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 × 10⁶ CFU/mL for bacterial suspension. Furthermore, pork meat artificially contaminated with serovar 4h L. monocytogenes in a concentration of 1.8 × 10³–1.8 × 10⁰ 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.

李斯特菌 （单核细胞增生李斯特菌）是一种普遍存在的食源性病原体，包含14种血清型，其中4h血清型是最近报道的新型血清型。血清素4h单核细胞增生李斯特菌属于杂合子系II的人表现出高毒力特征。常规生化测试和广泛使用的基于PCR的血清分组方案无法区分4h血清型。在这项研究中，我们开发了一种新的多重PCR检测法，可快速检测4h血清型单核细胞增生李斯特菌。基于靶基因LMxysn_1095的三对引物lmo1083和 smcL，被设计。多重PCR结果表明，可以从所有测试菌株中特异性鉴定出血清型4h菌株，包括单核细胞增生李斯特菌 血清型， 李斯特菌spp。和其他物种。多重PCR的检测限为基因组DNA为291 fg /μL ，细菌悬液为5.5×10 ⁶ CFU / mL。此外，猪肉被人血清型4小时人工污染单核细胞增生李斯特菌在10–16小时内成功检测到浓度为1.8×10 ³ –1.8×10 ⁰ CFU / 10 g的CFU。这些结果表明，具有高特异性和高灵敏度的多重PCR方法可用于快速检测肝癌。单核细胞增生李斯特菌 血清型4h菌株。