Identification and quantification of fecal pollutions by using genetic and chemical markers

粪便来源的微生物污染物的鉴定和定量是控制饮用水和娱乐用水质量的主要优先事项。在这项研究中，我们提出了一种新的PCR为基础的方法，利用肠道厌氧菌，拟杆菌-普雷沃氏菌属的16 S rRNA基因标记区分人，牛，猪粪便污染的环境沃茨，而无需培养指示生物。基于从每个粪便样品构建的16S rRNA基因克隆文库，分别鉴定了1个人特异性、3个牛特异性和2个猪特异性类杆菌-普雷沃氏菌16S rRNA遗传标记。宿主特异性标记表明，各宿主粪便中类杆菌-普雷沃菌群落的物种组成存在差异。此外，所有的主机特异性遗传标记，检测河水中收集的粪便污染频繁的地区。我们进一步开发了一种实时聚合酶链反应（qPCR）测定，以定量粪便和环境水样中的每个宿主特异性遗传标记。首先，我们设计了宿主特异性引物用于实时PCR检测，并验证了它们对人、牛和猪粪便样品的特异性。用实时荧光定量PCR检测脆弱B. fragilis纯培养物和质粒DNA的线性范围分别为4.0~4.0 × 10^5 copies/PCR和6.0 × 10^1~6.0 × 10^8 copies/PCR。使用简单的过滤方法，新开发的qPCR检测的定量限为10 - 70个遗传标记拷贝/100 mL。第二，我们将这种qPCR检测与每一个主机特异性引物集，以天然河流超过一年，并证实我们的qPCR检测可以区分人，牛，猪粪便污染源具有高度的特异性。这些结果表明，本文所述的qPCR测定应广泛适用于监测自然环境中特定粪便污染物的时空波动。

Identification and quantification of microbial contaminants of fecal origin are major priority in the control of drinking and recreational water qualities. In this study, we proposed a new PCR-based approach using 16S rRNA gene markers of enteric anaerobes, Bacteroides-Prevotella spp for discriminating human, cow, and pig fecal contamination in environmental waters without culturing indicator organisms. One human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers were identified based on 16S rRNA gene clone libraries constructed from each fecal samples, respectively. Host-specific markers suggested that there are species composition differences in Bacteroides-Prevotella community in feces of each host species. Additionally, all host-specific genetic markers were detected in river water collected from area frequently contaminated with fecal pollution. We further developed a real-time polymerase chain reaction (qPCR) assay to quantify each host-specific genetic marker in fecal and environmental water samples. First we designed host-specific primers for the real-time PCR assay and validated their specificity on human, cow, and pig fecal samples. The real-time PCR detection of serially diluted DNA extracted DNA from pure cultured B.fragilis and plasmid DNAs was linear ranging from 4.0 to 4.0×10^5 copies/PCR and 6.0×10^1 to 6.0×10^8 copies/PCR, respectively. Using a simple filtration method, the quantification limit of newly developed qPCR assay was 10-70 genetic marker copies/100mL. Second, we applied this qPCR assay with each host-specific primer set to ratural river for over one year and confirmed that our qPCR assay could discriminate human, cow, and pig sources of fecal contamination with high specificity. These results implied that the qPCR assay described here should be widely applicable for monitoring spatial and temporal fluctuations in specific fecal contaminations in natural environments.