Shanks Orin C, Atikovic Emina, Blackwood A Denene, Lu Jingrang, Noble Rachel T, Domingo Jorge Santo, Seifring Shawn, Sivaganesan Mano, Haugland Richard A
U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45268, USA.
Appl Environ Microbiol. 2008 Feb;74(3):745-52. doi: 10.1128/AEM.01843-07. Epub 2007 Dec 7.
Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10(6) copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.
准确评估与牛粪便污染相关的健康风险需要可靠的宿主特异性遗传标记和快速定量方法。我们报告了用于检测两种最近描述的牛粪便特异性遗传标记的定量PCR检测方法的开发，以及使用马尔可夫链蒙特卡罗方法对这些标记进行计数的方法。两种检测方法的定量范围均为25至2×10⁶个靶DNA拷贝，变异系数<2.1%。其中一种检测方法可与内部扩增对照进行多重检测，以同时检测牛特异性遗传靶标和扩增抑制剂的存在。当针对来自16种不同动物物种的204份粪便DNA提取物进行测试时，这些检测方法仅检测到牛粪便样本，并且在从五个地理上不同的地点收集的单个牛样本中也显示出广泛的分布（98%至100%）。在48个个体样本中测量了每种牛特异性遗传标记的丰度，并与定量PCR计数的同一标本中代表总拟杆菌、嗜热栖热放线菌和肠球菌的rRNA基因序列数量进行了比较。可接受的检测性能以及不同牛群中DNA靶标的普遍性提供了实验证据，表明这些定量检测方法将有助于监测环境水体中的牛粪便污染。
