Sensitive detection of sample interference in environmental qPCR

• Published in: Water research (Oxford) Vol. 46; no. 10; pp. 3251 - 3260
• Main Authors: Green, Hyatt C., Field, Katharine G.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 15.06.2012; Elsevier
• Subjects: Amplification; Applied sciences; Assaying; Bacteroides; DNA, Bacterial - genetics; Environmental Microbiology; Escherichia coli - genetics; Estimates; Ethanol; Ethanol - analysis; Ethyl alcohol; Exact sciences and technology; Green Fluorescent Proteins; Humans; Humic Substances - analysis; Inhibition; Inhibitors; Kinetic outlier detection; Microbial ecology; Microbial source tracking; Pollution; Polymerase Chain Reaction - methods; qPCR; Reference Standards; Water quality; Water treatment and pollution; Microbial ecology; qPCR; Kinetic outlier detection; Microbial source tracking; Water quality; Bacteroides; Chromosomal aberration; Microbiology; Environmental policy; Escherichia coli; Identification; Molecular marker; Sample preparation; Health and environment; Pathogenic; Bacteria; Control method; Biological contamination; Public health; Enterobacteriaceae; Chromosome insertion; Interference; Polymerase chain reaction; DNA; Green fluorescent protein; Pollution source; Abnormal chromosome; Regulation; Mutation
• ISSN: 0043-1354; 1879-2448
• DOI: 10.1016/j.watres.2012.03.041

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-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR. [Display omitted] ► A full-process control with kinetic outlier detection (KOD) methods detected qPCR inhibition. ► The approach estimated qPCR inhibition and DNA recovery with one control. ► Thus, the approach is simple and cheap. ► For water quality, the methods prevent under-estimation of contaminants due to sample interference. ► This will be broadly useful in environmental qPCR applications of all kinds.