本文關(guān)鍵詞：基于宏基因組學(xué)的豬—腸道微生物互作靶點(diǎn)發(fā)掘及其微生物源追溯（MST）研究　出處：《浙江工商大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 微生物源示蹤(MST) 競(jìng)爭(zhēng)性雜交富集特異性片段(GFE) 豬-腸道微生物互作基因 豬糞便特異性分子標(biāo)記 熒光定量PCR 糞便指示菌(FIB) 致病菌

【摘要】：水環(huán)境對(duì)水產(chǎn)品質(zhì)量與安全及公眾健康有著重要影響。然而,目前全球范圍的非點(diǎn)源污染(主要指人類和動(dòng)物的排泄物)已成為影響水體質(zhì)量、破壞海洋生態(tài)以及導(dǎo)致水產(chǎn)品重大安全隱患的主要原因。因此,建立一種靈敏度高、特異性強(qiáng)并能高效指示糞便污染源的MST方法顯得尤為迫切。腸道微生物群落與其宿主在共同進(jìn)化過(guò)程中會(huì)產(chǎn)生一些參與宿主-微生物互作的基因。這些基因具有一定的宿主腸道微生物特異性,利用其設(shè)計(jì)分子標(biāo)記能準(zhǔn)確識(shí)別糞便污染來(lái)源。本研究采用宏基因組學(xué)策略靶向篩選豬糞便特異性基因片段,設(shè)計(jì)豬糞便特異性分子標(biāo)記,建立相應(yīng)的熒光定量PCR檢測(cè)方法,評(píng)價(jià)其靈敏度、特異性及檢測(cè)限,最終對(duì)水環(huán)境未知糞便污染來(lái)源開(kāi)展微生物源示蹤(或追溯)研究。該研究為微生物示蹤(或追溯)技術(shù)在非點(diǎn)源污染方面的應(yīng)用提供一定的基礎(chǔ)數(shù)據(jù)。本研究的研究?jī)?nèi)容包括以下4個(gè)部分:1.本研究共采集9個(gè)物種260個(gè)糞便樣品,提取DNA后利用競(jìng)爭(zhēng)性雜交富集方法(GFE)靶向篩選參與豬-腸道微生物互作的有關(guān)特異性基因。經(jīng)BLASTx比對(duì)分析,GFE富集的82%豬特異性非冗余序列存在相似序列,以擬桿菌綱(Bacteroidetes)(43.2%)、梭菌綱(Clostridia)(19.5%)、芽孢桿菌綱(Bacilli)(8.6%)蛋白相似序列為主。所得的非冗余序列中61.5%功能明確,大部分與擬桿菌群(62.6%)與梭狀桿菌群(27.2%)蛋白序列相似。從蛋白功能方面分析,7.6%序列與信息貯存與加工有關(guān),12.8%序列與細(xì)胞加工及信息傳導(dǎo)有關(guān),22%序列與代謝有關(guān),結(jié)果發(fā)現(xiàn)能夠編碼優(yōu)勢(shì)菌群(Bacteroidetes、Clostridia等)信息傳導(dǎo)有關(guān)的表面蛋白、膜分泌蛋白及有些碳水化合物代謝蛋白的相關(guān)基因可作為豬特異性分子標(biāo)記篩選的靶點(diǎn)。2.從富集的豬糞便特異性基因組文庫(kù)中共篩選26個(gè)豬糞便特異性基因片段,分別設(shè)計(jì)引物后利用豬糞便混合DNA及對(duì)照組中單個(gè)物種各自的混合DNA為模板對(duì)所有引物進(jìn)行宿主特異性驗(yàn)證,發(fā)現(xiàn)1-38,2-95,2-109,3-53四條序列的引物能特異性擴(kuò)增豬糞便DNA,表明這四條序列可作為分子標(biāo)記用于豬糞便污染追溯的熒光定量PCR檢測(cè)方法的建立。3.針對(duì)以上四條分子標(biāo)記分別篩選引物和探針,通過(guò)優(yōu)化反應(yīng)條件、確立檢測(cè)限、分析方法的批內(nèi)重復(fù)性和批間穩(wěn)定性建立熒光定量PCR檢測(cè)方法,對(duì)72個(gè)豬糞樣DNA(包括豬糞廢水)及71個(gè)其他動(dòng)物糞便DNA進(jìn)行靈敏性、特異性評(píng)價(jià)發(fā)現(xiàn)豬糞便特異性分子標(biāo)記(1-38,2-95,2-109,3-53)特異性分別為 93%,73%,79%,90%,靈敏性分別為94%,88%,72%,90%。基于1-38和3-53的特異性熒光定量PCR檢測(cè)方法具有較好的檢測(cè)效果,具有應(yīng)用于MST的潛質(zhì)。4.利用膜過(guò)濾法和所建立熒光定量PCR檢測(cè)方法對(duì)浙江省內(nèi)27個(gè)不同地區(qū)所采集64個(gè)地表水樣分別進(jìn)行糞便指示菌(FIB)計(jì)數(shù)、豬糞便特異性分子標(biāo)記和致病菌檢測(cè)。豬糞便特異性分子標(biāo)記(1-38,3-53)在64個(gè)地表水樣的陽(yáng)性檢出率分別51.3%,53%,根據(jù)貝葉斯理論豬糞便特異性分子標(biāo)記(1-38,3-53)在檢測(cè)水樣受豬糞便污染而非其他動(dòng)物糞便污染的條件可能性分別為92.5%,91.7%,表明所建立的豬糞便特異性分子標(biāo)記熒光定量PCR檢測(cè)方法實(shí)際有效性;同時(shí),E.coli O157 rfbE,Campylobacter 16S rRNA,Salmonella invA 基因分別在70.3%,17.2%,51.5%的采集水樣DNA中檢出陽(yáng)性信號(hào)。通過(guò)一致性及相關(guān)性分析可知FIB中E.coli與腸道致病菌或特異性分子標(biāo)記的OR值均為1,說(shuō)明不存在相關(guān)性,Enterococcus spp.僅與特異性分子標(biāo)記3-53、Campylobacter 16S rRNA相關(guān)(OR值分別為0.419、0.365),說(shuō)明FIB不能指示腸道致病菌及特異性分子標(biāo)記的存在情況,與之相比糞便特異性分子標(biāo)記與腸道致病菌存在一定的相關(guān)性。因此,在水質(zhì)監(jiān)測(cè)時(shí)可利用特異性多分子標(biāo)記組合結(jié)合潛在致病菌進(jìn)行水樣中豬糞便污染程度的指示。
[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.

【學(xué)位授予單位】：浙江工商大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X830;S828

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本文編號(hào)：1404973