应用聚合酶链反应检测伪狂犬病病毒DNA

Use of Polymerase Chain Reaction to Detect PseudorabiesVirus DNA

选用标准的伪狂犬病病毒（ＰＲＶ）闽Ａ株，经ＢＨＫ２１细胞培养，提取ＰＲＶＤＮＡ作为摸板；合成１对２０－ｍｅｒ寡核苷酸作为引物；选择扩增序列由２６２个碱基对组成的位于编码多糖蛋白ｇｐ５０基因，用耐热的Ｔａｑ－ＤＮＡ聚合酶经３０个循环以后，扩增的产物直接用凝胶电泳检测，并用标准分子量确定。结果表明：以ＰＲＶＤＮＡ作为模板进行扩增，有扩增产物生成，以同科的疱疹病毒ＤＮＡ作为模板在相同的条件下进行ＰＣＲ扩增，无扩增产物生成，说明ＰＲＶＰＣＲ扩增是特异的。ＰＣＲ技术检测ＰＲＶＤＮＡ敏感度为２８．５ｐｇ．ＰＲＶＰＣＲ技术的建立，为伪狂犬病诊断和流行病学研究提供了更敏感、可靠的手段。

The pseudorabies virus DNA was detected by using PCR technique。We selected pseudorabies virus (PRV)Min-A strain and used BHK21 cells to propagate PRV and extracted PRV DNA as template,A pair of 20-mer oligonucleotides flanking the se-quence was synthesized as primer。The sequence selected for amplification consisted of 262 base pairs lying in the gene coding for the glycoprotein gp50.After 30 cycles of PCR by using the thermostable Taq DNA polymerase the amplified products were directly de- tected by gel electrophoresis and verified by molecular weight markers,The results showed that amplified products were produced by amplification of the PRV DNA as template and were not produced by amplification of other herpesviridae virus DNA astemplate under same conditions,and indicated the specificity of the PRV PCR tech- nique,The sensitivity of PRV DNA was 28.5 pg by this PCR technique,The estab-lished PRV PCR technique provided a more sensitive and reliable method for diagnosis and epizootic study of the pseudorabies。