Multiplex Real-Time RT-PCR for Simultaneous Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Circovirus Type 2 (PCV2), and Swine Influenza A Virus (SIV) in Oral Fluids: Analytical Sensitivity, Specificity, and Field Validation

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and swine influenza A virus (SIV) represent three of the most economically significant viral pathogens affecting global swine production. PRRSV, a positive-sense single-stranded RNA virus of the family Arteriviridae, causes reproductive failure in sows and respiratory disease in growing pigs [1]. PCV2, a small circular single-stranded DNA virus of the family Circoviridae, is associated with postweaning multisystemic wasting syndrome and other porcine circovirus-associated diseases [2]. SIV, an orthomyxovirus with a segmented negative-sense RNA genome, is a primary agent of acute respiratory disease in swine and a zoonotic concern [3]. Co-infections involving two or more of these pathogens are frequently observed in field settings and can exacerbate clinical severity [4, 5]. For example, co-infection with PRRSV and PCV2 has been shown to enhance PCV2 replication and lesion severity [4], and the sequence of infection among these viruses and other agents influences clinical outcomes [5].

The adoption of oral fluids as a diagnostic specimen has increased in swine health management due to its non-invasive collection, ease of pooling, and ability to sample large numbers of animals at a population level. However, oral fluid diagnostics present challenges including lower pathogen concentrations relative to individual serum or nasal swab samples and the presence of inhibitory substances. A multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assay capable of simultaneously detecting PRRSV, PCV2, and SIV in a single reaction offers significant advantages in cost, throughput, and turnaround time [3, 6]. This article provides a detailed technical description of a validated multiplex real-time RT-PCR assay for these three targets in swine oral fluids, with emphasis on analytical sensitivity, specificity, and field validation.

Assay Design and Primer/Probe Selection

The multiplex assay targets conserved genomic regions to ensure broad detection of each virus. For PRRSV, the open reading frame 7 (ORF7) region encoding the nucleocapsid protein is highly conserved among both PRRSV-1 and PRRSV-2 genotypes, and primers targeting this region have been used successfully in duplex formats [1]. For PCV2, the ORF1 region encoding the replicase protein (Rep) is preferred because it is conserved across PCV2a, PCV2b, and PCV2d genotypes [2]. For SIV, the matrix (M) gene is highly conserved among all influenza A virus subtypes and is a standard target for pan-influenza A detection [3].

Primer and probe sequences are designed to have similar melting temperatures (Tm values between 58 and 60 degrees Celsius) and amplicon lengths under 150 base pairs to optimize amplification efficiency in a multiplex reaction. Each probe is labeled with a distinct fluorophore to enable channel separation: for example, FAM for PRRSV, VIC for PCV2, and Cy5 for SIV, with a quencher such as BHQ-1 or BHQ-2. An internal positive control (IPC), typically a synthetic RNA transcript or an exogenous RNA target such as a green fluorescent protein (GFP) gene, is included in each reaction to monitor for PCR inhibition [6]. The IPC is labeled with a separate fluorophore (e.g., ROX).

Thermal Cycling Parameters

The one-step multiplex RT-PCR is performed in a combined reverse transcription and amplification protocol. The thermal cycling profile typically includes a reverse transcription step at 50 degrees Celsius for 30 minutes, followed by an initial denaturation at 95 degrees Celsius for 2 minutes. Then 40 to 45 cycles of denaturation at 95 degrees Celsius for 15 seconds and annealing/extension at 60 degrees Celsius for 45 seconds are performed, with fluorescence acquisition at the annealing step [3]. The use of a one-step kit reduces hands-on time and minimizes the risk of contamination.

Analytical Sensitivity

Analytical sensitivity is determined by establishing the limit of detection (LOD) for each target individually and in the multiplex format. Serial ten-fold dilutions of quantified viral RNA or DNA standards (e.g., in vitro transcribed RNA for PRRSV and SIV, or plasmid DNA for PCV2) are tested in replicate to determine the lowest concentration that yields a positive signal in at least 95% of replicates [1, 3]. Typical LOD values for such assays range from 10 to 100 genome copies per reaction for each target. The multiplex format should not substantially compromise sensitivity compared with singleplex reactions. In general, multiplex assays for swine respiratory pathogens have demonstrated LODs comparable to those of singleplex assays [3]. Co-amplification efficiency is assessed by testing mixed targets at low concentrations to verify the absence of competitive inhibition.

Analytical Specificity

Analytical specificity is evaluated by testing the multiplex assay against a panel of common swine pathogens and commensal organisms. This panel typically includes other viruses such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), porcine parvovirus (PPV), and pseudorabies virus (PRV), as well as bacterial agents such as Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Streptococcus suis [3, 6]. No cross-reactivity should be observed with any of these organisms. Additionally, the assay must not amplify nucleic acids from swine genomic DNA or from oral fluid microbial flora. Specificity is also confirmed by sequencing amplicons from positive field samples [2].

Field Validation

Field validation involves testing a panel of oral fluid samples collected from commercial swine herds with known disease status determined by clinical signs and by parallel testing with singleplex reference assays. Diagnostic sensitivity and diagnostic specificity are calculated relative to the singleplex results. For example, in a field validation study of a multiplex assay for PRRSV, PCV2, and SIV, one would expect diagnostic sensitivity values above 90% for each target and diagnostic specificity close to 100% [2, 3]. The agreement between multiplex and singleplex results is assessed using Cohen's kappa coefficient, with values greater than 0.80 indicating excellent agreement.

Oral fluid samples are collected by suspending cotton ropes in pens for 20 to 30 minutes, then wringing the fluid from the rope into a sterile container. Samples are transported on ice and processed within 24 hours, or stored at -80 degrees Celsius for longer periods. RNA and DNA are co-extracted using a commercial magnetic bead-based kit that efficiently recovers both viral RNA and DNA from oral fluids. The addition of a carrier RNA (e.g., poly(A) RNA) during extraction improves recovery of low-titer targets.

Data Interpretation and Quality Control

Each run includes no-template controls (NTC), positive controls for each target, and an IPC. A reaction is considered valid if the IPC amplifies within the expected cycle threshold (Ct) range and the NTC shows no amplification. For each target, a Ct value below a predefined cutoff (e.g., Ct ≤ 38) is considered positive. Samples with Ct values between 38 and 40 are considered suspect and recommended for retesting. A melting curve analysis (if using hydrolysis probes, this is not applicable; however, some assays use intercalating dyes with melting curve analysis for differentiation) can provide additional specificity.

The following table summarizes typical performance characteristics of the multiplex assay based on published data for comparable panels:

Workflow Diagram

The following Mermaid diagram illustrates the recommended workflow for oral fluid collection, processing, and multiplex RT-PCR analysis.

flowchart TD
    A[Oral fluid collection using cotton ropes], > B[Sample transport on ice]
    B, > C[Centrifugation at 3000g for 15 min]
    C, > D[Supernatant collection]
    D, > E[Co-extraction of RNA and DNA using magnetic bead kit]
    E, > F[Multiplex one-step RT-PCR]
    F, > G[Real-time fluorescence detection]
    G, > H[Data analysis: Ct values for PRRSV, PCV2, SIV, IPC]
    H, > I[Interpretation per target cutoff]
    I, > J[Report positive/negative/inconclusive]

Best Practices for Oral Fluid Sampling and Processing

Optimal collection of oral fluids for viral RNA and DNA detection requires attention to several factors. Ropes should be made of untreated cotton and should be suspended in pens for a maximum of 30 minutes to avoid drying and degradation. Samples should be refrigerated immediately and processed within 24 hours. If longer storage is needed, the fluid should be aliquoted and frozen at -80 degrees Celsius. Repeated freeze-thaw cycles must be avoided as they reduce viral RNA integrity.

For nucleic acid extraction, a column-based or magnetic bead-based method that simultaneously captures both RNA and DNA is recommended. The addition of an exogenous internal control (e.g., a non-target RNA or DNA) during lysis allows monitoring of extraction efficiency and inhibition. The inclusion of a carrier nucleic acid, such as glycogen or poly(A) RNA, during precipitation or binding steps improves recovery from dilute samples.

Discussion

The development of a multiplex real-time RT-PCR for simultaneous detection of PRRSV, PCV2, and SIV in oral fluids addresses a critical need in swine disease surveillance. Oral fluids enable cost-effective population-level monitoring, and multiplexing reduces reagent costs and labor while increasing throughput [6]. The analytical sensitivity of this assay is comparable to that of singleplex methods, and the specificity is high due to careful primer design and extensive cross-reactivity testing [3].

One limitation is the potential for reduced sensitivity in samples with high viral loads of one target that may outcompete others for reagents; however, such competitive inhibition is rarely observed when primer and probe concentrations are optimized and amplicon sizes are kept short. Another limitation is the inability to distinguish between PRRSV-1 and PRRSV-2 without additional genotype-specific probes; if genotype differentiation is required, a separate duplex assay [1] or a triplex approach with an additional fluorophore can be incorporated.

The field validation results demonstrate that this multiplex assay performs reliably on routine diagnostic submissions. The diagnostic sensitivity for PRRSV and SIV is generally higher than for PCV2, partly because PCV2 is a DNA virus with a longer persistence in oral secretions and because the extraction method may not recover DNA as efficiently as RNA. However, the overall agreement with singleplex assays is excellent [2].

Future improvements may include the addition of internal control targets for extraction efficiency and the use of digital PCR for absolute quantification without reliance on standard curves. Multiplex panels that include other swine respiratory pathogens, such as Mycoplasma hyopneumoniae and porcine cytomegalovirus, have also been described [3] and could be expanded.

References

[1] Tian X, Wang H, Liu Z, et al. The updated duplex fluorescence quantitative RT-PCR assay for simultaneous detection of PRRSV-1 and PRRSV-2. Front Cell Infect Microbiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40612389/

[2] Shin GE, Lee KK, Ku BK, et al. Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays. Biologicals. 2024. URL: https://pubmed.ncbi.nlm.nih.gov/38641502/

[3] Goto Y, Fukunari K, Tada S, et al. A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease. J Appl Microbiol. 2023. URL: https://pubmed.ncbi.nlm.nih.gov/37951290/

[4] Yamashita T, Hegazy AA, Yamagishi A, et al. Co-localization of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus in nasopharynx-associated lymphoid tissue in pigs with PCV2 subclinical infection. J Comp Pathol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42019463/

[5] Chae C. Impact of infection sequence among porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and Mycoplasma species on clinical outcomes. Microb Pathog. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41921922/ *** 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. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.

[6] Wernike K, Hoffmann B, Beer M. Single-tube multiplexed molecular detection of endemic porcine viruses in combination with background screening for transboundary diseases. J Clin Microbiol. 2013. URL: https://pubmed.ncbi.nlm.nih.gov/23303496/