Real-time PCR assay for detection and differentiation of < i > Coccidioides immitis < /i > and < i > Coccidioides posadasii < /i > from culture and clinical specimens

by Sudha Chaturvedi, Tanya R. Victor, Anuradha Marathe, Ketevan Sidamonidze, Kelly L. Crucillo, Vishnu Chaturvedi Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agentsCoccidioides immitis andC.posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation ofC.immitis andC.posadasii from culture and clinical specimens. TheRRA2 gene encoding proline-rich antigen 2, specific forCoccidioides genus, was the source for the first set of primers and probe.Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the correspondingC.posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies ofC.immitis orC.posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens asC.posadasii and 30 isolates asC.immitis from human coccidioidomycosis cases, while all eight primary...
Source: PLoS Neglected Tropical Diseases - Category: Tropical Medicine Authors: Source Type: research