鹅细小病毒和番鸭细小病毒双重PCR检测方法的建立

根据GenBank上登录的鹅细小病毒(GPV)和番鸭细小病毒(MDPV)基因序列,分别设计合成针对GPV非结构蛋白(NS)和MDPV NS2-VP1基因片段的2对引物GPV U/L和MDPV U/L,将GPV和MDPV提取核酸混合后作为模板,优化PCR反应条件,建立了能同时检测这2种病毒的双重PCR。特异性试验结果显示,引物GPV U/L仅特异性扩增出GPV-GZ1和GPV-GZ2株730bp核酸片段,引物MDPVU/L仅特异性扩增出MDPV的624bp核酸片段,双重PCR扩增出长度分别为730bp和624bp的2条特异性片段,而扩增鸭瘟病毒(DPV)和鹅副黏病毒(GPMV)的核酸扩增结果均为阴性。敏感性试验结果显示,双重PCR能同时检测到14.4pg的GPV核酸和28.8pg的MDPV核酸。结果表明,建立的双重PCR可用于GPV和MDPV的鉴别诊断和联合检测。

Development of duplex PCR for detections of goose parvovirus and muscovy duck parvovirus

Two pairs of primers GPV U/L and MDPV U/L were designed according to the genomic sequences of NS gene of goose parvovirus(GPV) and NS2-VP1 gene of muscovy duck parvovirus (MDPV) available in GenBank. Genomic DNA of GPV-GZ1 and MDPV strains were extracted respectively from allantoic fluids of goose and muscovy duck embryo,and then mixed for amplification and a duplex PCR was established successfully for detection of the two virus based on optimization of reaction conditions. Using the primer GPV U/L,only the 730 bp specific fragments were amplified from DNA samples of GPV-GZ1 and GPV-GZ2; using the primer MDPV U/L, only the 624 bp specific fragment was amplified from DNA samples of MDPV; using the two pairs of primers, the specific fragments 730 bp and 624 bp could be amplified from the mixed samples with GPV and MDPV,but not from duck plague virus and goose paramyxovirus. The sensitivity test showed that DNA used for the duplex PCR might be as low as 14.4 pg for GPV and 28.8 pg for MDPV,respectively. The results indicated that the duplex PCR assay could be used for the differential or simultaneous diagnosis of GPV and MDPV infections.