Revista iberoamericana de micologia · 2011
Selection and optimization of PCR-based methods for the detection of Histoplasma capsulatum var. capsulatum.
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Sampaio Ivanete De Lima, Freire Ana Karla Lima, Ogusko Mauricio Morishi, Salem Júlia Ignez, De Souza João Vicente Braga
Аннотация
BACKGROUND: Current methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.
OBJECTIVES: Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).
METHODS: Three DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.
RESULTS: The DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.
CONCLUSIONS: In our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.
OBJECTIVES: Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).
METHODS: Three DNA extraction methods and three PCR methods were evaluated. We optimised the concentration of the components of this PCR reaction and determined its sensitivity and specificity using blood samples to which H. capsulatum had been added.
RESULTS: The DNA extraction method that yielded the highest-quality DNA used silica membranes (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany), and the amplification method with the best detection capacity used a target gene encoding a 100-kDa protein. Our optimisation of the PCR conditions indicated that the reaction works over a significant range of component concentrations; in addition, it was able to detect H. capsulatum better than traditional culture techniques, with a detection limit of only 10 pg of DNA.
CONCLUSIONS: In our experimental conditions, the PCR method selected in this work (instead of nested-PCR) is a tool sensitive enough for the diagnosis of histoplasmosis.