The genomes (DNA or RNA) of PEDV, TGEV, PoRVA, pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine deltacoronavirus (PDCoV), Seneca virus A (SVA), Toxoplasma gondii, Leptospira interrogans, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Haemophilus parasuis, Streptococcus suis, Pasteurella multocida and Actinobacillus pleuropneumoniae were preserved by the State Key Laboratory for Animal Disease Control and Prevention of China or Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine. In addition, 256 anal swab samples from pigs with or without clinical diarrhea were obtained from Animal Health Testing Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The Institutional Review Board of Harbin Veterinary Research Institute or the Regulations on the Administration of Laboratory Animals of China did not require the study to be reviewed or approved by an ethics committee because the samples were collected from animals that were already dead or euthanized for other purposes, and no additional harm or intervention was imposed on the animals.

To ensure the detection performance of the primers and probes used in the triplex qRT-PCR, M gene of PEDV genome, N gene of TGEV genome and NSP3 gene of PoRVA genome were selected as the detection targets, based on their relative conservation. We obtained 86 PEDV M gene sequences, 46 TGEV N gene sequences and 20 PoRVA NSP3 gene sequences from GenBank database. We used MegAlign to align them and determine the most conserved regions of each target gene. Using Primer Express 3.0.1, we designed primers and probes for the three viruses with the following conditions: primer length 18–30 bp, primer melting temperature (T_m) 58–62°C, primer GC content 40–60%, product T_m 70–90°C and product size 70–150 bp. We ensured that the probe T_m value was higher than that of the primers. The specificity of the primers and probes was verified using the BLAST tool provided by the National Center for Biotechnology Information. For triplex detection, the probes for the three viral genes were labeled with different 5′-reporting dyes: Victoria Blue (VIC), Cyanine 5 (Cy5) and Fluorescein (FAM) and corresponding 3′-quenchers: Black Hole Quencher 1 (BHQ1), Black Hole Quencher 2 (BHQ2) and Minor Groove Binder (MGB). The triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018, 2018) was used to verify the accuracy of the results for clinical samples. The details of the primers and probes are provided in Table 1. Figure 1 shows the locations of the triplex qRT-PCR primers and probes for PEDV, TGEV and PoRVA in different reference strains.

A synthetic gene fragment (PEDV-M-TGEV-N-PoRVA-NSP3) containing partial sequences of PEDV M gene, TGEV N gene and PoRVA NSP3 gene was constructed in Sangon Biotech Co., Ltd. (Shanghai, China). This fragment was inserted into the pUC57 cloning vector, forming a standard plasmid (pUC57-PEDV M & TGEV N & PoRVA NSP3) for subsequent detection (Figure 2). The sequence of the synthetic gene fragment is shown in Supplementary Table S1. The plasmids were quantified by ultraviolet absorbance at 260 and 280 nm wavelengths using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) and their copy number was calculated based on the size of the standard plasmid template using the following formula: copies/μL = (A260 (ng/μL) × 10⁻⁹ × 6.02 × 10²³)/(DNA length × 650). The standard plasmids were serially diluted 10-fold to a concentration gradient of 10⁸–10⁰ copies/μL with EASY Dilution (TaKaRa, China, Dalian).

The unoptimized triplex qRT-PCR consisted of 10 μL 2× One Step U⁺ Mix (Vazyme, China, Nanjing), 1 μL One Step U⁺ Enzyme Mix (Vazyme), 0.4 μL 50× ROX Reference Dye II, 0.4 μL each primer (final concentration of 200 nM), 0.2 μL each probe (final concentration of 100 nM), 4 μL template and 1.6 μL RNase-free water in a total volume of 20 μL. The amplification was performed on an ABI QuantStudio5 real-time PCR system (Thermo Fisher Scientific) with the following program: 55°C for 15 min, 95°C for 30 s; 40 cycles of 95°C for 10 s and 60°C for 30 s. The fluorescence signal was automatically collected at the end of each cycle. To optimize the reaction system, we explored different primer volumes (10 μM) and probe volumes (10 μM). A range of primer volumes (0.3–1.2 μL) was assessed to achieve final concentrations spanning 150–600 nM. Additionally, probe volumes were varied from 0.1 to 0.6 μL, covering a concentration range of 50–300 nM. Recombinant plasmids (10⁷ copies/μL) served as the detection template for optimization. Finally, the fluorescence intensity and cycle threshold (Ct) values of each primer and probe concentration were compared to determine the optimal volumes. The annealing temperature was also optimized by setting six gradients from 56 to 61°C and comparing the fluorescence intensity and Ct values of each gradient.

On the basis of the optimized reaction and protocol, three replicates of plasmid samples with serial dilutions from 10⁸ to 10 copies/μL were detected using the triplex qRT-PCR and subjected to linear regression between Ct values and the logarithm of plasmid copy numbers. Eight-point standard curves were established for PEDV, TGEV and PoRVA, including negative controls.

To evaluate the specificity of the primer and probe sets, genomes (DNA or RNA) of PEDV, TGEV, PoRVA, PRV, PCV2, PPV, PDCoV, SVA, T. gondii, L. interrogans, M. hyopneumoniae, M. hyorhinis, H. parasuis, S. suis, P. multocida and A. pleuropneumoniae were tested using the triplex qRT-PCR. All nucleic acid samples were stored previously in our laboratory.

For sensitivity assessment, standard plasmids were serially diluted 10-fold to final concentrations ranging from 10⁸ to 1 copies/μL. These dilutions were used as templates to determine the limit of detection (LoD) for triplex qRT-PCR and each reaction was repeated three times in a single test. We tested the standard plasmids at 100, 10 and 1 copies/μL 20 times to ensure the LoD accuracy. We set the LoD as the lowest concentration of standard plasmids that gave positive results in 85% of the replicates and marked it on the amplification curves. The threshold was set in the middle of the exponential amplification phase in the logarithmic view. A positive test result was defined as an exponential fluorescence curve that crossed the threshold within 35 cycles [(Ct) <35]. According to this definition, we calculated the positive rates at 100, 10 and 1 copies/μL of standard plasmids.

The repeatability (intra-assay precision) and reproducibility (inter-assay precision) of the developed triplex qRT-PCR were determined using standard plasmids at three different concentrations (10⁶, 10⁴ and 100 copies/μL). We analyzed each dilution in triplicate on the same day for intra-assay variability and in six independent experiments by two different operators on different days for inter-assay variability. The coefficient of variation (CV) of the Ct values of the samples at different concentrations was calculated in both intra-assay and inter-assay tests to estimate the repeatability and reproducibility.

Viral RNA was extracted from 256 anal swab samples using AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit (Corning Life Sciences, China, Wujiang). The RNA samples were tested in triplicate by the optimized triplex qRT-PCR. Subsequently, the sample RNA was reverse transcribed into cDNA using PrimeScript^™ RT Master Mix (Perfect Real Time) (TaKaRa) and detected by the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018, 2018), to validate the clinical performance of the developed triplex qRT-PCR assay. For the triplex RT-PCR, the reaction mixture (25 μL) contained 12.5 μL 2× Taq PCR Star Mix (Genstar, China, Beijing), 0.2 μL PEDV primers (final concentration of 80 nM), 0.4 μL TGEV primers (final concentration of 160 nM), 1 μL PoRVA primers (final concentration of 400 nM), 4 μL cDNA template and 5.3 μL RNase-free water. We performed the triplex RT-PCR with the following parameters: pre-denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and final extension at 72°C for 10 min.

A plasmid with three conserved gene fragments of PEDV M (693 bp), TGEV N (649 bp) and PoRVA NSP3 (114 bp) was constructed, all containing their respective qRT-PCR amplicons. The plasmid was used as the standard for subsequent detection.

Using the standard plasmid pUC57-PEDV M & TGEV N & PoRVA NSP3 with the target fragments as the template, we optimized the reaction conditions of the triplex qRT-PCR. Orthogonal experiments determined the optimal annealing temperature and concentrations of primers and probes. For TGEV and PoRVA, the optimal volumes of probes and primers were 0.4 and 0.7 μL, respectively. For PEDV, both were 0.3 μL (Figure 3). The confirmed reaction system was listed in Table 2. The optimal annealing temperature was 60°C, which yielded the highest amplification efficiency (Figure 4).

The standard plasmid was diluted in a 10-fold series and eight standard samples (10⁸–10 copies/μL) were selected as templates to establish the standard curve of the triplex qRT-PCR. Figure 5 shows the correlation coefficients (R²), equation slopes and amplification efficiencies (E) for each virus: PEDV, 0.999, −3.272 and 102.118%; TGEV, 0.999, −3.294 and 101.179%; and PoRVA, 0.999, −3.22 and 104.441%. The initial template and Ct value had a good linear relationship, as indicated by R² and E.

Genomic DNA or RNA of 16 porcine pathogens (PEDV, TGEV, PoRVA, PRV, PCV2, PPV, PDCoV, SVA, T. gondii, L. interrogans, M. hyopneumoniae, M. hyorhinis, H. parasuis, S. suis, P. multocida and A. pleuropneumoniae) was used as a template for the triplex qRT-PCR. Amplification curves were obtained for PEDV, TGEV and PoRVA but not for the other porcine pathogens (Figure 6). Therefore, the triplex qRT-PCR assay was specific for detection of PEDV, TGEV and PoRVA, and had no cross-reaction with other porcine pathogens.

Different concentrations of standard plasmids were used as templates for the triplex qRT-PCR. Table 3 shows that a plasmid concentration of 100 copies/μL resulted in 100% positive detection rates for PEDV, TGEV and PoRVA. At 10 copies/μL, the positive detection rates were 100, 90 and 95% for PEDV, TGEV and PoRVA, respectively. At 1 copy/μL, the positive detection rates were 75, 5% and 0 for PEDV, TGEV and PoRVA, respectively. The LoD was defined as the lowest standard plasmid concentration with positive results in 85% of 20 replicates. Therefore, the triplex qRT-PCR showed high sensitivity, with a LoD of 10 copies/μL for PEDV, TGEV and PoRVA (Figure 7).

Three concentrations of standard plasmids, 10⁶, 10⁴ and 100 copies/μL, were used to assess the repeatability and reproducibility of the triplex qRT-PCR. The intra-and inter-assay CVs were 0.08–0.79% and 0.37–0.83%, respectively (Table 4), which indicated good repeatability and reproducibility.

A total of 256 porcine anal swab samples were tested using the triplex qRT-PCR. The positive rates for PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 19.14% (49/256), respectively. To verify the accuracy of the method, the clinical samples were also tested by the triplex RT-PCR recommended by the Chinese national standard (GB/T 36871-2018, 2018). The triplex RT-PCR results showed that the positive rates for PEDV, TGEV and PoRVA were 2.73% (7/256), 3.91% (10/256) and 14.84% (38/256), respectively. Both methods detected seven samples co-infected with PEDV and PoRVA. The new triplex qRT-PCR had 100% (PEDV), 100% (TGEV) and 95.70% (PoRVA) agreement with the triplex RT-PCR, indicating that the new approach was accurate, reliable and more sensitive (Table 5).