RPA-CRISPR nucleic acid detection system and method based on digital micro-fluidic chip

Abstract

The invention discloses an RPA-CRISPR nucleic acid detection system and method based on a digital micro-fluidic chip, the system comprises a micro-fluidic chip, a heating unit, a fluorescence detection unit and a control unit, the micro-fluidic chip comprises a first liquid injection zone, a first reaction zone, a quantitative liquid separation zone, a second reaction zone, a second liquid injection zone, a detection zone and an electrode array; the electrode array covers each area and drives liquid drops in the areas to move; the control unit is used for controlling the electrode array to drive the movement sequence and position of the liquid drops in each region according to a nucleic acid detection process; wherein the electrode array covered by the quantitative liquid separation area comprises a plurality of quantitative electrodes which are mutually spaced, and the RPA product in the quantitative liquid separation area is quantitatively controlled by controlling the on-off of the quantitative electrodes. The RPA and CRISPR reaction efficiency is high, and the result can be quickly obtained on the premise of ensuring high sensitivity and high specificity.

Description

Background Art

[0002] Nucleic acid detection technology is widely used in the field of disease screening and diagnosis. Real-time quantitative PCR and RT-PCR have become the gold standard for nucleic acid detection due to their high sensitivity and specificity. However, PCR detection mostly requires the use of sophisticated and expensive instruments and is carried out by professional technicians in special experimental sites, which greatly limits the application scenarios of nucleic acid detection. Some isothermal nucleic acid amplification technologies have developed rapidly due to their low requirements for equipment and users, such as recombinase polymerase amplification (RPA) technology, loop-mediated isothermal amplification technology (LAMP), rolling circle amplification technology (RCA), etc., especially when combined with nucleic acid detection technology based on CRISPR Cas proteins (Cas12a, Cas13a, etc.), after two rounds of signal amplification (one round of amplification, one round of CRISPR shearing) and two rounds of specific recognition, its advantages of high sensitivity and high specificity are more prominent. The RPA product and CRISPR reagent need to be mixed in a ratio of 1:9 for reaction, so it is necessary to perform quantitative operations on the RPA product and CRISPR reagent.

[0003] Most existing RPA-CRISPR detection methods use multiple manual quantitative operations. The transfer of RPA products in an open system increases the risk of contamination caused by residual amplification products, and also causes quantitative errors due to manual operations. In order to avoid the gradual development of quantitative errors, dedicated quantitative equipment has emerged. However, these quantitative equipment require complex pump and valve structures and high-precision micro-peristaltic pumps, which will greatly increase the cost of the equipment on the one hand, and the size of the equipment will also increase accordingly, which will limit its application in scenarios such as on-site detection. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide an RPA-CRISPR nucleic acid detection system and method, an electronic device and a computer-readable storage medium based on a digital microfluidic chip that has a high degree of automation and takes into account both nucleic acid detection efficiency and accuracy.

[0005] To achieve the above-mentioned objectives, the first aspect of the present invention provides an RPA-CRISPR nucleic acid detection system based on a digital microfluidic chip, comprising a microfluidic chip, a heating unit, a fluorescence detection unit and a control unit, wherein the microfluidic chip comprises a first liquid injection area, a first reaction area, a quantitative liquid separation area, a second reaction area, a second liquid injection area, a detection area and an electrode array; the heating unit is used to regulate the temperature of the first reaction area and the second reaction area, the fluorescence detection unit is used to perform fluorescence detection on the reaction products in the detection area, the electrode array covers each area and drives the movement of droplets in the area; the control unit is used to control the electrode array to drive the movement order and position of droplets in each area according to the nucleic acid detection process; wherein the electrode array covered by the quantitative liquid separation area comprises a plurality of mutually spaced quantitative electrodes, and the RPA products in the quantitative liquid separation area are quantitatively controlled by controlling the on and off power of the quantitative electrodes.

[0006] Furthermore, the quantitative electrode includes four quantitative sub-electrodes arranged at intervals, and the shape of the quantitative sub-electrodes is semicircular.

[0007] Furthermore, an RPA freeze-dried ball packaging structure is arranged above the first liquid injection area, and the RPA freeze-dried ball is sealed and stored above the first liquid injection area through the packaging structure.

[0008] Furthermore, a CRISPR freeze-dried ball packaging structure is provided above the second liquid injection area, and the CRISPR freeze-dried ball is sealed and stored above the second liquid injection area through the packaging structure.

[0009] Furthermore, the microfluidic chip includes a plurality of the first liquid injection areas, a plurality of the first reaction areas, a plurality of the quantitative liquid separation areas, a plurality of the second reaction areas, a plurality of the second liquid injection areas and a plurality of the detection areas.

[0010] The second aspect of the present invention provides an RPA-CRISPR nucleic acid detection method based on a digital microfluidic chip, using the above-mentioned nucleic acid detection system, the method comprises the following steps:

[0011] 1) Injecting the RPA reaction solution into the first injection area;

[0012] 2) driving the PRA reaction solution to move to the first reaction area for RPA amplification reaction;

[0013] 3) After the RPA amplification reaction is completed, the amplification product is driven to the quantitative separation area for quantitative operation;

[0014] 4) After the quantitative operation, the quantified portion of the RPA amplification product is moved to the second reaction area;

[0015] 5) Injecting the CRISPR reaction solution into the second injection area;

[0016] 6) driving the CRISPR reaction solution to move to the second reaction area to mix with the RPA amplification product and then perform a CRISPR reaction;

[0017] 7) After the CRISPR reaction is completed, the CRISPR reaction product is driven to the detection area for fluorescent nucleic acid detection.

[0018] Further, step 1) comprises:

[0019] Injecting the sample solution to be tested into the injection port of the first injection zone;

[0020] The sample solution dissolves the RPA freeze-dried beads encapsulated in the encapsulation structure to form an RPA reaction solution.

[0021] Further, step 2) comprises:

[0022] driving the RPA reaction solution to move to the first reaction zone;

[0023] driving the RPA reaction solution to continuously move in the first reaction zone to mix the droplets;

[0024] The RPA amplification reaction is performed after adjusting the reaction temperature of the first reaction zone.

[0025] Further, step 3) includes:

[0026] Drive the RPA amplification product to move to the quantitative liquid separation area and allow the droplets to fill the quantitative electrode;

[0027] The power is cut off for some of the quantitative electrodes, and the remaining droplets on the quantitative electrodes that are not cut off are part of the RPA amplification products after quantification.

[0028] Further, step 5) includes:

[0029] Injecting enzyme-free water into the injection port of the second injection zone;

[0030] The enzyme-free water dissolves the CRISPR freeze-dried beads encapsulated in the encapsulation structure to form a CRISPR reaction solution.

[0031] A third aspect of the present invention provides an electronic device, comprising:

[0032] Processor; and

[0033] A memory having computer-readable instructions stored therein, wherein the computer-readable instructions implement the above method when executed by the processor.

[0034] A fourth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the above method when executed by a processor.

[0035] The present invention automatically realizes a series of operations such as RPA amplification reaction, quantitative operation, CRISPR reaction, etc. by controlling the control unit in the first liquid injection area, the first reaction area, the quantitative liquid separation area, the second reaction area, the second liquid injection area, and the detection area in combination with the electrode arrays covered in each area. The RPA amplification product is quantified by the quantitative electrodes arranged in the quantitative liquid separation area, thereby ensuring the accurate ratio of the RPA amplification product and the CRISPR reagent and improving the accuracy of nucleic acid detection; and with the cooperation of the heating unit and the electrode arrays covered in the reaction area, the RPA and CRISPR reaction times are greatly shortened, thereby improving the efficiency of nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 This is a schematic diagram of an RPA-CRISPR nucleic acid detection system based on a digital microfluidic chip according to an embodiment of the present invention;

[0038] Figure 2 for Figure 1 Schematic diagram of RPA-CRISPR nucleic acid detection;

[0039] Figure 3 This is a schematic diagram of the overall structure of a digital microfluidic chip and housing for RPA-CRISPR nucleic acid detection according to an embodiment of the present invention;

[0040] Figure 4 This is a schematic diagram of the packaging structure of RPA and CRISPR freeze-dried balls according to an embodiment of the present invention;

