Multiple-locus variable-number tandem-repeat analysis as a tool for subtyping Listeria monocytogenes strains

Listeria monocytogenes, like many other food-borne bacteria, has certain strains that are commonly linked to outbreaks. Due to the relatively low numbers of affected individuals, outbreaks of L. monocytogenes can be difficult to detect. The current technique of molecular subtyping in PulseNet laboratories to identify genetically similar strains is pulsed-field gel electrophoresis (PFGE). While PFGE is state-of-the-art, interlaboratory comparisons are difficult because the results are highly susceptible to discrepancies due to even minor variations in experimental conditions and the subjectivity of band marking. This research was aimed at the development of a multiple-locus variable-number tandem-repeat analysis (MLVA) that can be implemented in PulseNet laboratories to replace or complement existing protocols. MLVA has proven to be a rapid and highly discriminatory tool for subtyping many bacteria. In this study, a novel MLVA method for L. monocytogenes strains was developed utilizing eight loci multiplexed into two PCRs. The PCR products were separated by capillary gel electrophoresis for high throughput and accurate sizing, and the fragment sizes were analyzed and clustered based on the number of repeats. When tested against a panel of 193 epidemiologically linked and nonlinked isolates, this MLVA for L. monocytogenes strains demonstrates strong epidemiological concordance. Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to perform, rapid, and reliable, it is well suited to interlaboratory comparisons during epidemiological investigations of food-borne illness.