Duplex TaqMan MGB Fluorescence Quantitative Real-Time PCR(FQ-PCR)Assay for the Differential Detection of Porcine Circoviruses 2 and 3
To develop a duplex TaqMan MGB fluorescence quantitative real-time PCR(FQ-PCR)assay for simultaneous and differential detection porcine circovirus 2(PCV2)and PCV3 in the same reaction system,two sets of specific primers for PCV2 and PCV3 and two TaqMan MGB probes were designed for development of a duplex FQ-PCR method according to PCV2 rep gene and PCV3 cap gene sequences.The duplex FQ-PCR method developed was then optimized for its reaction conditions.The results showed that the duplex FQ-PCR specifically detected PCV2 and PCV3 samples and had no cross-reaction with other 8 pathogens and negative controls,The minimum detection limit was 10 copies/μL nucleic acid,which was the same as the single PCV2 or PCV3 FQ-PCR.The coefficients of intra-batch and inter-batch variation were less than 3％,which indicated that the assay had high specificity,sensitivity and repeatability.The interference test demonstrated that the FQ-PCR method had no effect on the detection and accurate quantification of either virus nucleic acid when the initial concentration of the positive plasmids of these viruses were very different.Meanwhile,this method was used to test 16 PCV2,10 PCV3 and 6 PCV2/PCV3 double positive samples and the results were consistent with those of the gene sequencing.The duplex FQ-PCR method developed here had the advantages of high sensitivity,and specificity,simultaneously rapid identification of PCV2 and PCV3,which might be used for the monitoring and surveillance of virus infection.
国家科技期刊平台
NSTL
万方数据
维普
