{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:26:13Z","timestamp":1785432373188,"version":"3.56.0"},"reference-count":46,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T00:00:00Z","timestamp":1780358400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002322","name":"Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100002322","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/00x0ma614","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Background\/Objective: Leishmaniasis is a neglected tropical disease caused by species of the genus Leishmania, with a broad clinical spectrum that can overlap with other infectious and non-infectious conditions. Accurate species identification is critical for appropriate treatment and prognosis; however, parasitological methods are limited by suboptimal sensitivity, specificity, and inability to reliably differentiate species. This study aimed to develop and validate a real-time PCR assay based on melting-curve analysis (Leish-qPCR) for the detection of Leishmania spp. and the differentiation of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. Methods and Results: Genus-specific primers were designed based on the kDNA (kinetoplast DNA) minicircle consensus sequences of Leishmania species, while species-specific primers targeted the internal transcribed spacer 2 (ITS2) consensus regions of the ribosomal RNA locus of L. (L.) amazonensis and L. (V.) braziliensis. Analytical performance was evaluated in silico and in vitro using a panel of protozoa, fungi, and bacteria, exhibiting 100% specificity with no cross-amplification. The limit of detection was one copy per reaction for all targets using positive controls. Clinical validation was performed using skin biopsy specimens from patients with granulomatous lesions. The optimized Leish-qPCR assay, performed in separate reaction tubes within the same run, demonstrated reliable analytical specificity and sensitivity, with distinct and reproducible melting temperature (Tm) peaks across plasmid controls, parasite DNA, and clinical samples. Comparative analysis with histopathological examination demonstrated moderate agreement between the methods, supporting the applicability of the assay for sensitive detection and species-level discrimination of Leishmania spp. in clinical samples. Conclusions: The Leish-qPCR assay presented high sensitivity, specificity, and diagnostic accuracy, representing a promising tool for routine diagnosis of leishmaniasis and for the differentiation of L. (L.) amazonensis and L. (V.) braziliensis in clinical samples.<\/jats:p>","DOI":"10.3390\/diagnostics16111704","type":"journal-article","created":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T10:14:57Z","timestamp":1780395297000},"page":"1704","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Molecular Diagnosis of Leishmaniasis: Development of a qPCR Assay for Genus Detection and Differentiation of Leishmania (L.) amazonensis and Leishmania (V.) braziliensis"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6127-1475","authenticated-orcid":false,"given":"Guilherme Ferreira","family":"Correia","sequence":"first","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruna Terci","family":"Fernandes","sequence":"additional","affiliation":[{"name":"Departamento de Farm\u00e1cia, Faculdade Cristo Rei, Corn\u00e9lio Proc\u00f3pio CEP 86300-000, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paulo Henrique Guilherme","family":"Borges","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabela Madeira de","family":"Castro","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3497-5362","authenticated-orcid":false,"given":"Guilherme","family":"Bartolomeu-Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thiago Fran\u00e7a","family":"Soares","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eloiza Teles","family":"Caldart","sequence":"additional","affiliation":[{"name":"Departamento de Medicina Veterin\u00e1ria Preventiva, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1500-3998","authenticated-orcid":false,"given":"Phileno","family":"Pinge-Filho","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Patologia Experimental, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivete","family":"Conchon-Costa","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Patologia Experimental, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vitor","family":"Takashiba","sequence":"additional","affiliation":[{"name":"Departamento de Cl\u00ednica Cir\u00fargica, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nayara Anitelli","family":"Artero","sequence":"additional","affiliation":[{"name":"Departamento de Cl\u00ednica Cir\u00fargica, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5213-2337","authenticated-orcid":false,"given":"Marco Aur\u00e9lio","family":"Fornazieri","sequence":"additional","affiliation":[{"name":"Departamento de Cl\u00ednica Cir\u00fargica, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7778-8404","authenticated-orcid":false,"given":"Wander Rog\u00e9rio","family":"Pavanelli","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Patologia Experimental, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6234-1589","authenticated-orcid":false,"given":"Eliandro Reis","family":"Tavares","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"},{"name":"Laborat\u00f3rio de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2241-8814","authenticated-orcid":false,"given":"Lucy Megumi","family":"Yamauchi","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"},{"name":"Laborat\u00f3rio de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9911-7369","authenticated-orcid":false,"given":"Celso Vataru","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"},{"name":"Laborat\u00f3rio de Inova\u00e7\u00e3o Tecnol\u00f3gica no Desenvolvimento de F\u00e1rmacos e Cosm\u00e9ticos, Universidade Estadual de Maring\u00e1, Maring\u00e1 CEP 87020-900, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1183-146X","authenticated-orcid":false,"given":"Sueli Fumie","family":"Yamada-Ogatta","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86057-970, Paran\u00e1, Brazil"},{"name":"Laborat\u00f3rio de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina CEP 86038-350, Paran\u00e1, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,2]]},"reference":[{"key":"ref_1","unstructured":"(2026, January 07). World Health Organization\u2014Leishmaniasis. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/leishmaniasis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1038\/s41572-025-00663-w","article-title":"Leishmaniasis","volume":"11","author":"Pareyn","year":"2025","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","unstructured":"(2026, January 07). Minist\u00e9rio da Sa\u00fade\u2014Leishmaniose Tegumentar, Available online: https:\/\/www.gov.br\/saude\/pt-br\/assuntos\/saude-de-a-a-z\/l\/lt."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1051\/parasite\/1990653111","article-title":"Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classification","volume":"65","author":"Rioux","year":"1990","journal-title":"Ann. Parasitol. Hum. Comp."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"111671","DOI":"10.1016\/j.biopha.2021.111671","article-title":"Host immune response against leishmaniasis and parasite persistence strategies: A review and assessment of recent research","volume":"139","author":"Elmahallawy","year":"2021","journal-title":"Biomed. Pharmacother."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"951","DOI":"10.1016\/S0140-6736(18)31204-2","article-title":"Leishmaniasis","volume":"392","author":"Burza","year":"2018","journal-title":"Lancet"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1587","DOI":"10.1038\/s42003-024-07278-z","article-title":"Evolutionary genomics of Leishmania braziliensis across the neotropical realm","volume":"7","author":"Heeren","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1590\/abd1806-4841.20175794","article-title":"Final diagnosis of 86 cases included in differential diagnosis of American tegumentary leishmaniasis in a Brazilian sample: A retrospective cross-sectional study","volume":"92","author":"Tirelli","year":"2017","journal-title":"An. Bras. Dermatol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"107820","DOI":"10.1016\/j.actatropica.2025.107820","article-title":"Emerging trends and Innovative strategies for the diagnosis of Leishmaniasis: A quantum leap from classical to modern era","volume":"270","author":"Nharwal","year":"2025","journal-title":"Acta Trop."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3165","DOI":"10.1007\/s00436-020-06841-x","article-title":"Granulomas in parasitic diseases: The good and the bad","volume":"119","author":"Giorgio","year":"2020","journal-title":"Parasitol. Res."},{"key":"ref_11","unstructured":"(2026, January 06). World Health Organization Ending the Neglect to Attain the Sustainable Development Goals: A Roadmap for Neglected Tropical Diseases 2021\u20132030. Available online: https:\/\/apps.who.int\/gb\/ebwha\/pdf_files\/EB150\/B150_10-en.pdf."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Otaguiri, E.S., Morguette, A.E.B., Morey, A.T., Tavares, E.R., Kerbauy, G., de Almeida Torres, R.S.L., Chaves J\u00fanior, M., Tognim, M.C.B., G\u00f3es, V.M., and Krieger, M.A. (2018). Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides. BMC Pregnancy Childbirth, 18.","DOI":"10.1186\/s12884-018-1774-5"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Tavares, E.R., de Lima, T.F., Bartolomeu-Gon\u00e7alves, G., de Castro, I.M., de Lima, D.G., Borges, P.H.G., Nakazato, G., Kobayashi, R.K.T., Venancio, E.J., and Tarley, C.R.T. (2023). Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection. Microorganisms, 11.","DOI":"10.3390\/microorganisms11112692"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Candia-Puma, M.A., Roque-Pumahuanca, B.M., Machaca-Luque, L.Y., Pola-Romero, L., Galdino, A.S., Machado-de-\u00c1vila, R.A., Cordeiro Giunchetti, R., Ferraz Coelho, E.A., Adaui, V., and Ch\u00e1vez-Fumagalli, M.A. (2025). Global Landscape of Molecular and Immunological Diagnostic Tests for Human Leishmaniasis: A Systematic Review and Meta-Analysis. Pathogens, 14.","DOI":"10.1101\/2025.09.12.25335647"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1007\/s00436-025-08503-2","article-title":"Exploring real-time PCR techniques for diagnosing leishmaniasis: Key insights from a systematic review","volume":"124","author":"Rihs","year":"2025","journal-title":"Parasitol. Res."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Tavares, E.R., Santos, V.P., Castro, I.M., Borges, P.H.G., Silva-Rodrigues, G., Olak, A.P.S., Bartolomeu-Gon\u00e7alves, G., Santos, J.P., Morey, A.T., and Ishida, K. (2025). Development of Melting-Curve-Based Real-Time PCR for Differentiating Medically Important Candida Species. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26199411"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/S0074-7696(08)61426-6","article-title":"Kinetoplast DNA in trypanosomid flagellates","volume":"99","author":"Simpson","year":"1986","journal-title":"Int. Rev. Cytol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/S0167-7012(02)00099-4","article-title":"Rapid differentiation of Old World Leishmania species by LightCycler polymerase chain reaction and melting curve analysis","volume":"51","author":"Nicolas","year":"2002","journal-title":"J. Microbiol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3892","DOI":"10.1128\/JCM.r00764-11","article-title":"Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples","volume":"49","author":"Weirather","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/1756-3305-5-15","article-title":"SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases","volume":"5","author":"Lins","year":"2012","journal-title":"Parasit. Vectors"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.mcp.2013.01.003","article-title":"Quantitative real time PCR assays for the detection of Leishmania (Viannia) braziliensis in animals and humans","volume":"27","author":"Otranto","year":"2013","journal-title":"Mol. Cell. Probes"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ceccarelli, M., Galluzzi, L., Migliazzo, A., and Magnani, M. (2014). Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high-resolution melt analysis targeting kinetoplast minicircle DNA. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0088845"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1186\/s13071-017-2181-x","article-title":"The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis","volume":"10","author":"Ceccarelli","year":"2017","journal-title":"Parasit. Vectors"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1128\/JCM.01954-16","article-title":"Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples to Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic","volume":"55","author":"Gomes","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Wu, Y., Tian, X., Song, N., Huang, M., Wu, Z., Li, S., Waterfield, N.R., Zhan, B., Wang, L., and Yang, G. (2020). Application of Quantitative PCR in the Diagnosis and Evaluating Treatment Efficacy of Leishmaniasis. Front. Cell. Infect. Microbiol., 10.","DOI":"10.3389\/fcimb.2020.581639"},{"key":"ref_26","first-page":"137","article-title":"High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran","volume":"13","author":"Fayaz","year":"2021","journal-title":"Iran. J. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e2022006","DOI":"10.4322\/biori.00062022","article-title":"Real-Time PCR Assay for detection and quantification of Leishmania: Standardization, positive control, validation, and intra-laboratory assay","volume":"7","author":"Soccola","year":"2023","journal-title":"Biotech. Res. Innov."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1128\/EC.1.4.495-502.2002","article-title":"Kinetoplast DNA network: Evolution of an improbable structure","volume":"1","author":"Lukes","year":"2002","journal-title":"Eukaryot. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/S0147-619X(80)90030-X","article-title":"Sequence heterogeneity in kinetoplast DNA: Reassociation kinetics","volume":"3","author":"Steinert","year":"1980","journal-title":"Plasmid"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3143","DOI":"10.1128\/JCM.01177-11","article-title":"Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2","volume":"49","author":"Steurer","year":"2011","journal-title":"J. Clin. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1186\/s13071-020-04261-5","article-title":"Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis","volume":"13","author":"Darzi","year":"2020","journal-title":"Parasit. Vectors"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Paun, A., and Grigg, M.E. (2024). Rapid detection, quantification and speciation of Leishmania using real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2. bioRxiv.","DOI":"10.1101\/2024.05.20.595045"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/0166-6851(95)00108-D","article-title":"Intergenic region typing (IRT): A rapid molecular approach to the characterization and evolution of Leishmania","volume":"73","author":"Cupolillo","year":"1995","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"106093","DOI":"10.1016\/j.actatropica.2021.106093","article-title":"Full nucleotide sequencing of ribosomal DNA internal transcribed spacer of Leishmania species causing cutaneous leishmaniasis in Brazil and its potential for species typing","volume":"223","author":"Espada","year":"2021","journal-title":"Acta Trop."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1111\/j.1550-7408.1982.tb05428.x","article-title":"Ribosomal DNA of Leishmania brasiliensis: Number of ribosomal copies and gene isolation","volume":"29","author":"Villalba","year":"1982","journal-title":"J. Protozool."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/S0166-6851(98)00009-7","article-title":"Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia","volume":"92","author":"Inga","year":"1998","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/S0166-6851(01)00310-3","article-title":"Genomic organization and functional characterization of the Leishmania major Friedlin ribosomal RNA gene locus","volume":"116","author":"Sunkin","year":"2001","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_38","unstructured":"World Health Organization WHO (2022, June 01). WHO Information for the Molecular Detection of Influenza Viruses. Available online: https:\/\/cdn.who.int\/media\/docs\/default-source\/influenza\/molecular-detention-of-influenza-viruses\/protocols_influenza_virus_detection_2024.pdf?sfvrsn=df7d268a_8."},{"key":"ref_39","first-page":"191","article-title":"Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco","volume":"20","author":"Silva","year":"1953","journal-title":"Folia Clin. Biol."},{"key":"ref_40","first-page":"93","article-title":"Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media","volume":"6","author":"Camargo","year":"1964","journal-title":"Rev. Inst. Med. Trop. Sao Paulo"},{"key":"ref_41","unstructured":"Thermo Fisher Scientific (2008). Creating Standard Curves with Genomic DNA or Plasmid DNA Templates for Use in Quantitative PCR; Application Note, ThermoFisher Scientific. Available online: https:\/\/assets.thermofisher.com\/TFS-Assets\/LSG\/Application-Notes\/cms_042486.pdf."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"634","DOI":"10.1093\/clinchem\/hvaf043","article-title":"MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines","volume":"71","author":"Bustin","year":"2025","journal-title":"Clin. Chem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"159","DOI":"10.2307\/2529310","article-title":"The measurement of observer agreement for categorical data","volume":"33","author":"Landis","year":"1977","journal-title":"Biometrics"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"qzae016","DOI":"10.1093\/gpbjnl\/qzae016","article-title":"RNase P: Beyond Precursor tRNA Processing","volume":"22","author":"Wang","year":"2024","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1099\/jmm.0.001868","article-title":"An end-point multiplex RT-PCR for SARS-CoV-2, Influenza A and B detection, including simultaneous RNAse P amplification: A timely tool for more accessible differential diagnosis","volume":"73","author":"Lopes","year":"2024","journal-title":"J. Med. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Souza, C.F., Santos, C.A.D., Bevilacqua, P.D., Andrade Filho, J.D., and Brazil, R.P. (2024). Molecular Detection of Leishmania spp. and Blood Source of Female Sand Flies in the Parque Estadual do Rio Doce and Municipality of Tim\u00f3teo, Minas Gerais, Brazil. Trop. Med. Infect. Dis., 9.","DOI":"10.3390\/tropicalmed9060133"}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/11\/1704\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T04:45:41Z","timestamp":1780548341000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/11\/1704"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,2]]},"references-count":46,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["diagnostics16111704"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics16111704","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,2]]}}}