Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

K. E. Gibson, K. J. Schwab, S. K. Spencer, M. A. Borchardt

Research output: Contribution to journalArticle

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 3,193 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.

Original languageEnglish (US)
Pages (from-to)4281-4291
Number of pages11
JournalWater Research
Volume46
Issue number13
DOIs
StatePublished - Sep 1 2012

Keywords

  • Environmental sampling
  • PCR inhibition
  • QPCR
  • Waterborne viruses

ASJC Scopus subject areas

  • Ecological Modeling
  • Water Science and Technology
  • Waste Management and Disposal
  • Pollution

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