Sampaio Ivanete De Lima, Freire Ana Karla Lima, Ogusko Mauricio Morishi, Salem Júlia Ignez, De Souza João Vicente Braga
College of Health Sciences, Universidade do Estado do Amazonas, Manaus, AM, Brazil.
Rev Iberoam Micol. 2012 Jan-Mar;29(1):34-9. doi: 10.1016/j.riam.2011.03.008. Epub 2011 Apr 20.
Current methods for the laboratory diagnosis of histoplasmosis are problematic in terms of their sensitivity, specificity and runtime.
Thus, in this study, we sought to select and optimize methods for the detection of Histoplasma capsulatum var. capsulatum by polymerase chain reaction (PCR).
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.
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.
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.
目前组织胞浆菌病的实验室诊断方法在敏感性、特异性和检测时长方面存在问题。
因此，在本研究中，我们试图选择并优化通过聚合酶链反应（PCR）检测荚膜组织胞浆菌荚膜变种的方法。
评估了三种DNA提取方法和三种PCR方法。我们优化了该PCR反应组分的浓度，并使用添加了荚膜组织胞浆菌的血样确定其敏感性和特异性。
产生最高质量DNA的DNA提取方法使用硅胶膜（德国希尔德市Qiagen公司的DNeasy Blood & Tissue Kit），具有最佳检测能力的扩增方法使用编码100 kDa蛋白质的靶基因。我们对PCR条件的优化表明，该反应在相当大的组分浓度范围内有效；此外，它比传统培养技术能更好地检测荚膜组织胞浆菌，检测限仅为10 pg DNA。
在我们的实验条件下，本研究中选择的PCR方法（而非巢式PCR）是一种对组织胞浆菌病诊断足够敏感的工具。
