鉴别PCV2b与PCV2d基因亚型PCR方法的建立及应用

Development and application of PCR assay for differential detection of porcine circovirus type 2b from 2d

为建立可鉴定PCV2b和PCV2d基因亚型的PCR方法,通过分析Gen Bank内PCV2 ORF2基因序列,设计了可分别扩增ORF2基因和鉴别PCV2b、PCV2d的引物,并建立PCR方法。应用PCV2b和PCV2d引物扩增108份PCV2阳性样本,选择11份PCV2b和19份PCV2d单一阳性样本扩增ORF2基因序列,并用于遗传进化分析。结果显示,PCV2b和PCV2d引物扩增的敏感性较好,PCV2b和PCV2d最低可扩增基因拷贝数分别为19 copies/L和80 copies/L。108份PCV2阳性DNA中PCV2b阳性率为42.5%,PCV2d阳性率为69.4%,混合感染阳性率为12%。30条ORF2序列的遗传进化分析结果显示,11条为PCV2b基因亚型,19条为PCV2d基因亚型,遗传进化分析结果与PCR结果一致。本试验建立了一种鉴别PCV2b和PCV2d的PCR方法,可用于PCV2流行病学的调查。

Primers used to amplify the PCV2-ORF2 gene and differentiate PCV2b and PCV2d geno-subtype were designed based on analysis of ORF2downloaded from Genbank.The sensitivity and specificity were evaluated using p MD19-T plasmids with PCV2b and PCV2d complete genomes. One hundred and eight PCV2 positive DNA samples were amplified using PCV2b and PCV2d primers. Complete ORF2genome of 11 PCV2b and19 PCV2d single positive DNA were amplified and sequenced,and phylogenetic trees were reconstructed based on 30 obtained and 34 reference sequences. The specificity of PCV2b and PCV2d primer was well,and the minimum copies of PCV2b and PCV2d were 19 and 80 copies/μL,respectively.The positive rate of PCV2b and PCV2d in 108 PCV2DNA samples was 42.5% and 69.4%,respectively,and the coinfection rate of PCV2b and PCV2d was 12%. Phylogenetic analysis indicated that 11 ORF2sequences belonged to PCV2b,while 19 belonged to PCV2d,which was in agreement with PCR results.Conclusion： we designed two primers which could be used for specific amplification of PCV2b and PCV2d.