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. 1993 Jul;31(7):1777–1782. doi: 10.1128/jcm.31.7.1777-1782.1993

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization.

F S Nolte 1, B Metchock 1, J E McGowan Jr 1, A Edwards 1, O Okwumabua 1, C Thurmond 1, P S Mitchell 1, B Plikaytis 1, T Shinnick 1
PMCID: PMC265631  PMID: 8349753

Abstract

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Buck G. E., O'Hara L. C., Summersgill J. T. Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction. J Clin Microbiol. 1992 May;30(5):1331–1334. doi: 10.1128/jcm.30.5.1331-1334.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cimino G. D., Metchette K. C., Tessman J. W., Hearst J. E., Isaacs S. T. Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res. 1991 Jan 11;19(1):99–107. doi: 10.1093/nar/19.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Eisenach K. D., Crawford J. T., Bates J. H. Repetitive DNA sequences as probes for Mycobacterium tuberculosis. J Clin Microbiol. 1988 Nov;26(11):2240–2245. doi: 10.1128/jcm.26.11.2240-2245.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eisenach K. D., Sifford M. D., Cave M. D., Bates J. H., Crawford J. T. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis. 1991 Nov;144(5):1160–1163. doi: 10.1164/ajrccm/144.5.1160. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle. 1991 Mar;72(1):1–6. doi: 10.1016/0041-3879(91)90017-m. [DOI] [PubMed] [Google Scholar]
  10. Kolk A. H., Schuitema A. R., Kuijper S., van Leeuwen J., Hermans P. W., van Embden J. D., Hartskeerl R. A. Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system. J Clin Microbiol. 1992 Oct;30(10):2567–2575. doi: 10.1128/jcm.30.10.2567-2575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  12. Longo M. C., Berninger M. S., Hartley J. L. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990 Sep 1;93(1):125–128. doi: 10.1016/0378-1119(90)90145-h. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Plikaytis B. B., Eisenach K. D., Crawford J. T., Shinnick T. M. Differentiation of Mycobacterium tuberculosis and Mycobacterium bovis BCG by a polymerase chain reaction assay. Mol Cell Probes. 1991 Jun;5(3):215–219. doi: 10.1016/0890-8508(91)90043-j. [DOI] [PubMed] [Google Scholar]
  16. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  17. Shawar R. M., el-Zaatari F. A., Nataraj A., Clarridge J. E. Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods. J Clin Microbiol. 1993 Jan;31(1):61–65. doi: 10.1128/jcm.31.1.61-65.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  20. Sritharan V., Barker R. H., Jr A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol Cell Probes. 1991 Oct;5(5):385–395. doi: 10.1016/s0890-8508(06)80011-3. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Thierry D., Cave M. D., Eisenach K. D., Crawford J. T., Bates J. H., Gicquel B., Guesdon J. L. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 1990 Jan 11;18(1):188–188. doi: 10.1093/nar/18.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Victor T., du Toit R., van Helden P. D. Purification of sputum samples through sucrose improves detection of Mycobacterium tuberculosis by polymerase chain reaction. J Clin Microbiol. 1992 Jun;30(6):1514–1517. doi: 10.1128/jcm.30.6.1514-1517.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walker G. T., Fraiser M. S., Schram J. L., Little M. C., Nadeau J. G., Malinowski D. P. Strand displacement amplification--an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 1992 Apr 11;20(7):1691–1696. doi: 10.1093/nar/20.7.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]

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