猪粪便污染特异性分子标记筛选及其PCR检测方法的建立
发布时间：2018-12-31 19:34
【摘要】：肠道微生物群落在与其宿主长期协同进化过程中会形成大量的宿主-肠道微生物互作基因,这些互作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有效识别粪便污染源.本研究首次利用竞争性杂交的方法富集猪粪便特异性基因,从中筛选出具有猪粪便特异性的基因片段,以此设计引物并建立相应的PCR检测方法,并对采集样进行应用调查.竞争性杂交富集的猪粪便特异性基因文库以拟杆菌群(Bacteroidetes)(43.2%)和梭状杆菌群(Clostridia)(19.5%)相似序列为主,其蛋白功能主要分为3大类:与信息贮存与加工有关(7.6%),与细胞加工及信息传导有关(12.8%)以及与代谢有关(22.0%).进一步针对功能蛋白序列筛选宿主粪便特异性分子标记.分析表明序列3-53-2对应引物可作为猪粪便污染的特异性分子标记,针对其建立的常规PCR方法对粪便DNA的检出限可低至0.01 ng·μL-1,且对实际样品具有较高的应用灵敏性(97%)和特异性.进一步对可能受污染水样进行猪粪便污染特异性检测,结果显示不同地区的阳性检出率高达75%~100%,证明了此方法的有效性,可为研究微生物示踪技术在非点源污染方面的应用提供一定的基础数据.
[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.
【作者单位】： 浙江工商大学食品与生物工程学院;浙江省检验检疫科学技术研究院;
【基金】：国家自然科学基金(No.31301492) 浙江省科技厅重点研发计划项目(No.2015C02044)~~
【分类号】：X713;X830
本文编号：2397023
[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.
【作者单位】： 浙江工商大学食品与生物工程学院;浙江省检验检疫科学技术研究院;
【基金】：国家自然科学基金(No.31301492) 浙江省科技厅重点研发计划项目(No.2015C02044)~~
【分类号】：X713;X830
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1 樊丽华;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[D];浙江工商大学;2017年
，本文编号：2397023
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