Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay T. A. KOSCH and K. SUMMERS Department of Biology, East Carolina University, Howell Science Complex, Greenville, NC, USA Abstract Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its nonin- vasiveness, high sensitivity and ease of use. However, PCR is not without problems – to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean-up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detec- tion may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method. Keywords: amphibians, Batrachochytrium dendrobatidis, chytridiomycosis, DNA extraction, humic acid, PCR assay, PCR inhibition Received 7 September 2012; revision received 29 October 2012; accepted 2 November 2012 Introduction Chytridiomycosis is an infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendro- batidis (hereafter Bd). This disease has contributed to major declines and extinctions of amphibians worldwide (Fisher et al. 2009). Since its discovery and subsequent description, detection techniques for chytridiomycosis have improved considerably in reliability and sensitivity. However, as we will demonstrate, there are still many issues with the Bd assay that need to be resolved before it will be up to par with the quality of standard assays for most wildlife diseases. Despite the high sensitivity and relative ease of use of the PCR method for Bd detection, the reliability of this procedure is often plagued by a high probability of false negatives (e.g. up to 72.5% of reactions in one study, Gar- land et al. 2010). Wrongly diagnosing population status as Bd free can have devastating consequences, especially given that disease management decisions may be influ- enced by this type of data. One of the main contributors to PCR failure is reaction inhibition. Humic acids (HA) are cited as the main contributor to PCR inhibition in environmental samples (Wilson 1997), although other compounds such as fulvic acids, metals and polysaccha- rides can also have inhibitory effects (Tsai & Olson 1992). Humic acids are poly-phenolic compounds produced during the degradation of organic materials. These com- pounds are known to interfere with the binding of Taq polymerase (Wilson 1997), and have been shown to com- pletely inhibit PCR when present in quantities of as low as 1 ng per 50 lL reaction (Kermekchiev et al. 2009). Unfortunately, due to their physicochemical similarities to DNA (i.e. high molecular weight and negative charge), these compounds react in the same way as DNA mole- cules with the reagents used in most extraction proce- dures (e.g. DNeasy; Qiagen), ultimately leading to their copurification along with the target DNA (Tsai & Olson 1992). Correspondence: Tiffany A. Kosch, Fax: +1-252-328-4178; E-mail: koscht@ecu.edu © 2012 Blackwell Publishing Ltd Molecular Ecology Resources (2013) 13, 230–236 doi: 10.1111/1755-0998.12041