Location: Warmwater Aquaculture Research UnitTitle: A Multiplex PCR for Species- and Virulence-specific Determination of Listeria monocytogenes) Author
Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/21/2007
Publication Date: 11/1/2007
Citation: Liu, D., Lawrence, M.L., Austin, F.W., Ainsworth, A.J. 2007. A Multiplex PCR for Species- and Virulence-specific Determination of Listeria monocytogenes. Journal of Microbiological Methods. 71(2):133-140. Interpretive Summary: Listeria monocytogenes is an opportunistic foodborne pathogen that encompasses a diversity of strains with varied virulence. Rapid determination of the species and potential virulence of Listeria will be useful for maintaining food safety. A multiplex PCR assay based on genes that distinguish among Listeria species, and among avirulent and virulent strains of Listeria monocytogenes was developed and tested. A assay was developed that quickly and accurately confirms L. monocytogenes species identity and virulence. This assay will allow rapid determination of the pathogenic potential of Listeria and play an important role in the the control and prevention of listeriosis.
Technical Abstract: Listeria monocytogenes internalin gene inlJ has been described previously for differentiation of virulent from avirulent strains. However, a recent report indicated that there exist some unusual lineage IIIB strains (e.g., serotype 7 strain R2-142) that possess no inlJ gene but have the capacity to cause mouse mortality via intraperitoneal inoculation. Therefore, a multiplex PCR incorporating inlA, inlC and inlJ gene primers was developed in this study for rapid speciation and virulence determination of L. monocytogenes. Although inlB gene was also assessed for species-specific recognition, it was not included in the multiplex PCR due to the negative reaction observed between the inlB primers and serotypes 4a–e strains. The species identity of the 36 L. monocytogenes strains under investigation was verified through the amplification of an 800 bp fragment with the inlA primers and the virulence of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ primers, respectively. Whereas L. monocytogenes pathogenic strains with capacity to cause mortality (showing relative virulence of 30–100%) in A/J mice via the intraperitoneal route were invariably detected by the inlC and/or inlJ primers, naturally non-pathogenic strains (showing relative virulence of 0%) were negative with these primers. While 8 of the 10 L. ivanovii strains reacted with the inlC primers, they could be effectively excluded as non-L. monocytogenes through their negative reactions with the inlA primers in the multiplex PCR. Thus, the use of the multiplex PCR targeting inlA, inlC and inlJ genes facilitates simultaneous confirmation of L. monocytogenes species identity and virulence.