Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system

Abstract

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.