鸭圆环病毒套式PCR检测方法的建立

Establishment of Nested-PCR Assay for Detection of Duck Circovirus

根据GenBank发布的鸭圆环病毒(duck circovirus,DuCV)序列(AY228555),运用Primer Premier 5.0设计2对引物,建立了适合DuCV快速检测的套式PCR方法,采用该方法对从安徽省望江县采集的发病鸭肝脏、胸腺、法氏囊、肾脏和脾脏等内脏病料进行DuCV检测。结果显示,套式PCR对所有内脏病料均能扩增出340 bp的条带,而正常鸭胚、健康鸭肝脏、鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、网状内皮组织增生病毒、鸡传染性贫血病毒、鸭源大肠杆菌和鸭疫里氏杆菌的扩增结果均为阴性;该方法第1次扩增的敏感性是1 ng,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高106倍;表明本试验所建立的套式PCR方法可用于鸭圆环病毒(DuCV)感染的临床诊断和流行病学调查。

According to the sequence of duck circovirus（DuCV） in GenBank（AY228555）,two pairs of primers were designed by Primer Premier 5.0.A nested-PCR assay for rapid detection of DuCV was established.The results showed that a specific 340 bp fragment was amplified from liver,thymus gland,bursa of Fabricius,kidney,spleen collected from Wangjiang county of Anhui province,but no bands were amplified with templates extracted respectively from normal duck embryo,healthy duck liver tissue,duck plague virus（DPV）,avian influenza virus（AIV subtype H9）,Newcastle disease virus（NDV）,infectious bursal disease virus（IBDV）,reticuloendotheliosis virus（REV）,chicken infectious anemia virus（CAV）,Escherichia coli（E.coli） of duck origin and Riemerella anatipestifer（RA）.Sensitivity of the first and second amplifications by the nested-PCR assay was 1 ng and 1 fg,respectively.The sensitivity of the second amplifications was increased by 106 times.Results showed that the nested-PCR assay could be used as a method for the diagnosis and detection of clinical cases of DuCV and epidemiological investigation.