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J Clin Microbiol. Aug 1992; 30(8): 2025–2028.
PMCID: PMC265435

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

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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  • Bates JH. Diagnosis of tuberculosis. Chest. 1979 Dec;76(6 Suppl):757–763. [PubMed]
  • Böddinghaus B, Rogall T, Flohr T, Blöcker H, Böttger EC. Detection and identification of mycobacteria by amplification of rRNA. J Clin Microbiol. 1990 Aug;28(8):1751–1759. [PMC free article] [PubMed]
  • Borremans M, de Wit L, Volckaert G, Ooms J, de Bruyn J, Huygen K, van Vooren JP, 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Brisson-Noël A, Gicquel B, Lecossier D, Lévy-Frébault V, Nassif X, Hance AJ. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet. 1989 Nov 4;2(8671):1069–1071. [PubMed]
  • De Bruyn J, Bosmans R, Nyabenda J, Van Vooren JP. 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. [PubMed]
  • Del Portillo P, Murillo LA, Patarroyo ME. 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Drake TA, Hindler JA, Berlin OG, Bruckner DA. Rapid identification of Mycobacterium avium complex in culture using DNA probes. J Clin Microbiol. 1987 Aug;25(8):1442–1445. [PMC free article] [PubMed]
  • Eisenach KD, Cave MD, Bates JH, Crawford JT. Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J Infect Dis. 1990 May;161(5):977–981. [PubMed]
  • Hance AJ, 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. [PubMed]
  • Katila ML, Mattila J. Enhanced isolation of MOTT on egg media of low pH. APMIS. 1991 Sep;99(9):803–807. [PubMed]
  • Khan G, Kangro HO, Coates PJ, Heath RB. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol. 1991 May;44(5):360–365. [PMC free article] [PubMed]
  • Kiehn TE, Edwards FF. 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. [PMC free article] [PubMed]
  • Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet. 1988;22:631–677. [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Pao CC, Lin SS, Wu SY, Juang WM, Chang CH, Lin JY. The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes. Tubercle. 1988 Mar;69(1):27–36. [PubMed]
  • Pao CC, Yen TS, You JB, Maa JS, Fiss EH, Chang CH. Detection and identification of Mycobacterium tuberculosis by DNA amplification. J Clin Microbiol. 1990 Sep;28(9):1877–1880. [PMC free article] [PubMed]
  • Roberts MC, McMillan C, Coyle MB. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex. J Clin Microbiol. 1987 Jul;25(7):1239–1243. [PMC free article] [PubMed]
  • Sjöbring U, Mecklenburg M, Andersen AB, Miörner H. Polymerase chain reaction for detection of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Oct;28(10):2200–2204. [PMC free article] [PubMed]
  • Thierry D, Brisson-Noël A, Vincent-Lévy-Frébault V, Nguyen S, Guesdon JL, Gicquel B. Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J Clin Microbiol. 1990 Dec;28(12):2668–2673. [PMC free article] [PubMed]

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