Establishment and Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Porcine Circovirus Type 2

摘要

[Objective]To establish a real-time fluorescent quantitative polymerase chain reaction(PCR) method with SYBR Green I for the detection of porcine circovirus type 2(PCV2).[Methods]Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR.The amplified gene was cloned into the vector of pMD~18-T and transformed into DH5α to screen positive clones.After being extracted and purified,the recombinant plasmids pMD~ 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2,and the PCR reaction conditions were optimized.[Results]Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21×10~0-4.16×10~8 copies /μL,the correlation coefficient was 0.998 8 and the slope was-3.286.The method did not show any cross-reactions with the genomes of PRRSV,PCV1,CSFV,PRV,PPV and Escherichia coli.Sensitivity of this method was proved to be 3.21×10° copies/μL,which was 1 000 times higher as conventional PCR method.Variation coefficients of the repeated trials among same batch or different batches were both less than 3.00%.Positive rate of clinical samples detected by the established PCR method was 58.94%,which was significantly higher than the detection rate by conventional PCR.[Conclusions]A real-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established,which was better for conducting the quantitative analysis and the early diagnosis of PCV2 infection.