weak correlation between individual genetic and geographic distances. More interestingly, they found that adult *Phormia regina* that arrived together to the baits were closely related individuals compared to a random sample. They later applied the same method for investigating the population genetic structure of *Phaenicia sericata* from North America based on AFLP genotypes with 249 loci [38]. Although the study could not find any regional genetic variation, they nevertheless detected high local relatedness among the females in the samples. This led them to suggest that a pattern of local relatedness might support a genetic

We have conducted using similar methods a preliminary study of the population genetic variation among *Chrysomya megacephala* individuals in Malaysia. We tested the usefulness of COI gene for differentiating Malaysian *Chrysomya megacephala* individuals from four locations. Our results showed that the individuals could be put into two geographical groups based on a single nucleotide polymorphism (SNP) observed (unpublished results). It would appear possible then to infer if a corpse has been relocated from one location to another by comparing the SNP of larvae or pupae left behind at one place and those on the

Many studies found that human DNA can be recovered from insects found at the scene. The recovery of DNA provides useful information for forensic cases. For example, the identity of the suspect or the deceased could be identified from a fed mosquito, fly larvae or bed bugs [39-43]. The detection of insect gut content by PCR amplification is useful for forensic entomology. DNA is extracted from the collected insect, often from the insect gut contents. Then PCR amplification usually is conducted using either short tandem repeat (STR), human mtDNA hypervariable region (HVR) or insect mtDNA for profiling. STR and HVR

Coulson et al. [44] demonstrated the possibility of human DNA extraction, amplification and fingerprinting from *Anopheles gambiae* mosquitoes stored at different storing conditions. The results showed that it is possible to use PCR for the amplification of human DNA extracted from mosquitoes. A very interesting casework has been demonstrated in 2006 [41], where only a fresh mosquito blood stain from a smashed mosquito was found in a room of the death scene. DNA was successfully extracted from the blood stain, and PCR amplification and STRs profiling at 15 human genetic loci was then performed on the extracted DNA, using AmpFLSTR Identifiler. This produced a complete genetic profile

make a conclusion if a body had been relocated prior to its discovery.

test for inferring the post-mortem relocation of a corpse.

corpse which has been moved postmortem.

**6. Recovery of human DNA from insects** 

typing are commonly used for human profiling.

which aided the identification of the suspect.

With the advent of molecular techniques, forensic entomology has certainly come a long way since the days of Song Ci. Molecular technology has changed the manner by which forensic entomological investigations are being carried out, making it a sophisticated a science. This has resulted in quicker, more accurate determination of the species, as well as the age of specimens recovered from the corpse, and consequently a more accurate of PMI.

From the initial use simple PCR in forensic entomology, it has progressed to RFLP, then RAPD, ISSR, SCAR and finally to RT- PCR Assay.

Initially the molecular techniques were used mainly for species identification [3, 23, 24]. However, later works extended to ageing the pupa [33], which is very useful as the size does not change during metamorphosis, and physical ageing is not possible. The accuracy of PMI estimates increases if ageing of the immature stages becomes more precise.

It would appear that future research is in the direction of RT-PCR assay, as this is a faster and more accurate method. Similarly studies on the intraspecific genetic variation on forensic insect populations will result in more accurate methods of population identification, and aid in deciding if a victim's body has been relocated by the criminals for burial to avoid suspicion, or to mislead criminal investigation.

Another area which has great potential use is identification of suspect from the gut content of a fed mosquito [44] or body louse [47] at the crime scene. In the case where the body has been moved, the carrion fly larvae or pupae may help identify the deceased indicating the relocation of the corpse. Although the blood meal may be partially digestion and makes DNA extraction difficult, future research will likely to yield better technology for genotyping profile with degraded or low-copy DNA template.

Although molecular methods have advanced forensic entomology, the validity and reliability of the methods, and have also questioned the statistical basis of the sampling size have been questioned [18], and suggestions to improve have been offered. Among many things, it was suggested (a) the DNA extraction procedure should include a negative control, and the genotyping procedures should include both positive and negative controls, (b) a portion of the original tissue should be saved so that it is available for independent testing, (c) there must be an extensive record of reproducibility under specied working conditions, both when performed by the same analyst and by different analysts, (e) the analyst should have considerable experience with the particular genotyping method, and publications based on the same kind of analysis, (f) the analyst should provide a description

Role of Polymerase Chain Reaction in Forensic Entomology 61

[8] Harvey, M.L.; Dadour, I.R. & Gaudieri, S. (2003a). Mitochondrial DNA cytochrome

[9] Harvey, M.L; Mansell, M.W.; Villet, M.H. & Dadour, I.R. (2003b). Molecular

*International Journal of Legal Medicine*, Vol. 118, pp. 245-247, ISSN 0937-9827 [11] Chen, W-Y.; Hung, T-H. & Shiao, S.F. (2004). Molecular Identification of Forensically

[12] Ames, C.; Turner, B. & Daniel, B. (2006a). The use of mitochondrial cytochrome oxidase

[13] Wells, J.D. & Williams, D.W. (2007). Validation of a DNA-based method for identifying

[14] Park, S.H.; Zhang, Y.; Piao, H.; Yu, D.H.; Jeong, H.J.; Yoo, G.Y.; Chung, U.; Jo, T-H. &

[15] Cai, J-F.; Liu, M.; Ying, B-W.; Deng, R-L.;Dong, J-G.; Zhang, L.; Tao, T.; Pan, H-F; Yang,

[16] Mazzanti, M.; Alessandrini, F.; Tagliabracci, A.; Wells, J.D. & Campobasso, C.P. (2010).

[17] Dobler, S. & Muller, J. K. (2000). Resolving phylogeny at the family level by

[18] Wells, J. D. & Stevens, J. R. (2008). Application of DNA-Based Methods in Forensic Entomology. *Annual Review of Entomology*, Vol. 53, pp.103–20. ISSN 0066-4170 [19] Song, Z-K.; Wang, X-Z. & Liang, G-Q. (2008). Species identification of some common

*Entomologica Sinica*, Vol. 48, No. 3, pp. 380-385, ISSN 0454-6296

*International*, Vol. 131, pp. 134-139, ISSN 0379-073

*Entomology*, Vol. 41, No. 1, pp. 47-57, ISSN 0022-2585

*Review of Entomology*, Vol. 53, pp.103–20. ISSN 0066-4170

No. , pp. 1058-1063, ISSN 1011-8934

ISSN 0379-0738

390–402, ISSN

ISSN 0379-0738

0738

oxidase I gene: potential for distinction between immature satges of some forensically important fly species (Diptera) in Western Australia. *Forensic Science* 

identification of some forensically important blowflies of southern Africa and Australia. *Medical and Veterinary Entomology*, Vol. 17, pp. 363-369, ISSN 0269-283X [10] Zehner, R.;Amendt J.; Schutt S.; Sauer J.; Krettek, R. & Povolny, D. (2004). Genetic

identification of forensically important flesh flies (Diptera: Sarcophagidae).

Important Blow Fly Species (Diptera: Calliphoridae) in Taiwan. *Journal of Medical* 

I gene (COI) to differentiate two UK blowy species – *Calliphora vicina* and *Calliphora vomitoria*. *Forensic Science International*, Vol. 64, pp.179–182, ISSN 0379-

Chrysomyinae (Diptera: Calliphoridae) used in death investigation. *International Journal of Legal Medicine,* Vol. 121, pp.1–8, ISSN 0937-982718. Wells, J. D. & Stevens, J. R. (2008). Application of DNA-Based Methods in Forensic Entomology. *Annual* 

Hwang, J-J. (2009). Use of Cytochrome c Oxidase Subunit I (COI) Nucleotide Sequences for Identification of Korean Luciliinae Fly Species (Diptera: Calliphoridae) in Forensic Investigations. *Journal of Korean Medical Science*, Vol. 24,

H-T. & Liao, Z-G. (2005). The availability of mitochondrial DNA cytochrome oxidase I gene for the distinction of forensically important flies in China. *Acta* 

DNA Degradation and genetic analysis of empty puparia: Genetic identification limits in forensic entomology. *Forensic Science International,* Vol. 195, pp. 99-102,

mitochondrial cytochrome oxidase sequences: phylogeny of carrion beetles (Coleoptera: Silphidae). *Molecular Phylogenetics and Evolution*, Vol. 15, No. 3, pp.

necrophagous flies in Guangdong province, southern China based on the rDNA internal transcribed spacer 2 (ITS2). *Forensic Science International*, Vol. 175, pp. 17-22,

of all aspects of the laboratory protocol used (e.g., PCR primer sequences) in response to a reasonable request, and (g) a forensic insect species identication must include phylogenetic analysis of sequence data. They also asked (a) what research sample size is adequate for a species-diagnostic test to be used in court, (b) whether the DNA-Based species identication using BLAST search of the huge and easily queried GenBank /EMBL/DDBJ sequence database is critical enough, bearing in mind there are possible errors in some of these sequences, and (c) whether a taxonomic expert had confirmed the identification of the specimen the gene sequence of which was uploaded on the web.

These are important considerations as the analyst may need to testify in court about his findings, and above all, a forensic scientist must take great care to avoid a miscarriage of justice arising from careless interpretation of molecular data.

#### **8. Acknowledgements**

We thank Universiti Malaysia Sabah and Monash University Sunway for research facilities made available to us in the preparation of this paper.

#### **9. References**


of all aspects of the laboratory protocol used (e.g., PCR primer sequences) in response to a reasonable request, and (g) a forensic insect species identication must include phylogenetic analysis of sequence data. They also asked (a) what research sample size is adequate for a species-diagnostic test to be used in court, (b) whether the DNA-Based species identication using BLAST search of the huge and easily queried GenBank /EMBL/DDBJ sequence database is critical enough, bearing in mind there are possible errors in some of these sequences, and (c) whether a taxonomic expert had confirmed the identification of the

These are important considerations as the analyst may need to testify in court about his findings, and above all, a forensic scientist must take great care to avoid a miscarriage of

We thank Universiti Malaysia Sabah and Monash University Sunway for research facilities

[1] McKnight, B.E (1981). *The washing away of wrongs: Forensic medicine in thirteenth-century* 

[2] Gunn, A. (2006). *Essential Forensic Biology.* John Wiley & Sons, Ltd, ISBN -10: 0470012773 [3] Sperling, F.A.H.; Anderson, G.S. & Hickey, D.A. (1994). A DNA-based approach to the

[4] Benecke, M. (1998). Random amplified polymorphic DNA (RAPD) typing of

[5] Wallman, J.F. & Donnellan, S.C. (2001). The utility of mitochondrial DNA sequences for

[6] Wells, J.D. & Sperling, F.A.H. (2001). DNA-based identification of forensically important

[7] Wells, J.D.; Pape, T., & Sperling, F.A. (2001). DNA-based identification and molecular

*China* by Tz'u Sung*.* Translated by McKnight, B.E. University of Michigan, Ann

identification of insect species used for postmortem interval estimation. *Journal Forensic Science,* Vol*.* 39, No. , pp. 418–27. Erratum. 2000. *Journal of Forensic Sciences,* 

necrophageous insects (Diptera, Coleoptera) in criminal forensic studies: validation and use in practice. *Forensic Science International*, Vol. 98, No. , pp. 157-168, ISSN 0379-07385. Wallman, J.F. & Donnellan, S.C. (2001). The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia. *Forensic Science International*, Vol. 120,

the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia. *Forensic Science International*, Vol. 120, pp.60-67, ISSN 0379-

Chrysomyinae (Diptera: Calliphoridae). *Forensic Science International*, Vol. 120, pp.

systematic of forensically important Sarcophagidae (Diptera). *Journal of Forensic* 

specimen the gene sequence of which was uploaded on the web.

justice arising from careless interpretation of molecular data.

made available to us in the preparation of this paper.

Arbor,. 181 pp, ISSN 0892648007

Vol*.* 45, pp. 1358–59, ISSN 1556-4029

pp.60-67, ISSN 0379-0738

110-115, ISSN 0379-0738

*Sciences*, Vol. 46, pp. 1098-102, ISSN 1556-4029

0738

**8. Acknowledgements** 

**9. References** 


Role of Polymerase Chain Reaction in Forensic Entomology 63

[33] Ames, C.; Turner, B. & Daniel, B. (2006b). Estimating the post-mortem interval (II): The

[34] Zehner, R.; Mösch, S. & Amendt, J. (2006). Estimating the post-mortem interval by

[35] Hadrys, H., Balick, M. & Schierwater, B. (1992). Applications of random amplified

[36] Stevens, J. & Wall, R. (1995). The use of random amplified polymorphic DNA (RAPD)

[37] Picard, C.J. & Wells, J.D. (2009). Survey of the genetic diversity of *Phormia regina*

[38] Picard, C.J. & Wells, J.D. (2010). The population genetic structure of North American

[39] Kester, K.M.; Toothman, M.T.; Brown, B.L.; Street, W.S. & Cruz, T.D. (2010). Recovery

[40] Li, K.; Ye, G-Y.; Zhu, J-Y. & Hu, C. (2007). Detection of food source by PCR analysis of

[42] Szalanski, A.L.; Austin, J.W.; Mckern, J.A.; McCoy T.; Steelman, C.D. & Miller, D.M.

*Agricultural Urban Entomology*, Vol. 23, No. 4, pp. 237-241, ISSN 1523-5475 [43] Mumcuoglu, K.Y.; Gallili, N.; Reshef, A.; Brauber, P. & Grant, H. (2004). Use of Human

[44] Coulson, R.M.R.; Curtis, C.F.; Ready, P.D.; Hill, N. & Smith, D.F. (1990). Amplification

[45] Zehner, R.; Amendt, J. & Krettek, R. (2004). STR Typing of Human DNA from Fly

[46] Kondakci, G.O.; Bulbul, O.; Shahzad, M.S.; Polat, E.; Cakan, H.; Altuncul, H. & Filoglu,

*Veterinary Entomology*, Vol. 4, No. , pp. 357-366, ISSN 0269-283X

post-feeding period. *Insect Science*, Vol. 14, pp. 47-52, ISSN 1744-7917 [41] Spitaleri, S.; Romano, C.; Luise, E.D.; Ginestra, E. & Saravo, L. (2006). Genptyping of

*Medical Entomology*, Vol. 46, No. 3, pp. 664-670, ISSN 0022-2585

*International*, Vol. 195, pp. 63-67, ISSN 0379-0738

*Congress Series*, Vol. 1288, pp. 574-576, ISSN 0531-5131

*International Congress Series* 1288, pp. 861-863, ISSN 0531-5131

*Congress Series,* Vol. 1288, pp. 619-621, ISSN 0531-5131

63, ISSN 0962-1083

85, pp. 549–555, ISSN 0007-4853

pp. 1543-1551, ISSN 1556-4029

806, ISSN 0022-2585

1-4, ISSN 1556-4029

1875-1768, ISSN 1875-1768

use of differential temporal gene expression to determine the age of blowfly pupae.

determining the age of fly pupae: Are there any molecular tools? *International* 

polymorphic DNA (RAPD) in molecular ecology. *Molecular Ecology*, Vol. 1, pp. 55-

analysis for studies of genetic variation in populations of the blowfly *Lucilia sericata* (Diptera: Calliphoridae) in southern England. *Bulletin of Entomological Research*, Vol.

(Diptera: Calliphoridae) using amplified fragment length polymorphisms. *Journal of* 

*Lucillia sericata* (Diptera: Calliphoridae), and the utility of genetic assessment methods for reconstruction of post-mortem corpse relocation. *Forensic Science* 

of environmental human DNA by insects. *Journal of Forensic Sciences*, Vol. 55, No. 6,

the gut contents of *Aldrinchina graham* (Aldrich) (Diptera: Calliphoridae) during

human DNA recovered from mosquitoes found on a crime scene. *International* 

(2006). Time course analysis of bed bug, *Cimex lectularius* L., (Hemiptera: Cimicidae) blood meals with the use of polymerase chain reaction. *Journal of* 

Lice in Forensic Entomology. *Journal of Medical Entomology*, Vol. 41, No. 4, pp. 803-

and analysis of human DNA present in mosquito blood meals. *Medical and* 

Larvae Fed on Decomposing Bodies. *Journal of Forensic Sciences,* Vol. 49, No. 2, pp.

G. (2009). STR and SNP analysis of human DNA from *Lucillia sericata* larvae's gut contents. *Forensic Science International*, *Genetic Supplement Series,* Vol. 2, pp. 178-179,


[20] Wang, X.; Cai, J.; Guo, Y.; Chang, Y.; Wu, K.; Wang, J., Yang, L.; Lan, L.; Zhong, M.;

[22] Nelson, L.A.; Wallman, J.F. & Dowton, M. (2008). Identification of forensically

[23] Schroeder, H., Klotzbach, H., Elias, S., Augustin C. & Pueschel, K. (2003). Use of PCR-

[25] Lin, H.; Wang, S.B.; Miao, X.X.; Wu, H. & Huang, Y.P. (2007). Identication of

[26] Jarrett, S.; Morgan, J.A.T.; Wlodek, B.M.; Brown, G.W.; Urech, R.; Green, P.E. & Lew-

[27] Donovan, S.E.; Hall, M.J.R.; Turner, B.D. & Moncrieff, C.B. (2006). Larval growth rates

[28] Anderson, G.S. (2000). Minimum and maximum development rates of some forensically

[29] Clark, K.; Evans, L. & Wall, R. (2006). Growth rates of the blowfly, *Lucillia sericata*, on

[30] Gaudry, E.; Blais, C.; Maria, A. & Dauphin-Villemant, C. (2006). Study of

[31] Tarone, A.M.; Jennings, K.C. & Foran, D.R. (2007). Aging Blow Fly Eggs using Gene

[32] Tarone, A.M. & Foran, D.R. (2011). Gene expression during blow fly development:

corpses. *Forensic Science International*, Vol. 132, Pp.76-81, ISSN 0379-0738 [24] Chen, C-H. & Shih, C-J. (2003). Rapid identification of three species of blowflies

*Entomologist,* Vol. 23, No. , pp. 59–70, ISSN 1680-7650

*Entomology*, Vol. 24, pp. 227-235, ISSN 0269-283X

*Entomology*, Vol. 20, No.1, pp. 106-114, ISSN 0269-283X

*International,* Vol. 160, No. 1, pp. 27-34, ISSN 0379-0738

*Sciences*, Vol. 56, pp. S114-S122, ISSN 1556-4029

*International,* Vol. 168, No. 2-3, pp. 148–153,ISSN 0379-0738

2442

238-247, ISSN 0379-0738

842-832, ISSN 1556-4029

ISSN 0379-0738

1354, ISSN 1556-4029

Wang, X. ; Liu, Q.; Cheng, Y. S.; Liu, Y.; Chen, Y.; Li, J.; Zhang, J. & Xin, P. (2010). The availability of 16SrDNA gene for identifying forensically important blowflies in China. *Romanian Society of Legal Medicine,* Vol.1, pp. 43 – 50, ISSN 1221-8618 [21] Zaidi, F., Wei, S-j., Shi, M. & Chen, X-x. (2011). Utility of multi-geneloci for forensic

species diagnosis of blowflies. *Journal of Insect Sciences*, Vol. 11, pp. 59, ISSN 1536-

important *Chrysomya* (Diptera: Calliphoridae) species using the second ribosomal internal transcribed spacer (ITS2). *Forensic Science International*, Vol. 177, No. , pp.

RFLP for differentiation of calliphorid larvae (Diptera: Calliphoridae) on human

(Diptera: Calliphoridae) by PCR-RFLP and DNA sequencing analysis. *Formosan* 

necrophagous y species using ISSR and SCAR markers. *Forensic Science* 

Tabor A.E. (2010). Specific detection of Old World screwworm fly, *Chrysomya bezziana*, in bulk fly trap catches using real-time PCR. *Medical and Veterinary* 

of the blowfly, *Calliphora vicina*, over a range of temperatures. *Medical and Veterinary* 

important Calliphoridae (Diptera). *Journal of Forensic Sciences*, Vol. 45, No. 2, pp.

different body tissues. *Forensic Science International*, Vol. 156, No. 2-3, pp. 145-149,

steriodogenesis in pupae of the forensically important blow fly *Protophormia terraenovae* (Robineau-Desvoidy) (Diptera: Calliphoridae). *Forensic Science* 

expression: A Feasibility Study. *Journal of Forensic Sciences*, Vol. 52, No.6, pp. 1350-

improving the precision of age estimates in forensic entomology. *Journal of Forensic* 


**4** 

*México* 

**PCR for Screening Potential** 

*1Universidad Autónoma de Baja California Sur,* 

Maurilia Rojas-Contreras1,

*La Paz, Baja California Sur,* 

**Probiotic Lactobacilli for Piglets** 

María Esther Macías-Rodríguez2 and José Alfredo Guevara Franco1

*2Universidad de Guadalajara, Centro Universitario de Ciencias e Ingenierías,* 

*Área de Conocimientos Ciencias Agropecuarias, Food Science and Technology Laboratory,* 

*Department of Pharmacobiology, Sanitary Microbiology Laboratory, Guadalajara, Jalisco,* 

To continuously select probiotic bacteria, is needed to look for new strategies to make easy this task. In this chapter the characterization and identification by PCR of presumptive adhering lactobacilli to piglet gastrointestinal tract components is described and compared with previous reports. *Lactobacillus* is one of the major bacterial groups in the gastrointestinal tract of humans and animals (Smith, 1965; Dubos, 1965). Moreover, there is accumulating scientific evidence which strongly suggest that lactobacilli are associated with health (Bibel, 1988; Sanders, 2011). Consequently lactobacilli are frequently used as probiotics. This term refers to preparations of living microbes that can be added to the diet to improve health in humans and in farm animals (Fuller, 1989; Guilliland et al., 2001). The number of reports of health-promoting effects attributed to *Lactobacillus* strains has been increased in recent years where antagonistic activities against enteropathogens and modulation of immune system are well documented (Collado, 2006). The worldwide impact of advances in the scientific knowledge in this area is being enormous. For instance, diarrheal diseases affect millions of people throughout the world, having the greatest impact among children in developing countries (Guerrant et al., 1990; Guarino et al., 2011; Mondal et al., 2011). *Lactobacillus* have been shown to possess inhibitory activity toward the growth of pathogenic bacteria such as *Listeria monocytogenes* (Ashenafi 2005; Harris et al., 1989), *Escherichia coli*, *Salmonella* spp. (Chateau & Castellanos, 1993; Hudault et al., 1997), and others (Coconnier et al., 1997). When lactobacilli could be commonly used to prevent or alleviate some of the infections by enteropathogens, e. g. *E. coli*, *Salmonella*, *Shigella*, *Campylobacter*, etc. it could be an achievement for human beings. From an economical point of view, lactobacilli could reduce the risk for major economic losses due to decreased performance and health in the farm industry. For example, pig rising has become more industrialized and intestinal disturbances, e. g. diarrhea, affect significantly the piglet health and decrease intestinal performance (Goswami et al., 2011; Oostindjer et al., 2010).

**1. Introduction** 

**1.1 Screening of potential probiotic lactobacilli** 

[47] Lord, W.D.; DiZinno, J.A.; Wilson, M.R.; Budowle, B.; Taplin, D. & Meinking, T.L. (1998). Isolation, amplication, and sequencing of human mitochondrial DNA obtained from human crab louse, *Pthirus pubis* (L.), blood meals. *Journal of Forensic Sciences*, Vol. 43, No. 2, pp. 1097-1100, ISSN 1556-4029

## **PCR for Screening Potential Probiotic Lactobacilli for Piglets**

Maurilia Rojas-Contreras1,

María Esther Macías-Rodríguez2 and José Alfredo Guevara Franco1 *1Universidad Autónoma de Baja California Sur, Área de Conocimientos Ciencias Agropecuarias, Food Science and Technology Laboratory, La Paz, Baja California Sur, 2Universidad de Guadalajara, Centro Universitario de Ciencias e Ingenierías, Department of Pharmacobiology, Sanitary Microbiology Laboratory, Guadalajara, Jalisco, México* 

## **1. Introduction**

64 Polymerase Chain Reaction

[47] Lord, W.D.; DiZinno, J.A.; Wilson, M.R.; Budowle, B.; Taplin, D. & Meinking, T.L.

*Sciences*, Vol. 43, No. 2, pp. 1097-1100, ISSN 1556-4029

(1998). Isolation, amplication, and sequencing of human mitochondrial DNA obtained from human crab louse, *Pthirus pubis* (L.), blood meals. *Journal of Forensic* 

#### **1.1 Screening of potential probiotic lactobacilli**

To continuously select probiotic bacteria, is needed to look for new strategies to make easy this task. In this chapter the characterization and identification by PCR of presumptive adhering lactobacilli to piglet gastrointestinal tract components is described and compared with previous reports. *Lactobacillus* is one of the major bacterial groups in the gastrointestinal tract of humans and animals (Smith, 1965; Dubos, 1965). Moreover, there is accumulating scientific evidence which strongly suggest that lactobacilli are associated with health (Bibel, 1988; Sanders, 2011). Consequently lactobacilli are frequently used as probiotics. This term refers to preparations of living microbes that can be added to the diet to improve health in humans and in farm animals (Fuller, 1989; Guilliland et al., 2001). The number of reports of health-promoting effects attributed to *Lactobacillus* strains has been increased in recent years where antagonistic activities against enteropathogens and modulation of immune system are well documented (Collado, 2006). The worldwide impact of advances in the scientific knowledge in this area is being enormous. For instance, diarrheal diseases affect millions of people throughout the world, having the greatest impact among children in developing countries (Guerrant et al., 1990; Guarino et al., 2011; Mondal et al., 2011). *Lactobacillus* have been shown to possess inhibitory activity toward the growth of pathogenic bacteria such as *Listeria monocytogenes* (Ashenafi 2005; Harris et al., 1989), *Escherichia coli*, *Salmonella* spp. (Chateau & Castellanos, 1993; Hudault et al., 1997), and others (Coconnier et al., 1997). When lactobacilli could be commonly used to prevent or alleviate some of the infections by enteropathogens, e. g. *E. coli*, *Salmonella*, *Shigella*, *Campylobacter*, etc. it could be an achievement for human beings. From an economical point of view, lactobacilli could reduce the risk for major economic losses due to decreased performance and health in the farm industry. For example, pig rising has become more industrialized and intestinal disturbances, e. g. diarrhea, affect significantly the piglet health and decrease intestinal performance (Goswami et al., 2011; Oostindjer et al., 2010).

PCR for Screening Potential Probiotic Lactobacilli for Piglets 67

Colonization studies of lactobacilli to the gastrointestinal tract first were concentrated on the attachment to the non secretory epithelium from the stomach. Cell morphology by electron microscopy, viable counts and biochemical test have been very important tools to identify lactobacilli attached to the keratinized squamous epithelium of the stomach of mice (N. Suegara et al., 1975; Moser & Savage, 2001; Savage, 1992; Tannock & Savage, 1974; Conway & Adams, 1989) and pig (Fuller et al., 1978; Pedersen & Tannock, 1989; Tannock et al., 1987; Henriksson et al., 1991). Later other reports on colonization by lactobacilli to other regions in the intestinal tract were found. Colonization of lactic acid bacteria isolated from rats and humans in the gastrointestinal tract of gnotobiotic rats has been studied by performing viable counts of the contents and tissue homogenates from the different regions of the intestinal tract. It was observed that lactobacilli seem to be retained, and to multiply on the mucosal surfaces along the intestinal tract (Kawai et al., 1982). In other report lactobacilli were ingested by human volunteers and samples of jejunal fluid at varying intervals were cultured for lactobacilli (Robins-Browne & Levine, 1981). It was shown that lactobacilli entered the small intestine and persisted there for 3-6 h after which time, levels returned to the base-line (Dixon, 1960). Studies on the possible interaction of lactobacilli with mammalian extracellular proteins have been performed. It was shown that specific collagen

binding is common among lactobacilli of various origins (Aleljung et al., 1991).

Attention has been focused on interactions of lactobacilli with the mucosa of the intestinal tract. The gastrointestinal tract is covered by a protective mucus layer consisting of glycolipids and a complex mixture of large and highly glycosylated proteins called mucins as the main components. Mucus layer represents the first barrier of contact between bacteria contained in the lumen and the epithelial cell layer of the host (Tassell et al., 2011). Ability of commensal bacteria to adhere mucus is an important characteristic that is evaluated in probiotic bacteria (Ma et al., 2005). Adherence of lactobacilli to the intestinal epithelium and mucus is associated with stimulation of the immune system and inhibition of adhesion of pathogens (Herías et al., 1999). Caco-2 and HT-29 cells and a subpopulation of mucus secreting HT29-MTX cells have been used to study the adhesion of human isolated *L. acidophilus* BG2F04 strain. These studies showed scanning electron micrographs where mucus secreting HT29-MTX monolayer covered by the dense mucus gel produced by these typical goblet cells, bound to lactobacilli. In addition they proposed a model for the adherence of this *Lactobacillus* strain to human intestinal cells (Coconnier et al., 1997). Other workers used human colon mucosa in an *in vitro* assay, to test the capacity of five *Lactobacillus* strains to colonize; a dense population of lactobacilli was observed covering the whole mucosal surface of the colon tissue (Sarem-Daamerdji et al., 1995). Other contributions for understanding the interactions between gastrointestinal mucosa and lactobacilli have been reported. The diversity of *Lactobacillus spp* on healthy and diseased human intestinal mucosa biopsies has be studied (Molin, 1993). These workers assessed the potential of the *Lactobacillus* isolates for treating intestinal disorders, suggesting that there are no general differences in the type of dominating *Lactobacillus* microbiota between mucosa from different regions of the intestine. In another report, different *Lactobacillus* strains in fermented oatmeal soup were administered to healthy human volunteers. Biopsies were taken from both the upper jejunum region and the rectum before one and eleven days after administration. Results showed significantly increased counts of lactobacilli on the jejunum mucosa and high levels of all those strains that remained eleven days after

**1.2 Colonization by lactobacilli** 

Antibiotics have been used successfully against these infections, however there is an increasing concern consuming meat containing antibiotic residues as well as the potential hazards from spreading of resistance factors. Lactobacilli *Lactobacillus* is an alternative to maintain the health of growing pigs, mainly were environmental conditions are not controlled (Chiduwa et al., 2008). Under these conditions are a large number of pig farms worldwide. These conditions stress the animals, causing susceptibility to gastrointestinal diseases. It is well known that lactobacilli is a habitant of the intestinal tract of pigs and has been found as dominant microbiota. However confinement in small yards, large variations in temperatures, diet and other conditions, stress the animals, causing susceptibility to gastrointestinal diseases (Shimizu & Shimizu, 1978). Lactobacilli should retain special features to survive under these harsh conditions. At birth, piglets are exposed to a huge variety of microorganisms. Most of them come from the vagina, faeces, and skin of the mother as well as the environment (Jonsson & Conway, 1992). Composition of gut microbiota can be modulated by host, environmental, and bacterial factors (Thompson-Chagoyán et al., 2007). The colonization potential of lactobacilli has been investigated using small intestinal mucus extracts from 35 day old pigs. Numbers of lactobacilli in different portions of the small intestine of 35 days old pigs were enumerated. Mucus isolated from the small intestine of pigs was investigated for its capacity to support the growth of lactobacilli and results confirmed that *Lactobacillus* spp inhabit the mucus layer of the small intestine and can grow and adhere to ileal mucus (Rojas & Conway, 1996). The survivability and colonization of probiotics in the digestive tract are considered critical to ensure optimal functionality and expression of health promoting physiological functions. Muralidhara (Muralidhara, 1977) reported that viable counts of lactobacilli in tissue homogenates from the duodenum and upper jejunum of 3 weeks old pigs were 5.5-6.21 log10 per g mucosa. In addition, when segments of the small intestine of piglets, from the duodenum to the ileum were examined, it was found that lactobacilli increased from 6.4 to 8.2 log10 per g of mucosa (McAllister et al., 1979). From the total numbers of identified strict anaerobic organisms associated with the cecal mucosa, anaerobic lactobacilli were much lower (4.0-5.7 log10) per cm2 than the numbers of obligated anaerobes. Although differences in the counts of the different groups of organisms have been quite large for the various reports, *Lactobacillus* appears to be dominant group in cecal and colonic content.

Screening for functional and probiotic attributes in lactobacilli new isolates is commonly performed, following these assays: Gram stain, acid and bile salt tolerance, cell surface hydrophobicity, adhesion to mucus and mucin, autoaggregation, Caco-2 cell-binding as well as antibacterial activity against *E. coli, L. monocytogenes, S. typhi*, etc. and antioxidative activities (Jacobsen et al., 1999; Macías-Rodríguez et al., 2008; Kaushik et al., 2009). Recently a screening of predominant *Lactobacillus* strains from healthy piglets has been performed in order to select specific probiotics for arid land piglets. Among the 164 isolates, 27 adhesive strains were identified using comparisons with 16S rDNA and intergenic 16-23S sequences. Results indicated that *L. fermentum* and *L. reuteri* were the most common species in faeces and mucus, respectively (Macías-Rodríguez et al., 2009). Likewise probiotics are increasingly used as nutraceuticals, functional foods or prophylactics and considering that probiotics strains have shown to be populationspecific due to variation in gut microbiota, food habits and specific host-microbial interactions (Kaushik et al., 2009), screening of new indigenous probiotic strains in different region of the world is necessary.

#### **1.2 Colonization by lactobacilli**

66 Polymerase Chain Reaction

Antibiotics have been used successfully against these infections, however there is an increasing concern consuming meat containing antibiotic residues as well as the potential hazards from spreading of resistance factors. Lactobacilli *Lactobacillus* is an alternative to maintain the health of growing pigs, mainly were environmental conditions are not controlled (Chiduwa et al., 2008). Under these conditions are a large number of pig farms worldwide. These conditions stress the animals, causing susceptibility to gastrointestinal diseases. It is well known that lactobacilli is a habitant of the intestinal tract of pigs and has been found as dominant microbiota. However confinement in small yards, large variations in temperatures, diet and other conditions, stress the animals, causing susceptibility to gastrointestinal diseases (Shimizu & Shimizu, 1978). Lactobacilli should retain special features to survive under these harsh conditions. At birth, piglets are exposed to a huge variety of microorganisms. Most of them come from the vagina, faeces, and skin of the mother as well as the environment (Jonsson & Conway, 1992). Composition of gut microbiota can be modulated by host, environmental, and bacterial factors (Thompson-Chagoyán et al., 2007). The colonization potential of lactobacilli has been investigated using small intestinal mucus extracts from 35 day old pigs. Numbers of lactobacilli in different portions of the small intestine of 35 days old pigs were enumerated. Mucus isolated from the small intestine of pigs was investigated for its capacity to support the growth of lactobacilli and results confirmed that *Lactobacillus* spp inhabit the mucus layer of the small intestine and can grow and adhere to ileal mucus (Rojas & Conway, 1996). The survivability and colonization of probiotics in the digestive tract are considered critical to ensure optimal functionality and expression of health promoting physiological functions. Muralidhara (Muralidhara, 1977) reported that viable counts of lactobacilli in tissue homogenates from the duodenum and upper jejunum of 3 weeks old pigs were 5.5-6.21 log10 per g mucosa. In addition, when segments of the small intestine of piglets, from the duodenum to the ileum were examined, it was found that lactobacilli increased from 6.4 to 8.2 log10 per g of mucosa (McAllister et al., 1979). From the total numbers of identified strict anaerobic organisms associated with the cecal mucosa, anaerobic lactobacilli were much lower (4.0-5.7 log10) per cm2 than the numbers of obligated anaerobes. Although differences in the counts of the different groups of organisms have been quite large for the various reports, *Lactobacillus*

appears to be dominant group in cecal and colonic content.

different region of the world is necessary.

Screening for functional and probiotic attributes in lactobacilli new isolates is commonly performed, following these assays: Gram stain, acid and bile salt tolerance, cell surface hydrophobicity, adhesion to mucus and mucin, autoaggregation, Caco-2 cell-binding as well as antibacterial activity against *E. coli, L. monocytogenes, S. typhi*, etc. and antioxidative activities (Jacobsen et al., 1999; Macías-Rodríguez et al., 2008; Kaushik et al., 2009). Recently a screening of predominant *Lactobacillus* strains from healthy piglets has been performed in order to select specific probiotics for arid land piglets. Among the 164 isolates, 27 adhesive strains were identified using comparisons with 16S rDNA and intergenic 16-23S sequences. Results indicated that *L. fermentum* and *L. reuteri* were the most common species in faeces and mucus, respectively (Macías-Rodríguez et al., 2009). Likewise probiotics are increasingly used as nutraceuticals, functional foods or prophylactics and considering that probiotics strains have shown to be populationspecific due to variation in gut microbiota, food habits and specific host-microbial interactions (Kaushik et al., 2009), screening of new indigenous probiotic strains in Colonization studies of lactobacilli to the gastrointestinal tract first were concentrated on the attachment to the non secretory epithelium from the stomach. Cell morphology by electron microscopy, viable counts and biochemical test have been very important tools to identify lactobacilli attached to the keratinized squamous epithelium of the stomach of mice (N. Suegara et al., 1975; Moser & Savage, 2001; Savage, 1992; Tannock & Savage, 1974; Conway & Adams, 1989) and pig (Fuller et al., 1978; Pedersen & Tannock, 1989; Tannock et al., 1987; Henriksson et al., 1991). Later other reports on colonization by lactobacilli to other regions in the intestinal tract were found. Colonization of lactic acid bacteria isolated from rats and humans in the gastrointestinal tract of gnotobiotic rats has been studied by performing viable counts of the contents and tissue homogenates from the different regions of the intestinal tract. It was observed that lactobacilli seem to be retained, and to multiply on the mucosal surfaces along the intestinal tract (Kawai et al., 1982). In other report lactobacilli were ingested by human volunteers and samples of jejunal fluid at varying intervals were cultured for lactobacilli (Robins-Browne & Levine, 1981). It was shown that lactobacilli entered the small intestine and persisted there for 3-6 h after which time, levels returned to the base-line (Dixon, 1960). Studies on the possible interaction of lactobacilli with mammalian extracellular proteins have been performed. It was shown that specific collagen binding is common among lactobacilli of various origins (Aleljung et al., 1991).

Attention has been focused on interactions of lactobacilli with the mucosa of the intestinal tract. The gastrointestinal tract is covered by a protective mucus layer consisting of glycolipids and a complex mixture of large and highly glycosylated proteins called mucins as the main components. Mucus layer represents the first barrier of contact between bacteria contained in the lumen and the epithelial cell layer of the host (Tassell et al., 2011). Ability of commensal bacteria to adhere mucus is an important characteristic that is evaluated in probiotic bacteria (Ma et al., 2005). Adherence of lactobacilli to the intestinal epithelium and mucus is associated with stimulation of the immune system and inhibition of adhesion of pathogens (Herías et al., 1999). Caco-2 and HT-29 cells and a subpopulation of mucus secreting HT29-MTX cells have been used to study the adhesion of human isolated *L. acidophilus* BG2F04 strain. These studies showed scanning electron micrographs where mucus secreting HT29-MTX monolayer covered by the dense mucus gel produced by these typical goblet cells, bound to lactobacilli. In addition they proposed a model for the adherence of this *Lactobacillus* strain to human intestinal cells (Coconnier et al., 1997). Other workers used human colon mucosa in an *in vitro* assay, to test the capacity of five *Lactobacillus* strains to colonize; a dense population of lactobacilli was observed covering the whole mucosal surface of the colon tissue (Sarem-Daamerdji et al., 1995). Other contributions for understanding the interactions between gastrointestinal mucosa and lactobacilli have been reported. The diversity of *Lactobacillus spp* on healthy and diseased human intestinal mucosa biopsies has be studied (Molin, 1993). These workers assessed the potential of the *Lactobacillus* isolates for treating intestinal disorders, suggesting that there are no general differences in the type of dominating *Lactobacillus* microbiota between mucosa from different regions of the intestine. In another report, different *Lactobacillus* strains in fermented oatmeal soup were administered to healthy human volunteers. Biopsies were taken from both the upper jejunum region and the rectum before one and eleven days after administration. Results showed significantly increased counts of lactobacilli on the jejunum mucosa and high levels of all those strains that remained eleven days after

PCR for Screening Potential Probiotic Lactobacilli for Piglets 69

Cell wall of Gram positive bacteria is composed primarily of peptidoglycan, which often contains peptide interbridge and large amounts of teichoic acids (polymers of glycerol or ribitol joined by phosphate groups). Amino acids and sugars are attached to the glycerol and ribitol groups. These molecules are important for maintaining the structure of the wall. Capsules, slims S-layers, sheaths or even pili (fimbriae) can occur as superficial layers above the cell wall. They can occur singly or in combination. Distinction among them is based primarily on their structural attributes (Beveridge, 1989; Beveridge & Graham, 1991). The term adhesin has been used to denote functions that are involved in one or more of the three following activities: 1) they may promote attachment and then initiate colonization of surface habitats, 2) They may be responsible for the organization of microbial communities and assemblages, and 3) they may be instrumental in promoting cell to cell contact as a phase preceding the transfer of genetic information between cells. The term adhesion has been used to describe the relatively stable, irreversible attachment of bacteria to surfaces, and the term receptor has been used for both known and putative entities on surfaces to which adhesins bind to effect specific adhesion (Jones & Isaacson, 1984). While there is a considerable amount of information published about proteinaceous bacterial adhesins and their receptors on pathogenic bacteria (Jones & Isaacson, 1984; Klemm, 1994; Bonazzi & Cossart, 2011), there are fewer studies on the mechanisms of adhesion of lactobacilli to gastrointestinal mucosa. Adhesion of *L. acidophilus* to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer protein (S-layer) protein was reported (Schneitz & Lounatma, 1993), and the adhesion to collagen by *L. reuteri* NCIB 11951 was shown to be mediated by a 29 KDa protein (Aleljung et al., 1994) and to *L. crrispatus* JCM 5810 was mediated by a 120 KDa S-layer protein (Toba et al., 1995). Another interesting finding was a 32 KDa protein, an aggregation promoting factor on *L. plantarum* strain 4B2 which increased the frequency of conjugation (Reniero et al., 1992; Reniero et al., 1993). The ability of probiotic bacteria to aggregate should be considered a desirable characteristic because they potentially inhibit adherence of pathogenic bacteria to intestinal mucosa either by direct coaggregation with the pathogens to facilitate clearance, by forming a barrier via self-aggregation or coaggregation with commensal organisms on the intestinal mucosa. Surface proteins from lactobacilli have been reported to be affected by freeze drying (Ray & Johnson, 1986; Brennan et al., 1986) and by the composition of the culture media (Pavlova et

Purification and characterization of proteins from lactobacilli which promote the adhesion to mucus have been well studied. The purification of a mucus and mucin adhesion promoting protein (MAPP) from the surface of *L. reuteri* 104R was performed by using LiCl (1M). A variety of different agents to extract proteins have been used. EDTA (0.1M), urea (8M) or MgCl2 have been used to effectively release surface associated material from bacterial cells of various genera. Solutions of detergents such as sodium lauryl sarcosinate, triton X 100 (1% v/v final concentration), sonication and sodium dodecyl sulphate (SDS, 1%, w/v) have been shown to be effective in extracting proteins from *L. reuteri* strain 100-23 (Boot et al., 1993; Chagnaud et al., 1992). Guanidine hydrochloride (4M, GHCl) was used to extract regular arrays from the cell walls of different strains (Masuda & Kawata, 1983) and an S-layer protein from *L. acidophilus* ATCC 4356 (Boot et al., 1993). GHCl (2M) was used to extract a collagen bindig S-layer protein from *L. crispatus* JCM 5810 (Toba et al., 1995) while LiCl (1M) for 20 h at 20°C after treatment with lysozyme (2 mg per ml) for 1 h, was used to extract another collagen binding protein from *L. reuteri* NCIB 11951 (Aleljung et al., 1994).

al., 1993; Cook et al., 1988).

administration (Johansson et al., 1993). Colonization experiments in mice, also showed that the number of lactobacilli detected in samples collected from various regions of the gastrointestinal tract, two weeks after inoculation, were not statistically significant different, no matter which strain had been used to colonize mice. In addition, it was concluded that bile salt hydrolase production was not an essential attribute for lactobacilli to colonize the murine gastrointestinal tract. Furthermore, the growth rate of mice that consumed a nutritionally balanced diet were not affected by the presence of bile salt hydrolase producing or not lactobacilli in the gastrointestinal tract (Bateup et al., 1995). The capacity of different lactobacillus strains to grow in and adhere to small intestinal mucus as well as the characteristics of binding was studied. It was shown that six *Lactobacillus* strains isolated from porcine small intestinal mucosa, one isolated from faeces, one isolated from stomach and one more isolated from human feces, all grew equally well in intestinal mucus extract. Growth was monitored by enumerating the colony forming units. During growth in mucus, a visible precipitation was developed because lactobacilli formed clusters surrounded by mucus. In this study it was observed that when lactobacilli were grown in mucus, the ability to adhere to mucus was reduced from 35% to 10% of the adhesion. This could occur because adhesin(s) on the surface of the bacteria were being blocked by receptors or receptors-like components in the mucus (Rojas & Conway 1996). Adhesion assays of *Lactobacillus fermentum* 104R (Actually identified as *L. reuteri* 104R) indicated that this strain adhered to mucus when it was grown in synthetic media. Adhesion data were analyzed by Scatchard plot and it was noted that the binding of lactobacilli to mucus is not mediated by a single adhesin-receptor interaction. The quantitative interpretation of the binding data for this system was not possible to perform because the complexity of the system. These results correlate with other report suggesting that lactobacillus species adhere to intestinal cells via mechanisms which involve different combination of factors on the bacterial cell surface (Greene & Klaenhammer, 1994). Adhesion promoting compound(s) from *L. reuteri* 104R were found in the spent culture medium on the late stationary phase of growth. The spent culture fluid was used to inhibit adhesion to mucus of whole *L. reuteri* 104R strain, revealing that proteinaceous compound(s) were involved in the binding (Rojas & Conway, 1996).

#### **1.3** *Lactobacillus* **adhesins**

Bacteria can have many types of surfaces, including sheaths, S-layers, capsules and walls. In the laboratory certain surface types are usually expressed. For example, *E. coli* K12 contains only core polysaccharide plus lipid A in its lipopolisaccharide that was why this strain is restricted to a laboratory habitat since it cannot withstand the rigors of a natural environment. This strain possesses only an outer membrane as its surface component surfaces components, but a related strain, K-30, is enclosed in a capsule. Frequently, it is the natural environment and their intrinsic stress that elicit expression of the surface attributes of a bacterium (Costerton, 1988; Brown et al., 1988). A bacterium in its native habitat will often possess a wall overlaid by a multiplicity of superficial layers. After several subcultures in laboratory medium these layers are not longer required and are lost (Costerton, 1988). This surface character could makes difficult the correlation of laboratory studies on adhesins of the bacteria with the *In vivo* state. Intestinal mucus extract from the small intestine of pig was used for lactobacilli growth and for studying the production and expression of the mucus and mucin adhesion promoting proteins.

administration (Johansson et al., 1993). Colonization experiments in mice, also showed that the number of lactobacilli detected in samples collected from various regions of the gastrointestinal tract, two weeks after inoculation, were not statistically significant different, no matter which strain had been used to colonize mice. In addition, it was concluded that bile salt hydrolase production was not an essential attribute for lactobacilli to colonize the murine gastrointestinal tract. Furthermore, the growth rate of mice that consumed a nutritionally balanced diet were not affected by the presence of bile salt hydrolase producing or not lactobacilli in the gastrointestinal tract (Bateup et al., 1995). The capacity of different lactobacillus strains to grow in and adhere to small intestinal mucus as well as the characteristics of binding was studied. It was shown that six *Lactobacillus* strains isolated from porcine small intestinal mucosa, one isolated from faeces, one isolated from stomach and one more isolated from human feces, all grew equally well in intestinal mucus extract. Growth was monitored by enumerating the colony forming units. During growth in mucus, a visible precipitation was developed because lactobacilli formed clusters surrounded by mucus. In this study it was observed that when lactobacilli were grown in mucus, the ability to adhere to mucus was reduced from 35% to 10% of the adhesion. This could occur because adhesin(s) on the surface of the bacteria were being blocked by receptors or receptors-like components in the mucus (Rojas & Conway 1996). Adhesion assays of *Lactobacillus fermentum* 104R (Actually identified as *L. reuteri* 104R) indicated that this strain adhered to mucus when it was grown in synthetic media. Adhesion data were analyzed by Scatchard plot and it was noted that the binding of lactobacilli to mucus is not mediated by a single adhesin-receptor interaction. The quantitative interpretation of the binding data for this system was not possible to perform because the complexity of the system. These results correlate with other report suggesting that lactobacillus species adhere to intestinal cells via mechanisms which involve different combination of factors on the bacterial cell surface (Greene & Klaenhammer, 1994). Adhesion promoting compound(s) from *L. reuteri* 104R were found in the spent culture medium on the late stationary phase of growth. The spent culture fluid was used to inhibit adhesion to mucus of whole *L. reuteri* 104R strain, revealing that proteinaceous compound(s) were involved in the binding (Rojas &

Bacteria can have many types of surfaces, including sheaths, S-layers, capsules and walls. In the laboratory certain surface types are usually expressed. For example, *E. coli* K12 contains only core polysaccharide plus lipid A in its lipopolisaccharide that was why this strain is restricted to a laboratory habitat since it cannot withstand the rigors of a natural environment. This strain possesses only an outer membrane as its surface component surfaces components, but a related strain, K-30, is enclosed in a capsule. Frequently, it is the natural environment and their intrinsic stress that elicit expression of the surface attributes of a bacterium (Costerton, 1988; Brown et al., 1988). A bacterium in its native habitat will often possess a wall overlaid by a multiplicity of superficial layers. After several subcultures in laboratory medium these layers are not longer required and are lost (Costerton, 1988). This surface character could makes difficult the correlation of laboratory studies on adhesins of the bacteria with the *In vivo* state. Intestinal mucus extract from the small intestine of pig was used for lactobacilli growth and for studying the production and expression of the

Conway, 1996).

**1.3** *Lactobacillus* **adhesins** 

mucus and mucin adhesion promoting proteins.

Cell wall of Gram positive bacteria is composed primarily of peptidoglycan, which often contains peptide interbridge and large amounts of teichoic acids (polymers of glycerol or ribitol joined by phosphate groups). Amino acids and sugars are attached to the glycerol and ribitol groups. These molecules are important for maintaining the structure of the wall. Capsules, slims S-layers, sheaths or even pili (fimbriae) can occur as superficial layers above the cell wall. They can occur singly or in combination. Distinction among them is based primarily on their structural attributes (Beveridge, 1989; Beveridge & Graham, 1991). The term adhesin has been used to denote functions that are involved in one or more of the three following activities: 1) they may promote attachment and then initiate colonization of surface habitats, 2) They may be responsible for the organization of microbial communities and assemblages, and 3) they may be instrumental in promoting cell to cell contact as a phase preceding the transfer of genetic information between cells. The term adhesion has been used to describe the relatively stable, irreversible attachment of bacteria to surfaces, and the term receptor has been used for both known and putative entities on surfaces to which adhesins bind to effect specific adhesion (Jones & Isaacson, 1984). While there is a considerable amount of information published about proteinaceous bacterial adhesins and their receptors on pathogenic bacteria (Jones & Isaacson, 1984; Klemm, 1994; Bonazzi & Cossart, 2011), there are fewer studies on the mechanisms of adhesion of lactobacilli to gastrointestinal mucosa. Adhesion of *L. acidophilus* to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer protein (S-layer) protein was reported (Schneitz & Lounatma, 1993), and the adhesion to collagen by *L. reuteri* NCIB 11951 was shown to be mediated by a 29 KDa protein (Aleljung et al., 1994) and to *L. crrispatus* JCM 5810 was mediated by a 120 KDa S-layer protein (Toba et al., 1995). Another interesting finding was a 32 KDa protein, an aggregation promoting factor on *L. plantarum* strain 4B2 which increased the frequency of conjugation (Reniero et al., 1992; Reniero et al., 1993). The ability of probiotic bacteria to aggregate should be considered a desirable characteristic because they potentially inhibit adherence of pathogenic bacteria to intestinal mucosa either by direct coaggregation with the pathogens to facilitate clearance, by forming a barrier via self-aggregation or coaggregation with commensal organisms on the intestinal mucosa. Surface proteins from lactobacilli have been reported to be affected by freeze drying (Ray & Johnson, 1986; Brennan et al., 1986) and by the composition of the culture media (Pavlova et al., 1993; Cook et al., 1988).

Purification and characterization of proteins from lactobacilli which promote the adhesion to mucus have been well studied. The purification of a mucus and mucin adhesion promoting protein (MAPP) from the surface of *L. reuteri* 104R was performed by using LiCl (1M). A variety of different agents to extract proteins have been used. EDTA (0.1M), urea (8M) or MgCl2 have been used to effectively release surface associated material from bacterial cells of various genera. Solutions of detergents such as sodium lauryl sarcosinate, triton X 100 (1% v/v final concentration), sonication and sodium dodecyl sulphate (SDS, 1%, w/v) have been shown to be effective in extracting proteins from *L. reuteri* strain 100-23 (Boot et al., 1993; Chagnaud et al., 1992). Guanidine hydrochloride (4M, GHCl) was used to extract regular arrays from the cell walls of different strains (Masuda & Kawata, 1983) and an S-layer protein from *L. acidophilus* ATCC 4356 (Boot et al., 1993). GHCl (2M) was used to extract a collagen bindig S-layer protein from *L. crispatus* JCM 5810 (Toba et al., 1995) while LiCl (1M) for 20 h at 20°C after treatment with lysozyme (2 mg per ml) for 1 h, was used to extract another collagen binding protein from *L. reuteri* NCIB 11951 (Aleljung et al., 1994).

PCR for Screening Potential Probiotic Lactobacilli for Piglets 71

Additionally, it is recognized that resistance of potential probiotic to bile salts is a testable

The mechanisms used by lactobacilli to recognize and adhere to gastrointestinal components, until now is not completely understood. Protein and carbohydrate play an important role in mediating the adhesion to mucosal and or epithelial host surfaces. Some cell-surface biomolecules as exopolysaccharides and proteins have been recognized by their ability to bind gastrointestinal components (Vélez et al., 2007; Rojas et al., 2002; Sun et al., 2007). The best characterized are proteins present in the surface of lactobacilli that can be attached covalently or not to the cell wall (Vélez et al., 2007). Recently, proteins that adhere to mucus or mucins have been described and characterized. Adhering protein molecules characterized from *Lactobacillus* are Mucus-binding protein (Mub) of *L. reuteri* 1063 (Roos & Jonsson, 2002), the lectine-like mannose-specific adhesin (Msa) of *L. plantarum* WCFS1 (Pretzer et al., 2005), the mucus adhesion promoting protein (MAPP or MapA) from *L. reuteri* 104R reported by its ability to bind porcine mucus and mucin (Rojas et al., 2002) and Caco-2 cells (Miyoshi et al., 2006) and the Mub of *L. acidophilus* NCFM (Buck et al., 2005). Moreover, two proteins EF-Tu (Elongation Factor-Tu) and GroEL (a class of heat shock protein) of *L. johnsonii* La1 NCC533 showed abilities to adhere to mucins at specific conditions of pH (Granato et al., 2004; Bergonzelli et al., 2006). Recently a piglet mucus adhesion protein was completely characterized from the potential probiotic *L. fermentum*

Genetic research on *Lactobacillus* is underway in many laboratories around the world. Research has centered on 1) characterization and construction of vectors based on endogenos *Lactobacillus* plasmids which are capables to replicateof replicate and express molecules in specific lactobacilli strains, 2) molecular cloning of genes and operons from lactobacilli encoding important metabolic pathways, proteinases and adhesins 3) methods for introduction of genes *In vivo* and *In vitro* through conjugation, transfection and transformation (Chassy, 1987), and more recently 4) the global analysis of proteins and genes using the new tools of proteomic and genomic and the data base information of diferent species of *Lactobacillus* which are in public data bases. The development of cloning systems of *Lactobacillus* have increased in the last years. Methods for the introduction and stable maintenance of DNA into *Lactobacillus* are routine now and can be applied to almost any *Lactobacillus* species. Both broad host-range and narrow host range multi-copy plasmid vectors based on a variety of replicons have become available for the introduction and expression of homologous and heterologous genes (Pouwels & Leer, 1993). The sequenced genomes of lactobacilli are increasing and their availability might lead to the identification of the adhesin domain containing proteins in other species of *Lactobacillus* and in the specific functions of this surface proteins. Genes codifying for above adhesins are well known. The cloning and sequencing of the *L. reuteri* 104R gene encoding the adhesion promoting protein (MAPP) that binds to porcine gastrointestinal mucus was also studied. The sequence revealed one open reading frame consisting of 744 nucleotides corresponding to 244 aminoacids with deduced pI of 10.57, net charge at pH 7 of 16.23 and a molecular mass of 26.4 KDa. No putative promoter was found, however a start codon (ATG) appeared 6 bases downstream from the beginning of the sequence. The open reading frame ended with stop

and is a necessary property (Moser & Savage, 2001).

strain BCS87 (Macías-Rodríguez et al., 2009).

**1.5 Genes codifying for** *Lactobacillus* **adhesins** 

The MAPP protein from *L. reuteri* 104R was extracted from the surface, by treating the cells after 14-16 h growth in a semi-defined medium (LDM), with LiCl (1M) for 1 h with gently mixing at 4°C. However, when other lactobacilli strains isolated also from intestinal tract which presented binding to mucus and mucin were treated as above did not show the characteristic band of the MAPP adhesin as it was visualized by western blot with the horse radish peroxidase labeled mucin (Rojas et al., 2002). The adhesion of *L. reuteri* JCM1081 to HT-29 cells mediated by a cell surface protein was reported. Results showed a 29-kDa surface protein which displays significant peptide sequence similarity to the Lr0793 protein from *L. reuteri* ATCC55730 (71.1% identity), whereas the protein Lr0793 is homologous to the ABC transporter component CnBP, which previously has been described as a collagen binding protein. The 29-kDa surface protein of *L. reuteri* JCM1081 probably is classified as a member of the ABC transporter family, as well as CnBP from *L. reuteri* NCIB11951 and MapA from *L. reuteri* 104R (Wang et al., 2008). The mucus-binding properties of a large collection of *L. reuteri* strains isolated from a range of vertebrate hosts and the correlation of the adherence of a subset of strains to the presence and expression of MUB was performed by immunodetection, microscopic immunolocalization of MUB on the bacteria, characterization of cell-surface extracts and spent media by gel electrophoresis, Western blotting and mass spectrometry, quantification of *mub* gene expression by qRT-PCR, cell aggregation and cell-surface MUB quantification. Results revealed that the particular MUB investigated is highly specific to a very small set of closely related strains of *L. reuteri.* This was observed despite the fact that 17 proteins with a putative MucBP domain were found in the available genomes of *L. reuteri.* strains 100-23, DSM 20016T, MM2-3, MM4-1, ATCC 55730 and CF48-3A, nine of which were present in the rodent isolate 100-23 (Mackenzie et al., 2010).

#### **1.4 Adhering probiotic** *Lactobacillus*

Two requirements have been identified as desirable properties for *Lactobacillus* to be considered as an effective probiotic microorganism, these include the ability to adhere (Reid, 1999), and then to consequently colonize mucous surfaces. Mucus layer is the first physical barrier to host-cell stimulation by bacteria in the gut. Adhesion to mucus is therefore the first step required for probiotic organisms to interact with host cells and elicit any particular response. Adherence to intestinal mucus has been associated to competitive exclusion of pathogens (Gueimonde et al., 2006; Lee et al., 2008) considering it as a critical event for colonization not only for lactobacilli but also for pathogenic bacteria (Beachey, 1981; Soto & Hultgren, 1999). In the gastrointestinal tract, mucus is the outermost luminal layer, and is the first intestinal component of surface that microorganisms are likely to contact before they reach epithelial cells. Mackie (Mackie et al., 1999) suggested that during a colonization event, bacterial population remains stable in size, with no need of periodic reintroduction of bacteria by oral doses. This implies that colonizing bacteria multiply in a given intestinal niche at a rate that equals or exceeds their rate of washout or elimination from the intestinal site. However, in practical terms it is well known that external factors can arise such as antibiotic treatments or a change in the nutritional regime that can disrupt the equilibrium of the normal bacterial population (Jernberg et al., 2005). In these cases, it is necessary to supplement the feed with probiotics to restore the balance. Therefore, the ability to replicate in mucus represents an important parameter to evaluate in potential probiotic strains.

The MAPP protein from *L. reuteri* 104R was extracted from the surface, by treating the cells after 14-16 h growth in a semi-defined medium (LDM), with LiCl (1M) for 1 h with gently mixing at 4°C. However, when other lactobacilli strains isolated also from intestinal tract which presented binding to mucus and mucin were treated as above did not show the characteristic band of the MAPP adhesin as it was visualized by western blot with the horse radish peroxidase labeled mucin (Rojas et al., 2002). The adhesion of *L. reuteri* JCM1081 to HT-29 cells mediated by a cell surface protein was reported. Results showed a 29-kDa surface protein which displays significant peptide sequence similarity to the Lr0793 protein from *L. reuteri* ATCC55730 (71.1% identity), whereas the protein Lr0793 is homologous to the ABC transporter component CnBP, which previously has been described as a collagen binding protein. The 29-kDa surface protein of *L. reuteri* JCM1081 probably is classified as a member of the ABC transporter family, as well as CnBP from *L. reuteri* NCIB11951 and MapA from *L. reuteri* 104R (Wang et al., 2008). The mucus-binding properties of a large collection of *L. reuteri* strains isolated from a range of vertebrate hosts and the correlation of the adherence of a subset of strains to the presence and expression of MUB was performed by immunodetection, microscopic immunolocalization of MUB on the bacteria, characterization of cell-surface extracts and spent media by gel electrophoresis, Western blotting and mass spectrometry, quantification of *mub* gene expression by qRT-PCR, cell aggregation and cell-surface MUB quantification. Results revealed that the particular MUB investigated is highly specific to a very small set of closely related strains of *L. reuteri.* This was observed despite the fact that 17 proteins with a putative MucBP domain were found in the available genomes of *L. reuteri.* strains 100-23, DSM 20016T, MM2-3, MM4-1, ATCC 55730 and CF48-3A, nine of which were present in the rodent isolate 100-23 (Mackenzie et

Two requirements have been identified as desirable properties for *Lactobacillus* to be considered as an effective probiotic microorganism, these include the ability to adhere (Reid, 1999), and then to consequently colonize mucous surfaces. Mucus layer is the first physical barrier to host-cell stimulation by bacteria in the gut. Adhesion to mucus is therefore the first step required for probiotic organisms to interact with host cells and elicit any particular response. Adherence to intestinal mucus has been associated to competitive exclusion of pathogens (Gueimonde et al., 2006; Lee et al., 2008) considering it as a critical event for colonization not only for lactobacilli but also for pathogenic bacteria (Beachey, 1981; Soto & Hultgren, 1999). In the gastrointestinal tract, mucus is the outermost luminal layer, and is the first intestinal component of surface that microorganisms are likely to contact before they reach epithelial cells. Mackie (Mackie et al., 1999) suggested that during a colonization event, bacterial population remains stable in size, with no need of periodic reintroduction of bacteria by oral doses. This implies that colonizing bacteria multiply in a given intestinal niche at a rate that equals or exceeds their rate of washout or elimination from the intestinal site. However, in practical terms it is well known that external factors can arise such as antibiotic treatments or a change in the nutritional regime that can disrupt the equilibrium of the normal bacterial population (Jernberg et al., 2005). In these cases, it is necessary to supplement the feed with probiotics to restore the balance. Therefore, the ability to replicate in mucus represents an important parameter to evaluate in potential probiotic strains.

al., 2010).

**1.4 Adhering probiotic** *Lactobacillus* 

Additionally, it is recognized that resistance of potential probiotic to bile salts is a testable and is a necessary property (Moser & Savage, 2001).

The mechanisms used by lactobacilli to recognize and adhere to gastrointestinal components, until now is not completely understood. Protein and carbohydrate play an important role in mediating the adhesion to mucosal and or epithelial host surfaces. Some cell-surface biomolecules as exopolysaccharides and proteins have been recognized by their ability to bind gastrointestinal components (Vélez et al., 2007; Rojas et al., 2002; Sun et al., 2007). The best characterized are proteins present in the surface of lactobacilli that can be attached covalently or not to the cell wall (Vélez et al., 2007). Recently, proteins that adhere to mucus or mucins have been described and characterized. Adhering protein molecules characterized from *Lactobacillus* are Mucus-binding protein (Mub) of *L. reuteri* 1063 (Roos & Jonsson, 2002), the lectine-like mannose-specific adhesin (Msa) of *L. plantarum* WCFS1 (Pretzer et al., 2005), the mucus adhesion promoting protein (MAPP or MapA) from *L. reuteri* 104R reported by its ability to bind porcine mucus and mucin (Rojas et al., 2002) and Caco-2 cells (Miyoshi et al., 2006) and the Mub of *L. acidophilus* NCFM (Buck et al., 2005). Moreover, two proteins EF-Tu (Elongation Factor-Tu) and GroEL (a class of heat shock protein) of *L. johnsonii* La1 NCC533 showed abilities to adhere to mucins at specific conditions of pH (Granato et al., 2004; Bergonzelli et al., 2006). Recently a piglet mucus adhesion protein was completely characterized from the potential probiotic *L. fermentum* strain BCS87 (Macías-Rodríguez et al., 2009).

#### **1.5 Genes codifying for** *Lactobacillus* **adhesins**

Genetic research on *Lactobacillus* is underway in many laboratories around the world. Research has centered on 1) characterization and construction of vectors based on endogenos *Lactobacillus* plasmids which are capables to replicateof replicate and express molecules in specific lactobacilli strains, 2) molecular cloning of genes and operons from lactobacilli encoding important metabolic pathways, proteinases and adhesins 3) methods for introduction of genes *In vivo* and *In vitro* through conjugation, transfection and transformation (Chassy, 1987), and more recently 4) the global analysis of proteins and genes using the new tools of proteomic and genomic and the data base information of diferent species of *Lactobacillus* which are in public data bases. The development of cloning systems of *Lactobacillus* have increased in the last years. Methods for the introduction and stable maintenance of DNA into *Lactobacillus* are routine now and can be applied to almost any *Lactobacillus* species. Both broad host-range and narrow host range multi-copy plasmid vectors based on a variety of replicons have become available for the introduction and expression of homologous and heterologous genes (Pouwels & Leer, 1993). The sequenced genomes of lactobacilli are increasing and their availability might lead to the identification of the adhesin domain containing proteins in other species of *Lactobacillus* and in the specific functions of this surface proteins. Genes codifying for above adhesins are well known. The cloning and sequencing of the *L. reuteri* 104R gene encoding the adhesion promoting protein (MAPP) that binds to porcine gastrointestinal mucus was also studied. The sequence revealed one open reading frame consisting of 744 nucleotides corresponding to 244 aminoacids with deduced pI of 10.57, net charge at pH 7 of 16.23 and a molecular mass of 26.4 KDa. No putative promoter was found, however a start codon (ATG) appeared 6 bases downstream from the beginning of the sequence. The open reading frame ended with stop

PCR for Screening Potential Probiotic Lactobacilli for Piglets 73

SpaB may be involved in SpaCBA pilus-mediated adherence to intestinal mucus. It was established that the SpaF minor pilin is the only mucus binding component in the putative SpaFED pilus fiber (von Ossowski et al., 2010). Aggregation promoting factors (Apf) are secreted proteins that have been associated with a diverse number of functional roles in lactobacilli, including self aggregation, coaggregation with other commensal or pathogenic bacteria, maintenance of cell shape and the bridging of conjugal pairs. Genes encoding Apf´s have been characterized for several *Lactobacillus* species, including *L. crispatus, L. johnsonii, L. gasseri, L. paracasei* and *L. coryniformis*. Investigation of the functional role of the putative *apf* gene (LBA0493) in *L. acidophilus* NCFM by mutational analysis was performed. It was observed that survival rates mutant strain NCK2033 decreased when stationary phase cells were exposed to simulated small intestinal and gastric juices. Furthermore, NCK2033 in the stationary phase showed a reduction of *In vitro* adherence to Caco-2 intestinal epithelial cells, mucin glycoproteins and fibronectin. It was suggested that the Apf-like proteins may contributes to the survival of *L. acidophilus* during transit through the digestive tract and, potentially, participate in the interactions with the host intestinal mucosa (Goh & Klaenhammer, 2010). The ability to tolerate the toxic levels of bile salts accumulated therein is the essential requirement to survive in the gut and it is generally included among the criteria used for selection of the potential probiotic strains and their application as functional ingredients in foods and nutraceuticals. Expression of bile salt hydrolase and surface proteins were targeted to look at their expression profile in two putative probiotic *L. plantarum* Lp9 and Lp91, (compared with standard strain CSCC5276) by quantitative real time PCR (RT – qPCR). Expression ratio for *bsh, mub, mapA* and *EF-Tu* genes under *In vitro* simulated gut conditions was tested for significance by qBase-Plus software. Amongst the three probiotic strains used in that study, Lp91 showed the highest level of *bsh* gene expression when the medium was supplemented with 0.01% mucin along with 1% of both bile and pancreatin in all the three strains. Results suggested that the expression of *mub* is a characteristic of not only the specie but could also be strain specific. The highest level of expression of *mapA* gene was recorded when normal gut conditions (Mucin, 0.01% and 0.3% each of bile and pancreatin, 0.3% supplemented in MRS at pH 6.5) were used. The relative expression of EF-Tu gene was significantly up-regulated in Lp9 in presence of mucin along at 0.01 and 0.05%, respectively at pH 7.0. It was concluded that the efficacy of both Lp9 and Lp91 with regards to expression of *mub, mapA* and *EF-Tu* was found to be either superior or comparable to that of standard probiotic strain (Duary et al., 2011). To confirm the *MapA* results in this last report it is important to find if the *L. plantarum* genome contains this gene to probe then its functionality.

