High-Throughput Real-Time RT-PCR Panel for Simultaneous Detection and Subtyping of Avian Influenza Virus in Poultry Flocks

Introduction

Avian influenza virus (AIV) is a major pathogen affecting poultry production and trade worldwide. Rapid and accurate detection of AIV, combined with subtype identification for H5, H7, and H9, is essential for surveillance, outbreak response, and containment [1, 2]. Real-time reverse transcription polymerase chain reaction (RT-PCR) targeting conserved regions of the matrix (M) gene has become the standard molecular method for AIV detection, while subtype-specific hemagglutinin (HA) assays enable differentiation of epidemiologically relevant subtypes [2, 3]. A high-throughput multiplex panel that simultaneously screens for AIV and subtypes H5, H7, and H9 in a single reaction well offers significant advantages in cost, turnaround time, and sample throughput compared to singleplex assays or virus isolation [1, 3]. This article describes the design, analytical validation, and field performance of such a panel, emphasizing its utility in commercial poultry flocks and outbreak settings.

Assay Design and Primer/Probe Chemistry

The multiplex real-time RT-PCR panel employs a one-step, single-tube format with fluorogenic hydrolysis (TaqMan) probes. The panel includes four target channels: a universal AIV assay targeting a conserved region of the matrix (M) gene, and three subtype-specific assays targeting the hemagglutinin genes of H5, H7, and H9 [1, 2]. An internal control (e.g., a synthetic RNA transcript or avian housekeeping gene) is included to monitor RNA extraction and amplification efficiency [3]. Each probe is labeled with a distinct reporter fluorophore and a quencher, allowing simultaneous detection in a single well [1, 4].

The M gene primers and probe are designed from highly conserved sequences across all AIV subtypes, ensuring broad coverage of lineages including H5Nx, H7Nx, and H9N2 strains [1, 2]. The H5 primers are selected from a conserved region of the HA gene that detects both low pathogenicity and high pathogenicity H5 viruses, including panzootic H5Nx clade 2.3.4.4 [1, 2]. The H7 primers target a conserved domain of the H7 HA gene, validated against Eurasian and North American lineages [2]. The H9 primers are designed to detect H9N2 viruses, a subtype endemic in many Asian and Middle Eastern poultry populations [4, 3].

Primer and probe sequences are optimized to avoid cross-reactivity with other avian respiratory pathogens. Melting temperature differences, GC content, and amplicon length (typically 80-150 bp) are balanced to ensure equal amplification efficiency across all four targets [1, 3]. Primer concentrations are titrated to minimize competition and primer-dimer formation in the multiplex format [1]. The final optimized panel is formulated as a lyophilized or liquid master mix containing buffer, dNTPs, reverse transcriptase, DNA polymerase, and the four primer/probe sets.

Analytical Sensitivity and Specificity

The limit of detection (LOD) of the multiplex panel is determined using serial dilutions of quantified AIV RNA transcripts or live virus (e.g., egg-cultured allantoic fluid titrated by 50% egg infectious dose, EID50) [1, 2]. The LOD for the M gene assay is typically ≤10 RNA copies per reaction, while the subtype assays (H5, H7, H9) achieve LODs of 10-50 RNA copies per reaction [1, 2]. Coopersmith et al. (2025) [1] reported that a similar bird-side molecular assay could detect panzootic H5Nx with a sensitivity equivalent to real-time RT-PCR. In the multiplex panel, the presence of four reactions does not significantly impair the LOD compared to singleplex assays, provided primer concentrations are optimized [1, 3].

The analytical specificity of the panel is evaluated against a panel of common avian respiratory pathogens including Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian metapneumovirus, and Mycoplasma spp. [3]. No cross-amplification is observed with these agents [3]. The H5 assay does not cross-react with H7 or H9 viruses, and the M gene assay detects all AIV subtypes tested [1, 2]. Specificity is also confirmed by sequencing of amplicons from representative positive field samples [2].

Workflow and High-Throughput Implementation

The laboratory workflow is designed for processing large numbers of pooled oropharyngeal and cloacal swabs from poultry flocks. Table 1 summarizes the key steps.

Table 1. Step-by-step workflow for high-throughput multiplex RT-PCR.

Total time from RNA extraction to result: approximately 2.5-3.5 hours for a 384-well plate.

The high-throughput format using 384-well plates and automated liquid handling enables processing of up to 384 samples (including controls) in a single run, which is critical for large-scale surveillance [3]. Parvin et al. (2022) [3] demonstrated the utility of simultaneous detection assays in investigating respiratory disease outbreaks in Bangladesh, where high sample volumes are common. The multiplex panel reduces reagent costs and labor compared to running four separate singleplex reactions [1, 3].

Field Validation and Performance in Commercial Flocks

Field validation of the multiplex panel is conducted using samples collected from commercial poultry flocks during routine surveillance or outbreak investigations. Samples are tested in parallel with virus isolation (VI) in embryonated chicken eggs and/or conventional singleplex RT-PCR as reference methods [2, 3].

Diagnostic sensitivity and specificity are calculated. For example, Slomka et al. (2023) [2] reported that efficient laboratory testing using real-time RT-PCR rapidly confirmed H5N1 (clade 2.3.4.4) outbreaks in the United Kingdom, with high concordance between M gene and H5 assays. In field studies, the multiplex panel typically achieves a diagnostic sensitivity of >95% and specificity of >99% compared to virus isolation [2, 3]. The panel detects AIV in samples with Ct values ranging from 15 to 38, corresponding to viral loads from 10^6 to 10^1 EID50/mL [1, 2].

Discrepant results (e.g., M gene positive but subtype negative) are further investigated by sequencing of the HA gene [2]. These cases may represent unsubtyped AIV strains or insufficient viral RNA for subtype amplification. The inclusion of the M gene assay ensures that even if subtype fails, a presumptive AIV positive result is obtained, triggering further testing [1, 3].

Pooled sampling (e.g., five birds per pool) increases throughput without substantial loss of sensitivity. Parvin et al. (2022) [3] showed that pooling is effective for detecting AIV in respiratory disease outbreaks, and the multiplex panel retains the ability to detect low viral loads in pooled samples.

Advantages Over Virus Isolation and Conventional RT-PCR

Virus isolation in embryonated eggs remains a gold standard for AIV detection and subtyping, but it requires 2-7 days, specialized biocontainment (BSL-3 for HPAI), and skilled personnel [2]. The multiplex real-time RT-PCR panel produces results within hours, enabling immediate biosecurity actions and culling decisions [1, 2]. It also eliminates the need for egg supply and reduces biosafety risks because the RNA extraction step inactivates the virus [1].

Conventional singleplex RT-PCR requires separate reactions for M gene and each HA subtype, consuming more sample and reagents. The multiplex format reduces hands-on time and the probability of cross-contamination [3]. Furthermore, real-time detection with Ct values provides semiquantitative viral load information that can help differentiate high-pathogenicity (rapid Ct rise) from low-pathogenicity infections [1, 2].

Role in Surveillance and Rapid Containment

The ability to rapidly detect and subtype AIV in poultry is critical for outbreak containment. The World Organisation for Animal Health (WOAH) and national veterinary authorities recommend real-time RT-PCR as the primary molecular test for AIV surveillance [1, 2]. The multiplex panel described here aligns with these guidelines and can be deployed in field laboratories or centralized diagnostic facilities.

In the event of an outbreak, the panel enables high-throughput screening of large numbers of samples from infected premises and surveillance zones. Rapid identification of H5 or H7 subtypes triggers stamping-out policies and trade restrictions [2]. Detection of H9N2, though often low pathogenicity, is important for monitoring endemic circulation and preventing secondary bacterial complications [4, 3].

The panel also supports the investigation of mixed infections, where multiple AIV subtypes may circulate simultaneously [3]. Chen et al. (2024) [4] developed a CRISPR/Cas13a-based detection method for H9N2, but the multiplex RT-PCR panel remains the gold standard for simultaneous detection of multiple subtypes in high-throughput settings.

Conclusion

A high-throughput multiplex real-time RT-PCR panel targeting the AIV M gene and H5, H7, and H9 HA genes provides a rapid, sensitive, and specific tool for surveillance and outbreak response in poultry. The assay has been validated both analytically and in field conditions, with performance equal to or exceeding virus isolation. Its automation-friendly design makes it suitable for large-scale testing, and the simultaneous detection of multiple subtypes streamlines the diagnostic workflow. Integration of this panel into routine monitoring and emergency response plans enhances the capacity for early detection and containment of avian influenza in poultry flocks.

References

[1] Coopersmith M, Dijkman R, Bartlett ML, et al. Development and Laboratory Validation of Rapid, Bird-Side Molecular Diagnostic Assays for Avian Influenza Virus Including Panzootic H5Nx. Microorganisms. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40431263/

[2] Slomka MJ, Reid SM, Byrne AMP, et al. Efficient and Informative Laboratory Testing for Rapid Confirmation of H5N1 (Clade 2.3.4.4) High-Pathogenicity Avian Influenza Outbreaks in the United Kingdom. Viruses. 2023. URL: https://pubmed.ncbi.nlm.nih.gov/37376643/

[3] Parvin R, Kabiraj CK, Hossain I, et al. Investigation of respiratory disease outbreaks of poultry in Bangladesh using two real-time PCR-based simultaneous detection assays. Front Vet Sci. 2022. URL: https://pubmed.ncbi.nlm.nih.gov/36583036/ *** 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.

[4] Chen SS, Yang YL, Wang HY, et al. CRISPR/Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus. Poult Sci. 2024. URL: https://pubmed.ncbi.nlm.nih.gov/39096825/