Development of Droplet Digital PCR for Quantitative Detection of Porcine Circovirus Type 3
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Abstract:
Porcine circovirus type 3 is a novel porcine circovirus that can cause diseases associated with porcine infection. In this experiment, primer probes were designed based on the OPF2 gene sequence of porcine circovirus type 3, and a droplet digital PCR (ddPCR) method for accurate and quantitative detection was established. After optimization of the primer probe concentration and annealing temperature for the ddPCR reaction system, the optimal primer probe concentration ratio was 800 nM: 800 nM: 400 nM; and the optimal annealing temperature was 60 ℃. The correlation coefficient R2 of the ddPCR standard curve was 0.999, showing a good linear relationship. The sensitivity was high with the limit of detection being 4.4 copies/μL and good specificity (no cross-reaction with the common pig pathogens ). The detection results of the clinical samples revealed that the ddPCR method established in this study was more sensitive than the real-time fluorescent PCR method by one order of magnitude, and could generate results consistent with those obtained by the fluorescent PCR and analyze quantitatively suspicious samples. The results showed that the newly established ddPCR method in this study had high sensitivity and specificity, thus could be used for quantitative detection of porcine circovirus type 3.
