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ORIGINAL RESEARCH article

Front. Vet. Sci.
Sec. Veterinary Experimental and Diagnostic Pathology
Volume 12 - 2025 | doi: 10.3389/fvets.2025.1520349
This article is part of the Research Topic Emerging Diseases and Diagnostics in Poultry Production View all 7 articles

Rapid and specific on-site H5Nx avian influenza diagnosis via RPA and PAM-independent CRISPR-Cas12a assay combined with anti-NP antibody-based viral RNA purification

Provisionally accepted
JINHA SONG JINHA SONG 1Seung-Eun Son Seung-Eun Son 1Se-Hee An Se-Hee An 2Chung-Young Lee Chung-Young Lee 3Ho-Won Kim Ho-Won Kim 1Seung-Ji Kim Seung-Ji Kim 1Hyuk-Joon Kwon Hyuk-Joon Kwon 4,5,6,7*Kang-Seuk Choi Kang-Seuk Choi 1,4*
  • 1 Laboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of Korea
  • 2 Avian Influenza Research & Diagnostic Division, Animal and Plant Quarantine Agency, Gimcheon-si, Republic of Korea
  • 3 Department of Microbiology, College of Medicine, Kyungpook National University, Daegu, North Gyeongsang, Republic of Korea
  • 4 Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Seoul, Republic of Korea
  • 5 Laboratory of Poultry Medicine, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of Korea
  • 6 Farm Animal Clinical Training and Research Center (FACTRC), GBST, Seoul National University, Pyeongchang, Republic of Korea
  • 7 GeNiner Inc, Seoul, Republic of Korea

The final, formatted version of the article will be published soon.

    Rapid and accurate detection of H5Nx avian influenza viruses is critical for effective surveillance and control measures. Currently, RT-qPCR with spin column RNA extraction is the gold standard for HPAIV surveillance, but its long reaction time and need for specialized equipment limit its effectiveness for rapid response. In this study, we introduce a centrifuge-free, rapid detection method for on-site detection of H5Nx viruses that combines magnetic bead-based ribonucleoprotein (RNP) purification and concentration with a CRISPR-Cas12a system that is independent of the protospacer adjacent motif (PAM) sequence. Our approach employs anti-NP monoclonal antibodies for the targeted isolation of RNA bound to RNPs, facilitating a quick and specific RNA extraction process that negates the need for centrifugation. Additionally, by denaturing the RT-RPA amplicon using 60% DMSO, we activate the trans-ssDNA cleavage activity of the Cas12a protein without the need for a specific PAM (5’-TTTV-3’) sequence. This strategy increases flexibility in CRISPR RNA design, providing a significant advantage when targeting genes with high variability. We validated the efficacy of our magnetic RNP purification and concentration method in combined with an RT-RPA/PAM-independent Cas12a assay for detecting the H5 gene. The assay achieved a sensitivity threshold of 101 EID50 with fluorescent detection and 102 EID50 using lateral flow strips. It also exhibited high specificity, yielding positive results solely for H5Nx viruses among various influenza A virus subtypes. Furthermore, in clinical samples, the assay demonstrated 80% sensitivity and 100% specificity. These results highlight the advantages of using NP-specific antibodies for RNP purification and CRISPR-Cas12a with viral gene-specific crRNA to achieve exceptional diagnostic specificity.

    Keywords: Avian influenza virus, Magnetic beads, Ribonucleoprotein purification, CRISPR-Cas12a, PAM-independent, on-site detection

    Received: 31 Oct 2024; Accepted: 02 Jan 2025.

    Copyright: © 2025 SONG, Son, An, Lee, Kim, Kim, Kwon and Choi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Hyuk-Joon Kwon, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 151-747, Seoul, Republic of Korea
    Kang-Seuk Choi, Laboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of Korea

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