Interlaboratory validation trial report on multiplex real-time PCR method for molecular serotyping and identification of the 30 major clonal complexes of Listeria monocytogenes circulating in food in Europe

The performance of a new method developed in 2021 by the European Union Reference Laboratory (EURL) for Listeria monocytogenes based on 12 multiplex real-time PCR, allowing the identification of the molecular serotype and the 30 major L. monocytogenes multilocus sequence typing clonal complexes (CC), was assessed through a European interlaboratory validation trial (ILVT). This ILVT was adapted from ISO standard 16140 part 6. Overall, 98 blinded pure strains of Listeria (monocytogenes or spp.), previously characterized by the EURL, were sent to 15 laboratories distributed in 11 countries. The molecular serotype had to be identified for 20 strains of the ILVT panel, while CC identification had to be performed for the whole panel. The results of the 12 multiplex real-time PCR were reproducible between the participating laboratories with high individual concordance values for molecular serotyping (100%) and CC identification (90.8%-100%) irrespective of DNA extraction protocols, PCR master mixes, and thermocycler diversity. Master mixes identified as incompatible with some of the multiplex real-time PCR were excluded from the method. The overall concordance of the results was sufficient for the method to be confidently applied in other laboratories involved in L. monocytogenes typing.IMPORTANCEThis interlaboratory validation trial, coordinated by the European Union Reference Laboratory for Listeria monocytogenes, was the final step to assess the performance of the multiplex real-time PCR method developed and published by B. F & eacute;lix, K. Capitaine, S. Te, A. Felten, et al. (Microbiol Spectr 11:e0395422, 2023, https://doi.org/10.1128/spectrum.03954-22). Different combinations of parameter settings were applied in 15 French and European laboratories involved in L. monocytogenes typing. It was a prerequisite to establish this new real-time PCR method as a standard for rapid molecular serotyping and clonal complex identification. The accuracy and reproducibility of the results obtained on the panel of 98 strains of L. monocytogenes sent to the participants proved that the real-time PCR was suitable for use in their conditions. Rapid screening of strains is therefore now possible, and the method provides a valuable tool for epidemiological investigations to identify food-associated strains during listeriosis outbreaks.