Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

• Published in: Biosensors & bioelectronics Vol. 23; no. 12; pp. 1805 - 1811
• Main Authors: Lermo, A., Zacco, E., Barak, J., Delwiche, M., Campoy, S., Barbé, J., Alegret, S., Pividori, M.I.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.07.2008; Elsevier Science
• Subjects: (Strept)avidin; Biological and medical sciences; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Biosensors; Biotechnology; Colony Count, Microbial - instrumentation; Colony Count, Microbial - methods; Electrochemical DNA biosensor; Electrochemical genosensing; Electrochemistry - instrumentation; Escherichia coli; Escherichia coli O157 - genetics; Escherichia coli O157 - isolation & purification; Expressed Sequence Tags; Fundamental and applied biological sciences. Psychology; Magnetic beads; Methods. Procedures. Technologies; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Q-PCR; Various methods and equipments; Magnetic beads; Q-PCR; Electrochemical genosensing; Escherichia coli; Electrochemical DNA biosensor; (Strept)avidin; Avidin; Polymerase chain reaction; Biosensor; DNA; Pathogenic; Electrochemistry; Bacteria; Detection; Enterobacteriaceae
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2008.02.020

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng μl −1 and 0.45 ng μl −1 of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.