基于宏基因组学的畜禽粪便微生物污染来源识别研究

Livestock and poultry feces are one of the main factors leading to water pollution in China. For this reason, accurate source identification is particularly important in pollution control and management. In this project, we aim to identify fecal pollution source at Pearl River Delta region by using metagenomic technology. Fecal samples will be firstly collected around selected area. Following metagenomic sequencing, 16S rRNA gene clone libraries and microbial community diversity analysis, functional genes will be distinguished from dominant population and primers will be designed based on BLASTx match. For specific optimization of designed primer, PCR assay will be tested against DNA extract from target and nontarget fecal sample. After that, effect of selected antibiotics on community structures and host-specific fecal indicator will be quantitative investigated. Attenuation dynamic characteristics of genetic markers in simulated artificial river device will also be demonstrated by real-time PCR. This study will provide scientific support for quantitative identification of microbial source tracking in field.

畜禽粪便造成了地表水体普遍存在微生物污染，及时有效的针对性源头控制是解决问题的关键环节。微生物源解析技术为污染来源的快速识别提供新的思路，却存在特异性生物标记种类单一和地域性差异的局限。针对这一问题，本项目拟选取珠江三角洲地区为目标研究区域，将宏基因组学方法有机的运用到特异性生物标记探寻的整个过程中，高效提取样品DNA后以杂交法构建宏基因克隆文库，结合限制性片段长度多态性技术（T-RFLP）分析粪便中的微生物群落结构特征，继而针对优势菌群的16S rRNA基因进行多序列对位分析，识别具有宿主特征的特异性生物标记，以实时定量PCR法揭示不同宿主的特异性生物标记在自然水体中的入河衰减动态特征规律。本研究旨在研发多种兼具宿主特异性、灵敏性、稳定性和适应目标研究区域的畜禽粪便生物标记，为今后系统化研究水体环境的微生物源解析技术提供科学依据和支撑。

畜禽粪便造成了地表水体普遍存在微生物污染，及时有效的针对性源头控制是解决问题的关键环节。微生物源解析技术为污染来源的快速识别提供新的思路，却存在特异性生物标记种类单一、地域性差异，且其在水体中的稳定性直接影响到定量的准确性及应用的适应性。针对以上问题，本项目选取珠江三角洲地区为目标研究区域，广泛提取了流域内人及常见畜禽粪便的DNA样品，分别筛选并识别出用于定性和定量分析的猪、牛、鸡等常见畜禽和人的宿主特异性生物标记，揭示了溶解氧、营养水平等环境条件对宿主特异性生物标记在自然水体中的衰减动态特征规律，阐明了大肠埃希氏菌、拟杆菌及特异性生物标记等指标用于评价水体微生物污染的适用范围。项目研究成果可为今后系统化研究水体环境的微生物源解析技术提供科学依据和支撑。