• 关键词：竞争性杂交  宿主-微生物互作基因  宿主粪便特异性分子标记  常规PCR

• 基金项目：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）

• 樊丽华
• 浙江工商大学食品与生物工程学院, 杭州 310035

• 帅江冰
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 莫虹斐
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 曾若雪
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 金晨晨
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 张晓峰
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 陈青
• 浙江工商大学食品与生物工程学院, 杭州 310035

• 何永强
• 浙江省检验检疫科学技术研究院, 杭州 310016

• 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：Functional genes directly involved in host-microbial interactions are considered to be a potential pool of targets for host-specific genetic markers used for microbial source tracking (MST).This study applied a genome fragment enrichment (GFE) method to enrich for swine-specific metagenomic region that differed from fecal metagenome of other animal species.Based on top BLASTx hits in GenBank non-redundant database, the swine-specific sequences were putatively grouped into 19 bacterial classes including the predominant groups of Bacteroidetes-like sequences (43.2%) and Clostridia-like sequences (19.5%).Biochemical function annotation revealed that sequences were most frequently assigned to putative proteins associated with metabolism (22%), cellular processes (12.8%) and information storage and processing (7.6%).PCR assays based on enriched specific sequences were then developed and evaluated for tracking swine fecal pollution.One host-specific PCR assay restricted to swine was confirmed based on the genetic marker of 3-53-2.The detection limit of the PCR assay was determined as 0.01 ng·μL^-1of fecal DNA extracts.The PCR assay showed high specificity (81%) and sensitivity (97%) on clinical samples from different animal species.Moreover, in a panel of field samples of contaminated water, the high positive rates (75%~100%) of swine feces were reliably detected, indicating that the novel conventional PCR assay could be used for sensitive tracing of swine fecal pollution.

• Key words：genome fragment enrichment method  host-microbial interactions gene  host-specific maker  PCR assay

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