Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR
Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR
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DOI：
10.1016/j.syapm.2010.06.001
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发表时间：
2010-10-01
影响因子：
3.4
通讯作者：
Shanks, Orin C.
中科院分区：
文献类型：
--
作者：
Haugland, Richard A.;Varma, Manju;Shanks, Orin C.
从人胃肠道中定量确定的拟杆菌目物种的分子方法可能具有重要的临床和环境应用，范围从感染的诊断到地表沃茨中的粪便源追踪。在本研究中，在开发qPCR试验时靶向小亚基核糖体RNA基因V2区的序列，以定量6种拟杆菌属和1种普雷沃菌属的DNA。计算机模拟和实验分析表明，每种检测试剂在检测预期种属的DNA时具有高度区分性。通过在10至4 x 10（4）个靶序列拷贝范围内循环阈值测量的变异系数小于2%，证明了每种试验的分析灵敏度、精密度和定量范围。这些检测被应用于评估人类和五个动物群体粪便中靶序列的发生和相对丰度，以及来自13个不同处理设施的14个污水样本。在人类排泄物中检测到高水平（>10（3）拷贝/ng总提取DNA）的每个物种的序列。在所有的污水样品中，每种测定也检测到序列，除了普雷沃氏菌序列外，显示样品之间浓度的高度相关性（R-2 >= 0.7）变化。相比之下，这些序列的出现和相对丰度谱在来自每个动物组的粪便样品中显著不同。这些结果表明，多个单独的拟杆菌属物种的分析可能是有用的，在确定人类粪便污染的环境沃茨。由Elsevier GmbH出版。
Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4 x 10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3) copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R-2 >= 0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters. Published by Elsevier GmbH.