Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe.

Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans.