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[目的]建立重复序列聚合酶链反应(rep-PCR)微生物源追踪方法,应用于淮河流域某水库的粪便污染来源追踪。[方法]采集已知来源的人和动物粪便标本以及水样标本,分离并确认指示菌大肠杆菌,以BOXA1R为引物进行PCR扩增;用Bionumerics 4.0软件对rep-PCR基因指纹图进行判别分析和多元方差分析并计算各源种类的正确归类率。[结果]2分类、4分类和10分类的正确归类率分别为:85.60%、79.20%和70.98%。判别分析和多元方差分析结果显示rep-PCR基因指纹图能将已知源不同来源指示菌区分开。水样中主要粪便污染来源为猪、鸡和人。[结论]建立的rep-PCR微生物源追踪方法能较好地区分水体中指示菌的不同来源,该方法的建立为查找水体中粪便污染来源以及水体治理提供了新技术。
[Objective] The purpose of this study was to establish a method of trace-source PCR for rep-PCR and to trace the source of faecal contamination in a reservoir in the Huaihe River Basin. [Methods] Human and animal stool samples and water samples from known sources were collected. Escherichia coli was isolated and confirmed, and BOXA1R was used as a primer for PCR amplification. The fingerprint of rep-PCR gene was discriminated by Bionumerics 4.0 software and Multivariate analysis of variance and calculation of the correct classification of each source category. [Result] The correct classification rates of Category 2, Category 4 and Category 10 were 85.60%, 79.20% and 70.98% respectively. Discriminant analysis and multivariate analysis of variance showed that fingerprinting of the rep-PCR gene could distinguish between different sources of known sources. The main source of faecal contamination in water samples is pigs, chickens and humans. [Conclusion] The established rep-PCR method of microbial source tracking can distinguish different sources of indicator bacteria in water well. The establishment of this method provides a new technology for finding the source of faecal contamination in water body and the treatment of water body.