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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF DAIRY FORAGE AND MANURE MANAGEMENT TO REDUCE ENVIRONMENTAL RISK Title: Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

Authors
item Gibson, Kristen -
item Schwab, Kellogg -
item Spencer, Susan
item Borchardt, Mark

Submitted to: Water Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 19, 2012
Publication Date: May 16, 2012
Citation: Gibson, K.E., Schwab, K.J., Spencer, S.K., Borchardt, M.A. 2012. Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples. Water Research. 46:4281-4291.

Interpretive Summary: One approach for measuring human and livestock pathogens in the environment is to detect their DNA using a method called quantitative polymerase chain reaction (qPCR). This method is advantageous in that it is sensitive to low densities of pathogens and it is specific to pathogen type. However, its primary shortcoming with environmental samples, like water or soil, is the presence of molecules that block or inhibit the test. In this paper we describe an approach for measuring the level of qPCR inhibition in environmental samples. The approach is simple and can easily be transferred to other laboratories. In collaboration with scientists at Johns Hopkins University, we measured inhibition in 3,195 samples collected from groundwater, rivers and lakes, sewage, drinking water, and agricultural runoff. We found 33% of the samples were inhibited. Even at the same sample site, inhibition level changed over time, demonstrating that one test for inhibition is not sufficient; every sample needs to be checked. Inhibition can be reduced by diluting the concentrated sample with pure water just before the qPCR test. Our method calculates the precise dilution needed to remove the inhibitors without diluting so much that the target pathogen can no longer be detected. We show that if we had not made the correct dilution, depending on the source of the samples, 0.3% to 71% of the samples could have been called negative for human viruses when in fact they were positive. When sample inhibition causes a false-negative result, a potential disease transmission source, for example a swimming beach, could be incorrectly deemed safe when it is not. Measuring qPCR inhibition is important for accurately characterizing the risk of disease transmission from environmental sources, thereby protecting the health of people and livestock.

Technical Abstract: 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 (Cq) 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 Cq to indicate the specific volume of nuclease-free water necessary to dilute the effect of inhibitors. The standardized inhibition method was applied to 3,195 large-volume water (surface, groundwater, drinking water, agricultural run-off, sewage) concentrates of which 1,072 (33%) 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 and RT-qPCR results for viral pathogens in environmental waters, minimizing the likelihood of reporting false negatives or under-quantifying virus concentration.

Last Modified: 10/1/2014
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