Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.
OBJECTIVE
To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples.


METHODS
Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure.


RESULTS
The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative.


CONCLUSIONS
Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory.
D001431:Bacteriological Techniques; D015139:Blotting, Southern; D004587:Electrophoresis, Agar Gel; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D019914:Taq Polymerase; D014376:Tuberculosis / Q000175:diagnosis*
D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D019914:Taq Polymerase