Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Nov;33(11):2908–2912. doi: 10.1128/jcm.33.11.2908-2912.1995

Immunomagnetic PCR and DNA probe for detection and identification of Porphyromonas gingivalis.

R M Benkirane 1, E Guillot 1, C Mouton 1
PMCID: PMC228605  PMID: 8576344

Abstract

The aim of the study that we describe was to combine an immunomagnetic separation and a PCR followed by dot blot hybridization with a DNA probe for the detection and identification of Porphyromonas gingivalis. Immunomagnetic particles were coated with monoclonal antibody specific for P. gingivalis and were incubated with a suspension containing seven oral bacterial species spiked with various dilutions of P. gingivalis. Beads with their load of bound bacterial were boiled in water, and the target DNA in the supernatant was amplified with a primer pair to generate a 593-bp PCR fragment specific for P. gingivalis. Finally, the product of amplification was detected by dot blot hybridization with a digoxigenin-labeled 593-bp probe. The detection limit was determined to be 100 bacterial cells per ml. The immunomagnetic-PCR/DNA probe procedure described here should be useful for the rapid, specific, and sensitive detection and identification of P. gingivalis in clinical samples.

Full Text

The Full Text of this article is available as a PDF (326.2 KB).

Selected References

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

  1. Boondeekhun H. S., Gurtler V., Odd M. L., Wilson V. A., Mayall B. C. Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction. J Med Microbiol. 1993 May;38(5):384–387. doi: 10.1099/00222615-38-5-384. [DOI] [PubMed] [Google Scholar]
  2. Brakstad O. G., Aasbakk K., Maeland J. A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol. 1992 Jul;30(7):1654–1660. doi: 10.1128/jcm.30.7.1654-1660.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cudjoe K. S., Thorsen L. I., Sørensen T., Reseland J., Olsvik O., Granum P. E. Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA. Int J Food Microbiol. 1991 Apr;12(4):313–321. doi: 10.1016/0168-1605(91)90145-f. [DOI] [PubMed] [Google Scholar]
  4. Deslauriers M., Mouton C. Targetting the production of monoclonal antibodies to the hemagglutinating adhesin of Bacteroides (Porphyromonas) gingivalis by injection of an immunoprecipitate from crossed immunoelectrophoresis. J Biol Buccale. 1991 Jun;19(2):155–160. [PubMed] [Google Scholar]
  5. Fournier D., Mouton C. Phenotypic characterization of human and animal biotypes within the species Porphyromonas gingivalis. Res Microbiol. 1993 Jul-Aug;144(6):435–444. doi: 10.1016/0923-2508(93)90051-3. [DOI] [PubMed] [Google Scholar]
  6. Frankel G., Riley L., Giron J. A., Valmassoi J., Friedmann A., Strockbine N., Falkow S., Schoolnik G. K. Detection of Shigella in feces using DNA amplification. J Infect Dis. 1990 Jun;161(6):1252–1256. doi: 10.1093/infdis/161.6.1252. [DOI] [PubMed] [Google Scholar]
  7. French C. K., Savitt E. D., Simon S. L., Eklund S. M., Chen M. C., Klotz L. C., Vaccaro K. K. DNA probe detection of periodontal pathogens. Oral Microbiol Immunol. 1986 Nov;1(1):58–62. doi: 10.1111/j.1399-302x.1986.tb00320.x. [DOI] [PubMed] [Google Scholar]
  8. Gouvea V., Glass R. I., Woods P., Taniguchi K., Clark H. F., Forrester B., Fang Z. Y. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990 Feb;28(2):276–282. doi: 10.1128/jcm.28.2.276-282.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Greisen K., Loeffelholz M., Purohit A., Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol. 1994 Feb;32(2):335–351. doi: 10.1128/jcm.32.2.335-351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haffajee A. D., Socransky S. S. Microbial etiological agents of destructive periodontal diseases. Periodontol 2000. 1994 Jun;5:78–111. doi: 10.1111/j.1600-0757.1994.tb00020.x. [DOI] [PubMed] [Google Scholar]
