Elsevier

Journal of Food Protection

Volume 84, Issue 2, February 2021, Pages 333-344
Journal of Food Protection

RESEARCH PAPERS
Development and Validation of a Quantitative PCR Method for Species Verification and Serogroup Determination of Listeria monocytogenes Isolates

https://doi.org/10.4315/JFP-20-178Get rights and content
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open access

HIGHLIGHTS

  • The new qPCR method identified major members of Listeria spp. (excluding L. grayii) and members of the Lm species.

  • The new qPCR-based method identified major serogroups of Lm.

  • The method was at least equal to the BAM method when identifying Lm species.

  • Molecular serogroup determination was more accurate than the seroagglutination assay.

ABSTRACT

Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information.

Keywords

Listeria monocytogenes
qPCR characterization
Serotyping

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Present address: Office of Food Safety, Center for Food Safety and Applied Nutrition, College Park, MD 20740, USA.