Analytical and Bioanalytical Chemistry

, Volume 399, Issue 7, pp 2413–2420

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

Authors

  • Susana Campuzano
    • Departamento de Química Analítica, Facultad de CC. QuímicasUniversidad Complutense de Madrid
    • Departamento de Microbiología Molecular y Biología de las InfeccionesCentro de Investigaciones Biológicas, CSIC
    • Department of NanoengineeringUniversity of California San Diego
  • María Pedrero
    • Departamento de Química Analítica, Facultad de CC. QuímicasUniversidad Complutense de Madrid
  • José L. García
    • Departamento de Microbiología Molecular y Biología de las InfeccionesCentro de Investigaciones Biológicas, CSIC
  • Ernesto García
    • Departamento de Microbiología Molecular y Biología de las InfeccionesCentro de Investigaciones Biológicas, CSIC
    • Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES, initiative of Instituto de Salud Carlos III)
  • Pedro García
    • Departamento de Microbiología Molecular y Biología de las InfeccionesCentro de Investigaciones Biológicas, CSIC
    • Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES, initiative of Instituto de Salud Carlos III)
    • Departamento de Química Analítica, Facultad de CC. QuímicasUniversidad Complutense de Madrid
Original Paper

DOI: 10.1007/s00216-010-4645-0

Cite this article as:
Campuzano, S., Pedrero, M., García, J.L. et al. Anal Bioanal Chem (2011) 399: 2413. doi:10.1007/s00216-010-4645-0

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−1S. pneumoniae.

https://static-content.springer.com/image/art%3A10.1007%2Fs00216-010-4645-0/MediaObjects/216_2010_4645_Figa_HTML.gif
Figure

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)

Keywords

Streptococcus pneumoniae Biosensors Gene technology Bacterial identification lytA gene

Supplementary material

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

Copyright information

© Springer-Verlag 2011