应用聚合酶链式反应检测犬细小病毒

Detection of Canine Parvovirus by Polymerase Chain Reaction

将具有犬细小病毒病典型临床症状的病犬肠溶物经 SDS-酚裂解法提取总 DNA,并以此 DNA为模板 ,通过人工合成的两条 2 0 mer引物进行 PCR扩增 ,该对引物扩增的片段为犬细小病毒结构蛋白 VP2 基因中间 1 /3区段 ,PCR产物经琼脂糖凝胶电泳鉴定 .试验结果表明 :用 PCR扩增检测犬细小病毒具有良好的特异性 ,应用该方法检测的 2 7份病犬肠溶物样品均获得了预计长度的 DNA片段 ( 53 1 bp) ,而健康犬的肠溶物样品 PCR结果为阴性 .该方法的检测灵敏度很高 ,最高可将提取的总 DNA样品稀释 1 0 6倍 ,即可以检测到 1 .3 7pg总 DNA样品中所含的病毒 DNA.由于该方法直接利用病犬肠溶物提取总 DNA,检测方法快速、简便 ,结果可靠 ,可在 2 4 h内获得满意结果 。

The total DNA was isolated from the enteric lysate of virus infected dog showing enteritis symptoms.A pair of primers,20 mer oligonucleotides,spanning to the mid 1/3 region of VP 2 gene of canine parvovirus were synthesized.The total DNA was used as the template for PCR amplification,and the products of PCR were identified by 1% agarose gel electrophoresis.The results show that the PCR has a good specificity,being able to amplify DNA of expected length (531 bp) from 27 infected enteric lysate samples.But all the uninfected enteric lysate samples give negative results.Sensitivity test of PCR indicated that the maximum dilution of total DNA which gives positive reaction is 10 -6 ,corresponding to 1.37 pg total DNA.This diagnostic technique is rapid and simple because the samples are directly assayed from virus infected enteric lysate and the results could be obtained within 24 hours.This method can be used for clinical detection of the canine parvovirus with high sensitivity and specificity.