A total of 282 PCV complete genomes (175 PCV2, 82 PCV3, and 25 PCV4) were downloaded from the GenBank database. Using MEGA-X software, a multiple sequence alignment was performed to select specific conserved regions. The conserved region of Rep was chosen for designing the primer and probe for PCV2, and the Cap conserved region was chosen for PCV3 and PCV4. Specific primers and probes for each type were designed at the conserved sequences using primer 6.0 software, and qPCR amplification fragment sizes were as follows: PCV2 (116 bp), PCV3 (137 bp) and PCV4 (75 bp). All primer and probe sequences synthesized by Beijing Tsingke Biotech Co., Ltd. (Beijing, China) are shown in Table 1.

All viruses used for specificity validation were obtained from commercial vaccines purchased from Jilin Zhengye Biological Products Co., Ltd. (Jilin, China), including Porcine Reproductive and Respiratory Syndrome virus (PRRSV, HuN4-F112), Pseudorabies virus (PRV, Bartha-K61), Porcine Transmissible Gastroenteritis virus (PTGV), Porcine Epidemic Diarrhea virus (PEDV), Porcine Rotavirus (PoRV), and Classical Swine Fever virus (CSFV).

During 2020–2021, a total of 738 samples were collected from swine farms in Guangdong province and preserved in our laboratory, including 326 nasopharyngeal swab samples and 412 blood samples (Table 2). Molecular epidemiological investigation of the above samples was conducted using the established multiplex qPCR assay established in this study. Furthermore, complete genome amplification and genetic evolutionary analysis were also performed on the positive samples detected in 2020–2021 as described above and PCV-positive samples kept in our laboratory from 2015 to 2021.

RNA/DNA was extracted from samples or vaccines using a RaPure Viral RNA/DNA Kit (Magen, Guangzhou, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using extracted viral RNA and a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). DNA and cDNA products were stored at −80°C.

To construct the recombinant standard plasmid, the multiplex qPCR assay amplification products (PCV2-PCV3-PCV4) were connected together as a standard sequence for synthesis (Tsingke, Beijing, China). The synthesized sequence was cloned into the pUC57 vector and transformed into DH5α competent cells. Then, the bacterial cultures were shaken and grown for 16 h at 37°C. The recombinant plasmid was purified using a GeneJET Plasmid Miniprep Kit (Thermo Scientific, USA) and the concentration was determined using Qubit 4.0 (Thermo Scientific, USA). The final plasmid obtained was named PUC57-PCVs. The plasmid concentration was converted into a copy number using the following formula: y copies / μL = 6.02 × 10 23 × x ng / μL × 10 − 9 D N A length × 660

The multiplex qPCR conditions were optimized, including annealing temperature, primer and probe concentrations. The reaction system was 20 μL, including 10 μL of 2 × AceQ Universal U+ Probe Master Mix V2 (Vazyme, Nanjing, China), 2 μL of templates, different final concentrations of primers, probes, and nuclease-free water. The Optimization of the reaction conditions was performed utilizing a matrix approach with annealing temperatures ranging from 50°C to 60°C; final primer concentrations spanning from 100 nM to 500 nM in nine incremental dilutions; and final probe concentrations set at 50, 100, 150, and 200 nM, respectively. Amplification conditions were 37°C for 2 min, pre-denaturation at 95°C for 300 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The qPCR instrument fluorescence channels were configured as follows: channel 1, FAM; channel 2, HEX; and channel 3, Cy5. Fluorescence signals were collected by a LightCycler® 96 Instrument (Roche Applied Science, Penzberg, Germany). Standard plasmids were diluted from 3.8 × 10⁶ copies/μL to 3.8 × 10² copies/μL as amplification templates, and the system was optimized by generating the lowest threshold cycle (Ct) and the highest cycle increase (ΔRn) for each specific fluorescent signal.

Based on the optimal reaction system, the standard plasmid was diluted 10-fold from 3.8 × 10⁶ copies/μL to 3.8 × 10² copies/μL, and multiplex qPCR was performed with five concentrations of the standard plasmid as templates. The standard curve was plotted using Prism software.

To assess the specificity of the multiplex qPCR assay, the PUC57-PCVs standard plasmid was used as the positive control and ddH₂O as the negative control, DNA/cDNA of PRRSV, PEDV, CSFV, PTGV, PoRV and PRV extracted from the vaccine strains were used as templates for the qPCR assay detection. To determine the sensitivity, the standard plasmid was diluted 10-fold from 3.8 × 10⁶ copies/μL to 3.8 × 10⁰ copies/μL, qPCR amplification was performed in an optimal reaction system, and three replicates of each concentration were performed to confirm the detection limit of this multiplex qPCR assay. Intra-assay and inter-assay reproducibility was assessed using standard plasmids (3.8 × 10⁶ copies/μL to 3.8 × 10² copies/μL) as templates. For intra-assay reproducibility, three parallel replicates of each dilution were performed daily under the same conditions, and for inter-assay reproducibility, each dilution was tested by six independent experiments performed by two operators on different days according to the MIQE guidelines (22). Coefficients of variation of the Ct values were calculated based on the intra-assay or inter-assay results.

From 2020 to 2021, 738 clinical samples, including 326 nasopharyngeal swabs and 412 blood samples, were collected from swine farms in Guangdong Province, China. These samples were subsequently analyzed using an established multiplex qPCR assay to determine infection rates.

PCV2 (23) and PCV3 (20) complete genomes were amplified using the primers described previously. The reaction system was 50 μL, containing 25 μL of 2 × Platinum™ II Taq Hot-Start DNA Polymerase (Thermo Scientific, USA), 2 μL of template DNA, 200 nM primers, and the remaining was added to ddH₂O. The PCR reaction for PCV2 was executed by pre-denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 56°C for 15 s, and 68°C for 20 s. PCV3 amplification conditions were similar to those of PCV2, except that the PCV3 primer set was annealed at 57°C. The purified PCR products were cloned using a pMD18-T vector cloning kit (Takara, Dalian, China), and propagated in DH5α competent cells (Takara, Dalian, China) according to the manufacturer’s instructions. Positive clones were sequenced by Beijing Tsingke Biotech Co., Ltd. The obtained complete genome sequences were edited and assembled using DNAstar V7.1 software.

A total of 17 PCV2 and 4 PCV3 complete genomes were obtained in this study (Supplementary Table 1) and uploaded to NCBI GenBank with accession numbers MZ667312 (PCV2), MZ667314-MZ667329 (PCV2) and MZ667331-MZ667334 (PCV3). The complete PCV3 coding sequence (ORF1 + ORF2) was analyzed. Non-coding regions were deleted and the coding sequence was split into two parts, ORF1 (Rep) and ORF2 (Cap). Because ORF2 was in the opposite orientation, respective ORFs were downloaded and spliced separately. PCV3 subtypes were proposed by Li et al. (24). The PCV2 strain ORF2 sequence and the complete PCV3 coding sequence were aligned with the relevant reference sequences in GenBank using the ClustalW function of the Molecular Evolutionary Genetics Analysis software (MEGA, version 11.0), respectively. Phylogenetic trees were generated by the neighbor-joining (NJ) method in MEGA 11.0 with a p-distance model, and a bootstrap of 1,000 replicates.

Annealing temperatures of 50, 55, and 60°C, primer final concentrations of 100–500 nM and probe final concentrations of 100–250 nM were selected for multiplex qPCR system optimization. Based on the optimization results, the annealing temperature was determined to be 60°C (Table 3), final primer concentrations for PCV2, PCV3, and PCV4 were 300, 350 and 200 nM, respectively (Supplementary Tables 2–4), and final concentrations of all three probes were 100 nM (Supplementary Table 5). Therefore, the final amplification system was 10 μL of 2× AceQ Universal U+ Probe Master Mix V2, 300 nM PCV2 primer, 350 nM PCV3 primer, and 200 nM PCV4 primer, each with 100 nM for each probe, 2 μL of template, and ddH₂O was added to a final volume of 20 μL. Reaction conditions were 37°C for 2 min, pre-denaturation at 95°C for 300 s, followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s.

The recombinant standard plasmid was diluted to 3.8 × 10⁶ to 3.8 × 10² copies/μL in a 10-fold gradient, and multiplex qPCR assay was performed according to the optimal reaction system and procedure. The standard curve was established with the obtained Ct values as the y axis coordinates and the logarithm of the plasmid concentration as the x axis coordinates. All standard curves had good correlation coefficients and amplification efficiency. The slopes of PCV2, PCV3 and PCV4 were −3.383, −3.331 and −3.345, respectively; the correlation coefficient (R²) and amplification efficiency (Eff%) of the equation were as follows: PCV2 (R² = 0.9991, Eff% = 97.51), PCV3 (R² = 0.9992, Eff% = 99.62), PCV4 (R² = 0.9991, Eff% = 99.05) (Figure 1).

To assess the specificity of the multiplex qPCR, the recombinant standard plasmid PUC57-PCVs was used as a positive control. DNA/cDNA extracted from PRRSV, PEDV, CSFV, PTGV, PoRV, and PRV vaccines were used as templates, and ddH₂O was used as a negative control. qPCR was performed using an optimal reaction system. Specific fluorescent signals for FAM, HEX and Cy5 were detected only when the PUC57-PCVs standard plasmid was used as a template. However, DNA and cDNA of other pathogens such as PRRSV, PEDV CSFV, and PRV were not amplified (Figure 2). These results support high specificity of the multiplex qPCR assay.

To further assess the sensitivity of the multiplex qPCR assay, multiplex qPCR was performed using standard plasmids with concentrations ranging from 3.8 × 10⁶ to 3.8 × 10⁰ copies/μL. The results show that the detection limits of the assay were 3.8 × 10⁰ copies/μL for PCV2, PCV3 and PCV4 (Figure 3), suggesting that the multiplex qPCR assay has a good sensitivity.

To assess the reproducibility of the multiplex qPCR assay, standard plasmids with concentrations ranging from 3.8 × 10⁶ to 3.8 × 10² copies/μL were tested, respectively. Intra- and inter-assay coefficients of variation (CV) of Ct values for the multiplex qPCR assay ranged from 0.10 to 0.77% and 0.47 to 1.45%, respectively (Table 4). These results indicated that the multiplex qPCR assay established in this study has good reproducibility and reliability.

738 samples collected from different swine farms in Guangdong Province during 2020–2021 were tested using the method established in this study. Among the 738 samples, PCV2 infection rate was 65.18% (481/738), PCV3 infection rate was 29.27% (216/738), and PCV2/PCV3 co-infection rate was 23.17% (171/738) (Table 5). In the Pearl River Delta, western Guangdong, eastern Guangdong and northern Guangdong, PCV2 infection rate ranged from 50.44 to 71.95%, PCV3 infection rate ranged from 25.0 to 33.74%, and PCV2 and PCV3 co-infection rate ranged from 16.15 to 29.27%. For nasopharyngeal swabs, PCV2 infection rate was 70.25% (229/326), PCV3 infection rate was 25.77% (84/326), and PCV2/PCV3 co-infection rate was 21.17% (69/326). For blood samples, infection rate for PCV2 was 61.17% (252/412), 32.04% for PCV3 (132/412), and for PCV2/PCV3 co-infection rate was 24.76% (102/412) (Table 6). PCV4 was not detected in the above samples.

We attempted Complete genome amplification from the above samples and PCV-positive samples kept in our laboratory from 2015 to 2021. A total of 17 PCV2 complete genomes were obtained, each of which was 1767 nt in size. A phylogenetic tree was constructed based on the ORF2 sequences of the 17 PCV2 strains and 35 PCV2 reference sequences (PCV2a-h) (Figure 4A). 17 PCV2 sequences we obtained could be classified into PCV2b (6 strains) and PCV2d (11 strains) subtypes. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7–100% complete nucleotide identity and 94.0–100% ORF2 identity. Compared with the 35 PCV2 reference strains, the nucleotide identity of complete nucleotide and ORF2 varied 92.0 to 99.9% and 82.9 to 100%.

Similarly, we successfully obtained 4 PCV3 complete genomes, all of which was 2000 nt in size, which were submitted to NCBI GenBank. A phylogenetic tree was constructed based on the coding gene sequences (ORF1 + ORF2) of the 4 PCV3 strains and the reference sequences of 25 PCV3 strains (Figure 4B). Among the 4 PCV3 strains we obtained, two belonged to PCV3b and two belonged to PCV3a-1, which had different clustering levels. Nucleotide sequence analysis showed that the 4 PCV3 strains shared 98.9–99.4% complete nucleotide identity and 98.3–99.2% ORF2 identity. Compared with the 25 PCV3 reference strains, the nucleotide identity of complete nucleotide and ORF2 varied 98.8 to 99.7% and 97.8 to 99.7%.