A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing

LERMO, ANABEL; LIÉBANA, SUSANA; CAMPOY, SUSANA; FABIANO, SILVIA; GARCÍA, MARÍA INÉS; SOUTULLO, ADRIANA; ZUMÁRRAGA, MARTÍN JOSÉ; ALEGRET, SALVADOR; PIVIDORI, MARÍA ISABEL

INTERNATIONAL MICROBIOLOGY

VIGUERA EDITORES

Año: 2010 vol. 13 p. 91 - 97

1139-6709

A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developedbased on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification wascarried 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-HRPconjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promisingfeatures 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 tuberculinskin testing.