Study On Key Technology Of Automatic Preparation Of Universal Closed Microbial Sample Nucleic Acid Based On Cavitation Effect

Objective: Problems of food safety, outbreaks of infectious diseases, and bioterrorist attract caused by pathogenic microorganism occur one after another, not only posing threat to personal well-being and life safety, but also potentially causing panic and disorder in society. When such suspicious circumstances occur, the traditional way of identifying suspected microbial samples by detecting them in a biological laboratory would normally takes 2 to 4 days, which includes on-site sample collection, transfer, isolation and culture, extraction of nucleic acid, detection and identification. Apparently, the time span cannot meet the need for an on-site rapid and emergent response. In order to increase the efficiency of on-site emergent response, it is necessary to detect and identify microbial samples on the spot. The common solution is to conduct nucleic acid detection by using RT-PCR. Because of the specific and highly sensitive technique it provides for nucleic acid detection, RTPCR has become the “gold standard” for microbial detection and identification.Currently, portable RT-PCR instruments which can be brought to the spot are already available in the market. However, the preparation of nucleic acid from microbial samples is still operated manually. Not only it is time-wasting and energyconsuming due to its poor efficiency and consistency, but also it poses high risks of cross contamination. While the existing automatic preparation instrument of nucleic acid has achieved the “automatization” and “standardization” of nucleic acid preparation and has contributed to increase in efficiency and accuracy, it fails to resolve the problems of “versatility” and “closedness”, which makes it unable to be applied to on-site preparation of nucleic acid.Under such background, this project has conducted a research on key technologies of versatile closed on-site preparation of nucleic acid from microbial samples, in order to provide the theoretical support and solution for the development of an automatic, standardized, versatile, and closed system of nucleic acid preparation.Methods and contents: This project studies the key technologies of versatile closed on-site preparation of nucleic acid from microbial samples. The study includes 5 parts: study on the lysis mechanism for cells that lysed by ultrasonic cavitation; design and modeling analysis of non-contact ultrasonic system for microorganism lysis; demonstration of multi-mode resonance reinforcement theory and optimization of vibrating diaphragm parameter; establishment and optimization of the standardized solution for closed isolation and purification of nucleic acid; and design and development of versatile closed automatic preparation platform of nucleic acid. The technical routes and methodologies adopted in each part are, respectively, “observation – literature research – flow field modeling – lysis modeling – parameter calculation”, “analysis of present situation – schematic design – modeling analysis – analytical calculation – numerical calculation”, “theoretical proposal – theory demonstration – optimization of parameter – platform establishment – evaluation of experiment”, “analysis of present situation – schematic design – system optimization – platform establishment – evaluation of experiment”, “schematic design – structural design – platform design – prototype trial – evaluation of experiment”. The details are as followings:(1) Study on the lysis mechanism for cells that lysed by ultrasonic cavitationBased on observation and literature research, the study analyzed the factors of ultrasonic cavitation that cause the lysis of cells. Then, a flow field model of ultrasonic cavitation has been established in order to describe its characteristics of motion. A dynamical equation of cell lysis in ultrasonic cavitation has been built according to the mechanism of ruptured dropping liquid in the flow field model, and the relative parameters were calculated.(2) Design and modeling analysis of non-contact ultrasonic system formicroorganism lysisConsidering the various deficiencies in current contact ultrasonic lysis instruments, a non-contact ultrasonic system for microorganism lysis was designed. Regarding to the core problems of the research, a simplified model of non-contact ultrasonic lysis system was built. According to the basic theorization and equation of elastomer vibration, the study calculated the analytic solution of the mode parameter of vibrating diaphragm. Then, based on finite element analysis, the numerical solution of the mode parameter of vibrating diaphragm was calculated.(3) Demonstration of multi-mode resonance reinforcement theory andoptimization of vibrating diaphragm parameterAccording to the distribution regularities of vibrating diaphragm’s first ten natural frequencies in the given example, the multi-mode resonance reinforcement theory was put forward and demonstrated. In order to ensure the optimal efficiency of energy transfer, this study discussed and determined the operative step order and mode order of vibrating diaphragm. It also investigated the influences of curvature radius, thickness and preload force on the vibrating diaphragm’s second step order, and made a more optimal designs to each parameters. Then, this study set up a platform for lysis experiment, conducted the cell lysis experiment with microbial samples, and made a verification and judgment to the design schemes from the three aspects of morphology, lysis rates and nucleic acid detection.(4) Establishment and optimization of the standardized solution for closedisolation and purification of nucleic acidGiven that the existing methods of nucleic acid isolation and purification were unable to be used in the process of on-site nucleic acid automatic preparation, this study planed to design a supercharged and filtrated scheme for nucleic acid isolation and purification, so a isolation and purification system was built and optimized In accordance with the designed scheme and optimized system, this study carried out the microbial nucleic acid isolation and purification experiment, and through the PCR amplification and gel electrophoresis, it also made a verification and judgment to the designed scheme and optimized system.(5) Design and development of versatile closed automatic preparationplatform of nucleic acidOn the basis of non-contact ultrasonic lysis scheme and close nucleic acid isolation and purification scheme, this study designed a principle scheme for versatile closed preparation of the nucleic acid of microbial samples and also devised the physical structure of the cartridge. In order to achieve the automated function of the cartridge, it designed a versatile closed automated preparation platform for nucleic acid, which included the design and realization of mechanical module, circuit module and control module. After that, this study trial-produced a four-channel prototype to carry out the automated preparation experiment for microbial nucleic acid, so as to conduct a performance evaluation to this prototype from four perspectives, namely the cell’s concentration efficiency, the yield and quality of nucleic acid, the consistency(repetition) of nucleic acid and its detectability.Results: In light of the established technical routes and methods, this study researched every part in the subject of versatile closed on-site automated preparation for microbial nucleic acid, from which the expected results were obtained.(1) Study on the lysis mechanism for cells that lysed by ultrasonic cavitationBased on observation and literature research, this study made sure that the factors of ultrasonic cavitation causing the lysis of cells came from the mechanical forces. Then, a vortex flow field model was established in order to describe its characteristics of motion in this ultrasonic cavitation flow field. This study also created the dynamical conditions for cell lysis, deduced the mathematical expressions about the dynamic pressure difference occurring around the cells for large-scale vortex and small-scale vortex respectively. According to the critical conditions of cell lysis, the theoretical equation of cell’s largest steady-state scale in the ultrasonic cavitation flow field was deduced. In accordance with the distribution function of cell scale, the theoretical equation of cell lysis rate was also deduced.(2) Design and modeling analysis of non-contact ultrasonic system for microorganism lysisThis study designed a non-contact ultrasonic system for microorganism lysis to overcome the various deficiencies in current contact ultrasonic lysis instrument. It built a simplified model of non-contact ultrasonic lysis system and analyzed the core and “bottleneck” problems. Based on the fundamental principles of elastomer vibration, the study calculated the analytic solutions of vibrating diaphragm’s natural frequencies and natural mode of vibration. On account of the mode analysis of ANSYS, this study obtained the numerical solutions of special parameters(including width: W=12 mm, curvature radius: R=22 mm, thickness: T=0.4 mm and preload force from ultrasonic probe: F=3 N),vibrating diaphragm’s natural frequencies and natural mode of vibration.( 3) Demonstration of multi-mode resonance reinforcement theory and optimization of vibrating diaphragm parameterThe reinforcement theory of multi-mode resonance provided the follow-up optimization of vibrating diaphragm with the guiding references and basic principles. This theory showed that the vibrating diaphragm should be operated in the second step order while the multi-mode resonance took place in the third-step mode(M3), fourth-step mode(M4), fifth-step mode(M5) and sixth-step mode(M6). It also analyzed the influences of curvature radius, thickness and preload force on the vibrating diaphragm’s second step order, optimized every parameter, and found that the G2 step frequencies showed a slow decrease with the increase of curvature radius, a rapidly linear increase with the increase of thickness and a slowly linear decrease with the increase of preload force. In line with the optimized parameters, the study set up a platform for lysis experiment to conduct the cell lysis experiment with microbial samples, and from the three aspects of morphology, lysis rates and nucleic acid detection, it proved that the non-contact ultrasonic lysis scheme could effectively split different kinds of microbial sample cells, achieve the intracelluar release of nucleic acid and possess a great universality.(4)Establishment and optimization of the standardized solution for closed isolation and purification of nucleic acidThe supercharged and filtrated scheme for nucleic acid isolation and purification could meet the closed, automated, standardized, versatile and consistent demands in the isolation and purification of nucleic acid. The study comprehensively considered the yield and quality of nucleic acid and optimized the isolation and purification system, in which the adsorption carrier of nucleic acid should be the Affimag SLE series’ magnetic microsphere with its size of 1 μm, and the concentration of Na Cl should be controlled within 4~5 mol/L while the p H value within 3~4 in the combined liquid. According to the optimized system, the study used the simple filter apparatus in supercharged and filtrated scheme for nucleic acid isolation and purification, extracted the nucleic acids from escherichia coli, staphylococcus aureus and bacillus atrophaeus after lysis, and also conducted a effect evaluation through the PCR amplification and gel electrophoresis; the experiment results indicated that the supercharged and filtrated scheme for nucleic acid isolation and purification could achieve the isolation and purification of nucleic acid of microbial samples.(5)Design and development of versatile closed automatic preparation platformof nucleic acidThe study designed a versatile closed automated preparation platform for nucleic acid, completed the trial-production of four-channel prototype and carried out the automated preparation experiment for microbial nucleic acid. The experiment results showed that as for the concentration efficiency, the retention rate of microporous membrane exceeded 90%, and the maximum concentration multiple of the cartridge was 59.31. With regard to the quality(purity) of nucleic acid prepared by the fourchannel prototype, it’s a bit lower than the nucleic acid that was manually prepared by Tiangen Company’s TIANamp Bacteria DNA Kit, but its OD260/280 all surpassed 1.6, which indicated that the purity of nucleic acid was relatively high and could be used in the analysis of downstream detection. As for the yield(concentration) of nucleic acid, the four-channel prototype was inferior to TIANamp kit in the process of preparing the gram-negative bacteria of nucleic acid, but it’s superior to TIANamp kit in preparing the gram-positive bacteria and bacterial spore of nucleic acid, which demonstrated that this prototype had a fine capacity of cell lysis but needed a further optimization in the isolation and purification system for nucleic acid. In the aspect of the consistency of nucleic acid preparation, the four channels showed the variation coefficients of nucleic acid’s yield in the six-time preparation process, which were 5.02%, 4.96%, 4.74% and 5.73% respectively; the average variation coefficients(sixtime repetitions) of nucleic acid’s yield and prototype in the channels were 5.55% and 5.66% respectively, while the variation coefficient of TIANamp kit in six experiments reached to 17.14%, which displayed that the four-channel prototype enjoyed a good consistency(repetition) in the preparation of nucleic acid. As to the detecability of nucleic acid, the nucleic acid prepared by four-channel prototype could be directly used to the RT-PCR detection, and its lowest detection limit was 43 cfu/m L theoretically.Conclusions: This paper carried out the research about the key technology of versatile closed on-site automated preparation for nucleic acid of microbial samples, designed a serials of solutions to this technology, and developed a four-channel prototype for it which satisfied the “automated”, “standardized”, “versatile” and “closed” demands. Through experiences, this paper also conducted a performance evaluation to this prototype in various aspects, such as the cell’s concentration efficiency, the yield and quality of nucleic acid, the consistency(repetition) of nucleic acid and its detectability and so on. The experiment results showed that this prototype could be utilized in various kinds of automated preparation for nucleic acid of microbial samples. It has high concentration efficiency, and the nucleic acid prepared by it enjoys relatively high yield, quality and consistency and can be directly used in the later detection and identification. Therefore, the solutions designed in this paper and the four-channel prototype could both be adopted in the automated preparation of nucleic acid under any on-site conditions, which could help to solve the “bottleneck” problem in the on-site detection and identification for microorganisms.nucleic acid preparation; ultrasonic lysis; multi-mode resonance;...