title: 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 (alpha(s1)-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.
authors.affiliation: Agence Française de Sécurité Sanitaire des Aliments, Unité d'Etude Moléculaire des Contaminants Biologiques Alimentaires (EBA), 94700 Maisons-Alfort, France. l.petit@afssa.fr
authors.forename: Laetitia
authors.initials: L
authors.lastname: Petit
authors.forename: Fabienne
authors.initials: F
authors.lastname: Baraige
authors.forename: Yves
authors.initials: Y
authors.lastname: Bertheau
authors.forename: Philippe
authors.initials: P
authors.lastname: Brunschwig
authors.forename: Annick
authors.initials: A
authors.lastname: Diolez
authors.forename: Koenraad
authors.initials: K
authors.lastname: Duhem
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Duplan
authors.forename: Patrick
authors.initials: P
authors.lastname: Fach
authors.forename: André
authors.initials: A
authors.lastname: Kobilinsky
authors.forename: Stephen
authors.initials: S
authors.lastname: Lamart
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schattner
authors.forename: Patrice
authors.initials: P
authors.lastname: Martin
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000097:blood* / Q000235:genetics*; D001427:Bacterial Toxins / Q000097:blood* / Q000235:genetics*; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics* / Q000302:isolation & purification; D004731:Endotoxins / Q000097:blood* / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D030841:Food, Genetically Modified; D006460:Hemolysin Proteins; D010944:Plants / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D018744:DNA, Plant; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis