Rapid Detection of Hypervirulent Serovar 4h Listeria monocytogenes by Multiplex PCR

Abstract

Listeria monocytogenes (L. monocytogenes) is a ubiquitous foodborne pathogen that comprises 14 serotypes, of which serovar 4h is a novel serotype recently reported. Serovar 4h L. monocytogenes belonging to hybrid sub-lineage II exhibit hypervirulent features. Conventional biochemical tests and widely used PCR-based serogrouping schemes could not distinguish serovar 4h strains. In this study, we developed a new multiplex PCR assay for rapid detection of serotype 4h L. monocytogenes. Three primer pairs based on the target genes, LMxysn_1095, lmo1083, and smcL, were designed. The multiplex PCR results showed that serovar 4h strains could be specifically identified from all tested strains, including various L. monocytogenes serovars, Listeria spp., and other species. The detection limits of the multiplex PCR were 291 fg/µL for genomic DNA and 5.5 × 10 6 CFU/mL for bacterial suspension. Furthermore, pork meat artificially contaminated with serovar 4h L. monocytogenes in a concentration of 1.8 × 10 3-1.8 × 10 0 CFU/10 g were successfully detected within 10-16 h. These results demonstrate that the multiplex PCR with high specificity and sensitivity is applicable for the rapid detection of L. monocytogenes serotype 4h strains.

Application of Whole Genome Sequencing to Aid in Deciphering the Persistence Potential of Listeria monocytogenes in Food Production Environments

“…L. monocytogenes is a heterogenous group of bacteria. This bacterium can be differentiated into 14 different serotypes, with serovar 4 h being recently described [ 16 , 17 ]. These serotypes are divided into four lineages (denoted as I-IV) [ 18 ].…”

Assessment of the Prevalence and Drug Susceptibility of Listeria monocytogenes Strains Isolated from Various Types of Meat

“…Because of the genetically heterogeneous nature of L. monocytogenes, serotyping as the primary characterization in epidemiological surveillance has been widely accepted [46]. Feng et al [47] developed a new multiplex PCR assay for rapid detection of new serotype 4h L. monocytogenes. These results demonstrate that the multiplex PCR with high specificity and sensitivity is applicable for the rapid detection of L. monocytogenes serotypes.…”

Landscape of Stress Response and Virulence Genes Among Listeria monocytogenes Strains

“…It encompasses the smcL gene coding for sphingomyelinase (involved in phagosome disruption) and 10 genes coding for proteins of the internalin family ( Domínguez-Bernal et al, 2006 ). However, recent studies showed that L. monocytogenes isolates, belonging to a new sub-lineage of the major lineage II with hypervirulent features (SL626/CC33, serovar 4 h), contain a truncated LIPI-2, i.e., carrying only smcL and two internalins genes, namely i-inlF and i-inlE , likely acquired by transposon-mediated horizontal gene transfer from L. ivanovii ( Yin et al, 2019 ; Feng et al, 2020 ).…”