Detection of feed-derived Bt-maize DNA in goat milk and evaluation of possible horizontal gene transfer to bacteria
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The presence and the transforming capacity of feed-derived DNA in milk obtained from eight lactating goats fed on maize E176 silage were evaluated. The presence of single- and multi-copy maize genes was monitored by real-time PCR and conventional PCR. Chromosomal and plastid DNA extracted directly from maize flour and silage were readily amplifiable by conventional PCR, however, only chloroplast-specific gene fragments of 199 and 532 bp were detected in about 60 and 20%, respectively, of the milk samples analysed. Quantification by real time PCR yielded 9.5 (±6.7) × 102 plant gene copies/mL of milk sediment. In contrast, all milk samples were negative for the chromosomally located maize zein gene or the E176 specific cry1Ab transgene. The minimum concentration of plant DNA required for detection was 0.01 ng/mL raw milk for the chloroplast-specific fragment and 1 ng/mL for the cry1Ab transgene. The detection limit was determined by spiking milk samples with plant DNA prior to DNA extraction. The transformation capability of DNA in milk was evaluated after constructing a marker rescue system in Acinetobacter baylyi strain BD413 based on recombinational repair of the bla TEM gene. Two systems were developed that allowed the plant marker gene to recombine with the bacterial chromosome [A. baylyi BD413 (pUC-bla)] or plasmids [A. baylyi BD413 (pBBR1MCS-2Φ)]. The two systems showed the same efficiency of transformation, yielding 10−5 transformants per recipient cell (t/r) using plasmid pUC18 or a 1,873 bp fragment as donor DNA, and 3.5 × 10−11 t/r using DNA isolated from flour (E176). No transformants were detected when exposing the recipient bacterium to DNA extracted from maize (E176) silage or from milk obtained from goats feed maize (E176).