Dulplex PCR Identification And Molecular Typing Of Listeria Monocytogenes In Frozen Products

Listeria monocytogenes(LMO) is a sort of cold bacilli which can survive at 1-4℃,cause meningitis, septicemia in human, and abortion in both human and livestock, LMO can pollute almost all of the food and is classified as pathogenic bacteria from food source in 1980s and one of the four main pathogenic bacteria in 1990s.The freshwater life resources are abundant in our country, and the export of frozen freshwater products is rising year by year. To ensure the quality of exported frozen freshwater fish products, LMO detection is one of the compulsory items. To detect LMO, the method SN0184-93 needs 5-14d; the method mini-VIDAS equipment needs special reagents and costs a lot. Thus a rapid and cheap method is required for the customs inspection.Besides, in order to control the pollution of LMO effectively, it is equally important to research the sources and transmission of the pollution. The genotyping of LMO would provide the corresponging evidence. Therefore, in this thesis, we studied both the rapid detection and genotyping methods for LMO. The results were summarized as follows.1. iap and hly genes of LMO were used as target genes to establish a dulplex PCR to identify LMO in fish products;2. Specificity, sensitivity, interference and mimical contamination experiments were conducted to illustrate the specificity, sensitivity and stability of the dulplex PCR. The results indicated that the method had a high stability, specificity and sensitivity, and could resist the interruption by common pathogenic bacteria;3. 120 samples of the frozen fish products were detected by the dulplex PCR. The results of the dulplex PCR, SN0184-93 method and mini-VIDAS technique were in agreement. However, this dulplex PCR was faster than SN0184-93 method, cheaper than mini-VIDAS technique, and more pratical for detection of a large amount of samples at customs.4. The 16S～23S rRNA intergenic spacer regions of 47 LMO isolates from fish products in recent years in Hubei province were investigated for the genotyping pattern to provide data for the pollution sources and transmission model of LMO in fish products in Hubei.5. The 16S～23S rRNA intergenic spacer regions of all the islates were 93%～100% similarity to that of LMO reference strain 4b F2365 and could be classified 3 main types and 16 subtypes. The diversities of the isolates were associated with the types and origins of the samples.