Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis

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Abstract
The complexity, expense, and susceptibility to contamination of the polymerase chain reaction (PCR) are all issues which need to be overcome if PCR is to be used outside of research laboratories. We addressed these problems with respect to the diagnosis of tuberculosis. First, we simplified the procedure for extracting Mycobacterium tuberculosis DNA from sputum samples. Two methods of sample preparation were compared: the chaotrope-silica method and a novel, more simple chloroform method. Second, we developed a colorimetric method for product detection. This method was as sensitive and specific as agarose gel electrophoresis for detection of PCR product. By using a one-tube nested protocol, 5 to 50 genome equivalents of M. tuberculosis DNA were detected. The simplified colorimetric PCR was compared with microscopy and culture for detection of M. tuberculosis in clinical specimens of sputum. A total of 171 sputum samples were investigated from 108 patients, 12 of whom were subsequently found to have tuberculosis by culture and/or microscopy. PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100% whereas PCR of samples prepared by the chloroform method had a sensitivity of 92% and a specificity of 99% when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. The simplified colorimetric PCR in combination with the chloroform sample preparation method was at least as sensitive as microscopy but had a greater specificity because samples with atypical mycobacteria were not detected by PCR. The sensitivity of the method for detection of smear-negative and extrapulmonary tuberculosis remains to be investigated.