The Study Of Nucleic Acid Beacon Aptamer-pcr Detection Technology Based On The Separation By Magnetic Agar Microsphere

With the development of SELEX technology,aptamer,as a new type of immune molecules with high specificity,wide range of application and easy modification,plays a more and more important role in biological detection,disease diagnosis,medicine research and development.Molecular nucleic acid beacon aptamer as an extension of the aptamer technology,has both abilities of beacon and aptamer to recognize and detect molecules and signal amplification,this superiority makes it become a new detective molecule with easily structure,high specificity and sensitivity,wide range of application.Magnetic agar microsphere is a kind of magnetic polymer microspheres,which structure is the magnetic particles using chemical method wrapped with a layer of agarose on the surface to make a whole.It has high plasticity,easy workability and biological compatibility as an organic materials,also good rigidity and magnetic responsiveness as an inorganic material.These features allow magnetic agar microspheres to be widely used in enzyme immobilization,biological separation,medical and environmental research.All the characteristics of magnetic agar mi-crospheres,like responsiveness of magnetic,functional group,superficiality and biocompatibility makes it to be the ideal vehicle for the nucleic acid beacon aptamer detection.In this paper,the magnetic microspheres will combine with nucleic acid beacon aptamer as vehicle to bulid a new protein detection technology.For the success of this technology establishment,the experiment successfully made three kinds of magnetic agar microspheres:agarose magnetic microspheres,epoxy magnetic microspheres and carboxyl magnetic microspheres by using chemical coprecipitation method,reverse polymerization embedding technology and functional group modification.And by microscopically,titration analysis method and enzyme-linked immunoassay,these three magnetic microspheres were compared in characterization and active identification,finally chose carboxylic magnetic microsphere as the vehicle.This technique is to use carboxyl magnetic microspheres to fix target protein,then combined with nucleic acid beacon aptamers,and complete signal conversion from the target protein to the nucleic acid beacon by electrophoresis separation;Using real-time quantitative-PCR technology to take a rapid and quantitative detection of beacon aptamers.In this process,electrophoretic separation methods,electrophoresis temperature,time,voltage and current is optimized,and we carry on the experimental verification of the specificity,repeatability and accuracy of this method.The results shows that the method can be used to recognize the specific target protein,and can reflect the minute amount of target protein,so it is proved to be feasible.This technology has the signal amplification characteristics of nucleic acid beacon aptamer,vector characteristics of magnetic microspheres,and few artificial operation,high automatic degree of electrophoresis separation as a means of separation,then complete the indirect quantitative detection of target molecules by using real-time quantitative-PCR technology.This technique provides a rapid,sensitive and practical technical method for protein quantitative detection.