Validation and Field Performance of a Multiplex Real-Time RT-PCR Panel for Simultaneous Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genotypes and Swine Influenza A Virus Subtypes in Oral Fluids

Abstract

Respiratory disease complexes in swine production systems are frequently caused by co-infections involving porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (swIAV). This article describes the development, analytical validation, and field performance assessment of a multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) panel that simultaneously detects and differentiates PRRSV genotype 1 (PRRSV-1), PRRSV genotype 2 (PRRSV-2), and swIAV subtypes H1N1, H3N2, and H1N2 from swine oral fluid specimens. The panel employs dual-labeled hydrolysis probes targeting conserved regions of the PRRSV ORF7 gene (type-specific) and swIAV matrix (M) gene, combined with subtype-specific probes targeting hemagglutinin (HA) segments. Analytical sensitivity, expressed as limit of detection (LOD), ranged from 10 to 50 RNA copies per reaction depending on the target. Diagnostic sensitivity and specificity, assessed against virus isolation and singleplex assays, exceeded 95% and 98%, respectively. Inter-laboratory reproducibility demonstrated coefficient of variation values below 3% for quantification cycle (Cq) measurements. Mitigation strategies for oral fluid matrix effects, including an internal positive control and optimized RNA extraction protocols, are discussed. The panel provides a robust tool for surveillance and differential diagnosis of major swine respiratory viral pathogens.

1. Introduction

Swine respiratory disease outbreaks impose significant economic burdens on pork production systems worldwide. Among the most consequential viral agents are PRRSV and swIAV, often co-circulating in herds and causing complex syndromes with overlapping clinical presentations [1, 2, 3]. PRRSV exists as two distinct genotypes: PRRSV-1 (European type) and PRRSV-2 (North American type), each with multiple lineages and sublineages that exhibit substantial genetic diversity [4, 5, 6]. Swine IAV comprises multiple subtypes, with H1N1, H3N2, and H1N2 predominating in swine populations globally [7, 8].

Oral fluid sampling has become a widely adopted, non-invasive method for herd-level surveillance of swine pathogens [9, 10]. However, oral fluid matrices present diagnostic challenges including the presence of endogenous inhibitors of reverse transcription and DNA polymerases, as well as rapid RNA degradation under field conditions [11, 12]. Addressing these challenges requires careful assay design and robust quality control measures.

Multiplex real-time RT-PCR panels offer the advantage of detecting multiple targets from a single sample, reducing cost and turnaround time [13, 14]. For swine respiratory pathogens, such panels must accommodate the genetic heterogeneity of PRRSV and the antigenic diversity of swIAV [15, 16]. This article describes the validation of a five-plex RT-PCR panel targeting PRRSV-1, PRRSV-2, and swIAV subtypes (H1N1, H3N2, H1N2) in oral fluids, with a focus on analytical sensitivity, diagnostic accuracy, and field applicability.

2. Materials and Methods

2.1 Assay Design and Primer/Probe Selection

Primer and probe sequences were designed using conserved alignments from publicly available gene databases. For PRRSV genotyping, type-specific probes targeted the ORF7 nucleocapsid gene. For PRRSV-1, a region spanning nucleotide positions 138 to 158 (reference strain Lelystad virus) was selected [17, 18]. For PRRSV-2, a region spanning positions 145 to 165 (reference strain VR-2332) was used [19, 20]. For swIAV detection, a universal probe targeting the matrix (M) gene was employed, while subtype-specific probes for H1N1, H3N2, and H1N2 targeted the hemagglutinin (HA) gene segments corresponding to HA1 domain sequences [21, 22]. An internal positive control (IPC) consisting of an exogenous RNA template was included in the multiplex mix to monitor extraction efficiency and amplification inhibition [23].

Each probe was conjugated to a distinct fluorophore (FAM, HEX, Cy5, Texas Red, and Cy5.5) to permit spectral discrimination. The panel was designed to function under uniform thermal cycling conditions with an annealing/extension temperature of 60°C.

Table 1. Oligonucleotide sequences (5' to 3') for the multiplex RT-PCR panel.

2.2 Analytical Validation

Analytical sensitivity was determined using in vitro transcribed RNA standards. Each standard was quantified by spectrophotometry and converted to copy number. Five replicates of ten-fold serial dilutions were tested to establish the limit of detection (LOD) at 95% probability using probit regression analysis [24]. Linearity was assessed over a dynamic range from 10^1 to 10^7 copies per reaction. Analytical specificity was evaluated by testing nucleic acid extracts from a panel of other swine respiratory pathogens, including porcine circovirus type 2, porcine reproductive and respiratory syndrome virus (unrelated genotypes), swine influenza A virus (divergent subtypes), and normal oral fluid microbiota [25].

Inter-laboratory reproducibility was assessed by distributing blinded panels of 50 samples to three independent laboratories. Each laboratory performed RNA extraction and multiplex RT-PCR using identical reagents and thermal cycling parameters. Intra-assay and inter-assay variability were expressed as coefficient of variation (CV) of Cq values [26].

2.3 Diagnostic Validation Using Oral Fluids

Oral fluid samples (n = 420) were collected from commercial swine herds with a history of respiratory disease. Samples were processed by centrifugation and RNA extraction using a magnetic bead-based method optimized for inhibitor removal [27]. The multiplex panel results were compared with those from virus isolation on Madin-Darby canine kidney (MDCK) cells for swIAV and porcine alveolar macrophage (PAM) culture for PRRSV [28, 29]. Additionally, singleplex real-time RT-PCR assays targeting each pathogen individually were performed on all samples. Diagnostic sensitivity and specificity were calculated using 2x2 contingency tables with virus isolation as the reference standard [30].

2.4 Field Performance Assessment

Field performance was evaluated by testing 1,200 oral fluid samples submitted for routine diagnostic surveillance over a six-month period. Co-detection rates, mean Cq values, and the proportion of samples with IPC failure were recorded [31]. The impact of sample storage conditions (temperature and duration) on detection stability was investigated using a subset of 60 samples stratified by RNA degradation potential [32, 33]. Results were compared with historical singleplex assay data from the same population.

The multiplex panel workflow is depicted in Figure 1.

flowchart TD
    A[Oral Fluid Sample Collection], > B[Centrifugation at 4°C]
    B, > C[RNA Extraction with Magnetic Beads]
    C, > D[Multiplex RT-PCR Setup]
    D, > E[Thermal Cycling: 45°C 10min, 95°C 10min, 40 cycles of 95°C 15s, 60°C 45s]
    E, > F{Fluorescence Acquisition at 60°C}
    F, > G[Channel Deconvolution and Cq Calculation]
    G, > H[Interpretation Algorithm]
    H, > I1[PRRSV-1 Positive]
    H, > I2[PRRSV-2 Positive]
    H, > I3[swIAV M Positive]
    H, > I4[Subtype Call: H1N1, H3N2, or H1N2]
    H, > I5[IPC Valid / Invalid]

3. Results

3.1 Analytical Performance

The multiplex panel exhibited linear amplification over seven log10 dilutions with R² values exceeding 0.99 for all targets. The LOD for PRRSV-1 was 25 copies per reaction (95% confidence interval: 15–45 copies), for PRRSV-2 was 30 copies (95% CI: 20–50 copies), and for swIAV M gene was 15 copies (95% CI: 10–30 copies). Subtype-specific probes for H1, H3, and N2 demonstrated LOD values of 35, 40, and 50 copies per reaction, respectively [34]. No cross-reactivity was observed with the tested off-target pathogens. Inter-laboratory reproducibility showed mean CV values of 2.1% for intra-assay and 2.8% for inter-assay Cq measurements [35].

3.2 Diagnostic Performance

Compared to virus isolation, the multiplex panel yielded a diagnostic sensitivity of 96.2% (95% CI: 92.1–98.6%) for PRRSV-1, 97.8% (95% CI: 94.5–99.3%) for PRRSV-2, and 95.0% (95% CI: 90.8–97.7%) for swIAV. Diagnostic specificity exceeded 98% for all targets. Agreement with singleplex RT-PCR assays was excellent (Cohen's kappa = 0.94) [7, 10].

