The Study On The Detection Of Genetically Modified Soybean Using Loop-Mediated Isothermal Amplification

Labeling of food from transgenic crops become one of the key issues dominating public due to their safety concerns,and laws or regulations have been issued to label the GMOs and their derived products in more than thirty countries.At present,the genetically modified soybean has entered Chinese market without labeling and claiming, which makes it necessary to confirm the existence of transgenic ingredient and the appropriate analytical methodology to detect these GM crops is becoming urgent and important.Traditional method for routine detection of genetically modified soybean is complex and time-consuming. It takes from 5 to 7 days with low sensitivity.This method can not fulfil. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified food.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method developed by Eiken Chemical Co., Ltd., Japan, and has the potential to replace PCR because of its simplicity, rapidity, specificity, and cost-effectiveness. It is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature (about 65℃) using a DNA polymerase with strand displacement activity. It provides high amplification efficiency, with DNA being amplified 109-1010 times in less than 60 min. Therefore, LAMP is bound to become more popular throughout the detection domain.The specific LAMP primers of Genetically Modified Soybean were designed on the basis of the published sequence of strain CP4-EPSPS (GenBank accession number AY5966948) with the LAMP primer design support software program, using primer explorer software.The reaction conditions were optimized including temperature, time, the concentration of Mg2+, concentration of Bst DNA polymerase etc.The LAMP reaction was carried out in a 25μL total reaction mixture volume with containing 40 pmol each of inner primers FIP and BIP, 5 pmol each of outer primers F3 and B3, 1.0 mmol/L each deoxynucleoside triphosphate, 1.0 mmol/L betaine,4.0mmol/L MgSO4, 2.5μL Bst DNA Polymerase Buffer(20 mM Tris-HCl (pH 8.8, 25°C)), 10 mM KCl,10 mM (NH4)2SO4, 2 mM MgSO4, 0.1 %. Triton X-100), BstDNA polymerase, and the specified amounts of target DNA. The mixture was incubated at 61°C for 60 min in a heating block and then heated at 80°C for 2 min to terminate the reaction. LAMP products were electrophoresed in a 1% agarose gel. The size of the fragment of the LAMP product is in good agreement with the predicted ladderlike size.In addition, there were different samples to be detected by PCR and LAMP respectively in order to evaluate the specificity of primers. The result of Genetically Modified Soybean was positive and those of other samples were negative. The detection limits of PCR and LAMP assays using the CTAB method as templates were 0.2 % and 0.01 %, respectively. The LAMP assay was found to be 20-fold more sensitive than the traditional PCR assay.The LAMP assay enables Genetically Modified Soybean to be detected in less than 2.5 h (contain DNA purified,LAMP and Electrophoresis).If add to SYBR Green I,the LAMP assay enables Genetically Modified Soybean to be detected in less than 2 h . Under the optimum reaction conditions,a rapid technique for detection of Genetically Modified Soybean was initially established by LAMP.In conclusion, LAMP amplification provides a faster and more sensitive method for the detection of Genetically Modified Soybean and it has higher specificity. For the rapid detection of Genetically Modified crop build a technology platform.