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.
Yang
Miao
M
Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an 625014, China.
Cheng
Anchun
A
Wang
Mingshu
M
Xing
Hongyi
H
D000818:Animals; D002642:Chick Embryo; D002645:Chickens / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004372:Ducks / Q000821:virology; D004371:Hepatitis Virus, Duck / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase