猪圆环病毒2型基因型鉴别PCR检测方法的建立

Development and application of genotyping PCR assay for detection of porcine circovirus type 2

采用PCR方法从猪圆环病毒2型（PCV2）山东分离株中扩增ORF2基因片段,结合GenBank中已登录的PCV2基因序列进行同源性分析及基因型鉴定,确定山东省PCV2的流行优势基因型,探讨PCV2基因的遗传变异特性。依据PCV2基因型间的差异性序列,设计、合成特异性PCR引物,建立PCV2基因型鉴别PCR检测方法,并从敏感性、特异性、重复性等方面评价其性能。结果,获得了28株PCV2ORF2完整基因,其中4株为PCV2a型,21株为PCV2b型,3株为新基因型PCV2d型,显示山东省流行的PCV2优势基因型为PCV2b。基因同源性分析表明,2000-2012年国内不同地区分离株的基因同源性为90.5%~99.8%,不同基因型PCV2存在一定形式的特有氨基酸位点序列。用建立的PCV2基因型鉴别PCR方法扩增的片段大小分别为341bp和177bp,该方法最低能检测病毒DNA的量为5ng/μL;特异性试验结果显示,该方法只对PCV2a和PCV2b的DNA扩增阳性,而对猪圆环病毒1型（PCV1）、猪繁殖与呼吸综合征病毒（PRRSV）、伪狂犬病病毒（PRV）、猪细小病毒（PPV）、猪瘟病毒（CSFV）、乙型脑炎病毒（JEV）和大肠杆菌（Escherichia coli）的扩增结果均为阴性;重复检测10次的结果均一致。结果表明,建立的PCR方法具有良好的特异性和重复性,可用于PCV2基因型的鉴别检测。

To analysis the epidemic genotypes and genetic mutation of PCV2 in Shandong areas and establish a rapid detection method of different genotype porcine corcivirus type 2 by PCR,the gene fragment was amplified from PCV2 strain isolated in Shandong by using PCR. Compared with PCV2 gene sequences registered in OenBank, the homology and genotypes were analysed by MegAlin software. The genotypes i and genetic variation characteristics were determined by gene sequence analysis. To screen highly variable region genes and analyse the gene sequence diversity of PCV2, the specific primers were designed and the PCR detection method was established, which is able to distinguish different genotype PCV2. The PCR sensitivity,specificity and reproducibility was tested. The results showed that PCV20RF2 genes were amplified in the 28 strains by PCR and genetic analysis showed that 4 strains belonged to PCV2a,21 strains to PCV2b and 3 strains a new genotype PCV2d. This indicated that the main popular genotype was PCV2b. The homology analysis showed that gene homolog was 90.5%--99.8% among different strains in Chinain 2000-2012. The genotypes of PCV2 have specific amino acid sites. The specific detection PCR method for PCV2 genotype was successfully established,which gave 341 bp and 177 bp amplicons. The lowest detection of viral of the PCR method was 5 ngμL. These results demonstrate that the specificity and repeatability of the developed PCR are good and suitable for differentiation of PCV2 genotypes.