Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species
The parasite protozoan <i>Leishmania</i>, the causative agent of leishmaniasis, includes two subgenera of medical interest: <i>Leishmania</i> (<i>Leishmania</i>) and <i>Leishmania</i> (<i>Viannia</i>). Parasite species detection and charact...
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MDPI AG
2020-12-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/12/2006 |
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| author | Marcello Ceccarelli Gloria Buffi Aurora Diotallevi Francesca Andreoni Daniela Bencardino Fabrizio Vitale Germano Castelli Federica Bruno Mauro Magnani Luca Galluzzi |
| author_facet | Marcello Ceccarelli Gloria Buffi Aurora Diotallevi Francesca Andreoni Daniela Bencardino Fabrizio Vitale Germano Castelli Federica Bruno Mauro Magnani Luca Galluzzi |
| author_sort | Marcello Ceccarelli |
| collection | DOAJ |
| description | The parasite protozoan <i>Leishmania</i>, the causative agent of leishmaniasis, includes two subgenera of medical interest: <i>Leishmania</i> (<i>Leishmania</i>) and <i>Leishmania</i> (<i>Viannia</i>). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the <i>Leishmania</i> genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish <i>L.</i> (<i>L</i>.) <i>infantum</i> and <i>L.</i> (<i>L</i>.) <i>amazonensis</i> from <i>L. Viannia</i> species. Successively, this assay has been integrated with other qPCR assays, to differentiate <i>L.</i> (<i>L</i>.) <i>infantum</i>, <i>L.</i> (<i>L</i>.) <i>amazonensis</i> and <i>L.</i> (<i>L</i>.) <i>mexicana.</i> In this work, we tested the applicability of our qPCR-ML assay on <i>L.</i> (<i>L</i>.) <i>donovani</i>, <i>L.</i> (<i>L</i>.) <i>major</i>, <i>L.</i> (<i>L</i>.) <i>tropica</i> and <i>L.</i> (<i>L</i>.) <i>aethiopica</i>, showing that the qPCR-ML assay can also amplify Old World species, different from <i>L.</i> (<i>L</i>.) <i>infantum</i>, with good quantification limits (1 × 10<sup>−4</sup>–1 × 10<sup>−6</sup> ng/pcr tube). Moreover, we evaluated 11 <i>L.</i> (<i>L</i>.) <i>infantum</i> strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-<i>Leishmania</i> assay. |
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| issn | 2076-2607 |
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| spelling | doaj.art-70989dee3c654e8fbaa3248ed0977dc42023-11-21T01:00:29ZengMDPI AGMicroorganisms2076-26072020-12-01812200610.3390/microorganisms8122006Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> SpeciesMarcello Ceccarelli0Gloria Buffi1Aurora Diotallevi2Francesca Andreoni3Daniela Bencardino4Fabrizio Vitale5Germano Castelli6Federica Bruno7Mauro Magnani8Luca Galluzzi9Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyNational Reference Center for Leishmaniasis (C.Re.Na.L.), Istituto Zooprofilattico Sperimentale della Sicilia, 90129 Palermo, PU, ItalyNational Reference Center for Leishmaniasis (C.Re.Na.L.), Istituto Zooprofilattico Sperimentale della Sicilia, 90129 Palermo, PU, ItalyNational Reference Center for Leishmaniasis (C.Re.Na.L.), Istituto Zooprofilattico Sperimentale della Sicilia, 90129 Palermo, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyDepartment of Biomolecular Sciences, University of Urbino “Carlo Bo”, 61029 Urbino, PU, ItalyThe parasite protozoan <i>Leishmania</i>, the causative agent of leishmaniasis, includes two subgenera of medical interest: <i>Leishmania</i> (<i>Leishmania</i>) and <i>Leishmania</i> (<i>Viannia</i>). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the <i>Leishmania</i> genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish <i>L.</i> (<i>L</i>.) <i>infantum</i> and <i>L.</i> (<i>L</i>.) <i>amazonensis</i> from <i>L. Viannia</i> species. Successively, this assay has been integrated with other qPCR assays, to differentiate <i>L.</i> (<i>L</i>.) <i>infantum</i>, <i>L.</i> (<i>L</i>.) <i>amazonensis</i> and <i>L.</i> (<i>L</i>.) <i>mexicana.</i> In this work, we tested the applicability of our qPCR-ML assay on <i>L.</i> (<i>L</i>.) <i>donovani</i>, <i>L.</i> (<i>L</i>.) <i>major</i>, <i>L.</i> (<i>L</i>.) <i>tropica</i> and <i>L.</i> (<i>L</i>.) <i>aethiopica</i>, showing that the qPCR-ML assay can also amplify Old World species, different from <i>L.</i> (<i>L</i>.) <i>infantum</i>, with good quantification limits (1 × 10<sup>−4</sup>–1 × 10<sup>−6</sup> ng/pcr tube). Moreover, we evaluated 11 <i>L.</i> (<i>L</i>.) <i>infantum</i> strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-<i>Leishmania</i> assay.https://www.mdpi.com/2076-2607/8/12/2006<i>Leishmania</i><i>Viannia</i>qPCROld Worldhigh resolution meltingkDNA |
| spellingShingle | Marcello Ceccarelli Gloria Buffi Aurora Diotallevi Francesca Andreoni Daniela Bencardino Fabrizio Vitale Germano Castelli Federica Bruno Mauro Magnani Luca Galluzzi Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species Microorganisms <i>Leishmania</i> <i>Viannia</i> qPCR Old World high resolution melting kDNA |
| title | Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species |
| title_full | Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species |
| title_fullStr | Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species |
| title_full_unstemmed | Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species |
| title_short | Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World <i>Leishmania</i> Species |
| title_sort | evaluation of a kdna based qpcr assay for the detection and quantification of old world i leishmania i species |
| topic | <i>Leishmania</i> <i>Viannia</i> qPCR Old World high resolution melting kDNA |
| url | https://www.mdpi.com/2076-2607/8/12/2006 |
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