{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T10:52:07Z","timestamp":1782730327053,"version":"3.54.5"},"reference-count":31,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,5,29]],"date-time":"2020-05-29T00:00:00Z","timestamp":1590710400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100013394","name":"Instituto Nacional de Medicina Gen\u00f3mica","doi-asserted-by":"publisher","award":["INMEGEN-08\/2013\/I"],"award-info":[{"award-number":["INMEGEN-08\/2013\/I"]}],"id":[{"id":"10.13039\/501100013394","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify\/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.<\/jats:p>","DOI":"10.3390\/microorganisms8060818","type":"journal-article","created":{"date-parts":[[2020,6,1]],"date-time":"2020-06-01T11:49:21Z","timestamp":1591012161000},"page":"818","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis"],"prefix":"10.3390","volume":"8","author":[{"given":"Marcello","family":"Ceccarelli","sequence":"first","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5456-1821","authenticated-orcid":false,"given":"Aurora","family":"Diotallevi","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gloria","family":"Buffi","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2983-8344","authenticated-orcid":false,"given":"Mauro","family":"De Santi","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edith A.","family":"Fern\u00e1ndez-Figueroa","sequence":"additional","affiliation":[{"name":"Department of Population Genomics, Computational and Integrative Genomics, National Institute of Genomic Medicine, Mexico City 14610, Mexico"},{"name":"Centro de Medicina Tropical, Unidad de Investigaci\u00f3n en Medicina Experimental, Facultad de Medicina, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Hospital General de M\u00e9xico, M\u00e9xico City 04360, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1303-0834","authenticated-orcid":false,"given":"Claudia","family":"Rangel-Escare\u00f1o","sequence":"additional","affiliation":[{"name":"Department of Population Genomics, Computational and Integrative Genomics, National Institute of Genomic Medicine, Mexico City 14610, Mexico"},{"name":"School of Engineering and Sciences, Tecnologico de Monterrey, Queretaro 76130, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5122-5275","authenticated-orcid":false,"given":"Said A.","family":"Mu\u00f1oz-Montero","sequence":"additional","affiliation":[{"name":"Department of Population Genomics, Computational and Integrative Genomics, National Institute of Genomic Medicine, Mexico City 14610, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ingeborg","family":"Becker","sequence":"additional","affiliation":[{"name":"Centro de Medicina Tropical, Unidad de Investigaci\u00f3n en Medicina Experimental, Facultad de Medicina, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Hospital General de M\u00e9xico, M\u00e9xico City 04360, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6456-6626","authenticated-orcid":false,"given":"Mauro","family":"Magnani","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1747-526X","authenticated-orcid":false,"given":"Luca","family":"Galluzzi","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Sciences, University of Urbino \u201cCarlo Bo\u201d, 61029 Urbino (PU), Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,5,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Alvar, J., V\u00e9lez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., and den Boer, M. 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