Abstract:

This study aimed to establish a rapid and convenient triplex fluorescent quantitative PCR assay for simultaneous detection of porcine circovirus (PCV) types 2, 3, and 4, and to apply this method for the detection and molecular epidemiological analysis of PCV2, PCV3, and PCV4 in clinically suspected PCV-infected samples. Three pairs of specific primers and three TaqMan probes labeled with distinct fluorescent groups were designed and synthesized based on the Rep gene sequences of PCV2 and PCV3 and the Cap gene sequence of PCV4. The reaction system and conditions were optimized, and recombinant plasmids were constructed as positive controls to generate standard curves. The specificity, sensitivity, and reproducibility of the assay were systematically evaluated. The developed triplex fluorescent quantitative PCR assay demonstrated high specificity with no cross-reactivity among different PCV genotypes or common swine pathogens. The lower detection limit was 1.0×10² copies·μL^-1 for all targets, with intra-and inter-assay coefficients of variation both below 2.0%, indicating excellent reproducibility. Cap gene amplification, cloning, and sequencing of positive samples yielded 41 sequences, including 11 PCV2a, 8 PCV2b, 15 PCV2d, and 7 PCV3a strains. Sequence analysis revealed 96.7%-99.7% homology among 34 PCV2 Cap proteins, 97.2%-99.9% homology among 7 PCV3 Cap proteins. This study successfully established a triplex fluorescent quantitative PCR assay capable of detecting co-infections with different PCV genotypes. The results confirmed PCV2d and PCV3a as the predominant circulating genotypes in China. The assay provides a rapid and accurate diagnostic tool for clinical detection of PCV infections.

Key words: porcine circovirus, fluorescence quantitative PCR, Cap gene, Genetic evolutionary analysis