title: 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(220) and 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 the M antigen 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(230) SCAR marker and the M antigen 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 the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283(220) marker can be used to detect and identify H. capsulatum in samples from different sources.
authors.affiliation: Departamentos de Microbiología-Parasitologíaa and Fisiología,b Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: María Guadalupe
authors.initials: MG
authors.lastname: Frías De León
authors.forename: Gabina
authors.initials: G
authors.lastname: Arenas López
authors.forename: Maria Lucia
authors.initials: ML
authors.lastname: Taylor
authors.forename: Gustavo
authors.initials: G
authors.lastname: Acosta Altamirano
authors.forename: María Del Rocío
authors.initials: Mdel R
authors.lastname: Reyes-Montes
mesh_terms: D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004783:Environmental Microbiology; D006658:Histoplasma / Q000235:genetics / Q000302:isolation & purification*; D006660:Histoplasmosis / Q000382:microbiology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal