Preparation Of A Kind Of Magnetic Microsphere(to Extracte DNA ) And Four Encephalitis Virus Nucleic Acid Detection Kits(Fluorescent PCR)

Objective:1 To prepare a kind of high-performance Fe₃O₄@SiO₂ magnetic composite microsphere which has good dispersion,strong magnetic responsive capability,chemical stability and high mechanical strength.It is to be used to extract nucleic acids from different pathogenic microorganisms as well as to be applied to the nucleic acid extraction instrument to extract and purify DNA automatically.2.To improve the capabilities of prevention and detection in meningitis syndrome viruses,four kinds encephalitis virus nucleic acid detection kits（Fluorescent PCR）are developed for Eastern equine encephalitis virus,Western equine encephalitis virus,Venezuelan equine encephalitis virus and tick-borne encephalitis virus.Methods:1 Firstly,under the condition of high pressure and high temperature,iron oxide magnetic nano-microspheres could be synthesized by FeCl₃·6H₂O through a hydrothermal synthesis method with Na Ac as electrostatic stabilizer,ethylene glycol reducing agent,polyethylene glycol a surfactant.Secondly,under ultrasonic-assisted conditions,magnetic microspheres could be rapidly coated with a uniform silica shell layer by St?ber synthesis method.And by adjusting the input volume of TEOS of the reaction system,the thickness of silica shell could be well controlled,Thus a series of Fe₃O₄@SiO₂ magnetic composite microspheres with different silica shell thickness could be prepared.Then the basic properties of Fe₃O₄@SiO₂ composite microspheres were characterized by means of TEM and SQUID.Finally,by extracting DNA from the salmon sperm DNA solutions,hepatitis B virus samples and bacterial plasmid diluted samples,the effect of extraction of Fe₃O₄@SiO₂ composite microspheres could be valued.2.There are three parts in technical route for the development of Encephalitis Virus Nucleic Acid Detection Kits（real-time PCR method）: First,Design and screen primers as well as probes.Target genes of pathogens are determined by literature research.Login genbank and download target gene sequences of the corresponding gene and comparatively analysis.Primers and probes are designed for detection kits.Strains are Collected to establish standard plasmid,while artificial synthetic target genes are prepared for some deficient pathogens through overlap extension method.Specificity and sensitivity references are prepared.Primers and probes are screened.Second,optimize experimental conditions.It is necessary to optimize the annealing temperature and time of the PCR process.Third,evaluate the nucleic acid detection kits（Fluorescent PCR）.The specificity of the nucleic acid detection kits are assessed by positive references and negative references.The minimum detection limit are evaluated by sensitivity references.Eventually,clinical samples are used to evaluate nucleic acid detection kit.Results:1 Firstly,particle size of Fe₃O₄ magnetic microspheres were about 400⁵00nm and three kinds of silica shell thickness of Fe₃O₄@SiO₂ magnetic composite microspheres were prepared by further synthesis,namely T50,T100,T150.Secondly,transmission electron microscopy revealed that the silica shell thicknesses,from thin to thickness,were 20 nm,40nm,65 nm,respectively.SQUID detected that saturation magnetization of Fe₃O₄@SiO₂ magnetic composite microspheres could be up to 62.5 emu/g.HBV DNA extraction experiments showed that Ct value of Fe₃O₄@SiO₂ magnetic composite microsphere（T100）was the least,showing the best extraction of T100 beads.Salmon sperm DNA solution extraction experiments suggested that the extraction efficiency of T100 magnetic composite microsphere was up to 85%.The amount of beads and elution time experiments showed that when the usage of magnetic composite microsphere was 1mg and elution time 10 min,it extracted better.T100 magnetic composite microsphere also had a better extraction efficiency when compared with commercial bead kits.In addition,T100 magnetic composite microsphere also had a good extraction to plasmid DNA.2.Firstly,target genes of Eastern equine encephalitis virus,Western equine encephalitis virus,Venezuelan equine encephalitis virus and tick-borne encephalitis virus were determined by literature research.According to the results of the gene sequence alignment,2-3 pairs of primers and 3-5 of probes of each virus were designed.Plasmid references were prepared and in vitro transcribed RNA of four viruses were transcribed by plasmid references as sensitivity references.Secondly,primers and probes were screened.Furthermore,the annealing temperature and time of PCR process were optimized respectively in order to guarantee that every target gene was amplified well.Finally,the overall performance of nucleic acid detection kit was evaluated.The evaluation results indicated that the specific nucleic acid detection kits well distinguished the four viruses.Sensitivity evaluation results showed that the sensitivity limitations of the four insect-borne viruses were 10²copies/μL.Conclusion:1 A kind of high-performance Fe₃O₄@SiO₂ magnetic composite microsphere,which had good dispersion,strong magnetic responsive capability as well as chemical stability and high mechanical strength,had been prepared.It could both be applied to extract nucleic acids from different pathogenic microorganisms and to help the nucleic acid extraction instrument to extract and purify DNA automatically.2.Four encephalitis virus nucleic acid detection kits（Fluorescent PCR）were developed to diagnose Eastern equine encephalitis virus,Western equine encephalitis virus,Venezuelan equine encephalitis virus and tick-borne encephalitis virus.This study also provided new methods for prevention and diagnosis of the four insect-borne encephalitis viruses.