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. 1992 Aug;30(8):2025–2028. doi: 10.1128/jcm.30.8.2025-2028.1992

Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein.

H Soini 1, M Skurnik 1, K Liippo 1, E Tala 1, M K Viljanen 1
PMCID: PMC265435  PMID: 1500509

Abstract

A polymerase chain reaction (PCR) assay for the rapid detection of mycobacterial DNA is described. Oligonucleotide primers, derived from the sequence of a gene coding for the 32-kDa antigen of Mycobacterium tuberculosis, amplified DNA from all 28 species of mycobacteria tested. All nonmycobacterial species tested were negative. An oligonucleotide probe hybridized to the PCR products of the strains belonging to the M. tuberculosis complex. This method could detect as little as 50 fg, as tested with purified M. tuberculosis DNA. By this amplification method, 127 sputum specimens were tested, with 7.9% of the specimens proving to be inhibitory in PCR. The sensitivity of detection by PCR compared with that by culture was 55.9%; when the inhibitory specimens were excluded, the sensitivity was 70.4%. The specificity of PCR combined with hybridization was 100%.

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Selected References

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  1. Bates J. H. Diagnosis of tuberculosis. Chest. 1979 Dec;76(6 Suppl):757–763. doi: 10.1378/chest.76.6_supplement.757. [DOI] [PubMed] [Google Scholar]
  2. Borremans M., de Wit L., Volckaert G., Ooms J., de Bruyn J., Huygen K., van Vooren J. P., Stelandre M., Verhofstadt R., Content J. Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infect Immun. 1989 Oct;57(10):3123–3130. doi: 10.1128/iai.57.10.3123-3130.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brisson-Noel A., Aznar C., Chureau C., Nguyen S., Pierre C., Bartoli M., Bonete R., Pialoux G., Gicquel B., Garrigue G. Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet. 1991 Aug 10;338(8763):364–366. doi: 10.1016/0140-6736(91)90492-8. [DOI] [PubMed] [Google Scholar]
  4. Brisson-Noël A., Gicquel B., Lecossier D., Lévy-Frébault V., Nassif X., Hance A. J. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet. 1989 Nov 4;2(8671):1069–1071. doi: 10.1016/s0140-6736(89)91082-9. [DOI] [PubMed] [Google Scholar]
  5. Böddinghaus B., Rogall T., Flohr T., Blöcker H., Böttger E. C. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. doi: 10.1128/jcm.28.8.1751-1759.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Bruyn J., Bosmans R., Nyabenda J., Van Vooren J. P. Effect of zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in culture filtrates of mycobacteria. J Gen Microbiol. 1989 Jan;135(1):79–84. doi: 10.1099/00221287-135-1-79. [DOI] [PubMed] [Google Scholar]
  7. De Wit L., de la Cuvellerie A., Ooms J., Content J. Nucleotide sequence of the 32 kDa-protein gene (antigen 85 A) of Mycobacterium bovis BCG. Nucleic Acids Res. 1990 Jul 11;18(13):3995–3995. doi: 10.1093/nar/18.13.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Del Portillo P., Murillo L. A., Patarroyo M. E. Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis. J Clin Microbiol. 1991 Oct;29(10):2163–2168. doi: 10.1128/jcm.29.10.2163-2168.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drake T. A., Hindler J. A., Berlin O. G., Bruckner D. A. Rapid identification of Mycobacterium avium complex in culture using DNA probes. J Clin Microbiol. 1987 Aug;25(8):1442–1445. doi: 10.1128/jcm.25.8.1442-1445.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hance A. J., Grandchamp B., Lévy-Frébault V., Lecossier D., Rauzier J., Bocart D., Gicquel B. Detection and identification of mycobacteria by amplification of mycobacterial DNA. Mol Microbiol. 1989 Jul;3(7):843–849. doi: 10.1111/j.1365-2958.1989.tb00233.x. [DOI] [PubMed] [Google Scholar]
  12. Katila M. L., Mattila J. Enhanced isolation of MOTT on egg media of low pH. APMIS. 1991 Sep;99(9):803–807. doi: 10.1111/j.1699-0463.1991.tb01263.x. [DOI] [PubMed] [Google Scholar]
  13. Khan G., Kangro H. O., Coates P. J., Heath R. B. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 1991 May;44(5):360–365. doi: 10.1136/jcp.44.5.360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kiehn T. E., Edwards F. F. Rapid identification using a specific DNA probe of Mycobacterium avium complex from patients with acquired immunodeficiency syndrome. J Clin Microbiol. 1987 Aug;25(8):1551–1552. doi: 10.1128/jcm.25.8.1551-1552.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindquist S., Craig E. A. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. doi: 10.1146/annurev.ge.22.120188.003215. [DOI] [PubMed] [Google Scholar]
  16. Manjunath N., Shankar P., Rajan L., Bhargava A., Saluja S., Shriniwas Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis. Tubercle. 1991 Mar;72(1):21–27. doi: 10.1016/0041-3879(91)90020-s. [DOI] [PubMed] [Google Scholar]
  17. Mercier B., Gaucher C., Feugeas O., Mazurier C. Direct PCR from whole blood, without DNA extraction. Nucleic Acids Res. 1990 Oct 11;18(19):5908–5908. doi: 10.1093/nar/18.19.5908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Panaccio M., Lew A. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res. 1991 Mar 11;19(5):1151–1151. doi: 10.1093/nar/19.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pao C. C., Lin S. S., Wu S. Y., Juang W. M., Chang C. H., Lin J. Y. The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes. Tubercle. 1988 Mar;69(1):27–36. doi: 10.1016/0041-3879(88)90037-2. [DOI] [PubMed] [Google Scholar]
  20. Pao C. C., Yen T. S., You J. B., Maa J. S., Fiss E. H., Chang C. H. Detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin Microbiol. 1990 Sep;28(9):1877–1880. doi: 10.1128/jcm.28.9.1877-1880.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roberts M. C., McMillan C., Coyle M. B. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex. J Clin Microbiol. 1987 Jul;25(7):1239–1243. doi: 10.1128/jcm.25.7.1239-1243.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Thierry D., Brisson-Noël A., Vincent-Lévy-Frébault V., Nguyen S., Guesdon J. L., Gicquel B. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J Clin Microbiol. 1990 Dec;28(12):2668–2673. doi: 10.1128/jcm.28.12.2668-2673.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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