Analytical and Bioanalytical Chemistry

, Volume 399, Issue 7, pp 2413–2420 | Cite as

Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of Streptococcus pneumoniae

  • Susana Campuzano
  • María Pedrero
  • José L. García
  • Ernesto García
  • Pedro García
  • José M. Pingarrón
Original Paper


A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 105 times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 103 CFU mL−1S. pneumoniae.


A lytA-based magnetogenosensor for pneumococcal identification: The lytA gene, encoding the main pneumococcal autolysin, is a suitable target for an accurate diagnosis of the pneumococcal disease. Asymmetric PCR amplification with precisely designed primers together with amperometric measurements allows a rapid and accurate differentiation between S. pneumoniae and closely related streptococci (see picture)


Streptococcus pneumoniae Biosensors Gene technology Bacterial identification lytA gene 



This research was supported by a grant from the Dirección General de Investigación Científica y Técnica (SAF2009-10824). CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of Spanish Instituto de Salud Carlos III. The financial support of Santander/Complutense Research Project PR 27/05-13953, and of the Spanish Ministerio de Ciencia e Innovación Research Project CTQ2009-09351BQU, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are also gratefully acknowledged. S.C. acknowledges a “Juan de la Cierva” research contract.

Supplementary material

216_2010_4645_MOESM1_ESM.pdf (88 kb)
ESM 1 (PDF 87 kb)


  1. 1.
    WHO (2007) Wkly Epidemiol Rec 82:93–104Google Scholar
  2. 2.
    Lund E, Henrichsen J (1978) Methods Microbiol 12:241–262CrossRefGoogle Scholar
  3. 3.
    Dowell SF, Garman RL, Liu G, Levine OS, Yang YH (2001) Clin Infect Dis 32:824–825CrossRefGoogle Scholar
  4. 4.
    Samra Z, Shmuely H, Nahum E, Paghis D, Ben-Ari J (2003) Diagn Microbiol Infect Dis 45:237–240CrossRefGoogle Scholar
  5. 5.
    Moore NJ, Fent MK, Koulchin VA, Molokova EV (2004) US Patent No. US 6,824,997 B1.Google Scholar
  6. 6.
    0Ehara N, Fukushima K, Kakeya H, Mukae H, Akamatsu S, Kageyama A, Saito A, Kohno S (2008) J Med Microbiol 57:820–826CrossRefGoogle Scholar
  7. 7.
    Sheppard CL, Harrison TG, Morris R, Hogan A, George RC (2004) J Med Microbiol 53:189–195CrossRefGoogle Scholar
  8. 8.
    Harris KA, Turner P, Green EA, Hartley JC (2008) J Clin Microbiol 46:2751–2758CrossRefGoogle Scholar
  9. 9.
    Rouphael NG, Atwell-Melnick N, Longo D, Whaley M, Carlone GM, Sampson JS, Ades EW (2008) Diagn Microbiol Infect Dis 62:23–25CrossRefGoogle Scholar
  10. 10.
    Smith MD, Sheppard CL, Hogan A, Harrison TG, Dance DAB, Derrington P, George RC, on behalf of the South West Pneumococcus Study Group (2009) J Clin Microbiol 47:1046–1049CrossRefGoogle Scholar
  11. 11.
    Marriott HM, Mitchell TJ, Dockrell DH (2008) Curr Mol Med 8:497–509CrossRefGoogle Scholar
  12. 12.
    García P, García JL, García E, López R (1986) Gene 43:265–272CrossRefGoogle Scholar
  13. 13.
    López R, García E (2004) FEMS Microbiol Rev 28:553–580CrossRefGoogle Scholar
  14. 14.
    Jefferies J, Nieminen L, Kirkham LA, Johnston C, Smith A, Mitchell TJ (2007) J Bacteriol 189:627–632CrossRefGoogle Scholar
  15. 15.
    Simões AS, Sá-Leão R, Eleveld MJ, Tavares DA, Carriço JA, Bootsma HJ, Hermans PWM (2010) J Clin Microbiol 48:238–246CrossRefGoogle Scholar
  16. 16.
    Obregón V, García P, García E, Fenoll A, López R, García JL (2002) J Clin Microbiol 40:2545–2554CrossRefGoogle Scholar
  17. 17.
    Llull D, López R, García E (2006) J Clin Microbiol 44:1250–1256CrossRefGoogle Scholar
  18. 18.
    Lucarelli F, Tombelli S, Minunni M, Marrazza G, Mascini M (2008) Anal Chim Acta 609:139–159CrossRefGoogle Scholar
  19. 19.
    Drummond TG, Hill MG, Barton JK (2003) Nat Biotechnol 21:1192–1199CrossRefGoogle Scholar
  20. 20.
    Liao JC, Mastali M, Gau V, Suchard MA, Møller AK, Bruckner DA, Babbitt JT, Li Y, Gornbein J, Landaw EM, McCabe ERB, Churchill BM, Haake DA (2006) J Clin Microbiol 44:61–570Google Scholar
  21. 21.
    Mastali M, Babbitt JT, Li Y, Landaw EM, Gau V, Churchill BM, Haake DA (2008) J Clin Microbiol 46:2707–2716CrossRefGoogle Scholar
  22. 22.
    Paleček E, Fojta M (2007) Talanta 74:276–290CrossRefGoogle Scholar
  23. 23.
    Fuentes M, Mateo C, Rodriguez A, Casqueiro M, Tercero JC, Riese HH, Fernández-Lafuente R, Guisán JM (2006) Biosens Bioelectron 21:1574–1580CrossRefGoogle Scholar
  24. 24.
    Wang J, Flechsig GU, Erdem A, Korbut O, Gründler P (2004) Electroanal 16:928–931CrossRefGoogle Scholar
  25. 25.
    Morales M, García P, de la Campa AG, Liñares J, Ardanuy C, García E (2010) J Bacteriol 192:2624–2632CrossRefGoogle Scholar
  26. 26.
    Lacks S, Hotchkiss RD (1960) Biochim Biophys Acta 39:508–517CrossRefGoogle Scholar
  27. 27.
    Loaiza ÓA, Campuzano S, Pedrero M, Pividori MI, García P, Pingarrón JM (2008) Anal Chem 80:8239–8245CrossRefGoogle Scholar
  28. 28.
    Campuzano S, Pedrero M, Pingarrón JM (2005) Talanta 66:1310–1319CrossRefGoogle Scholar
  29. 29.
    Long GL, Winefordner JD (1983) Anal Chem 55:712A–724ACrossRefGoogle Scholar
  30. 30.
    Hasebe K, Osteryoung J (1975) Anal Chem 47:2412–2418CrossRefGoogle Scholar
  31. 31.
    Hernández-Santos D, González-García MB, Costa-García A (2005) Anal Chem 77:2868–2874CrossRefGoogle Scholar
  32. 32.
    Xu D, Huang K, Liu Z, Liu Y, Ma L (2001) Electroanal 13:882–887CrossRefGoogle Scholar
  33. 33.
    Carpini G, Lucarelli F, Marrazza G, Mascini M (2004) Biosens Bioelectron 20:167–175CrossRefGoogle Scholar
  34. 34.
    Del Giallo ML, Ariksoysal DO, Marrazza G, Mascini M, Ozsoz M (2005) Anal Lett 38:2509–2523CrossRefGoogle Scholar
  35. 35.
    Tang L, Zeng G, Shen G, Li Y, Liu C, Li Z, Luo J, Fan C, Yang C (2009) Biosens Bioelectron 24:1474–1479CrossRefGoogle Scholar
  36. 36.
    Hernández-Santos D, Díaz-González M, González-García MB, Costa-García A (2004) Anal Chem 76:6887–6893CrossRefGoogle Scholar
  37. 37.
    Fode-Vaughan KA, Wimpee CF, Remsen CC, Collins MLP (2001) Biotechniques 31:598–607Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Susana Campuzano
    • 1
    • 2
    • 4
  • María Pedrero
    • 1
  • José L. García
    • 2
  • Ernesto García
    • 2
    • 3
  • Pedro García
    • 2
    • 3
  • José M. Pingarrón
    • 1
  1. 1.Departamento de Química Analítica, Facultad de CC. QuímicasUniversidad Complutense de MadridMadridSpain
  2. 2.Departamento de Microbiología Molecular y Biología de las InfeccionesCentro de Investigaciones Biológicas, CSICMadridSpain
  3. 3.Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES, initiative of Instituto de Salud Carlos III)MallorcaSpain
  4. 4.Department of NanoengineeringUniversity of California San DiegoLa JollaUSA

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