**4.2.7 MLST (multilocus sequence typing)**

A recently developed methodology (Maiden et al., 1998) called multilocus sequence typing (MLST) may provide an ideal balance of high discriminatory power and a powerful data analysis capability requiring minimal human input. Multilocus sequence typing (MLST) is a molecular typing strategy that compares DNA sequences from portions of housekeeping or virulence genes and/or rRNA sequences which varies due to mutation or recombination events (Maiden et al., 1998). Nucleotide differences in the individual genes

Laboratory Typing Methods for Diagnostic of

(Chamberlain et al., 1989).

and hence sensitivity of the reaction (Raeymaekers, 1995).

resistance of the polymerase to denaturation (Hengen, 1997).

possibility of cross-contamination, and may complicate automation.

min (Kebelmann-Betzing et al., 1998).

Salmonella Strains, the "Old" Organism That Continued Challenges 363

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

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

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

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

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

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

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 isolates together.

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 statistical software
