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