ArticleFrontiers in veterinary science2025
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.
Article in Frontiers in veterinary science, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Streamlined CRISPR-based assays for detection and subtyping of H5 and H7 avian influenza.iScience · 2026Article
- Innovations, Applications, and Future Trends in Veterinary Diagnostic Technologies.Transboundary and emerging diseases · 2026Review
- Recent advances in lateral flow devices and point-of-care diagnostics for highly pathogenic avian influenza A viruses.Journal of virology · 2025Review
- Preventive Immunology for Livestock and Zoonotic Infectious Diseases in the One Health Era: From Mechanistic Insights to Innovative Interventions.Veterinary sciences · 2025Review
- A Host Cell Vector Model for Analyzing Viral Protective Antigens and Host Immunity.International journal of molecular sciences · 2025Article
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Authors and funding
8 authors.
Funding
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Abstract
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 10
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