Polymerase chain reaction for diagnosis of M. tuberculosis: comparison of simple boiling and a conventional method for DNA extraction.
Although the polymerase chain reaction (PCR) has been used increasingly for rapid diagnosis of tuberculosis (TB) in clinical specimens, no consensus exists regarding DNA extraction protocols. We compared a simple boiling method to a conventional, labor-intensive chemical method using lysozyme and silica particles. The boiling method was performed in less than 30 min; the chemical method required at least 6 h. A total of 82 clinical specimens (mostly respiratory) from 77 patients were obtained after routine processing from the microbiology laboratory. After DNA extraction by each method, PCR was performed to detect the 123-bp segment of IS6110, and results were compared to culture. Of 20 culture-positive specimens, 17 (85%) and 12 (60%) were positive by boiling and chemical methods, respectively. Of 62 culture-negative specimens, 61 (98%) and 57 (92%) were negative by boiling and chemical methods, respectively. The sensitivity was 100 and 92% for the boiling and chemical methods, respectively, for smears containing more than rare AFB. Our results suggest that boiling method of DNA extraction is more sensitive and no less specific than a conventional chemical method. Larger studies including a variety of clinical specimens are necessary to standardize the optimal conditions of PCR for diagnosis of M. tuberculosis.
Afghani
B
B
Pediatric Infectious Diseases, University of California, Irvine, California, 90806, USA.
Stutman
H R
HR
D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*
D004269:DNA, Bacterial