{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:35:58Z","timestamp":1760060158006,"version":"build-2065373602"},"reference-count":20,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,8,4]],"date-time":"2025-08-04T00:00:00Z","timestamp":1754265600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Conselho Nacional de Desenvolvimento Cient\u00edfico e Tecnol\u00f3gico-CNPq","award":["404565\/2021-2"],"award-info":[{"award-number":["404565\/2021-2"]}]},{"name":"Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior (CAPES)","award":["404565\/2021-2"],"award-info":[{"award-number":["404565\/2021-2"]}]},{"name":"Sistema Nacional de Laborat\u00f3rios em Nanotecnologia (SisNANO)","award":["404565\/2021-2"],"award-info":[{"award-number":["404565\/2021-2"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Infectious Disease Reports"],"abstract":"<jats:p>Background\/Objectives: Due to the significant overlap in symptoms between COVID-19 and other respiratory infections, a multiplex PCR-based platform was developed to simultaneously detect 22 respiratory pathogens. Target sequences were retrieved from the GenBank database and aligned using Clustal Omega 2.1 to identify conserved regions prioritized for primer design. Primers were designed using Primer Express\u00ae 3.0.1 and evaluated in Primer Explorer to ensure specificity and minimize secondary structures. A multiplex strategy organized primers into three groups, each labeled with distinct fluorophores (FAM, VIC, or NED), allowing for detection by conventional PCR or capillary electrophoresis (CE). Methods: After reverse transcription for RNA targets, amplification was performed in a single-tube reaction. A total of 340 clinical samples\u2014nasopharyngeal and saliva swabs\u2014were collected from patients, during the COVID-19 pandemic period. The automated analysis of electropherograms enabled precise pathogen identification. Results: Of the samples analyzed, 57.1% tested negative for all pathogens. SARS-CoV-2 was the most frequently detected pathogen (29%), followed by enterovirus (6.5%). Positive results were detected in both nasopharyngeal and saliva swabs, with SARS-CoV-2 predominating in saliva samples. Conclusions: This single-tube multiplex PCR-CE assay represents a cost-effective and robust approach for comprehensive respiratory pathogen detection. It enables rapid and simultaneous diagnosis, facilitating targeted treatment strategies and improved patient outcomes.<\/jats:p>","DOI":"10.3390\/idr17040093","type":"journal-article","created":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T10:58:26Z","timestamp":1754564306000},"page":"93","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Detection of Microorganisms Causing Human Respiratory Infection Using One-Tube Multiplex PCR"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0953-487X","authenticated-orcid":false,"given":"Isabela L.","family":"Lima","sequence":"first","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38405-302, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2299-2114","authenticated-orcid":false,"given":"Adriana F.","family":"Neves","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Catal\u00e3o, Catal\u00e3o 75704-020, Goi\u00e1s, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8453-3194","authenticated-orcid":false,"given":"Robson J.","family":"Oliveira-J\u00fanior","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38405-302, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lorrayne C. M. G.","family":"Hon\u00f3rio","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38405-302, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vit\u00f3ria O.","family":"Arruda","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Catal\u00e3o, Catal\u00e3o 75704-020, Goi\u00e1s, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juliana A.","family":"S\u00e3o Juli\u00e3o","sequence":"additional","affiliation":[{"name":"Technical Department, BioGenetics Tecnologia Molecular Ltda, Uberl\u00e2ndia 38400-446, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luiz Ricardo","family":"Goulart Filho","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38405-302, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2041-0053","authenticated-orcid":false,"given":"Vivian","family":"Alonso-Goulart","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia, Universidade Federal de Uberl\u00e2ndia, Uberl\u00e2ndia 38405-302, Minas Gerais, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s15010-025-02553-5","article-title":"Rapid diagnosis of acute pediatric respiratory infections with Point-of-Care and multiplex molecular testing","volume":"53","author":"Caldwell","year":"2025","journal-title":"Infection"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1038\/s41579-021-00535-6","article-title":"Transmissibility, and transmission of respiratory viruses","volume":"19","author":"Leung","year":"2021","journal-title":"Nat. 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