Decaplex and real-time PCR based detection of MON531 and MON15985 Bt cotton events.
The genetically modified (GM) Bt crops expressing delta-endotoxins from Bacillus thuringiensis provide protection against a wide range of lepidopteron insect pests throughout the growing season of the plant. Bt cotton is the only commercialized crop in India that is planted on an area of 7.6 million hectares. With the increase in development and commercialization of transgenic crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different transgenic events. The present study reports on the development of a decaplex polymerase chain reaction (PCR) assay for simultaneous detection of transgene sequences, specific transgene constructs, and endogenous stearoyl acyl desaturase (Sad1) gene in two events of Bt cotton, i.e., MON531 and MON15985. The decaplex PCR assay is an efficient tool to identify and discriminate the two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India. Real-time PCR assays were also developed for quantification of cry1Ac and cry2Ab genes being employed in these two events. The quantitative method was developed using seven serial dilutions containing different levels of Bt cotton DNA mixed with a non-Bt counterpart ranging from 0.01 to 100%. The results revealed that the biases from the true value and the relative standard deviations were all within the range of ±20%. The limit of quantification (LOQ) of the developed real-time PCR method has also been established up to 0.01%.
Randhawa
Gurinder Jit
GJ
Referral Centre for Molecular Diagnosis of Transgenic Planting Materials, National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110012, India. gurinder.randhawa@rediffmail.com
Chhabra
Rashmi
R
Singh
Monika
M
D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018556:Crops, Agricultural / Q000235:genetics; D018744:DNA, Plant / Q000302:isolation & purification / Q000378:metabolism; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism; D003368:Gossypium / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D010572:Pest Control, Biological; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods; D019076:Transgenes
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D018744:DNA, Plant; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis