Detection of Genetically Modified Corn (Bt176) in Spiked Cow Blood Samples by Polymerase Chain Reaction and Immunoassay Methods

Abstract

The fate of DNA and protein transgenic sequences in products derived from animals fed transgenic crops has recently raised public interest. Sensitive molecular tests targeting the Bt176 genetic construct and the transgenic Cry1Ab protein were developed to determine whether plant sequences, especially transgenic sequences, are present in animal products. A protocol for total DNA extraction and purification from cow whole blood samples was first drawn up and assessed by spiking with known amounts of DNA from Bt176 maize. The limit of detection for transgenic sequences (35S promoter and Bt176-specific junction sequence) was determined by both the polymerase chain reaction–enzyme-linked immunosorbent assay (PCR–ELISA) and the 5′-nuclease PCR assay. Four additional PCR systems were built to substantiate the results. The first detects a mono-copy maize-specific sequence (ADH promoter). Two others target multi-copy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The last one, used as a positive control, targets a mono-copy animal sequence (αs1-casein gene). Both methods detected a minimum spiking at 25 copies of Bt176 maize/mL in 10 mL whole blood samples. The sandwich ELISA kit used detected down to 1 ng transgenic Cry1Ab protein/mL spiked whole blood.

Persistence of Plant DNA Sequences in the Blood of Dairy Cows Fed with Genetically Modified (Bt176) and Conventional Corn Silage

“…Since Bt176 copy number is half of p35S, we cannot discard the hypothesis of GMO derived sequence being present in these 2 cow blood samples at the limit of detection level. In such a case, the discrepancy between p35S and Bt176 might be due to differences between PCR test efficiencies as previously observed .…”

“…In a first experimental set, 4 (p35S, adh , psaB, CSN1S1) qRT-PCR systems previously developed and validated ( , , CRL-GMFF validated methods ) were used to detect GMO and plant DNA sequences among animal DNA. Ct values lower than 45 cycles were considered as positive results.…”

“…A second set of PCR tests was performed on dilutions 1/10, 1/20 and 1/50 (to avoid any inhibitory effect) with a more specific GMO test (Bt176) and a nuclear (26S rRNA) multicopy plant test .…”

“…Given the methods’ LOD (multicopy target methods being several hundreds to 1,000 more sensitive than the oligo-copy target methods whose LOD is almost 20 copies ) and volume analyzed per PCR (5 μL of test portion corresponding to 125 μL of blood), GMO sequences might be found only when using at least 1,000-fold larger blood sample assuming the mean number of GMO sequence present per tube is at least of 5, i.e. more than 125 mL (see Appendix).…”

“…DNA was extracted in 3 independent triplicates from total blood samples (2.5 mL per extraction) and quantified by fluorimetry as described by Petit et al . Bovine and corn haploid equivalent genomes were assumed to be 3.3 and 2.5 pg DNA, respectively , .…”