Diagnosis of Meningococcal Infection Using Internally Controlled Multiplex Real-Time PCR

  • Ala-Eddine DeghmaneEmail author
  • Eva Hong
  • Muhamed-Kheir Taha
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)


Neisseria meningitidis (Nm) is a leading cause of invasive infections associated with high mortality and morbidity, notably meningitis and septicemia. Etiological rapid diagnosis is key for the preventive management of invasive meningococcal disease (IMD). However, conventional methods for diagnosis are time-consuming and could be hampered by the difficulties in culturing the isolates from clinical specimens especially due to early antibiotic treatment. Therefore, sensitive, specific and rapid non-culture-based methods are valuable for early diagnosis, effective therapy, and prevention. Here we describe a real-time PCR multiplex assays for the detection of Nm targeting the meningococcal-specific gene crgA, coding for a LysR-like transcriptional regulator, and six serogroup-specific (A, B, C, W, X, Y) Nm capsular genes, using a Qiagen column-based method for the optimum isolation of DNA from clinical specimens. Internal quality controls were included to monitor extraction of DNA, inhibition and the technical validation of the PCR as well.

Key words

Neisseria meningitidis Multiplex real-time PCR Clinical specimens DNA isolation Phocine herpes virus crgA Serogroups 



This work was supported by the Institut Pasteur, Paris.


  1. 1.
    Whittaker R, Dias JG, Ramliden M, Kodmon C, Economopoulou A, Beer N, Pastore Celentano L (2017) The epidemiology of invasive meningococcal disease in EU/EEA countries, 2004–2014. Vaccine 35(16):2034–2041CrossRefGoogle Scholar
  2. 2.
    Caugant DA, Maiden MC (2009) Meningococcal carriage and disease—population biology and evolution. Vaccine 27(Suppl 2):B64–B70CrossRefGoogle Scholar
  3. 3.
    Stephens DS, Greenwood B, Brandtzaeg P (2007) Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 369(9580):2196–2210Google Scholar
  4. 4.
    WHO (13 Aug 2015) Meningococcal Meningitis. Fact sheet no. 141Google Scholar
  5. 5.
    Edmond K, Clark A, Korczak VS, Sanderson C, Griffiths UK, Rudan I (2010) Global and regional risk of disabling sequelae from bacterial meningitis: a systematic review and meta-analysis. Lancet Infect Dis 10(5):317–328CrossRefGoogle Scholar
  6. 6.
    Thompson MJ, Ninis N, Perera R, Mayon-White R, Phillips C, Bailey L, Harnden A, Mant D, Levin M (2006) Clinical recognition of meningococcal disease in children and adolescents. Lancet 367(9508):397–403CrossRefGoogle Scholar
  7. 7.
    Nadel S (2016) Treatment of meningococcal disease. J Adolesc Health 59(2 Suppl):S21–S28CrossRefGoogle Scholar
  8. 8.
    CDC (2015) Meningococcal disease (Neisseria meningitidis). 2015 case definition. CDC website. http://www.cdcgov/nndss/conditions/meningococcal-disease/case-definition/2015. Accessed 19 June 2018
  9. 9.
    Ragunathan L, Ramsay M, Borrow R, Guiver M, Gray S, Kaczmarski EB (2000) Clinical features, laboratory findings and management of meningococcal meningitis in England and Wales: report of a 1997 survey. Meningococcal meningitis: 1997 survey report. J Inf Secur 40(1):74–79Google Scholar
  10. 10.
    Cartwright K, Reilly S, White D, Stuart J (1992) Early treatment with parenteral penicillin in meningococcal disease. BMJ 305(6846):143–147CrossRefGoogle Scholar
  11. 11.
    Sobanski MA, Barnes RA, Coakley WT (2001) Detection of meningococcal antigen by latex agglutination. Methods Mol Med 67:41–59PubMedGoogle Scholar
  12. 12.
    Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846CrossRefGoogle Scholar
  13. 13.
    Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88(16):7276–7280Google Scholar
  14. 14.
    Bryant PA, Li HY, Zaia A, Griffith J, Hogg G, Curtis N, Carapetis JR (2004) Prospective study of a real-time PCR that is highly sensitive, specific, and clinically useful for diagnosis of meningococcal disease in children. J Clin Microbiol 42(7):2919–2925CrossRefGoogle Scholar
  15. 15.
    Carrol ED, Thomson AP, Shears P, Gray SJ, Kaczmarski EB, Hart CA (2000) Performance characteristics of the polymerase chain reaction assay to confirm clinical meningococcal disease. Arch Dis Child 83(3):271–273CrossRefGoogle Scholar
  16. 16.
    Taha MK (2000) Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol 38(2):855–857Google Scholar
  17. 17.
    Niesters HG (2001) Quantitation of viral load using real-time amplification techniques. Methods 25(4):419–429CrossRefGoogle Scholar
  18. 18.
    Longo MC, Berninger MS, Hartley JL (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93(1):125–128CrossRefGoogle Scholar
  19. 19.
    Roth SJ, Tischer BK, Kovacs KM, Lydersen C, Osterrieder N, Tryland M (2013) Phocine herpesvirus 1 (PhHV-1) in harbor seals from Svalbard, Norway. Vet Microbiol 164(3–4):286–292CrossRefGoogle Scholar
  20. 20.
    Guiver M, Borrow R, Marsh J, Gray SJ, Kaczmarski EB, Howells D, Boseley P, Fox AJ (2000) Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA. FEMS Immunol Med Microbiol 28(2):173–179CrossRefGoogle Scholar
  21. 21.
    Tzeng YL, Noble C, Stephens DS (2003) Genetic basis for biosynthesis of the (alpha 1→4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X. Infect Immun 71(12):6712–6720CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ala-Eddine Deghmane
    • 1
    Email author
  • Eva Hong
    • 1
  • Muhamed-Kheir Taha
    • 1
  1. 1.Invasive Bacterial Infections UnitInstitut PasteurParis CedexFrance

Personalised recommendations