食源性单增李斯特菌数值鉴定系统及其遗传多样性研究

发布时间：2018-02-10 01:16

  本文关键词： 单核细胞增生李斯特菌 数值鉴定系统 污染调查 遗传多样性 蛋白质组　出处：《华南理工大学》2016年博士论文　论文类型：学位论文

【摘要】：李斯特菌属(Listeria spp.)为革兰氏阳性菌,兼性厌氧。目前,该属包含了16个种和2个亚种,其中6种李斯特菌与食品相关。单增细胞增生李斯特菌(Listeria monocytogenes)是李斯特菌属最具危害的一种食源性致病菌,它对人有致病性,能引起人和动物患脑膜炎、败血症、流产和局部的脓性损伤,造成孕妇流产、死胎等疾病,发病者死亡率可达20%~30%,被世界卫生组织定义为全球四大食源性病原菌之一。本论文基于细菌数值鉴定法,结合计算机辅助技术,自主研发了食源性李斯特菌数值鉴定系统(Numide-Listeria v1.0)。在此基础上,系统调查全国不同地区不同类型的食品中单增李斯特菌的分布情况,在验证本鉴定系统的同时,进一步研究了单增李斯特菌分离株的遗传多样性、血清型、毒力现状和耐药性等遗传特性。此外,本论文还通过iTRAQ蛋白组学技术建立了李斯特菌全菌蛋白数据库,为寻找种间差异物质,进一步完善鉴定系统提供了依据。本论文的主要内容和研究结果如下:1.利用李斯特菌种间生化反应的差异,筛选了11种不同于目前市面上同等产品的生化试验组合。对本实验室前期分离到近2000株李斯特菌进行相关实验,获得各生化反应阳性率,建立了这11种生化反应阳性率数据库。基于细菌数值鉴定法的原理,运用C++语言等计算机技术编写相关软件,初步构建了食源性李斯特菌数值鉴定系统(Numide-Listeria v1.0),可有效区分六种食源性李斯特菌。2.对1400份零售食品进行了全国单增李斯特菌污染调查研究,共检出232份阳性样品,平均污染率16.6%,平均污染水平15.72 MPN/g。七大类食品的检出率从高到低依次为:速冻食品(44.9%)食用菌(23.2%)肉与肉制品(20.9%)即食食品(8.2%)水产品(6.4%)蔬菜(5.7%)乳制品(2.4%)。从采样城市来看,北京市的污染率最高,为34%,污染水平也达到了42.7 MPN/g。以长江为界,北方几大城市的整体污染率和污染水平要显著高于南方地区。同时,对分离的880株疑似李斯特菌分别进行了MID-67、Numide-Listeria v1.0和分子鉴定,分子鉴定显示99.8%的菌株含有种特异性基因,而这其中99.3%的单增李斯特菌、96.3%的英诺克李斯特菌、100%的格式李斯特菌、100%的西式李斯特菌、100%的绵羊李斯特菌和80%威尔士李斯特菌显示两种鉴定系统结果一致,而本论文研制的Numide-Listeria v1.0在鉴定单增李斯特菌和英诺克李斯特菌方面表现出优于MID-67的能力。3.对全国食品污染调查中分离的单增李斯特菌分离株进行了遗传多样性和毒力特性等研究。耐药菌株普遍存在,仅32.5%的即食食品分离株和23.8%的生鲜分离株对所选的14种抗生素全部敏感,30.8%的菌株为多重耐药菌株,其中13株分离株对10种以上抗生素具有耐药性。菌株在血清型、ERIC-PCR、REP-PCR甚至是耐药性上均有着一定的关联性,表现出相似的遗传关系。本研究中选取的毒力基因,除llsX是Lineage I菌株所特有的毒力基因,大部分的菌株均携带有hly(100%)、inlA(99.7%)、inlB(99.1%)、inlC(98.5%)、inlJ(99.4%)。Epidemic Clones(ECs)作为食源性李斯特菌病流行暴发的可能标志物,也在26.3%的即食食品分离株和6.7%的生鲜分离株中检测到。对即食食品分离株进行分子遗传特性研究,也进一步诠释了相关菌株的毒力现状。MLST分型结果显示除新发现的ST型外(ST804,ST805,ST806,ST807),其余ST型均在临床菌株和李斯特菌病的暴发中出现过;毒力基因inlA的全长测序,显示91.4%的菌株具有编码InlA全长蛋白并侵染细胞的能力,发现的3个提前终止密码子(PMSC),其中1个型为首次出现,位于326号氨基酸位置(GAA→TAA)。4.运用iTRAQ蛋白组学技术,获得了L.monocytogenes ATCC19115和L.innocua ATCC33090全菌蛋白图谱。通过LC-MS/MS技术,分别在单增李斯特菌和英诺克李斯特菌中鉴定出1650和1732个可信蛋白。经蛋白丰度比分析后,鉴定出121个独有蛋白(包括49个单增李斯特菌特有蛋白和72个英诺克李斯特菌特有蛋白)。对这些差异蛋白进行Gene Ontology(GO)分析,发现它们主要参与了两种李斯特菌的细胞内碳氮化合物的生物合成、分子转运、糖类运输和代谢等生物学途径。进一步的生物学功能分析,发现大部分(75%)蛋白为功能未知的假设蛋白,其余则涉及酶、DNA结合蛋白和离子结合蛋白等相关蛋白。综上所述,本论文始终围绕食源性单增李斯特菌进行研究,构建了食源性李斯特菌数值鉴定系统(Numide-Listeria v1.0),首次全面掌握了我国不同地区不同类型食品中单增李斯特菌的污染情况和分布规律;建立在此基础上的单增李斯特菌遗传多样性和毒力特征研究,为我们进一步了解该菌在我国的流行趋势和风险识别提供了坚实的基础。通过比较蛋白组学获得的种间特异性酶,对于进一步研发特异性的酶底物有效区分有害和无害李斯特菌提供了依据。此外,其他独有蛋白的发现,也为研究单增李斯特菌的生物学功能提供了基础和依据。
[Abstract]:Lester (Listeria spp.) is a genus of gram positive bacteria, facultative anaerobic. At present, the genus contains 16 species and 2 subspecies, including 6 species of bacteria associated with food. Lester List Rand Listeria cell proliferation (Listeria monocytogenes) is a genus of Lester most foodborne pathogen, it has pathogenicity of human and animal, can cause meningitis, septicemia, abortion and purulent partial injury, cause miscarriage, stillbirth and other diseases, the incidence rate of up to 20%~30%, the WHO is defined as the world's four largest food source pathogenic bacteria of venereal disease. The bacteria identification method based on the combination of numerical and computer aided technology independent research and development of food borne bacteria Lester numerical identification system (Numide-Listeria v1.0). On this basis, the distribution of Listeria bacteria Lester of different types of food in different regions of the country in the survey system, verify the The identification system at the same time, further research on Listeria isolates from Lester genetic diversity, serotype, virulence and drug resistance status of genetic characteristics. In addition, this paper also through iTRAQ proteomics technology established Lester bacteria whole cell protein database, the difference of material for species, provide a basis to further improve the identification system. The main content of this thesis and the research results are as follows: 1. by Lester strains biochemical differences, screened 11 different from the currently available on the same products. The combination of biochemical tests on laboratory isolated 2000 strains of bacteria in Lester related experiments to obtain the positive rate of each of these 11 biochemical reactions. Biochemical reactions and the positive rate of bacteria identification database. Numerical method based on the principle, the use of C++ language programming and computer technology, constructs a foodborne bacteria Lester numerical discrimination The system (Numide-Listeria v1.0), which can effectively distinguish between six kinds of food borne bacteria Lester.2. for Listeria contamination of Lester national survey of 1400 retail food, were detected in 232 positive samples, the average pollution rate was 16.6%, the average pollution level of 15.72 MPN/g. seven kinds of food detection rate from high to low are: frozen food (44.9%) edible fungus (23.2%) meat and meat products (20.9%) instant food (8.2%) aquatic products (6.4%) and vegetables (5.7%) dairy products (2.4%). From the sampling of pollution in the city of Beijing City, the highest rate is 34%, the pollution level has reached 42.7 MPN/g. is bounded by the Yangtze River, the whole the contamination rate and the pollution level in several northern city was significantly higher than in the south. At the same time, the separation of the 880 strains of suspected Lester bacteria were analyzed by MID-67, v1.0 and Numide-Listeria molecular identification, molecular identification showed that 99.8% strains contain a specific gene, and this 鍏朵腑99.3%鐨勫崟澧炴潕鏂�鐗硅彛�

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