Paper-based microfluidic RPA (recombinase polymerase amplification) detection device based on sound wave vibration and rotary micro valve

Abstract

The invention relates to a paper-based micro-fluidic RPA detection device based on acoustic vibration and a rotary micro valve. The paper-based micro-fluidic RPA detection device comprises a shell; the micro-fluidic chip comprises a central chip and a peripheral chip, the central chip is provided with a reagent area, the peripheral chip can rotate around the central chip and is provided with a reaction area, and the peripheral chip is also internally provided with a micro-channel which is communicated with the reaction area and extends towards the reagent area; the heating assembly is arranged at the bottom of the micro-fluidic chip; the rotating assembly is fixed in the shell and is connected to the peripheral chip; the fluorescence detector is arranged on the shell and faces the reaction area of the micro-fluidic chip; and the control system is connected with the heating assembly, the vibration assembly, the rotating assembly and the fluorescence detector. The device has the characteristics of high automation degree, rapid chemical response, good reagent mixing effect, stable detection signal, portable detection and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of instant detection, in particular to a paper-based microfluidic RPA detection device based on acoustic wave vibration and rotating micro valve. BACKGROUND

[0002] African swine fever (ASF) and pseudorabies (PR) are two important viral infectious diseases that endanger the global pig industry, posing a fatal threat to the safe production and economic benefits of the pig breeding industry. Among them, ASF is caused by African swine fever virus (ASFV) and has extremely strong infectivity and lethality. At present, there is no effective preventive vaccine worldwide, and once it breaks out in a breeding farm, it often leads to 100% mortality of the pig population, not only causing direct breeding loss, but also triggering a chain reaction of regional pig transportation restrictions, market supply shortages, and other chain reactions, resulting in huge economic losses. PR is caused by pseudorabies virus (PRV), and piglets infected with PR often show neurological symptoms (such as ataxia and convulsions), and sows infected with PR are prone to respiratory difficulties, abortion, stillbirth, and other reproductive disorders, which seriously affect the reproductive efficiency and survival rate of piglets. In addition to direct breeding losses, both diseases will further exacerbate indirect losses in the industry through quarantine isolation, international trade restrictions, and control measures such as culling of infected pigs, which seriously restricts the sustainable development of the pig industry.

[0003] To effectively prevent and control the spread of ASF and PR, precise and efficient early detection technology is the key. Currently, the commonly used detection methods mainly include serological detection and nucleic acid amplification-based molecular diagnosis methods. Among them, polymerase chain reaction (PCR) and real-time fluorescent quantitative PCR (qPCR) have become the mainstream technology for ASFV and PRV nucleic acid detection due to their high detection sensitivity and strong specificity. However, such molecular diagnostic methods have significant application limitations: on the one hand, they rely on expensive thermal cycler instruments (such as PCR instruments and fluorescent quantitative PCR instruments) and professional laboratory operating environments, with high equipment procurement and maintenance costs, making it difficult to deploy in resource-limited scenarios such as primary breeding farms, slaughterhouses, and cold-chain quarantine points; on the other hand, the detection process requires multiple steps such as nucleic acid extraction, system preparation, thermal cycle amplification, and result interpretation, with a long overall detection period (usually 1-2 hours), and high requirements for the professional skills of the operators, which cannot meet the core needs of "instant, portable, and easy-to-operate" for point-of-care testing (POCT), and is difficult to adapt to the rapid screening and emergency prevention and control scenarios in the event of an outbreak.

[0004] To solve the adaptability of PCR / qPCR technology in POCT scenarios, in recent years, researchers have developed a variety of nucleic acid isothermal amplification technologies, among which loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are the most representative. Compared with other isothermal amplification technologies, RPA technology shows more prominent POCT adaptation advantages: it does not require complex temperature cycling process, only requires constant temperature conditions at 37-42°C to achieve rapid nucleic acid amplification, usually within 30 minutes to complete the amplification reaction; at the same time, RPA has high fault tolerance for primer design, and the specific binding mechanism during amplification can effectively reduce the risk of non-specific amplification, and the detection accuracy is stable. Compared with PCR, RPA does not need to rely on a thermal cycler, and the equipment demand is greatly simplified; compared with LAMP, RPA further reduces the false positive problem caused by non-specific amplification, so it becomes an ideal choice for animal disease POCT field.

[0005] In the design of POCT detection platform carriers, paper-based microfluidic analysis devices (μPADs) have become an important development direction for building low-cost and portable detection platforms in recent years due to their unique advantages. The porous fiber structure of paper can drive liquid transport autonomously through capillary action, without relying on external pumping equipment (such as peristaltic pumps, syringe pumps), which can achieve sample and reagent flow and mixing; at the same time, μPADs have the advantages of low manufacturing cost (can be prepared by printing, cutting, etc. Simple process), low operation threshold (no need for professional instrument operation skills), disposable use (avoid cross contamination), environmentally friendly (easy to degrade after disposal), etc. It is widely used in the exploration of on-site rapid detection scenarios such as pathogen detection and biochemical index analysis.

[0006] However, when combining RPA isothermal amplification technology with μPADs platform to realize the on-site rapid detection of ASFV and PRV, the existing technology still faces two major challenges: Firstly, the degree of automation is insufficient. Most existing paper-based RPA detection platforms rely on manual operation or external auxiliary equipment to complete the detection process, and it is difficult to achieve fully automated operation. For example, the folding paper-based RPA device proposed by Rohrman et al. in Analytical Chemistry (2012) requires manual folding of the paper to achieve mixing of reagents and samples. The operation process relies on the experience of personnel and is prone to poor reaction consistency due to differences in folding force and speed. The centrifugal-driven paper-based microfluidic system reported by Chen et al. in Lab on a Chip (2019) can achieve automatic reagent transport, but it relies on external electric centrifugal equipment for power supply. The equipment is bulky, costly, and cannot be used without power supply, making it difficult to adapt to basic field detection scenarios. The sliding sheet detector proposed by Cordray et al. in Analytical Chemistry (2015) simplifies the structural design, but still requires manual pushing of the sliding component to trigger reagent mixing and amplification reaction. The operation has poor repeatability and cannot avoid the pollution risk introduced by manual operation. The above schemes have not broken through the limitations of "manual dependence" or "external equipment dependence", and it is difficult to achieve truly automated detection.

[0007] Secondly, the integration degree is low. Most existing μPADs devices can only realize a single link in the detection process, and cannot integrate the "automatic reagent mixing-constant temperature amplification-real-time detection" whole process on a single chip. For example, some paper-based devices can only complete the RPA amplification reaction, and the amplified product needs to be transferred to other detection equipment (such as a fluorescence detector) for result interpretation. Some devices integrate the reaction and detection modules, but the reagent mixing still needs to be manually added and shaken after adding the sample, or relies on external equipment for assistance. This "separate link operation" mode not only prolongs the detection time, but also increases the risk of sample exposure and cross contamination, and requires additional equipment, greatly reducing the portability and application value of the POCT platform.

[0008] In summary, although the existing technology has made some progress in ASFV and PRV nucleic acid rapid detection (such as RPA technology) and POCT platform carriers (such as μPADs), there are still key defects such as insufficient automation and low detection integration. Therefore, it is urgent to develop a nucleic acid amplification and detection platform that can realize "reagent storage-automatic mixing-constant temperature amplification-result detection" integration and automation, to meet the actual needs of ASFV and PRV on-site rapid diagnosis and provide technical support for disease prevention and control in pig farming. SUMMARY

[0009] The present application aims to solve the problems of poor portability and low automation of existing RPA detection devices.

[0010] In a first aspect, the application provides a paper-based microfluidic RPA detection device based on acoustic wave vibration and rotating microvalve, comprising: a shell as a main body; a microfluidic chip comprising a center chip provided with a reagent area and a peripheral chip rotatable around the center chip and provided with a reaction area, and a microfluidic channel in the peripheral chip, which is in communication with the reaction area and extends towards the reagent area; the center chip is fixedly connected to the shell; a heating assembly arranged below the microfluidic chip for heating the reaction area; a rotating assembly fixed in the shell and connected to the peripheral chip; the rotating assembly is used to connect or disconnect the microfluidic channel to the reagent area; a vibration assembly comprising a piezoelectric transducer arranged below the microfluidic chip and a signal generator connected to the piezoelectric transducer; a fluorescence detector arranged on the shell and facing the reaction area of the microfluidic chip, for detecting the fluorescence signal generated by the microfluidic chip; a control system connected to the heating assembly, rotating assembly, vibration assembly and fluorescence detector.

[0011] Paper-based microfluidic analysis devices (μPADs) rely on the porous structure of paper and capillary driving, and can realize liquid transport without pump, with advantages of low cost, disposability and intuitive reading, and have been widely used in POCT and other scenes. However, the existing μPADs still have common pain points in reagent mixing: first, the colorimetric detection is not uniform. Droplet drying can easily cause "coffee ring effect", causing product enrichment at the edge of the paper, making it difficult to obtain uniform signal in space, affecting quantitative accuracy; second, sample processing is limited. The mechanical strength of the paper substrate is limited and the channels are irregular, making it difficult to quickly and stably enrich particles and cells and realize signal amplification; third, the mixing efficiency is low. The passive mixing method relies on multi-layer stacking or tortuous flow channel, and the process is complex, and the end area is prone to unmixed phenomenon, which is difficult to meet the high-throughput and uniform mixing demand. The existing μPADs drive liquid through capillary action, but the flow speed and mixing effect are difficult to meet the RPA reaction demand. Although micro-pump or mechanical stirring can improve the mixing effect, but this goes against the low-cost and external device-free advantages of the paper-based platform. The application provides a paper-based RPA platform based on low-frequency acoustic wave driving, which solves the problem of insufficient mixing of traditional paper-based RPA chips through acoustic wave assisted mixing, and realizes rapid, low-cost and high-sensitivity nucleic acid detection.

[0012] Further specifically, the peripheral chip comprises, from bottom to top, a first chip shell provided with a heat transfer port, a paper-based microfluidic chip provided with the reaction area and the microfluidic channel, and a second chip shell, the heat transfer port corresponding to the heating assembly.

[0013] Further, a substrate paper chip is arranged between the first chip shell and the paper-based microfluidic chip.

[0014] Further, a third chip shell is arranged, and the central chip is connected to the housing through the third chip shell.

[0015] Further, the microfluidic channel is subjected to surface modification treatment, and the surface modification treatment method is specifically as follows: after oxygen plasma treatment is performed on the surface of the microfluidic channel, PVP solution is added for modification.

[0016] Further, the heating assembly comprises a metal shell and an electric heating wire surrounding the outer surface of the metal shell.

[0017] Further, the surface of the metal shell close to the microfluidic chip is a plane.

[0018] Further, the microfluidic channel and the reaction area have three connection ports which are geometrically symmetrically distributed.

[0019] Further, the rotating assembly comprises a motor and a transmission rod connected to the rotating shaft of the motor, and the transmission rod is connected to the peripheral chip.

[0020] In a second aspect, the application further provides an RPA detection method, which is performed by using the RPA detection device, and specifically comprises the following steps: S1, placing a sample and a corresponding RPA reaction enzyme powder into the reaction area; S2, controlling the heating assembly to heat the reaction area and maintain constant temperature for a preset time; S3, controlling the rotating assembly to rotate the peripheral chip around the central chip, and make the microfluidic channel communicate with the reagent area where the fluorescent activator is placed; S4, after the microfluidic channel communicates with the reagent area for a set time, controlling the rotating assembly to rotate the peripheral chip to disconnect the microfluidic channel from the reagent area, and rotate the reaction area to above the heating assembly, and controlling the heating assembly to continue heating the reaction area and maintaining constant temperature; S5, after the reaction is completed, controlling the rotating assembly to rotate the peripheral chip to rotate the reaction area to directly below the fluorescent detector, and the fluorescent detector detects the RPA fluorescent signal.

[0021] The application has the beneficial effects that: through the internal control system, the rotating assembly can drive the microfluidic chip to rotate accurately, can automatically control the connection or disconnection of the microfluidic channel and the reagent area, the approach or distance of the reaction area and the heating assembly, and the approach or distance of the reaction area and the fluorescence detector. The application can realize the timed and quantitative addition of reagents, the efficient mixing of reagents, the start or stop of constant temperature amplification reaction, and can automatically detect the RPA fluorescence signal. The application has the characteristics of high automation degree, rapid chemical response, good reagent mixing effect, stable detection signal, portable detection and the like. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a perspective view of the RPA detection device in the application.

[0023] Figure 2 is a top view of the microfluidic chip in the application.

[0024] Figure 3 is a bottom view of the microfluidic chip in the application.

[0025] Figure 4 is an exploded schematic view of the peripheral chip in the application.

[0026] Figure 5 is an internal connection section view of the RPA detection device in the application.

[0027] Figure 6 is a schematic view of the connection of each component in the application.

[0028] Figure 7 is a perspective view of the heating assembly in the application.

[0029] Figure 8 is a distance optimization test diagram between the fluorescence detector and the microfluidic chip in the application.

[0030] Figure 9 is an RPA detection flowchart in the application.

[0031] Figure 10 is an ASFV test data diagram in the application.

[0032] Figure 11 is a PRV test data diagram in the application.

[0033] Figure 12 is a fluorescence activation liquid volume optimization test diagram in the application.

[0034] Figure 13 is a colorimetric experiment in the application. The colorimetric mixing effect comparison diagram.

[0035] In the figure: 100, the shell; 200, the microfluidic chip; 21, the microfluidic channel; 22, the reaction area; 23, the reagent area; 210, the peripheral chip; 211, the first chip shell; 212, the heat transfer port; 213, the substrate paper chip; 214, the paper-based microfluidic chip; 215, the second chip shell; 216, the detection port; 220, the center chip; 221, the third chip shell; 300, the heating assembly; 310, the metal shell; 320, the heating wire; 400, the rotating assembly; 500, the fluorescence detector. DETAILED DESCRIPTION

[0036] For the purpose, technical solutions and advantages of the implementation of the present application, the specific embodiments of the present application are described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0037] The paper-based microfluidic RPA detection device designed in the present application is shown in Figure 1 . After the user adds the sample to the designated area of the microfluidic chip 200, covers the cover body, starts the device and presets the temperature and other adjustment parameters, the RPA detection of the sample can be automatically completed, and the detection result is finally displayed on the front display screen.

[0038] In some specific embodiments, the specific structure of the microfluidic chip 200 is shown in Figure 2 and Figure 3 . The microfluidic chip 200 includes a center chip 220 located at the rotation center and a peripheral chip 210 capable of rotating around the center chip 220. The center chip 220 is provided with a reagent area 23, and the peripheral chip 210 is provided with a plurality of reaction areas 22 and a plurality of microfluidic channels 21 corresponding to the reaction areas 22. The microfluidic channels 21 are connected to the reaction areas 22 and extend towards the reagent area 23. The reagent area 23 also has interfaces corresponding to the plurality of reaction areas 22, respectively.

[0039] The center chip 220 is fixedly connected to the shell 100 through the third chip shell 221, and the microfluidic channel 21 can be controlled to be connected or disconnected with the reagent area 23 by rotating the peripheral chip 210. When the microfluidic channel 21 connects the reagent area 23 and the reaction area 22, the reagent stored in the reagent area 23 will be driven to flow to the reaction area 22 by capillary action due to the porous fiber structure of the paper. Through this rotation control, the device can automatically complete the timed and quantitative addition of reagents in the RPA detection, without the user's complex sample addition operation.

[0040] As shown in Figure 4As shown, the peripheral chip 210 specifically comprises a first chip shell 211, a paper-based microfluidic chip 214 and a second chip shell 215. The microfluidic channel 21 and the reaction zone 22 are arranged on the paper-based microfluidic chip 214. The first chip shell 211 is provided with a heat transfer port 212 corresponding to the heating assembly 300, and the second chip shell 215 is provided with a detection port 216 corresponding to the fluorescence detector 500. The first chip shell 211 and the second chip shell 215 are both made of hard material (such as PMMA, PDMS, etc.), and are provided with a plurality of positioning holes. The first chip shell 211, the second chip shell 215 and the paper-based microfluidic chip 214 are rotated together by the rotating assembly 400. The hard shell can ensure the reliability of the rotation of the paper-based microfluidic chip 214. When the device is used, the first chip shell 211 and the second chip shell 215 can be reused, and the user only needs to replace the disposable paper-based microfluidic chip 214. In a feasible scheme, the reagent zone 23 is designed to store sufficient reagents, which can meet the needs of multiple paper-based microfluidic chips 214 for detection reagents. In the further improvement of the present application, in order to avoid contamination caused by reagent penetration, a substrate paper chip 213 is further arranged between the first chip shell 211 and the paper-based microfluidic chip 214.

[0041] In the further improvement of the present application, in order to accelerate the speed of reagent flow to the reaction zone 22 and reduce the reaction waiting time, especially for high viscosity reagents (such as enzyme reagents and primer mixtures in RPA reaction system, which have high viscosity due to the presence of PEG in the reagents), the present application further performs surface modification treatment on the microfluidic channel 21. Specifically, the paper-based microfluidic channel is subjected to oxygen plasma treatment for 2-6 min, and a PVP solution with a concentration of 1%-17% is added to the microfluidic channel 21 after oxygen plasma treatment, and after drying, a microfluidic channel 21 with improved fluid transmission rate is obtained. In order to further improve the reagent mixing efficiency and avoid the problem of inaccurate reaction results caused by uneven distribution of reagents, the present application designs multiple connection positions between the microfluidic channel 21 and the reaction zone 22, which are geometrically symmetrically distributed, and the reagents can enter the reaction zone 22 from multiple angles and uniformly fill the reaction zone 22.

[0042] In some embodiments, as shown in Figure 5 In the present application, the microfluidic chip 200, the heating assembly 300 and the rotating assembly 400 are connected and fixed by a cylindrical structure, which is only an example, and other convenient connection methods can also be used. As shown in Figure 6 The heating assembly 300 is arranged at the bottom of the microfluidic chip 200, and the rotating assembly 400 controls the peripheral chip 210 to move away from or close to the heating assembly 300. As shown in Figure 6As shown, the heating assembly 300 can consist of a metal shell 310 and a heating wire 320 surrounding the outer surface of the metal shell 310. The heating wire 320 heats the metal shell 310, so that the uniform heat distribution on the surface of the metal shell 310 can uniformly heat the reaction zone 22. The heating assembly 300 can also be a heating plate or other uniform heating device that can control the temperature. In one feasible solution, the heating assembly 300 can be fixed on the side of the first chip shell 211 away from the paper-based microfluidic chip 214, and the heating assembly 300 can be directly activated when the reaction zone 22 needs to be heated.

[0043] In some specific embodiments, to improve the reagent mixing effect within the reaction zone 22, an ultrasonic vibration component is also provided between the microfluidic chip 200 and the heating component 300. The ultrasonic vibration component includes a piezoelectric transducer (PZT, model SMBA4510T05, 1mm thick) adhered to the first chip housing 211. The transducer is connected to a function signal generator (Agilent 33500B series) and driven by a power amplifier (Aigtek ATA-3080). Preferably, the acoustic wave driving frequency is set to 500~1500Hz, and the voltage range is 20~60V; a Faraday wave is generated by low-frequency sine wave excitation, driving the reagents within the paper-based microfluidic chip 214 to generate uniform convective current, thereby accelerating mixing.

[0044] In some specific embodiments, the rotating assembly 400 includes a rotating motor and a rotating rod connected to the motor shaft. The end of the rotating rod is fixed to a positioning hole on the peripheral chip 210. The rotating assembly 400 controls the connection or disconnection of the microchannel 21 to the reagent area 23. In actual testing, the volume of reagent entering the reaction area 22 can be controlled by adjusting the connection time. When the microchannel 21 undergoes the aforementioned surface modification treatment, a shorter connection time for the microchannel 21 is sufficient for the reaction area 22 to obtain a sufficient amount of reagent. The rotating assembly 400 is also used to align the reaction area 22 with the fluorescence detector 500. When a fluorescence signal is generated, the rotating assembly 400 moves the reaction area 22 to the optimal detection position of the fluorescence detector 500. If multiple reaction areas 22 are provided within the peripheral chip 210, the rotating assembly 400 can rotate and adjust the peripheral chip 210 sequentially as needed. Figure 8 As shown, the fluorescence signal intensity obtained at different intervals under the same sample concentration is detected. In order to obtain the best fluorescence detection signal, when the device cover is closed, the distance between the fluorescence detector 500 and the microfluidic chip 200 is preferably 6 mm.

[0045] In some embodiments, the device also has a display screen connected to the control system, and the user can independently adjust the heating temperature, chip rotation angle, rotation speed, waiting time of each step, and other parameters. The final RPA signal can be directly displayed on the display screen, and the user can perform RPA detection on multiple samples and intuitively obtain the RPA detection result without the aid of large and complex equipment and without complex operation.

[0046] The specific use method of the device is as shown in Figure 9 S1, place the sample and the RPA reaction enzyme powder corresponding to the sample into the reaction area 22. Different reaction areas 22 can place different samples and RPA reaction enzyme powders corresponding to the samples. For example, as shown in the chip structure Figure 9 When the paper-based microfluidic chip 214 has two reaction areas 22, ASF samples and PRV samples can be placed respectively.

[0047] S2, rotate the peripheral chip 210 around the central chip 220 by the rotating assembly 400, control the reaction area 22 to rotate to the upper side corresponding to the heating assembly 300, start the heating assembly 300 to heat the reaction area 22 and keep constant temperature for a period of time, and the reaction area 22 performs preliminary amplification reaction.

[0048] S3, after a period of reaction, rotate the peripheral chip 210 around the central chip 220 by the rotating assembly 400, control the microfluidic channel 21 to communicate with the reagent area 23 where the fluorescence activator is placed, and the activator enters the reaction area 22 through the microfluidic channel 21.

[0049] S4, after the microfluidic channel 21 communicates with the reagent area 23 for a period of time, the reaction area 22 obtains sufficient activator, the rotating assembly 400 is controlled to rotate the peripheral chip 210 to disconnect the microfluidic channel 21 from the reagent area 23. Start the vibration assembly to vibrate the chip, and make the reaction area 22 rotate to the upper side of the heating assembly 300 again, control the heating assembly 300 to continue heating the reaction area 22 and keep constant temperature, and the reagent performs constant temperature amplification reaction.

[0050] S5, after the reaction is completed, the rotating assembly 400 is controlled to rotate the peripheral chip 210 to make the reaction area 22 rotate to the position directly below the fluorescence detector 500, and the fluorescence detector 500 detects the RPA fluorescence signal.

[0051] ​Preparation of paper-based microfluidic chip 214: The chip pattern was drawn using software (flow channel 21 width 2 mm, reaction zone 22 diameter 4 mm). The hydrophilic region and hydrophobic barrier region were printed on Whatman No. 4 filter paper using a Xerox 8580DN wax jet printer. The printed paper was placed on a CS15956-31 hot plate (Cole-Parmer) and heated at 120°C for 120 seconds to allow the wax to fully melt and penetrate the paper to form a stable hydrophobic barrier. The laser cutting machine was used to cut according to the preset pattern, laser intensity 10%, cutting speed 50 mm / s, to obtain the paper-based microfluidic chip 214 as shown in Figure 4 .

[0052] Example 1: RPA detection of African swine fever virus (ASFV). The freeze-dried enzyme powder (recombinant enzyme 5 U, DNA polymerase 2 U, single-stranded binding protein 1 μg) was pre-stored in the reaction zone 22 of the paper-based microfluidic chip 214, and 25 μL of ASFV fluorescent isothermal amplification detection reagent (containing 10% PEG-20000) was added to the reagent zone 23. The test was carried out using the flow as shown in Figure 9 . First, the sample ASFV specific primer (final concentration 0.4 μM) was added to the reaction zone 22, and the heating assembly 300 was started (at this time the heating assembly 300 was opposite to the reaction zone 22), the temperature of the reaction zone was controlled at 42±0.3°C, and the isothermal nucleic acid amplification was carried out for 1 min. After amplification, the peripheral chip 210 was rotated by the rotating assembly 400 to connect the microfluidic channel 21 with the reagent zone 23, and the fluorescent activator stored in the reagent zone 23 entered the reagent zone 23 (the average time was 22 s). Then the peripheral chip 210 was rotated to make the reaction zone 22 return to the heating zone position, and the amplification reaction was continued for 12 min, finally the fluorophore was hydrolyzed to release, and the fluorescence detector 500 detected the fluorescence signal. The fluorescence signals of different concentrations and different amplification times were detected by repeating the experiment, and the results are shown in Figure 10 . The detection limit of the device for ASFV was 5 copies / μL.

[0053] Example 2: RPA detection of pseudorabies virus (PRV). The test was carried out using the flow as shown in Figure 8The same detection process, the difference is that the addition of fluorescent activated liquid after the amplification reaction time is different. In the reagent area 23, 17 μL of PRV fluorescent constant temperature amplification detection reagent is added, 8 μL of sample is added to the reaction area, and freeze-dried enzyme powder (recombinant enzyme 5 U, DNA polymerase 2 U, single-stranded binding protein 1 μg) is added. By vibrating the assembly at a frequency of 500 Hz for 10 seconds, the reagent and sample are fully mixed. After 1 minute, the assembly is rotated 400 times to rotate the peripheral chip 210, connecting the microfluidic channel 21 to the reagent area 23, and the fluorescent amplification reagent enters the reaction area 22. The temperature is controlled at 42±0.3°C for constant temperature amplification, and the PRV fluorescent signal can be stably detected after 16 minutes of amplification. Repeat the experiment, detect the fluorescent signal of different concentrations and different amplification times, and the results are shown in Figure 11 The detection limit of the device for PRV is 10 copies / μL.

[0054] Example 3: Optimal fluorescent activation liquid volume detection. After rotating the assembly 400 to connect the microfluidic channel 21 to the reagent area 23, the fluorescent activation liquid will flow into the reaction area 22 on its own, and the transmission time of the reagent in the microfluidic channel 21 is about 22 seconds. In order to ensure that the activation liquid reaches the reaction area 22 in sufficient quantity, the time for connecting the microfluidic channel 21 to the reagent area 23 is designed to be 1 minute. Different volumes of fluorescent activation liquid are added to the reagent area 23, and PRV and ASFV samples are detected in the surface-modified microfluidic channel 21 and the ordinary microfluidic channel 21 without modification, respectively, and the results are shown in Figure 12 Due to the high viscosity of the fluorescent activation liquid, the transmission speed of the activation liquid in the ordinary microfluidic channel 21 without modification is slow, and only a small amount of activation liquid reaches the reaction area, resulting in weak fluorescent signal. The surface-modified microfluidic channel 21 significantly improves the transmission speed of high-viscosity reagents, and in less waiting time, the activation liquid can quickly fill the reaction area, resulting in strong fluorescent signal. In the microfluidic chip 200 designed in the device, the consumption of fluorescent activation liquid for a single detection is less than 30 μL.

[0055] Example 4: By comparing the RGB signal distribution of the liquid in the paper-based chip before and after mixing, the effect of low-frequency sound waves on liquid mixing can be directly observed. As shown in Figure 13 Before mixing, the R channel pixel distribution is wide, ranging from 100 to 180, and showing a large dispersion; although the G channel has a main peak, the tail distribution is long, and the signal is obviously uneven; the B channel even shows a double-peak structure, concentrated in the 70-90 and 130-150 intervals, respectively, indicating that the liquid has not been fully mixed, and the color components differ significantly in different areas. After mixing, as shown in Figure 13(R) shows that all the three channels of signals are converted into single sharp peaks, and the distribution range is significantly narrowed: R value is concentrated around 120, G value is mainly concentrated in the interval of 120-130, B value is stable in the range of 80-90, and the tail distribution is obviously weakened. The results show that after being driven by low-frequency sound waves, the liquid in the paper-based chip is fully mixed, the consistency of RGB signals is significantly improved, the overall uniformity is enhanced by about 2-3 times, and the coffee ring effect is effectively avoided, which provides a stable and uniform reaction environment for RPA reaction.

[0056] The above describes the embodiments of the present application in detail, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A paper-based microfluidic RPA detection device based on acoustic vibration and a rotary microvalve, characterized in that, include: The shell serves as the main body; A microfluidic chip includes a central chip with a reagent zone and a peripheral chip that can rotate around the central chip and has a reaction zone. The peripheral chip also has microchannels that communicate with the reaction zone and extend toward the reagent zone. The central chip is fixedly connected to a housing. A heating component, located below the microfluidic chip, is used to heat the reaction zone; A rotating component is fixed inside the housing and connected to the peripheral chip; the rotating component is used to connect or disconnect the reagent area in the microchannel. The vibration assembly includes a piezoelectric transducer disposed below the microfluidic chip and a signal generator connected to the piezoelectric transducer; A fluorescence detector is disposed on the housing and faces the reaction area of ​​the microfluidic chip, for detecting the fluorescence signal generated by the microfluidic chip; The control system connects the heating component, the rotating component, the vibrating component, and the fluorescence detector.

2. The RPA detection device according to claim 1, characterized in that, The peripheral chip, from bottom to top, includes: a first chip shell with a heat transfer port, a paper-based microfluidic chip with the reaction zone and microchannels, and a second chip shell, wherein the heat transfer port corresponds to the heating component.

3. The RPA detection device according to claim 2, characterized in that, A substrate paper chip is also disposed between the first chip shell and the paper-based microfluidic chip.

4. The RPA detection device according to claim 1, characterized in that, It also includes a third chip housing, through which the central chip is connected to the housing.

5. The RPA detection device according to claim 2, characterized in that, The microchannels undergo surface modification treatment, specifically by subjecting the microchannels to oxygen plasma treatment followed by the addition of PVP solution for modification.

6. The RPA detection device according to claim 1, characterized in that, The heating assembly includes a metal shell and heating wires surrounding the outer surface of the metal shell.

7. The RPA detection device according to claim 6, characterized in that, The vibration component is disposed between the heating component and the microfluidic chip.

8. The RPA detection device according to claim 1, characterized in that, The microchannel and the reaction zone have three geometrically symmetrical connection points.

9. The RPA detection device according to claim 1, characterized in that, The rotating assembly includes a motor and a transmission rod connected to the rotating shaft of the motor, the transmission rod being connected to an external chip.

10. An RPA detection method, characterized in that, The RPA detection device described in any one of claims 1 to 9 is used, specifically including the following steps: S1. Place the sample and the corresponding RPA reaction enzyme powder into the reaction area; S2. Control the heating component to heat the reaction zone and maintain a constant temperature for a preset time; S3. Control the rotating component to make the peripheral chip rotate around the central chip, and make the microchannel connect to the reagent area where the fluorescent activator is placed; S4. After the microchannel connects to the reagent area for a set time, the rotating component is controlled to rotate the peripheral chip to disconnect the microchannel from the reagent area, the vibration component is started to mix thoroughly, and the reaction area is rotated above the heating component. The heating component is then controlled to continue heating the reaction area and maintain a constant temperature. S5. After the reaction is completed, control the rotating component to rotate the peripheral chip so that the reaction area is directly below the fluorescence detector, and the fluorescence detector detects the RPA fluorescence signal.

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