A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing.
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.
Lermo
Anabel
A
Autonomous University of Barcelona, Bellaterra, Spain.
Liébana
Susana
S
Campoy
Susana
S
Fabiano
Silvia
S
García
M Inés
MI
Soutullo
Adriana
A
Zumárraga
Martín J
MJ
Alegret
Salvador
S
Pividori
M Isabel
MI
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
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D001710:Biotin; D004076:Digoxigenin