ArticlePloS one2014
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.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers, 1 of them a synthesis that pooled it.
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Who cites it
50 citing papers in PubMed, 1 synthesis or guideline pooled it, 99 citations in OpenAlex.
- Sensitivity of PCR in conjunctival swab samples for the diagnosis of canine visceral leishmaniasis: a systematic review and meta-analysis.Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria · 2023Pooled it
- Molecular Diagnosis of Leishmaniasis: Development of a qPCR Assay for Genus Detection and Differentiation ofDiagnostics (Basel, Switzerland) · 2026Article
- Zoonotic leishmaniasis in China: Current status and challenges to elimination.PLoS neglected tropical diseases · 2026Review
- From Skin to Sequence: Pan-species Detection and Speciation of Leishmania.The Journal of infectious diseases · 2026Article
- Advances and Challenges in the Diagnosis of Leishmaniasis.Molecular diagnosis & therapy · 2025Review
- Occurrence of Leishmania spp. in Roadkilled Wild Mammals on Highways in the State of São Paulo, Brazil.Revista da Sociedade Brasileira de Medicina Tropical · 2025Article
- Instrument-Free Point-of-Care Diagnostic for Leishmania Parasites.Diagnostics (Basel, Switzerland) · 2024Article
- Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis.Scientific reports · 2023Article
- HRM Accuracy and Limitations as a Species Typing Tool forInternational journal of molecular sciences · 2023Article
- High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping.Parasites & vectors · 2023Article
- Evaluation of Molecular Methods to Identify Chagas Disease and Leishmaniasis in Blood Donation Candidates in Two Brazilian Centers.Pathogens (Basel, Switzerland) · 2023Article
- Novel CRISPR-based detection ofFrontiers in microbiology · 2022Article
- Equine infection with Leishmania spp. in Costa Rica: Study of five cases.Veterinary medicine and science · 2021Article
- Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity.Pathogens (Basel, Switzerland) · 2021Article
- Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection ofPathogens (Basel, Switzerland) · 2021Article
- In Vitro Reduced Susceptibility to Pentavalent Antimonials of aMicroorganisms · 2021Article
- Nanodiagnostics in leishmaniasis: A new frontiers for early elimination.Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology · 2021Review
- High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran.Iranian journal of microbiology · 2021Article
- Article
- Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old WorldMicroorganisms · 2020Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples, must be taken into account in quantitative PCR-based applications; however, it might also be used to differentiate between Leishmania subgenera.
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