USDA-ARS, US Arid Land Agricultural Research Center, Maricopa, AZ 85138, USA.
FEMS Microbiol Lett. 2009 Oct;299(1):38-43. doi: 10.1111/j.1574-6968.2009.01745.x. Epub 2009 Aug 1.
Methods focused on members of the genus Bacteroides have been increasingly utilized in microbial source-tracking studies for identifying and quantifying sources of nonpoint fecal contamination. We present results using standard and real-time PCR to show cross-amplification of Bacteroides 16S rRNA gene molecular assays targeting human fecal pollution with fecal DNA from freshwater fish species. All except one of the presumptively human-specific assays amplified fecal DNA from at least one fish species, and one real-time PCR assay amplified DNA from all fish species tested. Sequencing of PCR amplicons generated from fish fecal DNA using primers from the real-time assay revealed no mismatches to the human-specific probe sequences, but the nucleotide sequences of clones from fish fecal samples differed markedly from those of human feces, suggesting that the fish-related bacteria may be different strains. Our results strongly demonstrate the potential for cross-amplification of human-specific PCR assays with fish feces, and may call into question the results of studies in which these Bacteroides-specific molecular markers are used to quantify human fecal contamination in waters where fish contribute to fecal inputs.
方法集中在拟杆菌属的成员已被越来越多地用于微生物源追踪研究，以识别和量化非点源粪便污染的来源。我们使用标准和实时 PCR 显示跨扩增的结果细菌 16S rRNA 基因分子检测针对人类粪便污染的淡水鱼类粪便 DNA。除了一个假定的人类特异性检测之外，所有的检测都从至少一种鱼类中扩增了粪便 DNA，并且一个实时 PCR 检测从所有测试的鱼类中扩增了 DNA。使用实时检测的引物从鱼粪便 DNA 中扩增的 PCR 产物的测序显示，与人类特异性探针序列没有错配，但来自鱼类粪便样本的克隆的核苷酸序列与人类粪便的明显不同，表明鱼类相关细菌可能是不同的菌株。我们的结果强烈证明了人类特异性 PCR 检测与鱼类粪便交叉扩增的可能性，这可能使人们对在鱼类粪便对粪便输入有贡献的水域中使用这些拟杆菌特异性分子标记来量化人类粪便污染的研究结果产生质疑。
