A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing Authors Anabel Lermo Autonomous University of Barcelona, Bellaterra, Spain Susana Liébana Autonomous University of Barcelona, Bellaterra, Spain Susana Campoy Autonomous University of Barcelona, Bellaterra, Spain Silvia Fabiano National University of the Littoral, Santa Fe, Argentina M. Inés García National University of the Littoral, Santa Fe, Argentina Adriana Soutullo General Management of Animal Health, Ministry of Production, Santa Fe, Argentina Martín J. Zumárraga CNIA-INTA Castelar, Buenos Aires, Argentina Salvador Alegret Autonomous University of Barcelona, Bellaterra, Spain M. Isabel Pividori Autonomous University of Barcelona, Bellaterra, Spain Keywords: Mycobacterium bovis, electrochemical DNA biosensor, avidin, magnetic beads, double-tagging PCR Abstract A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium’s DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing. [Int Microbiol 2010; 13(2):91-97] Author Biographies Anabel Lermo, Autonomous University of Barcelona, Bellaterra, Spain Autonomous University of Barcelona, Bellaterra, Spain Susana Liébana, Autonomous University of Barcelona, Bellaterra, Spain Autonomous University of Barcelona, Bellaterra, Spain Susana Campoy, Autonomous University of Barcelona, Bellaterra, Spain Autonomous University of Barcelona, Bellaterra, Spain Silvia Fabiano, National University of the Littoral, Santa Fe, Argentina National University of the Littoral, Santa Fe, Argentina M. Inés García, National University of the Littoral, Santa Fe, Argentina National University of the Littoral, Santa Fe, Argentina Adriana Soutullo, General Management of Animal Health, Ministry of Production, Santa Fe, Argentina General Management of Animal Health, Ministry of Production, Santa Fe, Argentina Martín J. Zumárraga, CNIA-INTA Castelar, Buenos Aires, Argentina CNIA-INTA Castelar, Buenos Aires, Argentina Salvador Alegret, Autonomous University of Barcelona, Bellaterra, Spain Autonomous University of Barcelona, Bellaterra, Spain M. Isabel Pividori, Autonomous University of Barcelona, Bellaterra, Spain Autonomous University of Barcelona, Bellaterra, Spain Downloads PDF Published 2010-09-20 Issue Vol. 13 No. 2 (2010) Section Research Articles License Submission of a manuscript to International Microbiology implies: that the work described has not been published before, including publication in the World Wide Web (except in the form of an Abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that all the coauthors have agreed to its publication. 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