Preparation Of Magnetic And Magnetic Fluorescent Microspheres For Genetically Modified Food Detection

Nowadays, the main ways of detecting genetically modified food are polymerasechain reaction (PCR) and real-time PCR (qRT-PCR). The key problem of thedetection is how to extract high-quality DNA from food samples withhigh-through-put and fast speed. The main DNA-extraction methods are hexadecyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and kindsof kits. But the CTAB and SDS methods need complex operations and large amountof organic solvent, so they cannot meet the demand of auto-extraction. And the kitsare always expensive and cannot be used in the routine detection of the geneticallymodified food. Thus, we introduced magnetic separation to the extraction of DNA,and prepared magnetic microspheres with uniform particle size, well dispersion, andhigh magnetic susceptibility. Meanwhile, multi-detection is becoming popular in thegenetically modified food detection. So it is also necessary to prepare differentfluorescent microspheres.In order to solve above problems, we performed following work consisting ofthree-folds:(1) Preparation and characterization of magnetic microspheres;(2)Magnetic microspheres for DNA extraction from soybean foods and withcomprehensive evaluation; extraction of specific complementary strand ssDNA withDNA probe modified magnetic microspheres;(3) Preparation and characterization ofdifferent fluorescent microspheres. The details ofthe work are as follows:A solvothermal reduction method is described for the preparation of magneticresponsive particles. The effects of dispersant, reducing agent and precursors on themagnetic microspheres were investigated. After beingcoated withsilica bythe sol-gelapproach, the particles were modified withdifferent chemical groups.Then we applied these magnetic microspheres in the extraction of DNA fromsoybean derived foods, and compared this method with traditional methods likeCTAB and commercial kits such as Omega and Wizard kits. Then, we modifiedthis amino-magnetic microsphere with avidin, which were then coupled withbiotin-ssDNA. In the mean time, we studied the specific extractionefficiencyof thesessDNA-biotin-avidin mangentic microspheres with complementarystrand ssDNAandnon-complementarystrand ssDNA.Multi-detection can detect more than one target genes in genetically modifiedfoods. This method can not only reduce the amount of DNA for the detection, but canalso accelerate detecting speed. We prepared different fluorescent microspheres by layer-by-layer method using quantum dots as fluorescent labels. The emissionwavelengths of microspheres canbe adjusted bydifferent sizes ofquantumdots. Also,we prepared fluorescent microspheres with magnetic susceptibility.In summary, this thesis laid a basis for the rapid extraction of nucleic acid basedon magnetic separation technology from genetically modified foods as well as themultiplex assay. And this basic research work can promote the automation of nucleicacid extractionof genetically modified foods, high-throughput and multi-detection.