Multiplex PCR For Detection Of Genetically Modified Crops

With the development of genetic technology and application of Genetically Modified Organisms (GMOs) crops, many countries began to pay attention to the adverse impacts of GMOs on human health, biodiversity and environmental safety. To this end, EU, Japan and other countries applied compulsive label requirement on GMOs crops. Stricter requirements have been proposed for the specificity and accuracy of transgenic detection technology, transgenic detection technology has become a hot spot. "Identity management approach for agricultural genetically modified organisms" has been enforced since March 20,2002 in China, five categories of genetically modified organisms involving 17 kinds of genetically modified products require to be identitied, which include genetically modified cotton, transgenic rapeseed, transgenic maize, transgenic soybean. Therefore the establishment of a convenient, fast GMO detection technology is an important prerequisite for implementing the identification system.In this study, four kinds of Multiplex PCR detection system are estabished for detecting transgenic components for four kinds of crops, based on the multiple PCR primer design method.1. To estabish four-plex PCR detection system for detecting transgenic components in genetically modified soybean, endogenous gene Lectin, exogenous herbicide gene EPSPS, CaMV35S promoter and Nos terminator were chosen for the targeted fragment, then 4 pairs of primers are designed, the PCR conditions for the multiplex PCR analysis were optimized according to the primer concentration and annealing temperature. The results show that four-Plex PCR system can detect endogenous gene and transgenic components with good stability.2. To estabish Seven-plex PCR detection system for detecting transgenic components in genetically modified Maize, endogenous gene IVR, exogenous herbicide gene and PAT, exogenous insect resistant gene CrylAb, screening gene NPTII, CaMV35S promoter and Nos terminator were chosen for the targeted fragment, then 7 pairs of primers are designed, the PCR conditions for the multiplex PCR analysis were optimized according to the primer concentration and annealing temperature. The results show that Seven-Plex PCR system can detect endogenous gene and transgenic components with high efficiency and good stability.3. Multiplex PCR detection system for rape is established with targeted genes including FMV35S, CaMV35S promoter, NOS terminator, NPTII, PAT, and an endogenous gene PEP. The PCR conditions for the multiplex PCR analysis were optimized according to the primer concentration and annealing temperature. Results showed that this multiplex PCR system can detect the genetically modified components from rapeseed meal and other crops(soybean, corn, rice and cotton seeds), with the advantages of simple, accurate and highly specific.4. To detect transgenic components in genetically modified cotton, endogenous gene sad1, reporter gene GUS, exogenous insect resistant gene Cry1Ab/Ac, screening gene NPTII, NOS terminator and CaMV35S promoter were chosen for establishing a multiplex polymerase chain reaction method. The designed 6 pairs of primers can amplify proper fragments and avoid primer dimer. The PCR conditions for the multiplex PCR analysis were optimized based on the primer concentration and annealing temperature, then the Six-Plex PCR detection system for transgenic components in cotton is established. The results showed that the Six-Plex PCR can successfully detect the transgenic components in genetically modified cotton, from even a small quantity sample as little as a cotton seed using a modified genomic DNA extraction method for cotton seeds in this study.5. A set of convenient, practical and excellent effect multiple PCR primer design methods was established. This method adopted as many as 45 primers authentication and adjustment for a number of primers to overcome the multiple PCR primer design difficulties,which offer a good reference.In this study, based on the multiplex PCR detection system used in our group, multiplex PCR detection kits for detecting 4 different transgenic crops which verified by repeated multiple PCR reaction were developed. The Kits were of high efficiencies for the promotion and application, which can be used as a rapid detection method for GM plants and other GM alternative products.