Characterizing Quantitative PCR Inhibition Due to Melanin and Humic Acid for the Detection of Ophidiomyces ophiodiicola
Ophidiomycosis, first described in 2006, poses a threat to the health of wild snake populations in the United States.2 Currently, quantitative PCR (qPCR) of cutaneous swabs is the preferred method for detection of the causative fungus, Ophidiomyces ophiodiicola;1 however, swabs may be contaminated with substances that inhibit qPCR. For example, humic acid (found in the environment) and melanin (found within snake skin) have been shown to yield falsely low DNA quantities when present in qPCR reactions.3,6 The objective of this study was to characterize the inhibition of an Ophidiomyces-specific qPCR assay by humic acid and melanin. We ran qPCR reactions with DNA concentrations ranging from 101 to 107 fungal copies, and melanin or humic acid concentrations ranging from 0.5–50 ng/µl. Melanin concentrations of 4–10 ng/µl completely inhibited qPCR only at the lowest DNA dilution, while concentrations above 10 ng/µl were completely inhibitory across all DNA concentrations. Humic acid concentrations 5–15 ng/µl completely inhibited qPCR at the lowest DNA dilution, while concentrations >15 ng/µl were inhibitory across all DNA concentrations. Partial inhibition was observed at the lowest Ophidiomyces DNA dilution by 1.5 ng/µl of melanin and 2.5 ng/µl of humic acid, and qPCR efficiency declined as inhibitor concentration increased. These results demonstrate that inhibitors in environmental samples, such as melanin and humic acid, can alter interpretation of qPCR results for O. ophiodiicola. This inhibition can result in underestimation of the prevalence and distribution of Ophidiomycosis, leading to significant management consequences.4,5
1. Allender M, Bunick D, Dzhaman E, Burrus L, Maddox C. Development and use of a real-time polymerase chain reaction assay for the detection of Ophidiomyces ophiodiicola in snakes. J Vet Diagn Invest. 2015;27(2):217–220.
2. Allender M, Raudabaugh D, Gleason F, Miller A. The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on free-ranging snake populations. Fungal Ecol. 2015;17:187–196.
3. Eckhart L, Bach J, Ban J, Tschachler E. Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. Biochem Biophys Res Commun. 2000;271:726–730.
4. Hileman ET, Allender MC, Bradke DR, Faust LJ, Moore JA, Ravesi MJ, Tetzlaff SJ. Estimation of Ophidiomyces prevalence to evaluate snake fungal disease risk. J Wildl Manage. 2018;82:173–181.
5. Kosch T, Summers K. Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay. Mol Ecol Resour. 2013;13(2):230–236.
6. Sutlovic D, Gamulin S, Deﬁnis-Gojanovic M, Gugic D, Andjelinovic S. Interaction of humic acids with human DNA: proposed mechanisms and kinetics. Electrophoresis. 2008;29:1467–1472.