title: A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing.
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.
authors.affiliation: Autonomous University of Barcelona, Bellaterra, Spain.
authors.forename: Anabel
authors.initials: A
authors.lastname: Lermo
authors.forename: Susana
authors.initials: S
authors.lastname: Liébana
authors.forename: Susana
authors.initials: S
authors.lastname: Campoy
authors.forename: Silvia
authors.initials: S
authors.lastname: Fabiano
authors.forename: M Inés
authors.initials: MI
authors.lastname: García
authors.forename: Adriana
authors.initials: A
authors.lastname: Soutullo
authors.forename: Martín J
authors.initials: MJ
authors.lastname: Zumárraga
authors.forename: Salvador
authors.initials: S
authors.lastname: Alegret
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Pividori
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004076:Digoxigenin / Q000737:chemistry; D055664:Electrochemical Techniques / Q000379:methods*; D008403:Mass Screening / Q000379:methods*; D008892:Milk / Q000382:microbiology*; D009163:Mycobacterium bovis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D001710:Biotin; D004076:Digoxigenin