[0041] Figure 5 This is a flow chart of an RPA-CRISPR nucleic acid detection method based on a digital microfluidic chip according to an embodiment of the present invention. DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0043] like Figure 1 and 3As shown, the first aspect of the present invention provides an RPA-CRISPR nucleic acid detection system based on a digital microfluidic chip, including a microfluidic chip 7, a heating unit, a fluorescence detection unit and a control unit, wherein the microfluidic chip 7 includes a first injection area 1, a first reaction area 2, a quantitative liquid separation area 3, a second reaction area 4, a second injection area 5, a detection area 6 and an electrode array; the heating unit is used to regulate the temperature of the first reaction area 2 and the second reaction area 4, the fluorescence detection unit is used to perform fluorescence detection on the reaction product in the detection area 6, the electrode array covers each area and drives the movement of droplets in the area; the control unit is used to control the movement sequence and position of the droplets driven by the electrode array in each area according to the nucleic acid detection process; wherein the electrode array covered by the quantitative liquid separation area 3 includes a plurality of quantitative electrodes spaced apart from each other, and the RPA product of the quantitative liquid separation area 3 is quantitatively controlled by controlling the on and off of the quantitative electrodes. Specifically, an injection port is set above the first injection area and the second injection area, and the injection port can realize the injection of RPA reaction solution and enzyme-free water and other solutions into the microfluidic chip 7. The heating unit includes a heat spreader and a temperature control module. The heating unit can be set at the bottom of the chip. The heat spreader of the heating unit is in direct contact with the chip. The temperature of the heat spreader is adjusted by the temperature control module, and then the temperature of the first reaction zone and the second reaction zone is adjusted to the RPA and CRISPR reaction temperatures. The fluorescence detection unit includes an optical fiber probe and a fluorescence signal amplification system. The optical fiber probe is fixed on the top of the detection area. A large beam diameter quartz multimode optical fiber with a numerical aperture of 0.23NA is used for excitation light transmission and emission light collection. The optical fiber is small in size and easy to install. While meeting the requirements of small and portable equipment, it cooperates with the fluorescence signal amplification system to achieve highly sensitive nucleic acid detection. The electrode array includes multiple electrodes laid in each area. The multiple electrodes are laid according to the movement path of the droplets in each area to ensure that the droplets injected into the chip can move between areas under the drive of the electrode array.

[0044] In one embodiment of the present invention, the RPA-CRISPR nucleic acid detection system based on the digital microfluidic chip includes an upper cover 8, a microfluidic chip 7 and a lower base plate 9. The upper cover 8 is provided with a sealing cover on the lower base plate 9, and there is a space for accommodating the microfluidic chip 7 between the upper cover 8 and the lower base plate 9, and the microfluidic chip 7 is sealed therein. The RPA freeze-dried ball packaging structure 81 and the CRISPR freeze-dried ball packaging structure 82 are provided at both ends of the top of the upper cover 8, and the RPA freeze-dried ball packaging structure 81 encapsulates the RPA freeze-dried ball, and the CRISPR freeze-dried ball packaging structure 82 encapsulates the CRISPR freeze-dried ball.

[0045] like Figure 2As shown, the present invention adopts a method for nucleic acid detection by combining RPA with CRISPR. Taking the detection of RNA as an example (the reaction system contains CRISPR Cas13a protein), the principle of RPA-CRISPR nucleic acid detection is as follows: the reverse transcriptase in the RPA reaction system reverse transcribes RNA into cDNA, and the recombinase and primer DNA are tightly bound to form a recombinase / primer complex, which invades the RNA-cDNA double-stranded nucleic acid template. At the invasion site, the recombinase opens the double strand, and at the same time, the single-stranded binding protein binds to the single strand opened by the recombinase, maintaining the double-stranded template in an open-strand state. The recombinase / primer complex begins to scan the double strand. When the primer searches for a complementary sequence that completely matches it on the template, the recombinase / primer complex solves the problem, and the DNA polymerase binds to the 3' end of the primer to start synthesizing a new chain. The synthesized new chain can be used as a template, and the final amplification product grows exponentially. The RPA amplification product is transcribed into RNA under the action of T7 RNA polymerase in the CRISPR reaction system. The Cas13a protein binds to crRNA and, under the guidance of crRNA, identifies the specific RNA sequence in the sample. The Cas13a protein, crRNA and target RNA form a ternary complex, activating the "non-specific cleavage" activity of the Cas13a protein and randomly cleaving the reporter probe in the solution. One end of the reporter probe is a fluorescent group and the other end is a quenching group. After cleavage, the fluorescent group and the quenching group are separated. Under the irradiation of the excitation light, the sample fluoresces; when there is no target RNA in the system, the "non-specific cleavage" activity of the Cas13a protein is not activated, the reporter probe is not cleaved, and no fluorescent signal is generated. This enables the determination of positive and negative, and qualitative detection of unknown samples.

[0046] In one embodiment of the present invention, the quantitative electrode includes four quantitative sub-electrodes arranged at intervals, and the shape of the quantitative sub-electrodes is semicircular. The content of the RPA amplification product after the RPA amplification reaction from the first reaction zone is relatively high. Due to the ratio requirements of the RPA amplification product and the CRISPR reaction reagent, it is necessary to quantify the content of the RPA amplification product passing into the CRISPR reaction zone. In this embodiment, the RPA amplification product is quantitatively separated by four quantitative sub-electrodes arranged at intervals. Since the four quantitative sub-electrodes are arranged at intervals, the quantitative sub-electrode on the far left is far away from the second reaction zone, so the quantitative portion of the RPA droplet on it cannot be moved to the second reaction zone. By controlling the upper and lower two quantitative sub-electrodes to be powered off, the quantitative sub-electrode on the far right near the second reaction zone remains powered on, and the quantitative portion of the RPA droplet on the rightmost quantitative sub-electrode can be driven to the second reaction zone. In this embodiment, quantitative control can be achieved by simply turning on and off the quantitative sub-electrode, without the need to design a responsible control algorithm, reducing the requirements for electrode control accuracy and quantitative accuracy. It should be noted that the number and shape of the quantitative sub-electrodes of the present invention are not limited to this, and they can be adjusted accordingly as needed.

[0047] In one embodiment of the present invention, Figure 4 As shown, an RPA freeze-dried ball packaging structure is provided above the first injection area, and the RPA freeze-dried ball is sealed and stored above the first injection area through the packaging structure. A CRISPR freeze-dried ball packaging structure is provided above the second injection area, and the CRISPR freeze-dried ball is sealed and stored above the second injection area through the packaging structure. The RPA injection area and the CRISPR injection area correspond to the chip shell injection port, which is wide at the top and narrow at the bottom. The upper opening diameter is larger than the freeze-dried ball diameter, and the lower injection port diameter is smaller than the freeze-dried ball diameter. After the freeze-dried ball is packaged, the surface is coated, and the outer packaging is vacuumed or filled with nitrogen to ensure that the chip is in a closed and dry state after the freeze-dried ball is packaged, which is convenient for transportation and long-term storage. After the detection starts, the sample solution and enzyme-free water are injected into the RPA injection area and the CRISPR injection area respectively, the freeze-dried ball is dissolved, and the reaction system is injected into the chip.

[0048] In one embodiment of the present invention, the microfluidic chip 7 includes a plurality of the first injection areas 1, a plurality of the first reaction areas 2, a plurality of the quantitative liquid separation areas 3, a plurality of the second reaction areas 4, a plurality of the second injection areas 5 and a plurality of the detection areas 6. For example, Figure 1 The embodiment includes 4 first liquid injection areas 1, 4 first reaction areas 2, 4 quantitative liquid separation areas 3, 4 second reaction areas 4, 4 second liquid injection areas 5 and 4 detection areas 6, so that nucleic acid detection can be performed on 4 samples to be tested simultaneously through 4 channel experiments, further improving the efficiency of immune detection.

[0049] like Figure 5 As shown, the RPA-CRISPR nucleic acid detection method based on a digital microfluidic chip of the present invention utilizes the above-mentioned nucleic acid detection system, and the method comprises the following steps:

[0050] Step S110: injecting the RPA reaction solution into the first liquid injection area;

[0051] Step S120: driving the PRA reaction solution to move to the first reaction area to perform RPA amplification reaction;

[0052] Step S130: After the RPA amplification reaction is completed, the amplification product is driven to the quantitative liquid separation area for quantitative operation;

[0053] Step S140: after the quantitative operation, moving the quantified portion of the RPA amplification product to the second reaction area;

[0054] Step S150: injecting a CRISPR reaction solution into the second injection area;

[0055] Step S160: driving the CRISPR reaction solution to move to the second reaction area to mix with the RPA amplification product and then perform a CRISPR reaction;

[0056] Step S170: After the CRISPR reaction is completed, the CRISPR reaction product is driven to the detection area for fluorescent nucleic acid detection.

[0057] In one embodiment of the present invention, step S110 includes:

[0058] Injecting the sample solution to be tested into the injection port of the first injection zone;

[0059] The sample solution dissolves the RPA freeze-dried beads encapsulated in the encapsulation structure to form an RPA reaction solution.

[0060] In one embodiment of the present invention, step S120 includes:

[0061] driving the RPA reaction solution to move to the first reaction zone;

[0062] driving the RPA reaction solution to continuously move in the first reaction zone to mix the droplets;

[0063] The RPA amplification reaction is performed after adjusting the reaction temperature of the first reaction zone.

[0064] In one embodiment of the present invention, step S130 includes:

[0065] Drive the RPA amplification product to move to the quantitative liquid separation area and allow the droplets to fill the quantitative electrode;

[0066] The power is cut off for some of the quantitative electrodes, and the remaining droplets on the quantitative electrodes that are not cut off are the partial RPA amplification products after quantification.

[0067] In one embodiment of the present invention, step S150 includes:

[0068] Injecting enzyme-free water into the injection port of the second injection zone;

[0069] The enzyme-free water dissolves the CRISPR freeze-dried beads encapsulated in the encapsulation structure to form a CRISPR reaction solution.

[0070] In order to enable those skilled in the art to more clearly understand the scheme of the present invention, an example of nucleic acid detection is now cited for illustration.

[0071] Example 1: Simultaneous detection of the novel coronavirus N gene in four samples

[0072] The digital microfluidic chip RPA-CRISPR nucleic acid detection system is connected to an external power supply for power supply, the upper computer software is opened, 20 μL of the sample to be tested is injected into each of the four injection holes, the sample dissolves the N gene detection RPA freeze-dried ball encapsulated in the injection hole, and the RPA reaction solution is driven to the RPA reaction area;

[0073] The control software drives the droplets to move in the RPA reaction area, mixes the droplets, turns on the 42°C temperature control, and starts the RPA amplification reaction for 20 minutes;

[0074] After the RPA reaction is finished, the control software drives the droplet (amplification product) to the liquid separation area, allowing the droplet to cover the four semicircular electrode structures and the electrode structure on the left, disconnecting the upper and lower semicircular electrodes, separating the droplet on the right semicircular structure (about 2 μL), and driving the droplet to the CRISPR reaction area;

[0075] Inject 18 μL of enzyme-free water into the corresponding injection ports of the CRISPR injection area to dissolve the N gene detection CRISPR freeze-dried balls, energize the electrodes in the CRISPR injection area, drive the dissolved CRISPR reaction system to the CRISPR reaction area, and mix it with the RPA product droplets separated from the separation area to form a 20 μL reaction system;

[0076] The CRISPR reaction area was set to 37°C and the CRISPR reaction was started for 15 minutes.

[0077] After the CRISPR reaction is completed, the CRISPR reaction product droplets are driven to the signal detection area for fluorescent signal detection to complete the RPA-CRISPR COVID-19 N gene detection.

[0078] The chip is divided into 6 areas: RPA injection area / loading area, RPA reaction area, liquid separation area, CRISPR reaction area, CRISPR injection area and signal detection area. RPA freeze-dried ball reagents and CRISPR freeze-dried ball reagents are respectively encapsulated in the RPA injection area and the CRISPR injection area corresponding to the chip shell injection port. One chip can complete the simultaneous detection of 4 samples or four targets of a single sample. At the beginning of the detection, 4 samples are injected into the four RPA injection holes respectively. The liquid sample dissolves the RPA freeze-dried ball. The control software powers on the loading area, injects the RPA reaction system dissolved in the sample into the chip, and drives the droplets to the RPA reaction area; the control software drives the droplets to move in the RPA reaction area, mixes the droplets, turns on the 42°C temperature control, and starts the RPA amplification reaction; when the RPA reaction is finished, the control software drives the droplets (amplification products) to the liquid separation area, allowing the droplets to cover the four semicircular electrode structures and the electrode structure on the left, and the upper and lower semicircular electrodes. The electrode is powered off, the droplets on the right semicircular structure are separated, and the droplets are driven to the CRISPR reaction area; enzyme-free water is injected into the injection port corresponding to the CRISPR injection area to dissolve the CRISPR freeze-dried ball, and the electrode of the CRISPR injection area is powered to drive the dissolved CRISPR reaction system to the CRISPR reaction area to mix with the RPA product droplets separated from the separation area; the CRISPR reaction area starts 37°C temperature control to start the CRISPR reaction; after the CRISPR reaction is completed, the CRISPR reaction product droplets are driven to the signal detection area for fluorescence signal detection to complete the RPA-CRISPR detection process. All droplet operation actions (driving, temperature control, mixing, separation, fluorescence signal detection) are written in a fixed process through software. According to the above steps, the droplet operation software process is called and started with one button to realize the automation of the entire RPA-CRISPR detection process.

[0079] In summary, the present invention can realize automated RPA-CRISPR nucleic acid detection. On the one hand, it does not rely on any professional detection personnel, precision detection instruments and professional laboratories, and has flexible usage scenarios, and can achieve on-site detection and instant testing; on the other hand, the fully enclosed chip and equipment system can not only avoid the leakage of pathogenic microorganisms in the sample, protect the safety of operators and the environment, but also avoid false positive results caused by the leakage of amplification products during the detection process; the system also supports the pre-packaging of RPA freeze-dried ball reagents and CRISPR freeze-dried ball reagents, simplifies the experimental process, and does not require reagent injection, only samples and enzyme-free water need to be added; and the system and method are based on a small system reaction on the chip, and the RPA and CRISPR reactions are highly efficient, and the detection can be completed within 40 minutes while ensuring high sensitivity and high specificity.

[0080] In addition, in an exemplary embodiment of the present disclosure, an electronic device capable of implementing the above method is also provided, including a processor; and a memory, wherein computer-readable instructions are stored in the memory, and the computer-readable instructions implement the above method when executed by the processor.

[0081] It will be appreciated by those skilled in the art that various aspects of the present invention may be implemented as systems, methods or program products. Therefore, various aspects of the present invention may be specifically implemented in the following forms, namely: complete hardware embodiments, complete software embodiments (including firmware, microcode, etc.), or embodiments combining hardware and software aspects, which may be collectively referred to herein as "circuits", "modules" or "systems".

[0082] The electronic device is presented in the form of a general-purpose computing device. The components of the electronic device may include, but are not limited to: at least one processing unit, at least one storage unit, a bus connecting different system components (including the storage unit and the processing unit), and a display unit.

[0083] The storage unit stores program codes, which can be executed by the processing unit, so that the processing unit performs the steps according to various exemplary embodiments of the present invention described in the above “Exemplary Method” section of this specification. For example, the processing unit can perform the following steps: Figure 3 Steps S110 to S170 shown in FIG.

[0084] The storage unit may include a readable medium in the form of a volatile memory unit, such as a random access memory unit (RAM) and / or a cache memory unit, and may further include a read-only memory unit (ROM).

[0085] The storage unit may also include a program / utility having a set (at least one) of program modules, such program modules including but not limited to: an operating system, one or more application programs, other program modules, and program data, each of which or some combination may include an implementation of a network environment.

[0086] The bus may represent one or more of several types of bus structures, including a memory unit bus or memory unit controller, a peripheral bus, an accelerated graphics port, a processing unit, or a local bus using any of a variety of bus architectures.

[0087] The electronic device can also communicate with one or more external devices (such as keyboards, pointing devices, Bluetooth devices, etc.), and can also communicate with one or more devices that enable users to interact with the electronic device, and / or communicate with any device (such as routers, modems, etc.) that enables the electronic device to communicate with one or more other computing devices. This communication can be carried out through an input / output (I / O) interface. In addition, the electronic device can also communicate with one or more networks (such as local area networks (LANs), wide area networks (WANs) and / or public networks, such as the Internet) through a network adapter. As shown in the figure, the network adapter communicates with other modules of the electronic device through a bus. It should be understood that, although not shown in the figure, other hardware and / or software modules can be used in conjunction with the electronic device, including but not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data backup storage systems, etc.

[0088] Through the description of the above embodiments, it is easy for those skilled in the art to understand that the example embodiments described here can be implemented by software, or by software combined with necessary hardware. Therefore, the technical solution according to the embodiment of the present disclosure can be embodied in the form of a software product, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.) or on a network, and includes several instructions to enable a computing device (which can be a personal computer, a server, a terminal device, or a network device, etc.) to execute the method according to the embodiment of the present disclosure.

[0089] In an exemplary embodiment of the present disclosure, a computer-readable storage medium is also provided, on which a program product capable of implementing the above method of the present specification is stored. In some possible embodiments, various aspects of the present invention can also be implemented in the form of a program product, which includes a program code, and when the program product is run on a terminal device, the program code is used to enable the terminal device to perform the steps according to various exemplary embodiments of the present invention described in the above "Exemplary Method" section of the present specification.

[0090] The program product for implementing the above method according to an embodiment of the present invention may adopt a portable compact disk read-only memory (CD-ROM) and include program code, and may be run on a terminal device, such as a personal computer. However, the program product of the present invention is not limited thereto, and in this document, a readable storage medium may be any tangible medium containing or storing a program, which may be used by or in combination with an instruction execution system, apparatus, or device.

[0091] The program product may use any combination of one or more readable media. The readable medium may be a readable signal medium or a readable storage medium. The readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination of the above. More specific examples (non-exhaustive list) of readable storage media include: an electrical connection with one or more wires, a portable disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the above.

[0092] Computer readable signal media may include data signals propagated in baseband or as part of a carrier wave, in which readable program code is carried. Such propagated data signals may take a variety of forms, including but not limited to electromagnetic signals, optical signals, or any suitable combination of the above. Readable signal media may also be any readable medium other than a readable storage medium, which may send, propagate, or transmit a program for use by or in conjunction with an instruction execution system, apparatus, or device.

[0093] The program code embodied on the readable medium may be transmitted using any appropriate medium, including but not limited to wireless, wired, optical cable, RF, etc., or any suitable combination of the foregoing.

[0094] Program code for performing the operations of the present invention may be written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Java, C++, etc., and conventional procedural programming languages ​​such as "C" or similar programming languages. The program code may be executed entirely on the user computing device, partially on the user device, as a separate software package, partially on the user computing device and partially on a remote computing device, or entirely on a remote computing device or server. In cases involving a remote computing device, the remote computing device may be connected to the user computing device through any type of network, including a local area network (LAN) or a wide area network (WAN), or may be connected to an external computing device (e.g., through the Internet using an Internet service provider).

[0095] In addition, the above-mentioned figures are only schematic illustrations of the processes included in the method according to an exemplary embodiment of the present invention, and are not intended to be limiting. It is easy to understand that the processes shown in the above-mentioned figures do not indicate or limit the time sequence of these processes. In addition, it is also easy to understand that these processes can be performed synchronously or asynchronously, for example, in multiple modules.

[0096] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present disclosure that follow the general principles of the present disclosure and include common knowledge or customary technical means in the art that are not disclosed in the present disclosure. The specification and examples are to be considered as exemplary only, and the true scope and spirit of the present disclosure are indicated by the claims.

[0097] It should be understood that the present disclosure is not limited to the exact structures that have been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A RPA-CRISPR nucleic acid detection system based on a digital microfluidic chip, characterized in that: The invention comprises a microfluidic chip, a heating unit, a fluorescence detection unit and a control unit, wherein the microfluidic chip comprises a first liquid injection area, a first reaction area, a quantitative liquid separation area, a second reaction area, a second liquid injection area, a detection area and an electrode array; the heating unit is used to regulate the temperature of the first reaction area and the second reaction area, the fluorescence detection unit is used to perform fluorescence detection on the reaction products in the detection area, the electrode array covers each area and drives the movement of droplets in the area; the control unit is used to control the movement sequence and position of droplets driven by the electrode array in each area according to the nucleic acid detection process; wherein the electrode array covered by the quantitative liquid separation area comprises a plurality of mutually spaced quantitative electrodes, and the RPA products in the quantitative liquid separation area are quantitatively controlled by controlling the on and off of the quantitative electrodes.

2. The nucleic acid detection system according to claim 1, characterized in that: The quantitative electrode comprises four quantitative sub-electrodes arranged at intervals, and the shape of the quantitative sub-electrodes is semicircular.

3. The nucleic acid detection system according to claim 1, characterized in that: An RPA freeze-dried ball packaging structure is arranged above the first liquid injection area, and the RPA freeze-dried ball is sealed and stored above the first liquid injection area through the packaging structure.

4. The nucleic acid detection system according to claim 1, characterized in that: A CRISPR freeze-dried ball packaging structure is arranged above the second liquid injection area, and the CRISPR freeze-dried ball is sealed and stored above the second liquid injection area through the packaging structure.

5. The nucleic acid detection system according to claim 1, characterized in that: The microfluidic chip includes a plurality of the first liquid injection areas, a plurality of the first reaction areas, a plurality of the quantitative liquid separation areas, a plurality of the second reaction areas, a plurality of the second liquid injection areas and a plurality of the detection areas.

6. A RPA-CRISPR nucleic acid detection method based on a digital microfluidic chip, characterized in that: Utilizing the nucleic acid detection system according to any one of claims 1 to 5, the method comprises the following steps: 1) Injecting the RPA reaction solution into the first injection area; 2) driving the PRA reaction solution to move to the first reaction area for RPA amplification reaction; 3) After the RPA amplification reaction is completed, the amplification product is driven to the quantitative separation area for quantitative operation; 4) After the quantitative operation, the quantified portion of the RPA amplification product is moved to the second reaction area; 5) Injecting the CRISPR reaction solution into the second injection area; 6) driving the CRISPR reaction solution to move to the second reaction area to mix with the RPA amplification product and then perform a CRISPR reaction; 7) After the CRISPR reaction is completed, the CRISPR reaction product is driven to the detection area for fluorescent nucleic acid detection.

7. The nucleic acid detection method according to claim 6, characterized in that: Step 1) includes: Injecting the sample solution to be tested into the injection port of the first injection zone; The sample solution dissolves the RPA freeze-dried beads encapsulated in the encapsulation structure to form an RPA reaction solution.

8. The nucleic acid detection method according to claim 6, characterized in that: Step 2) includes: driving the RPA reaction solution to move to the first reaction zone; driving the RPA reaction solution to continuously move in the first reaction zone to mix the droplets; The RPA amplification reaction is performed after adjusting the reaction temperature of the first reaction zone.

9. The nucleic acid detection method according to claim 6, characterized in that: Step 3) includes: Drive the RPA amplification product to move to the quantitative liquid separation area and allow the droplets to fill the quantitative electrode; The power is cut off for some of the quantitative electrodes, and the remaining droplets on the quantitative electrodes that are not cut off are part of the RPA amplification products after quantification.

10. The nucleic acid detection method according to claim 6, characterized in that: Step 5) includes: Injecting enzyme-free water into the injection port of the second injection zone; The enzyme-free water dissolves the CRISPR freeze-dried beads encapsulated in the encapsulation structure to form a CRISPR reaction solution.

11. An electronic device, characterized in that: include: processor; as well as A memory having computer-readable instructions stored thereon, wherein the computer-readable instructions, when executed by the processor, implement the method according to any one of claims 6 to 10.

12. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the method according to any one of claims 6 to 10 is implemented.

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