**1.6 Methods for screening mucus or mucin adhering bacteria** 

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoprotein forms a framework to which microbial population can adhere, including probiotic *Lactobacillus* strains. Numerous factors have been shown to influence binding of lactobacilli to mucus *in vitro*. Experimental methods should be reviewed and compared to get a better understanding of the bacteria-mucosa interaction. The mechanism of this interaction could help to determine the degree of probiotic functionality imparted by adhesion (Tassell et al., 2011). Different methods to measure adhesion to mucus have been reported. Mucus contains about 80% of carbohydrates which occur as oligosaccharides and most of the glycans are present in

codons in all three reading frames (TGA A TAA T TAA). Computer search of the nucleotide and aminoacid sequences, showed that this adhesin is related to proteins encoding adherence factors from several pathogenic bacteria, as well as amino acid transporter

binding protein precursors (Rojas, 1996; E. Satoh et al., 2000). Expression by real time PCR of the genes *Mub* and *MapA*, adhesion-like factor *EF-Tu* and bacteriocin gene *plaA* by *L. plantarum* 423 grown in the presence of bile, pancreatin and at low pH, was reported. It was found that under normal physiological concentration of bile and pancreatin, expression of the *Mub* gene was affected, the *MapA* gene was over expressed and the *EF-Tu* gene remained stable, suggesting that whilst the expression of certain mucus genes may be affected by bile and pancreatin, others mucus genes are switched on, enabling the strain to adapt to physiological conditions and adhere to the gastrointestinal tract (Ramiah et al., 2007). To confirm the *MapA* results will be interesting to search in *L. plantarum* genome the compete sequence of this gene and find the adhering function in the reported specie or strain. By searching bacterial genome sequences and the UniProt protein data base for potential mucus binding proteins based on the sequence of the Mub domains of *L. reuteri* and *L. plantarum*. Boaekhorst et al, 2006. found that MUB domain is variable in size and sequence, making it difficult to determine precise domain boundaries. However the high variability in the number of MUB domain in putative mucus-binding proteins suggested that the MUB domain is often duplicated or deleted in evolution and appears to be only present in lactic acid bacteria, with the highest abundance in lactobacilli of the gastrointestinal tract, fulfilling an important function in host-microbe interactions (Boekhorst et al., 2006). Characterization of 32 Mmubp and *32-mmubp* gene from the potential probiotic strain previously isolated from piglet *L. fermentum* BCS87 was reported (Macías-Rodríguez et al., 2008). In the adhesion of this wild type strain to mucus and mucin, two proteins were identified, one of them, the 32Mmubp was characterized and the gene that codes for it was reported. Results indicate that the gene encoding this adhesin is conserved for *L. fermentum.* Other results suggested that 32Mmubp is released to the medium, but it could be anchored to cell wall by electrostatic interactions with acidic groups. It was indicated that Mmubp protein is a member of an ABC transporter system and is part of the OpuAC family. Based on homology and sequence domain search and in a phylogenetic tree with sequences of a seed group of the OpuAC family were shown conserved sequences between prokaryotic proteins of substrate-binding region on ABC type glycine/betaine transport systems. Some members of the corresponding taxa having similar ecological niches to those occupied by lactobacilli (gastrointestinal and respiratory tracts), i.e. *Helicobacter pylori* and *Mycobacterium tuberculosis,* did not group together suggesting that adhesion mechanisms is not a phylogenetic associated trait (Macías-Rodríguez et al., 2009). Recently was discovered only in the genome of the probiotic *Lactobacillus rhamnosus* GG, two different pilus fiber in the *spaCBA* and *spaFED* gene clusters. Moreover the expression and localization of intact SpaCBA pili on the cell surface of this strain were confirmed by immunoblotting and immunogold-labeled electron microscopy using antiserum specific for the Spa pilin. SpaCBA pilus-mediated binding of *L. rhamnosus* GG cells to human intestinal mucus was revealed (Kankainen et al., 2009). More recently pilin subunits SpaA, SpaB, SpaD, SpaE and SpaF encoded by genes in the *spaCBA* and *spaFED* genes clusters were cloned in *E. coli*. Recombinant, overproduced proteins were purified and assessment of the adherence to human intestinal mucus was performed. Results suggested that SpaC and

codons in all three reading frames (TGA A TAA T TAA). Computer search of the nucleotide and aminoacid sequences, showed that this adhesin is related to proteins encoding

binding protein precursors (Rojas, 1996; E. Satoh et al., 2000). Expression by real time PCR of the genes *Mub* and *MapA*, adhesion-like factor *EF-Tu* and bacteriocin gene *plaA* by *L. plantarum* 423 grown in the presence of bile, pancreatin and at low pH, was reported. It was found that under normal physiological concentration of bile and pancreatin, expression of the *Mub* gene was affected, the *MapA* gene was over expressed and the *EF-Tu* gene remained stable, suggesting that whilst the expression of certain mucus genes may be affected by bile and pancreatin, others mucus genes are switched on, enabling the strain to adapt to physiological conditions and adhere to the gastrointestinal tract (Ramiah et al., 2007). To confirm the *MapA* results will be interesting to search in *L. plantarum* genome the compete sequence of this gene and find the adhering function in the reported specie or strain. By searching bacterial genome sequences and the UniProt protein data base for potential mucus binding proteins based on the sequence of the Mub domains of *L. reuteri* and *L. plantarum*. Boaekhorst et al, 2006. found that MUB domain is variable in size and sequence, making it difficult to determine precise domain boundaries. However the high variability in the number of MUB domain in putative mucus-binding proteins suggested that the MUB domain is often duplicated or deleted in evolution and appears to be only present in lactic acid bacteria, with the highest abundance in lactobacilli of the gastrointestinal tract, fulfilling an important function in host-microbe interactions (Boekhorst et al., 2006). Characterization of 32 Mmubp and *32-mmubp* gene from the potential probiotic strain previously isolated from piglet *L. fermentum* BCS87 was reported (Macías-Rodríguez et al., 2008). In the adhesion of this wild type strain to mucus and mucin, two proteins were identified, one of them, the 32Mmubp was characterized and the gene that codes for it was reported. Results indicate that the gene encoding this adhesin is conserved for *L. fermentum.* Other results suggested that 32Mmubp is released to the medium, but it could be anchored to cell wall by electrostatic interactions with acidic groups. It was indicated that Mmubp protein is a member of an ABC transporter system and is part of the OpuAC family. Based on homology and sequence domain search and in a phylogenetic tree with sequences of a seed group of the OpuAC family were shown conserved sequences between prokaryotic proteins of substrate-binding region on ABC type glycine/betaine transport systems. Some members of the corresponding taxa having similar ecological niches to those occupied by lactobacilli (gastrointestinal and respiratory tracts), i.e. *Helicobacter pylori* and *Mycobacterium tuberculosis,* did not group together suggesting that adhesion mechanisms is not a phylogenetic associated trait (Macías-Rodríguez et al., 2009). Recently was discovered only in the genome of the probiotic *Lactobacillus rhamnosus* GG, two different pilus fiber in the *spaCBA* and *spaFED* gene clusters. Moreover the expression and localization of intact SpaCBA pili on the cell surface of this strain were confirmed by immunoblotting and immunogold-labeled electron microscopy using antiserum specific for the Spa pilin. SpaCBA pilus-mediated binding of *L. rhamnosus* GG cells to human intestinal mucus was revealed (Kankainen et al., 2009). More recently pilin subunits SpaA, SpaB, SpaD, SpaE and SpaF encoded by genes in the *spaCBA* and *spaFED* genes clusters were cloned in *E. coli*. Recombinant, overproduced proteins were purified and assessment of the adherence to human intestinal mucus was performed. Results suggested that SpaC and

adherence factors from several pathogenic bacteria, as well as amino acid transporter

SpaB may be involved in SpaCBA pilus-mediated adherence to intestinal mucus. It was established that the SpaF minor pilin is the only mucus binding component in the putative SpaFED pilus fiber (von Ossowski et al., 2010). Aggregation promoting factors (Apf) are secreted proteins that have been associated with a diverse number of functional roles in lactobacilli, including self aggregation, coaggregation with other commensal or pathogenic bacteria, maintenance of cell shape and the bridging of conjugal pairs. Genes encoding Apf´s have been characterized for several *Lactobacillus* species, including *L. crispatus, L. johnsonii, L. gasseri, L. paracasei* and *L. coryniformis*. Investigation of the functional role of the putative *apf* gene (LBA0493) in *L. acidophilus* NCFM by mutational analysis was performed. It was observed that survival rates mutant strain NCK2033 decreased when stationary phase cells were exposed to simulated small intestinal and gastric juices. Furthermore, NCK2033 in the stationary phase showed a reduction of *In vitro* adherence to Caco-2 intestinal epithelial cells, mucin glycoproteins and fibronectin. It was suggested that the Apf-like proteins may contributes to the survival of *L. acidophilus* during transit through the digestive tract and, potentially, participate in the interactions with the host intestinal mucosa (Goh & Klaenhammer, 2010). The ability to tolerate the toxic levels of bile salts accumulated therein is the essential requirement to survive in the gut and it is generally included among the criteria used for selection of the potential probiotic strains and their application as functional ingredients in foods and nutraceuticals. Expression of bile salt hydrolase and surface proteins were targeted to look at their expression profile in two putative probiotic *L. plantarum* Lp9 and Lp91, (compared with standard strain CSCC5276) by quantitative real time PCR (RT – qPCR). Expression ratio for *bsh, mub, mapA* and *EF-Tu* genes under *In vitro* simulated gut conditions was tested for significance by qBase-Plus software. Amongst the three probiotic strains used in that study, Lp91 showed the highest level of *bsh* gene expression when the medium was supplemented with 0.01% mucin along with 1% of both bile and pancreatin in all the three strains. Results suggested that the expression of *mub* is a characteristic of not only the specie but could also be strain specific. The highest level of expression of *mapA* gene was recorded when normal gut conditions (Mucin, 0.01% and 0.3% each of bile and pancreatin, 0.3% supplemented in MRS at pH 6.5) were used. The relative expression of EF-Tu gene was significantly up-regulated in Lp9 in presence of mucin along at 0.01 and 0.05%, respectively at pH 7.0. It was concluded that the efficacy of both Lp9 and Lp91 with regards to expression of *mub, mapA* and *EF-Tu* was found to be either superior or comparable to that of standard probiotic strain (Duary et al., 2011). To confirm the *MapA* results in this last report it is important to find if the *L. plantarum* genome contains this gene to probe then its functionality.

#### **1.6 Methods for screening mucus or mucin adhering bacteria**

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoprotein forms a framework to which microbial population can adhere, including probiotic *Lactobacillus* strains. Numerous factors have been shown to influence binding of lactobacilli to mucus *in vitro*. Experimental methods should be reviewed and compared to get a better understanding of the bacteria-mucosa interaction. The mechanism of this interaction could help to determine the degree of probiotic functionality imparted by adhesion (Tassell et al., 2011). Different methods to measure adhesion to mucus have been reported. Mucus contains about 80% of carbohydrates which occur as oligosaccharides and most of the glycans are present in

PCR for Screening Potential Probiotic Lactobacilli for Piglets 75

utmost importance in the unraveling of modes of action of lactobacilli as they can often directly relate genotype to phenotype. Nevertheless the number of currently identified genetic loci hypothesized to encode features supporting probiotic action confirmed by mutant analysis is still limited (Lebeer et al., 2008). Although the availability of genome sequences will certainly advance the field, they need to be complemented with functional studies. Methods that start to be applied for differential gene expression analysis of lactobacilli under relevant conditions are genome-wide comparisons of RNA profiles using microarrays, comparison of protein profiles with two dimensional (2D) difference gel electrophoresis, *In vivo* expression technology (IVET) using a promoter probe library and

**2. Materials and methods for screening probiotic potential lactobacilli** 

stress caused by high temperatures, piglets were bathed every day at midday.

Newborn piglets (*Landrace-Duroc)* from a pig farm were maintained with their mothers in maternity cages with grid floors during 23 days before weaned. Piglets received an intramuscular Fe injection (100 mg Fe, VITALECHON DEXTRAN) the second day after birth. Mother's milk fed piglets were given free access to commercial starter feed (17.5% crude protein, 2.5% crude fat, 5% crude fiber, 12% moisture, salts, vitamins, and minerals) and water (<900 ppm) 2-5 days before weaning. Maternity cages were maintained at room temperature and warmed up with lamps during the night when needed. To avoid excessive

Faecal samples of healthy 23-day-old preweaned piglets from different cages with weights of 10 to 12 Kg were collected in sterile falcon tubes just at the time of defecating and transported to the laboratory at 4 °C. Piglets randomly selected, were sacrificed by a humanitarian method in the laboratory and immediately the small intestine and cecum were removed and sectioned with a sterile dissection kit. These pieces were opened and rinsed with sterile ice-cold phosphate-buffer saline (PBS) (145 mM NaCl, 2.87 mM KH2PO4, and 6.95 mM K2HPO4, pH 7.2) in order to remove loosely associated intestinal material. Mucus was then released by gently scraping the small intestine and cecum with a spatula and used

Isolation and characterization of bacteria was previously performed as reported before (Rojas & Conway, 1996; Macías-Rodríguez et al., 2008) . Briefly, lactic acid bacteria from faeces and from associated small intestine and cecum mucus of healthy preweaned piglets were isolated. Both faecal and mucosal samples were diluted in PBS and serial dilutions were plated on Rogosa SL agar (Difco). Plates were incubated at 37 °C for 24 h in an anaerobic jar with a Gaspack system. Counts of colony forming units (CFU) per gram and for cm2 were reported. Colonies from each faecal or mucosal piglet sample were randomly selected from the last dilutions, purified on Rogosa SL plates and grown in MRS broth (Mann, Rogosa and Sharpe, Difco). Aliquots of each strain were kept in 1.5 ml tubes with

50% of glycerol at -85° C. Fresh cultures were used to perform the adhesion assay.

differential-display PCR (DD-PCR) (Lebeer et al., 2008).

**2.1 Animals** 

**2.2 Sampling** 

to isolate lactic acid bacteria.

**2.3 Isolation of bacteria** 

clusters flanked by naked regions of the protein core (Clamp & Sheehan, 1978). Since mucins from different sources could be substituted with different oligosaccharides, properties such as the linear charge density could vary considerably. Porcine and rat mucin differ markedly in glycosylation and charge density (Malmsten et al., 1992). This characteristic of mucin, need to be considered when performing experiment to test the interaction between bacteria and mucus or mucin. A common method used to test *E. coli* adhesion to mucus extract prepared from the large and small intestine of mice involved immobilizing the mucus extracts on polystyrene. Radioactively labeled bacterial suspensions were added to the immobilized mucus compound and after a short inoculation time, the unbound cells were removed and adhesive cells were enumerated by measuring the amount of radioactivity (Laux et al., 1984; Laux, 1986). This method was adapted for studying *E. coli* adhesion to ileal mucus extracts from pigs (Conway et al., 1990; Blomberg & Conway, 1989). It has also been used to study the adhesion of *L. reuteri* 104R to small intestinal mucus extracts from pig (Rojas & Conway, 1996). This method still is used with some modifications (Mackenzie et al., 2010), however, it was not suitable for studying adhesion to mucin since it bound poorly to the polystyrene. In a control experiment where horse radish peroxidase labeled mucus and mucin were used, it was shown that mucin adhered to polystyrene a less extent than mucus. These results are consistent with other finding where rat and pig mucin layers on hydrophobic surfaces were studied. It was found by ellipsometry and surface force measurements, by using mica and silica surfaces, that the adsorption equilibrium of rat gastric mucin was reached after 5 hours, however for pig gastric mucin equilibrium it was not reached. It was demonstrated that for such layers, as the repulsive forces become weaker the slower the surfaces are brought together (Malmsten et al., 1992). Dot blot assay, a qualitative *In vitro* assay to detect the binding of bacterial cell surface components to mucus extracts was developed whereby extracts containing bacterial components and fractionated proteins were immobilized in a solid phase matrix and then blotted with enzymatically labelled mucus (Rojas and Conway, 2001). Results were compared to those obtained using the inhibition assay. In addition, whole cells of *Lactobacillus* and *E. coli* were tested in the dot blot assay and results compared with a modification of the method of Laux and coworkers (Conway et al., 1990). The results obtained using the dot blot assay provided further information about the binding of *Lactobacillus* and *E. coli* to gastrointestinal mucus, not only because adhesion promoting compounds could be detected in fractionated extracts but also because porcine gastric mucin as well as small intestinal mucus could be used for blotting (Rojas & Conway, 2001). Other methods have used to study adhesion to mucosa. Cultured cells have been suggested to be the best available models to study intestinal attachment of bacteria and viruses (Coconnier et al., 1997). Particularly, mucus secreting cells could be the best to study *Lactobacillus*-mucus and mucin interactions. Unfortunately this method has the same limitations as the mucus immobilization method of Laux et al. (Conway et al., 1990) for studing adhesins in soluble extracts.

#### **1.7 Genetic tools to study the expression of genes encoding adhesins**

The number of genetic tools that have been developed has increased tremendously during the last 20 years. Genetic analysis is made possible for several lactobacilli strains of known probiotic action, such as *L. plantarum* WCFS1, *L. acidophilus* NCFM*, L. johonsonii* NCC533, *L. salivarius* UCC118*, L. reuteri* ATCC 55730 and *L. rhamnosus* GG. Mutant studies are of the utmost importance in the unraveling of modes of action of lactobacilli as they can often directly relate genotype to phenotype. Nevertheless the number of currently identified genetic loci hypothesized to encode features supporting probiotic action confirmed by mutant analysis is still limited (Lebeer et al., 2008). Although the availability of genome sequences will certainly advance the field, they need to be complemented with functional studies. Methods that start to be applied for differential gene expression analysis of lactobacilli under relevant conditions are genome-wide comparisons of RNA profiles using microarrays, comparison of protein profiles with two dimensional (2D) difference gel electrophoresis, *In vivo* expression technology (IVET) using a promoter probe library and differential-display PCR (DD-PCR) (Lebeer et al., 2008).

## **2. Materials and methods for screening probiotic potential lactobacilli**

### **2.1 Animals**

74 Polymerase Chain Reaction

clusters flanked by naked regions of the protein core (Clamp & Sheehan, 1978). Since mucins from different sources could be substituted with different oligosaccharides, properties such as the linear charge density could vary considerably. Porcine and rat mucin differ markedly in glycosylation and charge density (Malmsten et al., 1992). This characteristic of mucin, need to be considered when performing experiment to test the interaction between bacteria and mucus or mucin. A common method used to test *E. coli* adhesion to mucus extract prepared from the large and small intestine of mice involved immobilizing the mucus extracts on polystyrene. Radioactively labeled bacterial suspensions were added to the immobilized mucus compound and after a short inoculation time, the unbound cells were removed and adhesive cells were enumerated by measuring the amount of radioactivity (Laux et al., 1984; Laux, 1986). This method was adapted for studying *E. coli* adhesion to ileal mucus extracts from pigs (Conway et al., 1990; Blomberg & Conway, 1989). It has also been used to study the adhesion of *L. reuteri* 104R to small intestinal mucus extracts from pig (Rojas & Conway, 1996). This method still is used with some modifications (Mackenzie et al., 2010), however, it was not suitable for studying adhesion to mucin since it bound poorly to the polystyrene. In a control experiment where horse radish peroxidase labeled mucus and mucin were used, it was shown that mucin adhered to polystyrene a less extent than mucus. These results are consistent with other finding where rat and pig mucin layers on hydrophobic surfaces were studied. It was found by ellipsometry and surface force measurements, by using mica and silica surfaces, that the adsorption equilibrium of rat gastric mucin was reached after 5 hours, however for pig gastric mucin equilibrium it was not reached. It was demonstrated that for such layers, as the repulsive forces become weaker the slower the surfaces are brought together (Malmsten et al., 1992). Dot blot assay, a qualitative *In vitro* assay to detect the binding of bacterial cell surface components to mucus extracts was developed whereby extracts containing bacterial components and fractionated proteins were immobilized in a solid phase matrix and then blotted with enzymatically labelled mucus (Rojas and Conway, 2001). Results were compared to those obtained using the inhibition assay. In addition, whole cells of *Lactobacillus* and *E. coli* were tested in the dot blot assay and results compared with a modification of the method of Laux and coworkers (Conway et al., 1990). The results obtained using the dot blot assay provided further information about the binding of *Lactobacillus* and *E. coli* to gastrointestinal mucus, not only because adhesion promoting compounds could be detected in fractionated extracts but also because porcine gastric mucin as well as small intestinal mucus could be used for blotting (Rojas & Conway, 2001). Other methods have used to study adhesion to mucosa. Cultured cells have been suggested to be the best available models to study intestinal attachment of bacteria and viruses (Coconnier et al., 1997). Particularly, mucus secreting cells could be the best to study *Lactobacillus*-mucus and mucin interactions. Unfortunately this method has the same limitations as the mucus immobilization method of Laux et al. (Conway et al., 1990)

for studing adhesins in soluble extracts.

**1.7 Genetic tools to study the expression of genes encoding adhesins** 

The number of genetic tools that have been developed has increased tremendously during the last 20 years. Genetic analysis is made possible for several lactobacilli strains of known probiotic action, such as *L. plantarum* WCFS1, *L. acidophilus* NCFM*, L. johonsonii* NCC533, *L. salivarius* UCC118*, L. reuteri* ATCC 55730 and *L. rhamnosus* GG. Mutant studies are of the Newborn piglets (*Landrace-Duroc)* from a pig farm were maintained with their mothers in maternity cages with grid floors during 23 days before weaned. Piglets received an intramuscular Fe injection (100 mg Fe, VITALECHON DEXTRAN) the second day after birth. Mother's milk fed piglets were given free access to commercial starter feed (17.5% crude protein, 2.5% crude fat, 5% crude fiber, 12% moisture, salts, vitamins, and minerals) and water (<900 ppm) 2-5 days before weaning. Maternity cages were maintained at room temperature and warmed up with lamps during the night when needed. To avoid excessive stress caused by high temperatures, piglets were bathed every day at midday.

## **2.2 Sampling**

Faecal samples of healthy 23-day-old preweaned piglets from different cages with weights of 10 to 12 Kg were collected in sterile falcon tubes just at the time of defecating and transported to the laboratory at 4 °C. Piglets randomly selected, were sacrificed by a humanitarian method in the laboratory and immediately the small intestine and cecum were removed and sectioned with a sterile dissection kit. These pieces were opened and rinsed with sterile ice-cold phosphate-buffer saline (PBS) (145 mM NaCl, 2.87 mM KH2PO4, and 6.95 mM K2HPO4, pH 7.2) in order to remove loosely associated intestinal material. Mucus was then released by gently scraping the small intestine and cecum with a spatula and used to isolate lactic acid bacteria.

## **2.3 Isolation of bacteria**

Isolation and characterization of bacteria was previously performed as reported before (Rojas & Conway, 1996; Macías-Rodríguez et al., 2008) . Briefly, lactic acid bacteria from faeces and from associated small intestine and cecum mucus of healthy preweaned piglets were isolated. Both faecal and mucosal samples were diluted in PBS and serial dilutions were plated on Rogosa SL agar (Difco). Plates were incubated at 37 °C for 24 h in an anaerobic jar with a Gaspack system. Counts of colony forming units (CFU) per gram and for cm2 were reported. Colonies from each faecal or mucosal piglet sample were randomly selected from the last dilutions, purified on Rogosa SL plates and grown in MRS broth (Mann, Rogosa and Sharpe, Difco). Aliquots of each strain were kept in 1.5 ml tubes with 50% of glycerol at -85° C. Fresh cultures were used to perform the adhesion assay.

PCR for Screening Potential Probiotic Lactobacilli for Piglets 77

Oligonucleotides used for PCR amplifications were designed with the Primer Select tool of the Laser gene software (Version 5) and synthesized at the Instituto de Biotecnología,

**Orientation Sequence** 

Amplification of the *32-Mmubp* gene of *L. fermentum* previously reported by (Macías-Rodríguez et al., 2009) was performed using as template the chromosomal DNA of *Lactobacillus* strains previously characterized as potential probiotic by traditional methods (Table 1). A combination of gene specific oligonucleotides for an internal fragment MEF7 and MER9 was used to perform the amplification. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 3mmol l-1 MgCl2, 0.4mmol l-1 for each dNTP, 120 pmol of each primer, 250 ng chromosomal DNA and 1 U of Taq DNA polymerase in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 30 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 55°C for 1 min and an extension step at 72° C for 1 min. A final extension was performed at 72° C for 5 min.

Amplification of the gene *mapp* or *mapA* (Genebank accession number AJ293860) previously described (Rojas 1996, Satoh et al., 2000 and Miyoshi et al., 2006) was performed using as template the same chromosomal DNA of *Lactobacillus* strains used for amplification of the 32*Mmubp* gene. A combination of gene specific oligonucleotides for an internal fragment of the open reading frame MAP1F and MAP1R (Table 2) was used. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 3mmol MgCl2, 0.4mmol for each dNTP, 60 pmol of each primer, 300 ng chromosomal DNA and 1 U of Taq DNA polymerase in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 28 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 49°C for 1 min and an extension step at 72° C for 2 min. A final extension was performed at 72° C for 5 min. PCR products were analyzed in a 1.5% agarose gel.

MAP1F Forward 5' ATGCCTGCAGGAATCACAA 3' MAP1R Reverse 5' AGTAATATCTGCACCGAAGTA 3' MEF7 Forward 5´ ATTTACGCCCTGGCCCTGGAAAAG-3´ MER9 Reverse 5´ AGAGGGTGTATTTGTTGCCATTGG-3´ MAP2F Forward 5' TCTTATGCGACCCACAGTTTG 3' MAP2R Reverse 5' CTAAGAGCCCCGTCGTTC 3'

**2.4 Oligonucleotide design and synthesis** 

UNAM (Mexico). All are listed in Table 2.

Table 2. Oligonucleotides used for PCR amplifications

PCR products were then analyzed in a 1.5% agarose gel.

**2.6 PCR amplification of the** *mapp* **or** *mapA* **gene** 

**2.5 PCR amplification of the** *32-Mmubp* **gene** 

**Oligonucleotide name** 


\*Intestinal tract mucus, \*\* Cecum mucus

Table 1. Strains isolated from faeces and mucus of healthy piglets used in this study (Macías-Rodríguez et al., 2008).

## **2.4 Oligonucleotide design and synthesis**

76 Polymerase Chain Reaction

Faeces BCS9 EF113967/ EF113958 *99% to Lactobacillus fermentum* Faeces BCS10 EF113968/ EF113959 *99% to Lactobacillus fermentum* Faeces BCS12 EF113969/ EF113960 *99% to Lactobacillus fermentum* Faeces BCS13 EF113970/ EF113961 *99% to Lactobacillus fermentum* Faeces BCS14 EF113971/ EF113962 *99% to Lactobacillus fermentum* Faeces BCS21 EU547278/ EU547296 *99% to Lactobacillus fermentum* Faeces BCS24 E113972/ EF113963 *99 % to Lactobacillus fermentum* Faeces BCS25 EU547279/ EU547297 *99% to Lactobacillus fermentum* Faeces BCS27 EU547280/ EU547298 *99% to Lactobacillus fermentum* Faeces BCS30 EU547281/ EU547299 *99% to Lactobacillus fermentum* Faeces BCS36 EU547282/ EU547300 *100 %Lactobacillus fermentum* Faeces BCS41 EU547283/ EU547301 *100% Lactobacillus johnsonii*; Faeces BCS46 EF113973/ EF113964 99% to *Lactobacillus fermentum* Faeces BCS68 EU547284/ EU547302 99% to *Lactobacillus vaginalis* Faeces BCS75 EF113974/ EF113965 99% to *Lactobacillus fermentum* Faeces BCS80 EU547285/ EU547303 99% to *Lactobacillus fermentum* Faeces BCS81 EU547286/ EU547304 99% to *Lactobacillus fermentum* Faeces BCS82 EU547287/ EU547305 99% to *Lactobacillus fermentum* Faeces BCS87 EF113975/ EF113966 99% to *Lactobacillus fermentum* SI mucus\* BCS113 EU547288/ EU547306 92 % to *Lactobacillus delbrueckii* 

SI mucus\* BCS125 EU547289/ EU547307 99% to *Lactobacillus crispatus* SI mucus\* BCS127 EU547290/ EU547308 99% to *Lactobacillus reuteri* SI mucus\* BCS154 EU547294/ EU547312 99% to *Lactobacillus vaginalis* C mucus\*\* BCS134 EU547291/ EU547309 99% to *Lactobacillus reuteri* C mucus\*\* BCS136 EU547292/ EU547310 99% to *Lactobacillus reuteri* C mucus\*\* BCS142 EU547293/ EU547311 99% to *Lactobacillus reuteri* C mucus\*\* BCS159 EU547294/ EU547313 99% to *Lactobacillus reuteri*

Table 1. Strains isolated from faeces and mucus of healthy piglets used in this study

\*Intestinal tract mucus, \*\* Cecum mucus

(Macías-Rodríguez et al., 2008).

**% identity** 

**Based on 16S rDNA sequence** 

*subsp. bulgaricus*

**(16-23S/ 16Sr DNA)** 

**Source Strain Accession numbers** 

Oligonucleotides used for PCR amplifications were designed with the Primer Select tool of the Laser gene software (Version 5) and synthesized at the Instituto de Biotecnología, UNAM (Mexico). All are listed in Table 2.


Table 2. Oligonucleotides used for PCR amplifications

## **2.5 PCR amplification of the** *32-Mmubp* **gene**

Amplification of the *32-Mmubp* gene of *L. fermentum* previously reported by (Macías-Rodríguez et al., 2009) was performed using as template the chromosomal DNA of *Lactobacillus* strains previously characterized as potential probiotic by traditional methods (Table 1). A combination of gene specific oligonucleotides for an internal fragment MEF7 and MER9 was used to perform the amplification. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 3mmol l-1 MgCl2, 0.4mmol l-1 for each dNTP, 120 pmol of each primer, 250 ng chromosomal DNA and 1 U of Taq DNA polymerase in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 30 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 55°C for 1 min and an extension step at 72° C for 1 min. A final extension was performed at 72° C for 5 min. PCR products were then analyzed in a 1.5% agarose gel.

#### **2.6 PCR amplification of the** *mapp* **or** *mapA* **gene**

Amplification of the gene *mapp* or *mapA* (Genebank accession number AJ293860) previously described (Rojas 1996, Satoh et al., 2000 and Miyoshi et al., 2006) was performed using as template the same chromosomal DNA of *Lactobacillus* strains used for amplification of the 32*Mmubp* gene. A combination of gene specific oligonucleotides for an internal fragment of the open reading frame MAP1F and MAP1R (Table 2) was used. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 3mmol MgCl2, 0.4mmol for each dNTP, 60 pmol of each primer, 300 ng chromosomal DNA and 1 U of Taq DNA polymerase in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 28 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 49°C for 1 min and an extension step at 72° C for 2 min. A final extension was performed at 72° C for 5 min. PCR products were analyzed in a 1.5% agarose gel.

PCR for Screening Potential Probiotic Lactobacilli for Piglets 79

presented adhesion ability respectively. These results showed the highest percentage of adhesive strains in the cecum and intestine compared with faeces. Adhesive strains isolated from faeces could be released to the lumen during the renewal of mucus. Different adhesive abilities between faecal and mucosal strains could be also explained if is considered that microbiota in the intestine differs from that in faeces (Marteau, 2002). Moreover, adhesive properties are strain-dependent and differences exist even if strains were isolated from the

For molecular identification the most common amplified sequences by PCR are the 16-23S intergenic region and 16S rDNA gene. The 27 strains used in this work were identified by these methods. Analysis of 16S rDNA gene sequences showed that 17 strains belong to *L. fermentum* specie (between 98 to 100% identity), one strain *to L. johnsonii*, 2 strains to *L. vaginalis*, one strain to *L. crispatus* and 5 strains *to L. reuteri* species (Table 1). Except strain BCS113 that showed 92% identity to 16S rDNA of *L. delbrueckii* subsp. *bulgaricus*. These results showed that *L. fermentum* was predominant in faecal adhesive isolates whereas *L. reuteri* was the principal in mucus of cecum. In small intestinal mucus there was not predominant specie. These observations agree with previously reported by Lin *et al*. (Lin et al., 2007) and (De Angelis et al., 2006) who found both species in faeces and mucus of pigs. This result confirmed the relevance of these species in the intestinal tract of pigs. Moreover, *L. fermentum* and *L. reuteri* species have been reported as good candidates as probiotics (De Angelis et al., 2007; Zoumpopoulou et al., 2008). Another species identified as *L. johnsonii*, *L. delbrueckii* subsp. *bulgaricus*, *L. vaginalis* and *L. crispatus* have been reported by their probiotic potential in humans and animals (Chen et al., 2007; Matijasic et al., 2006; Ohashi et al., 2007). To understand the relevance of surface proteins in the adhesion of *Lactobacillus* to mucus and mucin, the purification and characterization of the adhesins should be performed. In previous reports proteins have been obtained by treatment with chaotropic agents as LiCl. From the spent, centrifuged growth medium and from soluble cytoplasmic extracts. A western blot assay using labelled mucus and mucin has been usually performed to show the protein bands with their relative molecular weight (MW) and in order to characterize them, N-terminal and internal peptide sequences has been determined. The MAPP adhesin of *L. reuteri* and the *Mmubp of L. fermentum* have been characterized in that manner (Rojas et al., 2002; Macías-Rodríguez et al., 2008). Recently the mucus-binding proteins (MUBs) have been revealed as one of the effectors molecules involved in mechanisms of the adherence of lactobacilli to the host; *mub*, or *mub*-like, genes were found in all of the six genomes of *L. reuteri* that are available but the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein (Mackenzie et al.,

The complete process to get new strains of probiotic potential lactobacilli has been long and complex. Above a review of the different methods and results used was exposed and the

The strains listed in Table 1 were selected because they were the predominant cultivable lactic acid bacteria in a selective medium (Rogosa agar, DIFCO); attached strongly to mucus and mucin when tested by the Dot Blot adhesion assay; grew in mucus, in presence of bile salt and in a broad range of temperatures. Likewise the molecular identification confirmed that *L. fermentum* and *L. reuteri* were the main isolates with probiotic potential for piglets (Macías-Rodríguez et al., 2008). In addition the genes *mapp* or *mapA* of *L. reuteri* and *mmub* of

same source (Kinoshita et al., 2007).

2010).

results of a proposal are described.

#### **2.7 PCR amplification of the operon containing the** *MapA* **gene**

Polymerase Chain Reactions was performed with primers MAP2F and MAP2R (Table 2) for an internal fragment of the operon containing the *mapA* gene (Genebank LOCUS AJ293860) using as template the chromosomal DNA of *Lactobacillus* strains *map*A positive. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 2mmol MgSO4, 0.4mmol for each dNTP, 100 pmol of each primer, 300 ng chromosomal DNA and 2 U of Platinum *Taq* DNA Polymerase (Invitrogene), in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 30 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 62°C for 1 min and an extension step at 68° C for 2 min. A final extension was performed at 68° C for 5 min. PCR products were analyzed in a 1.0% agarose gel.

## **3. Results and discussion**

The association of lactobacilli with the epithelial and mucosal surfaces and their presence in faeces in pigs has been well studied (Rojas & Conway 1996; Macías-Rodríguez et al., 2008). It was shown that *Lactobacillus* population in faeces ranged between 107 and 109 CFU gr-1. Likewise, in intestinal mucosa, counts of 3.8x106 and 3.2 x106 CFU per cm2 of small intestine and cecum respectively were reported. Cultivable *Lactobacillus* strains has been found in similar amounts in faeces and intestinal mucus of pigs that inhabit different environmental conditions, cool countries (Rojas and Conway, 1996) and warm arid coasts (Macías-Rodríguez et al., 2008). It was found too that *L. fermentum* and *L. reuteri* are the major strains which colonize the gastrointestinal tract of pigs. Therefore the screening of *Lactobacillus* with probiotic potential for piglets with the ability to interact with the host should be addressed to this species. It has been reported that species of *Lactobacillus* which colonize humans, differ in number and specie from one region to other in the world. Likewise *In vivo* trials have been shown that probiotic effect of one strain in one region of the world could produce confused results in other. This finding supports the idea to look for a new generation of specific probiotics for animals and humans inhabiting specific region in the world.

Traditionally the screening of *Lactobacillus* with probiotic potential involve the isolation and purification of many colonies of lactic acid bacteria, confirmation that correspond to presumptive lactobacilli (grown in selective medium, Gram stain, catalasa reaction, etc), selection according to adhesion profile, growth in mucus, bile salt resistance, growth in broad range of temperature and salt concentration, bacteriocin production, growth and adhesion inhibition of enteropathogens, molecular identification, etc. Previously, more than 150 strains were isolated from mucus and feaces of piglets. Results showed that 64% of presumptive *Lactobacillus* presented abilities to grow in the presence of 680 mM of NaCl. Additionally 75% of the isolates were able to grow at 50 ºC. These abilities are important considering that probiotic bacteria are exposed to high temperatures and presence of NaCl during their technological preparation as pelleted or dried feed for pigs. The adhesion assay of the 164 isolates to porcine mucus and mucin allowed visualize strains that bind mucus or gastric mucin in a qualitative manner. Results indicated that 88 isolates representing 53.7% of the 164 strains, presented adhesion to both mucus and gastric mucin similar to the positive control *L. reuteri* 104R, (Rojas et al., 2002). From the total of faecal strains 45% showed binding ability, whereas from intestinal and cecal mucus strains, 64 and 78%

Polymerase Chain Reactions was performed with primers MAP2F and MAP2R (Table 2) for an internal fragment of the operon containing the *mapA* gene (Genebank LOCUS AJ293860) using as template the chromosomal DNA of *Lactobacillus* strains *map*A positive. The PCR solution contained a final concentration of 1× Taq polymerase buffer, 2mmol MgSO4, 0.4mmol for each dNTP, 100 pmol of each primer, 300 ng chromosomal DNA and 2 U of Platinum *Taq* DNA Polymerase (Invitrogene), in a total volume of 25 μl. Amplification reaction was performed in a thermocycler (Perkin-Elmer mod. GeneAmp 2400) with the following temperature program: 1 cycle at 94°C for 5 min; 30 cycles consisted in a denaturation step at 94° C for 1 min, an annealing step at 62°C for 1 min and an extension step at 68° C for 2 min. A final extension was performed at 68° C for 5 min. PCR products

The association of lactobacilli with the epithelial and mucosal surfaces and their presence in faeces in pigs has been well studied (Rojas & Conway 1996; Macías-Rodríguez et al., 2008). It was shown that *Lactobacillus* population in faeces ranged between 107 and 109 CFU gr-1. Likewise, in intestinal mucosa, counts of 3.8x106 and 3.2 x106 CFU per cm2 of small intestine and cecum respectively were reported. Cultivable *Lactobacillus* strains has been found in similar amounts in faeces and intestinal mucus of pigs that inhabit different environmental conditions, cool countries (Rojas and Conway, 1996) and warm arid coasts (Macías-Rodríguez et al., 2008). It was found too that *L. fermentum* and *L. reuteri* are the major strains which colonize the gastrointestinal tract of pigs. Therefore the screening of *Lactobacillus* with probiotic potential for piglets with the ability to interact with the host should be addressed to this species. It has been reported that species of *Lactobacillus* which colonize humans, differ in number and specie from one region to other in the world. Likewise *In vivo* trials have been shown that probiotic effect of one strain in one region of the world could produce confused results in other. This finding supports the idea to look for a new generation of

specific probiotics for animals and humans inhabiting specific region in the world.

Traditionally the screening of *Lactobacillus* with probiotic potential involve the isolation and purification of many colonies of lactic acid bacteria, confirmation that correspond to presumptive lactobacilli (grown in selective medium, Gram stain, catalasa reaction, etc), selection according to adhesion profile, growth in mucus, bile salt resistance, growth in broad range of temperature and salt concentration, bacteriocin production, growth and adhesion inhibition of enteropathogens, molecular identification, etc. Previously, more than 150 strains were isolated from mucus and feaces of piglets. Results showed that 64% of presumptive *Lactobacillus* presented abilities to grow in the presence of 680 mM of NaCl. Additionally 75% of the isolates were able to grow at 50 ºC. These abilities are important considering that probiotic bacteria are exposed to high temperatures and presence of NaCl during their technological preparation as pelleted or dried feed for pigs. The adhesion assay of the 164 isolates to porcine mucus and mucin allowed visualize strains that bind mucus or gastric mucin in a qualitative manner. Results indicated that 88 isolates representing 53.7% of the 164 strains, presented adhesion to both mucus and gastric mucin similar to the positive control *L. reuteri* 104R, (Rojas et al., 2002). From the total of faecal strains 45% showed binding ability, whereas from intestinal and cecal mucus strains, 64 and 78%

**2.7 PCR amplification of the operon containing the** *MapA* **gene** 

were analyzed in a 1.0% agarose gel.

**3. Results and discussion** 

presented adhesion ability respectively. These results showed the highest percentage of adhesive strains in the cecum and intestine compared with faeces. Adhesive strains isolated from faeces could be released to the lumen during the renewal of mucus. Different adhesive abilities between faecal and mucosal strains could be also explained if is considered that microbiota in the intestine differs from that in faeces (Marteau, 2002). Moreover, adhesive properties are strain-dependent and differences exist even if strains were isolated from the same source (Kinoshita et al., 2007).

For molecular identification the most common amplified sequences by PCR are the 16-23S intergenic region and 16S rDNA gene. The 27 strains used in this work were identified by these methods. Analysis of 16S rDNA gene sequences showed that 17 strains belong to *L. fermentum* specie (between 98 to 100% identity), one strain *to L. johnsonii*, 2 strains to *L. vaginalis*, one strain to *L. crispatus* and 5 strains *to L. reuteri* species (Table 1). Except strain BCS113 that showed 92% identity to 16S rDNA of *L. delbrueckii* subsp. *bulgaricus*. These results showed that *L. fermentum* was predominant in faecal adhesive isolates whereas *L. reuteri* was the principal in mucus of cecum. In small intestinal mucus there was not predominant specie. These observations agree with previously reported by Lin *et al*. (Lin et al., 2007) and (De Angelis et al., 2006) who found both species in faeces and mucus of pigs. This result confirmed the relevance of these species in the intestinal tract of pigs. Moreover, *L. fermentum* and *L. reuteri* species have been reported as good candidates as probiotics (De Angelis et al., 2007; Zoumpopoulou et al., 2008). Another species identified as *L. johnsonii*, *L. delbrueckii* subsp. *bulgaricus*, *L. vaginalis* and *L. crispatus* have been reported by their probiotic potential in humans and animals (Chen et al., 2007; Matijasic et al., 2006; Ohashi et al., 2007).

To understand the relevance of surface proteins in the adhesion of *Lactobacillus* to mucus and mucin, the purification and characterization of the adhesins should be performed. In previous reports proteins have been obtained by treatment with chaotropic agents as LiCl. From the spent, centrifuged growth medium and from soluble cytoplasmic extracts. A western blot assay using labelled mucus and mucin has been usually performed to show the protein bands with their relative molecular weight (MW) and in order to characterize them, N-terminal and internal peptide sequences has been determined. The MAPP adhesin of *L. reuteri* and the *Mmubp of L. fermentum* have been characterized in that manner (Rojas et al., 2002; Macías-Rodríguez et al., 2008). Recently the mucus-binding proteins (MUBs) have been revealed as one of the effectors molecules involved in mechanisms of the adherence of lactobacilli to the host; *mub*, or *mub*-like, genes were found in all of the six genomes of *L. reuteri* that are available but the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein (Mackenzie et al., 2010).

The complete process to get new strains of probiotic potential lactobacilli has been long and complex. Above a review of the different methods and results used was exposed and the results of a proposal are described.

The strains listed in Table 1 were selected because they were the predominant cultivable lactic acid bacteria in a selective medium (Rogosa agar, DIFCO); attached strongly to mucus and mucin when tested by the Dot Blot adhesion assay; grew in mucus, in presence of bile salt and in a broad range of temperatures. Likewise the molecular identification confirmed that *L. fermentum* and *L. reuteri* were the main isolates with probiotic potential for piglets (Macías-Rodríguez et al., 2008). In addition the genes *mapp* or *mapA* of *L. reuteri* and *mmub* of

PCR for Screening Potential Probiotic Lactobacilli for Piglets 81

A mucus adhesion promoting protein (MAPP) from *L. reuteri* 104R was reported (Rojas et al., 2002; Rojas, 1996). The gene encoding this MAPP adhesin (*mapp* gene) was found by using a PCR strategy were peptide derived oligonucletides were carefully devised and PCR reactions performed using chromosomal DNA of *L. reuteri* 104R as template. A PCR product was cloning and sequencing. Southern blotting of digested chromosomal DNA with selected enzyme mixtures was performed by using a 189 bp PCR product as a probe. Then a subgenomic DNA library of the hybridized fragment approximately of 4600 bp was running out. DNA fragments in this region were ligated in the pGEM3 vector and cloned in *E. coli*. Hybridization with the same probe showed a 4500 bp fragment containing the *mapp* gene. A subcloning and sequencing strategy (Figure 2) was used to determine the nucleotide sequence of the *mapp* gene. Nucleotide sequence analysis and search of the nucleotide and deduced aminoacid sequenced were searched in different data bases (NCBI). The complete gene *mapp* was sequenced. The sequence revealed one open reading frame which consists of 744 nucleotides corresponding to a protein of 244 aminoacids with a deduced pI of 10.57

Fig. 2. Schematic drawing of the subcloning and sequencing strategy to determine the nucleotide sequence of the *mapp* gene. A) The stippled box represents the pGEM-3 vector used to clone the chromosomal DNA fragment (4500 bp) from *L. reuteri* 104R and to subclone the fragments a, b, c and d. (inside boxes): a)189 bp, PCR fragment b)610 *Bgl*II-*Bgl*II fragment, c)146 bp *Bgl* II-*Bgl* II fragment and d) vector plus fragment without the two *Bgl* II fragments. B) The largest box represent the 4500bp fragment and the inside box represent the *mapp* gene. Universal primers are indicated, arrows indicate nucleotides determined and the heads of the arrow indicate the transcription direction. C) The box represents the 744bp open reading frame of the *mapp* gene. Universal and sequence specific primers are indicates, arrows indicate nucleotides determined and the heads of the arrow

indicate the transcription direction.

**3.2 Amplification and sequencing of the** *mapp* **or** *mapA* **gene** 

*L. fermentum*, which codified for mucus adhesins have been well characterized. Here these genes are described and the results of this proposal are discussed.

#### **3.1 Amplification and sequencing of the** *32-Mmubp* **encoding gene (***32-mmub***)**

Primers MEF7 and MER9 were previously deduced from the complete nucleotide sequence of *32-mmub* gene. The gene presented an ORF (open reading frame) of 903 bp encoding a predicted primary protein of 300 amino acids. This protein presented a signal peptide of 28 amino acids. Cleavage site between residues 28 and 29 were detected with the Signal P 3.0 prediction software. The prediction of transmembrane helices showed that the first 1 to 7 amino acids are predicted to be inside of the cell whereas residues 7 to 29 could be in the membrane and finally the region encompassing amino acids 30 to 300 could be outside. The mature protein consists of 272 residues with a molecular mass of 29,974 Da, an isoelectric point of 9.78 and a positive net charge of 21.22 at pH 7.0. This adhesin protein showed high identity only to *L. fermentum* (BAG27284). A search of homology (BLAST) with the genome of *L. fermentum* IFO 3956 recently published (Morita et al., 2008) showed that 32-Mmubp in *L. fermentum* BCS87 is part of an ABC transporter system and belongs to the PBPb superfamily. It showed to be conserved between prokaryotic protein sequences of substrate binding domains on the ABC-type glycine/betaine transport systems of the OpuAc familiy (PF04069). This family is part of a high-affinity multicomponent binding proteins-dependent transport system involved in bacterial osmoregulation and members of this family are often integral membrane proteins or predicted to be attached to the membrane by a lipid anchor. Some members of the corresponding taxa having similar ecological niches to those occupied by lactobacilli (gastrointestinal and respiratory tracts), i.e. *Helicobacter pylori* and *Mycobacterium tuberculosis*, do not group together suggesting that adhesion mechanisms is not a phylogenetic associated trait.

To confirm that 32-Mmubp of *L. fermentum* BCS87 is specific for this especie, a PCR using the MEF7 and MER9 oligonucleotides was performed. Chromosomal DNA of the 26 adhering strains of Table 1 was used as template to amplify an internal product of *32-mmub* gene. PCR products of the same size (550 bp) were observed in *L. fermentum* strain BCS87 and in all strains which belong to the same specie (Figure 1). Moreover a weak band was also observed in species *L. johnsonii* BCS41, *L. vaginalis* strains BCS68 and BCS154, *L. delbrueckii* subsp. *bulgaricus* BCS113, *L. crispatus* BCS125 and *L. reuteri* strains BCS127, BCS134, BCS136, BCS142 and BCS159 (Figure 1) suggesting *32-mmub* gene is conserved in piglets adhesive *L. fermentum*.

Fig. 1. Amplification of internal fragment of the *32-Mmubp* gene in adhesive strains of *L. fermentum* isolated from piglets intestinal tract. Lane MW, Molecular weight. Numbers 9 to 159 represents the identification code for each *Lactobacillus* strains from Table 1.

*L. fermentum*, which codified for mucus adhesins have been well characterized. Here these

Primers MEF7 and MER9 were previously deduced from the complete nucleotide sequence of *32-mmub* gene. The gene presented an ORF (open reading frame) of 903 bp encoding a predicted primary protein of 300 amino acids. This protein presented a signal peptide of 28 amino acids. Cleavage site between residues 28 and 29 were detected with the Signal P 3.0 prediction software. The prediction of transmembrane helices showed that the first 1 to 7 amino acids are predicted to be inside of the cell whereas residues 7 to 29 could be in the membrane and finally the region encompassing amino acids 30 to 300 could be outside. The mature protein consists of 272 residues with a molecular mass of 29,974 Da, an isoelectric point of 9.78 and a positive net charge of 21.22 at pH 7.0. This adhesin protein showed high identity only to *L. fermentum* (BAG27284). A search of homology (BLAST) with the genome of *L. fermentum* IFO 3956 recently published (Morita et al., 2008) showed that 32-Mmubp in *L. fermentum* BCS87 is part of an ABC transporter system and belongs to the PBPb superfamily. It showed to be conserved between prokaryotic protein sequences of substrate binding domains on the ABC-type glycine/betaine transport systems of the OpuAc familiy (PF04069). This family is part of a high-affinity multicomponent binding proteins-dependent transport system involved in bacterial osmoregulation and members of this family are often integral membrane proteins or predicted to be attached to the membrane by a lipid anchor. Some members of the corresponding taxa having similar ecological niches to those occupied by lactobacilli (gastrointestinal and respiratory tracts), i.e. *Helicobacter pylori* and *Mycobacterium tuberculosis*, do not group together suggesting that adhesion mechanisms is

To confirm that 32-Mmubp of *L. fermentum* BCS87 is specific for this especie, a PCR using the MEF7 and MER9 oligonucleotides was performed. Chromosomal DNA of the 26 adhering strains of Table 1 was used as template to amplify an internal product of *32-mmub* gene. PCR products of the same size (550 bp) were observed in *L. fermentum* strain BCS87 and in all strains which belong to the same specie (Figure 1). Moreover a weak band was also observed in species *L. johnsonii* BCS41, *L. vaginalis* strains BCS68 and BCS154, *L. delbrueckii* subsp. *bulgaricus* BCS113, *L. crispatus* BCS125 and *L. reuteri* strains BCS127, BCS134, BCS136, BCS142 and BCS159 (Figure 1) suggesting *32-mmub* gene is conserved in

Fig. 1. Amplification of internal fragment of the *32-Mmubp* gene in adhesive strains of *L. fermentum* isolated from piglets intestinal tract. Lane MW, Molecular weight. Numbers 9 to

159 represents the identification code for each *Lactobacillus* strains from Table 1.

**3.1 Amplification and sequencing of the** *32-Mmubp* **encoding gene (***32-mmub***)** 

genes are described and the results of this proposal are discussed.

not a phylogenetic associated trait.

piglets adhesive *L. fermentum*.

#### **3.2 Amplification and sequencing of the** *mapp* **or** *mapA* **gene**

A mucus adhesion promoting protein (MAPP) from *L. reuteri* 104R was reported (Rojas et al., 2002; Rojas, 1996). The gene encoding this MAPP adhesin (*mapp* gene) was found by using a PCR strategy were peptide derived oligonucletides were carefully devised and PCR reactions performed using chromosomal DNA of *L. reuteri* 104R as template. A PCR product was cloning and sequencing. Southern blotting of digested chromosomal DNA with selected enzyme mixtures was performed by using a 189 bp PCR product as a probe. Then a subgenomic DNA library of the hybridized fragment approximately of 4600 bp was running out. DNA fragments in this region were ligated in the pGEM3 vector and cloned in *E. coli*. Hybridization with the same probe showed a 4500 bp fragment containing the *mapp* gene. A subcloning and sequencing strategy (Figure 2) was used to determine the nucleotide sequence of the *mapp* gene. Nucleotide sequence analysis and search of the nucleotide and deduced aminoacid sequenced were searched in different data bases (NCBI). The complete gene *mapp* was sequenced. The sequence revealed one open reading frame which consists of 744 nucleotides corresponding to a protein of 244 aminoacids with a deduced pI of 10.57

Fig. 2. Schematic drawing of the subcloning and sequencing strategy to determine the nucleotide sequence of the *mapp* gene. A) The stippled box represents the pGEM-3 vector used to clone the chromosomal DNA fragment (4500 bp) from *L. reuteri* 104R and to subclone the fragments a, b, c and d. (inside boxes): a)189 bp, PCR fragment b)610 *Bgl*II-*Bgl*II fragment, c)146 bp *Bgl* II-*Bgl* II fragment and d) vector plus fragment without the two *Bgl* II fragments. B) The largest box represent the 4500bp fragment and the inside box represent the *mapp* gene. Universal primers are indicated, arrows indicate nucleotides determined and the heads of the arrow indicate the transcription direction. C) The box represents the 744bp open reading frame of the *mapp* gene. Universal and sequence specific primers are indicates, arrows indicate nucleotides determined and the heads of the arrow indicate the transcription direction.

PCR for Screening Potential Probiotic Lactobacilli for Piglets 83

operon *MapA* were not found in *L. plantarum* by a nucleotide data base search in blastn suite (NCBI). However results in this work suggested that functional MapA gene is specific for at

Mucus-binding proteins (MUBs) are molecules involved in mechanisms of the adherence of lactobacilli to the host (Roos & Jonsson, 2002). It was suggested that MUB domain is an LAB –specific functional unit that performs its task in various domain contexts and could fulfils an important role in host-microbe interactions in the gastrointestinal tract (Boekhorst et al., 2006). Recently was reported that in spite that *mub*, or *mub*-like, genes are found in all of the six genomes of *L. reuteri* and further demonstrated that MUB and MUB-like proteins are present in many *L. reuteri* isolates, MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion *in vitro* among *L. reuteri* strains, showing a high genetic heterogeneity among strains (Mackenzie et al., 2010). Different results were observed for the *MapA* gene which was present in all the adhesive *L. reuteri* 

Recently was reported a well-defined degradation product with antimicrobial activity obtained from the mucus adhesion-promoting protein (MapA) termed AP48-MapA from *L. reuteri* strain. The peptide was purified and characterized. This finding gave a new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota (Bøhle et al., 2010). This finding correlate with a report where trypsin digestion of the MapA protein resulted in peptides that bound to mucin suggesting that MapA protein could be involved in colonization of the intestinal mucosa of piglet, since the adhesive capacity could be retained in the intestinal mieleu

To find if *L. reuteri* strains which contain the *MapA* gene present the same operon as strain

Fig. 4. Amplification of the *MapA* operon (3.9Kb) from different adhesive *L. reuteri* strains isolated from piglets intestinal tract. Lane MW) 500-5000 bp ladder lane 1) Control strain, *L. fermentum* BCS87 lane 2) *L. reuteri* BCS136, lane 3) *L. reuteri* BCS127, lane 4) *L. reuteri* BCS159

These results together with the review of adhesins from *L. fermentum* and *L. reuteri* and their genes indicate that *Mmubp* and *MapA* genes are conserved in these species, at least in

least adhesive *L. reuteri* strains.

strains used to amplify this gene.

104R, amplification was run out (Figure 4).

(Rojas et al., 2002).

and lane 5) *L. reuteri* BCS142

and a molecular mass of 26380.90 Da. No putative promoter was found, however, a start codon (ATG) was noted 6 bases downstream of the beginning of the sequence and 30 bases upstream of the first N terminal aminoacid derived codon. The open reading frame ends with stop codons in all three reading frames (TGA A TAA T TAA) (Rojas, 1996).

The *mapp* gene described in Rojas, 2006, was later reported in Gene Bank as *MapA* and as part of one operon whose expression is controlled by a mechanism of transcription attenuation involved cysteine, with accession number AJ 293860 (Satoh et al., 2000). The relation between MapA and adhesion of *L. reuteri* to human intestinal (Caco 2) cells was reported. Quantitative analysis of adhesion of *L. reuteri* strains to Caco 2 cells showed that various strains bind also intestinal epithelial cells. In addition purified MapA bound to Caco 2 cells and this binding inhibited the adhesion of *L. reuteri* in a concentration dependent manner. Additionally it was concluded that multiple receptor-like molecules are involved in the MapA binding to Caco 2 cells (Miyoshi et al., 2006).

To confirm that *MapA* gene is specific for adhesive *L. reuteri* strains, a PCR using the MAPF1 and MAPR1 oligonucleotides was performed. Chromosomal DNA of the 26 adhering strains of Table 1 was also used as template to amplify the *MapA* gene. PCR products of the same size were observed only in the *L. reuteri* strains tested (Figure 3) but not in other species. This result strongly suggests that *MapA* gene is conserved in piglet adhesive *L. reutri* strains.

Fig. 3. Amplification of the gene *MapA* in adhesive *L. reuteri* strains isolated from piglets intestinal tract. KB Kilobases. MW; Molecular weight. Names on the lanes represent the identification code for each *Lactobacillus* strains from Table 1.

Expression of the mucus adhesion genes *Mub* and *MapA,* adhesion-like factor *EF-Tu* and bacteriocin gene *plaA* by *L. plantarum* 423 was reported. Growth in the presence of bile, pancreatin and at low pH, was studied by real-time PCR. It was found that *Mub, MapA* and *EF-Tu* were up-regulated in the presence of mucus, proportional to increasing concentrations. Expression of *Mub* and *MapA* remained unchanged at pH 4.0, whilst expression of *EF-Tu* and *plaA* were up-regulated. Expression of *MapA* was down-regulated in the presence of 1.0 g/l l-cysteine HCl, confirming that the gene is regulated by transcription attenuation that involves cysteine (Ramiah et al., 2007). However the gene and

and a molecular mass of 26380.90 Da. No putative promoter was found, however, a start codon (ATG) was noted 6 bases downstream of the beginning of the sequence and 30 bases upstream of the first N terminal aminoacid derived codon. The open reading frame ends

The *mapp* gene described in Rojas, 2006, was later reported in Gene Bank as *MapA* and as part of one operon whose expression is controlled by a mechanism of transcription attenuation involved cysteine, with accession number AJ 293860 (Satoh et al., 2000). The relation between MapA and adhesion of *L. reuteri* to human intestinal (Caco 2) cells was reported. Quantitative analysis of adhesion of *L. reuteri* strains to Caco 2 cells showed that various strains bind also intestinal epithelial cells. In addition purified MapA bound to Caco 2 cells and this binding inhibited the adhesion of *L. reuteri* in a concentration dependent manner. Additionally it was concluded that multiple receptor-like molecules are involved in

To confirm that *MapA* gene is specific for adhesive *L. reuteri* strains, a PCR using the MAPF1 and MAPR1 oligonucleotides was performed. Chromosomal DNA of the 26 adhering strains of Table 1 was also used as template to amplify the *MapA* gene. PCR products of the same size were observed only in the *L. reuteri* strains tested (Figure 3) but not in other species. This result strongly suggests that *MapA* gene is conserved in piglet adhesive *L. reutri* strains.

Fig. 3. Amplification of the gene *MapA* in adhesive *L. reuteri* strains isolated from piglets intestinal tract. KB Kilobases. MW; Molecular weight. Names on the lanes represent the

Expression of the mucus adhesion genes *Mub* and *MapA,* adhesion-like factor *EF-Tu* and bacteriocin gene *plaA* by *L. plantarum* 423 was reported. Growth in the presence of bile, pancreatin and at low pH, was studied by real-time PCR. It was found that *Mub, MapA* and *EF-Tu* were up-regulated in the presence of mucus, proportional to increasing concentrations. Expression of *Mub* and *MapA* remained unchanged at pH 4.0, whilst expression of *EF-Tu* and *plaA* were up-regulated. Expression of *MapA* was down-regulated in the presence of 1.0 g/l l-cysteine HCl, confirming that the gene is regulated by transcription attenuation that involves cysteine (Ramiah et al., 2007). However the gene and

identification code for each *Lactobacillus* strains from Table 1.

with stop codons in all three reading frames (TGA A TAA T TAA) (Rojas, 1996).

the MapA binding to Caco 2 cells (Miyoshi et al., 2006).

operon *MapA* were not found in *L. plantarum* by a nucleotide data base search in blastn suite (NCBI). However results in this work suggested that functional MapA gene is specific for at least adhesive *L. reuteri* strains.

Mucus-binding proteins (MUBs) are molecules involved in mechanisms of the adherence of lactobacilli to the host (Roos & Jonsson, 2002). It was suggested that MUB domain is an LAB –specific functional unit that performs its task in various domain contexts and could fulfils an important role in host-microbe interactions in the gastrointestinal tract (Boekhorst et al., 2006). Recently was reported that in spite that *mub*, or *mub*-like, genes are found in all of the six genomes of *L. reuteri* and further demonstrated that MUB and MUB-like proteins are present in many *L. reuteri* isolates, MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion *in vitro* among *L. reuteri* strains, showing a high genetic heterogeneity among strains (Mackenzie et al., 2010). Different results were observed for the *MapA* gene which was present in all the adhesive *L. reuteri*  strains used to amplify this gene.

Recently was reported a well-defined degradation product with antimicrobial activity obtained from the mucus adhesion-promoting protein (MapA) termed AP48-MapA from *L. reuteri* strain. The peptide was purified and characterized. This finding gave a new perspective on how some probiotic bacteria may successfully compete in this environment and thereby contribute to a healthy microbiota (Bøhle et al., 2010). This finding correlate with a report where trypsin digestion of the MapA protein resulted in peptides that bound to mucin suggesting that MapA protein could be involved in colonization of the intestinal mucosa of piglet, since the adhesive capacity could be retained in the intestinal mieleu (Rojas et al., 2002).

To find if *L. reuteri* strains which contain the *MapA* gene present the same operon as strain 104R, amplification was run out (Figure 4).

Fig. 4. Amplification of the *MapA* operon (3.9Kb) from different adhesive *L. reuteri* strains isolated from piglets intestinal tract. Lane MW) 500-5000 bp ladder lane 1) Control strain, *L. fermentum* BCS87 lane 2) *L. reuteri* BCS136, lane 3) *L. reuteri* BCS127, lane 4) *L. reuteri* BCS159 and lane 5) *L. reuteri* BCS142

These results together with the review of adhesins from *L. fermentum* and *L. reuteri* and their genes indicate that *Mmubp* and *MapA* genes are conserved in these species, at least in

PCR for Screening Potential Probiotic Lactobacilli for Piglets 85

Blomberg, L. & Conway, P L, 1989. An *In vitro* study of ileal colonisation resistance to

Boekhorst, J. et al., 2006. Comparative analysis of proteins with a mucus-binding domain

Bonazzi, M. & Cossart, P., 2011. Host-pathogen interactions: Impenetrable barriers or entry

Boot, H.J. et al., 1993. S-Layer protein of *Lactobacillus acidophilus* ATCC 4356: purification,

Brennan, M. et al., 1986. Cellular damage in dried *Lactobacillus acidophilus. Journal of Food* 

Brown, M.R.W., Anwar, H. & Casterton, J.W., 1988. Surface antigens *In vivo*: a mirror for vaccin development. *Canadian Journal of Microbiology*, 34, pp.494-498. Buck, B.L. et al., 2005. Functional Analysis of Putative Adhesion Factors in *Lactobacillus acidophilus* NCFM. *Applied and Environmental Microbiology*, 71(12), pp.8344-8351. Bøhle, L.A. et al., 2010. Specific degradation of the mucus adhesion-promoting protein

Chagnaud, P., Jenkinnson, H.F. & Tannock, G. W., 1992. Cell surface associated proteins of

Chassy, B.M., 1987. Pospects for the genetic manipulation of lactobacilli. *FEMS Microbiol* 

Chateau N, Castellanos I, D.A., 1993. Distribution of pathogen inhibition in the *Lactobacillus*

Chen, X. et al., 2007. The S-layer proteins of *Lactobacillus crispatus* strain ZJ001 is responsible

Clamp, J.R. & Sheehan, J.K., 1978. Chemical aspects of mucus. *British Medical Bulletin*, 34(1),

Coconnier, M.H., Liévin, V. & Hudault, S, 1997. Antibacterial effect of the adhering human

Collado, M.C. et al., 2006. Protection mechanism of probiotic combination against human

Conway, P L, Welin, A. & Cohen, P.S., 1990. Presence of K88-specific receptors in porcine ileal mucus is age dependent. *Infection and Immunity*, 58(10), pp.3178-3182. Conway, P L & Adams, R.F., 1989. Role of erythrosine in the inhibition of adhesion of

*Lactobacillus acidophilus* strain LB. *Microbiology*, 41(5), pp.1046-1052.

*typhimurium*. *International Journal of Food Microbiology.* 115, pp.307-312. Chiduwa, G. et al., 2008. Herd dynamics and contribution of indigenous pigs to the

*Health and Disease*, 2, pp.285-291.

*Journal of bacteriology*, 175(19), pp.6089-6096.

*Environmental Microbiology*, 76(21), pp.7306-9.

pp.273-80.

pp.121-131.

pp.36-40.

pp.25-41.

*Rev*, 46, pp.297-312.

*and Production*, 40(2), pp.125-36.

*Clinical Nutrition.* 15(4), pp.570-575.

195(3), pp.349-358.

*Protection*, 49, pp.47-53.

*Escherichia coli* K88 to piglet ileal mucus by *Lactobacillus* spp. *Microbial Ecology in* 

found exclusively in lactic acid bacteria. *Microbiology (Reading, England)*, 152(Pt 1),

portals? The role of cell-cell adhesion during infection. *The Journal of Cell Biology*,

expression in *Escherichia coli*, and nucleotide sequence of the corresponding gene.

(MapA) of *Lactobacillus reuteri* to an antimicrobial peptide. *Applied and* 

gastrointestinal strains of lactobacilli. *Microbial Ecology in Health and Disease.* 5(3),

isolates of a commercial probiotic consortium. *Journal of Applied Bacteriology.* 74(1),

for competitive exclusion against *Escherichia coli* O157:H7 and *Salmonella* 

livelihoods of rural farmers in a semi-arid area of Zimbabwe. *Tropical Animal Health* 

pathogens: in vitro adhesion to human intestinal mucus. *Asia Pacific Journal of* 

*Lactobacillus fermentum* strain 737 to mouse stomach tissue. *Journal of General* 

adhesive strains isolated from intestinal tract of piglets. In Addition these strains are considered the main *Lactobacillus* species which colonize the intestinal tract of piglets. Therefore the traditional methods for screening new probiotic strains for piglets could be reduced as described.

Take faeces and intestinal tract mucus samples from healthy piglets and make a viable count in a selective medium (Rogosa Agar, DIFCO). Incubate at 36°C in anaerobic conditions for 24-48 h and select colonies from the plates with the more diluted samples to grow and purify the DNA. Perform a PCR reaction using the specific primers for the *Mmub* and *MapA* genes. Strains which amplify a fragment with the size mentioned above should be *L. fermentum* for the *Mmub* gene and *L. reuteri* for the *MapA* gene.

## **4. Conclusion**

Bacteria cultivated in the laboratory for long time could mutate and lost probiotic attributes, therefore it is important to look for an easy strategy to routinely screening for probiotics. Screening for new probiotic *Lactobacillus fermentum* and *Lactobacillus reuteri*, which are the dominant microbiota in healthy piglets and present the ability to adhere the intestinal tract mucus is described in this chapter. The main advantage of this method is the expend time.

## **5. Acknowledgment**

This study was supported by Universidad Autónoma de Baja California Sur, México and Conacyt, Project No. 29410-B

## **6. References**

Aleljung, P. et al., 1991. Collagen Binding by Lactobacilli. *Current Microbiology*, 23, pp.33-38.


adhesive strains isolated from intestinal tract of piglets. In Addition these strains are considered the main *Lactobacillus* species which colonize the intestinal tract of piglets. Therefore the traditional methods for screening new probiotic strains for piglets could be

Take faeces and intestinal tract mucus samples from healthy piglets and make a viable count in a selective medium (Rogosa Agar, DIFCO). Incubate at 36°C in anaerobic conditions for 24-48 h and select colonies from the plates with the more diluted samples to grow and purify the DNA. Perform a PCR reaction using the specific primers for the *Mmub* and *MapA* genes. Strains which amplify a fragment with the size mentioned above should be *L.* 

Bacteria cultivated in the laboratory for long time could mutate and lost probiotic attributes, therefore it is important to look for an easy strategy to routinely screening for probiotics. Screening for new probiotic *Lactobacillus fermentum* and *Lactobacillus reuteri*, which are the dominant microbiota in healthy piglets and present the ability to adhere the intestinal tract mucus is described in this chapter. The main advantage of this method is the expend time.

This study was supported by Universidad Autónoma de Baja California Sur, México and

Aleljung, P. et al., 1991. Collagen Binding by Lactobacilli. *Current Microbiology*, 23, pp.33-38. Aleljung, P. et al., 1994. Purification of collagen-binding proteins of *Lactobacillus reuteri* NCIB

Ashenafi, M., 2005. Growth of *Listeria monocytogenes* in fermenting tempeh made of various

Bateup, J.M. et al., 1995. Comparison of *Lactobacillus* strains with respect to bile salt

murine host. *Applied and Environmental Microbiology*, 61(3), pp.1147-11499. Beachey, E.H., 1981. Bacterial adherence: Adhesin-receptor interactions mediating the

Bergonzelli, G.E. et al., 2006. GroEL of *Lactobacillus johnsonii* La1 ( NCC 533 ) is cell surface

Beveridge, T.J., 1989. Role of cellular design in bacterial metal accumulation and

Beveridge, T.J. & Graham, L.L., 1991. Surface layers of bacteria. *Microbiological Reviews*, 55(4),

Bibel, D.J., 1988. Elie Metchnikoff's Bacillus of long life. *ASM News*, 54(12), pp.661-665.

*Helicobacter pylori. Infection and immunity*, 74(1), pp.425-434.

mineralization. *Annual Review of Microbiology*, 43, pp.147-171.

beans and its inhibition by *Lactobacillus plantarum*. *Food Microbiology*, 8(4), pp.1991-

hydrolase activity, colonization of the gastrointestinal tract, and growth rate of the

attachment of bacteria to mucosal surface. *Journal of Infectious Diseases.*, 143, pp.325-

associated: potential role in interactions with the host and the gastric pathogen

*fermentum* for the *Mmub* gene and *L. reuteri* for the *MapA* gene.

11951. *Current Microbiology*, 28, pp.231-236.

reduced as described.

**4. Conclusion** 

**5. Acknowledgment** 

1991.

345.

pp.684-705.

**6. References** 

Conacyt, Project No. 29410-B


PCR for Screening Potential Probiotic Lactobacilli for Piglets 87

Harris, L. J., et al., 1989. Antimicrobial activity of lactic acid bacteria against *Listeria* 

Henriksson, A.R., Szewzyk, R. & Conway, P. L., 1991. Characteristics of the adhesive

Herías M.V. et al., 1999. Immunomodulatory effects of *Lactobacillus plantarum* colonizing the intestine of gnotobiotic rats. *Clinical & Experimental Immunology*, 116, pp.283-290. Hudault, S et al., 1997. Antagonistic activity exerted *In vitro* and *In vivo* by *Lactobacillus casei*

Jacobsen, C.N. et al., 1999. Screening of probiotic activities of forty-seven strains of

Jernberg, C. et al., 2005. Monitoring of antibiotic-induced alterations in the human intestinal

indigenous flora. *Applied and Environmental Microbiology*, 59(1), pp.15-20. Jones, G.W. & Isaacson, R.E., 1984. Proteinaceous bacterial adhesins and their receptors.

Jonsson, E. & Conway, P.L., 1992. Probiotics for pigs. In R. Fuler, ed. *Probiotics, the scientific* 

Kankainen, M. et al., 2009. Comparative genomic analysis of *Lactobacillus rhamnosus* GG

Kawai, Y., Suegara, Y.N. & Shimohashi, H., 1982. Colonization of lactic acid bacteria isolated

Klemm, P., 1994. *Fibriae: adhesion, genetics, biogenesis and vaccines* Per Klemm, ed., London:

Laux, D.C., 1986. Identification and characterization of mouse small intestine mucosal receptors for *Escherichia coli* K-12(K88ab). *Infection and Immunity*, 52(1), pp.18-25. Laux, D.C., McSweegan, E.F. & Cohen, P.S., 1984. Adhesion of enterotoxigenic *Escherichia* 

mucosal surface components. *Journal of Microbiological Methods*, 2, pp.27-39. Lebeer, S., Vanderleyden, J. & De Keersmaecker, S.C.J., 2008. Genes and molecules of

Lee, N.K. et al., 2008. Screening of Lactobacilli derived from chicken feces and partial

*Academy of Sciences of the United States of America*, 106(40), pp.17193-8. Kaushik, J.K. et al., 2009. Functional and probiotic attributes of an indigenous isolate of

*Critical Reviews in Microbiology*, 10(3), pp.229-260.

*basis*. London: Chapman Press, pp. 260-314.

*Lactobacillus plantarum*. *PloS one*, 4(12), pp.1-11.

*Microbiology and Biotechnology*, 18(2), pp.338-342.

*Immunology*, 26(5), pp.363-373.

CRC Press.

72(4), pp.728-64.

determinants of *Lactobacillus fermentum* 104. *Applied and Environmental Microbiology*,

(strain GG) against *Salmonella typhimurium* C5 infection. *Microbiology*, 63(2), pp.513-

*Lactobacillus* spp. by *In vitro* techniques and evaluation of the colonization ability of five selected strains in humans. *Applied and Environmental Microbiology*, 65(11),

microflora and detection of probiotic strains by se of Terminal Restriction Fragment Length Polymorphism. *Applied and Environmental Microbiology*, 71(1), pp.501-506. Johansson, M.L. et al., 1993. Administration of different *Lactobacillus* strains in fermented

oatmeal soup: *In vivo* colonization of human intestinal mucosa and effect on the

reveals pili containing a human- mucus binding protein. *Proceedings of the National* 

from rats and humans in the gastrointestinal tract of rats. *Microbiology and* 

*coli* to immobilized intestinal mucosal preparations: a model for adhesion to

lactobacilli supporting probiotic action. *Microbiology and Molecular Biology Reviews*,

characterization of *Lactobacillus acidophilus* A12 as an animal probiotics. *Journal of* 

*monocytogenes. Journal of Food Protection*, 52, pp.384-387.

57(2), pp.499-502.

518.

pp.4949-56.

*Microbiology*, 135(5), pp.1167-73. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/2559943.


