禽黄病毒套式RT-PCR检测方法的建立及应用

禽黄病毒病是新发的一种疫病,可引起鸭、鹅和鸡等家禽产蛋和采食量下降及死亡。本研究旨在建立一种快速诊断方法用于临床诊断及流行病学调查。根据GenBank发表的鹅黄病毒JS804株全基因序列,应用Primer Premier 5.0软件设计了2对特异性引物,建立了禽黄病毒套式RT-PCR检测方法。结果:该套式RT-PCR方法具有特异性强、敏感性高的特点,最低病毒检测量为101.89TCID50/0.1 mL,比普通RT-PCR方法敏感性高1 000倍。应用该方法对江苏地区疑似禽黄病毒病的70份鹅病料、4份鸭病料、12份鸡病料进行检测,总阳性率为58.14%,而用普通RT-PCR方法检测的阳性率仅为17.44%。结果表明,禽黄病毒套式RT-PCR检测方法具有快速、特异、敏感的特点,可用于禽黄病毒感染的临床诊断和流行病学调查。

Establishment and application of a nested RT-PCR method for detection of avian flavivirus

The avian flavivirus,a newly emerged virus,caused egg drop,feed uptake decline and even death among domestic fowls including ducks,geese and chickens in China.This study was aimed at developing a reliable method to diagnose and detect the virus in clinical cases.According to the sequence of goose flavivirus strain JS804 in GenBank,two pairs of primers were designed using Primer Premier5.0.A nested RT-PCR method for rapid detection of avian flavivirus was established.The results showed that the sensitivity of the nested RT-PCR was 101.89 TCID50/0.1mL,which was 103 times higher than that of the conventional RT-PCR.Then this method was used to detect the clinical samples in Jiangsu area,including 70 samples from geese,4 from ducks and 12 from chickens.The positive rate for avian flavivirus was 58.14% nested RT-PCR,while the positive rate was only 17.44% by conventional RT-PCR.The results confirmed that the nested RT-PCR was a fast,sensitive,specific and reliable method for detecting avian flavivirus in clinical cases and epidemiological investigation.