Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8.
Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10 mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200 ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (ΔC(T)) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring.
Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
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Schriewer
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Wehlmann
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Wuertz
D005591:Chemical Fractionation / Q000379:methods; D004783:Environmental Microbiology; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006812:Humic Substances; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D006812:Humic Substances