Evaluación de cuatro métodos de extracción del ADN de Histoplasma capsulatum y su uso en reacciones de PCR
Histoplasma capsulatum is a fungus endemic from Cuba and it is the etiologic agent of histoplasmosis. This mycosis appears as epidemic outbreaks and affects people regardless their immunological status. Diagnosis of histoplasmosis is based on microbiological, histopathological and serological methods, which show moderated sensitivity or are time-consuming. Rapid and efficient molecular tools are highly demanding. Four DNA extraction methods (enzymatic, mechanic, and two chemistry: guanidine tiocianate and TX-100) were evaluated from a H. capsulatum strain in the mold phase. DNA concentration, purity, integrity and yield were estimated by spectrophotometry and agarose gel electrophoresis. Two PCR techniques were tested using specific and arbitrary primers. The best DNA extraction method results were obtained with the enzymatic procedure. Sixteen different Cuban H. capsulatum strains were confirmed by the PCR standardized with specific primers. Different sequences were amplified using the arbitrary primer. The enzymatic protocol is the best option for obtaining a good quality DNA for molecular analysis. This work has created a platform a faster and reliable diagnosis of H. capsulatum in Cuba, and provides the ability to identify strains with genetically related structure of pathogenicity and virulence markers useful as vaccine immunogen.