“Implementación y evaluación de un ensayo de PCR para la detección del agente causal de la quitridiomicosis”

Abstract

Chytridiomycosis is an emerging disease that has caused worldwide decline of at least 500 species of amphibians. Since the description of the causative agent of this mycosis, Batrachochytrium dendrobatidis, several diagnostic techniques have been developed, among them the most used is the polymerase chain reaction (PCR) test. In Ecuador, there are few institutions in which reliable diagnosis of this pathogen can be made. In the present work a multiple conventional PCR test was developed, which detects genetic material of the fungus and amphibians. Several tests were conducted to verify the performance of the universal amphibian primers and the fungus from frog samples and thus establish a protocol for diagnosis. The ANFFor/ANFRev oligonucleotide pair was used for amplification of amphibian DNA and Bd1a/Bd2a for the fungus The amplification of amphibian genetic material functioned as a process control, allowing us to know if the sample was taken properly and if the DNA extraction, prior to amplification, was appropriate. The diagnostic test was conducted in 85 frog samples from five locations in Azuay. The diagnostic sensitivity and specificity of the assay was evaluated, comparing the results with those obtained under the real-time PCR test, in which ITS1-3Bd/5.8SBd primers were used. A 14.29% sensitivity and 95% specificity were obtained. The prevalence of the disease was obtained according to the results of the real-time PCR and was 25.93%.