应用PCR快速鉴别番鸭和鹅细小病毒

Rapid Differentiation of Muscovy Duck and Goose Parvoviruses by Polymerase Chain Reaction

根据已发表的番鸭和鹅细小病毒基因组全序列 ,设计并合成了 1对通用引物 (PC1/PC2 )和 1对MPV特异引物 (PS1/PS2 )。以MPV_DNA、MPV尿囊液、MPV尿囊液和GPV尿囊液混合液、GPV_DNA、GPV尿囊液和GPV细胞培养物、DHV尿囊液和H2 O为模板在同一条件下分别以 2对引物进行扩增 ,PCR产物经 1 5 %琼脂糖凝胶电泳分析 ,结果显示 :在PC1/PC2系统中 ,除DHV尿囊液和H2 O外 ,所有样品均出现长约 4 80bp的特异核酸带 ,对MPV_DNA的敏感性为 0 2pg ,对GPV_DNA的敏感性为2pg ;对MPV尿囊液的敏感性为 10 0ELD50 / 2 μl;PS1/PS2系统中 ,只有MPV_DNA、MPV尿囊液及MPV尿囊液和GPV尿囊液混合液出现预期约 110 0bp特异扩增产物 ,对MPV_DNA的敏感性为 0 2ng ,对MPV尿囊液的敏感性为 10 0 0ELD50 / 2 μl,而GPV_DNA、GPV尿囊液、GPV细胞培养物、DHV尿囊液和和空白对照均未见到条带。分别以 1ngMPV_DNA、1ngGPV_DNA、MPV尿囊液、GPV尿囊液、DHV尿囊液和空白对照样品进行扩增 ,3次重复实验结果完全一致。表明该PCR检测技术可准确、快速鉴别番鸭和鹅细小病毒 ,为临床上准确。

s: Two paris of Primers PC1/PC2 and PS1/PS2 were designed based on the complete nucleotide sequences of Muscovy Duck and Goose Parvoviruses to differentiate them. Two PCRs were amplified under same condition from MPV_DNA, MPV allantoic fluids, the mixture of MPV and GPV allantoic fluids, GPV_DNA, GPV allantoic fluids, GPV cell cultures, DHV allantoic fluids and H 2O, only one fragment with size of 1100bp from PMV_DNA, MPV allantoic fluids and the mixture of MPV and GPV allantoic fluids with PS1/PS2, the detectbale DNA amount was 10 3 ELD 50 /2μl for MPV allantoic fluids, 0.2ng for MPV_DNA, only one fragment with size of 480bp from all the samples except DHV allantoic fluids and H 2O with PC1/PC2, the detectable DNA amount was 0.2pg for MPV_DNA, 10 2ELD 50 /2μl for MPV allantoic fluids, 2pg for GPV_DNA. The results of 3 individual times by PCRs were consistant. Therefore, the PCRs will be usefull in the cliniclly diagnosis of Muscovy Duck and Goose Parvovirusis.