{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T00:52:06Z","timestamp":1774659126865,"version":"3.50.1"},"reference-count":54,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,12,19]],"date-time":"2023-12-19T00:00:00Z","timestamp":1702944000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses can cause respiratory illnesses with similar clinical symptoms, making their differential diagnoses challenging. Additionally, in critically ill SARS-CoV-2\u2013infected patients, co-infections with other respiratory pathogens can lead to severe cytokine storm and serious complications. Therefore, a method for simultaneous detection of SARS-CoV-2 and influenza A and B viruses will be clinically beneficial.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We designed an assay to detect five gene targets simultaneously via asymmetric PCR-mediated melting curve analysis in a single tube. We used specific probes that hybridize to corresponding single-stranded amplicons at low temperature and dissociate at high temperature, creating different detection peaks representing the targets. The entire reaction was conducted in a closed tube, which minimizes the risk of contamination. The limit of detection, specificity, precision, and accuracy were determined.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The assay exhibited a limit of detection of &amp;lt;20 copies\/\u03bcL for SARS-CoV-2 and influenza A and &amp;lt;30 copies\/\u03bcL for influenza B, with high reliability as demonstrated by a coefficient of variation for melting temperature of &amp;lt;1.16% across three virus concentrations. The performance of our developed assay and the pre-determined assay showed excellent agreement for clinical samples, with kappa coefficients ranging from 0.98 (for influenza A) to 1.00 (for SARS-CoV-2 and influenza B). No false-positive, and no cross-reactivity was observed with six common non-influenza respiratory viruses.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>The newly developed assay offers a straightforward, cost-effective and nucleic acid contamination-free approach for simultaneous detection of the SARS-CoV-2, influenza A, and influenza B viruses. The method offers high analytical sensitivity, reliability, specificity, and accuracy. Its use will streamline testing for co-infections, increase testing throughput, and improve laboratory efficacy.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2023.1249085","type":"journal-article","created":{"date-parts":[[2023,12,19]],"date-time":"2023-12-19T11:08:40Z","timestamp":1702984120000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A\/B viruses"],"prefix":"10.3389","volume":"14","author":[{"given":"Peng","family":"Gao","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanyan","family":"Fan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaomu","family":"Kong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lida","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongwei","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meimei","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guoxiong","family":"Deng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yongtong","family":"Cao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liang","family":"Ma","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2023,12,19]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sjbs.2022.103465","article-title":"SARS-CoV-2 detection methods: a comprehensive review.","volume":"29","author":"Alhamid","year":"2022","journal-title":"Saudi J. 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