{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T18:07:25Z","timestamp":1788977245449,"version":"build-2803163510"},"reference-count":43,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science and Technology Development Program of Jinan Municipal Health Commission","award":["2023-1-20"],"award-info":[{"award-number":["2023-1-20"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Pathogens causing respiratory infections have reemerged globally since the COVID-19 pandemic. Rapid and accurate identification of respiratory pathogens is critical for diagnosis and treatment. In this study, we developed and evaluated a multiplex real-time PCR assay with a fluorescence probe melting curve (mqPCR-PMC) assay to simultaneously detect six common pathogens, namely, influenza virus A and B (IFV-A and -B), respiratory syncytial virus (RSV), human rhinovirus (HRV), human adenovirus (HAdV) and\n                    <jats:italic>Mycoplasma pneumoniae<\/jats:italic>\n                    (MP). The primers and probes optimized in this assay exhibited a specificity of 100% with a panel of 57 pathogens. The detection limits of the assay ranged from 248 copies\/ml to 394 copies\/ml. Moreover, the coefficients of variation ranged from 0.11% to 0.30% for Tm and from 1.59% to 4.10% for Rm. The clinical accuracy of the assay was validated using 760 pharyngeal samples. Compared with a commercial kit based on conventional fluorescence RT-PCR, the assay showed more than 87% sensitivity and 99% specificity for each pathogen. The kappa values ranged from 0.927 to 1.000. Moreover, the assay was more precise for HRV and more sensitive on IFV-A, HAdV, MP and RSV. In summary, the mqPCR-PMC assay was demonstrated to be highly sensitive, highly specific and high throughput, and has potential for clinical use in respiratory infectious disease diagnosis.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0357377","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T17:25:50Z","timestamp":1788974750000},"page":"e0357377","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a multiplex real-time PCR assay with fluorescence probe-melting-curve analysis for one-tube detection of respiratory pathogens"],"prefix":"10.1371","volume":"21","author":[{"given":"Mingyu","family":"Ji","sequence":"first","affiliation":[{"name":"Medical Integration and Practice Center, Shandong University","place":["Jinan, Shandong, China"]},{"name":"Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University","place":["Jinan, Shandong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongjiao","family":"Dong","sequence":"additional","affiliation":[{"name":"GuoRan Gene Technology (Shandong) Company Limited","place":["Jinan, Shandong, China"],"department":["Research and Development Department"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yihui","family":"Xu","sequence":"additional","affiliation":[{"name":"Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University","place":["Jinan, Shandong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3767-6728","authenticated-orcid":true,"given":"Yunshan","family":"Wang","sequence":"additional","affiliation":[{"name":"Medical Integration and Practice Center, Shandong University","place":["Jinan, Shandong, China"]},{"name":"Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University","place":["Jinan, Shandong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-7018-970X","authenticated-orcid":true,"given":"Qianqian","family":"Zhao","sequence":"additional","affiliation":[{"name":"Medical Integration and Practice Center, Shandong University","place":["Jinan, Shandong, China"]},{"name":"Medical Research and Laboratory Diagnostic Center, Jinan Central Hospital Affiliated to Shandong First Medical University","place":["Jinan, Shandong, China"]},{"name":"Shandong LaiBo Biotechnology Company Limited","place":["Jinan, Shandong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,9]]},"reference":[{"issue":"166","key":"pone.0357377.ref001","doi-asserted-by":"crossref","first-page":"220150","DOI":"10.1183\/16000617.0150-2022","article-title":"Respiratory 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