RCA-CRISPR virus nucleic acid detection system based on programmable digital micro-fluidic chip and use method of RCA-CRISPR virus nucleic acid detection system

Abstract

The invention discloses an RCA-CRISPR virus nucleic acid detection system based on a programmable digital micro-fluidic chip and a use method.The system comprises the digital micro-fluidic chip, a control unit, a heating and temperature control unit and a fluorescence detection unit, the micro-fluidic chip comprises an RCA area, a CRISPR reagent storage area, a mixed reaction and detection area and an electrode array, and when the system is used, the RCA area and the CRISPR reagent storage area are separated from each other; the liquid in the RCA area and the liquid in the CRISPR reagent storage area are mixed and reacted through the control unit, and then are detected through the fluorescence detection unit. The system is miniaturized, automatic, low in cost, efficient and rapid.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochips, in particular to a RCA-CRISPR virus nucleic acid detection system based on a programmable digital microfluidic chip and a use method thereof. BACKGROUND

[0002] Virus rapid detection (POCT, Point-of-Care Testing) can provide instant diagnostic results on site, providing a clinically relevant detection method, which has significantly improved the management and monitoring of infectious diseases on site in recent years (Xiao M, et al. Adv Sci (Weinh), 2022 Jun; 9(17): e2105904). Traditional virus detection methods such as RT-PCR and antibody detection, although highly accurate, rely on complex laboratory equipment, long operation processes and professional personnel operation, which are not suitable for use in scenarios requiring rapid response, and are also costly, which cannot meet the demand for large-scale detection. Therefore, it is particularly urgent to develop new, simple, rapid and on-site detection techniques.

[0003] CRISPR technology, as a new nucleic acid detection technology, has become a powerful tool for virus detection due to its advantages in high sensitivity, specificity and accuracy. The advantage of the CRISPR system is that it can accurately identify target nucleic acid sequences, and can achieve high-sensitivity detection of low-concentration virus samples through pre-amplification signal amplification (Kaminski MM, et al. Nat Biomed Eng, 2021 Jul; 5(7): 643-656): e2105904). Compared with PCR, isothermal amplification does not rely on temperature cycling, and by amplifying at a single temperature, it simplifies the equipment requirements and improves the convenience of detection. Isothermal amplification technology (such as RCA rolling circle amplification) has the advantages of high sensitivity and rapid reaction, and can achieve efficient amplification of virus nucleic acids in a short time, which is suitable for on-site detection (Wei Z, et al. Crit Rev Biotechnol, 2023 May; 43(3): 415-432)

[0004] Digital microfluidic chip as a new emerging miniaturized analysis platform, can control the micro liquid volume accurately, digital microfluidic chip can realize high-throughput, low-cost, rapid virus detection (Lehnert T, et al. LabChip, 2024 Feb 27; 24(5): 1441-1493). Its miniaturization and automation make it very suitable for on-site virus detection, especially in complex environments, it can achieve low-cost and efficient rapid virus diagnosis.

[0005] Therefore, those skilled in the art are dedicated to developing a multiplex virus detection method that combines CRISPR with rolling circle amplification on a digital microfluidic chip. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a miniaturized, automated, low-cost, efficient and rapid virus detection system.

[0007] To achieve the above objectives, the present invention provides an RCA-CRISPR multiplex virus detection system based on a digital microfluidic chip, characterized in that it includes a digital microfluidic chip, a control unit, a heating and temperature control unit, and a fluorescence detection unit.

[0008] In a preferred embodiment of the present invention, the digital microfluidic chip includes an RCA region, a CRISPR reagent storage region, a mixing reaction and detection region, and an electrode array.

[0009] In another preferred embodiment of the present invention, the RCA region and the CRISPR reagent storage region are not connected to each other, but are respectively connected to the mixing reaction and detection region, and the RCA region and the CRISPR reagent storage region extend outward from the edge of the mixing reaction and detection region.

[0010] In another preferred embodiment of the present invention, the digital microfluidic chip includes a plurality of the RCA regions, a CRISPR reagent storage region capable of storing multiple different / identical reagents, and a plurality of the mixed reaction and detection regions.

[0011] In another preferred embodiment of the present invention, the control unit controls the movement sequence, movement position and amount of the electrode array driven droplets in each region according to the nucleic acid detection process.

[0012] In another preferred embodiment of the present invention, the fluorescence detection unit includes a camera and a laser emitter, the laser emitter being located on a slide rail, and the fluorescence detection unit being located below the digital microfluidic chip for detecting the fluorescence of reaction droplets in the chip.

[0013] In another preferred embodiment of the present invention, the heating and temperature control unit includes a heating element, a thermistor, and a temperature controller, wherein the heating element and the thermistor are bonded to a digital microfluidic chip.

[0014] In another preferred embodiment of the invention, the system further includes a power supply and a display screen.

[0015] This invention also provides a method for using the RCA-CRISPR multiplex virus detection system based on a digital microfluidic chip, characterized in that the method includes the following steps: Step 1: Inject a ring-shaped sample and RCA reaction solution into the RCA region; Step 2: Perform RCA amplification reaction; Step 3: After the RCA amplification reaction is complete, drive the quantitative RCA amplification product to the mixing reaction and detection area; Step 4: Inject CRISPR reaction solution into the CRISPR reagent storage area to drive the CRISPR reaction solution to the mixing reaction and detection area; Step 5: After mixing the RCA amplification product with the CRISPR reaction solution, perform the CRISPR reaction; Step 6: Perform fluorescence detection after the CRISPR reaction is complete.

[0016] In a preferred embodiment of the present invention, in step 5, the CRISPR reaction solution and the RCA amplification product are driven to move continuously in the mixing reaction and detection zone to mix the droplets.

[0017] Technical effect

[0018] 1. RCA-CRISPR detection of viral nucleic acid relies on laboratory equipment and professional personnel, and has drawbacks such as long operation time and susceptibility to aerosol contamination during the step-by-step operation process, making it unsuitable as a POCT method. This invention combines the RCA-CRISPR viral nucleic acid detection method with a digital microfluidic chip. The digital microfluidic chip can precisely control the amount of liquid, enabling RCA-CRISPR viral nucleic acid detection to be completed on the digital microfluidic chip, which is anti-contamination, time-saving, and provides high-throughput rapid detection.

[0019] 2. Both RCA and CRISPR reactions require a certain temperature. Existing heating and temperature control devices are relatively complex, and there is no simple device for heating chips. This invention provides a simple and portable heating and temperature control device. It consists of a heating element, a thermistor, a power supply, and a display screen, which can accurately control the reaction temperature. The heating device is simple and portable and can accurately control the reaction temperature inside the chip.

[0020] 3. Current fluorescence detection devices are relatively complex, and there is no simple detection device for fluorescence intensity within a chip. This invention provides a simple fluorescence unit, which consists of a fluorescence detector, a slide rail, and a camera. It is set inside the digital microfluidic control unit, below the digital microfluidic chip, and can detect whether the reaction droplets in the chip are fluorescent. The fluorescence detection device is simple and portable, and can detect whether the reaction liquid in the chip is fluorescent, thereby obtaining the detection result.

[0021] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the digital microfluidic chip in the RCA-CRISPR nucleic acid detection system based on a preferred embodiment of the present invention. Figure 2 This is a schematic diagram of the RCA-CRISPR nucleic acid detection principle according to a preferred embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of an RCA-CRISPR nucleic acid detection system based on a digital microfluidic chip according to a preferred embodiment of the present invention; Figure 4 This is a flowchart illustrating the usage method of an RCA-CRISPR nucleic acid detection system based on a digital microfluidic chip, according to a preferred embodiment of the present invention. Figure 5 This is a fluorescence detection result diagram of a preferred embodiment of the present invention. Detailed Implementation

[0023] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0024] like Figure 1 As shown, the microfluidic chip includes an RCA region, a CRISPR reagent storage region, a mixing reaction and detection region, and an electrode array; further, the microfluidic chip includes multiple RCA regions, multiple CRISPR reagent storage regions, and multiple mixing reaction and detection regions. The RCA regions and CRISPR reagent storage regions are not connected to each other, but are respectively connected to the mixing reaction and detection regions, and the RCA regions and CRISPR reagent storage regions extend outward from the edge of the mixing region.

[0025] like Figure 2 As shown, in this invention, the circular sample and the corresponding primers bind through complementary pairing, and the amplification reaction is initiated under the action of DNA polymerase, rapidly generating a large amount of target DNA.

[0026] Cas12a binds to crRNA to produce an inactive Cas12a-crRNA complex.

[0027] Cas12a recognizes and binds to target DNA through its crRNA, activates its endonuclease activity, cuts the target DNA, and releases a fluorescent signal.

[0028] If the sample contains the target DNA, the cleavage signal will be activated; otherwise, if the sample does not contain the target DNA, the cleavage will not be activated and no fluorescent signal will be generated.

[0029] like Figure 3 The RCA-CRISPR multiplex virus detection system based on a digital microfluidic chip of the present invention shown includes a digital microfluidic chip, a heating and temperature control unit, a fluorescence detection unit, and a control unit.

[0030] The heating and temperature control unit includes a heating element, a thermistor, and a temperature controller. The heating element and the thermistor are bonded to the digital microfluidic chip.

[0031] The fluorescence detection unit includes a camera and a laser emitter. The laser emitter is located on a slide rail, and the fluorescence detection unit is located below the digital microfluidic chip to detect the fluorescence of the reaction droplets in the chip.

[0032] The control unit is used to control the movement sequence, position, and amount of droplets in each region of the electrode array according to the nucleic acid detection process.

[0033] In addition, the system also includes a power supply and a display screen.

[0034] like Figure 4 The method of using the RCA-CRISPR multiplex viral nucleic acid detection system based on a digital microfluidic chip of the present invention, shown herein, includes the following steps: 1) Inject a mixture of a ring-shaped sample and the RCA reaction solution into the RCA region; 2) Perform RCA amplification reaction; 3) After the RCA amplification reaction is completed, the program controls the electrodes at different positions to drive the quantitative RCA amplification products to the mixing reaction and detection area; 4) Inject CRISPR reaction solution into the CRISPR reagent storage area to drive the CRISPR reaction solution to the corresponding mixing reaction and detection area; 5) The RCA amplification product is mixed with the CRISPR reaction solution and then subjected to a CRISPR reaction; 6) After the CRISPR reaction is completed, the laser emitter is moved to each mixing reaction and detection area and the results are interpreted by using a camera.

[0035] Furthermore, in step 5)

[0036] The CRISPR reaction solution and RCA amplification products are continuously moved in the mixing reaction and detection zone to mix the droplets.

[0037] This invention automatically performs a series of operations, including RCA amplification, liquid separation, mixing, and CRISPR reaction, by combining the electrode arrays covering each region in the RCA region, CRISPR reagent storage region, and mixing reaction and detection region, and through control unit control.

[0038] Example 1: Detection of Nipah virus

[0039] The digital microfluidic chip RCA-CRISPR viral nucleic acid detection system is connected to an external computer and the control software is turned on. A mixture of the test sample and the RCA reaction solution, totaling 6 μL, is injected into the RCA region.

[0040] Turn on the heating and temperature control unit, set the temperature control to 37℃, and start the RCA amplification reaction for 30 minutes. After the RCA reaction is complete, the control software drives the amplification product to be separated into 2 μL and mixed in the mixing zone. Add 4 μL of CRISPR reaction solution to the CRISPR reagent storage area, and use the control software to drive the CRISPR reaction solution to the mixing reaction and detection area, where it is mixed with the RCA amplification product to form a 6 μL reaction system. The mixing and detection zone is set to 37°C for temperature control, and the CRISPR reaction begins after 30 minutes. After the CRISPR reaction is complete, the laser emitter is moved to the mixed reaction and detection area to detect the fluorescence signal, thus completing the RCA-CRISPR Nipah virus nucleic acid detection.

[0041] like Figure 5 As shown, the camera captured green fluorescence, indicating that the sample was positive for Nipah virus.

[0042] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A digital microfluidic chip-based RCA-CRISPR multiplex virus detection system, characterized in that, The chip comprises a digital microfluidic chip, a control unit, a heating and temperature control unit, and a fluorescence detection unit.

2. The viral detection system of claim 1, wherein, The digital microfluidic chip comprises RCA zones, CRISPR reagent storage zones, mixing and detection zones, and an electrode array.

3. The viral detection system of claim 2, wherein the viral detection system is configured to detect the presence of the virus in the sample by detecting the presence of the virus-specific nucleic acid sequence in the sample. The RCA zones and the CRISPR reagent storage zones are not connected to each other, but are connected to the mixing and detection zones respectively, and the RCA zones and the CRISPR reagent storage zones extend outward from the edges of the mixing and detection zones respectively.

4. The viral detection system of claim 2, wherein the viral detection system is configured to detect the presence of the virus in the sample by detecting the presence of the virus-specific nucleic acid sequence in the sample. The digital microfluidic chip comprises multiple RCA zones, CRISPR reagent storage zones capable of storing multiple different / same reagents, and multiple mixing and detection zones.

5. The viral detection system of claim 2, wherein the viral detection system is configured to detect the presence of a virus in the sample by detecting the presence of a nucleic acid sequence of the virus in the sample. The control unit controls the movement sequence, movement position, and movement amount of the droplets in each zone according to the nucleic acid detection process.

6. The viral detection system of claim 1, wherein, The fluorescence detection unit comprises a camera and a laser emitter, and the laser emitter is located on a slide rail.

7. The viral detection system of claim 1, wherein the viral detection system is configured to detect the presence of a virus in the sample by detecting the presence of a nucleic acid sequence of the virus in the sample. The heating and temperature control unit comprises a heating sheet, a thermal sensor, and a temperature controller.

8. The viral detection system of claim 1, wherein, The system further comprises a power supply and a display screen.

9. The use of a RCA-CRISPR multiplex virus detection system based on digital microfluidic chip according to any one of claims 2-5, wherein, The method comprises the following steps: Step 1: injecting a circular sample and RCA reaction solution into the RCA zone; Step 2: performing RCA amplification reaction; Step 3: driving the quantitative RCA amplification product to the mixing and detection zone after the RCA amplification reaction is completed; Step 4: injecting a CRISPR reaction solution into the CRISPR reagent storage zone and driving the CRISPR reaction solution to the mixing and detection zone; Step 5: mixing the RCA amplification product with the CRISPR reaction solution and performing CRISPR reaction; Step 6: performing fluorescence detection after the CRISPR reaction is completed.

10. The method of use of claim 9, wherein, In step 5, the CRISPR reaction solution and the RCA amplification product are continuously driven to move in the mixing and detection zone to mix the droplets.

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