Research On Multiplex Real-time PCR Of Genetically Modified Cotton Detection Based On The Locked Nucleic Acid Technology And Development And Validation Of Reference Molecules

In this study, we developed a quadruplex real-time PCR method based on the locked nucleic acid technique for simultaneous detection and quantification of GM cotton events GHB119, T304-40and the insect-resistance gene Cry2Ae of GHB119, which has not been authorized by the Ministry of Agriculture of China. Compared with the simple real-time PCR, we have tested the established quadruplex real-time quantitative PCR detection methods. The results show that the method has highly specificity to GHB119, T304-40and Cry2Ae gene. The limit of detection (LOD) was all lower25initial template copies of DNA for GHB119, T304-40and Cry2Ae gene. The limit of quantification (LOQ) was all lower50copies for GHB119, T304-40and Cry2Ae gene. Meanwhile, pUC57reference molecules suitable for10kinds of genetically modified cotton (MON531,281-24-236,3006-210-2, MON15985, GBH614, MON1445. GHB119, LLcotton25, MON88913and T304-40) event-specific detection method were developed. And the suitability of pUC57reference molecules used in the qualitative and quantitative real-time PCR assay was tested and validated in our laboratory as a positive reference material. The results show that the conventional matrix reference materials can be replaced by plasmid reference molecular pUC57, used to real-time qualitative and quantitative PCR analysis of GM cotton component.In addition, the value of three target fragments in pEASY-RT73reference molecular was further tested and optimized by digital PCR (ddPCR) for Genetically Modified rapeseed event RT73. The primers and probes were selected and optimized to enhance the reliability and stability in quantitative analysis of GM rapeseed RT73component when using pEASY-RT73reference molecular as positive reference materials.