Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens.
BACKGROUND
The diagnosis of pulmonary tuberculosis is still a major challenge. Using a polymerase chain reaction (PCR), one can detect Mycobacterium tuberculosis in clinical samples within a few hours. However, single gene targets may result in false negativity due to the absence of target DNA in some M. tuberculosis isolates. The objective of this study was to develop and evaluate a multiplex PCR (M-PCR) using IS6110 and devR primers for the detection of M. tuberculosis in sputum samples.


METHODS
Sputum samples were collected from: (1) 200 confirmed cases of tuberculosis; (2) 100 suspected cases of tuberculosis diagnosed on the basis of clinical and radiological findings; (3) 200 non-tubercular patients suffering from respiratory diseases other than tuberculosis, in whom tuberculosis had been excluded. All 500 sputum samples were subjected to PCR using IS6110 primers, and M-PCR using IS6110 and devR primers; results were compared with conventional techniques.


RESULTS
It was found that M-PCR was 97.5% successful in detecting the presence of tuberculosis in the confirmed tuberculosis group as compared to 84.5% by IS6110-based PCR. In the suspected tuberculosis group, M-PCR could detect 45% of cases as compared to 40% by IS6110-based PCR. Overall, the specificities of both the PCR and M-PCR were found to be 96.5%.


CONCLUSIONS
This study demonstrated that the M-PCR assay is more sensitive than the IS6110-based PCR for the detection of M. tuberculosis in sputum specimens and could be applied in situations of highly suspected tuberculosis when all others tests including IS6110 PCR are negative.
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Shiv Kumar
SK
Sharma
Sunil
S
Sethi
Meera
M
Sharma
Sandeep K
SK
Meharwal
V M
VM
Katoch
S K
SK
Jindal
Rupinder
R
Tewari
D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial