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

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 10(5) 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 10(3) CFU mL(-1) S. pneumoniae.

Magnetic Beads‐Based Electrochemical Sensors Applied to the Detection and Quantification of Bioterrorism/Biohazard Agents

“…These results demonstrated the usefulness of the developed sensors as screening or alarm devices indicating the possible presence of coliforms, together with their applicability to drinking water quality assurance (no coliform bacteria per 100 mL of water). The same research group employed similar strategies in the development of DNA sensors for the detection of a characteristic 235-bp region of the gene coding for autolysin (LytA), a specific pneumococcus virulent factor [48]. The obtained results demonstrated the usefulness of the developed methodology for rapid, simple, specific, quantitative, and sensitive detection of aPCR amplicons with a detection limit of 1.1 nM.…”

“…The amplified products obtained by daPCR from bacteria attached to specific capture antibody-modified MBs were detected similarly to that commented above using the electrochemical genosensor [48]. The developed methodology allowed an easy discrimination between only 100 cfu mL À1 S. pneumoniae cultures and high concentrations (~1.0 10 8 cfu mL…”

“…An electrochemical magnetoimmuno-PCR approach has been recently described for direct and highly sensitive detection of S. pneumoniae [51]. The amplified products obtained by daPCR from bacteria attached to specific capture antibody-modified MBs were detected similarly to that commented above using the electrochemical genosensor [48].…”

Magnetic Particles Coupled to Disposable Screen Printed Transducers for Electrochemical Biosensing

“…A disposable amperometric MMPs-based DNA sensor coupled to asymmetric PCR (aPCR) was developed for the ultrasensitive determination of Streptococcus pneumoniae [47]. This approach was based on the selective hybridization of specific biotynilated capture DNA probe, complementary to a specific region of the pneumococcal lytA gene.…”

An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification

“…Moreover, the content of target in real samples is low. To solve this difficulty, some detection methods based on polymerase chain reaction (PCR) derived techniques are developed, such as asymmetric PCR (Campuzano et al, 2011;Chen et al, 2013), magnetic separation (Stevenson et al, 2008;Ma et al, 2012), size separation on denaturing urea polyacrylamide gel electrophoresis (Marimuthu et al, 2012;Liang et al, 2015) and toehold-mediated DNA strand displacement (Khodakov et al, 2013). Although these methods can generate ssDNA or singlestranded toehold, there are also different weaknesses to limit their use in bioanalysis.…”