Development of specific sequence-characterized amplified region markers for detecting Histoplasma capsulatum in clinical and environmental samples

Abstract

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283 220and 1281-1283 230. The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283 220 and 1281-1283 230 as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and theMantigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283 220 SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283 230SCAR marker and theMantigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than theMantigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283220 marker can be used to detect and identify H. capsulatum in samples from different sources. Copyright © 2012, American Society for Microbiology. All Rights Reserved.