Validation and cost-effectiveness of an alternative method to quantify Batrachochytrium dendrobatidis infection in amphibian samples using real-time PCR

Abstract

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is considered one of the main causes of amphibian declines worldwide. Detection of Bd infection relies on both histological and molecular techniques, but only quantitative real-time PCR (qPCR) provides quantitative information on intensity of infection. To quantify Bd infection, Boyle et al. (2004) developed a standard method using TaqMan qPCR assay: however, the high costs of this method can hinder its applicability for studies with limited resources. Starting from the method by Boyle et al. (2004), we set up and fully validated a qPCR assay based on modified primers and SYBR Green chemistry. The modified SYBR Green has high sensitivity and specificity, as well as the ability to quantify Bd infection loads as well as the standard TaqMan qPCR, but it is more than 60% cheaper. This is obtained both by intrinsic cost-effectiveness of the SYBR Green technique and by reducing reaction volumes of qPCR. Therefore, this modified qPCR assay constitutes a cheaper molecular method to quantify Bd in amphibians.