{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T03:11:33Z","timestamp":1788923493709,"version":"build-2803163510"},"reference-count":34,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2023,11,11]],"date-time":"2023-11-11T00:00:00Z","timestamp":1699660800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2023,11,11]],"date-time":"2023-11-11T00:00:00Z","timestamp":1699660800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Microbiol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      During the coronavirus disease 2019 (COVID-19) pandemic, the simultaneous detection of severe acute respiratory syndrome coronavirus 2\n                      <jats:italic>(SARS-CoV-2<\/jats:italic>\n                      ) and\n                      <jats:italic>Influenza A, and Influenza B<\/jats:italic>\n                      viruses is essential for rapid differential diagnosis in patients with similar symptoms, especially during \u201c\n                      <jats:italic>flu<\/jats:italic>\n                      season\u201d in the post-pandemic era. So far, several multiplex methods have been approved for the simultaneous detection of\n                      <jats:italic>SARS-CoV-2, Influenza A, and Influenza B<\/jats:italic>\n                      . However, due to the rapid mutation rate of the\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      genome and the emergence of new variants, existing methods must be improved and updated.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      To identify a highly conserved region in the\n                      <jats:italic>SARS-CoV-2\u00a0N-gene<\/jats:italic>\n                      , a genomic survey was performed to increase the sensitivity and specificity of primer and probe sets targeting the\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      genome. The 95% LLOD (95% lower limits of detection) were calculated by probit analysis. A total of 70 predetermined clinical samples using singleplex RT-qPCR assays, were included. The clinical performance of the multiplex RT-qPCR assay was determined and compared with a commercial multiplex kit. The Cohen\u2019s kappa coefficient,\n                      <jats:italic>P<\/jats:italic>\n                      -value (McNemar\u2019s test), Passing-Bablok regression, and Bland Altman agreement analysis were determined to monitor the agreement of the assays.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The novel\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      primer and probe set designed in this assay was able to detect all variants of concern (VOCs) and variants of interest (VOIs) with high analytical and clinical performance. The 95% LLOD for the multiplex RT-qPCR was 20 copies per reaction for the\n                      <jats:italic>N<\/jats:italic>\n                      gene of\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      , 2 copies per reaction for\n                      <jats:italic>M1<\/jats:italic>\n                      gene of\n                      <jats:italic>Influenza A<\/jats:italic>\n                      and\n                      <jats:italic>NS1<\/jats:italic>\n                      gene of\n                      <jats:italic>Influenza B<\/jats:italic>\n                      . The diagnostic sensitivity of the multiplex RT-qPCR was 94.4%, 93.7%, and 100% for the detection of\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      ,\n                      <jats:italic>Influenza A<\/jats:italic>\n                      , and\n                      <jats:italic>Influenza B<\/jats:italic>\n                      genomes, respectively. Moreover, the specificity was identical (100%) in both assays. According to the agreement analysis results, there was no statistical difference between our multiplex assay and the commercial kit.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      In this study, we developed a novel in-house made multiplex RT-qPCR assay, with high sensitivity, specificity, and reliability for the diagnosis of\n                      <jats:italic>SARS-CoV-2<\/jats:italic>\n                      infection in clinical samples. This is valuable during\n                      <jats:italic>Influenza<\/jats:italic>\n                      seasons when influenza co-circulates with SARS-CoV-2, as it saves costs, time, and thus specific and timely treatment of patients.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12866-023-03048-9","type":"journal-article","created":{"date-parts":[[2023,11,11]],"date-time":"2023-11-11T10:01:32Z","timestamp":1699696892000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Development of a robust TaqMan probe-based one-step multiplex RT-qPCR for simultaneous detection of SARS-CoV-2 and Influenza A\/B viruses"],"prefix":"10.1186","volume":"23","author":[{"given":"Hamidreza","family":"Abbasi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadi Razavi","family":"Nikoo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fatemeh","family":"Fotouhi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayyoob","family":"Khosravi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,11,11]]},"reference":[{"issue":"3","key":"3048_CR1","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1159\/000520234","volume":"65","author":"A Fallah","year":"2022","unstructured":"Fallah A, et al. 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The covid-19 patients were not directly enrolled in this study. The covid-19 positive samples were prepared from the covid-19 bank of the Golestan University of Medical Science that was diagnosed and stored previously at -80\u00b0c.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"335"}}