MORE RELIABLE PCR FOR DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN CLINICAL-SAMPLES

Diagnostic techniques based on PCR have two major problems: false-posi tive reactions due to contamination with DNA fragments from previous P CRs (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 tubercul osis complex-specific insertion element IS6110 with respect to both pr oblems. False-positive reactions caused bg 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 f ormerly used primers to avoid false-positive reactions caused by dTTP- containing amplicons still present in the laboratory. With this new pr imer 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 inhi bitors of the Tag polymerase, which may cause false-negative reactions , part of each sample was spiked with M. tuberculosis DNA. The DNA pur ification method using guanidinium thiocyanate and diatoms effectively removed most or ah inhibitors of the PCR. However, this was not suita ble for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detec tion of 20 M. tuberculosis bacteria per mi of whale blood. Various lab oratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different cl inical specimens obtained from patients suspected of having tuberculos is. The samples included sputum (Ir = 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 = I). The results obtained by PCR were consistent with those obtain ed with culture, which is the ''gold standard.'' We demonstrate that P CR is a useful technique for the rapid diagnosis of tuberculosis at va rious sites.