Design and evaluation of an AFLP molecular marker for the detection of Coccidioides spp. in biological samples
Por:
del Rocio Reyes-Montes, Maria, Guadalupe Frias-De-Leon, Maria, Victoriano-Pastelin, Isai, Acosta-Altamirano, Gustavo, Duarte-Escalante, Esperanza
Publicada:
1 sep 2019
Resumen:
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 (R) -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. (C) 2019
Sociedade Brasileira de Infectologia. Published by Elsevier Espana,
S.L.U.
Filiaciones:
del Rocio Reyes-Montes, Maria:
UNAM, Dept Microbiol & Parasitol, Lab Micol Mol, Fac Med, Ciudad De Mexico, Mexico
Guadalupe Frias-De-Leon, Maria:
Hosp Reg Alta Especialidad Ixtapaluca, Direcc Invest, Ixtapaluca, Mexico
Victoriano-Pastelin, Isai:
UNAM, Dept Microbiol & Parasitol, Lab Micol Mol, Fac Med, Ciudad De Mexico, Mexico
Acosta-Altamirano, Gustavo:
Hosp Reg Alta Especialidad Ixtapaluca, Direcc Invest, Ixtapaluca, Mexico
Duarte-Escalante, Esperanza:
UNAM, Dept Microbiol & Parasitol, Lab Micol Mol, Fac Med, Ciudad De Mexico, Mexico
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