Method for regulating compatibility of hcr and cas12a based on gradient magnetic force and application thereof

Abstract

The application belongs to the technical field of detection, and discloses a method for regulating compatibility of HCR and Cas12a based on gradient magnetic force and application thereof. The application provides a nucleic acid detection kit containing a large magnetic bead-RNP conjugate and a small magnetic bead-FAM-ssDNA-bio probe complex. The nucleic acid detection kit can realize the compatibility of nucleic acid isothermal amplification and CRISPR technology through a two-step spatial isolation strategy of 'large magnetic bead sedimentation-small magnetic bead covering', and further achieve the purpose of accurate, high-sensitivity and high-specificity nucleic acid detection. Further, the components in the nucleic acid detection kit provided by the application are pre-packaged in a single reaction tube / microfluidic chip channel, and only the nucleic acid sample to be detected needs to be added during detection, without the need for opening operation. The 'amplification-isolation-detection' whole process can be completed through gradient magnetic force regulation, and the detection of nucleic acid is simply and quickly realized.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a method for HCR and Cas12a compatibility based on gradient magnetic force modulation and its application. Background Technology

[0002] The CRISPR-Cas system, with its unique target recognition mechanism and highly efficient trans-cleavage activity, has become a core technology in the field of point-of-care testing (POCT). Integrating isothermal amplification of nucleic acids with CRISPR technology can synergistically combine the high-sensitivity pre-amplification of isothermal amplification with the high-specificity recognition and signal amplification of CRISPR, developing nucleic acid detection methods that significantly improve the detection limit, reduce false positives, simplify the operation process, and adapt to the needs of rapid on-site testing. Currently, most mainstream detection schemes couple Cas12a with isothermal amplification technologies (such as RPA and LAMP) to achieve high-sensitivity and high-specificity detection. However, there is an inherent reaction compatibility bottleneck between the two: Cas12a can be activated by template nucleic acid or early amplicons in the early stages of amplification, leading to cis-cleavage of the template and amplification failure, while trans-cleavage of amplification primers significantly reduces amplification efficiency.

[0003] To address the aforementioned issues, existing technologies primarily employ strategies such as physical isolation, component optimization, or Cas protein / crRNA modification to achieve single-tube detection. For instance, microfluidic chips separate amplification and enzymatic digestion reactions through multi-chamber design; however, this technology originates from patent literature in the field of microfluidic detection and suffers from drawbacks such as complex consumables and high costs. While photocontrolled CRISPR technology utilizes caged crRNA or photolytic groups to block Cas activity, achieving time-series regulation, it requires additional illumination equipment, and the reagent modification process is cumbersome and its stability is limited; related technologies can be found in some journal articles. Summary of the Invention

[0004] To address the poor reaction compatibility of existing technologies when CRISPR-Cas is used in conjunction with nucleic acid isothermal amplification, as well as the shortcomings of existing solutions such as complex consumables, high cost, need for additional equipment, and insufficient reagent stability, the technical problem to be solved by this invention is to develop a simple, highly compatible, low-cost, and highly stable one-tube nucleic acid detection method that can achieve compatibility between isothermal amplification and CRISPR detection, thereby promoting its clinical translation and POCT application.

[0005] The first objective of this invention is to provide a nucleic acid detection kit.

[0006] The second aspect of the present invention is to provide the application of the nucleic acid detection kit of the first aspect of the present invention in detecting nucleic acids or preparing products for detecting nucleic acids.

[0007] A third aspect of the present invention is to provide a method for detecting nucleic acids.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a nucleic acid detection kit comprising a large magnetic bead-RNP conjugate and a small magnetic bead-FAM-ssDNA-bio probe complex; The large magnetic bead-RNP conjugate comprises magnetic beads with a particle size of 1000-8000 nm, crRNA attached to the surface of the magnetic beads, and Cas protein bound to the crRNA; The small magnetic bead-FAM-ssDNA-bio probe complex includes magnetic beads with a particle size of 100-700 nm and a signal reporting probe attached to the surface of the magnetic beads. The crRNA includes an anchor sequence and a guide sequence. The anchor sequence can bind to the Cas protein, and the guide sequence is specifically complementary to the nucleic acid sequence to be tested.

[0009] In some embodiments of the present invention, the particle size of the large magnetic beads is 1000-6000 nm, such as any value or a range formed by any combination of 1000, 2000, 3000, 4000, 5000 or 6000 nm.

[0010] In some embodiments of the present invention, the particle size of the small magnetic beads is 100-500 nm, such as any value of 100, 200, 300, 400 or 500 nm or a range formed by any two of them.

[0011] In some embodiments of the invention, the connection includes the use of specific binding of avidin and biotin.

[0012] In some embodiments of the present invention, the surface of the magnetic beads is modified with an affinity element.

[0013] In some embodiments of the present invention, the magnetic beads are superparamagnetic affinity-modified magnetic beads.

[0014] Superparamagnetic avidin-modified magnetic beads were selected as carriers, and two sizes were screened based on differences in magnetic response: large magnetic beads (1000nm-6000nm in diameter) were used to load Cas proteins (such as Cas12a) to ensure strong magnetic responsiveness and achieve rapid sedimentation; small magnetic beads (100-500nm in diameter) were used to modify signal reporter probes (fluorescent reporter molecules plus single-stranded nucleic acid probes), which have excellent dispersibility and large specific surface area, enabling high loading. Both types of magnetic beads exhibit good physicochemical stability and low non-specific adsorption characteristics after nucleic acid modification, and the avidin on the surface can achieve specific and efficient coupling with biotin-modified biomolecules.

[0015] In some embodiments of the present invention, the crRNA and the signal reporter probe are modified with biotin.

[0016] In some embodiments of the present invention, the biotin is modified at the 5' end of the crRNA.

[0017] In some embodiments of the present invention, the biotin is modified at the 5' end of the signal reporting probe.

[0018] In some embodiments of the present invention, the 3' end of the signal reporting probe is modified with a fluorescent gene.

[0019] In some embodiments of the present invention, the large magnetic bead-RNP conjugate is prepared by a method comprising the following steps: Avidin-modified magnetic beads were mixed with a solution containing biotin-modified crRNA and reacted to obtain crRNA-modified magnetic beads. The crRNA-modified magnetic beads were mixed with Cas protein and reacted again to obtain the large magnetic bead-RNP conjugate.

[0020] In some preferred embodiments of the present invention, the reaction conditions are: 35-39°C (such as any value of 35, 36, 37, 38, or 39°C, or a range formed by both) with shaking incubation for 20-40 min (such as any value of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 min, or a range formed by both). The crRNA is firmly immobilized on the surface of magnetic beads by utilizing the specific binding of biotin and avidin.

[0021] In some preferred embodiments of the invention, the re-reaction is performed under the following conditions: 35-39°C (e.g., any value of 35, 36, 37, 38, or 39°C, or a range thereof) with shaking incubation for 20-30 min (e.g., any value of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 min, or a range thereof). This allows the Cas protein to specifically bind to the crRNA immobilized on the magnetic bead surface, forming a complex and trapping it on the magnetic bead surface.

[0022] In some preferred embodiments of the present invention, the reaction system for preparing the crRNA-modified magnetic beads contains 0.01-0.1 mg / μL of avidin-modified magnetic beads, such as any value or range of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 mg / μL.

[0023] In some preferred embodiments of the present invention, the reaction system for preparing the crRNA-modified magnetic beads contains 5-15 μM of biotin-modified crRNA, such as any value or range of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μM.

[0024] In some preferred embodiments of the present invention, the magnetic beads, which are surface-modified with avidin, are washed to remove impurities from the surface of the magnetic beads before mixing with a solution containing biotin-modified crRNA. The washing solution is Tris buffer.

[0025] In some preferred embodiments of the present invention, the crRNA-modified magnetic beads are purified before being mixed with Cas protein, including washing with Tris buffer to remove unbound free crRNA.

[0026] In some preferred embodiments of the present invention, in the mixture of crRNA-modified magnetic beads and Cas protein, the concentration of the Cas protein is 5-15 μM, such as any value or range formed by any combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μM.

[0027] In some preferred embodiments of the present invention, the preparation method further includes a purification step, comprising washing the large magnetic bead-RNP conjugate with Tris buffer to remove unbound Cas protein.

[0028] In some preferred embodiments of the present invention, the large magnetic bead-RNP conjugate is resuspended in 1-10 mM appropriate buffer (low salt type), the magnetic bead concentration is adjusted to any concentration between 1-10 mg / mL, and it is sealed and stored at 2-8℃ for later use.

[0029] In some embodiments of the present invention, the small magnetic bead-FAM-ssDNA-bio probe complex is prepared by a method comprising the following steps: Avidin-modified magnetic beads are mixed with a solution containing a biotin-modified signal reporter probe, and the reaction yields a small magnetic bead-FAM-ssDNA-bio probe complex.

[0030] In some preferred embodiments of the present invention, the reaction conditions are: 35-39°C (such as any value of 35, 36, 37, 38, or 39°C, or a range formed by both) with shaking incubation for 20-40 min (such as any value of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 min, or a range formed by both). The signal reporting probe is firmly fixed to the surface of the small magnetic bead by utilizing the specific binding of biotin and avidin.

[0031] In some embodiments of the present invention, the Cas protein is selected from at least one of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas13a, Cas13b, Cas13c, Cas13d, Cas13X / Cas13Y, or Cas13j.

[0032] In some embodiments of the present invention, the nucleic acid detection kit further includes reagents for HCR amplification.

[0033] In some embodiments of the present invention, the reagents used for HCR amplification include DNA hairpin probes and amplification buffer.

[0034] In some embodiments of the present invention, the amplification buffer includes MgCl2, NaCl, and Tris-HCl.

[0035] In some embodiments of the present invention, the amplification buffer comprises 8-15 mM MgCl2 and 150-250 mM NaCl and 8-15 mM Tris-HCl (pH=7.4).

[0036] In some embodiments of the present invention, the crRNA and DNA hairpin probes can be adaptively designed according to different detection targets. Corresponding crRNA and DNA hairpin probes are designed according to conventional knowledge in the art.

[0037] In some embodiments of the present invention, when the nucleic acid to be tested is PRRSV (SEQ ID NO:1), the nucleotide sequence of the crRNA is as shown in SEQ ID NO:2.

[0038] In some embodiments of the present invention, when the nucleic acid to be tested is PRRSV, the DNA hairpin probe includes DNA hairpin probe H1a with nucleotide sequence as shown in SEQ ID NO:3, DNA hairpin probe Ha1 with nucleotide sequence as shown in SEQ ID NO:4, and DNA hairpin probe Ha2 with nucleotide sequence as shown in SEQ ID NO:5.

[0039] In some embodiments of the present invention, when the nucleic acid to be tested is PRRSV, the nucleotide sequence of the signal reporting probe is 5'-TATTATT-3'.

[0040] In some embodiments of the present invention, the kit further includes a reaction solution.

[0041] In some embodiments of the present invention, the reaction solution is a phosphate buffer (pH 7.2) with 0.01%-0.1% Tween 20 added, and the concentration of the phosphate buffer is 10-200 mM.

[0042] A 10mM-200mM phosphate buffer solution (pH 7.0-8.5) was used as the reaction solution, with 0.01%-0.1% Tween 20 added to improve the dispersibility of the magnetic beads. This also ensures compatibility with the amplification reaction and the activity requirements of Cas12a, avoiding interference between components. Fine-tuning within the above range can achieve similar results.

[0043] A second aspect of the present invention provides the application of the nucleic acid detection kit of the first aspect of the present invention in detecting nucleic acids or preparing products for detecting nucleic acids.

[0044] In some embodiments of the present invention, the application is for purposes other than disease diagnosis.

[0045] A third aspect of the present invention provides a method for detecting nucleic acids, comprising the steps of using a nucleic acid detection kit according to the first aspect of the present invention.

[0046] In some embodiments of the present invention, the method includes the following steps: Mix the large magnetic bead-RNP conjugate, the small magnetic bead-FAM-ssDNA-bio probe complex, the reagents for HCR amplification, and the nucleic acid of the sample to be tested. Apply a magnetic field of 15-22V and let stand for 4-10 min; apply a magnetic field of 23-30V and let stand for 1-4 min; react; remove the magnetic field and detect the fluorescence signal.

[0047] In some embodiments of the present invention, when a magnetic field with a voltage of 15-22V (such as any value of 15, 16, 17, 18, 19, 20, 21 or 22V or a range formed by both) is applied, the resting time is 4-8 min, such as any value of 4, 5, 6, 7 or 8 min or a range formed by both.

[0048] In some embodiments of the present invention, when a magnetic field with a voltage of 23-30V (such as any value of 23, 24, 25, 26, 27, 28, 29 or 30V or a range formed by both) is applied, the resting time is 1-4 min, such as any value of 1, 2, 3 or 4 min or a range formed by both.

[0049] In some embodiments of the present invention, the reaction conditions are 35-39°C for 10-30 min.

[0050] In some preferred embodiments of the present invention, the reaction conditions are 35-38°C (such as any value of 35, 36, 37 or 38°C or a range formed by both) and 15-25 min (such as any value of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 min or a range formed by both).

[0051] In some embodiments of the present invention, after the magnetic field is removed, a slight oscillation (about 10-15 min) is applied to fully disintegrate the bilayer magnetic bead structure, allowing Cas12a to bind to the amplification product and activate trans-cleavage activity.

[0052] In some embodiments of the present invention, a fluorescence detector / portable detection device is used to detect the fluorescence signal, with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0053] The method provided by this invention utilizes the difference in magnetic response between two-sized magnetic beads to achieve two-step spatial isolation and activity control through gradient adjustment of the external magnetic field strength, as detailed below: Step 1: Low-intensity magnetic sedimentation of large magnetic beads (Cas protein). In the initial detection stage, add the large magnetic bead-RNP conjugate, the small magnetic bead-FAM-ssDNA-bio probe complex, amplification reagent (HCR amplified DNA hairpin), and the nucleic acid template to be detected to the reaction tube. After mixing thoroughly, apply a low-intensity magnetic field (15-22V). This magnetic field strength range is crucial; within this range, rapid sedimentation of the large magnetic beads is achieved. Exceeding this range may result in incomplete sedimentation of the large magnetic beads or simultaneous sedimentation of the small magnetic beads. Due to their larger particle size and stronger magnetic responsiveness, the large magnetic beads are quickly adsorbed to the bottom of the reaction tube, forming a dense precipitate. The small magnetic beads, however, remain uniformly dispersed in the reaction system due to insufficient magnetic force to drive sedimentation.

[0054] Step 2: High-intensity magnetic force drives the small magnetic beads to cover and isolate the area. The magnetic field strength is increased to ensure rapid sedimentation of the small magnetic beads and the formation of a covering layer. At this point, the dispersed small magnetic beads are rapidly adsorbed, settling from top to bottom and covering the surface of the large magnetic bead deposit, forming a bilayer structure of "small magnetic bead-FAM-ssDNA-bio probe complex covering layer + large magnetic bead-RNP conjugate bottom layer". This covering layer physically isolates the Cas protein on the surface of the large magnetic beads from contact with the amplification products in the system. The Cas protein at the bottom is physically restricted, unable to contact the nucleic acid template and amplification products in the system, thus avoiding interference with the amplification reaction and ensuring efficient nucleic acid amplification. After the amplification reaction is complete (usually 15-25 minutes), the magnetic field is removed, the bilayer magnetic bead structure disintegrates, the Cas protein is released and binds to the amplification products, activating trans-cleavage activity, efficiently cleaving the FAM-ssDNA-bio probe (signal reporter probe) on the surface of the small magnetic beads, releasing a fluorescent signal, and the results are read by a fluorescence detector / portable detection device.

[0055] The beneficial effects of this invention are: This invention provides a nucleic acid detection kit containing a large magnetic bead-RNP conjugate and a small magnetic bead-FAM-ssDNA-bio probe complex. Using this nucleic acid detection kit, a two-step spatial isolation strategy of "large magnetic bead sedimentation-small magnetic bead coverage" can be adopted to achieve compatibility between nucleic acid isothermal amplification and CRISPR technology, thereby achieving the purpose of accurate, highly sensitive and highly specific nucleic acid detection.

[0056] Furthermore, the components of the nucleic acid detection kit provided by this invention (such as modified dual-size magnetic beads (large magnetic bead-RNP conjugate and small magnetic bead-FAM-ssDNA-bio probe complex), reagents for HCR amplification, reaction solution, and all other components) are pre-encapsulated in a single reaction tube / microfluidic chip channel. During detection, only the sample to be tested needs to be added, without opening the cap. The entire process of "amplification-isolation-detection" can be completed through gradient magnetic control, achieving nucleic acid detection simply and quickly.

[0057] This invention provides a method for detecting nucleic acids. This method utilizes the difference in magnetic response between two-sized magnetic beads and achieves two-step spatial isolation and activity regulation by adjusting the gradient of the external magnetic field strength. It has the advantages of simple operation, strong compatibility, low cost and high stability, and can realize one-tube nucleic acid detection compatible with isothermal amplification and CRISPR technology.

[0058] Specifically, the method provided by the present invention has the following beneficial effects: Significant technical effects and excellent detection performance: This invention completely solves the degradation interference of Cas proteins (such as Cas12a) on amplification templates and primers through a two-step spatial isolation strategy of "large magnetic bead sedimentation - small magnetic bead coverage".

[0059] Easy to operate and suitable for POCT scenarios: Abandoning traditional chemical modification or light control methods, it is the first to use gradient magnetic force to regulate the spatial distribution of dual-size magnetic beads, realizing the temporal control of Cas protein (such as Cas12a) activity. No special equipment is required (only a conventional fluorescence detector and magnetic field generator are needed). The operation steps are simple and can be completed without professional technicians, making it fully suitable for the application needs of POCT scenarios.

[0060] Low cost and highly practical: Compared to microfluidic chip technology, no custom consumables are required; compared to light-controlled CRISPR technology, no lighting equipment is required.

[0061] High compatibility and wide applicability: Based on the universal biotin / avidin conjugation technology, it can flexibly adapt to different target nucleic acids (such as pathogenic microorganisms, tumor nucleic acid markers, and nucleic acids related to hereditary diseases) by changing the biotin-modified crRNA with different sequences. It does not require complex system optimization for different targets, and has high versatility and wide applicability. Attached Figure Description

[0062] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is the verification result of the ability of the electromagnet to precisely control the magnetic force to achieve the separate sinking of large and small magnetic probes in Embodiment 2 of the present invention; wherein, A is the sinking of large and small magnetic probes under the applied voltage of 21V, and B is the sinking of large and small magnetic probes under the applied voltage of 24V.

[0063] Figure 2 This is the technical route of the Cas12a one-tube nucleic acid detection technology based on magnetic regulation of dual-size magnetic beads in Embodiment 3 of the present invention. 1. First, a low-intensity magnetic field is applied; the larger magnetic beads, with their stronger paramagnetism, are preferentially adsorbed to the bottom layer. 2. The magnetic field strength is increased, and the smaller magnetic beads are also adsorbed to the bottom layer. Simultaneously, a deposition layer is formed on the surface of the Cas protein-modified magnetic beads, creating a physical barrier that separates Cas12a from the hairpin required for HCR. 3. Amplification is performed by heating. 4. After amplification, the magnetic field is removed, causing the magnetic beads to disperse, and Cas12a begins cleavage. 5. After a specific time, a low-intensity magnetic field is applied again, stopping Cas protein cleavage and achieving controllable start and stop of activity. 6. The magnetic field strength is increased to excite and extract the fluorescence signal.

[0064] Figure 3 The results validate the feasibility of precisely magnetically controlling the on / off state of Cas12a enzyme activity. Detailed Implementation

[0065] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0066] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0067] Terminology Explanation: The CRISPR-Cas system, a system of clustered regularly spaced short palindromic repeats and their associated proteins, is an adaptive immune system of bacteria and archaea. It is now widely used in gene editing and nucleic acid detection, playing a role through its specific target recognition mechanism and highly efficient trans-cleavage activity.

[0068] Cas12a: A Cas protein in the CRISPR-Cas system, characterized by its ability to directly process precursor crRNA (pre-crRNA) and its strong trans-cleavage activity, making it a core functional protein in nucleic acid detection.

[0069] POCT: Point-of-Care Testing refers to rapid testing performed near the patient, providing quick results without the need for complex laboratory equipment or specialized operating environments.

[0070] Nucleic acid isothermal amplification technology: This technology achieves specific amplification of nucleic acid fragments under isothermal conditions. Common examples include RPA (recombinase polymerase amplification), LAMP (loop-mediated isothermal amplification), and HCR (hybridization chain reaction). It has advantages such as fast amplification speed and no need for a PCR instrument.

[0071] crRNA: CRISPR RNA is an RNA molecule in the CRISPR-Cas system responsible for recognizing target nucleic acids and guiding Cas proteins to specifically bind to and cleave target nucleic acids.

[0072] Trans-cleavage activity: After recognizing and binding to target nucleic acids (cis-cleavage), Cas proteins activate their cleavage activity on non-target nucleic acids, which is a key characteristic of CRISPR technology for nucleic acid detection.

[0073] FAM-ssDNA-bio probe: A nucleic acid probe with fluorescent and coupling groups modified at both ends. It can be coupled to the surface of magnetic beads of different sizes according to functional requirements. The magnetic beads can effectively quench the fluorescent group after coupling. After being cleaved by the trans-cleavage activity of Cas protein, it can release a fluorescent signal for reading the detection results.

[0074] Biotin / avidin coupling: a highly specific and high-affinity biomolecule binding mechanism. The binding constant between biotin and avidin is extremely high, and the resulting complex is stable. It is widely used in the fixation, capture and coupling reactions of biomolecules.

[0075] DNA hairpin probes: Probes with DNA hairpin structures in HCR can trigger a chain hybridization reaction in the presence of target nucleic acids, thereby amplifying nucleic acid signals.

[0076] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0077] Example 1: Synthesis method of dual-size magnetic nanoprobes (1) Raw material preparation: Large magnetic beads: superparamagnetic avidin-modified magnetic beads, 5000 nm in diameter, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., Cat. #22306-10; Small magnetic beads: Superparamagnetic avidin-modified magnetic beads, 300nm in diameter, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., Cat. #22308C-1; Cas12a protein: purity ≥95%, purchased from Shenzhen Yizhi Biotechnology Co., Ltd., catalog number CAS-12E-001; crRNA: The sequence is designed based on the target nucleic acid, modified with biotin, and synthesized by Sangon Biotech (Shanghai) Co., Ltd.; for example, when the target nucleic acid is PRRSV (5'-AGGCAAACAAGGAGTACCATTCCAACGGG-3', SEQ ID NO:1), the crRNA sequence is 5'-AAAUAAUUUCUACUAAGUGUAGAUCCUAGAGUUCCAUGUCAGUGU-3' (SEQ ID NO:2), with biotin modification at the 5' end of the crRNA; FAM-ssDNA-bio probe: A FAM-modified nucleic acid probe with a sequence designed to trans-cleave the Cas12a substrate. It is biotin-modified and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the FAM-ssDNA-bio probe is 5'-TATTATT-3', with biotin modified at the 5' end and FAM fluorescent group modified at the 3' end. Tris buffer, Tween 20, etc., were all of analytical grade and purchased from a regular chemical reagent supplier.

[0078] (2) Instrument preparation: Electromagnets, vortex shakers, thermostatic shakers, pipettes and tips, and suitable centrifuge tubes.

[0079] (3) Modification of magnetic bead function: Large magnetic bead-RNP conjugate coupling: A bottle containing 5000 nm superparamagnetic avidin-modified magnetic beads (hereinafter referred to as large magnetic beads) was placed on a vortex mixer and vortexed for 20 s to fully resuspend the beads. 100 μL of 10 mg / mL large magnetic beads (in 0.01 M PBS) was precisely pipetted into a new centrifuge tube. The centrifuge tube was placed on an electromagnet and allowed to stand for 1 min for magnetic separation. After complete adsorption of the beads, the supernatant was removed by pipetting, and the centrifuge tube was then removed from the electromagnet. 1 mL of Tris-HCl buffer was added, the centrifuge tube was capped, and the beads were thoroughly vortexed to resuspend them. Magnetic separation was performed again, and the supernatant was removed. This washing step was repeated 3 times to thoroughly remove impurities from the surface of the magnetic beads. After washing, add 20 μL of 10 μM biotin-modified crRNA (solvent: 0.001 M Tris-HCl buffer + 0.01 M NaCl) to the centrifuge tube, and thoroughly vortex to resuspend the magnetic beads, ensuring the concentration is suitable for the reaction system. Place the centrifuge tube on a constant-temperature shaker and incubate at 37°C for 30 min. Utilizing the specific binding of biotin and avidin, the crRNA is firmly immobilized onto the surface of the large magnetic beads. After incubation, place the centrifuge tube on an electromagnet for 1 min for magnetic separation, and transfer the supernatant to a new centrifuge tube. Then, add Tris-HCl buffer again to the magnetic beads, vortex to resuspend, perform magnetic separation, and aspirate the supernatant. Repeat the washing step three times to thoroughly remove unbound free crRNA. Add 50 μL of 5 μM Cas12a protein solution (using the diluent provided with CAS-12E-001) to the washed magnetic beads, vortex thoroughly to resuspend the beads, and incubate the centrifuge tube at 37°C for 20 min using a rotary mixer. This allows the Cas12a protein to specifically bind to the crRNA immobilized on the magnetic bead surface, forming an RNP complex that is then captured onto the bead surface. After incubation, wash the magnetic beads (repeat the Tris buffer washing procedure three times) to remove unbound Cas12a protein. Resuspend the beads in a suitable adaptation buffer (low-salt type, here 0.001 M Tris-HCl buffer) according to subsequent experimental requirements, adjusting the final concentration of the magnetic beads to 5 mg / mL to obtain the large magnetic bead-RNP conjugate. Store the conjugate at 4°C for later use.

[0080] (4) Coupling of the magnetic beads-FAM-ssDNA-bio probe complex: Place the bottle containing 300 nm superparamagnetic avidin-modified magnetic beads (hereinafter referred to as small magnetic beads) on a vortex mixer and shake for 20 s to fully resuspend the magnetic beads. Accurately transfer 100 μL of small magnetic beads with a concentration of 10 mg / mL (0.01 M Tris-HCl buffer) into a new centrifuge tube. Place the centrifuge tube on an electromagnet and let it stand for 1 min for magnetic separation. After the magnetic beads are completely adsorbed, remove the supernatant with a pipette and then remove the centrifuge tube from the electromagnet. Add 1 mL of Tris-HCl buffer (0.01 M), cap the centrifuge tube, shake well to resuspend the magnetic beads, perform magnetic separation again and remove the supernatant. Repeat this washing step 3 times to thoroughly remove impurities from the surface of the magnetic beads. After washing, add 20 μL of 10 μM biotin-modified FAM-ssDNA-bio probe to the centrifuge tube, and resuspend the magnetic beads by thorough shaking to ensure the concentration is suitable for the reaction system. Place the centrifuge tube on a constant temperature shaker and incubate at 37°C for 30 min. Utilize the specific binding of biotin and avidin to firmly immobilize the FAM-ssDNA-bio probe onto the surface of the small magnetic beads. After incubation, place the centrifuge tube on an electromagnet for 1 min for magnetic separation, and transfer the supernatant to a new centrifuge tube for later use. Then, add Tris buffer to the magnetic beads again, shake to resuspend, perform magnetic separation, and aspirate the supernatant. Repeat the washing step 3 times to thoroughly remove unbound free FAM-ssDNA-bio probe. According to the requirements of subsequent experiments, add a suitable adaptation buffer (low-salt type, 0.001 M Tris-HCl buffer in this case) to resuspend the magnetic beads, adjusting the final concentration of the magnetic beads to 5 mg / mL to obtain the small magnetic bead-FAM-ssDNA-bio probe complex, which is then sealed and stored at 4°C for later use.

[0081] Example 2: Verification of the ability of an electromagnet to precisely control magnetic force to achieve the separate sinking of magnetic probes of different sizes. Take two clean 1.5 mL enzyme-free centrifuge tubes and label them "Large Magnetic Bead" and "Small Magnetic Bead," respectively. Place each tube on a vortex mixer and vortex for 20 seconds to fully resuspend the beads. Use a pipette to transfer 1 μL of 10 mg / mL large magnetic beads into the "Large Magnetic Bead" tube; add 1 μL of small magnetic beads (with the same concentration as the large beads) into the "Small Magnetic Bead" tube. Add 99 μL of 0.01 M Tris-HCl buffer to each tube, cap them, and vortex for 10 seconds to evenly disperse the beads in the buffer.

[0082] Adjust the electromagnet to a stable state and set the output voltage to 21V (corresponding to a low-intensity magnetic field, capable of driving the large magnetic bead to settle without affecting the small magnetic bead). Fix the two centrifuge tubes, one with the large magnetic bead and the other with the small magnetic bead, parallel to each other within the magnetic field area corresponding to the electromagnet, ensuring both tubes are in the same magnetic field environment to avoid magnetic interference due to positional differences. Maintain a stable voltage, observe and record the settling of the magnetic beads in both centrifuge tubes for 1 minute.

[0083] The results are as follows Figure 1 As shown, significant magnetic bead sedimentation was observed in centrifuge tubes containing "large magnetic beads," while no magnetic bead sedimentation was observed in centrifuge tubes containing "small magnetic beads." Figure 1 (A). After completing the observation of the 21V voltage group, the input voltage of the electromagnet was further increased and adjusted to 24V (corresponding to a high-intensity magnetic field, which can simultaneously drive the sedimentation of large and small magnetic beads). After the voltage stabilized, it was observed that the sedimentation of the large magnetic bead in the centrifuge tube remained unchanged. At this time, obvious sedimentation of the small magnetic bead also appeared in the centrifuge tube. Figure 1 (B)

[0084] Example 3: Construction of a Cas12a One-Tube Nucleic Acid Detection Method Based on Magnetic Control of Dual-Size Magnetic Beads A single-tube nucleic acid detection method for Cas12a based on magnetic control of dual-size magnetic beads (see schematic diagram of the detection process). Figure 2 (As shown), including the following steps: (1) Preparation of reaction system: Add the following to the reaction tube in sequence: 2 μL of large magnetic bead-RNP conjugate (about 5 mg / mL, prepared in Example 1), 1.5 μL of small magnetic bead-FAM-ssDNA-bio probe complex (about 8 mg / mL, prepared in Example 1), 25 μL of HCR amplification reagent, and 20 μL of reaction buffer (100mM phosphate buffer (pH 7.2) with 0.05% Tween 20 added), mix well to obtain the reaction system; The HCR amplification reagent was an HCR reaction buffer containing 250 nM DNA hairpin probe H1a, 500 nM DNA hairpin probe Ha1, and 500 nM DNA hairpin probe Ha2 (a mixed solution of 10 mM MgCl2, 10 mM Tris-HCl (pH=7.4), and 200 mM NaCl, respectively). DNA hairpin primers are adaptively designed according to different detection targets. For example, when the detection target is PRRSV, The nucleotide sequence of the DNA hairpin probe H1a is: 5'-CCCGTTGGAATGGTACTCCTTGTTTGCCTAGAGTTCCATGTCAGTGTGGCGGCGTGGAACTCTAGGGTTTGTAGGAGTACCATTCC-3' (SEQ ID NO:3); The nucleotide sequence of the DNA hairpin probe Ha1 is: 5'-CCTAGAGTTCCATGTCAGTGTGGCGGCGTTAACCCGCCGCCACACTGACATGGAACTCTAGGCAAACA-3' (SEQ ID NO:4); The nucleotide sequence of the DNA hairpin probe Ha2 is: 5'-GGTTAACGCCGCCACACTGACATGGAACTCTAGGTGTTTGCCTAGAGTTCCATGTCAGTGTGGCGGCG-3' (SEQ ID NO:5); (2) Add 20 μL of the nucleic acid sample to be tested (such as the PRRSV gene simulation sample shown in SEQ ID NO:1, with a concentration of 1 mM) to the reaction system of step (1) and mix well; (3) Adjust the electromagnet to a stable state, apply a voltage of 21V to the electromagnet, and let it stand at room temperature for 5 min to allow the large magnetic bead-RNP conjugate to settle to the bottom of the tube; then apply a voltage of 24V to the electromagnet, and let it stand at room temperature for 3 min to allow the small magnetic bead-FAM-ssDNA-bio probe complex to settle and cover the surface of the large magnetic bead precipitate; place the reaction tube in a constant temperature environment of 37℃ and perform the amplification reaction for 20 min; (4) After the reaction is complete, remove the magnetic field and apply slight shaking for 10-15 min to fully disintegrate the double-layer magnetic bead structure, so that Cas12a binds to the amplification product and activates the trans-cleavage activity. (5) Use a fluorescence detector / portable detection device to detect the fluorescence signal, with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Record and analyze the fluorescence intensity values.

[0085] The HCR process described above works as follows: The conserved sequence first binds to H1a, causing it to open and exposing the trigger sequence located in the loop. The trigger sequence then binds to Ha1, exposing the sequence that binds to Ha2, which in turn opens Ha2. The opened Ha2 then triggers the opening of Ha1, and the cycle repeats until the amplification product is obtained. Both Ha1 and Ha2 contain the PAM sequence necessary for Cas12a recognition, and Cas12a also produces crRNA necessary for its cleavage activity.

[0086] Example 4: Feasibility verification of precise magnetic regulation of Cas12a enzyme activity on / off Take a 200 μL EP tube and use a pipette to add 5 μL of the nucleic acid sample to be tested and 15 μL of DNA hairpin probe pre-dissolved in reaction buffer to the experimental group as the positive group. Add 5 μL of deionized water and 15 μL of HCR hairpin probe pre-dissolved in reaction buffer to the experimental group as the negative group. Gently shake to mix the sample and DNA hairpin probe evenly. Continue to add pre-synthesized dual-size magnetic probes (i.e., add 2 μL of large magnetic bead-RNP conjugate (approximately 5 mg / mL) and 1.5 μL of small magnetic bead-FAM-ssDNA-bio probe complex (approximately 8 mg / mL)) to the EP tube. Fix the reaction tube in the electromagnet area and apply a low-intensity magnetic field (21 V) to maintain magnetic separation for 1 min (to allow the large magnetic beads to settle). Apply a high-intensity magnetic field (24 V) to maintain magnetic separation for 1 min (to cover and isolate with small magnetic beads). After amplification at 37℃ for 40 min, a portable device combined with a smartphone was used to take pictures (dark box UV analyzer, Shanghai Jiapeng Technology Co., Ltd., ZF-8) to detect the fluorescence intensity in the tube, and the fluorescence signal values ​​of the positive and negative groups during the "magnetic separation stage" were analyzed and recorded respectively.

[0087] The electromagnet was then turned off (corresponding to the magnetic field removal stage), and the reaction tube was placed on a vortex oscillator and gently oscillated for 5 seconds to disintegrate the double-layer magnetic bead structure and ensure uniform mixing of the system. After standing at 37°C for 15 minutes, the fluorescence signal value during the "magnetic field removal" stage was analyzed and recorded again using a portable device combined with a smartphone.

[0088] Finally, a low-intensity magnetic field (21 V) is reapplied, maintaining magnetic separation for 1 minute (allowing the large magnetic beads to settle). A high-intensity magnetic field (24 V) is then applied, maintaining magnetic separation for 1 minute (with small magnetic beads providing coverage and isolation). Immediately, fluorescence images of the tube are taken, and the fluorescence signal values ​​of the negative and positive groups during the "re-magnetic separation" phase are analyzed and recorded.

[0089] Finally, a low-intensity magnetic field (21 V) is reapplied, maintaining magnetic separation for 1 min (allowing the large magnetic beads to settle). A high-intensity magnetic field (24 V) is then applied, maintaining magnetic separation for 1 min (with small magnetic beads providing coverage and isolation). Immediately, fluorescent images of the tube are taken, and the fluorescence signal values ​​of the negative and positive groups during the "re-magnetic separation" phase are analyzed and recorded.

[0090] The above experiment was repeated three times to calculate the error, and the results are as follows: Figure 3As shown, the fluorescence signal value of the positive group during the magnetic separation stage was extremely low, with almost no significant difference from the negative group, indicating that Cas12a cleavage activity could be inhibited by magnetic separation. During the magnetic field removal stage, the fluorescence signal of the positive group increased significantly, while the increase in the negative group was not significant, showing a significant difference between the two groups (more than 10 times the difference in fluorescence signal value). This indicates that Cas12a cleavage activity could be activated by removing magnetic separation, and the target (PRRSV) could be correctly identified, demonstrating good detection performance. Finally, after re-magnetic separation, the fluorescence signal remained almost unchanged, indicating that the Cas12a cleavage reaction could be stopped by magnetic separation. This example verifies the feasibility of precisely controlling the on / off state of Cas12a enzyme activity using magnetic force. Furthermore, using the fluorescence signal from the "re-magnetic separation" stage as the final experimental result, the RSD of the three repeated experiments in the positive group was 2.66%, indicating good stability of the separation and detection protocol.

[0091] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nucleic acid detection kit, comprising a large magnetic bead-RNP conjugate and a small magnetic bead-FAM-ssDNA-bio probe complex; The large magnetic bead-RNP conjugate comprises magnetic beads with a particle size of 1000-8000 nm, crRNA attached to the surface of the magnetic beads, and Cas protein bound to the crRNA; The small magnetic bead-FAM-ssDNA-bio probe complex includes magnetic beads with a particle size of 100-700 nm and a signal reporting probe attached to the surface of the magnetic beads. The crRNA includes an anchor sequence and a guide sequence. The anchor sequence can bind to the Cas protein, and the guide sequence is specifically complementary to the nucleic acid sequence to be tested.

2. The nucleic acid detection kit according to claim 1, characterized in that, The connection includes the use of specific binding of avidin and biotin; Preferably, the surface of the magnetic beads is modified with an affinity element; Preferably, the crRNA and the signal reporter probe are modified with biotin; Preferably, the biotin is modified at the 5' end of the crRNA; Preferably, the biotin is modified at the 5' end of the signal reporting probe.

3. The nucleic acid detection kit according to claim 2, characterized in that, The large magnetic bead-RNP conjugate was prepared by the following method: Avidin-modified magnetic beads were mixed with a solution containing biotin-modified crRNA and reacted to obtain crRNA-modified magnetic beads. The crRNA-modified magnetic beads were mixed with Cas protein and reacted again to obtain the large magnetic bead-RNP conjugate. And / or, the small magnetic bead-FAM-ssDNA-bio probe complex is prepared by a method comprising the following: Avidin-modified magnetic beads are mixed with a solution containing a biotin-modified signal reporter probe, and the reaction yields a small magnetic bead-FAM-ssDNA-bio probe complex.

4. The nucleic acid detection kit according to any one of claims 1-3, characterized in that, The nucleic acid detection kit also includes reagents for HCR amplification.

5. The nucleic acid detection kit according to claim 4, characterized in that, The reagents used for HCR amplification include DNA hairpin probes and amplification buffer; Preferably, the amplification buffer comprises MgCl2, NaCl, and Tris-HCl.

6. The nucleic acid detection kit according to claim 5, characterized in that, When the nucleic acid to be tested is PRRSV, the nucleotide sequence of the crRNA is shown in SEQ ID NO:2; Preferably, the DNA hairpin probe includes DNA hairpin probe H1a with nucleotide sequence as shown in SEQ ID NO:3, DNA hairpin probe Ha1 with nucleotide sequence as shown in SEQ ID NO:4, and DNA hairpin probe Ha2 with nucleotide sequence as shown in SEQ ID NO:5; Preferably, the nucleotide sequence of the signal reporting probe is 5'-TATTATT-3'; Preferably, the 3' end of the signal reporting probe is modified with a fluorescent gene.

7. The use of the nucleic acid detection kit according to any one of claims 1-6 in detecting nucleic acids or preparing products for detecting nucleic acids.

8. A method for detecting nucleic acid, comprising the step of using the nucleic acid detection kit according to any one of claims 5-6.

9. The method according to claim 8, characterized in that, The method includes the following steps: Mix the large magnetic bead-RNP conjugate, the small magnetic bead-FAM-ssDNA-bio probe complex, the reagents for HCR amplification, and the nucleic acid of the sample to be tested. Apply a magnetic field of 15-22V and let stand for 4-10 min; apply a magnetic field of 23-30V and let stand for 1-4 min; react; remove the magnetic field and detect the fluorescence signal.

10. The method according to claim 9, characterized in that, The reaction conditions are 35-39℃ for 10-30 minutes.

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