Research On Nucleic Acid Amplification System For Field Detection

Objective: PCR technology was a significant achievement of the 20 th century in the field of molecular biology. Because of its specificity, high sensitivity, low purity requirements for samples and some other advantages, it is widely used in the researches on genetic diseases, the pathogenesis of cancer and life sciences. In the early 21 st century, PCR technology was wildly adopted to detect unknown samples in the detection and response to emerging and emergent infectious diseases, safety detection of food and drinking water after disasters, anti-bioterrorism, etc. What is complementary to PCR technology is the research and development of nucleic acid amplification systems. As a major part of sample amplification, nucleic acid amplification systems affect the rate, efficiency and accuracy of the amplification with their operating parameters. The traditional nucleic acid amplification systems tend to be laboratorial life science researches-oriented. They are mainly characterized by good stability and high capacity. Nevertheless, they have defects in the portable degree, heating and cooling rate and accuracy of results in field applications such as the safety detection of food and drinking water and anti-bioterrorism. In addition, in order to detect the unknown samples in field applications, the amplification programs are needed to be selected. However, the traditional nucleic acid amplification systems do not have the corresponding designs or functions at all.In order to promote the application of PCR technology in rapid field detection, it is necessary to research and improve the existing nucleic acid amplification systems and realize the portability of devices, rapid and accurate amplification as well as the automatic selecting of amplification programs.Method and Contents: To solve the problems of the traditional nucleic acid amplification systems in field detection, this paper starts from the operating principle of these systems to research a portable nucleic acid amplification system that is used for rapid detection and in favor of the rapid and accurate amplification and filtering. The research contents are as follows: 1. The heating and cooling rate of amplification systems were taken as the primary indicators to conduct thermodynamic analysis on the heat sources and heat transfer modes of different nucleic acid amplification systems.The heat transfer coefficient in the heat transfer process and actual temperature-raising and cooling parameters of the systems were considered to be the indicators to select reasonable heat transfer modes and heat sources; 2. In accordance with the selection of heat transfer modes and heat sources, the chambers were desgined by aiming at portability. During the design process, it is necessary to consider the degree of integration, ensure that its internal structure is conducive to heat transfer and guarantee enough heat intensity; 3. The heat transfer modules of the system was selected. The ANSYS software was used to simulate the temperature uniformity in reaction chambers and temperature experiments were carried on different regions of the actual nucleic acid amplification system. The temperature uniformity, heating and cooling rate and accuracy of the system were evaluated by combining the simulation results with the experimental results, so as to ensure that the system meets the design requirements; 4. The theoretical time constant of transfer heat from the heat sources to the samples was analyzed to against the prevailing problem of temperature latency in nucleic acid amplification systems. Tthe theoretical temperature latency of individual module samples and heat sources was calculated by basing on the nucleic acid amplification system designed in this paper.The actual temperature latency of the system was computed through concrete temperature experimental measurement. The latency of system was optimized by optimization of the heat transfer mechanism to transfer heat from heat sources to samples in accordance with the theoretical latency analysis and actual latency, thereby improving the overall rate of amplification; 5. Amplification test was detected by the nucleic acid amplification system designed in this paper. Electrophoresis was carried on the amplification results of the system and standard PCR. The amplification effects of the nucleic acid amplification system in this paper was evaluated.Results: 1. By comparing the heat transfer coefficient of air-bath nucleic acid amplification systems and that of temperature-dependent-metal nucleic acid amplification systems, the following conclusions can be drawn: the heat transfer process of air-bath nucleic acid amplification systems is simpler. In addition, the heat transfer coefficient of air-bath systems is larger. Taking into account practical operating parameters of the PCR instrument, this paper selects air bath as the heat transfer mode of the nucleic acid amplification system in this paper; 2. With air bath as the main mode of heat transfer, the paper respectively calculates the heat transfer intensity between air and heat sources of the system which takes heating wires and infrared lights as the heat sources. Comprehensively considering the heating and cooling rate of different heat sources and their volume parameters, the paper selects heating wires as the heat sources of the nucleic acid amplification system in this paper; 3. The paper proposes to utilize multiple independent micro reaction chambers to carry out independent amplification, thereby filtering the amplification programs. It designs the structure of micro reaction chambers while ensuring the heat transfer intensity between air in the reaction chambers and reaction tubes. Eventually, rectangular-section channels are selected as the reaction chambers, with the specifications of 80 mm × 40 mm × 20mm(length × height × width); 4. The paper abandons the circulating fan design of traditional air-bath nucleic acid amplification systems and selects three-millimeter-thick foamy copper(a porosity of 90%, the same as the rectangular section) as the heat transfer module to uniform air temperature in the channels. The CFD simulation and experimental results show that the air uniformity is ± 0.52 ℃, the maximum heating rate is 10 ℃ / s, the maximum cooling rate is 12 ℃ / s and the temperature accuracy is ± 0.55 ℃: they meet the design requirements; 5. The paper conducts thermodynamic analysis on nucleic acid amplification systems against the prevailing temperature latency between samples and heat sources in nucleic acid amplification. It computes the theoretical time constant in different stages of heat transfer from heat sources to samples in accordance with the time constant calculation in unsteady heat conduction. Furthermore, it optimizes the temperature latency on the basis of the analysis and calculation. It takes 19.4s for samples in the optimized micro reaction chambers to be annealed from the moment they are denatured and cooled down, 8.1s to enter the extending stage from annealing and heating up and 6.4s to enter the denaturing stage from extending and heating up. In comparison with the previous heating and cooling rate of samples, the entire amplification process has been improved significantly; 6. According to the electrophoregram comparison of products of Bacillus subtilis and Listeria monocytogenes amplified by the micro reaction chambers and standard PCR instrument, the micro reaction chambers work well; they meet the design requirements. The paper adopts micro reaction chambers before and after optimization to amplify Listeria monocytogenes. It can be seen from the electrophoregram of amplified products before and after optimization that amplification effects of the micro reaction chambers are improved by changing samples with optimized temperature difference and air temperature latency.Conclusion: This paper designs a nucleic acid amplification system facing field detection and put forward the project that thoughing the intergrated of reaction chamber complete the screening of amplification procedure. The copper foma is used to replace fan to even air temperature and increase thermal efficiency, the problem of portable and intergration is also sloved by using copper foam. The size of a single micro reaction chamber is 80 mm × 40 mm × 20mm(length × height × width); the maximum heating rate is 10 ℃ / s and the maximum cooling rate is 12 ℃ / s; the temperature accuracy is ± 0.55 ℃; the air uniformity is ± 0.52 ℃. The results of amplification of the samples show that the system works well and meets the initial design requirements.