Development Of The Rapid And High Throughput Detection Methods For GMOS

With the rapid development of genetically modified organisms (GMOs) and theirapplications in food/feeds, stricter safety regulations have been issued and driven theneed for development of rapid, simple effective and high throughput GMO detectionmethods. In this paper, we focused on these trends and designed new methods todetect GMOs.1. We managed to develop a simple system for GMOs detection based on thegas pressure and silica membrane. The system included a low cost DNA extractiondevice and modified visual loop-mediated isothermal amplification (vLAMP) assay,which was well-adapted to both on-field testing and/or routine laboratory analysis ofGMOs.(1) The whole detection process just needs1h including15mins for DNAextraction and40mins for vLAMP reaction.(2) The designed DNA extraction device and its standard operating procedurehave advantages of low cost, time-saving (1015mins), no need of specific labinstruments, and yet good DNA quality. Despite of the lower DNA yields comparedwith those using the two commercial DNA extraction kits, this device should havegood applicability in rapid on-field nucleic acids extraction.(3) We repeated two previously reported LAMP assays for GM soybean eventsGTS40-3-2and MON89788to test the limit of detection (LOD) using our system, theresults showed that the lowest detection level was5copies haploid genome perreaction, which is more sensitive than LOD of conventional PCR assays.(4) We explored to apply the system to analysis of GM maize and soybeansamples from the GIPSA proficiency test. The results showed that our system waswell suited for practical analysis of GM maize and soybean samples. (5) We tested the applicability of this system for on-site GMO analysis of GMrice T1c-19, TT51-1, and Kefeng No.6. Three sets of LAMP primers were designed toamplify common transgenic elements (CaMV35S promoter and NOS terminator) andbar gene. The results indicated that the developed DNA extraction and analysis systemcould be successfully applied in field test of GMOs.2. We explored high throughput GMO detection method by applying multiplexPCR on a hydrophobically and hydrophilically patterned microarray (MPH&HPM).(1) We designed the hydrophobically and hydrophilically patterned microarraysuitable for the amplification of complex plant genomic DNA.(2) After searching for GMO database, we collected more than120targetingsequences including events, elements and endogenous genes.(3) We designed the corresponding primers and probes based on the sequences.And after selecting, there were97targets can be used for detection: including48events,41elements and8endogenous genes. The coverage of known GMOs was96.9%.(4) The high throughput detection system was set up for detection GMOs byoptimizing the conditions of two round of chip PCR and hybridization.(5) The system was explored to test practical samples and this work is to becontinued.