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Title: Variation in inhibitor effects on qPCR assays and implications for eDNA surveys
Authors: Lance, Richard F., 1967-
Guan, Xin.
Keywords: Microbial ecology
Molecular microbiology
Environmental monitoring
Polymerase chain reaction
Aquatic habitats
Bighead carp
Silver carp
Publisher: Environmental Laboratory (U.S.)
Engineer Research and Development Center (U.S.)
Series/Report no.: Miscellaneous Paper (Engineer Research and Development Center (U.S.)) ; no. ERDC/EL MP-21-9
Is Version Of: Lance, Richard F., and Xin Guan. "Variation in inhibitor effects on qPCR assays and implications for eDNA surveys." Canadian Journal of Fisheries and Aquatic Sciences 77, no. 1 (2020): 23-33.
Abstract: Aquatic environmental DNA (eDNA) surveys are sometimes impacted by polymerase chain reaction (PCR) inhibitors. We tested varying concentrations of different inhibitors (humic, phytic, and tannic acids; crude leaf extracts) for impacts on quantitative PCR (qPCR) assays designed for eDNA surveys of bighead and silver carp (Hypophthalmichthys nobilis and Hypophthalmichthys molitrix). We also tested for inhibition by high concentrations of exogenous DNA, hypothesizing that DNA from increasingly closely related species would be increasingly inhibitory. All tested inhibitors impacted qPCR, though only at very high concentrations — likely a function, in part, of having used an inhibitor-resistant qPCR solution. Closer phylogenetic relatedness resulted in inhibition at lower exogenous DNA concentrations, but not at relatively close phylogenetic scales. Inhibition was also influenced by the qPCR reporter dye used. Importantly, different qPCR assays responded differently to the same inhibitor concentrations. Implications of these results are that the inclusion of more than one assay for the same target taxa in an eDNA survey may be an important countermeasure against false negatives and that internal positive controls may not, in the absence of efforts to maximize inhibition compatibility, provide useful information about the inhibition of an eDNA assay.
Description: Miscellaneous Paper
Gov't Doc #: ERDC/EL MP-21-9
Rights: Approved for Public Release; Distribution is Unlimited
Size: 16 pages / 1.04 MB
Types of Materials: PDF/A
Appears in Collections:Miscellaneous Paper

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