{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T05:04:46Z","timestamp":1789535086757,"version":"build-2803163510"},"reference-count":24,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T00:00:00Z","timestamp":1789516800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background\/objectives<\/jats:title>\n                    <jats:p>Novel influenza A viruses (NIAV) infection is a zoonotic acute respiratory disease. The H5N1 and H7N9 subtypes cause high mortality, posing major public health threats. This study aimed to establish rapid, field-deployable subtype-specific nucleic acid detection assays for H5 and H7 by integrating reverse-transcription enzymatic recombinase amplification (RT-ERA) with CRISPR\/Cas12a. Two separate reactions are required per sample.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Conserved hemagglutinin (H) sequences were retrieved from the NCBI database, and primers, probes, and crRNAs were designed using Primer Premier 5 and Primer-BLAST. The sensitivity of RT-ERA alone was compared with that of the ERA-CRISPR\/Cas12a using serially diluted virus strain nucleic acid. The specificity was verified against other respiratory pathogens with similar clinical manifestations.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimal primers were F1R2 for H5 and F2R3 for H7. The limit of detection (LOD) of RT-ERA alone was 2.63\u00d710\u00b3 copies\/\u00b5L for H5 and 1.86\u00d710\u00b3 copies\/\u00b5L for H7; the ERA-CRISPR\/Cas12a assay improved the LOD to 2.63\u00d710\u00b9 copies\/\u00b5L for H5 and 1.86 copies\/\u00b5L for H7, representing 100-fold and 1000-fold enhancements in sensitivity, respectively. Specificity testing showed that the assay exclusively detected H5N1 and H7N9 with no cross-reactivity to other tested pathogens.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The ERA-CRISPR\/Cas12a method exhibits enhanced sensitivity for H5 and H7 subtypes detection, along with high specificity, rapidity, and minimal equipment requirements. It offers a promising screening tool for NIAV infections.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2026.1922968","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T04:34:00Z","timestamp":1789533240000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment and preliminary evaluation of an RT-ERA combined with CRISPR\/Cas12a assay for detection of H5 and H7 influenza viruses"],"prefix":"10.3389","volume":"16","author":[{"given":"Zhenghan","family":"Luo","sequence":"first","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]},{"name":"Department of Microbiology, Faculty of Naval Medicine, Naval Medical University","place":["Shanghai, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minzhi","family":"Xu","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Letian","family":"Zhang","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifang","family":"Han","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anqi","family":"Tan","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taiwu","family":"Wang","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fuqiang","family":"Ye","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunhui","family":"Wang","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinhai","family":"Zhang","sequence":"additional","affiliation":[{"name":"Huadong Research Institute for Medicine and Biotechnics","place":["Nanjing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"870","DOI":"10.1002\/phar.4621","article-title":"Update and narrative review of avian influenza (H5N1) infection in adult patients","volume":"44","author":"Aldhaeefi","year":"2024","journal-title":"Pharmacotherapy"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.diagmicrobio.2025.117223","article-title":"Molecular diagnostics in clinical microbiology: Advances, applications, and future directions","volume":"114","author":"Asokan","year":"2026","journal-title":"Diagn. 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