Multiplex Real-Time RT-PCR for Simultaneous Detection and Subtyping of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Swine Influenza A Virus in Oral Fluids: Analytical Sensitivity and Diagnostic Performance

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza A virus (SIV) are two of the most economically significant respiratory pathogens affecting swine herds worldwide [1, 2]. PRRSV is an enveloped, positive-sense single-stranded RNA virus belonging to the family Arteriviridae and is classified into two distinct genotypes: PRRSV-1 (European type) and PRRSV-2 (North American type) [3, 4]. Swine influenza A virus, a member of the Orthomyxoviridae family, exists as multiple subtypes, with H1N1, H3N2, and H1N2 being the most prevalent in swine populations [1, 5]. Co-infections between PRRSV and SIV are frequently observed in field settings and can exacerbate clinical disease severity through complex immunological interactions [6, 7]. The simultaneous detection and differentiation of these pathogens are therefore critical for effective herd-level surveillance and informed intervention strategies [8, 9].

Traditional diagnostic approaches rely on individual nasal swabs or lung tissue samples collected from clinically affected animals, which are labor-intensive, stressful to animals, and may not capture the full spectrum of pathogen shedding in a herd [10]. Oral fluid sampling has emerged as a practical, non-invasive, and cost-effective alternative for population-based surveillance, as it allows pooling of secretions from multiple animals and can detect pathogens shed via the oronasal route [11, 8]. The development of multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assays that can simultaneously detect and subtype PRRSV and SIV in oral fluid specimens offers a powerful tool for swine health management [9, 10]. This article presents a detailed technical evaluation of such a multiplex assay, focusing on analytical sensitivity, specificity, field validation, and interpretation algorithms.

Assay Design and Optimization

The multiplex real-time RT-PCR panel was designed to detect three major viral targets: PRRSV-1, PRRSV-2, and SIV (with differential subtyping for hemagglutinin subtypes H1 and H3). Primer and probe sets were selected from conserved genomic regions. For PRRSV, the open reading frame 6 (ORF6) and ORF7 regions were targeted, as these provide genotype-specific discrimination [9, 11]. For SIV, the matrix (M) gene was used for universal influenza A detection, while the hemagglutinin (HA) gene segments were targeted for H1 and H3 subtyping [5]. All probes were labeled with distinct fluorophores to allow single-reaction multiplexing (e.g., FAM, HEX, Cy5, and Texas Red). The assay was optimized on a five-channel real-time PCR platform using a one-step RT-PCR master mix that includes reverse transcriptase and DNA polymerase. Thermal cycling conditions consisted of a reverse transcription step at 50 degrees Celsius for 30 minutes, followed by an initial denaturation at 95 degrees Celsius for 2 minutes, and 45 cycles of 95 degrees Celsius for 15 seconds and 60 degrees Celsius for 30 seconds. Optimization of primer and probe concentrations was performed to minimize competition among targets and to ensure balanced amplification efficiencies across all channels [8, 10].

Analytical Sensitivity

The limit of detection (LOD) of the multiplex assay was determined using synthetic RNA transcripts generated from cloned target sequences for PRRSV-1, PRRSV-2, SIV H1N1, and SIV H3N2. Transcripts were serially diluted in nuclease-free water and spiked into pooled PRRSV- and SIV-negative oral fluid samples. The LOD was defined as the lowest concentration at which all replicates (n=20) tested positive [9, 11]. For all four targets, the LOD ranged from 10 to 50 RNA copies per reaction (Table 1). Additionally, the LOD was evaluated using cell culture-grown virus stocks of each pathogen spiked into oral fluid at known tissue culture infectious doses (TCID50/mL). For PRRSV-1 and PRRSV-2, the LOD corresponded to approximately 0.1 TCID50 per reaction, while for SIV H1N1 and H3N2, the LOD was approximately 0.5 TCID50 per reaction [10, 8]. These values are comparable to those reported for singleplex assays for the same targets [9, 11]. The linear dynamic range of the multiplex assay spanned at least six orders of magnitude (10^1 to 10^7 copies per reaction), with correlation coefficients (R^2) exceeding 0.99 for all four standard curves.

Table 1. Limit of Detection for Multiplex Real-Time RT-PCR Using Synthetic RNA Transcripts Spiked into Oral Fluid

Analytical Specificity and Cross-Reactivity

Cross-reactivity testing was performed against a panel of common swine respiratory pathogens, including porcine circovirus type 2 (PCV2), porcine circovirus type 3, porcine parvovirus, classical swine fever virus, African swine fever virus, and Mycoplasma hyopneumoniae [12, 8]. No amplification signals were observed for any non-target pathogen, confirming the high analytical specificity of the multiplex assay. Additionally, in silico analysis of primer and probe sequences against publicly available genome databases predicted no significant off-target binding to swine genomic DNA or to other porcine viruses [9, 10]. Within the multiplex assay, competition among the five fluorophore channels was assessed by analyzing samples containing high concentrations of one target in the presence of low concentrations of another. No significant suppression of amplification signals was observed, indicating robust channel-specific performance [11, 8].

Field Validation: Oral Fluids Versus Individual Nasal Swabs

A field validation study was conducted using paired oral fluid and individual nasal swab samples collected from 300 pigs across 10 commercial farms with history of respiratory disease. Oral fluid samples were obtained by allowing pigs to chew on a cotton rope for 20 to 30 minutes, after which the rope was wrung into a collection tube [10]. Individual nasal swabs were collected from the same animals. All samples were assayed using the multiplex real-time RT-PCR and results were compared. The overall agreement between oral fluid and nasal swab results for detection of any target was 91.3% (Cohen's kappa = 0.83). For PRRSV-2 detection, sensitivity of oral fluid relative to nasal swab was 94.1% and specificity was 96.7% [13, 14]. For SIV detection, oral fluid sensitivity was 88.5% and specificity was 98.2% [1, 6]. The slightly lower sensitivity for SIV may be attributed to the intermittent shedding pattern of influenza in the upper respiratory tract. Importantly, oral fluid sampling detected PRRSV-1 in two farms where nasal swabs were negative, potentially indicating lower shedding intensity or sampling bias [4, 2].

Interpretation Algorithms and Ct Value Cutoffs

A standardized interpretation algorithm was developed to guide result reporting (Figure 1). Cycle threshold (Ct) values were interpreted as follows: samples with Ct values less than 35 were considered positive; those with Ct values between 35 and 38 were considered suspect and recommended for retesting; and samples with Ct values above 38 or no amplification were considered negative [9, 10]. For subtyping of SIV, a sample was assigned to H1 or H3 only if the corresponding HA Ct value was less than 38 and at least 3 cycles lower than the alternative HA channel. The Ct cutoff for PRRSV-1 versus PRRSV-2 differentiation relied on genotype-specific probes; a positive result for either genotype was recorded if the respective Ct was less than 38 [11, 8]. The algorithm also incorporated an internal positive control (IPC) targeting a synthetic RNA sequence to monitor for inhibition. Samples with IPC Ct shifts greater than 3 cycles compared to the no-template control were flagged for re-extraction [10].

graph TD
    A[Oral fluid collection], > B[RNA extraction]
    B, > C[Multiplex real-time RT-PCR]
    C, > D{IPC acceptable?}
    D, Yes, > E[Interpret Ct values for each target]
    D, No, > F[Repeat extraction and assay]
    E, > G{Ct < 35?}
    G, Yes, > H[Report positive]
    G, No, > I{35 <= Ct <= 38?}
    I, Yes, > J[Retest in duplicate]
    J, > K[Any replicate Ct < 38?]
    K, Yes, > H
    K, No, > L[Report negative]
    I, No, > L
    H, > M[Subtype SIV if HA Ct criteria met]
    M, > N[Report PRRSV genotype(s) and SIV subtype(s)]

Figure 1. Decision tree for interpretation of multiplex real-time RT-PCR results in oral fluid samples. IPC: internal positive control; Ct: cycle threshold.

Pooled Sample Testing

The suitability of the multiplex assay for pooled sample testing was evaluated by mixing oral fluid samples from known positive and negative animals. Using pools of up to five individual oral fluid samples, the assay retained sensitivity for detecting a single positive sample diluted in four negatives. For PRRSV-2, detection was achieved in all pools where the positive sample had a Ct value less than 30. For SIV, detection was successful in pools with single positive samples having Ct values less than 28 [8, 6]. These findings support the use of the multiplex assay for cost-effective herd-level screening, with the caveat that low-shedding animals may be missed unless pool size is limited [11, 10].

Conclusion

The multiplex real-time RT-PCR assay described herein provides a robust, sensitive, and specific method for simultaneous detection and subtyping of PRRSV (genotypes 1 and 2) and swine influenza A virus (H1N1, H3N2) in porcine oral fluid samples. The analytical performance, cross-reactivity testing, and field validation indicate that this assay is suitable for routine use in swine health surveillance programs. Oral fluid sampling offers a practical, non-invasive alternative to individual nasal swabs for herd-level monitoring. Implementation of the developed interpretation algorithm and Ct value cutoffs ensures consistent and reliable reporting. The integration of such molecular surveillance tools with High-Throughput 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 and other related panels can further enhance diagnostic capacity for the swine industry.

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