Rapid detection of mycobacterial DNA in clinical samples by multiplex PCR

Triplex-polymerase chain reaction technique (PCR) was developed for the detection and identification of mycobacterial DNA sequences in uncultured clinical samples. A 123 bp fragment corresponding to a specific Mycobacterium tuberculosis sequence complex, a 383 bp DNA fragment encoding for part of the 65 kD mycobacterial surface antigen, and a 268 bp fragment of the human beta-globin gene to demonstrate the presence of suitable DNA were amplified by triplex PCR. To demonstrate the applicability of this method, 206 alcohol-fixed, paraffin-embedded sputum samples from 47 patients with culture-proven tuberculosis were investigated. Of 206 samples, 157 were PCR positive, resulting in correct diagnosis of tuberculosis in 46 of 47 (97.8%) patients. Furthermore, 165 alcohol-fixed, auramin-stained sputum smears were examined in a blind trial. Triplex PCR revealed tuberculosis in 20 of 21 samples from patients with tuberculosis. In comparison, cultures were positive in 20 of 21 samples, and acid-fast organisms were found by microscopy in 18 of 21 samples. We conclude that triplex PCR is a rapid and sensitive technique for the detection of mycobacterial DNA in uncultured clinical samples and offers equivalent sensitivity (95.2%) and specificity (98.6%) as do culture methods.