Study On Loop-mediated Isothermal Amplification(LAMP) Method For Detection Of Cry1Ab Gene In KMD1

More GMOs were imported into China market，which were potential to beharmful to ecology and human health in recent years. Many countries have takenmeasures to strengthen inspection and labeling of GMOs and GM food. It hasbecome more and more urgent and important to establish rapid and sensitivedetection methods. This paper aims to set up LAMP method for rapiddetection of cry1Ab gene for research purposes, and provide technical supportfor rapid detection of GM plants and GM foods.Loop-mediated isothermal amplification （LAMP） assay is a novel nucleic acidamplification method with high specificity and rapidity under isothermal conditions，our study focus on partial sequence of cry1Ab gene to design two sets ofLAMP primers. At the same position, the specific primers were designed forreal-time PCR and regular PCR in order to construct recombinant plasmid aspositive control. Optimizing the reaction condition to determine its reactionsystem:0.2mmol/L FIP&BIP，0.8mmol/L F3&B3，1.4mmol/L dNTPs，0.6MBetaine，8mmol/L MgSO₄，10mmol/L KCl，20mmol/L Tris-HCl，10mmol/L（NH₄）₂SO₄，0.1%Triton X-100，8U Bst DNAPolymerase，1μL of DNATemplate，add ddH₂O to25μL。Incubation at65℃for60min. The results of detection can beevaluated by three methods below: directly observe the white precipitate or turbidity;observe the fluorescence intensity under ultraviolet irradiation by adding1μL ofSYBR Green I dye; trapezoid bands of amplified products after gelelectrophoresis.In the specificity test of the LAMP assay, the results showed that KMD1cultivars displayed positive reaction to the detections while all of the non-cry1Abgene cultivars and other transgenic cultivars were negative, which was the same asreal-time quantitative PCR and regular PCR, indicating high specificity of LAMPprimers. In the sensitivity test of the LAMP assay, the results showed thatcompared with that of regular PCR and real-time quantitative PCR the sensitivity ofthe LAMP assay was3×10²copies, which was the same as real-time quantitative PCR and was higher than regular PCR. In experiments of comparing4rapidextraction methods of plant genomic DNA, Triton X-100method was better than3other rapid extraction methods in aspects of integrity of bands of genomic DNA andoperation complexity and was the best method suitable for rapid detection. Duringthe actual sample detection, LAMP detection method could detect at least0.25%transgenic content of mixed genomic DNA, and successfully detectcry1Ab gene only from different tissues of KMD1cultivars rather than non-cry1Abgene cultivars, which was the same as real-time quantitative PCR.A visual LAMP detection method for cry1Ab gene was established by addingcalcein and MnCl₂into LAMP reaction system. Optimized final concentrations are:10μM calcein，0.5mM MnCl₂。Incubation at65℃for60min. The results ofdetection can be evaluated by directly observing the colorimetric change of reactionsolution. In the specificity test of the visual LAMP assay, the results showedthat KMD1cultivars displayed high specificity. In the sensitivity test of the visualLAMP assay, the results showed that the sensitivity of the LAMP assay was3×10³copies. We developed the visual LAMP detection kit for cry1Ab gene withexpiration of more than4months.During the actual sample detection, the visual LAMP detection methodcould detect at least0.5%transgenic content of mixed genomic DNA and wasapplied to detect cry1Ab gene from4different transgenic powder samples. Theresults showed that BT11maize powder contained about2.5%content of cry1Abgene, GT73rapeseed powder contained no cry1Ab gene content, MON810maizepowder contained about1-2.5%content of cry1Ab gene and BT1761maize powdercontained about1%content of cry1Ab gene, which was the same as real-timequantitative PCR and regular PCR.In this study, we have established the LAMP method and visual LAMPmethod for detecting cry1Ab gene and developed visual LAMP detection kitfor cry1Ab gene，which basically achieves the goal of rapid detection. Thismethod provides a novel technology platform of detecting cry1Ab gene in GMplants and GM foods and provides technical reference for testingorganizations.