Rapid detection of Listeria monocytogenes sequence type 121 strains using a novel multiplex PCR assay

Abstract

Listeria monocytogenes is a hazardous foodborne causative agent, commonly found in foods and food production environments. The persistent contamination of L. monocytogenes sequence type (ST) 121 in food production environments is one of the challenging issues. The objective of this study was to develop a multiplex PCR for identification of L. monocytogenes ST121 strains. A total of 50 specific candidate genes of ST121 were screened by the comparative genomics approach. A novel multiplex PCR for ST121 identification, which comprised of ST121 strain-specific primers (LM6179_0609 and LM6179_0171) and L. monocytogenes-specific primers (prfA), was designed. The specificity of this multiplex PCR was robustly verified by other STs of L. monocytogenes and non-L. monocytogenes strains. The detection limit of the multiplex PCR was 253 fg/μL genomic DNA. This multiplex PCR could avoid the interference of L. monocytogenes ST9, ST8, ST87, and ST155 in different ratios, which are common contaminants in food items. In addition, this multiplex PCR method could successfully detect 2.5 × 104–2.5 × 100 cfu/10 mL of L. monocytogenes ST121 in artificially contaminated milk after 4–12 h enrichment. These results suggested that the newly developed multiplex PCR in this study offers a promising approach for specific, rapid, and accurate detection of L. monocytogenes ST121 strains.