Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

Abstract

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.