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Diagn Microbiol Infect Dis. 2014 Jun;79(2):214-21. doi: 10.1016/j.diagmicrobio.2014.01.029. Epub 2014 Feb 20.

Development and validation of a quantitative real-time PCR assay for the early diagnosis of coccidioidomycosis.

Author information

1
Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
2
California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA; Department of Medicine, Stanford University, Stanford, CA, USA. Electronic address: Clemons@cimr.org.
3
California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA.
4
California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA; Department of Medicine, Stanford University, Stanford, CA, USA.

Abstract

A new real-time polymerase chain reaction (RT-PCR) assay based on a Coccidioides genus-specific molecular beacon probe was developed for the detection of coccidioidomycosis and validated with tissues from animal models and clinical samples. The assay showed high analytic reproducibility (r(2) > 0.99) and specificity for cultured strains (100%); the lower limit of detection was 1 fg of genomic DNA/μL of reaction. Fungal burdens in the organs of mice infected with Coccidioides posadasii strain Silveira were more accurately quantified by RT-PCR compared to colony-forming unit for all tissues. The RT-PCR assay was positive for 97.7% of spleen and 100% of liver or lung. Progression of infection in all organs was similar by both methods (P > 0.05). The sensitivity of the assay also was 100% for paraffin-embedded samples and samples from patients with positive cultures. Our RT-PCR assay is effective for the diagnosis and monitoring of Coccidioides infection, and its use also avoids the biohazard and time delay of identifying cultures in the clinical setting.

KEYWORDS:

Coccidioidomycosis; Diagnosis; RT-PCR

[Indexed for MEDLINE]

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