Sensitive detection of sample interference in environmental qPCR

Water Research
Hyatt C Green, Katharine G Field

Abstract

Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inh...Continue Reading

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Citations

Apr 29, 2015·Molecular Ecology Resources·Jessica J EichmillerPeter W Sorensen
Aug 5, 2014·Analytical Chemistry·David KovářPetr Skládal
Feb 24, 2016·Environmental Science & Technology·Dan WangAlexandria B Boehm
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Sep 10, 2014·Environmental Science & Technology·Hyatt C GreenOrin C Shanks
Jan 28, 2016·PloS One·Irene Adrian-Kalchhauser, Patricia Burkhardt-Holm
Aug 11, 2017·Environmental Health and Toxicology·Hyun Jeong LimAhjeong Son
Aug 18, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Madalitso TsakamaXiaofeng Dai
Oct 13, 2018·Genome Génome / Conseil National De Recherches Canada·Bastian EgeterGuillermo Velo-Antón
Feb 12, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Yiqi CaoBaiyu Zhang
Aug 19, 2018·EcoHealth·Sydney P RudkoPatrick C Hanington
Jun 3, 2021·Life·Adrián Ruiz-VillalbaMaurice J B van den Hoff
Aug 13, 2021·Analytical Chemistry·Cong LiuChuixiu Huang

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