【作者】 骆笑凯；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2009， 硕士

【摘要】 李斯特菌属包括七个种,其中单核细胞增多性李斯特菌(Listeria monocytogenes,以下简称Lm或单增李斯特菌)是能引起人畜共患病的食源性致病菌。单增李斯特菌为短小的革兰氏阳性无芽胞杆菌,能引起人的败血症、脑炎、脑膜炎和胃肠炎,在主要食源性致病菌中引起的死亡率最高。该菌在食品加工过程中可以通过多种途径污染食品,对消费者健康构成潜在威胁。在食品中发现任何一种李斯特菌都被认为是卫生状况不良。因此,建立敏感、特异的方法用于食品中李斯特菌属细菌的快速检测,对控制和提高食品的卫生质量具有重要意义。本实验室研制了李斯特菌种别检测试剂盒,通过多重PCR快速鉴别李斯特菌属细菌并区分单增李斯特菌毒力强弱。本研究优化该试剂盒后进行冻干,同时每个月进行特异性,敏感性及稳定性试验,结果表明冻干试剂盒具有良好的特异性,只有李斯特菌属细菌可以扩增出相应条带,而马链球菌兽疫亚种、金黄色葡萄球菌、多杀性巴氏杆菌、猪胸膜肺炎放线杆菌、支气管败血博代氏菌、大肠杆菌O157：H7等不能扩增出相应的条带。冻干试剂盒对单增李斯特菌的最低检测下限2.58×103CFU,而未冻干试剂盒的检测下限为102CFU。将冻干试剂盒用于人工污染牛奶的模拟试验,当牛奶中含有约1.43×102CFU/ml的Lm时可以通过直接预增菌6h检出。冻干试剂盒在-20℃保存八个月后依然保持稳定。本试验选择单增李斯特菌胞内感染相关毒力基因plcB和mpl为研究对象,设计其编码基因的特异性引物,PCR扩增目的片段后插入原核表达载体pET30a,重组质粒分别命名为pET30a-mpl和pET30a-plcB。重组质粒转化大肠杆菌BL21感受态细胞,筛选阳性克隆,诱导表达并分析其产物。结果表明,plcB和mpl基因片段获得了融合表达。可溶性分析发现该蛋白以包涵体形式存在。以纯化的Mpl和PlcB蛋白作为包被抗原,鼠源抗单增李斯特菌全菌多抗作为一抗,ELISA和免疫印迹试验结果显示PlcB蛋白与全菌多抗具有良好的免疫反应性,而Mpl不能与抗Lm阳性血清反应。本研究为开发Lm PCR快速检测冻干试剂盒、建立基于PlcB蛋白抗体的免疫磁珠集菌方法提供了基础。更多还原

【Abstract】 The genus Listeria consists of seven species, one of which named Listeria monocytogenes, short for Lm, is a zoonosis pathogen. Listeria monocytogenes is a Gram-positive, rod-shaped, facultative anaerobic, non-spore-forming bacterium. It can cause septicaemia, encephalitis, meningitis and gastroenteritis in humans. Listeia monocytogenes was reported to cause the highest mortality among the food-borne pathogens.Lm can contaminate food through many routes along the processing line, thus becoming a potential hazard to the consumers. Any Listeia spp. found in foods is a index of disqualification. Thus, it is important to set up a rapid, sensitive, and differential detection method to supervise and elevate the hygienic quality of food.Our laboratory estabished a PCR Identification Kit for Listeria species. In This study the PCR components were determined after the PCR amplification conditions were serially optimized. Then the PCR Identification Kit was freeze dried and stored at -20℃. A series of tests were conducted to assess its specificity, sensitivity, stability and reproducibility of freeze-dried PCR component. The results showed that DNA products was amplified from Listeria species, while Streptococcus equi subsp, zooepidemicus, Staphylococcus aureus, Pasteurellamul tocida, Actnobacillus suis, Bordetella bronchiseptica,E. coli etc. were not. 2.58×103 of Lm was the lowest number that could be detected by freeze dried Kit while102 CFU for unfreeze-dried Kit.Lower level of L.monocytogenes cell in milk(1.43×102 CFU/ml) could be detectable after cultured directly at 30℃for 6hrs. The PCR component still worked efficiently for the amplification after being stored at -20℃for eight months.In this study, we selected two of genes which mediating listerial intracellular infection cycle, PlcB and Mpl, as the targets based. The gene fragments of plcB and mpl were amplified from Listeia monocytogenes genome and were inserted between EcoR I and Xho I sites of prokaryotic expression vector pET30a to construct the recombinant plasmids named pET30a-mpl and pET30a-plcB. Then they were transferred into E.coli BL21. Expression of the recombinant proteins were induced by IPTG and analysed by SDS-PAGE. Prokaryotic expression product PlcB reacted with the antibody of the whole Listeria monocytogenes from mice, but not Mpl. The result indicated PlcB had immunoreactivity.Our study provides some foundations for development of a freeze-dried PCR Identification Kit for Listeria species and enrichment of listerial cells using specific antibodies based on PlcB for quick identification of Listeria monocytogenes.更多还原