水库水非点源粪便污染微生物源追踪法鉴定

Use of microbial source tracking to identify non-point fecal pollution in reservior water

目的应用细菌基因组重复序列PCR技术(rep-PCR)微生物源追踪方法对江苏省盱眙县桂五水库中粪便污染来源进行追踪。方法分别采集不同季节水库周边已知来源粪便标本,分离大肠埃希菌作为指示菌,建立已知污染源指示菌rep-PCR特征指纹库,用基因聚类分析软件计算平均正确归类率,进行判别分析和多元方差分析;同期采集水库水样,分离并确认指示菌,进行rep-PCR扩增,与已知污染源数据库进行对比,判断水样中指示菌污染来源。结果将已知源数据库分为2,3,4,5和9类时,平均正确归类率分别为89.19%,77.58%,76.69%,75.25%和70.92%;已知源数据库可区分指示菌的不同来源。对534株水库水样指示菌微生物源追踪结果显示,人、鸡、鸭、鹅、狗、猪、牛、羊和野生动物各占26.49%,14.74%,7.77%,5.78%,3.98%,7.97%,10.96%,8.76%和8.57%。结论桂五水库粪便污染来源种类繁多,其中人、鸡和牛为主要污染源;该方法为查找水体粪便污染来源及污染整改效果评估提供了新技术。

Objective To track fecal pollution sources in Guiwu reservoir at Xuyu,Jiangsu province with repetitive extragenic palindromic-polymerase chain reaction（rep-PCR） microbial source tracking method（MST）.Methods Fresh fecal samples from human and animals（poultry,livestock,dogs,and wild animals）were collected in villages around Guiwu reservoir in four seasons in a year.Escherichia coli were isolated and identified by API20E as the indicator.A known-source fingerprinting library containing all kinds of animal and human feces possibly polluting the water of the reservoir was developed.Then the library with Bionumerics 4.0 was used to calculate average rate of correct classification.Multivariate analysis of variance（MANOVA）and discriminant analysis were also conducted to evaluate the ability of discrimination of the library.Water samples were collected once a month,and Escherichia coli were isolated with membrane filtration and identified with API20E.The Escherichia coli wase subject to rep-PCR amplification.The fecal pollution sources were identified statistically by compared the rep-PCR fingerprinting of each Escherichia coli from water sample with those from the known-source library.Results When the known-source fingerprint library was classified as 2,3,4,5 and 9 groups,the average rates of correct classification were 89.19%,77.58%,76.69%,75.25%,and 70.92%,respectively.MANOVA and discriminant analysis showed that the division of different groups was clearly.Five hundred and thirty-four indicators from water samples were identified for theirs pollution sources from human（26.49%）,chicken（14.74%）,duck（7.77%）,goose（5.78%）,dog（3.98%）,pig（7.97%）,cattle（10.96%）,goat（8.76%）,and wild animals（8.57%）,respectively.Conclusion The results showed that there are lots of sources of fecal pollution in water of Guiwu reservoir with human,chicken and cattle as the main contributors of the pollution.The MST is a new and useful method for tracking and evaluating the source of fecal pollution in nature water.