Paper-based microfluidic RPA (recombinase polymerase amplification) detection device based on surface modification and rotary micro valve

Abstract

The invention relates to a paper-based microfluidic RPA detection device based on surface modification and a rotary micro valve. The paper-based microfluidic RPA detection device comprises a shell; the micro-fluidic chip comprises a central chip provided with a reagent area and a peripheral chip which can rotate around the central chip and is provided with a reaction area, and a micro-channel which is communicated with the reaction area and extends towards the reagent area is further arranged in the peripheral chip; the central chip is fixedly connected to the shell; the heating assembly is arranged below 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 rotating assembly and the fluorescence detector. The device has the characteristics of high automation degree, rapid chemical response, portable detection and the like.

Description

Technical Field

[0001] This invention relates to the field of point-of-care testing, and in particular to a paper-based microfluidic RPA testing device based on surface modification and rotary microvalve. Background Technology

[0002] African swine fever (ASF) and pseudorabies (PR) are two major viral infectious diseases that threaten the global pig industry, posing a deadly threat to the safe production and economic benefits of pig farming. ASF, caused by the African swine fever virus (ASFV), is highly contagious and has a high mortality rate. Currently, there is no effective vaccine available globally. Once an outbreak occurs on a farm, it often leads to a 100% mortality rate in the pig herd, causing not only direct losses in pig stocks but also triggering a chain reaction of restricted pig transportation and tight market supply within the region, resulting in huge economic losses. Pseudorabies, caused by the pseudorabies virus (PRV), often manifests as neurological symptoms in infected piglets (such as ataxia and convulsions), while infected sows are prone to respiratory distress, abortion, stillbirth, and other reproductive problems, severely impacting pig reproductive efficiency and piglet survival rates. In addition to direct losses in pig farming, the two diseases can further exacerbate indirect losses to the industry through control measures such as quarantine, international trade restrictions, and culling of infected pigs, severely restricting the sustainable development of the pig farming industry.

[0003] To effectively control the spread of ASF and PR, accurate and efficient early detection technologies are crucial. Currently, commonly used clinical detection methods are mainly divided into two categories: serological testing and molecular diagnostic methods based on nucleic acid amplification. Among them, polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) have become the mainstream technologies for ASFV and PRV nucleic acid detection due to their high detection sensitivity and strong specificity. However, these molecular diagnostic methods have significant limitations: on the one hand, they rely on expensive thermal cycling instruments (such as PCR instruments and quantitative real-time PCR instruments) and professional laboratory operating environments, resulting in high equipment procurement and maintenance costs, making them difficult to deploy in resource-limited scenarios such as grassroots farms, slaughterhouses, and cold chain quarantine points; on the other hand, the detection process involves multiple steps such as nucleic acid extraction, system preparation, thermal cycling amplification, and result interpretation, resulting in a long overall detection cycle (usually 1-2 hours) and high requirements for the professional skills of operators. This fails to meet the core requirements of "immediate, portable, and easy to operate" for point-of-care testing (POCT), making it unsuitable for rapid screening and emergency control scenarios during outbreaks of infectious diseases.

[0004] To address the compatibility issues of PCR / qPCR technologies in point-of-care testing (POCT), researchers have developed various isothermal nucleic acid amplification techniques in recent years, among which loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are the two most representative. Compared to other isothermal amplification techniques, RPA exhibits more prominent advantages in POCT adaptation: it eliminates the need for complex temperature cycling processes, achieving rapid nucleic acid amplification under isothermal conditions of 37–42°C, typically completing the amplification reaction within 30 minutes; simultaneously, RPA offers higher tolerance for primer design errors, and its specific binding mechanism during amplification effectively reduces the risk of non-specific amplification, resulting in stable detection accuracy. Compared to PCR, RPA does not rely on thermal cyclers, significantly simplifying equipment requirements; compared to LAMP, RPA further reduces false positives caused by non-specific amplification, thus becoming an ideal technology choice in the field of animal disease POCT.

[0005] In the design of carriers for POCT testing platforms, paper-based microfluidic analytical devices (μPADs) have become an important development direction for building low-cost, portable testing platforms in recent years due to their unique advantages. The porous fiber structure of paper can autonomously drive liquid transport through capillary action, achieving sample and reagent flow and mixing without relying on external pumping equipment (such as peristaltic pumps or syringe pumps). At the same time, μPADs have advantages such as low manufacturing cost (can be prepared using simple processes such as printing and cutting), low operating threshold (no professional instrument operation skills required), single use (avoiding cross-contamination), and environmental friendliness (easily degradable after disposal), and are widely used in the exploration of rapid on-site testing scenarios such as pathogen detection and biochemical index analysis.

[0006] However, when combining RPA isothermal amplification technology with the μPADs platform to achieve rapid on-site detection of ASFV and PRV, existing technologies still face two major challenges: First, the level of automation is insufficient. Most existing paper-based RPA detection platforms rely on manual operation or external auxiliary equipment to complete the detection process, making it difficult to achieve fully automated operation. For example, the foldable paper-based RPA device proposed by Rohrman et al. in Analytical Chemistry (2012) requires manual folding of paper to mix reagents and samples. The operation process depends on human experience and is prone to poor reaction consistency due to differences in folding force and speed. The centrifuge-driven paper-based microfluidic system reported by Chen et al. in Labona Chip (2019) can automate reagent transfer, but it requires external electric centrifuge equipment for power. The equipment is bulky, expensive, and cannot be used without a power source, making it difficult to adapt to grassroots field detection scenarios. The sliding plate detector proposed by Cordray et al. in Analytical Chemistry (2015), although simplifying the structural design, still requires manual pushing of the sliding component to trigger reagent mixing and amplification reactions. The operation has poor repeatability and cannot avoid the risk of contamination introduced by manual operation. None of the above solutions have been able to overcome the limitations of "manual dependence" or "external equipment dependence", making it difficult to achieve truly fully automated testing.

[0007] Secondly, the integration level is low. Most existing μPADs devices can only complete a single step in the detection process, and cannot integrate the entire process of "automatic reagent mixing - isothermal amplification - real-time detection" on a single chip. For example, some paper-based devices can only complete the RPA amplification reaction, and the amplified products need to be transferred to other detection devices (such as fluorescence detectors) for result interpretation; other devices, although integrating reaction and detection modules, still require manual addition of samples and shaking after reagent mixing, or rely on external equipment assistance. This "segmented operation" mode not only prolongs the detection time, but also increases the risk of sample exposure and cross-contamination, and requires additional equipment, significantly reducing the portability and application value of the POCT platform.

[0008] In summary, while existing technologies have made some progress in the rapid nucleic acid detection of ASFV and PRV (such as RPA technology) and POCT platform carriers (such as μPADs), key shortcomings remain, including insufficient automation and low detection integration. Therefore, there is an urgent need to develop an integrated and automated nucleic acid amplification and detection platform capable of simultaneously achieving "reagent storage-automatic mixing-isothermal amplification-result detection" to meet the practical needs of rapid on-site diagnosis of ASFV and PRV and provide technical support for disease prevention and control in the swine industry. Summary of the Invention

[0009] The purpose of this invention is to solve the problems of poor portability and low automation of existing RPA detection devices.

[0010] In a first aspect, this application provides a paper-based microfluidic RPA detection device based on surface modification and a rotary microvalve, comprising: 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. 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; A control system, which connects the heating assembly, the rotating assembly, and the fluorescence detector.

[0011] More specifically, 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.

[0012] More specifically, a substrate paper chip is also disposed between the first chip shell and the paper-based microfluidic chip.

[0013] More specifically, it also includes a third chip housing, through which the central chip is connected to the housing.

[0014] More specifically, the microchannel undergoes surface modification treatment, which specifically involves treating the surface of the microchannel with oxygen plasma and then adding a PVP solution for modification.

[0015] More specifically, the heating assembly includes a metal shell and heating wires surrounding the outer surface of the metal shell.

[0016] More specifically, the surface of the metal shell near the microfluidic chip is planar.

[0017] More specifically, the microchannel and the reaction zone have three geometrically symmetrical connection points.

[0018] More specifically, 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.

[0019] Secondly, this application also provides an RPA detection method, which uses the above-mentioned RPA detection device and specifically includes 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, and the reaction area is rotated to the top of 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.

[0020] The beneficial effects of this invention are: through the internal control system, the rotating component can drive the microfluidic chip to rotate precisely, automatically controlling the connection or disconnection between the microchannel and the reagent area, the proximity or distance between the reaction area and the heating component, and the proximity or distance between the reaction area and the fluorescence detector. This application can realize the timed and quantitative addition of reagents, the start or stop of the isothermal amplification reaction, and the automatic detection of RPA fluorescence signals. This application features high automation, rapid chemical response, and portable detection capabilities. Attached Figure Description

[0021] Figure 1 This is a three-dimensional schematic diagram of the RPA detection device in this application.

[0022] Figure 2 This is a top view of the microfluidic chip in this application.

[0023] Figure 3 This is a bottom view of the microfluidic chip in this application.

[0024] Figure 4 This is an exploded view of the peripheral chip in this application.

[0025] Figure 5 This is a cross-sectional view of the internal connections of the RPA detection device in this application.

[0026] Figure 6 This is a schematic diagram of the connections between the components in this application.

[0027] Figure 7 This is a three-dimensional schematic diagram of the heating component in this application.

[0028] Figure 8 This is a test diagram showing the optimized spacing between the fluorescence detector and the microfluidic chip in this application.

[0029] Figure 9 This is the RPA testing flowchart in this application.

[0030] Figure 10 This is a graph of ASFV test data from this application.

[0031] Figure 11 This is a graph of PRV test data from this application.

[0032] Figure 12 This is a test diagram showing the optimized volume of the fluorescence activation liquid in this application.

[0033] In the diagram: 100, shell; 200, microfluidic chip; 21, microchannel; 22, reaction zone; 23, reagent zone; 210, peripheral chip; 211, first chip shell; 212, heat transfer port; 213, substrate paper chip; 214, paper-based microfluidic chip; 215, second chip shell; 216, detection port; 220, central chip; 221, third chip shell; 300, heating assembly; 310, metal shell; 320, heating wire; 400, rotating assembly; 500, fluorescence detector. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments of this invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] The paper-based microfluidic RPA detection device designed in this application is as follows: Figure 1 As shown, after the user adds the sample to the designated area of ​​the microfluidic chip 200, closes the cover, starts the device, and presets the temperature and other adjustment parameters, the RPA detection of the sample can be completed automatically, and the detection results will be displayed on the front display screen.

[0036] In some specific embodiments, the specific structure of the microfluidic chip 200 is as follows: Figure 2 and Figure 3 As shown, the microfluidic chip 200 includes a central chip 220 located at the center of rotation and peripheral chips 210 capable of rotating around the central chip 220. The central chip 220 contains a reagent zone 23, and the peripheral chip 210 contains several reaction zones 22 and microchannels 21 corresponding to the reaction zones 22. The microchannels 21 are connected to the reaction zones 22 and extend towards the reagent zones 23. The reagent zones 23 also have interfaces corresponding to the various reaction zones 22.

[0037] The central chip 220 is fixedly connected to the housing 100 via the third chip housing 221. Rotating the peripheral chip 210 controls the connection or disconnection of the microchannel 21 to the reagent zone 23. When the microchannel 21 connects the reagent zone 23 and the reaction zone 22, the reagents stored in the reagent zone 23 are driven to the reaction zone 22 via capillary action due to the porous fiber structure of the paper. This rotational control allows the device to automatically add reagents at precise times and in precise quantities during RPA detection, eliminating the need for complex sample addition operations by the user.

[0038] like Figure 4 As shown, the peripheral chip 210 specifically includes a first chip shell 211, a paper-based microfluidic chip 214, and a second chip shell 215. Microchannels 21 and reaction zones 22 are disposed on the paper-based microfluidic chip 214. The first chip shell 211 has a heat transfer port 212 corresponding to the heating component 300, and the second chip shell 215 has a detection port 216 corresponding to the fluorescence detector 500. Both the first chip shell 211 and the second chip shell 215 are made of rigid materials (such as PMMA, PDMS, etc.) and have several positioning holes. The rotating component 400 drives the first chip shell 211, the second chip shell 215, and the paper-based microfluidic chip 214 to rotate together. The rigid shells ensure the reliability of the rotation of the paper-based microfluidic chip 214. In use, the first chip shell 211 and the second chip shell 215 are reusable; the user only needs to replace the disposable paper-based microfluidic chip 214. In one feasible solution, the reagent area 23 is designed to store a sufficient amount of reagent to meet the detection reagent needs of multiple paper-based microfluidic chips 214. In a further improvement of this application, to avoid reagent permeation and contamination, a substrate paper chip 213 is also provided between the first chip shell 211 and the paper-based microfluidic chip 214.

[0039] In a further improvement of this application, to accelerate the flow of reagents to the reaction zone 22 and reduce reaction waiting time, especially for high-viscosity reagents (such as enzyme reagents and primer mixtures in the RPA reaction system, which have high viscosity due to the presence of PEG), this application further modifies the surface of the microchannel 21. Specifically, the paper-based microchannel is first subjected to oxygen plasma treatment for 2-6 minutes. A 1%-17% PVP solution is then added to the oxygen plasma-treated microchannel 21, and after drying, a microchannel 21 with improved fluid transport rate is obtained. To further improve reagent mixing efficiency and avoid inaccurate reaction results due to uneven reagent distribution, this application designs multiple connections between the microchannel 21 and the reaction zone 22, with the connections being geometrically symmetrically distributed, allowing reagents to enter from multiple angles and uniformly fill the reaction zone 22.

[0040] In some specific embodiments, such as Figure 5As shown, in this application, the microfluidic chip 200, heating component 300, and rotating component 400 are connected and fixed by a cylindrical structure. This fixing method is only an example, and other connection methods that are easy to install can also be used. Figure 6 As shown, the heating component 300 is disposed at the bottom of the microfluidic chip 200, and the rotating component 400 controls the peripheral chip 210 to move away from or closer to the heating component 300. Figure 6 As 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.

[0041] 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.

[0042] In some specific embodiments, the device also has a display screen connected to the control system, allowing users to independently adjust parameters such as heating temperature, chip rotation angle, rotation speed, and waiting time for each step. The final RPA signal can be directly displayed on the screen, enabling users to perform RPA detection on various samples and obtain intuitive RPA results without the need for large, complex equipment or complicated operations.

[0043] The specific usage method of this device is as follows: Figure 9 As shown: S1. Place the sample and the corresponding RPA enzyme powder into the reaction zone 22. Different reaction zones 22 can hold different samples and their corresponding RPA enzyme powders, such as... Figure 9 The chip structure shown allows for the placement of ASF and PRV samples when the paper-based microfluidic chip 214 has two reaction regions 22.

[0044] S2. The peripheral chip 210 is rotated around the central chip 220 by the rotating component 400, and the reaction zone 22 is rotated to the position above the heating component 300. The heating component 300 is then activated to heat the reaction zone 22 and maintain a constant temperature for a period of time, and the initial amplification reaction takes place in the reaction zone 22.

[0045] S3. After a period of reaction, the outer chip 210 is rotated around the central chip 220 by the rotating component 400, and the microchannel 21 is connected to the reagent area 23 containing the fluorescent activator. The activator enters the reaction area 22 through the microchannel 21.

[0046] S4. After the microchannel 21 is connected to the reagent area 23 for a period of time, and the reaction area 22 obtains sufficient activation solution, the rotating component 400 is controlled to rotate the peripheral chip 210 to disconnect the microchannel 21 from the reagent area 23, and the reaction area 22 is rotated back to above the heating component 300. The heating component 300 is controlled to continue heating the reaction area 22 and maintain a constant temperature, and the reagent undergoes a constant temperature amplification reaction.

[0047] S5. After the reaction is complete, control the rotating component 400 to rotate the peripheral chip 210 so that the reaction area 22 is rotated directly below the fluorescence detector 500, and the fluorescence detector 500 detects the RPA fluorescence signal.

[0048] Fabrication of paper-based microfluidic chip 214: The chip pattern (channel 21 width 2mm, reaction zone 22 diameter 4mm) was drawn using software. Hydrophilic and hydrophobic barrier regions were printed on Whatman No.4 filter paper using a Xerox 8580DN wax printer. The printed paper was placed on a CS15956-31 Cole-Parmer heating plate and heated at 120℃ for 120 seconds to allow the wax to fully melt and penetrate the paper, forming a stable hydrophobic barrier. The chip was then cut according to the preset pattern using a YueMing laser cutter at a laser intensity of 10% and a cutting speed of 50mm / s, yielding the desired result. Figure 4 The paper-based microfluidic chip 214 is shown.

[0049] Example 1: RPA detection of African swine fever virus (ASFV). Lyophilized enzyme powder (5U recombinant enzyme, 2U DNA polymerase, and 1μg single-stranded binding protein) was pre-stored in reaction zone 22 of a paper-based microfluidic chip 214. 25μL of ASFV fluorescence isothermal amplification detection reagent (containing 10% PEG-20000) was added to reagent zone 23. The assay was performed as follows: Figure 9 The following procedure was followed for testing: First, ASFV-specific primers (final concentration 0.4 μM) were added to reaction zone 22. Heating assembly 300 was then activated (at this point, heating assembly 300 was directly facing reaction zone 22), and the temperature of the reaction zone was controlled at 42 ± 0.3 °C. Isothermal nucleic acid amplification was performed for 1 min. After amplification, rotating assembly 400 rotated the peripheral chip 210, connecting microchannel 21 with reagent zone 23, allowing the fluorescent activation solution stored in reagent zone 23 to enter (average time 22 s). Then, the peripheral chip 210 was rotated again, returning reaction zone 22 to the heating zone position, and the amplification reaction continued for 12 min. Finally, the fluorophore was hydrolyzed and released, and the fluorescence detector 500 detected the fluorescence signal. The experiment was repeated, and fluorescence signals at different concentrations and amplification times were detected. The results are as follows: Figure 10 As shown in the figure. This device achieves a detection limit of 5 copies / μL for ASFV.

[0050] Example 2: RPA detection of pseudorabies virus (PRV). Using [method / approach]... Figure 8 The detection procedure is the same, the difference lies in the amplification reaction time after adding the fluorescent activation solution. 17 μL of PRV fluorescent isothermal amplification detection reagent is added to reagent zone 23, followed by 8 μL of sample and lyophilized enzyme powder (5 U recombinant enzyme, 2 U DNA polymerase, and 1 μg single-strand binding protein). After 1 minute, the rotating component rotates 400 degrees to rotate the peripheral chip 210, connecting the microchannel 21 with reagent zone 23, allowing the fluorescent amplification reagent to enter reaction zone 22. Amplification is maintained at 42 ± 0.3℃, and the PRV fluorescence signal can be stably detected after 16 minutes of amplification. The experiment was repeated, and fluorescence signals at different concentrations and amplification times were detected. The results are as follows: Figure 11 As shown in the figure. This device achieves a detection limit of 10 copies / μL for PRV.

