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Literature Sharing: Establishment of Two Rapid RT-RAA Assays for H5 Subtype Avian Influenza Virus Detection
Release date:
2025-12-29
Avian influenza virus (AIV) poses a serious threat to the global poultry industry and public health, especially the highly pathogenic H5 subtype (H5-AIV). Between 2021 and 2022, the number of poultry deaths due to avian influenza exceeded 138 million, highlighting the urgent need to develop rapid detection technologies. Based on RT-RAA technology, this study developed two novel detection methods: RF-RT-RAA and RT-RAA-LFD, which can achieve rapid detection of H5-AIV within 15-30 minutes.
Source: Li Y, Shang J, Wang Y, et al. Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus. Microbiol Spectr. 2023;11(6):e02186-23. doi:10.1128/spectrum.02186-23
Background
Avian influenza virus (AIV) poses a serious threat to the global poultry industry and public health, especially the highly pathogenic H5 subtype (H5-AIV). Between 2021 and 2022, the number of poultry deaths due to avian influenza exceeded 138 million, highlighting the urgent need to develop rapid detection technologies. Based on RT-RAA technology, this study developed two novel detection methods: RF-RT-RAA and RT-RAA-LFD, which can achieve rapid detection of H5-AIV within 15-30 minutes.
Existing Detection Methods and Their Limitations
• Virus isolation: High sensitivity but requires BSL-3 laboratories, making it unsuitable for large-scale testing.
•HA and hemagglutination inhibition (HI) assays: Simple result interpretation but time-consuming and labor-intensive.
• Real-time quantitative RT-PCR: Accurate results but requires expensive equipment and professional operation.
The emergence of isothermal amplification techniques such as RT-RAA provides a new approach to overcoming these limitations.
Study Design and Detection Method Establishment
In this study, researchers designed primers and probes targeting the conserved region of the H5-AIV hemagglutinin (HA) gene. After screening six primer pairs, F3/R1 was selected for subsequent experiments. The optimal reaction conditions for the two methods were: RF-RT-RAA at 39°C for 20 minutes, and RT-RAA-LFD at 37°C for 15 minutes. The detection process includes nucleic acid extraction, RT-RAA amplification, and result interpretation via fluorescence (RF-RT-RAA) or test strip color development (RT-RAA-LFD).
Primer and Probe Design
Sequence alignment ensured that the primers and probes specifically bound only to H5-AIV strains, with no cross-reactivity with other subtypes (e.g., H3, H7, H9) or avian pathogens. Probes were modified with fluorophores (FAM/BHQ1) and fluorescein/biotin for convenient detection.
Performance of the RF-RT-RAA Detection Method
• Specificity: The RF-RT-RAA method produced fluorescence signals only for H5-AIV, with no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), group A rotavirus (RVA), or duck astrovirus (DAstV).
• Sensitivity: The limit of detection reached 1 copy/μL, which is 100 times more sensitive than conventional RT-PCR (100 copies/μL). The LOD95 was 188 copies/μL.
Performance of the RT-RAA-LFD Detection Method
• Optimization: Reaction condition optimization showed that 37°C for 15 minutes yielded the best results, with clear detection line signals and no non-specific signals.
• Specificity: Similar to RF-RT-RAA, RT-RAA-LFD detected only H5-AIV, with no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, IBV, NDV, RVA, or DAstV.
• Sensitivity: The limit of detection was 1 copy/μL, comparable to RF-RT-RAA. It was 100 times more sensitive than RT-PCR (100 copies/μL) but 10 times less sensitive than real-time quantitative RT-PCR (0.1 copy/μL). The LOD95 was 406 copies/μL.
Validation with Clinical and Experimental Samples
• Clinical Samples (350 samples): Compared with virus isolation (60 positive samples):
• RF-RT-RAA: 55 positive samples detected, sensitivity 91.67%, specificity 100%, Kappa value 0.948.
• RT-RAA-LFD: 51 positive samples detected, sensitivity 85%, specificity 100%, Kappa value 0.904.
• Experimental Samples (26 samples):
• RF-RT-RAA: Fully consistent with virus isolation (19/19), Kappa value 1.
• RT-RAA-LFD: 18 positive samples detected, sensitivity 94.7%, Kappa value 0.906.
Discussion and Conclusion
The RF-RT-RAA and RT-RAA-LFD detection methods outperform existing methods in speed (15-30 minutes), sensitivity (1 copy/μL), and ease of operation, requiring no complex instrumentation and making them particularly suitable for grassroots-level surveillance. Compared to techniques like LAMP or biosensors, these methods strike a good balance between speed, accuracy, and accessibility.
Conclusion: This study provides reliable and scalable technical tools for H5-AIV detection, with the potential to enhance global influenza surveillance and outbreak response capabilities. Future work could focus on multiplex detection for other subtypes or integration with digital health platforms.
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