Detection Methods Of Genetically Modified Products Based On Digital PCR And Isothermal Amplification

The planting area and the number of genetically modified organisms(GMOs)has been remarkablely increasing in the last two decades.With the development of the global trade liberalization and commercialization of GMOs,the requirements of analytical approaches for GMOs also enhances unceasingly.The composition of the inserted fragments is increasingly complicated and diversified.Meanwhile,illegal circulation of unauthorised GMOs and crops or products mixed with low level of GMOs or ingredients occasionally happen in global market.Due to the increasing complexity and diversity of genetic modifications in GMOs and occasionally happened cases of crops or products mixed with low level of GMOs or ingredients,analytical methods with high sensitivity,accuracy and rapidity are urgently required.The appearance and application of digital PCR and LAMP show that they may have a good potential in GMO detection.This thesis focuses on to developing accurate and rapid detection methods of GM products employing digital PCR and LAMP technology.The first part of this thesis is the application of digital PCR in GM contents quantification,transgene copy number analysis and characterization of certified reference materials.We compare three main digital PCR platforms with different principle to provide reference for their applicability in GMOs.Digital PCR is a new absolute quantitation method for nucleic acids based on Poisson distribution.Compared with the quantitative real-time PCR(qRT-PCR),digital PCR(dPCR)realizes the accurate and absolute quantification of DNA,thus have been applied in several fields.Our results suggest that the absolute DNA quantitation with high accuracy can be obtained from the three dPCR platforms.The results from ddPCR and 3DdPCR platforms show wider dynamic range in absolute and relative quantitation than cdPCR due to more partitions.We also compare the applicability of these three platforms on GM contents quantification,characterization of certified reference materials,and transgene copy number analysis,demonstrating that dPCR is an ideal and suitable tool for GMO detection.The first part of this thesis is the development of a Capillary Arraybased Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids(CALM)platform for on-field GMO detection.CALM combines visual LAMP technology with a capillary array,which is high-throughput,compact-size,low-cost,and easy-to-operate.In CALM,Loop-mediated isothermal amplification(LAMP)primer sets are pre-fixed in the inner surface of capillaries.The out surface of the capillary array is hydrophobic while the inner surface of the capillaries is hydrophilic,enabling simultaneous loading and separation of the LAMP reaction mixture into each capillary by capillary forces.LAMP reactions are performed in parallel in the capillaries,and the results are visually read out following illumination with a hand-held UV flashlight.Using CALM,we successfully detected eight important GMO-related genes with high specificity and sensitivity.Moreover,multiplex measurements of the practical samples by CALM are completely consistent with results obtained by independent real-time PCRs.Thus,we believe that CALM will be widely applied in routine GMO monitoring.