SUBSTANCES INTERFERING WITH DIRECT-DETECTION OF MYCOBACTERIUM-TUBERCULOSIS IN CLINICAL SPECIMENS BY PCR - EFFECTS OF BOVINE SERUM-ALBUMIN

Interfering substances have been reported to inhibit PCR assays for th e direct detection of Mycobacterium tuberculosis in clinical specimens . Using an internal control, we determined that 52% of respiratory spe cimens interfered with our PCR assay, On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sedimen ts. With sediment from a routinely processed sputum known to be inhibi tory to PCR, one aliquot was prepared in a routine manner for PCR. Rem aining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12 B medium including PANTA reagent (polymyxin B, amphotericin Il, nalidi xic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% gl ycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were test ed in a similar manner. Only 0.05% BSA resulted in amplification of th e internal control DNA, Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR, Internal control was detected in all reaction mixtures containing 0.0 0038 to 0.1% BSA. To determine the ability of BSA to override inhibiti on, respiratory specimens were run in triplicate: undiluted, diluted 1 :2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 in hibitory specimens, BSA was able to override the presence of interferi ng substances, These data suggest that the presence of BSA in a PCR as say is critical for the direct detection of M. tuberculosis in respira tory specimens.