title: Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.
abstract: To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach can be adopted as a standard procedure for complete molecular characterization of Bt cotton. These assays will be of interest and use to importers, breeders, research laboratories, safety regulators, and food processors for detection of cry1Ac bearing GMOs.
authors.affiliation: Industrial Toxicology Research Centre, Post Box No. 80, M.G. Marg Lucknow-226001 U.P., India.
authors.forename: Chandra K
authors.initials: CK
authors.lastname: Singh
authors.forename: Abhishek
authors.initials: A
authors.lastname: Ojha
authors.forename: Devendra N
authors.initials: DN
authors.lastname: Kachru
mesh_terms: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000737:chemistry; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004731:Endotoxins / Q000235:genetics*; D005784:Gene Amplification; D005819:Genetic Markers; D003368:Gossypium / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified / Q000235:genetics*; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D018744:DNA, Plant; D004731:Endotoxins; D005819:Genetic Markers; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D004262:DNA Restriction Enzymes