Nucleic acid detection method based on gel self-interference and microfluidic detection platform

Abstract

The invention provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform. The method comprises the following steps: after a to-be-detected sample is added, mixing the to-be-detected sample with a lysis solution and magnetic beads, a magnetic bead cleaning solution and a nucleic acid eluent in sequence through a digital micro-fluidic chip so as to extract nucleic acid liquid drops of the sample; mixing the sample nucleic acid liquid drops with the first round of RPA reagent and the second round of RPA reagent through a digital micro-fluidic chip, and carrying out two rounds of RPA amplification to obtain sample product liquid drops; mixing the sample product liquid drops with a CRISPR reagent through a digital micro-fluidic chip, and moving to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction; and carrying out self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction through an optical detection system, and outputting a nucleic acid detection result that whether the to-be-detected sample contains the to-be-detected nucleic acid sequence or not. According to the method, high-specificity and full-automatic label-free PRA detection is realized.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection technology, and particularly to a nucleic acid detection method based on gel self-interference and a microfluidic detection platform. Background Art

[0002] Nucleic acid detection is a method for analyzing whether a specific nucleic acid sequence of an object to be detected exists in a biological sample (such as a blood sample) under laboratory conditions. Currently, the mainstream RPA nucleic acid detection technology mainly uses fluorescent probes or lateral flow test strips to characterize amplicons. For example, exo fluorescent probes are used to detect amplicons, and the minimum detectable amount is 7.74 copies of RNA molecules in 1 μL of template solution; or, the RPA amplification products labeled with FAM and biotin are combined with anti-FAM gold particles, and then the biotin ligand and anti-FAM probe on the lateral flow test strip are used to detect the amplification products, with a detection limit of 1.0 copy / μL. However, currently, most RPA nucleic acid detection technologies use fluorescence or other markers to read amplicons, which increases the detection cost and the complexity of the detection process. The development of highly integrated non-labeled RPA amplicon detection is yet to be developed. Summary of the Invention

[0003] This application provides a nucleic acid detection method based on gel self-interference and a microfluidic detection platform, realizing a highly specific and fully automatic non-labeled PRA detection general method. The technical solution is as follows:

[0004] According to one aspect of this application, a nucleic acid detection method based on gel self-interference is provided. The method is applied to a microfluidic detection platform, which includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and a lysis solution, magnetic beads, magnetic bead washing solution, nucleic acid elution solution, first-round RPA reagent, second-round RPA reagent, and CRISPR reagent are stored in the lower chip. The digital microfluidic chip is placed on the stage of the optical detection system. The method includes:

[0005] After adding a sample to be tested into the lower chip, the sample to be tested is sequentially mixed with the lysis solution and magnetic beads, the magnetic bead washing solution, and the nucleic acid elution solution through the digital microfluidic chip to extract a sample nucleic acid droplet from the sample to be tested;

[0006] The sample nucleic acid droplet is mixed with the first-round RPA reagent and the second-round RPA reagent through the digital microfluidic chip for two rounds of RPA amplification to obtain a sample product droplet containing two rounds of nucleic acid amplification products;

[0007] Mix the sample product droplets with the CRISPR reagent through the digital microfluidic chip, and move them to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction;

[0008] Detect the self-interference signal of the solid-phase gel in the solution after the cleavage reaction through the optical detection system, and determine and output the nucleic acid detection result of whether the sample to be tested contains the nucleic acid sequence to be tested.

[0009] According to another aspect of the present application, a microfluidic detection platform is provided. The microfluidic detection platform includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and a lysis solution and magnetic beads, a magnetic bead washing solution, a nucleic acid elution solution, a first-round RPA reagent, a second-round RPA reagent, and a CRISPR reagent are stored in the lower chip. The digital microfluidic chip is placed on the stage of the optical detection system;

[0010] The digital microfluidic chip is used to, after adding the sample to be tested into the lower chip, mix the sample to be tested with the lysis solution and magnetic beads, the magnetic bead washing solution, and the nucleic acid elution solution in sequence to extract sample nucleic acid droplets from the sample to be tested;

[0011] The digital microfluidic chip is further used to mix the sample nucleic acid droplets with the first-round RPA reagent and the second-round RPA reagent for two rounds of RPA amplification to obtain sample product droplets containing two rounds of nucleic acid amplification products;

[0012] The digital microfluidic chip is further used to mix the sample product droplets with the CRISPR reagent, and move them to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction;

[0013] The optical detection system is used to detect the self-interference signal of the solid-phase gel after the cleavage reaction, and determine and output the nucleic acid detection result of whether the sample to be tested contains the nucleic acid sequence to be tested.

[0014] According to one aspect of the present application, a nucleic acid detection device is provided, including: a processor and a memory storing a program. The program includes instructions that, when executed by the processor, cause the processor to execute the nucleic acid detection method based on gel self-interference as described above.

[0015] According to another aspect of the present application, a non-transitory computer-readable storage medium storing computer instructions is provided. The computer instructions are used to cause the computer to execute the nucleic acid detection method based on gel self-interference as described above.

[0016] According to another aspect of the present application, there is provided a computer program product, which includes computer instructions stored in a computer-readable storage medium. A processor of a nucleic acid detection device reads the computer instructions from the computer-readable storage medium, and the processor executes the computer instructions, so that the computer device executes the above-mentioned nucleic acid detection method based on gel self-interference.

[0017] The beneficial effects brought by the technical solution provided by the embodiments of the present application at least include:

[0018] By integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded in the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be detected; in the nucleic acid detection process, exponential amplification of the nucleic acid sequence to be detected is achieved through nested RPA amplification, and Cas12a is combined to cut the solid-phase gel, so as to realize judging whether the sample to be detected contains the nucleic acid sequence to be detected by detecting the self-interference signal of the solid-phase gel. While achieving high-sensitivity nucleic acid detection, a high-specificity non-labeled PRA detection general method is also realized. In addition, a new automated microfluidic detection platform is constructed by integrating an optical detection system and a digital microfluidic chip, further improving the automation and integration of the nucleic acid detection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the following description of exemplary embodiments with reference to the accompanying drawings, more details, features and advantages of the present application are disclosed. In the drawings:

[0020] Figure 1 A flowchart of a nucleic acid detection method based on gel self-interference according to an exemplary embodiment of the present application is shown;

[0021] Figure 2 is the construction process of the solid-phase gel provided by an exemplary embodiment of the present application;

[0022] Figure 3 A flowchart of another nucleic acid detection method based on gel self-interference according to an exemplary embodiment of the present application is shown;

[0023] Figure 4 is a schematic structural diagram of a digital microfluidic chip provided by an exemplary embodiment of the present application;

[0024] Figure 5 is a schematic structural diagram of an optical detection system provided by an exemplary embodiment of the present application;

[0025] Figure 6 is an optical principle diagram of gel self-interference provided by an exemplary embodiment of the present application;

[0026] Figure 7It is a schematic structural diagram of a microfluidic detection platform provided by an embodiment of the present application. Detailed implementation manners

[0027] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although some embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided to more thoroughly and completely understand the present application. It should be understood that the drawings and embodiments of the present application are only for exemplary purposes and are not used to limit the protection scope of the present application.

[0028] It should be understood that the various steps recorded in the method embodiments of the present application can be executed in different orders and / or executed in parallel. In addition, the method embodiments may include additional steps and / or omit the steps shown. The scope of the present application is not limited in this regard.

[0029] The term "including" and its variants used herein are open-ended, that is, "including but not limited to". The term "based on" is "at least partially based on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first", "second", etc. mentioned in the present application are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence relationship of the functions performed by these devices, modules or units. It should be noted that the modifications of "one" and "multiple" mentioned in the present application are illustrative rather than restrictive. Those skilled in the art should understand that unless clearly indicated otherwise in the context, it should be understood as "one or more". The names of the messages or information exchanged between multiple devices in the embodiments of the present application are only for illustrative purposes and are not used to limit the scope of these messages or information.

[0030] The solution of the present invention will be described below with reference to the accompanying drawings. The technical solutions provided by the embodiments of the present invention will be described in detail through specific embodiments and their application scenarios.

[0031] Currently, the mainstream RPA (Recombinase polymerase amplification) nucleic acid detection technology mainly uses fluorescent probes or lateral flow test strips to characterize amplicons. For example, exo fluorescent probes are used to detect amplicons, and the minimum detectable amount is 7.74 copies of RNA (Ribonucleic Acid) molecules in 1 μL of template solution. Or, the RPA amplification products labeled with FAM (carboxyfluorescein) and biotin are bound to anti-FAM gold particles, and then the biotin ligand and anti-FAM probe on the lateral flow test strip are used to detect the amplification products, with a detection limit of 1.0 copy / μL. However, currently, most RPA nucleic acid detection technologies use fluorescence or other markers to read amplicons, which increases the detection cost and the complexity of the detection process. The development of highly integrated label-free RPA amplicon detection is awaited.

[0032] In order to achieve highly sensitive, highly specific, and highly integrated label-free RPA nucleic acid detection, the embodiments of the present application provide a microfluidic detection platform that integrates interferometric sensing and RPA nucleic acid detection, and realizes the detection of amplification products through interferometric sensing without using other markers. Please refer to Figure 1 , which shows a flowchart of a nucleic acid detection method based on gel self-interference according to an exemplary embodiment of the present application. This method will be described by taking its application to the microfluidic detection platform as an example. As Figure 1 shown, this method includes:

[0033] Step 101, after adding the sample to be tested into the lower chip, the sample to be tested is successively mixed with the lysis solution, magnetic beads, magnetic bead washing solution, and nucleic acid elution solution through the digital microfluidic chip to extract the sample nucleic acid droplet from the sample to be tested.

[0034] The microfluidic detection platform provided by this application includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip (the upper chip can also be called the upper electrode plate) and a lower chip (the lower chip can also be called the lower electrode plate). A solid-phase gel is embedded in the upper chip and contacts the strip-shaped control electrodes on both sides of the lower chip through conductive tapes. The gel surface of the solid-phase gel is suspended above the lower electrode plate. When the droplet passes through the lower electrode, the solid-phase gel contacts the droplet. The lower chip includes a liquid storage electrode, a sample addition electrode, a waste liquid storage electrode, and an electrode array for droplet manipulation. The liquid storage electrode is used to store lysis solution and magnetic beads, magnetic bead washing solution, nucleic acid elution solution, the first round of RPA reagent, the second round of RPA reagent, and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) reagent; when the digital microfluidic chip is placed on the stage of the optical detection system, the optical acquisition module of the optical detection system can collect the interference signal of the solid-phase gel through the lower electrode plate.

[0035] Exemplarily, the upper chip in the digital microfluidic chip can use ITO (Indium Tin Oxide) coated glass, and the lower chip prepares a liquid storage electrode, a sample addition electrode, a waste liquid storage electrode, and an electrode array for droplet manipulation through ITO electrodes, etc.

[0036] Different from the related technology that uses fluorescence or other markers to read the nucleic acid sequence to be detected, this application uses a solid-phase gel (which can also be called a solid-phase DNA gel) as the sensing unit of the nucleic acid sequence to be detected, and characterizes the detected nucleic acid sequence through the thickness change of the solid-phase gel. As Figure 2 shown, it is the construction process of the solid-phase gel provided by an exemplary embodiment of this application. As Figure 2 shown, first, in the liquid phase, the synthesized cyclizable template ( Figure 2 long chain 2 in) forms a circular template precursor with the primer primer 1 ( Figure 2 short chain 1 in). Under the action of T4 enzyme ( Figure 2 3 in), the circular template precursor is ligated into a ring. Then the circular template is added to the surface of the silicon wafer ( Figure 2 4 in) modified with solid-phase primer 1. Since the 3' end of primer 1 used in forming the circular template is modified with C3 Spacer, it will not compete with the solid-phase primer. The immobilized primer 1 binds to the circular template and, under the action of RCA (Rolling Circle Amplification) reaction, forms a long-chain amplification primer ( Figure 2 5 in). Thereafter, the silicon wafer is immersed in a solution containing primer2 (Figure 2 in 6), primer 3( Figure 2 in the RCA reaction system of 7), primer 2 binds to multiple sites of the long DNA single strand formed by the long-chain amplification primer 5 and amplifies again to form a double-stranded product with a single-stranded branched structure. The sequences of these branched structures repeat the circular template sequence. Under the action of primer 3, further amplification occurs, causing the single-stranded branched structure to form a double strand and carry a secondary single-stranded branched structure. Alternating in this way, the DNA (DeoxyriboNucleic Acid) molecules on the silicon wafer continuously increase, and each round of RCA will generate the single-stranded template for the next round of RPA, ultimately forming a solid-phase DNA gel( Figure 2 in 9), Figure 2 8 in is the RCA reaction system including 6 and 7. It should be noted that the thickness of the generated solid-phase gel should be not less than 20 nm. In this way, after CRISPR cutting, the change in the thickness of the solid-phase gel is sufficiently obvious compared to the sub-nanometer sensitivity of optical interference, thereby ensuring the signal-to-noise ratio of nucleic acid detection.

[0037] In a possible implementation, after constructing the solid-phase gel, the solid-phase gel is embedded in the upper chip of the digital microfluidic chip. Lysis solution, magnetic beads, magnetic bead cleaning solution, nucleic acid elution solution, the first-round RPA reagent, the second-round RPA reagent, and the CRISPR reagent are respectively added to the liquid storage electrodes of the lower chip of the digital microfluidic chip. After adding the test sample to be detected to the sample addition electrode, the subsequent nucleic acid amplification and nucleic acid detection processes are started. First, the digital microfluidic chip is used to mix the test sample with the lysis solution and magnetic beads, the magnetic bead cleaning solution, and the nucleic acid elution solution in sequence, so as to extract the sample nucleic acid from the test sample to obtain a sample nucleic acid droplet containing the sample nucleic acid.

[0038] Exemplarily, the test sample can be a test blood sample or other biological products from which nucleic acids can be extracted.

[0039] Step 102, the digital microfluidic chip is used to mix the sample nucleic acid droplet with the first-round RPA reagent and the second RPA reagent for two rounds of RPA amplification to obtain a sample product droplet containing the nucleic acid amplification products of two rounds.

[0040] To achieve high-sensitivity detection of the nucleic acid sequence to be detected, the present application realizes exponential amplification of the nucleic acid sequence to be detected through nested RPA amplification. Correspondingly, after extracting the sample nucleic acid from the test blood sample, the digital microfluidic chip is used to mix the sample nucleic acid droplet with the first-round RPA reagent and the second-round RPA reagent respectively for two rounds of RPA amplification to obtain a sample product droplet containing the nucleic acid amplification products of two rounds. Through two rounds of RPA amplification, a sufficient concentration of recognition products can be amplified for subsequent nucleic acid detection.

[0041] Step 103: Mix the sample product droplets with the CRISPR reagent through a digital microfluidic chip and move them to the solid-phase gel for sequence recognition and cleavage reaction, obtaining the solid-phase gel after the cleavage reaction.

[0042] The nucleic acid detection principle of this application is as follows: The change in the self-interference thickness of the solid-phase DNA gel is achieved through the CRISPR / Cas12a protein. First, determine the specific nucleic acid fragment of the object to be detected and determine the crRNA recognition sequence according to the technical requirements of the Cas12a protein. Then, through two nested rounds of RPA amplification, exponential amplification of the nucleic acid sequence to be detected is achieved. The two rounds of nucleic acid amplification products contain the crRNA recognition region of the CRISPR system. When the two rounds of RPA amplify the crRNA recognition products with sufficient concentration, the Cas12a protein in the CRISPR system can highly specifically recognize the nucleic acid sequence to be detected, activate the molecular mechanism for cleaving DNA, and cleave the solid-phase DNA gel on the silicon wafer. After the solid DNA gel molecules are cleaved, the physical thickness decreases, resulting in a decrease in the self-interference phase of the solid-phase DNA gel, enabling the detection of the self-interference signal of the solid-phase DNA gel to determine whether the sample to be detected contains the nucleic acid sequence to be detected.

[0043] Based on this nucleic acid detection principle, after obtaining the sample product droplets through two rounds of RPA amplification, the sample product droplets are continuously mixed with the CRISPR reagent through a digital microfluidic chip. The crRNA recognizes the specific sequence, and the mixed droplets are transferred to the solid-phase gel for sequence recognition and cleavage reaction. After incubation, the droplets are moved to the waste liquid electrode, and the solid-phase gel after the cleavage reaction is detected to determine whether the nucleic acid sequence to be detected exists.

[0044] Step 104: Detect the self-interference signal of the solid-phase gel in the solution after the cleavage reaction through an optical detection system, and determine and output the nucleic acid detection result of whether the sample to be detected contains the nucleic acid sequence to be detected.

[0045] In a possible implementation, the digital microfluidic chip is placed on the stage. The light source in the optical detection system irradiates the surface of the solid-phase DNA gel in the digital microfluidic chip through a beam splitter and an objective lens. The reflected light is focused on the end face of a multimode optical fiber by an imaging lens and then connected to a spectrometer and a data analysis device to detect the self-interference signal of the solid-phase gel in the solution after the cleavage reaction. Since the self-interference signal is related to the thickness of the solid-phase gel, and whether the thickness of the solid-phase gel changes is related to whether the sample to be detected contains the nucleic acid sequence to be detected, according to this correlation, the nucleic acid detection result of whether the sample to be detected contains the nucleic acid sequence to be detected can be determined and output based on the detection result of the self-interference signal.

[0046] In summary, the embodiment of the present application provides a nucleic acid detection method based on gel self-interference: by integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded in the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be detected; so that in the nucleic acid detection process, exponential amplification of the nucleic acid sequence to be detected is achieved through nested RPA amplification, and Cas12a is combined to cut the solid-phase gel, so as to determine whether the sample to be detected contains the nucleic acid sequence to be detected by detecting the self-interference signal of the solid-phase gel. While achieving high-sensitivity nucleic acid detection, a general method for high-specificity label-free PRA detection is also realized. In addition, a new automated microfluidic detection platform is constructed by integrating an optical detection system and a digital microfluidic chip, further improving the automation and integration of the nucleic acid detection process.

[0047] The digital microfluidic chip controls the microdroplets of samples and reagents by means of electro-wetting. By applying a potential to the electrodes, operations such as merging, mixing, dispensing, and separating of microliter-sized droplets can be performed on the electrode array coated with a hydrophobic layer, realizing the automation, miniaturization, and integration of the detection process.

[0048] Please refer to Figure 3 , which shows a flowchart of another nucleic acid detection method based on gel self-interference according to an exemplary embodiment of the present application. This method is described by taking its application to a microfluidic detection platform as an example. As Figure 3 shown, this method includes:

[0049] Step 301, after adding the sample to be detected into the lower chip, the sample to be detected, the lysis solution, and magnetic beads are mixed through the electrode array to release the sample nucleic acid in the sample to be detected, and the sample nucleic acid is enriched on the magnetic beads.

[0050] Step 302, the magnetic beads are mixed with the magnetic bead washing solution through the electrode array for magnetic bead washing.

[0051] Step 303, the washed magnetic beads are mixed with the nucleic acid elution solution through the electrode array to obtain a sample nucleic acid droplet.

[0052] Step 304, the first-round RPA reagent is mixed with the sample nucleic acid droplet through the electrode array to obtain the first-round amplification product.

[0053] Step 305, the second-round RPA reagent is mixed with the first-round amplification product through the electrode array to obtain a sample product droplet containing the nucleic acid amplification products of two rounds.

[0054] Step 306, the sample product droplet is mixed with the CRISPR reagent through the electrode array and moved to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction.

[0055] Step 307: Use an optical detection system to detect the self-interference signal of the solid-phase gel in the solution after the cutting reaction, and determine whether the thickness of the solid-phase gel has changed.

[0056] In the embodiment of the present application, the lower chip (lower electrode plate) of the digital microfluidic chip is provided with a sample-loading electrode, a waste liquid storage electrode, an electrode array, and multiple liquid storage electrodes through ITO electrodes. Among them, the number of electrodes in the electrode array can be increased according to the detection throughput by adding electrodes or solid-phase gels. Among the multiple liquid storage electrodes, some are respectively used to store lysis solution and magnetic beads, magnetic bead washing solution, nucleic acid elution solution, the first round of RPA reagent, the second round of RPA reagent, and CRISPR reagent. The sample-loading electrode is used to store the sample to be tested, and the electrode array is used to control the movement of the sample to be tested, the sample nucleic acid droplet, and the sample product droplet.

[0057] Exemplarily, as Figure 4 shown, it is a schematic structural diagram of a digital microfluidic chip provided by an exemplary embodiment of the present application. As Figure 4 shown, Figure 4 18 in Figure 4 is the sample-loading electrode, and a blood sample can be added thereto; Figure 4 19, 20, 21, 22, 23, 24 in Figure 4 are 6 liquid storage electrodes. Lysis solution and magnetic beads can be added to 19, magnetic bead washing solution can be added to 20, nucleic acid elution solution can be added to 21, the first round of RPA reagent can be added to 22, the second round of RPA reagent can be added to 23, and CRISPR reagent can be added to 24; Figure 4 25 in

[0058] is an embedded gel silicon wafer;

[0059] Based on the above description of the structure of the digital microfluidic chip, the control process of droplets in the nucleic acid detection test includes the following steps: (1) After adding the sample to be tested into the lower-layer chip, separate one droplet from the sample to be tested, lysate, and magnetic beads respectively through the electrode array; (2) Mix one droplet of the sample to be tested with 1 droplet of lysate and magnetic beads to release the sample nucleic acid in the sample to be tested through the lysate, and the released sample nucleic acid is incubated and enriched on the magnetic beads; (3) Fix the magnetic beads by magnetic control, remove the solution, and control the mixing of the magnetic bead cleaning solution and the magnetic beads through the electrode array, that is, add the magnetic bead cleaning solution to the remaining magnetic beads to clean the magnetic beads and wash away the excess solution; (4) After the magnetic beads are cleaned, continue to retain the magnetic beads, and move the magnetic bead cleaning solution to the waste liquid storage electrode through the electrode array; (5) Separate a droplet from the nucleic acid eluent through the electrode array, and mix the droplet of the nucleic acid eluent with the washed magnetic beads for incubation to separate the sample nucleic acid enriched on the magnetic beads from the magnetic beads to obtain a sample nucleic acid droplet; (6) Move out the sample nucleic acid droplet through the electrode array, and mix the first-round RPA reagent with the sample nucleic acid droplet for amplification to obtain the first-round amplification product. The droplet can be moved to improve the amplification efficiency; (7) Mix the second-round RPA reagent with the first-round amplification product through the electrode array to obtain a sample product droplet containing two rounds of nucleic acid amplification products. The amplification efficiency can also be improved by moving the droplet; (8) Mix the sample product droplet with the CRISPR reagent through the electrode array, and the crRNA recognizes the specific sequence; (9) Move the mixed droplet to the solid-phase gel through the electrode array for sequence recognition and cleavage reaction, and after incubation, move the droplet to the waste liquid storage electrode to obtain the solid-phase gel after the cleavage reaction; (10) Detect the self-interference signal of the solid-phase gel in the solution after the cleavage reaction through the optical detection system to determine whether the thickness of the solid-phase gel changes, so as to obtain the nucleic acid detection result of the nucleic acid sequence to be tested.

[0060] Please refer to Figure 5 , which is a schematic structural diagram of the optical detection system provided by an exemplary embodiment of the present application. As Figure 5 shown, the light source is collimated by the collimating lens 12 and then irradiates the surface of the solid-phase gel in the solution through the beam splitter 14 and the objective lens 15 of the optical detection system. The solid-phase gel is embedded in the digital microfluidic chip 16. The reflected interference signal is converged by the imaging lens 13 to the end face of the multimode optical fiber 17 through the beam splitter 14. The optical fiber is externally connected to a spectrometer and connected to a computer for data analysis.

[0061] In order to infer the nucleic acid detection result of the nucleic acid sequence to be tested based on the detection result of the self-interference signal, it is first necessary to analyze the optical principle of the self-interference of the solid-phase gel. Exemplarily, please refer to Figure 6 , Figure 6 which is an optical principle diagram of the gel self-interference provided by an exemplary embodiment of the present application. As Figure 6As shown in (a), when light with unit intensity is incident vertically on the silicon wafer, it will be reflected by the upper surface of the solid-phase DNA gel 9 and then reflected by the interface between the silica layer 10 and the silicon substrate 11 through the solid-phase DNA gel 9. The two reflected light beams are denoted as U1(k) and U2(k) respectively, where k is the vacuum wave number (since the refractive indices of silica and DNA molecules are not very different, the reflection between these two layers is often ignored in self-interference sensors). The two reflected light beams form an interference, and the interference phase can be expressed as: 2n0k(d + δ). Among them, n0 represents the average refractive index of silica and the solid-phase DNA gel (≈1.47), d is the thickness of the silica layer, and δ is the thickness of the gel layer of the solid-phase gel.

