The Study On The Detection Of The Cry1A Gene In Transgenic Insect-resistant Rice And Cotton Using Loop-mediated Isothermal Amplification

Since1980s, genetic engineering made a rapid development, and it brings moreconvenient for our life. Nowadays, many genetically modified crops have appeared in ourlives. GM crops offer distinctive advantages like the resistance against insects, weed,disease and drought, higher yield, better nutritional value, etc. While bringing the infiniteprofit to human beings, the security of transgenic technique was still questioned by people.Since the transgenic technique emerged, people were all thinking and arguing about itsfood safety, ecollgy security. Considering the GM food’s safety, relevant law andregulations have been issued to label the genetically modified organisms （GMOs） and theirderived products in many counties. It is necessary to confirm the existence of transgenicingredient and the appropriate analytical methodology to detect these GM crops isbecoming urgent and important.Loop-mediated isothermal amplification （LAMP） is a nucleic acid amplificationmethod developed by Eiken Chemical Co., Ltd., Japan. It has the potential advantages incomparison with the PCR for detection of a gene because of its simplicity, rapidity,specificity and cost-effectiveness. It is characterized by the use of4different primersspecifically designed to recognize6distinct regions on the target gene and the reactionprocess proceeds at a constant temperature （about65oC） using a DNA polymerase withstrand displacement activity. It provides high amplification efficiency, with DNA beingamplified109-1010times in less than60min.A LAMP method was established for detection of the artificially modified cry1A genein transgenic insect-resistant rice and cotton. The specific LAMP primers of GM rice andGM cotton were designed on the basis of the sequence of the modified cry1A gene（according to the patent of Sandui Guo, Wanchao Ni, Qiongfang Xu, the Chinese academyof agricultural sciences biological technology research center, application number95119563.8, patent number CN1134981A） using the LAMP primer design supportsoftware program, the primer explorer software. The reaction conditions including theconcentration of Mg²⁺, concentration of dNTPs, concentration of Bst DNA polymerase,and temperature were optimized.The LAMP reaction for detection of the modified cry1A gene in transgenicinsect-resistant rice was carried out in a25μL of total reaction mixture volume containing 0.5mmol/L of each outer primers F3and B3,3.0mmol/L of each inner primers FIP andBIP,2.5μL Bst DNA polymerase buffer （20mM Tris-HCl（pH8.8,25oC）,10mM KCl,10mM （NH₄）₂SO₄,2mM MgSO₄,0.1%Triton X-100）,0.5mmol/L Mg²⁺,1.5mmol/L ofeach deoxynucleoside triphosphate,0.7μL of8U Bst DNA polymerase, and50ng thetarget DNA. The mixture was incubated at65oC for60min in a heating block and thenheated at80oC for2min to terminate the reaction. The LAMP for detection of the GMcotton was carried out in a25μL of total reaction is same as rice except1.0mmol/L Mg²⁺,and5mmol/L dNTPs. LAMP products were electrophoresed in a2%agarose gel. The sizeof the fragment of the LAMP product is in good agreement with the predicted ladderlikesize.The detection limits of PCR and LAMP assays using the DNA templates extractedwith CTAB method were0.1%and0.01%, respectively. The LAMP assay was found to be10-fold more sensitive than the traditional PCR assay. In addition, there were differentsamples （contain three kinds of non-GM rice, three kinds of cotton, two kinds of non-GMsoybean, two kinds of non-GM tomato, and3kinds of positive Bt strains containing cry1Agene） to be detected by PCR and LAMP respectively in order to evaluate the specificity ofprimers. The results of the one GM cotton and two non-GM cottons were positive andthose of other samples were negative. The PCR results are consistent with LAMP results,indicating that the both LAMP and PCR method have a high specificity. Moreover, bothmotheds can detect the cry1A gene in two kinds of non-GM cottons imply that cry1A genemay migrate from GM cotton to non-GM cotton during cultivation in the field.The time of LAMP assay for detection of the modified cry1A gene in transgenicinsect-resistant rice and cotton was less than2.5h （including the procedures of DNApurification, LAMP and electrophoresis assay）. If add SYBR Green I to the reactionproduct, the LAMP assay enabled GM rice and GM cotton to be detected in less than2h.In conclusion, LAMP amplification provides a faster and more sensitive method forthe detection of GM rice and GM cotton and it has higher specificity. It established a soundtechnology platform for the rapid detection of genetically modified and laid an importantfoundation for further development of the industry or national standards of GM cropsassay.