Triplex PCR Identification And Molecular Typing Of Listeria Monocytogenes

Listeria monocytogenes, the causative agent of the zoonotic disease listeriosis, is a small Gram-positive rod that can proliferate in a number of mammalian cells including macrophages. The bacterium is widely distributed in the environments and can survive at refrigeration temperatures. It has been considered as a food-borne pathogen since the early 1980s, thus becoming a major concern for public health authorities and food industries. L. monocytogenes can present in raw foodstuff and processing environments. Thus, methods for rapid identification and typing are need to trace the source of contamination along the food chain.In this study, a triplex PCR procedure based on genes iap and hly of L. monocytogenes was used to investigate the status of its contamination along the major food-processing environments. With triplex PCR, all L. monocytogenes strains had a short iap fragment of about 700bp in addition to the 402bp hly fragment, and other listerial spp had a long iap fragment about 1250bp. The triplex PCR procedure detected as low as 3.2X101 cfu/ml of listerial cells even in the presence of L. innocua (108cfu/ml). It could detect 1.4x102 listerial cells per ml directly from milk artificially contaminated with the bacterium. Lower levels of L. monocytogenes cells in milk (1.45X101 - 1.45x10?cfu/ml) could be detectable if the inoculated milk samples were cultured for 3-6 hrs. Presence of L. innocua or other bacteria in broth or milk did not interfere with specific and sensitive detection. Nor did other non-listerial species such as E. coli or Staph. aureus affect the detection.We approached two methods of molecular typing for L. monocytogenes. With actA-based DNA sequence typing procedure, a pair of primers was designed for PCR amplification of a 820bp DNAfragment. The products from 21 L. monocytogenes strains were submitted directly for sequencing. The sequences of 506 bp within the amplified fragments were aligned by Clustal V for phylogenetic analysis. The strains were also typed by PFGE in parallel. All the tested L. monocytogenes strains were grouped into five clusters by both methods. Clusters A, D and E were related to milk including raw milk, homogenized milk and pasteurized milk, while the strains isolated from sewages and other processing environments were in an independent cluster. These results indicate that L.monocytogenes strains contaminating the pasteurized milk products most probably came from raw milk but not from processing environments.In conclusion, the present studies laid the technological foundation for rapid and sensitive molecular identification of L. monocytogenes from foods as well as for tracking of sources of its contamination on the food products during production and processing.