Converse Reagan R, Blackwood A Denene, Kirs Marek, Griffith John F, Noble Rachel T
Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell St., Morehead City, NC 28557, USA.
Water Res. 2009 Nov;43(19):4828-37. doi: 10.1016/j.watres.2009.06.036. Epub 2009 Jun 24.
Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.
粪便指示菌（FIB，如大肠杆菌和肠球菌属）在确定娱乐用水中粪便污染来源方面的应用有限，因为它们无法区分人类粪便污染和非人类粪便污染。几种拟杆菌属已被提议作为潜在的替代指标。我们开发了一种快速、无需培养的方法，使用针对16S rRNA基因的定量PCR（QPCR）来定量粪便中的拟杆菌属。该检测方法专门针对并定量最常见的人类拟杆菌属。本文介绍了该方法的详细信息，包括对来自不同生物体的多种粪便样本的分析。还通过实验室实验测试了QPCR检测方法的特异性和性能，在该实验中，将人类污水和鸥粪接种到一系列环境水样中。使用QPCR测量粪便拟杆菌属、总肠球菌属、粪肠球菌、屎肠球菌和格氏肠球菌的浓度，通过膜过滤（MF）对总肠球菌属和大肠杆菌进行定量。添加了鸥粪的样本中总肠球菌属、大肠杆菌、粪肠球菌和格氏肠球菌高度浓缩，表明这些指标在动物粪便中很突出。另一方面，含有污水的样本中粪便拟杆菌属浓度较高，而添加了鸥粪的样本中该浓度相对较低。敏感性和特异性结果表明，快速粪便拟杆菌属QPCR检测方法可能是有效预测人类特异性粪便污染的存在和浓度的有用工具。
