Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates
ABSTRACT Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.
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Instituto de Medicina Tropical de São Paulo
2019
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oai:scielo:S0036-466520190050002192019-08-05Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolatesReyes-Montes,María del RocíoAcosta-Altamirano,GustavoDuarte-Escalante,EsperanzaSalazar,Eduardo GarcíaMartínez-Herrera,ErickArenas,RobertoGonzález,GloriaFrías-De-León,María Guadalupe Candida glabrata complex Candida bracarensis Candida nivariensis Multiplex PCR Candidiasis Mexico ABSTRACT Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.info:eu-repo/semantics/openAccessInstituto de Medicina Tropical de São PauloRevista do Instituto de Medicina Tropical de São Paulo v.61 20192019-01-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652019005000219en10.1590/s1678-9946201961037 |
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Reyes-Montes,María del Rocío Acosta-Altamirano,Gustavo Duarte-Escalante,Esperanza Salazar,Eduardo García Martínez-Herrera,Erick Arenas,Roberto González,Gloria Frías-De-León,María Guadalupe |
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Reyes-Montes,María del Rocío Acosta-Altamirano,Gustavo Duarte-Escalante,Esperanza Salazar,Eduardo García Martínez-Herrera,Erick Arenas,Roberto González,Gloria Frías-De-León,María Guadalupe Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
author_facet |
Reyes-Montes,María del Rocío Acosta-Altamirano,Gustavo Duarte-Escalante,Esperanza Salazar,Eduardo García Martínez-Herrera,Erick Arenas,Roberto González,Gloria Frías-De-León,María Guadalupe |
author_sort |
Reyes-Montes,María del Rocío |
title |
Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
title_short |
Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
title_full |
Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
title_fullStr |
Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
title_full_unstemmed |
Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates |
title_sort |
usefulness of a multiplex pcr for the rapid identification of candida glabrata species complex in mexican clinical isolates |
description |
ABSTRACT Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species. |
publisher |
Instituto de Medicina Tropical de São Paulo |
publishDate |
2019 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652019005000219 |
work_keys_str_mv |
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