基于宏基因组学的猪—肠道微生物互作靶点发掘及其微生物源追溯（MST）研究

发布时间：2018-01-10 10:38

本文关键词：基于宏基因组学的猪—肠道微生物互作靶点发掘及其微生物源追溯（MST）研究　出处：《浙江工商大学》2017年硕士论文　论文类型：学位论文

【摘要】：水环境对水产品质量与安全及公众健康有着重要影响。然而,目前全球范围的非点源污染(主要指人类和动物的排泄物)已成为影响水体质量、破坏海洋生态以及导致水产品重大安全隐患的主要原因。因此,建立一种灵敏度高、特异性强并能高效指示粪便污染源的MST方法显得尤为迫切。肠道微生物群落与其宿主在共同进化过程中会产生一些参与宿主-微生物互作的基因。这些基因具有一定的宿主肠道微生物特异性,利用其设计分子标记能准确识别粪便污染来源。本研究采用宏基因组学策略靶向筛选猪粪便特异性基因片段,设计猪粪便特异性分子标记,建立相应的荧光定量PCR检测方法,评价其灵敏度、特异性及检测限,最终对水环境未知粪便污染来源开展微生物源示踪(或追溯)研究。该研究为微生物示踪(或追溯)技术在非点源污染方面的应用提供一定的基础数据。本研究的研究内容包括以下4个部分:1.本研究共采集9个物种260个粪便样品,提取DNA后利用竞争性杂交富集方法(GFE)靶向筛选参与猪-肠道微生物互作的有关特异性基因。经BLASTx比对分析,GFE富集的82%猪特异性非冗余序列存在相似序列,以拟杆菌纲(Bacteroidetes)(43.2%)、梭菌纲(Clostridia)(19.5%)、芽孢杆菌纲(Bacilli)(8.6%)蛋白相似序列为主。所得的非冗余序列中61.5%功能明确,大部分与拟杆菌群(62.6%)与梭状杆菌群(27.2%)蛋白序列相似。从蛋白功能方面分析,7.6%序列与信息贮存与加工有关,12.8%序列与细胞加工及信息传导有关,22%序列与代谢有关,结果发现能够编码优势菌群(Bacteroidetes、Clostridia等)信息传导有关的表面蛋白、膜分泌蛋白及有些碳水化合物代谢蛋白的相关基因可作为猪特异性分子标记筛选的靶点。2.从富集的猪粪便特异性基因组文库中共筛选26个猪粪便特异性基因片段,分别设计引物后利用猪粪便混合DNA及对照组中单个物种各自的混合DNA为模板对所有引物进行宿主特异性验证,发现1-38,2-95,2-109,3-53四条序列的引物能特异性扩增猪粪便DNA,表明这四条序列可作为分子标记用于猪粪便污染追溯的荧光定量PCR检测方法的建立。3.针对以上四条分子标记分别筛选引物和探针,通过优化反应条件、确立检测限、分析方法的批内重复性和批间稳定性建立荧光定量PCR检测方法,对72个猪粪样DNA(包括猪粪废水)及71个其他动物粪便DNA进行灵敏性、特异性评价发现猪粪便特异性分子标记(1-38,2-95,2-109,3-53)特异性分别为 93%,73%,79%,90%,灵敏性分别为94%,88%,72%,90%。基于1-38和3-53的特异性荧光定量PCR检测方法具有较好的检测效果,具有应用于MST的潜质。4.利用膜过滤法和所建立荧光定量PCR检测方法对浙江省内27个不同地区所采集64个地表水样分别进行粪便指示菌(FIB)计数、猪粪便特异性分子标记和致病菌检测。猪粪便特异性分子标记(1-38,3-53)在64个地表水样的阳性检出率分别51.3%,53%,根据贝叶斯理论猪粪便特异性分子标记(1-38,3-53)在检测水样受猪粪便污染而非其他动物粪便污染的条件可能性分别为92.5%,91.7%,表明所建立的猪粪便特异性分子标记荧光定量PCR检测方法实际有效性;同时,E.coli O157 rfbE,Campylobacter 16S rRNA,Salmonella invA 基因分别在70.3%,17.2%,51.5%的采集水样DNA中检出阳性信号。通过一致性及相关性分析可知FIB中E.coli与肠道致病菌或特异性分子标记的OR值均为1,说明不存在相关性,Enterococcus spp.仅与特异性分子标记3-53、Campylobacter 16S rRNA相关(OR值分别为0.419、0.365),说明FIB不能指示肠道致病菌及特异性分子标记的存在情况,与之相比粪便特异性分子标记与肠道致病菌存在一定的相关性。因此,在水质监测时可利用特异性多分子标记组合结合潜在致病菌进行水样中猪粪便污染程度的指示。
[Abstract]:Water environment of aquatic product quality and safety and public health has an important influence. However, at present the global scope of non-point source pollution (mainly refers to human and animal excreta) has an impact on water quality, marine ecological damage and the main reason that causes significant security risks of the aquatic products. Therefore, the establishment of a high sensitivity. The MST method has strong specificity and efficient indicator of fecal pollution is particularly urgent. The intestinal microbial community and its host will produce some involved in host microbe interaction genes in a co evolutionary process. These genes have certain intestinal microbial specificity, using the design of molecular markers can accurately identify the sources of fecal pollution in this research. The metagenomic strategy targeted screening and specific gene fragment of pig manure, pig manure and the design of specific markers, establish corresponding fluorescence quantitative PCR detection method, To evaluate the sensitivity, specificity and detection limit, and ultimately to the water environment of unknown fecal pollution sources to carry out microbial source tracking (or back). The study of microbial tracer (or back) provides some basic data application technology in non point source pollution. The 4 part of the research contents of this study include the following: 1. this study collected 9 species of 260 fecal samples, competitive hybridization using enrichment method after extraction of DNA (GFE) gene targeting screening in pig intestinal microbial interaction. Analysis by BLASTx comparison, there are similar sequence redundancy sequences of GFE enriched 82% pigs specific to Bacteroides (gang Bacteroidetes) (43.2%) (Clostridia), clostridia, bacilli (19.5%) (Bacilli) (8.6%) was similar to sequences. 61.5% non redundant sequences in the function is clear, and most Bacteroide (62.6%) and Clostridium group (27.2% Protein sequence similarity analysis). From the 7.6% aspects of protein function, sequence and information storage and processing, 12.8% sequences related to the cell processing and information transmission, 22% sequence and metabolism, the result shows that encoding dominant bacteria (Bacteroidetes, Clostridia) surface protein information transmission on the target.2. gene related to membrane protein secretion and some carbohydrate metabolism proteins can be used as pig specific molecular markers screened from enriched pig fecal specific genomic library screened 26 pig fecal specific gene fragment, primers were designed using single pig manure mixed with DNA and control group in each species mixed DNA as the template for host specificity validation on all primers found, primer 1-38,2-95,2-109,3-53 four sequences to pig fecal DNA specific amplification showed that four sequences can be used as molecular markers for pig manure Fluorescence quantitative PCR detection method of pollution back to establish.3. for the above four molecular markers were screened primers and probes, by optimizing the reaction conditions, the establishment of the detection limit, detection method of fluorescent quantitative PCR analysis method in batch repeatability and inter batch stability established in 72 swine fecal samples of DNA (including piggery wastewater) and 71 other animal feces DNA sensitivity and specificity evaluation found that pig fecal specific molecular markers (1-38,2-95,2-109,3-53) were 93%, 73%, 79%, 90%, 88%, 94% respectively. The sensitivity and specificity of 72%, based on the 90%. fluorescence quantitative PCR method for detection of 3-53 and 1-38 has better detection effect, has applied to MST potential.4. using membrane filtration method and the method of fluorescence quantitative PCR detection of fecal indicator bacteria were 64 surface water samples collected in 27 different regions of Zhejiang province (FIB) count, then the specificity of pig manure Molecular markers and detection of pathogenic bacteria. Pig fecal specific molecular marker (1-38,3-53) in 64 surface water samples the positive rate were 51.3%, 53%, according to the Bias theory of pig fecal molecular marker (1-38,3-53) in the detection of water by pig manure pollution and other animal feces pollution probability conditions were 92.5%, 91.7% that is, specific molecular markers of fluorescence quantitative PCR method for detection of swine manure is effective; at the same time, E.coli O157 rfbE, Campylobacter 16S rRNA and Salmonella invA gene respectively in 70.3%, 17.2%, 51.5%. The positive signal in water DNA. Through the consistency and correlation analysis of E.coli and FIB in the intestinal pathogens or the specific molecular marker of OR value was 1, indicating there was no correlation between spp. and Enterococcus, only specific molecular markers of 3-53, Campylobacter 16S rRNA (OR = 0.419,0 .365), indicating the presence of FIB can indicate intestinal pathogens and specific molecular markers, compared with fecal specific molecular markers and intestinal pathogens have certain correlation. Therefore, the water quality monitoring can be used for specific molecular markers combined with potential pathogenic bacteria pollution degree of pig manure in water indicator.

【学位授予单位】：浙江工商大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X830;S828

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