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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.
Intestinal microflora in the course of its long-term co-evolution with its host will form a large number of host-gut microbiota interaction genes, which have a certain host intestine microbial specificity, the use of its molecular markers can effectively identify stool contamination source. In this study, we first used competitive hybridization method to enrich pig feces-specific genes, and then screened out specific fragments of pig feces, designed primers and established corresponding PCR detection methods, and applied the sample survey. The pig feces-specific gene library enriched by sexual hybridization is mainly composed of Bacteroidestes (43.2%) and Clostridia (19.5%), and the protein functions are mainly divided into three categories: Information storage and processing related to processing (7.6%), related to cell processing and information transduction (12.8%) and metabolic (22.0%) .Furthermore, screening of host-specific stool-specific molecular markers for functional protein sequences showed that the sequence 3-53- The corresponding primers can be used as specific molecular markers for pig manure contamination. The detection limit of the standard PCR method for stool DNA can be as low as 0.01 ng · μL-1, The product has high sensitivity (97%) and specificity.Furthermore, the specific detection of pig feces contamination may be carried out on contaminated water samples, the results show that the positive detection rate in different regions is as high as 75% to 100%, proving this The validity of the method can provide some basic data for studying the application of microbial tracing technology in non-point source pollution.