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Development and Application of a One-step Real-time TaqMan RT-PCR Assay to Detect duck hepatitis virus type 1

Development and Application of a One-step Real-time TaqMan R...

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第四届第九次全国学术研讨会暨饲料和动物源食品安全战略论...

作者： Miao Yang Anchun Cheng Mingshu Wang (Avian Disease Research Center,College of Veterinary Medicine,Sichuan Agricultural University,Yaan 625014,PR China The Key Laboratory of Animal Disease and Human Health of Sichuan Province,Yaan 625014,PR China)

A one-step real-time reverse-transcription polymerase chain reaction assay (rRT-PCR) was developed for efficient detection of duck hepatitis virus type 1 (DHV-1).A pair of specific primers which showed 87bp fragment was designed against the conserved region in the 3D gene with a single conserved TaqManTM *** could obtain excellent linear when the DHV-1 RNA concentration between 1×10 and 1×10 *** 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 less-virulent strain in chicken *** results revealed that the copy number of DHV-1 reached a peak in duck embryos and chicken embryos at 28-40h,44-56h post inoculation respectively,maintaining 10 copies/g level in embryoid bodies and 10-10 copies/ml level in allantoic *** RNA copy number in chorioallantoic membrance and muscle was higher than that in allantoic fluid,heart,liver,spleen,brain,intestine from dead duck and chicken embryos inoculated with *** this method and neutralization test to detect 10 liver samples taking from ducklings after artificially infected and 40 clinical liver samples taking from suspected ducklings showed that the positive results of artificially infected samples were the same,while the rRT-PCR method was more sensitive than neutralization test for clinical samples detection,and the positive rate was significant difference (P<0.01).In conclusion,the rRT-PCR assay is rapid,sensitive, specific,reliable and *** will be a powerful tool for DHV-1 suspected case detection,distribution pattern of DHV-1 in vivo and molecular epidemiological screening.

关键词： duck hepatitis virus type1 rRT-PCR duck embryo Chicken embryo Distribution

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