Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 May;32(5):1354–1356. doi: 10.1128/jcm.32.5.1354-1356.1994

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

A H Kolk 1, G T Noordhoek 1, O de Leeuw 1, S Kuijper 1, J D van Embden 1
PMCID: PMC263700  PMID: 8051267

Abstract

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample.

Full text

PDF
1354

Images in this article

1355Image
on p.1355
1356Image
on p.1356

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. 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]
  3. Hermans P. W., van Soolingen D., Bik E. M., de Haas P. E., Dale J. W., van Embden J. D. Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun. 1991 Aug;59(8):2695–2705. doi: 10.1128/iai.59.8.2695-2705.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Lee M. H., Pascopella L., Jacobs W. R., Jr, Hatfull G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3111–3115. doi: 10.1073/pnas.88.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Noordhoek G. T., Kolk A. H., Bjune G., Catty D., Dale J. W., Fine P. E., Godfrey-Faussett P., Cho S. N., Shinnick T., Svenson S. B. Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol. 1994 Feb;32(2):277–284. doi: 10.1128/jcm.32.2.277-284.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 1990 Nov;4(11):1911–1919. doi: 10.1111/j.1365-2958.1990.tb02040.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. deWit D., Wootton M., Allan B., Steyn L. Simple method for production of internal control DNA for Mycobacterium tuberculosis polymerase chain reaction assays. J Clin Microbiol. 1993 Aug;31(8):2204–2207. doi: 10.1128/jcm.31.8.2204-2207.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES