【作者】 胡玉山；

【导师】 陈晓光；

【作者基本信息】 南方医科大学 ， 病原生物学， 2011， 博士

【摘要】 背景食源性疾病是由于食用或饮用了被致病因素污染的食物或饮料引起的疾病。常见的致病因素有病原微生物、天然毒素、寄生虫和有毒有害化学物质等。虽然毒素、有毒有害化学物质等是食源性疾病的重要原因,但是病原菌才是引起食源性感染疾病发生的主要因素。由于全球化进程的加快,全球贸易和人员来往日益频繁,食源性感染已成为当前一项重要的公共卫生问题。无论发展中国家还是发达国家,每年都有很多爆发病例。流行病学监测数据表明食源性感染疾病的发病率持续上升,同时引起食源性感染的病原菌也逐渐表现出多样化。除肠道病原菌外,近年来许多致病力很弱的菌株也成为食源性感染的病原菌。但许多公共卫生部门并未充分认识到食品安全的重要性,食源性感染疾病的预防控制仍面临着巨大挑战。食源性感染病原菌的特征主要表现在菌株生物学特性的改变,如革兰染色、菌落形态、血清学特性的变化等,但这些生物学特性改变往往使常规鉴定方法有时难以准确的对病原菌进行分类鉴定。食源性感染的另一特点是发病人数往往较多、发病较快,如果疫情没有及时处理,会有进一步蔓延的可能性。如何对食源性感染病原菌进行溯源以及快速鉴定是控制食源性感染的关键。随着对细菌感染研究的日益深入,传统的细菌鉴定技术已不能很好地满足细菌感染的诊断和流行病学溯源的需要,从型、亚型、株,甚至分子水平上去鉴别细菌变得愈来愈重要,近年来随着分子生物学理论和技术的发展,使得细菌鉴定、耐药基因的检测、分子流行病学溯源变得更加准确、简洁和快速。多位点序列分型(Multilocus sequence typing, MLST)通过直接测定菌株的几个看守基因的序列,与标准克隆株的等位基因图谱比较,从而确定细菌的型别。其优点在于可直接对标本的基因片段进行扩增,无需培养病原菌,结果明确,易于标准化,不同实验室之间的结果可以互相比较。目前在万维网上已经建立了脑膜炎奈瑟菌、肺炎链球菌、幽门螺杆菌等细菌的MLST全球数据库。脉冲场凝胶电泳(Pulse-field gel electrophoresis, PFGE)是近年兴起的DNA指纹图新技术,它是对细菌的整个基因组进行分析,能够分辨20-500 kb的DNA片断,最大分辨率可达5000kb,具有重复性好、分辨率高和容易标准化等特点,可用于实验室室间比较。基因芯片技术(microarray)是一种反向固相杂交技术,它将大量探针分子固定于支持物上,然后与标记的样品进行杂交,通过检测杂交信号的强弱进而判断样品中靶分子的数量。由于用该技术可以将大量的探针同时固定于支持物上,因而基因芯片技术具有高通量、并行处理、微型化和易于自动化等优点。目前,基因芯片技术在医学、分子生物学领域应用已经越来越广泛,它可实现大量序列的平行鉴定,迅速敏感地检测基因表达。基因芯片技术在病原微生物研究中已得到广泛应用,但是往往需要昂贵的点样仪和扫描仪,给该技术的广泛应用带来了不便。本项研究的目的是建立新的寡核苷酸膜阵列技术,它不需要昂贵的检测设备,同时保持检测的敏感度和特异性。选择同---基因片段可同时进行多种致病菌的鉴别。该基因必须是所有病原菌共有的基因片段,有一定的保守性,其序列的变异性又适合进行属种的鉴定。因此,病原微生物的保守基因常用作鉴别诊断的靶基因,如16S rRNA、23SrRNA、16-23S rRNA间隔区等是相对保守的基因。其中,16-23S rRNA间隔区虽种属间变异较大,但片段较短；23S rRNA在细菌间的变异性较大,但序列不全,数据库资料不完整；16S rRNA基因在种属内保守性较强,并且序列齐全,常用于细菌的种系分类。热休克蛋白60是一种高度保守的蛋白质,其编码基因groEL基因是生物进化中的保守成分。groEL基因数据库资料比较完整,国外已有科研人员选取其作为靶基因用于沙门氏菌、空肠弯曲菌及葡萄球菌等的分型鉴定。本研究选取groEL基因的一个变异区对引起食源性感染的17属(种)常见病原菌,运用寡核苷酸膜阵列技术进行鉴别诊断。本学位论文主要研究：(1)运用MLST和PFGE进行食源性感染病原菌分子分型,用于溯源研究；(2)常见病原菌groEL基因序列同源与变异分析,为细菌鉴别诊断和相关研究提供科学依据；(3)应用寡核苷酸膜阵列技术,建立食源性感染病原菌的快速检测方法。材料和方法材料1.标准菌株：选取引起食源性感染的17种(属)病原菌,它们是：大肠埃希菌、沙门菌、志贺菌、霍乱弧菌、副溶血弧菌、溶藻弧菌、金黄色葡萄球菌、蜡样芽胞杆菌、单核细胞增生性李斯特菌、小肠结肠炎耶尔森菌、变形杆菌、溶血性链球菌、肉毒梭菌、产气荚膜梭菌和空肠弯曲菌,肺炎链球菌、铜绿假单胞菌和脑膜炎双球菌为对照菌株。以上菌株均为本室保存的标准菌株。2.食源性感染模拟标本：本研究将纯种细菌培养物按10倍稀释法从106稀释混匀至100作为食源性感染模拟标本。其中食源性感染模拟标本1含有大肠埃希菌；食源性感染模拟标本2含有肠炎沙门菌；食源性感染模拟标本3含有单核细胞增生性李斯特菌；食源性感染模拟标本4含有空肠弯曲菌；食源性感染模拟标本5含有副溶血弧菌；食源性感染模拟标本6含有普通变形杆菌。3.食源性感染临床标本：2000年6月至2010年6月从广州疾病预防控制中心收集20份食源性感染爆发临床标本。4.生物信息学软件：Bioedit软件,DNA star软件包,Cluster W软件,BioNumerics4.0软件,Primer 5.0引物设计软件和Primer express3.0荧光定量PCR设计软件。方法1.根据管家基因序列设计引物进行MLST分型和在细菌整个染色体基因组上进行PFGE分型,用于溯源分析。2.运用GenBank数据库和生物信息分析软件,并通过PCR扩增groEL基因片段,分析不同种属细菌groEL基因的保守序列、变异规律及菌株间种系进化关系。3.用groEL基因片段作为病原菌荧光定量PCR检测的靶区进行检测。4.运用带正电荷尼龙膜作为芯片载体,合成寡核苷酸探针,点样于载体制成寡核苷酸芯片,对食源性感染常见病原菌、食源性感染模拟标本和食源性感染临床标本细菌groEL基因片段扩增产物进行杂交检测。5.使用地高辛显色系统对杂交结果进行显色分析。结果1.MLST分型中获得了沙门菌8个血清群中的14个MLST型,食品来源沙门菌菌株的血清群有7个,ST型为10个,患者来源菌株血清群为5个,ST型为10个,实验中还发现2个新的ST型,分别命名为Newl-New2。沙门菌09群有4个ST型,其中以ST13最多；沙门菌010群和011群都有2个ST型；沙门菌02群、03群、07群、08群都只有1个ST型。PFGE分型中沙门菌04群、07群、09群、011群分离株属于A组；08群分离株属于B组；02群、03群属于C组。A组相似程度最高,B组其次,C组相似性最差。2.通过PCR扩增和生物信息学比较分析,获得34个groEL的通用保守序列,groEL基因保守序列与变异区在种系间呈间隔分布,大量变异区呈“马赛克”式分布。种系间进化关系与16S rRNA分析结果大致一致。3.建立了用groEL基因片段作为检测靶区病原菌荧光定量PCR检测方法。4.在同一条件下运用设计的通用引物扩增出17种(属)细菌groEL基因目标片段。寡核苷酸芯片杂交结果表明,沙门菌、霍乱弧菌、副溶血弧菌、溶藻弧菌、变形杆菌、单核细胞增生李斯特菌、蜡样芽胞杆菌、小肠结肠炎耶尔森菌、溶血性链球菌、产气荚膜梭菌、肉毒梭菌和空肠弯曲菌种(属)细菌杂交结果显示出高灵敏度和特异性。志贺菌与大肠埃希菌的出现交叉反应。肺炎链球菌、铜绿假单胞菌和脑膜炎双球菌对照菌株没有杂交反应。本方法可以应用于食源性感染模拟标本和食源性感染临床标本的检测。检出水平可以达到101cfu/mL。5.建立了符合在基层推广使用的地高辛显色检测结果分析方法。结论1.运用管家基因序列设计引物进行MLST分型和运用细菌整个基因组进行PFGE分型,可用于食源性感染病原菌溯源分析。2.groEL基因序列变异性规律为进行细菌的鉴别诊断提供了重要的理论和实验基础。3.对于食源性感染模拟标本,本方法可以获得预期的结果。对食源性感染临床标本检测的结果表明,本方法可以在属及部分种水平进行鉴定,但在亚种及血清型水平的鉴定需要结合传统方法进行鉴别。4.本研究只对细菌性食源性感染常见病原菌进行膜阵列杂交的研究,该研究为制备高密度的功能齐全的芯片奠定了基础,下一步将进行扩大芯片容量和检测范围的研究,同时进一步提高检测的特异性。更多还原

