Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin

Abstract

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining 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 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the 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 0.00038 to 0.1% BSA.To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.

Sensitivity of the Cobas Amplicor system for detection of Mycobacterium tuberculosis in respiratory and extrapulmonary specimens

“…This protocol can also elicit false‐negative results following dilution of DNA template in the samples. Other factors that may affect PCR reliability have been reported, such as decontamination/fluidification, especially when a high concentration (4%) of NaOH is used, inefficiency in the liquefaction procedure, formation of aggregates by M. tuberculosis , and heterogeneous repartition of bacilli in specimens [3,11]. Most specimens that remained inhibited were sputa ( n = 34).…”

Widespread occurrence of Mycobacterium tuberculosis DNA from 18th–19th century Hungarians

“…The final composition of the PCR mixture (50 μl) was: 75 mM Tris‐HCl (pH 8.8); 20 mM (NH 4 ) 2 SO 4 ; 1.5 mM MgCl 2 ; 0.01% (v/v) Tween‐20; 200 μM (each) dATP, dCTP, dGTP, and dTTP; primer, 0.5 μM (each); and 1.25 units Taq DNA polymerase. Bovine serum albumin (BSA) was added to give a final concentration of 10 mM, as this has been shown to improve the yield (Forbes and Hicks, 1996; Abu Al‐Soud and Rådström, 2000). The primer pair and DNA preparation (5.0 μl) were added to each prealiquoted tube, plus sufficient water to bring the volume to 50 μl.…”

PCR amplification of shorter fragments from thedevR(Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis

“…The sensitivity of the devR assay was only 55–68% in paucibacillary lymph node biopsies and fine needle aspirates (Singh et al , 2000). PCR sensitivity is determined primarily by the bacterial load and the presence of PCR inhibitors in the specimen, which can oftentimes result in false negative reporting (Forbes & Hicks, 1996; Boddinghaus et al , 2001; Suffys, 2001). We developed a highly efficient method to extract mycobacterial DNA that is free of PCR inhibitors from clinical specimens (Chakravorty & Tyagi, 2005).…”