This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forbes, B. A.
Right arrow Articles by Hicks, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forbes, B. A.
Right arrow Articles by Hicks, K. E.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, 09 1996, 2125-2128, Vol 34, No. 9
Copyright © 1996 by the American Society for Microbiology. All rights reserved.

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

BA Forbes and KE Hicks
Department of Clinical Pathology, SUNY Health Science Center 13210, USA.

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.


This article has been cited by other articles:

  • Donoghue, H. D., Lee, O. Y.-C., Minnikin, D. E., Besra, G. S., Taylor, J. H., Spigelman, M. (2009). Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis. Proc R Soc B 0: rspb.2009.1484v1-rspb20091484 [Abstract] [Full Text]  
  • Kermekchiev, M. B., Kirilova, L. I., Vail, E. E., Barnes, W. M. (2009). Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res 37: e40-e40 [Abstract] [Full Text]  
  • Seagar, A.-L., Prendergast, C., Emmanuel, F. X., Rayner, A., Thomson, S., Laurenson, I. F. (2008). Evaluation of the GenoType Mycobacteria Direct assay for the simultaneous detection of the Mycobacterium tuberculosis complex and four atypical mycobacterial species in smear-positive respiratory specimens. J Med Microbiol 57: 605-611 [Abstract] [Full Text]  
  • BAUER, O., BANETH, G., ESHKOL, T., SHAW, S. E., HARRUS, S. (2006). POLYGENIC DETECTION OF RICKETTSIA FELIS IN CAT FLEAS (CTENOCEPHALIDES FELIS) FROM ISRAEL.. Am J Trop Med Hyg 74: 444-448 [Abstract] [Full Text]  
  • Chakravorty, S., Tyagi, J. S. (2005). Novel Multipurpose Methodology for Detection of Mycobacteria in Pulmonary and Extrapulmonary Specimens by Smear Microscopy, Culture, and PCR. J. Clin. Microbiol. 43: 2697-2702 [Abstract] [Full Text]  
  • Honore-Bouakline, S., Vincensini, J. P., Giacuzzo, V., Lagrange, P. H., Herrmann, J. L. (2003). Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction. J. Clin. Microbiol. 41: 2323-2329 [Abstract] [Full Text]  
  • Winther, B., Hayden, F. G., Arruda, E., Dutkowski, R., Ward, P., Hendley, J. O. (2002). Viral Respiratory Infection in Schoolchildren: Effects on Middle Ear Pressure. Pediatrics 109: 826-832 [Abstract] [Full Text]  
  • Qian, Q., Tang, Y.-W., Kolbert, C. P., Torgerson, C. A., Hughes, J. G., Vetter, E. A., Harmsen, W. S., Montgomery, S. O., Cockerill, F. R. III, Persing, D. H. (2001). Direct Identification of Bacteria from Positive Blood Cultures by Amplification and Sequencing of the 16S rRNA Gene: Evaluation of BACTEC 9240 Instrument True- Positive and False-Positive Results. J. Clin. Microbiol. 39: 3578-3582 [Abstract] [Full Text]  
  • Boddinghaus, B., Wichelhaus, T. A., Brade, V., Bittner, T. (2001). Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor Mycobacterium tuberculosis Kit. J. Clin. Microbiol. 39: 3750-3752 [Abstract] [Full Text]  
  • Desmond, E. P., Loretz, K. (2001). Use of the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test for Early Detection of Mycobacterium tuberculosis in BACTEC 12B Medium. J. Clin. Microbiol. 39: 1993-1995 [Abstract] [Full Text]  
  • DONOGHUE, H.D., HOLTON, J., SPIGELMAN, M. (2001). PCR primers that can detect low levels of Mycobacterium leprae DNA. J Med Microbiol 50: 177-182 [Abstract] [Full Text]  
  • Pietilä, J., He, Q., Oksi, J., Viljanen, M. K. (2000). Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene. J. Clin. Microbiol. 38: 2756-2759 [Abstract] [Full Text]  
  • Mahony, J., Chong, S., Jang, D., Luinstra, K., Faught, M., Dalby, D., Sellors, J., Chernesky, M. (1998). Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of Chlamydia trachomatis Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription-Mediated Amplification: Identification of Urinary Substances Associated with Inhibition and Removal of Inhibitory Activity. J. Clin. Microbiol. 36: 3122-3126 [Abstract] [Full Text]  
  • Leckie, G. W., Erickson, D. D., He, Q., Facey, I. E., Lin, B.-C., Cao, J., Halaka, F. G. (1998). Method for Reduction of Inhibition in a Mycobacterium tuberculosis-Specific Ligase Chain Reaction DNA Amplification Assay. J. Clin. Microbiol. 36: 764-767 [Abstract] [Full Text]  
  • Kearns, A. M., Freeman, R., Steward;, M., Chapin, K., Lauderdale, T.-l. (1998). Evaluation of a Rapid Air Thermal Cycler for Detection of Mycobacterium tuberculosis. J. Clin. Microbiol. 36: 604-605 [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»