Establishment of Dipstick Development Technology for Detection of Cry1Ac in Transgenic Plants ( Vol-2,Issue-6,November - December 2017 )
Author: Muhammad Irfan, Muhammad Asif, Aftab Bashir, Kauser Abdullah Malik
Keyword: Nitrocellulose membrane, Cry1Ac, Dipstick, Nano-colloidal gold particles.
Abstract: The insecticidal Bt Cry1Ac protein is, currently used for transgene expression in numerous crops or deliberating resistance against lepidopteron pests. Since the introduction of Bt cotton in Pakistan. It has been demonstrated that the technology has achieved the goal of providing an effective tool for lepidopteron control. In this study, single step, sensitive and specific dipstick strip test for the revealing of recombinant Cry1Ac protein in the transgenic plants was established. Anti-Bt-Cry1Ac antibodies and goat anti-rabbit IgG antibodies were used in test and control lines, respectively. The distance betweenthese lines were optimized as 0.5 cm. Polyclonal rabbit anti Bt-Cry1Ac antibody conjugated to nanocolloidal gold (20 nm of OD 15 and 40 nm; OD 1 in separate experiments) at pH 9.2 was used to serve as a probe for detecting Cry1Ac protein in transgenic Bt cotton samples. Both conjugate solutions were coated on separate polyester conjugate pads (0.7 cm × 0.5 cm). The total size of strip was 7.5 cm × 0.5 cm. For 20 nm gold conjugated strip, purple color test line and for 40 nm gold red color test line indicated the binding of gold labeled antibodies to antigen. The assay was corroborated with transgenic cotton samples with protein extraction buffer 1X PBS of pH 7. This on-site test offers fast screening for any genetically modified crop devouring Cry1Ac transgenic protein.
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