title: 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 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.
  
  
  RESULTS
  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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Molecular Biology and Pathology, School of Medicine, Buenos Aires University, Argentina.
authors.forename: Juan
authors.initials: J
authors.lastname: Garberi
authors.forename: Jorge
authors.initials: J
authors.lastname: Labrador
authors.forename: Federico
authors.initials: F
authors.lastname: Garberi
authors.forename: Juan Ezequiel
authors.initials: JE
authors.lastname: Garberi
authors.forename: Julian
authors.initials: J
authors.lastname: Peneipil
authors.forename: Miguel
authors.initials: M
authors.lastname: Garberi
authors.forename: Luis
authors.initials: L
authors.lastname: Scigliano
authors.forename: Alcides
authors.initials: A
authors.lastname: Troncoso
mesh_terms: D003955:Diagnostic Tests, Routine / Q000379:methods*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
keywords: Low cost; Microbiological culture; Molecular biology technology; Molecular diagnosis; Mycobacterium tuberculosis; Real-time PCR; Sputum; Sputum sample; Tuberculosis