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Duplex Electrochemical DNA Sensor to Detect Bacillus anthracis CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP

Affiliation
INTERFIBIO Consolidated Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona, Spain
Magriñá, Ivan;
Affiliation
INTERFIBIO Consolidated Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona, Spain
Jauset-Rubio, Miriam;
Affiliation
INTERFIBIO Consolidated Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona, Spain
Ortiz, Mayreli;
GND
1019579781
Affiliation
Friedrich-Loeffler-Institut, Naumburger Str. 96a, Jena, Germany
Tomaso, Herbert;
Affiliation
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, Prague, Czech Republic
Simonova, Anna;
Affiliation
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, Prague, Czech Republic
Hocek, Michal;
Affiliation
INTERFIBIO Consolidated Research Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, Tarragona, Spain
O'Sullivan, Ciara K.

We report the duplex amplification of two plasmid DNA markers involved in the virulence of Bacillus anthracis, CAP and PAG, and the direct electrochemical detection of these amplicons. The method consists of the simultaneous amplification of the two targets in a single-pot reaction via polymerase chain reaction (PCR) using tailed primers and ferrocene-labeled dATP. Following amplification, the PCR products hybridize to probes immobilized on electrodes in a microfabricated electrode array chip. The incorporated ferrocene labeled dATP is then detected using square wave voltammetry. We evaluated the effect of electrolyte cations, anions, and concentration to condense, bend, and shrink double-stranded DNA and their effect on the intensity of the ferrocene signal. We obtained detection limits of 0.8 and 3.4 fM for CAP and PAG targets, respectively. We successfully developed a method to detect the presence of both targets in genomic DNA extracted from real samples.

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