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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #236147
Title: Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

Author
item LERMO, A - UNIV. DE BARCELONA, SPAIN
item ZACCO, E - UNIV. DE BARCELONA, SPAIN
item BARAK-CUNNINGHAM, JERI - UNV. WISCONSIN MADISON
item DELWICHE, M - UNV. CALIF. DAVIS
item CAMPOY, S - UNIV. DE BARCELONA, SPAIN
item BABE, J - UNIV. DE BARCELONA, SPAIN
item ALAGRET, S - UNIV. DE BARCELONA, SPAIN
item PIVIDORI, M - UNIV. DE BARCELONA, SPAIN

Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2008
Publication Date: 7/15/2009
Citation: Lermo, A., Zacco, E., Barak-Cunningham, J., Delwiche, M., Campoy, S., Babe, J., Alagret, S., Pividori, M. 2009. Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection. Biosensors and Bioelectronics. 23:1805-1811.

Interpretive Summary: 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_l-1 and 0.45 ng_l-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.

Technical Abstract: 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_l-1 and 0.45 ng_l-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.