[go: up one dir, main page]
More Web Proxy on the site http://driver.im/ Skip to main content

Advertisement

Springer Nature Link
Log in

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

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

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−1  S. 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)

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
£29.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (United Kingdom)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. WHO (2007) Wkly Epidemiol Rec 82:93–104

    Google Scholar 

  2. Lund E, Henrichsen J (1978) Methods Microbiol 12:241–262

    Article  Google Scholar 

  3. Dowell SF, Garman RL, Liu G, Levine OS, Yang YH (2001) Clin Infect Dis 32:824–825

    Article  CAS  Google Scholar 

  4. Samra Z, Shmuely H, Nahum E, Paghis D, Ben-Ari J (2003) Diagn Microbiol Infect Dis 45:237–240

    Article  CAS  Google Scholar 

  5. Moore NJ, Fent MK, Koulchin VA, Molokova EV (2004) US Patent No. US 6,824,997 B1.

  6. 0Ehara N, Fukushima K, Kakeya H, Mukae H, Akamatsu S, Kageyama A, Saito A, Kohno S (2008) J Med Microbiol 57:820–826

    Article  CAS  Google Scholar 

  7. Sheppard CL, Harrison TG, Morris R, Hogan A, George RC (2004) J Med Microbiol 53:189–195

    Article  CAS  Google Scholar 

  8. Harris KA, Turner P, Green EA, Hartley JC (2008) J Clin Microbiol 46:2751–2758

    Article  CAS  Google Scholar 

  9. Rouphael NG, Atwell-Melnick N, Longo D, Whaley M, Carlone GM, Sampson JS, Ades EW (2008) Diagn Microbiol Infect Dis 62:23–25

    Article  CAS  Google Scholar 

  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 47:1046–1049

    Article  CAS  Google Scholar 

  11. Marriott HM, Mitchell TJ, Dockrell DH (2008) Curr Mol Med 8:497–509

    Article  CAS  Google Scholar 

  12. García P, García JL, García E, López R (1986) Gene 43:265–272

    Article  Google Scholar 

  13. López R, García E (2004) FEMS Microbiol Rev 28:553–580

    Article  Google Scholar 

  14. Jefferies J, Nieminen L, Kirkham LA, Johnston C, Smith A, Mitchell TJ (2007) J Bacteriol 189:627–632

    Article  CAS  Google Scholar 

  15. Simões AS, Sá-Leão R, Eleveld MJ, Tavares DA, Carriço JA, Bootsma HJ, Hermans PWM (2010) J Clin Microbiol 48:238–246

    Article  Google Scholar 

  16. Obregón V, García P, García E, Fenoll A, López R, García JL (2002) J Clin Microbiol 40:2545–2554

    Article  Google Scholar 

  17. Llull D, López R, García E (2006) J Clin Microbiol 44:1250–1256

    Article  CAS  Google Scholar 

  18. Lucarelli F, Tombelli S, Minunni M, Marrazza G, Mascini M (2008) Anal Chim Acta 609:139–159

    Article  CAS  Google Scholar 

  19. Drummond TG, Hill MG, Barton JK (2003) Nat Biotechnol 21:1192–1199

    Article  CAS  Google Scholar 

  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 44:61–570

    Google Scholar 

  21. Mastali M, Babbitt JT, Li Y, Landaw EM, Gau V, Churchill BM, Haake DA (2008) J Clin Microbiol 46:2707–2716

    Article  CAS  Google Scholar 

  22. Paleček E, Fojta M (2007) Talanta 74:276–290

    Article  Google Scholar 

  23. Fuentes M, Mateo C, Rodriguez A, Casqueiro M, Tercero JC, Riese HH, Fernández-Lafuente R, Guisán JM (2006) Biosens Bioelectron 21:1574–1580

    Article  CAS  Google Scholar 

  24. Wang J, Flechsig GU, Erdem A, Korbut O, Gründler P (2004) Electroanal 16:928–931

    Article  CAS  Google Scholar 

  25. Morales M, García P, de la Campa AG, Liñares J, Ardanuy C, García E (2010) J Bacteriol 192:2624–2632

    Article  CAS  Google Scholar 

  26. Lacks S, Hotchkiss RD (1960) Biochim Biophys Acta 39:508–517

    Article  CAS  Google Scholar 

  27. Loaiza ÓA, Campuzano S, Pedrero M, Pividori MI, García P, Pingarrón JM (2008) Anal Chem 80:8239–8245

    Article  CAS  Google Scholar 

  28. Campuzano S, Pedrero M, Pingarrón JM (2005) Talanta 66:1310–1319

    Article  CAS  Google Scholar 

  29. Long GL, Winefordner JD (1983) Anal Chem 55:712A–724A

    Article  CAS  Google Scholar 

  30. Hasebe K, Osteryoung J (1975) Anal Chem 47:2412–2418

    Article  CAS  Google Scholar 

  31. Hernández-Santos D, González-García MB, Costa-García A (2005) Anal Chem 77:2868–2874

    Article  Google Scholar 

  32. Xu D, Huang K, Liu Z, Liu Y, Ma L (2001) Electroanal 13:882–887

    Article  CAS  Google Scholar 

  33. Carpini G, Lucarelli F, Marrazza G, Mascini M (2004) Biosens Bioelectron 20:167–175

    Article  CAS  Google Scholar 

  34. Del Giallo ML, Ariksoysal DO, Marrazza G, Mascini M, Ozsoz M (2005) Anal Lett 38:2509–2523

    Article  CAS  Google Scholar 

  35. Tang L, Zeng G, Shen G, Li Y, Liu C, Li Z, Luo J, Fan C, Yang C (2009) Biosens Bioelectron 24:1474–1479

    Article  CAS  Google Scholar 

  36. Hernández-Santos D, Díaz-González M, González-García MB, Costa-García A (2004) Anal Chem 76:6887–6893

    Article  Google Scholar 

  37. Fode-Vaughan KA, Wimpee CF, Remsen CC, Collins MLP (2001) Biotechniques 31:598–607

    CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Author notes

  1. Susana Campuzano

    Present address: Department of Nanoengineering, University of California San Diego, La Jolla, CA, 92093, USA

Authors and Affiliations

  1. Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, 28040, Madrid, Spain

    Susana Campuzano, María Pedrero & José M. Pingarrón

  2. Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain

    Susana Campuzano, José L. García, Ernesto García & Pedro García

  3. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES, initiative of Instituto de Salud Carlos III), Carretera Soller Km 12, Bunyola, 07110, Mallorca, Illes Balears, Spain

    Ernesto García & Pedro García

Authors
  1. Susana Campuzano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Pedrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José L. García
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ernesto García
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Pedro García
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. José M. Pingarrón
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José M. Pingarrón.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 87 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Campuzano, S., Pedrero, M., García, J.L. et al. Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of Streptococcus pneumoniae . Anal Bioanal Chem 399, 2413–2420 (2011). https://doi.org/10.1007/s00216-010-4645-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-010-4645-0

Keywords

Search

Navigation