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.
Full Text
The Full Text of this article is available as a PDF (295.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akane A., Matsubara K., Nakamura H., Takahashi S., Kimura K. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci. 1994 Mar;39(2):362–372. [PubMed] [Google Scholar]
- Amicosante M., Richeldi L., Trenti G., Paone G., Campa M., Bisetti A., Saltini C. Inactivation of polymerase inhibitors for Mycobacterium tuberculosis DNA amplification in sputum by using capture resin. J Clin Microbiol. 1995 Mar;33(3):629–630. doi: 10.1128/jcm.33.3.629-630.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buck G. E., O'Hara L. C., Summersgill J. T. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol. 1992 May;30(5):1331–1334. doi: 10.1128/jcm.30.5.1331-1334.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Clarridge J. E., 3rd, Shawar R. M., Shinnick T. M., Plikaytis B. B. Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J Clin Microbiol. 1993 Aug;31(8):2049–2056. doi: 10.1128/jcm.31.8.2049-2056.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eisenach K. D., Cave M. D., Bates J. H., Crawford J. T. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis. 1990 May;161(5):977–981. doi: 10.1093/infdis/161.5.977. [DOI] [PubMed] [Google Scholar]
- Eisenach K. D., Sifford M. D., Cave M. D., Bates J. H., Crawford J. T. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis. 1991 Nov;144(5):1160–1163. doi: 10.1164/ajrccm/144.5.1160. [DOI] [PubMed] [Google Scholar]
- Forbes B. A., Hicks K. E. Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction. J Clin Microbiol. 1993 Jul;31(7):1688–1694. doi: 10.1128/jcm.31.7.1688-1694.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschner P., Rosenau J., Springer B., Teschner K., Feldmann K., Böttger E. C. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study. J Clin Microbiol. 1996 Feb;34(2):304–312. doi: 10.1128/jcm.34.2.304-312.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kox L. F., Rhienthong D., Miranda A. M., Udomsantisuk N., Ellis K., van Leeuwen J., van Heusden S., Kuijper S., Kolk A. H. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol. 1994 Mar;32(3):672–678. doi: 10.1128/jcm.32.3.672-678.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sjöbring U., Mecklenburg M., Andersen A. B., Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Oct;28(10):2200–2204. doi: 10.1128/jcm.28.10.2200-2204.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soini H., Skurnik M., Liippo K., Tala E., Viljanen M. K. Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. J Clin Microbiol. 1992 Aug;30(8):2025–2028. doi: 10.1128/jcm.30.8.2025-2028.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Victor T., du Toit R., van Helden P. D. Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction. J Clin Microbiol. 1992 Jun;30(6):1514–1517. doi: 10.1128/jcm.30.6.1514-1517.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yuen K. Y., Chan K. S., Chan C. M., Ho B. S., Dai L. K., Chau P. Y., Ng M. H. Use of PCR in routine diagnosis of treated and untreated pulmonary tuberculosis. J Clin Pathol. 1993 Apr;46(4):318–322. doi: 10.1136/jcp.46.4.318. [DOI] [PMC free article] [PubMed] [Google Scholar]