Skip to main content
NCBI home page
Emerging Infectious Diseases logoLink to Emerging Infectious Diseases
. 2001 Nov-Dec;7(6):952–958. doi: 10.3201/eid0706.010606

Rapid identification of Bordetella pertussis pertactin gene variants using LightCycler real-time polymerase chain reaction combined with melting curve analysis and gel electrophoresis.

J Mäkinen 1, M K Viljanen 1, J Mertsola 1, H Arvilommi 1, Q He 1
PMCID: PMC2631898  PMID: 11747721

Abstract

Recently, eight allelic variants of the pertactin gene (prn1-8) have been characterized in Bordetella pertussis strains isolated in Europe and the United States. It has been suggested that the divergence of the pertactin types of clinical isolates from those of the B. pertussis vaccine strains is a result of vaccine-driven evolution. Sequencing of the prn, which is relatively time-consuming, has so far been the only method for the differentiation of prn types. We have developed a rapid real-time polymerase chain reaction assay suitable for large-scale screening of the prn type of the circulating strains. This method correctly identified the prn type of all tested 41 clinical isolates and two Finnish vaccine strains. The method is simple and reliable and provides an alternative for sequencing in pertussis research.

Full Text

The Full Text of this article is available as a PDF (110.9 KB).

Selected References

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

  1. Andrews R., Herceg A., Roberts C. Pertussis notifications in Australia, 1991 to 1997. Commun Dis Intell. 1997 May 29;21(11):145–148. doi: 10.33321/cdi.1997.21.30. [DOI] [PubMed] [Google Scholar]
  2. Bass J. W., Stephenson S. R. The return of pertussis. Pediatr Infect Dis J. 1987 Feb;6(2):141–144. doi: 10.1097/00006454-198702000-00001. [DOI] [PubMed] [Google Scholar]
  3. Bass J. W., Wittler R. R. Return of epidemic pertussis in the United States. Pediatr Infect Dis J. 1994 May;13(5):343–345. doi: 10.1097/00006454-199405000-00002. [DOI] [PubMed] [Google Scholar]
  4. Bohling S. D., Wittwer C. T., King T. C., Elenitoba-Johnson K. S. Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma. Lab Invest. 1999 Mar;79(3):337–345. [PubMed] [Google Scholar]
  5. Brennan M. J., Li Z. M., Cowell J. L., Bisher M. E., Steven A. C., Novotny P., Manclark C. R. Identification of a 69-kilodalton nonfimbrial protein as an agglutinogen of Bordetella pertussis. Infect Immun. 1988 Dec;56(12):3189–3195. doi: 10.1128/iai.56.12.3189-3195.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cassiday P., Sanden G., Heuvelman K., Mooi F., Bisgard K. M., Popovic T. Polymorphism in Bordetella pertussis pertactin and pertussis toxin virulence factors in the United States, 1935-1999. J Infect Dis. 2000 Oct 9;182(5):1402–1408. doi: 10.1086/315881. [DOI] [PubMed] [Google Scholar]
  7. Cebula T. A., Payne W. L., Feng P. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol. 1995 Jan;33(1):248–250. doi: 10.1128/jcm.33.1.248-250.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charles I. G., Dougan G., Pickard D., Chatfield S., Smith M., Novotny P., Morrissey P., Fairweather N. F. Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A. 1989 May;86(10):3554–3558. doi: 10.1073/pnas.86.10.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charles I. G., Li J. L., Roberts M., Beesley K., Romanos M., Pickard D. J., Francis M., Campbell D., Dougan G., Brennan M. J. Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis. Eur J Immunol. 1991 May;21(5):1147–1153. doi: 10.1002/eji.1830210509. [DOI] [PubMed] [Google Scholar]
  10. Cherry J. D., Gornbein J., Heininger U., Stehr K. A search for serologic correlates of immunity to Bordetella pertussis cough illnesses. Vaccine. 1998 Dec;16(20):1901–1906. doi: 10.1016/s0264-410x(98)00226-6. [DOI] [PubMed] [Google Scholar]
  11. De Serres G., Boulianne N., Douville Fradet M., Duval B. Pertussis in Quebec: ongoing epidemic since the late 1980s. Can Commun Dis Rep. 1995 Mar 15;21(5):45–48. [PubMed] [Google Scholar]
  12. Espinosa de los Monteros L. E., Galán J. C., Gutiérrez M., Samper S., García Marín J. F., Martín C., Domínguez L., de Rafael L., Baquero F., Gómez-Mampaso E. Allele-specific PCR method based on pncA and oxyR sequences for distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: intraspecific M. bovis pncA sequence polymorphism. J Clin Microbiol. 1998 Jan;36(1):239–242. doi: 10.1128/jcm.36.1.239-242.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. He Q., Mertsola J., Soini H., Skurnik M., Ruuskanen O., Viljanen M. K. Comparison of polymerase chain reaction with culture and enzyme immunoassay for diagnosis of pertussis. J Clin Microbiol. 1993 Mar;31(3):642–645. doi: 10.1128/jcm.31.3.642-645.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mastrantonio P., Spigaglia P., van Oirschot H., van der Heide H. G., Heuvelman K., Stefanelli P., Mooi F. R. Antigenic variants in Bordetella pertussis strains isolated from vaccinated and unvaccinated children. Microbiology. 1999 Aug;145(Pt 8):2069–2075. doi: 10.1099/13500872-145-8-2069. [DOI] [PubMed] [Google Scholar]
  15. Mooi F. R., Hallander H., Wirsing von König C. H., Hoet B., Guiso N. Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Eur J Clin Microbiol Infect Dis. 2000 Mar;19(3):174–181. doi: 10.1007/s100960050455. [DOI] [PubMed] [Google Scholar]
  16. Mooi F. R., He Q., van Oirschot H., Mertsola J. Variation in the Bordetella pertussis virulence factors pertussis toxin and pertactin in vaccine strains and clinical isolates in Finland. Infect Immun. 1999 Jun;67(6):3133–3134. doi: 10.1128/iai.67.6.3133-3134.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mooi F. R., van Oirschot H., Heuvelman K., van der Heide H. G., Gaastra W., Willems R. J. Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution. Infect Immun. 1998 Feb;66(2):670–675. doi: 10.1128/iai.66.2.670-675.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nitsche A., Steuer N., Schmidt C. A., Landt O., Siegert W. Different real-time PCR formats compared for the quantitative detection of human cytomegalovirus DNA. Clin Chem. 1999 Nov;45(11):1932–1937. [PubMed] [Google Scholar]
  19. Pietilä J., He Q., Oksi J., Viljanen M. K. Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J Clin Microbiol. 2000 Jul;38(7):2756–2759. doi: 10.1128/jcm.38.7.2756-2759.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ririe K. M., Rasmussen R. P., Wittwer C. T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem. 1997 Feb 15;245(2):154–160. doi: 10.1006/abio.1996.9916. [DOI] [PubMed] [Google Scholar]
  21. Shahin R. D., Brennan M. J., Li Z. M., Meade B. D., Manclark C. R. Characterization of the protective capacity and immunogenicity of the 69-kD outer membrane protein of Bordetella pertussis. J Exp Med. 1990 Jan 1;171(1):63–73. doi: 10.1084/jem.171.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Storsaeter J., Hallander H. O., Gustafsson L., Olin P. Levels of anti-pertussis antibodies related to protection after household exposure to Bordetella pertussis. Vaccine. 1998 Dec;16(20):1907–1916. doi: 10.1016/s0264-410x(98)00227-8. [DOI] [PubMed] [Google Scholar]
  23. Wittwer C. T., Herrmann M. G., Moss A. A., Rasmussen R. P. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques. 1997 Jan;22(1):130-1, 134-8. doi: 10.2144/97221bi01. [DOI] [PubMed] [Google Scholar]
  24. Wittwer C. T., Ririe K. M., Andrew R. V., David D. A., Gundry R. A., Balis U. J. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques. 1997 Jan;22(1):176–181. doi: 10.2144/97221pf02. [DOI] [PubMed] [Google Scholar]
  25. de Melker H. E., Conyn-van Spaendonck M. A., Rümke H. C., van Wijngaarden J. K., Mooi F. R., Schellekens J. F. Pertussis in The Netherlands: an outbreak despite high levels of immunization with whole-cell vaccine. Emerg Infect Dis. 1997 Apr-Jun;3(2):175–178. doi: 10.3201/eid0302.970211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. von Ahsen N., Oellerich M., Armstrong V. W., Schütz E. Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler. Clin Chem. 1999 Dec;45(12):2094–2101. [PubMed] [Google Scholar]

Articles from Emerging Infectious Diseases are provided here courtesy of Centers for Disease Control and Prevention

RESOURCES