High‑throughput multiplex real‑time RT‑PCR for simultaneous detection and subtyping of swine influenza A virus in oral fluids: assay design, validation, and field performance

Swine influenza A virus (swIAV) is a major respiratory pathogen of swine that circulates globally with three predominant hemagglutinin (HA) and neuraminidase (NA) subtype combinations: H1N1, H1N2, and H3N2 [1, 2, 3, 4]. The genetic diversity of these subtypes is shaped by continuous antigenic drift and occasional reassortment events, which are documented in both commercial production systems and live animal markets [5, 6, 7]. Diagnostic surveillance for swIAV has increasingly shifted from individual nasal swabs to pen‑based oral fluid sampling, because oral fluids are logistically simpler to collect, less invasive, and representative of group‑level infection dynamics [8, 9, 10, 11, 12]. Oral fluid specimens contain virus shed from the respiratory tract via saliva, and the presence of swIAV RNA in oral fluids correlates well with active infection as measured by virus isolation and real‑time RT‑PCR from paired nasal swabs [13, 9, 11]. The matrix effect of oral fluids (mucins, enzymes, feed debris) can inhibit reverse transcription and amplification, but appropriate RNA extraction methods and internal controls mitigate this issue [14, 15, 16, 12]. Stabilizers added to oral fluid collection devices further preserve viral RNA integrity during transport and storage [14, 15].

This article describes a high‑throughput multiplex real‑time RT‑PCR assay that simultaneously detects a universal influenza A virus (IAV) target (matrix gene) and subtypes the HA and NA genes of the three major circulating swIAV subtypes. The assay is designed for processing large numbers of oral fluid samples in a 384‑well format using a robotic liquid handler and a five‑channel real‑time PCR platform. Detailed steps include primer and probe design, multiplex reaction optimization, analytical sensitivity and specificity testing using synthetic RNA transcripts and spiked oral fluids, cross‑reactivity evaluation against other swine respiratory viruses, and field validation against virus isolation and next‑generation sequencing.

Assay design and primer/probe selection

The universal IAV detection component targets a conserved region of the matrix (M) gene, which is present in all influenza A viruses regardless of HA/NA subtype [17, 18, 16]. For subtyping, two separate multiplex reactions were developed: Reaction 1 targets H1 (hemagglutinin of H1N1 and H1N2), H3 (hemagglutinin of H3N2), and M (universal); Reaction 2 targets N1 (neuraminidase from H1N1), N2 (neuraminidase from H1N2 and H3N2), and M (universal). This split design reduces competition between HA and NA primers in the same tube and allows independent optimization of annealing temperatures. Primers and probes were designed based on alignments of publicly available swIAV sequences (>1000 per subtype) from global databases, with emphasis on the North American and European lineages that predominate in commercial swine populations [1, 2, 4]. Each probe was conjugated to a distinct fluorophore (e.g., FAM for M, HEX for H1, Cy5 for H3) with a matched quencher (e.g., BHQ‑1 or BHQ‑3) to enable multiplex detection [17, 18]. The amplicon lengths were kept below 150 bp to enhance amplification efficiency in the presence of degraded RNA that may be present in oral fluids [16, 12].

Multiplex reaction optimization

Reaction conditions were optimized using a central composite design to minimize competition among primer pairs and maximize signal intensity for each target. Factors varied included primer concentration (50–400 nM each), probe concentration (50–250 nM), annealing temperature (55–62 °C), and MgCl₂ concentration (3–6 mM). The final master mix contained 1× reaction buffer (with ROX passive reference), 5 mM MgCl₂, 300 µM each dNTP, 200 nM each primer, 150 nM each probe, 1.6 U of reverse transcriptase, and 2 U of DNA polymerase per 25 µL reaction. Cycling conditions on a high‑throughput thermal cycler were: reverse transcription at 50 °C for 15 min, initial denaturation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 10 s and 58 °C for 30 s (with fluorescence acquisition at the annealing‑extension step). These conditions were validated on synthetic RNA controls (see below) and replicated across three independent runs with no evidence of cross‑talk between fluorophore channels. Multiplex balance was confirmed by comparing cycle threshold (Ct) values from monoplex reactions versus the corresponding multiplex reaction; a shift of less than 1.5 was considered acceptable.

Analytical validation

Synthetic RNA controls and limit of detection

Full‑length coding sequences of the M gene and the HA and NA genes from contemporary swIAV strains (H1N1, H1N2, H3N2) were cloned into an SP6‑based transcription vector and transcribed in vitro. Single‑stranded RNA transcripts were purified by DNase treatment and silica‑column cleanup, and quantified using UV spectrophotometry and digital PCR. The copy number of each transcript was calculated based on molecular weight. Serial ten‑fold dilutions (10⁶ to 1 copies/reaction) were prepared in nuclease‑free water and in a pool of swIAV‑negative oral fluid matrix to assess matrix interference. The limit of detection (LOD) was defined as the lowest concentration at which 95% of replicates (n = 24) were detected. For the M target, the LOD was 10 copies/reaction in both water and oral fluid matrix. For the subtype‑specific HA and NA targets, the LOD ranged from 10 to 50 copies/reaction, depending on the primer‑probe set. Table 1 summarizes the LOD values for each target.

Table 1. Analytical limit of detection (LOD) for each target in the multiplex assay.

Analytical specificity and cross‑reactivity

The assay was tested against a panel of common swine respiratory and enteric viruses to verify that no nonspecific amplification occurred. The panel included porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis virus (TGEV). RNA or DNA from these pathogens (10⁴ to 10⁶ genome copies/reaction) was spiked into the multiplex reactions. No amplification signals above background were observed for any of the non‑target pathogens, confirming high analytical specificity [17, 18]. Additionally, the subtype‑specific primers and probes were tested against heterologous swIAV subtype templates (e.g., H1 primers against H3 templates, N1 primers against N2 templates) at 10⁶ copies/reaction; no cross‑reaction was detected. These specificity results are consistent with those reported for other multiplex influenza A virus assays [17, 18, 2].

Repeatability and reproducibility

Intra‑assay repeatability was assessed by testing three concentrations of synthetic RNA (10², 10⁴, and 10⁶ copies/reaction) in 10 replicates on the same run. Inter‑assay reproducibility was evaluated by running the same panel on three separate days with different reagent lots. The coefficient of variation (CV) for Ct values was less than 3% for intra‑assay and less than 5% for inter‑assay determinations for all targets.

Field validation

Sample collection and processing

A total of 1,200 oral fluid samples were collected from growing‑finishing pigs across 30 commercial swine farms in North America. Collections were performed using cotton ropes suspended in pens for 20–30 minutes as described in standard protocols [10, 11]. After collection, oral fluids were transferred to sterile tubes containing RNA stabilizer [14, 15] and transported on ice to the laboratory within 24 h. RNA was extracted from 200 µL of oral fluid using a silica‑column‑based method [16] and eluted in 50 µL of nuclease‑free water. An external RNA control (synthetic armored RNA) was added to each lysis buffer to monitor extraction efficiency and the presence of inhibitors.

Comparison with virus isolation and sequencing

All 1,200 samples were tested with the multiplex assay. A subset of 200 samples (50 positive by multiplex and 150 negative) were processed for virus isolation in MDCK cells, followed by hemagglutination assay and confirmation by conventional RT‑PCR and Sanger sequencing [13, 9]. Another subset of 50 multiplex‑positive samples underwent whole‑genome sequencing using an amplicon‑based Nanopore approach [19] to independently confirm the subtype. The multiplex assay showed a sensitivity of 96.2% (50/52) and specificity of 98.0% (145/148) when virus isolation was used as the reference standard. Discrepant results were further resolved by sequencing: two samples positive by multiplex but negative by virus isolation were confirmed as swIAV RNA by amplicon sequencing, suggesting the presence of non‑viable virus or RNA degradation that prevented culture. In all 50 sequenced samples, the subtype assigned by the multiplex assay (H1N1, H1N2, or H3N2) matched the subtype determined by genomic sequencing, yielding 100% concordance for subtyping.

Detection duration in oral fluids

Longitudinal oral fluid samples were collected from 10 pens of experimentally infected pigs (n = 5 pigs per pen) over 21 days post‑infection with a prototypic H1N2 strain. The multiplex assay detected swIAV RNA in oral fluids from day 1 through day 14 post‑infection, with peak Ct values (lowest Ct) occurring at day 3–5. This detection window aligns with previously reported kinetics [20, 7]. In parallel, virus isolation from matched nasal swabs was positive only between days 2 and 7, confirming that RNA detection by multiplex RT‑PCR extends the window of surveillance beyond the period of active shedding of infectious virus [20, 21, 9].

Workflow for high‑throughput implementation

The assay was formatted for a 384‑well plate to accommodate large surveillance studies. The entire process, from RNA extraction to final Ct output, is summarized in the following workflow diagram.

graph TD
    A[Pen-based oral fluid collection], > B[RNA extraction with internal control]
    B, > C[Multiplex RT-PCR set-up in 384-well plate]
    C, > D[Thermal cycling and real-time fluorescence acquisition]
    D, > E[Automated calling of Ct values and subtype assignment]
    E, > F[Data export to laboratory information management system]
    F, > G[Reporting results: positive/negative and subtype]

The use of liquid‑handling robots for master mix dispensing and sample addition reduces manual error and increases throughput. Each run includes positive controls (synthetic RNA cocktails representing each subtype) and no‑template controls. Results are considered valid if the external RNA control is detected (Ct < 35) and no amplification is seen in no‑template controls.

Discussion

The multiplex real‑time RT‑PCR assay described here provides a high‑throughput, cost‑effective tool for simultaneous detection and subtyping of swIAV in oral fluids. The use of oral fluid specimens instead of individual nasal swabs reduces labor costs and pig handling stress, while still providing reliable group‑level surveillance data [8, 10, 11, 12]. The assay’s analytical sensitivity (10–50 copies/reaction) is comparable to other published mono‑ and multiplex real‑time RT‑PCR assays for influenza A virus [17, 18, 16]. Field validation showed excellent agreement with virus isolation and sequencing, confirming that the multiplex assay can replace multiple singleplex reactions without loss of accuracy. Cross‑reactivity testing against PRRSV, PCV2, PEDV, and TGEV was negative, which is essential for differential diagnosis in swine respiratory disease panels. Existing articles on this site provide complementary information on similar multiplex approaches for PRRSV and PCV2 in conjunction with swIAV (see 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: Assay Design and Field Validation and related articles). The current assay can be integrated into broader respiratory pathogen panels, providing a modular platform for swine herd health monitoring.

One limitation is the genetic diversity of swIAV, particularly within the H1 and N2 lineages [3, 4]. Primer‑probe sets may lose sensitivity as new antigenic variants emerge. Regular monitoring of circulating strains through targeted sequencing [19] and periodic redesign of oligonucleotides are recommended. Incorporation of degenerate bases or multiple forward/reverse primer mixes can help maintain coverage. Additionally, the assay does not distinguish between human‑origin pandemic H1N1 (pdm09) and classical swine H1N1; a separate primer‑probe set targeting the pdm09 HA gene could be included if differentiation is required [2, 7].

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