The Loop-mediated Isothermal Amplification For The Detection Of Genetically Modified Rapeseed RT73

Since the transgenic calona frequently come into China, the lack of strictmanagement on genetically modified plants can easily lead to food unsafety andenvirionmental problem. Nowadays, there are many measures for (GeneticallyModified Organism) GMO detection,among which,Polymerase Chain Reaction (PCR)and Real-time PCR are the most generally used for quality inspection. Since theTaqMan Rea-time PCR which needs high technical and equipment requirements is notsuitable to those areas with relatively lower level of regulation,a rapid,convenient andaccurate standard detection measurd is in urgently needed in the first-line laboratoryof inspection and quarantine.In2000,Doctor Notimi T, from Eiken Chemical Co., Ltd., Japan,invited a newamplification method,the loop-mediated isothermal amnlification(LAMP)techenology on the basis of PCR.The LAMP assay is performed with a set of fourspecific frimers recognizing6distinct regions of target DNA.The significantadvantage of LAMP is that it can amplify DNA isothermally(60~65℃) based onautocycling strand displacement synthesis of DNA by Bst DNA polymerase largefragment with high strand displacement activity. Also, it is time-saving for DNAamplifcation (within60min) with high efficiency,reaching109～1010times theoriginal,compared to2hours PCR reaction. Moreover, the presence or absence ofLAMP products can be visually judged with naked eyes. All these above indicateLAMP assays are more convenient, rapid, efficient and low-cost for GMO routineanalysis in basic laboratory,also has a promising application prospect.Based on preliminary study,we applied LAMP assay to the detection of GM rape-RT74strain. The event-specific LAMP primers RT73used for RT73rapeseeddetection were designed based on the3’ junction region between host DNA andexogenous sequence originating from E9gene terminator.For the rapeseedendogenous genomic DNA detection,HMG primers were designed from HMG I/Ygene.After optimizing the established detection system,we detected the amplifiedlimit of detection (LODs),sensitivity,specificity and stability of the system.In indoor verification section,we compared LAMP with the classical Real-time PCR assay inLOD,specificity,stability,measure time and interpretation means.The LAMP method owned good stability when GMO RT73content no less than0.5%.Its sensitivity is0.01%(equal to0.01ng DNA),and its LOD is0.1%. The resultswere observed by direct visual observation using SybrGreen Ⅰ in10min or real-timeturbidimeter in no more than40min with perfect specificity.these three identificationmeans—using turbidimeter, direct visual observation depending on the magnesiumpyrophosphate precipitate and using SybrGreen Ⅰ, are complementary and mutualverification.Real-time PCR has a perfect repeatability and specificity with a highsensibility of0.01%,as same as LAMP assay.However,it is much more timeconsuming with100min than LAMP of40min,and we judge the reaction through theamplification curve.LAMP meathod has an advantage of speediness and convenience over Real-timePCR,and its sensitivity is parallel with Real-time PCR,while when come to low GMOantent samples(≤0.01%),its stability and repeatability need further development andimprovement.The high sensitivity of this techenology is both the advantage and disadvantage. Avery small amounts of gene pollution will cause false positive result, so we need to becareful enough while walking LAMP experiment in the lab so as to put an end toamplification product contamination.It’s hopefully for LAMP techenology to replace the costly Real-time PCRmeathod and become a new meathod of GMO detection in entry and exit inspectionand quarantine department.