Garberi Juan, Labrador Jorge, Garberi Federico, Garberi Juan Ezequiel, Peneipil Julian, Garberi Miguel, Scigliano Luis, Troncoso Alcides
Laboratory of Molecular Biology and Pathology, School of Medicine, Buenos Aires University, Argentina.
Asian Pac J Trop Biomed. 2011 Apr;1(2):89-93. doi: 10.1016/S2221-1691(11)60002-6.
To present an integrated molecular biology dedicated system for tuberculosis diagnosis.
One hundred and five sputum specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl-Neelsen staining, by cultivation on solid medium and by a balanced heminested fluorometric PCR system (Orange G3TB) that could preserve worker safety and produce a rather pure material free of potential inhibitors. DNA amplification was performed in a low cost tuberculosis termocycler-fluorometer. Produced double stranded DNA was flurometrically detected. The whole reaction was conducted in one single tube which would not be opened after adding the processed sample in order to minimize the risk of cross contamination with amplicons.
The assay was able to detect 30 bacillus per sample mL with 99.8% interassay variation coefficient. PCR was positive in 23 (21.9%) tested samples (21 of them were smear negative). In our study it showed a preliminary sensitivity of 94.5% for sputum and an overall specificity of 98.7%.
Total run time of the test is 4 h with 2.5 real working time. All PCR positive samples are also positive by microbiological culture and clinical criteria. Results show that it could be a very useful tool to increase detection efficiency of tuberculosis disease in low bacilus load samples. Furthermore, its low cost and friendly using make it feasible to run in poor regions.
介绍一种用于结核病诊断的集成分子生物学专用系统。
对105份临床高度怀疑为结核病患者的痰标本进行萋-尼氏染色、固体培养基培养，以及使用一种能保障工作人员安全且能产生无潜在抑制剂的较纯物质的平衡半巢式荧光定量PCR系统（橙色G3TB）进行研究。DNA扩增在低成本的结核热循环荧光仪中进行。对产生的双链DNA进行荧光检测。整个反应在一个单管中进行，加入处理后的样本后不再打开，以尽量降低与扩增子交叉污染的风险。
该检测方法能够检测出每毫升样本中有30条杆菌，批间变异系数为99.8%。在23份（21.9%）检测样本中PCR呈阳性（其中21份涂片阴性）。在我们的研究中，其对痰标本的初步敏感性为94.5%，总体特异性为98.7%。
该检测的总运行时间为4小时，实际有效工作时间为2.5小时。所有PCR阳性样本经微生物培养和临床标准检测也呈阳性。结果表明，它可能是提高低杆菌载量样本中结核病检测效率的非常有用的工具。此外，其低成本和使用方便使其在贫困地区运行成为可能。
