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. 1995 Nov;33(11):2994–2998. doi: 10.1128/jcm.33.11.2994-2998.1995

Detection and identification of mycobacteria by DNA amplification and oligonucleotide-specific capture plate hybridization.

H De Beenhouwer 1, Z Liang 1, P De Rijk 1, C Van Eekeren 1, F Portaels 1
PMCID: PMC228621  PMID: 8576360

Abstract

We have developed an easy and rapid detection and identification system for the diagnosis of mycobacterial diseases. The system is based on selective amplification by PCR of mycobacteria with primers based on the genes coding for 16S rRNA. During PCR, a label (digoxigenin-11-dUTP) is incorporated with biotinylated species-specific oligonucleotides (oligonucleotide-specific capture plate hybridization [OSCPH]. One oligonucleotide specific for the genus Mycobacterium and seven species-specific (Mycobacterium tuberculosis, M. avium, M. intracellulare, M. scrofulaceum, M. xenopi, M. genavense, and M. chelonae) oligonucleotides were designed as capturing probes. After specific hybridization, an enzyme immunoassay reveals the specifically bound complexes and thus permits identification of the mycobacterium. A total of 70 mycobacterial strains were tested. For 69 strains, results concordant with conventional identification were obtained. One M. chelonae strain was negative with the M. chelonae probe and was later reidentified as M. fortuitum. Moreover, for 15 clinical samples suspected of harboring nontuberculous mycobacteria, OSCPH was able to confirm all culture results and could identify one M. genavense infection for which standard culture results were negative. PCR-OSCPH is easily applicable and much faster than culture. It could become a valuable alternative approach for the diagnosis of mycobacterial infections.

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Selected References

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  1. American Thoracic Society. Diagnostic standards and classification of tuberculosis and other mycobacterial diseases (14th edition). Am Rev Respir Dis. 1981 Mar;123(3):343–358. doi: 10.1164/arrd.1981.123.3.343. [DOI] [PubMed] [Google Scholar]
  2. Bosquée L., Böttger E. C., De Beenhouwer H., Fonteyne P. A., Hirschel B., Larsson L., Meyers W. M., Palomino J. C., Realini L., Rigouts L. Cervical lymphadenitis caused by a fastidious mycobacterium closely related to Mycobacterium genavense in an apparently immunocompetent woman: diagnosis by culture-free microbiological methods. J Clin Microbiol. 1995 Oct;33(10):2670–2674. doi: 10.1128/jcm.33.10.2670-2674.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Böddinghaus B., Wolters J., Heikens W., Böttger E. C. Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level. FEMS Microbiol Lett. 1990 Jul;58(2):197–203. doi: 10.1111/j.1574-6968.1990.tb13978.x. [DOI] [PubMed] [Google Scholar]
  5. Böttger E. C., Teske A., Kirschner P., Bost S., Chang H. R., Beer V., Hirschel B. Disseminated "Mycobacterium genavense" infection in patients with AIDS. Lancet. 1992 Jul 11;340(8811):76–80. doi: 10.1016/0140-6736(92)90397-l. [DOI] [PubMed] [Google Scholar]
  6. Cox R. A., Kempsell K., Fairclough L., Colston M. J. The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction. J Med Microbiol. 1991 Nov;35(5):284–290. doi: 10.1099/00222615-35-5-284. [DOI] [PubMed] [Google Scholar]
  7. Debrunner M., Salfinger M., Brändli O., von Graevenitz A. Epidemiology and clinical significance of nontuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin Infect Dis. 1992 Aug;15(2):330–345. doi: 10.1093/clinids/15.2.330. [DOI] [PubMed] [Google Scholar]
  8. Fiss E. H., Chehab F. F., Brooks G. F. DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory. J Clin Microbiol. 1992 May;30(5):1220–1224. doi: 10.1128/jcm.30.5.1220-1224.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frothingham R., Wilson K. H. Sequence-based differentiation of strains in the Mycobacterium avium complex. J Bacteriol. 1993 May;175(10):2818–2825. doi: 10.1128/jb.175.10.2818-2825.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Havlik J. A., Jr, Horsburgh C. R., Jr, Metchock B., Williams P. P., Fann S. A., Thompson S. E., 3rd Disseminated Mycobacterium avium complex infection: clinical identification and epidemiologic trends. J Infect Dis. 1992 Mar;165(3):577–580. doi: 10.1093/infdis/165.3.577. [DOI] [PubMed] [Google Scholar]
  11. Hirschel B., Chang H. R., Mach N., Piguet P. F., Cox J., Piguet J. D., Silva M. T., Larsson L., Klatser P. R., Thole J. E. Fatal infection with a novel, unidentified mycobacterium in a man with the acquired immunodeficiency syndrome. N Engl J Med. 1990 Jul 12;323(2):109–113. doi: 10.1056/NEJM199007123230207. [DOI] [PubMed] [Google Scholar]
  12. Horsburgh C. R., Jr Mycobacterium avium complex infection in the acquired immunodeficiency syndrome. N Engl J Med. 1991 May 9;324(19):1332–1338. doi: 10.1056/NEJM199105093241906. [DOI] [PubMed] [Google Scholar]
  13. Jonas V., Alden M. J., Curry J. I., Kamisango K., Knott C. A., Lankford R., Wolfe J. M., Moore D. F. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J Clin Microbiol. 1993 Sep;31(9):2410–2416. doi: 10.1128/jcm.31.9.2410-2416.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kirschner P., Kiekenbeck M., Meissner D., Wolters J., Böttger E. C. Genetic heterogeneity within Mycobacterium fortuitum complex species: genotypic criteria for identification. J Clin Microbiol. 1992 Nov;30(11):2772–2775. doi: 10.1128/jcm.30.11.2772-2775.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kirschner P., Springer B., Vogel U., Meier A., Wrede A., Kiekenbeck M., Bange F. C., Böttger E. C. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol. 1993 Nov;31(11):2882–2889. doi: 10.1128/jcm.31.11.2882-2889.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolk A. H., Evers R., Groothuis D. G., Gilis H., Kuijper S. Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex. Infect Immun. 1989 Aug;57(8):2514–2521. doi: 10.1128/iai.57.8.2514-2521.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Musial C. E., Tice L. S., Stockman L., Roberts G. D. Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex. J Clin Microbiol. 1988 Oct;26(10):2120–2123. doi: 10.1128/jcm.26.10.2120-2123.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Neefs J. M., Van de Peer Y., De Rijk P., Chapelle S., De Wachter R. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res. 1993 Jul 1;21(13):3025–3049. doi: 10.1093/nar/21.13.3025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Plikaytis B. B., Plikaytis B. D., Yakrus M. A., Butler W. R., Woodley C. L., Silcox V. A., Shinnick T. M. Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis. J Clin Microbiol. 1992 Jul;30(7):1815–1822. doi: 10.1128/jcm.30.7.1815-1822.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Portaels F. Epidemiology of mycobacterial diseases. Clin Dermatol. 1995 May-Jun;13(3):207–222. doi: 10.1016/0738-081x(95)00004-y. [DOI] [PubMed] [Google Scholar]
  21. Reisner B. S., Gatson A. M., Woods G. L. Use of Gen-Probe AccuProbes to identify Mycobacterium avium complex, Mycobacterium tuberculosis complex, Mycobacterium kansasii, and Mycobacterium gordonae directly from BACTEC TB broth cultures. J Clin Microbiol. 1994 Dec;32(12):2995–2998. doi: 10.1128/jcm.32.12.2995-2998.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rogall T., Flohr T., Böttger E. C. Differentiation of Mycobacterium species by direct sequencing of amplified DNA. J Gen Microbiol. 1990 Sep;136(9):1915–1920. doi: 10.1099/00221287-136-9-1915. [DOI] [PubMed] [Google Scholar]
  23. Rogall T., Wolters J., Flohr T., Böttger E. C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol. 1990 Oct;40(4):323–330. doi: 10.1099/00207713-40-4-323. [DOI] [PubMed] [Google Scholar]
  24. Saito H., Tomioka H., Sato K., Tasaka H., Dawson D. J. Identification of various serovar strains of Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol. 1990 Aug;28(8):1694–1697. doi: 10.1128/jcm.28.8.1694-1697.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suzuki Y., Yoshinaga K., Ono Y., Nagata A., Yamada T. Organization of rRNA genes in Mycobacterium bovis BCG. J Bacteriol. 1987 Feb;169(2):839–843. doi: 10.1128/jb.169.2.839-843.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Telenti A., Marchesi F., Balz M., Bally F., Böttger E. C., Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993 Feb;31(2):175–178. doi: 10.1128/jcm.31.2.175-178.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vaneechoutte M., De Beenhouwer H., Claeys G., Verschraegen G., De Rouck A., Paepe N., Elaichouni A., Portaels F. Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis. J Clin Microbiol. 1993 Aug;31(8):2061–2065. doi: 10.1128/jcm.31.8.2061-2065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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