Realization Of A Real-time Fluorescence Quantitative PCR Instrument Which Based On Thermal Convection

The most widely used method of quantifying the initial DNA template at home and abroad is real-time fluorescence quantitative PCR(qPCR).In view of the existing market of qPCR instruments,most of which use the semiconductor thermal cycler to repeat the reaction tube heating and cooling.But qPCR instruments are expensive and require a longer experimental time.Therefore this paper studies a time-saving and inexpensive real-time fluorescence quantitative PCR instrument based on thermal convection technology.The paper focuses on the four parts of thermal convection real-time fluorescence quantitative PCR instrument,including hardware module,thermal cycle module,fluorescence detection system,software design and data processing module.First,the author design and produce hardware circuit of thermal convection real-time fluorescence quantitative PCR instrument,including LED drive circuit,servo drive circuit,power supply circuit design and serial communication module circuit;Then,in the design of thermal cycle module,the temperature controller with parameter self-tuning fuzzy PID algorithm is used to control the upper and lower aluminum blocks at a certain temperature.And the plug tube is designed to be inserted into the reaction tube,greatly reducing the experimental time while improving the amplification efficiency and specificity;As an important module for stimulating fluorescent labeling substances and weak fluorescence signal detection,the working contents of the fluorescence detection system include excitation light source,LED constant current source circuit,optical system and photoelectric detector design and selection.The module achieve two channels simultaneously amplified and collect two different wavelengths of fluorescence signal;Finally,in the software design and data processing links,the author develope the MCU program and the host computer program,to achieve instrument control and fluorescence signal data acquisition and processing.At the same time,the internal standard calibration target method is proposed to reduce the final data processing error resulting from the difference between the holes.There are three points of innovation in this paper.The first one is that the design of the plug tube in the reaction tube to improve the amplification efficiency and specificity.The second one is that the use of thermal convection mechanism for PCR amplification to improve the experimental efficiency.The last one is that dual-channel simultaneous amply internal target gene and propose internal standard calibration target method to reduce the final data processing error resulting from the difference between the holes.The test of the thermal convection real-time fluorescence quantitative PCR instrument shows that the instrument for PCR qualitative analysis accuracy of up to 100%;instrument linear relationship is good and the difference between tube is small,but the repeatability is poor,and the accurate quantification can not be achieved.Finally,the paper analyzes the possible factors leading to poor reproducibility and puts forward the optimization direction of the instrument.Although the instrument has not yet reached the commercial requirements,it has important scientific research value and promoting significance in the field of quantitative PCR instrument.