Research On Optimization Of Structure And Function Of Polymerase Chain Reaction System And Its Application

In the detection of environmental microorganism,clinical detection and large public health events,portable PCR system has been widely concerned for its high efficiency,portability and easy operation.In these tests,we need to amplify the trace of target Deoxyribonucleic Acid(DNA)or Ribonucleic Acid(RNA).The in-vitro amplification technology of nucleic acid is called the polymerase chain reaction(PCR)technology(divided into three generations),which has the characteristics of high sensitivity,high specificity and low requirement on the quality and quantity of raw materialsThis paper describes the background,significance and research status of the three generations of PCR system,mainly optimizes the structure and function of the system,and studies its application.The main research work is as follows1.It's been focused on the problem that the inaccurate thermal cycle temperature controlling method of the traditional PCR microreactor affects the efficiency of PCR reaction,and the large size of power supply block the portability of the system.Based on the single constant temperature cycle controlling method,this paper proposes a trapezoidal structure spatial PCR microreactor instead of a rectangular structure spatial PCR microreactor for the first time.The optimization of the temperature controlling method for continuous flow PCR system is realized by changing the structure of the microreactor.The ratio of the trapezoidal structure between the upper and the bottom can be used to control the denaturation and annealing time of PCR reaction flexibly,so as to avoid the same denaturation and annealing time of the rectangular structure spatial PCR microreactor.The heating device of the system adopts metal heating plate carrying out the temperature feedback regulation by small temperature controller and temperature sensor.The sample solution is fed by a self pressure gas diffusion micropump made up of disposable medical syringes.The power of the whole system is supplied by portable lithium battery.The volume of the whole system is 55×100×110 mm3,which can be placed on the palm of one hand.The system can work continuously for 4.5 hours with full power.It was used to amplify the plasmids of hepatitis B virus(HBV),avian influenza virus(H7N9),Escherichia coli by PCR performing multiplex PCR amplification,and the results were similar to those of conventional bench PCR2.Real-time quantitative PCR(qPCR)system needs high sensitivity and high speed fluorescence imaging.PVC(PVC)material is used in real-time fluorescence detection of qPCR system for the first time.The sensitivity of the system is improved by changing the material of spatial PCR microreactor.Meanwhile,the smart phone is used to image solving the problem of missed shooting in the continuous photographing process by only using software.In this paper,PVC,a high light transmittance polymer material,was used to build a spatial PCR microreactor for the first time,which improved the light transmittance of the spatial PCR microreactor.Spatial PCR microreactors were built respectively.Two typical dyes(EVA green and SYBR green)and a probe(FAM)were used to verify that the fluorescence image obtained by the system with PVC spatial PCR microreactor was clearer than that obtained by the system with PTFE spatial PCR microreactor.The automatic cycle counting device developed by ourselves is used to take photos that the micro manipulator controlled by the steering gear is used to take photos externally,which can effectively avoid the phenomenon of missed shooting.In order to carry it easily,the system adopts self pressure gas diffusion micropump to propel sample,and with the integration of temperature controlling and detection all powered by lithium battery.Finally,it was used for qPCR detection for the DNA plasmid of H7N9 avian influenza virus.The results showed a good dynamic range,covering 104-107 copies/?L,which exceeded four orders of magnitude3.For the droplet digital PCR(ddPCR),the liquid controlling method of droplet preparation method is complex,the cost of drop preparation device is high,and the existing methods are not easy to integrate ddPCR system.Based on the step emulsification principle,this paper proposes a method of preparing the off-chip step emulsion droplets composed of capillary and centrifugal tube,and sets up a set of droplet preparation device.When the continuous separated phase fluid in capillary is injected into the static continuous phase fluid in the centrifugal tube at a certain speed,the continuous dispersed phase fluid emulsifies into stable droplets due to the change of interfacial tension.The size of the droplets can be precisely controlled by controlling the distance between the outlet end of capillary and the bottom of the inner wall of centrifugal tube by screw mechanism.The factors affecting the droplet size,such as the height of the step,the size of the nozzle and the velocity of the dispersed phase,are discussed with experiments.At the same time,we used the droplet obtained from the preparation device to carry out ddPCR reaction with high dynamic range.The results show that the device not only has the advantages of low cost and simple operation without complex controlling structure,but also provides a possible development scheme for rapid preparation of high flux and strong stability dropletsIn conclusion,this paper solves the problems encountered in the miniaturization of PCR system,realizes the development of portable continuous flow PCR system and the real-time fluorescence quantitative PCR system of hand-held continuous flow.At the same time,a method of liquid drop preparation easy to integrate is proposed to be used in the research of miniaturized digital PCR system.