Cook, R.L., Harris, R.J. & Reid, G, 1988. Effect of culture media and growth phase on the

Costerton, J.W., 1988. Structure and plasticity at various organization levels in the bacterial

De Angelis, M., et. al., 2006. Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. *Research in Microbiology*, 157, pp.792-801. Dixon, J.M.S., 1960. The fate of bacteria in the small intestine. *Journal of Pathology* 

Duary, R.K., Batish, V.K. & Grover, S., 2011. Relative gene expression of bile salt hydrolase

Dubos, R., et al., 1965. Indigenous, normal and autochthonous flora of the Gastrointestinal

Fuller, R., 1989. Probiotics in man and animals. *Journal of Applied Bacteriology,* 66, pp.365-378. Fuller, R., Barrow, P.A. & Brooker, M.E., 1978. Bacteria associated with the gastric epithelium of neonatal pigs. *Applied and Environmental Microbiology*, 35(3), pp.582-591. Goh, Y.J. & Klaenhammer, T.R, 2010. Functional roles of aggregation-promoting-like factor

Goswami, P.S. et al., 2011. Preliminary investigations of the distribution of *Escherichia coli* 

Granato, D. et al., 2004. Cell surface-associated elongation factor Tu mediates the attachment

Greene, J.D. & Klaenhammer, T R, 1994. Factors involved in adherence of lactobacilli to human Caco-2 cells. *Applied and Environmental Microbiology*, 60(12), pp.4487-94. Guarino, A. et al., 2011. The management of acute diarrhea in children in developed and

Gueimonde M, Sakata S, Kalliomaki M, Isolauri E, Benno Y, S.S., 2006. Effect of maternal

Guerrant, R. L., Hughes, J. M., Lima, N. L., Crane, J., 1990. Diarrhea in developed and

Guilliland, S. E., Morelli, L., R.G., 2001. Health and nutritional properties of probiotics in

H, Kinoshita et al., 2007. Quantitative evaluation of adhesion of lactobacilli isolated from

(BIACORE assay). *Journal of Applied Microbiology,* 102, pp.116-123.

and surface proteins in two putative indigenous *Lactobacillus plantarum* strains under *In vitro* gut conditions. *Molecular Biology Reports*, DOI: 10.1007/s11033-011-

in stress tolerance and adherence of *Lactobacillus acidophilus* NCFM. *Applied and* 

O149 in sows, piglets, and their environment. *Canadian Journal of Veterinary* 

of *Lactobacillus johnsonii* NCC533 ( La1 ) to human intestinal cells and mucins.

developing areas: from evidence base to clinical practice. *Expert Opinion on* 

consumption of *Lactobacillus* GG on transfer and establishment of fecal bifidobacterial microbiota in neonates. *Journal of Pediatric Gastroenterology and* 

developing countries: magnitude, special settings, and etiologies. *Reviews of* 

food including powder milk with live lactic acid bacteria. . In *Joint FAO/WHO* 

human intestinal tissues to human colonic mucin using surface plasmon resonance

morphology of lactobacilli and on their ability to adhere to epithelial cells. *Current* 

*Microbiology*, 135(5), pp.1167-73. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2559943.

cell. *Canadian Journal of Microbiology*, 34, pp.513-521.

Tract. *Journal of Experimental Medicine*, 122, pp.67-76.

*Environmental Microbiology*, 76(15), pp.5005-12.

*Infection and Immunity*, 72(4), pp.2160-2169.

*Pharmacotherpy*, pp.22106840-22106840.

*Nutrition*, 42(2), pp.266-270.

*Infectious Diseases*, 12(1), pp.41-50.

*expert consultation, Cordova Argentina*.

*Microbiology*, 17, pp.159-166.

*&Bacterology*, 79, pp.131-141.

*Research,* 75(1), pp.57-60.

1006-9.


PCR for Screening Potential Probiotic Lactobacilli for Piglets 89

Oostindjer, M. et al., 2010. Effects of environmental enrichment and loose housing of

von Ossowski, I. et al., 2010. Mucosal adhesion properties of the probiotic *Lactobacillus* 

Pavlova, S.I. et al., 1993. Effect of medium composition on the ultrastructure of *Lactobacillus*

Pedersen, K. & Tannock, G W, 1989. Colonization of the porcine gastrointestinal tract by lactobacilli. *Applied and Environmental Microbiology*, 55(2), pp.279-83. Pouwels, P H & Leer, R J, 1993. Genetics of lactobacilli: Plasmids and gene expression.

Pretzer, G. et al., 2005. Biodiversity-based identification and functional characterization of

Ramiah, K., van Reenen, C. a & Dicks, L.M.T., 2007. Expression of the mucus adhesion genes

Ray, B. & Johnson, M.C., 1986. Freeze drying injury of surface layer protein and its

Reid, G., 1999. The scientific basis for probiotic strains of *Lactobacillus*. *Applied and* 

Reniero, R. et al., 1992. High frequency of conjugation in *Lactobacillus* mediated by an aggregation-promoting factor. *Journal of General Microbiology*, 138(4), pp.763-768. Reniero, R. et al., 1993. Purification of *Lactobacillus* secreted proteins. *Biotechnology* 

Robins-Browne, R.M. & Levine, M.M., 1981. The fate of ingested lactobacilli in the proximal small intestine. *American Journal of Clinical Nutrition*, 34, pp.514-519. Rojas, M & Conway, P L, 1996. Colonization by lactobacilli of piglet small intestinal mucus.

Rojas, M. and Conway, P., 2001. A dot blot assay for adhesive components relative to

Rojas, M., 1996. Studies on an adhesion promoting protein from *Lactobacillus* and its role in

Roos, S. & Jonsson, H., 2002. A high-molecular-mass cell-surface protein from *Lactobacillus* 

*Goteborg, Sweden.* I. Maurilia Rojas, ed., Goteborg: Goteborg University. Rojas, M., Ascencio, F. & Conway, P.L., 2002. Purification and characterization of a surface

probiotics. In R. J. Doyl, ed. *Methods of enziyology Vol. 336. Microbial Growth and Biofilms. Part A. Developmental and Molecular Biological Aspects*. San Diego,

the colonisation of the gastrointestinal tract*. PhD thesis Goteborg University,* 

protein from *Lactobacillus fermentum* 104R that binds to porcine small intestinal mucus and gastric mucin. *Applied and Environmental Microbiology*, 68(5), pp.2330-

*reuteri* 1063 adheres to mucus components. *Microbiology (Reading, England)*, 148(Pt

protection in *Lactobacillus acidophilus*. *CryoLetters*, 7, pp.210-217.

the mannose-specific adhesin of *Lactobacillus plantarum*. *Journal of Bacteriology*,

Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of *Lactobacillus plantarum* 423, monitored with real-time PCR. *International Journal of* 

*Science*, 88(11), pp.3554-62.

187(17), pp.6128-6136.

*Microbiology*, 76(7), pp.2049-57.

strains. *Archives of Microbiology*, 160, pp.132-136.

*Antonie van Leeuwenhoek*, 64(2), pp.85-107.

*Food Microbiology*, 116(3), pp.405-409.

*Techniques*, 7(7), pp.401-406.

2336.

2), pp.433-42.

*Environmental Microbiology*, 65(9), pp. 3763-3766.

*The Journal of Applied Bacteriology*, 81(5), pp.474-80.

California, U.S.A.: Academic Press, pp. 389-402.

lactating sows on piglet performance before and after weaning. *Journal of Animal* 

*rhamnosus* GG SpaCBA and SpaFED pilin subunits. *Applied and Environmental* 


Lin, W.H. et al., 2007. Different probiotic properties for *Lactobacillus fermentum* strains

Ma, Y.L. et al., 2005. Effect of *Lactobacillus* isolates on the adhesion of pathogens to chicken intestinal mucus *in vitro. Letters in Applied Microbiology*, 42, pp.369-374. Mackenzie, D. et al., 2010. Strain-specific diversity of mucus-binding proteins in the

Mackie, R.I., Sghir, a & Gaskins, H.R., 1999. Developmental microbial ecology of the

Macías-Rodríguez, M.E. et al., 2009. *Lactobacillus fermentum* BCS87 expresses mucus- and

Macías-Rodríguez, M.E. et al., 2008. Potential probiotic *Lactobacillus* strains for piglets from

Malmsten, M. et al., 1992. Mucin layers on hydrophobic surfaces studied with ellipsometry and surface force measurements. *Journal of Colloid and Interface*, 151, pp.579-590. Marteau, P.R., 2002. Probiotics in clinical conditions. *Clinical reviews in Allergy & Immunology*, 22(3), pp.255-73. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12043384. Masuda, K. & Kawata, T., 1983. Distribution and chemical characterization of regular arrays

Matijasic, B., Stojkovic, S. & Rogelj, I., 2006. Survival and *In vivo* adhesion of human isolates

McAllister, J.S., Kurtz, H.J. & Short, E.C., 1979. Changes in the intestinal flora of young pigs

Miyoshi, Y. et al., 2006. A Mucus Adhesion Promoting Protein, MapA, Mediates the

Molin, G., 1993. Numerical taxonomy of *Lactobacillus* spp. associated with healthy and

Mondal, D. et al., 2011. Contribution of enteric infection, altered intestinal barrier function,

Moser, S.A. & Savage, D.C., 2001. Bile salt hydrolase activity and resistance to toxicity of

Muralidhara, K.S., 1977. Effect of feeding lactobacilli on the coliform and lactobacillus flora of intestinal tissue and feces from piglets. *Journal of Food Protection*, 40(5), pp.288-295. Ohashi, Y. et al., 2007. Stimulation of indigenous lactobacilli by fermented milk prepared

*Bioscience, Biotechnology, and Biochemistry*, 70(7), pp.1622-1628.

adhesion and aggregation properties of *Lactobacillus reuteri*. *Microbiology (Reading,* 

neonatal gastrointestinal tract. *The American Journal of Clinical Nutrition*, 69(5),

mucin-binding proteins on the cell surface. *Journal of Applied Microbiology*, 107(6),

in the cell walls of strains of the genus *Lactobacillus*. *FEMS Microbiology Letters*, 20,

*Lactobacillus gasseri* LF221 and K7 in weaned piglets and their effects on coliforms, clostridia and lactobacilli viable counts in faeces and mucosa. *Journal of Dairy* 

with postweaning diarrhea or edema disease. *Journal of Animal Science*, 49, pp.868-

Adhesion of *Lactobacillus reuteri* to Caco-2 Human Intestinal Epithelial Cells.

diseased mucosa of the human intestines. *Journal of Applied Bacteriology*, 74, pp.314-

and maternal malnutrition to infant malnutrition in Bangladesh. *Clinical Infectious* 

conjugated bile salts are unrelated properties in Lactobacilli. *Applied and* 

with probiotic bacterium, *Lactobacillus delbrueckii subsp. bulgaricus* strain 2038, in the pigs. *Journal of Nutritional Science and Vitaminology (Tokyo) 53:82-86*, 53(82-86).

isolated from swine and poultry. *Anaerobe*, 13, pp.107-113.

an arid coast. *Annals of Microbiology*, 58(4), pp.641-648.

*England)*, 156(Pt 11), pp.3368-78.

p.1035S-1045S.

pp.1866-74.

pp.145-150.

679.

323.

*Research*, 73, pp.417-422.

*Diseases*, doi:10.1093/cid/cir807

*Environmental Microbiology*, 67(8), pp.3476-3480.


**1. Introduction** 

devising of pathogen management.

pathogens in plants or insect vectors (65).

**5** 

Duška Delić

*Bosnia and Herzegovina* 

**Polymerase Chain Reaction** 

 **for Phytoplasmas Detection** 

This chapter inspired treat caused by phytoplasmas diseases in food production, and increased need for sensitive and accurate detection of these microorganisms. Early and sensitive detection and diagnosis of phytoplasmas is of paramount importance for effective prevention strategies and it is prerequisite for study of the diseases epidemiology and

Phytoplasmas are prokaryotes lacking cell walls that are currently classified in the class *Mollicutes* (2). To the class *Mollicutes* (cell wall-less prokaryotes) belonging both pathogenic groups: mycoplasma-like organisms (MLOs) and mycoplasmas. However, in contrast to mycoplasmas, which cause an array of disorders in animals and humans, the phytopathogenic MLOs resisted all attempts to culture them *in vitro* in cell free media (89). Following the application of molecular technologies the enigmatic status of MLOs amongst the prokaryotes was resolved and led to the new trivial name of "phytoplasma", and

Diseases associated with phytoplasma presence occur worldwide in many crops, although individual phytoplasmas may be limited in their host range or distribution. There are more than 300 distinct plant diseases attributed to phytoplasmas, affecting hundreds of plant genera (70). Many of the economically important diseases are those of woody plants, including coconut lethal yellowing, peach X-disease, grapevine yellows, and apple proliferation. Following their discovery, phytoplasmas have been difficult to detect due to their low concentration especially in woody hosts and their erratic distribution in the sieve tubes of the infected plants (15). First detection technique which indicated presence of some intercellular disorder was based on graft transmission of the pathogen to healthy indicator plants. The establishment of electron microscopy (EM) based techniques represents an alternative approach to the traditional indexing procedure for phytoplasmas. EM observation (17, 33) and less frequently scanning EM (59) were the only diagnostic techniques until staining with DNA-specific dyes such as DAPI (148) was developed. Lately, protocols for the production of enriched phytoplasma-specific antigens have been developed, thus introducing serological-based detection techniques for the study of these

Phytoplasma detection is now routinely done by different nucleic acid techniques based on polymerase chain reaction (PCR) (144, 12, 52, 165). The procedures developed in the last 20

eventually to the designation of a new taxon named 'Candidatus phytoplasma' (73).

*University of Banjaluka, Faculty of Agriculture* 


## **Polymerase Chain Reaction for Phytoplasmas Detection**

## Duška Delić

*University of Banjaluka, Faculty of Agriculture Bosnia and Herzegovina* 

## **1. Introduction**

90 Polymerase Chain Reaction

Sanders, M.E., 2011. Impact of probiotics on colonizing microbiota of the gut. *Journal of* 

Sarem-Daamerdji, L. et al., 1995. *In vitro* colonization ability of human colon mucosa by exogenous *Lactobacillus* strains. *FEMS Microbiology Letters*, 131(2), pp.133-137. Satoh, E. et al., 2000. The gene encoding the adhesion promoting protein MapA from

Savage, D C, 1992. Growth phase, cellular hydrophobicity, and adhesion in vitro of

Schneitz, C.L. & Lounatma, K., 1993. Adhesion of *Lactobacillus acidophilus* to avian intestinal

Shimizu M, Shimizu Y, K.Y., 1978. Effects of ambient temperatures on induction of transmissible gastroenteritis in feeder pigs. *Infection and Immunity*, 21, pp.747-752. Smith, H.W., 1965. Observations on the flora of the alimentary tract of animals and factors affecting its composition. *Journal of Pathology Bacteriology*, 89, pp.95-122. Soto, G.E. & Hultgren, S.J., 1999. Bacterial adhesins: common themes and variations in architecture and assembly. *Journal of Bacteriology*, 181(4), pp.1059-1071. Suegara, N. et al., 1975. Behavior of microflora in the rat stomach: adhesion of lactobacilli to

Sun, J. et al., 2007. Factors involved in binding of *Lactobacillus plantarum* Lp6 to rat small

Tannock, G. W., Blumershine, R. & Archibald, R., 1987. Demonstration of epithelium-

Tannock, G. W. & Savage, D. C., 1974. Influences of dietary and environmental stress on

Van Tassell, M.L. & Miller, M.J., 2011. *Lactobacillus* adhesion to mucus. *Nutrients*, 3(5),

Thompson-Chagoyán, O.C., Maldonado, J. & Gil, A., 2007. Colonization and impact of

Toba, T. et al., 1995. A Collagen-binding S-Layer protein in *Lactobacillus crispatus*. *Applied and* 

Vélez, M.P., De Keersmaecker, S.C.J. & Vanderleyden, J., 2007. Adherence factors of

Wang, B. et al., 2008. Identification of a surface protein from *Lactobacillus reuteri* JCM1081

intestinal mucus. *Letters in Applied Microbiology*, 44(1), pp.79-85.

*Lactobacillus reuteri* 104R is part of one operon whose expression is controlled by a mechanism of transcription attenuation, involving cysteine. Gene Bank Accession

lactobacilli colonizing the keratinizing gastric epithelium in the mouse. *Applied and* 

epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer).

the keratinized epithelial cells of the rat stomach *In vitro*. *Infection and Immunity*,

associated microbes in the oesophagus of pigs, cattle, rats and deer. *FEMS* 

microbial populations in the murine gastrointestinal tract. *Infection and Immunity*,

disease and other factors on intestinal microbiota. *Digestive Diseases and Sciences*,

*Lactobacillus* in the human gastrointestinal tract. *FEMS Microbiology Letters*, 276(2),

that adheres to porcine gastric mucin and human enterocyte-like HT-29 cells.

*Clinical Gastroenterology*, 45(5), pp.115-119.

*Environmental Microbiology*, 58(6), pp.1992-5.

*Journal of Applied Bacteriology*, 74, pp.290-294.

Number AJ 293860.

12(1), pp.173-179.

9(3), pp.591-598.

52(9), pp.2069-2077.

pp.613-636.

pp.140-148.

*Microbiology Ecology*, 45, pp.199-203.

*Environmental Microbiology*, 61(7), pp.2467-2471.

*Current Microbiology*, 57(1), pp.33-38.

This chapter inspired treat caused by phytoplasmas diseases in food production, and increased need for sensitive and accurate detection of these microorganisms. Early and sensitive detection and diagnosis of phytoplasmas is of paramount importance for effective prevention strategies and it is prerequisite for study of the diseases epidemiology and devising of pathogen management.

Phytoplasmas are prokaryotes lacking cell walls that are currently classified in the class *Mollicutes* (2). To the class *Mollicutes* (cell wall-less prokaryotes) belonging both pathogenic groups: mycoplasma-like organisms (MLOs) and mycoplasmas. However, in contrast to mycoplasmas, which cause an array of disorders in animals and humans, the phytopathogenic MLOs resisted all attempts to culture them *in vitro* in cell free media (89). Following the application of molecular technologies the enigmatic status of MLOs amongst the prokaryotes was resolved and led to the new trivial name of "phytoplasma", and eventually to the designation of a new taxon named 'Candidatus phytoplasma' (73).

Diseases associated with phytoplasma presence occur worldwide in many crops, although individual phytoplasmas may be limited in their host range or distribution. There are more than 300 distinct plant diseases attributed to phytoplasmas, affecting hundreds of plant genera (70). Many of the economically important diseases are those of woody plants, including coconut lethal yellowing, peach X-disease, grapevine yellows, and apple proliferation. Following their discovery, phytoplasmas have been difficult to detect due to their low concentration especially in woody hosts and their erratic distribution in the sieve tubes of the infected plants (15). First detection technique which indicated presence of some intercellular disorder was based on graft transmission of the pathogen to healthy indicator plants. The establishment of electron microscopy (EM) based techniques represents an alternative approach to the traditional indexing procedure for phytoplasmas. EM observation (17, 33) and less frequently scanning EM (59) were the only diagnostic techniques until staining with DNA-specific dyes such as DAPI (148) was developed. Lately, protocols for the production of enriched phytoplasma-specific antigens have been developed, thus introducing serological-based detection techniques for the study of these pathogens in plants or insect vectors (65).

Phytoplasma detection is now routinely done by different nucleic acid techniques based on polymerase chain reaction (PCR) (144, 12, 52, 165). The procedures developed in the last 20

Polymerase Chain Reaction for Phytoplasmas Detection 93

Not all plant species infected with phytoplasmas have disease symptoms, but infected plants normally show symptoms such as virescence, phyllody, yellowing, witches' broom, leaf rool and generalized decline (19). The most common symptoms of the infected plants are yellowing caused by the breakdown of chlorophyll and carotenoids, whose biosynthesis is also inhibited (21). Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants grown in the field was also recently

Phytoplasmas are mainly spread by insects of the families *Cicadellidae* (leafhoppers), *Fulgoridae* (planthoppers), and *Psyllidae*, which feed on the phloem tissues of infected plants acquiring the phytoplasmas and transmitting them to the next plant they feed on (136, 2). They enter the insect's body through the stylet and then move through the intestine and been absorbed into the haemolymph. From here they proceeded to colonize the salivary glands, a process that can take up to some weeks (5, 80). Another pathway of phytoplasma survival and transmission is vegetative propagating plant material. As it mentioned phytoplasma invading phloem tissue and it is mostly find that in woody plants they disappear from aerial parts of trees during the winter and survive in the root system to re-

In time when phytoplasmas were discovered as plant pathogens diagnostic was difficult since detection was based on symptoms observation insect or dodder/graft transmission to host plant and electron microscopy of ultra-thin sections of the phloem tissue. Serological diagnostic techniques for the detection of phytoplasma began to emerge in the 1980's with ELISA based methods. However, serological methods weren't always sensitive enough to detect various phytoplasmas (13, 47). Finally, in the early 1990's PCR coupled with RFLP analysis allowed the accurate identification of different strains and species of phytoplasma (127, 91, 145). Nowadays, diagnosis of phytoplasmas is routinely done by PCR and can be divided into three phases: total DNA extraction from symptomatic tissue or insects; PCR amplification of phytoplasma-specific DNA; characterization of the amplified DNA by

For the DNA extraction of known phytoplasma, several protocols for isolation from infected plant material and insects have been developed. Control samples are drowning from plants commonly infected by phytoplasmas. Reference phytoplasma strain collections are maintained in experimentally infected periwinkle (*Catharanthus roseus*) which is available for

In the second stage of the testing, DNA extracted from plants or insects is amplifying by using the polymerase chain reaction or PCR. PCR is a standardised technique in gene analysis to provide sufficient genetic material for detection (153). It works through the use of short lengths of DNA called primers that have a known sequence. Double stranded DNA is melting in a heating step exposing two single strands to which the primer can anneal. For the final stage, study of genetic variability is performing in order to differentiate between

In adition to sequencing, there are several strategies which allow study of genetic variability in PCR products: Restriction fragment length polymorphism (RFLP) (93, 162); Terminal

sequencing, RFLP analysis or nested PCR with group-specific primers (117).

demonstrated (72).

colonize the stem and branches in spring (149, 150, 58).

**1.2 Laboratory diagnostic of phytoplasmas** 

research and classification purposes (18, 26).

gene sequences from different phytoplasma.

years are now used routinely and are adequate for detecting phytoplasma infection in plant propagation material and identifying insect vectors, thus helping in preventing the spread of the diseases and their economical impact.

Therefore, aim of this chapter is to provide an overview of the PCR-based techniques for detection, identification and characterisation of this plant-pathogenic *Mollicutes* (cell wallless prokaryotes).

## **1.1 Relevant features of phytoplasmas**

Phytoplasmas, previosly known as 'Mycoplasma-like organisms' or MLOs, are wall-less bacteria obligate parasites of plant phloem tissue, and of several insect species (Fig. 1). Phytoplasma-type diseases of plants for long time were believed to be caused by viruses considering their infective spreading, symptomatology, and transmission by insects (84, 85, 86, 119, 90). Etiology of these pathogens was explored accidentally by group of Japanese sciences (45). They demonstrated that the causes agent of the yellows-type diseases are wallless prokaryotes related to bacteria, pleomorphic incredibly resembling to mycoplasmas.

Phytoplasmas have diverged from gram-positive bacteria, and belong to the '*Candidatus* Phytoplasma' genus within the Class *Mollicutes* (73). Through evolution the genomes of phytoplasmas became greatly reduced in size and they also lack several biosynthetic pathways for the synthesis of compounds necessary for their survival, and they must obtain those substances from plants and insects in which they are parasites (11) thus they can't be cultured *in vitro* in cell-free media.

Fig. 1. Electron microscopy: of cross sections: A) of the vector leafhopper muscle cells around the midgut; B) sieve tubes of phytoplasmas infecting plants. http://www.jic.ac.uk/staff/saskia-hogenhout/insect.htm

years are now used routinely and are adequate for detecting phytoplasma infection in plant propagation material and identifying insect vectors, thus helping in preventing the spread

Therefore, aim of this chapter is to provide an overview of the PCR-based techniques for detection, identification and characterisation of this plant-pathogenic *Mollicutes* (cell wall-

Phytoplasmas, previosly known as 'Mycoplasma-like organisms' or MLOs, are wall-less bacteria obligate parasites of plant phloem tissue, and of several insect species (Fig. 1). Phytoplasma-type diseases of plants for long time were believed to be caused by viruses considering their infective spreading, symptomatology, and transmission by insects (84, 85, 86, 119, 90). Etiology of these pathogens was explored accidentally by group of Japanese sciences (45). They demonstrated that the causes agent of the yellows-type diseases are wallless prokaryotes related to bacteria, pleomorphic incredibly resembling to mycoplasmas.

Phytoplasmas have diverged from gram-positive bacteria, and belong to the '*Candidatus* Phytoplasma' genus within the Class *Mollicutes* (73). Through evolution the genomes of phytoplasmas became greatly reduced in size and they also lack several biosynthetic pathways for the synthesis of compounds necessary for their survival, and they must obtain those substances from plants and insects in which they are parasites (11) thus they can't be

Fig. 1. Electron microscopy: of cross sections: A) of the vector leafhopper muscle cells

around the midgut; B) sieve tubes of phytoplasmas infecting plants.

http://www.jic.ac.uk/staff/saskia-hogenhout/insect.htm

of the diseases and their economical impact.

**1.1 Relevant features of phytoplasmas** 

cultured *in vitro* in cell-free media.

less prokaryotes).

Not all plant species infected with phytoplasmas have disease symptoms, but infected plants normally show symptoms such as virescence, phyllody, yellowing, witches' broom, leaf rool and generalized decline (19). The most common symptoms of the infected plants are yellowing caused by the breakdown of chlorophyll and carotenoids, whose biosynthesis is also inhibited (21). Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants grown in the field was also recently demonstrated (72).

Phytoplasmas are mainly spread by insects of the families *Cicadellidae* (leafhoppers), *Fulgoridae* (planthoppers), and *Psyllidae*, which feed on the phloem tissues of infected plants acquiring the phytoplasmas and transmitting them to the next plant they feed on (136, 2). They enter the insect's body through the stylet and then move through the intestine and been absorbed into the haemolymph. From here they proceeded to colonize the salivary glands, a process that can take up to some weeks (5, 80). Another pathway of phytoplasma survival and transmission is vegetative propagating plant material. As it mentioned phytoplasma invading phloem tissue and it is mostly find that in woody plants they disappear from aerial parts of trees during the winter and survive in the root system to recolonize the stem and branches in spring (149, 150, 58).

#### **1.2 Laboratory diagnostic of phytoplasmas**

In time when phytoplasmas were discovered as plant pathogens diagnostic was difficult since detection was based on symptoms observation insect or dodder/graft transmission to host plant and electron microscopy of ultra-thin sections of the phloem tissue. Serological diagnostic techniques for the detection of phytoplasma began to emerge in the 1980's with ELISA based methods. However, serological methods weren't always sensitive enough to detect various phytoplasmas (13, 47). Finally, in the early 1990's PCR coupled with RFLP analysis allowed the accurate identification of different strains and species of phytoplasma (127, 91, 145). Nowadays, diagnosis of phytoplasmas is routinely done by PCR and can be divided into three phases: total DNA extraction from symptomatic tissue or insects; PCR amplification of phytoplasma-specific DNA; characterization of the amplified DNA by sequencing, RFLP analysis or nested PCR with group-specific primers (117).

For the DNA extraction of known phytoplasma, several protocols for isolation from infected plant material and insects have been developed. Control samples are drowning from plants commonly infected by phytoplasmas. Reference phytoplasma strain collections are maintained in experimentally infected periwinkle (*Catharanthus roseus*) which is available for research and classification purposes (18, 26).

In the second stage of the testing, DNA extracted from plants or insects is amplifying by using the polymerase chain reaction or PCR. PCR is a standardised technique in gene analysis to provide sufficient genetic material for detection (153). It works through the use of short lengths of DNA called primers that have a known sequence. Double stranded DNA is melting in a heating step exposing two single strands to which the primer can anneal. For the final stage, study of genetic variability is performing in order to differentiate between gene sequences from different phytoplasma.

In adition to sequencing, there are several strategies which allow study of genetic variability in PCR products: Restriction fragment length polymorphism (RFLP) (93, 162); Terminal

Polymerase Chain Reaction for Phytoplasmas Detection 95

Prior to start extraction from collected plant samples, leaf midribs and/or phloem shaves are preparing for homogenization. Homogenization in liquid nitrogen with mortar and pestles is the most used method although some automatic homogenizers such as Fast Prep (MP Biomedicals, USA) (137) and Homex 6 (Bioreba, Switzerland) (52, 131) are available as

Accuracy of molecular analysis for pathogen detection in plant material requires efficient and reproducible methods to access nucleic acids. The preparation of samples is critical and target DNA should be made as available as possible for applying the different molecular techniques. However the suitability of most of the molecular methods depends closely on the amount of phytoplasma cells or nucleic acid in the extract. Approximately, 1% of phytoplasma DNA is extracted from tissue of total DNA (20). Since the concentration of this phloem-inhabiting pathogens is subjected to significant variations according to season (151), and is very low especially in woody hosts (79, 88), the importance of obtaining phytoplasma

There are a great many published methods for preparing the plant tissues or other type of samples before molecular detection of phytoplasmas; however, they all pursue access the nucleic acid, avoiding the presence of inhibitory compounds that compromise the detection systems. Target sequences are usually purified or treated to remove DNA polymerase inhibitors, such as polysaccharides, phenolic compounds or humic substances from plants

Depending on the material to be analyzed the extraction methods can be quite simple or more complex. Generally there are three main approaches for obtaining of DNA template: protocols including a phytoplasma enrichment step, CTAB (cetyltrimethylammonium

Phytoplasma enrichment extraction protocols (1, 138, 108) including preparation of plant extract in the phytoplasma enrichment buffer (PGB), after one or two centrifugations the obtained pellet is dissolving in the CTAB buffer following chloroform and/or phenol

Simple laboratory protocols based on preparation of plant extract in CTAB-buffer have also been published by several authors (35, 46, 6, 106, 165, 120, 152) with few steps and minimal handling, reducing the risk of cross contamination, cost and time, with similar results to

CTAB based-protocols were also adopted for extraction of phytoplasmas DNA from

The use of commercial kits, either general or specifically designed for plant material or for insect individuals, in some cases with magnetic separation has gained acceptance for extraction, given the ease of use and avoidance of toxic reagents during the purification process. Among those: DNeasy Plant kits, Qiagen (52, 42); Genomic DNA Purification kit,

DNA at a concentration and purity high enough for precise analysis is aparent.

bromide) buffer-extraction protocols and DNA extraction using commercial kits.

**3. Preparation of DNA templates** 

**3.1 Samples preparation for homogenization** 

faster alternative for the standard method.

extraction and precipitation in isopropanol.

those of longer and more expensive protocols.

hemipterian vectors (107, 46, 116, 50, 51).

**3.2 DNA extraction** 

(121, 63, 164, 122).

restriction fragment length polymorphism (T-RFLP) (66); Heteroduplex Mobility Assays (HMAs) (160); Single Strand Conformation Polymorphisms (SSCP) (126).

Alternative diagnostic methods have been established such as real-time PCR (12, 71, 161) and recently developed method for rapid detection of several phytoplasma species called loop-mediated isothermal amplification (LAMP) (155, 68).

## **2. Sampling procedure**

Quality of DNA is of key importance in molecular diagnostics, since it can affect the final result. On other hand, for preparations of good quality and enriched in phytoplasma DNA, sampling material is of essential importance. Nevertheless, the quality of DNA depends on which plant tissue is examined.

## **2.1 Sampling of plants**

It is generally more accurate sampling in the growing season, and although it can be used in the dormant season, this is not appropriate for the plant health inspections under the certification scheme. Due to the seasonal variation the optimal time for the diagnosis of phytoplasmas is from June to late autumn (30). Phytoplasmas could be detected using the polymerase chain reaction (PCR) from leaf midribs or phloem shaves from shoots, cordons, trunks and roots (117). Phytoplasmas were not always detected in samples from the same sampling area, from one sampling period to the next, firstly due to the uneven distribution, seasonal movement. Having this in mind, when collecting samples the best is to take leaves from different part of plant if it is possible symptomatic one, total amount should be around 20 g. If symptoms are absent phytoplasma detection by PCR can be improved by sampling from shoots, cordons and trunks, especially during October or early spring. In this case the best is to sample roots near to the plant bases though small feeding roots are the best tissue for extraction. Sampling of dry and rotted plant parts is not recommended since phytoplasmas are obligatory parasites. Palmano (2001) (134) demonstrated importance of proper identification of plant parts sampling; in this case the leaves have to show obvious symptoms but without being necrotic or completely yellow. In addition, variance in phytoplasma titters between infected plants of the same species has been observed by Berges et al. (2000) (15) and may be caused by different stages of development and age of plants.

It is recommended to record sampling area and plants by GPS device taking the coordinates and keep samples on cold (4 °C) till laboratory delivery.

#### **2.2 Sampling of insects**

Collection of the insect vectors for phytoplasma PCR analyses should be done in period where insects carry phytoplasma, furthermore knowledge about insects host plants and habitats are crucial things for successful collection.

Different traps and sampling techniques can be applied to collect and monitor phytoplasma vectors according to the objective of the study. The most common trapping techniques are sticky chromotropic traps, emergence traps, sweep net and vacuum insect collectors (107, 40). Collected insects should be place in ethanol and/or frozen.

## **3. Preparation of DNA templates**

## **3.1 Samples preparation for homogenization**

Prior to start extraction from collected plant samples, leaf midribs and/or phloem shaves are preparing for homogenization. Homogenization in liquid nitrogen with mortar and pestles is the most used method although some automatic homogenizers such as Fast Prep (MP Biomedicals, USA) (137) and Homex 6 (Bioreba, Switzerland) (52, 131) are available as faster alternative for the standard method.

#### **3.2 DNA extraction**

94 Polymerase Chain Reaction

restriction fragment length polymorphism (T-RFLP) (66); Heteroduplex Mobility Assays

Alternative diagnostic methods have been established such as real-time PCR (12, 71, 161) and recently developed method for rapid detection of several phytoplasma species called

Quality of DNA is of key importance in molecular diagnostics, since it can affect the final result. On other hand, for preparations of good quality and enriched in phytoplasma DNA, sampling material is of essential importance. Nevertheless, the quality of DNA depends on

It is generally more accurate sampling in the growing season, and although it can be used in the dormant season, this is not appropriate for the plant health inspections under the certification scheme. Due to the seasonal variation the optimal time for the diagnosis of phytoplasmas is from June to late autumn (30). Phytoplasmas could be detected using the polymerase chain reaction (PCR) from leaf midribs or phloem shaves from shoots, cordons, trunks and roots (117). Phytoplasmas were not always detected in samples from the same sampling area, from one sampling period to the next, firstly due to the uneven distribution, seasonal movement. Having this in mind, when collecting samples the best is to take leaves from different part of plant if it is possible symptomatic one, total amount should be around 20 g. If symptoms are absent phytoplasma detection by PCR can be improved by sampling from shoots, cordons and trunks, especially during October or early spring. In this case the best is to sample roots near to the plant bases though small feeding roots are the best tissue for extraction. Sampling of dry and rotted plant parts is not recommended since phytoplasmas are obligatory parasites. Palmano (2001) (134) demonstrated importance of proper identification of plant parts sampling; in this case the leaves have to show obvious symptoms but without being necrotic or completely yellow. In addition, variance in phytoplasma titters between infected plants of the same species has been observed by Berges et al. (2000) (15) and may be caused by different stages of development and age of

It is recommended to record sampling area and plants by GPS device taking the coordinates

Collection of the insect vectors for phytoplasma PCR analyses should be done in period where insects carry phytoplasma, furthermore knowledge about insects host plants and

Different traps and sampling techniques can be applied to collect and monitor phytoplasma vectors according to the objective of the study. The most common trapping techniques are sticky chromotropic traps, emergence traps, sweep net and vacuum insect collectors (107,

(HMAs) (160); Single Strand Conformation Polymorphisms (SSCP) (126).

loop-mediated isothermal amplification (LAMP) (155, 68).

and keep samples on cold (4 °C) till laboratory delivery.

habitats are crucial things for successful collection.

40). Collected insects should be place in ethanol and/or frozen.

**2. Sampling procedure** 

which plant tissue is examined.

**2.1 Sampling of plants** 

plants.

**2.2 Sampling of insects** 

Accuracy of molecular analysis for pathogen detection in plant material requires efficient and reproducible methods to access nucleic acids. The preparation of samples is critical and target DNA should be made as available as possible for applying the different molecular techniques. However the suitability of most of the molecular methods depends closely on the amount of phytoplasma cells or nucleic acid in the extract. Approximately, 1% of phytoplasma DNA is extracted from tissue of total DNA (20). Since the concentration of this phloem-inhabiting pathogens is subjected to significant variations according to season (151), and is very low especially in woody hosts (79, 88), the importance of obtaining phytoplasma DNA at a concentration and purity high enough for precise analysis is aparent.

There are a great many published methods for preparing the plant tissues or other type of samples before molecular detection of phytoplasmas; however, they all pursue access the nucleic acid, avoiding the presence of inhibitory compounds that compromise the detection systems. Target sequences are usually purified or treated to remove DNA polymerase inhibitors, such as polysaccharides, phenolic compounds or humic substances from plants (121, 63, 164, 122).

Depending on the material to be analyzed the extraction methods can be quite simple or more complex. Generally there are three main approaches for obtaining of DNA template: protocols including a phytoplasma enrichment step, CTAB (cetyltrimethylammonium bromide) buffer-extraction protocols and DNA extraction using commercial kits.

Phytoplasma enrichment extraction protocols (1, 138, 108) including preparation of plant extract in the phytoplasma enrichment buffer (PGB), after one or two centrifugations the obtained pellet is dissolving in the CTAB buffer following chloroform and/or phenol extraction and precipitation in isopropanol.

Simple laboratory protocols based on preparation of plant extract in CTAB-buffer have also been published by several authors (35, 46, 6, 106, 165, 120, 152) with few steps and minimal handling, reducing the risk of cross contamination, cost and time, with similar results to those of longer and more expensive protocols.

CTAB based-protocols were also adopted for extraction of phytoplasmas DNA from hemipterian vectors (107, 46, 116, 50, 51).

The use of commercial kits, either general or specifically designed for plant material or for insect individuals, in some cases with magnetic separation has gained acceptance for extraction, given the ease of use and avoidance of toxic reagents during the purification process. Among those: DNeasy Plant kits, Qiagen (52, 42); Genomic DNA Purification kit,

Polymerase Chain Reaction for Phytoplasmas Detection 97

sequence. These differences can be compared and used as a diagnostic test for a particular phytoplasma. Phytoplasma diagnostics has been routinely based on phytoplasma-specific universal (generic) (Table 1) or phytoplasma group specific (Table 2) Polymerase Chain Reaction (PCR) primers designed on the basis of the highly conserved 16S ribosomal RNA (rRNA) gene sequences (1, 38, 44, 61, 77, 144, 153). Nevertheless, to detect phytoplasmas in DNA samples universal phytoplasma primers designed on sequences of the 16S-23S rRNA

Nested-PCR is performing by preliminary amplification using a universal primers pair followed by second amplification using a second universal primer pair. By using a universal primer pair followed by PCR using a group specific primer pair, nested-PCR is capable of detection of dual or multiple phytoplasmas present in the infected tissues in case of mixed infection (92). Until the reliability of universal primers detecting phytoplasmas is determined, it is advisable to use at least 2 different primer pairs to test a sample (eg P1/P7 (44) and R16F2/R16R2 (91); 6F/7R (146) and fU5/rU3 (102). Unfortunately, some of the primers can induce dimers or unspecific bands. They also have sequence homology in the 16S-spacer region to chloroplasts and plastids increasing the risk of false positives (64). Therefore, more specific universal phytoplasma primers are currently being developed (66,

**Reaction References** 

Nested PCR

Nested PCR

PCR

PCR

(153)

(91) (55)

(38) (133)

(67)

112) and it may be that these will be more suitable for diagnostics from samples.

16S 1050 bp semi-nested

sec A gene 480 bp semi-nested

elongation factor Tu) (Table 2) (56, 67, 109, 147, 87,).

Table 1. PCR universal primers commonly used for the detection of phytoplasma

16S/23SR 1800 bp Direct PCR (44)

16S/23 1700 bp Direct PCR (146)

16S 880 bp Nested PCR (102)

secA gene 840 bp Direct PCR (67)

Phytoplasma group-specific primers have also been designed on ribosomal protein gene, SecA, SecY genes (coding for the translocase protein) (28, 98), *vmp*1 gene (stolbur phytoplasma membrane protein) (28), *imp* gene (coding immunodominant membrane protein (112, 36), non-ribosomal gene *aceF* (115) and *tuf* gen (encoding the translation

**length** 

1245 bp

1240 bp

spacer region (SR) (153) are generally using.

**Primer set Location PCR product** 

16S/IS

16S/IS

P1 P7

F1 B6

6F 7R

fU3 fU5

SecAfor 1 SecArev 3

SecAfor 2 SecArev 3

R16F2 R16R2 R16F2n R6R2

Fermentas (143, 77); High Pure PCR Template Preparation kit, Roche (132); Wizard Genomic DNA Purification kit, Promega (104); NucleoSpin PlantII kit, Macherey-Nagel (135); FastDNA spin kit MP, Biomedicals (10); while InviMag Plant DNA Mini kit, Invitek; and QuickPick Plant DNA kit, Bio Nobile are optimized for extraction with a King Fisher mL Thermo Science workstation (137, 24, 99, 41).

Recently a new method (LFD) (37, 155) has been developed for rapid DNA extraction which processing DNA in loop-mediated isothermal amplification (LAMP) procedure for the detection of phytoplasmas from infected plant material. LFD method allows DNA extraction from leaf and wood material just in two minutes. Plant extract prepared in commercial buffer supplied with the LFD (Forsite Diagnostics Ltd) commercial kit is placing onto LFD membranes of lateral flow devices, and small sections of these membranes are then adding directly into the LAMP reaction mixture and incubating for 45 min at 65 °C. Moreover, Hodgetts et al. (2011) (68) obtained also satisfied results with LAMP using DNA prepared with an alkaline polyethylene glycol (PEG). This DNA extraction method (31) involves gently maceration of a small amount of plant tissue in the PEG buffer and then transfer of the macerate to the LAMP reaction.

Nevertheless, the choice of one or another system for nucleic acid extraction relies in practice on the phytoplasma to be detected and the nature of the sample, the experience of the personnel, the number of analyses to be performed per day, and the type of technique. As there are no universally validated nucleic-acid extraction protocols for all kinds of material and phytoplasma pathogens, those available should be compared before selecting one method for routine.

## **4. Nucleic acid amplification method**

Detection and identification of phytoplasmas is necessary for accurate disease diagnosis. Sensitive methods need to be implemented in order to monitor the presence and spread of phytoplasma infections. Hence, it is necessary to devise a rapid, effective and efficient mechanism for detecting and identifying these microorganisms. Molecular diagnostic techniques for the detection of phytoplasma introduced during the last two decades have proven to be more accurate and reliable than biological criteria long used for phytoplasma identification (95). Polymerase Chain Reaction (PCR) is the most versatile tool for detecting phytoplasmas in their plant and insect hosts (153). One of the most utilized protocols for phytoplasma detection and characterization encompasses nested-PCR and RFLP analyses.

## **4.1 Nested PCR**

Nested-PCR assay, designed to increase both sensitivity and specificity, is the leading method for the amplification of phytoplasmas from samples in which unusually low titer, or inhibitors are present that may interfere the PCR efficacy (56). The use of nested-PCR has been reported for diagnostic purposes particularly in plants when phytoplasmas occur in low titer in the phloem vessels of their host-plants and their concentration may be subjected to seasonal fluctuation (57, 75, 100, 117).

DNA consists of long sequences of paired bases called genes which code for a particular trait. Some of these gene sequences are consistent across bacteria but vary in their detailed

Fermentas (143, 77); High Pure PCR Template Preparation kit, Roche (132); Wizard Genomic DNA Purification kit, Promega (104); NucleoSpin PlantII kit, Macherey-Nagel (135); FastDNA spin kit MP, Biomedicals (10); while InviMag Plant DNA Mini kit, Invitek; and QuickPick Plant DNA kit, Bio Nobile are optimized for extraction with a King Fisher mL

Recently a new method (LFD) (37, 155) has been developed for rapid DNA extraction which processing DNA in loop-mediated isothermal amplification (LAMP) procedure for the detection of phytoplasmas from infected plant material. LFD method allows DNA extraction from leaf and wood material just in two minutes. Plant extract prepared in commercial buffer supplied with the LFD (Forsite Diagnostics Ltd) commercial kit is placing onto LFD membranes of lateral flow devices, and small sections of these membranes are then adding directly into the LAMP reaction mixture and incubating for 45 min at 65 °C. Moreover, Hodgetts et al. (2011) (68) obtained also satisfied results with LAMP using DNA prepared with an alkaline polyethylene glycol (PEG). This DNA extraction method (31) involves gently maceration of a small amount of plant tissue in the PEG buffer and then transfer of

Nevertheless, the choice of one or another system for nucleic acid extraction relies in practice on the phytoplasma to be detected and the nature of the sample, the experience of the personnel, the number of analyses to be performed per day, and the type of technique. As there are no universally validated nucleic-acid extraction protocols for all kinds of material and phytoplasma pathogens, those available should be compared before selecting

Detection and identification of phytoplasmas is necessary for accurate disease diagnosis. Sensitive methods need to be implemented in order to monitor the presence and spread of phytoplasma infections. Hence, it is necessary to devise a rapid, effective and efficient mechanism for detecting and identifying these microorganisms. Molecular diagnostic techniques for the detection of phytoplasma introduced during the last two decades have proven to be more accurate and reliable than biological criteria long used for phytoplasma identification (95). Polymerase Chain Reaction (PCR) is the most versatile tool for detecting phytoplasmas in their plant and insect hosts (153). One of the most utilized protocols for phytoplasma detection and characterization encompasses nested-PCR and RFLP analyses.

Nested-PCR assay, designed to increase both sensitivity and specificity, is the leading method for the amplification of phytoplasmas from samples in which unusually low titer, or inhibitors are present that may interfere the PCR efficacy (56). The use of nested-PCR has been reported for diagnostic purposes particularly in plants when phytoplasmas occur in low titer in the phloem vessels of their host-plants and their concentration may be subjected

DNA consists of long sequences of paired bases called genes which code for a particular trait. Some of these gene sequences are consistent across bacteria but vary in their detailed

Thermo Science workstation (137, 24, 99, 41).

the macerate to the LAMP reaction.

**4. Nucleic acid amplification method** 

to seasonal fluctuation (57, 75, 100, 117).

one method for routine.

**4.1 Nested PCR** 

sequence. These differences can be compared and used as a diagnostic test for a particular phytoplasma. Phytoplasma diagnostics has been routinely based on phytoplasma-specific universal (generic) (Table 1) or phytoplasma group specific (Table 2) Polymerase Chain Reaction (PCR) primers designed on the basis of the highly conserved 16S ribosomal RNA (rRNA) gene sequences (1, 38, 44, 61, 77, 144, 153). Nevertheless, to detect phytoplasmas in DNA samples universal phytoplasma primers designed on sequences of the 16S-23S rRNA spacer region (SR) (153) are generally using.

Nested-PCR is performing by preliminary amplification using a universal primers pair followed by second amplification using a second universal primer pair. By using a universal primer pair followed by PCR using a group specific primer pair, nested-PCR is capable of detection of dual or multiple phytoplasmas present in the infected tissues in case of mixed infection (92). Until the reliability of universal primers detecting phytoplasmas is determined, it is advisable to use at least 2 different primer pairs to test a sample (eg P1/P7 (44) and R16F2/R16R2 (91); 6F/7R (146) and fU5/rU3 (102). Unfortunately, some of the primers can induce dimers or unspecific bands. They also have sequence homology in the 16S-spacer region to chloroplasts and plastids increasing the risk of false positives (64). Therefore, more specific universal phytoplasma primers are currently being developed (66, 112) and it may be that these will be more suitable for diagnostics from samples.


Table 1. PCR universal primers commonly used for the detection of phytoplasma

Phytoplasma group-specific primers have also been designed on ribosomal protein gene, SecA, SecY genes (coding for the translocase protein) (28, 98), *vmp*1 gene (stolbur phytoplasma membrane protein) (28), *imp* gene (coding immunodominant membrane protein (112, 36), non-ribosomal gene *aceF* (115) and *tuf* gen (encoding the translation elongation factor Tu) (Table 2) (56, 67, 109, 147, 87,).

Polymerase Chain Reaction for Phytoplasmas Detection 99

The search for phytoplasma-specific primers has led to evaluation of primers based on these regions appears to offer more variation than that of the 16S gene. Nevertheless, design of primers based on various conserved sequences such as 16S rRNA gene, ribosomal protein gene operon, *tuf* and *SecY* genes was the major breakthrough in detection, identification,

Primers previously designed for specific amplification of DNA from stolbur phytoplasma were recently found to prime amplification of DNA from other phytoplasmas (39, 77); therefore, it may be advisable to supplement use of phytoplasma-specific primers with

The choice of primer sets for phytoplasma diagnosis by nested PCR mostly depends on the phytoplasma we are looking for. Nested-PCR with a combination of different universal primers (Table 1) can improve the diagnosis of unknown phytoplasmas present with low titter in the symptomatic host. Universal ribosomal primers followed with nested with group-specific primers (Table 2) are extremely useful when the phytoplasma to be

PCR products are usually visualised on 1% agarose gel prepared in 1xTAE buffer, stained

The efficiency of nested-PCR has shown that it can reamplify the direct PCR product in dilution of 1: 60 000 (81). However, the system has not yet been devised to identify all the taxonomic groups, and this approach requires more than one PCR step, increasing the chances of contamination between samples, and does not provide the rapid and simple

For identification of all detected phytoplasmas as well as for molecular characterisation of certain phytoplasmas strains Restriction Fragment Length Polymorphism, or RFLP is commonly used. RFLP is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory

Phytoplasma amplified PCR products are cutting into fragments at specific sites using enzymes. More specific detection methods involve using phytoplasma-specific primers or differentiation on the basis of phylogenetic RFLP analysis of PCR amplified sequences (91, 145). RFLP analysis of PCR amplified DNA sequences using a number of endonuclease restriction enzymes (93). The pattern of cut DNA is viewing using 5% polyacrilamid gel (95) or 2,5% to 3% agarose gel electrophoresis. Analysis of a known genomic sequence can show what size of fragments to expect depending upon the enzymes chosen for the cuts e.g., providing that 6 or more frequently cutting restriction enzymes are used in the RFLP

Moreover this analysis is very useful for identification of new phytoplasmas, or phytoplasmas from a poorly studied region or crop. Because the RFLP patterns characteristics of each phytoplasmas are conserved, unknown phytoplasmas can be identified by comparing the patterns of the unknown with the available RFLP patterns for

and classification of phytoplasmas (57, 147, 109, 161, 111, 112).

diagnosed belongs to a well-defined taxonomic group (117).

**4.1.1 Restriction fragment length polymorphism (RFLP)** 

technique by which these segments can be illustrated.

analysis, specific identification of the phytoplasma may be obtained.

RFLP analysis of amplified DNA sequences.

with ethidium bromide (40).

diagnostic tool required.


Table 2. Several group specific primers used for phytoplasma detection

**Primer set Specificity Location Expected size of PCR product References** 

16SrI Ribosomal

16SrII Ribosomal

16SrIII Ribosomal

16SrV Ribosomal

16SrVI Ribosomal

16SrIX Ribosomal

16SrXII-A Ribosomal

16SrX-A Ribosomal

16SrVII, 16SrVIII protein

protein

protein

protein

protein

protein

protein

protein

Table 2. Several group specific primers used for phytoplasma detection

Ribosomal protein

16SrI tuf gene 940 bp (147)

16SrI secY gene 1400 bp (98)

16SrIV 16S 1400 bp (62)

16SrIV nonribosomal 1000 bp (60)

16SrX-A nonribosomal 776 bp (27)

16SrX 16S 1100 bp (102)

16SrX aceF 500 bp (115)

16SrV secY 1300 (35)

16SrV secY 1300 bp (29)

16SrXII secY 990 bp (35)

16SrXII secY 720 bp (29)

16SrXII-A 16S/SR 570 bp (106)

16S 16SrV 300 bp (1)

1200 bp (96)

1200 bp (112)

1200 bp (112)

1200 bp (97)

1000 bp (112)

1000 bp (112)

800 bp (112)

1372 bp (112)

1000 bp (114)

(6)

fTufAy rTufAy

AysecYF1 AysecYR1

rp(I)F1A rp(I)R1A

rp(II)F1 rp(II)R1

rp(III)F1 rp(III)R1

LY 16Sf LY16Sr

LYC24F LYC24R

rp(V)F1A rp(V)R1A

rp(VI)F2 rp(VI)R2

rp(VIII)F2 rp(VIII)R2

rp(IX)F2 rp(IX)R2

rpStolIF rpStolIR

rpAP15f rp/AP15r

f01 r01

FD9R FD9F

FD9F3b FD9R2

STOL11R1 STOL11F2

STOL11R2 STOL11F3

fStol rStol

fAY rEY

AP13/AP10 AP14/AP15

AceFf1/AceFr1 AceFf2/AceFr2 The search for phytoplasma-specific primers has led to evaluation of primers based on these regions appears to offer more variation than that of the 16S gene. Nevertheless, design of primers based on various conserved sequences such as 16S rRNA gene, ribosomal protein gene operon, *tuf* and *SecY* genes was the major breakthrough in detection, identification, and classification of phytoplasmas (57, 147, 109, 161, 111, 112).

Primers previously designed for specific amplification of DNA from stolbur phytoplasma were recently found to prime amplification of DNA from other phytoplasmas (39, 77); therefore, it may be advisable to supplement use of phytoplasma-specific primers with RFLP analysis of amplified DNA sequences.

The choice of primer sets for phytoplasma diagnosis by nested PCR mostly depends on the phytoplasma we are looking for. Nested-PCR with a combination of different universal primers (Table 1) can improve the diagnosis of unknown phytoplasmas present with low titter in the symptomatic host. Universal ribosomal primers followed with nested with group-specific primers (Table 2) are extremely useful when the phytoplasma to be diagnosed belongs to a well-defined taxonomic group (117).

PCR products are usually visualised on 1% agarose gel prepared in 1xTAE buffer, stained with ethidium bromide (40).

The efficiency of nested-PCR has shown that it can reamplify the direct PCR product in dilution of 1: 60 000 (81). However, the system has not yet been devised to identify all the taxonomic groups, and this approach requires more than one PCR step, increasing the chances of contamination between samples, and does not provide the rapid and simple diagnostic tool required.

## **4.1.1 Restriction fragment length polymorphism (RFLP)**

For identification of all detected phytoplasmas as well as for molecular characterisation of certain phytoplasmas strains Restriction Fragment Length Polymorphism, or RFLP is commonly used. RFLP is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated.

Phytoplasma amplified PCR products are cutting into fragments at specific sites using enzymes. More specific detection methods involve using phytoplasma-specific primers or differentiation on the basis of phylogenetic RFLP analysis of PCR amplified sequences (91, 145). RFLP analysis of PCR amplified DNA sequences using a number of endonuclease restriction enzymes (93). The pattern of cut DNA is viewing using 5% polyacrilamid gel (95) or 2,5% to 3% agarose gel electrophoresis. Analysis of a known genomic sequence can show what size of fragments to expect depending upon the enzymes chosen for the cuts e.g., providing that 6 or more frequently cutting restriction enzymes are used in the RFLP analysis, specific identification of the phytoplasma may be obtained.

Moreover this analysis is very useful for identification of new phytoplasmas, or phytoplasmas from a poorly studied region or crop. Because the RFLP patterns characteristics of each phytoplasmas are conserved, unknown phytoplasmas can be identified by comparing the patterns of the unknown with the available RFLP patterns for

Polymerase Chain Reaction for Phytoplasmas Detection 101

Heteroduplex mobility assay (HMA) has been recently developed as fast and inexpensive method for determining relatedness between phytoplasmas DNA sequences. Initially, it was developed by Delwart et al. (1993) (43) to evaluate viral heterogeneity and for genetic typing

So far, HMA was used in studies for differentiation of phytoplasmas in the aster yellows group and clover proliferation group (159) determination of genetic variability among isolates of Australian grapevine phytoplasmas (32); study of the genetic diversity of 62 phytoplasma isolates from North America, Europe and Asia (160); for phylogenetic relationships among flavescence dorée strains and related phytoplasmas belonging to the elm yellows group (7); and to determine genomic diversity among African isolates of coconut lethal yellowing phytoplasmas causing Cape St. Paul wilt disease (CSPD, Ghana),

Amplified PCR products from positive phytoplasma strains are combining with the amplified products of reference strain mixing with annealing buffer and submitting to HMA analyses (110, 160) following visualization of HMA products on polyacrylamide gel. Heteroduplexes migrate more slowly than a homoduplex in polyacrylamide gel electrophoresis. The extent of the retardation has been shown to be proportional to the degree of divergence between the two DNA sequences. It was noticed, that presence of an unpaired base influence the mobility of a heteroduplex more than a mismatched nucleotide (158, 157). Performing HMA, Marihno et al (2008) (110) succeeded to identified three groups of phytoplasmas associated with various coconut lethal yellowing diseases. Moreover, this grouping was consistent with the genetic diversity described in the coconut yellowing-associated phytoplasmas detected after cloning, sequencing, and

Further optimisations of this approach could facilitate phylogenetic study and diagnosis of many other phytoplasmas and development of a comprehensive PCR-based classification system. Considering simplicity and rapidness of the method, HMA could be used for initial screening among a large number of isolates and rapid identification of phytoplasmas as well

Immuno-capture PCR assay, in which the phytoplasma of interest is first selectively captured by specific antibody adsorbed on microtiter plates, and then the phytoplasma DNA is released and amplified using specific or universal primers, can be an alternative method to increase detection sensitivity (139, 64). This method is aimed at avoiding the lengthy extraction procedures to prepare target DNAs. Nonetheless, this method is not

Since the most universal as well as specific diagnostic protocols rely on nested PCR which, although extremely sensitive, is also time-consuming and posses risk in terms of carry-over

suitable for detection of fruit tree and grapevine phytoplasmas.

lethal disease (LD, Tanzania), and lethal yellowing (LYM, Mozambique) (110).

**4.1.4 Heteroduplex Mobility Assay (HMA)** 

of human immunodeficiency virus (HIV).

phylogenetic analyses.

as other organisms.

**4.3 Real-time PCR** 

**4.2 Immuno-capture PCR** 

known phytoplasmas without co-analyses of all reference representative phytoplasmas (94, 162, 163, 25). In this case it is preferable to use bigger number of enzymes to achieve identification (38). Enzymes found valuable for these analyses include AluI, BamHI, BfaI, DraI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, TaqI and ThaI.

Phytoplasma has not been cultured in cell-free medium, thus cannot be differentiated and classified by the traditional methods which are applied to culturable prokaryotes. The highly conserved 16S rRNA gene sequence has been widely used as the very useful primary molecular tool for preliminary classification of phytoplasmas. A total of 19 distinct groups, termed 16S rRNA groups (16Sr groups), based on actual RFLP analysis of PCR-amplified 16S rDNA sequences or 29 groups based on RFLP with new computer-simulated RFLP *in silico* analysis have been identified (93, 162).

## **4.1.2 Terminal restriction fragment length polymorphism (T-RFLP)**

A protocol based on the Terminal Restriction Fragment Length Polymorphism (T-RLFP) analysis of 23S rDNA sequence using a DNA sequence analysis system has been developed to provide the simultaneous detection and taxonomic grouping of phytoplasmas (66). Terminal-restriction fragment length polymorphism (T-RFLP) analysis is a direct DNA-profiling method that usually targets rRNA (82). This genetic fingerprinting method uses a fluorescently labelled oligonucleotide primer for PCR amplification and the digestion of the PCR products with one or more restriction enzymes. This generates labelled terminal restriction fragments (TRFs) of various lengths depending on the DNA sequence of the bacteria present and the enzyme used to cut the sequence. The results of T-RFLP are obtaining through TRF separation by high-resolution gel electrophoresis on automated DNA sequencers. The laser scanning system of the DNA sequencer detects the labelled primer (141) and from this signal the sequencer can record corresponding fragment sizes and relative abundances. Resulting data is very easy to analyse, being presented as figures for statistical analysis and graphically for rapid visual interpretation.

The method was also designed to allow simple and easy testing of phytoplasmas and at the same time gave indication of their taxonomic group (9, 66). Comparing with the conventional nested-PCR/RFLP, method is less time-consuming and the approach is less expensive than sequencing.

## **4.1.3 Single Strand Conformation Polymorphisms (SSCP)**

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detection of polymorphism in PCR-amplified fragments. SSCP was also assessed for the application in detection of the molecular variability phytoplasmas (125, 126). Amplified phytoplasma regions (16S rDNA, *tuf* gene, and *dnaB* gene), respectively are mixing with denaturing buffer after incubation, results of the SSCP are visualising on a non-denaturing polyacrylamide gel, optimized for each fragment length. SSCP revealed the presence of polymorphism undetected by routine RFLP analyses in all analyzed phytoplasma regions. Advantages of the SSCP in comparison with RFLP are sensitivity, time and cost consumption as well as suitability when large number of samples are screening for molecular variability.

## **4.1.4 Heteroduplex Mobility Assay (HMA)**

100 Polymerase Chain Reaction

known phytoplasmas without co-analyses of all reference representative phytoplasmas (94, 162, 163, 25). In this case it is preferable to use bigger number of enzymes to achieve identification (38). Enzymes found valuable for these analyses include AluI, BamHI, BfaI,

Phytoplasma has not been cultured in cell-free medium, thus cannot be differentiated and classified by the traditional methods which are applied to culturable prokaryotes. The highly conserved 16S rRNA gene sequence has been widely used as the very useful primary molecular tool for preliminary classification of phytoplasmas. A total of 19 distinct groups, termed 16S rRNA groups (16Sr groups), based on actual RFLP analysis of PCR-amplified 16S rDNA sequences or 29 groups based on RFLP with new computer-simulated RFLP *in* 

A protocol based on the Terminal Restriction Fragment Length Polymorphism (T-RLFP) analysis of 23S rDNA sequence using a DNA sequence analysis system has been developed to provide the simultaneous detection and taxonomic grouping of phytoplasmas (66). Terminal-restriction fragment length polymorphism (T-RFLP) analysis is a direct DNA-profiling method that usually targets rRNA (82). This genetic fingerprinting method uses a fluorescently labelled oligonucleotide primer for PCR amplification and the digestion of the PCR products with one or more restriction enzymes. This generates labelled terminal restriction fragments (TRFs) of various lengths depending on the DNA sequence of the bacteria present and the enzyme used to cut the sequence. The results of T-RFLP are obtaining through TRF separation by high-resolution gel electrophoresis on automated DNA sequencers. The laser scanning system of the DNA sequencer detects the labelled primer (141) and from this signal the sequencer can record corresponding fragment sizes and relative abundances. Resulting data is very easy to analyse, being presented as figures for statistical analysis and graphically for rapid visual

The method was also designed to allow simple and easy testing of phytoplasmas and at the same time gave indication of their taxonomic group (9, 66). Comparing with the conventional nested-PCR/RFLP, method is less time-consuming and the approach is less

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detection of polymorphism in PCR-amplified fragments. SSCP was also assessed for the application in detection of the molecular variability phytoplasmas (125, 126). Amplified phytoplasma regions (16S rDNA, *tuf* gene, and *dnaB* gene), respectively are mixing with denaturing buffer after incubation, results of the SSCP are visualising on a non-denaturing polyacrylamide gel, optimized for each fragment length. SSCP revealed the presence of polymorphism undetected by routine RFLP analyses in all analyzed phytoplasma regions. Advantages of the SSCP in comparison with RFLP are sensitivity, time and cost consumption as well as suitability when large number of samples are screening for

DraI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, TaqI and ThaI.

**4.1.2 Terminal restriction fragment length polymorphism (T-RFLP)** 

**4.1.3 Single Strand Conformation Polymorphisms (SSCP)** 

*silico* analysis have been identified (93, 162).

interpretation.

expensive than sequencing.

molecular variability.

Heteroduplex mobility assay (HMA) has been recently developed as fast and inexpensive method for determining relatedness between phytoplasmas DNA sequences. Initially, it was developed by Delwart et al. (1993) (43) to evaluate viral heterogeneity and for genetic typing of human immunodeficiency virus (HIV).

So far, HMA was used in studies for differentiation of phytoplasmas in the aster yellows group and clover proliferation group (159) determination of genetic variability among isolates of Australian grapevine phytoplasmas (32); study of the genetic diversity of 62 phytoplasma isolates from North America, Europe and Asia (160); for phylogenetic relationships among flavescence dorée strains and related phytoplasmas belonging to the elm yellows group (7); and to determine genomic diversity among African isolates of coconut lethal yellowing phytoplasmas causing Cape St. Paul wilt disease (CSPD, Ghana), lethal disease (LD, Tanzania), and lethal yellowing (LYM, Mozambique) (110).

Amplified PCR products from positive phytoplasma strains are combining with the amplified products of reference strain mixing with annealing buffer and submitting to HMA analyses (110, 160) following visualization of HMA products on polyacrylamide gel. Heteroduplexes migrate more slowly than a homoduplex in polyacrylamide gel electrophoresis. The extent of the retardation has been shown to be proportional to the degree of divergence between the two DNA sequences. It was noticed, that presence of an unpaired base influence the mobility of a heteroduplex more than a mismatched nucleotide (158, 157). Performing HMA, Marihno et al (2008) (110) succeeded to identified three groups of phytoplasmas associated with various coconut lethal yellowing diseases. Moreover, this grouping was consistent with the genetic diversity described in the coconut yellowing-associated phytoplasmas detected after cloning, sequencing, and phylogenetic analyses.

Further optimisations of this approach could facilitate phylogenetic study and diagnosis of many other phytoplasmas and development of a comprehensive PCR-based classification system. Considering simplicity and rapidness of the method, HMA could be used for initial screening among a large number of isolates and rapid identification of phytoplasmas as well as other organisms.

## **4.2 Immuno-capture PCR**

Immuno-capture PCR assay, in which the phytoplasma of interest is first selectively captured by specific antibody adsorbed on microtiter plates, and then the phytoplasma DNA is released and amplified using specific or universal primers, can be an alternative method to increase detection sensitivity (139, 64). This method is aimed at avoiding the lengthy extraction procedures to prepare target DNAs. Nonetheless, this method is not suitable for detection of fruit tree and grapevine phytoplasmas.

#### **4.3 Real-time PCR**

Since the most universal as well as specific diagnostic protocols rely on nested PCR which, although extremely sensitive, is also time-consuming and posses risk in terms of carry-over

Polymerase Chain Reaction for Phytoplasmas Detection 103

mismatched (83, 128). Furthermore, applying the same protocols, phytoplasmas DNA could be also detected in insect samples (113, 76, 48, 71) what is also decisive in the search for

16S rDNA (34)

Table 3. Oligonucleotide primers and probes used for phytoplasma detection by real-time

A well-optimized reaction is essential for accurate results, which must be further analysed. As it is mentioned before, diagnosis of the pathogens in woody plants is often hampered by the presence of PCR inhibitors such as polyphenolics, polysaccharides and other molecules that may produce false negative results even from heavily infected samples. Additional problem may be also caused by amplification of other bacteria with universal phytoplasma primers/probe which could be present on the surface of some plants (49). Therefore, to avoid false positives specific probe can be included. So far, several sequence-specific detection tools are available: the chloroplast chaperonin 21 gene (8); cytochrome oxidase gene (71); the chloroplast gene for tRNA leucine (12); and the 18S rDNA gene (30, 118, 113,

**Specificity Target gene References** 

Universal 16S rDNA (30) Universal 16S rDNA (48) Universal 16S rDNA (71) FD 16S rDNA (48) FD 16S rDNA (8) FD Sec Y (71) FD 16S rDNA (22) BN Genomic fragment (48) BN 16S rDNA (8) BN Genomic fragment (71) AP Nitro reductase (48) AP Genomic fragment (76) AP 16S rDNA (12) AP 16S rDNA (4) AP 16S rDNA (23) AP 16S–23S rRNA (128) PD 16S–23S rRNA (128) ESFY 16S–23S rRNA (128) ESFY Ribosomal protein (113) 'Ca. P. asteris'(onion yellows) tuf (161) 'Ca. P. asteris'(aster yellows) 16S rDNA (8) 'Ca. P. asteris'(aster yellows) 16S rDNA (69)

other potential vectors.

Beet leafhopper transmitted

virescence virus

PCR

contamination between the two rounds of amplification, real-time PCR has recently replaced the traditional PCR in efforts to increase the speed and sensitivity of detection for mass screening.

The main principle of real-time PCR is based on fluorescent chemistries for labelling of the amplicons. During a real-time PCR run, accumulation of newly generated amplicons is monitored by each cycle by fluorescent detection methods, and so there is no need for post-PCR manipulation such as electrophoresis, which is required at the end of regular PCR. Moreover, the amount of fluorescent, monitored at each cycle is proportional to the log of concentration of the PCR target, and for this reason real-time PCR is also powerful technique for quantification of specific DNA. There are several labelling techniques, most of which specially bind to a target sequence on the amplicon, while others aspecifically stain double-stranded (ds) DNA amplicons. In addition, numbers of protocols have been developed for real-time PCR universal and specific detection phytoplasma.

For preliminary screening, 16S rDNA gene were adapted for the universal diagnosis of phytoplasmas using direct real-time PCR amplification (30, 48, 71) (Table 3) and all of them exploited a TaqMan probe for detection. TaqMan probes are labelled at the 5'end with reporter dye and at the 3' end with a quenching molecule; during each PCR cycle in the presence of the specific target DNA, the TaqMan probe, bound to its target sequence, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase as it extend the primer. The fluorescence moiety of the probe is therefore freed from its quencher-labelled portion and the fluorescence is detected by the optical system of the apparatus. The sensitivity of the 16S rDNA-based primer/probe system can be used to detect phytoplasmas belonging to several ribosomal subgroups and they showed sensitivity similar to that of conventional nested-PCR.

Group specific phytoplasma primers and probes for real-time PCR system have been designed to overcome problem with the time-consuming methods for phytoplasma strains identification and to further enhance the specificity of detection. Several laboratories have proposed rapid, specific and sensitive diagnostic protocols for detection of quarantine and economically important phytoplasmas of fruit trees and grapevine such as flavescance dorée (FD) and bois noir (BN) phytoplasmas infecting grapevine (22, 48, 8, 53, 71, 14); '*Ca*. Phytoplasma mali' (apple proliferation, AP), '*Ca*. Phytoplasma pyri' (pear decline, PD), '*Ca*. Phytoplasma pruni' (European stone fruit yellows, ESFY) important pathogens of fruit trees (12, 76, 48, 156, 3,4, 113, 23, 128, 41). Most of the primer/probe systems are targeting 16S rDNA gene though some others genes or even randomly cloned DNA fragments to which no specific function is assigned have been used (Table 3). For fluorescent detection SYBR Green I has been applied for the diagnosis of AP, PD, ESFY and FD, all quarantine phytoplasmas of fruit trees and grapevine in Europe. Real-time PCR assays were also developed using TaqMan minor groove binding (MGB) probe to detect AP in plant material (12, 3) as well as for FD, BN and other phytoplasmas less frequently infecting grapevines (71, 128). MGB (minor groove binding) probe has an MGB ligand and non-fluorescent quencher conjugated to the 3' end, plus a fluorescent reporter dye at the 5' end. The MGB ligand allows the use of shorter and more specific probes by increasing the stability of the probe-target bond. This property allows the use of shorter probes, with higher specificity than conventional TaqMan ones and the discrimination of even single nucleotide

contamination between the two rounds of amplification, real-time PCR has recently replaced the traditional PCR in efforts to increase the speed and sensitivity of detection for

The main principle of real-time PCR is based on fluorescent chemistries for labelling of the amplicons. During a real-time PCR run, accumulation of newly generated amplicons is monitored by each cycle by fluorescent detection methods, and so there is no need for post-PCR manipulation such as electrophoresis, which is required at the end of regular PCR. Moreover, the amount of fluorescent, monitored at each cycle is proportional to the log of concentration of the PCR target, and for this reason real-time PCR is also powerful technique for quantification of specific DNA. There are several labelling techniques, most of which specially bind to a target sequence on the amplicon, while others aspecifically stain double-stranded (ds) DNA amplicons. In addition, numbers of protocols have been

For preliminary screening, 16S rDNA gene were adapted for the universal diagnosis of phytoplasmas using direct real-time PCR amplification (30, 48, 71) (Table 3) and all of them exploited a TaqMan probe for detection. TaqMan probes are labelled at the 5'end with reporter dye and at the 3' end with a quenching molecule; during each PCR cycle in the presence of the specific target DNA, the TaqMan probe, bound to its target sequence, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase as it extend the primer. The fluorescence moiety of the probe is therefore freed from its quencher-labelled portion and the fluorescence is detected by the optical system of the apparatus. The sensitivity of the 16S rDNA-based primer/probe system can be used to detect phytoplasmas belonging to several ribosomal subgroups and they showed sensitivity similar to that of

Group specific phytoplasma primers and probes for real-time PCR system have been designed to overcome problem with the time-consuming methods for phytoplasma strains identification and to further enhance the specificity of detection. Several laboratories have proposed rapid, specific and sensitive diagnostic protocols for detection of quarantine and economically important phytoplasmas of fruit trees and grapevine such as flavescance dorée (FD) and bois noir (BN) phytoplasmas infecting grapevine (22, 48, 8, 53, 71, 14); '*Ca*. Phytoplasma mali' (apple proliferation, AP), '*Ca*. Phytoplasma pyri' (pear decline, PD), '*Ca*. Phytoplasma pruni' (European stone fruit yellows, ESFY) important pathogens of fruit trees (12, 76, 48, 156, 3,4, 113, 23, 128, 41). Most of the primer/probe systems are targeting 16S rDNA gene though some others genes or even randomly cloned DNA fragments to which no specific function is assigned have been used (Table 3). For fluorescent detection SYBR Green I has been applied for the diagnosis of AP, PD, ESFY and FD, all quarantine phytoplasmas of fruit trees and grapevine in Europe. Real-time PCR assays were also developed using TaqMan minor groove binding (MGB) probe to detect AP in plant material (12, 3) as well as for FD, BN and other phytoplasmas less frequently infecting grapevines (71, 128). MGB (minor groove binding) probe has an MGB ligand and non-fluorescent quencher conjugated to the 3' end, plus a fluorescent reporter dye at the 5' end. The MGB ligand allows the use of shorter and more specific probes by increasing the stability of the probe-target bond. This property allows the use of shorter probes, with higher specificity than conventional TaqMan ones and the discrimination of even single nucleotide

developed for real-time PCR universal and specific detection phytoplasma.

mass screening.

conventional nested-PCR.

mismatched (83, 128). Furthermore, applying the same protocols, phytoplasmas DNA could be also detected in insect samples (113, 76, 48, 71) what is also decisive in the search for other potential vectors.


Table 3. Oligonucleotide primers and probes used for phytoplasma detection by real-time PCR

A well-optimized reaction is essential for accurate results, which must be further analysed. As it is mentioned before, diagnosis of the pathogens in woody plants is often hampered by the presence of PCR inhibitors such as polyphenolics, polysaccharides and other molecules that may produce false negative results even from heavily infected samples. Additional problem may be also caused by amplification of other bacteria with universal phytoplasma primers/probe which could be present on the surface of some plants (49). Therefore, to avoid false positives specific probe can be included. So far, several sequence-specific detection tools are available: the chloroplast chaperonin 21 gene (8); cytochrome oxidase gene (71); the chloroplast gene for tRNA leucine (12); and the 18S rDNA gene (30, 118, 113,

Polymerase Chain Reaction for Phytoplasmas Detection 105

DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops, and produces up to 109 copies of the target in less than 1 h at 65°C. Further, LAMP products can be detected by conventional agarose gel electrophoresis; using spectrophotometric equipment to measure turbidity (124); in real-time using intercalating fluorescent dyes (105); or by visual inspection of turbidity or colour changes (123, 74).

For the routine diagnosis colorimetric assay that uses hydroxyl napthol blue to detect the magnesium pyrophosphate by-product in successful LAMP amplification (54) showed the best suitability. The hydroxyl napthol blue can be incorporated into the LAMP reaction and the colour change visualized immediately after amplification has been completed, and amplification can subsequently be confirmed by agarose gel electrophoresis when necessary. Two methods for extraction of nucleic acid from plant material were adopted for LAMP application: LFD (37, 155) and an alkaline polyethylene glycol (PEG) DNA extraction

Primers for the LAMP assays were designed as described in Tomlison et al. (2010) (155) and Bekele et al (2011) (16) based on the 16S-23S intergenic spacer region. In addition *cox* gene primers were used to confirm that all DNA extractions supported LAMP (16). Primers for LAMP assays were designed against range of ribosomal group (16SrI, 16SrII, 16SrIII, 16SrIV,

Developed protocol for LAMP-based diagnostic for a range of phytoplasmas can be conducted in the field and used to provide diagnosis within 1-hour of DNA extraction (68). According to the same author, PEG extraction method showed several advantages such is rapidness and requires less equipment than the LFD-based method, reducing the likelihood of sample contamination though the disadvantage of this method is that the DNA cannot be stored reliable long-term. Further efforts are doing to develop a hand held device capable of performing extraction, set-up and real-time detection for grapevine phytoplasmas. The device will make a single step homogeneous system from sampling to result, further reducing the risk of sample-to-sample contamination and enabling testing by non-specialists

In this review, molecular approaches for phytoplasma detection, identification and characterisation have been discussed. Before molecular techniques were developed, the diagnosis of phytoplasma diseases was difficult because they could not be cultured. Thus classical diagnostic techniques, such as observation of symptoms, were used. Ultrathin sections were also examined for the presence of phytoplasmas in the phloem tissue of suspected infected plants. Treating infected plants with antibiotics such as tetracycline to see if this cured the plant was another diagnostic technique employed. Diagnostic techniques such as ELISA test which allowed the specific detection of the phytoplasma began to emerge in the 1980s. In the early 1990s, PCR-based methods were developed that were far more sensitive than those that used ELISA, and RFLP analysis allowed the accurate identification of different strains and species of phytoplasma. Restriction fragments length polymorphism (RFLP) analysis together with the sequencing of 16Sr phytoplasma genes was the first step on this way enabling the construction of phylogenetic trees. Nowadays, polymerase chain

method (31, 68).

in the field (68).

**5. Conclussions** 

16SrV, 16SrXI, 16SrXII, 16SrXXII) (68).

128) addressed as targets to control the quality of total DNA extracted. SYBR Green I is one of the cheapest chemistry for real-time PCR detection, but the specificity of the reaction is extremely low, and needs to be checked. SYBR Green I dye chemistry will detect all doublestranded DNA, including non-specific reaction products. Therefore, amplification of non specific DNA may occur and analyses of melting curve is usually indispensible (48, 156).

One of the biggest advantages of real-time PCR is suitability of the method for quantification of nucleic acids of many plant pathogens, including phytoplasmas. In past competitive PCR was applied to monitor multiplication of '*Candidatus* Phytoplasma asteris' in vector *Macrosteles quadrinlineatus* (101). Quantification was achieved following coamplification of phytoplasma DNA and several dilutions of an appropriate internal standard. This approach was complex, several steps, such as electrophoresis, image analysis of gel, compensating for differences in intensity due to the different sizes of the product from the pathogen target and the internal standard, were required before the band intensities could be plotted for linear regression analysis. However, nowadays absolute quantification of phytoplasma DNA was achieved per gram of extracted tissue (161, 23) or per insect vector (76). Possibility of the method to quantify amount of phytoplasma DNA in plant tissue and insect vectors gave opportunity to better understand biology and epidemiology of the pathogens, to allow examination of different multiplication rates and to calculate the concentration in their plant and vector host (161, 142, 23) as well as to study interactions of different phytoplasma species or strains present in mixed infection (100, 19). These results will find application in development of resistant plant varieties, a hot topic for economically important woody crops such as palms, fruits and grapevines.

#### **4.4 Loop-mediated isothermal amplification assay (LAMP)**

Methods described above require relatively expensive equipment for amplification of the phytoplasma DNA and/or analysis of the results. In addition, standard methods for DNA extraction involve buffers, such as a CTAB buffer combined with phenol chloroform extraction and isopropanol precipitation (46, 165), which are time-consuming and cannot be performed in the field. Whilst leaf tissue is usually used as the source of DNA for detection of many phytoplasmas, in other cases, such as coconuts, trunk borings or roots are often used, and DNA is then extracted from this woody tissue either by grinding in liquid nitrogen, or when this is unavailable, the sawdust is left in the CTAB extraction buffer for 48 h before the subsequent phenol chloroform extraction and alcohol precipitation (129). For that reason there is increase need for development of the method for a more rapid diagnostic assay for phytoplasmas that can be used to produce a diagnosis within an hour of sampling in the field or on site in case of imported material in quarantine stations.

Several attempts to produce field-based systems, e.g. using phytoplasma-specific antibodies and ELISA-based or lateral flow devices (LFD)-based systems, fall down because of a lack of sensitivity, and whilst a phytoplasma IgG antibody based system is commercially available for few phytoplasmas (103). Recently, Fera (Food and Environment Research Agency) developed isothermal amplification assays, such as the Loop-Mediated Isothermal Amplification (LAMP) procedure for detection of several human and plant pathogens including phytoplasmas (130, 140, 37, 154). In the method the cycling accumulates stem-loop

128) addressed as targets to control the quality of total DNA extracted. SYBR Green I is one of the cheapest chemistry for real-time PCR detection, but the specificity of the reaction is extremely low, and needs to be checked. SYBR Green I dye chemistry will detect all doublestranded DNA, including non-specific reaction products. Therefore, amplification of non specific DNA may occur and analyses of melting curve is usually indispensible (48, 156).

One of the biggest advantages of real-time PCR is suitability of the method for quantification of nucleic acids of many plant pathogens, including phytoplasmas. In past competitive PCR was applied to monitor multiplication of '*Candidatus* Phytoplasma asteris' in vector *Macrosteles quadrinlineatus* (101). Quantification was achieved following coamplification of phytoplasma DNA and several dilutions of an appropriate internal standard. This approach was complex, several steps, such as electrophoresis, image analysis of gel, compensating for differences in intensity due to the different sizes of the product from the pathogen target and the internal standard, were required before the band intensities could be plotted for linear regression analysis. However, nowadays absolute quantification of phytoplasma DNA was achieved per gram of extracted tissue (161, 23) or per insect vector (76). Possibility of the method to quantify amount of phytoplasma DNA in plant tissue and insect vectors gave opportunity to better understand biology and epidemiology of the pathogens, to allow examination of different multiplication rates and to calculate the concentration in their plant and vector host (161, 142, 23) as well as to study interactions of different phytoplasma species or strains present in mixed infection (100, 19). These results will find application in development of resistant plant varieties, a hot topic for

economically important woody crops such as palms, fruits and grapevines.

Methods described above require relatively expensive equipment for amplification of the phytoplasma DNA and/or analysis of the results. In addition, standard methods for DNA extraction involve buffers, such as a CTAB buffer combined with phenol chloroform extraction and isopropanol precipitation (46, 165), which are time-consuming and cannot be performed in the field. Whilst leaf tissue is usually used as the source of DNA for detection of many phytoplasmas, in other cases, such as coconuts, trunk borings or roots are often used, and DNA is then extracted from this woody tissue either by grinding in liquid nitrogen, or when this is unavailable, the sawdust is left in the CTAB extraction buffer for 48 h before the subsequent phenol chloroform extraction and alcohol precipitation (129). For that reason there is increase need for development of the method for a more rapid diagnostic assay for phytoplasmas that can be used to produce a diagnosis within an hour of sampling in the field or on site in case of imported material in

Several attempts to produce field-based systems, e.g. using phytoplasma-specific antibodies and ELISA-based or lateral flow devices (LFD)-based systems, fall down because of a lack of sensitivity, and whilst a phytoplasma IgG antibody based system is commercially available for few phytoplasmas (103). Recently, Fera (Food and Environment Research Agency) developed isothermal amplification assays, such as the Loop-Mediated Isothermal Amplification (LAMP) procedure for detection of several human and plant pathogens including phytoplasmas (130, 140, 37, 154). In the method the cycling accumulates stem-loop

**4.4 Loop-mediated isothermal amplification assay (LAMP)** 

quarantine stations.

DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops, and produces up to 109 copies of the target in less than 1 h at 65°C. Further, LAMP products can be detected by conventional agarose gel electrophoresis; using spectrophotometric equipment to measure turbidity (124); in real-time using intercalating fluorescent dyes (105); or by visual inspection of turbidity or colour changes (123, 74).

For the routine diagnosis colorimetric assay that uses hydroxyl napthol blue to detect the magnesium pyrophosphate by-product in successful LAMP amplification (54) showed the best suitability. The hydroxyl napthol blue can be incorporated into the LAMP reaction and the colour change visualized immediately after amplification has been completed, and amplification can subsequently be confirmed by agarose gel electrophoresis when necessary.

Two methods for extraction of nucleic acid from plant material were adopted for LAMP application: LFD (37, 155) and an alkaline polyethylene glycol (PEG) DNA extraction method (31, 68).

Primers for the LAMP assays were designed as described in Tomlison et al. (2010) (155) and Bekele et al (2011) (16) based on the 16S-23S intergenic spacer region. In addition *cox* gene primers were used to confirm that all DNA extractions supported LAMP (16). Primers for LAMP assays were designed against range of ribosomal group (16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrXI, 16SrXII, 16SrXXII) (68).

Developed protocol for LAMP-based diagnostic for a range of phytoplasmas can be conducted in the field and used to provide diagnosis within 1-hour of DNA extraction (68). According to the same author, PEG extraction method showed several advantages such is rapidness and requires less equipment than the LFD-based method, reducing the likelihood of sample contamination though the disadvantage of this method is that the DNA cannot be stored reliable long-term. Further efforts are doing to develop a hand held device capable of performing extraction, set-up and real-time detection for grapevine phytoplasmas. The device will make a single step homogeneous system from sampling to result, further reducing the risk of sample-to-sample contamination and enabling testing by non-specialists in the field (68).

## **5. Conclussions**

In this review, molecular approaches for phytoplasma detection, identification and characterisation have been discussed. Before molecular techniques were developed, the diagnosis of phytoplasma diseases was difficult because they could not be cultured. Thus classical diagnostic techniques, such as observation of symptoms, were used. Ultrathin sections were also examined for the presence of phytoplasmas in the phloem tissue of suspected infected plants. Treating infected plants with antibiotics such as tetracycline to see if this cured the plant was another diagnostic technique employed. Diagnostic techniques such as ELISA test which allowed the specific detection of the phytoplasma began to emerge in the 1980s. In the early 1990s, PCR-based methods were developed that were far more sensitive than those that used ELISA, and RFLP analysis allowed the accurate identification of different strains and species of phytoplasma. Restriction fragments length polymorphism (RFLP) analysis together with the sequencing of 16Sr phytoplasma genes was the first step on this way enabling the construction of phylogenetic trees. Nowadays, polymerase chain

Polymerase Chain Reaction for Phytoplasmas Detection 107

[5] Alma, A., Bosco, D., Danielli, A., Bertaccini, A., Vibrio, M., and Arzone, A. 1997.

[6] Angelini E., Clair D., Borgo M., Bertaccini A., Boudon-Padieu E. 2001. Flavescence dorée

[7] Angelini E., Negrisolo E., Clair D., Borgo M., Boudon-Padieu E. 2003. Phylogenetic

[8] Angelini E., Bianchi G. L., Filippin L., Morassutti C.,Borgo M. 2007. A new TaqMan

[10] Arocha-Rosete Y., Kent P., Agrawal V., Hunt D., Hamilton A., Bertaccini A., Scott J.,

[11] Bai X, Zhang J, Ewing A, Miller S.A., Radek A.J., Shevchenko D.V., Tsukerman K.,

their insect and plant hosts. Journal of Bacteriology, 188: 3682–3696. [12] Baric S., Dalla Via J. 2004. A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. Journal of Microbiological Methods, 57: 135-145. [13] Batlle A., Laviña A., García-Chapa, M., Sabaté J., Folch C., Asin L. 2004. Comparative

and apple certification program. Acta Horticulturae (ISHS), 657:71-77. [14] Berger J., SchweiIgkoFfler W., Kerschbamer C., Roschatt C., Dalls Via J., Baric S. 2009.

byreal-time PCR assay.- Journal of Microbiological Methods*,*68: 613-622. [9] Anthony RM*,* Brown TJ*,* French GL*,* 2000*.* Rapid diagnosis of bacteremia by universal

oligonucleotide array*.* Journal of Clinical Microbiology 38*,* 781*–*8*.* 

Ball reared on healthy plants. *Insects Molecular Biology,* 6: 115-121.

phytoplasma. Vitis, 40: 79-86.

Phytopathology, 90: 1145-1152.

345-355.

1041- 1043.

nonribosomal DNA. Plant Pathology, 52:663-672.

Bulletin of Insectology, 64 (supplement), pp. 133-134.

Identification of phytoplasmas in eggs, nymphs, and adults of Scaphoideus titanus

in France and Italy - occurrence of closely related phytoplasma isolates and their near 101 relationships to palatinate grapevine yellows and an alder yellows

relationships among Flavescence dorée strains and related phytoplasmas determined by heteroduplex mobility assay and sequence of ribosomal and

method for the identificationof phytoplasmas associated with grapevine yellows

amplification of 23S ribosomal DNA followed by hybridisation to an

Crosby W., Michelutti R. 2011. Preliminary investigations on Graminella nigrifrons as a potential vector for phytoplasmas identified at the Canadian Clonal Genebank.

Walunas T., Lapidus A., Campbell J.W., and Hogenhout S.A. 2006. Living with genome instability: the adaptation of phytoplasmas to diverse environments of

results between different detection methods of virus and phytoplasmas for pear

Occurrence of Stolbur phytoplasma in the vector *Hyalesthes obsoletus*, herbaceous host plants and grapevine in South Tyrol (Northern Italy). Vitis, 48 (4), 185–192. [15] Berges R., Rott M., Semüller E. 2000. Range of phytoplasmas concentrations in various

plant hosts as determined by competitive polymerase chain reaction.

2011. Use of a real-time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathology, 60:

organisms in lilies with deformed flowers. Phytopathologia mediterranea, 21: 8-14.

mycoplasmalike organisms in infected plant tissues. Horticultural Science, 27(9):

[16] Bekele B., Hodgetts J., Tomlinson J., Boonham N.,Nikolic P., Swarbrick P., Dickinson M.

[17] Bertaccini A., Marani F. 1982. Electron microscopy of two viruses and mycoplasma-like

[18] Bertaccini A., Davis R.E., Lee I.-M. 1992. In vitro micropropagation for maintenance of

reaction with primers from sequencing of randomly cloned phytoplasma DNA, from 16S rRNA, from ribosomal protein gene sequences, from SecY and Tuf genes, and from membrane associated protein genes opened new paths for research on phytoplasma identification and classification.

Nested PCR has been applied to overcome problems related to sensitivity of phytoplasma detection, although this approach is more time consuming and subject to template. Unfortunately, nested-PCR also meets some difficulties: unspecific bands, false positives or negatives caused by DNA and contamination of single or nested PCR. Therefore, confirmation of PCR results by using different primer pairs combinations (generic and group-specific) with subsequent RFLP and/or sequencing of PCR amplicons seems to be the way for correct phytoplasma identification in the examined samples.

More recently, real-time PCR has replaced the traditional PCR in efforts to increase the speed and sensitivity of detection and improve techniques for mass screening as well as to bypass post-PCR manipulations. Moreover, the techniques as quantitative real-time PCR (QPCR) have been developed to allow assessment of the level of infection in plants and vectors.

T-RFLP, SSCP and HMA analyses provide simultaneous detection and group characterisation of phytoplasmas.

Isothermal amplification of nucleic acid has recently been described as an alternative to PCR and applied for specific detection of several phytoplasmas. This method has potential for testing in field or in under equipped laboratories.

Despite the developments of all protocols which overcome most of the difficulties of phytoplasma diagnosis, the detection of these pathogens is still quite laborious. Therefore, future work is needed to develop quicker procedures to extract phytoplasma-enriched nucleic acids, giving accent on automation which involving silica or magnetic beads. Furthermore, developments for phytoplasma detection should be stressed on improvements of methods which enable simultaneous detection and taxonomic grouping of phytoplasmas. Use of high-throughput, sensitive, rapid and quantitative techniques will help to understand how phytoplasmas exploit their unique ecological niches.

## **6. References**


reaction with primers from sequencing of randomly cloned phytoplasma DNA, from 16S rRNA, from ribosomal protein gene sequences, from SecY and Tuf genes, and from membrane associated protein genes opened new paths for research on phytoplasma

Nested PCR has been applied to overcome problems related to sensitivity of phytoplasma detection, although this approach is more time consuming and subject to template. Unfortunately, nested-PCR also meets some difficulties: unspecific bands, false positives or negatives caused by DNA and contamination of single or nested PCR. Therefore, confirmation of PCR results by using different primer pairs combinations (generic and group-specific) with subsequent RFLP and/or sequencing of PCR amplicons seems to be the

More recently, real-time PCR has replaced the traditional PCR in efforts to increase the speed and sensitivity of detection and improve techniques for mass screening as well as to bypass post-PCR manipulations. Moreover, the techniques as quantitative real-time PCR (QPCR) have been developed to allow assessment of the level of infection in plants and

T-RFLP, SSCP and HMA analyses provide simultaneous detection and group

Isothermal amplification of nucleic acid has recently been described as an alternative to PCR and applied for specific detection of several phytoplasmas. This method has potential for

Despite the developments of all protocols which overcome most of the difficulties of phytoplasma diagnosis, the detection of these pathogens is still quite laborious. Therefore, future work is needed to develop quicker procedures to extract phytoplasma-enriched nucleic acids, giving accent on automation which involving silica or magnetic beads. Furthermore, developments for phytoplasma detection should be stressed on improvements of methods which enable simultaneous detection and taxonomic grouping of phytoplasmas. Use of high-throughput, sensitive, rapid and quantitative techniques will help to

[1] Ahrens U., Seemüller E. 1992. Detection of plant pathogenic mycoplasmalike organisms

[2] Agrios G. N. 1997. Plant diseases caused by *Mollicutes*: phytoplasmas and spiroplasmas.

[3] Aldaghi M., Massert S., Roussel S., Jijaki M.H. 2007. Development of a new probe for

[4] Aldaghi M., Massert S., Roussel S., Dutrecq O., Jijaki, M.H. 2008. Adaptation of real-time

by a polymerase chain reaction that amplify a sequence of the 16S rRNA gene.

*In* Plant Pathology, 4th, pp. 457-470. Edited by G. N. Agrios. New York: Academic

specific and sensitive detection of *Candidatus* Phytoplasma mali in inoculated apple

PCR assay for specific detection of apple proliferation phytoplasma. Acta

way for correct phytoplasma identification in the examined samples.

understand how phytoplasmas exploit their unique ecological niches.

trees. Annals of Applied Biology 151: 251-258.

identification and classification.

characterisation of phytoplasmas.

testing in field or in under equipped laboratories.

Phytopathology, 82: 828-832.

Horticulturae 781: 387-393.

vectors.

**6. References** 

Press.


Polymerase Chain Reaction for Phytoplasmas Detection 109

[33] Cousin M.T., Sharma A.K., Isra S. 1986. Correlation between light and electron

consecutive 350 nm think sections. Journal of Phytopathology, 115: 368-374. [34] Crosslin J. M., Vandemark G. J., Munyaneza J. E. 2006. Development of a real-time,

phytoplasma in plants and beet leafhoppers. Plant Diseases 90: 663–667. [35] Daire X., Clair D., Larrue J., Boudon-Padieu E. 1997. Survey for grapevine yellows phytoplasmas in diverse European countries and Israel. Vitis, 36: 53-54. [36] Da Rold G., Filippin L., Malembic-Maher S., Forte V., Borgo M., Foissac X., Angelini E.

Mycoplasmology, Chianciano Terme (SI, Italy), 11th-16th July, p. 101. [37] Danks C. and Boonham N. 2007. PurificationMethod and Kits. Patent WO 2007

[38] Davis R.E., Lee I.-M. 1993. Cluster-specific polymerase chain reaction amplification of

[39] Davis R.E., Dally E.L., Gundersen D.E., Lee I-M., Habili N. 1997. '*Candidatus* 

[41] Delić D., Mehle N., Lolić B., Ravnikar M. Đurić G. 2010. Current status of European

[42] Delić D., Contaldo N., Paltrinieri S., Lolić B., Đurić Z., Hrnčić S., Bertaccini A. 2011.

[43] Delwart EL. and Gordon CJ. 1997. Tracking changes in HIV-1 envelope quasispecies

[44] Deng S., Hiruki C. 1991. Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. Journal of Microbiology Methods, 14: 53-61. [45] Doi Y., Teranaka M., Yora K., Asuyama H. 1967. Mycoplasma or PLT grouplike

[46] Doyle, J.J., J.L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15. [47] Fos A., Danet J.L., Zreik J., Garnier M., Bové J.M. 1992. Use of a monoclonal antibody to

[48] Galetto L., Bosco D., Marzachi C. 2005. Universal and group-specific real-time PCR

using DNA heteroduplex analysis. Methods, 12:348-354.

Phytopathological Society Japan, 33: 259-266.

a vector in France. Plant Disease, 76: 1092-1096.

104962.

417.

245-246.

Phytopathology, 83: 1008-1011.

*Insectology*, 60 (2), pp. 369-370

microscopic observations and identification of mycoplasmalikeorganisms using

quantitative PCR for detection of the Columbia Basin potato purple top

2010. The imp gene in flavescence dorée and related phytoplasmas from grapevine, Clematis sp. and alder. In: 18th Congress of the International Organization for

16SrDNA sequences for detection and identification of mycoplasmalike organisms.

Phytoplasma australiense', a new phytoplasma taxon associated with Australian grapevine yellows*.* International Journal of Systematic Bacteriology, 47: 262-269. [40] Delić, D., Seljak, G., Martini, M., Ermacora, P., Carraro, L., Myrta, A. Đurić, G. 2007.

Surveys for grapevine yellows phytoplasmas in Bosnia and Herzegovina. *Bulletin of* 

stone fruit yellows in Bosnia and Herzegovina: Julius-Kühn-Archiv , Procedeeng of *21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops,* July 5-10 2009, Neustadt, Germany, Neustadt: Julius Kühn-Institut, 427: 415-

Grapevine yellows in Bosnia and Herzegovina: surveys to identify phytoplasmas in grapevine, weeds and insect vectors. Bulletin of Insectology, 64 (supplement), pp.

microrganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches' broom, aster yellows or pawlonia witches'broom*.* Annals of

detect the stolbur mycoplasma –like organism in plants and insects and to identify

diagnosis of flavescance dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation


[19] Bertaccini A., Franova J., Botti S., Tabanelli D. 2005. Molecular characterization of

[20] Bertaccini A. 2007. Phytoplasmas: diversity, taxonomy, and epidemiology. Frontieres in

[21] Bertamini M., Nedunchezhian N. 2001. Effect of phytoplasma, stolbur-subgroup (Bois

[22] Bianco P.A., Casati P., Marziliano, N. 2004. Detection of phytoplasmas associated with

[23] Bisognin C., Schneider B., Salm H., Grando M.S., Jarausch W., Moll E., Seemüller E.

[24] Boben J., Mehle N., Ravnikar M. 2007. Optimization of extraction procedure can improve phytoplasma diagnostic. Bulletin of Insectology, 60 (2), pp. 249-250 [25] Cai H., Wei W., Davis R.E., Chen H., Zhao Y. 2008. Genetic diversity among

[26] Carraro., Rugero O., Refatti E., Poggi P. 1988. Transmission of the possible agent of

[27] Casati P., Quaglino F., Tedeschi R., Spiga F.M., Alma A., Spadone P., Bianco P.A. 2010.

isolates in North-western Italy. Journal of Phytopathology, 158, 81-87. [28] Cimerman A., Pacifico D., Salar P., Marzachì C., Foissac X. 2009. The striking diversity

phytoplasma. Applied and Environmental Microbiology, 75: 2951-2957 [29] Clair, D., Larrue, J., Aubert, G., Gillet, J., Cloquemin, G., Boudon-Padieu, E. 2003. A

[30] Christensen N.M., Nicolaisen M., Hansen M., Schultz A. 2004. Distribution of

[31] Chomczynski P. and Rymaszewski M. 2006. Alkaline polyethylene glycol-based

[32] Constable FE., Symons RH. 2004. Genetic variability amongst isolates of Australian

grapevine phytoplasmas. Australasian Plant Pathology 33:115-119.

use in survey of grapevine yellows in France. Vitis, 42, 151–157.

Molecular Plant–Microbe Interactions, 17: 1175-1184.

BioTechniques, 40: 454-458.

of Systematic and Evolutionary Microbiology, 58(6): 1448-1457.

Letters, 249: 79-85.

119-122.

98, 153-158.

52.

Bioscience, 12: 673-689.

Pathology, 86:257-261.

phytoplasmas in lilies with fasciations in the Czech Republic. Fems Microbiology

noir- BN)] of photosynthetic pigments, saccarides, ribulose-1,5-bisphosphate carboxylase, nitrate and nitrite reductases and photosynthetic activities in fieldgrow grapevine (*Vitis vinifera* L. cv Chardonnay) leaves. Photosynthetica, 39(1):

grapevine flavescance dorée disease using real-time PCR. Journal of Plant

2008. Apple proliferation resistance in apomictic rootstocks and its relationship to phytoplasma concentration and simple sequence repeat genotypes. Phytopathology

phytoplasmas infecting *Opuntia* species: virtual RFLP analysis identifies new subgroups in the peanut witches'-broom phytoplasma group. International Journal

apple proliferation to *Vinca rosea* by dodder. Rivista di Patologia Vegetale, 24: 43-

Identification and molecular characterisation of 'Candidatus Phytoplasma mali'

of *Vmp*1, a gene encoding a variable putative membrane protein of the Stolbur

multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of phytoplasma in the Elm yellows group and stolbur group and its

phytoplasmas in infected plants as revealed by real time PCR and bioimaging.

method for direct PCR from bacteria eukaryotic tissue samples and whole blood.


Polymerase Chain Reaction for Phytoplasmas Detection 111

[62] Harrison N.A., Womack M., Carpio M.L. 2002. Detection and characterization of a

[63] Hartung J.S., Pruvost, O.P., Villemot I., Alvarez A. 1996. Rapid and sensitive

[65] Hobbs H.A., Reddy D.V.R., Reddy A.S. 1987. Detection of a mycoplasma-lke organism

[66] Hodgetts J., Ball T., Boonham N., Mumford R , Dickinson M. 2007. Use of terminal

[67] Hodgetts J., Boonham N., Mumford R., Harrison N., Dickinson M. 2008. Phytoplasma

[69] Hollingsworth C.R., Atkinson L.M., Samac D.A., Larsen L.E., Motteberg C.D.,

[70] Hoshi A., Ishii Y., Kakizawa S., Oshima K., Namba S. 2007. Host-parasite interaction of

[71] Hren M., Boben J., Rotter A., Kralj P., Gruden K., Ravnikar M. 2007. Real-time PCR

[72] Hren I.M., Ravnikar M., Brzin J., Ermacora P., Carraro L., Bianco P.A., Casati P., Borgo

[73] IRPCM. 2004. '*Candidatus* Phytoplasma', a taxon for the wall-less, non-helical

[74] Iwamoto T., Sonobe T., Hayashi K. 2003. Loop-mediated isothermal amplification for

grown in the field. Plant Pathology, 58(1): 170-180.

Systematic and Evolutionary Microbiology, 54: 1243-1255.

Journal of Systematic and Evolutionary Microbiology. 58: 1826-1837. [68] Hodgetts J., Tomlison J., Boonham N., Gonzales-Martin I., Nikolić P., Swarbrick P.,

a nested-polymerase chain reaction assay. Phytopathology 86, 95–101. [64] Heinrich M., Botti SCaprara., L., Arthofer W., Strommer S. 2001. Improved detection

by lethal decline in Texas. Plant Disease, 86: 676-681.

assay (ELISA). Plant Pathology, 36: 164-167.

64:41-42.

623-630.

1145-1152.

Pathology, 56: 785-796.

Illinois. Plant Disease, 87: 241-246.

phytoplasmas in plants. Plant Pathology, 56: 357-365.

lethal yellowing (16SrIV) group phytoplasma in Canary Island date palms affected

colorimetric detection of *Xanthomonas ax*- - *onopodis* pv. *citri* by immunocapture and

methods for fruit tree phytoplasmas. Plant Molecular Biology Reporter, 19: 169-179.

in peanut plants with witches' broom using indirect enzyme-linked immunosorben

restriction fragment length polymorphism (T-RFLP) for identification of

phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of *Candidatus* Phytoplasma. International

Yankey E.N., Dickinson M. 2011. Development of rapid in-field loop mediated isothermal amplification (LAMP) assays for phytoplasmas. Bulletin of Insectology,

Abrahamson M.D., Glogoza P., MacRae I.V. 2008. Region and field level distributions of aster yellows phytoplasma in small grain crops. Plant Disease, 92:

hosts as determined by competitive polymerase chain reaction. Phytopathology, 90:

detection systems for Flavescence doree and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics. Plant

M., Angelini E., Rotter A., Gruden K. 2009. Induced expression of sucrose synthase and alcohol dehydrogenase I genes in phytoplasma-infected grapevine plants

prokaryotesthat colonise plant phloem and insects. International Journal of

direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. Journal of Clinical Microbiology 41: 2616–22. [75] Jacobs K.A., Lee I.M., Griffiths H.M., Miller F.D., Bottner K.D. 2003. A new member of

the clover proliferation phytoplasma group (16SrVI) associated with elm yellows in

(16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Annals of Applied Biology 147: 191-201.


[49] Galetto L. Marzachi C. 2010. Real-time PCR Diagnosis and Quantification of

[50] Garcia-Chapa M., Sabate J., Lavina A., Battle A.2005. Role of Cacopsylla pyri in the

[51] Gatineau F., Larruae J., Clair D, Lortoin F., Richard-Molard M., Boudon-Padieu E. 2001:

[52] Green M. J., Thompson D. A., MacKenzie D. J. 1999. Easy and efficient DNA extraction

[53] Gori M., Monnanni R, Buiatti M., Goti E., Carnevale S., Da Prato L., Bertaccina A.,

[54] Goto M., Honda E., Ogura A., Nomoto A., Hanaki KI. 2009. Colorimetric detection of

[55] Gundersen D.E., Lee I.-M. 1996. Ultrasensitive detection of phytoplasmas by nested-

[56] Gundersen D.E, Lee I.-M., Rehner S.A., Davis R.E, Kingsbury D.T. 1994. Phylogeny of

[57] Gundersen D.E., Lee I.-M., Schaff D.A., Harrison N.A., Chang C.J., Davis R.E., Kinsbury

phytoplasmas). International Journal of Systematic Bacteriology, 46: 64-75. [58] Guthrie J.N., White D.T., Walsh K.B., Scott P.T. 1998. Epidemiology of phytoplasma

[59] Haggis G.H., Sinha R.C. 1978. Scanning electron microscopy of mycoplasmalike

[60] Harrison N.A., Richardson P.A., Kramer J.B., Tsai J.H. 1994. Detection of the

Florida by polymerase chain reaction. Plant Pathology, 43: 998-1008. [61] Harrison N.A., Richardson, P.A., Tsai J.H., Ebbert M.A., Kramer J.B. 1996. PCR assay

P.G. and P. Jones (Eds.). CABI Publishers, USA., pp: 1-19.

Applied Biology 147: 191-201.

reaction. Plant Dis 83, 482–485.

BioTechniques 46, 167–72.

of Bacteriology, 176: 5244-5254

aster yellows. Phytopathology, 68: 677-680.

17.

271.

(2): 255-256.

144-151.

1111.

Disease, 80: 263-269.

(16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Annals of

Phytoplasmas. In: Phytoplasmas Genomes, Plant Hosts and Vectors, Weintraub,

epidemiology of pear decline in Spain. European Journal of Plant Pathology, 111: 9-

A new natural planthopper vector of stolbur phytoplasma in the genus *Pentastiridius* (Hemiptera: Cixiidae). European. Journal of Plant Pathology, 107: 263-

from woody plants for the detection of phytoplasmas by polymerase chain

Biricolti S. 2007. Establishing a real-time PCR detection procedure of "flavescence dorée" and "bois noir" phytoplasmas for mass screening. Bulletin of Insectology 60

loop-mediated isothermal amplification reaction by using hydroxyl napthol blue.

PCR assays using two universal primer pairs. Phytopathologia Mediterranea, 35:

Mycoplasmalike organisms (Phytoplasmas): a basis for their classification. Journal

D.T. 1996. Genomic diversity among phytoplasma strains in 16S rRNA Group I (Aster Yellows and related phytoplasmas) and III (X-Disease and related

associated papaya diseases in Queensland, Australia. Plant Disease, 82: 1107-

organisms after freeze fracture of plant tissues affected with clover phyllody and

mycoplasmalike organism associated with lethal yellowing disease of palms in

for detection of the phytoplasma associated with maize bushy stunt disease. Plant


Polymerase Chain Reaction for Phytoplasmas Detection 113

[92] Lee I.-M., Gundersen D.E., Hammond R.W., Davis R.E. 1994. Use of mycoplasmalike

[94] Lee I.-M., Gundersen-Rindal D.E., Bertaccini A. 1998. Phytoplasma: ecology and

[95] Lee I.M., Davis R.E., Gundersen-Rindal D.E. 2000. Phytoplasma: Phytopathogenic

[96] Lee I.-M., Gundersen-Rindal D., Davis R.E., Bottner K.D., Marcone C., Seemueller E.

[97] Lee I.-M., Martini M., Marcone C., Zhu S.F. 2004. Classification of phytoplasma strains

[98] Lee I.-M., Zhao Y., Bottner K.D. 2006*.* SecY gene sequence analysis for finer

[99] Leifting L., Veerakone S., Gerrard R.G., Clover L., Ward L.I. 2011. An update on

[100] Leyva-Lopez N.E., Ochoa-Sanchez J.C., Leal-Klevezas D.S., Martinez-Soriano J.P. 2002.

[101] Liu H.W., Goodwin P.H., Kuske C.R. 1994. Quantificationof DNA from the aster

[102] Lorenz K.H., Schneider B., Ahrens U., Seemüller E. 1995. Detection of the apple

[103] Loi N., Ermacora P., Carraro L., Osler R., Tsen An C. 2001. Production of monoclonal

[104] Ludvikova H., Lauterer P., Sucha J., Franova J. 2011. Monitoring of psyllid species

[105] Maeda H., Kokeguchi S., Fujimoto C. 2005. Detection of periodontal pathogen

genomic diversity. Phytopathology, 88: 1359-1366.

mollicutes. Annual Review of Microbiology, 54: 221-555.

Systematic and Evolutionary Microbiology, 54(2): 337-347.

and nonribosomal DNA. Phytopathology, 85: 771-776.

detection. European Journal of Plant Pathology, 108: 81-86.

FEMS Immunology and Medical Microbiology, 43: 233–239.

and Cellular Probes, 20(2): 87-91.

Journal of Microbiology, 48: 1062-1068.

Insectology, 64 (supplement), pp. 121-122.

1169.

1048.

pp. 93-94.

17: 274-280.

organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology, 84: 559-566. [93] Lee I.M., Gundersen-Rindal D.E., Davis R.E., Bartoszyk I.M. 1998. Revised classification

scheme of phytoplasma based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences International Journal of Systematic Bacteriology, 48: 1153-

2004.'*Candidatus* Phytoplasma asteris', a novel taxon associated with aster yellows and related diseases. International Journal of Systematic Bacteriology, 54: 1037-

in the elm yellows group (16SrV) and proposal of '*Candidatus* Phytoplasma ulmi' for the phytoplasma associated with elm yellows. International Journal of

differentiation of diverse strains in the aster yellows phytoplasma group*.* Molecular

phytoplasma diseases in New Zealand. Bulletin of Insectology, 64 (supplement),

Multiple phytoplasmas associated with potato diseases in Mexico. Canadian

yellows mycoplasma like organism in aster leafhoppers (*Macrosteles fascifrons* Stål) by a competitive polymerase chain reaction. Systematic and Applied Microbiology,

proliferation and pear decline phytoplasmas by PCR amplification of ribosomal

antibodies against apple proliferation phytoplasma and their use in serological

(Hemiptera, Psylloidea) in apple and pear orchards in East Bohemia. Bulletin of

Porphyromonas gingivalis by loopmediated isothermal amplification method.


[76] Jarausch W., Peccerella T., Schwind N., Jarausch B., Krczal G. 2004. Establishment of a

[78] Jomantiene R., Valiunas D., Ivanauskas A., Urbanaviciene L., Staniulis J., Davis R.E.

[79] Kartte S., Seemüller E. 1991. Susceptibility of grafted *Malus* taxa and hybrids to apple

[80] Kawakita, H., Saiki, T., Wei, W., Mitsuhashi, W., Watanabe, K. and Sato, M. 2000.

[83] Kostina E.V., Ryabinin V.A., Maksakova G.A., Sinyakov A.N. 2007. TaqMan probes

[86] Kunkel L.O. 1955. Cross protection between strains of aster yellow-type viruses.

[87] Langer M., Maixner M. 2004. Molecular characterization of grapevine yellows

[88] Lederer W., Seemüller E. 1991. Occurrence of mycoplasmalikeorganisms in diseased

[89] Lee I.M. and Davis R.E. 1986. Prospects for *in vitro* culture of plant-pathogenic mycoplasmalike organisms. Annual Review of Phytopathology, 24: 339-354. [90] Lee G.T.N., Golino D.A., Hackett K.J., Kirkptrick B.C., Marwitz R., Petzold H., Shina

(eds. Whitcomb R.F. e Tully J.G.). Academic Press, New York: 545-640. [91] Lee I.-M., Hammond R.W., Davis R.E., Gundersen D.E. 1993. Universal amplification

mycoplasmalike organisms. Phytopathology, 83: 834-842.

associated phytoplasmas of the Stolbur-group based on RFLP-analysis of non-

and non-symptomatic alder trees (*Alnus* spp.).- European Journal of Forest

R.H., Sugiura M., Whitcomb R.F., Yang I.L., Zhu B.M., Seemüller E. 1989. Plant Diseases Associated with Mycoplasmalike Organisms. In: The Mycoplasmas, vol. 5.

and analysis of pathogen 16S rDNA for classification and identification of

[84] Kunkel L.O. 1926. Studies on aster yellows. American Journal of Botany, 13: 646-705. [85] Kunkel L.O. 1931. Celery yellows of California not identical with the aster yellows of

leafhopper Hishimonoides sellatiformis *. Phytopathology,* 90: 909-914. [81] Khan JA, Srivastava P, Singh SK. 2004. Efficacy of nested-PCR for the detection of phytoplasma causing spike disease of sandal. Current Science 86: 1530–1533. [82] Klamer M*,* Roberts MS*,* Levine LH*,* Drake BG*,* Garland JL*.* 2002*.* Influence of elevated

phytoplasmas in plants and insects. Acta Horticulturae 657, 415–20. [77] Jomantiene R., Davis R.E., Maas J., Dally E.L. 1998. Classification of new phytoplasmas

Bulletin of Insectology, 64 (supplement), pp. 101-102.

Environmental Microbiology 68: 4370*–*6*.* 

New York. Boyce Thompson Institute, 4: 405-414.

of Bioorganic Chemistry 33, 614-616.

Advances in Virus Research, 3: 251-273.

ribosomal DNA. Vitis, 43: 191-199.

Pathology, 21: 90-96.

proliferation disease. Journal of Phytopathology, 131: 137-148.

Bacteriology, 48: 269-277.

quantitative real-time PCR assayfor the quantification of apple proliferation

associated with diseases of strawberry in Florida, based on analysis of 16S rRNA and ribosomal protein gene operon sequences. International Journal of Systematic

2011. Larch is a new host for a group 16SrI, subgroup B, phytoplasma in Ukraine.

Identification of mulberry dwarf phytoplasmas in genital organs and eggs of the

CO2 on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis. Applied and

based on oligonucleotide-hairpin minor groove binder conjugates. Russian Journal


Polymerase Chain Reaction for Phytoplasmas Detection 115

[120] Mikec I., Križanac I., Budinščak Ž., Šeruga Musić M., Krajačić M., Škorić D. 2006.

[121] Minsavage G.V., Thompson C.M., Hopkins D.L., Leite RMVBC, Stall RE. 1994.

[122] Munford R.A., Boonham N., Tomlinson J., Barker I. 2006. Advances in molecular

[123] Mori Y., Nagamine K., Tomita N., Notomi T. 2001. Detection of loop-mediated

[124] Mori Y., Kitao M., Tomita N., Notomi T. 2004. Real-time turbidimetry of LAMP

[125] Musić M.S., Krajačić M., Škorić D. 2007. Evaluation of SSCP analysis as a tool for

[126] Musić M.S., Krajačić M., Škorić D. 2008. The use of SSCP analysis in the assessment of phytoplasma gene variability. Journal of Microbiological Methods, 73: 69-72. [127] Namba S., Kato S., Iwanami S., Oyaizu H., Shiozawa H., Tsuchizaki T. 1993. Detection

[128] Nikolić P., Mehle N., Gruden K., Ravnikar M., Dermastia M. 2010. A panel of real-time

[129] Nipah J.O., Jones P., Dickinson M.J. 2007. Detection of lethal yellowing phytoplasma in

[130] Notomi T, Okayama H, Masubuchi H. 2000. Loop-mediated isothermal amplification

[131] Oberhänsli T., Altenbach D., Bitterlin W. 2011. Development of a duplex TaqMan real-

[132] Orsagova H., Brezikova M., Schlesingerova G. 2011. Presence of phytoplasmas in

[133] Padovan A.C., Gibb K.S., Bertaccini A., Vibio M., Bonfiglioli R.E., Magarey P.A., Sears

polymerase chain reaction. Phytopathology, 83: 786-791.

proliferation group. Molecular and Cellular Probes, 24, 303-309.

fruit trees and other plants. Bulletin of Insectology, 64:37-38.

Italy. Australian Journal of Grape and Wine Research, 1: 25 31.

fastidiosa in plant-tissue. Phytopathology*,* 84:456-461

545-640.

257.

Pathology, 116: 1-19.

Methods, 59: 145–157.

Plant Pathology, 56: 777-784.

of DNA. Nucleic Acids Research 28: 63.

289:150–154.

246.

120.

E. 1989. Plant Diseases Associated with Mycoplasmalike Organisms. In: The Mycoplasmas, vol. 5. (eds. Whitcomb R.F. e Tully J.G.). Academic Press, New York:

Phytoplasmas and their potential vectors in vineyards of indigenous Croatian varieties. *Extended Abstracts 15th Meeting of the ICVG, Stellenbosch, South Africa*: 255-

Development of a polymerase chain-reaction protocol for detection of Xylella-

phytodiagnostics – new solutions for old problems. European Journal of Plant

isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochemical and Biophysical Research Communications,

reaction for quantifying template DNA. Journal of Biochemistry and Biophysical

detection of phytoplasma molecular variability. Bulletin of Insectology, 60: 245-

and differentiation of plant-pathogenic mycoplasmalike organisms using

PCR assays for specific detection of three phytoplasmas from the apple

embryos from coconut palms infected with Cape St Paul wilt disease in Ghana.

time PCR for the general detection of phytoplasmas and 18S rRNA host genes in

hemipterans in Czech vineyards. Bulletin of Insectology, 64 (supplement), pp. 119-

B.B. 1995. Molecular detection of the Australian grapevine yellows phytoplasma and comparison with a grapevine yellows phytoplasma from Emilia-Romagna in


[106] Maixner M., Ahrens U., Seemüller E. 1995. Detection of the German grapevine yellows

specific PCR procedure. European Journal of Plant Pathology, 101: 241-250. [107] Maixner M. 2010. Grapevine yellows vector sampling and monitoring training school.-

http://costphytoplasma.eu/PDF%20files/Proceedings\_Vector\_TS.pdf [108] Malisano G., Firrao G., Locci R. 1996. 16S rDNA-derived oligonucleotide probes for the

[109] Marcone C., Lee I.-M., Davis R.E., Ragozzino A., Seemüller E. 2000. *C*lassification of

[110] Marinho V L A., Sandrine F., Michel D. 2008. Genetic variability among isolates of

[111] Martini M., Botti S., Marcone C., Marzachì C., Casati P., Bianco P.A., Benedetti R.,

Journal of Systematic and Evolutionary Microbiology, 57: 2037-2051. [113] Martini M., Loi N., Ermacora P., Carraro L., Pastore M. 2007. A real-time PCR method

Giulia region (North- East Italy). Acta Horticulturae, 781: 395-402.

EPPO Bulletin 26, 421–428.

Microbiology, 50(5): 1703-1713.

Institut, 427: 386-391.

Mediterranean, 43: 228-231.

(HMA). Tropical Plant Pathology, 33 (5): 377-380.

natural host. Bulletin of Insectology 69(2): 251-252.

reaction. Molecular Biotechnology, 30: 117-127.

(Vergilbungskrankheit) MLO in grapevine, alternative hosts and a vector by a

*COST Action FA0807, Working group 2-* Grapevine Yellows Vector Sampling and Monitoring Training School*, Bernkastel-Kues Germany,* 5th to 9th of July, 2010. From:

differential diagnosis of plum leptonecrosis and apple proliferation phytoplasmas.

aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences. International Journal of Systematic and Evolutionary

*Coconut lethal yellowing phytoplasmas* determined by Heteroduplex Mobility Assay

Bertaccini A. 2002. Genetic variability among Flavescence dorée phytoplasmas from different origins in Italy and France. Molecular and Cellular probes, 16(3): 197-208. [112] Martini M., Lee I.-M. Bottner K.D., Zhao Y., Botti S., Bertaccini A., Harrison N.A.,

Carraro L., Marcone C., Khan J., Osler R. 2007. Ribosomal protein gene-based filogeny for finer differentiation and classification of phytoplasmas. International

for detection and quantification of '*Candidatus* Phytoplasma prunorum' in its

differentiation of '*Candidatus* Phytoplasma mali' and its spreading in Friuli Venezia

Carraro L. 2010: PCR/RFLP based method for molecular characterization of "*Candidatus* Phytoplasma prunirum" strains using *aceF* gene: Julius-Kühn-Archiv , Procedeeng of *21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops,* July 5-10 2009, Neustadt, Germany, Neustadt: Julius Kühn-

[114] Martini M., Ermacora P., Falginella L., Loi N., Carraro L. 2008. Molecular

[115] Martini M., Ferrini F., Danet J.L., Ermacora P., Gülşen S., Delić D., Nazia L.,Xavier F.,

[116] Marzachi C., Verrati F., Bosco D. 1998. Direct PCR detection of phytoplasmas in experimentally infected insects. Annals of Applied Biology, 133: 45-54. [117] Marzachi C. 2004. Molecular diagnosis of phytoplasmas. Phytopathology

[118] Marzachi C. and Bosco D. 2005. Relative quantification of chrysanthemum yellows

[119] Mc Coy R.E., Caudwell A., Chang C.J., Chen T.A., Chiykowskyi L.N., Cousin M.T.,

(16SrI) phytoplasma in its plantand insect host using real-time polymerase chain

Dale de Leeuw G.T.N., Golino D.A., Hackett K.J., Kirkptrick B.C., Marwitz R., Petzold H., Shina R.H., Sugiura M., Whitcomb R.F., Yang I.L., Zhu B.M., Seemüller E. 1989. Plant Diseases Associated with Mycoplasmalike Organisms. In: The Mycoplasmas, vol. 5. (eds. Whitcomb R.F. e Tully J.G.). Academic Press, New York: 545-640.


Polymerase Chain Reaction for Phytoplasmas Detection 117

[147] Schneider B., Gibb K.S., Seeümller E. 1997. Sequence and RFLP analysis of the

[148] Seemüller E. 1976. Investigation to demostrate mycoplasmalike organism in diseases

[149] Seemüller E. Kunze L., Schaper U. 1984. Colonization behaviour of MLO and symptom

[151] Seemüller, E., Stolz, E., Kison, H. 1998. Persistence of European stone fruit yellows

[152] Seemüller E., Moll E., Schneider B. 2007. *Malus sieboldii*-based rootstocks mediate apple proliferation resistance to grafted trees. Bulletin of Insectology, 60(2): 301-302. [153] Smart, C.D., B. Schneider, C.L. Blomquist, L.J. Guerra and N.A. Harrison *et al*., 1996.

[154] Tomlinson JA., Barker I., Boonham N. 2007. Faster, simpler, more-specific methods for

[155] Tomlinson J. A., Boonham N., Dickinson M. 2010. Development and evaluation of a

[156] Torres E., Bertolini E., Cambra M., Montón C., Martín MP. 2005. Real-time PCR for

[159] Wang K., Hiruki C. 2001. Use of heteroduplex mobility assay for identification and

[160] Wang K., Hiruki C. 2005. Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay. Plant Pathology, 54: 625-633. [161] Wei W., Kakizawa S., Suzuki S., Jung H.Y., H. Nishigawa H., Miyata S., Oshima K.,

[162] Wei W., Davis R.E., Lee I.M., Zhao Y. 2007. Computer-simulated RFLP analysis of 16S

[163] Wei W., Lee I.-M., Davis R.E., Suo X., Zhao Y. 2008. Automated RFLP pattern

proliferation (16SrX) group. Molecular and Cellular Probes 19, 334–40. [157] Upchurch DA, Shankarappa R, Mullins JI. 2000. Position and degree of mismatches and the mobility of DNA heteroduplexes. Nucleic Acids Research, 28 (12), 69-69. [158] Wang YH., Griffith J. 1991. Effects of bulge composition and flanking sequence on the

rapid detection of phytoplasmas. Plant Pathology, 59, 465–471.

kinking of DNA by bulged bases. Biochemistry, 30:1358-1363.

of Systematic and Evolutionary Microbiology, 57: 1855-1867.

proliferation group. Phytopathology 91:546-552.

94(3): 244-250.

plants by fluorescence microscopy. Acta Horticulturae, 67: 109-112.

pear decline. Z. Pflanzenkrankeiten Pflanzenschutz, 91: 371-382.

phytoplasmas. Microbiology, 143: 3381-3389.

Phytopathology, 146: 407–410.

region. Phytopathology, 62: 2988-2993.

Environmental Microbiology 73: 4040–4047.

elongation factor Tu gene used in differentiation and classification of

expression of proliferation in diseased apple trees and decline-diseased pear trees over a period of several years. Journal of Plant Disease Protection, 91: 525-532. [150] Seemüller E., Schaper U., Zimbelmann F. 1984. Seasonal variations in the colonization

patterns of mycoplasma-like organisms associated with apple proliferation and

phytoplasma in aerial parts of *Prunus* taxa during the dormant season. Journal of

Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer

improved molecular detection of Phytophthoraramorum in the field. Applied and

one-hour DNA extraction and loop-mediated isothermal amplification assay for

simultaneous and quantitative detection of quarantine phytoplasmas from apple

differentiation of phytoplasmas in the aster yellows group and the clover

Ugaki M., Hibi T., Namba S. 2004. *In planta* dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission. Phytopathology,

rRNA genes: Identification of ten new phytoplasma groups. International Journal

comparison and similarity coefficient calculation for rapid delineation of new and


[134] Palmano S. 2001. A comparison of different phytoplasma DNA extraction methods

[135] Petrzik K., Sarkisova T., Čurnova L. 2011. Universal primers for plasmid detection and

[136] Ploaie, P. G. 1981. Mycoplasma-like organisms and plant diseases in Europe. Pages 61-

[137] Prezelj N., Mehle N., Nikolić P., Ravnikar M., Dermastia M. 2010. Rapid diagnostic for

[138] Prince J.P., Davies. R.E., Wolf T.K., Lee I.-M., Mogen B.D., Dally E.L., Bertaccini A.,

yellows, X-disease and elm yellows MLOs. Phytopathology, 83: 1130-1137. [139] Rajan J., Clark M.F. 1995. Detection of apple proliferation and other MLOs by

[140] Saito R., Misawa Y., Moriya K., Koike K., Ubukata K., Okamura N. 2005. Development

[141] Sakai M*,* Matsuka A*,* Komura T*,* Kanazawa S*.* 2004*.* Application of a new PCR primer

communities in plant roots. Journal of Microbiological Methods*,* 59: 81*–8*9. [142] Saracco P., Bosco D., Veratti F., Marzachi C. 2006. Quantification over time of

[143] Samuitiene M. and Navalinskiene M. 2006. Molecular detection and characterization of

[144] Schaff D.A., Lee I.-M., Davis R.E. 1992. Sensitive detection and identification of

[145] Schneider B., Ahrens U., Kirkpatrick B.C., Seemüller E. 1993. Classification of plant

amplified 16S rDNA*.* Journal of General Microbiology, 139: 519-527. [146] Schneider B., Seemuller E., Smart C.D., Kirkpatrick B.C. 1995. Phylogenetic

immunocapture PCR (IC-PCR). Acta Horticulturae, 386: 511-514.

vector. Physiological and Molecular Plant Pathology, 67: 212-219.

Biophysics Research Communications, 186: 1503-1509.

(Eds.). Academic Press, San Diego, pp: 369-380.

method for their relative quantification in phytoplasma-infected plants. Bulletin of

104 *In* Plant Diseases and Vectors: Ecology and Epidemiology. Maramorosch, K.,

economically important phytoplasmas in grapevine and fruit trees. Knjiga povzetkov = Book of abstracts / [5. slovenski simpozij o rastlinski biologiji z mednarodno udeležbo, 6.-9. september 2010, Ljubljana = 5th Slovenian Symposium on Plant Biology with International Participation, September 6-9 2010, Ljubljana, Slovenia]; [organizator] Slovensko društvo za biologijo rastlin = [organized by]

Credi R., Barba M. 1993. Molecular detection of diverse mycoplasmalike organisms (MLOs) associated with grapevine yellows and their classification with aster

and evaluation of a loop-mediatedisothermal amplification assay for rapid detection of Mycoplasma pneumoniae. Journal of Medical Microbiology, 54:1037–

for terminal restriction fragment length polymorphism analysis of the bacterial

chrysanthemum yellows phytoplasma (16Sr-I) in leaves and roots of the host plant Chrysanthemum carinatum (Schousboe) following inoculation with its insect

phytoplasma infecting *Celosia argentea* L. plants in Lithuania. Agronomy-Research,

mycoplasmalike organisms by polymerase chain reactions. Biochemistry

pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-

Classification of Plant Pathogenic Mycoplasmalike Organisms or Phytoplasmas. In: Molecular and Diagnostic Procedures in Mycoplasmology, Razin, S. and J.G. Tully

using competitive PCR. Phytopathology Mediterranean, 40:99-107.

Insectology, 64 (supplement), pp. 25-26.

Slovenian Society of Plant Biology; p, 94.

41.

4: 345-348.

and Harris, K. F., eds. Academic Press, New York.


**6** 

*México* 

**Molecular Diagnostics of Mycoplasmas:** 

Saúl Flores-Medina1,2,\*, Diana Mercedes Soriano-Becerril1

*1Departamento de Infectología, Instituto Nacional de Perinatología, D.F.,* 

*3Departamento de Salud Pública, Facultad de Medicina, UNAM, D.F.,* 

and Francisco Javier Díaz-García3

*2CECyT No. 15 "DAE", IPN, D.F.,* 

**Perspectives from the Microbiology Standpoint** 

Some of the smallest self-replicating bacteria, the wall-less mycoplasmas belonging to Class Mollicutes, are pathogenic for mammals and humans, showing tissue and host-specificity. In humans, the pathogenic species of the *Mycoplasma* or *Ureaplasma genus* cause covert infections that tend to chronic diseases. At present, 7 species of *Mycoplasma*, 2 species of *Ureaplasma* and 1 of *Acholeplasma* have been consistently isolated/detected from several specimens from diseased subjects, specially through the use of molecular detection

Current laboratory diagnosis of these infections relies on cultural methods, however this is complicated and emission of results may delay up to 5 weeks. Thus development and application of molecular methods, such as polymerase chain reaction (PCR), have allowed direct detection in clinical specimens and shortened the time to get the final results. Nevertheless some pitfalls still hampers the widespread use of these technologies, mainly due to technical difficulties in collecting representative specimens and optimizing sample preparation. There are countless reports on new nucleic acid-based tests (NATs) for mycoplasma detection, however there is a great variation between methods from study to study, including variability of target gene sequences, assay format and technologic platform

The processing of the clinical samples is crucial for the improvement of PCR assays as part of routine diagnostic approaches. In general, for the strength of performance of any diagnostic PCR, the overall setting-up of the assay should consider the following four basic steps: 1) sampling, 2) sample preparation, 3) nucleic acid amplification, and 4) detection of

As occurred with much of the emerging or reemerging pathogens, the molecular detection plays a key role in the discovery, identification and association or such pathogens with human disease [Relman & Persing, 1996]. Nevertheless, routine clinical microbiology

techniques [Mendoza *et al.,* 2011; Waites & Talkington, 2005; Waites, 2006].

**1. Introduction** 

[Waites *et al.,* 2000; Waites , 2006;].

PCR products [Rådström *et al.,* 2004].

Corresponding Author

 \*

distinct phytoplasma 16Sr subgroup lineages. International Journal of Systematic and Evolutionary Microbiology, 58(10): 2368-2377.


## **Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint**

Saúl Flores-Medina1,2,\*, Diana Mercedes Soriano-Becerril1 and Francisco Javier Díaz-García3 *1Departamento de Infectología, Instituto Nacional de Perinatología, D.F., 2CECyT No. 15 "DAE", IPN, D.F., 3Departamento de Salud Pública, Facultad de Medicina, UNAM, D.F., México* 

## **1. Introduction**

118 Polymerase Chain Reaction

[164] Wilson IG. 1997. Inhibition and facilitation of nucleic acid amplification. Applied and

[165] Zhang Y., Uyemoto J.K., Kirkpatrick B.C. 1998. A small-scale procedure for extracting

and Evolutionary Microbiology, 58(10): 2368-2377.

Environmental Microbiology, 63: 3741–3751.

assay. Journal of Virological Methods, 71: 45-50.

distinct phytoplasma 16Sr subgroup lineages. International Journal of Systematic

nucleic acids from woody plants infected with various phytopathogens for PCR

Some of the smallest self-replicating bacteria, the wall-less mycoplasmas belonging to Class Mollicutes, are pathogenic for mammals and humans, showing tissue and host-specificity. In humans, the pathogenic species of the *Mycoplasma* or *Ureaplasma genus* cause covert infections that tend to chronic diseases. At present, 7 species of *Mycoplasma*, 2 species of *Ureaplasma* and 1 of *Acholeplasma* have been consistently isolated/detected from several specimens from diseased subjects, specially through the use of molecular detection techniques [Mendoza *et al.,* 2011; Waites & Talkington, 2005; Waites, 2006].

Current laboratory diagnosis of these infections relies on cultural methods, however this is complicated and emission of results may delay up to 5 weeks. Thus development and application of molecular methods, such as polymerase chain reaction (PCR), have allowed direct detection in clinical specimens and shortened the time to get the final results. Nevertheless some pitfalls still hampers the widespread use of these technologies, mainly due to technical difficulties in collecting representative specimens and optimizing sample preparation. There are countless reports on new nucleic acid-based tests (NATs) for mycoplasma detection, however there is a great variation between methods from study to study, including variability of target gene sequences, assay format and technologic platform [Waites *et al.,* 2000; Waites , 2006;].

The processing of the clinical samples is crucial for the improvement of PCR assays as part of routine diagnostic approaches. In general, for the strength of performance of any diagnostic PCR, the overall setting-up of the assay should consider the following four basic steps: 1) sampling, 2) sample preparation, 3) nucleic acid amplification, and 4) detection of PCR products [Rådström *et al.,* 2004].

As occurred with much of the emerging or reemerging pathogens, the molecular detection plays a key role in the discovery, identification and association or such pathogens with human disease [Relman & Persing, 1996]. Nevertheless, routine clinical microbiology

<sup>\*</sup> Corresponding Author

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 121

and *M. penetrans* are primarily urogenital residents, but exceptionally they can be isolated from other unusual tissues and organs, especially in immunocompromised patients or in patients undergoing solid organ transplantation [Cassel *et al*., 1993; Waites & Talkington,

Most of mycoplasmal diseases are underdiagnosed because the specific laboratory diagnostic strategies are quite different than those for fast-growing bacteria. It is noteworthy that mycoplasmal etiology of diseases in humans is considered only after failure of diagnosis of other common bacterial etiologies. In addition, outside their hosts, the mycoplasmas are highly labile to environmental factors, such as changing osmotic pressure and temperature, desiccation and/or alkaline or acidic conditions [Cassell *et al.,* 1994a; Waites *et al.,* 2000]. Noteworthy, there are few specialized or reference laboratories for diagnosis of mycoplasmal

Several different detection techniques of mycoplasmal infections have been developed, each one of which has its advantages and limitations with respect to cost, time, reliability, specificity, and sensitivity. According to the laboratory's infrastructure, the most common methods include: a) culture-based isolation/detection/identification/antimicrobial susceptibility profile; b) antigen detection, c) mycoplasmal-specific serologic responses; and, d) PCR and other NATs. [Razin *et al*., 1998; Talkington & Waites, 2009; Waites *et al*., 2000;

Relationship between mycoplasmas as etiologic agents and diseases in humans remains doubtful due to unsuccessful isolation/detection of these microorganisms in specimens from affected persons, as compared with healthy carriers [Taylor-Robinson, 1996]. Demonstration of growth of mycoplasma, by means of *in vitro* culture from clinical specimens, is still required to link the pathogen with the disease; thus culture is considered as the Gold Standard. However, current culture methods for detection of mycoplasmas in clinical specimens are arduous and emission of results may delay up to 5 weeks, which even

In this context, detection or isolation of mycoplasmas from clinical specimens requires careful consideration of the type of specimen available and the organism (species) sought [Cassell *et al*., 1994a]. The adequate specimens for culture include: a) normally sterile body fluids (sinovial, amniotic, cerebrospinal, urine, peritoneal, pleural, etc., b) secretions exudates or swabs from sites with associated flora (from nasopharinx, pharinx, cervix, vagina, urethra, surgical wounds, prostate, sputum, etc.), and c) cell-rich fluids (including blood and semen) or tissue biopsies. Overall, specimen collection should reflect the site of infection and/or the disease process. [Atkinson *et al*., 2008; Waites & Talkington, 2004].

Liquid specimens or tissues do not require special transport media if culture can be performed within 1 hour, otherwise specimens should be placed in transport media, such as SP-4, 10B or 2SP broths. When swabbing is required, aluminum- or plastic-shafted calcium alginate or dacron swabs should be used, taking care to obtain as many cells as possible

then may be inconclusive or inaccurate [Cassell *et al*., 1994a; Waites *et al*., 2000].

diseases and therefore, limited skilled laboratory personnel [Cassell *et al.,* 1994a].

2004; Waites *et al*, 2005; Waites *et al.,* 2008].

**3. Routine laboratory diagnostic approaches** 

[Atkinson *et al*., 2008; Cassell *et al*., 1994a; Waites, 2006].

Yoshida *et al*., 2002].

**3.1 Culture** 

laboratories still lack of skilled personnel in molecular detection techniques, and consequently in the appropriate sample preparation procedures [Cassell *et al*, 1994a; Talkington & Waites, 2009]. Unlike other fast-growing pathogens, the pathogenic Mollicutes species exhibit unique features that make them the last link in the diagnostic chain, only sought after failure in other diagnostic approaches [Cassell *et al.,* 1994a].

## **2. Relevant features of mycoplasmas**

The term mycoplasmas will be used to refer to any member of the Class Mollicutes. The mycoplasmas are the smallest microorganisms (0.3 - 0.8 μm diameter) capable of selfreplication, which lack a rigid cell wall. These bacteria also incorporate exogenous cholesterol into their own plasma membrane and use the UGA codon to encode tryptophan. Due to their reduced cell dimensions, they possess small genome sizes (0.58-2.20 Mb) and exhibit restricted metabolic alternatives for replication and survival. As a result of the above mentioned, the mycoplasmas show a strict dependence to their hosts for acquisition of biosynthetic precursors (aminoacids, nucleotides, lipids and sterols), in a host- and tissuerestricted manner, reflecting their nutritional demands and parasitic lifestyle [Baseman & Tully, 1997; Razin, 1992; Razin *et al.,* 1998].

Mycoplasmas infecting humans mainly colonize the mucosal surfaces of the respiratory and genitourinary tracts [Cassell *et al.,* 1994, Patel & Nyirjesy, 2010; Taylor-Robinson, 1996]. The mycoplasma species commonly isolated from humans and their attributes are listed in Table 1. Of the pathogenic species, *Mycoplasma pneumoniae* is found principally in the respiratory tract, whereas *M. genitalium, Ureaplasma parvum, U. urealyticum., M. hominis, M. fermentans* 


aLo *et al*,1993; bLo *et al*, 1992; cYáñez *et al*., 1999.

Table 1. Mycoplasmas which infect humans. Adapted from: Taylor-Robinson, 1996.

and *M. penetrans* are primarily urogenital residents, but exceptionally they can be isolated from other unusual tissues and organs, especially in immunocompromised patients or in patients undergoing solid organ transplantation [Cassel *et al*., 1993; Waites & Talkington, 2004; Waites *et al*, 2005; Waites *et al.,* 2008].

Most of mycoplasmal diseases are underdiagnosed because the specific laboratory diagnostic strategies are quite different than those for fast-growing bacteria. It is noteworthy that mycoplasmal etiology of diseases in humans is considered only after failure of diagnosis of other common bacterial etiologies. In addition, outside their hosts, the mycoplasmas are highly labile to environmental factors, such as changing osmotic pressure and temperature, desiccation and/or alkaline or acidic conditions [Cassell *et al.,* 1994a; Waites *et al.,* 2000]. Noteworthy, there are few specialized or reference laboratories for diagnosis of mycoplasmal diseases and therefore, limited skilled laboratory personnel [Cassell *et al.,* 1994a].

## **3. Routine laboratory diagnostic approaches**

Several different detection techniques of mycoplasmal infections have been developed, each one of which has its advantages and limitations with respect to cost, time, reliability, specificity, and sensitivity. According to the laboratory's infrastructure, the most common methods include: a) culture-based isolation/detection/identification/antimicrobial susceptibility profile; b) antigen detection, c) mycoplasmal-specific serologic responses; and, d) PCR and other NATs. [Razin *et al*., 1998; Talkington & Waites, 2009; Waites *et al*., 2000; Yoshida *et al*., 2002].

## **3.1 Culture**

120 Polymerase Chain Reaction

laboratories still lack of skilled personnel in molecular detection techniques, and consequently in the appropriate sample preparation procedures [Cassell *et al*, 1994a; Talkington & Waites, 2009]. Unlike other fast-growing pathogens, the pathogenic Mollicutes species exhibit unique features that make them the last link in the diagnostic chain, only

The term mycoplasmas will be used to refer to any member of the Class Mollicutes. The mycoplasmas are the smallest microorganisms (0.3 - 0.8 μm diameter) capable of selfreplication, which lack a rigid cell wall. These bacteria also incorporate exogenous cholesterol into their own plasma membrane and use the UGA codon to encode tryptophan. Due to their reduced cell dimensions, they possess small genome sizes (0.58-2.20 Mb) and exhibit restricted metabolic alternatives for replication and survival. As a result of the above mentioned, the mycoplasmas show a strict dependence to their hosts for acquisition of biosynthetic precursors (aminoacids, nucleotides, lipids and sterols), in a host- and tissuerestricted manner, reflecting their nutritional demands and parasitic lifestyle [Baseman &

Mycoplasmas infecting humans mainly colonize the mucosal surfaces of the respiratory and genitourinary tracts [Cassell *et al.,* 1994, Patel & Nyirjesy, 2010; Taylor-Robinson, 1996]. The mycoplasma species commonly isolated from humans and their attributes are listed in Table 1. Of the pathogenic species, *Mycoplasma pneumoniae* is found principally in the respiratory tract, whereas *M. genitalium, Ureaplasma parvum, U. urealyticum., M. hominis, M. fermentans* 

*Primary colonization sites Main metabolic substrates* 

*Urogenital* 


*Pathogenicity Respiratory* 


*tract Glucose Arginine Urea* 

+ + + + + - + - + + + + + - - - -



sought after failure in other diagnostic approaches [Cassell *et al.,* 1994a].

**2. Relevant features of mycoplasmas** 

Tully, 1997; Razin, 1992; Razin *et al.,* 1998].

*tract* 

+ + + + + + + + + - - ¿? - + + + ¿?

*Species* 

*M. orale M. buccale M. faucium M. lipophilum M. pneumoniae M. hominis M. genitalium M. fermentans M. primatum M. spermatophilum* 

*M. pirum M. penetrans* 

*U. parvum* 

*A. oculi* 

*Ureaplasma urealyticum*

*Acholeplasma laidlawii* 

aLo *et al*,1993; bLo *et al*, 1992; cYáñez *et al*., 1999. Table 1. Mycoplasmas which infect humans. Adapted from: Taylor-Robinson, 1996.

*Mycoplasma. salivarium* 

Relationship between mycoplasmas as etiologic agents and diseases in humans remains doubtful due to unsuccessful isolation/detection of these microorganisms in specimens from affected persons, as compared with healthy carriers [Taylor-Robinson, 1996]. Demonstration of growth of mycoplasma, by means of *in vitro* culture from clinical specimens, is still required to link the pathogen with the disease; thus culture is considered as the Gold Standard. However, current culture methods for detection of mycoplasmas in clinical specimens are arduous and emission of results may delay up to 5 weeks, which even then may be inconclusive or inaccurate [Cassell *et al*., 1994a; Waites *et al*., 2000].

In this context, detection or isolation of mycoplasmas from clinical specimens requires careful consideration of the type of specimen available and the organism (species) sought [Cassell *et al*., 1994a]. The adequate specimens for culture include: a) normally sterile body fluids (sinovial, amniotic, cerebrospinal, urine, peritoneal, pleural, etc., b) secretions exudates or swabs from sites with associated flora (from nasopharinx, pharinx, cervix, vagina, urethra, surgical wounds, prostate, sputum, etc.), and c) cell-rich fluids (including blood and semen) or tissue biopsies. Overall, specimen collection should reflect the site of infection and/or the disease process. [Atkinson *et al*., 2008; Waites & Talkington, 2004].

Liquid specimens or tissues do not require special transport media if culture can be performed within 1 hour, otherwise specimens should be placed in transport media, such as SP-4, 10B or 2SP broths. When swabbing is required, aluminum- or plastic-shafted calcium alginate or dacron swabs should be used, taking care to obtain as many cells as possible [Atkinson *et al*., 2008; Cassell *et al*., 1994a; Waites, 2006].

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 123

diagnostics. Selection of a variety of target sequences, starting with highly conserved regions of the genes, allowed design of primers of wide specificity ("universal primers") (Table 2) for detection of mycoplasmal infections in anatomic sites where at least 2 or 3 species are frequently found. The use of a single Mollicutes universal primer set in cases of life-threatening infections has the advantage of allowing a rapid positive or negative report to clinicians, and in turn to establish as soon as possible the appropriate treatment [Razin, 1994]. The approach of using *Mollicutes*-specific and *Ureaplasma* spp-specific universal primers allowed better discrimination between organisms of the *Mycoplasma* and *Ureaplasma* genus, and subsequent identification by species-specific primers in urine specimens from

**Targets Applications References** 

Screening for any mycoplasma species in clinical specimens and cell cultures.

> Species-specific detection.

MgPa adhesin gene: *M. genitalium* Selective detection Palmer *et al*., 1991;

Ureasa genes: *Ureaplasma spp.* Genus-specific detection Blanchard *et al*., 1993b

Is-like elements: *M. fermentans* Selective detection Wang *et al*., 1992 Rep elements of P1: *M. pneumoniae* Selective detection Ursi *et al*., 1992

*pneumoniae* Selective detection Lüneberg *et al*., 1993

Identification of tetracycline-resistant strains

van Kuppeveld *et al*., 1992; van Kuppeveld *et al*., 1994; Yoshida *et al*., 2001.

Blanchard *et al*., 1993a; Grau *et al*., 1994; van Kuppeveld *et al*., 1992.

Jensen *et al*., 1991.

Blanchard *et al*., 1992

contamination. Harasawa *et al*., 1993.

typing Bernet *et al*., 1989

detection and typing. Kong *et al*., 1996

HIV-infected patients [Díaz-García *et al*., 2004].

**16S rRNA gene sequence** 

Conserved regions of mycoplasmal 16S rRNA genes.

Variable regions of mycoplasmal 16S rRNA genes:

**Mycoplasmal protein genes** 

Elongation factor *tuf* gene of *M.*

*tet M* gene (tetracycline-resistance determinant)

**Repetitive genomic sequences** 

Adapted from: Razin, 1994.

The 16S-23S intergenic regions Detection of cell culture

P1 adhesin gene: *M. pneumoniae* Selective detection and

*Mba gene* Species-specific

Table 2. Nucleic acid sequences suitable for PCR-based mycoplasma testing

There is no ideal formulation of culture media for all pathogenic species, mainly due to their different substrate and pH requirements [Waites *et al*., 2000]. Modified SP-4 media (broth and agar) [Lo *et al*., 1993a], containing both glucose and arginine, can support the growth of all human pathogenic *Mycoplasma* species, including the fastidious *M. pneumoniae* and *M. genitalium*. A set of Shepard´s 10B broth and A8 agar can be used for cultivation of *Ureaplasma* species and *M. hominis*. For cultivation, specimens in transport media should be thoroughly mixed, and then should be 10-fold serially diluted in broth (usually up to 10-6) in order to allow semiquantitative estimation of mycoplasmal load, but subcultures in agar media should also be performed [Cassel *et al*., 1994a]. Inoculated media should be incubated under microaerophilic atmosphere at 37 °C.

Detection of *M. pneumoniae* in broth culture is based on its ability to ferment glucose, causing an acidic shift after 4 or more weeks, readily visualized by the presence of the phenol red pH indicator. Broths with any color change, and subsequent blind broth passages, should be subcultured to SP4 agar, incubated, and examined under the low-power objective of the light microscope in order to look for development of typical "fried egg"-like colonies of up to 100 μm in diameter. Examination of agar plates must be done on a daily basis during the first week, and thereafter every 3 to 4 days until completing 5 weeks or until growth is observed [Waites *et al*., 2000; Waites & Talkington, 2004]. *M. genitalium*, *M. fermentans* and *M. penetrans* are also glucose-fermenting and form colonies morphologically indistinguishable from those of *M. pneumoniae*, thus serologic-based definitive identification can be done by growth inhibition, metabolic inhibition, and mycoplamacidal tests [Atkinson *et al*., 2008].

Hidrolysis of urea by Ureaplasma and hidrolysis of arginine by *M. hominis* cause an alkaline shift, turning the colour of 10B broth from yellow to pink. Tiny brown or black irregular colonies of *Ureaplasma* species develop between 1-5 days on A8 agar plates, due to urease activity in the presence of manganese sulfate. Typical fried egg colonies are produced by *M. hominis* in this medium [Cassell *et al*, 1994a; Waites *et al*., 2000].

## **3.2 Molecular assays**

The nucleic acid-based techniques have several advantages over culture-based methods, including rapid results, low detection limits (theoretically a single copy of target sequence), and specific organism detection. This is critical in a hospital setting, since rapid pathogen detection is important for faster and improved patient treatment and consequently for shortening hospitalization time [Mothershed & Whitney, 2006].

In particular, for PCR-based detection tests, selection of the appropriate target sequences for amplification appears to be of major concern. Mycoplasmal sequences to be amplified can be chosen from published gene sequences or from a mycoplasma-specific cloned DNA fragments [Kovacic *et al*., 1996; Razin, 2002]. The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence [Jaffe *et al*., 2004; Razin 2002]. Besides complete or almost complete sequences of the 16S rRNA genes for almost all the established mycoplasma species, the published full genome sequences of the human pathogenic mycoplasma species [Fraser *et al*., 1995; Glass *et al*., 2000; Himmelreich *et al.*, 1996] will accelerate the process of identification of novel target sequences for PCR

There is no ideal formulation of culture media for all pathogenic species, mainly due to their different substrate and pH requirements [Waites *et al*., 2000]. Modified SP-4 media (broth and agar) [Lo *et al*., 1993a], containing both glucose and arginine, can support the growth of all human pathogenic *Mycoplasma* species, including the fastidious *M. pneumoniae* and *M. genitalium*. A set of Shepard´s 10B broth and A8 agar can be used for cultivation of *Ureaplasma* species and *M. hominis*. For cultivation, specimens in transport media should be thoroughly mixed, and then should be 10-fold serially diluted in broth (usually up to 10-6) in order to allow semiquantitative estimation of mycoplasmal load, but subcultures in agar media should also be performed [Cassel *et al*., 1994a]. Inoculated media should be incubated

Detection of *M. pneumoniae* in broth culture is based on its ability to ferment glucose, causing an acidic shift after 4 or more weeks, readily visualized by the presence of the phenol red pH indicator. Broths with any color change, and subsequent blind broth passages, should be subcultured to SP4 agar, incubated, and examined under the low-power objective of the light microscope in order to look for development of typical "fried egg"-like colonies of up to 100 μm in diameter. Examination of agar plates must be done on a daily basis during the first week, and thereafter every 3 to 4 days until completing 5 weeks or until growth is observed [Waites *et al*., 2000; Waites & Talkington, 2004]. *M. genitalium*, *M. fermentans* and *M. penetrans* are also glucose-fermenting and form colonies morphologically indistinguishable from those of *M. pneumoniae*, thus serologic-based definitive identification can be done by growth inhibition, metabolic inhibition, and mycoplamacidal tests [Atkinson

Hidrolysis of urea by Ureaplasma and hidrolysis of arginine by *M. hominis* cause an alkaline shift, turning the colour of 10B broth from yellow to pink. Tiny brown or black irregular colonies of *Ureaplasma* species develop between 1-5 days on A8 agar plates, due to urease activity in the presence of manganese sulfate. Typical fried egg colonies are produced by *M.* 

The nucleic acid-based techniques have several advantages over culture-based methods, including rapid results, low detection limits (theoretically a single copy of target sequence), and specific organism detection. This is critical in a hospital setting, since rapid pathogen detection is important for faster and improved patient treatment and consequently for

In particular, for PCR-based detection tests, selection of the appropriate target sequences for amplification appears to be of major concern. Mycoplasmal sequences to be amplified can be chosen from published gene sequences or from a mycoplasma-specific cloned DNA fragments [Kovacic *et al*., 1996; Razin, 2002]. The accelerated rate of genomic sequencing has led to an abundance of completely sequenced genomes. Annotation of the open reading frames (ORFs) (i.e., gene prediction) in these genomes is an important task and is most often performed computationally based on features in the nucleic acid sequence [Jaffe *et al*., 2004; Razin 2002]. Besides complete or almost complete sequences of the 16S rRNA genes for almost all the established mycoplasma species, the published full genome sequences of the human pathogenic mycoplasma species [Fraser *et al*., 1995; Glass *et al*., 2000; Himmelreich *et al.*, 1996] will accelerate the process of identification of novel target sequences for PCR

*hominis* in this medium [Cassell *et al*, 1994a; Waites *et al*., 2000].

shortening hospitalization time [Mothershed & Whitney, 2006].

under microaerophilic atmosphere at 37 °C.

*et al*., 2008].

**3.2 Molecular assays** 

diagnostics. Selection of a variety of target sequences, starting with highly conserved regions of the genes, allowed design of primers of wide specificity ("universal primers") (Table 2) for detection of mycoplasmal infections in anatomic sites where at least 2 or 3 species are frequently found. The use of a single Mollicutes universal primer set in cases of life-threatening infections has the advantage of allowing a rapid positive or negative report to clinicians, and in turn to establish as soon as possible the appropriate treatment [Razin, 1994]. The approach of using *Mollicutes*-specific and *Ureaplasma* spp-specific universal primers allowed better discrimination between organisms of the *Mycoplasma* and *Ureaplasma* genus, and subsequent identification by species-specific primers in urine specimens from HIV-infected patients [Díaz-García *et al*., 2004].


Table 2. Nucleic acid sequences suitable for PCR-based mycoplasma testing Adapted from: Razin, 1994.

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 125

The usefulness of a PCR assay for diagnostic purposes is rather limited; this is partially explained by the presence of inhibitory substances in complex biological samples, which then provoke a significant reduction or even blockage of the amplification activity of DNA polymerases in comparison with that obtained with the use of pure solutions of nucleic acids. This in consequence affects the performance and the analytical sensitivity of the PCR

*Advantages Disadvantages*

• Presence of undefined inhibitors of DNA polymerases may yield false-

discriminates between disease or

• Risk of false-positive results due to carryover contamination with amplicons from previous reactions.

• Setting-up quantitative determination of bacteria in clinical specimens may

• Performance of PCR assays for routine diagnostic purposes in microbiology laboratories is still complex and

• Skilled personnel are required to carry out tests and analysis of results.

• Depending on the target sequences used, cross reactivity with closely related bacteria may occur.

be a very complicated task.

negative results.

carriage

expensive.

Table 3. Considerations for using PCR assays in diagnosis of mycoplasma infections.

Kam, 2006; Rådström *et al*., 2004; Vaneechoute & Van Eldere, 1997].

Due to the above mentioned, improvement of PCR assays for routine diagnostic purposes clearly should begin with optimal processing of clinical specimens prior to amplification reaction, thus successful amplification of the target DNA sequence can be obtained in the context of trace amounts of sample-associated inhibitory substances [Horz *et al*, 2010; Lo &

• Upon detection, PCR poorly

assays [Lo & Kam, 2006; Vaneechoute & Van Eldere, 1997].

• Overcomes the need for mycoplasma

• Emission of results is faster than culture (less than 24 h vs. up to 5

• Allows detection of antibiotic-

clinical specimens

PCR results

serology).

carriers.

inhibited or uncultivable species in

• Selective detection. Presence of nucleic acids from the host or from other microorganisms usually do not affect

• Higher sensitivity than other non-

molecular diagnostic assays (culture,

• Use as an epidemiological tool since it allows detection of asymptomatic

• Allows detection of mycoplasmas at the level of Family, Genus, Species,

Subspecies and/or Type.

Adapted from: Razin 1994.

cultivation

weeks)

When differentiation of the mycoplasmas is required, a multiplex PCR system consisting of a universal set of primers along with primer sets specific for the mycoplasma species commonly involved in a given disease process can be successfully applied [Razin, 2002, Choppa *et al*., 1998]. Moreover, both conserved and variable regions within the mycoplasmal 16S rRNA genes can also be selected for detection at cluster-, genus- species-, subspecies-, biovar- or serovar-specific levels [Kong *et al*., 2000; Razin, 2002].

For diagnostic purposes in mycoplasmology, the nucleic acid tests are more sensitive than culture, and showing a fair to good correlation with serology. PCR testing for speciesspecific mycoplasmal infection are suitable for both urogenital and respiratory samples [Povlsen *et al*., 2001, 2002]. Interestingly, sample processing prior amplification must be optimized depending of the type of specimen to overcome the presence of undefined inhibitory substances for DNA polymerases, avoiding false negative results. For example, nasopharyngeal samples have a higher rate of PCR inhibition than throat swabs. In general, results obtained by means of NATs will be as good as the quality of the nucleic acid used for the test [Mothersehed & Whitney, 2006; Maeda *et al*., 2004].

Early in the past decade, Loens *et al*., 2003b, stated that the development and application of new nucleic acid tests (NATs) in diagnostic mycoplasmology required proper validation and standardization, and performance of different NATs must be compared with each other in order to define the most sensitive and specific tests. The NATs have demonstrated their potential to produce rapid, sensitive and specific results, and are now considered the methods of choice for direct detection of *M. pneumoniae*, *M. genitalium,* and *M. fermentans* [Cassell *et al*., 1994a; Loens *et al*., 2003b]. There is a great variation in methods used from study to study, including variability of target gene sequences (P1, 16S RNA, ATPase, *tuf*), assay format (single, multiplex) or technologies (end-point PCR, Realtime PCR, NASBA) [Loens *et al*., 2003a, 2003b; 2010]. Also, target DNA has been obtained from different specimens, such as sputum, nasopharyngeal or pharyngeal swabs, brochoalveolar lavages or pleural fluid, and then comparisons of performance between these assays are difficult. For comprehensive understanding of the use of NATs for the detection of *M. pneumoniae*, genital mycoplasmas and other respiratory pathogens in clinical specimens, see the reviews done by Ieven, 2007; ; Lo & Kam, 2006; Loens *et al*., 2003b, 2010.

As with any other diagnostic test, PCR assays designed for mycoplasma detection in the clinical setting offer several advantages over other non-molecular tests, but still have several drawbacks to take into account (Table 3). Notwithstanding, there are several primer sets that have been successfully applied for diagnosis of mycoplasmal diseases in humans (Table 4).

## **4. Importance of the specimen collection and processing**

Clinical specimens must be collected with use of strict aseptic techniques from anatomic sites likely to yield pathogenic microorganisms [Taylor, 1998; Wilson, 1996]. In the case of mycoplasmal infections, these are clinically silent or covert, thus it is important to differentiate between asymptomatic carriage and disease. In this context, sampling of representative diseased body sites is critical for successful diagnosis.

When differentiation of the mycoplasmas is required, a multiplex PCR system consisting of a universal set of primers along with primer sets specific for the mycoplasma species commonly involved in a given disease process can be successfully applied [Razin, 2002, Choppa *et al*., 1998]. Moreover, both conserved and variable regions within the mycoplasmal 16S rRNA genes can also be selected for detection at cluster-, genus- species-,

For diagnostic purposes in mycoplasmology, the nucleic acid tests are more sensitive than culture, and showing a fair to good correlation with serology. PCR testing for speciesspecific mycoplasmal infection are suitable for both urogenital and respiratory samples [Povlsen *et al*., 2001, 2002]. Interestingly, sample processing prior amplification must be optimized depending of the type of specimen to overcome the presence of undefined inhibitory substances for DNA polymerases, avoiding false negative results. For example, nasopharyngeal samples have a higher rate of PCR inhibition than throat swabs. In general, results obtained by means of NATs will be as good as the quality of the nucleic acid used for

Early in the past decade, Loens *et al*., 2003b, stated that the development and application of new nucleic acid tests (NATs) in diagnostic mycoplasmology required proper validation and standardization, and performance of different NATs must be compared with each other in order to define the most sensitive and specific tests. The NATs have demonstrated their potential to produce rapid, sensitive and specific results, and are now considered the methods of choice for direct detection of *M. pneumoniae*, *M. genitalium,* and *M. fermentans* [Cassell *et al*., 1994a; Loens *et al*., 2003b]. There is a great variation in methods used from study to study, including variability of target gene sequences (P1, 16S RNA, ATPase, *tuf*), assay format (single, multiplex) or technologies (end-point PCR, Realtime PCR, NASBA) [Loens *et al*., 2003a, 2003b; 2010]. Also, target DNA has been obtained from different specimens, such as sputum, nasopharyngeal or pharyngeal swabs, brochoalveolar lavages or pleural fluid, and then comparisons of performance between these assays are difficult. For comprehensive understanding of the use of NATs for the detection of *M. pneumoniae*, genital mycoplasmas and other respiratory pathogens in clinical specimens, see the reviews done by Ieven, 2007; ; Lo & Kam, 2006; Loens *et al*.,

As with any other diagnostic test, PCR assays designed for mycoplasma detection in the clinical setting offer several advantages over other non-molecular tests, but still have several drawbacks to take into account (Table 3). Notwithstanding, there are several primer sets that have been successfully applied for diagnosis of mycoplasmal diseases in

Clinical specimens must be collected with use of strict aseptic techniques from anatomic sites likely to yield pathogenic microorganisms [Taylor, 1998; Wilson, 1996]. In the case of mycoplasmal infections, these are clinically silent or covert, thus it is important to differentiate between asymptomatic carriage and disease. In this context, sampling of

**4. Importance of the specimen collection and processing** 

representative diseased body sites is critical for successful diagnosis.

subspecies-, biovar- or serovar-specific levels [Kong *et al*., 2000; Razin, 2002].

the test [Mothersehed & Whitney, 2006; Maeda *et al*., 2004].

2003b, 2010.

humans (Table 4).

The usefulness of a PCR assay for diagnostic purposes is rather limited; this is partially explained by the presence of inhibitory substances in complex biological samples, which then provoke a significant reduction or even blockage of the amplification activity of DNA polymerases in comparison with that obtained with the use of pure solutions of nucleic acids. This in consequence affects the performance and the analytical sensitivity of the PCR assays [Lo & Kam, 2006; Vaneechoute & Van Eldere, 1997].


Table 3. Considerations for using PCR assays in diagnosis of mycoplasma infections. Adapted from: Razin 1994.

Due to the above mentioned, improvement of PCR assays for routine diagnostic purposes clearly should begin with optimal processing of clinical specimens prior to amplification reaction, thus successful amplification of the target DNA sequence can be obtained in the context of trace amounts of sample-associated inhibitory substances [Horz *et al*, 2010; Lo & Kam, 2006; Rådström *et al*., 2004; Vaneechoute & Van Eldere, 1997].


Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 127

bp, Base pairs; Sen, sense or downstream; Antisen, antisense or upstream; IP, internal probe.

Table 4. Primer sets used for end-point PCR detection of mycoplasmas in clinical specimens

Sen: Antisen: UMS-125 UMA-226- GTA TTT GCA ATC TTT ATA TGT TTT CG CAG CTG ATG TAA GTG CAG CAT TAA ATT C *mba* gene 402,403 (Up) 443 (Uu) Kong *et al*., 2000

**Group or species Primer sets. Sequence (5'**→**3') Target Amplicon size (bp) Refs.** *M. pneumoniae* Sen: Antisen: IP: MP5-1 MP5-2 MP5-4 GAA GCT TAT GGT ACA GGT TGG ATT ACC ATC CTT GTT GTA AGG CGT AAG CTA TCA GCT ACA TGG AGG Unknown gene 144 Bernet *et al*., 1989 Sen: Antisen: IP: P1-F P1-R PI-P GCC ACC CTC GGG GGC AGT CAG- GAG TCG GGA TTC CCC GCG GAG G CTG AAC GGG GGC GGG GTG AAG G- P1 adhesin gene 209 Ieven *et al*., 1996 Sen: Antisen: IP: 16S-F 16S-R 16S-P AAG GAC CTG CAA GGG TTC GT CTC TAG CCA TTA CCT GCT AA ACT CCT ACG GGA GGC AGC AGT A 16S rDNA 277 Ieven *et al*., 1996 Sen: Antisen: IP: MP-P11 MP-P12 MP-I TGC CAT CAA CCC GCG CTT AAC CCT TTG CAA CTG CTC ATA GTA CAA ACC GGG CAG ATC ACC TTT P1 Adhesin gene 466 De Barbeyrac *et al*., 1993 *Ureaplasma spp.* Sen: Antisen: IP: U5 U4 U9 CAA TCT GCT CGT GAA GTA TTA C ACG ACG TCC ATA AGC AAC T GAG ATA ATG ATT ATA TGT CAG GAT CA Urease locus 429 Blanchard *et al*., 1993a Sen: Antisen: IP: Uu-1 Uu-2 UUSO TAA ATG TCG GCT CGA ACG AG GCA GTA TCG CTA GAA AAG CAA C CAT CTA TTG CGA CGC TA 16s rDNA 311 van Kuppeveld *et al*., 1992 *U. parvum U. urealyticum*


**Group or species Primer sets. Sequence (5'**

Sen:

GPO-1

ACT CCT ACG GGA GGC AGC AGT A

TGC ACC ATC TGT CAC TCT GTT AAC CTC 16S rDNA 715 van Kuppeveld *et al.,*

1992

Antisen:

Sen:

My-Ins

GTAATACATAGGTCGCAAGCGTTATC

TGC ACC ATC TGT CAC TCT GTT AAC CTC 16S rDNA 520 Yoshida *et al.,* 2001

Antisen:

Sen:

RW005

GGT TAT TCG ATT TCT AAA TCG CCT

Insertion

sequence-like

206 Wang *et al.,* 1992

rDNA 16s 272 van Kuppeveld *et al*., 1992

element

GGA CTA TTG TCT AAA CAA TTT CCC

GCT GTG GCC ATT CTC TTC TAC GTT

GAA GCC TTT CTT CGC TGG AG

ACA AAA TCA TTT CCT ATT CTG TC

ACT CCT ACG GGA GGC AGC AGT A

GAG CCT TTC TAA CCG CTG C

MgPa

Adhesin

673 de Barbeyrac *et al*., 1993

gene

MgPa

Adhesin

371 de Barbeyrac *et al*., 1993

gene

16s rDNA 170 Grau *et al*., 1994

16s rDNA 281 van Kuppeveld *et al*., 1992

GTG GGG TTG AAG GAT GAT TG

AAG CAA CGT AGT AGC GTG AGC

GAG CCT TTC TAA CCG CTG C

GTT GTT ATC ATA CCT TCT GAT

AAG CAA CGT AGT AGC GTG AGC

ATA CAT GCA TGT CGA GCG AG

CAT CTT TTA GTG GCG CCT TAC

CGC ATG GAA CCG CAT GGT TCC GTT G

TGA AAG GCG CTG TAA GGC GC

GTC TGC AAT CAT TTC CTA TTG CAA A

ACT CCT ACG GGA GGC AGC AGT A

Antisen:

RW004

IP:

Sen:

Mf-1

Antisen:

Mf-2

IP:

Sen:

MGS-1

Antisen:

MGS-2

IP:

Sen:

MGS-1

Antisen:

MGS-4

IP:

Sen:

MYCHOMP

Antisen:

MYCHOMN

IP:

Sen:

Mh-1

Antisen:

Mh-2

IP:

Sen:

RNAH1

CAATGGCTAATGGCCGGATACGC

GGTACCGTCAGTCTGCAAT 16S rDNA 334 Blanchard *et al*., 1993b

Antisen:

Sen:

MYCPENETP

CAT GCA AGT CGG ACG AAG CA

AGC ATT TCC TCT TCT TAC AA

16s rDNA 407 Grau *et al*., 1994

CAT GAG AAA ATG TTT AAA GTC TGT TTG

Antisen:

MYCPENETN

IP:

MYCPENETS

*M. penetrans* 

RNAH2

GPO-1

MYCHOMS

*M. hominis* 

MGS-I

MGS-I

*M. genitalium* 

GPO-1

RW006

*M. fermentans* 

MGSO

MGSO

*Mollicutes-specífic*

→**3') Target Amplicon** 

**size (bp)** 

**Refs.** 

bp, Base pairs; Sen, sense or downstream; Antisen, antisense or upstream; IP, internal probe.

*M. pneumoniae* 

Table 4. Primer sets used for end-point PCR detection of mycoplasmas in clinical specimens

*Ureaplasma spp.* 

*U. parvum* 

*U. urealyticum*

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 129

*pneumoniae*-specific 16S rDNA PCR, they obtained positive amplification frequencies of 69%, 50% and 37.5% for sputum, nasopharyngeal aspirate and throat swab specimens,

Since genital tract mycoplasmas are closely associated to live epithelial cells, collection of exudates must be avoided and vigorous scraping of epithelia must be done to obtain as many cells as possible. In this case, a higher associated flora is frequently present in the samples; therefore use of transport liquid media (for culture) of buffered solutions (for DNA

Collection of normally sterile body fluids is made through invasive procedures, usually performed by physicians under aseptic conditions [Wilson, 1996]. When specimens are going to be collected through puncture, careful disinfection of the skin spot must be done, this is crucial to both avoid contamination of the specimen with the skin's associated flora and to minimize the risk of introduction of bacteria into patient's body. Clinically, access of mycoplasmas to sterile body sites may be associated with an underlying immune compromise, and probably the bacteria spread from pulmonary or genital infectious foci [Cassell *et al*., 1994b; Waites & Talkington, 2004]. Ureaplasmas and mycoplasmas should always be sought from synovial fluid when hypogammaglobulinemic patients develop

Since mycoplasma-containing body fluids rarely became turbid; these specimens should be concentrated 10-fold by high-speed centrifugation (aprox. 12,000 x g) and immediately resuspended in one tenth of the original supernatant if culture will be performed. Prior to DNA extraction, the resulting pellet can be washed 1-2 times with Hank's balanced salt

Unlike normally sterile body fluids, blood and semen are cell-rich fluid specimens, thus processing for culture or PCR is quite different. It is important to note that mycoplasmas have the ability to invade several cell types, including leukocytes and spermatozoa [Andreev *et al*., 1995; Baseman *et al*., 1995; Díaz-García *et al*., 2006; Girón *et al*., 1996; Jensen *et al*., 1994; Lo *et al*., 1993b, Rottem, 2003; Taylor-Robinson *et al*., 1991; Yavlovich *et al*., 2004], consequently a high input of cells into culture media may result in a higher probability of

In contrast, when DNA extraction must be performed for PCR assays, depuration of the sample must be done, (i.e. erythrocyte lysis and selective enrichment for leukocytes in blood; density gradient-based purification of spermatozoa). Noteworthy, the average content of leukocyte DNA per milliliter of blood ranges from 32 to 76 µg, therefore surpasses considerably the amount of bacterial DNA in a specimen from an infected subject. [Greenfield & White, 1993], so a high amount of sample DNA should be added to the PCR

reaction mixture to raise the chances to detect bacterial target sequences.

respectively.

**4.1.2 Urogenital tract** 

**4.2 Sterile body fluids** 

acute arthritis [Waites *et al*., 2000].

**4.3 Cell-rich fluids and tissues** 

solution or PBS, pH 7.4.

detection.

extraction) is required immediately after sampling.

Under certain conditions PCR detection/identification/confirmation of mycoplasmas could be attempted from culture broths used for primary isolation, whether or not it have bacterial growth. According to broth turbidity boiling of small aliquots can be sufficient to release the DNA, but presence of precipitated material may inhibit the amplification assay.

In our experience, an alkaline shift around pH 8 frequently results in bacterial lysis, mainly of ureaplasmas, therefore concentration of insoluble material by ultracentifugation prior to DNA extraction is unproductive. This is due to spontaneous release of mycoplasmal DNA that easily dissolves in the aqueous phase and cannot be sedimentated by centrifugation. In such cases, one can take advantage of the alkaline condition to precipitate the dissolved DNA by adding one tenth of 1M NaCl and twice the volume of cold 100% ethanol, and proceed with conventional DNA extraction protocols (unpublished data).

## **4.1 Exudates and secretions**

These types of specimens are fluids closely associated with mucosal surfaces, in low quantities, so collection should be done with the aid of swabs, cytological brush or small syringes. Secretions and exudates can be taken from upper respiratory airways and from lower genital tract, and exceptionally from surgical wounds [Waites, 2006].

## **4.1.1 Respiratory tract**

Respiratory *M. pneumoniae* infection can be assessed by culture and PCR in nasopharyngeal and oropharyngeal secretions, sputa, bronchoalveolar lavage and lung tissue obtained by biopsy. There are reports that nasopharyngeal and oropharyngeal specimens are equally effective for detection of *M. pneumoniae* by PCR, although it is desirable that both sites are screened in parallel for better diagnostic yield [Waites *et al*., 2008].

When neonatal mycoplasmal infections are suspected, endotracheal, nasopharyngeal and throat secretions are appropriate to evaluate respiratory infection., though specimens for culture should be transported quickly to laboratory since they are likely to contain at least a few contaminating microorganisms [Waites *et al*., 2005].

Presence of mucous material in this kind of specimens frequently hampers appropriate processing for culture or PCR. Use of aggressive mucolytic agents (NaOH, n-acetyl-cisteine) can damage as well the mycoplasma cells, thus thorough homogenization by wide-bore pippeting is required prior to culture attempt. For nucleic acid extraction, addition of starch has been of help to enhance recovery of total genomic DNA from sputum samples [Harasawa *et al*., 1993]. In other study, dithiotreitol was used as the mucolytic agent without any apparent detrimental effect on mycoplasmal DNA integrity [Raty *et al*., 2005].

It this worthy to note that differential sample preparation from the same specimen may be necessary when testing separate single-species PCRs on BAL, as described by [de Barbeyrac *et al*., 1993]. In that report, freeze-thawing cycles were applied for sample preparation for *M. genitalium* detection, while standard DNA extraction was needed for *M. pneumoniae* detection. During a study of Finnish patients with radiologically confirmed pneumonia, [Raty *et al*., 2005], evidence further supported the notion that selection of the appropriate specimen is crucial for diagnosis of *M. pneumoniae* infection. By means of a *M.*  *pneumoniae*-specific 16S rDNA PCR, they obtained positive amplification frequencies of 69%, 50% and 37.5% for sputum, nasopharyngeal aspirate and throat swab specimens, respectively.

## **4.1.2 Urogenital tract**

128 Polymerase Chain Reaction

Under certain conditions PCR detection/identification/confirmation of mycoplasmas could be attempted from culture broths used for primary isolation, whether or not it have bacterial growth. According to broth turbidity boiling of small aliquots can be sufficient to release the

In our experience, an alkaline shift around pH 8 frequently results in bacterial lysis, mainly of ureaplasmas, therefore concentration of insoluble material by ultracentifugation prior to DNA extraction is unproductive. This is due to spontaneous release of mycoplasmal DNA that easily dissolves in the aqueous phase and cannot be sedimentated by centrifugation. In such cases, one can take advantage of the alkaline condition to precipitate the dissolved DNA by adding one tenth of 1M NaCl and twice the volume of cold 100% ethanol, and

These types of specimens are fluids closely associated with mucosal surfaces, in low quantities, so collection should be done with the aid of swabs, cytological brush or small syringes. Secretions and exudates can be taken from upper respiratory airways and from

Respiratory *M. pneumoniae* infection can be assessed by culture and PCR in nasopharyngeal and oropharyngeal secretions, sputa, bronchoalveolar lavage and lung tissue obtained by biopsy. There are reports that nasopharyngeal and oropharyngeal specimens are equally effective for detection of *M. pneumoniae* by PCR, although it is desirable that both sites are

When neonatal mycoplasmal infections are suspected, endotracheal, nasopharyngeal and throat secretions are appropriate to evaluate respiratory infection., though specimens for culture should be transported quickly to laboratory since they are likely to contain at least a

Presence of mucous material in this kind of specimens frequently hampers appropriate processing for culture or PCR. Use of aggressive mucolytic agents (NaOH, n-acetyl-cisteine) can damage as well the mycoplasma cells, thus thorough homogenization by wide-bore pippeting is required prior to culture attempt. For nucleic acid extraction, addition of starch has been of help to enhance recovery of total genomic DNA from sputum samples [Harasawa *et al*., 1993]. In other study, dithiotreitol was used as the mucolytic agent without

It this worthy to note that differential sample preparation from the same specimen may be necessary when testing separate single-species PCRs on BAL, as described by [de Barbeyrac *et al*., 1993]. In that report, freeze-thawing cycles were applied for sample preparation for *M. genitalium* detection, while standard DNA extraction was needed for *M. pneumoniae* detection. During a study of Finnish patients with radiologically confirmed pneumonia, [Raty *et al*., 2005], evidence further supported the notion that selection of the appropriate specimen is crucial for diagnosis of *M. pneumoniae* infection. By means of a *M.* 

any apparent detrimental effect on mycoplasmal DNA integrity [Raty *et al*., 2005].

DNA, but presence of precipitated material may inhibit the amplification assay.

proceed with conventional DNA extraction protocols (unpublished data).

lower genital tract, and exceptionally from surgical wounds [Waites, 2006].

screened in parallel for better diagnostic yield [Waites *et al*., 2008].

few contaminating microorganisms [Waites *et al*., 2005].

**4.1 Exudates and secretions** 

**4.1.1 Respiratory tract** 

Since genital tract mycoplasmas are closely associated to live epithelial cells, collection of exudates must be avoided and vigorous scraping of epithelia must be done to obtain as many cells as possible. In this case, a higher associated flora is frequently present in the samples; therefore use of transport liquid media (for culture) of buffered solutions (for DNA extraction) is required immediately after sampling.

## **4.2 Sterile body fluids**

Collection of normally sterile body fluids is made through invasive procedures, usually performed by physicians under aseptic conditions [Wilson, 1996]. When specimens are going to be collected through puncture, careful disinfection of the skin spot must be done, this is crucial to both avoid contamination of the specimen with the skin's associated flora and to minimize the risk of introduction of bacteria into patient's body. Clinically, access of mycoplasmas to sterile body sites may be associated with an underlying immune compromise, and probably the bacteria spread from pulmonary or genital infectious foci [Cassell *et al*., 1994b; Waites & Talkington, 2004]. Ureaplasmas and mycoplasmas should always be sought from synovial fluid when hypogammaglobulinemic patients develop acute arthritis [Waites *et al*., 2000].

Since mycoplasma-containing body fluids rarely became turbid; these specimens should be concentrated 10-fold by high-speed centrifugation (aprox. 12,000 x g) and immediately resuspended in one tenth of the original supernatant if culture will be performed. Prior to DNA extraction, the resulting pellet can be washed 1-2 times with Hank's balanced salt solution or PBS, pH 7.4.

#### **4.3 Cell-rich fluids and tissues**

Unlike normally sterile body fluids, blood and semen are cell-rich fluid specimens, thus processing for culture or PCR is quite different. It is important to note that mycoplasmas have the ability to invade several cell types, including leukocytes and spermatozoa [Andreev *et al*., 1995; Baseman *et al*., 1995; Díaz-García *et al*., 2006; Girón *et al*., 1996; Jensen *et al*., 1994; Lo *et al*., 1993b, Rottem, 2003; Taylor-Robinson *et al*., 1991; Yavlovich *et al*., 2004], consequently a high input of cells into culture media may result in a higher probability of detection.

In contrast, when DNA extraction must be performed for PCR assays, depuration of the sample must be done, (i.e. erythrocyte lysis and selective enrichment for leukocytes in blood; density gradient-based purification of spermatozoa). Noteworthy, the average content of leukocyte DNA per milliliter of blood ranges from 32 to 76 µg, therefore surpasses considerably the amount of bacterial DNA in a specimen from an infected subject. [Greenfield & White, 1993], so a high amount of sample DNA should be added to the PCR reaction mixture to raise the chances to detect bacterial target sequences.

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 131

Since culture rely on viability of mycoplasmas to give a positive result, analytical comparisons between culture and PCR invariably will regard the second as less sensitive and less specific, which by any means is wrong. The PCR assay ideally detect target DNA sequences present in the sample, whether it comes from live, dead or uncultivable bacteria

Performance of non culture-based detection tests is frequently evaluated against culture, as the gold standard. Indirect assays measuring serologic responses as correlates of mycoplasma infections, have shown poor sensitivity and required at least 2 serum samples

When very few mycoplasma cells are present in a given specimen there is a high probability of obtaining false-negative results, even when the sensitivity of the specific PCR assay is high. To overcome this, several authors have developed culture-based pre-enrichment protocols for mycoplasmas, mycobacteria and *Actinobacillus* [Abele-Horn *et al.,* 1998; Díaz-García *et al.,* 2004; Flemmig *et al.,* 1995; Noussair *et al.,* 2009]. The effects of this procedure are, on one hand the dilution of potential undefined inhibitors, and on the other hand the promotion of short-term bacterial multiplication. This experimental approach has been termed as Culture-enhanced PCR (CE-PCR) [Abele-Horn *et al*., 1998]. The genomic DNA content in overnight enriched mycoplasma cultures are extracted by standard or commercial techniques, and then subjected to broad-range or species-specific PCR assays. Under this approach, improved detection of *M. pneumoniae* has been achieved in respiratory specimens [Abele-Horn

*et al.,* 1998], and of genital mycoplasmas in urine specimens [Díaz-García *et al.,* 2004].

Another culture-based enrichment approach for improvement of PCR detection of mycoplasmas is the cocultivation of these bacteria with permissive immortalized mammalian and/or insect cell lines [Kong *et al*., 2007; Volokhov *et al*., 2008]. Although this approach has been design for intentional screening of cell-derived biological and pharmaceutical products, including vaccines and cell culture substrates, it is a potential tool for biological enrichment of normally-sterile clinical specimens such as CFS, sera, synovial

Interestingly, strains of mycoplasma-free *Trichomonas vaginalis* are readily infected *in vitro* by *M. hominis* isolates, but not by other urogenital mycoplasmas. The infection can be detected by a *M. hominis*-specific PCR assay after long-term incubation, since the mycoplasma can be transmitted between the protozoan cells [Dessi *et al*., 2006; Rapelli *et al*., 2001]. The symbiotic interplay between *M. hominis* and *T. vaginalis* has been well established, as well a significant correlation between detection of both microorganisms in vaginal specimens from infected women [Dessi *et al*., 2006]. Thus it is likely to take advantage of such symbiosis and employ mycoplasma-free *T. vaginalis* cultures for specific enrichment of *M. hominis*-containing

Unlike the in-house PCR assays for diagnostic purposes, developed by several researchers, the commercial PCR kits are well standardized in terms of sensitivity and specificity,

taken several days apart, to be informative [Waites, 2000].

**5.2 Culture-enhanced PCR approach** 

clinical specimens prior to PCR detection tests.

**6. Commercial molecular diagnostic kits** 

[Persing, 1993].

fluid, etc.

In the case of solid tissues, mechanical homogenization is required to release single cells, either for culture or DNA extraction. A challenge for DNA extraction is when tissues have been formalin-fixed and/or paraffin-embedded since there is high risk of DNA damage [Shi *et al*., 2004].

A summary of the processing of different specimen types for intended mycoplasma detection is depicted in Figure 1.

Fig. 1. Differential specimen processing for mycoplasma detection.

## **5. Culture vs. nucleic acid amplification methods**

## **5.1 The gold standard for mycoplasmal infections**

Some genital mycoplasmas, *Ureaplasma spp*. and *M. hominis*, are the fastest growing species among the *Mollicutes*, and due to this, culture-based detection is still the first-line diagnostic approach. However, extrapolating this particular feature to all pathogenic human mycoplasmas is inaccurate. PCR amplification has become essential if fastidious, slow-growing, mycoplasma species are sought in certain clinical conditions, especially in patients with high risk of invasive infections (neonates) or when invasive methods of sampling are required [Waites *et al*., 2005]. It is well recognized that culture techniques are of poor or null value for detection of some mycoplasma species (i.e. *M. genitalium*) [Razin *et al*., 1998].

Since culture rely on viability of mycoplasmas to give a positive result, analytical comparisons between culture and PCR invariably will regard the second as less sensitive and less specific, which by any means is wrong. The PCR assay ideally detect target DNA sequences present in the sample, whether it comes from live, dead or uncultivable bacteria [Persing, 1993].

Performance of non culture-based detection tests is frequently evaluated against culture, as the gold standard. Indirect assays measuring serologic responses as correlates of mycoplasma infections, have shown poor sensitivity and required at least 2 serum samples taken several days apart, to be informative [Waites, 2000].

## **5.2 Culture-enhanced PCR approach**

130 Polymerase Chain Reaction

In the case of solid tissues, mechanical homogenization is required to release single cells, either for culture or DNA extraction. A challenge for DNA extraction is when tissues have been formalin-fixed and/or paraffin-embedded since there is high risk of DNA damage [Shi

A summary of the processing of different specimen types for intended mycoplasma

**Direct inoculacion**

**10-fold concentration by centrifugation (12,000 x g) Inoculationat 1:10 ratio (reconstitution)**

**Mince sample if required Inoculationat 1:10 ratio**

Fig. 1. Differential specimen processing for mycoplasma detection.

Some genital mycoplasmas, *Ureaplasma spp*. and *M. hominis*, are the fastest growing species among the *Mollicutes*, and due to this, culture-based detection is still the first-line diagnostic approach. However, extrapolating this particular feature to all pathogenic human mycoplasmas is inaccurate. PCR amplification has become essential if fastidious, slow-growing, mycoplasma species are sought in certain clinical conditions, especially in patients with high risk of invasive infections (neonates) or when invasive methods of sampling are required [Waites *et al*., 2005]. It is well recognized that culture techniques are of poor or null value for detection of some mycoplasma species (i.e. *M. genitalium*) [Razin

**Transport / growth broth or buffered solutions.** 

**5. Culture vs. nucleic acid amplification methods** 

**5.1 The gold standard for mycoplasmal infections** 

*et al*., 2004].

*et al*., 1998].

detection is depicted in Figure 1.

**Exudates and secretions (sputum, nasopharingeal swab, Cervical and vaginal swabs, etc.)** 

**Normally sterile fluids (CSF, Pleural, synovial, ammniotic, etc.)**

**Cell-rich fluids (blood, semen) and Solid-organ biopsies.**

When very few mycoplasma cells are present in a given specimen there is a high probability of obtaining false-negative results, even when the sensitivity of the specific PCR assay is high. To overcome this, several authors have developed culture-based pre-enrichment protocols for mycoplasmas, mycobacteria and *Actinobacillus* [Abele-Horn *et al.,* 1998; Díaz-García *et al.,* 2004; Flemmig *et al.,* 1995; Noussair *et al.,* 2009]. The effects of this procedure are, on one hand the dilution of potential undefined inhibitors, and on the other hand the promotion of short-term bacterial multiplication. This experimental approach has been termed as Culture-enhanced PCR (CE-PCR) [Abele-Horn *et al*., 1998]. The genomic DNA content in overnight enriched mycoplasma cultures are extracted by standard or commercial techniques, and then subjected to broad-range or species-specific PCR assays. Under this approach, improved detection of *M. pneumoniae* has been achieved in respiratory specimens [Abele-Horn *et al.,* 1998], and of genital mycoplasmas in urine specimens [Díaz-García *et al.,* 2004].

Another culture-based enrichment approach for improvement of PCR detection of mycoplasmas is the cocultivation of these bacteria with permissive immortalized mammalian and/or insect cell lines [Kong *et al*., 2007; Volokhov *et al*., 2008]. Although this approach has been design for intentional screening of cell-derived biological and pharmaceutical products, including vaccines and cell culture substrates, it is a potential tool for biological enrichment of normally-sterile clinical specimens such as CFS, sera, synovial fluid, etc.

Interestingly, strains of mycoplasma-free *Trichomonas vaginalis* are readily infected *in vitro* by *M. hominis* isolates, but not by other urogenital mycoplasmas. The infection can be detected by a *M. hominis*-specific PCR assay after long-term incubation, since the mycoplasma can be transmitted between the protozoan cells [Dessi *et al*., 2006; Rapelli *et al*., 2001]. The symbiotic interplay between *M. hominis* and *T. vaginalis* has been well established, as well a significant correlation between detection of both microorganisms in vaginal specimens from infected women [Dessi *et al*., 2006]. Thus it is likely to take advantage of such symbiosis and employ mycoplasma-free *T. vaginalis* cultures for specific enrichment of *M. hominis*-containing clinical specimens prior to PCR detection tests.

## **6. Commercial molecular diagnostic kits**

Unlike the in-house PCR assays for diagnostic purposes, developed by several researchers, the commercial PCR kits are well standardized in terms of sensitivity and specificity,

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 133

genes, the 16S-23S rRNA intergenic sequences, etc, have been already used as complementary comparative data, thus there is no unique phylogenetic tree for Mollicutes

There are several in-house species-specific end-point or real-time PCR assays developed to detect mycoplasmas in diverse respiratory and urogenital tract infections [Blanchard *et al*., 1993b; Loens *et al*., 2003b; Sung *et al*., 2006; van Kuppeveld *et al*., 1992; Wang *et al*, 1992]. Of those clinical entities, more than one mycoplasma species are commonly associated as etiologic agents, i.e. urethritis, infertility, pelvic inflammatory disease, etc. [Cassell *et al*, 1994b; Taylor-Robinson, 1996]. Thus, simultaneous testing of several species-specific or multiplex PCRs to determine all possible pathogenic mycoplasmas associated with a particular clinical entity would be very complicated. Combination of PCR amplification of a given highly conserved target genome sequence with determination of its nucleotide sequences and phylogenetic analysis has been successfully applied for diagnosis and identification of mycoplasmal etiologies in male urethritis cases [Hashimoto *et al*., 2006;

Due to their fastidious growth conditions and frequent cross-reactive antigenic profile, identification and typing of human mycoplasmas is a very difficult task. Other approaches termed "Random Amplified Polymorphic DNA" (RAPD) or "Arbitrarily Primed PCR" (AP-PCR), and "Amplified-Fragment Length Polimorphism" (AFLP), are PCR-based typing methods used for intra- and inter-species differentiation of mycoplasma isolates. The RAPD / AP-PCR method involves PCR amplification with a single arbitrary primer at low stringency, while AFLP method selectively amplifies restriction fragments from whole genome. These PCR-based genotyping techniques have allowed faster and reproducible typing of mycoplasmas for epidemiologic studies [Cousin-Allery *et al*., 2000; Geary & Forsyth, 1996; Grattard *et al*., 1995; Iverson-Cabral, *et al*., 2006; Kokotovic *et al*., 1999; Rawadi,

In today's clinical microbiology laboratory, introduction of PCR and other NATs has the potential to increase the speed and accuracy of bacterial detection/identification, especially of those fastidious microorganisms such as mycoplasmas. However, those molecular assays still have serious drawbacks that arise from inadequate acquisition, handling and processing of representative clinical specimens. False negative results ultimately can have a significant

It is widely accepted that molecular methods are more sensitive and specific than cultureand serology-based diagnostic approaches but, what does a ''positive'' test result mean clinically?. This issue is a matter of controversy for genital mycoplasmas since the duality of their relationship with their host: Is it a commensal or is it a pathogen?. The answer depends of an integral clinical evaluation of patients, where a "signs and symptoms"-focused

In the clinical setting, when negative results after mycoplasma-specific PCR assays are reported, the type and quality of the specimen, history of antibiotic treatment of the patient, and how representative was the specimen used for the assay, should be taken into account.

[Razin *et al*., 1998].

Yoshida *et al*., 2002].

1998; Schwartz *et al*., 2009].

impact on patient management.

sampling will improve laboratory diagnosis.

**8. Conclusion** 

allowing their global use in clinical microbiology laboratories. Thus inter-laboratory performance comparisons of such kits are suitable, including testing of several specimen types. Indeed, according to the *In Vitro Diagnostic Medical Devices Directive* 98/79/EC, all commercial diagnostic kits used in European countries must have the CE (*Conformité Européene*) label [Dosà *et al*., 1999].

Among commercially available real-time PCR kits are intended for *M. pneumoniae* detection, mainly targeting the P1 cytadhesin gene, including Nanogen Mycoplasma pn Q-PCR Alert kit (Nanogen Advanced Diagnostics); the Simplexa *Mycoplasma pneumoniae* kit (Focus Diagnostics, California); the Diagenode detection kit for *Mycoplasma pneumoniae*/*Chlamydophila pneumoniae* (Diagenode SA, Liège, Belgium); the Cepheid *Mycoplasma pneumoniae* ASR kit (Cepheid, Paris, France), and the Venor Mp-Qp PCR detection kit (Minerva Biolabs GmbH). It has been shown that these commercial kits had acceptable analytical sensitivity and performance with clinical specimens [Touati *et al*., 2009].

Interestingly, many commercially available extraction kits incorporate a buffer to lyses the bacteria and a silica matrix membrane (typically in column format) to trap the DNA or RNA. Several wash steps are required to remove protein and other macromolecules, and the purified DNA and RNA is then eluted from the membrane. Many of the manual extraction methods require several centrifugation steps. To reduce hands-on time, operator error, and sample contamination, semi-automated DNA or RNA extraction kits and equipment have been designed and are commercially available.

## **7. PCR, sequencing, phylogeny and molecular epidemiology**

The mycoplasmas may have evolved through regressive evolution from closely related Gram positive bacteria with low content of guanine plus cytosine (G+C), probably the Clostridia or Erysipelothrix [Bove, 1993; Brown *et al*., 2007; Razin *et al*., 1998]. The massive gene losses (i.e. genes involved in cell wall and aminoacid biosynthesis) had left mycoplasmas with a coding repertoire of 500 to 2000 genes [Sirand-Pugnet *et al*., 2007]. The G+C content in DNA of mycoplasmas varies from 23 to 40 mol%, while genome size range is 580–2200 Kbp, much smaller than those of most walled bacteria [Razin *et al*., 1998].

After PCR amplification and sequencing of the conserved 16S rDNA gene sequences from representative members of the Mollicutes, the resulting phylogenetic tree was shown to be monophyletic, arising from a single branch of the Clostridium ramosum branch [International Committee on Systematics of Prokaryotes- Subcommittee on the taxonomy of Mollicutes (ICSP-STM), 2010]. The Mollicutes split into two major branches: the AAP branch, containing the *Acholeplasma*, *Anaeroplasma* and *Asteroleplasma* genera, and the Candidatus *Phytoplasma* phyla; the other is the SEM branch that includes the *Spiroplasma*, *Entomoplasma*, *Mesoplasma*, *Ureaplasma* and *Mycoplasma* genera [Johansson *et al*., 1998; Maniloff, 1992; Razin *et al*., 1998]. Interestingly, the genus Mycoplasma is polyphyletic, with species clustering within the Spiroplasma, Pneumoniae and Hominis phylogenetic groups [Behbahani *et al*., 1993; Johansson *et al*., 1998; Maniloff, 1992]. Nevertheless, additional phylogenetic markers such as the elongation factor EF-Tu (tuf) gene, ribosomal protein genes, the 16S-23S rRNA intergenic sequences, etc, have been already used as complementary comparative data, thus there is no unique phylogenetic tree for Mollicutes [Razin *et al*., 1998].

There are several in-house species-specific end-point or real-time PCR assays developed to detect mycoplasmas in diverse respiratory and urogenital tract infections [Blanchard *et al*., 1993b; Loens *et al*., 2003b; Sung *et al*., 2006; van Kuppeveld *et al*., 1992; Wang *et al*, 1992]. Of those clinical entities, more than one mycoplasma species are commonly associated as etiologic agents, i.e. urethritis, infertility, pelvic inflammatory disease, etc. [Cassell *et al*, 1994b; Taylor-Robinson, 1996]. Thus, simultaneous testing of several species-specific or multiplex PCRs to determine all possible pathogenic mycoplasmas associated with a particular clinical entity would be very complicated. Combination of PCR amplification of a given highly conserved target genome sequence with determination of its nucleotide sequences and phylogenetic analysis has been successfully applied for diagnosis and identification of mycoplasmal etiologies in male urethritis cases [Hashimoto *et al*., 2006; Yoshida *et al*., 2002].

Due to their fastidious growth conditions and frequent cross-reactive antigenic profile, identification and typing of human mycoplasmas is a very difficult task. Other approaches termed "Random Amplified Polymorphic DNA" (RAPD) or "Arbitrarily Primed PCR" (AP-PCR), and "Amplified-Fragment Length Polimorphism" (AFLP), are PCR-based typing methods used for intra- and inter-species differentiation of mycoplasma isolates. The RAPD / AP-PCR method involves PCR amplification with a single arbitrary primer at low stringency, while AFLP method selectively amplifies restriction fragments from whole genome. These PCR-based genotyping techniques have allowed faster and reproducible typing of mycoplasmas for epidemiologic studies [Cousin-Allery *et al*., 2000; Geary & Forsyth, 1996; Grattard *et al*., 1995; Iverson-Cabral, *et al*., 2006; Kokotovic *et al*., 1999; Rawadi, 1998; Schwartz *et al*., 2009].

## **8. Conclusion**

132 Polymerase Chain Reaction

allowing their global use in clinical microbiology laboratories. Thus inter-laboratory performance comparisons of such kits are suitable, including testing of several specimen types. Indeed, according to the *In Vitro Diagnostic Medical Devices Directive* 98/79/EC, all commercial diagnostic kits used in European countries must have the CE (*Conformité* 

Among commercially available real-time PCR kits are intended for *M. pneumoniae* detection, mainly targeting the P1 cytadhesin gene, including Nanogen Mycoplasma pn Q-PCR Alert kit (Nanogen Advanced Diagnostics); the Simplexa *Mycoplasma pneumoniae* kit (Focus Diagnostics, California); the Diagenode detection kit for *Mycoplasma pneumoniae*/*Chlamydophila pneumoniae* (Diagenode SA, Liège, Belgium); the Cepheid *Mycoplasma pneumoniae* ASR kit (Cepheid, Paris, France), and the Venor Mp-Qp PCR detection kit (Minerva Biolabs GmbH). It has been shown that these commercial kits had acceptable analytical sensitivity and performance with clinical specimens [Touati *et al*.,

Interestingly, many commercially available extraction kits incorporate a buffer to lyses the bacteria and a silica matrix membrane (typically in column format) to trap the DNA or RNA. Several wash steps are required to remove protein and other macromolecules, and the purified DNA and RNA is then eluted from the membrane. Many of the manual extraction methods require several centrifugation steps. To reduce hands-on time, operator error, and sample contamination, semi-automated DNA or RNA extraction kits and equipment have

The mycoplasmas may have evolved through regressive evolution from closely related Gram positive bacteria with low content of guanine plus cytosine (G+C), probably the Clostridia or Erysipelothrix [Bove, 1993; Brown *et al*., 2007; Razin *et al*., 1998]. The massive gene losses (i.e. genes involved in cell wall and aminoacid biosynthesis) had left mycoplasmas with a coding repertoire of 500 to 2000 genes [Sirand-Pugnet *et al*., 2007]. The G+C content in DNA of mycoplasmas varies from 23 to 40 mol%, while genome size range is 580–2200 Kbp, much smaller than those of most walled bacteria [Razin *et al*.,

After PCR amplification and sequencing of the conserved 16S rDNA gene sequences from representative members of the Mollicutes, the resulting phylogenetic tree was shown to be monophyletic, arising from a single branch of the Clostridium ramosum branch [International Committee on Systematics of Prokaryotes- Subcommittee on the taxonomy of Mollicutes (ICSP-STM), 2010]. The Mollicutes split into two major branches: the AAP branch, containing the *Acholeplasma*, *Anaeroplasma* and *Asteroleplasma* genera, and the Candidatus *Phytoplasma* phyla; the other is the SEM branch that includes the *Spiroplasma*, *Entomoplasma*, *Mesoplasma*, *Ureaplasma* and *Mycoplasma* genera [Johansson *et al*., 1998; Maniloff, 1992; Razin *et al*., 1998]. Interestingly, the genus Mycoplasma is polyphyletic, with species clustering within the Spiroplasma, Pneumoniae and Hominis phylogenetic groups [Behbahani *et al*., 1993; Johansson *et al*., 1998; Maniloff, 1992]. Nevertheless, additional phylogenetic markers such as the elongation factor EF-Tu (tuf) gene, ribosomal protein

*Européene*) label [Dosà *et al*., 1999].

been designed and are commercially available.

**7. PCR, sequencing, phylogeny and molecular epidemiology** 

2009].

1998].

In today's clinical microbiology laboratory, introduction of PCR and other NATs has the potential to increase the speed and accuracy of bacterial detection/identification, especially of those fastidious microorganisms such as mycoplasmas. However, those molecular assays still have serious drawbacks that arise from inadequate acquisition, handling and processing of representative clinical specimens. False negative results ultimately can have a significant impact on patient management.

It is widely accepted that molecular methods are more sensitive and specific than cultureand serology-based diagnostic approaches but, what does a ''positive'' test result mean clinically?. This issue is a matter of controversy for genital mycoplasmas since the duality of their relationship with their host: Is it a commensal or is it a pathogen?. The answer depends of an integral clinical evaluation of patients, where a "signs and symptoms"-focused sampling will improve laboratory diagnosis.

In the clinical setting, when negative results after mycoplasma-specific PCR assays are reported, the type and quality of the specimen, history of antibiotic treatment of the patient, and how representative was the specimen used for the assay, should be taken into account.

Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 135

Blanchard A, Hentschel J, Duffy L, Baldus K, Cassell GH. (1993a). Detection of *Ureaplasma* 

*Diseases*, Vol. 17, Suppl. 1 (August 1993), pp. S148-S153, ISSN 1058-4838 Blanchard, A., Yáñez, A., Dybvig, K., Watson, H.L., Griffiths, G., & Cassell, G.H. (1993b).

*Microbiology*, Vol. 31, No. 5, (May 1993), pp. 1358-1361, ISSN 0095-1137 Bové, J.M. (1993). Molecular Features of *Mollicutes*. *Clinical Infectious Diseases,* Vol. 17, Suppl

Brown, D.R., Whitcomb, R.F., & Bradbury, J.M. (2007). Revised Minimal Standards for

Cassell, G.H., Blanchard, A., Duffy, L., Crabb, D., & Waites, K.B. (1994a). *Mycoplasma*s, In:

Cassell, G.H., Yáñez, A., Duffy, L. B., Moyer, J., Cedillo, L., Hammerschlag, M.R., Rank,

Choppa, P.C., Vojdani, A., Tagle, C., Andrin, R., & Magtoto, L. (1998). Multiplex PCR for

Cousin-Allery, A., Charron, A., de Barbeyrac, B., Fremy, G., Jensen, J.S., Renaudin, H., &

de Barbeyrac, B., Bernet-Poggi, C., Febrer, F., Renaudin, H., Dupon, M., & Bebear, C. (1993).

Dessì, D., Delogu, G., Emonte, E., Catania, M.R., Fiori, P.L., & Rappelli, P. (2005). Long-Term

1, (August 1993), pp. S10-S31, ISSN 1058-4838

(November 2007), pp. 2703–2719, ISSN 1466-5026

*Letters* Vol, 3, p. 456, ISSN 1023-1226

1993), pp. S83-S89, ISSN 1058-4838

0893-8512

0890-8508

9567

111, ISSN 0950-2688

*urealyticum* by Polymerase Chain Reaction in the Urogenital Tract of Adults, in Amniotic Fluid, and in the Respiratory Tract of Newborns. *Clinical Infectious* 

Evaluation of Intraspecies Genetic Variation Within The 16S rRNA Gene of *Mycoplasma hominis* and Detection by Polymerase Chain Reaction. *Journal of Clinical* 

Description of New Species of the Class *Mollicutes* (Division Tenericutes). *International Journal of Systematic and Evolutionary Microbiology*, Vol. 57, No. 11,

*Clinical and Pathogenic Microbiology*, B.J. Howard, J.F., Keiser, A.S. Weissfeld, T.F. Smith, & R.C. Tilton (Eds.), 491-502, Mosby, ISBN 978-0801664267, Boston, USA. Cassell, G.H., Waites, K.B., Watson, H.L., Crouse, D.T., & Harasawa, R. (1993). *Ureaplasma* 

*urealyticum* Intrauterine Infection: Role in Prematurity and Disease in Newborns. *Clinical Microbiology Reviews*, Vol. 6, No. 1, (January-March 1993), pp. 69-87, ISSN

R.G., & Glass, J.I. (1994b). Detection of *Mycoplasma fermentans* in the Respiratory Tract of Children with Pneumonia. 10th International Congress of the International Organization for Mycoplasmology (IOM), Bordeaux, France, July 1994. In *IOM* 

the Detection of *Mycoplasma fermentans, M. hominis* and *M. penetrans* in Cell Cultures and Blood Samples of Patients With Chronic Fatigue Syndrome. *Molecular and Cellular Probes*, Vol. 12, No. 5, (October 1998), pp. 301-308, ISSN

Bebear, C. (2000). Molecular Typing of *Mycoplasma pneumoniae* Strains by PCR-Based Methods and Pulse-Field Gel Electrophoresis. Application to French and Danish Isolates. *Epidemiology and Infection*, Vol. 124, No. 1, (February 2000), pp. 103-

Detection of *Mycoplasma pneumoniae* and *Mycoplasma genitalium* in Clinical Samples by Polymerase Chain Reaction. *Clinical Infectious Diseases*, Vol. 17, Supp 1 (August

Survival and Intracellular Replication of *Mycoplasma hominis* in *Trichomonas vaginalis* Cells: Potential Role of the Protozoon in Transmitting Bacterial Infection. *Infection and Immunity*, Vol. 73, No. 2 (February 2005), pp. 1180–1186, ISSN 0019-

Therefore, any set of diagnostic results must be reviewed and critically interpreted before diagnosis and intervention measures are made.

#### **9. Acknowledgements**

The authors want to give a very special thanks to the Instituto Nacional de Perinatología-SS, Grant; 2122250-077261 and the Facultad de Medicina, UNAM, for their support.

SFM is a Doctoral Student. Postgraduate Program; Molecular Biomedicine and Biotechnology at the Escuela Nacional de Ciencias Biológicas, IPN, México, D.F.; FJDG has been granted with the National Research Fellowship I of the CONACyT, México, D.F.

Corresponding author: M. Sc. Saúl Flores-Medina. Laboratorio de Biología Molecular, Departamento de Infectología, INPer. Montes Urales # 800, Col. Lomas de Virreyes. C.P. 11,000, México, D.F., México. Phone: +52(55)55209900 ext. 520. Email: s.flores@inper.mx

#### **10. References**


Therefore, any set of diagnostic results must be reviewed and critically interpreted before

The authors want to give a very special thanks to the Instituto Nacional de Perinatología-SS,

SFM is a Doctoral Student. Postgraduate Program; Molecular Biomedicine and Biotechnology at the Escuela Nacional de Ciencias Biológicas, IPN, México, D.F.; FJDG has been granted with the National Research Fellowship I of the CONACyT, México, D.F.

Corresponding author: M. Sc. Saúl Flores-Medina. Laboratorio de Biología Molecular, Departamento de Infectología, INPer. Montes Urales # 800, Col. Lomas de Virreyes. C.P. 11,000, México, D.F., México. Phone: +52(55)55209900 ext. 520. Email: s.flores@inper.mx

Abele-Horn, M., Busch, U., Nitschko, H., Jacobs, E., Bax, R., Pfaff, F., Schaffer, B., &

Andreev, J., Borovsky, Z., Rosenshine, I., & Rottem, S. (1995). Invasion of HeLa Cells by

Atkinson, T.P., Balish, M.F., & Waites, K.B. (2008). Epidemiology, Clinical Manifestations,

Baseman, J.B., & Tully, J.G. (1997). Mycoplasmas: Sophisticated Reemerging and Burdened

Baseman, J.B., Lange, M., Criscimagna, N.L., Girón, J.A., & Thomas, C.A. (1995). Interplay

Behbahani, N., Blanchard, A., Cassell, G.H., & Montagnier, L. (1993). Phylogenetic Analysis

Bernet, C., Garret, M., de Barbeyrac, B., Bebear, C., & Bonnet, J. (1989). Detection of

*Letters*, Vol. 109, No. 1, (May 1993), pp. 63-6, ISSN 0378-1097

No. 2-3 (August 1992): pp. 277-281, ISSN 0378-1097

Heesemann, J. (1998). Molecular Approaches to Diagnosis of Pulmonary Diseases Due to *Mycoplasma pneumoniae*. *Journal of Clinical Microbiology,* Vol. 36, No. 2

*Mycoplasma penetrans* and the Induction of Tyrosine Phosphorylation of a 145 kDa Host Cell Protein. *FEMS Microbiology Letters,* Vol. 132, No. 3, (October 1995), pp.

Pathogenesis and Laboratory Detection of *Mycoplasma pneumoniae* Infections. *FEMS Microbiology Reviews*, Vol. 32, No. 6, (November 2008), pp. 956–973, ISSN

by Their Notoriety. *Emerging Infectious Diseases*, Vol. 3, No. 1, (January-March 1997),

Between *Mycoplasma*s and Host Target Cells. *Microbial Pathogenesis,* Vol. 19, No. 2,

of *Mycoplasma penetrans*, Isolated From HIV-Infected Patients. *FEMS Microbiology* 

*Mycoplasma pneumoniae* by Using Polymerase Chain Reaction. *Journal of Clinical Microbiology*, Vol. 27, No. 11, (November 1989), pp. 2492-2496, ISSN 0095-1137 Blanchard A, Crabb DM, Dybvig K, Duffy LB, Cassell GH. (1992). Rapid Detection of tetM in

*Mycoplasma hominis* and *Ureaplasma urealyticum* by PCR: *tetM* Confers Resistance to Tetracycline But not Necessarily to Doxycycline. *FEMS Microbiology Letters,* Vol. 74,

Grant; 2122250-077261 and the Facultad de Medicina, UNAM, for their support.

(February 1998), pp. 548-551, ISSN 0095-1137

(August 1995), pp. 105-116, ISSN 0882-4010

189-194, ISSN 0378-1097

pp. 21-32, ISSN 1080-6040

0168-6445

diagnosis and intervention measures are made.

**9. Acknowledgements** 

**10. References** 


Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 137

Harasawa R., Misuazawa H., & Nakagawa.T. (1993). Detection and Tentative Identification

Hashimoto, O., Yoshida, T., Ishiko, H., Ido, M., & Deguchi, T. (2006). Quantitative Detection

*Chemotherapy*, Vol. 12, No. 1, (February 2006), pp. 25-30, ISSN 1341-321X Himmelreich R., Hilbert, H., Plagens, H., Pirkl, E., Li, B.C., Herrmann, R. (1996). Complete

(July-August 1993), pp. 489-493, ISSN 0923-2508

2010), pp. 47-53, ISSN 2009.04.009

3715-26, ISSN 0019-9567

pp. 59-77, ISSN 1615-9853

ISBN 0-89603-525-5, Totowa, NJ

X, U.S.A.

1899

T. White (Eds.), pp. 122-137. American Society for Microbiology, ISBN 1-55581-056-

of Dominant Mycoplasma Species in Cell Cultures by Restriction Analysis of the 16S-23S rRNA Intergenic Spacer Regions. *Research in Microbiology*, Vol. 144, No. 6,

and Phylogeny-Based Identification of Mycoplasmas and Ureaplasmas from Human Immunodeficiency VirusType 1-Positive Patients. *Journal of Infection and* 

Sequence Analysis of the Genome of the Bacterium *Mycoplasma pneumoniae*. *Nucleic Acids Research*, Vol. 24, No. 22, (November 1996), pp. 4420-4449. ISSN: 0305-1048 Horz, H-P., Scheer, S., Vianna, M.E., & Conrads G. (2010). New Methods for Selective

Isolation of Bacterial DNA from Human Clinical. *Anaerobe*, Vol. 16, No. 1, (February

Viruses and Atypicals in Acute Respiratory Infections. *Journal of Clinical Virology*,

Detection of *Mycoplasma pneumoniae* by Two Polymerase Chain Reactions and Role of *M. pneumoniae* in Acute Respiratory Tract Infections in Pediatric Patients. *Journal of Infectious Diseases*, Vol. 173, No. 6 (June 1996), pp. 1445-1452, ISSN 0022-

Mollicutes (ICSP-STM). (2011). *International Journal of Systematic and Evolutionary* 

Heterogeneity of the mgpB Gene in *Mycoplasma genitalium* is Extensive *in vitro* and *in vivo* and Suggests that Variation is Generated via Recombination With Repetitive Chromosomal Sequences. *Infection and Immunity*, Vol. 74, No. 7, (July 2006), pp.

Method to Perform Genome Annotation. *Proteomics*, Vol. 4, No. 1, (January 2004),

Cultured Vero Cells as Demonstrated by Electron Microscopy. *International Journal of Experimental Pathology,* Vol. 75, No. 2, (April 1994), pp. 91-98, ISSN 0959-9673 Jensen, J.S., Uldum, S.A., Søndergård-Andersen, J., Vuust, J., & Lind, K. (1991). Polymerase

Chain Reaction for Detection of *Mycoplasma genitalium* in Clinical Samples. *Journal of Clinical Microbiology*, Vol. 29, No. 1, (January 1991), pp. 46-50. ISSN 0095-1137 Johansson, K.E., Heldtander, M.U.K., & Petterson, B. (1998). Characterization of

Mycoplasmas by PCR and Sequence Analysis with Universal 16S rDNA Primers, In: *Mycoplasma protocols*, R. Miles, & R. Nicholas (Eds.), 145-165, Humana Press,

Ieven, M. (2007). Currently Used Nucleic Acid Amplification Tests for the Detection of

Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H.G., & Goossens, H. (1996).

International Committee on Systematics of Prokaryotes- Subcommittee on the taxonomy of

Jaffe, J.D., Berg, H.C., & Church, G.M. (2004). Proteogenomic Mapping as a Complementary

Jensen, J.S., Blom, J., & Lind, K. (1994). Intracellular Location of *Mycoplasma genitalium* in

*Microbiology*, Vol. 61, No. 3 (March 2011), pp. 695–697, ISSN 1466-5026 Iverson-Cabral, S.L., Astete, S.G., Cohen, C.R., Rocha, E.P., & Totten, P.A. (2006). Intrastrain

Vol.40, No.4, (December 2007), pp. 259-276, ISSN 1386-6532


Dessi, D., Rappelli, P., Diaz, N., Cappuccinelli, P., & Fiori, P.L. (2006). *Mycoplasma hominis*

Díaz-García, F.J., Giono-Cerezo, S., Tapia, J.L., Flores-Medina, S., López-Hurtado, M., &

Díaz-García, F.J., Herrera-Mendoza, A.P., Giono-Cerezo, S., & Guerra-Infante, F. (2006).

Dosá, E., Nagy, E., Falk, W., Szöke, I., & Ballies, U. (1999). Evaluation of the Etest for

Flemmig, T.F., Rüdiger, S., Hofmann, U., Schmidt, H., Plaschke, B., Strätz, A., Klaiber, B., &

Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., Fleischmann, R.D.,*et al*.

