RESUMO
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.
Assuntos
Proteínas de Bactérias/sangue , Proteínas de Bactérias/genética , Toxinas Bacterianas/sangue , Toxinas Bacterianas/genética , DNA de Plantas/genética , Endotoxinas/sangue , Endotoxinas/genética , Alimentos Geneticamente Modificados , Zea mays/química , Zea mays/genética , Animais , Toxinas de Bacillus thuringiensis , Bovinos , Núcleo Celular/química , Cloroplastos/química , Primers do DNA , DNA de Plantas/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas , Plantas/química , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.
Assuntos
Alimentos Geneticamente Modificados , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Zea mays/química , Zea mays/genética , Região 3'-Flanqueadora/genética , Região 5'-Flanqueadora/genética , Sequência de Bases , Primers do DNA , DNA de Plantas/genética , União Europeia , Legislação sobre Alimentos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Sementes/químicaRESUMO
A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.
Assuntos
DNA de Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Zea mays/genética , Primers do DNA , DNA de Plantas/química , DNA Viral/química , DNA Viral/genética , Sondas de Oligonucleotídeos , Vírus de Plantas/genética , Reprodutibilidade dos Testes , Sementes/química , Vírus/químicaRESUMO
Four real-time polymerase chain reaction systems aiming at the specific detection and quantification of maize DNA are described. They have been developed in four independent laboratories targeting different maize sequences, i.e., alcohol dehydrogenase (Adh1), high mobility group protein (hmga), invertase A (ivr1), and zein, respectively. They were all fully specific, showing a very similar quantification accuracy along a number of distantly related maize cultivars and being either single or low copy number genes. They were highly sensitive and exhibited limits of quantification below 100 maize genomic copies. In consequence, they are considered suitable for use as maize specific endogenous reference genes in DNA analyses, including GMO quantitative tests.
