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Rapid Detection of Hypervirulent Serovar 4h Listeria monocytogenes by Multiplex PCR
Youwei Feng1,2,3, Hao Yao1,2,3, Sisi Chen1,2,3
1Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, China.
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 × 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.
Insights
A new multiplex PCR assay rapidly detects the novel, hypervirulent Listeria monocytogenes serovar 4h. This method accurately identifies serovar 4h strains in food samples, improving food safety.
Area of Science:
- Food Microbiology
- Molecular Biology
- Pathogen Detection
Background:
- Listeria monocytogenes is a common foodborne pathogen.
- A novel, hypervirulent serovar 4h has been identified.
- Existing detection methods cannot distinguish serovar 4h.
Purpose of the Study:
- To develop a rapid multiplex PCR assay for Listeria monocytogenes serovar 4h detection.
- To validate the assay's specificity and sensitivity.
Main Methods:
- Designed three primer pairs targeting specific genes (LMxysn_1095, lmo1083, smcL).
- Tested the multiplex PCR assay against various Listeria strains and other species.
- Determined the assay's limit of detection using genomic DNA and bacterial suspensions.
- Validated the assay on artificially contaminated pork meat samples.
Main Results:
- The multiplex PCR assay specifically identified Listeria monocytogenes serovar 4h.
- The assay demonstrated high specificity, distinguishing serovar 4h from other Listeria serovars and species.
- Achieved low detection limits for genomic DNA (291 fg/μL) and bacterial suspensions (5.5 × 10^6 CFU/mL).
- Successfully detected serovar 4h in contaminated pork within 10-16 hours.
Conclusions:
- The developed multiplex PCR assay is a specific and sensitive tool.
- This assay enables rapid and accurate detection of Listeria monocytogenes serovar 4h.
- The method is applicable for routine food safety monitoring and surveillance.
