Optimization and applications of nested - Polymerase Chain reaction for rapid detection of Histoplasma capsulatum based on hpc100 gene

Abstract

This study aims to optimize and apply nested – polymerase chain reaction for rapid detection of Histoplasma capsulatum based on hpc100 gene. A nested PCR was developed to detect Histoplasma capsulatum in two reactions based on Hcp100 gene of H. capsulatum. The results showed that the nested –PCR method with HcI, HcII primers (PCR1) and HcIII, HcIV (PCR2)
allows simple, rapid and reliable detection to identify H. capsulatum with 2,0mM Mg2+ and annealing temperature 50oC for the frst round and 1,5mM Mg2+ and 65oC for the second round PCR. The minimum PCR detection limit was 10fg of H. capsulatum plasmid DNA. In order to evaluate the developed nested PCR, 96 DNA samples of bronchoalveolar lavage fluid were examined for detection of H. capsulatum infections. H. capsulatum infection did not reveal in bronchoalveolar lavage fluid. This result were similar to those of culture on Sabouraud dextrose agar. This assay provides a rapid and reliable solution compared to conventional methods to diagnose H. capsulatum.