Ring - Mediated Isothermal Amplification Technology For Exogenous Transgenic Components Detection And Its Cooperative Verification

The safety of genetically modified food (GM food) in recent years has been the hot topic in the public. Therefore, the genetically modified food safety regulation has becomes more important. For the detection of exogenous transgenic ingredients of genetically modified (GM) food, the detection tend to not only for the conventional single target nucleic acid detection but also for rapid, accurate and high-throughput multiple targets detection analysis at the same time.Conventional GM food detection method is mainly polymerase chain reaction (PCR) technology, real-time fluorescent quantitative PCR technology, but these transgenic detection technology requires sophisticated temperature cycle instrument and spends more time, thus greatly restricts their application of rapid detection on the spot or the field. But the isothermal nucleic acid amplification technique has obtained the rapid development in recent years, the loop-mediated isothermal amplification techniques (LAMP) has been well developed with the advantages of high specificity and sensitivity, simple instruments and operation, high-efficiency in the detection of bacteria, virus and genetically modified foodThis research has developed LAMP detection method for the transgenic exogenous genetically modified ingredients in food, we mainly choose three common genetically modified ingredients among genetically modified food.According to the three common genetically modified ingredients (CP4 epsps, pat ,Npt II) sequence,we designed special LAMP primers, optimized LAMP reaction system, and established visible identification with SYBR Green I (1000 x) fluorescent dye through the loop-mediated isothermal amplification detection method.Experimental results proved that the LAMP detection method for these 3 common exogenous transgenic ingredients in GM food is of high specificity, sensitivity and limit of detection can achieve 5 copies, is higher than the detection limit of traditional PCR, and the LAMP testing just need about one hour. In addition to observed the change of the color by naked eye identification directly, it could also detected through agarose gel electrophoresis.In order to verify the practicability and validity of loop-isothermal amplification method, we had invited ten research institutions to help us validate the LAMP methods. After the validation, these methods have been applied for the practical sample detection. Therefore loop-mediated isothermal amplification technique provides a strong technical support for the rapid, convenient detection field in the future.