A Novel Sero-Specific-Gene Dependent Multiplex PCR Enhances the Discrimination of Major <i>Listeria monocytogenes</i> Serovars

<i>Listeria monocytogenes</i> is a foodborne bacterial pathogen distributed worldwide. Serotyping is extensively applied in the classification of <i>L. monocytogenes</i> and is crucial in the early stage of epidemiological tracing. Among the 13 serovars, 1/2a, 1/2b, 1/2c, and 4b are the ones most frequently isolated. Numerous PCR-based methods have been presented, however, their target genes, <i>lmo0737</i>, <i>ORF2110</i> and <i>ORF2819</i>, are prone to horizontal transfer or loss in certain strains, thus leading to incorrect serovar designation. Herein, we selected novel sero-specific genes and developed an improved multiplex PCR assay. The specificity of our assay was confirmed by the use of target and nontarget <i>Listeria</i> reference strains, as well as by the use of isolates yielding incorrect profiles in previous studies. Sensitivity tests indicated that a minimum of 5 ng of genomic DNA or approximately 3 × 10⁶ CFU of pure culture could be detected. Many collected isolates and genomes of global isolates were used to evaluate the specificity and reproducibility of our assay. The agreement between our assay and the agglutination test was 95%, and the one between our assay and the Doumith scheme was 97%. However, our assay overcomes the drawbacks of currently used PCR-based approaches by exhibiting 100% accuracy for certain strains and clones, for instance, ST782 within the hypervirulent CC2 and ST218 that were incorrectly assigned by the Doumith scheme. In conclusion, the developed assay herein will be a powerful tool and an alternative for the classification of <i>L. monocytogenes</i> strains in foodborne outbreak investigations and surveillance programs.