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Sixteen PCV3 strains were retrieved from GenBank and aligned to design the primers and probes (Supplementary Table S1). The primers and probes were optimized to contain the strongest fluorescence and the lowest threshold cycle with different concentrations. The final optimized concentrations of the primers and probes were 1 μL primers (1 μmol/L) and 1 μL probe (0.5 μmol/L) for both PCV2 and PCV3.
Table S1. Access numbers of the PCV3 strains retrieved from GenBank for primer and probe design
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To assess the specificity of the duplex real-time PCR, the DNAs or cDNAs from PCV1, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, classical swine fever virus, and porcine epidemic diarrhea virus were used as templates in the established PCV2/3 duplex real-time PCR. As shown in Fig. 2, the duplex real-time PCR assay was able to detect and differentiate between PCV2 and PCV3 independently and simultaneously. In contrast, the other swine pathogens were not detected, demonstrating the high specificity of the established PCV2/3 duplex real-time PCR.
Figure 2. Specificity of the PCV2/3 duplex real-time PCR. A Specificity of the duplex real-time PCR for PCV2. B Specificity of the duplex real-time PCR for PCV3. C Specificity of the duplex real-time PCR for PCV2 and PCV3. The DNAs or cDNAs of swine viruses were used as templates. Other pathogens included PCV1, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine parvovirus, classical swine fever virus, and porcine epidemic diarrhea virus.
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The sensitivity of the PCV2/3 duplex real-time PCR was tested using serial tenfold dilutions of the constructed pMD19-PCV2 and pMD19-PCV3 plasmids. As shown in Fig. 3, the detection limits of the duplex real-time PCR were 2.9 copies for PCV2 and 22.5 copies for PCV3. The reproducibility of the assay was determined by testing three concentrations of plasmids five different times. The intraassay CVs of 107, 105, and 103 copy numbers of PCV2 were 0.31%, 0.44%, and 0.41%, and the respective interassay CVs were 1.33%, 1.52%, and 1.14%. For PCV3, the intra-assay CVs of 107, 105, and 103 copy numbers were 0.53%, 0.48%, and 0.67%, and the respective inter-assay CVs were 1.69%, 1.47%, and 1.54%.
Figure 3. Sensitivity of the PCV2/3 duplex real-time PCR. A Sensitivity of the duplex real-time PCR for PCV2. (1–8): 103–1010 dilutions of pMD19-PCV2 plasmid were used as the templates for real-time PCR. B Sensitivity of the duplex real-time PCR for PCV3. (1–9): 101–109 dilutions of pMD19-PCV3 plasmid were used as the templates for real-time PCR.
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The established PCV2/3 duplex real-time PCR was applied to detect PCV2 and PCV3 DNAs in 340 clinical samples. The results showed that 68.2% of clinical samples (232/340) were PCV2 PCR positive, 44.4% of clinical samples (151/340) were PCV3 PCR positive, and 27.6% of clinical samples (94/340) were both PCV2 and PCV3 PCR positive (Table 1). The positive PCR products were confirmed by gene sequencing (Genewiz, Suzhou, China). Among the different types of tissues, the highest frequency of PCV2-positive clinical tissues was found in tonsil samples, followed by lung and lymph node samples. In contrast, the highest frequency of PCV3-positive clinical tissues was found in lung samples, followed by lymph node and tonsil samples (Table 1).
Table 1. Summary of PCV2/3 detection in 340 clinical samples using the established real-time PCR.