**4.2.8 Multiplex PCR**

#### **Theoretical basis of multiplex PCR method: Critical Parameters**

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more loci are simultaneously amplified in the same reaction. Since its first description in 1988 (Chamberlain et al., 1988), this method has been successfully applied in many areas of DNA testing, including analyses of deletions (Henegariu et al., 1994), mutations (Shuber et al., 1993) and polymorphisms (Mutirangura et al., 1993), or quantitative assays (Mansfield et al., 1993) and reverse transcription PCR (Crisan, 1994).

The role of various parameters that may influence the performance of standard (uniplex) PCR has been discussed (Robertson & J., 1998). However, fewer publications discuss multiplex PCR (Henegariu et al., 1997).

The optimization of multiplex PCRs can pose several difficulties, including poor sensitivity or specificity and/or preferential amplification of certain specific targets (Polz & C. M., 1998). The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of primer dimers (Brownie et al., 1997). These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. Thus, the optimization of multiplex PCR should aim to minimize or reduce such non-specific interactions.

Compatibility among the primers within the reaction mixture such that there is no interference, is of great technical importance. Primer selection followed simple rules (i) primer length of 18–24 bp or higher and (ii) a GC content of 35%–60%, thus having an annealing temperature of 55 °C-58 °C or higher. Longer primers (28-30 bp) allowed the reaction to be performed at a higher annealing temperature and yielded less unspecific products.

Combining the primers in various mixtures and amplifying many loci simultaneously required alteration/optimization of some of the parameters of the reaction. When the

are combined and used to determine the differentiation of strains (Yan et al., 2003). MLST provides data similar to those obtained by multilocus enzyme electrophoresis, but in substantively greater detail, because it has the ability to assess individual nucleotide changes rather than to screen for changes in the overall charge and expression of the

This method is extremely useful for long-term epidemiological studies or phylogenetic analyses. Over 230 *Salmonella* isolates were recently characterized by MLST based on sequences from the 16S RNA, pduF, glnA and manB genes (Kotetishvili et al., 2002). These results were compared to PFGE and serotype analysis. MLST was able to differentiate strains better than PFGE, though not all genes performed equally. Among the four loci, only manB demonstrated clusters among the clinical and environmental strains. As expected, the 16S rRNA locus showed significant homogeneity among the isolates and grouped most

MLST shows great promise for accurate strain discrimination with data that can be accurately shared between laboratories. However, like FAFLP, the universal appeal of this technique will be improved when automated sequence machinery becomes more affordable and labs can develop familiarity with complicated DNA sequence analysis and

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more loci are simultaneously amplified in the same reaction. Since its first description in 1988 (Chamberlain et al., 1988), this method has been successfully applied in many areas of DNA testing, including analyses of deletions (Henegariu et al., 1994), mutations (Shuber et al., 1993) and polymorphisms (Mutirangura et al., 1993), or quantitative assays (Mansfield et al.,

The role of various parameters that may influence the performance of standard (uniplex) PCR has been discussed (Robertson & J., 1998). However, fewer publications discuss

The optimization of multiplex PCRs can pose several difficulties, including poor sensitivity or specificity and/or preferential amplification of certain specific targets (Polz & C. M., 1998). The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of primer dimers (Brownie et al., 1997). These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. Thus, the optimization of multiplex PCR should aim to minimize

Compatibility among the primers within the reaction mixture such that there is no interference, is of great technical importance. Primer selection followed simple rules (i) primer length of 18–24 bp or higher and (ii) a GC content of 35%–60%, thus having an annealing temperature of 55 °C-58 °C or higher. Longer primers (28-30 bp) allowed the reaction to be

Combining the primers in various mixtures and amplifying many loci simultaneously required alteration/optimization of some of the parameters of the reaction. When the

performed at a higher annealing temperature and yielded less unspecific products.

**Theoretical basis of multiplex PCR method: Critical Parameters** 

1993) and reverse transcription PCR (Crisan, 1994).

multiplex PCR (Henegariu et al., 1997).

or reduce such non-specific interactions.

enzyme under study (Maiden et al., 1998).

isolates together.

statistical software

**4.2.8 Multiplex PCR** 

multiplex reaction is performed for the first time, it is useful to add the primers in equimolar amounts. The results will suggest how the individual primer concentration and other parameters need to be changed. Special attention to primer design parameters such as homology of primers with their target nucleic acid sequences, their length, the GC content, and their concentration have to be considered (Robertson & J., 1998). Ideally, all the primer pairs in a multiplex PCR should enable similar amplification efficiencies for their respective target. This may be achieved through the utilization of primers with nearly identical optimum annealing temperatures and should not display significant homology either internally or to one another (Henegariu et al., 1997). Also, the extension rate of specific primer-target hybrids depends on the activity of the enzyme, availability of essential components such as deoxyribonucleoside triphosphates (dNTPs), and the nature of the target DNA. Thus, the majority of modifications to improve PCR performance have been directed towards the factors affecting annealing and/or extension rates. Therefore, in multiplex PCR, as more loci are simultaneously amplified, the pool of enzyme concentrations, PCR buffer constituents and nucleotides becomes a limiting factor and more time is necessary for the polymerase molecules to complete synthesis of all the products (Chamberlain et al., 1989).

Variation in concentrations of reaction components above those used in uniplex PCR probably reflects the competitive nature of the PCR process. The desired target DNA can be outcompeted by the more efficient amplification of other targets (including nonspecific products), leading to decreases in the efficiency of the amplification of the desired targets and hence sensitivity of the reaction (Raeymaekers, 1995).

Various authors recommend dimethyl sulfoxide (DMSO) and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5%–10% (vol/vol) (Innis & D.H., 1990). Also bovine serum albumin, or betaine, has been reported to be of benefit in multiplex PCRs (Jackson et al., 1996). The components may act to prevent the stalling of DNA polymerization, which can occur through the formation of secondary structures within regions of template DNA during the extension process (Hengen, 1997). Also it can act as destabilizing agents, reducing the melting temperature of GC-rich sequences, or as osmoprotectants, increasing the resistance of the polymerase to denaturation (Hengen, 1997).

A straightforward solution to difficulties encountered in the development of multiplex PCR has been the use of hot start PCR (Chou et al., 1992) and/or nested PCR (Zheng et al., 1995). The former often eliminates nonspecific reactions (particularly production of primer dimers) caused by primer annealing at low temperature (4 to 25°C) before commencement of thermocycling (Chou et al., 1992). The procedure has recently been made more practicable through the use of a nonmechanical hot start methodology which involves the use of a form of Taq polymerase, for example, Ampli Taq Gold (Roche Diagnostics), which is activated only if the reaction mixture is heated in first denaturation step at approximately 94°C for 10 min (Kebelmann-Betzing et al., 1998).

Nested PCR increases the sensitivity and specificity of the test through two independent rounds of amplification using two discrete primer sets. Although this adaptation is undoubtedly effective in most cases, it also considerably complicates the practical application of PCR. The second round of amplification delays results, increases the possibility of cross-contamination, and may complicate automation.

Laboratory Typing Methods for Diagnostic of

**5. Conclusions and perspectives** 

effective in the field.

250.

*Microbiol*, 80, 85-98.

*Scand*, 35(4), 349-54.

249, 82–90.

**6. References** 

Salmonella Strains, the "Old" Organism That Continued Challenges 365

and food isolates of *S. enterica* subsp. *enterica* in our region. This technique can be used as an

Overall the *Salmonella* demonstrate significant phenotypic diversity. Several phenotypic typing techniques have been developed and have been used successfully for decades. Over the years, serotype and phage type analyses have been particularly useful as evidenced by the success of the National *Salmonella* Surveillance System, and many other national surveillance projects throughout the world. However, these techniques have often been relegated to reference laboratories making rapid analysis by an individual laboratory difficult. An ideal typing method should fulfil the following six criteria: typeability, reproducibility, discriminatory power, and ease of interpretation, easy to use, and low cost. It is clear, that any method used currently for typing of *Salmonella* strains is an ideal method alone in terms of these criteria, but all methods exhibit benefits and also limitations. It is obvious that it is difficult to find a single method, which is most suitable for typing of *Salmonella* strains. As a consequence, the best discrimination has resulted from combinations of techniques, often a combination of phenotypic and genotypic techniques. At this time, major reference institutions rely on serotype analysis followed by PFGE as the gold standard for strain discrimination. PCR-based techniques, though, are more rapid and within a particular laboratory can be used as a primary screening tool for strain discrimination. Better standardization between laboratories will be required before any of the PCR techniques can become the method of choice. Additionally, validation in outbreak situations involving varied serotypes will be required to prove these techniques

Agarwal, A.; Makker, A. & Goel, SK. (2002). Application of the PCR technique for a rapid,

Ahmed, R.; Soule, G.; Demczuk, W. H.; Clark, C.; Khakhria, R.; Ratnam, S.; Marshall, S.; Ng,

Baggesen, DL. & Wegener, HC. (1994). Phage types of *Salmonella enterica ssp. enterica* serovar

Ballvora, A., Hesselbach, J., Niewo¨hner, J., Leister, D., Salamini, F. and Gebhardt, C. (1995)

gastroenteritis due to contaminated cheese. *J Clin Microbiol*, 38, 2403-6. Amavisit, P.; Markham, P. F.; Lightfoot, D.; Whithear, K. G. & Browning, G. F. (2001).

specific and sensitive detection of *Salmonella spp.* in foods. *Mol Cell Probes*, 16, 243–

L. K.; Woodward, D. L.; Johnson, W. M. & Rodgers, F. G. (2000). Epidemiologic typing of *Salmonella enterica* serotype *enteritidis* in a Canada-wide outbreak of

Molecular epidemiology of *Salmonella Heidelberg* in an equine hospital. *Vet* 

*typhimurium* isolated from production animals and humans in Denmark. *Acta Vet* 

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1. Molecular General Genetics

alternative method of standard serotyping in many clinical laboratories.
