猪圆环病毒2型和3型双重PCR方法的建立与优化

Establishment and optimization of a duplex PCR assay for simultaneous detection of Porcine circovirus types 2 and 3

为了能够同时检测猪圆环病毒2型(Porcine circovirus type 2,PCV-2)和猪圆环病毒3型(Porcine circovirus type 3,PCV-3),试验根据国内报道的PCV-2和PCV-3毒株全基因序列设计6对特异性引物(P3~P8),并构建标准质粒进行引物特异性试验,然后通过优化扩增条件建立检测PCV-2和PCV-3混合感染的双重PCR方法。最后用PCV-2和PCV-3的感染性克隆同时转染PK-15细胞,盲传5代后用建立的双重PCR方法检测细胞培养液中的病毒核酸。结果表明:构建的PCV-2和PCV-3标准质粒浓度分别为290 ng/μL和380 ng/μL,双重PCR方法的最佳引物组合为P3/P6和P4/P8,最佳DNA聚合酶浓度为0.02 U/μL。使用引物组合P3/P6时,扩增得到的目的片段大小分别为308 bp和608 bp,最佳退火温度为58℃,最佳循环次数为28个;使用引物组合P4/P8时,扩增得到的目的片段大小分别为291 bp和645 bp,最佳退火温度为60℃,最佳循环次数为26个。运用建立好的双重PCR方法评估感染细胞模型,能有效鉴别PCV-2和PCV-3的单一感染和混合感染。说明优化得到的双重PCR检测方法可用于PCV-2和PCV-3的同时检测。

In order to simultaneously detect porcine circovirus type 2(PCV-2)and porcine circovirus type 3(PCV-3),six pairs of specific primers(P3-P8)were designed based on the published sequence of the complete genome of PCV-2 and PCV-3 in China,and standard plasmids were constructed for specificity test of primers,then duplex PCR methods were established by optimizing the amplification conditions to detect the co-infection of PCV-2 and PCV-3.Finally,the infectious clones of PCV-2 and PCV-3 were transfected into PK-15 cells simultaneously,after 5 passages of cell culture,the established duplex PCR methods were used for detection of viral nucleic acid in cell-culture medium.The results showed that the concentrations of constructed standard plasmids of PCV-2 and PCV-3 were 290 ng/μL and 380 ng/μL,respectively.Furthermore,the optimal combinations of primers were P3/P6 and P4/P8,and.the optimal concentration of the DNA polymerase used for duplex PCR was 0.02 U/μL.The amplified fragments of the two target genes were 308 bp and 608 bp by using primers(P3/P6),the optimal annealing temperature was 58℃and the optimal number of cycles was 28.The amplified fragments of the two target genes were 291 bp and 645 bp by using primers(P4/P8),the optimal annealing temperature was 60℃and the optimal number of cycles was 26.Two types of duplex PCR methods were used for effectively distinguish between the single infection and the co-infection of PCV-2 and PCV-3 by evaluating the infected cell model.It is indicated that the optimized duplex PCR methods could be used for simultaneous detection of PCV-2 and PCV-3.