Tewari, shikha , Goel, Sudhir .
Detection and Quantitation of Genetically Modified Foods using Real Time PCR.
In India, genetically modified foods/organisms are regulated under the purview of the 1986 Indian Environment (Protection) Act (henceforth, the EP Act). These rules and regulations cover the areas of research as well as large-scale applications of GMOs and products, throughout the country involving manufacture, use, import, export, storage and research. The target substances covered are all genetically engineered organisms including microorganisms, plants and animals. Indian Government is following a policy of case-by-case approval of transgenic crops. Henceforth, Bt. Cotton is the first and the only transgenic crop approved by GEAC (regulatory body of DBT, New Delhi) for commercial cultivation in 6 States of India namely Andhra Pradesh, Gujarat, Karnataka, Madhya Pradesh, Maharashtra and Tamil Nadu. Over the last years, there has been a dramatic and steady increase in the surface area planted with transgenic crops globally. The major transgenic foodstuffs are maize, soybean, oilseed rape (canola) cotton, and tomato etcetera. There are many laboratories in India which are also in different stages of developing variety of GM crops. Regulations for the use of genetically modified organisms (GMO) and their labeling are being implemented worldwide, which demand reliable and accurate methods to detect the transgenes in raw materials and food products. Current E.U. legislation limits for the accidental presence of Genetically Modified Organism (GMO) derived products in foodstuff are 0.9 % w/w. As reported by Hepeng jia (Nature biotechnology, 2003) China has opened the free entry of GM crops in the country with regulatory norms. To implement the regulatory norms they needed to import detection kits costing handsomely as they could not foresee this problem of not having any cost effective indigenous methodologies. The present study is aimed with the objective to develop the facility and standardization of protocols for detection and quantitation of GM Foods / organisms keeping in view the present scenario of restrictions in import of GM foods/ Products etc. in India and also to avoid situation in near future, with changes in regulatory norms in our country as faced by China. To quantitatively determine the level of GMO derived products found in foodstuff, the most sensitive method currently available is Real-Time Polymerase Chain Reaction (PCR).There are two general approaches for the quantification of nucleic acids, the comparative Ct approach and the standard curve approach, which are currently in use for GMO detection. We have applied both the approaches for quantitation of Genetically Modified Foods by Real Time PCR usin standards of GM food supplied by SD Fine Chemicals, Geel Belgium. As per the earlier reports where plasmid DNA was used for quantative purposes, the present work focuses using genomic DNA for the quantitation of GM genes to be used with reproducible results. Quantitative PCR (qPCR) strategy involved dye SYBR Green I and dual labeled probes. The results of RR Soybean concentration will be discussed along with MON810. It was observed that PCR efficiency of the housekeeping and GM gene was close to 0.99. The sensitivity of detection was up to the level of single copy of gene. Therefore, our attempt to develop detection protocols for GM Foods/products and attempting the use of genomic DNA for quantitation purposes is duly achieved and this strategy of using genomic DNA for quantitative purposes might give an alternative to the use of plasmid DNA thus avoiding many steps and time saving put for the cloning of the specific genes.
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1 - Industrial Toxicology Research Centre, Lucknow, India, Petroleum Toxicology Division
2 - Industrial Toxicology Research Centre, Lucknow, India, Petroleum Toxicology Division, Mahatama Gandhi Marg,, PO. Box #80, Lucknow,, UP, 226001, India
Real time PCR
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM