A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples
- 1 March 1994
- journal article
- research article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 32 (3), 672-678
- https://doi.org/10.1128/jcm.32.3.672-678.1994
Abstract
Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.Keywords
This publication has 11 references indexed in Scilit:
- PCR: how to kill unwanted DNA.1992
- Detection ofMycobacterium tuberculosisin Sputum Samples Using a Polymerase Chain ReactionAmerican Review of Respiratory Disease, 1991
- A simple method for diagnosing M. tuberculosis infection in clinical samples using PCRMolecular and Cellular Probes, 1991
- Diagnosis of tuberculosis by DNA amplification in clinical practice evaluationThe Lancet, 1991
- Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferaseAnalytical Biochemistry, 1991
- Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactionsGene, 1990
- Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 familyMolecular Microbiology, 1990
- IS6110, an IS-like element ofMycobacterium tuberculosiscomplexNucleic Acids Research, 1990
- RAPID DIAGNOSIS OF TUBERCULOSIS BY AMPLIFICATION OF MYCOBACTERIAL DNA IN CLINICAL SAMPLESThe Lancet, 1989
- SPUTUM DIGESTION AND DECONTAMINATION WITH N-ACETYL-L-CYSTEINE - SODIUM HYDROXIDE FOR CULTURE OF MYCOBACTERIAPublished by Elsevier ,1963