An improved real-time quantitative PCR assay for the differentiation of major Listeria monocytogenes serovars.

Listeria monocytogenes, the causative agent of listeriosis, is one of the most important foodborne pathogens with a significant impact on public health worldwide. Among the subtyping methods used for epidemiological purposes, serotyping is extensively utilized in the early stage of source tracking for L. monocytogenes infections. Many PCR-based methods have been established based on lineage-specific genes as sero-specific markers. However, these genetic elements are prone to horizontal transfer, thus leading to incorrect serovar designation. To overcome the drawbacks of currently used methods, we developed an improved real-time quantitative PCR method for differentiating the predominant serovars of L. monocytogenes. The specificity of this method was measured using target, non-target Listeria, and other foodborne strains. A minimum of 0.25 ng of genomic DNA or less than 10 bacterial cells per 2.5 g of milk powder sample could be detected, suggesting the robustness of our assay. The specificity and reproducibility of the method were also confirmed via the use of 81 collected foodborne isolates, which indicated that 1/2c strains are highly abundant (57 %). Moreover, our in silico analysis revealed a unique serovar pattern among the foodborne isolates from North America: 54.8 % serovar 1/2a and 41.3 % serovars 1/2b and 4b. Lastly, we revealed novel underlying mechanisms that lead to incorrect serovar designations via a commonly employed PCR method. In general, our assay exhibited greater reliability and could be a powerful tool for the classification and epidemiological monitoring of L. monocytogenes.