{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T18:27:55Z","timestamp":1767205675227,"version":"build-2238731810"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T00:00:00Z","timestamp":1763424000000},"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. Cell. Infect. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      Community-acquired pneumonia (CAP) remains a leading cause of mortality in children under five years of age worldwide.\n                      <jats:italic>Streptococcus pneumoniae<\/jats:italic>\n                      and\n                      <jats:italic>Haemophilus influenzae<\/jats:italic>\n                      are the most common bacterial pathogens causing CAP that requires hospitalization, highlighting the critical need for a simple, low-cost, and highly sensitive method for rapid diagnosis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      We developed a duplex recombinase-aided amplification (RAA) assay for the simultaneous detection of\n                      <jats:italic>S. pneumoniae<\/jats:italic>\n                      and\n                      <jats:italic>H. influenzae<\/jats:italic>\n                      . Following comparative genomic analysis, the conserved lytA gene and omp6 gene were selected as the specific targets for\n                      <jats:italic>S. pneumoniae<\/jats:italic>\n                      and\n                      <jats:italic>H. influenzae<\/jats:italic>\n                      , respectively. The reaction conditions, including temperature and probe concentration, were optimized.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The established duplex RAA assay can be completed within 10 minutes at a constant temperature of 39\u00b0C, with an optimal probe concentration combination of 0.6 \u03bcM for\n                      <jats:italic>S. pneumoniae<\/jats:italic>\n                      and 0.8 \u03bcM for\n                      <jats:italic>H. influenzae<\/jats:italic>\n                      . The assay demonstrated high sensitivity, with a limit of detection of 72 copies per reaction for\n                      <jats:italic>S. pneumoniae<\/jats:italic>\n                      and 35 copies per reaction for\n                      <jats:italic>H. influenzae<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>This study presents a rapid and accurate nucleic acid amplification assay for the concurrent detection of two major bacterial pathogens in childhood CAP. The speed, simplicity, and sensitivity of the duplex RAA assay make it a promising tool for early and rapid etiological diagnosis in clinical settings.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fcimb.2025.1631633","type":"journal-article","created":{"date-parts":[[2025,11,18]],"date-time":"2025-11-18T06:23:26Z","timestamp":1763447006000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid detection of Hemophilus influenzae and Streptococcus pneumoniae simultaneously using a duplex recombinase-aided amplification assay directly from invasive clinical samples"],"prefix":"10.3389","volume":"15","author":[{"given":"Lin","family":"Zhou","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuyan","family":"Xia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanling","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bing","family":"Du","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xianyi","family":"Huang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenjian","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fang","family":"Fang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chun","family":"Meng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lijuan","family":"Ma","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guanhua","family":"Xue","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jing","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,11,18]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"366","DOI":"10.1016\/j.diagmicrobio.2009.03.030","article-title":"Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction","volume":"64","author":"Abdeldaim","year":"2009","journal-title":"Diagn. 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