【Abstract】 Background:Intaking the contaminated food or water causes foodborne disease. The usual nosogenetic factors are as follows:pathogens, natural toxins, parasites and poisonous chemicals. Although natural toxins and poisonous chemicals could cause foodborne disease, the pathogens are main factors.Foodborne infection has become a very important hygienic problem because of the rapid expansion of food trade and highly increased mobility of today’s populations. Hundreds of outbreaks of foodborne infection cases occur every year around the world.The epidemic surveillance data show that the incidence of foodborne infective disease is increasing and the species of bacteria causing foodborne infection have become more diversified continuously. In addition, many kinds of bacteria with weak toxigenicity have also become foodborne pathogens except intestinal pathogenic bacteria in recent years. But many public health governments still can not wake up to the importance of the food safety. There is big challenge for prevention and control of the foodborne infection disease. The major property of many foodborne pathogens is the alteration in their biological characters, such as Gram staining, colony formation and antigenicity. These changes often make conventional detection methods inefficient. Another property of foodborne infection is that it often involve quite a large number of affected individual and can spread rapidly in schools, factories and other institutions if the foodborne infection disease could not be controlled in time.Rapid trace to the source, detection and identification of the pathogens are the key issues to control foodborne infections. With more study in pathogens, many conventional microbiological assay such as Gram staining, culturial, biochemical properities, biotype- or serotype- identification is often inefficient and time consuming. It is more important to identify the bacteria on type, subtype, strain and molecule. The advent of genetic-based technologies makes feasible to develop a sensitive and specific screening test for the detection of microbial pathogens and their drug-resistence. The development in molecular biological technologies provides an efficient tool for bacteria classification. It makes bacteria division to molecular level and provides more details for picking out pathogens and transmission routes. We cannot only rapidly identify pathogens but also study their heredity relations in subtype, serotype and gene level. Multilocus sequence typing (MLST) is based on determination of the DNA sequence of a series of selected housekeeping. MLST may provide an ideal balance of high discriminatory power and a powerful data analysis capability requiring minimal human input. This technique, made possible by the increased availability of robotic sequencers, is based on determinationof the nucleotide sequences of a series of predetermined housekeeping gene. Now the MLST data of several pathogenic bacteria, such as Neisseria meningitides, Streptococcus pneumoniae have been based in worldwide web. Pulsed-field gel electrophoresis (PFGE) is a new DNA finger technique in recent years. The technique is based on all genome of bacteria and can discriminate 20～500 kb DNA segment. The data of MLST and PFGE can be compared among different labs. Gene chip technology is a reverse solid hybridization technology. Firstly a large number of probes are fixed onto the supporting material, then are hybridized with marked sample. At last, targets of sample are detected by the intensity of hybridization signal. Because more probes are fixed on the supporting material at the same time, microarray technology is characterized by high