【摘要】：腸道微生物群落在與其宿主長期協(xié)同進化過程中會形成大量的宿主-腸道微生物互作基因,這些互作基因具有一定的宿主腸道微生物特異性,利用其設計分子標記能有效識別糞便污染源.本研究首次利用競爭性雜交的方法富集豬糞便特異性基因,從中篩選出具有豬糞便特異性的基因片段,以此設計引物并建立相應的PCR檢測方法,并對采集樣進行應用調(diào)查.競爭性雜交富集的豬糞便特異性基因文庫以擬桿菌群(Bacteroidetes)(43.2%)和梭狀桿菌群(Clostridia)(19.5%)相似序列為主,其蛋白功能主要分為3大類:與信息貯存與加工有關(guān)(7.6%),與細胞加工及信息傳導有關(guān)(12.8%)以及與代謝有關(guān)(22.0%).進一步針對功能蛋白序列篩選宿主糞便特異性分子標記.分析表明序列3-53-2對應引物可作為豬糞便污染的特異性分子標記,針對其建立的常規(guī)PCR方法對糞便DNA的檢出限可低至0.01 ng·μL-1,且對實際樣品具有較高的應用靈敏性(97%)和特異性.進一步對可能受污染水樣進行豬糞便污染特異性檢測,結(jié)果顯示不同地區(qū)的陽性檢出率高達75%~100%,證明了此方法的有效性,可為研究微生物示蹤技術(shù)在非點源污染方面的應用提供一定的基礎(chǔ)數(shù)據(jù).
[Abstract]:A large number of host-intestinal microorganism interaction genes will be formed in the long-term coevolution of intestinal microbial communities with their host, and these interaction genes have a certain host intestinal microorganism specificity. By using the designed molecular markers, the fecal pollution sources can be effectively identified. For the first time, competitive hybridization was used to enrich the porcine fecal specific gene, and the specific gene fragment was screened out from it. The primers were designed and the corresponding PCR detection method was established, and the applied investigation was carried out on the collected samples. In the porcine fecal specific gene library enriched by competitive hybridization, the similar sequences of Bacteroides (Bacteroidetes) () and Clostridium (Clostridia) () were 43.2% and 19.5%, respectively. Its protein function can be divided into three categories: information storage and processing (7.6%), cell processing and information transduction (12.8%) and metabolism (22.0%). Further screening the fecal specific molecular markers for the functional protein sequence. The results showed that the corresponding primer 3-53-2 could be used as a specific molecular marker for porcine fecal contamination. The detection limit of fecal DNA by conventional PCR method was as low as 0.01 ng 渭 L -1. And it has high application sensitivity (97%) and specificity to the actual samples. Furthermore, the specific detection of pig feces contamination in potentially polluted water samples was carried out. The results showed that the positive rate in different areas was as high as 75% and 100%, which proved the effectiveness of this method. It can provide some basic data for the application of microbial tracer technology in non-point source pollution.
【作者單位】： 浙江工商大學食品與生物工程學院;浙江省檢驗檢疫科學技術(shù)研究院;
【基金】：國家自然科學基金(No.31301492) 浙江省科技廳重點研發(fā)計劃項目(No.2015C02044)~~
【分類號】：X713;X830

【相似文獻】

相關(guān)碩士學位論文 前1條

1 樊麗華;基于宏基因組學的豬—腸道微生物互作靶點發(fā)掘及其微生物源追溯（MST）研究[D];浙江工商大學;2017年

，

本文編號：2397023