沙门氏菌流行病学调查及生物被膜形成能力研究

发布时间：2018-05-09 11:04

本文选题：沙门氏菌 + 多位点序列分析　；参考：《吉林农业大学》2012年硕士论文

【摘要】：沙门氏菌(Salmonella)是一种重要的人畜共患型食源性致病菌,在全世界范围内广泛流行,严重威胁人类健康,并给畜牧业及食品加工业带来巨大的经济损失。沙门氏菌在食品加工业的仪器设备中常以生物被膜的形式存在,给食品生产过程中的消毒灭菌工作带来很大难度。因此,对沙门氏菌的流行病学进行调查,以及研究野生分离株沙门氏菌的生物被膜形成能力具有重要意义。本研究通过在猪肉食品生产加工的不同阶段采集样品,先采用我国沙门氏菌检测的国家标准方法中规定的选择性增菌液和选择性平板分离得到疑似沙门氏菌后,再采用PCR方法鉴定疑似菌株。共采集得到15株沙门氏菌。通过研究野生分离菌株的来源情况,得出结论认为猪肉食品中沙门氏菌的污染主要来自于后期屠宰和加工阶段,养殖场中生猪的带菌率很低。将分离得到的沙门氏菌采用多位点序列分析(MLST)方法进行分子分型,然后与血清学分型结果进行对比。发现MLST分型方法对沙门氏菌的分型能力要优于血清学分型方法。本研究通过结晶紫染色法结合细胞微孔培养板,定量研究了野生分离菌株在模式培养基胰蛋白胨大豆肉汤(TSB)中的生物被膜的形成能力,并且研究了其中两株具有强生物被膜形成能力的菌株在不同培养条件下生物被膜形成能力的变化。发现高渗溶液对沙门氏菌生物被膜的形成有抑制作用,并且氯化钠(NaCl)溶液的抑制作用强于蔗糖溶液。本研究通过将沙门氏菌黏附素蛋白基因misL包含其启动子及转录调控序列与pUC18质粒构建重组质粒,并转入模式菌株大肠杆菌MG1655和AAEC189中,分析该基因对细菌形成生物被膜能力的影响,发现该基因对细菌的生物被膜形成能力具有促进作用。本研究构建了pET-28a-MisL重组表达载体,转入大肠杆菌BL21中,利用原核表达系统成功表达出沙门氏菌黏附素蛋白MisL。为深入研究该蛋白的功能奠定了基础。
[Abstract]:Salmonellais is an important zoonotic foodborne pathogen, which is widespread in the world. It is a serious threat to human health and brings great economic losses to animal husbandry and food processing industry. Salmonella often exists in the form of biofilm in the equipment of food processing industry, which makes it difficult to disinfect and sterilize food. Therefore, it is of great significance to investigate the epidemiology of Salmonella and to study the biofilm forming ability of wild isolates of Salmonella. In this study, samples were collected at different stages of pork food production and processing, and then the suspected salmonella was isolated by selective bacteriostasis and selective plate, which was stipulated in the national standard method for the detection of salmonella in China. The suspected strain was identified by PCR method. A total of 15 Salmonella strains were collected. By studying the source of wild isolated strains, it is concluded that the contamination of salmonella in pork food mainly comes from the later stage of slaughter and processing, and the rate of carrying bacteria in live pigs in breeding farm is very low. The isolated Salmonella was classified by multilocus sequence analysis (MLST) and compared with serological typing. It was found that the MLST typing method was superior to the serological typing method in the typing of Salmonella. In this study, the biofilm forming ability of wild isolated strains in the model medium of tryptone soybean broth (TSBs) was quantitatively studied by means of crystal violet staining and cell micropore culture plate. The changes of biofilm formation ability of two strains with strong biofilm formation ability under different culture conditions were studied. It was found that hypertonic solution inhibited the formation of salmonella biofilm, and the inhibitory effect of sodium chloride solution was stronger than that of sucrose solution. In this study, recombinant plasmids were constructed from Salmonella adhesion protein gene misL containing its promoter and transcriptional regulatory sequence and pUC18 plasmid. The recombinant plasmids were transformed into Escherichia coli MG1655 and AAEC189. By analyzing the effect of the gene on the biofilm formation ability of bacteria, it was found that the gene could promote the biofilm formation ability of bacteria. In this study, the recombinant pET-28a-MisL expression vector was constructed and transferred into Escherichia coli BL21, and the Salmonella adhesion protein Misl was successfully expressed by prokaryotic expression system. It lays a foundation for further study of the function of the protein.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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