番鸭和鹅细小病毒PCR鉴别方法的建立

The Rapid Differentiation of Muscovy duck and goose parvoviruses by Polymerse Chain Reaction

根据基因库中番鸭细小病毒 (MDPV)FM株和鹅细小病毒 (GPV)的 B株的基因序列设计了 3个引物 PVC、MDPVdn、GPVdn,分别用 MDPV-DNA、GPV-DNA、MDPV 尿囊液、GPV尿囊液、鸭瘟病毒尿囊液、传染性喉气管炎病毒尿囊液和无离子水对照 ,以 PVC/ GPVdn和 PVC/ MDPVdn特异引物在同一条件下进行PCR扩增 ,产物经 1 0 g/ L的琼脂糖凝胶电泳分析。在 PVC/ GPVdn系统中 ,MDPV-DNA、MD-PV尿囊液、DP、IL T和无离子水对照无条带 ,其余样品均出现约 460 bp的条带 ,在 PVC/ MD-PVdn系统中 ,只有 MDPV-DNA、MDPV-GPV混合 DNA、MDPV尿囊液中出现约 90 0 bp的条带 ,其余无特异性核酸带 ,对 MDPV-DNA的敏感性达 0 .5pg,对 MDPV尿囊液敏感性为 1 0 0 0EL D50 / 5μL ,对 GPV-DNA的敏感性为 0 .5pg,对 GPV尿囊液敏感性为 1 0 EL D50 / 5μL。分别将不同时间提取 0 .5ng/ μL MDPV-DNA、0 .5ng/μL GPV-DNA、MDPV尿囊液、GPV尿囊液和无离子水对照样 ,以 PVC/ GPVdn和 PVC/MDPVdn特异引物进行 PCR扩增 3次 ,结果稳定。表明该 PCR检测技术可灵敏。

s:Three primers PVC, MDPVdn, GPVdn were designed according to the sequence of GPV and MDPV published in the gene bank to differentiate Muscovy Duck and Goose Parvoviruses. Two PCRs were amplified under the same condition from MDPV DNA,MDPV allantois fluids,GPV DNA,GPV allantoi ses fluids,ILT allantois fluids, DP allantois fluids and H 2O.Only one fragment 900 bp was detected from MDPV DNA,MDPV allantois fluids by PVC/MDPVdn and 460 bp fragment from GPV DNA,GPV allantoises fluids by PVC/GPVdn.The detectable DNA amount was 0.5 pg for MDPV DNA,1 000 ELD 50 /5μL for MDPV allantois fluids;0.5 pg fg for GPV DNA,10 ELD 50 /5 μL for GPV allantois fluids. The results of amplification for three individual times by PCRs were consistent,and therefore,the PCR detection methods could be used as clinical diagnosis tool.