Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples

Comparative Proteomic Analysis of Two Varieties of Genetically Modified (GM) Embrapa 5.1 Common Bean (Phaseolus vulgaris L.) and Their Non-GM Counterparts

The genetically modified (GM) common bean event Embrapa 5.1 was commercially approved in Brazil in 2011; it is resistant to golden mosaic virus infection. In the present work grain proteome profiles of two Embrapa 5.1 common bean varieties, Pérola and Pontal, and their non-GM counterparts were compared by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). Analyses detected 23 spots differentially accumulated between GM Pérola and non-GM Pérola and 21 spots between GM Pontal and non-GM Pontal, although they were not the same proteins in Pérola and Pontal varieties, indicating that the variability observed may not be due to the genetic transformation. Among them, eight proteins were identified in Pérola varieties, and four proteins were identified in Pontal. Moreover, we applied principal component analysis (PCA) on 2-DE data, and variation between varieties was explained in the first two principal components. This work provides a first 2-DE-MS/MS-based analysis of Embrapa 5.1 common bean grains

Development of Plasmid DNA Reference Material for the Quantification of Genetically Modified Common Bean Embrapa 5.1

The genetically modified (GM) common bean Embrapa 5.1 was recently approved for commercialization. The reliable detection and quantification of GM organisms is strongly dependent on validated methods as well as calibration systems. This work presents the development of a calibrant plasmid for Embrapa 5.1 common bean detection. The reaction parameters were determined and compared for both the plasmid DNA (pDNA) and the genomic DNA (gDNA). PCR efficiencies for pDNA were 81% for the construction-specific assays and 76% for the taxon-specific assay, whereas for gDNA efficiencies were 94 and 93%, respectively. The limits of detection (LOD) in both qPCR assays were 10² and 10³ copies of gDNA and pDNA per PCR reaction, respectively. This is sufficient to detect 0.067 and 0.67% of GM common bean in 100 ng of DNA, respectively, which is in agreement with detecting the 1% GM content required by the Brazilian legislation