[0062] When the solid-phase DNA gel reacts in the solution, part of the incident light U0(k) will also be interfered by the weak scattering medium in the solution, as Figure 6 shown in (b). The weak scattering medium is equivalently regarded as a spherical particle with a radius of a and a refractive index of n, and the distribution f (q) of its scattering amplitude can be expressed as where q is the scattering wave vector, and its physical meaning is the vector difference between the scattering wave and the incident wave. Therefore, the scattering wave vector generated by part of the incident light can be expressed as Since the molecular size of the scattering medium is much smaller than the wavelength, and it can be approximately obtained that u s (k)≈C k U0(k)(n 2 -1)V, where C k is a constant coefficient generated in the calculation, and V is the volume of the scattering medium.

[0063] From this, it can be obtained that due to the existence of the weak scattering medium in the solution, the objective lens can collect a backscattered light beam, and the wave vector can be expressed as u3(k) = α k C k U0(k)(n 2 -1)V, where α k represents the ratio of the collection angle of the objective lens to the 4π space. Scattering also weakens the illumination light intensity on the solid-phase gel. When the incident light passes through the solution and irradiates on the solid-phase DNA gel, the reflected light beams U1(k) and U2(k) are scattered by the medium again when passing through the solution, and the amplitude further decreases. The amplitudes of the two reflected light beams collected by the objective lens are denoted as A1(k) and A2(k) respectively, and the amplitude of the wave vector u3(k) is denoted as A3(k). The synthesized light beam is composed of the two reflected light beams and the scattered light, so the synthesized light beam can be expressed as where n wis the average refractive index of the solution, and z is the axial distance of the scattering medium relative to the upper surface of the gel. The reflection intensity I(k) is the sum of squares of the synthesized light beams, and the interference term contains the phase difference. Therefore, the reflection intensity of the self-interference spectrum can be expressed as: I(k) = |U(k)| 2 ≈

[0064] A1(k) 2 +A2(k) 2 +A3(k) 2 +2A2(k)A3(k)cos[2n0k(d + δ)] + 2a1(k)A2(k)cos[2n0k(d + δ) + 2n w kz]. According to the common method of optical field reconstruction, the multiple interferences with relatively small intensities and the interference between U1(k) and U3(k) are theoretically ignored.

[0065] In the analytical method, the interference spectrum presents a cosine function distribution along the wave number k dimension. Using the fast Fourier transform (FFT), the conversion of spectral frequency domain information to axial spatial domain information can be realized, so that the interference information represented by cos[2n0k(d + δ)] and cos[2n0k(d + δ) + 2n w kz] can be effectively separated, that is, the self-interference signal of the solid-phase gel can be separated from the scattering signal in the solution by FFT. Since the FFT result of the digital spectral signal is discrete, its sensitivity is not sufficient to resolve the nanoscale δ value. In this application, δ is resolved through the phase information of FFT. During the analysis process, the reflectance spectrum R0(k) of a standard silicon wafer with an oxide layer thickness of d is pre-recorded, and the reflectance spectrum of the solid-phase gel in the solution is recorded as R1(k). Perform FFT on R0(k) - R1(k). Because 2n0k(d + δ) ≈ 2n0k d +2n0k c δ(k c is the average wave number of the spectrum), divide the phase information of the peak corresponding to 2n0k d after FFT by the coefficient 2n0k c , and the value of δ can be obtained.

[0066] Based on the above-described analytical principle of the self-interference signal, the thickness change (δ) of the solid-phase gel will cause a change in the phase (2n0k(d + δ)) of the self-interference signal. Correspondingly, by detecting the phase change of the self-interference signal, it can be determined whether the thickness of the solid-phase gel has changed. Correspondingly, in an exemplary example, step 307 may further include step 307A and step 307B.

[0067] Step 307A, detecting the self-interference signal of the solid-phase gel in the solution after the cutting reaction through an optical detection system to obtain the target interference signal.

[0068] Step 307B, if the phase of the target interference signal changes, determine the thickness change of the solid-phase gel through the optical detection system.

[0069] In a possible implementation, the white light LED is collimated and then irradiated on the surface of the solid-phase gel in the solution through the beam splitter and objective lens of the optical detection system. The reflected interference signal is converged by the imaging lens onto the end face of the multimode optical fiber through the beam splitter. The optical fiber is externally connected to a spectrometer and then connected to a computer. The target interference signal is separated by FFT, and the phase of the target interference signal is analyzed to determine whether the phase of the target interference signal changes. If the phase of the target interference signal changes, it is determined that the thickness of the solid-phase gel has changed. On the contrary, if the phase of the target interference signal does not change, it is determined that the thickness of the solid-phase gel has not changed.

[0070] Due to the influence of the scattering medium in the solution, the optical detection system initially receives a composite beam, which is composed of an interference signal and a scattering signal. Therefore, before analyzing the interference signal, it is necessary to separate the interference signal from it. In a possible implementation, after the self-interference signal of the solid-phase gel in the solution after the cleavage reaction is detected by the optical detection system and the composite reflection signal (i.e., the composite beam) in the microfluidic chip is obtained, considering that the interference signal and the scattering signal in the solution have different characteristics in the frequency domain, the target interference signal can be separated from the composite reflection signal in the microfluidic chip by fast Fourier transform (FFT). Specifically, through FFT, the spatial domain information of the reflection spectrum can be converted into frequency domain information. The low-frequency part corresponds to the interference signal, and the high-frequency part corresponds to the scattering signal in the solution. By extracting the low-frequency peak from the FFT conversion result, the target interference signal can be extracted.

[0071] Step 308, if the thickness of the solid-phase gel changes, output the nucleic acid detection result of the nucleic acid sequence to be detected in the sample to be detected through the optical detection system.

[0072] If the sample to be detected contains the nucleic acid sequence to be detected, after nested RPA amplification, the nucleic acid concentration of the nucleic acid sequence to be detected can be increased. After the CRISPR reagent recognition and cleavage reaction, the physical thickness of the solid-phase gel can be reduced, resulting in a decrease in the phase of the self-interference signal. Therefore, by analyzing whether the phase of the self-interference signal changes through the optical detection system, it can be determined whether the thickness of the solid-phase gel has changed (i.e., whether the thickness has decreased); if the thickness of the solid-phase gel has changed, it can be determined that the sample to be detected contains the nucleic acid sequence to be detected, and the nucleic acid detection result of the nucleic acid sequence to be detected in the sample to be detected is output accordingly.

[0073] Conversely, if the sample to be tested does not contain the nucleic acid sequence to be tested, nested RPA amplification will not increase the nucleic acid concentration of the nucleic acid sequence to be tested, nor will there be subsequent recognition and cleavage reactions. The physical thickness of the solid-phase gel will not change, and the phase of the self-interference signal detected by the optical detection system will not change. Therefore, if the thickness of the solid-phase gel does not change, it can be determined that the sample to be tested does not contain the nucleic acid sequence to be tested.

[0074] Step 309, if the sample to be tested contains the nucleic acid sequence to be tested, analyze the phase change value of the target interference signal through the optical detection system.

[0075] Step 310, based on the phase change value through the optical detection system, determine and output the nucleic acid concentration of the nucleic acid sequence to be tested.

[0076] In addition to being able to output the nucleic acid detection result of the nucleic acid sequence to be tested (i.e., whether the sample to be tested contains the nucleic acid sequence to be tested), after the sample to be tested contains the nucleic acid sequence to be tested, the thickness (δ) of the solid-phase gel and the nucleic acid concentration (C) of the nucleic acid sequence to be tested can also be determined by analyzing the phase change value of the target interference signal.

[0077] In an exemplary example, the method for analyzing the thickness of the solid-phase gel may include the following steps:

[0078] I. Analyze the interference signal after FFT: After FFT conversion, the frequency-domain representation of the interference term is:

[0079] 2n0k0(d + δ) = 2n0k0d + 2n0k0cδ

[0080] Where: c is the average wave number of the spectrum; n0 is the average refractive index of silica and DNA gel, d represents the thickness of the silica layer, δ represents the thickness of the solid-phase gel, and k0 represents the wave number corresponding to the low-frequency peak.

[0081] II. Extract phase information: Extract the phase information of the low-frequency peak from the FFT result to obtain the phase of the interference peak. Assuming the phase of this peak is Φ(k0), then δ can be calculated through the following steps:

[0082] (1) Calculate the phase information of the interference term: Φ(k0) = 2n0k0(d + δ)

[0083] (2) Solve for δ: By comparing with the known reflectivity spectrum R0(k) of the standard silicon wafer, we get:

[0084]

[0085] Where, Phase ofΔR(k)peak atk0 represents 2n0k after performing FFT on the standard silicon wafer reflectivity spectrum R0(k) - R1(k)d Phase information of the corresponding peak.

[0086] There is a direct linear relationship between the thickness change of the solid-phase gel and the nucleic acid concentration of the nucleic acid sequence to be measured. As the nucleic acid concentration of the nucleic acid sequence to be measured increases, the amplification products increase, resulting in an increase in the CRISPR / Cas12a reaction products, thereby causing a thickness change in the solid-phase gel. By performing Fourier analysis on the interference signal, the thickness change of the solid-phase gel can be accurately resolved, and then the quantitative detection of the nucleic acid concentration of the nucleic acid sequence to be measured can be achieved. In another exemplary example, the method for analyzing the nucleic acid concentration of the nucleic acid sequence to be measured may include the following steps:

[0087] I. Analyze the relationship between the thickness of the solid-phase gel and the amplification products: Assume that there is a linear relationship between the thickness change δ of the solid-phase gel and the nucleic acid concentration c of the nucleic acid sequence to be measured. That is:

[0088] δ = αC + β (1)

[0089] Where: δ is the thickness change of the solid-phase gel layer, C is the nucleic acid concentration of the nucleic acid sequence to be measured, α is a coefficient related to the reaction efficiency and gel characteristics, representing the ratio of the thickness change of the solid-phase gel caused by the change in the nucleic acid concentration of the nucleic acid sequence to be measured; β is a constant, representing the baseline of the solid-phase gel thickness at zero nucleic acid concentration. In practical applications, it is usually assumed that β = 0 because there is no thickness change in the solid-phase gel at zero nucleic acid concentration.

[0090] II. Analyze the relationship between the interference signal and the gel thickness: The reflection intensity I(k) is expressed as: I(k) = |U(k)| 2 ≈ A1(k) 2 + A2(k) 2 + A3(k) 2 + 2A2(k)A3(k)cos(2n0k(d + δ)) + 2A1(k)A2(k)cos(2n0k(d + δ) + 2n w kz), where the influence of the solid-phase gel thickness change on the interference signal is reflected by the phase term 2n0k(d + δ), and δ in the phase term will directly affect the intensity of the interference signal. Correspondingly, by analyzing the phase change of the interference signal, that is, the influence of the solid-phase gel thickness change δ on the interference signal, it can be expressed as:

[0091] Δφ = 2n0kδ (2)

[0092] Where, Δφ is the phase change caused by the gel thickness change.

[0093] III. Analyze the relationship between the solid-phase gel thickness and the nucleic acid concentration of the nucleic acid sequence to be measured: Based on formula (1) and formula (2), a linear relationship - formula (3) can be obtained:

[0094] Δφ = 2n0k(αC) (3)

[0095] Simplification can yield formula (4):

[0096] Δφ = γC (4)

[0097] Where γ = 2n0kα is a constant related to experimental settings (such as wavelength λ, refractive index n0, etc.) and the characteristics of the solid-phase gel.

[0098] IV. Analyze the quantitative relationship between the concentration of the nucleic acid to be detected and the change in gel thickness (or phase change): By performing linear regression on the experimental data, the value of γ can be further determined, and a specific linear relationship can be obtained. By measuring the reflection signal I(k) or the phase change Δφ at different nucleic acid concentrations C, the quantitative relationship between the nucleic acid concentration of the nucleic acid sequence to be detected and the change in the thickness (or phase change) of the solid-phase gel layer can be obtained. Therefore, the final linear relationship formula is as shown in formula (5):

[0099]

[0100] Where δ represents the change in the thickness of the solid-phase gel layer, n0 is the average refractive index of silica and the DNA gel, k represents the wave number, γ = 2n0kα is a constant value, and C represents the nucleic acid concentration of the nucleic acid sequence to be detected.

