Development of a multiplex SNP-typing method to identify the four epidemic clones of Listeria monocytogenes

The concept of epidemic clone (EC) is used to describe closely related strains of L.monocytogenes belonging to geographically and temporally distinct outbreaks. Currently, four ECs have been identified: ECI, ECII, ECIII, and ECIV (also ECIa). The current classification of ECs is based on results from different subtyping techniques such as RFLP analysis, hybridization assays, PFGE and ribotyping. Recently, a multiplex PCR able to identify ECI, ECII and ECIII was developed. In addition, sequence-based methods, such as multivirulence-locus sequence typing, were able to further subtype EC strains and also to highlight potentially informative single nucleotide polymorphisms (SNPs). The aim of the present study was to develop an alternative method for the identification of all four ECs by developing a SNP-typing method based on minisequencing.This technique simultaneously analyses several SNPs based on a Primer-Extension Reaction (PER). Extension primers are designed immediately adjacent to the SNPs and extended by a single [F]ddNTP. Results are then visualized with a genetic analyzer as specific patterns. Six SNPs, yielding a specific pattern for each EC, were selected. These SNPs were located on 4 virulence genes (inlA,inlJ,inlB andsrtA). Gene fragments were preliminary amplified with a multiplex PCR and then used as template for the PER reaction. Analyzed samples were 39 ECs strains, 25 strains from the ILSI Diversity Subset and 22 non ECstrains. All ECs gave a clone-specific pattern. Moreover, 9 strains not previously classified as ECs showed the same profile as ECI, ECII and ECIV. These findings were confirmed with other biomolecular tests (e.g. ECs-specific PCR) and by considering available epidemiological data for these strains. The proposed SNP-typing assay is potentially high-throughput and amenable to automation, allowing a rapid identification of all four ECs and can therefore be used as a screening method in the analysis ofL. monocytogenes strains.