Rapid and Biosecure Diagnostic Test for Tuberculosis

• Published in: Cell biochemistry and biophysics 2013-03, Vol.65 (2), p.173-179
• Main Authors: Garberi, Juan, Labrador, Jorge, Garberi, Federico, Garberi, Juan Ezequiel, Peneipil, Julián, Garberi, Miguel, Scigliano, Luis, Troncoso, Alcides
• Format: Article
• Language: English
• Subjects: Bacilli; Bacillus; Biochemistry; Biological and Medical Physics; Biomedical and Life Sciences; Biophysics; Biotechnology; Cell Biology; Cell culture; Coefficient of variation; Contamination; Deoxyribonucleic acid; Development; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Fluorometry - methods; Humans; Inoculum; Life Sciences; Microbiological culture; Molecular biology; Molecular Diagnostic Techniques - instrumentation; Molecular Diagnostic Techniques - methods; Mycobacterium; Mycobacterium tuberculosis - genetics; Occupational safety; Original Paper; Pharmacology/Toxicology; Polymerase chain reaction; Polymerase Chain Reaction - methods; Reproducibility of Results; Risk reduction; Sensitivity and Specificity; Tuberculosis; Tuberculosis - diagnosis; Tuberculosis - microbiology
• ISSN: 1085-9195; 1559-0283
• DOI: 10.1007/s12013-012-9413-7

Early and rapid detection of the causative organism is necessary in tuberculosis. We present here an integrated and dedicated molecular biology system for tuberculosis diagnosis. One hundred and eighty-nine (189) biologic specimens from patients strongly suspected by clinical parameters of tuberculosis were studied by Ziehl–Neelsen staining, cultivation on a solid medium, and by a balanced heminested fluorometric PCR system (Orange G3TB) that preserves worker safety and produces a rather pure material free of potential inhibitors. DNA amplification was carried out in a low cost using a tuberculosis thermocycler-fluorometer. The double stranded DNA produced is fluorometrically detected. The whole reaction is carried out in one single tube which is never opened after adding the processed sample, thus minimizing the risk of cross contamination with amplicons. The assay is able to detect 30 bacilli/ml of sample having a 99.8 % inter-assay coefficient of variation. PCR was positive in 36 (18.9 %) tested samples (33 of them were smear-negative). In our study, it yields a preliminary overall sensitivity of 97.4 %. In addition, its overall specificity is 98.7 %. The total run time of the test is 4 h with two and a half real working hours. All PCR-positive samples also had a positive result by microbiological culture and clinical criteria. The results obtained showed that it could be a very useful tool to increase efficiency in detecting the tuberculosis disease in low bacillus inoculum samples. Furthermore, its low cost and friendly usage make it feasible to be used in regions with poor development.