throughput, parallel disposal, micro miniaturization and easy automatization. At present, microarray technology is largely applied in medicine and molecular biology. It can parallelly identify many sequences and rapidly detect gene expression. Microarray technology can be largely used in the pathogens.Recently a DNA and oligonucleotide microarray technique has been applied to screen multiple microbial organism isolates in diagnostic assays. But such gene chips require sophisticated instruments to prepare and the results can be read only by laser scanner, thus limiting its broad application in common laboratories. Therefore we tried to establish an oligonucleotide array method in order to eliminate the need for sophisticated equipments but retain the sensitivity and specificity of the technique.To select a common gene fragment for identification of multiple pathogens, such gene must contain conservative regions common to these pathogens, and on the other hand sufficient sequence diversity for species identification. In literature, the 16S rRNA,23S rRNA, and 16S-23S rRNA spacer region have been selected as identification targets of bacteria. The conservation of 16S rRNA makes it suitable for identification of different species (genera) of bacteria. The extent of mutation is great in the 16S-23S rRNA spacer region among bacterial species or genera, but this region is too short to identify certain species of bacteria. Heat shock protein is a highly conserved protein, whose encoding gene groEL constitutes to be the most conserved component in evolution. The groEL gene has been used as the target gene in the typing and identification of Salmonella, Campylobacter jejuni and Staphylococcus on account of its complete database. In this study a mutation region of groEL was selected to prepare oligonucleotide membrane array on a nylon membrane to detect 17common bacterial pathogens causing foodborne infections.Our studies are as follows:(1) To study MLST and PFGE typing of foodborne infection pathogens for epidemiologic investigation. (2) To study the mutagenecity of pathogenic bacteria groEL gene and provide the materials to discriminative diagnosis of pathogen. (3) To construct a rapid and accurate detection method for common pathogenic bacteria in foodborne diseases with the technology of oligonucleotide membrane array. Materials and methods:Materials:1. Standard bacteria species:Seventeen common bacteria species causing foodborne infections were selected in our study. They were Escherichia coli, Escherichia coli, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, Streptococcus hemolyticus, Yersinia enterocolitica,Proteus vulgaris, Bacillus cereus, Salmonella enterica, Salmonella typhimurium,Listeria monocytogenes, Shigella dysenteriae, Shigella flexneri,Clostridium perfringens,Clostridium botulinum Streptococcus pneumoniae, Klebsiella pneumoniae and Neisseria gonorrhoeaeserved as control species unrelated to foodborne infections. All strains above are stored in our lab.2. Foodborne infection mock samples:Pure cultures of two or three species of other bacteria were mixed and diluted as foodborne infection mock samples. Mock sample No.1 contains bacteria species of E. coli; mock sample No.2 contains bacteria species of Salmonella enterica; mock sample No.3 contains bacteria species of Listeria monocytogenes; mock sample No.4 contains bacteria species of Campylobacter jejuni; mock sample No.5contains bacteria species of Vibrio parahaemolyticus; mock sample No.6contains bacteria species of Proteus vulgaris3. Foodborne infection true samples:Ten foodborne infection true samples were collected from