The Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, Division of Environmental Health Engineering, and Johns Hopkins Center for Water and Health, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Water Res. 2012 Sep 1;46(13):4281-91. doi: 10.1016/j.watres.2012.04.030. Epub 2012 May 16.
Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, the method 1) employs a commercially available standard RNA control; 2) defines inhibition by the change in the quantification cycle (C(q)) of the standard RNA control when added to the sample concentrate; and 3) calculates a dilution factor using a mathematical formula applied to the change in C(q) to indicate the specific volume of nuclease-free water necessary to dilute the effect of inhibitors. The standardized inhibition method was applied to 3,193 large-volume water (surface, groundwater, drinking water, agricultural runoff, sewage) concentrates of which 1,074 (34%) were inhibited. Inhibition level was not related to sample volume. Samples collected from the same locations over a one to two year period had widely variable inhibition levels. The proportion of samples that could have been reported as false negatives if inhibition had not been mitigated was between 0.3% and 71%, depending on water source. These findings emphasize the importance of measuring and mitigating inhibition when reporting qPCR results for viral pathogens in environmental waters to minimize the likelihood of reporting false negatives and under-quantifying virus concentration.
在环境水样，特别是大容量水样的 qPCR 和 RT-qPCR 分析中，天然存在的抑制化合物是一个主要关注点。这里描述了一种用于测量和减轻环境水样浓缩物中样品抑制的标准化方法。具体来说，该方法 1）采用市售的标准 RNA 对照；2）通过将标准 RNA 对照添加到样品浓缩物时定量循环 (C(q)) 的变化来定义抑制作用；3）使用应用于 C(q)变化的数学公式计算稀释因子，以指示用无核酸酶水稀释抑制剂影响所需的特定体积。标准化抑制方法应用于 3193 个大容量水（地表水、地下水、饮用水、农业径流、污水）浓缩物，其中 1074 个（34%）受到抑制。抑制水平与样品体积无关。在一年到两年的时间内从同一地点采集的样本具有广泛变化的抑制水平。如果没有减轻抑制作用，可能被报告为假阴性的样本比例在 0.3%到 71%之间，具体取决于水源。这些发现强调了在报告环境水中病毒病原体的 qPCR 结果时测量和减轻抑制作用的重要性，以最大程度地减少报告假阴性和低估病毒浓度的可能性。
