地高辛标记核酸探针检测牛粘膜病病毒

Development of DNA Probe Labelled with Digoxigenin for Detection of BVDV

本课题建立了地高辛标记核酸探针检测牛病毒性腹泻 -粘膜病病毒 ( BVDV)的诊断方法。根据 BVDV基因序列的结构特点 ,在编码非结构蛋白 p1 2 5高度保守基因区内 ,设计合成一对特异性引物。以 PCR技术从 BVDV基因重组质粒中扩增出了 BVDV特异的、保守的 40 0 bp核苷酸片段 ,经纯化后 ,地高辛标记 ,制备牛病毒性腹泻病毒的特异性核酸探针。通过检测 BVDV细胞培养物、相关病毒及核酸载体和 BVDV细胞培养物的总 RNA、反转录产物及 RT- PCR产物 ,结果证明 ,该探针特异性强、敏感性高 ,适于病毒反转录产物的检测。本试验用所制备的核酸探针对某奶牛场的腹泻犊牛粪便进行了 BVD的临床诊断的初步应用试验。

A DNA probe labelled with digoxigenin was used to detect bovine viral diarrhea viruses.According to gene sequences of BVDV, a pair of primers coming from the highly conserved gene region coding for the p125 were designed and synthetied,and about 400bp cDNA fragment was amplified by polymerase chain reaction(PCR) from recombinant plasmid.Then the purified PCR products was labelled with digoxigenin and random primers as DNA probes for BVDV.The results showed that the probe is of high specificity and sensitivity and the probe is suitable to detect the RT products by detecting RNA,RT and RT\|PCR products from cell cultures infected with BVDV,correlative virus and genic vector.The probe was preliminarily applied to detect diarrheal calf feaes of some dairy farm.