Development and Validation of a Multiplex TaqMan Real-Time PCR Assay for Simultaneous Detection of PEDV Genotypes G1, S-INDEL, and G2

TL;DR

A new real-time PCR test was developed to quickly detect and differentiate three types of PEDV in pigs, improving disease control and monitoring.

Contribution

A novel triplex TaqMan real-time PCR assay for rapid detection and differentiation of three PEDV genotypes.

Findings

The assay detected as few as 102 copies/μL of PEDV with no cross-reactivity to other swine pathogens.

Clinical validation showed 100% agreement with traditional sequencing methods.

Co-infections with G2 and S-INDEL strains were identified in 160 clinical samples.

Abstract

Porcine epidemic diarrhea virus (PEDV) is a major pathogen responsible for severe diarrhea, dehydration, and high mortality in neonatal piglets, continually threatening global swine production. Rapid differentiation of its major genotypes (classical G1, variant G2, and recombinant S-INDEL) is vital for molecular epidemiology and effective disease control, yet existing approaches rely mainly on time-consuming sequencing and phylogenetic analysis of the S gene. To overcome this limitation, we developed a novel triplex TaqMan-based real-time PCR assay for rapid detection and differentiation of the three PEDV genotypes. The assay demonstrated high sensitivity, with the lowest detection limit of 102 copies/μL, and strong specificity, showing no cross-reactivity with six other common swine pathogens (TGEV, PDCoV, PoRV, PRRSV, CSFV, and PRV). It also exhibited excellent reproducibility, with both intra- and inter-assay coefficients of variation maintained below 1.5%. In clinical validation, the assay showed 100% concordance with results obtained from S gene sequencing and phylogenetic analysis. Furthermore, testing of 160 clinical samples revealed cases of co-infection involving G2 and S-INDEL strains. In conclusion, this rapid, specific, and reproducible assay provides a reliable tool for routine molecular diagnosis, facilitating large-scale epidemiological surveillance and enabling genotype-informed control strategies against PEDV.

Figures

Full text

1. Introduction

Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus within the family Coronaviridae, is the etiological agent of porcine epidemic diarrhea (PED). This acute enteric disease is characterized by severe watery diarrhea, vomiting, and dehydration in swine, with mortality rates in neonatal piglets reaching up to 100%, leading to substantial economic losses and remaining a major persistent threat to the global swine industry [1,2,3]. The viral genome consists of a single-stranded, positive-sense RNA of approximately 28 kb, encoding four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) [4,5]. Among these, the S glycoprotein determines viral antigenicity, host cell tropism, and immune evasion, thereby serving as the primary genetic marker for phylogenetic classification [6,7,8].

Based on S gene variations, PEDV strains are generally categorized into three genotypes: the classical G1 (e.g., CV777, G1a), the highly virulent variant G2 (e.g., AH2012, G2a, domain strain in Asia), and the recombinant S-INDEL (e.g., OH851 and EJS6) strains [9,10,11,12]. A major epidemiological shift has occurred over the past decades. While classical G1 strains were once predominant, the emergence of G2 variants around 2010 has drastically altered the landscape. These G2 strains, now dominant globally and particularly in China, exhibit extreme virulence, causing near-universal morbidity and mortality rates of 50–100% in neonatal piglets [13,14,15]. Critically, commercial vaccines derived from classical G1 strains offer only limited cross-protection against these prevalent G2 variants [16,17]. The ongoing evolution of porcine epidemic diarrhea virus (PEDV) has generated distinct internal lineages (G1a, G1b, G2a, G2b, G2c, and S-INDEL), complicating its control and prevention [18,19]. Although vaccines based on the G2b subtype were subsequently introduced in China, emerging evidence in recent years suggests their protective efficacy against the G2a and G2c subtypes may also be limited [20]. Concurrently, S-INDEL strains, which likely arose from recombination events between G1 and G2 lineages, display an intermediate pathogenic profile, more virulent than classical G1 but less so than the emergent G2 strains [11,21,22,23,24]. Recent epidemiological data from China underscore this dynamic. Epidemiological surveillance from 2011 to 2021 revealed that 89.9% of circulating strains were G2, with G1 comprising only 10.1% [25]. More recent monitoring across six provinces (2020–2023) confirmed the ongoing dominance of G2 (83.3%) and the stable circulation of S-INDEL strains (16.7%), whereas G1 strains were not detected [26]. Following the initial circulation and identification of PEDV G2 and S-INDEL strains, these variants have subsequently emerged as predominant strains in multiple regions beyond Asia, including the United States and various European countries, where they similarly pose a substantial threat to piglet health [24,27,28]. This co-circulation of genetically distinct genotypes with divergent pathogenic potential complicates disease control and underscores an urgent need for rapid, accurate genotyping to inform targeted intervention strategies.

Currently, S gene sequencing coupled with phylogenetic analysis remains the “gold standard” for definitive PEDV classification. However, this method is time-consuming, labor-intensive, and impractical for rapid decision-making during acute outbreaks. While real-time reverse transcription polymerase chain reaction (RT-qPCR) has become a mainstay for rapid detection, most available assays fail to differentiate all major PEDV genotypes (G1, G2, and S-INDEL) [29,30,31]. Specifically, many assays cannot resolve the recombinant S-INDEL genotype, limiting their utility for comprehensive surveillance and rapid response.

To address this diagnostic gap, we developed and validated a novel triplex TaqMan probe-based real-time RT-PCR assay for rapid detection and differentiation of the three major PEDV genotypes: G1, G2, and S-INDEL. This assay enables precise genotyping within 90 min, providing a practical tool to facilitate timely vaccination decisions, enhance epidemiological monitoring, and mitigate the economic impact of PEDV outbreaks.

2. Materials and Methods

2.1. Viruses and Clinical Samples

The PEDV CV777 vaccine strain (G1) and field isolates of PEDV SH2501 (G2), PEDV AH2408 (S-INDEL), transmissible gastroenteritis virus (TGEV, Purdue), porcine rotavirus (PoRV, SL/2025), and porcine deltacoronavirus (PDCoV, WX/2025) were maintained in our laboratory. Vaccine strains of porcine reproductive and respiratory syndrome virus (PRRSV, JXA1-R), classical swine fever virus (CSFV, C strain) and pseudorabies virus (PRV, JS-A1) were obtained from Chengdu SG-Biotech Co., Ltd. (Chengdu, China), Guangdong Winsun Bio-pharmaceutical Co., Ltd. (Guangzhou, China), and Shiji Biological Pharmaceutical Co., Ltd. (Wuhan, China), respectively.

From 2021 to 2025, a total of 160 clinical samples (intestinal contents or feces) were collected from diarrheic piglets across nine Chinese provinces, including Anhui, Guangxi, Hebei, Heilongjiang, Hubei, Jiangsu, Shandong, Shanxi, and Sichuan. All swabs were immersed in 1 mL of PBS, vortexed, and centrifuged to collect the supernatant, which was then either used directly for nucleic acid extraction or stored at −80 °C. Additionally, 25 PEDV-positive fecal samples with pre-sequenced S genes were selected for the development of the PEDV subtyping qPCR assay.

2.2. RNA Extraction and cDNA Synthesis

Total nucleic acid was extracted from 200 μL of viral culture stock or clinical specimens using the Viral DNA/RNA Extraction Kit 2.0 (Vazyme, Nanjing, China, Cat. No. RM 401), yielding a final volume of 50 μL. Following extraction, nucleic acid (RNA/DNA) concentration was measured, and integrity was verified by spectrophotometric assessment of A260/A280 (~1.8–2.0) and A260/A230 (>2.0) ratios prior to downstream use. For RNA viral nucleic acids, 16 μL of the extracted nucleic acid was aliquoted, and complementary DNA (cDNA) was synthesized by reverse transcription using the HiScript IV All-in-One Ultra RT SuperMix (Vazyme, China, Cat. No. R433) in a total reaction volume of 20 μL. All extracted nucleic acids and synthesized cDNA were stored at −80 °C until further use.

2.3. Phylogenetic Analysis

In total, 30 reference S gene sequences of identified PEDV genotypes (G1, G2, and S-INDEL) were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (https://www.ncbi.nlm.nih.gov/, 10 June 2025) and combined with 25 complete S gene sequences from PEDV-positive samples (Table S1). Multiple sequence alignment was performed with the ClustalW algorithm in MEGA 11 [32]. Phylogenetic analysis was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates. Evolutionary distances were calculated using the p-distance method with a constant rate. The phylogenetic tree was visualized and annotated using MEGA 11 [32] software and the Interactive Tree of Life (iTOL) platform [33].

2.4. Primer and Probe Design

Multiple sequence alignment of all sequences sorted according to genotypes was performed using the built-in MAFFT algorithm in MegAlign Pro 17 to better identify and visualize genotype-specific conserved regions within the S gene. Based on the conserved regions, primers and probes were designed using Primer 5 and Oligo 7 software, with attention to minimizing self-complementarity (especially at the 3′- end) and cross-complementarity between primer pairs. Stretches of ≥3 consecutive complementary bases were excluded to prevent dimer formation. Specificity for PEDV was verified by NCBI BLAST analysis to reduce potential cross-reactivity. All primers and probes were synthesized by General Biol Co., Ltd. (Chuzhou, China). Primers were PAGE-purified, and probes were HPLC-purified to ensure fluorescence signal stability.

2.5. Preparation of Standard Plasmids

The cDNA of PEDV reference strains CV 777 (G1), SH 2501 (G2), and AH 2408 (S-INDEL) was amplified via conventional PCR employing genotype-specific primers (Table 1) and 2 × Taq Master Mix (Dye Plus) (Vazyme, China, Cat. No. P112). Amplification products were resolved by electrophoresis on a 1% agarose LE gel (Generay, China, Cat. No. RA 1011), and target bands were visualized under UV light. The corresponding DNA fragments were excised, purified using a DNA Gel Extraction Kit (Tiangen, Cat. No. DP204), and subsequently cloned into the pMD 18-T vector (Takara, Cat. No. 6011). Recombinant plasmids were transformed into E. coli DH5α competent cells (Sangon, China, Cat. No. B528411) and plated on LB agar (Beyotime, Shanghai, China, Cat. No. ST158) supplemented with 100 μg/mL ampicillin (Beyotime, China, Cat. No. ST008). Following overnight incubation, positive colonies were selected, inoculated into LB liquid medium (Beyotime, China, Cat. No. ST156), and cultured at 37 °C with shaking at 180 rpm for 12 h. Plasmid DNA was extracted using a commercial plasmid extraction kit (Tiangen, China, Cat. No. DP103) and verified by Sanger sequencing. Aliquots of the resulting standard plasmids were stored at −80 °C for subsequent use. Before each detection, the concentration of plasmid DNA (ng/μL) was determined by spectrophotometry, and copy numbers (copies/μL) were calculated using the formula:

$$[eqn]$$

2.6. Optimization of the qPCR Assay

A single-variable control method was used to optimize qPCR detection parameters, including annealing temperature and concentrations of primer/probe. The final qPCR was performed in a 20 μL reaction mixture consisting of 10 μL of 2 × Taq Pro HS Probe Master Mix (Vazyme, China, Cat. No: QN111), 1 μL of template pDNA (1 × 10^8^ copies/μL), forward and reverse primers (100 to 400 nM each), probe (40 to 160 nM), and nuclease-free ddH_2_O. The prepared qPCR master mix did not contain an ROX reference dye. Amplifications were carried out on a LineGene 9600 Plus Real-Time Fluorescence Quantitative PCR Detection System (Bioer, Hangzhou, China, Product Model: FQD-96A) with the following cycling protocol: initial denaturation at 95 °C for 5 min; 40 cycles of denaturation at 95 °C for 15 s and annealing at 50 °C to 60 °C (in 2 °C increments) for 20 s. Fluorescence signals were collected at the end of each annealing step. The validity threshold for qPCR assessment in this study was defined as a Ct value of 40 cycles.

2.7. Generation of Standard Curves

Recombinant plasmids of each PEDV genotype (G1, G2, and S-INDEL) were quantified and diluted to a standard concentration of 1 × 10^10^ copies/μL, followed by ten-fold serial dilution (1 × 10^10^ to 1 × 10^3^ copies/μL) to generate templates for standard curve construction. qPCR amplification was performed under optimized conditions. Standard curves were plotted with the logarithm of plasmid concentration on the x-axis versus the cycle threshold (Ct value) on the y-axis. The linearity of the detection method for each genotype was assessed by analyzing the slope and correlation coefficient (R^2^) of each standard curve.

2.8. Specificity Test

The specificity of the optimized qPCR assay was assessed using full-length DNA or cDNA templates from three target PEDV genotypes (G1, G2, and S-INDEL) and several other non-target porcine pathogens, including: TGEV, PDCoV, PoRV, PRV, PCV3, CSFV, and PRRSV. Nuclease-free ddH_2_O was used as a negative control. The assay demonstrated high specificity, with no cross-reactivity observed with any of the non-target pathogens with non-target pathogens.

2.9. Sensitivity Test

Ten-fold serial dilutions of recombinant plasmids of each PEDV genotype (G1, G2, and S-INDEL) were prepared, spanning a concentration range of 1 × 10^10^ to 1 × 10^0^ copies/μL. qPCR was performed under optimized conditions using these dilutions as templates, with nuclease-free water serving as a negative control. The limit of detection (LOD) for each genotype was defined as the lowest plasmid concentration at which reproducible fluorescence signals could be detected in all replicates, encompassing a minimum of three separate experimental runs.

2.10. Repeatability Test

The repeatability of the three qPCR detection methods was evaluated using standard plasmids with concentrations of 1 × 10^6^, 1 × 10^5^, and 1 × 10^4^ copies/μL as templates, under optimized reaction conditions. Intra-assay repeatability was assessed by performing three replicates within the same experiment, while inter-assay reproducibility was determined across three independent experiments. Precision was evaluated by calculating the coefficient of variation (CV) of Ct values. Repeatability (Intra-assay CV) = (SD of Ct replicates/Mean Ct) × 100%. Reproducibility (Inter-assay CV) = (SD of mean Cts across runs/Overall mean Ct) × 100%.

2.11. Clinical Sample Testing

The established TaqMan probe-based qPCR assay was first validated using 25 clinical samples with previously determined genotypes of S gene sequences. The accuracy of this assay was evaluated by comparing the qPCR genotyping results with the known classifications. Subsequently, the assay was applied to screen 160 clinical samples collected from diarrheic piglets. Reference strains (CV777, SH2501, and AH2408) served as positive controls, while nuclease-free ddH_2_O was used as the negative control in each detection.

3. Results

3.1. Phylogenetic Analysis

Phylogenetic analysis of the S gene revealed that PEDV can be classified into three genotypes: G1 (represented by the CV777), G2 (represented by the AJ1102), and S-INDEL (represented by the OH851) (Figure 1). Among the 25 clinical samples sequenced for the PEDV S gene, 15 belonged to the G2 genotype and 10 to the S-INDEL genotype.

3.2. Primer and Probe Design

Based on the phylogenetic classification results (Figure 1), 30 complete S gene sequences representing the three major PEDV genotypes were compiled and analyzed. Comparative nucleotide homology analysis of the S, S1, and S2 regions revealed that the S1 subunit exhibited notably low homology between genotypes (91.03–95.56%), and high homology within the same genotype (95.78–100%) (Table S2). This high intra-genotype conservation coupled with inter-genotype divergence in the S1 region enabled the identification of genotype-specific conserved sequences, which were subsequently used to design discriminating primers and TaqMan probes (Table 1). The probe sequences exhibited high specificity and conservation across the 203 reference sequences in the alignment analysis (Figure S1).

3.3. Validation of Standard Plasmid

The standard plasmids of the three target PEDV genotypes (CV777/G1, SH2501/G2, and AH2408/S-INDEL) were verified by multiple approaches. RT-PCR yielded amplicons of the expected sizes: 236 bp (G1), 108 bp (G2), and 117 bp (S-INDEL) (Figure 2A). Sequencing results also confirmed the expected sequences. Furthermore, qPCR analysis of each standard plasmid generated strong, genotype-specific amplification curves in their respective fluorescence channels (Figure 2B). These results collectively confirmed the successful construction of the plasmids.

3.4. Optimization of Reaction Conditions

In the qPCR system, the initial probe concentration was set to 40% of the primer concentration. Using the cDNA of each genotype as the template, annealing temperature optimization was performed across a gradient from 50–60 °C (in 2 °C increments) with the primer concentration fixed at 300 nM. As shown in Figure 3A, the Ct values of the three genotypes all reached the minimum at 54 °C, which was therefore selected as the optimal annealing temperature. Subsequently, with the annealing temperature fixed at 54 °C, the optimal primer concentration was tested from 100 to 400 nM (in 50 nM increments). A primer concentration of 300 nM yielded the lowest Ct values and the highest fluorescence signals (Figure 3B), and thus this concentration was chosen as the optimal concentration for subsequent detection.

3.5. Specificity Analysis

The specificity of each qPCR assay was assessed, using nucleic acids of three target PEDV genotypes and six non-target swine pathogens (TGEV, PoRV, PDCoV, PRRSV, CSFV, and PRV), all maintained at or above 10^7^ copies/μL. As shown in Figure 4, specific amplification curves were observed only for the three PEDV genotypes, while no signal was detected for any of the non-target pathogens or the negative control. These results confirmed that the detection method has high specificity and no cross-reactivity with other common porcine pathogens.

3.6. Construction of Standard Curves

Strand curves of each genotype were generated, using ten-fold serial dilutions of each related plasmid, under the optimized qPCR conditions. Linear regression analysis (Figure 5) showed the following relationships:

G1, R^2^ = 0.9976, Y = −3.3639X + 45.097, E = 98.29%;

G2, R^2^ = 0.9994, Y = −3.3817X + 44.206, E = 97.54%;

S-INDEL, R^2^ = 0.9990, Y = −3.4135X + 45.469, E = 96.27%.

All R^2^ values were greater than 0.99, indicating a strong linear correlation between the Ct value and the logarithm of the plasmid copy number, which confirmed that the method has high quantitative accuracy over a wide dynamic range (Table S3).

3.7. Sensitivity Analysis

To evaluate the sensitivity of the method, ten-fold serial dilutions of each genotype-specific plasmid (1 × 10^10^ to 1 × 10^0^ copies/μL) were analyzed. The results showed that the limit of detection (LOD) for all three PEDV genotypes stably reached 1 × 10^2^ copies/μL (Figure 6). This LOD was confirmed across repeated independent experiments.

3.8. Repeatability Analysis

The repeatability of the assay was evaluated using 10-fold serial dilutions (1 × 10^6^ to 1 × 10^4^ copies/μL) of each genotype-specific plasmid. Three independent runs were performed, each containing three replicates. As summarized in Table 2, the intra-assay CV range was 0.442% to 1.057% for G1, 0.458% to 1.007% for G2, and 0.461% to 0.708% for S-INDEL. The inter-assay CV range was 0.677% to 0.807% for G1, 0.721% to 1.494% for G2, and 0.352% to 1.404% for S-INDEL. These low CV values indicate that the method has high stability and excellent repeatability across different concentrations and experimental batches.

3.9. Clinical Sample Detection

To evaluate the accuracy of the established qPCR assay, we analyzed 25 samples with previously confirmed S gene sequences. Complete concordance was observed between the qPCR genotyping results and those derived from S gene sequencing and phylogenetic analysis. Following validation, the assay was employed to analyze 160 clinical samples collected from diarrheic piglets across nine provinces in China. Of these, 130 samples (81.25%) tested positive for PEDV, while the remaining 30 (18.75%) were negative. Among the positive samples, the G2 genotype predominated, accounting for 84.62% (110/130), followed by the S-INDEL genotype at 22.31% (29/130). Co-infection with both G2 and S-INDEL strains was identified in nine samples (6.92%). No G1 genotype was detected in any sample (Table 3).

4. Discussion

PED continues to pose a significant threat to global swine production [2,34,35]. The etiologic agent, PEDV, has evolved into three major genotypes: the classical G1, the variant G2, and the recombinant S-INDEL strains [35,36]. Currently, G2 variants represent the dominant strains circulating worldwide, while S-INDEL strains co-circulate as an epidemiologically significant lineage. In contrast, the classical G1 is now rarely detected [25,26]. Existing methods, including conventional PCR and certain qPCR assays, either cannot reliably differentiate S-INDEL strains or depend on time-consuming Sanger sequencing and phylogenetic analysis, which are impractical for routine diagnostics and timely intervention [37,38,39]. To address this critical gap, we developed a TaqMan probe-based real-time PCR assay for the rapid differentiation of PEDV genotypes G1, G2, and S-INDEL. Traditional antibody-based assays such as ELISA and LFIA are suitable for rapid and economical assessment of herd immunity and infection history. Meanwhile, PEDV genotyping via PCR amplification followed by Sanger sequencing remains a well-established and reliable approach and is still the preferred method when time allows. In comparison, the assay developed in this study offers a faster and more cost-effective alternative for PEDV genotyping. It enables simultaneous PEDV detection and genotype identification within 90 min for small batches of samples, without requiring a sequencing step. This significantly reduces turnaround time and overall cost, making it suitable for large-scale surveillance and rapid outbreak screening. Moreover, its ability to accurately discriminate among the three main genotypes allows for the monitoring of multi-strain co-infections, which is important for guiding targeted immunization strategies, evaluating biosecurity measures, and clarifying transmission dynamics.

The selection of an appropriate target gene is critical for assay design. The PEDV genome encodes four major structural proteins (S, M, E, and N), among which M and N genes are highly conserved and commonly targeted for universal PEDV detection [40,41,42]. For instance, Chen et al. established a qRT-PCR assay capable of detecting PEDV with a LOD of 1 × 10^1^ copies/μL [43]. While assays based on these M- or N-gene demonstrate high sensitivity for confirming PEDV infection, their high genetic conservation limits their utility for genotype discrimination. In contrast, the S glycoprotein, which is essential for viral entry and host neutralization, exhibits high genetic variability and serves as the primary basis for PEDV genotyping [14,44]. Consequently, the S gene is the optimal target for developing molecular assays capable of differentiating PEDV subtypes. Previous studies have employed S-gene targets to distinguish between G1 and G2 genotypes. Su et al. and Wang et al. targeted the S gene to develop qPCR assays for differentiating PEDV G1 and G2 genotypes, respectively [30,45]. Advancing these approaches, our study identified and targeted genotype-specific conserved regions within the S gene to develop a novel qPCR assay that is free of an ROX reference dye and capable of discriminating all three major genotypes—G1, G2, and the recombinant S-INDEL.

The assay demonstrated robust performance in validation tests. It showed extremely high specificity, with no cross-reactivity with six common swine pathogens (TGEV, PoRV, PDCoV, PRRSV, CSFV, PRV). The assay was also highly reproducible, with both intra-assay and inter-assay CV below 1.5%. In a clinical validation using 25 samples of predetermined genotypes, the qPCR results aligned completely with S gene sequencing. However, the high cost of Sanger sequencing followed by phylogenetic analysis limited the number of samples available for comparison. While these initial findings are promising, confirming the assay’s reliability in practice for worldwide samples will require validation with larger sample sets in future studies.

The application of this assay provided novel insights into the molecular epidemiology of PEDV in China. Analysis of 160 clinical samples from nine provinces revealed that 6.9% of PEDV-positive cases involved mixed G2 and S-INDEL infections. This demonstrates that co-circulation of distinct genotypes within herds is not uncommon. Such co-infections are epidemiologically significant, as they provide the biological environment necessary for homologous recombination, which is a primary driver of coronavirus evolution and the emergence of novel variants [46,47].

While the assay demonstrates promising performance, several limitations should be noted. Firstly, plasmid concentration was quantified by spectrophotometry, a method with recognized limitations in accuracy and stability compared to fluorescence-based quantification (e.g., Qubit). This inherent variability may introduce uncertainty in the derived detection limit and standard curve, an aspect identified for future methodological refinement [48,49]. Secondly, its sensitivity, currently at 10^2^ copies/μL, is moderately lower than that of some non-differentiating diagnostic assays, which typically achieve 10^1^ copies/μL. This limit was established using plasmid templates; in clinical fecal samples, the presence of amplification inhibitors in extracted nucleic acids may raise the effective detection limit further. While this sensitivity remains adequate for diagnosing clinically affected animals with typically high viral loads [50,51,52,53], further optimization of primer-probe design or reaction conditions is essential to improve detection of subclinical or low-level shedding. Thirdly, owing to the limited number of circulating G1 strains in China in recent years, the collection of clinical samples for validating the G1 genotyping assay has been challenging [25,36,54,55,56]; therefore, the current validation primarily relied on the CV777 vaccine strain. Future testing with archived or newly collected field G1 samples would provide a more robust evaluation of the assay’s performance for this genotype. Finally, given the high mutation rate of the PEDV S gene, continuous monitoring of circulating strains is necessary. Periodic reevaluation of primer-probe matching against emerging variants will be essential to maintain the long-term accuracy and reliability of the assay [57,58].

Overall, this study successfully established a robust real-time RT-qPCR platform that enables the rapid detection and differentiation of PEDV genotypes G1, G2, and S-INDEL within 90 min for small batches of samples. By effectively addressing the diagnostic challenge posed by the virus’s complex evolution, this assay enables precise epidemiological surveillance and supports genotype-informed disease control. Future efforts will focus on large-scale field validation and the potential adaptation of this assay for point-of-care testing, thereby enhancing its practical application in diverse swine production systems.