Abstract: According to genome sequences of ORF4 of porcine circovirus type 2（PCV2）published in GenBank, a pair of primers was designed. The ORF4 gene was amplified with traditional PCR. The PCR product was cloned into pMD18-T vector and sequenced to construct positive recombinant plasmid. The recombinant plasmid was used as template for EvaGreen real-time PCR to generate standard curve and melt curve. The results showed that EvaGreen real-time PCR in the present study was highly specific while used to detect other nontarget viruses, and its sensitivity was proved to be 10 copies/μL and reproducibility for both intra-assay and inter-assay were less than 2.5%. Then the established method was utilized to test 118 clinical samples. The result indicated that the PCV2 positive rate was 25/118, which was 99.2% consistent with that of conventional PCR tests, except one sample that was positive for PCV2 by real-time PCR but negative by conventional PCR. Thus, this method in the current study showed the characteristics of specificity, sensitivity, reproducibility and simple operation, which could be used in subclinical diagnosis and epidemiological investigation of PCV2.

Key words: Porcine circovirus type 2, EvaGreen, Real-time, PCR, Detection