Study On Rapid Analysis Of Genetically Modified Organizes By The Multiplex PCR-capillary Electrophoresis System With Laser-induced Fluorescence Detection

Objective: In order to effectively enforce the regulation regarding genetically modified food labeling and safeguard the human health, the rapid analytical methods for the identification and detection of genetically modified soybean resistant to glyphosate and maize event M0N810 were developed accurately, resulting from the worldwide concerns about the genetically modified food safety issues. This study also can be considered as an valuable effort to develop the standard detection method of the genetically modified organisms in the future.Methods: Non-gel sieving capillary electrophoresis with laser induced fluorescence detection was developed to analysis the polymerase chain reaction(PCR) products rapidly. Accordingto the exogenous gene characteristics of genetically modified soybean resistant to glyphosate and maize event MON810, the suitable primers were designed and used for multiplex PCR systems. The Multiplex PCR systems were optimized and performed to rapidly detect the two above-mentioned genetically modified soybean and maize.Conclusions: 1. A setup of capillary electrophoresis with laser induced fluorescence detection was developed. For Sulforhdamine B, its detection limit was 2. 1×10^-9mol/L(S/N=3) and equal to that of the other similar setup, but it was developed more uncomplexly and cheaply. The setup offers wide equipment flat for development of the method of rapid and sensitive detection of biological macro-materials. The effect of separate conditions on the resolution of pUC19DNA/MspI (HpaII) Marker 23 was studied. When using Hydroxypropylmethylcellulose(HPMC-4000) as the sieving medium, the resolution of dsDNA fragments 147/190bp is best at the concentration of 0. 8% and the resolution of dsDNA fragments 331/404bp at the concentration of 1.2%. The CE inner wall has an obvious influence on the separation of DNA. The modified CE column with linear polyacrylamide can greatly improve the electrophoresis and has more than 100 times life-span. Although the increase of electric field strength would lead to the decrease of migration time of DNA fragments, the efficiency of dsDNA fragments separation also decreased, thus the choice of electric field strength should lie on theintegrated consideration of analysis time and resolution. The resolution of dsDNA fragments would be best at the 3. 75 uM of intercalating dye EB. Column temperature has also an effect on resolution of dsDNA fragments. For separation of less than 400bp dsDNA fragments, the best available temperature is 20°C. The relative standard deviation of the migration times of pUC19 DNA/MspI DNA molecular weight Marker 23 were 1. 53%-2. 28% under the optimized electrophoresis conditions. In one word, a rapid and sensitive non-gel sieving capillary electrophoresis analysis method with laser-induced fluorescence detection has been developed and would provide better choice for rapid detection of genetically modified organisms.2. The study was performed on the relation of appearance mobility logarithmQg u iip) and concentration of HPMO4000 , the result has shown that there was linear relationship between lg U MP and concentration of HPMC only for the 147 base-pairs. DNA fragment The Ogston model can describe satisfactorily the mobilities of fragments of sizes ranging from 34bp to 147bp with a correlation coefficient of 0.999. By plotting the mobilities as a function of 1/N for the fragments with sizes ranging from 147 to 404bp, the linear relations were obtained with correlation coefficients of 0.998> 0. 99K 0.999 under 0.8%, 1.0%, 1.2% of HPMC solutions. It indicated the reptation model can been applied for fragments of more than 150bp. It was also found that the relative fluorescence strength of dsDNA fragmentincreased linearly with its concentration. Using the function the dsDNA fragment might be quantified comparatively.3. The multiplex PCR with three primer pairs were used to detect the genetically modified Roundup Ready soybean. The primers lectinl/2 targeting to soybean lectin housekeeping gene was used as internal positive control to confirm the presence and quality of the DNA extracted from soybean and assess the availability of PCR system with an amplicon of 118bp. The primers NOS1/2 targeting to NOS terminator was used for genetically modified food sieving with an amplicon of 180bp. The primers P35S/CTP4 targeting to the joined regions of the CPT4 gene and CaMV 35S promoter was designed for the spcific detection of the transgene of Roundup Ready soybean with an amplicon of 142bp. The optimized PCR system included annealing temperature 60"C, MgCL concentration 2. Ommol/L, primers lectinl/2 0. 1 u mol/L, primers P35S/CTP4 0.4 u mol/L, primers N0S1/2 0.9 u mol/L. Sequencing the PCR products indicated the high alignment with the reported sequences to confirm the reliability of the PCR system. Using the proposed method to analysis the mostly grown genetically modified maize lines (M0N8KK Btl76> BtlK GA2K T14/25 > CBH351) and other common non-transgenic foodstuff (soybean> maize , canola), the results had great agreement with the expected. Capillary non-gel sieving electrophoresis was used to separate the multiplex PCR products. The analysis time decreased about 40min compared with the conventional agarose gelelectrophoresis. The detection limit of 0. 025% was obtained with laser-induced fluorescence detection and reduced about 20 times than with the conventional agarose gel electrophoresis. The real samples were detected by the proposed method and sensitive real-time quantitative PCR. The analysis results had no disagreement to further confirm the accuracy of the proposed method in this study.4. Three primer sets were chosen to detect the maize invertase housekeeping gene -. the sequence between hsp70 intronl gene and cryla(b) gene specific for transgenic maize MON810 and CaMV 35S promoter. Four different groups of multiplex PCR were assessed to choose the best primer pairs. There were non-specific amplicons expect B group. The optimized PCR system were annealing temperature 58°C, MgCl2 concentration 3.0 mmol / L, primers P35S1/2 0. 84 u mol/L, primers HSOl/CryOl 0. 4 u mol/L, primers IVR1/2 0. 1 u mol/L. Sequencing the PCR products indicated the high alignment with the reported sequences to confirm the reliability of the PCR system. Using the proposed method to analysis the mostly grown genetically modified maize lines (Btl76, BtlK GA2K T14/25, CBH351) and transgenic Roundup Ready soybean and other common non-transgenic foodstuff (soybean, maize , canola), the results had great agreement with the expected. Capillary non-gel sieving electrophoresis was used to separate the multiplex PCR products. The analysis time decreased about 45min compared with the conventional agarose gelelectrophoresis. The detection limit of 0. 05% was obtained with laser-induced fluorescence detection and reduced 10 times than with the conventional agarose gel electrophoresis. The real samples were detected by the proposed method and sensitive real-time quantitative PCR. The analysis results had no disagreement to further confirm the accuracy of the proposed method in this study.In this study, multiples PCR was performed for genetically modified organisms testing of food in stead of common step-by-step PCR procedure, which significantly shortened the analysis time and reduced the occurrence of operating error to avoid the false positive results. The capillary non-gel sieving electrophoresis with laser-induce fluorescence detection was used to analysis the PCR produces, which greatly improved the sensitivity and cut down the analysis time compared with the conventional agarose gel electrophoresis. The presented methods support effectively the implementation of any transgenic labeling agency. The presented methods offer the experimental bases for establishment of full-automated and rapid standard detection of genetically modified organisms in food.