Development and application of a reverse transcriptase polymerase chain reaction to detect Chinese isolates of duck...

We developed a reverse transcriptase polymerase chain reaction (RT-PCR) method for the detection of duck hepatitis virus type 1 (DHV-1) in the tissues of infected and clinically affected ducks and in chick and duck embryos. We found the assay to be effective in detecting the virus in China, where it is being used in studies on the epidemiology of the disease. We applied this simple and rapid diagnostic method to the detection of DHV isolates grown in chick and duck embryos and in tissues obtained from infected birds. The assay also proved useful for the differentiation of DVH from the duck plague virus (DPV), muscovy parvovirus (MPV), gosling parvovirus (GPV), avian influenza virus (AIV/H5N1), Pasteurella multocida (PA/5:A), Riemerella anatipestifer (RA/serotype 1), and Salmonella enteritidis (SE). The limit of the sensitivity of this method for the detection of DHV-1 RNA was 3 pg/10 microl. As compared to Dot-ELISA and virus isolation, the rate of agreement for the detection of experimentally infected livers was 100%; moreover, the RT-PCR method was also capable of detecting DHV-1 RNA from the livers that had been infected and stored at -20 degrees C for 22 years; in contrast, Dot-ELISA and virus isolation method could only detect DHV-1 from the livers that had been infected and stored at -20 degrees C for 13 and 11 years, respectively. The rate of positivity in 185 clinically suspected diseased livers subjected to detection by RT-PCR, Dot-ELISA, and virus isolation was 89.2%, 69.2%, and 55.7%, respectively. These results indicated that the RT-PCR approach is rapid, sensitive, and reliable for the detection and differentiation of DHV-1 from the other clinical samples and suspected isolates.