  11. Hofstad T. Utility of newer techniques for classification and identification of pathogenic anaerobic bacteria. Clin Infect Dis. 1994 May;18 (Suppl 4):S250–S252. doi: 10.1093/clinids/18.supplement_4.s250. [DOI] [PubMed] [Google Scholar]
  12. Jensen J. S., Uldum S. A., Søndergård-Andersen J., Vuust J., Lind K. Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples. J Clin Microbiol. 1991 Jan;29(1):46–50. doi: 10.1128/jcm.29.1.46-50.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loesche W. J., Lopatin D. E., Stoll J., van Poperin N., Hujoel P. P. Comparison of various detection methods for periodontopathic bacteria: can culture be considered the primary reference standard? J Clin Microbiol. 1992 Feb;30(2):418–426. doi: 10.1128/jcm.30.2.418-426.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Luk J. M., Nnalue N. A., Lindberg A. A. Efficient production of mouse and rat monoclonal antibodies against the O antigens of Salmonella serogroup C1, using LPS-coated bacteria as immunogen. J Immunol Methods. 1990 May 25;129(2):243–250. doi: 10.1016/0022-1759(90)90445-2. [DOI] [PubMed] [Google Scholar]
  15. Lund A., Hellemann A. L., Vartdal F. Rapid isolation of K88+ Escherichia coli by using immunomagnetic particles. J Clin Microbiol. 1988 Dec;26(12):2572–2575. doi: 10.1128/jcm.26.12.2572-2575.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Muir P., Nicholson F., Jhetam M., Neogi S., Banatvala J. E. Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens. J Clin Microbiol. 1993 Jan;31(1):31–38. doi: 10.1128/jcm.31.1.31-38.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ménard C., Gosselin P., Duhaime J. F., Mouton C. Polymerase chain reaction using arbitrary primer for the design and construction of a DNA probe specific for Porphyromonas gingivalis. Res Microbiol. 1994 Oct;145(8):595–602. doi: 10.1016/0923-2508(94)90076-0. [DOI] [PubMed] [Google Scholar]
  18. Savitt E. D., Keville M. W., Peros W. J. DNA probes in the diagnosis of periodontal microorganisms. Arch Oral Biol. 1990;35 (Suppl):153S–159S. doi: 10.1016/0003-9969(90)90147-3. [DOI] [PubMed] [Google Scholar]
  19. Skjerve E., Rørvik L. M., Olsvik O. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl Environ Microbiol. 1990 Nov;56(11):3478–3481. doi: 10.1128/aem.56.11.3478-3481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Socransky S. S., Haffajee A. D. Evidence of bacterial etiology: a historical perspective. Periodontol 2000. 1994 Jun;5:7–25. doi: 10.1111/j.1600-0757.1994.tb00016.x. [DOI] [PubMed] [Google Scholar]
  21. Syed S. A., Loesche W. J. Survival of human dental plaque flora in various transport media. Appl Microbiol. 1972 Oct;24(4):638–644. doi: 10.1128/am.24.4.638-644.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Widjojoatmodjo M. N., Fluit A. C., Torensma R., Keller B. H., Verhoef J. Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella. Eur J Clin Microbiol Infect Dis. 1991 Nov;10(11):935–938. doi: 10.1007/BF02005447. [DOI] [PubMed] [Google Scholar]
  23. Widjojoatmodjo M. N., Fluit A. C., Torensma R., Verdonk G. P., Verhoef J. The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J Clin Microbiol. 1992 Dec;30(12):3195–3199. doi: 10.1128/jcm.30.12.3195-3199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wilde J., Eiden J., Yolken R. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J Clin Microbiol. 1990 Jun;28(6):1300–1307. doi: 10.1128/jcm.28.6.1300-1307.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zambon J. J., Bochacki V., Genco R. J. Immunological assays for putative periodontal pathogens. Oral Microbiol Immunol. 1986 Nov;1(1):39–47. doi: 10.1111/j.1399-302x.1986.tb00316.x. [DOI] [PubMed] [Google Scholar]
  26. Zappa U., Reinking-Zappa M., Graf H., Gmür R., Savitt E. Comparison of serological and DNA probe analyses for detection of suspected periodontal pathogens in subgingival plaque samples. Arch Oral Biol. 1990;35 (Suppl):161S–164S. doi: 10.1016/0003-9969(90)90148-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES