title: 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.
authors.affiliation: National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110 002, India. gjr@nbpgr.ernet.in
authors.forename: Gurinder Jit
authors.initials: GJ
authors.lastname: Randhawa
authors.forename: Monika
authors.initials: M
authors.lastname: Singh
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D017796:Caulimovirus / Q000235:genetics; D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004731:Endotoxins / Q000096:biosynthesis / Q000235:genetics*; D005819:Genetic Markers; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics*; D019868:Kanamycin Kinase; D057230:Limit of Detection; D030781:Organisms, Genetically Modified; D012275:Oryza / Q000737:chemistry* / Q000235:genetics*; D018515:Plant Leaves / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D019076:Transgenes; D014404:Tubulin / Q000235:genetics
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D017931:DNA Primers; D018744:DNA, Plant; D004731:Endotoxins; D005819:Genetic Markers; D006460:Hemolysin Proteins; D014404:Tubulin; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D019868:Kanamycin Kinase