鉴别猪圆环病毒1型和2型的二重PCR方法的建立

Establishment of a duplex PCR for the distinction of porcine circovirus type 1 and type 2

根据GenBank上发表的PCV-1和PCV-2序列,设计2对引物分别扩增PCV-1和PCV-2的独特区域,长度为382 bp和222 bp。将PCR产物分别克隆至pMD-18T vector,经转化、PCR鉴定和序列测定,构建了PCV-1和PCV-2的阳性质粒,并等量混合作为二重PCR阳性模板。将2对PCR引物混合后,进行退火温度优化,敏感性和特异性试验,并用于临床检测。结果表明,该二重PCR方法可以特异性地鉴别PCV-1和PCV-2,最低检出量分别为0.1 ng和0.01 ng,敏感性较高。应用该方法可对临床样品进行准确、快速地诊断,为猪圆环病毒的临床诊断与流行病学调查等研究奠定了基础。

According to the sequences of porcine circovirus type 1 （PCV-1） and type 2 （PCV-2） genome pub- lished in GenBank,two pairs of primers were designed to amplify the unique fragments,the products were 382 bp and 222 bp in size, respectively. PCR products were cloned to pMD-18T vector, constructing the recombinant plasmids containing PCV-1 and PCV-2 gene identified with PCR and sequencing. The positive plasmids were mixed and taken as positive template for duplex PCR. The two pairs of primers were mixed to optimize annealing temperature, test sensitivity and specificity,then used for detection of clinical samples. The results showed that the duplex PCR could distinguish specially PCV-1 from PCV-2,and the limits of detection was 0.1 ng for PCV-1 and 0.01 ng,for PCV-2, with relatively high sensitivity. The duplex PCR arrays can be used for PCV detection of clinical samples.