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[Detection of rpoB gene mutation in Mycobacterium tuberculosis by PCR "cold" SSCP]
Objective:
To evaluate the applicability of detection of rpoB gene mutation in M. tuberculosis susceptibility testing.
Methods:
87 M. tuberculosis isolates and 22 sputum specimens from patients with active pulmonary tuberculosis were detected by PCR-SSCP.
Results:
The sensitivity of PCR for rpoB gene amplification was 100 pg DNA and 5000 organisms. The rpoB gene could be detected in the all isolates tested. In comparison with conventional susceptibility testing methods, the sensitivity and specificity of PCR-"cold" SSCP analysis for detecting rifampin resistance in 87 M. tuberculosis isolates was 89.6% and 100%, respectively. Among 22 smear- and culture-positive sputum specimens, only 1 (4.5%) was positive by PCR, however, 6 (27.3%) of them were positive by nested-PCR. The "cold" SSCP results of these 6 specimens were corresponding to that of the susceptibility testing.
Conclusions:
The PCR-"cold" SSCP described here can easily and rapidly detect rifampin resistance of M. tuberculosis. After increasing the primer specificity and amplification sensitivity, the technique might be used for detection of M. tuberculosis rifampin resistance in clinical specimen directly.