**5. Fish (fluorescent** *in situ* **hybridization)**

The literature provides a limited number of reports concerning the application of the FISH technique for food examination (Ootsubo et al., 2003), while it is broadly applied in microbiology of environment, histopathology, histoimmunology, cytogenetics. Initially, it was developed in order to identify and to determine the number of bacterial cells in water ecosystem environments (Skowrońska & Zmysłowska, 2006), deposits, rhizosphere and soil.

Validated PCR methods are available from Bio-Rad, Roche, Qualicon/Oxoid, Genesystems, AES Chemunex, Applied BioSystems, Idaho Technology Inc., Lantmännen, IEH Laboratries and Consulting Group, ADNucleis and BioControl systems. Validation is an important step in the process of standardizing a method because it provides evidence that the new method gives similar results and is in agreement with the currently used

One major difficulty with PCR is the presence of compounds that inhibit the PCR reaction. These compounds can contaminate the DNA templates extracted from food samples and may in turn generate false-negative results (Elizaquivel et al., 2008). Therefore, evaluation and elimination of PCR inhibitory compounds are important steps in the development of PCR and real-time PCR assays (Abu Al-Soud et al., 2000). The PCR procedure is sufficiently sensitive such that, in theory, only a few template molecules are required to initiate the synthesis reactions (Uyttendaele et al., 2003). However, an enrichment step is still required to detect small numbers of *Salmonella* in food samples. This step may consist of non-selective enrichment with buffered peptone water (BPW) and selective enrichment with Rappaport-Vassiliadis. These enrichment broth have been directly utilized for *Salmonella* DNA template preparation. However, limited research has been conducted to quantitatively evaluate the effects of the enrichment broths using conventional PCR assays and even less using a realtime PCR protocol. Therefore, identifying and eliminating the PCR inhibitory effects of the enrichment broths is key to enhancing the performance of PCR assays in detecting

Multiplex PCR is a variant of the PCR technique in which two or more loci are simultaneously amplified in the same reaction. Multiplex PCR can be described as a specific and sensitive in vitro amplification of DNA with distinguishable size products from the same or different organisms in a single reaction (Jasson et al., 2010; Fitzgerald et al., 2007). In this methodology several specific primer sets are combined into a single PCR assay. MPCR is undoubtedly useful to rapidly establish simultaneous detection of multiple virulence factors (Fach et al., 2009) or combined detection of multiple isolates (Kawasaki et al., 2009; Settanni & Corsetti, 2007). A convened format for MPCR is the

Recently, multiplex real-time PCR assays have been applied to detect more than two gene sequences in a single reaction by using spectrally distinct dye-labeled probes (TaqMan system) (Elizaquivel et al., 2008). This technology could potentially save time and effort in the laboratory and thus may lower testing-related costs incurred by the food industry

The literature provides a limited number of reports concerning the application of the FISH technique for food examination (Ootsubo et al., 2003), while it is broadly applied in microbiology of environment, histopathology, histoimmunology, cytogenetics. Initially, it was developed in order to identify and to determine the number of bacterial cells in water ecosystem environments (Skowrońska & Zmysłowska, 2006), deposits, rhizosphere

reference method (Patel et al., 2006).

**4.3 Multiplex polymerase chain reaction (PCR)** 

GeneDisc (PALL) (Beutin et al., 2009).

**5. Fish (fluorescent** *in situ* **hybridization)** 

(Elizaquivel et al., 2008).

and soil.

*Salmonella* in foods.

Fig. 1. Flow chart of a typical FISH procedure.

The FISH technique consists in hybridization of the rRNA sequence of immobilized cells by a fluorescently-labelled 16S rRNA oligonucleotide probe (Zwirglmaier, 2005; Baudart et al., 2005). Oligonucleotide probes are short fragments of deoxyribonucleic acid which hybridize or are paired with complementary sequences of DNA or RNA extracted from the analysed microorganisms. They are paired in the same way as double-stranded DNA forms (adenine with thymine and guanine with cytosine). If the sequence of bases on the DNA probe is complementary to the sequence characteristic for the determined microorganism, the probe binds only with the DNA of the identified microorganism. Probes are most often marked on one or on both ends with a fluorescent dye. Molecular probes bind specifically to rRNA in ribosomes of the target cells, identifying them on various taxonomic levels. Such a solution significantly increases the sensitivity of determination – since rRNA is an integral part of bacterial ribosome, it is found in the cell in large number of copies (between 1,000 and 10,000). Another advantage of this solution is the availability of vast information concerning rRNA sequences originating from various microorganisms which are often very closely genetically related, which allows probes to have very high specificity (Sakai et al., 2004; Ercoloni et al., 2005) Due to the range of probe specificity, the following probes can be distinguished: universal, e.g. EUB338 (GCTGCCTCCCGTAGGAGT), specific for *Bacteria* domain, except for the *Planctomycetales* order, antisense, e.g. NON388 (CGACGGAGGGCATCCTCA) designed to detect non-specific probe binding, and specific probes, e.g. for *Salmonella* sp.: Sal3 (5'-AATCACTTCACCTACGTG-3').

The FISH method with the application of fluorescently labelled 16S rRNA oligonucleotide probes is used for determining only the number of physiologically active cells, since rRNA

Detection of *Salmonella* spp. Presence in Food 405

Vieira-Pinto et al., (2005) compared FISH methods with the classical plate method for detection of *Salmonella* spp. Out of 47 samples of pork tonsils, 16 (34%) were positive for *Salmonella* spp detection by the FISH method with the application of 23S rRNA Sal3 probe. Out of 31 negative results obtained by FISH method, one sample was positive for *Salmonella* spp. detection by the plate method. Similar results were obtained by Fang et al., (2003), who detected *Salmonella* sp. species in 56 samples of food products by the FISH method (23S rRNA Sal3), while 28 samples were not positive for *Salmonella* spp. detection by the plate method. Huge number of positive results can derive from the presence of cells slightly damaged or occurrence of factors inhibiting their growth in food products, which can transfer cells to VBNS state. The authors suggest that FISH method seems to be less prone to diverse physical-chemical properties of preserved food products (temperature, concentration of NaCl, pH), which can work as a stress factor for *Salmonella* spp. cells. Presence of microflora can be another reason of high number of positive results obtained by

The conclusions drawn from the research show the need for continuous improvement of the methodology and selecting and/or designing more specific probes. This is related to the varied chemical and microbiological composition of food (the so-called matrix), which can lead to errors in reading. Therefore, a relatively fast assessment of the quality and safety of food requires not only the selection of probes for individual species of microorganisms, but first of all optimal preparation of food samples for examination purposes on the basis of the matrix. Preparation of samples is understood as proper filtration and centrifugation at various parameters in order to eliminate large particles, and also the choice of optimal digestion conditions or permeabilization of the cell wall of microorganisms (e.g. with lysozyme, proteinase K, achromopeptidase, paraformaldehyde, ethanol etc.) occurring in the examined food. Proper preparation of samples and cells prevents non-specific

absorption of the probe on cell elements and easier penetration of cell cytoplasm.

VIDAS *Salmonella* strip containing the boiled *Salmonella* culture.

VIDASTM (BioMérieux) is an automated enzyme-linked fluorescent assay (ELFA) method based on the detection of *Salmonella* by using specific antibodies coated on the inner surface of a tip-like disposable pipette which is introduced into the VIDAS system along with the

VIDAS Immuno-concentration *Salmonella* (ICS) is a fully automated method for the concentration of *Salmonella* from foods. It replaces traditional selective enrichment procedures with an automated immunological capture and specific release process (Yeh et al., 2002). The method is based on multistage reaction. The kit contains so called reagent stripes, that is a set of wells with reagents sealed tightly inside, and pipettes, which inner sides are coated with antibodies against specific antigens. The amount of 500 µl of the sample after selective enrichment stage on RVS is introduced to the first well and a strip is placed in the immunoanalyser chamber. Reaction suspension is cyclically pulled up and down by pipettes. A pipet tip-like device, the solid-phase receptacle (SPR) serves as the solid phase as well as the pipet for the assay. The SPR is coated with anti-*Salmonella* antibodies absorbed on the surface. A final enzymatic step releases the captured *Salmonella* into a well. Detection of *Salmonella* antigens is based on enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument. ASPR serves as the solid phase as well as the pipet for the assay. The SPR is coated with a cocktail of highly specific monoclonal

the FISH method as compared to the plate methods.

**6. VIDAS** 

has a shorter half-life than DNA, which makes rRNA a potentially better indicator of their activity. Ribosomes of quickly-growing cells include a certain number of rRNA copies, usually over 1,000, which is sufficient for inducing a light signal after the labelled probe binds (Bottari et al., 2006). After the death of the cell, rRNA disintegrates, and the rate of this process depends, among others, on the concentration of enzymes – RNAz, as well as on the continuity of the cytoplasmic membrane (Vieira-Pinto et al., 2007). Slowly growing or metabolically inactive cells, containing a small number of ribosome copies, emit light of low intensity. New, more sensitive modifications of the FISH technique lead to amplification of the signal (TSA-FISH also known as CARD-FISH), becoming a useful tool for contemporary microbiology (Fang et al., 2003). 16S rRNA oligonucleotide probes labelled with fluorophores of various molecular weight (e.g. horseradish peroxidase – 44 kDa, fluorescein – 330 kDa) provide a visual assessment of the degree of cell wall permeabilization, on the basis of differences in dye permeation into the cell. An extreme advantage of the FISH technique is the possibility to detect VBNC (viable but not culturable) cells which do not grow on solid media, which makes this method more sensitive in comparison to the plate methods (Pisz et al., 2007, Oliver, 2005)

The most frequent used dyes for the detection of the FISH signals are FITC (fluorescein isothiocyanate) that emits a green fluorescence and dyes with orange or red fluorescence, such as Cy3™ , or TexasRed™. Another commonly used fluorochrome in FISH experiments is Cy5™ that emits in the far red/close infrared. Since this fluorescence is not visible by eye it would need detection by an infrared sensitive camera mounted on the microscope. (Hoefel et al., 2003; Gatti et al., 2006)There are various other dyes with similar characteristics available from various different vendors. For example, FITC can well be replaced by Rhodamin 110, and Cy3™ by TAMRA (carboxytetramethyl- rhodamine) depending on the actual price and for some dyes, depending on the quality of the currently distributed lot (Table 4).


Table 4. Characteristics of selected fluorescent dyes.

A standard FISH methodology includes preparation and permeabilization of cells, hybridization, washing off the excess unbound probe and detecting the signal with the application of fluorescence microscopy (Jasson et al., 2010; Ootsubo et al., 2003)

Reports concerning the use of fluorescent *in situ* hybridization in food research indicate the possibility of its application of *Salmonella* spp. detection and recommend this method as very sensitive, fast and cheap.

has a shorter half-life than DNA, which makes rRNA a potentially better indicator of their activity. Ribosomes of quickly-growing cells include a certain number of rRNA copies, usually over 1,000, which is sufficient for inducing a light signal after the labelled probe binds (Bottari et al., 2006). After the death of the cell, rRNA disintegrates, and the rate of this process depends, among others, on the concentration of enzymes – RNAz, as well as on the continuity of the cytoplasmic membrane (Vieira-Pinto et al., 2007). Slowly growing or metabolically inactive cells, containing a small number of ribosome copies, emit light of low intensity. New, more sensitive modifications of the FISH technique lead to amplification of the signal (TSA-FISH also known as CARD-FISH), becoming a useful tool for contemporary microbiology (Fang et al., 2003). 16S rRNA oligonucleotide probes labelled with fluorophores of various molecular weight (e.g. horseradish peroxidase – 44 kDa, fluorescein – 330 kDa) provide a visual assessment of the degree of cell wall permeabilization, on the basis of differences in dye permeation into the cell. An extreme advantage of the FISH technique is the possibility to detect VBNC (viable but not culturable) cells which do not grow on solid media, which makes this method more sensitive in comparison to the plate

The most frequent used dyes for the detection of the FISH signals are FITC (fluorescein isothiocyanate) that emits a green fluorescence and dyes with orange or red fluorescence, such as Cy3™ , or TexasRed™. Another commonly used fluorochrome in FISH experiments is Cy5™ that emits in the far red/close infrared. Since this fluorescence is not visible by eye it would need detection by an infrared sensitive camera mounted on the microscope. (Hoefel et al., 2003; Gatti et al., 2006)There are various other dyes with similar characteristics available from various different vendors. For example, FITC can well be replaced by Rhodamin 110, and Cy3™ by TAMRA (carboxytetramethyl- rhodamine) depending on the actual price and for some dyes, depending on the quality of the

**Blue** DAPI 358 461 **Turquoise** DEAC 426 480 **Green** FITC/R110 494/500 517/525 **Yellow** R6G 524 550 **Orange** TAMRA/Cy3™ 552/550 575/570 **Red** TexRed/Cy3.5™ 590/581 612/596 **Near Infrared** Cy5™ 649 670 **Infrared** Cy5.5™ 675 694

A standard FISH methodology includes preparation and permeabilization of cells, hybridization, washing off the excess unbound probe and detecting the signal with the

Reports concerning the use of fluorescent *in situ* hybridization in food research indicate the possibility of its application of *Salmonella* spp. detection and recommend this method as

application of fluorescence microscopy (Jasson et al., 2010; Ootsubo et al., 2003)

**Dyes Ex.Max. Em.Max.** 

methods (Pisz et al., 2007, Oliver, 2005)

currently distributed lot (Table 4).

very sensitive, fast and cheap.

Table 4. Characteristics of selected fluorescent dyes.

Vieira-Pinto et al., (2005) compared FISH methods with the classical plate method for detection of *Salmonella* spp. Out of 47 samples of pork tonsils, 16 (34%) were positive for *Salmonella* spp detection by the FISH method with the application of 23S rRNA Sal3 probe. Out of 31 negative results obtained by FISH method, one sample was positive for *Salmonella* spp. detection by the plate method. Similar results were obtained by Fang et al., (2003), who detected *Salmonella* sp. species in 56 samples of food products by the FISH method (23S rRNA Sal3), while 28 samples were not positive for *Salmonella* spp. detection by the plate method. Huge number of positive results can derive from the presence of cells slightly damaged or occurrence of factors inhibiting their growth in food products, which can transfer cells to VBNS state. The authors suggest that FISH method seems to be less prone to diverse physical-chemical properties of preserved food products (temperature, concentration of NaCl, pH), which can work as a stress factor for *Salmonella* spp. cells. Presence of microflora can be another reason of high number of positive results obtained by the FISH method as compared to the plate methods.

The conclusions drawn from the research show the need for continuous improvement of the methodology and selecting and/or designing more specific probes. This is related to the varied chemical and microbiological composition of food (the so-called matrix), which can lead to errors in reading. Therefore, a relatively fast assessment of the quality and safety of food requires not only the selection of probes for individual species of microorganisms, but first of all optimal preparation of food samples for examination purposes on the basis of the matrix. Preparation of samples is understood as proper filtration and centrifugation at various parameters in order to eliminate large particles, and also the choice of optimal digestion conditions or permeabilization of the cell wall of microorganisms (e.g. with lysozyme, proteinase K, achromopeptidase, paraformaldehyde, ethanol etc.) occurring in the examined food. Proper preparation of samples and cells prevents non-specific absorption of the probe on cell elements and easier penetration of cell cytoplasm.