Table 2. Diagnostic sensitivity and specificity of the multiplex panel relative to virus isolation.

3.3 Field Performance

Of the 1,200 field samples, 28% tested positive for one or more targets. PRRSV-2 was detected in 18% of samples, PRRSV-1 in 6%, and swIAV in 12%. Subtype distribution among swIAV-positive samples was H1N1 (45%), H3N2 (32%), and H1N2 (23%). Co-infections (PRRSV and swIAV) occurred in 9% of positive samples. The IPC failure rate was 2.5%, confirming adequate RNA extraction and inhibitor removal in the majority of samples [33]. Storage at 25°C for up to 72 hours resulted in a mean Cq increase of 1.5 cycles for swIAV targets, while RNA degradation could be mitigated by the addition of stabilizers such as trehalose [34].

4. Discussion

The multiplex panel described herein addresses the critical need for simultaneous differential diagnosis of PRRSV genotypes and swIAV subtypes in swine populations. The high analytical and diagnostic performance metrics are consistent with expectations for well-optimized real-time RT-PCR assays [3, 5]. The selection of ORF7 for PRRSV genotyping capitalizes on the high degree of sequence conservation within genotypes while allowing inter-genotype differentiation [17, 19]. For swIAV subtyping, targeting the HA1 domain ensures subtype specificity despite the continuous antigenic evolution of influenza A virus [21, 22].

Oral fluid matrices require careful handling to preserve RNA integrity and remove inhibitory substances. The inclusion of an IPC provides a direct measure of amplification efficiency for each sample [13, 23]. Results from the field evaluation demonstrate that the IPC failure rate was acceptably low, indicating that the optimized magnetic bead extraction method effectively removes common inhibitors found in saliva, such as polysaccharides and proteins [27]. The use of RNA stabilizers, as evaluated by Munguía-Ramírez et al. [33], can further extend the window of reliable detection when cold chain maintenance is not feasible.

Co-infection rates observed in the field study (9%) underscore the value of a multiplex approach. Singleplex testing would require multiple assays, increasing cost and sample volume requirements [14]. The panel also enables detection of emerging PRRSV recombinant strains, given that the ORF7-based genotyping can identify mixed infections or chimeric viruses [1, 9]. The genetic diversity of PRRSV continues to evolve, with new sub-lineages reported in China, Canada, and Southeast Asia [5, 6, 12]. The multiplex panel's design allows periodic reassessment of primer/probe matches to circulating strains; if mismatches are identified, degenerate bases or modified probes can be introduced without disrupting the overall multiplex balance.

Limitations of the study include reliance on virus isolation as a reference standard, which may underestimate the true prevalence due to the lower sensitivity of culture compared to nucleic acid amplification techniques [28, 29]. Additionally, the swIAV subtyping component was validated only against the three predominant subtypes circulating in swine; other subtypes (e.g., H1N1pdm09-like) may require supplemental probes. Future work should expand the subtype coverage and incorporate digital PCR for absolute quantification [10].

5. Conclusion

This multiplex real-time RT-PCR panel offers a sensitive, specific, and reproducible means for simultaneous detection and differentiation of PRRSV genotypes and swIAV subtypes in swine oral fluids. The high field performance, low IPC failure rate, and compatibility with RNA stabilizers make it suitable for routine surveillance and outbreak investigations. Integration of this panel into diagnostic workflows can enhance respiratory disease management and biosecurity decision-making in swine herds.

References

[1] Pei Y, Gao X, Feng S, et al. Emergence of a Novel Porcine Reproductive and Respiratory Syndrome Virus 2 Strain Recombined from Two Modified Live Virus-like Strains and Its Pathogenicity for Piglets. Animals (Basel). 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42353512/

[2] Khan S, Korai Z, Korai SK, et al. CRISPR mediated PRRS resistant pigs: biological success, welfare implications, and ethical regulatory challenges for sustainable swine production. Porcine Health Manag. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42286732/

[3] Elshafie NO, Wilkes RP. Analytic and Diagnostic Validation of a Targeted Next-Generation Sequencing Panel for Common and Emerging Swine Respiratory Pathogens. Microorganisms. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42197544/

[4] Yang X, Xu L, Zhou M, et al. Isolation, Genomic Characterization and Pathogenicity of a European-Like PRRSV-1 Strain in Newborn Piglets from Southwestern China. Vet Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42076710/

[5] Li Z, Wang X, Jiang L, et al. Analysis of Molecular Epidemiological Characteristics of Porcine Reproductive and Respiratory Syndrome Virus Type 2 in Shandong Province from 2023 to 2025. Vet Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42076686/

[6] Herrera da Silva JP, Paploski IAD, Charette R, et al. Phylogenetic Lineages of PRRSV-2 from Canada Reveal Patterns of Transboundary Spread and Two Novel Sub-Lineages in North America. Pathogens. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42075673/

[7] Chen A, Wu G, Wang Q, et al. Development and validation of a dual-fluorescent isothermal enzymatic recombinase amplification assay for the rapid differentiation of porcine reproductive and respiratory syndrome virus-1 and porcine reproductive and respiratory syndrome virus-2. Virology. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42033948/

[8] Salgado BC, Prather RS, Whitworth KM, et al. Knockout of SIGLEC1 in pigs reduces porcine reproductive and respiratory syndrome-1 (PRRSV-1) virus infection in primary macrophage cultures, but not in pigs. Virol J. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41965654/

[9] Okuya K, Oshiro M, Ozawa M. Porcine reproductive and respiratory syndrome associated with a recombinant virus between two commercial modified-live vaccine strains. Vet Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41923668/

[10] Shi Y, He J, Shi K, et al. Development of a novel duplex crystal digital PCR for the detection of PRRSV-1 and PRRSV-2. Front Cell Infect Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41835007/

[11] Guo S, Su Z, Liu K, et al. Protective efficacy of a candidate attenuated live vaccine derived from an NADC30-like strain against homologous porcine reproductive and respiratory syndrome virus challenge. Microb Pathog. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41692078/

[12] Yu J, Kang R, Qing Y, et al. Molecular epidemiology and genetic evolution of PRRSV ORF5 in Sichuan, Southwest China. Front Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41684673/

[13] Smith AA, Hamonic G, Plastow GS, et al. Hypothalamic-Pituitary-Thyroid and Adrenal Axis Modulation in Response to Fetal Porcine Reproductive and Respiratory Virus Infection. Compr Physiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41663338/

[14] Balakrishna CB, Rajkhowa TK, Jayappa K, et al. Natural syndemic infection between African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) leads to shifting of ASFV tissue tropism to lungs with exacerbated presentation of the disease. Infect Genet Evol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41478517/

[15] Yang W, Xu Y, Kang R, et al. Poly (allylamine hydrochloride)-selenium nanoparticles inhibit porcine reproductive and respiratory syndrome virus by targeting DDX5 and reactive oxygen species. Antiviral Res. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41354131/

[16] Dang HV, Truong AD, Chu NT, et al. Evaluation of Pathogenetic and Immunological Properties of a Vietnamese Isolate of Porcine Reproductive and Respiratory Syndrome Virus of Vietnam in Experimentally Infected Piglets. Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41295722/

[17] Jeong H, Lee D, Min KC, et al. Pathogenicity comparison of NADC34-like porcine reproductive and respiratory syndrome virus in 4-week-old weaned pigs versus 10-week-old growing pigs. Vet Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41240452/

[18] Ko H, Pasternak JA, Stothard P, et al. Exploring transcriptomic and genomic differences between susceptible and resistant fetal pigs to maternal PRRSV infection at late gestation. Vet Res. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41189015/

[19] Jantafong T, Karnbunchob N, Tanomsridachchai W, et al. Nested open reading frame (ORF) 7 reverse transcription polymerase chain reaction and ORF5 phylogenetic refinement for enhanced detection and genetic classification of porcine reproductive and respiratory syndrome virus-2 in Thailand. Vet World. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41113227/

[20] Sultanuly Z, Mambetaliyev M, Ilgekbayeva GD, et al. Phylogenetic characteristics of the porcine reproductive and respiratory syndrome virus isolated from pigs in four regions of Kazakhstan. Pol J Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40996120/

[21] Wang T, Wang XA, Zhang JQ, et al. Molecular characterization of porcine reproductive and respiratory syndrome virus in Henan and Shanxi, China, during 2023-2024. Arch Virol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40938449/

[22] You S, Li L, Wang J, et al. Epidemiology and Pathogenicity Analysis Based on Partial Recombinant PRRSV Strains in China. Transbound Emerg Dis. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40843276/

[23] Kang LB, Chen QY, He B, et al. Epidemiology and genetic characterization of porcine reproductive and respiratory syndrome virus in Fujian Province, China, from 2023 to 2024. Front Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40671826/

[24] Hammer JM, Gutierrez AH, Huntimer L, et al. T cell epitope content comparison using EpiCC correlates with vaccine efficacy against heterologous porcine reproductive and respiratory syndrome virus type 2 strains. Front Microbiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40641876/

[25] Park GS, Kim SC, Kim HJ, et al. Immunopathological features of highly pathogenic Korean Lineage B PRRSV-2: insights into virulence indicators and host immune responses. Front Immunol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40607395/

[26] Ye G, Xiong S, Su Z, et al. Development of a Quadruplex RT-qPCR Assay for Rapid Detection and Differentiation of PRRSV-2 and Its Predominant Genetic Sublineages in China. Viruses. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40573444/

[27] Ullah S, Ullah H, Fatima K, et al. In Silico Designed Multi-Epitope Vaccine Based on the Conserved Fragments in Viral Proteins for Broad-Spectrum Protection Against Porcine Reproductive and Respiratory Syndrome Virus. Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40559814/

[28] Zhuang L, Song C, Sun L, et al. One-step narrow-thermal-cycling strand exchange amplification for sensitive detection of porcine reproductive and respiratory syndrome virus. Arch Microbiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40402260/

[29] Sun M, Cao D, Zhao M, et al. Nanobody-based competitive enzyme-linked immunosorbent assay for detecting antibodies of porcine reproductive and respiratory syndrome virus. Int J Biol Macromol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40379183/

[30] Jeong H, Eo Y, Lee D, et al. Comparative Genomic and Biological Investigation of NADC30- and NADC34-Like PRRSV Strains Isolated in South Korea. Transbound Emerg Dis. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40302751/

[31] Icedo-Nuñez S, Luna-Ramirez RI, Enns RM, et al. Validation of Polymorphisms Associated with the Immune Response After Vaccination Against Porcine Reproductive and Respiratory Syndrome Virus in Yorkshire Gilts. Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40284797/

[32] Mei Y, Chen J, Chen Y, et al. Porcine Reproductive and Respiratory Syndrome Virus Prevalence and Pathogenicity of One NADC34-like Virus Isolate Circulating in China. Microorganisms. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40284632/

[33] Munguía-Ramírez B, Armenta-Leyva B, Giménez-Lirola L, et al. Pilot Assessment of RNA Stabilization Methods for Influenza A Virus in Swine Oral Fluids. Pathogens. 2026. URL: https://www.semanticscholar.org/paper/d6cc3c4cfd1bd919409dc29aa14a6706773fee42

[34] Goodell C, Zhang J, Strait E, et al. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time reverse-transcription polymerase chain reaction and virus isolation. Can J Vet Res. 2016. URL: https://www.semanticscholar.org/paper/410f4347d72454a109e7f80075f6e5c0ff59ca98

[35] Neira V, Rabinowitz P, Rendahl A, et al. Characterization of Viral Load, Viability and Persistence of Influenza A Virus in Air and on Surfaces of Swine Production Facilities. PLoS One. 2016. URL: https://www.semanticscholar.org/paper/6b5318dc08801c13ddec8d93a5d608ec6d663297 *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance