A reverse transcription-insulated isothermal PCR assay for the detection of duck hepatitis A virus type 3 based on the POCKIT™ system

Abstract Duck hepatitis A virus type 3 (DHAV-3) infection is characterized by severe hepatitis. In recent years, DHAV-3 has become widespread in Asia and has led to economic losses. Conventional methods for DHAV-3 detection usually depend on the use of larger equipment that is not portable and is not fit for on-site diagnoses. In this study, a rapid reverse transcription insulated isothermal (RT-iiPCR) technique was developed for the on-site detection of DHAV-3 based on the POCKIT™ system in a convenient device. The concentration of primer pairs and probes were optimized for amplification of the DHAV-3 VP3 gene of DHAV-3, with no amplification of 12 other duck pathogens. The detection limit of viral RNA was 3.85 × 10 1 copies/μL, and the analytical sensitivity and specificity levels were both 100% in the detection of 40 liver samples. Furthermore, 97.5% of the RT-iiPCR results were in agreement with those of rRT-PCR, with a kappa value of 0.93. This method is time-saving and better suited to field diagnoses because of its portable device.