Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Sep;34(9):2117–2120. doi: 10.1128/jcm.34.9.2117-2120.1996

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

L F Kox 1, G T Noordhoek 1, M Kunakorn 1, S Mulder 1, M Sterrenburg 1, A H Kolk 1
PMCID: PMC229200  PMID: 8862568

Abstract

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization.

Full Text

The Full Text of this article is available as a PDF (363.8 KB).

Selected References

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

  1. Boom R., Sol C. J., Salimans M. M., Jansen C. L., Wertheim-van Dillen P. M., van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. doi: 10.1128/jcm.28.3.495-503.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cho S. N., van der Vliet G. M., Park S., Baik S. H., Kim S. K., Chong Y., Kolk A. H., Klatser P. R., Kim J. D. Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples. J Clin Microbiol. 1995 Mar;33(3):752–754. doi: 10.1128/jcm.33.3.752-754.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clarridge J. E., 3rd, Shawar R. M., Shinnick T. M., Plikaytis B. B. Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory. J Clin Microbiol. 1993 Aug;31(8):2049–2056. doi: 10.1128/jcm.31.8.2049-2056.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cousins D. V., Wilton S. D., Francis B. R., Gow B. L. Use of polymerase chain reaction for rapid diagnosis of tuberculosis. J Clin Microbiol. 1992 Jan;30(1):255–258. doi: 10.1128/jcm.30.1.255-258.1992. [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. Hermans P. W., van Soolingen D., Dale J. W., Schuitema A. R., McAdam R. A., Catty D., van Embden J. D. Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J Clin Microbiol. 1990 Sep;28(9):2051–2058. doi: 10.1128/jcm.28.9.2051-2058.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kolk A. H., Noordhoek G. T., de Leeuw O., Kuijper S., van Embden J. D. Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria. J Clin Microbiol. 1994 May;32(5):1354–1356. doi: 10.1128/jcm.32.5.1354-1356.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kox L. F., Kuijper S., Kolk A. H. Early diagnosis of tuberculous meningitis by polymerase chain reaction. Neurology. 1995 Dec;45(12):2228–2232. doi: 10.1212/wnl.45.12.2228. [DOI] [PubMed] [Google Scholar]
  11. Kox L. F., Rhienthong D., Miranda A. M., Udomsantisuk N., Ellis K., van Leeuwen J., van Heusden S., Kuijper S., Kolk A. H. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol. 1994 Mar;32(3):672–678. doi: 10.1128/jcm.32.3.672-678.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nolte F. S., Metchock B., McGowan J. E., Jr, Edwards A., Okwumabua O., Thurmond C., Mitchell P. S., Plikaytis B., Shinnick T. Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization. J Clin Microbiol. 1993 Jul;31(7):1777–1782. doi: 10.1128/jcm.31.7.1777-1782.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Noordhoek G. T., Kaan J. A., Mulder S., Wilke H., Kolk A. H. Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples. J Clin Pathol. 1995 Sep;48(9):810–814. doi: 10.1136/jcp.48.9.810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ossewaarde J. M., Rieffe M., van Doornum G. J., Henquet C. J., van Loon A. M. Detection of amplified Chlamydia trachomatis DNA using a microtiter plate-based enzyme immunoassay. Eur J Clin Microbiol Infect Dis. 1994 Sep;13(9):732–740. doi: 10.1007/BF02276056. [DOI] [PubMed] [Google Scholar]
  15. Wilson S. M., McNerney R., Nye P. M., Godfrey-Faussett P. D., Stoker N. G., Voller A. Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. J Clin Microbiol. 1993 Apr;31(4):776–782. doi: 10.1128/jcm.31.4.776-782.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zambardi G., Druetta A., Roure C., Fouqué B., Girardo P., Chypre C., Marchand J., Freney J., Fleurette J. Rapid diagnosis of Mycobacterium tuberculosis infections by an ELISA-like detection of polymerase chain reaction products. Mol Cell Probes. 1995 Apr;9(2):91–99. doi: 10.1016/s0890-8508(95)80033-6. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES