The nucleic acid of various viruses and bacteria, including respiratory syndrome virus (PRRSV), pseudorabies virus (PrV), porcine circovirus (PCV1), PCV2, classical swine fever virus (CSFV), African swine fever virus (ASFV), Streptococcus suis (SS), Salmonella enteritidis (SE), PEDV, PoRV, and PDCoV were stored at −80°C in our laboratory until needed. At least 30 genome sequences of PEDV, PoRV, and PDCoV were downloaded from NCBI for analysis. After comparison, the M gene of PEDV and PDCoV, and the NSP3 gene of PoRV were found to be the most conserved gene sequences. Primers and probes were designed using Primer Premier 5 software (Premier, Canada) based on the most conserved region. TaqMan probes for PEDV, PoRV, and PDCoV were labeled with FAM, VIC, and Cy5 at the 5′ end, respectively, with all quenchers at the 3′ end being BHQ. The sequences of the primers and probes designed in this study are presented in Table 1. Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

The target fragments of PEDV, PoRV, and PDCoV were amplified separately using the HiScript II One Step RT-PCR Kit (Dye Plus; Nanjing Vazyme Biotech Co., Ltd.). The PCR fragments were purified and cloned into the pMD18-T vector (Takara Biomedical Technology (Beijing) Co., Ltd.). The transformed clones were then introduced into the E.coli DH5α strain. Positive clones were cultured and plasmid extraction was performed using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit [Takara Biomedical Technology (Beijing) Co., Ltd.]. The constructed plasmid was confirmed by DNA sequencing and used as the standard positive control. The copy number of recombination plasmids was calculated using the following formula (Li et al., 2022):

A 10-fold series dilution was conducted for each plasmid, ranging from 1.0 × 10⁸ to 1.0 × 10¹ copies/μL. Singleplex real-time PCR was performed for each virus using the 10-fold diluted plasmids to generate standard curve. For multiplex standard curves, each plasmid was diluted to 3.0 × 10⁹ copies/μL and pooled in equal volume to make a concentration of 1.0 × 10⁹ copies/μL for each plasmid. The pooled plasmid was then diluted in a 10-fold series, ranging from 1.0 × 10⁸ to 1.0 × 10³ copies/μL, to establish multiplex standard curves.

All real-time PCR reaction systems were set to a volume of 20 μL. For singleplex real-time PCR of PDCoV, RV, and PEDV, the reaction system included 10 μL 2× PerfectStart Probe One-Step qPCR SuperMix, 0.4 μL TransScript Probe One-Step RT Enzyme Mix (Transgen, Beijing), 0.4 μL each of forward/reverse primer (10 μM), 0.4 μL TaqMan probe (10 μM), 5.5 μL RNA template, and 2.9 μL nuclease-free water.

Amplification was performed on a Bio-Rad CFX96™ Real-time System (Bio-Rad, Hercules, CA, USA) using the following program: 45°C for 5 min, 94°C for 30 s, 40 cycles of 94°C for 5 s, and 52°C for 30 s. Fluorescence signal was automatically collected at the end of each cycle. All qPCR results were analyzed using CFX Manager™ software.

The concentrations of primers and probes were optimized as previously described (Pan et al., 2020). After repeated tests, the optimal reaction conditions for multiplex real-time PCR were determined as follows: 10 μL 2× PerfectStart Probe One-Step qPCR SuperMix, 0.4 μL TransScript Probe One-Step RT Enzyme Mix (Transgen, Beijing), 0.4 μL each of forward/reverse primer and probe (10 μM) for PoRV and PDCoV, 0.2 μL each of forward/reverse primer and probe (10 μM) for PEDV, 5.5 μL RNA template, 1.1 μL nuclease-free water, with a total reaction volume of 20 μL. The same instrument and real-time PCR program were used as described above.

To determine the limit of detection (LOD) for the multiplex real-time PCR method, the aforementioned pooled standard plasmids were diluted in a 10-fold serial manner, ranging from 1.0 × 10³ to 1.0 × 10⁰ copies/μL in nuclease-free water. These diluted standard plasmids were then used as templates for multiplex real-time PCR amplification. The reliable LOD was the lowest concentration that achieved a 95% positive detection rate.

To avoid false positives caused by other viruses or bacteria that may be present in the samples, two RNA viruses (PRRSV and CSFV), three DNA viruses (PCV1, PCV2, and ASFV), and two bacterias (S. suis and S. enteritidis) were used for specificity test of multiplex real-time PCR assay. Nucleic acids were extracted using the VAMNE Virus DNA/RNA Extraction Kit (Nanjing Vazyme Biotech Co., Ltd.). For RNA viruses, the cDNA was generated with the TransScript Probe One-Step qRT-PCR SuperMix (Beijing Transgen Biotech Co., Ltd.). These DNA and cDNA samples were first detected using commercial kits, and positive samples with CT values <25 were selected as template for specificity test of the multiplex real-time PCR assay. The standard plasmids of PEDV, PoRV, and PDCoV were used as positive controls, while nuclease-free water served as the negative control.

To assess its repeatability of the multiplex real-time PCR, a 10-fold serially diluted standard template was used, ranging from 1.0 × 10⁸ to 1.0 × 10³ copies/μL. Each reaction was performed with three replicates. Intra-assay repeatability was determined by conducting three simultaneous detections of each plasmid under identical conditions. Inter-assay repeatability was evaluated by repeating the assays three times individually at different time points. The coefficient of variation (CV) of the Cq values from the three experiments was calculated to estimate the level of repeatability. Data analysis was conducted using Microsoft Excel.

Our multiplex real-time PCR method and a commercial singleplex real-time PCR Kit were used to simultaneously detect 30 clinical samples with diarrhea symptoms. The detection results obtained from our established methods were then compared with those obtained from singleplex real-time PCR.

A total of 4,800 samples including fecal samples (n = 2,341), rectal swabs (n = 1,874), oral fluid (n = 145), and oropharyngeal swabs (n = 440), were used to investigate the prevalence of PEDV, PoRV, and PDCoV using the multiplex real-time PCR assay. These samples were collected from pig farms located in numerous provinces of China, including Gansu, Shanxi, Shaanxi, Shandong, Henan, Hubei, Anhui, Zhejiang, Jiangsu, Guangdong, Guangxi, Guizhou, Sichuan, Hunan, Chongqing, and Jiangxi, during the period from November 2021 to July 2022.

Clinical samples were treated with phosphate buffer saline (PBS), and the supernatant was collected after vortexing and centrifugation. Nucleic acids were extracted using the VAMNE Virus DNA/RNA Extraction Kit (Nanjing Vazyme Biotech Co., Ltd.). Reverse transcription was done with the TransScript Probe One-Step qRT-PCR SuperMix (Beijing Transgen Biotech Co., Ltd.). The constructed plasmid and nuclease-free were employed as positive and negative controls, respectively. All samples were tested with the multiplex real-time PCR assay to determine virus positivity. Infection rates were analyzed after obtaining assay results for all clinical samples.

To develop a multiplex real-time PCR, we first established singleplex real-time PCR for each virus using different fluorescence-labeled target probes. Standard curve for each virus was created using 10-fold serial dilutions of plasmids ranging from 1.0 × 10⁸ to 1.0 × 10¹ copies/μL. All the standard curves showed excellent correlation coefficients and amplification efficacy, with PEDV (R² = 1.000; E = 101.3%), PoRV (R² = 0.999; E = 103.9%), PDCoV (R² = 1.000; E = 100.5%), respectively. This indicates that our plasmid standards were qualified, and the designed primers and probes were efficient (Figures 1A–C).

Serial dilutions of mixed viral standard plasmids were tested with the using the optimized multiplex assay. The results demonstrated that the multiplex real-time PCR successfully detected all target genes of these three viruses (Figure 1D). The standard curves exhibited excellent correlation coefficients and amplification efficacy for each virus, for details, PEDV (R² = 1.000; E = 98.2%), PoRV (R² = 0.999; E = 100.7%), and PDCoV (R² = 0.998; E = 98.5%; Figure 1D), indicating the validity and reliability of the multiplex real-time PCR.

To evaluate the specificity of the multiplex real-time PCR, the positive samples of PRRSV, PRV, PCV1, PCV2, CSFV, ASFV, SS, and SE were used as templates for amplification with this multiplex system. The standard plasmids of PEDV, PoRV, and PDCoV were tested as the positive controls, while nuclease-free water was tested as negative control. As shown Figure 2A, all the target pathogens were successfully detected, while no positive signal was observed from other eight viruses and negative control. Additionally, three clinical samples from healthy pigs were further tested to confirm the specificity. Amplification curves also demonstrated that only target genes from the standard plasmids were detected, with no positive signal detected in the clinical samples from healthy pigs (Figure 2B). These findings indicated that the multiplex real-time PCR assay was highly specific.

To access the sensitivity of this multiplex real-time PCR assay, we tested pooled standard plasmids with concentrations ranging from 1.0 × 10³ to 1.0 × 10⁰ copies/μL under optimized reaction conditions. The method was able to detect positive samples with concentrations as low as 1.0 × 10¹ copies/μL (Table 2). However, further experiments showed the detection rate for samples at 1.0 × 10¹ copies/μL was < 95% of replicates (Table 2). Therefore, the reliable limit of detection for this method is 1 × 10² copies/μL.

The multiplex real-time PCR assay was confirmed to be repeatable by detecting standard plasmids at concentrations ranging from 1.0 × 10⁸ to 1.0 × 10³ copies/μL. As shown in Table 3, the variation coefficients (CVs) of Cq values in the intra-group and inter-group reproducibility tests were between 0.11 and 0.87% and between 0.11 and 1.63%, respectively. These results indicated that the multiplex real-time PCR assay established in this study had satisfactory repeatability.

Thirty clinical samples were used to compare the results of the multiplex real-time PCR with commercial singleplex real-time PCR kit. As shown in Supplementary Table 1, the results of the multiplex real-time PCR were consistent with the commercial singleplex real-time PCR, suggesting that it can effectively replace the commercial singleplex real-time PCR for simultaneous detection of PEDV, PoRV, and PDCoV.

A total of 4,800 clinical samples were detected using the multiplex real-time PCR assay established in this study. As shown in Figure 3A, the single infection rates for PEDV, PoRV, and PDCoV were 6.56% (315/4,800), 21.69% (1,041/4,800), and 6.65% (319/4,800), respectively. The co-infection rates for PEDV + PoRV, PEDV + PDCoV, and PoRV + PDCoV were 11.83% (568/4,800), 0.29% (14/4,800), and 5.71% (274/4,800), respectively. The mixed infection rate for PEDV + PDCoV + PoRV was 1.29% (62/4,800).

These 4,800 clinical samples were collected from 16 provinces which were divided into three regions based on the geographical distribution: Northern China (Gansu, Shanxi, and Shaanxi), Mideastern China (Shandong, Henan, Hubei, Anhui, Zhejiang, and Jiangsu), and Southern China (Guangdong, Guangxi, Guizhou, Sichuan, Hunan, Chongqing, and Jiangxi). In the Northern China, PEDV, PoRV, and PDCoV had infection rates of 10.33% (224/2,169), 14.71% (319/2,169), and 11.66% (253/2,169), respectively (Figure 3B). Co-infection rates were 18.63% (404/2,169) for PEDV + PoRV, 0.60% (13/2,169) for PEDV + PDCoV, 10.28% (223/2,169) for PoRV + PDCoV, 0.55% (12/2,169) for PEDV + PDCoV + PoRV, respectively (Figure 3B). In the Mideastern China, PEDV, PoRV, and PDCoV had infection rates of 2.44% (40/1,642), 26.79% (440/1,642), and 3.59% (59/1,642), respectively (Figure 3B). Co-infection rates were 5.66% (93/1,642) for PEDV + PoRV, 0.06% (1/1,642) for PEDV + PDCoV, 2.68% (44/1,642) for PoRV + PDCoV, 1.22% (20/1,642) for PEDV + PDCoV + PoRV, respectively (Figure 3B). In the Southern China, PEDV, PoRV, and PDCoV had infection rates of were 5.16% (51/989), 28.51% (282/989), and 0.71% (7/989), respectively (Figure 3B). Co-infection rates were 7.18% (71/989) for PEDV + PoRV, 0 (0/989) for PEDV + PDCoV, 0.71% (7/989) for PoRV + PDCoV, 3.03% (30/989) for PEDV + PDCoV + PoRV, respectively (Figure 3B).