Polymerase chain reaction targeting of single- and multiple-copy genes of mycobacteria in the diagnosis of tuberculosis.
Tuberculosis is a worldwide health problem of major concern. Direct detection of Mycobacterium tuberculosis in clinical specimens is the best approach to identify the causative agent. Identification of M. tuberculosis by culture is the gold standard, but the results are delayed for days to weeks. Microscopic examination of smears is quite fast, but a sample must contain a large number of M. tuberculosis (> 7.5 x 10(3) organisms/ml) for smear positivity. To diagnose tuberculosis specifically within 1 d of receiving clinical specimens, we have established multiplex polymerase chain reaction (MPCR) assays by targeting DNA fragments in the genes present in single or multiple copies in the M. tuberculosis genome. The MPCR results are available within a few hours, and the detection limit for different targets ranges between 2 and 200 organisms. The targets selected in the MPCRs could differentiate between M. tuberculosis complex and other mycobacteria from culture-grown specimens. The MPCRs were compared with microscopic examination of smears and culture in the diagnosis of tuberculosis. Coded sputum samples from suspected tuberculosis patients were tested. The codes were broken at the end of the study and the results were compared. All the samples negative for smear and/or culture were also negative by multiple-copy gene MPCRs (specificity = 100%), whereas the single-copy gene MPCR showed 98% specificity. With respect to sensitivity, compared with culture, the single-copy gene MPCR showed a sensitivity of 92%, whereas the three- and two-band multiple-copy gene MPCRs exhibited sensitivities of 87% and 93%, respectively. These results suggest that the MPCRs could be helpful in early and specific diagnosis of pulmonary tuberculosis.
Mustafa
A S
AS
Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait.
Das Chugh
T
T
Abal
A T
AT
D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
D004269:DNA, Bacterial