Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species
In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total o...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2012-08-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000500014&lng=en&tlng=en |
| _version_ | 1827845028328767488 |
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| author | Grazielle Cardoso da Graça Angela Cristina Volpini Gustavo Adolfo Sierra Romero Manoel Paes de Oliveira Neto Marcia Hueb Renato Porrozzi Mariana Côrtes Boité Elisa Cupolillo |
| author_facet | Grazielle Cardoso da Graça Angela Cristina Volpini Gustavo Adolfo Sierra Romero Manoel Paes de Oliveira Neto Marcia Hueb Renato Porrozzi Mariana Côrtes Boité Elisa Cupolillo |
| author_sort | Grazielle Cardoso da Graça |
| collection | DOAJ |
| description | In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol. |
| first_indexed | 2024-03-12T08:51:35Z |
| format | Article |
| id | doaj.art-1336adb6b13e4d94b5ffe225e2068aa5 |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T08:51:35Z |
| publishDate | 2012-08-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-1336adb6b13e4d94b5ffe225e2068aa52023-09-02T16:17:52ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602012-08-01107566467410.1590/S0074-02762012000500014S0074-02762012000500014Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite speciesGrazielle Cardoso da Graça0Angela Cristina Volpini1Gustavo Adolfo Sierra Romero2Manoel Paes de Oliveira Neto3Marcia Hueb4Renato Porrozzi5Mariana Côrtes Boité6Elisa Cupolillo7Fundação Oswaldo CruzFundação Oswaldo CruzUniversidade de BrasíliaFundação Oswaldo CruzUniversidade Federal de Mato GrossoFundação Oswaldo CruzFundação Oswaldo CruzFundação Oswaldo CruzIn this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000500014&lng=en&tlng=enleishmaniasismolecular diagnosisspecies identificationpolymerase chain reactionRFLPshsp70 |
| spellingShingle | Grazielle Cardoso da Graça Angela Cristina Volpini Gustavo Adolfo Sierra Romero Manoel Paes de Oliveira Neto Marcia Hueb Renato Porrozzi Mariana Côrtes Boité Elisa Cupolillo Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species Memorias do Instituto Oswaldo Cruz leishmaniasis molecular diagnosis species identification polymerase chain reaction RFLPs hsp70 |
| title | Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species |
| title_full | Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species |
| title_fullStr | Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species |
| title_full_unstemmed | Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species |
| title_short | Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species |
| title_sort | development and validation of pcr based assays for diagnosis of american cutaneous leishmaniasis and identificatio nof the parasite species |
| topic | leishmaniasis molecular diagnosis species identification polymerase chain reaction RFLPs hsp70 |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762012000500014&lng=en&tlng=en |
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