猪圆环病毒复合PCR检测方法的建立

Establishment of the multiplex PCR for detection of porcine circovirus

根据已发表的PCV 1和PCV 2全基因组序列 ,设计了 3条引物 ,组成PCV 2的特有引物对和PCV 1、PCV 2的共有引物对 ,分别扩增长度为 4 72bp和 339bp的片段。为验证PCR扩增的特异性 ,将 4 72bp的扩增产物纯化后 ,克隆到 pMD 18 T载体 ,转化大肠埃希氏菌TG1,对阳性克隆进行酶切及PCR鉴定 ,并测序。将该序列递交NCBI进行BLAST同源序列比较 ,发现它与美国、加拿大及我国台湾的PCV 2毒株核苷酸序列的同源性均在 99%以上 ,与PCV 1毒株的同源性为 86 %。结果显示 ,该方法最少可用于 3μg病料组织和 0 .75 pgDNA的PCV 2检出 ,具有很高的灵敏性。应用该PCR方法检测了浙江省和上海市 4 7份“猪高热综合征”病料 ,PCV 2感染阳性率为 36 .17% ,PCV 1单独感染阳性率为 34.0 4 %。

According to the published genome sequences of porcine circovirus type 1 (PCV-1) and type 2(PCV-2), three primers were designed to amplify a 472 bp PCV-2 specific DNA and a 339 bp DNA of both PCV-1 and PCV-2. In order to verify the specificity of PCR product, the 472 bp fragment was extracted and cloned into pMD 18-T vector. After the recombination plasmids were transformed into Escherichia coli TG1, they were identified by restriction enzymes digestion and by PCR. A selected clone was then (sequenced). The BLAST result showed that the cloned 472 bp sequence has 99% homology with respect (sequence) of other PCV-2 isolates, and about 86% with that of PCV-1 isolates. The sensitive test showed that the detection limitation of PCV-2 was about 0.75 pg DNA and 3 μg tissue. The epidemic investigation of 47 samples with hyperpyrexia syndrome from Zhejiang and Shanghai was carried out, the results (revealed) that PCV-2 (both 339bp and 472bp ) positive rate and PCV-1 (339 bp) positive rate were 36.17% (17/47) and 36.04% (16/47), respectively.