Development and application of a one-step real-time Taqman RT-PCR assay for detection of Duck hepatitis virus type1

A one-step real-time RT-PCR assay (rRT-PCR) was developed for efficient detection of Duck hepatitis virus type1 (DHV-1). A pair of specific primers was designed against the conserved region in the 3D gene that encodes the RNA dependent RNA polymerase with a single conserved TaqMan probe. The detection limit of this assay was 10 viral genomic copies per reaction and it was highly specific to DHV-1. The rRT-PCR assay was used to determine the distribution and concentration of DHV-1 virulent strain in duck embryos as well as the DHV-1 attenuated vaccine strain in chicken embryos. The results revealed that the copy numbers of DHV-1 reached a peak in duck embryos and chicken embryos at 28-40h, 44-56h postinoculation respectively. The comparative tests for ducklings infected artificially and clinical samples between neutralization test and rRT-PCR showed that the positive results of infected samples were the same, while the rRT-PCR method was more sensitive than neutralization test for detection of clinical samples. The rapid, sensitive and specific rRT-PCR assay will be a powerful tool for detection of suspected cases of DHV-1, distribution pattern of DHV-1 in vivo and molecular epidemiological screening.