[0051] Example 3: Optimal fluorescence activation solution volume detection. After the rotating component 400 connects the microchannel 21 to the reagent zone 23, the fluorescence activation solution will flow autonomously to the reaction zone 22. The reagent transport time in the microchannel 21 is approximately 22 seconds. To ensure sufficient activation solution reaches the reaction zone 22, the connection time between the microchannel 21 and the reagent zone 23 is designed to be 1 minute. Different volumes of fluorescence activation solution were added to the reagent zone 23 sequentially. PRV and ASFV samples were detected in both surface-modified microchannel 21 and unmodified ordinary microchannel 21. The results are as follows. Figure 12 As shown. Due to the high viscosity of the fluorescent activation solution, the transmission speed of the activation solution in the unmodified ordinary microfluidic channel 21 is slow, with only a small amount of activation solution reaching the reaction zone, resulting in a weak fluorescence signal. The surface-modified microfluidic channel 21 significantly improves the transmission speed of high-viscosity reagents, allowing the activation solution to rapidly fill the reaction zone with less waiting time, resulting in a strong fluorescence signal. In the microfluidic chip 200 designed in this device, the consumption of fluorescent activation solution per detection is less than 30 μL.

[0052] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A paper-based microfluidic RPA detection device based on surface modification and rotary microvalve, characterized in that, The application relates to a RPA detection device. The device comprises a shell, a micro-fluidic chip, a heating assembly, a rotating assembly, a fluorescent detector and a control system. The micro-fluidic chip comprises a center chip provided with a reagent area and a peripheral chip rotatable around the center chip and provided with a reaction area, and the peripheral chip is further provided with a micro-fluid channel communicating with the reaction area and extending towards the reagent area. The heating assembly is arranged below the micro-fluidic chip and used for heating the reaction area. The rotating assembly is fixed in the shell and connected to the peripheral chip. The fluorescent detector is arranged on the shell and faces the reaction area of the micro-fluidic chip and is used for detecting the fluorescent signal generated by the micro-fluidic chip. The control system is connected to the heating assembly, the rotating assembly and the fluorescent detector.

2. The RPA detection device of claim 1, wherein, The peripheral chip comprises, from bottom to top, a first chip shell provided with a heat transfer port, a paper-based micro-fluidic chip provided with the reaction area and the micro-fluid channel and a second chip shell.

3. The RPA detection device of claim 2, wherein, A substrate paper chip is further arranged between the first chip shell and the paper-based micro-fluidic chip.

4. The RPA detection apparatus of claim 1, wherein, A third chip shell is further arranged, and the center chip is connected to the shell through the third chip shell.

5. The RPA detection device of claim 2, wherein, The micro-fluid channel is subjected to surface modification treatment, and the surface modification treatment method is that PVP solution is added after oxygen plasma treatment of the surface of the micro-fluid channel.

6. The RPA detection apparatus of claim 1, wherein, The heating assembly comprises a metal shell and an electric heating wire surrounding the outer surface of the metal shell.

7. The RPA detection device of claim 6, wherein, The surface of the metal shell close to the micro-fluidic chip is a plane.

8. The RPA detection apparatus of claim 1, wherein, The micro-fluid channel and the reaction area have three connection ports in geometrically symmetrical distribution.

9. The RPA detection apparatus of claim 1, wherein, 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.

10. A method of detecting RPA, characterized by, The RPA detection device is used to perform the following steps. S1, the sample and the RPA reaction enzyme powder corresponding to the sample are placed in the reaction area. S2, the heating assembly is controlled to heat the reaction area and keep constant temperature for a preset time. S3, the rotating assembly is controlled to rotate the peripheral chip around the center chip, and the micro-fluid channel is connected to the reagent area provided with a fluorescent activator. S4, after the micro-fluid channel is connected to the reagent area for a set time, the rotating assembly is controlled to rotate the peripheral chip to disconnect the micro-fluid channel from the reagent area, and the reaction area is rotated above the heating assembly, and the heating assembly is controlled to continue heating the reaction area and keeping constant temperature. S5, after the reaction is completed, the rotating assembly is controlled to rotate the peripheral chip to rotate the reaction area directly below the fluorescent detector, and the fluorescent detector detects the RPA fluorescent signal.

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