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J Clin Microbiol. Oct 1992; 30(10): 2567–2575.
PMCID: PMC270480

Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system.

Abstract

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

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  • Boom R, Sol CJ, Heijtink R, Wertheim-van Dillen PM, van der Noordaa J. Rapid purification of hepatitis B virus DNA from serum. J Clin Microbiol. 1991 Sep;29(9):1804–1811. [PMC free article] [PubMed]
  • Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. [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]
  • Eisenach KD, Sifford MD, Cave MD, Bates JH, Crawford JT. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis. 1991 Nov;144(5):1160–1163. [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]
  • Hermans PW, Schuitema AR, Van Soolingen D, Verstynen CP, Bik EM, Thole JE, Kolk AH, van Embden JD. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J Clin Microbiol. 1990 Jun;28(6):1204–1213. [PMC free article] [PubMed]
  • Hermans PW, van Soolingen D, Dale JW, Schuitema AR, McAdam RA, Catty D, van Embden JD. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol. 1990 Sep;28(9):2051–2058. [PMC free article] [PubMed]
  • KUBICA GP, DYE WE, COHN ML, MIDDLEBROOK G. Sputum digestion and decontamination with N-acetyl-L-cysteine-sodium hydroxide for culture of mycobacteria. Am Rev Respir Dis. 1963 May;87:775–779. [PubMed]
  • Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1;93(1):125–128. [PubMed]
  • McAdam RA, Hermans PW, van Soolingen D, Zainuddin ZF, Catty D, van Embden JD, Dale JW. Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol Microbiol. 1990 Sep;4(9):1607–1613. [PubMed]
  • Middlebrook G, Reggiardo Z, Tigertt WD. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. Am Rev Respir Dis. 1977 Jun;115(6):1066–1069. [PubMed]
  • Nunn P, Kibuga D, Gathua S, Brindle R, Imalingat A, Wasunna K, Lucas S, Gilks C, Omwega M, Were J, et al. Cutaneous hypersensitivity reactions due to thiacetazone in HIV-1 seropositive patients treated for tuberculosis. Lancet. 1991 Mar 16;337(8742):627–630. [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]
  • Persing DH. Polymerase chain reaction: trenches to benches. J Clin Microbiol. 1991 Jul;29(7):1281–1285. [PMC free article] [PubMed]
  • Pitchenik AE. Tuberculosis control and the AIDS epidemic in developing countries. Ann Intern Med. 1990 Jul 15;113(2):89–91. [PubMed]
  • Schöningh R, Verstijnen CP, Kuijper S, Kolk AH. Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures. J Clin Microbiol. 1990 Apr;28(4):708–713. [PMC free article] [PubMed]
  • Shankar P, Manjunath N, Lakshmi R, Aditi B, Seth P, Shriniwas Identification of Mycobacterium tuberculosis by polymerase chain reaction. Lancet. 1990 Feb 17;335(8686):423–423. [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]
  • Tala E, Kochi A. Elimination of tuberculosis from Europe and the world. Eur Respir J. 1991 Nov;4(10):1159–1160. [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]
  • Thierry D, Cave MD, Eisenach KD, Crawford JT, Bates JH, Gicquel B, Guesdon JL. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 1990 Jan 11;18(1):188–188. [PMC free article] [PubMed]
  • van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 1991 Nov;29(11):2578–2586. [PMC free article] [PubMed]
  • Verstijnen CP, Ly HM, Polman K, Richter C, Smits SP, Maselle SY, Peerbooms P, Rienthong D, Montreewasuwat N, Koanjanart S, et al. Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures. J Clin Microbiol. 1991 Jul;29(7):1372–1375. [PMC free article] [PubMed]

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