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
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 effectiven...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2025.1520349/full |
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| author | Jin-Ha Song Seung-Eun Son Ho-Won Kim Seung-Ji Kim Se-Hee An Chung-Young Lee Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Kang-Seuk Choi Kang-Seuk Choi |
| author_facet | Jin-Ha Song Seung-Eun Son Ho-Won Kim Seung-Ji Kim Se-Hee An Chung-Young Lee Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Kang-Seuk Choi Kang-Seuk Choi |
| author_sort | Jin-Ha Song |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-5a618ac777f846008553f122c36409c3 |
| institution | Kabale University |
| issn | 2297-1769 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Veterinary Science |
| spelling | doaj-art-5a618ac777f846008553f122c36409c32025-01-17T06:51:05ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692025-01-011210.3389/fvets.2025.15203491520349Rapid and specific on-site H5Nx avian influenza diagnosis via RPA and PAM-independent CRISPR-Cas12a assay combined with anti-NP antibody-based viral RNA purificationJin-Ha Song0Seung-Eun Son1Ho-Won Kim2Seung-Ji Kim3Se-Hee An4Chung-Young Lee5Hyuk-Joon Kwon6Hyuk-Joon Kwon7Hyuk-Joon Kwon8Hyuk-Joon Kwon9Kang-Seuk Choi10Kang-Seuk Choi11Laboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaLaboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaLaboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaLaboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaAvian Influenza Research and Diagnostic Division, Animal and Plant Quarantine Agency, Gimcheon-si, Republic of KoreaDepartment of Microbiology, College of Medicine, Kyungpook National University, Daegu, Republic of KoreaResearch Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of KoreaLaboratory of Poultry Medicine, Department of Farm Animal Medicine, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaFarm Animal Clinical Training and Research Center (FACTRC), GBST, Seoul National University, Pyeongchang, Republic of KoreaGeNiner Inc., Seoul, Republic of KoreaLaboratory of Avian Diseases, College of Veterinary Medicine and BK21 PLUS for Veterinary Science, Seoul National University, Seoul, Republic of KoreaResearch Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of KoreaRapid 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.https://www.frontiersin.org/articles/10.3389/fvets.2025.1520349/fullavian influenza virusmagnetic beadsribonucleoprotein purificationCRISPR-Cas12aPAM-independenton-site detection |
| spellingShingle | Jin-Ha Song Seung-Eun Son Ho-Won Kim Seung-Ji Kim Se-Hee An Chung-Young Lee Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Hyuk-Joon Kwon Kang-Seuk Choi Kang-Seuk Choi 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 Frontiers in Veterinary Science avian influenza virus magnetic beads ribonucleoprotein purification CRISPR-Cas12a PAM-independent on-site detection |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | avian influenza virus magnetic beads ribonucleoprotein purification CRISPR-Cas12a PAM-independent on-site detection |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2025.1520349/full |
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