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Multiplex, construct-specific, and real-time PCR-based analytical methods for Bt rice with cry1Ac gene.

Abstract

Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptLL) marker gene, and an endogenous a-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise.

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  • Publisher Full Text
  • Authors

    Randhawa GJ, Singh M

    Institution

    National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110 002, India. gjr@nbpgr.ernet.in

    Source

    Journal of AOAC International 95:1 pg 186-94

    MeSH

    Agrobacterium tumefaciens
    Bacillus thuringiensis
    Bacterial Proteins
    Caulimovirus
    DNA Primers
    DNA, Plant
    Endotoxins
    Genetic Markers
    Hemolysin Proteins
    Kanamycin Kinase
    Limit of Detection
    Organisms, Genetically Modified
    Oryza sativa
    Plant Leaves
    Plants, Genetically Modified
    Plasmids
    Real-Time Polymerase Chain Reaction
    Reproducibility of Results
    Transgenes
    Tubulin

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    22468358