Abstract: A real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines was developed based on a set of primers and TaqMan probe designed targeting the Cap gene sequence of porcine circovirus type 2 from GenBank. After optimizing the reaction system, the specificity, sensitivity and repeatability of the established assay were assessed. Subsequently, the impacts of adding amount of inactivator and inactivation time on detection performance of the established assay were investigated. The results showed that positive amplification product was exclusively obtained for target gene of porcine circovirus type 2 by using the established real-time fluorescence quantitative PCR assay, while the other three kinds of porcine origin viruses had no expected amplification products; the sensitivity of the established assay reached up to 10^2.0 TCID₅₀/mL, which was 100 times higher than that of the ordinary PCR assay; the established assay had good repeatability with the variable coefficient of 2.28% in 10 repeated tests for the same one sample; the different adding amount of inactivator and inactivation time had limited impacts on detection performance of the established assay, in other words, the creditability of the inactivated vaccine comparison test was not affected by the adding amount of inactivator recommended by varied vaccine producers and different inactivation time. In this study, a real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines is successfully developed. This established assay can quantitatively discriminate the antigen concentration of different inactivated porcine circovirus type 2 vaccine samples, providing novel insight into the development of quantitative evaluation method for antigen concentration of inactivated porcine circovirus type 2 vaccines.

Key words: real-time fluorescence quantitative PCR assay, porcine circovirus type 2, inactivated vaccines, Cap gene