The Application Of G-quadruplex Cascade Signals Amplification Visual In Transgenic Detection

With the development of biotechnology,the wide cultivation of transgenic cropsin the worldhas made great contributions to solving the food crisis.The transgenic technology has broken the restriction of reproductive isolation,which made the transgenic crops be able to integrate many kinds of genes.Although there is no direct proof of scientific data,the transferred exogenous genes still posea potential threat to the environment and food safety.Therefore,the evaluation,supervision and detection of safety are very important to thetransgenic cropsand related products.In this paper,a method for the detection of transgenic components was established with a novel nucleotide probecoupled with DNA isothermal amplification.In the establishment process,the combined use of the probe and a variety of DNA amplification methods was tried to develop an appropriate system and the universal usability of the combined system in different conditions was studied.Through a specific probe containing the complementary sequence of G-quardruplex was designed for the isothermal amplification,and the single stranded DNA moleculecontainingpartial sequences of Bt gene was visually detected.And 1?L 10 X Isothermal Amplification,0.5?L 10 X NEBuffer 3.1,10 mM Mg²⁺,4mM Hemin,5U Nb.BbvCI,4U Bst 2.0 WarmStart is the best condition,and use probe contains three G-tetrads intramolecular G-quadruplex to detection for 30 min incubation time.we develped a simple label-free G-quadruplex isothermal amplification detection system.Through the combined use of G-quardruplexnucleotide probe and DNA amplification.The genomic DNA of transgenic rice Kefeng 6 was used as the target DNA of the detection of cry1Ab/cry1 Ac gene.We use primer contains intramolecular G-quadruplex with 1?M initial concentration to perform PCR program,In order to reduce the influence of the background signal is the result of the test.When the plant genomic DNA containing cry1Ab/cry1 Ac gene exists in the system,large numbers of G-quardruplex will be accumulated in the system through the reaction processes of PCR and isothermal amplification,and the detection results can be then obtained by the chromogenic system with the colorless solution into green.Based on this,a visualizeddetection method that could directly detect cry1Ab/cry1 Ac gene from the transgenic samples was established.Besides,The effect of the cascade method in detecting samples of othertransgenic crops especiallytransgenic maize MON89034 with a complex genome structure was further explored.We use 5?L and 2?L primers of corresponding to their respective system in Nb.BbvCI and Nt.BstNBI detection system to perform PCR,then incubation at 37? and 55? respectively for 30 min.last,The Nb.BbvCI and Nt.Bst NBI detection system has 100 and 50 copy detection limit respectively.Based on this,a kind of visualized detection method for transgenic crops with good selectivity and universal usability was established in the paper.Finally,the work of the paper is summarized and our own views and expectationson the future research work are put forward.