U.S. Environmental Protection Agency, Office of Research and Development, National Exposure Research Laboratory, 26 West Martin Luther King Drive, Cincinnati, OH 45268, USA.
Water Res. 2012 Nov 15;46(18):5989-6001. doi: 10.1016/j.watres.2012.08.017. Epub 2012 Aug 27.
A quantitative polymerase chain reaction (qPCR) method for the detection of enterococci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of water quality at recreational beaches. In this study we further examined the applicability of the method for analyses of diverse inland waters as well as tropical marine waters from Puerto Rico based on the frequencies of samples showing presumptive PCR interference. Interference was assessed by salmon DNA sample processing control (SPC) and internal amplification control (IAC) assay analysis results and pre-established acceptance criteria of <3.0 and <1.5 cycle threshold (Ct) offsets from control samples, respectively. SPC assay results were accepted in analyses of 93% of the inland water samples whereas the criterion was met at frequencies of 60% and 97% in analyses of samples from Puerto Rico in two different years of sampling. The functionality of the control assays and their acceptance criteria was assessed on the basis of relative recovery estimates of spiked enterococci target organisms extracted in the presence of water sample filters and sample-free control filters and was supported by observations that recovery estimates from the water sample and control filters were substantially different for samples that failed these criteria. Through the combined use of the SPC and IAC assays, two presumptive types of interference were identified. One type, observed in the tropical marine water samples, appeared to primarily affect the availability of the DNA templates for detection. The second type, observed in river water samples, appeared to primarily affect PCR amplification efficiency. In the presence of DNA template interference, adjustments from SPC assay results by the ΔΔCt comparative Ct calculation method decreased the variability of spiked enterococci recovery estimates and increased the similarity with control filters as compared to unadjusted recovery estimates obtained by the ΔCt calculation method. Use of a higher salmon DNA concentration in the extraction buffer also reduced this type of interference. The effects of amplification interference were largely reversed by dilution of the DNA extracts and even more effectively by the use of an alternative, commercial PCR reagent, designed for the analysis of environmental samples.
一种定量聚合酶链反应（qPCR）方法已被证明可用于检测粪便指示菌肠球菌，适用于分析温带淡水（大湖）和沿海水域，并可根据娱乐海滩的水质风险进行评估。在这项研究中，我们进一步研究了该方法在分析不同内陆水域以及波多黎各热带沿海水域时的适用性，方法是根据显示推定 PCR 干扰的样品频率进行评估。干扰通过鲑鱼 DNA 样品处理对照（SPC）和内部扩增对照（IAC）分析结果以及分别来自对照样品的<3.0 和<1.5 循环阈值（Ct）偏移的预先建立的可接受标准来评估。在分析 93%的内陆水样本时，SPC 分析结果是可接受的，而在两年的采样中，波多黎各样本的分析结果的符合率分别为 60%和 97%。控制分析的功能及其可接受标准是基于在存在水样过滤器和无样品控制过滤器的情况下提取的目标肠球菌目标生物的相对回收率估算来评估的，并得到了以下观察结果的支持：对于不符合这些标准的样品，水样和对照过滤器的回收率估算值有很大差异。通过同时使用 SPC 和 IAC 分析，确定了两种推定的干扰类型。一种类型，在热带海洋水样中观察到，似乎主要影响检测的 DNA 模板的可用性。第二种类型，在河水样本中观察到，似乎主要影响 PCR 扩增效率。在存在 DNA 模板干扰的情况下，通过 ΔΔCt 比较 Ct 计算方法对 SPC 分析结果进行调整，降低了添加肠球菌回收率估算值的变异性，并与通过 ΔCt 计算方法获得的未调整回收率估算值相比，增加了与对照过滤器的相似性。在提取缓冲液中使用更高浓度的鲑鱼 DNA 也减少了这种类型的干扰。通过稀释 DNA 提取物，甚至通过使用专为环境样本分析设计的替代商业 PCR 试剂，可以在很大程度上逆转扩增干扰的影响。
