Listeria monocytogenes (Lm) is one of the leading causes of death due to foodborne illness, affecting the elderly, pregnant women, neonates and the immune compromised. Serologically, Lm can be classified into 13 serotypes, though only four are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks was observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel qPCR method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species, and the second for Lm molecular serogrouping. This method was compared with the FDA Bacteriological Analytical Manual (BAM) method for Lm and the sero-agglutination method, using a 208-strain panel. Comparison of the genus/species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), as compared to the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the sero-agglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using sero-agglutination. The qPCR could not identify lineages III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III/IV strains. The qPCR method performed genus identification for Lm, L. innocua, L.  welshimeri, L. ivanovii, and L. seeligeri. Additionally, the method performed species identification for Lm, and serogrouped Lm into six molecular serogroups, 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling WGS analysis based on broad phylogeny, independent of other information.

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