Geary, S.J., & M.H. Forsyth. (1996). PCR: Random Amplified Polymorphic DNA

Girón, J.A., Lange, M., & Baseman, J.B. (1996). Adherence, Fibronectin Binding, and

Glass, J.I., Lefkowitz, E.J., Glass, J.S., Heiner, C.R., Chen, E.Y., & Cassell, G.H. (2000). The

Grattard, F., Pozzetto, B., de Barbeyrac, B., Renaudin, H., Clerc, M., Gaudin, O.G., & Bébéar

Grau, R., Kovacic, R., Griffais, R., Launay, V., & Montagnier, L. (1994). Development of PCR-

Greenfield, L., & White, T.J. (1993). Sample Preparation Methods. In: *Diagnostic Molecular* 

(December1995), pp. 3102-3105, ISSN 0095-1137

No. 5235, (October 1995), pp. 397-404, ISSN 0036-8075

407, No. 6805, (October 2000), pp. 757-62, ISSN 0028-0836

(December 1995), pp. 383-389, ISSN 0890-8508

139–148. ISSN 0890-8508

2028-2034, ISSN 1093-9946

ISSN 1201-9712

0268-1161

San Diego, Ca.

0019-9567

7453

and *Trichomonas vaginalis*: A Unique Case of Symbiotic Relationship Between Two Obligate Human Parasites. *Frontiers in Bioscience*, Vol. 11, (September 2006), pp.

Guerra-Infante, F.M. (2004). Overnight Enrichment Culture Improves PCR-Based Detection of Genital Mycoplasmas in Urine Samples. 11th. ICID Abstracts. *International Journal of Infectious Diseases*, Vol. 8, Suppl. 1, (March 2004), pp. S130,

*Mycoplasma hominis* Attaches to and Locates Intracellularly On Human Spermatozoa. *Human Reproduction,* Vol. 21, No. 6, (June 2006), pp. 1591-1598, ISSN

susceptibility testing of *Mycoplasma hominis* and *Ureaplasma urealyticum*. *The Journal of Antimicrobial Chemotherapy,* Vol. 43, No. 4, (April 1999), pp. 575-578, ISSN 0305-

Karch, H. (1995). Identification of *Actinobacillus actinomycetemcomitans* in Subgingival Plaque by PCR. *Journal of Clinical Microbiology*, Vol. 33, No. 12

(1995). The minimal gene complement of *Mycoplasma genitalium*. *Science*, Vol. 270,

Fingerprinting. In: *Molecular and Diagnostic Procedures in Mycoplasmology*, J.G. Tully & S. Razin (Eds.), 81-85, Diagnostic procedures. Academic Press, ISBN 0125838069,

Induction of Cytoskeleton Reorganization in Cultured Human Cells by *Mycoplasma penetrans*. *Infection and Immunity,* Vol. 64, No. 1, (January 1996), pp. 197-208, ISSN

Complete Sequence of the Mucosal Pathogen *Ureaplasma urealyticum*. *Nature*, Vol.

C. (1995). Arbitrarily-Primed PCR Confirms the Differentiation of Strains of *Ureaplasma urealyticum* Into Two Biovars. *Molecular and Cellular Probes*, Vol. 9, No. 6,

Based Assays for the Detection of Two Human Mollicute species, *Mycoplasma penetrans and M. hominis*. *Molecular and Cellular Probes*, Vol. 8, No. 2 (April 1994), pp.

*Microbiology, Principles and Applications*, D. H. Persing, T. F. Smith; F. C. Tenover, &

T. White (Eds.), pp. 122-137. American Society for Microbiology, ISBN 1-55581-056- X, U.S.A.


Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 139

Maeda, S., Deguchi, T., Ishiko, H., Matsumoto, T., Naito, S., Kumon, H., Tsukamoto, T.,

Maniloff, J. (1992). Phylogeny of Mycoplasmas. In: *Mycoplasmas: Molecular biology and* 

Mothershed, E.A., & Whitney, A.M. (2006). Nucleic Acid-Based Methods for the Detection of

Palmer, H.M., Gilroy, C.B., Furr, P.M., & Taylor-Robinson, D. (1991). Development and

Patel, M.A., & Nyirjesy, P. (2010). Role of *Mycoplasma* and *Ureaplasma* Species in Female

Povlsen, K,. Thorsen, P., & Lind, I. (2001). Relationship of *Ureaplasma urealyticum* Biovars to

Povlsen, K., Bjørnelius, E., Lidbrink, P., & Lind, I. (2002). Relationship of *Ureaplasma* 

Rådström, P., Knutsson, R., Wolffs, P., Lövenklev, M. & Löfström, C. (2004). Pre-PCR

*Biotechnology*, Vol. 26, No. 2, (February 2004), pp. 133-146, ISSN 1073–6085 Rappelli, P.,· Carta, F., Delogu, G., Addis, M.F.,· Dessì, D., Cappuccinelli, P., & Fiori, P.

549-559, American Society for Microbiology, ISBN 1-55581-050-0, U.S.A. Mendoza N., Ravanfar, P., Shetty, A.K., Pellicane, B.L., Creed, R., Goel, S., & Tyring, S.K.

9, (September 2004), pp. 750-754. ISSN: 0919-8172

Verlag, ISBN 978-3-642-14663-3, Heidelberg, Berlin

(November 2010), pp. 417–422, ISSN 1523-3847

No. 1, (January 2001), pp. 65-67, ISSN 0934-9723

Microbiology (2001) 175 :70–74.

1452-1457, ISSN 0095-1137

1097

9723

1137

*Journal of Clinical Microbiology*, Vol. 31, No. 5, (May 1993), pp. 1088-1094. ISSN: 0095-

Onodera, S., & Kamidono, S. (2004). Detection of *Mycoplasma genitalium*, *Mycoplasma hominis, Ureaplasma parvum* (Biovar 1) and *Ureaplasma urealyticum* (Biovar 2) in Patients with Non-gonococcal Urethritis Using Polymerase Chain Reaction-Microtiter Plate Hybridization. *International Journal of Urology,* Vol. 11, No.

*Pathogenesis*, J. Maniloff, R.N. McElhaney, L.R. Finch , & J.B. Baseman (Eds.), pp.

(2011). Genital Mycoplasma Infection, In: *Sexually Transmitted Infections and Sexually Transmitted Diseases*, G. Gross, & S.K. Tyring (Eds.), 197-201, Springer-

Bacterial Pathogens: Present and Future Considerations for the Clinical Laboratory. *Clinica Chimica Acta*, Vol. 363, No. 1-2, (January 2006), pp. 206–220, ISSN 0009-8981 Noussair L, Bert F, Leflon-Guibout V, Gayet N, Nicolas-Chanoine MH. (2009) Early

Diagnosis of Extrapulmonary Tuberculosis by a New Procedure Combining Broth Culture and PCR. *Journal of Clinical Microbiology*, Vol. 47, No. 5 (May 2009), pp.

Evaluation of the Polymerase Chain Reaction to Detect *Mycoplasma genitalium*. *FEMS Microbiology Letters*, Vol. 77, No. 2-3, (January 1991), pp. 199-204. ISSN 0378-

Lower Genital Tract Infections. *Current Infectious Diseases Report*, Vol. 12, No. 6,

the Presence or Absence of Bacterial Vaginosis in Pregnant Women and to the Time of Delivery. *European Journal of Clinical Microbiology and Infectious Diseases*, Vol. 20,

*urealyticum* Biovar 2 to Nongonococcal Urethritis. *European Journal of Clinical Microbiology and Infectious Diseases,* Vol. 21, No. 2, (February) pp. 97-101, ISSN: 0934-

Processing Strategies to Generate PCR-Compatible Samples. *Molecular* 

(2001). *Mycoplasma hominis* and *Trichomonas vaginalis* Symbiosis: Multiplicity of Infection and Transmissibility of *M. hominis* to Human Cells. Archives of


Kokotovic, B., Friis, N.F., Jensen, J.S., & Ahrens, P. (1999). Amplified-Fragment Length

Kong, F., Ma, Z., James, G., Gordon, S., & Gilbert, G.L. (2000). Species Identification and

Kong, H., Volokhov, D.V., George, J., Ikonomi, P., Chandler, D., Anderson, C., & Chizhikov,

Kovacic, R., Grau, O., & Blanchard, A. (1996). PCR: Selection of Target Sequences. In:

Lo, A.C.T., & Kam, K.M. (2006). Review of Molecular Techniques for Sexually Transmitted

Lo, S.C., Hayes, M.M., Tully, J.G., Wang, R.Y., Kotani, H., Pierce, P.F., Rose, D.L., & Shih,

Lo, S.C., Hayes, M.M., & Kotani, H. Pierce PF, Wear DJ, Newton PB 3rd, Tully JG, Shih JW.

Lo, S.C., Wear DJ, Green SL, Jones PG, Legier JF. (1993b). Adult Respiratory Distress

Loens, K., Goossens, H., & Ieven. M. (2010). Acute Respiratory Infection Due to *Mycoplasma* 

Loens, K., Ieven, M., Ursi, D., Beck, T., Overdijk, M., Sillekens, P., & Goossens, H. (2003a).

Loens, K., Ursi, D., Goossens, H., & Ieven, M. (2003b). Molecular Diagnosis of *Mycoplasma* 

Lüneberg, E., Jensen, J.S., & Frosch, M. (1993). Detection of *Mycoplasma pneumoniae* by

11, (November 2003), pp. 4915-4923, ISSN 0095-1137

*Pathology,* Vol. 6, No. 3, (May 1993), pp. 276-280, ISSN 0893-39520

No. 10, (October 1999), pp. 3300-3307, ISSN 0095-1137

& C.W. Stratton (Ed.), 353-386, ISBN 0387-32892

ISSN 0095-1137

Diego, Cal

pp. 223-232, ISSN 0175-7598

pp. 357-364, ISSN 0020-7713

pp. S259-S263, ISSN 1058-4838

4448-4450, ISSN 0095-1137

ISSN 0934-9723

Polymorphism Fingerprinting of *Mycoplasma* Species. J. Clin. Microbiol, Vol. 37,

Subtyping of *Ureaplasma parvum* and *Ureaplasma urealyticum* Using PCR-Based Assays. *Journal of Clinical Microbiology*, Vol. 38, No. 3 (March 2000), pp. 1175–1179.

V. (2007). Application of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT). Applied Microbiology and Biotechnology, Vol. 77, No. 1 (November 2007),

*Molecular and Diagnostic Procedures in Mycoplasmology*, J.G. Tully & S. Razin (Eds.), 53-60, Diagnostic procedures. Academic Press, ISBN 012-583806-9, San

Diseases Diagnosis, In: *Advanced Techniques in Diagnostic Microbiology*, Y-W. Tang,

J.W. (1992). *Mycoplasma penetrans* sp. nov., From the Urogenital Tract of Patients With AIDS. *International Journal of Systematic Bacteriology,* Vol. 42, No. 3 (July 1992),

(1993a). Adhesion Onto and Invasion Into Mammalian Cells by *Mycoplasma penetrans* - A Newly Isolated Mycoplasma From Patients with AIDS. *Modern* 

Syndrome With or Without Systemic Disease Associated With Infections Due to *Mycoplasma fermentans*. *Clinical Infectious Diseases*, Vol. 17, Suppl. 1 (August 1993),

*pneumoniae*: Current Status of Diagnostic Methods. *European Journal of Clinical Microbiology and Infectious Diseases*, Vol. 29, No. 9, (September 2010), pp. 1055–1069,

Detection of *Mycoplasma pneumoniae* by Real-Time Nucleic Acid Sequence-Based Amplification. *Journal of Clinical Microbiology*, Vol. 41, No. 9, (September 2003), pp.

*pneumoniae* Respiratory Tract Infections*. Journal of Clinical Microbiology*, Vol. 41, No.

Polymerase Chain Reaction and Nonradioactive Hybridization in Microtiter Plates.

*Journal of Clinical Microbiology*, Vol. 31, No. 5, (May 1993), pp. 1088-1094. ISSN: 0095- 1137


Molecular Diagnostics of Mycoplasmas: Perspectives from the Microbiology Standpoint 141

Taylor-Robinson, D., Davies, H. A., Sarathchandra, P., & Furr, P. M. (1991). Intracellular

Touati, A., Benard, A., Hassen, A.B., Bébéar, C.M., & Pereyre, S. (2009). Evaluation of Five

Ursi, J.P., Ursi, D., Ieven, M., & Pattyn, S.R. (1992). Utility of an Internal Control for the

van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bölske G, van der Logt JT, Melchers

van Kuppeveld, F.J.M., van der Logt, J.T.M., Angulo, A.F., van Zoest, M.J., Quint, W.G.,

Vaneechoutte, M., & Van eldere, J. (1997). The Possibilities and Limitations of Nucleic Acid

Waites, K.B. (2006). Mycoplasma and Ureaplasma, In: *Congenital and Perinatal Infections: A* 

Waites, K.B., & Talkington, D.F. (2004). *Mycoplasma pneumoniae* and Its Role as a Human

Waites, K.B., & Talkington, D.F. (2005). New Developments in Human Diseases Due to

Waites, K.B., Balish M.F., & Atkinson, T. P. (2008). New Insights Into the Pathogenesis and

Waites, K.B., Bebear, C.M., Robertson, J.A., Talkington, D.F., & Kenny, G.E. (2000).

*Nolte*. Washington: American Society for Microbiology.

*Microbiology*, Vol. 46, No. 3, (March 1997), pp. 188-194, ISSN 0022-2615 Volokhov, D.V., Kong, H., George, J., Anderson, C., & Chizhikov, V.E. (2008). Biological

6, (December 1991), pp. 705-714, ISSN 0959-9673

100, No. 7 (July 1992), pp. 635-639, ISSN 0903-4641

2009), pp. 2269-2271, ISSN 0095-1137

1994), pp. 149-152, ISSN 0099-2240

2240

5391, ISSN 0099-2240

ISSN 0893-8512

58829-297-5, Totowa, NJ.

ISBN 0849398614, Norwich, U.K

pp. 635–648, ISSN 1746-0913

Location of *Mycoplasma*s in Cultured Cells Demonstrated by Immunocytochemistry and Electron Microscopy. *International Journal of Experimental Pathology,* Vol. 72, No.

Commercial Real-Time PCR Assays for Detection of *Mycoplasma pneumoniae* in Respiratory Tract Specimens. *Journal of Clinical Microbiology*, Vol. 47, No. 7, (July

Polymerase Chain Reaction. Application to Detection of *Mycoplasma pneumoniae* in Clinical Specimens. *Acta Pathologica Microbiologica et Immunologica Scandinavica*, Vol.

WJ. Detection of Mycoplasma Contamination in Cell Cultures by a Mycoplasma Group-Specific PCR. *Applied and Environmental Microbiology*, Vol. 60, No. 1 (January

Niesters, H.G., Galama, J.M., & Melchers, W.J. (1992). Genus-and Species-Specific Identification of Mycoplasmas by 16S rRNA Amplification. *Applied and Environmental Microbiology,* Vol. 58, No. 8, (August 1992), pp. 2606-2615, ISSN 0099-

Amplification Technology in Diagnostic Microbiology. *Journal of Medical* 

Enrichment of *Mycoplasma* Agents by Cocultivation with Permissive Cell Cultures. *Applied and Environmental Microbiology*, Vol. 74, No. 17 (September 2008), pp. 5383-

*Concise Guide to Diagnosis*, C. Hutto (Ed.), 271-288, Humana Press Inc., ISBN 1-

Pathogen. *Clinical Microbiology Reviews*, Vol. 17, No. 4, (October 2004), pp. 697–728,

Mycoplasmas. In: *Mycoplasmas: pathogenesis, molecular biology, and emerging strategies for control*, A. Blanchard, & G. Browning (Eds.), 289-354, Horizon Scientific Press,

Detection of *Mycoplasma pneumoniae* Infections. *Future Microbiology*, Vol. 3, No. 6,

Laboratory Diagnosis of Mycoplasmal Infections. Cumitech 34. *Coordinating ed. FS* 


Raty, R., Ronkko, E. & Kleemola, M. (2005). Sample Type is Crucial to the Diagnosis of

Rawadi, G.A. (1998). Characterization of Mycoplasmas by RAPD Fingerprinting. In:

Razin, S. (1992). Peculiar Properties of Mycoplasmas: The Smallest Self-Replicating

Razin, S. (1994). DNA Probes and PCR in diagnosis of Mycoplasma Infections. *Molecular and Cellular Probes*, Vol. 8, No. 6, (December 1994), pp. 497-511, ISSN 0890-8508 Razin, S. (2002). Diagnosis of Mycoplasmal Infections. In: *Molecular Biology and Pathogenicity* 

Razin, S., Yoguev, D., & Naot, Y. (1998). Molecular Biology and Pathogenicity of

Relman, D.A., & Persing, D.H. (1996). Genotypic Methods for Microbial Identification. In:

Rottem, S. (2003). Interaction of Mycoplasmas with Host Cells. *Physiology Reviews*, Vol. 83,

Schwartz, S.B., Thurman, K.A., Mitchell, S.L., Wolff, B.J., &Winchell, J.M. (2009). Genotyping

Shi, S-R., Datar, R., Liu, C., Wu, L., Zhang, Z., Cote, R.J., & Taylor, C.R. (2004). DNA

Sirand-Pugnet, P., Citti, C., Barré, A., & Blanchard, A. (2007), Evolution of Mollicutes: Down

Sung, H., Kang, S.H., Bae, Y.J., Hong, J.T., Chung, Y.B., Lee, C.-K., & Song, S. (2006). PCR-

Talkington, D.F., & Waites, K.B. (2009). *Mycoplasma pneumoniae* and Other Human

Taylor, P. (1998). Recovery of Human Mycoplasmas, In: *Mycoplasma protocols*, R. Miles, & R. Nicholas (Eds.), 25-35, Humana Press, ISBN 0-89603-525-5, Totowa, NJ Taylor-Robinson, D. (1996). Infections Due to Species of *Mycoplasma* and *Ureaplasma*: an

No. (Pt 3), pp. 287–291, ISSN 0022-2615

Publishers, ISBN 0-306-47287-2, New York, NY

1998), pp. 1094-1156, ISSN 1092-2172

ISBN 1-55581-108-6, Washington, D.C.

No. 2 (Apr 2003), pp. 417-432, ISSN 0031-9333

No. 3, (September 2004), pp. 211–218, ISSN 0948-6143

(December 2007), pp. 754-766, ISSN 0923-2508

(February 2006), pp. 42-49, ISSN 1225-8873

0-89603-525-5, Totowa, NJ

432, ISSN 0378-1097

ISSN 1198-743X

NY.

1058-4838

*Mycoplasma pneumoniae* Pneumonia by PCR. *Journal of Medical Microbiology*, Vol. 54,

*Mycoplasma protocols*, R. Miles, & R. Nicholas (Eds.), 179-187, Humana Press, ISBN

Prokariotes. *FEMS Microbiology Letters*, Vol. 79, No. 1-3, (December 1992) pp. 423-

*of Mycoplasmas*, S. Razin & R. Herrmann (Eds.), 531-544, Kluwer Academic/Plenum

Mycoplasmas. *Microbiology and Molecular Biology Reviews,* Vol. 62, No. 4, (December

*PCR Protocols for Emerging Infectious Diseases*, Persing, D.H. (Ed), 3-31, ASM Press,

of *Mycoplasma pneumoniae* Isolates Using Real-Time PCR and High-Resolution Melt Analysis. *Clinical Microbiology Infection*. Vol. 15, No. 8, (August 2009), pp. 756-62,

Extraction from Archival Formalin-Fixed, Paraffin-Embedded Tissues: Heat-Induced Retrieval in Alkaline Solution. *Histochemistry and Cellular Biology*, Vol. 122,

a Bumpy Road with Twists and Turns. *Research in Microbiology*, Vol. 158, No. 10,

Based Detection of Mycoplasma Species. *The Journal of Microbiology*, Vol. 44, No. 1,

Mycoplasmas, In: *Bacterial Infections of Humans*, A.S. Evans, & P.S., Brachman (Eds.), 519-541, Springer-Science+Business, ISBN 978-0-387-09843-2, New York,

Update. *Clinical Infectious Diseases,* Vol. 23, No. 4, (October 1996), pp. 671-684, ISSN


**7** 

*Australia* 

**BRAF V600E Mutation Detection Using** 

Joseph W. Wrin1, Aravind Shivasami1, Irene Kanter1,

Jennifer E. Hardingham1,2, Ann Chua1,

Niall C. Tebbutt3 and Timothy J. Price1,2 *1The Queen Elizabeth Hospital, Adelaide, SA, 5011* 

*2University of Adelaide, SA, 5005* 

**High Resolution Probe Melting Analysis** 

*3Ludwig Institute for Cancer Research, Austin Health, Melbourne, VIC, 3084* 

Activation of oncogenic proteins is an important mechanism in carcinogenesis. The BRAF gene, located on chromosome 7q34, encodes a serine-threonine kinase that acts downstream of RAS in the RAS/RAF/MEK/ERK signaling pathway involved in regulating cell proliferation and survival. On activation of RAS, the BRAF kinase is activated and sequentially phosphorylates and activates MEK and ERK. A mutation in BRAF leads to constitutive hyperactivation of this pathway through evasion of the inhibitory feedback loop resulting in increased ERK signaling output which drives proliferative and anti-apoptotic signaling (Pratilas et al. 2009). Mutations in BRAF have been reported to occur at high frequency (66%) in melanoma with lower frequencies in colon and other tumours (Davies et al. 2002); BRAF is thus considered to be an important therapeutic target in melanoma (Bollag et al. 2010; Flaherty et al. 2010; Paraiso et al. 2011). Although over 30 single site missense mutations have been identified, 90% occur at nucleotide 1799 resulting in a T-A transition and an amino acid substitution at residue 600 (V600E) in the activation segment

In colorectal cancer (CRC) mutations in BRAF have been found in about 9-12% of tumours overall (Di Nicolantonio et al. 2008); (Deng et al. 2004; Jensen et al. 2008). However there is a distinct difference in frequency of BRAF mutations between mismatch repair (MMR) deficient (the microsatellite unstable (MSI-H) tumours) and the mismatch repair intact, microsatellite stable (MSS) tumours (Jensen et al. 2008). This is important clinically as tumours that are MSI-H have a better prognosis (Popat, Hubner, and Houlston 2005). BRAF is mutated in almost all sporadic CRCs with MSI-H (Jensen et al. 2008) but not in tumours arising in patients with an inherited form of MMR deficiency, hereditary nonpolyposis colon cancer (HNPCC), known as Lynch syndrome. Thus a major indication for BRAF mutation testing is for a differential diagnosis of Lynch Syndrome in a CRC that is MSI-H. If BRAF is mutated, the tumour is more likely to be sporadic, rather than the heritable type

**1. Introduction** 

(Wan et al. 2004).

(Sharma and Gulley 2010).


## **BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis**

Jennifer E. Hardingham1,2, Ann Chua1, Joseph W. Wrin1, Aravind Shivasami1, Irene Kanter1, Niall C. Tebbutt3 and Timothy J. Price1,2 *1The Queen Elizabeth Hospital, Adelaide, SA, 5011 2University of Adelaide, SA, 5005 3Ludwig Institute for Cancer Research, Austin Health, Melbourne, VIC, 3084 Australia* 

#### **1. Introduction**

142 Polymerase Chain Reaction

Waites, K.B., Katz, B., & Schelonka, R. (2005). Mycoplasmas and Ureaplasmas as Neonatal

Wang, R.Y., Hu, W.S., Dawson, M.S., Shih, J.W., & Lo, S.C. (1992). Selective Detection of

*Microbiology*, Vol. 30, No. 1 (January 1992), pp. 245-248, ISSN 0095-1137 Wang, R.Y.-H., Hu, W.S., Dawson, M.S., Shih, J.W.-K., Lo, S.-C. (1992) Selective Detection of

*Microbiology*, Vol. 30, No. 1 (January 1992), pp. 245-248, ISSN 0095-1137 Wilson, M.L. (1996). General Principles of Specimen Collection and Transport. *Clinical Infectious Diseases*, Vol. 22, No. 5, (May 1996), pp. 766-77, ISSN 1058-4838 Yáñez A, Cedillo L, Neyrolles O, Alonso E, Prévost MC, Rojas R, Watson HL, Blanchard A,

Yavlovich, A., Tarshis, M., & Rottem, S. (2004). Internalization and Intracellular Survival of

Yoshida, T., Maeda, S., Deguchi, T., & Ishiko, H. (2002). Phylogeny-Based Rapid

233, No. 2, (April 2004), pp. 241-246, ISSN: 1574-6968

ISSN 0893-8512

ISSN 1080-6040

Pathogens. *Clinical Microbiology Reviews,* Vol. 18, No. 4, (October 2005), pp. 757–789,

*Mycoplasma fermentans* by Polymerase Chain Reaction and By Using a Nucleotide Sequence Within the Insertion Sequence-Like Element. *Journal of Clinical* 

*Mycoplasma fermentans* by Polymerase Chain Reaction and by Using a Nucleotide Sequence Within the Insertion Sequence-Like Element. *Journal of Clinical* 

Cassell GH. 1999. *Mycoplasma penetrans* Bacteremia and Primary Antiphospholipid Syndrome. *Emerging Infectious Diseases*, Vol. 5, No. 1 (January 1999), pp. 164-167,

*Mycoplasma pneumoniae* by Non-Phagocytic Cells. *FEMS Microbiology Letters*, Vol.

Identification of Mycoplasmas and Ureaplasmas from Urethritis Patients. *Journal of Clinical Microbiology*, Vol. 40, No. 1, (January 2002), pp. 105-10, ISSN 0095-1137

Activation of oncogenic proteins is an important mechanism in carcinogenesis. The BRAF gene, located on chromosome 7q34, encodes a serine-threonine kinase that acts downstream of RAS in the RAS/RAF/MEK/ERK signaling pathway involved in regulating cell proliferation and survival. On activation of RAS, the BRAF kinase is activated and sequentially phosphorylates and activates MEK and ERK. A mutation in BRAF leads to constitutive hyperactivation of this pathway through evasion of the inhibitory feedback loop resulting in increased ERK signaling output which drives proliferative and anti-apoptotic signaling (Pratilas et al. 2009). Mutations in BRAF have been reported to occur at high frequency (66%) in melanoma with lower frequencies in colon and other tumours (Davies et al. 2002); BRAF is thus considered to be an important therapeutic target in melanoma (Bollag et al. 2010; Flaherty et al. 2010; Paraiso et al. 2011). Although over 30 single site missense mutations have been identified, 90% occur at nucleotide 1799 resulting in a T-A transition and an amino acid substitution at residue 600 (V600E) in the activation segment (Wan et al. 2004).

In colorectal cancer (CRC) mutations in BRAF have been found in about 9-12% of tumours overall (Di Nicolantonio et al. 2008); (Deng et al. 2004; Jensen et al. 2008). However there is a distinct difference in frequency of BRAF mutations between mismatch repair (MMR) deficient (the microsatellite unstable (MSI-H) tumours) and the mismatch repair intact, microsatellite stable (MSS) tumours (Jensen et al. 2008). This is important clinically as tumours that are MSI-H have a better prognosis (Popat, Hubner, and Houlston 2005). BRAF is mutated in almost all sporadic CRCs with MSI-H (Jensen et al. 2008) but not in tumours arising in patients with an inherited form of MMR deficiency, hereditary nonpolyposis colon cancer (HNPCC), known as Lynch syndrome. Thus a major indication for BRAF mutation testing is for a differential diagnosis of Lynch Syndrome in a CRC that is MSI-H. If BRAF is mutated, the tumour is more likely to be sporadic, rather than the heritable type (Sharma and Gulley 2010).

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 145

using the QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. DNA was quantified using the Nanodrop (Thermo Scientific,

Median 67 71 68 0.27

**Sex** Male 63 58 64 0.47

0-1 94 88 94 0.11

2000mg/m2/day 67 60 68 0.38

months 27 18 30 0.17

chemotherapy 22 9 23 0.06 Prior Radiotherapy 13 6 10 0.47

Caecum 10 21 9 0.02 Ascending colon 10 24 11 0.04 Transverse colon 6 15 5 0.02 Descending colon 3 6 4 0.48 Sigmoid colon 30 18 32 0.11 Recto-sigmoid colon 11 3 13 0.1 Rectum 23 6 22 0.03 Primary tumour resected 79 91 86 0.47 Any metastases resected 10 3 9 0.23

rectum) 36 15 33 0.03 Liver metastases 75 62 75 0.19 Lymph node metastases 47 59 45 0.09 Lung metastases 39 21 41 0.03 Bone metastases 4 0 4 0.23 Peritoneal metastases 18 21 16 0.49 Other metastases 10 24 10 0.01

BRAF MUT (%) (n=33)

BRAF WT (%)

(n=280) P

All patients (%) (n=471)

Range 32-86 36-85 32-86

2 6 12 6

Wilmington, DE, USA), ensuring the ratio 260/280 was >1.7.

Baseline characteristic

**ECOG performance status** 

**Capecitabine dosage** 

Primary site of cancer

**Extent of disease at** 

Local disease (colon or

Table 1. Patient demographic and clinical characteristics

(Reproduced with permission from the Journal of Clinical Oncology).

**baseline** 

Prior adjuvant

Disease-free interval > 12

**Age (years)** 

Mutated BRAF has also been associated with non response to anti-EGFR monoclonal antibody therapy (cetuximab or panitumamab) in metastatic CRC (mCRC) patients (Cappuzzo et al. 2008). In a larger study it was reported that 0/11 patients with a BRAF mutation responded to cetuximab or panitumumab, conversely none of the responders carried BRAF mutations (Di Nicolantonio et al. 2008). BRAF mutation has also been found to be a prognostic factor for poorer outcome in mCRC (Di Nicolantonio et al. 2008); (Price et al. 2011); (Samowitz et al. 2005); (Saridaki et al. 2010); (Souglakos et al. 2009; Tol, Nagtegaal, and Punt 2009); (Van Cutsem et al. 2011).

Although PCR-sequencing to detect BRAF mutations has been the gold standard technique, the improvement in instrumentation for high resolution analysis of PCR amplicon melt curves has opened up the way for the detection of single-base changes in short (approximately 100-200 bp) amplicons (Wittwer et al. 2003). Subsequently an improved method was developed, using melt curve analysis of an oligo-probe, annealing across the region of the mutation (Zhou et al. 2004). As the BRAF mutation is a class IV (T-A) change, we opted for this improved method using commercially available primer and probe sequences. Here we describe the optimisation and validation of this technique for the detection of the BRAF V600E mutation in formalin-fixed paraffin-embedded (FFPE) colorectal tumour tissue and, using the Kaplan-Meier method, the impact of this mutation on survival in the study cohort.

## **2. Materials and methods**

## **2.1 Tumour collection and processing**

Patient samples were obtained from the MAX phase III clinical trial colorectal tumour cohort, described in Price et al. (Price et al. 2011). The MAX study design and eligibility criteria have been reported previously (Tebbutt et al. 2010). Eligible patients were enrolled in this trial between July 2005 and June 2007. After enrollment, patients were randomly assigned to receive capecitabine (C), capecitabine and bevacizumab (CB), and capecitabine, bevacizumab and mitomycin C (CBM). Patient demographic and clinical characteristics are shown in Table 1. Patients in these three groups were evaluated for tumour response or progression every 6 weeks by means of radiologic imaging. Treatment was continued until the disease progressed or until the patient could not tolerate the toxic effects. Samples of tumour tissue from archived FFPE specimens collected at the time of diagnosis were retrieved from storage at participating hospital pathology departments. All patients participating in biomarker studies provided written informed consent at the time of study enrolment. Ethics approval was obtained centrally (Ethics Committee, Cancer Institute of NSW, Australia).

#### **2.2 DNA extraction**

DNA was extracted from 1-2 FFPE tissue sections (10 μm) mounted on plain glass slides, with an adjacent section stained with haematoxylin and eosin for reference. In cases that were deemed to have <50% presence of malignant crypts in the section (reviewed by a histopathologist), the tissue was manually dissected to ensure a high proportion of tumour cells. We used a single 10 μm section unless the size of the tissue section was <1 cm, in which case 2 10 μm sections were used. Paraffin was removed by xylene and DNA extracted

Mutated BRAF has also been associated with non response to anti-EGFR monoclonal antibody therapy (cetuximab or panitumamab) in metastatic CRC (mCRC) patients (Cappuzzo et al. 2008). In a larger study it was reported that 0/11 patients with a BRAF mutation responded to cetuximab or panitumumab, conversely none of the responders carried BRAF mutations (Di Nicolantonio et al. 2008). BRAF mutation has also been found to be a prognostic factor for poorer outcome in mCRC (Di Nicolantonio et al. 2008); (Price et al. 2011); (Samowitz et al. 2005); (Saridaki et al. 2010); (Souglakos et al. 2009; Tol, Nagtegaal,

Although PCR-sequencing to detect BRAF mutations has been the gold standard technique, the improvement in instrumentation for high resolution analysis of PCR amplicon melt curves has opened up the way for the detection of single-base changes in short (approximately 100-200 bp) amplicons (Wittwer et al. 2003). Subsequently an improved method was developed, using melt curve analysis of an oligo-probe, annealing across the region of the mutation (Zhou et al. 2004). As the BRAF mutation is a class IV (T-A) change, we opted for this improved method using commercially available primer and probe sequences. Here we describe the optimisation and validation of this technique for the detection of the BRAF V600E mutation in formalin-fixed paraffin-embedded (FFPE) colorectal tumour tissue and, using the Kaplan-Meier method, the impact of this mutation

Patient samples were obtained from the MAX phase III clinical trial colorectal tumour cohort, described in Price et al. (Price et al. 2011). The MAX study design and eligibility criteria have been reported previously (Tebbutt et al. 2010). Eligible patients were enrolled in this trial between July 2005 and June 2007. After enrollment, patients were randomly assigned to receive capecitabine (C), capecitabine and bevacizumab (CB), and capecitabine, bevacizumab and mitomycin C (CBM). Patient demographic and clinical characteristics are shown in Table 1. Patients in these three groups were evaluated for tumour response or progression every 6 weeks by means of radiologic imaging. Treatment was continued until the disease progressed or until the patient could not tolerate the toxic effects. Samples of tumour tissue from archived FFPE specimens collected at the time of diagnosis were retrieved from storage at participating hospital pathology departments. All patients participating in biomarker studies provided written informed consent at the time of study enrolment. Ethics approval was obtained centrally (Ethics Committee, Cancer Institute of

DNA was extracted from 1-2 FFPE tissue sections (10 μm) mounted on plain glass slides, with an adjacent section stained with haematoxylin and eosin for reference. In cases that were deemed to have <50% presence of malignant crypts in the section (reviewed by a histopathologist), the tissue was manually dissected to ensure a high proportion of tumour cells. We used a single 10 μm section unless the size of the tissue section was <1 cm, in which case 2 10 μm sections were used. Paraffin was removed by xylene and DNA extracted

and Punt 2009); (Van Cutsem et al. 2011).

on survival in the study cohort.

**2. Materials and methods** 

NSW, Australia).

**2.2 DNA extraction** 

**2.1 Tumour collection and processing**

using the QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. DNA was quantified using the Nanodrop (Thermo Scientific, Wilmington, DE, USA), ensuring the ratio 260/280 was >1.7.


Table 1. Patient demographic and clinical characteristics

(Reproduced with permission from the Journal of Clinical Oncology).

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 147

Samples that show poor amplification with late Ct values may give erroneous results on HRM as shown in Figure 2B. The samples in the boxed area need to be excluded from the analysis to avoid misinterpretation of the difference plot as mutant calls. The poor amplification of a DNA sample may be due to the presence of inhibitors, and we have found that subsequent isolation of DNA from microdissected sections gave much better, more reproducible amplification results. This also suggests that minimising the amount of paraffin in the DNA preparation may be contributing to the improvement in PCR

**whole sections microdissected**

Fig. 1. Dot plot of DNA yields. The average amount of DNA obtained from whole sections

The positioning of the normalisation regions 1 and 2 in the first HRM analysis window is also a very important parameter in the correct calling of genotypes. This is user-defined and performed separately for HRM analysis of the probe region or the amplicon region. The correct positioning may be determined by monitoring the normalised graph to show the

To determine the level of sensitivity of detection, serial doubling dilutions of a tumour sample carrying a homozygous BRAF V600E mutation were tested. The difference graph, normalised to the WT control, shows that the mutation could be detected down to a dilution of 6.25% mutant DNA in WT DNA (Figure 3A). Although there is a distinct difference between the WT control used for normalisation and the 6.25% and 12.5% dilutions, in practice the software cannot call these with any confidence. From the normalised graph and the melt curves graph (Figure 3B and 3C), 25% mutant DNA appears to be the lower limit of detection. However to increase the probability of correctly assigning a genotype we aimed for at least 50% epithelial tumour cells, hence all of the tumour tissue in the cohort was reviewed to ensure at least 50% epithelial tumour cells were present. Manual microdissection was performed in 1/5 of the cohort to ensure >50% enrichment of tumour cells, relative to muscularis mucosa and other cell types such as lymphoid aggregates, in the

was 6.5±0.25 μg and from manually microdissected sections 4.2±0.48 μg.

performance.

sample.

**0.01**

best separation of mutant versus WT curves.

**0.1**

**DNA (**

μ**g)**

**1**

**10**

**100**

### **2.3 Mutation analyses**

Mutation status of BRAF was determined using high resolution melting analysis (HRM) PCR on the Rotorgene 6000 real-time instrument (Qiagen). BRAF HRM PCR (119 bp amplicon) was performed on 10 ng DNA in triplicate reactions using SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories Inc., Hercules, USA) and a primer/probe combination (RaZor® probe HRM assay, PrimerDesign, Southampton, UK). The sequences were 5'ATGAAGACCTCACAGTAAAAATAGG (sense), CTCAATTCTTACCATCCACAAAATG (antisense) and 5'GTGAAATCTGGATGGAGTGGGTCCCATCA (probe). Appropriate mutant and wild type (WT) controls were included. A 'touch-down' PCR cycling protocol was used for the first 9 cycles to avoid primer mis-priming events and, due to the asymmetric design, 50 cycles were performed according to the manufacturer's protocol. The sensitivity of detection of mutant sequences was determined by assaying dilutions (100%, 50%, 25%, 12.5%, 6.25%) of a tumour DNA sample, with known homozygous BRAF mutation status, in BRAF WT cell line DNA. Using the Rotor Gene 6000 (Qiagen) software analysis features for HRM, patient samples (n=315) were classified as having mutated (MUT) or WT BRAF respectively. Direct PCR sequencing was used to validate all mutant BRAF results and an additional 106 randomly chosen samples (45% of samples in total). The primers for BRAF sequencing reactions were designed in-house and obtained commercially (Geneworks, Thebarton, SA, Australia): 5'AATGCTTGCTCTGATAGGAAAA (sense) and 5'AGTAACTCAGCAGCATCTCAGG (antisense). PCR products were purified using ExoSAP-IT (GE Healthcare, Buckinghamshire, UK) to remove unwanted deoxynucleotides and primers according to the manufacturer's protocol. Sequencing was performed by Flinders Sequencing Facility (Flinders Medical Centre, Bedford Park, SA, Australia) using BigDye Terminator v3.1 chemistry and the Applied Biosystems 3130xl Genetic Analyser (Life Technologies, Carlsbad, CA, USA).

### **2.4 Statistical analyses**

All randomly assigned patients for whom data on BRAF mutation status were available were included in the analysis (n=313). PFS, the primary endpoint, was defined as the time from randomisation until documented evidence of disease progression, the occurrence of new disease or death from any cause. The secondary endpoint was overall survival (OS), defined as the time from randomisation until death from any cause. The PFS and OS of patients according to BRAF status were summarised with the use of Kaplan–Meier curves, and the difference between these groups was compared with the use of the log-rank test. All reported P values were two-sided.

## **3. Results and discussion**

Although significantly less DNA was isolated from the microdissected sections (P=0.0001), the range of values obtained overall, 60 ng -31.3 µg, meant that all samples were well within the amount required for the PCR (30 ng) (Figure 1).

In interpreting the HRM results, the first criterion of robust PCR amplification must be met (Figure 2A), so that the duplicates must show close Ct values (standard deviation <0.5) otherwise samples must be excluded from the HRM analysis and the PCR repeated.

Mutation status of BRAF was determined using high resolution melting analysis (HRM) PCR on the Rotorgene 6000 real-time instrument (Qiagen). BRAF HRM PCR (119 bp amplicon) was performed on 10 ng DNA in triplicate reactions using SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories Inc., Hercules, USA) and a primer/probe combination (RaZor® probe HRM assay, PrimerDesign, Southampton, UK). The sequences were 5'ATGAAGACCTCACAGTAAAAATAGG (sense), CTCAATTCTTACCATCCACAAAATG (antisense) and 5'GTGAAATCTGGATGGAGTGGGTCCCATCA (probe). Appropriate mutant and wild type (WT) controls were included. A 'touch-down' PCR cycling protocol was used for the first 9 cycles to avoid primer mis-priming events and, due to the asymmetric design, 50 cycles were performed according to the manufacturer's protocol. The sensitivity of detection of mutant sequences was determined by assaying dilutions (100%, 50%, 25%, 12.5%, 6.25%) of a tumour DNA sample, with known homozygous BRAF mutation status, in BRAF WT cell line DNA. Using the Rotor Gene 6000 (Qiagen) software analysis features for HRM, patient samples (n=315) were classified as having mutated (MUT) or WT BRAF respectively. Direct PCR sequencing was used to validate all mutant BRAF results and an additional 106 randomly chosen samples (45% of samples in total). The primers for BRAF sequencing reactions were designed in-house and obtained commercially (Geneworks, Thebarton, SA, Australia): 5'AATGCTTGCTCTGATAGGAAAA (sense) and 5'AGTAACTCAGCAGCATCTCAGG (antisense). PCR products were purified using ExoSAP-IT (GE Healthcare, Buckinghamshire, UK) to remove unwanted deoxynucleotides and primers according to the manufacturer's protocol. Sequencing was performed by Flinders Sequencing Facility (Flinders Medical Centre, Bedford Park, SA, Australia) using BigDye Terminator v3.1 chemistry and the Applied Biosystems 3130xl Genetic Analyser

All randomly assigned patients for whom data on BRAF mutation status were available were included in the analysis (n=313). PFS, the primary endpoint, was defined as the time from randomisation until documented evidence of disease progression, the occurrence of new disease or death from any cause. The secondary endpoint was overall survival (OS), defined as the time from randomisation until death from any cause. The PFS and OS of patients according to BRAF status were summarised with the use of Kaplan–Meier curves, and the difference between these groups was compared with the use of the log-rank test. All

Although significantly less DNA was isolated from the microdissected sections (P=0.0001), the range of values obtained overall, 60 ng -31.3 µg, meant that all samples were well within

In interpreting the HRM results, the first criterion of robust PCR amplification must be met (Figure 2A), so that the duplicates must show close Ct values (standard deviation <0.5) otherwise samples must be excluded from the HRM analysis and the PCR repeated.

**2.3 Mutation analyses** 

(Life Technologies, Carlsbad, CA, USA).

reported P values were two-sided.

the amount required for the PCR (30 ng) (Figure 1).

**3. Results and discussion** 

**2.4 Statistical analyses** 

Samples that show poor amplification with late Ct values may give erroneous results on HRM as shown in Figure 2B. The samples in the boxed area need to be excluded from the analysis to avoid misinterpretation of the difference plot as mutant calls. The poor amplification of a DNA sample may be due to the presence of inhibitors, and we have found that subsequent isolation of DNA from microdissected sections gave much better, more reproducible amplification results. This also suggests that minimising the amount of paraffin in the DNA preparation may be contributing to the improvement in PCR performance.

Fig. 1. Dot plot of DNA yields. The average amount of DNA obtained from whole sections was 6.5±0.25 μg and from manually microdissected sections 4.2±0.48 μg.

The positioning of the normalisation regions 1 and 2 in the first HRM analysis window is also a very important parameter in the correct calling of genotypes. This is user-defined and performed separately for HRM analysis of the probe region or the amplicon region. The correct positioning may be determined by monitoring the normalised graph to show the best separation of mutant versus WT curves.

To determine the level of sensitivity of detection, serial doubling dilutions of a tumour sample carrying a homozygous BRAF V600E mutation were tested. The difference graph, normalised to the WT control, shows that the mutation could be detected down to a dilution of 6.25% mutant DNA in WT DNA (Figure 3A). Although there is a distinct difference between the WT control used for normalisation and the 6.25% and 12.5% dilutions, in practice the software cannot call these with any confidence. From the normalised graph and the melt curves graph (Figure 3B and 3C), 25% mutant DNA appears to be the lower limit of detection. However to increase the probability of correctly assigning a genotype we aimed for at least 50% epithelial tumour cells, hence all of the tumour tissue in the cohort was reviewed to ensure at least 50% epithelial tumour cells were present. Manual microdissection was performed in 1/5 of the cohort to ensure >50% enrichment of tumour cells, relative to muscularis mucosa and other cell types such as lymphoid aggregates, in the sample.

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 149

Fig. 2. A: amplification curves; B: difference plot normalised to WT. The boxed curves in A show samples with aberrant late amplification. The same samples boxed in B show the abnormal difference plots that could be incorrectly interpreted as mutant. Black arrow in B points to the heterozygous mutant control, red arrow shows the homozygous mutant control.

Fig. 2. A: amplification curves; B: difference plot normalised to WT. The boxed curves in A show samples with aberrant late amplification. The same samples boxed in B show the abnormal difference plots that could be incorrectly interpreted as mutant. Black arrow in B points to the heterozygous mutant control, red arrow shows the homozygous mutant

control.

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 151

Fig. 4. Sequencing result and corresponding HRM analysis. A: Difference graph normalized to WT control (duplicates), blue arrow shows heterozygous control, green arrow pt 109, red arrow homozygous MUT control. Boxed area shows WT samples. Inset is the sequencing trace (Chromas Lite software) of patient (pt) 109, red arrow showing mutant (A) amongst WT (T) sequence. B: Normalised melt curve of probe region; black boxed area shows homozygous mutant control and 2 samples including pt 109, arrow points to the

heterozygote mutant control, blue box shows WT control and WT samples. C: Melt curve

analysis, red trace WT, black trace homozygous MUT control, purple trace pt 109.

Fig. 3. A: Difference plot normalised to WT, with dilutions of homozygous MUT control DNA in WT DNA shown in replicate view (average of 3 for each dilution). Arrows indicate the plots for the dilutions of MUT control DNA in WT DNA from 100% MUT to 6.25%MUT; B: Normalised melting curves of the probe region. From this view it was not possible to distinguish the 12.5% or 6.25% dilutions of mutant sequence from WT; C: Melt curve showing Tm's for both the probe region and amplicon. The probe region HRM analysis was much easier to interpret than the amplicon HRM, however the 12.5% and 6.25%dilutions were indistinguishable from WT pattern.

We have found that it was of critical importance to select the control genotypes (WT or mutant) for the normalisation carefully. The DNA of these controls needed to be extracted from a similar tissue (i.e. colonic tissue FFPE), and be processed in exactly the same way as the test samples. Using cell line derived DNA as the controls resulted in too many mutation calls with low confidence (false positives), however when we used tumour samples of known *BRAF* status as the controls, the confidence of the software calls of the test samples reached >99%. Often we found it was more informative to look at the shape of the curves in the difference plot, even if a curve deflected away from the horizontal normalised line, the angle of deflection was much greater for mutant genotypes and shallower for WT (Figure 4). This visual interpretation usually correlated with the software calls and was a useful adjunct in interpretation where the confidence of the software calls was low.

Sequencing was used to validate the results and correlated with the HRM results. In some cases though sequencing showed a very small A peak which could be overlooked whereas HRM showed a very convincing shift and was called as a mutation with 99% confidence. An example is shown in Figure 5.

Fig. 3. A: Difference plot normalised to WT, with dilutions of homozygous MUT control DNA in WT DNA shown in replicate view (average of 3 for each dilution). Arrows indicate the plots for the dilutions of MUT control DNA in WT DNA from 100% MUT to 6.25%MUT; B: Normalised melting curves of the probe region. From this view it was not possible to distinguish the 12.5% or 6.25% dilutions of mutant sequence from WT; C: Melt curve showing Tm's for both the probe region and amplicon. The probe region HRM analysis was much easier to interpret than the amplicon HRM, however the 12.5% and 6.25%dilutions

We have found that it was of critical importance to select the control genotypes (WT or mutant) for the normalisation carefully. The DNA of these controls needed to be extracted from a similar tissue (i.e. colonic tissue FFPE), and be processed in exactly the same way as the test samples. Using cell line derived DNA as the controls resulted in too many mutation calls with low confidence (false positives), however when we used tumour samples of known *BRAF* status as the controls, the confidence of the software calls of the test samples reached >99%. Often we found it was more informative to look at the shape of the curves in the difference plot, even if a curve deflected away from the horizontal normalised line, the angle of deflection was much greater for mutant genotypes and shallower for WT (Figure 4). This visual interpretation usually correlated with the software calls and was a useful adjunct

Sequencing was used to validate the results and correlated with the HRM results. In some cases though sequencing showed a very small A peak which could be overlooked whereas HRM showed a very convincing shift and was called as a mutation with 99% confidence. An

in interpretation where the confidence of the software calls was low.

were indistinguishable from WT pattern.

example is shown in Figure 5.

Fig. 4. Sequencing result and corresponding HRM analysis. A: Difference graph normalized to WT control (duplicates), blue arrow shows heterozygous control, green arrow pt 109, red arrow homozygous MUT control. Boxed area shows WT samples. Inset is the sequencing trace (Chromas Lite software) of patient (pt) 109, red arrow showing mutant (A) amongst WT (T) sequence. B: Normalised melt curve of probe region; black boxed area shows homozygous mutant control and 2 samples including pt 109, arrow points to the heterozygote mutant control, blue box shows WT control and WT samples. C: Melt curve analysis, red trace WT, black trace homozygous MUT control, purple trace pt 109.

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 153

including age, sex, ECOG performance status, inoperable local disease, and prior

Fig. 6. Kaplan-Meier analysis for overall survival comparing patients WT or MUT for *BRAF* . The curves are significantly different (P=0.001, log-rank test). Reproduced with permission

HRM analysis is a useful fast technique to determine BRAF mutations using the platform of real-time PCR. It is both reproducible and reliable provided the preceding guidelines are followed and rigorous attention is given to the PCR performance as well as to the use of the software analysis package. Here we have described how the technique can be applied to the analysis of DNA extracted from archived FFPE tissue sections, which in many cases is the only source of tumour tissue available for retrospective analyses. The survival analysis showed that metastatic CRC patients with tumours carrying the V600E mutation had significantly poorer overall survival outcomes compared to those without the mutation. This HRM analysis could equally be applied to the assessment of tumours from patients

diagnosed with other diseases known to have a significant BRAF mutation rate.

from the Journal of Clinical Oncology.

**4. Conclusion** 

chemotherapy (HR: BRAF WT *vs* MUT, 0.45; 95% CI, 0.30 to 0.68; P<0.0001).

Fig. 5. An example of a sequencing result (pt 269) called WT (T) by the sequencing software that did in fact show a small A peak. The difference plot of the HRM analysis (normalized to WT control) showed a definite downward shifted curve (green arrow) between the homozygous BRAF MUT control (red arrow) and the heterozygous control (blue arrow). The boxed curves show the WT samples.

Of 471 patients who underwent random assignment, a total of 315 tumour specimens (n=103 from the capecitabine group, n=111 from the CB group, and n=101 from the CBM group, accounting for 66.9% of the total study population) were examined for *BRAF* mutation status by HRM. *BRAF* V600E mutations were detected in 10.5% of 313 tumours (2 samples were not evaluable). A proportion of samples were also genotyped using sequencing and showed 100% correlation with the HRM result.

A total of 313 patients were included in the survival analysis with a median follow-up time of 26.5 months (range, 0.4 to 37.6 months). There was no significant difference in PFS between patients with WT tumours and those with mutated tumours. The median PFS was 4.5 months among the patients with V600E tumours as compared with 8.2 months among those with WT tumours (HR: BRAF WT *vs* MUT, 0.80; 95% CI, 0.54 to 1.18; P=0.26). In contrast, there was a significant difference in OS between patients with WT tumours and those with V600E tumours. The median OS was 8.6 months among the patients with mutated BRAF tumours as compared with 20.8 months among those with WT tumours (HR: BRAF WT *vs* MUT, 0.49; 95% CI, 0.33 to 0.73; P=0.001) (Figure 6). BRAF status remained prognostically significant after adjustment of pre-defined baseline prognostic factors including age, sex, ECOG performance status, inoperable local disease, and prior chemotherapy (HR: BRAF WT *vs* MUT, 0.45; 95% CI, 0.30 to 0.68; P<0.0001).

Fig. 6. Kaplan-Meier analysis for overall survival comparing patients WT or MUT for *BRAF* . The curves are significantly different (P=0.001, log-rank test). Reproduced with permission from the Journal of Clinical Oncology.

## **4. Conclusion**

152 Polymerase Chain Reaction

Fig. 5. An example of a sequencing result (pt 269) called WT (T) by the sequencing software that did in fact show a small A peak. The difference plot of the HRM analysis (normalized to

Of 471 patients who underwent random assignment, a total of 315 tumour specimens (n=103 from the capecitabine group, n=111 from the CB group, and n=101 from the CBM group, accounting for 66.9% of the total study population) were examined for *BRAF* mutation status by HRM. *BRAF* V600E mutations were detected in 10.5% of 313 tumours (2 samples were not evaluable). A proportion of samples were also genotyped using sequencing and

A total of 313 patients were included in the survival analysis with a median follow-up time of 26.5 months (range, 0.4 to 37.6 months). There was no significant difference in PFS between patients with WT tumours and those with mutated tumours. The median PFS was 4.5 months among the patients with V600E tumours as compared with 8.2 months among those with WT tumours (HR: BRAF WT *vs* MUT, 0.80; 95% CI, 0.54 to 1.18; P=0.26). In contrast, there was a significant difference in OS between patients with WT tumours and those with V600E tumours. The median OS was 8.6 months among the patients with mutated BRAF tumours as compared with 20.8 months among those with WT tumours (HR: BRAF WT *vs* MUT, 0.49; 95% CI, 0.33 to 0.73; P=0.001) (Figure 6). BRAF status remained prognostically significant after adjustment of pre-defined baseline prognostic factors

WT control) showed a definite downward shifted curve (green arrow) between the homozygous BRAF MUT control (red arrow) and the heterozygous control (blue arrow).

The boxed curves show the WT samples.

showed 100% correlation with the HRM result.

HRM analysis is a useful fast technique to determine BRAF mutations using the platform of real-time PCR. It is both reproducible and reliable provided the preceding guidelines are followed and rigorous attention is given to the PCR performance as well as to the use of the software analysis package. Here we have described how the technique can be applied to the analysis of DNA extracted from archived FFPE tissue sections, which in many cases is the only source of tumour tissue available for retrospective analyses. The survival analysis showed that metastatic CRC patients with tumours carrying the V600E mutation had significantly poorer overall survival outcomes compared to those without the mutation. This HRM analysis could equally be applied to the assessment of tumours from patients diagnosed with other diseases known to have a significant BRAF mutation rate.

BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis 155

Pratilas, Christine A., Barry S. Taylor, Qing Ye, Agnes Viale, Chris Sander, David B. Solit,

Price, T. J., J. E. Hardingham, C. K. Lee, A. Weickhardt, A. R. Townsend, J. W. Wrin, A.

Mitomycin in Advanced Colorectal Cancer. *J Clin Oncol* 29 (19):2675-82. Samowitz, W. S., C. Sweeney, J. Herrick, H. Albertsen, T. R. Levin, M. A. Murtaugh, R. K.

mutation in microsatellite-stable colon cancers. *Cancer Res* 65 (14):6063-9. Saridaki, Z., D. Papadatos-Pastos, M. Tzardi, D. Mavroudis, E. Bairaktari, H. Arvanity, E.

Sharma, Shree G., and Margaret L. Gulley. 2010. BRAF Mutation Testing in Colorectal Cancer. *Archives of Pathology & Laboratory Medicine* 134 (8):1225-1228. Souglakos, J., J. Philips, R. Wang, S. Marwah, M. Silver, M. Tzardi, J. Silver, S. Ogino, S.

patients with metastatic colorectal cancer. *Br J Cancer* 101 (3):465-72. Tebbutt, N. C., K. Wilson, V. J. Gebski, M. M. Cummins, D. Zannino, G. A. van Hazel, B.

Randomized Phase III MAX Study. *J Clin Oncol* 28 (19):3191-8.

*N Engl J Med* 361 (1):98-9.

Status. *J Clin Oncol* 29 (15):2011-9.

Mutations of B-RAF. *Cell* 116 (6):855-867.

LCGreen. *Clin Chem* 49 (6):853-860.

Tol, J., I. D. Nagtegaal, and C. J. Punt. 2009. BRAF mutation in metastatic colorectal cancer.

Van Cutsem, E., C. H. Kohne, I. Lang, G. Folprecht, M. P. Nowacki, S. Cascinu, I.

Wan, Paul T. C., Mathew J. Garnett, S. Mark Roe, Sharlene Lee, Dan Niculescu-Duvaz,

Wittwer, Carl T., Gudrun H. Reed, Cameron N. Gundry, Joshua G. Vandersteen, and Robert

colorectal patients' outcome. *Br J Cancer* 102 (12):1762-8.

*Proceedings of the National Academy of Sciences* 106 (11):4519-4524.

and Neal Rosen. 2009. V600EBRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway.

Chua, A. Shivasami, M. M. Cummins, C. Murone, and N. C. Tebbutt. 2011. Impact of KRAS and BRAF Gene Mutation Status on Outcomes From the Phase III AGITG MAX Trial of Capecitabine Alone or in Combination With Bevacizumab and

Wolff, and M. L. Slattery. 2005. Poor survival associated with the BRAF V600E

Stathopoulos, V. Georgoulias, and J. Souglakos. 2010. BRAF mutations, microsatellite instability status and cyclin D1 expression predict metastatic

Hooshmand, E. Kwak, E. Freed, J. A. Meyerhardt, Z. Saridaki, V. Georgoulias, D. Finkelstein, C. S. Fuchs, M. H. Kulke, and R. A. Shivdasani. 2009. Prognostic and predictive value of common mutations for treatment response and survival in

Robinson, A. Broad, V. Ganju, S. P. Ackland, G. Forgeson, D. Cunningham, M. P. Saunders, M. R. Stockler, Y. Chua, J. R. Zalcberg, R. J. Simes, and T. J. Price. 2010. Capecitabine, bevacizumab, and mitomycin in first-line treatment of metastatic colorectal cancer: results of the Australasian Gastrointestinal Trials Group

Shchepotin, J. Maurel, D. Cunningham, S. Tejpar, M. Schlichting, A. Zubel, I. Celik, P. Rougier, and F. Ciardiello. 2011. Cetuximab Plus Irinotecan, Fluorouracil, and Leucovorin As First-Line Treatment for Metastatic Colorectal Cancer: Updated Analysis of Overall Survival According to Tumor KRAS and BRAF Mutation

Valerie M. Good, Cancer Genome Project, C. Michael Jones, Christopher J. Marshall, Caroline J. Springer, David Barford, and Richard Marais. 2004. Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic

J. Pryor. 2003. High-Resolution Genotyping by Amplicon Melting Analysis Using

#### **5. References**


Bollag, Gideon, Peter Hirth, James Tsai, Jiazhong Zhang, Prabha N. Ibrahim, Hanna Cho,

Cappuzzo, F., M. Varella-Garcia, G. Finocchiaro, M. Skokan, S. Gajapathy, C. Carnaghi, L.

Davies, Helen, Graham R. Bignell, Charles Cox, Philip Stephens, Sarah Edkins, Sheila Clegg,

Deng, Guoren, Ian Bell, Suzanne Crawley, James Gum, Jonathan P. Terdiman, Brian A.

Di Nicolantonio, F., M. Martini, F. Molinari, A. Sartore-Bianchi, S. Arena, P. Saletti, S. De

Flaherty, Keith T., Igor Puzanov, Kevin B. Kim, Antoni Ribas, Grant A. McArthur, Jeffrey A.

Jensen, L. H., J. Lindebjerg, L. Byriel, S. Kolvraa, and D. G. Cruger. 2008. Strategy in clinical

Paraiso, Kim H. T., Yun Xiang, Vito W. Rebecca, Ethan V. Abel, Y. Ann Chen, A. Cecilia

Popat, S., R. Hubner, and R. S. Houlston. 2005. Systematic review of microsatellite instability

Rimassa, E. Rossi, C. Ligorio, L. Di Tommaso, A. J. Holmes, L. Toschi, G. Tallini, A. Destro, M. Roncalli, A. Santoro, and P. A. Janne. 2008. Primary resistance to cetuximab therapy in EGFR FISH-positive colorectal cancer patients. *Br J Cancer* 99

Jon Teague, Hayley Woffendin, Mathew J. Garnett, William Bottomley, Neil Davis, Ed Dicks, Rebecca Ewing, Yvonne Floyd, Kristian Gray, Sarah Hall, Rachel Hawes, Jaime Hughes, and Vivian Kosmidou. 2002. Mutations of the BRAF gene in human

Allen, Brindusa Truta, Marvin H. Sleisenger, and Young S. Kim. 2004. BRAF Mutation Is Frequently Present in Sporadic Colorectal Cancer with Methylated hMLH1, But Not in Hereditary Nonpolyposis Colorectal Cancer. *Clin Cancer Res* 10

Dosso, L. Mazzucchelli, M. Frattini, S. Siena, and A. Bardelli. 2008. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal

Sosman, Peter J. O'Dwyer, Richard J. Lee, Joseph F. Grippo, Keith Nolop, and Paul B. Chapman. 2010. Inhibition of Mutated, Activated BRAF in Metastatic Melanoma.

practice for classification of unselected colorectal tumours based on mismatch

Munko, Elizabeth Wood, Inna V. Fedorenko, Vernon K. Sondak, Alexander R. A. Anderson, Antoni Ribas, Maurizia Dalla Palma, Katherine L. Nathanson, John M. Koomen, Jane L. Messina, and Keiran S. M. Smalley. 2011. PTEN Loss Confers BRAF Inhibitor Resistance to Melanoma Cells through the Suppression of BIM

Wayne Spevak, Chao Zhang, Ying Zhang, Gaston Habets, Elizabeth A. Burton, Bernice Wong, Garson Tsang, Brian L. West, Ben Powell, Rafe Shellooe, Adhirai Marimuthu, Hoa Nguyen, Kam Y. J. Zhang, Dean R. Artis, Joseph Schlessinger, Fei Su, Brian Higgins, Raman Iyer, Kurt D/'Andrea, Astrid Koehler, Michael Stumm, Paul S. Lin, Richard J. Lee, Joseph Grippo, Igor Puzanov, Kevin B. Kim, Antoni Ribas, Grant A. McArthur, Jeffrey A. Sosman, Paul B. Chapman, Keith T. Flaherty, Xiaowei Xu, Katherine L. Nathanson, and Keith Nolop. 2010. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. *Nature* 467

**5. References** 

(7315):596-599.

(1):83-89.

(1):191-195.

cancer. *Nature* 417 (6892):949.

cancer. *J Clin Oncol* 26 (35):5705-12.

*New England Journal of Medicine* 363 (9):809-819.

repair deficiency. *Colorectal Disease* 10 (5):490-497.

Expression. *Cancer Research* 71 (7):2750-2760.

and colorectal cancer prognosis. *J Clin Oncol* 23 (3):609-18.


**8** 

*Colombia* 

**Polymerase Chain Reaction:** 

**Types, Utilities and Limitations** 

Patricia Hernández-Rodríguez1 and Arlen Gomez Ramirez2 *1Molecular Biology and Immunogenetics Research Group (BIOMIGEN),* 

*Reproduction Research Center (CIMRA), Universidad de La Salle, Bogotá* 

 *Department of Basic Sciences, Biology Program, Universidad de La Salle, Bogotá 2Faculty of Agricultural Sciences, Veterinary Medicine Program, Animal Medicine and* 

Nowadays, advances and applications of research in biochemistry and genetic play an important role in the field of health sciences. This has become necessary a molecular approach of the disease for a better interpretation of processes and as horizon in the development of new diagnostic and therapeutic strategies. Therefore, techniques in molecular biology have modified diagnosis, prevention and control of diseases in living beings. Molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production and health, environment and the industry related to these areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. This technique was developed by Kary Mullis in the mid 80's [1, 2, 3, 4] and since then it has been considered as an essential tool in molecular biology which allows amplification of nucleic acid sequences (DNA and RNA) through repetitive cycles *in vitro*. The mechanisms involved in this methodology are similar to those occurring *in vivo* during DNA replication. Each cycle had three temperature patterns carried out by a thermocycler. The first pattern of temperature is 94 °C (denaturation), the second one is 45 - 55 °C (alignment of the specific primers) and the third one is 72 °C (final extension). The amplification of specific nucleic acid sequences, even in the presence of millions of other DNA molecules, is achieved by thermostable DNA polymerase enzyme (as the name of this technique suggests: "polymerase chain reaction") and specific primers. Primers are short sequences of DNA or RNA (oligonucleotides) that initiate DNA synthesis. These are complementary to the template strand of DNA. The total duration of PCR reaction is around two hours; this depends on the specific conditions of the reaction. Therefore, the DNA polymerase enzyme is capable of producing a complementary strand of a template DNA. In summary, the requirements of PCR are as follows: i. Template DNA; ii. Four deoxyribonucleotides (dNTPs: dATP, dTTP, dGTP and dCTP) which are the

**1. Introduction** 

**1.1 Types, utilities and limitations of PCR** 

*Animal Medicine and Reproduction Research Center (CIMRA),* 

Zhou, Luming, Alexander N. Myers, Joshua G. Vandersteen, Lesi Wang, and Carl T. Wittwer. 2004. Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye. *Clin Chem* 50 (8):1328-1335.

## **Polymerase Chain Reaction: Types, Utilities and Limitations**

Patricia Hernández-Rodríguez1 and Arlen Gomez Ramirez2 *1Molecular Biology and Immunogenetics Research Group (BIOMIGEN),* 

*Animal Medicine and Reproduction Research Center (CIMRA), Department of Basic Sciences, Biology Program, Universidad de La Salle, Bogotá 2Faculty of Agricultural Sciences, Veterinary Medicine Program, Animal Medicine and Reproduction Research Center (CIMRA), Universidad de La Salle, Bogotá Colombia* 

#### **1. Introduction**

156 Polymerase Chain Reaction

Zhou, Luming, Alexander N. Myers, Joshua G. Vandersteen, Lesi Wang, and Carl T.

and a Saturating DNA Dye. *Clin Chem* 50 (8):1328-1335.

Wittwer. 2004. Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes

#### **1.1 Types, utilities and limitations of PCR**

Nowadays, advances and applications of research in biochemistry and genetic play an important role in the field of health sciences. This has become necessary a molecular approach of the disease for a better interpretation of processes and as horizon in the development of new diagnostic and therapeutic strategies. Therefore, techniques in molecular biology have modified diagnosis, prevention and control of diseases in living beings. Molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production and health, environment and the industry related to these areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. This technique was developed by Kary Mullis in the mid 80's [1, 2, 3, 4] and since then it has been considered as an essential tool in molecular biology which allows amplification of nucleic acid sequences (DNA and RNA) through repetitive cycles *in vitro*. The mechanisms involved in this methodology are similar to those occurring *in vivo* during DNA replication. Each cycle had three temperature patterns carried out by a thermocycler. The first pattern of temperature is 94 °C (denaturation), the second one is 45 - 55 °C (alignment of the specific primers) and the third one is 72 °C (final extension). The amplification of specific nucleic acid sequences, even in the presence of millions of other DNA molecules, is achieved by thermostable DNA polymerase enzyme (as the name of this technique suggests: "polymerase chain reaction") and specific primers. Primers are short sequences of DNA or RNA (oligonucleotides) that initiate DNA synthesis. These are complementary to the template strand of DNA. The total duration of PCR reaction is around two hours; this depends on the specific conditions of the reaction. Therefore, the DNA polymerase enzyme is capable of producing a complementary strand of a template DNA. In summary, the requirements of PCR are as follows: i. Template DNA; ii. Four deoxyribonucleotides (dNTPs: dATP, dTTP, dGTP and dCTP) which are the

Polymerase Chain Reaction: Types, Utilities and Limitations 159

Multiplex PCR is an adaptation of PCR which allows simultaneous amplification of many sequences. This technique is used for diagnosis of different diseases in the same sample [8, 9]. Multiplex PCR can detect different pathogens in a single sample [10, 11, 12]. Also it can be used to identify exonic and intronic sequences in specific genes [13] (figure 2) and determination of gene dosage (figure 2, 3 and 4). This is achieved when in a single tube

> **267 234**

**Sequencer**

**Electrophoretogram**

**547 (45)**

Ex**E**o**x**n**ón**

**506 (48)**

**459 (19) 388 (51) 360 ( 8 )**

Fig. 2. Results of a multiplex PCR in a patient with Duchenne Muscular Dystrophy. Dystrophy gene has different mutations in exons; this is the cause of disease. In lane 7 is shown the absence of a band corresponding to exon 48 (506 bp) of the dystrophy gene

**1 2 3 4 5 6 7 8 9 10**

Fig. 3. A: Requirements for multiplex PCR. This molecular method is useful for identification of deletion and duplication mutations. B: Electrophoretogram showing duplication (area under the curve amplified compared to normal) and deletion (area under the curve reduced compared

**Computational Analysis**

to normal) obtained by analysis of gene dosage. Results are accompanied by a statistical analysis, established by software, which determines areas under curve obtained by a sequencer.

(Hernández-Rodríguez et al., 2000; Hernández-Rodríguez & Restrepo, 2002).

**DNA amplification. Maximum 20 cycles**

**1.2 Multiplex PCR** 

**A**

**B**

**EDTA Blood**

**DNA Extraction and Quantification**

**Normal**

**Deletion**

**Duplication**

base material to make the new strand from template DNA; iii. Two primers or oligonucleotides; iv. Mg2+ which joins to nucleotides to be recognized by the polymerase enzyme; and, v. Thermostable DNA polymerase enzyme. The synthesized product in each cycle can serve as a template in the next issue of copies of DNA, creating a chain reaction that can amplify a specific fragment of DNA. Requirements and purpose of PCR are showed in figure 1.

Fig. 1. Requirements and purpose of amplification cycles (denaturation, annealing and extension) in a polymerase chain reaction (PCR).

PCR is a relatively simple technique that can detect a nucleic acid fragment and amplify this sequence. In addition, this technique has other advantages that are described below. This technique offers *sensitivity* because from small amounts of genetic material can be detected target sequences in a sample. Also this offers *specificity* due to a specific sequence of DNA is amplified through strict conditions. It is considered a fast technique compared with other methods to detect microorganisms such as bacteria, fungus or virus, which require isolation and culture using culture media or cell lines. Finally we can mention that offers *versatility* due to the genetic sequences from various microorganisms can be identified with the same reaction conditions for diagnosis of different pathologies [4, 5, 6, 7].

In recent years, modifications or variants have been developed from the basic PCR method to improve performance and specificity, and to achieve the amplification of other molecules of interest in research as RNA. Some of these variants are: i. Multiplex PCR which simultaneously amplified several DNA sequences (usually exonic sequences); ii. Nested PCR increases the specificity of the amplified product for a second PCR with new primers that hybridize within the amplified fragment in the first PCR; iii. Semiquantitative PCR which allows an approximation to the relative amount of nucleic acids present in a sample; iv. RT-PCR which generates amplification of RNA by synthesis of cDNA (DNA complementary to RNA) that is then amplified by PCR; and, v. Real time PCR which performs absolute or relative quantification of nucleic acid copies obtained by PCR. The principles of each of the above techniques are described following.

## **1.2 Multiplex PCR**

158 Polymerase Chain Reaction

base material to make the new strand from template DNA; iii. Two primers or oligonucleotides; iv. Mg2+ which joins to nucleotides to be recognized by the polymerase enzyme; and, v. Thermostable DNA polymerase enzyme. The synthesized product in each cycle can serve as a template in the next issue of copies of DNA, creating a chain reaction that can amplify a specific fragment of DNA. Requirements and purpose of PCR are showed

DENATURATION

FIRST AMPLIFICATION CYCLE ORIGINAL DNA MOLECULE

ANNEALING

EXTENSION

AMPLIFIED DNA

Fig. 1. Requirements and purpose of amplification cycles (denaturation, annealing and

PCR is a relatively simple technique that can detect a nucleic acid fragment and amplify this sequence. In addition, this technique has other advantages that are described below. This technique offers *sensitivity* because from small amounts of genetic material can be detected target sequences in a sample. Also this offers *specificity* due to a specific sequence of DNA is amplified through strict conditions. It is considered a fast technique compared with other methods to detect microorganisms such as bacteria, fungus or virus, which require isolation and culture using culture media or cell lines. Finally we can mention that offers *versatility* due to the genetic sequences from various microorganisms can be identified with the same

In recent years, modifications or variants have been developed from the basic PCR method to improve performance and specificity, and to achieve the amplification of other molecules of interest in research as RNA. Some of these variants are: i. Multiplex PCR which simultaneously amplified several DNA sequences (usually exonic sequences); ii. Nested PCR increases the specificity of the amplified product for a second PCR with new primers that hybridize within the amplified fragment in the first PCR; iii. Semiquantitative PCR which allows an approximation to the relative amount of nucleic acids present in a sample; iv. RT-PCR which generates amplification of RNA by synthesis of cDNA (DNA complementary to RNA) that is then amplified by PCR; and, v. Real time PCR which performs absolute or relative quantification of nucleic acid copies obtained by PCR. The

extension) in a polymerase chain reaction (PCR).

• Buffer10X • Primers • dNTPs • MgCl2 • Taq Polymerase • DNA

AMPLIFICATION CYCLES

reaction conditions for diagnosis of different pathologies [4, 5, 6, 7].

principles of each of the above techniques are described following.

in figure 1.

Multiplex PCR is an adaptation of PCR which allows simultaneous amplification of many sequences. This technique is used for diagnosis of different diseases in the same sample [8, 9]. Multiplex PCR can detect different pathogens in a single sample [10, 11, 12]. Also it can be used to identify exonic and intronic sequences in specific genes [13] (figure 2) and determination of gene dosage (figure 2, 3 and 4). This is achieved when in a single tube

Fig. 2. Results of a multiplex PCR in a patient with Duchenne Muscular Dystrophy. Dystrophy gene has different mutations in exons; this is the cause of disease. In lane 7 is shown the absence of a band corresponding to exon 48 (506 bp) of the dystrophy gene (Hernández-Rodríguez et al., 2000; Hernández-Rodríguez & Restrepo, 2002).

Fig. 3. A: Requirements for multiplex PCR. This molecular method is useful for identification of deletion and duplication mutations. B: Electrophoretogram showing duplication (area under the curve amplified compared to normal) and deletion (area under the curve reduced compared to normal) obtained by analysis of gene dosage. Results are accompanied by a statistical analysis, established by software, which determines areas under curve obtained by a sequencer.

Polymerase Chain Reaction: Types, Utilities and Limitations 161

This PCR was designed to amplify RNA sequences (especially mRNA) through synthesis of cDNA by reverse transcriptase (RT). Subsequently, this cDNA is amplified using PCR. This type of PCR has been useful for diagnosis of RNA viruses, as well as for evaluation of antimicrobial therapy [18, 19, 20, 21]. It has also been used to study gene expression *in vitro*, due to the obtained cDNA retains the original RNA sequence. The main challenge of using this technique is the sample of mRNA, because this is considered difficult to handle by low level and concentration of mRNA of interest and low stability at room temperature together

This technique allows an approximation to the relative amount of nucleic acids present in a sample, as mentioned above. cDNA is obtained by RT-PCR when sample is RNA. Then, internal controls (that are used as markers) are amplified. The markers commonly used are Apo A1 and B actin. Amplification product is separated by electrophoresis. Agarose gel is photographed after ethidium bromide staining, and optical density is calculated by a densitometer. The disadvantage of the technique is possibility of nonspecific hybridizations, generating unsatisfactory results. Control of specificity is performed using highly specific

Real time PCR or quantitative PCR (qPCR) is other adaptation of the PCR method to quantify the number of copies of nucleic acids during PCR. Thus, qPCR is used to quantify DNA o cDNA, determining gene or transcript numbers present within different samples [25, 26, 27]. qPCR offers advantages such as speed in the result, the reduced risk of contamination and the ease in handling technology [28, 29]. This PCR uses fluorescence detection systems which are generally of two types: intercalating agents and labeled probes

Intercalating agents such as SYBR Green are fluorochromes that dramatically increase the fluorescence by binding to a double-stranded DNA [30, 31, 32]. Thus, the increase of DNA in each cycle reflects a proportional increase in the emitted fluorescence. However, it is considered that intercalating agents offer a low specificity because they can be bind to nonspecific products or primer dimers. Several studies have shown that careful selection of primers and using of optimal PCR conditions may minimize this nonspecificity [28, 32, 33]. The use of a high temperature to start the synthesis reaction (hot-start PCR) decreases the risk of nonspecific amplification. Another detection system used in real time PCR are specific hybridization probes labeled with two types of fluorochromes, a donor and an acceptor. The most commonly used probes are hydrolysis or TaqMan probes, molecular beacons probes, and FRET (fluorescent resonance energy transfer) [32, 33, 34]. The increase of DNA in each cycle is proportional to hybridization of probes, which in turn is proportional to the increase in the emitted fluorescence. The use of probes allows identifying polymorphisms and mutations; however, these are more complex and expensive

with sensitivity to action of ribonucleases and pH change [20, 21, 22].

**1.4 Reverse Transcriptase PCR (RT-PCR)** 

probes for hybridization [23, 24] (figure 5).

than intercalating agents [35, 36, 37].

**1.5 Semiquantitative PCR** 

**1.6 Real time PCR** 

with fluorophores.

include sets of specific primers for different targets. In this PCR is important the design of primers because they must be characterized by adherence to specific DNA sequences at similar temperatures. However, it may require several trials to achieve the standardization of the procedure [8, 9].

Fig. 4. Electrophoretogram which shows deletions associated with Duchenne Muscular Dystrophy (DMD). In this figure is noted the absence of peaks in men with deletions. Area under the curve in women with DMD is reduced compared to normal control [13, 14].

## **1.3 Nested PCR**

This PCR increases the sensitivity due to small amounts of the target are detected by using two sets of primers, involving a double process of amplification [15, 16]. The first set of primers allows a first amplification. The product of this PCR is subjected to a second PCR using the second set of primers. These primers used in the second PCR are specific to an internal amplified sequence in the first PCR. Therefore, specificity of the first PCR product is verified with the second one. The disadvantage of this technique is the probability of contamination during transfer from the first amplified product into the tube in which the second amplification will be performed. Contamination can be controlled using primers designed to anneal at different temperatures. Contamination can also be controlled by adding ultra-pure oil to make a physical separation of two mixtures of amplification [15, 17, 18].

## **1.4 Reverse Transcriptase PCR (RT-PCR)**

This PCR was designed to amplify RNA sequences (especially mRNA) through synthesis of cDNA by reverse transcriptase (RT). Subsequently, this cDNA is amplified using PCR. This type of PCR has been useful for diagnosis of RNA viruses, as well as for evaluation of antimicrobial therapy [18, 19, 20, 21]. It has also been used to study gene expression *in vitro*, due to the obtained cDNA retains the original RNA sequence. The main challenge of using this technique is the sample of mRNA, because this is considered difficult to handle by low level and concentration of mRNA of interest and low stability at room temperature together with sensitivity to action of ribonucleases and pH change [20, 21, 22].

## **1.5 Semiquantitative PCR**

160 Polymerase Chain Reaction

include sets of specific primers for different targets. In this PCR is important the design of primers because they must be characterized by adherence to specific DNA sequences at similar temperatures. However, it may require several trials to achieve the standardization

*Del 48*

*Del 48*

*Del 19 Del 48 Del 45*

*Del 19 Del 48 Del 45*

Fig. 4. Electrophoretogram which shows deletions associated with Duchenne Muscular Dystrophy (DMD). In this figure is noted the absence of peaks in men with deletions. Area under the curve in women with DMD is reduced compared to normal control

400 420 440 460 480 500 520 **51 19 48 45**

400 420 440 460 480 500 520 **51 19 48 45**

**Exones** 300 320 340 360 380

**34 Af ect ada 38**

**Exones**

**34 Af ect ada 38**

**M adre 3 8**

**Carrier Female Affected Male Exons of the Dystrophin Gene**

**He rma na 7**

**Madre 3 8**

**He rma na 7**

**31 Af ect ado 7**

**31**

**31**

**32**

**32**

540 560

540 560

**Af ect ado 3**

**30 M adre 3**

**Af ect ado 3**

**Affected Male Carrier Female Affected Male Carrier Female**

**30 Madre 3**

**31 Af ect ado 7**

**Muj er Control Hombre Control**

**M uj er Control Hombre Control**

**M arca dor**

**Marker**

**Control Female Control Male**

**27**

**27**

**29**

**26**

**20**

**29**

**26**

**20**

**Marca dor**

This PCR increases the sensitivity due to small amounts of the target are detected by using two sets of primers, involving a double process of amplification [15, 16]. The first set of primers allows a first amplification. The product of this PCR is subjected to a second PCR using the second set of primers. These primers used in the second PCR are specific to an internal amplified sequence in the first PCR. Therefore, specificity of the first PCR product is verified with the second one. The disadvantage of this technique is the probability of contamination during transfer from the first amplified product into the tube in which the second amplification will be performed. Contamination can be controlled using primers designed to anneal at different temperatures. Contamination can also be controlled by adding ultra-pure oil to make a physical separation of two mixtures of amplification

of the procedure [8, 9].

**Auto-Scaled Data Size (Bases)**

**( )**

*Del 43 Del 19*

*Del 43 Del 19*

[13, 14].

**1.3 Nested PCR** 

**43**

**43**

300 320 340 360 380

[15, 17, 18].

This technique allows an approximation to the relative amount of nucleic acids present in a sample, as mentioned above. cDNA is obtained by RT-PCR when sample is RNA. Then, internal controls (that are used as markers) are amplified. The markers commonly used are Apo A1 and B actin. Amplification product is separated by electrophoresis. Agarose gel is photographed after ethidium bromide staining, and optical density is calculated by a densitometer. The disadvantage of the technique is possibility of nonspecific hybridizations, generating unsatisfactory results. Control of specificity is performed using highly specific probes for hybridization [23, 24] (figure 5).

## **1.6 Real time PCR**

Real time PCR or quantitative PCR (qPCR) is other adaptation of the PCR method to quantify the number of copies of nucleic acids during PCR. Thus, qPCR is used to quantify DNA o cDNA, determining gene or transcript numbers present within different samples [25, 26, 27]. qPCR offers advantages such as speed in the result, the reduced risk of contamination and the ease in handling technology [28, 29]. This PCR uses fluorescence detection systems which are generally of two types: intercalating agents and labeled probes with fluorophores.

Intercalating agents such as SYBR Green are fluorochromes that dramatically increase the fluorescence by binding to a double-stranded DNA [30, 31, 32]. Thus, the increase of DNA in each cycle reflects a proportional increase in the emitted fluorescence. However, it is considered that intercalating agents offer a low specificity because they can be bind to nonspecific products or primer dimers. Several studies have shown that careful selection of primers and using of optimal PCR conditions may minimize this nonspecificity [28, 32, 33]. The use of a high temperature to start the synthesis reaction (hot-start PCR) decreases the risk of nonspecific amplification. Another detection system used in real time PCR are specific hybridization probes labeled with two types of fluorochromes, a donor and an acceptor. The most commonly used probes are hydrolysis or TaqMan probes, molecular beacons probes, and FRET (fluorescent resonance energy transfer) [32, 33, 34]. The increase of DNA in each cycle is proportional to hybridization of probes, which in turn is proportional to the increase in the emitted fluorescence. The use of probes allows identifying polymorphisms and mutations; however, these are more complex and expensive than intercalating agents [35, 36, 37].

Polymerase Chain Reaction: Types, Utilities and Limitations 163

During the past 30 years molecular techniques have been under development, however these have had a rapid and tremendous progress in recent year [38]. Among molecular techniques, PCR and its different variations are highlighted as the most commonly used in laboratories and research institutes. Thus, these have contributed to identification and characterization of several organisms and understanding of physiopathology of diverse diseases in human, animal and plant [39, 40]. Also these have provided clues for future research directions in specific topics with impact in public health such as genetics and biochemistry of antimicrobial resistance [41, 42]. The following describes some applications of PCR and its variants in studies in human medicine, forensic sciences, and agricultural

Molecular biology techniques, particularly PCR, have had a major impact on medicine. The versatility of molecular techniques has allowed advances and changes in all fields of medicine. The following is an overview of the main impacts generated for molecular biology

Clinical microbiology has been transformed with the use of molecular technology because it has generated a benefit to the patient affected by infectious diseases. Molecular biology has allowed the development of clinical microbiology because it has been possible to identify microorganisms that are difficult to culture, that have many requirements of laboratory or dangerous for laboratory personnel. These problems have been reduced with the implementation of molecular diagnosis that provides high sensitivity, specificity, precision and speed with one small sample. These applications are transforming and complementing the work of biochemists, immunologists, microbiologists and other health professionals who see in the molecular tools new alternatives for a rapid diagnosis of microorganisms as well as for the determination of multiple factors associated with antibiotic resistance thus expanding the knowledge of microbial epidemiology and surveillance at the genetic level

The usefulness of PCR in identification of microorganisms has led to the selection and quality assurance of blood that blood banks are using for patients with different pathologies [46]. The incorporation of molecular techniques has been of great importance in the identification and characterization of many viruses, including influenza, which through a rapid, sensitive, and effective molecular diagnosis has allowed inclusion of early treatment

The implementation of molecular tools has allowed a transformation of pathological studies and has changed the clinical practice. This is how the diagnosis and treatment of complex diseases that require a multidisciplinary clinical team currently has a base of molecular biology due to histopathological evaluation of tissues, which is an important part in the morphological assessment, is insufficient by itself. Thus, the ability to define molecular alterations associated with the disease is increasingly required to clarify the diagnosis and therapeutic guidance [48]. At pathological level, molecular biology has allowed the identification of mutations and carriers of diseases as in diabetes, obesity, neurological,

to benefit patients and control of a high impact infection [47, 48].

**2. Applications of PCR and impact on science** 

science and environment.

**2.1 Medicine** 

[43, 44, 45].

in medical sciences.

Fig. 5. Semiquantitative PCR procedure. This technique is useful for identifying small amounts of nucleic acids.

## **2. Applications of PCR and impact on science**

During the past 30 years molecular techniques have been under development, however these have had a rapid and tremendous progress in recent year [38]. Among molecular techniques, PCR and its different variations are highlighted as the most commonly used in laboratories and research institutes. Thus, these have contributed to identification and characterization of several organisms and understanding of physiopathology of diverse diseases in human, animal and plant [39, 40]. Also these have provided clues for future research directions in specific topics with impact in public health such as genetics and biochemistry of antimicrobial resistance [41, 42]. The following describes some applications of PCR and its variants in studies in human medicine, forensic sciences, and agricultural science and environment.

#### **2.1 Medicine**

162 Polymerase Chain Reaction

**RT RNA**

**Total cDNA Total RNA**

**Agarose Gel**

**dNTPs**

Fig. 5. Semiquantitative PCR procedure. This technique is useful for identifying small

**Densitograms 1 2**

**Agarose Gel**

**Apo A-1** β **Actin**

**Photography**

**Apo A-1 β Actin**

**Densitometer**

**Negative**

**MgCl2** *Primers* **of Apo A-1**

**PCR**

**Amplified Product**

**Total cDNA**

*Primers* **of β Actin**

amounts of nucleic acids.

**DNA ethidium bromide-stained**

**Product Quantification**

**β Actin Apo A-1** Molecular biology techniques, particularly PCR, have had a major impact on medicine. The versatility of molecular techniques has allowed advances and changes in all fields of medicine. The following is an overview of the main impacts generated for molecular biology in medical sciences.

Clinical microbiology has been transformed with the use of molecular technology because it has generated a benefit to the patient affected by infectious diseases. Molecular biology has allowed the development of clinical microbiology because it has been possible to identify microorganisms that are difficult to culture, that have many requirements of laboratory or dangerous for laboratory personnel. These problems have been reduced with the implementation of molecular diagnosis that provides high sensitivity, specificity, precision and speed with one small sample. These applications are transforming and complementing the work of biochemists, immunologists, microbiologists and other health professionals who see in the molecular tools new alternatives for a rapid diagnosis of microorganisms as well as for the determination of multiple factors associated with antibiotic resistance thus expanding the knowledge of microbial epidemiology and surveillance at the genetic level [43, 44, 45].

The usefulness of PCR in identification of microorganisms has led to the selection and quality assurance of blood that blood banks are using for patients with different pathologies [46]. The incorporation of molecular techniques has been of great importance in the identification and characterization of many viruses, including influenza, which through a rapid, sensitive, and effective molecular diagnosis has allowed inclusion of early treatment to benefit patients and control of a high impact infection [47, 48].

The implementation of molecular tools has allowed a transformation of pathological studies and has changed the clinical practice. This is how the diagnosis and treatment of complex diseases that require a multidisciplinary clinical team currently has a base of molecular biology due to histopathological evaluation of tissues, which is an important part in the morphological assessment, is insufficient by itself. Thus, the ability to define molecular alterations associated with the disease is increasingly required to clarify the diagnosis and therapeutic guidance [48]. At pathological level, molecular biology has allowed the identification of mutations and carriers of diseases as in diabetes, obesity, neurological,

[62].

discrimination [64, 65].

research are described below.

[70]; among others.

**2.3 Agricultural sciences and environment** 

Polymerase Chain Reaction: Types, Utilities and Limitations 165

research systemic changes involved in the pathophysiological process of death that cannot be detected by morphology. In addition, genetic basis of diseases with sudden death can also be investigated with molecular methods. Practical application of RNA analysis has not been accepted for post-mortem research, due to rapid decomposition after death. However, recent studies using variants of conventional PCR (qPCR and RT-PCR) have suggested that relative quantification of RNA transcripts can be applied in molecular pathology to research deaths ("molecular autopsy"). In a broad sense, forensic molecular pathology involves application of molecular biology in medical science to investigate the genetic basis of pathophysiology of diseases that lead to death. Therefore, molecular tools support and reinforce the morphological and physiological evidence in research of unexplained death

Molecular methods are used in forensic science to establish the filiations of a person (paternity testing) or to obtain evidence from minimal samples of saliva, semen or other tissue debris [63]. Genetic profile of the alleles identified in different regions of DNA is performed in paternity tests using a genetic marker STR (Short Tandem Repeat). Each region has an allele contributed by mother and one from father. This profile is virtually unique to each individual, offering a high power molecular evidence of genetic

Applications of molecular techniques in research in agricultural sciences and environment have been very numerous and varied. It is possible that one of the most important contributions of the applications of some molecular techniques such as PCR has been the identification and characterization of multiple infectious agents that have great impact on human and animal health. Some applications in agricultural science and environment

Currently, the genome of most domestic animals and major infectious agents that affect animals is known through the use of molecular tools, facilitating the study of mutations associated with disease (http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi). Some of the most recent reports are listed below: i. the identification of polymorphisms in ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies [66]; ii. A mutation of EDA gene associated with anhidrotic ectodermal dysplasia in Holstein cattle [67]; iii. The deletion of Meq gene which significantly decreases immunosuppression in chickens caused by Marek's disease virus [68]; iv. The MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers [69]; v. An insertion mutation in ABCB4 associated with gallbladder mucocele formation in dogs

Molecular techniques such as conventional PCR or qPCR have also facilitated research in detection of pathogens in plants, animals, and the environment; understanding of their epidemiology; and, development of new diagnostic tests, treatments or vaccines. Conventional PCR or PCR based methods are being applied to identification and characterization of specific pathogens of animals, e.g., infectious bursal disease virus in avian samples [71]; bovine respiratory syncytial virus [72]; *Actinobacillus pleuropneumoniae*

muscular, cardiac, metabolic, and congenital diseases and pathologies associated with sensory organs. At the ocular level, implementation of molecular biology has generated enormous advances in knowledge, diagnosis and treatment of ophthalmic diseases [49, 50]. The usefulness of this technique in the identification of mutations associated with ocular diseases has been widely used for the study of families at risk. Several reports show how PCR has allowed expanding the knowledge of certain diseases; thus, Woloschak et al. 1994 [51] showed the loss of heterozygosity in the retinoblastoma gene in pituitary human tumors. It was possible to demonstrate genetic heterogeneity in congenital fibrosis of extraocular muscles. With the advent of molecular technology, it has been possible to understand certain aspects of diseases as in retinitis pigmentosa, microftalmia, retinoblastoma, open-angle glaucoma, ocular diseases due to alterations in mitochondrial DNA and various types of corneal dystrophy, among others [50, 52, 53]. Also, genes that cause ocular diseases have been cloned at the anterior and posterior segment. In anterior segment basically aniridia and Peter's anomaly, autosomal dominant diseases in which have been identified candidate genes [54]. In posterior segment, the number of cloned genes has been higher; these are associated with different pathologies described as following. Retinitis pigmentosa with autosomal dominant inheritance pattern in most cases; however, it can also be found in a recessive or digenic form [55]. Congenital Stationary Night Blindness, a disease whose pattern of inheritance is autosomal dominant. Retinoblastoma which the genetic defect affects the retinoblastoma protein (Rb) whose gene rb has been cloned [56]. Cones degeneration inherited pattern linked to X, this means that the disease is transmitted by a carrier mother, where 50% of boys are likely to get the disease and 50% of their daughters are likely to pass it. Its alteration affects synthesis of red opsin [57, 58]. Leber hereditary optic neuropathy associated with alteration of mitochondrial DNA whose defect involves activity of mitochondrial enzymes [57]. These findings have strong implications for the understanding of physiopathology of these genetic entities and generate a new concept of ocular clinical practice due to advances in molecular biology not only can classify better the pathology but the diagnosis becomes specific and safe. On the other hand, in those ocular diseases attributed to mutations in genes located on chromosome X, it is possible to identify mothers or women on the mother line and to generate secondary prevention measures when inform the carrier or not carrier status of them [49].

Molecular tools have also allowed to perform preimplantation genetic diagnosis (PGD) being used for genetic analysis of embryos before transfer into the uterus. It was first developed in England in 1990, as part of the advances in reproductive medicine, genetics and molecular biology. PGD offers couples at risk of having an affected child the opportunity to have normal child by assisted fertilization. The molecular genetic analysis is performed on one or two blastomeres, and only unaffected embryos are transferred into the uterus. It is important to note that in many countries the using of this reproductive procedure has caused controversy. However, this technique provides an opportunity for couples whose children have shown earlier genetic abnormalities [59, 60, 61].

#### **2.2 Forensic science**

In forensic pathology, classic morphology remains as a basic procedure to investigate deaths, but recent advances in molecular biology have provided a very useful tool to

muscular, cardiac, metabolic, and congenital diseases and pathologies associated with sensory organs. At the ocular level, implementation of molecular biology has generated enormous advances in knowledge, diagnosis and treatment of ophthalmic diseases [49, 50]. The usefulness of this technique in the identification of mutations associated with ocular diseases has been widely used for the study of families at risk. Several reports show how PCR has allowed expanding the knowledge of certain diseases; thus, Woloschak et al. 1994 [51] showed the loss of heterozygosity in the retinoblastoma gene in pituitary human tumors. It was possible to demonstrate genetic heterogeneity in congenital fibrosis of extraocular muscles. With the advent of molecular technology, it has been possible to understand certain aspects of diseases as in retinitis pigmentosa, microftalmia, retinoblastoma, open-angle glaucoma, ocular diseases due to alterations in mitochondrial DNA and various types of corneal dystrophy, among others [50, 52, 53]. Also, genes that cause ocular diseases have been cloned at the anterior and posterior segment. In anterior segment basically aniridia and Peter's anomaly, autosomal dominant diseases in which have been identified candidate genes [54]. In posterior segment, the number of cloned genes has been higher; these are associated with different pathologies described as following. Retinitis pigmentosa with autosomal dominant inheritance pattern in most cases; however, it can also be found in a recessive or digenic form [55]. Congenital Stationary Night Blindness, a disease whose pattern of inheritance is autosomal dominant. Retinoblastoma which the genetic defect affects the retinoblastoma protein (Rb) whose gene rb has been cloned [56]. Cones degeneration inherited pattern linked to X, this means that the disease is transmitted by a carrier mother, where 50% of boys are likely to get the disease and 50% of their daughters are likely to pass it. Its alteration affects synthesis of red opsin [57, 58]. Leber hereditary optic neuropathy associated with alteration of mitochondrial DNA whose defect involves activity of mitochondrial enzymes [57]. These findings have strong implications for the understanding of physiopathology of these genetic entities and generate a new concept of ocular clinical practice due to advances in molecular biology not only can classify better the pathology but the diagnosis becomes specific and safe. On the other hand, in those ocular diseases attributed to mutations in genes located on chromosome X, it is possible to identify mothers or women on the mother line and to generate secondary prevention

measures when inform the carrier or not carrier status of them [49].

couples whose children have shown earlier genetic abnormalities [59, 60, 61].

**2.2 Forensic science** 

Molecular tools have also allowed to perform preimplantation genetic diagnosis (PGD) being used for genetic analysis of embryos before transfer into the uterus. It was first developed in England in 1990, as part of the advances in reproductive medicine, genetics and molecular biology. PGD offers couples at risk of having an affected child the opportunity to have normal child by assisted fertilization. The molecular genetic analysis is performed on one or two blastomeres, and only unaffected embryos are transferred into the uterus. It is important to note that in many countries the using of this reproductive procedure has caused controversy. However, this technique provides an opportunity for

In forensic pathology, classic morphology remains as a basic procedure to investigate deaths, but recent advances in molecular biology have provided a very useful tool to research systemic changes involved in the pathophysiological process of death that cannot be detected by morphology. In addition, genetic basis of diseases with sudden death can also be investigated with molecular methods. Practical application of RNA analysis has not been accepted for post-mortem research, due to rapid decomposition after death. However, recent studies using variants of conventional PCR (qPCR and RT-PCR) have suggested that relative quantification of RNA transcripts can be applied in molecular pathology to research deaths ("molecular autopsy"). In a broad sense, forensic molecular pathology involves application of molecular biology in medical science to investigate the genetic basis of pathophysiology of diseases that lead to death. Therefore, molecular tools support and reinforce the morphological and physiological evidence in research of unexplained death [62].

Molecular methods are used in forensic science to establish the filiations of a person (paternity testing) or to obtain evidence from minimal samples of saliva, semen or other tissue debris [63]. Genetic profile of the alleles identified in different regions of DNA is performed in paternity tests using a genetic marker STR (Short Tandem Repeat). Each region has an allele contributed by mother and one from father. This profile is virtually unique to each individual, offering a high power molecular evidence of genetic discrimination [64, 65].

#### **2.3 Agricultural sciences and environment**

Applications of molecular techniques in research in agricultural sciences and environment have been very numerous and varied. It is possible that one of the most important contributions of the applications of some molecular techniques such as PCR has been the identification and characterization of multiple infectious agents that have great impact on human and animal health. Some applications in agricultural science and environment research are described below.

Currently, the genome of most domestic animals and major infectious agents that affect animals is known through the use of molecular tools, facilitating the study of mutations associated with disease (http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi). Some of the most recent reports are listed below: i. the identification of polymorphisms in ABCB1 gene in phenobarbital responsive and resistant idiopathic epileptic Border Collies [66]; ii. A mutation of EDA gene associated with anhidrotic ectodermal dysplasia in Holstein cattle [67]; iii. The deletion of Meq gene which significantly decreases immunosuppression in chickens caused by Marek's disease virus [68]; iv. The MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers [69]; v. An insertion mutation in ABCB4 associated with gallbladder mucocele formation in dogs [70]; among others.

Molecular techniques such as conventional PCR or qPCR have also facilitated research in detection of pathogens in plants, animals, and the environment; understanding of their epidemiology; and, development of new diagnostic tests, treatments or vaccines. Conventional PCR or PCR based methods are being applied to identification and characterization of specific pathogens of animals, e.g., infectious bursal disease virus in avian samples [71]; bovine respiratory syncytial virus [72]; *Actinobacillus pleuropneumoniae*

Polymerase Chain Reaction: Types, Utilities and Limitations 167

[2] Mullis, K; Faloona, F; Scharf S; Saiki, RK; Horn, GT; Erlich, HA. (1986). Specific

[3] Mullis, K and Faloona, F. (1987). Specific synthesis of DNA *in vitro* via a polymerase-

[4] Louie, M; Louie, L; Simor, AE. (2000). The role of DNA amplification of technology in

[5] Fredriscks, DN and Relman, DA. (1999). Application polymerase chain reactions to the

[6] Erlich, HA; Gelfand, D; Snisky, JJ. (1991). Recent advances in the polymerase chain

[7] Tang, YM; Procop, GW; Persong, DH. (1997). Molecular diagnostics of infectious

[8] Jackson, CR; Fedorka-Cray, PJ; Barret, JB. (2004). Use of a Genus and Species Specific Multiplex PCR for amplification of Enterococci. *J. Clin Microbiol*. 42: 3558-3565. [9] Toma, C; Lu, Y; Higa, N; Nakasome, N; Chinen, I; Baschkier, A; Rivas, M; Iwanaga, M.

[10] Pehler, K; Khanna, M; Water, CR; Henrickson, KJ. (2004). Detection and amplification of

[11] Echeverria, JE; Erdman, DD; Swierkosz, EM; Holloway, BP; Anderson, LJ. (1998).

pneumophila and other Legionella species. *J. Clin. Microbiol*. 41: 4016-4021. [13] Hernández-Rodríguez, P; Gómez, Y and Restrepo, CM. (2000). Identification of carries

[14] Hernández-Rodríguez, P and Restrepo, CM. (2002). Identification of Deletions in

[15] Jann-Yuan, W; Li-Na, N; Chin-Sheng, C; Chung-Yi, H; Shu-Kuan, W; Hsin-Chih, L; Po-

[16] Zeaiter, Z; Fournier, PE; Greub, G; Raoult, D. (2003). Diagnosis of Bordertella pertussisand Bordertella parapertussis infections. *J. Clin. Microbiol*. 41: 919-25. [17] Kitagawa, Y; Ueda, M; Ando, N; Endo, M; Ishibiki, K; Kobayashi, Y. (1996). Rapid

tuberculosis in clinical specimens. *J. Clin. Microbiol.* 42: 4599-4603.

Polymerase Chain Reaction. *Ann. Surg*. 224: 665-71.

3 from clinical samples by multiplex PCR. *J. Clin. Microbiol*. 36: 1388-91. [12] Templeton, KE; Scheltinga, SA; Sillekens, P; Crielaard, JW; van Dam, AP; Goenssens, H;

(2003). Multiplex PCR Assay for identification of Human Diarrheagenic Eschericha

human adenovirus species by adenoplex, a multiplex PCR enzyme hybridization

Simultaneous detection and identification of human parainfluenza viruses 1,2 and

Claas, EC. (2003). Development and clinical evaluation of an internally controlled, single tube multiplex Real Time PCR assay for detection of Legionella

Duchenne and Becker Muscular Dystrophy through gene dosage and DNA

Affected of Duchenne and Becker Muscular Dystrophy (DMD/DMB) and Diagnostic of carrier for Molecular Methodologies. *Universitas Scientiarum*. 7 (1): 31-

Ren, H; Kwen-Tay, L. (2004). Performance assessment of a Nested-PCR assay (the RAPID BAP-MTB) and the BD ProbeTec ET system for detection of Micobacterium

diagnosis of methicillin-resistan Staphylococcus aureus bacteremia by Nested

Spring Harbor Symp. *Quant. Biol.* 51: 263-273.

reaction. *Science*. 252: 1643-51.

diseases. *Clin. Chem*. 43: 2021-38.

coli. *J. Clin. Microbiol*. 41: 2669-2671.

assay. *J. Clin. Microbiol*. 42:4070-4076.

polymorphisms. *Biomedica.* 20 (3): 228-237.

42.

catalyzed chain reaction. *Methods Enzymol*. 155:338-350.

the diagnosis of infection diseases. *CMAJ.* 163(3): 301-9.

diagnosis of infectious diseases. *Clin. Infect. Dis*. 29: 475-88.

enzymatic amplification of DNA *in vitro*: the polymerase chain reaction. Cold

from samples of pigs [73]; canine parvovirus type 2 (CPV 2) in faecal samples of dogs [74]; feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) [75]; among others. Nucleic acid based detection methods are also important to identification of foodborne pathogens, such as *Listeria monocytogenes* [76]; *Campylobacter* spp., *Salmonella enterica*, and *Escherichia coli* O157:H7 [77].

Despite these important applications of molecular methods, one of the purposes with the greatest impact is the detection and characterization of agents with zoonotic potential, such as pandemic (H1N1) influenza [78]; leptospirosis [39]; Canine visceral leishmaniasis [79]; among others.

In summary, PCR has advantages as a diagnostic tool in conventional microbiology, particularly in the detection of slow growing or difficult to cultivate microorganisms, or under special situations in which conventional methods are expensive or hazardous. Due to the stability of DNA, nucleic acid based detection methods can be also used when inhibitory substances, such as antimicrobials or formalin, are present [80]. Therefore, through the use of molecular techniques has been able to identify different pathogens, to elucidate its epidemiology, to achieve standardization of diagnostic methods, and to establish strategies of prevention and control of diseases, advancing in sanitary regulations in different countries.

## **3. Conclusions**

New knowledge has been generated in different fields of science with invention of PCR 25 years ago. The applications of molecular biology have transformed diagnosis, prognosis and treatment of many diseases. Likewise, molecular methodologies to measure and evaluate gene expression have become the key techniques of the post-genomic era. This correlates with the increasing number of reports of molecular technologies to identify and characterize multiple infectious agents and diseases affecting humans, plants, and animals. The above mentioned justifies the establishment of clear regulations and statistical models for evaluation and adoption of these protocols in laboratories of diagnosis [81]. Despite the continuing evolution of molecular biology, future efforts should continue to increase understanding of advantages and disadvantages of molecular methods in diagnosis, and its interpretation within the clinical context. In addition, it is necessary to increase research for the development of guideline for standardization, validation and comparison new molecular diagnostic methods with existing techniques regarding to sample type, sample preparation, PCR amplification, and reporting of results [80]. In conclusion, the development of molecular biology techniques such as PCR and its variants has led to advances in medicine, agriculture, animal science, forensic science and environment, among others; transforming the society and economy, and influencing the quality of life of people and the development of science and countries.

## **4. References**

[1] Saiki, RK; Scharf S; Faloona F; Mullis, KB; Horn, GT; Erlich, HA; and Arnheim, N. (1985). Enzymatic amplification of β-globin genomic sequences and restriction siteanalysis for diagnosis of sickle cell anemia. *Science* 230: 1350-1354.

from samples of pigs [73]; canine parvovirus type 2 (CPV 2) in faecal samples of dogs [74]; feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) [75]; among others. Nucleic acid based detection methods are also important to identification of foodborne pathogens, such as *Listeria monocytogenes* [76]; *Campylobacter* spp., *Salmonella enterica*, and

Despite these important applications of molecular methods, one of the purposes with the greatest impact is the detection and characterization of agents with zoonotic potential, such as pandemic (H1N1) influenza [78]; leptospirosis [39]; Canine visceral leishmaniasis [79];

In summary, PCR has advantages as a diagnostic tool in conventional microbiology, particularly in the detection of slow growing or difficult to cultivate microorganisms, or under special situations in which conventional methods are expensive or hazardous. Due to the stability of DNA, nucleic acid based detection methods can be also used when inhibitory substances, such as antimicrobials or formalin, are present [80]. Therefore, through the use of molecular techniques has been able to identify different pathogens, to elucidate its epidemiology, to achieve standardization of diagnostic methods, and to establish strategies of prevention and control of diseases, advancing in sanitary regulations in different

New knowledge has been generated in different fields of science with invention of PCR 25 years ago. The applications of molecular biology have transformed diagnosis, prognosis and treatment of many diseases. Likewise, molecular methodologies to measure and evaluate gene expression have become the key techniques of the post-genomic era. This correlates with the increasing number of reports of molecular technologies to identify and characterize multiple infectious agents and diseases affecting humans, plants, and animals. The above mentioned justifies the establishment of clear regulations and statistical models for evaluation and adoption of these protocols in laboratories of diagnosis [81]. Despite the continuing evolution of molecular biology, future efforts should continue to increase understanding of advantages and disadvantages of molecular methods in diagnosis, and its interpretation within the clinical context. In addition, it is necessary to increase research for the development of guideline for standardization, validation and comparison new molecular diagnostic methods with existing techniques regarding to sample type, sample preparation, PCR amplification, and reporting of results [80]. In conclusion, the development of molecular biology techniques such as PCR and its variants has led to advances in medicine, agriculture, animal science, forensic science and environment, among others; transforming the society and economy, and influencing the quality of life of people

[1] Saiki, RK; Scharf S; Faloona F; Mullis, KB; Horn, GT; Erlich, HA; and Arnheim, N. (1985).

for diagnosis of sickle cell anemia. *Science* 230: 1350-1354.

Enzymatic amplification of β-globin genomic sequences and restriction siteanalysis

*Escherichia coli* O157:H7 [77].

among others.

countries.

**3. Conclusions** 

**4. References** 

and the development of science and countries.


Polymerase Chain Reaction: Types, Utilities and Limitations 169

[34] Demeke, T and Jenkins GR (2010). Influence of DNA extraction methods, PCR

[35] Chagovetz, A and Blair, S. (2009). Real-time DNA microarrays: reality check. *Biochem.* 

[36] Smith, CJ and Osborn, AM. (2009). Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. *FEMS Microbiol. Ecol*. 67(1):6-20. [37] Giasuddin, AS; Jhuma, KA; Haq AM. (2008). Applications of free circulating nucleic

[38] Fluit, AC; Visser, MR; Schmitz, FJ. (2001). Molecular detection of antimicrobial

[39] Hernández-Rodríguez, P; Díaz, C; Dalmau, E; Quintero, G. (2011). A comparison

[40] Gomez, AP; Moreno, MJ; Baldrich, RM; Hernández, A. (2008). Endothelin-1 messenger

[41] Sundsfjord, A; Simonsen, GS; Haldorsen, BC; Haaheim, H; Hjelmevoll, SO; Littauer, P;

[42] Courvalin, P and Trieu-Cuot P. Minimizing potential resistance: the molecular view.

[43] Weile, J and Knabbe, C. (2009). Current applications and future trends of molecular diagnostics in clinical bacteriology. *Anal. Bioanal. Chem.* 394(3):731-42. [44] Dreier, J; Störmer, M; Kleesiek K. (2007). Real-time polymerase chain reaction in

[45] Hutchins, GG and Grabsch, HI. (2009). Molecular pathology the future?. *Surgeon*.

[46] Dreier J, Störmer M, Kleesiek K. (2007). Real-time polymerase chain reaction in

[47] Dale, SE. (2010). The role of rapid antigen testing for influenza in the era of molecular

[48] Wyczałkowska-Tomasik A, Zegarska J. (2009). Real-time polymerase chain reaction

[49] Hernández-Rodríguez, P. (2003). Technical molecular: an advance in the diagnosis and

[50] Mahdy MA.(2010a). Gene therapy in glaucoma-part 2: Genetic etiology and gene

[51] Woloschak, M; Roberts, J; and Kalmon, D. (1994). Loos of heterozygosity at the retinoblastoma locus in human pituitary tumors. *Cancer.* 74(2): 693-96. 49.

derived traits. *Anal. Bioanal. Chem.* 396(6):1977-90.

resistance. *Clin. Microbiol*. 14(4):836-71.

*Clin. Infect. Dis*. 2001 Sep 15;33 Suppl 3:S138-46.

products. *Transfus. Med. Rev.* 21(3):237-54.

products. *Transfus. Med. Rev.* 21(3):237-54.

mapping. *Oman J. Ophthalmol*. 3(2):51-9. 48.

diagnostics. *Mol. Diagn. Ther.* 2010 Aug 1;14(4):205-14.

*Soc. Trans.* 37(Pt 2):471-5.

34(1):26-32.

Aug;87(8):1689.

112(11-12):815-37.

7(6):366-77.

46.

*Health*. 1(1): 113-122. 47.

inhibitors and quantification methods on real-time PCR assay of biotechnology-

acids in clinical medicine: recent advances. *Bangladesh Med. Res. Counc. Bull.*

between Polymerase Chain Reaction (PCR) and traditional techniques for the diagnosis of leptospirosis in bovines. *Journal of Microbiological Methods.* 2011. 84: 1-7.

[corrected] ribonucleic acid expression in pulmonary hypertensive and nonhypertensive chickens. Poult Sci. 87(7):1395-401. *Erratum in: Poult Sci.* 2008

Dahl, KH. (2004). Genetic methods for detection of antimicrobial resistance. *APMIS.*

transfusion medicine: applications for detection of bacterial contamination in blood

transfusion medicine: applications for detection of bacterial contamination in blood

applications in research and clinical molecular diagnostics. *Przegl. Lek*. 66(4):209-12.

knowledge of ocular pathologies. *Journal Science and Technology for Vision and Eye* 


[18] Jou, NT; Yoshimori, RB; Mason, GR; Louei, JS; Liebling, MR. (2003). Single tube, nested,

[19] Salomon, RN. (1995). Introduction to quantitative reverse transcription polymerase

[20] Moon, SH; Lee, YJ; Park, SY; Song, KY; Kong, MH; Kim, JH. (2011). The Combined

[22] Puustinen, L; Blazevic, V; Huhti, L; Szakal, ED; Halkosalo, A; Salminen, M; Vesikari, T.

[24] Panitsas, FP and Mouzaki, A (2004). Effect of splenectomy on type-1/type-2 cytokine

[25] Higuchi, R; Fokler, C; Dollinger, G; Watson, R. (1993). Kinetic PCR analysis Real Time monitoring of DNA amplification reactions. *BioTechnology.* 11: 1026-30. [26] Marty, A; Greiner, O; Day, PJR; Gunziger, S; Muhlemann, K; Nadal, D. (2004). Detection Haemophylus influenza Type b by real Time PCR. *J. Clin. Microbiol.* 42: 3813-3815. [27] Lobert, S; Hiser, L; Correia, JJ. (2010). Expression profiling of tubulin isotypes and

[28] Mackay, IM; Arden, KE; Nitsche, A. (2002). Real Time PCR in virology. *Nucleic Acids* 

[29] Maibach, RC and Altwegg, M. (2003). Cloning and sequencing an unknown gene of

[30] Ke, D; Menard, C; Picard, FJ; Boissinot, M; Ouellette, M; Roy, PH. (2000). Development

[31] Vlková, B; Szemes, T; Minárik, G; Turna, J; Celec P. (2010). Advances in the research of

[32] Moretti, T; Koons, B; Budowle, B. (1998). Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. *Biotechniques.* 25: 716-22. [33] Kellogg, DE; Rybalkin, I; Chen, S; Mukhamedova, N; Vlasic, T. (1994). TaqStart

directed against Taq DNA polymerase. *Biotechniques.* 16: 1134-7.

children in Finland between 1994 and 2007. . *Epidemiol. Infect*. 14:1-8. [23] Wang, J; Zhao, ZH; Luo, SJ; Fan, YB. Expression of osteoclast differentiation factor and

*Neuroblastoma Cell Line in Hypoxia and Reperfusion Condition*. 49(1):13-19. [21] Li, J; Huang, X; Xie, X; Wang, J; Duan, M. (2011). Human telomerase reverse

*Clin. Microbiol.* 35: 1161-1165.

23(3):240-3.

B*MC Blood Disord*. 4(1):4.

*Methods Cell Biol.* 95:47-58.

*Microbiol. Infect Dis*. 46:181-7.

*Monit.* 16(4):RA85-91.

streptococci. *Clin. Chem*. 46: 324-31.

*Res*. 30: 1292-305.

chain reaction. *Diag. Mol. Pathol*. 4:82-84.

carcinoma. *Acta Otolaryngol.* 131(5):546-551.

reverse transcriptase PCR for detection of viable Micobacterium tuberculosis. *J.* 

Effects of Ginkgo Biloba Extracts and Aspirin on Viability of SK-N-MC,

transcriptase regulates cyclin D1 and G1/S phase transition in laryngeal squamous

(2011). Norovirus genotypes in endemic acute gastroenteritis of infants and

intercellular adhesion molecule-1 of bone marrow mesenchymal stem cells enhanced with osteogenic differentiation]. Hua Xi Kou Qiang Yi Xue Za Zhi.

gene expression in a patient with adult idiopathic thrombocytopenic purpura (ITP).

microtubule-interacting proteins using real-time polymerase chain reaction.

Tropheryma whipplei and deveploment of two LightCycler PCR assay. *Diagn.* 

of conventional and Real Time PCR assays for the rapid detection of group B

fetal DNA in maternal plasma for noninvasive prenatal diagnostics. *Med. Sci.* 

antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody


Polymerase Chain Reaction: Types, Utilities and Limitations 171

[68] Li, Y; Sun, A; Su, S; Zhao, P; Cui, Z; Zhu, H. (2011). Deletion of the Meq gene

[69] Beggs, AH; Böhm, J; Snead, E; Kozlowski, M; Maurer, M; Minor, K; Childers, MK;

[70] Mealey, KL; Minch, JD; White, SN; Snekvik, KR; Mattoon, JS. (2010). An insertion

[71] Cardoso, TC; Rosa, AC; Astolphi, RD; Vincente, RM; Novais, JB; Hirata, KY; Luvizotto,

[72] Hakhverdyan, M; Hägglund, S; Larsen, LE; Belák, S. (2005). Evaluation of a single-tube

[73] Savoye, C; Jobert, JL; Berthelot-Hérault, F; Keribin, AM; Cariolet, R; Morvan, H; Madec,

[74] Kumar, M and Nandi, S. (2010). Development of a SYBR Green based real-time PCR

[75] Arjona, A; Barquero, N; Doménech, A; Tejerizo, G; Collado, VM; Toural, C; Martín, D;

[76] Leclercq, A; Chenal-Francisque, V; Dieye, H; Cantinelli, T; Drali, R; Brisse, S; Lecuit, M.

[78] Slomka, MJ; Densham, AL; Coward, VJ; Essen, S; Brookes, SM; Irvine, RM; Spackman,

[79] Travi, BL; Tabares, CJ; Cadena, H; Ferro, C; Osorio, Y. (2001). Canine visceral

and infectivity for sand flies. *Am. J. Trop. Med. Hyg.* 64(3-4):119-24. 77.

profile IVb-v1. *Int. J. Food Microbiol.* Mar 21. [Epub ahead of print]. 74. [77] Jokinen, C; Edge, TA; Ho, S; Koning, W; Laing, C; Mauro, W; Medeiros, D; Miller, J;

watershed, Alberta, Canada. *Water Res.* 45(3):1247-57. 75.

infections in pigs. *Influenza Other Respi Viruses*. 4(5):277-93. 76.

clinical samples. *J. Virol. Methods.* 123(2):195-202.70.

conditions. *Vet. Microbiol*. 73(4):337-47. 71.

*Methods*. 169(1):198-201. 72.

*Med. Surg.* 9(1):14-22. 73

Marek's disease virus. *Virol. J.* 8:2. 66.

702. 67.

*Comp. Hepatol*. 9:6. 68.

37(4):457-61. 69.

significantly decreases immunosuppression in chickens caused by pathogenic

Taylor, SM; Hitte, C; Mickelson, JR; Guo, LT; Mizisin, AP; Buj-Bello, A; Tiret, L; Laporte, J; Shelton, GD. (2010). MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers. *Proc. Natl. Acad. Sci.* 107(33):14697-

mutation in ABCB4 is associated with gallbladder mucocele formation in dogs.

MC. (2008). Direct detection of infectious bursal disease virus from clinical samples by in situ reverse transcriptase-linked polymerase chain reaction. *Avian. Pathol*.

fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in

F; Kobisch, M. (2000). A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental

assay for detection and quantitation of canine parvovirus in faecal samples. *J. Virol.* 

Gomez-Lucia, E. (2006). Evaluation of a novel nested PCR for the routine diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). *J. Feline* 

(2011). Characterization of the novel Listeria monocytogenes PCR serogrouping

Robertson, W; Taboada, E; Thomas, JE; Topp, E;, Ziebell, K; Gannon, VP. (2011). Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River

E; Ridgeon, J; Gardner, R; Hanna, A; Suarez, DL; Brown, IH. (2009). Real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus

leishmaniasis in Colombia: relationship between clinical and parasitologic status


[52] Mahdy MA. (2010b). Gene therapy in glaucoma-3: Therapeutic approaches. *Oman J.* 

[53] Traboulsi, E; Bjorn, A; Lee, AB;, Khamis, AR and Engle, E. (2000). Evidence of genetic

[54] Jordan, T; Hanson, Y and Zaletayev, D. (1992). The human PAX6 gene is mutated in

[55] Farrar, GJ; Kenna, PF and Humphries, P. (2002). On the genetics of retinitis pigmentosa

[56] Bogdanici, C; Miron, I and Gherghel, D. (2000). Actualities in retinoblastoma´s

[57] Serratrice, J; Desnuelle, C ; Granel, B ; De Roux-Serratrice, C and Weiller, P. (2001). Mitochondrial diseases in adults. *Rev. Med. Interne.* 22 suppl 3 : 356-66 55. [58] McLaughing, ME; Lin, D; Berson, EL and Dryja, TP. (1993). Recessive mutations in the

[59] Giasuddin, AS; Jhuma, KA; Haq, AM. (2008). Applications of free circulating nucleic

[60] Basille, C; Frydman, R; El Aly, A; Hesters, L; Fanchin, R; Tachdjian, G; Steffann, J;

[61] Vlková, B; Szemes, T; Minárik, G; Turna, J; Celec, P. (2010). Advances in the research of

[62] Maeda, H; Zhu, BL; Ishikawa, T; Michiue, T. (2010). Forensic molecular pathology of

[63] Butler, JM. Forensic DNA Typing: *Biology, Technology, and Genetics of STR.* 1 Edition.

[64] Liu P, Li X, Greenspoon SA, Scherer JR, Mathies RA.. (2011). Integrated DNA

[65] Hurth C, Smith SD, Nordquist AR, Lenigk R, Duane B, Nguyen D, Surve A, Hopwood

[66] Alves, L; Hülsmeyer, V; Jaggy, A; Fischer, A; Leeb, T; Drögemüller M. (2011).

[67] Ogino, A; Kohama, N; Ishikawa, S; Tomita, K; Nonaka, S; Shimizu, K; Tanabe, Y;

of the art. *Eur. J. Obstet. Gynecol. Reprod. Biol.* 145(1):9-13. 58.

forensic human identification. *Lab. Chip.* 21;11(6):1041-8. 62.

violent deaths. *Forensic. Sci. Int.* 203(1-3):83-92. 60.

1676.2011.0718.x. [Epub ahead of print]. 64.

*American journal of Ophthalmology.* 129(5): 658-662. 51.

two patients with aniridia. *Nat. Genet*. 1: 328-32.52.

treatment. *Ophthalmology*. 50(4): 48-54. 54.

pigmentosa. *Nat. Genet*. 4: 130-34. 56.

heterogeneity in autosomal recessive congenital fibrosis of the extraocular muscles.

and on mutation independent approaches to therapeutic intervention. *EMBO J.*

gene encoding the beta subunit of rod phosphodiesterase in patients with retinitis

acids in clinical medicine: recent advances. *Bangladesh Med. Res. Counc. Bull.*

LeLorc'h, M; Achour-Frydman, N. (2009). Preimplantation genetic diagnosis: state

fetal DNA in maternal plasma for noninvasive prenatal diagnostics. *Med. Sci.* 

purification, PCR, sample cleanup, and capillary electrophoresis microchip for

AJ, Estes MD, Yang J, Cai Z, Chen X, Lee-Edghill JG, Moran N, Elliott K, Tully G, Zenhausern F. (2010). An automated instrument for human STR identification: design, characterization, and experimental validation. *Electrophoresis.* 31(21):3510-

Polymorphisms in the ABCB1 Gene in Phenobarbital Responsive and Resistant Idiopathic Epileptic Border Collies. *J. Vet. Intern. Med.* doi: 10.1111/j.1939-

Okawa, H; Morita, M. (2011). A novel mutation of the bovine EDA gene associated with anhidrotic ectodermal dysplasia in Holstein cattle. *Hereditas*.

*Ophthalmol.* 3(3):109-16. 50.

21[5]: 857-64. 53.

34(1):26-32. 57.

3517. 63.

148(1):46-9. 65.

*Monit.* 1;16(4):RA85-91. 59.

NY. Academic Press (2005). 61.


**9** 

 *Germany* 

**The Application of PCR-Based Methods** 

In food control laboratories the world over, molecular biological techniques play an increasingly central role in the analysis of food and food ingredients. Although the classical methods employing cultural, biochemical, cytological and immunological procedures are still being commonly practiced, molecular biological tools employing polymerase chain reaction (PCR) have become an increasingly popular alternative in many food control agencies in recent years. Factors responsible for the popularity of PCR-based detection assays include rapidity, specificity and enhanced sensitivity of the assays. With regard to the latter, often highly denatured food samples and ingredients can still be processed for PCR detection assays because the DNA may still be reliably amplified, as opposed to loss of

Microbial source tracking (MST) which involves the ability to trace microbes, particularly food-borne pathogens, poses unique challenges to the food industry and food regulatory agencies (Santo Domingo and Sadowsky, 2007). Such information would assist regulatory agencies in localizing food producers or vendors responsible for supplying foods involved in human infections. Additionally, such knowledge would afford public health investigators the opportunity to track food-borne disease outbreaks to their point of origin, thereby preventing future occurrences. In providing such crucial information reliably and within the shortest possible time frame, MST employs a number of PCR-based detection assays. The recent outbreak of EHEC infections arising from verocytotoxin-producing *Escherichia coli* EHEC O104:H4, predominantly in Germany furnishes a good example of the importance of a rapid screening tool for the prompt identification of an infectious agent and surveillance monitoring. More than 16 countries in Europe and North America reported a total of 4,075 cases and 50 deaths as of July 21 2011, two months after the first reported case at the beginning of May 2011 (WHO International Health Regulations, Outbreaks of E. coli

In this and other similar cases, PCR-based molecular biological methods are usually employed in the rapid and initial screening of samples, while complementing this approach with the classical cultural technique for reliable end-identification of the isolate. While not replacing the classical methodologies that have stood the test of time, PCR-based molecular approaches are rapidly becoming the initial screening tools in diverse food analytical processes. Commonly the molecular biological methods are supplemented with classical

processing material in detection methods relying on protein analytical tools.

**1. Introduction** 

O104:H4 infection, Update 30) .

 **in Food Control Agencies – A Review** 

Azuka Iwobi, Ingrid Huber and Ulrich Busch

*Bavarian Health and Food Safety Authority, Oberschleissheim* 


## **The Application of PCR-Based Methods in Food Control Agencies – A Review**

Azuka Iwobi, Ingrid Huber and Ulrich Busch *Bavarian Health and Food Safety Authority, Oberschleissheim Germany* 

## **1. Introduction**

172 Polymerase Chain Reaction

[80] Pusterla, N; Madigan, JE; Leutenegger, CM. (2006). Real-time polymerase chain

[81] Niedrig, M; Schmitz, H; Becker, S; Günter, S; Meulen, J; Meter, H; Ellerbrok, H; Nitsche,

*Intern. Med*. 20(1):3-12. 78.

79.

reaction: a novel molecular diagnostic tool for equine infectious diseases. *J. Vet.* 

A; Gelderblom, HR; Drosten, C. (2004). First international quality assurance study on the rapid detection of viral agents of bioterrorism. *J. Clin. Microbiol*. 42: 1753–5.

> In food control laboratories the world over, molecular biological techniques play an increasingly central role in the analysis of food and food ingredients. Although the classical methods employing cultural, biochemical, cytological and immunological procedures are still being commonly practiced, molecular biological tools employing polymerase chain reaction (PCR) have become an increasingly popular alternative in many food control agencies in recent years. Factors responsible for the popularity of PCR-based detection assays include rapidity, specificity and enhanced sensitivity of the assays. With regard to the latter, often highly denatured food samples and ingredients can still be processed for PCR detection assays because the DNA may still be reliably amplified, as opposed to loss of processing material in detection methods relying on protein analytical tools.

> Microbial source tracking (MST) which involves the ability to trace microbes, particularly food-borne pathogens, poses unique challenges to the food industry and food regulatory agencies (Santo Domingo and Sadowsky, 2007). Such information would assist regulatory agencies in localizing food producers or vendors responsible for supplying foods involved in human infections. Additionally, such knowledge would afford public health investigators the opportunity to track food-borne disease outbreaks to their point of origin, thereby preventing future occurrences. In providing such crucial information reliably and within the shortest possible time frame, MST employs a number of PCR-based detection assays. The recent outbreak of EHEC infections arising from verocytotoxin-producing *Escherichia coli* EHEC O104:H4, predominantly in Germany furnishes a good example of the importance of a rapid screening tool for the prompt identification of an infectious agent and surveillance monitoring. More than 16 countries in Europe and North America reported a total of 4,075 cases and 50 deaths as of July 21 2011, two months after the first reported case at the beginning of May 2011 (WHO International Health Regulations, Outbreaks of E. coli O104:H4 infection, Update 30) .

> In this and other similar cases, PCR-based molecular biological methods are usually employed in the rapid and initial screening of samples, while complementing this approach with the classical cultural technique for reliable end-identification of the isolate. While not replacing the classical methodologies that have stood the test of time, PCR-based molecular approaches are rapidly becoming the initial screening tools in diverse food analytical processes. Commonly the molecular biological methods are supplemented with classical

The Application of PCR-Based Methods in Food Control Agencies – A Review 175

only the production of all foods of animal origin (including meat, milk, eggs, fish and other products from aquaculture), but fruits and vegetables as well. Applying this approach means that food safety is not solely a matter of inspection at the slaughterhouse or processing plants as has traditionally been the case. On the contrary, this system emphasises the need for interaction between all participants in the entire food chain, from the animal

In Europe, a Rapid Alert System for Food and Feed (RASFF) was implemented in 1979, to provide food and feed control authorities an effective tool to exchange information about measures taken in responding to serious risks detected in relation to food or feed. This exchange of information helps Member States to act more rapidly and in a coordinated manner in response to a health threat caused by food or feed. In 2010, more than 3,358 notifications were transmitted through the RASFF, with cases of food poisoning accounting for 60 of such reports (Rapid Alert Systems for Food and Feed (RASFF) Annual Report 2010).

A major advantage in the application of PCR-based methodologies lies in the fact that such assays are generally more specific, sensitive, and faster than conventional microbiological assays. However the inherent complexities and composition of food matrices hampers the direct application of PCR detection assays, requiring a pre-enrichment step, thus increasing the processing time for the analysis of the food sample. Nevertheless the simplicity and time saving feature of the PCR reaction has made it increasingly applicable for detection of bacterial pathogens in food. For reliable detection of possible contaminants in the PCR reaction, it is essential to include appropriate negative controls, both during DNA extraction procedures (extraction control) and during the PCR reaction (master mix control). Additionally, it is essential to monitor or detect possible inhibitors that could hamper the efficiency of the PCR reaction. There are a number of possibilities to detect such PCR inhibitors, the commonest of which is to include in each PCR run, an inhibition control, or an internal amplifications control (IAC). The requirement for inclusion of an appropriate IAC for each PCR run is non-negotiable and is in fact jointly stipulated by the International Standard Organization (ISO) and the European Standardization Committee (CEN) in a general guiding policy for PCR reactions in food analytical procedures (EN ISO22174). The choice of the IAC may vary from an artificial DNA molecule which is co-amplified with the same primers for the target DNA (competitive IAC), to a foreign DNA molecule which is coamplified in the PCR

reaction with a different primer set (non-competitive) (Hoorfar *et al.,* 2004).

Presumptive *Salmonella* colonies are then confirmed by serotyping.

An example of a typical real-time PCR based approach for the detection of *Salmonella*, against the backdrop of the traditional cultural enumeration is outlined below. For the routine or traditional culture-based enumeration, an appropriate amount of the probe is inoculated in buffered peptone water. The culture is incubated at 37 °C for 18 – 24 h, followed by subculture in parallel, on a semi-solid MSRV plate (Rappaport-Vassilidis-Medium) and in Rappaport-Bouillon for 18-24 h at 43±1°C. On day 3, *Salmonella* suspects are then subcultured in parallel on XLD and Rambach agar, according to standard procedures.

With the traditional culture enumeration, outlined above, up to 5 days must be allowed for a definite identification of the bacteria. Sometimes, *Salmonella* positive probes can be completely missed with the conventional cultural enumeration due to strong growth of accompanying flora as mentioned previously. In contrast, a real-time PCR assay for *Salmonella* detection can be completed in less than 2 days, with an initial and shortened pre-

feed manufacturer down to the individual consumer.

diagnostic tools to reach a definitive consensus before prosecution for negligent practice or falsified declaration by food producers and processors is effected by food control agencies. This review looks at the plethora of PCR-based approaches in food control laboratories, from pathogen detection and control, food allergen and GMO detection and quantitative determination, to animal species verification.

## **2. Molecular biology tools for detection of foodborne pathogens**

In many food control agencies worldwide, continuous effort is devoted to risk monitoring assessments and evolvement of novel strategies for more rapid and reliable detection of the medically relevant enteropathogens. Although the ultimate goal is a zero-reduction of the pathogens in food, especially meat products and fresh produce, the quantitative microbiological risk-assessment has become an increasingly important parameter in predicting the infectious potential of a given food matrix (FAO/WHO, 2002). The medically relevant species are usually bacterial in origin, and include among others thermophilic *Campylobacter* spp.*, Salmonella* spp. *,* enterohaemorrhagic *Escherichia coli* (EHEC*), Listeria monocytogenes, Bacillus cereus, Clostridium* spp. and *Shigellla* spp. Typical clinical symptoms include diarrhea, which could be self-limiting, invasive or bloody, and vomiting. In Europe, salmonellosis and campylobacteriosis account for the most cases of notified bacterial infections, while listeriosis, although less commonly reported accounts for the most mortalities. In the USA, bacterial pathogens like *Salmonella* and *Campylobacter* are also prevalent, but surveillance of food borne illness is complicated by underreporting (European Food Safety Authority, EFSA 2009, Mead *et al.,* 1999).

The traditional culture-based enumeration of the bacteria is often laborious and timeconsuming. A typical detection assay for *Campylobacter* for example, requires up to 5 days, with enrichment. Additionally, the bacterial strain of interest can be frequently overlooked when only culture-based enumeration techniques are employed, due to a strong background of microflora that obscure the accurate detection and quantitative estimation of the pathogen. PCR-based detection of pathogens has therefore become increasingly popular in recent times. Effective PCR-detection assays have been successfully designed and implemented for a broad range of these bacterial food- borne pathogens such as *Salmonella*, *Campylobacter*, *Bacillus cereus*, pathogenic *Escherichia coli* (EHEC) and others (Anderson *et al*, 2010, Lehmann et al., 2010, Josefsen *et al*., 2010, Fratamico *et al*., 2011, Wang *et al.,* 2011).

#### **2.1 PCR-based food - borne pathogen (bacteria) detection**

On a global scale, the food sector remains a major player in the lives and well being of the general human population, and considerable trust and confidence is invested in it by consumers. When food-borne related illnesses or epidemics hit the headlines, the public is understandably disturbed and clamour for tighter regulations and more effective surveillance of food products. The food distribution chain is however a very complex one and tracing the origin of a food outbreak can be very difficult to achieve. In an attempt to address the challenges facing the food sector as regards protecting consumer trust and confidence, the Federation of Veterinarians of Europe (FVE) introduced the "stable to table approach" of food safety (FVE Food safety report). The concept involves a holistic approach embracing all elements, which may have an impact on the safety of food, at every level of the food chain from the stable to the table. Accordingly, the phrase is used to encompass not

diagnostic tools to reach a definitive consensus before prosecution for negligent practice or falsified declaration by food producers and processors is effected by food control agencies. This review looks at the plethora of PCR-based approaches in food control laboratories, from pathogen detection and control, food allergen and GMO detection and quantitative

In many food control agencies worldwide, continuous effort is devoted to risk monitoring assessments and evolvement of novel strategies for more rapid and reliable detection of the medically relevant enteropathogens. Although the ultimate goal is a zero-reduction of the pathogens in food, especially meat products and fresh produce, the quantitative microbiological risk-assessment has become an increasingly important parameter in predicting the infectious potential of a given food matrix (FAO/WHO, 2002). The medically relevant species are usually bacterial in origin, and include among others thermophilic *Campylobacter* spp.*, Salmonella* spp. *,* enterohaemorrhagic *Escherichia coli* (EHEC*), Listeria monocytogenes, Bacillus cereus, Clostridium* spp. and *Shigellla* spp. Typical clinical symptoms include diarrhea, which could be self-limiting, invasive or bloody, and vomiting. In Europe, salmonellosis and campylobacteriosis account for the most cases of notified bacterial infections, while listeriosis, although less commonly reported accounts for the most mortalities. In the USA, bacterial pathogens like *Salmonella* and *Campylobacter* are also prevalent, but surveillance of food borne illness is complicated by underreporting

The traditional culture-based enumeration of the bacteria is often laborious and timeconsuming. A typical detection assay for *Campylobacter* for example, requires up to 5 days, with enrichment. Additionally, the bacterial strain of interest can be frequently overlooked when only culture-based enumeration techniques are employed, due to a strong background of microflora that obscure the accurate detection and quantitative estimation of the pathogen. PCR-based detection of pathogens has therefore become increasingly popular in recent times. Effective PCR-detection assays have been successfully designed and implemented for a broad range of these bacterial food- borne pathogens such as *Salmonella*, *Campylobacter*, *Bacillus cereus*, pathogenic *Escherichia coli* (EHEC) and others (Anderson *et al*, 2010, Lehmann et al., 2010, Josefsen *et al*., 2010, Fratamico *et al*., 2011, Wang *et al.,* 2011).

On a global scale, the food sector remains a major player in the lives and well being of the general human population, and considerable trust and confidence is invested in it by consumers. When food-borne related illnesses or epidemics hit the headlines, the public is understandably disturbed and clamour for tighter regulations and more effective surveillance of food products. The food distribution chain is however a very complex one and tracing the origin of a food outbreak can be very difficult to achieve. In an attempt to address the challenges facing the food sector as regards protecting consumer trust and confidence, the Federation of Veterinarians of Europe (FVE) introduced the "stable to table approach" of food safety (FVE Food safety report). The concept involves a holistic approach embracing all elements, which may have an impact on the safety of food, at every level of the food chain from the stable to the table. Accordingly, the phrase is used to encompass not

**2. Molecular biology tools for detection of foodborne pathogens** 

(European Food Safety Authority, EFSA 2009, Mead *et al.,* 1999).

**2.1 PCR-based food - borne pathogen (bacteria) detection** 

determination, to animal species verification.

only the production of all foods of animal origin (including meat, milk, eggs, fish and other products from aquaculture), but fruits and vegetables as well. Applying this approach means that food safety is not solely a matter of inspection at the slaughterhouse or processing plants as has traditionally been the case. On the contrary, this system emphasises the need for interaction between all participants in the entire food chain, from the animal feed manufacturer down to the individual consumer.

In Europe, a Rapid Alert System for Food and Feed (RASFF) was implemented in 1979, to provide food and feed control authorities an effective tool to exchange information about measures taken in responding to serious risks detected in relation to food or feed. This exchange of information helps Member States to act more rapidly and in a coordinated manner in response to a health threat caused by food or feed. In 2010, more than 3,358 notifications were transmitted through the RASFF, with cases of food poisoning accounting for 60 of such reports (Rapid Alert Systems for Food and Feed (RASFF) Annual Report 2010).

A major advantage in the application of PCR-based methodologies lies in the fact that such assays are generally more specific, sensitive, and faster than conventional microbiological assays. However the inherent complexities and composition of food matrices hampers the direct application of PCR detection assays, requiring a pre-enrichment step, thus increasing the processing time for the analysis of the food sample. Nevertheless the simplicity and time saving feature of the PCR reaction has made it increasingly applicable for detection of bacterial pathogens in food. For reliable detection of possible contaminants in the PCR reaction, it is essential to include appropriate negative controls, both during DNA extraction procedures (extraction control) and during the PCR reaction (master mix control). Additionally, it is essential to monitor or detect possible inhibitors that could hamper the efficiency of the PCR reaction. There are a number of possibilities to detect such PCR inhibitors, the commonest of which is to include in each PCR run, an inhibition control, or an internal amplifications control (IAC). The requirement for inclusion of an appropriate IAC for each PCR run is non-negotiable and is in fact jointly stipulated by the International Standard Organization (ISO) and the European Standardization Committee (CEN) in a general guiding policy for PCR reactions in food analytical procedures (EN ISO22174). The choice of the IAC may vary from an artificial DNA molecule which is co-amplified with the same primers for the target DNA (competitive IAC), to a foreign DNA molecule which is coamplified in the PCR reaction with a different primer set (non-competitive) (Hoorfar *et al.,* 2004).

An example of a typical real-time PCR based approach for the detection of *Salmonella*, against the backdrop of the traditional cultural enumeration is outlined below. For the routine or traditional culture-based enumeration, an appropriate amount of the probe is inoculated in buffered peptone water. The culture is incubated at 37 °C for 18 – 24 h, followed by subculture in parallel, on a semi-solid MSRV plate (Rappaport-Vassilidis-Medium) and in Rappaport-Bouillon for 18-24 h at 43±1°C. On day 3, *Salmonella* suspects are then subcultured in parallel on XLD and Rambach agar, according to standard procedures. Presumptive *Salmonella* colonies are then confirmed by serotyping.

With the traditional culture enumeration, outlined above, up to 5 days must be allowed for a definite identification of the bacteria. Sometimes, *Salmonella* positive probes can be completely missed with the conventional cultural enumeration due to strong growth of accompanying flora as mentioned previously. In contrast, a real-time PCR assay for *Salmonella* detection can be completed in less than 2 days, with an initial and shortened pre-

The Application of PCR-Based Methods in Food Control Agencies – A Review 177

often observed with PMA-treated cells (Josefsen *et al.,* 2010). The PMA approach is currently being developed and validated in our laboratory for the reliable identification and

quantification of viable and live bacterial pathogens in various food matrices.

1ng/µl 100pg/µl 10pg/µl 1pg/µl 100fg/µl 10fg/µl

Fig. 1. Principle behind the quantitative PCR approach. A serial dilution of bacterial genomic DNA (fig. 1a) or DNA extracted from a dilution series of appropriate bacterial CFUs (fig. 1b) forms the basis for the calculation of a standard curve for quantification.\*

the 101 to 103 virus particle/g food range (Koopmans und Duizer, 2004).

cycles at which the fluorescence exceeds the threshold is called the cycle threshold).

a threshold for detection of DNA-based fluorescence is set slightly above background. The number of

A number of viruses associated with food infections are increasingly becoming important in recent years. The most relevant species are the norovirus, hepatitis-A virus, sapovirus, adenovirus, rotavirus, enterovirus and others. One category of implicated foods is those that are minimally processed, such as fresh produce and vegetables and bivalve molluscs. These are typically contaminated with viruses in the primary production environment. In addition, many of the documented outbreaks of foodborne viral illness have been linked to contamination of prepared, ready-to-eat food by an infected food handler. While in many countries viruses are now considered to be an extremely common cause of foodborne illness, they are rarely diagnosed as the analytical and diagnostic tools for such viruses are not widely available (Microbiological risk assessment series 13, 2008, WHO). Attempts have been made to implement PCR approaches in detection of food-bone viruses. While the overwhelming majority of food-associated viruses are RNA viruses, the RT-PCR (reverse transcription-PCR in which a reverse transcription step converting the viral RNA to template DNA precedes the PCR reaction) is the gold standard for analysis (Höhne and Schreier, 2004). Transferring the traditional and established methods for medical viral diagnosis to a food analytical setting is not readily implementable. While the viral particle load in human and animal tissues or organs is considerably great, the viral load in food samples is usually quite low – in some cases only 10-100 virions may be present in a food probe. Visualization of such a very low viral presence with electron microscopic means and detection of the viral protein through ELISA or latex tests would be impossible where the detection limits of such methods lie within the 105 to 106 virus particle range pro gram food. The PCR approach is in this regard the most promising of all techniques because the detection limit with RT-PCR lies in

**2.2 PCR detection of food-borne viruses** 

 (\*

enrichment step. In a comprehensive study by Anderson *et al.,* 2010, such a real-time PCR assay was described for the qualitative detection of *Salmonella* in several food samples. More than 1,900 natural food samples were analyzed in this study and the method was found to be robust and resulted in reliable identification of the bacteria in as little as 28 hr, in contrast to 4 or 5 days with conventional *Salmonella* diagnostics. An internal amplification control, which is co-amplified in a duplex PCR reaction, was included in the assay.

As mentioned previously, a number of real-time PCR assays have been published for several important food pathogens. Fricker *et al.,* (2007) reported on the successful application of real-time PCR in the detection of *B. cereus,* which together with the closely associated *S. aureus* are the two most important bacteria responsible for food-associated intoxications. The traditional detection of the emetic toxin associated with these bacteria is often difficult, time consuming and expensive. With the described real-time PCR assay, a first diagnosis can be achieved within 30 hours, greatly accelerating the potential for rapidly implementing risk assessment studies for different food products or matrices. In another study, the successful implementation of multiplex real-time PCR assays in the detection of neurotoxin producing *Clostridium botulinum* in clinical, food and environmental samples was described (De Medici *et al.,* 2009, Messelhäusser *et al.,* 2011a and b).

A more recent approach is the quantitative real-time PCR assay. Various possibilities exist for quantification strategies, one of which is the employment of a CFU-based standard curve for quantification. Briefly, the bacteria of interest are grown or cultivated according to standard procedures and a serial dilution of the bacteria, spanning a representative colony concentration (say 101 to 108 cells) is plotted as a standard curve. With this curve, the unknown concentration of bacteria in a food sample can be calculated. A second possibility is the employment of a serial dilution of bacterial DNA for the generation of a standard curve for quantification (see fig. 3). In a recent study by Josefsen et al., 2010, a CFU-based standard curve was utilized in the quantitative determination of *Campylobacter* in chicken rinse (Josefsen *et al.,* 2010). In this work, the quantification method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT)\* values with a quantification range of 1 x 102 to 1 x 107 CFU/ml). In a previous study, Yang *et al.,* (2003) also successfully applied a real-time PCR assay for quantitative detection of *C. jejuni* in poultry, milk and environmental water. Such quantification strategies are increasingly in demand and a number of commercial products are now available for such purposes.

Although the PCR method has evolved as a very powerful analytical tool indeed, a limitation of such methods is that the DNA analysis will generate results of all the bacteria present in the food sample or probe, irrespective of the status of the cells – whether the cells are live and viable or dead. Thus data for dead or inactivated bacteria which might not be significant from an epidemiological viewpoint are invariably included in such quantitative assays. An improvement in such analysis is the use of an appropriate DNA intercalating dye to distinguish dead from viable and viable, but non-culturable (VBNC) bacteria. Propidium monoazide (PMA) is one such chemical which selectively penetrates only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes (Nocker *et al*., 2006, 2009, Pan and Breidt, 2007). PMA possesses an azide group whch permits cross-linking of the dye to DNA after exposure to strong visible light. When the PMA-treated cells are subjected to DNA extraction procedures and subsequently PCR for detection of the bacteria of interest, a reduction in the number of detectable bacteria is often observed with PMA-treated cells (Josefsen *et al.,* 2010). The PMA approach is currently being developed and validated in our laboratory for the reliable identification and quantification of viable and live bacterial pathogens in various food matrices.

Fig. 1. Principle behind the quantitative PCR approach. A serial dilution of bacterial genomic DNA (fig. 1a) or DNA extracted from a dilution series of appropriate bacterial CFUs (fig. 1b) forms the basis for the calculation of a standard curve for quantification.\*

## **2.2 PCR detection of food-borne viruses**

176 Polymerase Chain Reaction

enrichment step. In a comprehensive study by Anderson *et al.,* 2010, such a real-time PCR assay was described for the qualitative detection of *Salmonella* in several food samples. More than 1,900 natural food samples were analyzed in this study and the method was found to be robust and resulted in reliable identification of the bacteria in as little as 28 hr, in contrast to 4 or 5 days with conventional *Salmonella* diagnostics. An internal amplification control,

As mentioned previously, a number of real-time PCR assays have been published for several important food pathogens. Fricker *et al.,* (2007) reported on the successful application of real-time PCR in the detection of *B. cereus,* which together with the closely associated *S. aureus* are the two most important bacteria responsible for food-associated intoxications. The traditional detection of the emetic toxin associated with these bacteria is often difficult, time consuming and expensive. With the described real-time PCR assay, a first diagnosis can be achieved within 30 hours, greatly accelerating the potential for rapidly implementing risk assessment studies for different food products or matrices. In another study, the successful implementation of multiplex real-time PCR assays in the detection of neurotoxin producing *Clostridium botulinum* in clinical, food and environmental samples

A more recent approach is the quantitative real-time PCR assay. Various possibilities exist for quantification strategies, one of which is the employment of a CFU-based standard curve for quantification. Briefly, the bacteria of interest are grown or cultivated according to standard procedures and a serial dilution of the bacteria, spanning a representative colony concentration (say 101 to 108 cells) is plotted as a standard curve. With this curve, the unknown concentration of bacteria in a food sample can be calculated. A second possibility is the employment of a serial dilution of bacterial DNA for the generation of a standard curve for quantification (see fig. 3). In a recent study by Josefsen et al., 2010, a CFU-based standard curve was utilized in the quantitative determination of *Campylobacter* in chicken rinse (Josefsen *et al.,* 2010). In this work, the quantification method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT)\* values with a quantification range of 1 x 102 to 1 x 107 CFU/ml). In a previous study, Yang *et al.,* (2003) also successfully applied a real-time PCR assay for quantitative detection of *C. jejuni* in poultry, milk and environmental water. Such quantification strategies are increasingly in demand and a number of commercial products

Although the PCR method has evolved as a very powerful analytical tool indeed, a limitation of such methods is that the DNA analysis will generate results of all the bacteria present in the food sample or probe, irrespective of the status of the cells – whether the cells are live and viable or dead. Thus data for dead or inactivated bacteria which might not be significant from an epidemiological viewpoint are invariably included in such quantitative assays. An improvement in such analysis is the use of an appropriate DNA intercalating dye to distinguish dead from viable and viable, but non-culturable (VBNC) bacteria. Propidium monoazide (PMA) is one such chemical which selectively penetrates only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes (Nocker *et al*., 2006, 2009, Pan and Breidt, 2007). PMA possesses an azide group whch permits cross-linking of the dye to DNA after exposure to strong visible light. When the PMA-treated cells are subjected to DNA extraction procedures and subsequently PCR for detection of the bacteria of interest, a reduction in the number of detectable bacteria is

which is co-amplified in a duplex PCR reaction, was included in the assay.

was described (De Medici *et al.,* 2009, Messelhäusser *et al.,* 2011a and b).

are now available for such purposes.

A number of viruses associated with food infections are increasingly becoming important in recent years. The most relevant species are the norovirus, hepatitis-A virus, sapovirus, adenovirus, rotavirus, enterovirus and others. One category of implicated foods is those that are minimally processed, such as fresh produce and vegetables and bivalve molluscs. These are typically contaminated with viruses in the primary production environment. In addition, many of the documented outbreaks of foodborne viral illness have been linked to contamination of prepared, ready-to-eat food by an infected food handler. While in many countries viruses are now considered to be an extremely common cause of foodborne illness, they are rarely diagnosed as the analytical and diagnostic tools for such viruses are not widely available (Microbiological risk assessment series 13, 2008, WHO). Attempts have been made to implement PCR approaches in detection of food-bone viruses. While the overwhelming majority of food-associated viruses are RNA viruses, the RT-PCR (reverse transcription-PCR in which a reverse transcription step converting the viral RNA to template DNA precedes the PCR reaction) is the gold standard for analysis (Höhne and Schreier, 2004). Transferring the traditional and established methods for medical viral diagnosis to a food analytical setting is not readily implementable. While the viral particle load in human and animal tissues or organs is considerably great, the viral load in food samples is usually quite low – in some cases only 10-100 virions may be present in a food probe. Visualization of such a very low viral presence with electron microscopic means and detection of the viral protein through ELISA or latex tests would be impossible where the detection limits of such methods lie within the 105 to 106 virus particle range pro gram food. The PCR approach is in this regard the most promising of all techniques because the detection limit with RT-PCR lies in the 101 to 103 virus particle/g food range (Koopmans und Duizer, 2004).

 (\* a threshold for detection of DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called the cycle threshold).

The Application of PCR-Based Methods in Food Control Agencies – A Review 179

immunosorbent assay, Hefle *et a*l., 2001 and Hlywka *et al.,* 2000). The ELISA method is by far the most common and is routinely employed in various food analysis labs due to its high precision, simple handling and good potential for standardization. Additionally, quantitative data are possible with the ELISA technique (Shim and Wanasundara, 2008). However results generated with the ELISA method must be sometimes taken with caution as substantial differences in the detectable protein from the standard on which the test is based, resulting for example from variations in the processing of the food matrix, might lead to false results. Recently, PCR-based detection of allergens has become increasingly popular. A major advantage in the employment of PCR-based methods lies in the high specificity of the reaction. Additionally, proteins in foods that have been harshly processed, might not be detectable in the classical ELISA based approach for example, while the target DNA might be nevertheless efficiently extracted under such denaturing conditions. Another advantage that the PCR holds out against the classical protein-based analytical methods is its stability against the backdrop of geographical and seasonal variations in fruits and nuts for example,

Hupfer and colleagues have developed and validated a number of molecular-biology based methods for the detection of a number of allergens, notably celery, lupine and cashew nut (Demmel *et al.,* 2008, Hupfer *et al.,* 2006, Ehlert *et al.,* 2008). A typical scheme for the development and validation of an allergen, with celery as an example is described below (Fig. 2). Other studies have successfully identified and quantified allergens in various food matrices such as the work by Hirao and colleagues who developed a PCR method for quantification of buckwheat by using a unique internal standard. Food-labelling regulations in Japan require that buckwheat must be declared on the food label if its protein is present at concentrations higher than a few micrograms per gram, thus the relevance of this study (Hirao et al., 2006). More recently, Mujico and colleagues developed a highly sensitive realtime PCR for quantification of wheat contamination in gluten-free food for celiac patients. The method compared well with the ELISA in efficiency, with a quantification limit of 20 pg DNA/mg food sample (Mujico *et al.,* 2011). In addition to the conventional singleplex PCR or real-time PCR reactions for allergenic qualitative detection, attempts have also been made to detect simultaneously more than one allergenic event in a food matrix. This multiplex approach was recently demonstrated by Köppel and colleagues and allows the parallel detection of peanuts, hazelnuts, celery and soya in one multiplex reaction, and the quantitative detection of egg, milk, almond and sesame in another multiplex reaction. The tests exhibited good specificity and sensitivity in the 0.01 % range. Due to comparatively lower DNA content in milk and eggs, the authors reported lower sensitivities for these allergens. Initial comparisons of the generated results with conventional ELISA suggested a qualitative accordance, with low correlation of quantitative data (Köppel *et al*., 2010a).

Another PCR-based approach partly developed and validated by our laboratory is the simultaneous detection of DNA from various food allergens by ligation-dependent probe amplification (LPA). Ligation dependent PCR is a technique originally used for detection of nucleic acids (Hsuih *et al.,* 1996). Briefly this method employs the ligation of bipartite hybridization probes that bind to a target DNA derived from the foodmatrix under investigation. The target DNA is first denatured according to standard protocols, and then incubated with the LPA probes, allowing binding of the LPA probes to the DNA strand, following which the two probes are ligated in a simple ligation reaction. The resulting

with accompanying variance in protein composition (Poms *et al.,* 2007).

Adequate care has to be however taken while subjecting the sample to extraction procedures for optimal yield of high quality nucleic acid (Croci *et al.,* 2003, De Husman *et al.,* 2007). Examples of successful application of the RT-PCR technique include the detection of norovirus in raspberries associated with a gastroenteritis outbreak, and the detection of the virus in oysters from China and Japan (Phan *et al.,* 2007). Other PCR-based methods that have been developed include a nested RT-PCR approach, real-time RT-PCR, and the limited application of nucleic acid sequence-based amplification, among others (Jean *et al.,* 2001, Kojima *et al.,* 2002, Nishida *et al.*, 2003, Beuret *et al.,* 2004).

## **3. PCR-based allergen detection and quantification in food matrices**

Globally, millions of people suffer from allergic reactions to food, which fortunately in most cases range from mild to minor symptoms. In some extreme cases however, food allergies can trigger moderate to more severe life threatening reactions. In contrast to food intolerance, which is also a common form of an adverse reaction to food arising for example from an enzymatic deficiency, such as lactose intolerance, food allergies are immunemediated. Usually a protein in the food is mistakenly recognized as harmful, triggering the recruitment of IgE antibody with a subsequent allergic reaction (Bush and Hefle, 1996). Symptoms may vary from dermatitis, gastrointestinal and respiratory distress to lifethreatening anaphylactic shock. The most common food substances, accounting for almost 90 % of all allergic food reactions are milk, egg, peanut, tree nuts, fish, shellfish, soy, and wheat.

In order to protect consumer safety and health, the EU Labelling Directive (Directive 2000/13/EC) and its later amendments specifically mandate the labelling of allergenic foods. The Labelling Directive requires food manufacturers to declare all ingredients present in pre-packaged foods sold in the EU allowing very few exceptions. In order to respond to our rapidly changing times, this directive has been amended a number of times with regard to allergens. The two most important amendments are: Directive 2003/89/EC introduced Annex IIIa, which is a list of allergenic foods that must always be labelled when present as ingredient in a product, and Directive 2007/68/EC which contains the most recent amendment of Annex IIIa. The latter lists all the allergenic foods that must be labelled as well as a few products derived from those foods for which allergen labelling is not required (European Commission, 2000, 2003, and 2006).

Food allergies are present in about 1-3 % of the global adult population, while in children, a slightly higher incidence (4-6 %) has been documented (Bock et al., 2001). While some of these allergies may be shed when children approach adolescence and adulthood, a few of them are present for life, such as peanut and shellfish allergies. A need for careful labelling of food and food ingredients is strongly underscored by the fact that in some cases, even very minute amounts of an allergen can trigger such life-threatening anaphylactic responses like biphasic anaphylaxis and vasodilation, requiring immediate emergency intervention. Threshold doses for peanut allergic reactions have been found to range from as low as 100 µg up to 1g of peanut protein (Hourihane *et al.,* 1997, Poms *et al.,* 2007).

A variety of techniques have evolved over the years for the detection and possible quantification of the most common food allergens. Protein-based methods that have been employed include the RAST (radio-allergosorbent test, Holgate *et al.,* 2001), RIE (rocket immuno-electrophoresis, Malmheden, *et al.,* 1994) and the ELISA (enzyme-linked

Adequate care has to be however taken while subjecting the sample to extraction procedures for optimal yield of high quality nucleic acid (Croci *et al.,* 2003, De Husman *et al.,* 2007). Examples of successful application of the RT-PCR technique include the detection of norovirus in raspberries associated with a gastroenteritis outbreak, and the detection of the virus in oysters from China and Japan (Phan *et al.,* 2007). Other PCR-based methods that have been developed include a nested RT-PCR approach, real-time RT-PCR, and the limited application of nucleic acid sequence-based amplification, among others (Jean *et al.,* 2001,

Globally, millions of people suffer from allergic reactions to food, which fortunately in most cases range from mild to minor symptoms. In some extreme cases however, food allergies can trigger moderate to more severe life threatening reactions. In contrast to food intolerance, which is also a common form of an adverse reaction to food arising for example from an enzymatic deficiency, such as lactose intolerance, food allergies are immunemediated. Usually a protein in the food is mistakenly recognized as harmful, triggering the recruitment of IgE antibody with a subsequent allergic reaction (Bush and Hefle, 1996). Symptoms may vary from dermatitis, gastrointestinal and respiratory distress to lifethreatening anaphylactic shock. The most common food substances, accounting for almost 90 % of all allergic food reactions are milk, egg, peanut, tree nuts, fish, shellfish, soy, and wheat. In order to protect consumer safety and health, the EU Labelling Directive (Directive 2000/13/EC) and its later amendments specifically mandate the labelling of allergenic foods. The Labelling Directive requires food manufacturers to declare all ingredients present in pre-packaged foods sold in the EU allowing very few exceptions. In order to respond to our rapidly changing times, this directive has been amended a number of times with regard to allergens. The two most important amendments are: Directive 2003/89/EC introduced Annex IIIa, which is a list of allergenic foods that must always be labelled when present as ingredient in a product, and Directive 2007/68/EC which contains the most recent amendment of Annex IIIa. The latter lists all the allergenic foods that must be labelled as well as a few products derived from those foods for which allergen labelling is not required

Food allergies are present in about 1-3 % of the global adult population, while in children, a slightly higher incidence (4-6 %) has been documented (Bock et al., 2001). While some of these allergies may be shed when children approach adolescence and adulthood, a few of them are present for life, such as peanut and shellfish allergies. A need for careful labelling of food and food ingredients is strongly underscored by the fact that in some cases, even very minute amounts of an allergen can trigger such life-threatening anaphylactic responses like biphasic anaphylaxis and vasodilation, requiring immediate emergency intervention. Threshold doses for peanut allergic reactions have been found to range from as low as 100

A variety of techniques have evolved over the years for the detection and possible quantification of the most common food allergens. Protein-based methods that have been employed include the RAST (radio-allergosorbent test, Holgate *et al.,* 2001), RIE (rocket immuno-electrophoresis, Malmheden, *et al.,* 1994) and the ELISA (enzyme-linked

µg up to 1g of peanut protein (Hourihane *et al.,* 1997, Poms *et al.,* 2007).

**3. PCR-based allergen detection and quantification in food matrices** 

Kojima *et al.,* 2002, Nishida *et al.*, 2003, Beuret *et al.,* 2004).

(European Commission, 2000, 2003, and 2006).

immunosorbent assay, Hefle *et a*l., 2001 and Hlywka *et al.,* 2000). The ELISA method is by far the most common and is routinely employed in various food analysis labs due to its high precision, simple handling and good potential for standardization. Additionally, quantitative data are possible with the ELISA technique (Shim and Wanasundara, 2008). However results generated with the ELISA method must be sometimes taken with caution as substantial differences in the detectable protein from the standard on which the test is based, resulting for example from variations in the processing of the food matrix, might lead to false results. Recently, PCR-based detection of allergens has become increasingly popular. A major advantage in the employment of PCR-based methods lies in the high specificity of the reaction. Additionally, proteins in foods that have been harshly processed, might not be detectable in the classical ELISA based approach for example, while the target DNA might be nevertheless efficiently extracted under such denaturing conditions. Another advantage that the PCR holds out against the classical protein-based analytical methods is its stability against the backdrop of geographical and seasonal variations in fruits and nuts for example, with accompanying variance in protein composition (Poms *et al.,* 2007).

Hupfer and colleagues have developed and validated a number of molecular-biology based methods for the detection of a number of allergens, notably celery, lupine and cashew nut (Demmel *et al.,* 2008, Hupfer *et al.,* 2006, Ehlert *et al.,* 2008). A typical scheme for the development and validation of an allergen, with celery as an example is described below (Fig. 2). Other studies have successfully identified and quantified allergens in various food matrices such as the work by Hirao and colleagues who developed a PCR method for quantification of buckwheat by using a unique internal standard. Food-labelling regulations in Japan require that buckwheat must be declared on the food label if its protein is present at concentrations higher than a few micrograms per gram, thus the relevance of this study (Hirao et al., 2006). More recently, Mujico and colleagues developed a highly sensitive realtime PCR for quantification of wheat contamination in gluten-free food for celiac patients. The method compared well with the ELISA in efficiency, with a quantification limit of 20 pg DNA/mg food sample (Mujico *et al.,* 2011). In addition to the conventional singleplex PCR or real-time PCR reactions for allergenic qualitative detection, attempts have also been made to detect simultaneously more than one allergenic event in a food matrix. This multiplex approach was recently demonstrated by Köppel and colleagues and allows the parallel detection of peanuts, hazelnuts, celery and soya in one multiplex reaction, and the quantitative detection of egg, milk, almond and sesame in another multiplex reaction. The tests exhibited good specificity and sensitivity in the 0.01 % range. Due to comparatively lower DNA content in milk and eggs, the authors reported lower sensitivities for these allergens. Initial comparisons of the generated results with conventional ELISA suggested a qualitative accordance, with low correlation of quantitative data (Köppel *et al*., 2010a).

Another PCR-based approach partly developed and validated by our laboratory is the simultaneous detection of DNA from various food allergens by ligation-dependent probe amplification (LPA). Ligation dependent PCR is a technique originally used for detection of nucleic acids (Hsuih *et al.,* 1996). Briefly this method employs the ligation of bipartite hybridization probes that bind to a target DNA derived from the foodmatrix under investigation. The target DNA is first denatured according to standard protocols, and then incubated with the LPA probes, allowing binding of the LPA probes to the DNA strand, following which the two probes are ligated in a simple ligation reaction. The resulting

The Application of PCR-Based Methods in Food Control Agencies – A Review 181

**4. Application of PCR in animal species detection and differentiation in meat** 

A major challenge for food control agencies worldwide is the accurate determination of declared meat components for food and feed ingredients. For protection of consumer trust and confidence and to ensure the quality of meat produce, the verification of declared animal species is important for the following reasons: a) ethical considerations of some might reject the consumption of certain meat products, b) the underlying health condition of some might preclude consuming certain meat products, and c) possible economic loss from the fraudulent substitution of expensive meat components with inferior products

A rapid and dependable detection system is therefore indispensable in a food control agency for protection of consumer trust. In the past, the traditional method for determination of animal species in food relied heavily on immunochemical and electrophoretic analysis of proteins. Although these protein-based analytical methods are still important tools in the food analytical industry, a major drawback in such applications is that in the case of highly processed food, the resulting protein denaturation affects the sensitivity of the procedure. Additionally, such methods may not enable the fine discrimination between closely related animal species like chicken and turkey, or sheep and goat. DNA-based detection systems have thus become increasingly popular in recent times. The distinct advantage of DNAbased detection lies in (1) the increased specificity (generally unambiguous identification of target sequences) and (2) relative stability of the DNA molecule, allowing detection of animal species even in food that have been seriously compromised by excessive processing. In the early stages, molecular biological methods in species identification were largely based on the use of hybridization of homologous sequences, employing genomic DNA as a species-specific probe, hybridized to DNA extracted from meat samples (Lenstra *et al.,* 2001). Later improvements saw the development of probes derived from species-specific satellite repetitive DNA sequences, making detection of admixtures that account for less than 5 % of the product possible. These methods are however time consuming and quite laborious, with reduced sensitivity in some cases. PCR-based methods have thus become increasingly important in recent times, allowing enhanced sensitivity and specificity of the assays. In most PCR-based approaches, species-specific primers are employed that bind to sequences unique to the species under investigation. Another approach is the employment of universal primers that bind to consensus sequences in all the animal species present in the meat sample. Following amplification, the resulting DNA fragments are subjected to differing analytical procedures for accurate determination of the present species. A popular approach is the use of restriction fragment length polymorphism (RFLP, Fig. 4), which commonly employs restriction digestion assays to generate fragments that are unique to the different animal species present in the sample. Each species is then recognised by its unique restriction fragment pattern (Ong *et al.,* 2007, Girish *et al.,* 2005, Gupta *et al.,* 2008, Meyer *et al*., 1995). In order to achieve a high level of sensitivity in these assays, especially when universal primers are employed for simultaneous amplification of all present meat species, genes present in multiple copies are usually employed as targets. Prime candidate genes are usually mitochondrial rRNA (12S or 18S) or the phylogenetically robust and highly

(Commission Directive 2002/86/EC, Commission Recommendation 2004/787/EC).

conserved *cyt b* gene (Kocher *et al.,* 1989, Jain *et al.,* 2007).

In an attempt to simultaneously detect several meat species present in a food sample, several multiplex real-time PCR assays for species differentiation have been described in recent

**products** 

oligonucleotide is turn subjected to PCR amplification. The arising PCR amplicon is then subjected to capillary electrophoresis and visualized with laser-induced fluorescence. With this method, the simultaneous detection of DNA from 10 allergens, notably peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts was possible (Ehlert *et al.,* 2009). Fig. 3 below outlines the principle of the LPA methodology.

Fig. 2. Development and Validation of a Real-time PCR Detection Method for Celery in Food (Hupfer *et al*., 2006)

(Demmel *et al*., 2011, Personal communication)

Fig. 3. Diagrammatic representation of the ligation dependent probe amplification (LPA) approach

oligonucleotide is turn subjected to PCR amplification. The arising PCR amplicon is then subjected to capillary electrophoresis and visualized with laser-induced fluorescence. With this method, the simultaneous detection of DNA from 10 allergens, notably peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts was possible (Ehlert *et al.,* 2009). Fig. 3 below outlines the principle of the

Fig. 2. Development and Validation of a Real-time PCR Detection Method for Celery in Food

**3´**

Relative Fluoreszenz

Fluorescence

Produkt A

Product A

Zeit

**Capillary electrophoresis**

Zeit

Time

Produkt B

Product B

Produkt C

Product C

(Demmel *et al*., 2011, Personal communication)

**3´**Ligation PCR

Target DNA **3´ 5´**

OH P

universal primer binding site spacer sequence

Fig. 3. Diagrammatic representation of the ligation dependent probe amplification (LPA)

LPA methodology.

(Hupfer *et al*., 2006)

approach

**5´**

## **4. Application of PCR in animal species detection and differentiation in meat products**

A major challenge for food control agencies worldwide is the accurate determination of declared meat components for food and feed ingredients. For protection of consumer trust and confidence and to ensure the quality of meat produce, the verification of declared animal species is important for the following reasons: a) ethical considerations of some might reject the consumption of certain meat products, b) the underlying health condition of some might preclude consuming certain meat products, and c) possible economic loss from the fraudulent substitution of expensive meat components with inferior products (Commission Directive 2002/86/EC, Commission Recommendation 2004/787/EC).

A rapid and dependable detection system is therefore indispensable in a food control agency for protection of consumer trust. In the past, the traditional method for determination of animal species in food relied heavily on immunochemical and electrophoretic analysis of proteins. Although these protein-based analytical methods are still important tools in the food analytical industry, a major drawback in such applications is that in the case of highly processed food, the resulting protein denaturation affects the sensitivity of the procedure. Additionally, such methods may not enable the fine discrimination between closely related animal species like chicken and turkey, or sheep and goat. DNA-based detection systems have thus become increasingly popular in recent times. The distinct advantage of DNAbased detection lies in (1) the increased specificity (generally unambiguous identification of target sequences) and (2) relative stability of the DNA molecule, allowing detection of animal species even in food that have been seriously compromised by excessive processing.

In the early stages, molecular biological methods in species identification were largely based on the use of hybridization of homologous sequences, employing genomic DNA as a species-specific probe, hybridized to DNA extracted from meat samples (Lenstra *et al.,* 2001). Later improvements saw the development of probes derived from species-specific satellite repetitive DNA sequences, making detection of admixtures that account for less than 5 % of the product possible. These methods are however time consuming and quite laborious, with reduced sensitivity in some cases. PCR-based methods have thus become increasingly important in recent times, allowing enhanced sensitivity and specificity of the assays. In most PCR-based approaches, species-specific primers are employed that bind to sequences unique to the species under investigation. Another approach is the employment of universal primers that bind to consensus sequences in all the animal species present in the meat sample. Following amplification, the resulting DNA fragments are subjected to differing analytical procedures for accurate determination of the present species. A popular approach is the use of restriction fragment length polymorphism (RFLP, Fig. 4), which commonly employs restriction digestion assays to generate fragments that are unique to the different animal species present in the sample. Each species is then recognised by its unique restriction fragment pattern (Ong *et al.,* 2007, Girish *et al.,* 2005, Gupta *et al.,* 2008, Meyer *et al*., 1995). In order to achieve a high level of sensitivity in these assays, especially when universal primers are employed for simultaneous amplification of all present meat species, genes present in multiple copies are usually employed as targets. Prime candidate genes are usually mitochondrial rRNA (12S or 18S) or the phylogenetically robust and highly conserved *cyt b* gene (Kocher *et al.,* 1989, Jain *et al.,* 2007).

In an attempt to simultaneously detect several meat species present in a food sample, several multiplex real-time PCR assays for species differentiation have been described in recent

The Application of PCR-Based Methods in Food Control Agencies – A Review 183

times. Köppel et al. (2009) have for example described the implementation of a heptaplex Realtime PCR assay for the simultaneous identification and quantification of DNA from beef, pork, chicken, turkey, horse meat, sheep (mutton) and goat. Although such multiplex approaches will greatly accelerate meat species identification, results generated must be taken with caution as several meat products are produced with widely varying fat and tissue composition, thus

As regards the accurate differentiation of fish species, several PCR assays have been developed. The majority of these assays rely on the application of universal primers for the generation of consensus sequences among various fish species and the subsequent use of restriction digestion to identify restriction fragments or patterns unique to various fish species. Here, as with meat species differentiation, molecular fish identification methods aim at ensuring that consumers get their money's worth when more expensive fish varieties are bought – substitution of expensive fish with much cheaper varieties can be unravelled by such techniques. Additionally, certain individuals are allergic to certain fish proteins and accurate identification of such potential fish allergens is another argument in favour of a

The 20th century saw an explosion of computer technology on all fronts. During the 1990s, molecular biology techniques met with computer electronics to see the birth of a DNA Microarray or DNA chip. One of the earliest attempts at microarray technology for global gene expression was reported by Shena et al., 1995, who designed a quantitative highcapacity system for monitoring of gene expression patterns with a complementary DNA microarray for *Arabidopsis*. Today microarray analyses are widely implemented in molecular biology laboratories, offering the unique advantage of simultaneous analysis of a variety of genetic events in an organism. In food control agencies, the biochip system has also come of age, enabling the quick and efficient analysis of meat products for answers as to their origin

The first commercial DNA-Chip for the detection of animal constituents in food products is the CarnoCheck Chip (Greiner Biosciences, http://www.greinerbioone.com). The chip allows the simultaneous identification of up to 8 different animal species in processed food and meat products with complex composition. The eight animal species detected by the CarnoCheck Test kit are pig, cattle, sheep, turkey, chicken, horse, donkey, and goat. Following sample homogenization and DNA extraction, a 389-bp fragment of the *cyt b* gene of all the animal species present in the food sample is amplified through polymerase chain reaction. By coupling the fluorophore Cy5 onto one of the primers, the amplified fragments are subsequently labelled in the applied PCR reaction. The labelled fragments are then hybridized to complementary oligonucleotide probes fixed as targets on the bottom of the biochip. The target probes themselves are coupled with the Cy3 fluorophore. Due to the use of fluorophore-labeled PCR primers (Cy5) and fluorophore-labeled target probes for the onchip control system (Cy3), the analysis of the biochips is performed by microarray scanners

Another Biochip test system for species differentiation is the LCD-Array from Chipron. The LCD Array (Chipon Germany, http://chipron.com/index.html) allows the simultaneous detection of up to 14 animal species in food: cattle, buffalo, pig, sheep, goat, horse, donkey,

the DNA extractable from similar meat products might vary greatly (Laube *et al.,* 2003).

robust fish differentiation method.

and composition.

**4.1 DNA Chip Technology in meat species differentiation** 

using wavelengths of ~532 nm (Cy3) and ~635 nm (Cy5).

Fig. 4. PCR-Restriction Fragment Length-Polymorphism (PCR-RFLP)

Fig. 4. PCR-Restriction Fragment Length-Polymorphism (PCR-RFLP)

times. Köppel et al. (2009) have for example described the implementation of a heptaplex Realtime PCR assay for the simultaneous identification and quantification of DNA from beef, pork, chicken, turkey, horse meat, sheep (mutton) and goat. Although such multiplex approaches will greatly accelerate meat species identification, results generated must be taken with caution as several meat products are produced with widely varying fat and tissue composition, thus the DNA extractable from similar meat products might vary greatly (Laube *et al.,* 2003).

As regards the accurate differentiation of fish species, several PCR assays have been developed. The majority of these assays rely on the application of universal primers for the generation of consensus sequences among various fish species and the subsequent use of restriction digestion to identify restriction fragments or patterns unique to various fish species. Here, as with meat species differentiation, molecular fish identification methods aim at ensuring that consumers get their money's worth when more expensive fish varieties are bought – substitution of expensive fish with much cheaper varieties can be unravelled by such techniques. Additionally, certain individuals are allergic to certain fish proteins and accurate identification of such potential fish allergens is another argument in favour of a robust fish differentiation method.

## **4.1 DNA Chip Technology in meat species differentiation**

The 20th century saw an explosion of computer technology on all fronts. During the 1990s, molecular biology techniques met with computer electronics to see the birth of a DNA Microarray or DNA chip. One of the earliest attempts at microarray technology for global gene expression was reported by Shena et al., 1995, who designed a quantitative highcapacity system for monitoring of gene expression patterns with a complementary DNA microarray for *Arabidopsis*. Today microarray analyses are widely implemented in molecular biology laboratories, offering the unique advantage of simultaneous analysis of a variety of genetic events in an organism. In food control agencies, the biochip system has also come of age, enabling the quick and efficient analysis of meat products for answers as to their origin and composition.

The first commercial DNA-Chip for the detection of animal constituents in food products is the CarnoCheck Chip (Greiner Biosciences, http://www.greinerbioone.com). The chip allows the simultaneous identification of up to 8 different animal species in processed food and meat products with complex composition. The eight animal species detected by the CarnoCheck Test kit are pig, cattle, sheep, turkey, chicken, horse, donkey, and goat. Following sample homogenization and DNA extraction, a 389-bp fragment of the *cyt b* gene of all the animal species present in the food sample is amplified through polymerase chain reaction. By coupling the fluorophore Cy5 onto one of the primers, the amplified fragments are subsequently labelled in the applied PCR reaction. The labelled fragments are then hybridized to complementary oligonucleotide probes fixed as targets on the bottom of the biochip. The target probes themselves are coupled with the Cy3 fluorophore. Due to the use of fluorophore-labeled PCR primers (Cy5) and fluorophore-labeled target probes for the onchip control system (Cy3), the analysis of the biochips is performed by microarray scanners using wavelengths of ~532 nm (Cy3) and ~635 nm (Cy5).

Another Biochip test system for species differentiation is the LCD-Array from Chipron. The LCD Array (Chipon Germany, http://chipron.com/index.html) allows the simultaneous detection of up to 14 animal species in food: cattle, buffalo, pig, sheep, goat, horse, donkey,

The Application of PCR-Based Methods in Food Control Agencies – A Review 185

primer mix provided with the test kit generates biotinylated amplicons of the animal mtDNA present in the food sample. The labelled PCR fragments are then hybridized to the corresponding capture sequences on the individual array fields. The strong affinity between Biotin and streptavidin is exploited by the LCD Array test principle, and positive samples can be visually identified or by employing the scanner and software provided by the kit

manufacturer. Figure 5 provides a schematic representation of the two test systems.

No Probe Specificity No Probe Specificity

01 Beef *Bos taurus* 08 Rabbit *Oryctolagus cuniculus*  02 Buffalo *Bubalus bubalis* 09 Hare *Lepus europaeus*  03 Pork *Sus scrofa* 10 Chicken *Gallus gallus*  04 Sheep *Ovis aries* 11 Turkey *Meleagris gallopavo*  05 Goat *Capra hircus* 12 Goose *Ansa albifrons*  06 Horse *Equus caballus 1)* 13 Mall. Duck *Anas platyrhyncos*  07 Donkey *Equus asinus 1)* 14 Musc. Duck *Cairina moschata* 

Fig. 5b. LCD Array Meat 1.6 Test System for meat species identification.

immobilized on each array (Data Sheet MeatSpecies 1.6, V-I-08, Chipron)

C Hyb-Contr. Functional controls (Hybridization +

The figure shows the spotting pattern of the array while the table lists the capture probes

In a recent study, these two biochip test systems were thoroughly validated and approved for routine use in the meat labour of a food control agency (Iwobi *et al.*, 2011). In this study, the two animal species differentiation biochip methods compared well in efficiency and detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Although such a simultaneous PCR approach will lead to accelerated analysis of meat species origin in food, while concomitantly revealing possible sources of deliberate adulteration or contamination, the efficiency of the approach is greatly influenced by the overall proficiency of the PCR reaction. In cases where very small amounts of a meat species is present in the

stain)

Capture probes

Fig. 5a. CarnoCheck Test kit for the detection of animal species in food. The small table above shows the order of the measurement points for the animal species while the figure below depicts the on-chip control systems for exact quality determination (orientation controls in red, printing controls in green). (CarnoCheck Handbook, Manual version: BQ-020-00, Greiner Bio-one).

rabbit, hare, chicken, turkey, goose, and two duck varieties. The test system here relies on the detection of specific sites within the 16S rRNA mitochondrial locus of all the meat species present in the tested food sample. Included in the test system is a consensus primer pair that amplifies the desired region of the animal species in a PCR. The pre-labeled PCR primer mix provided with the test kit generates biotinylated amplicons of the animal mtDNA present in the food sample. The labelled PCR fragments are then hybridized to the corresponding capture sequences on the individual array fields. The strong affinity between Biotin and streptavidin is exploited by the LCD Array test principle, and positive samples can be visually identified or by employing the scanner and software provided by the kit manufacturer. Figure 5 provides a schematic representation of the two test systems.

Capture probes

184 Polymerase Chain Reaction

Fig. 5a. CarnoCheck Test kit for the detection of animal species in food. The small table above shows the order of the measurement points for the animal species while the figure below depicts the on-chip control systems for exact quality determination (orientation controls in red, printing controls in green). (CarnoCheck Handbook, Manual version: BQ-

rabbit, hare, chicken, turkey, goose, and two duck varieties. The test system here relies on the detection of specific sites within the 16S rRNA mitochondrial locus of all the meat species present in the tested food sample. Included in the test system is a consensus primer pair that amplifies the desired region of the animal species in a PCR. The pre-labeled PCR

020-00, Greiner Bio-one).


Fig. 5b. LCD Array Meat 1.6 Test System for meat species identification. The figure shows the spotting pattern of the array while the table lists the capture probes immobilized on each array (Data Sheet MeatSpecies 1.6, V-I-08, Chipron)

In a recent study, these two biochip test systems were thoroughly validated and approved for routine use in the meat labour of a food control agency (Iwobi *et al.*, 2011). In this study, the two animal species differentiation biochip methods compared well in efficiency and detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Although such a simultaneous PCR approach will lead to accelerated analysis of meat species origin in food, while concomitantly revealing possible sources of deliberate adulteration or contamination, the efficiency of the approach is greatly influenced by the overall proficiency of the PCR reaction. In cases where very small amounts of a meat species is present in the

The Application of PCR-Based Methods in Food Control Agencies – A Review 187

provisions of regulation EC no. 1829/2003 and EC No. 1830/2003 (EC 2003a and b). In the EU appropriate thresholds have been set for both unintentional presence of GMOs in non-GMO food backgrounds (0.9 % per ingredient), and zero tolerance for non-approved varieties.

Detection of GMOs usually relies on the identification of the altered genotypic locus or the detection of the novel trait or phenotype arising from the genetic modification event. The genetic modification event will usually result in a new phenotypic trait, arising from the production of a new protein of the modified organism. In the context of plants, which account for the greatest number of GM events, such traits could include resistance to herbicides or pests. For detection of the altered phenotypic traits, a number of immunological assays, typically ELISA tests have been developed and even marketed commercially (Anklam *et al.*, 2002, Stave, 2002). For detection of the genotypic trait, the PCR reaction is the most important approach in use. In this context, real-time PCR detection is the preferred method of choice because of its high specificity, its closed amplification system, resulting in fewer contamination incidents, and its potential for quantification of GMO events. For a reliable PCR, good quality sample DNA is a prerequisite. Adequate care must be taken to ensure that the sample to be tested is truly representative of the matrix and that it has been adequately homogenized. Failure in extraction of adequate amounts of DNA for the PCR can be most readily overcome by increasing the volume of the sampling pool. Care however has to be taken in this regard as increasing the sample pool will also lead to an increased concentration of contaminants or inhibitors that could negatively hamper the PCR

In the event of a genetic transformation in an organism, not only the gene encoding the novel and desired trait is transferred, but also other important genetic control elements such as for example the strong 35S – Promoter from cauliflower mosaic virus (CaMV), which promotes high-level expression of the encoded trait, and *Agrobacterium tumefaciens nos*  terminator (*nos3*′). Additionally, for easier identification of the transformed plant cells, reporter genes are included in the design of the transformation event. Because the abovementioned markers are commonly found in many GMOs, they are readily employed for the routine screening of GMO events in food. However, the detection of these GMO markers is only an indication that the analyzed sample contains DNA from a GM plant, but does not provide unequivocal information on the specific trait that has been transformed in the plant. To achieve this, target sequences carrying the gene of interest that are characteristic for the transgenic organism must be reliably determined at their junctures with appropriate regulatory sequences (construct-specific detection). However this complete gene construct may have been transformed into different crops. To provide unambiguous verification of the transformation event in the particular plant under study, PCR reactions targeting the junction at the integration site between the plant genome and the inserted DNA or transgene provide the highest level of specificity (event-specific detection). An example of the principle behind the PCR-based detection of genetically modified plant is depicted below (Fig 6).

Several real-time PCR reactions for the detection of GMOs in food have been published in recent times (Gaudron *et al.,* 2009, Kluga *et al.*, 2011, Pansiot *et al.,* 2011). Reiting *et al.,* (2010) for example recently published a testing cascade for the real-time PCR detection of the genetically modified rice Kefeng6 which is unauthorized in Europe. While this work was

**5.2 PCR-based detection and quantification of GMOs** 

(Holst-Jensen, 2007, Anklam *et al*., 2002).

food matrix, the amplification of such sequences might be hampered by the presence of other meat species present in more abundance in the sample, leading to possible false negative results. Bai *et al.* (2009) cited the inherent complexity, low amplification efficiency, and unequal amplification efficiency on different templates as major drawbacks of currently described multiplex PCR reactions, thus precluding their commercial application. The biochips here described nevertheless hold great promise in the parallel identification of meat species in food products or samples.

## **5. GM Food and Feed detection using PCR methods**

Genetically modified organisms (GMOs) can be defined as organisms in which the DNA has been altered in a way that does not occur naturally. The technology used is often through recombinant DNA procedures and mainly involves the transfer of genetic material, usually from a microbe as donor to another host, in the context of this review, a plant. The resulting GM plants are then used to grow GM food crops. Generally, all GM crops available on the international market today have been designed to confer one of three basic traits to the plant: resistance to insect damage, resistance to viral infections and tolerance towards certain herbicides. Less common are genetic modifications resulting in plant varieties with altered nutritional values, or longer shelf lives (Holst-Jensen, 2007).

Although the DNA elements of interest mostly derive from microbes, such as the *cry* genes from *Bacillus thuringiensis*, which confer resistance to insects and the *cp4 EPSPS* gene encoded by *Agrobacterium* sp., other eukaryotic hosts may play a role, such as the plant *Petunia hybrida*, which is the source of a chloroplast transit peptide (CTP4). Transformation of the recipient plant cell might be characterized by one or more events or genetic rearrangements. Because current plant transformation procedures do not target specific locations in the recipient's genome, a second transformation event will be directed to a different location within the plant cell, thus making complex, detection of the genetic modification (Holst-Jensen *et al.,* 2006).

From its relatively small beginnings, GM plants have seen a recent explosion in recent times. Worldwide, more than 70 % of all soybeans cultivated are genetically modified, with genetically modified maize accounting for more than a quarter of global outputs. In 2009, for example, genetically modified corn was cultivated in approximately 91 % of all corn fields in the USA. In the most recent report on the Global Status of Commercialized Biotech/GM Crops in 2010, a total of 15.4 million farmers planted biotech crops on an estimated 148 million hectares in 29 countries (James, 2010). Detection and appropriate monitoring strategies are therefore indispensable in many food control agencies.

#### **5.1 Regulation of GMOs**

Worldwide, more than 100 genetically modified organisms (GMO) have received authorization for commercial use as food or feed.

Generally, GMOs are regulated by diverse legislation, aimed at protection of consumer safety and health. In the USA, the authorization process is simple and there is no requirement for traceability or labelling of de-regulated (approved) GMOs. In the EU, the GM legislation covering regulatory issues in the approval, detection and monitoring of GMOs is more complex. The authorization and use of genetically modified food and feed is covered by the provisions of regulation EC no. 1829/2003 and EC No. 1830/2003 (EC 2003a and b). In the EU appropriate thresholds have been set for both unintentional presence of GMOs in non-GMO food backgrounds (0.9 % per ingredient), and zero tolerance for non-approved varieties.

### **5.2 PCR-based detection and quantification of GMOs**

186 Polymerase Chain Reaction

food matrix, the amplification of such sequences might be hampered by the presence of other meat species present in more abundance in the sample, leading to possible false negative results. Bai *et al.* (2009) cited the inherent complexity, low amplification efficiency, and unequal amplification efficiency on different templates as major drawbacks of currently described multiplex PCR reactions, thus precluding their commercial application. The biochips here described nevertheless hold great promise in the parallel identification of meat

Genetically modified organisms (GMOs) can be defined as organisms in which the DNA has been altered in a way that does not occur naturally. The technology used is often through recombinant DNA procedures and mainly involves the transfer of genetic material, usually from a microbe as donor to another host, in the context of this review, a plant. The resulting GM plants are then used to grow GM food crops. Generally, all GM crops available on the international market today have been designed to confer one of three basic traits to the plant: resistance to insect damage, resistance to viral infections and tolerance towards certain herbicides. Less common are genetic modifications resulting in plant varieties with

Although the DNA elements of interest mostly derive from microbes, such as the *cry* genes from *Bacillus thuringiensis*, which confer resistance to insects and the *cp4 EPSPS* gene encoded by *Agrobacterium* sp., other eukaryotic hosts may play a role, such as the plant *Petunia hybrida*, which is the source of a chloroplast transit peptide (CTP4). Transformation of the recipient plant cell might be characterized by one or more events or genetic rearrangements. Because current plant transformation procedures do not target specific locations in the recipient's genome, a second transformation event will be directed to a different location within the plant cell, thus making complex, detection of the genetic

From its relatively small beginnings, GM plants have seen a recent explosion in recent times. Worldwide, more than 70 % of all soybeans cultivated are genetically modified, with genetically modified maize accounting for more than a quarter of global outputs. In 2009, for example, genetically modified corn was cultivated in approximately 91 % of all corn fields in the USA. In the most recent report on the Global Status of Commercialized Biotech/GM Crops in 2010, a total of 15.4 million farmers planted biotech crops on an estimated 148 million hectares in 29 countries (James, 2010). Detection and appropriate monitoring

Worldwide, more than 100 genetically modified organisms (GMO) have received

Generally, GMOs are regulated by diverse legislation, aimed at protection of consumer safety and health. In the USA, the authorization process is simple and there is no requirement for traceability or labelling of de-regulated (approved) GMOs. In the EU, the GM legislation covering regulatory issues in the approval, detection and monitoring of GMOs is more complex. The authorization and use of genetically modified food and feed is covered by the

species in food products or samples.

modification (Holst-Jensen *et al.,* 2006).

**5.1 Regulation of GMOs** 

**5. GM Food and Feed detection using PCR methods** 

altered nutritional values, or longer shelf lives (Holst-Jensen, 2007).

strategies are therefore indispensable in many food control agencies.

authorization for commercial use as food or feed.

Detection of GMOs usually relies on the identification of the altered genotypic locus or the detection of the novel trait or phenotype arising from the genetic modification event. The genetic modification event will usually result in a new phenotypic trait, arising from the production of a new protein of the modified organism. In the context of plants, which account for the greatest number of GM events, such traits could include resistance to herbicides or pests. For detection of the altered phenotypic traits, a number of immunological assays, typically ELISA tests have been developed and even marketed commercially (Anklam *et al.*, 2002, Stave, 2002). For detection of the genotypic trait, the PCR reaction is the most important approach in use. In this context, real-time PCR detection is the preferred method of choice because of its high specificity, its closed amplification system, resulting in fewer contamination incidents, and its potential for quantification of GMO events.

For a reliable PCR, good quality sample DNA is a prerequisite. Adequate care must be taken to ensure that the sample to be tested is truly representative of the matrix and that it has been adequately homogenized. Failure in extraction of adequate amounts of DNA for the PCR can be most readily overcome by increasing the volume of the sampling pool. Care however has to be taken in this regard as increasing the sample pool will also lead to an increased concentration of contaminants or inhibitors that could negatively hamper the PCR (Holst-Jensen, 2007, Anklam *et al*., 2002).

In the event of a genetic transformation in an organism, not only the gene encoding the novel and desired trait is transferred, but also other important genetic control elements such as for example the strong 35S – Promoter from cauliflower mosaic virus (CaMV), which promotes high-level expression of the encoded trait, and *Agrobacterium tumefaciens nos*  terminator (*nos3*′). Additionally, for easier identification of the transformed plant cells, reporter genes are included in the design of the transformation event. Because the abovementioned markers are commonly found in many GMOs, they are readily employed for the routine screening of GMO events in food. However, the detection of these GMO markers is only an indication that the analyzed sample contains DNA from a GM plant, but does not provide unequivocal information on the specific trait that has been transformed in the plant. To achieve this, target sequences carrying the gene of interest that are characteristic for the transgenic organism must be reliably determined at their junctures with appropriate regulatory sequences (construct-specific detection). However this complete gene construct may have been transformed into different crops. To provide unambiguous verification of the transformation event in the particular plant under study, PCR reactions targeting the junction at the integration site between the plant genome and the inserted DNA or transgene provide the highest level of specificity (event-specific detection). An example of the principle behind the PCR-based detection of genetically modified plant is depicted below (Fig 6).

Several real-time PCR reactions for the detection of GMOs in food have been published in recent times (Gaudron *et al.,* 2009, Kluga *et al.*, 2011, Pansiot *et al.,* 2011). Reiting *et al.,* (2010) for example recently published a testing cascade for the real-time PCR detection of the genetically modified rice Kefeng6 which is unauthorized in Europe. While this work was

The Application of PCR-Based Methods in Food Control Agencies – A Review 189

**hmgA 59122 3272**

**Bt11 Bt176 GA21**

**MIR604 M 810 M 863**

**NK603 TC1507**

**LY038 88017 T25**

**89034 CBH-351**

**Lectin RRS**

Two chocolate bar samples were analysed with the maize module on the Mx3005P. An overview of the recorded FAM fluorescence (R) of all 96 wells is shown. Positive control reactions are enclosed by green, negative control reactions by red, and samples by blue boundaries, respectively. Positive reactions were marked with a coloured dot in the upper left corner. All samples reacted positive for hmgA thus confirming that amplifiable DNA was present. One sample tested positive for eight maize events, the other was positive for

PCR-based applications in food control agencies have seen a tremendous boost in recent years. The simplicity, specificity and rapidity inherent in molecular-based approaches continue to make them increasingly attractive in a wide spectrum of food analytical procedures. Multiplexing applications will continue to see an increase in the near future as the demand for simultaneous detection and quantification of various events in food matrices grows. Additionally, it is expected that increased instrumental development will push the drive toward automation of various analytical procedures commonly employed in food

Anderson, A., Pietsch, K., Zucker, R., Mayr, A., Müller-Hohe, E., Messelhäusser, U., Sing, A.,

*Salmonella* spp. in Different Food Products. Food Anal Meth. 2010. 4: 259-267 Anklam, E., Gadani, F., Heinze, P., Pijnenburg, H., Van den Eede, G. 2002. Analytical

Busch, U., and Huber, I. Validation of a Duplex Real-Time PCR for the Detection of

methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. Eur Food Res Technol 214:3-26

Fig. 7. Analysis of samples with the maize module

four maize events, and RoundupReady soy (RRS).

**6. Conclusion** 

diagnostics.

**7. References** 

based on the construct-specific detection of this rice line, our lab recently published and validated an event-specific detection of this rice line, allowing greater specificity in its identification (Guertler *et al.*, 2011, in Press). Additionally, we currently developed a modular approach allowing the simultaneous and parallel detection of several GMOs in a food matrix. With this approach, the detection systems for 15 transgenic maize events were combined in one setup, with additional detection of maize and soybean reference genes (see Fig. 7). The reactions are based on validated single detection systems and are run in parallel with identical temperature profiles, thereby allowing the simultaneous detection of all relevant transgenic events together with corresponding controls for DNA quality, reaction setup and contamination (Gerdes *et al*., 2011, in Press).

Fig. 6. Principle behind the molecular biological PCR-based detection of a genetic modification event in rice LL601. The commonly employed genetic elements CaMV 35S promoter, the bar gene (encoding herbicide resistance) and *nos* terminator are here depicted for rapid detection of a genetic modification event. The point of integration of the newly inserted genetic element is the basis for the event-specific detection (adapted from Waiblinger, 2010).

Presently, a major challenge in PCR approaches is the development of multiplex assays for the simultaneous quantification of several targets in the same sample. Multiplexing offers the advantage of lower costs and expenditure, and higher throughput compared to singletarget assays. Kalogianni *et al.*, (2007) recently reported on a multiplex quantitative PCR based on a multianalyte hybridization assay performed on spectrally encoded microspheres. While these endpoint PCR approaches hold great promises, one major drawback is the requirement of separate steps for DNA amplification and detection of the products. Quantitative real-time PCR which allows continuous monitoring of the amplification products by a homogeneous fluorometric assay account therefore for the most widely used approach in GMO testing (Su *et al.,* 2011, Xu et al., 2011, ). In this regard, Köppel and colleagues reported on the development of a multiplex real-time PCR assay for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference material. Their test exhibited good specificity and sensitivity for the transgenes was in the range of 0.01-1% (Köppel *et al.*, 2010b). In summary, real-time PCR assays remain the gold standard in the analysis of GMO events in food. Because of the trend toward multiple detection events, multiplexing, with microarray-based methods will most likely continue to see greater applications in the future.

Fig. 7. Analysis of samples with the maize module

Two chocolate bar samples were analysed with the maize module on the Mx3005P. An overview of the recorded FAM fluorescence (R) of all 96 wells is shown. Positive control reactions are enclosed by green, negative control reactions by red, and samples by blue boundaries, respectively. Positive reactions were marked with a coloured dot in the upper left corner. All samples reacted positive for hmgA thus confirming that amplifiable DNA was present. One sample tested positive for eight maize events, the other was positive for four maize events, and RoundupReady soy (RRS).

## **6. Conclusion**

188 Polymerase Chain Reaction

based on the construct-specific detection of this rice line, our lab recently published and validated an event-specific detection of this rice line, allowing greater specificity in its identification (Guertler *et al.*, 2011, in Press). Additionally, we currently developed a modular approach allowing the simultaneous and parallel detection of several GMOs in a food matrix. With this approach, the detection systems for 15 transgenic maize events were combined in one setup, with additional detection of maize and soybean reference genes (see Fig. 7). The reactions are based on validated single detection systems and are run in parallel with identical temperature profiles, thereby allowing the simultaneous detection of all relevant transgenic events together with corresponding controls for DNA quality, reaction

Fig. 6. Principle behind the molecular biological PCR-based detection of a genetic modification event in rice LL601. The commonly employed genetic elements CaMV 35S promoter, the bar gene (encoding herbicide resistance) and *nos* terminator are here depicted for rapid detection of a genetic modification event. The point of integration of the newly inserted genetic element

Presently, a major challenge in PCR approaches is the development of multiplex assays for the simultaneous quantification of several targets in the same sample. Multiplexing offers the advantage of lower costs and expenditure, and higher throughput compared to singletarget assays. Kalogianni *et al.*, (2007) recently reported on a multiplex quantitative PCR based on a multianalyte hybridization assay performed on spectrally encoded microspheres. While these endpoint PCR approaches hold great promises, one major drawback is the requirement of separate steps for DNA amplification and detection of the products. Quantitative real-time PCR which allows continuous monitoring of the amplification products by a homogeneous fluorometric assay account therefore for the most widely used approach in GMO testing (Su *et al.,* 2011, Xu et al., 2011, ). In this regard, Köppel and colleagues reported on the development of a multiplex real-time PCR assay for the simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference material. Their test exhibited good specificity and sensitivity for the transgenes was in the range of 0.01-1% (Köppel *et al.*, 2010b). In summary, real-time PCR assays remain the gold standard in the analysis of GMO events in food. Because of the trend toward multiple detection events, multiplexing, with microarray-based methods will most likely continue to see greater

is the basis for the event-specific detection (adapted from Waiblinger, 2010).

applications in the future.

setup and contamination (Gerdes *et al*., 2011, in Press).

PCR-based applications in food control agencies have seen a tremendous boost in recent years. The simplicity, specificity and rapidity inherent in molecular-based approaches continue to make them increasingly attractive in a wide spectrum of food analytical procedures. Multiplexing applications will continue to see an increase in the near future as the demand for simultaneous detection and quantification of various events in food matrices grows. Additionally, it is expected that increased instrumental development will push the drive toward automation of various analytical procedures commonly employed in food diagnostics.

## **7. References**


The Application of PCR-Based Methods in Food Control Agencies – A Review 191

and O145 in Ground Beef. Foodborne Pathogens and Disease. 8: 601-607 Fricker, M., Messelhäußer, U., Busch, U., Scherer, S., and Ehling-Schulz, M. 2007. Diagnostic

Gerdes, L., Busch,U. and Pecoraro,S. 2011. Parallelised real-time PCR for identification of

Girish, P., Anjaneyulu, A., Viswas, K., Shivakumar,B., Anand, M., Patel, M., Sharma, B. 2005.

Gupta, A. R., Patra, R.C., Das, D.K., Gupta, P.K., Swarup, D., Saini, M. 2008. Sequence

Gürtler, P., Huber, I., Pecoraro, S., and Busch, U. 2011. Development of an event-specific

Hirao, T., Hiramoto, M., Imai, S., and Kato, H. 2006. A novel PCR method for quantification

Hlywka, J.J., Hefle, S.L., and Taylor, S.L. 2000. A sandwich enzyme-linked immunosorbent assay for the detection of almonds in foods. Journal of Food Protection 63: 252-257 Höhne, M., Schreier, E. 2004. Detection and characterization of norovirus outbreaks in

Holgate, S.T., Church, M.K., and Lichtenstein, L.M. 2001. Allergy. 2nd edn. (St Louis, Mosby) Holst-Jensen, A. Sampling, detection, identification and quantification of genetically

Holst-Jensen, A., De Loose, M. and Van den Eede, G. 2006. Coherence between legal

Hourihane, J. O'B., Kilburn, S.A., Nordlee, J.A:, Hefle, S.L., Taylor, S.L., and Warner, J. O.

challenge study. Journal of Allergy and Clinical Immunology. 100: 596-600

(GMOs) and their derived products. J. Agric. Food Chem. 54: 2799-2809 Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N., Wagner, M., Fach, P. 2004. Practical

real-time PCR. Journal of consumer protection and food safety. In Press. Hefle, S.L., Jeanniton, E., and Taylor, S.L. 2001. Development of a sandwich enzyme-linked

recent food-borne outbreaks. Appl Environ Microbiol. 73: 1892-1898 Gaudron, T., Peters, C., Boland, E., Steinmetz, A. and Moris, G.2009. Development of a

maize GMO events. Eur. Food Res. Technol*.* In Press. DOI:

species differentiation of deer. In Mitochondrial DNA

Eur Food Res Technol 229:295-305

http://10.0.3.239/s00217-011-1634-2

of Food Protection 64: 1812-1816

system. J Med Virol 72:312-319

ISBN-13:978-0-444-52843-8. Chapter 8, pp. 231-268

Protection 69: 2478-2486

Microbiol 42:1863-1868

of Shiga Toxin–Producing *Escherichia coli* Serogroups O26, O45, O103, O111, O121,

Real-time PCR assays for the detection of emetic *Bacillus cereus* strains in foods and

quadruplex-real-time PCR for screening food for genetically modified organisms.

Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Science 70: 107-112

characterization and polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial DNA 12S rRNA gene provides a method for

detection method for genetically modified rice (Kefeng 6) by means of quantitative

immunosorbent assay for the detection of egg residues in processed foods. Journal

of buckwheat by using a unique internal standard material. Journal of Food

Germany: application of a one-tube RT-PCR using a fluorogenic real-time detection

modified organisms (GMOs). In: Pico, Y. (ed.) 2007. Food toxicants analysis. Techniques, Strategies and Developments. Elsevier, Amsterdam, Netherlands.

requirements and approaches for detection of genetically modified organisms

considerations in design of internal amplification control for diagnostic PCR. J.Clin

1997. An evaluation of the sensitivity of subjects with peanut allergy to very low doses of peanut protein: a randomised, double-blind, placebo-controlled food


Bai, W., Xu, W., Huang, Y., Cao S., Luo, Y. 2009. A novel common primer multiplex PCR

Beuret, C. 2004. Simultaneous detection of enteric viruses by multiplex real-time RT-PCR. J

Bock, S.A., Muñoz-Furlong, A., Sampson, H.A.2001. Fatalities due to anaphylactic reactions

Bush, R.K., and Hefle, S. 1996. Food allergens. Critical reviews in food science and nutrition.

Croci, L., De Medici, D., Ciccozzi, M., Di Pasquale, S., Suffredini, E., Toti, L. 2003.

Commission Directive 2002/86/EC. L 305/19. 2002. Official Journal of the European

Commission Recommendation 2004/787/EC. L 348/18. 2004. Official Journal of the

De Husman, A.M., Lodder-Verschoor, F., van den Merg, H.H., Le Guyader, F. S., van Pelt, H.,

tracing of shellfish associated with disease outbreaks. J. Food Prot. 70:967-974 Demmel, A., Hupfer, C., Ilg Hampe, E., Busch, U., and Engel, K.H. 2008. Development of a

De Medici, D., Anniballi, F., Wyatt, G., Lindström, M., Messelhäußer, U., Aldus, C.,

Ehlert, A., Hupfer, C., Demmel, A., Engel, K-H., and Busch, U 2008. Detection of cashew nut in foods by a specific real-time PCR method. Food Anal Methods. 1: 136-143 Ehlert, A., Demmel, A., Hupfer, C., Busch, U., and Engel, K-H. 2009. Simultaneous detection

European Food Safety Authority (EFSA) (2009) Trend and sources of zoonoses and zoonotic

European Commission. Regulation (EC) No 1829/2003 of the European Parliament and of

European Commission. Regulation (EC) No 1830/2003(b) of the European Parliament and of

FAO/WHO. 2002. Risk assessment of *Salmonella* in eggs and broiler chickens. Microbiological Risk Assessment series no. 2. Switzerland (6446): 566–8. FVE Food Safety Report. http://www.fve.org/news/publications/pdf/stabletotable.pdf Fratamico, P.M., Bagi, L.K., Cray Jr.,W.C., Narang, N., Yan, X., Medina, M., and Liu, Y. 2011.

2001/18/EC. Official Journal of the European Union L 268/24

Contamination of mussels by hepatitis A virus: a public health problem in southern

van der Poel, W.H., Rutjes, S.A., 2007. Rapid virus detection procedure for molecular

real-time PCR for the detection of lupine DNA (*Lupinus* species) in foods. J Agric

Delibato, E., Korkeala, H., Peck, M.W., Fenicia, L. 2009. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food and environmental

of DNA from 10 food allergens by ligation-dependent probe amplification. Food

the Council of 22 September 2003 on genetically modified food and feed. Off. J. Eur.

the Council of 22 September 2003 concerning the traceability and labeling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive

Detection by Multiplex Real-Time Polymerase Chain Reaction Assays and Isolation

20: 366-370

36: 119-163

Communities

European Union

Food Chem 56:4328-4332

Union L 268 (2003a) 1-23

Virol Methods. 115:1-8

Italy. Food Control 14: 559-563

to foods. J Allergy Clin Immunol 107: 191-193

samples. Appl Environ Microbiol 75: 6457-6461

agents in the European Union in 2007. EFSA J. 223

Additives and Contaminants. 26: 409-418

(CP-M-PCR) method for the simultaneous detection of meat species. Food Control

of Shiga Toxin–Producing *Escherichia coli* Serogroups O26, O45, O103, O111, O121, and O145 in Ground Beef. Foodborne Pathogens and Disease. 8: 601-607


The Application of PCR-Based Methods in Food Control Agencies – A Review 193

Lehmann, L.E., Hunfeld, K.P., Steinbrucker, M., Brade, V., Book, M., Seifert, H., Bingold, T.,

Malmheden, Y.I., Eriksson, A., Everitt, G., Yman, L., and Karlsson, T. 1994. Analysis of food

Mead, P.S., Slutsker, L., Dietz, V., McCaig, L. F., Bresee, J, S., Shapiro, J., Griffin, P.M., and

Messelhäusser, U., Kämpf, P., Hörmansdorfer, S., Wagner, B., Schalch, B., Busch, U., Höller,

Messelhäusser, U., Kämpf, P., Colditz, J., Bauer, H., Schreiner, H., Höller, C., and Busch, U.

Meyer, R., Hofelein, C., Lüthy, J., and Candrian, U. 1995. Polymerase chain reaction-

Microbiological Risk assessments series 13: Viruses in food: Scientific advice to support risk

Mujico, J.R., Lombardía, Mena, M., C., Méndez, E., Albar, J.P. 2011. A highly sensitive real-

Nocker, A., Ceung, C.-Y., Camper, A.K. 2006. Comparison of propidium monoazide with

Nocker, A., and Camper, A.K. 2009. Novel approaches toward preferential detection of

Ong, S.B., Zuraini, M.I., Jurin, W.G., Cheah, Y.K., Tunung, R., Chai, L.C., Haryani, Y.,

differentiation of meat from animal origin. ASEAN Food Journal 14: 51-59 Pan, Y., and Breidt, Jr. 2007. Enumeration of viable *Listeria monocytogenes* cells by real-time

celiac patients. Food Chemistry DOI: 10.1016/j.foodchem.2011.03.061 Nishida, T., Kimura, H., Saitoh, M., Shinohara, M., Kato, M., Fukuda, S., Munemura, T.,

Japanese oysters. Appl Environ Microbiol 69:5782-5786

cells. Appl Environ Microbiol 73: 8028-8031

removal of DNA from dead cells. J Microbiol Meth 67:310-320.

identification in food. J Assoc Off Anal Chem Int 78: 1542-1551

2001

Press

Dis 1: 39-44.

management:

291:137-142

Immunology 6: 167-172

Dis 5: 607-625

Hoeft, A., Wissing, H., and Stüber, F. 2010. Improved detection of blood stream pathogens by real-time PCR in severe sepsis. Intensive care medicine 36: 49-56 Lenstra, J.A., Buntjer, J.B., and Janssen, F.W. 2001. On the origin of meat – DNA techniques

for species identification in meat products. Veterinary Sciences Tomorrow -15 May

proteins for verification of contamination or mislabelling. Food and Agricultural

Tauxe R. V. 1999. Food-related illness and death in the United States. Emerg Infect

C., Wallner, P., Barth, G., Rampp, A. 2011a. Cultural and molecular method for detection of *Mycobacterium tuberculosis* complex und *Mycobacterium avium* ssp. paratuberculosis in milk and dairy products. Appl Environ Microbiol Nov. 4. In

2011b. Qualitative and quantitative detection of human pathogenic *Yersinia enterocolitica* in different food matrices at retail level in Bavaria. Foodborne Pathog

restriction fragment length polymorphism analysis: a simple method for species

http://www.who.int/foodsafety/publications/micro/Viruses\_in\_food\_MRA.pdf

time PCR system for quantification of wheat contamination in gluten-free food for

Mikami, T., Kawamoto, A., Akijama, M., Kato, Y., Nishi, K., Kozawa, K., Nishio, O. 2003. Detection, quantification and phylogenetic analysis of noroviruses in

ethidium monoazide for differentiation of live vs. dead bacteria by selective

viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett

Ghazali, F.M., and Son, R. 2007. Meat molecular detection: sensitivity of polymerase chain reaction-restriction fragment length polymorphism in species

PCR with propidium monoazide and ethidium monoazide in the presence of dead


Hsuih, T.C., Park, Y.N., Zaretsky, C., Wu, F., Tyagi, S., Kramer, F.R., Sperling, R., Zhang,

Hupfer, C., Waiblinger, H.U., Busch, U. 2006. Development and validation of a real-time PCR detection method for celery in food. Eur Food Res Technol. 225: 329-335 Kalogianni DP, Elenis DS, Christopoulos TK, Ioannou PC (2007) Multiplex Quantitative

serum. J Clin Microbiol 34: 501-507

Animal Sciences 77:880-881

42: ISAAA: Ithaca, NY.

D.Y. 1996. Novel ligation-dependent PCR assay for detection of hepatitis C in

Competitive Polymerase Chain Reaction Based on a Multianalyte Hybridization Assay Performed on Spectrally Encoded Microspheres. *Anal Chem* 79:6655–6661 Iwobi, A.N., Huber, H., Hauner, G., Miller, A., and Busch, U. 2011. Biochip technology for the detection of animal species in meat products. Food Analytical Methods. 4: 389-398 Jain, S., Brahmbhait, M.N., Rank, D.N., Joshi, C.G. and Solank, J.V. 2007. Use of *cytochrome b*

gene variability in detecting meat species by multiplex PCR assay. Indian Journal of

sequence-based amplification technique and comparison with reverse

2010. Rapid quantification of viable *Campylobacter* bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative

A.C. 1989. Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86: 6196-6200 Köppel, R., Dvorak, V., Zimmerli, F., Breitenmoser, A., Eugster, A., Waiblinger, H.-U. 2010a.

Two tetraplex real-time PCR for the detection and quantification of DNA from

simultaneous detection and quantification of DNA from three transgenic rice species and construction and application of an artificial oligonucleotide as reference

identification and quantification of DNA from beef, pork, chicken, turkey, horse

''Real-Time PCR-Based Ready-to-Use Multi-Target Analytical System for GMO Detection in processed maize matrices. 2011. Eur Food Res Technol. DOI

PCR primers for the detection of Norwalk-like viruses. J Virol Methods 100:107-114

Methods for the detection of beef and pork in foods using real-time polymerase

James C. 2010. Global Status of Commercialized Biotech/GM Crops. 2010. ISAAA Brief No.

Jean, J., Blais, B., Darveau, A., Fliss, I. 2001. Detection of hepatitis A virus by the nucleic acid

Josefsen, M.H., Löfström, C., Hansen, T.B., Christensen, L.S., Olsen, J.E., and Hoorfar, J.

Kocher, T.D:, Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson,

Köppel, R., Zimmerli, F., Breitenmoser, A. 2010b. Multiplex real-time PCR for the

Köppel, R., Zimmerli, F., Breitenmoser, A. 2009. Heptaplex real-time PCR for the

Kluga, L., Folloni, S., Van den Bulcke, M., Van den Eede, G., Querci, M. Applicability of the

Kojima, S., Kageyama, T., Fukushi, S., Hoshino, F., Katayama, K.2002. Genogroup-specific

Koopmans, M., Duizer, E. 2004. Foodborne viruses: an emerging problem. Inter J Food

Laube, I., Spiegelberg, A., Butschke, A., Zagon, J., Schauzu, M., Kroh, L., Broll, H. 2003.

meat, sheep (mutton) and goat. Eur Food Res Technol 230: 125-133

transcription-PCR. Appl. Environ Microbiol 67: 5593-5600

risk assessment. Appl. Environ. Microbiol.76: 5097-5104

eight allergens in food. Eur Food Res Tech. 230: 367-374

molecule. Eur Food Res Technol 230:731-736

chain reaction. Int J Food Sci Technol. 38: 111-118

10.1007/s00217-011-1615-5

Microbiol 90: 23-41


http://www.who.int/foodsafety/publications/micro/Viruses\_in\_food\_MRA.pdf


**10** 

*Slovenia* 

**PCR in Food Analysis** 

Nataša Toplak2, Saša Piskernik1 and Barbara Jeršek1

*1Dept. of Food Science and Technology, Biotechnical Faculty, University of Ljubljana,* 

The aim of this chapter is to briefly present polymerase chain reaction (PCR)-based technologies for use in the detection and quantification of different microorganisms in foods, with an emphasis on sample preparation and evaluation of results. Furthermore, we indicate the PCR-based methods that are most commonly used for the typing of bacteria, and in the final section we provide examples of PCR application in the detection of

The microbiological safety of food production is a significant concern of regulatory agencies and the food industry. The most important aspect is to avoid potential negative consequences to human health and economic losses, as well as the loss of consumer

What is PCR? PCR is a technique that is used to amplify a single or a few copies of a piece of nucleic acid, to generate thousands to millions of copies of a particular nucleic acid. It allows much easier characterisation and comparisons of genetic material from different individuals and organisms. Simply stated, it is a "copying machine for DNA molecules". PCR represented a revolution in biological techniques when it was first developed in 1983 by Kary Mullis (Saiki et al., 1985). Mullis won the Nobel Prize for Chemistry in 1993 for his work on the use and development of PCR. PCR allows the biochemist to mimic the natural

DNA replication is a biological process in living cells that starts with one double-stranded DNA (dsDNA) molecule and produces two identical (double-stranded) copies of the original dsDNA. Each strand of the original dsDNA serves as a template for the production

PCR is now a common, simple and inexpensive tool that is used in many different areas, from medical and biological research, to veterinary medicine, hospital analyses, forensic sciences, and paternity testing, and in the food and beverage, biotechnology and

of the complementary strand. PCR is thus simply the *in-vitro* replication of dsDNA.

**1. Introduction** 

confidence.

**2.1 The basics of PCR** 

unwanted components in foods.

**2. PCR in the analysis of foods** 

DNA replication process of a cell in the test-tube.

Anja Klančnik1, Minka Kovač2,

*2Omega d.o.o., Ljubljana* 

