多重荧光定量PCR检测猪圆环病毒2a、2b和2d基因型方法的建立与应用

Establishment and Application of Multiple Fluorescence Quantitative PCR for Detection of Porcine Circovirus 2a,2b and 2d Genotype

为建立一种检测猪圆环病毒2型多重实时荧光定量PCR方法,可对3种基因型PCV2a、PCV2b、PCV2d检出并分型。针对3种基因型ORF2基因,在保守序列设计通用引物并分别设计特异Taq Man探针,3条探针分别标记NED、JOE和FAM荧光报告基团,建立多重实时荧光定量PCR反应体系,检测其灵敏性、重复性和特异性。经条件优化多重荧光定量PCR的反应体系为30μL,各型探针加样为0.6μL,退火温度为60℃;最低检测值为PCV2a 100 copies/μL、PCV2b 57 copies/μL和PCV2d 100 copies/μL,而且该方法对猪细小病毒、猪繁殖与呼吸综合征病毒等其他6种猪源主要病毒核酸检测均为阴性,具有良好的特异性;组内和组间的检测变异系数均小于等于3.6%;通过检测218份阳性样本,结果表明该方法能快速检测猪圆环病毒2型三种主要流行型并同时分型,为PCV2快速分型的诊断提供新方法。

In order to establish a multiple real-time fluorescent quantitative PCR method for detecting porcine circovirus type 2(PCV2)and genotyping PCV2a,PCV2b,and PCV2d.For the three genotypes of ORF2 genes,universal primers were designed in conservative sequences and specific Taqman probes were designed respectively,and the three probes were labeled with NED,JOE and FAM fluorescent reporter groups respectively,to establish and optimize multiple real-time fluorescent quantitative PCR reaction system and detect its sensitivity,repeatability and specificity.The results showed that the established multiple fluorescence quantitative PCR reaction system was 30 μL,and the lowest detection value was PCV2a 100 copies/μL,PCV2b 57 copies/μL and PCV2d 100 copies/μL when each type of probe was added 0.6 μL and the annealing temperature was 60°C,and this method is negative for other major pathogenic nucleic acids in pigs,and has good specificity.The coefficients of variability for intra-assay and inter-assay were both less than 3.6%.Through the detection of 218 positive samples,the results show that this method can quickly detect the three major epidemic genotypes of PCV2 and type them at the same time,providing a new method for the rapid diagnosis of PCV2.