Diagnosis of Mycobacterium tuberculosis using molecular biology technology

ABSTRACT

Objective:To present an integrated molecular biology dedicated system for tuberculosis diagnosis.Methods: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 fluorometricPCR system (OrangeG3TB) 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.Results: The assay was able to detect30 bacillus per sample mL with99.8% interassay variation coefficient.PCR was positive in23 (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 of98.7%.Conclusions:Total run time of the test is4 h with2.5 real working time. AllPCR 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.