SENSITIVITY OF 2-STAGE PCR-AMPLIFICATION FOR DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN PARAFFIN-EMBEDDED TISSUES

In order to improve the sensitivity of polymerase chain reaction (PCR) for the detection of mycobacterial DNA in paraffin-embedded tissues, a new approach with two sets of specific primers in two-stage PCR was employed in specimens obtained from tuberculosis patients and controls . Thirty-nine paraffin blocks selected from patients who had been diag nosed as having tuberculosis by radiological evaluations, histopatholo gical findings,land clinical symptoms and signs including response to antituberculous treatment were examined. The control group consisted o f 10 specimens from individuals that were proved to be negative for tu berculosis. After deparaffinization, lysis, phenol-chloroform extracti on, and ethanol precipitation, the isolated DNA was amplified by PCR. Initially, all specimens were examined by the one-stage PCR using spec ific primers for 123-base pair (bp) fragment in IS6110 of mycobacteria l DNA which yielded positive results only in 3 out of 39 (7.7%). In th e two-stage PCR technique, 245-bp fragment of mycobacterial DNA was am plified at the first-step, then the PCR products were reamplified usin g the second specific primer pairs for 123-bp fragment. The true posit ivity of the two-stage PCR was 84.6% (33/39). The results indicate tha t two-stage PCR is more sensitive than one-stage (84.6% vs. 7.7%). All control specimens were negative by both PCR amplification methods, in dicating that specificity of both methods was high. When the two-stage amplification was used, PCR positivity in the specimens obtained from different tissues was as follows: peritoneal and omental biopsies, 4/ 4; bone biopsies, 3/3; lymph node biopsies, 12/14; genito-urinary biop sies, 7/9; skin biopsies, 4/6; and one from each lung, breast, and ple ural biopsies. PCR showed a good correlation with the granulomatous ti ssue reaction resulting in a 83.8% (31/37) positivity. The results ind icate that the two-stage PCR amplification can be used for detection o f M. tuberculosis in paraffin-embedded tissues and is a useful techniq ue in confirming tuberculosis in patients with clinically suspected di sease who have acid-fast stain-negative. (C) 1997 Elsevier Science B.V .