Abstract
A method is described for the routine preparation of sputum samples for detection of Mycobacterium tuberculosis by using polymerase chain reaction amplification. Liquefaction of sputum samples with NaOH and subsequent removal of inhibitors of the polymerase reaction with a 50% sucrose centrifugation step (5 min) in a desktop centrifuge allow direct amplification of a 123-bp repetitive region of the M. tuberculosis genome. We have evaluated the sensitivity and specificity of the sucrose method with 155 sputum specimens from patients suspected of having tuberculosis and from normal healthy volunteers. This method, in our opinion, is reproducible, sensitive, and reliable. Multiple samples can be handled simultaneously, and results can be obtained in one working day.
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- Anargyros P., Astill D. S., Lim I. S. Comparison of improved BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical specimens. J Clin Microbiol. 1990 Jun;28(6):1288–1291. doi: 10.1128/jcm.28.6.1288-1291.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- De Wit D., Steyn L., Shoemaker S., Sogin M. Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification. J Clin Microbiol. 1990 Nov;28(11):2437–2441. doi: 10.1128/jcm.28.11.2437-2441.1990. [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]
- Gonzalez R., Hanna B. A. Evaluation of Gen-Probe DNA hybridization systems for the identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. Diagn Microbiol Infect Dis. 1987 Oct;8(2):69–77. doi: 10.1016/0732-8893(87)90152-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Hartskeerl R. A., de Wit M. Y., Klatser P. R. Polymerase chain reaction for the detection of Mycobacterium leprae. J Gen Microbiol. 1989 Sep;135(9):2357–2364. doi: 10.1099/00221287-135-9-2357. [DOI] [PubMed] [Google Scholar]
- Hermans P. W., Schuitema A. R., Van Soolingen D., Verstynen C. P., Bik E. M., Thole J. E., Kolk A. H., van Embden J. D. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J Clin Microbiol. 1990 Jun;28(6):1204–1213. doi: 10.1128/jcm.28.6.1204-1213.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Noordhoek G. T., Wieles B., van der Sluis J. J., van Embden J. D. Polymerase chain reaction and synthetic DNA probes: a means of distinguishing the causative agents of syphilis and yaws? Infect Immun. 1990 Jun;58(6):2011–2013. doi: 10.1128/iai.58.6.2011-2013.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Patel R. J., Fries J. W., Piessens W. F., Wirth D. F. Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis. J Clin Microbiol. 1990 Mar;28(3):513–518. doi: 10.1128/jcm.28.3.513-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierre C., Lecossier D., Boussougant Y., Bocart D., Joly V., Yeni P., Hance A. J. Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol. 1991 Apr;29(4):712–717. doi: 10.1128/jcm.29.4.712-717.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plikaytis B. B., Gelber R. H., Shinnick T. M. Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay. J Clin Microbiol. 1990 Sep;28(9):1913–1917. doi: 10.1128/jcm.28.9.1913-1917.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shankar P., Manjunath N., Mohan K. K., Prasad K., Behari M., Shriniwas, Ahuja G. K. Rapid diagnosis of tuberculous meningitis by polymerase chain reaction. Lancet. 1991 Jan 5;337(8732):5–7. doi: 10.1016/0140-6736(91)93328-7. [DOI] [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]
- 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]
- Vary P. H., Andersen P. R., Green E., Hermon-Taylor J., McFadden J. J. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease. J Clin Microbiol. 1990 May;28(5):933–937. doi: 10.1128/jcm.28.5.933-937.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Victor T., du Toit R., van Zyl J., Bester A. J., van Helden P. D. Improved method for the routine identification of toxigenic Escherichia coli by DNA amplification of a conserved region of the heat-labile toxin A subunit. J Clin Microbiol. 1991 Jan;29(1):158–161. doi: 10.1128/jcm.29.1.158-161.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]