A novel multiplex PCR method for simultaneous identification of hypervirulent Listeria monocytogenes clonal complex 87 and CC88 strains in China

Listeria monocytogenes is an important foodborne pathogen worldwide, with 20-30% fatality rate in vulnerable persons. The hypervirulent L. monocytogenes clonal complex (CC) 87 strains have emerging both in food production environments and clinic cases. The objective of this study was to develop a multiplex PCR to simultaneously detect L. monocytogenes CC87 and CC88 strains based on pan-genome analysis. A novel multiplex PCR comprised of genes A6K41_13255 (specific for CC87 and 88), BCW_4260_01987 group_8135 (specific for CC88) and 02-1103_01073 group_5869 (specific for L. monocytogenes) were designed. The specificity of this multiplex PCR was robust verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR for CC87 and CC88 were 1.7 x 10(4) cfu/mL and 2.1 x 10(4) cfu/mL, respectively. This multiplex PCR could accurately detect CC87 and CC88 strains with the interference of different ratios of L. monocytogenes CC8, CC9, CC121, CC155, and L. innocua strains. Furthermore, this multiplex PCR method could successfully detect 1.9 x 10(4) cfu/mL of L. monocytogenes CC87 and 1.7 x 10(4) cfu/mL CC88 strains in artificially contaminated milk after 9 h enrichment, respectively. In addition, this multiplex PCR could accurately detect CC87 isolates in food samples within 48 h, which was faster than the routine MLST analysis. In conclusion, this novel multiplex PCR offers a promising approach for accurate, inexpensive, and rapid detection of L. monocytogenes CC87 and CC88 strains simultaneously, which could apply to surveillance the prevalence of CC87 and CC88 strains in both food and food production environments and to evaluate the effect of disinfection measures for controlling the persistent L. monocytogenes contamination.