鸭瘟病毒UL35基因PCR方法的建立与初步应用

Development and application of DPV UL35 gene PCR assay

本研究建立了检测鸭瘟病毒(Duck pl ague vi rus,DPV)的PCR方法,并运用建立的检测方法对分离毒株和人工感染样品进行临床应用检测。根据GenBank(登录号为EF643558)中的DPV UL35基因保守区域,设计合成了一对引物,以DPV疫苗株为模板,优化PCR反应条件,建立了一种快速、有效的DPVPCR方法。结果显示:该方法能从DPV中扩增到与预期大小相符,长度为354 bp的特异性片段,而对禽流感病毒(AIV)、番鸭呼肠孤病毒(MDRV)、新城疫病毒(NDV)、番鸭细小病毒(MPV)、鹅细小病毒(GPV)等样品的扩增结果均为阴性;检测灵敏度达到470 ng病毒DNA。应用该方法对3株DPV分离株和6份由DPV BL8毒株人工感染鸭的肝脏和脾脏等组织进行PCR检测均为阳性。表明所建立的DPV PCR方法特异性强、灵敏度高,可用于DPV的临床诊断和流行病学调查。

The PCR assay for the detection of Duck plague virus （DPV） was developed and was applied to detect the isolated strains and the artificial infected samples. According to the sequences ofDPV UL35 gene available in GenBank （GenBank： EF643558）, a pair of primers were designed and synthesized for the detection of DPV. PCR method for the rapid detection of DPV was developed by optimizing some reaction factors. The results showed that a 354 bp specific fragment could be amplified from the DPV DNA and the sensitivity of PCR was 470 ng DPV-DNA. The negative results were detected from other viruses including AIV-H9, MDRV, NDV, MPV and GPV. The positive rate of PCR method for detecting viral pathogens in 3 field isolates and 6 samples of the liver and spleen tissues of ducklings which were artificially infected by DPV BL8 strain was 100%. The results indicated that this PCR method was sensitive and specific for detecting DPV and could be used for DPV clinical diagnosis and epidemiology investigation.