Ⅰ型鸭肝炎病毒PCR-DHPLC检测方法的建立

Establishment of the PCR-DHPLC Method for Identification of Duck Hepatitis Virus TypeⅠ

用RT-PCR结合变性高效液相色谱技术(DHPLC),建立Ⅰ型鸭肝炎病毒的快速检测方法。根据获得的Ⅰ型鸭肝炎病毒(DHV I)RNA聚合酶基因序列,应用Primer Premier5.0软件设计了1对引物,RT-PCR扩增产物经变性高效液相色谱进行快速检测。以正常鸭胚尿囊液、正常鸭肝组织、鸭瘟病毒、番鸭细小病毒、鹅细小病毒、H5N1亚型禽流感病毒、鸭源新城疫病毒、传染性法氏囊病病毒作对照进行特异性检测;将强毒株核酸稀释成不同梯度,做灵敏度检测;同时将该方法与病毒分离和荧光定量RT-PCR方法做人工感染样品的平行检测。结果表明,该方法有很好的特异性,且灵敏度高,检测限可达到4 pg的DHV I核酸模板,与荧光定量PCR检测结果一致。PCR-DHPLC对DHVⅠ强毒株人工感染鸭组织脏器的检测结果与荧光定量PCR检测结果的阳性检出率均为100%,对脑、脾、肺病料的检出率显著(P≤0.01)高于病毒分离,PCR-DHPLC与荧光RT-PCR检测方法相比较,两种方法检测DHVⅠ的符合率为100%。研究表明该方法可用于诊断Ⅰ型鸭肝炎病毒,是一种有效的新方法。

A new molecular method for rapid detection of duck hepatitis virus typeⅠ（DHVⅠ） was established by using denaturing high performance liquid chromatography（DHPLC） combined with nucleic acid amplification in this study.According to the sequence of RNA polymerase gene of duck hepatitis virus typeⅠ（DHVⅠ）,one pair of primers were designed by using Primer Premier 5.0.The PCR fragments were analysised by DHPLC compared to normal duck embryo allantoic fluid,duck plague virus（DPV）,muscovy parvovirus（MPV）,Avian influenza virus（AIV）,Newcastle disease virus（NDV）.They were tested to confirm the specificity of the PCR-DHPLC assay and no PCR products were amplified.The sensitivity of the assay DHVⅠ was as low as 4 pg.All results were 100 percent positive when the livers of dead ducks which were experimentally infected with DHVⅠvirulence were detected by the PCR-DHPLC and Real-time PCR.The positive rates by PCR-DHPLC were significantly higher（P≤0.01） than those by the virus isolation when samples were from spleen,lung and brain.The samples from the ducks experimentally infected with DHVⅠ were tested by PCR-DHPLC and real-time PCR,showing 100% agreement for DHVⅠ.Results showed that the PCR-DHPLC is specific and sensitive,and can be used as a method to detect clinical samples.