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.
Document Type: Research Article
Affiliations: 1: Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d'Etudes et de Recherches sur la Qualité des Aliments et sur les Procédés Agro-Alimentaires, Unité d'Etude Moléculaire des Contaminants Biologiques Alimentaires (EBA), 1-5 Rue de Belfort, 94700 Maisons-Alfort, France. 2: INRA, Unité Phytopathologie et Méthodologies de la Détection de Versailles (PMDV)/MDO, Route de St Cyr, 78026 Versailles Cedex, France. 3: Institut de l'Elevage, 9, Rue André Brouard, BP 70510, 49105 Angers Cedex 02, France. 4: CNIEL, 42 Rue de Châteaudun, 75314 Paris Cedex 09, France. 5: INRA, Unité de Biométrie, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France. 6: ARILAIT Recherches, 42 Rue de Châteaudun, 75009 Paris, France. 7: INRA, Unité Génomique et Physiologie de la Lactation (GPL), Département de Génétique Animale, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
Publication date: March 1, 2005
- The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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