User-friendly Taqman probe coupled-insulated isothermal PCR (iiPCR) for rapid detection of emerging Ambystoma tigrinum virus (ATV) in western tiger salamanders (Ambystoma mavortium) on a compact, portable instrument

Abstract

Portable user-friendly diagnostic tests can benefit detection and surveillance of wildlife diseases. Here, the performance of a compact POCKIT™ Nucleic Acid Analyzer for detection of Ambystoma tigrinum virus (ATV), an emerging Iridovirus that is associated with high host mortality in the western tiger salamander (Ambystoma mavortium) in North America was assessed. Tissue samples from 188 larval tiger salamanders collected from sites in Alberta, Canada were tested by both iiPCR and by conventional PCR. Results of the two assays showed 96.3% agreement. All 176 samples that tested positive by conventional PCR were also positive by iiPCR, while 12 of the samples that were negative by conventional PCR were positive by iiPCR. Comparison of the limits of detection of the two assays shows that the iiPCR assay was more sensitive than conventional PCR and had a LOD95 of 20 copies per reaction. The instrument automatically analyzes and displays results within 40min following nucleic acid extraction. The novel technology could enhance detection of, and response to, wildlife pathogens, particularly those that occur sporadically, cause rapid outbreaks, and/or occur within isolated geographical regions.