Guangzhou center for disease control and prevention between June 2000 and June 2010.4. Molecular biology software:Bioedit, DNA star, Cluster W, GeneDoc, Primer 5.0 and Primer express3.0Methods:1. To apply MLST based on housekeeping gene and PFGE based on all genome to type pathogens for epidemiologic investigation.2. With material of GenBank and biosoft and PCR amplification of groEL gene fragment of different bacteria, the groEL conservative regions, variable regions and their relation to the genetic development of bacteria strains were studied. 3. groEL gene was selected as target fragment for real-time PCR identification foodborne infection pathogens.4. Nylon membrane was used as array base. Oligonucleotide probes were synthesized and spotted onto nylon membrane as oligonucleotide array. The groEL gene amplifiaction products of different pathogenic bacteria from pure cultures, foodborne infection mock samples and foodborne samples were hybridized on oligonucleotide array.5. To apply digoxigenin developing color system for analysis the hybridization results.Results:1. To have acquired the data of Salmonella MLST and PFGE analysis.2. With gene amplfication and bioinformatic analysis,34 conservative regions were obtained. Conservative and mutant regions are distributed separately and most small mutant regions distributed like morsac among conservative regions. The phylogenetic trees among different bacteria drawn from the full length of groEL gene or partial fragment were the same from 16S rRNA.3. To have developed the real-time PCR based on groEL gene for identification of foodborne infection pathogens.4. With the same amplification and hybridization conditions, the groEL gene fragments of 17 species of pathogenic bacteria were amplified and hybridized onto the oligonucleotide array. Hybrizdization results manifested that 15 species {Escherichia coli, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, Streptococcus hemolyticus, Yersinia enterocolitica, Proteus vulgaris, Bacillus cereus, Salmonella enterica, Salmonella typhimurium, Listeria monocytogenes, Clostridium perfringens and Clostridium botulinum.) showed high sensitivity and specificity. As to Shigella dysenteriae and Shigella flexneri, we found cross-reaction with the E. coli species- specific probe. Three species irrelated to foodborne infection disease showed no hybridization signal on the oligonucleotide array. The new method could also be applied in rapid identification of pathogen in foodborne infection mock samples and foodborne infection samples. Sensitivity in the assay mounts to 10 cfu/ml.5. To have developed digoxigenin developing color system for analysis the hybridization results.Conclusion:1. MLST based on housekeeping gene and PFGE based on all genome can type pathogens for epidemiologic investigation.2. The mutagenecity of groEL gene would provide suitable resolution for bacteria identification.3. Anticipated results could be obtained from foodbore infection mock samples with the oligonucleotide array. With foodbore infection samples, the new method could only identified to genus or some species level. Identification in subspecies or serotype level must be applied with traditional methods and antimicrobial resistance test. The method of oligonucleotide array has the priority of rapidity and accuracy in the diagnosis of foodborne diseases, and provides the efficiency way in the diagnosis, therapy and control of foodborne infection.4. Much study should be carried out on increasing hybridization probes and broadening the detection scope, and also on improving the specificity of the detection method.更多还原