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Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific\n                    <jats:italic>Mycobacterium leprae<\/jats:italic>\n                    targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the\n                    <jats:italic>M<\/jats:italic>\n                    .\n                    <jats:italic>leprae<\/jats:italic>\n                    genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and\n                    <jats:italic>Mycobacteria<\/jats:italic>\n                    , showing no cross-reactivity. Intra- and inter-operator C\n                    <jats:sub>p<\/jats:sub>\n                    variation was evaluated using dilution curves of\n                    <jats:italic>M<\/jats:italic>\n                    .\n                    <jats:italic>leprae<\/jats:italic>\n                    DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects\n                    <jats:italic>M<\/jats:italic>\n                    .\n                    <jats:italic>leprae<\/jats:italic>\n                    DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.\n                  <\/jats:p>","DOI":"10.1371\/journal.pntd.0009850","type":"journal-article","created":{"date-parts":[[2022,2,18]],"date-time":"2022-02-18T13:42:31Z","timestamp":1645191751000},"page":"e0009850","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":17,"title":["Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis"],"prefix":"10.1371","volume":"16","author":[{"given":"Fernanda Saloum de Neves","family":"Manta","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1348-1523","authenticated-orcid":true,"given":"Thiago","family":"Jacomasso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8367-999X","authenticated-orcid":true,"given":"Rita de C\u00e1ssia Pontello","family":"Rampazzo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suelen Justo Maria","family":"Moreira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8086-2441","authenticated-orcid":true,"given":"Najua M.","family":"Zahra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stewart T.","family":"Cole","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlotte","family":"Avanzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thyago","family":"Leal-Calvo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sidra Ezidio Gon\u00e7alves","family":"Vasconcellos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phillip","family":"Suffys","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8663-3364","authenticated-orcid":true,"given":"Marcelo","family":"Ribeiro-Alves","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco Aurelio","family":"Krieger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8434-2152","authenticated-orcid":true,"given":"Alexandre Dias Tavares","family":"Costa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milton Oz\u00f3rio","family":"Moraes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,2,18]]},"reference":[{"key":"pntd.0009850.ref001","first-page":"2017","author":"WHO SEARO","journal-title":"A guide for surveillance of antimicrobial resistance in leprosy"},{"issue":"8","key":"pntd.0009850.ref002","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1590\/S0074-02762012000800015","article-title":"Leprosy exposure, infection and disease: a 25-year surveillance study of leprosy patient contacts","volume":"107","author":"EN Sarno","year":"2012","journal-title":"Mem Inst Oswaldo Cruz"},{"issue":"1","key":"pntd.0009850.ref003","first-page":"78","article-title":"A method for counting acid-fast bacteria","volume":"36","author":"CC Shepard","year":"2019","journal-title":"Int J Lepr Other Mycobact Dis"},{"issue":"3","key":"pntd.0009850.ref004","doi-asserted-by":"crossref","first-page":"e0007147","DOI":"10.1371\/journal.pntd.0007147","article-title":"Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study","volume":"13","author":"RR Barbieri","year":"2019","journal-title":"PLoS Negl Trop Dis"},{"issue":"9","key":"pntd.0009850.ref005","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1016\/j.medmal.2015.09.002","article-title":"Update on the epidemiology, diagnosis, and treatment of leprosy","volume":"45","author":"F Reibel","year":"2015","journal-title":"Med Mal Infect"},{"issue":"1","key":"pntd.0009850.ref006","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1128\/CMR.00079-13","article-title":"Leprosy in the 21st century","volume":"28","author":"C White","year":"2015","journal-title":"Clinical Microbiology Reviews. 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