Immuno‐analytical method development for detection of transgenic Cry1Ac protein and its validation

Abstract

BACKGROUND Bacillus thuringiensis (Bt) synthesizes Cry1Ac protein, which is toxic to many lepidopteran pests, and the cry1ac gene has been expressed in several transgenic crop plants. The Cry1Ac protein has been isolated from Bt kurstaki HD73 and purified to homogeneity. Polyclonal antibodies were raised against purified Cry1Ac in rabbits and goat. Sandwich ELISA was developed for Cry1Ac using goat IgG as a coating antibody, and affinity‐purified rabbit IgG as the primary antibody. RESULTS The sensitivity of the assay was in the range of 0.47–1000 ng. It was subsequently employed in validating biological samples. Fifteen different cotton‐seed samples were screened: 12 were found to be Bt positive and 3 Bt negative. The CS7 seeds showed the highest Bt content of 8.51 ± 0.45 μg g−1, followed by CS8 (6.0 ± 0.02 μg g−1), CS15 (5.9 ± 0.03 μg g−1), CS9 (5.5 ± 0.05 μg g−1), and CS10 (4.83 ± 0.013 μg g−1). The CS5 seeds showed Bt content of 3.6 ± 0.21 μg g−1. The F2 generation, CS6 (Kaveri seeds) showed lower Bt content (2.9 ± 0.06 μg g−1). The CL5 samples showed Cry1Ac content of 0.99 ± 0.009 μg g−1. The amount of Cry1Ac protein in leaves, stem, and roots of germinated Bt cotton plants (CS10 and CS4) were 1.76 ± 0.15 μg g−1, 1.9 ± 0.01 μg g−1, 2.0 ± 0.1 μg g−1, and 1.6 ± 0.15 μg g−1, 1.9 ± 0.01 μg g−1, and 2.0 ± 0.01 μg g−1 dry tissue, respectively. CONCLUSION The method developed can be used for screening the expression levels of Cry1Ac in different transgenic Bt cultivars and also spurious Bt cotton seeds procured by farmers. © 2019 Society of Chemical Industry

Advanced Technologies in On-Site Detection of Genetically Modified Products

“…With the increased emergence of new genetically modified (GM) products, the discussion about their safety is also increasing, and people are more and more concerned about whether a product is GM, which makes the demand for transgenic testing higher, and people's requirements for testing is growing as well [2,3]. The cornerstone of breeding, safety evaluation, supervision, and the healthy development of GM technology is the detection of GM substances, and the proteins [4], nucleic acids [5], and metabolite levels of these can be the biomarkers of detection. The testing of GM products has developed greatly with the growing demand for field testing and has become increasingly simple to use, rapid, and portable in recent years, which has aided in the preservation of people's right to know.…”