【作者】 张晓峰；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 食品科学， 2002， 硕士

【摘要】 单核细胞增多性李斯特菌(Listeria monocytogenes,Lm)是人畜共患病李斯特菌病的病原菌，为一革兰氏阳性小杆菌，可以在多种细胞内生存和繁殖。此菌有如下特性：1)在自然界中普遍存在；2)易感动物多，除人外还可感染多种哺乳动物，以牛、羊、犬最易感染；3)可在低温下生长(4-8℃)，对热有一定抵抗力。此病原菌主要经消化道感染，也可经眼结膜及破损皮肤接触感染。临床上有脑膜炎、败血症等表现形式，死亡率30％左右。孕妇或孕畜还可发生流产、早产、死胎等。近二十年来，世界各地食源性人的李斯特菌病日渐增多，流行病学调查结果表明，该病主要是由于人食用了污染Lm的食品而引起。但是由于该菌在多种动物和环境中广泛存在，在食品生产和加工过程中可通过多种途径进入成品食品中，对消费者健康构成潜在威胁。因此，建立Lm的快速鉴定和分子分型方法对于开展食品(特别是消毒牛奶)加工过程中Lm污染来源和途径的调查和控制，提高食品的微生物卫生质量具有重要意义。 本项目首先对FDA法、NGFIS法和国标法三种李斯特菌选择性增菌肉汤进行对比试验，结果表明：改良的NGFIS法对环境样品李斯特菌的分离率最高，FDA法对消毒牛奶中李斯特菌的检出率最高。本试验以具有李斯特菌种、属特异性的iap基因和单核细胞增多性李斯特菌特异的溶血素基因(hlyA)作为靶目标，分别设计单一PCR、双重PCR和三重PCR来鉴定李斯特菌，并比较了三种PCR方法的特异性。结果表明：建立的单一PCR只能鉴定Lm，而双重PCR和三重PCR方法对于李斯特菌属和Lm标准株均具有良好的特异性，但是双重PCR不能鉴定部分Lm食品分离株，而三重PCR不仅可用于李斯特菌属细菌的特异性鉴定还可用于混合培养物中Lm的特异性鉴别。本研究还建立了用于追踪牛奶加工过程中Lm污染源和污染途径的RAPD分型方法，为了提高RAPD分型法的重复性和分辨力，本研究采用两条随机引物分别进行RAPD，综合分析结果的策略。结果表明：建立的RAPD方法对17株不同来源的Lm分离株均具有良好的分型性和分辨力，并对L.innocua亦有良好的分辨力和可分型性。 总之，本试验所建立的三重PCR和RAPD分型方法为进一步开展牛奶生产加工过程中李斯特菌污染的监控奠定了基础。更多还原

【Abstract】 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.更多还原