Development and evaluation of reverse transcription-insulated isothermal PCR assay to detect duck hepatitis A virus type A in liver samples using the POCKITTM system

Abstract

Duck hepatitis A virus (DHAV) infection is characterized by severe hepatitis. In recent years, DHAV-A has become widespread in Asia and has led to economic losses. Conventional methods of DHAV-A detection must often be performed in the laboratory with inconvenience equipment. We have developed a rapid reverse transcription insulated isothermal (RT-iiPCR) technique for the on-site detection of DHAV-A based on the POCKIT^TM system in a convenient minitype device. We optimized the PCR primers and probes for the amplification of the DHAV-A 3C/3D genes, and successfully amplified a specific fragment of DHAV-A, but no fragment from 18 other duck pathogens. The limit of detection for viral RNA was 49 copies per reaction, and the sensitivity and specificity were each 100% in the analysis of 60 liver samples. By comparison, the sensitivities of RT-iiPCR was comparable in sensitivity to existing rRT-PCR. Furthermore, the RT-iiPCR results were 98.3% in agreement with those of the rRT-PCR, with a kappa value of 0.938. In conclusion, this new method not only offers a higher sensitivity and specificity than existing techniques, but also time-saving and better suited to field diagnoses because device is portable.

Keywords: detection; duck hepatitis A virus; insulated isothermal RT-PCR; real-time RT-PCR.

Fig. 1.

The linear relationship of every dilution detected by rRT-PCR.