Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Apr;34(4):778–784. doi: 10.1128/jcm.34.4.778-784.1996

Immunomagnetic separation and solid-phase detection of Bordetella pertussis.

M Stark 1, E Reizenstein 1, M Uhlén 1, J Lundeberg 1
PMCID: PMC228892  PMID: 8815083

Abstract

In the present study, novel solid-phase methods were used for both sample preparation and PCR detection of Bordetella pertussis. The sample preparation was performed by immunomagnetic separation with paramagnetic beads coated with polyclonal antibodies directed toward the surface antigens of the bacteria. The precoated immunobeads were directly used on nasopharyngeal aspirates to capture the bacteria on the solid support and were subsequently transferred to the PCR tube with no further manipulations. The region encompassing the pertussis toxin promoter was analyzed to allow direct discrimination between the three major Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica). The resulting amplicons were captured on a second magnetic solid phase, allowing detection and restriction analysis of the target sequence. A colorimetric detection system based on a DNA binding fusion protein enabled the use of standardized enzyme-linked immunosorbent format tests both for the detection of Bordetella spp. and for species evaluation. When the optimized system was evaluated on 55 clinical aspirate samples, 21 of 22 (95%) culture-positive samples were positive by the system that we developed. In addition, two samples were positive by the PCR-based assay, while the culture assay was negative. The implications of these results are discussed.

Full Text

The Full Text of this article is available as a PDF (774.6 KB).

Selected References

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

  1. Birkebaek N. H., Heron I., Skjødt K. Bordetella pertussis diagnosed by polymerase chain reaction. APMIS. 1994 Apr;102(4):291–294. [PubMed] [Google Scholar]
  2. Brinchmann J. E., Albert J., Vartdal F. Few infected CD4+ T cells but a high proportion of replication-competent provirus copies in asymptomatic human immunodeficiency virus type 1 infection. J Virol. 1991 Apr;65(4):2019–2023. doi: 10.1128/jvi.65.4.2019-2023.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bäckman A., Johansson B., Olcén P. Nested PCR optimized for detection of Bordetella pertussis in clinical nasopharyngeal samples. J Clin Microbiol. 1994 Oct;32(10):2544–2548. doi: 10.1128/jcm.32.10.2544-2548.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  5. Douglas E., Coote J. G., Parton R., McPheat W. Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene. J Med Microbiol. 1993 Feb;38(2):140–144. doi: 10.1099/00222615-38-2-140. [DOI] [PubMed] [Google Scholar]
  6. Glare E. M., Paton J. C., Premier R. R., Lawrence A. J., Nisbet I. T. Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction. J Clin Microbiol. 1990 Sep;28(9):1982–1987. doi: 10.1128/jcm.28.9.1982-1987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grimprel E., Bégué P., Anjak I., Betsou F., Guiso N. Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection. J Clin Microbiol. 1993 Oct;31(10):2745–2750. doi: 10.1128/jcm.31.10.2745-2750.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  9. Gustafsson L., Hallander H. O., Olin P., Reizenstein E., Storsaeter J. A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med. 1996 Feb 8;334(6):349–355. doi: 10.1056/NEJM199602083340602. [DOI] [PubMed] [Google Scholar]
  10. Hallander H. O., Reizenstein E., Renemar B., Rasmuson G., Mardin L., Olin P. Comparison of nasopharyngeal aspirates with swabs for culture of Bordetella pertussis. J Clin Microbiol. 1993 Jan;31(1):50–52. doi: 10.1128/jcm.31.1.50-52.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hallander H. O., Storsaeter J., Möllby R. Evaluation of serology and nasopharyngeal cultures for diagnosis of pertussis in a vaccine efficacy trial. J Infect Dis. 1991 May;163(5):1046–1054. doi: 10.1093/infdis/163.5.1046. [DOI] [PubMed] [Google Scholar]
  12. He Q., Mertsola J., Soini H., Viljanen M. K. Sensitive and specific polymerase chain reaction assays for detection of Bordetella pertussis in nasopharyngeal specimens. J Pediatr. 1994 Mar;124(3):421–426. doi: 10.1016/s0022-3476(94)70365-5. [DOI] [PubMed] [Google Scholar]
  13. Hedrum A., Lundeberg J., Påhlson C., Uhlén M. Immunomagnetic recovery of Chlamydia trachomatis from urine with subsequent colorimetric DNA detection. PCR Methods Appl. 1992 Nov;2(2):167–171. doi: 10.1101/gr.2.2.167. [DOI] [PubMed] [Google Scholar]
  14. Houard S., Hackel C., Herzog A., Bollen A. Specific identification of Bordetella pertussis by the polymerase chain reaction. Res Microbiol. 1989 Sep;140(7):477–487. doi: 10.1016/0923-2508(89)90069-7. [DOI] [PubMed] [Google Scholar]
  15. Hultman T., Bergh S., Moks T., Uhlén M. Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. Biotechniques. 1991 Jan;10(1):84–93. [PubMed] [Google Scholar]
  16. Islam D., Lindberg A. A. Detection of Shigella dysenteriae type 1 and Shigella flexneri in feces by immunomagnetic isolation and polymerase chain reaction. J Clin Microbiol. 1992 Nov;30(11):2801–2806. doi: 10.1128/jcm.30.11.2801-2806.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li Z., Jansen D. L., Finn T. M., Halperin S. A., Kasina A., O'Connor S. P., Aoyama T., Manclark C. R., Brennan M. J. Identification of Bordetella pertussis infection by shared-primer PCR. J Clin Microbiol. 1994 Mar;32(3):783–789. doi: 10.1128/jcm.32.3.783-789.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lichtinghagen R., Diedrich-Glaubitz R., von Hörsten B. Identification of Bordetella pertussis in nasopharyngeal swabs using the polymerase chain reaction: evaluation of detection methods. Eur J Clin Chem Clin Biochem. 1994 Mar;32(3):161–167. doi: 10.1515/cclm.1994.32.3.161. [DOI] [PubMed] [Google Scholar]
  19. Locht C., Keith J. M. Pertussis toxin gene: nucleotide sequence and genetic organization. Science. 1986 Jun 6;232(4755):1258–1264. doi: 10.1126/science.3704651. [DOI] [PubMed] [Google Scholar]
  20. Lundeberg J., Wahlberg J., Uhlén M. Affinity purification of specific DNA fragments using a lac repressor fusion protein. Genet Anal Tech Appl. 1990 May;7(3):47–52. doi: 10.1016/0735-0651(90)90040-m. [DOI] [PubMed] [Google Scholar]
  21. Meade B. D., Bollen A. Recommendations for use of the polymerase chain reaction in the diagnosis of Bordetella pertussis infections. J Med Microbiol. 1994 Jul;41(1):51–55. doi: 10.1099/00222615-41-1-51. [DOI] [PubMed] [Google Scholar]
  22. Olsvik O., Popovic T., Skjerve E., Cudjoe K. S., Hornes E., Ugelstad J., Uhlén M. Magnetic separation techniques in diagnostic microbiology. Clin Microbiol Rev. 1994 Jan;7(1):43–54. doi: 10.1128/cmr.7.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Onorato I. M., Wassilak S. G. Laboratory diagnosis of pertussis: the state of the art. Pediatr Infect Dis J. 1987 Feb;6(2):145–151. doi: 10.1097/00006454-198702000-00002. [DOI] [PubMed] [Google Scholar]
  24. Reizenstein E., Johansson B., Mardin L., Abens J., Möllby R., Hallander H. O. Diagnostic evaluation of polymerase chain reaction discriminative for Bordetella pertussis, B. parapertussis, and B. bronchiseptica. Diagn Microbiol Infect Dis. 1993 Oct;17(3):185–191. doi: 10.1016/0732-8893(93)90094-n. [DOI] [PubMed] [Google Scholar]
  25. Reizenstein E., Lindberg L., Möllby R., Hallander H. O. Validation of nested Bordetella PCR in pertussis vaccine trial. J Clin Microbiol. 1996 Apr;34(4):810–815. doi: 10.1128/jcm.34.4.810-815.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schläpfer G., Cherry J. D., Heininger U., Uberall M., Schmitt-Grohé S., Laussucq S., Just M., Stehr K. Polymerase chain reaction identification of Bordetella pertussis infections in vaccinees and family members in a pertussis vaccine efficacy trial in Germany. Pediatr Infect Dis J. 1995 Mar;14(3):209–214. doi: 10.1097/00006454-199503000-00008. [DOI] [PubMed] [Google Scholar]
  27. Seesod N., Lunderberg J., Hedrum A., Aslund L., Holder A., Thaithong S., Uhlén M. Immunomagnetic purification to facilitate DNA diagnosis of Plasmodium falciparum. J Clin Microbiol. 1993 Oct;31(10):2715–2719. doi: 10.1128/jcm.31.10.2715-2719.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wadowsky R. M., Laus S., Libert T., States S. J., Ehrlich G. D. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab. J Clin Microbiol. 1994 Apr;32(4):1054–1057. doi: 10.1128/jcm.32.4.1054-1057.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wahlberg J., Lundeberg J., Hultman T., Uhlén M. General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6569–6573. doi: 10.1073/pnas.87.17.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Woolfrey B. F., Moody J. A. Human infections associated with Bordetella bronchiseptica. Clin Microbiol Rev. 1991 Jul;4(3):243–255. doi: 10.1128/cmr.4.3.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van der Zee A., Agterberg C., Peeters M., Schellekens J., Mooi F. R. Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis. J Clin Microbiol. 1993 Aug;31(8):2134–2140. doi: 10.1128/jcm.31.8.2134-2140.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES