**2. PCR techniques and clinical applications in diagnostic of infectious disease**

#### **2.1 Multiplex PCR**

In diagnostic laboratories, the use of PCR is often limited by its cost and sometimes by the availability of adequate sample volume. To overcome these issues and

**139**

*PCR and Infectious Diseases*

especially mPCR methods [10].

tivity, and selectivity of reactions.

**2.2 Loop-mediated isothermal amplification**

final products are the accumulation of 109

*parvum*, and *Ureaplasma urealyticum* [15]*.*

technologies have been developed subsequently [11].

*DOI: http://dx.doi.org/10.5772/intechopen.85630*

also to increase the diagnostic capacity of PCR, there is a type termed multiplex PCR (mPCR). The mPCR refers to the use of different pairs of primers to simultaneously amplify multiple regions of the nucleic acid of the sample with visualization of the amplified products by gel electrophoresis. The use of multiple primer pairs in mPCRs is an innovation that offers significant benefits in cost, time, and exact diagnosis. The main advantage of this technology is to minimize the number of separate reactions, for example, to detect several pathogens at the same time in a single specimen such as sexually transmitted pathogens [6–8]. This technique makes it possible to diagnose several diseases with a single diagnostic test, with sensitivity,

This technique has become a mainstay of research and clinical diagnostic applications, such as sexually transmitted infections (STIs). Considering a major public health problem, the STIs are common everywhere from developed countries and developing countries. It is estimated that each year more than 340 million new cases of bacterial STI arise, including gonorrhea, chlamydia, and syphilis, and the incidence is increased worldwide in adults of 15–49 years of age [9]. The susceptibility to sexually transmitted infections, including the human immunodeficiency virus (HIV), and the high cost of treatment have led to the need for fast and reliable laboratory techniques for the identification of pathogens. Procedures for nucleic acid amplification to detect sexually transmitted pathogens have been developed,

The multiplex PCR has the potential to analyze many samples in a single reaction, and it is useful for diagnostic of multipathogenic infection. However, it has some limitations such as the nonspecific products generated through primer-primer interactions that may interfere with the amplification of targets, decreasing sensi-

Nucleic acid amplification is commonly used in the field of life science research.

The loop-mediated isothermal amplification (LAMP-PCR) was first developed over 15 years ago, and it has emerged as powerful method to concurrently detect multiple pathogens [12, 13]. The method employs a DNA polymerase with strand displacement activity and a set of four inner and outer primers that recognize a total of six distinct sequences of the target DNA. Moreover, the method involves two successive steps of amplification, with the first step comprising mPCR and the second step LAMP. Amplicons of the first step serve as templates in the second step. The amplification protocol requires only a single temperature for the reaction, and the amplification is diagnosed without the need for electrophoretic techniques, using in situ detection process with colorimetric dye or with a fluorescent dye. The

copies of target DNA in less than an hour.

With the development of molecular biology, many new molecular diagnostic

The LAMP-PCR has been regarded as an innovative technology and emerged as an alternative to PCR-based methodologies in clinical laboratory with significant increase of detection limits, efficiency, selectivity, and specificity over single-stage. With more and more scientists focusing their attention on the application of LAMP technology, the range of its use is not limited to the bacteria detection and identification any more [14]. The LAMP-PCR was developed and employed to detect species that cause chorioamnionitis and premature labor, *Ureaplasma* 

It was also applied to the parasite and virus detection [16–18]. Recently, Kurosaki et al. in their study [19] in 2017 developed a LAMP-PCR assay for the detection of

specificity, and speed, indispensable values in diagnostic tests.

#### *PCR and Infectious Diseases DOI: http://dx.doi.org/10.5772/intechopen.85630*

*Synthetic Biology - New Interdisciplinary Science*

diagnosis of infectious diseases.

cerebrospinal fluid (CSF).

been successfully eliminated.

digital PCR (dPCR), and real-time PCR.

Scientific studies of infectious agents and diseases provide a knowledge for the development of diagnostic tests for such diseases, drugs to treat, and vaccines for prevention. Earlier, specific and effective diagnosis is one of the most appropriate forms and strategies for managing. According to [3], in American hospitals, about 5 million cases of infectious disease are reported annually. Besides that, most of the cases are unreported, resulting in substantial morbidity and mortality [3]. In the last 20 years, analysis methods based on the detection and sequencing of 16S rDNA

Unfortunately, despite in clinical laboratories, the diagnosis of infectious diseases is directly associated with time of pathogen identification by conventional culture methods as these tests suffer from long turnaround times, from hours to days. A technical difficulty encountered in these methods performed before the advent of the molecular techniques was that not all pathogens are cultivable, and culture conditions ordinarily are not known. Other limitations of the traditional diagnostic include requirements for additional testing and wait times for characterizing detected pathogens (i.e., discern-

Nucleic acid amplification is one of the most valuable tools in virtually all life science fields, including application-oriented fields such as clinical medicine, for

The serological methods are limited by the cross-reactions between the types of agents and by the fact that some infectious agents have no clearly identified epitopes that are sufficiently specific, requiring them as a stringent clinic diagnostic. In addition, the specific IgM antibodies are detected only in acute phase of infection, and for detecting infections, the serological tests are inaccurate, labor-intensive, and unreliable. In the last 20 years, analysis methods based on the nucleic acid amplification have been widely used in all life science fields as a new way for the diagnosis

The use of amplification techniques such as Polymerase Chain Reaction (PCR) has long been used to detection, genotyping, and quantification of virus and bacteria in various clinical specimens, such as serum, plasma, urine, semen, and liquid

PCR-based diagnostics have been effectively developed for a wide range of microorganisms. Due to its incredible sensitivity, specificity, reproducibility, broad dynamic range, and speed of amplification, PCR has been championed by infectious disease experts for identifying organisms that cannot be grown in vitro, or in instances where existing culture techniques are insensitive and/or need prolonged incubation times [5]. Advances in development of molecular technology and diagnostics have enhanced understanding IDs' etiology, pathogenesis, and molecular epidemiology, which provide basis for appropriate detection, quantification, prevention, and control measures as well as rational design of vaccine, by which some diseases have

Since 1985, many PCR amplification-based techniques have been designed for detection and identification, including: multiplex PCR (M-PCR), LAMP-PCR,

**2. PCR techniques and clinical applications in diagnostic of infectious** 

In diagnostic laboratories, the use of PCR is often limited by its cost and sometimes by the availability of adequate sample volume. To overcome these issues and

have been widely used in place of conventional culture methods.

ment of species, virulence factors, and antimicrobial resistance) [3, 4].

of human pathogens like virus, bacteria, and parasites.

**138**

**disease**

**2.1 Multiplex PCR**

also to increase the diagnostic capacity of PCR, there is a type termed multiplex PCR (mPCR). The mPCR refers to the use of different pairs of primers to simultaneously amplify multiple regions of the nucleic acid of the sample with visualization of the amplified products by gel electrophoresis. The use of multiple primer pairs in mPCRs is an innovation that offers significant benefits in cost, time, and exact diagnosis. The main advantage of this technology is to minimize the number of separate reactions, for example, to detect several pathogens at the same time in a single specimen such as sexually transmitted pathogens [6–8]. This technique makes it possible to diagnose several diseases with a single diagnostic test, with sensitivity, specificity, and speed, indispensable values in diagnostic tests.

This technique has become a mainstay of research and clinical diagnostic applications, such as sexually transmitted infections (STIs). Considering a major public health problem, the STIs are common everywhere from developed countries and developing countries. It is estimated that each year more than 340 million new cases of bacterial STI arise, including gonorrhea, chlamydia, and syphilis, and the incidence is increased worldwide in adults of 15–49 years of age [9]. The susceptibility to sexually transmitted infections, including the human immunodeficiency virus (HIV), and the high cost of treatment have led to the need for fast and reliable laboratory techniques for the identification of pathogens. Procedures for nucleic acid amplification to detect sexually transmitted pathogens have been developed, especially mPCR methods [10].

The multiplex PCR has the potential to analyze many samples in a single reaction, and it is useful for diagnostic of multipathogenic infection. However, it has some limitations such as the nonspecific products generated through primer-primer interactions that may interfere with the amplification of targets, decreasing sensitivity, and selectivity of reactions.
