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°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%o). This PCR assay incorporates several innovations that make application of this new technology feasible in clinical microbiology laboratories. A recent World Health Organization survey estimated that in 1990 there were 8 million new cases of tuberculosis worldwide, with 2.9 million deaths directly attributable to the disease (10). Renewed interest in tuberculosis in the United States was prompted by recent increases in the number of new cases and by outbreaks of multiple-drugresistant strains. The lack of simple, rapid, and reliable tests

Comparison of two different PCR detection methods. Application to the diagnosis of pulmonary tuberculosis

“…For example, the results of one multicenter study, in which one of these techniques was evaluated in six different laboratories, show a sensitivity of 91.4% for samples with positive staining and 60.9%) for those with negative staining, with a specificity Reamplification techniques produce an increase in sensitivity and have been used to diagnose extrapulmonary tuberculosis (26), but they increase the possibility of cross-reactions since each PCR tube may contain up to 10l2 copies of an amplicon, and the smallest aerosolized drop may contain as many as lo5 copies of that amplicon. Since each amplicon can serve as a template for subsequent PCRs, considerable effort has been devoted to devising ways to limit amplicon carryover (19). The sensitivity of nested PCR was pointed out by Pierre et al., who detected the presence of DNA in 18/18 samples with more than 100 DFU/ml in culture and with positive staining, while obtaining positives results in 1/6 samples with less than 100 CFU/ml in culture with a specificity of 91% (24).…”

Identification of Acid Fast Bacteria From Caseous Lesions in Cattle in Sudan

“…The IS6110 was amplified using the primers TB41 and TB42 (Eisenach et al, 1990;Clarridge et al, 1993). The method for DNA isolation and amplification was done as described by Nolte et al (1993).…”

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

“…With respect to DNA extraction, boiling of the samples has been shown to be a simpler and economical method for releasing DNA from bacteria [9]. It also appears to be the most attractive and e⁄cient method for the extraction of S. suis DNA due to its rapidity and simplicity, and it compared unequivocally with other DNA extraction methods [5,9] that we evaluated (data not shown).…”