A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes

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.