| As the global GM products is increasing, and more and more specific strains of genetically modified crops for commercialization production, detection of specificity strains has become the development trend of the international Genetically Modified Organisms. Currently ordinary PCR and real-time fluorescence PCR method are the main detection methods for specific strains of genetically modified, but have their own shortcomings. Therefore, it is urgent needed to establish a simple, rapid, accurate detection method, that is for specific strains of genetically modified generally applicable to the front-line of laboratory inspection and quarantine. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. Compare to the ordinary PCR, its sensitivity and specificity are much better, because of its simplicity, efficiently, specific and rapidity. The LAMP can high-throughput detect rapidly without relying on any special equipment, so its detection cost is far lower than the real-time fluorescent PCR. LAMP has been widely used in the rapid detection of foodborne pathogenic microorganisms, but in genetically modified products detection, especially about the specific strains, there are rarely researches.7 sets of LAMP primers designed from specific sequent of GM soybean A2704-12, and 3 sets of LAMP primers designed from GM maize GA21, respectively screening a set of LAMP primer whose specific is better. The LAMP detection method of GM soybean A2704-12 and GM maize GA21 were established through optimizing the reaction condition and system, and applied to practical, built a good technology platform for the rapid detection of specific strains of genetically modified products.Design 7 sets of LAMP primers from exogenous gene of GM soybean A2704-12 and boundary sequence of soybean. Through optimizing reaction temperature and testing specific, screen a set of primer which has better specific, including one pair each of the inner primers, the outer primers and loop primers. The reaction condition and system of the LAMP system were optimized about the screened primer, with adding the SYBR Green I fluorescence dye, real-time monitoring by the real-time quantitative PCR apparatus. The optimal reaction condition was at 63℃ for lhour and then at 80℃ for 5min. The optimal LAMP reaction system was carried out, including 2.5μL 10×Thermo Pol buffer,0.5μL each of outer primers F3 (10 μmol/L) and B3 (10μmol/L),0.5 μL each of loop primers LF (40 μmol/L) and LB (40 μmol/L), 1.0 μL each of inner primers FIP (40μmol/L) and BIP (40 μmol/L),4.0 μL dNTPs (10 mmol/L),6.0 μL betaine (5 mol/L),1.0μL MgSO4 (150 mmol/L), 1.0μL Bst DNA polymerase buffer (8 U/μL),2.0 μL target DNA (50 ng/μL),4.5 μL ddH2O. There is no false positive result of the LAMP detection, according to the optimal reaction conditions and system, with the target DNA from different strains of GM soybean and plant, it showed that the specificity of the LAMP detection method established well. The result of sensitivity test of LAMP showed that the detection limit of LAMP can reach 0.1%, though slightly less than 0.05% of the real-time fluorescent PCR, but it has reached the genetically requirement of the limit for identifying GM products. So it can satisfy the daily detection. The LAMP detection respectively repeats 20 times with the target DNA from 5% A2704-12 and Non-GMO soybean, and the results of 20 repetitions exactly the same showed that well establish the stability of the LAMP detection method. This LAMP detection method was applied to detect A2704-12 from the soybean and its products, its detection accuracy rate can reach 100%, compared with the results of real-time PCR detection.Design 3 sets of LAMP primers from exogenous gene of GM maize GA21. Through optimizing reaction temperature and testing specific, screen a set of primer with better specific, including one pair each of the inner primers, the outer primers and loop primers. The reaction condition and system of the LAMP system were optimized about the screened primer. The optimal reaction condition was at 63℃ for 1hour and then at 80℃ for 5 min. The optimal LAMP reaction system was carried out, including 2.5 μL 10× Thermo Pol buffer,0.5μL each of outer primers F3 (10 μmol/L) and B3 (10 μmol/L),0.5μL each of loop primers LF (40 μmol/L) and LB (40 μmol/L),1.0 μL each of inner primers FIP (40 μmol/L) and BIP (40 μmol/L),3.5 μL dNTPs (10 mmol/L),4.0μL betaine (5 mol/L),1.0μL MgSO4(150 mmol/L),1.0 μL Bst DNA polymerase buffer (8U/μL),2.0 μL target DNA (50 ng/μL),7.0 μL ddH2O. There is no false positive result of the LAMP detection, according to the optimal reaction conditions and system, with the target DNA from different strains of GM maize and plant, it showed that the specificity of the LAMP detection method established well. The result of sensitivity test of LAMP showed that the detection limit of LAMP can reach 0.05%, though slightly less than 0.05% of the real-time fluorescent PCR, but it has reached the genetically requirement of the limit for identifying GM products. So it can satisfy the daily detection. The LAMP detection respectively repeats 20 times with the target DNA from 5% GA21 and Non-GMO maize, and the results of 20 repetitions exactly the same showed that well establish the stability of the LAMP detection method. This LAMP detection method was applied to detect GA21 from the maize and its products, its detection accuracy rate can reach 100%, compared with the results of real-time PCR detection. |
PDF Full Text Request