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Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples

At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and s...

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Autores principales: Reyes-Montes, María del Rocío, Frías-De-León, María Guadalupe, Victoriano-Pastelín, Isai, Acosta-Altamirano, Gustavo, Duarte-Escalante, Esperanza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9428003/
https://www.ncbi.nlm.nih.gov/pubmed/31539511
http://dx.doi.org/10.1016/j.bjid.2019.08.002
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author Reyes-Montes, María del Rocío
Frías-De-León, María Guadalupe
Victoriano-Pastelín, Isai
Acosta-Altamirano, Gustavo
Duarte-Escalante, Esperanza
author_facet Reyes-Montes, María del Rocío
Frías-De-León, María Guadalupe
Victoriano-Pastelín, Isai
Acosta-Altamirano, Gustavo
Duarte-Escalante, Esperanza
author_sort Reyes-Montes, María del Rocío
collection PubMed
description At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and sensitivity in fungal DNA-spiked blood and sputum samples, and compare it with previously described molecular markers. Specific amplified fragment length polymorphism (AFLP) amplicons for Coccidioides immitis and Coccidioides posadasii were cloned into the vector pGEM® -T Easy vector and sequenced to develop a SCAR marker. Oligonucleotides were designed to identify Coccidioides spp. by polymerase chain reaction (PCR), and the specificity and sensitivity of these oligonucleotides were tested with the DNA from related pathogens. The specificity and sensitivity of the SCAR marker was evaluated with blood and sputum samples spiked with Coccidioides DNA and compared with other previously described markers (621, GAC2, and Ag2/PRA). In addition, the conditions for its use were established using biological samples. A specific marker named SCAR(300) was obtained to identify Coccidioides spp. that exhibited good sensitivity and specificity. The results showed that all of the markers tested in this study can identify Coccidioides spp. However, the SCAR(300) and 621 markers were the most sensitive, whereas the SCAR(300) marker was the most specific. Thus, the SCAR(300) marker is a useful tool to identify Coccidioides spp.
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spelling pubmed-94280032022-09-01 Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples Reyes-Montes, María del Rocío Frías-De-León, María Guadalupe Victoriano-Pastelín, Isai Acosta-Altamirano, Gustavo Duarte-Escalante, Esperanza Braz J Infect Dis Original Article At present, there is no standardized marker that is routinely used in clinical laboratories to diagnose coccidioidomycosis. Thus, the goals of this study were to obtain a sequence characterized amplified region (SCAR) marker for the identification of Coccidioides spp., evaluate its specificity and sensitivity in fungal DNA-spiked blood and sputum samples, and compare it with previously described molecular markers. Specific amplified fragment length polymorphism (AFLP) amplicons for Coccidioides immitis and Coccidioides posadasii were cloned into the vector pGEM® -T Easy vector and sequenced to develop a SCAR marker. Oligonucleotides were designed to identify Coccidioides spp. by polymerase chain reaction (PCR), and the specificity and sensitivity of these oligonucleotides were tested with the DNA from related pathogens. The specificity and sensitivity of the SCAR marker was evaluated with blood and sputum samples spiked with Coccidioides DNA and compared with other previously described markers (621, GAC2, and Ag2/PRA). In addition, the conditions for its use were established using biological samples. A specific marker named SCAR(300) was obtained to identify Coccidioides spp. that exhibited good sensitivity and specificity. The results showed that all of the markers tested in this study can identify Coccidioides spp. However, the SCAR(300) and 621 markers were the most sensitive, whereas the SCAR(300) marker was the most specific. Thus, the SCAR(300) marker is a useful tool to identify Coccidioides spp. Elsevier 2019-09-17 /pmc/articles/PMC9428003/ /pubmed/31539511 http://dx.doi.org/10.1016/j.bjid.2019.08.002 Text en © 2019 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Reyes-Montes, María del Rocío
Frías-De-León, María Guadalupe
Victoriano-Pastelín, Isai
Acosta-Altamirano, Gustavo
Duarte-Escalante, Esperanza
Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title_full Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title_fullStr Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title_full_unstemmed Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title_short Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
title_sort design and evaluation of an aflp molecular marker for the detection of coccidioides spp. in biological samples
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9428003/
https://www.ncbi.nlm.nih.gov/pubmed/31539511
http://dx.doi.org/10.1016/j.bjid.2019.08.002
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