[0101] In this embodiment, by analyzing the detected interference signal through an optical detection system, it is possible to determine whether the thickness of the solid-phase gel changes based on whether the phase change of the interference signal changes, and further determine whether the sample to be detected contains the nucleic acid sequence to be detected based on whether the thickness of the solid-phase gel changes. Non-labeled nucleic acid detection is achieved. Moreover, by analyzing the phase change value, the change value of the thickness of the solid-phase gel and the nucleic acid concentration of the nucleic acid sequence to be detected can be obtained, realizing non-labeled nucleic acid concentration detection.

[0102] Please refer to Figure 7 , which is a schematic structural diagram of a microfluidic detection platform provided by an embodiment of the present application. Exemplarily, as Figure 7 shown, the microfluidic detection platform 700 includes a digital microfluidic chip 701 and an optical detection system 702. The digital microfluidic chip 701 includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and a lysis solution, magnetic beads, a magnetic bead cleaning solution, a nucleic acid elution solution, a first-round RPA reagent, a second-round RPA reagent, and a CRISPR reagent are stored in the lower chip. The digital microfluidic chip 701 is placed on the stage of the optical detection system 702.

[0103] The digital microfluidic chip 701 is used to add a sample to be tested into the lower-layer chip, and then sequentially mix the sample to be tested with the lysis solution and magnetic beads, the magnetic bead cleaning solution, and the nucleic acid elution solution, so as to extract a sample nucleic acid droplet from the sample to be tested;

[0104] The digital microfluidic chip 701 is further used to mix the sample nucleic acid droplet with the first-round RPA reagent and the second-round RPA reagent for two rounds of RPA amplification to obtain a sample product droplet containing nucleic acid amplification products of two rounds;

[0105] The digital microfluidic chip 701 is further used to mix the sample product droplet with the CRISPR reagent, and move it to the solid-phase gel for sequence recognition and cleavage reaction to obtain a solid-phase gel after the cleavage reaction;

[0106] The optical detection system 702 is used to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction, and determine and output a nucleic acid detection result on whether the sample to be tested contains a nucleic acid sequence to be tested.

[0107] Optionally, the optical detection system 702 is further used for:

[0108] Performing self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to determine whether the thickness of the solid-phase gel changes;

[0109] If the thickness of the solid-phase gel changes, output the nucleic acid detection result that the sample to be tested contains the nucleic acid sequence to be tested.

[0110] Optionally, the optical detection system 702 is further used for:

[0111] Performing self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to obtain a target interference signal;

[0112] If the phase of the target interference signal changes, determine that the thickness of the solid-phase gel changes.

[0113] Optionally, the optical detection system 702 is further used for:

[0114] Performing self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to obtain a synthetic reflection signal;

[0115] Performing a fast Fourier transform on the synthetic reflection signal to separate the target interference signal from the synthetic reflection signal in the microfluidic chip.

[0116] Optionally, the optical detection system 702 is further used for:

[0117] If the sample to be tested contains the nucleic acid sequence to be tested, analyze the phase change value of the target interference signal;

[0118] Based on the phase change value, determine and output the nucleic acid concentration of the nucleic acid sequence to be tested.

[0119] Optionally, the lower chip includes a sample loading electrode, a waste liquid storage electrode, an electrode array, and a plurality of liquid storage electrodes. The plurality of liquid storage electrodes are respectively used to store the lysis solution and magnetic beads, the magnetic bead washing solution, the nucleic acid elution solution, the first round of RPA reagent, the second round of RPA reagent, and the CRISPR reagent. The sample loading electrode is used to store the sample to be tested, and the electrode array is used to control the movement of the sample to be tested, the sample nucleic acid droplet, and the sample product droplet.

[0120] Optionally, the digital microfluidic chip 701 is further configured to:

[0121] Mix the sample to be tested with the lysis solution and magnetic beads to release the sample nucleic acid in the sample to be tested, and the sample nucleic acid is enriched on the magnetic beads;

[0122] Mix the magnetic beads with the magnetic bead washing solution for magnetic bead washing;

[0123] Mix the washed magnetic beads with the nucleic acid elution solution to obtain the sample nucleic acid droplet;

[0124] The digital microfluidic chip 701 is further configured to:

[0125] Mix the first round of RPA reagent with the sample nucleic acid droplet to obtain a first-round amplification product;

[0126] Mix the second round of RPA reagent with the first-round amplification product to obtain the sample product droplet containing the nucleic acid amplification products of two rounds.

[0127] In summary, the embodiment of the present application provides a nucleic acid detection method based on gel self-interference: by integrating an optical detection system and a digital microfluidic chip, a solid-phase gel is embedded in the digital microfluidic chip as a sensing unit for the nucleic acid sequence to be tested; so that in the nucleic acid detection process, exponential amplification of the nucleic acid sequence to be tested is achieved through nested RPA amplification, and Cas12a is combined to cut the solid-phase gel to realize judging whether the sample to be tested contains the nucleic acid sequence to be tested by detecting the self-interference signal of the solid-phase gel. While achieving high-sensitivity nucleic acid detection, a high-specificity non-labeled PRA detection general method is also realized. In addition, a new automated microfluidic detection platform is constructed by integrating the optical detection system and the digital microfluidic chip, further improving the automation and integration of the nucleic acid detection process.

[0128] An exemplary embodiment of the present application further provides a nucleic acid detection device, including: at least one processor; and a memory communicatively connected to the at least one processor. The memory stores a computer program executable by the at least one processor, and when the computer program is executed by the at least one processor, it is used to cause the nucleic acid detection device to execute the nucleic acid detection method based on gel self-interference according to the embodiment of the present application.

[0129] It should be noted that, in addition to the processor and the memory, the nucleic acid detection device may further include a digital microfluidic chip and an optical detection system.

[0130] An exemplary embodiment of the present application further provides a non-transitory computer-readable storage medium storing a computer program, wherein when the computer program is executed by a processor of a nucleic acid detection device, it is used to cause the computer to execute the nucleic acid detection method based on gel self-interference according to the embodiment of the present application.

[0131] An exemplary embodiment of the present application further provides a computer program product, including a computer program, wherein when the computer program is executed by a processor of a computer, it is used to cause the computer to execute the nucleic acid detection method based on gel self-interference according to the embodiment of the present application.

Claims

1. A nucleic acid detection method based on gel self-interference, characterized in that, The method is applied to a microfluidic detection platform, which includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper chip and a lower chip. A solid-phase gel is embedded in the upper chip, and a lysis solution, magnetic beads, a magnetic bead washing solution, a nucleic acid elution solution, a first-round RPA reagent, a second-round RPA reagent, and a CRISPR reagent are stored in the lower chip. The digital microfluidic chip is placed on the stage of the optical detection system. The method includes: After adding a sample to be tested into the lower chip, the digital microfluidic chip is used to sequentially mix the sample to be tested with the lysis solution and magnetic beads, the magnetic bead washing solution, and the nucleic acid elution solution, so as to extract a sample nucleic acid droplet from the sample to be tested; The digital microfluidic chip is used to mix the sample nucleic acid droplet with the first-round RPA reagent and the second-round RPA reagent for two rounds of RPA amplification to obtain a sample product droplet containing nucleic acid amplification products of two rounds; The digital microfluidic chip is used to mix the sample product droplet with the CRISPR reagent and move it to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction; The optical detection system is used to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction, and determine and output a nucleic acid detection result on whether the sample to be tested contains a nucleic acid sequence to be tested; 2. The method according to claim 1, wherein The step of using the optical detection system to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction, and determine and output a nucleic acid detection result on whether the sample to be tested contains a nucleic acid sequence to be tested includes: The optical detection system is used to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to determine whether the thickness of the solid-phase gel has changed; If the thickness of the solid-phase gel has changed, the optical detection system outputs the nucleic acid detection result that the sample to be tested contains the nucleic acid sequence to be tested; 3. The method according to claim 2, wherein The step of using the optical detection system to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to determine whether the thickness of the solid-phase gel has changed includes: The optical detection system is used to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to obtain a target interference signal; If the phase of the target interference signal has changed, the optical detection system determines that the thickness of the solid-phase gel has changed; 4. The method according to claim 3, wherein The step of using the optical detection system to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to obtain a target interference signal includes: The optical detection system is used to perform self-interference signal detection on the solid-phase gel in the solution after the cleavage reaction to obtain a synthetic reflection signal; The optical detection system performs a fast Fourier transform on the synthetic reflection signal to separate the target interference signal from the synthetic reflection signal in the microfluidic chip; 5. The method according to claim 3, characterized in that, The method further includes: If the sample to be tested contains the nucleic acid sequence to be tested, the optical detection system analyzes the phase change value of the target interference signal; Based on the phase change value, the optical detection system determines and outputs the nucleic acid concentration of the nucleic acid sequence to be measured.

6. The method according to any one of claims 1 to 5, characterized in that, The lower-layer chip includes a sample-adding electrode, a waste liquid storage electrode, an electrode array, and multiple liquid storage electrodes. The multiple liquid storage electrodes are respectively used to store the lysis solution and magnetic beads, the magnetic bead cleaning solution, the nucleic acid elution solution, the first-round RPA reagent, the second-round RPA reagent, and the CRISPR reagent. The sample-adding electrode is used to store the sample to be measured, and the electrode array is used to control the movement of the sample to be measured, the sample nucleic acid droplet, and the sample product droplet.

7. The method according to claim 6, wherein The step of mixing the sample to be measured with the lysis solution and magnetic beads, the magnetic bead cleaning solution, and the nucleic acid elution solution in sequence by the digital microfluidic chip to extract a sample nucleic acid droplet from the sample to be measured includes: Mixing the sample to be measured and the lysis solution and magnetic beads by the electrode array to release the sample nucleic acid in the sample to be measured, and the sample nucleic acid is enriched on the magnetic beads; Mixing the magnetic beads and the magnetic bead cleaning solution by the electrode array for magnetic bead cleaning; Mixing the cleaned magnetic beads and the nucleic acid elution solution by the electrode array to obtain the sample nucleic acid droplet; The step of mixing the sample nucleic acid droplet with the first-round RPA reagent and the second-round RPA reagent by the digital microfluidic chip for two-round RPA amplification to obtain a sample product droplet containing two-round nucleic acid amplification products includes: Mixing the first-round RPA reagent and the sample nucleic acid droplet by the electrode array to obtain a first-round amplification product; Mixing the second-round RPA reagent and the first-round amplification product by the electrode array to obtain the sample product droplet containing two-round nucleic acid amplification products.

8. A microfluidic detection platform, characterized in that, The microfluidic detection platform includes a digital microfluidic chip and an optical detection system. The digital microfluidic chip includes an upper-layer chip and a lower-layer chip. A solid-phase gel is embedded in the upper-layer chip. The lower-layer chip stores the lysis solution and magnetic beads, the magnetic bead cleaning solution, the nucleic acid elution solution, the first-round RPA reagent, the second-round RPA reagent, and the CRISPR reagent. The digital microfluidic chip is placed on the stage of the optical detection system; The digital microfluidic chip is used to, after adding the sample to be measured into the lower-layer chip, mix the sample to be measured with the lysis solution and magnetic beads, the magnetic bead cleaning solution, and the nucleic acid elution solution in sequence to extract a sample nucleic acid droplet from the sample to be measured; The digital microfluidic chip is further used to mix the sample nucleic acid droplet with the first-round RPA reagent and the second-round RPA reagent for two-round RPA amplification to obtain a sample product droplet containing two-round nucleic acid amplification products; The digital microfluidic chip is further used to mix the sample product droplet with the CRISPR reagent and move it to the solid-phase gel for sequence recognition and cleavage reaction to obtain the solid-phase gel after the cleavage reaction; The optical detection system is used to detect the self-interference signal of the solid-phase gel after the cleavage reaction, and determine and output the nucleic acid detection result of whether the sample to be tested contains the nucleic acid sequence to be detected.

9. A nucleic acid detection device, comprising: a processor; and a memory storing a program, wherein the program includes instructions that, when executed by the processor, cause the processor to execute the nucleic acid detection method based on gel self-interference according to any one of claims 1-7.

10. A non-transitory computer-readable storage medium storing computer instructions, wherein, The computer instructions are used to cause the computer to execute the nucleic acid detection method based on gel self-interference according to any one of claims 1-7.

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