Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
J Microbiol Biotechnol. 2010 Feb;20(2):245-53.
The base sequences representing human and cow-specific 16S rRNA gene markers identified in the T-RFLP analysis were recovered from clone libraries. The human and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. AllBac primer set showed positive results for all human, cow, and pig samples, while human-specific primer set showed positive result only for human sample but not for cow or pig samples. Likewise, cow-specific primer set showed positive results only for cow sample but not for human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human and cow-specific markers were detected in the order of 9 log(10) copies per gram wet feces which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in 1.7-6.2 log(10) copies/100 ml water which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.
从克隆文库中回收了在 T-RFLP 分析中确定的代表人类和牛特有的 16S rRNA 基因标记的碱基序列。从这些序列设计了人类和牛特异性引物，并用人、牛和猪的粪便 DNA 分析了它们的特异性。AllBac 引物组对所有人类、牛和猪样本均呈阳性结果，而人类特异性引物组仅对人类样本呈阳性结果，而对牛或猪样本则呈阴性。同样，牛特异性引物组仅对牛样本呈阳性结果，而对人类或猪样本则呈阴性。使用这些引物开发了实时 PCR 检测法，用于鉴定和量化河水中的粪便污染。以每克湿粪便 9 个对数（10）拷贝的顺序检测到人类和牛特异性标记物，这比总拟杆菌的数量低两个数量级。对于河水样本，人类特异性标记物的检测范围为 1.7-6.2 个对数（10）拷贝/100 毫升水，这比总拟杆菌的数量低两个数量级。总拟杆菌与传统粪便指标之间没有显著相关性，但拟杆菌与人类特异性标记物之间存在高度相关性。该检测法可可靠地识别和量化粪便污染源，从而在流域内采取有效措施并促进水质管理。
