Cao Yiping, Raith Meredith R, Griffith John F
Department of Microbiology, Southern California Costal Water Research Project Authority;
Department of Microbiology, Southern California Costal Water Research Project Authority.
J Vis Exp. 2016 Mar 9(109):53611. doi: 10.3791/53611.
This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e., simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.
本手稿描述了一种用于同时定量肠球菌属和人粪便相关HF183标志物的双重数字PCR检测方法（EntHF183 dPCR）。EntHF183双重dPCR检测方法（以下简称EntHF183 dPCR）使用与其已发表的单个定量PCR（qPCR）对应方法相同的引物和探针序列。同样，在进行dPCR之前，遵循与qPCR之前相同的水过滤和DNA提取程序。然而，双重dPCR检测方法相对于qPCR检测方法具有几个优点。最重要的是，dPCR检测方法通过直接对其靶标进行定量，无需绘制标准曲线，从而消除了相关的偏差和变异性。此外，虽然在qPCR中对肠球菌和HF183进行双重定量（即同时定量）通常会导致对样品中丰度较低的靶标严重低估，但dPCR能够对两个靶标进行一致的定量，无论是在同一反应中单独定量还是同时定量。dPCR检测方法还能够耐受比qPCR高1至2个数量级的PCR抑制剂浓度。这些优点使得EntHF183 dPCR检测方法特别有吸引力，因为它无需进行两次单独的qPCR检测，就能同时提供有关环境水体中一般和人相关粪便污染的准确且可重复的信息。尽管dPCR相对于qPCR具有优势，但目前可用仪器的dPCR检测方法的定量上限比qPCR可达到的定量上限低约4个数量级。因此，对于高浓度靶标生物（如污水泄漏后通常观察到的那些）的测量需要进行稀释。
