Molecular Identification And Typing Of Listeria Monocytogenes And Their Applications In Producing Safer Milk

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. It has been considered as one of the most important food-borne pathogens for a number of reasons. 1) The bacterium has wide distribution in thenature;2) It can infect a variety of mammalian species including human being;3) It can grow at refrigeration temperatures from 4-8C,survive freezing and is relatively resistant to heat. L. monocytogenes could infect the hosts through the gastrointestinal tract or injured skin. The common syndromes of listeriosis are encephalo-meningitis,septicemia,etc,with the mortality as high as 30%. If women and female animals are affected during pregnancy,there would be premature labors or delivery of stillborn. Epidemiological studies have implicated the involvement of foods as vehicles for human listeriosis. Apparently,L. monocytogenes is a pathogen of great concern to the food industry as well as to the consumers because of the diversity of its presence in the animals and food-processing environments. Therefore,rapid and convenient methods for identification and typing of L. monocytogens isolates from foods and processing environments are crucial to identify the principal contamination points along the processing lines and establish the critical control points thereafter.We first compared the suitability of different enrichment methods for L. monocytogens and found that the FDA method is suitable for pasteurized milk,while the NGIFS method suitable for environmental samples. In this study,single,duplex and triplex PCR techniques were examined for identification of reference strains and field isolates of L. monocytogenes. Primers targeting the iap and hly genes specific for listerial species and L. monocytogens were designed. Single PCR could detect L monocytogene only,while duplex PCR and triplex PCR were specific to all listerial reference strains. However,triplex PCR had better discriminative power than duplex PCR by identifying a smaller iap gene fragment (700 bp) specific to L. monocytogenes than that at 1200 bp general to listerial species. Triplex PCR could thus be used todifferentiate L monocytogenes from other listerial species in mixed suspensions. We also establish a RAPD (random amplified polymorphic DNA) protocol to type L monocytogenes isolates from foods. The profiles of 17 L. monocytogens isolates from different sources showed good discriminatory power of the typing method. It could also be used to type Listeria innocua isolated from different steps of the milk processing line.In conclusion,the present studies on triplex PCR identification and RAPD typing have provided good means for further work on the control of the listerial contamination in pasteurized milk.