A CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, a preparation method and application thereof

Abstract

The application belongs to the technical field of nucleic acid detection, and particularly relates to a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, a preparation method and application thereof, which comprises a DNA signal amplification reactor, a Cas13a recognition module and a Cas12a signal output module, the DNA signal amplification reactor is internally packaged with a fluorescently labeled probe and a Cas12a activation substrate; the Cas13a recognition module is used for triggering cutting of a connecting chain after recognizing a target RNA, and releasing the DNA signal amplification reactor; the Cas12a signal output module is used for recognizing the released DNA signal amplification reactor and generating a fluorescent signal, the Cas12a signal output module activates trans-cleavage activity after recognizing the released DNA signal amplification reactor, cuts a fluorescent reporter probe, and realizes double-stage signal amplification, the application combines the DNA reactor with a double CRISPR / Cas cascade system for non-amplification detection of pathogens RNA. The application is suitable for high-sensitivity, high-specificity and non-amplification instant detection of pathogens RNA.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, its preparation method, and its application. Background Technology

[0002] Pathogen infection is a significant threat to human health and public health security, characterized by rapid onset, rapid progression, and high mortality. Taking sepsis as an example, early and rapid diagnosis is crucial for reducing the rate of severe illness and mortality. However, traditional detection methods have a positive rate of less than 50% in the early stages of infection when the pathogen load is low, and the process takes 24-72 hours, easily leading to missed diagnoses and resulting in a mortality rate as high as 30%-50%.

[0003] RNA, as a direct biomarker of pathogen activity, plays a crucial role in early diagnosis; however, its low abundance and easy degradation place higher demands on detection technologies. Although PCR and isothermal amplification techniques have improved detection sensitivity and speed, they still suffer from operational complexity, reliance on specialized instruments, and detection times of 2-4 hours, failing to meet the clinical need for rapid diagnosis within the "golden hour." CRISPR / Cas systems, with their high specificity and efficient nucleic acid cleavage capabilities, have been widely used in pathogen detection, but still face challenges such as reliance on nucleic acid amplification, off-target risks, and complex sample processing. Summary of the Invention

[0004] To address the problems in existing technologies, this invention provides a CRISPR-cascaded nucleic acid detection system based on a DNA signal amplification reactor, its preparation method, and its applications. This nucleic acid detection platform, based on a DNA signal amplification reactor and a CRISPR / Cas13a-Cas12a dual-enzyme cascade signal amplification system, combines the DNA reactor with a dual CRISPR / Cas cascade system for amplification-free detection of pathogen RNA. This achieves highly sensitive, highly specific, amplification-free, real-time detection of pathogen RNA, with simple operation and rapid detection.

[0005] The DNA signal amplification reactor, as a novel DNA self-assembly structure, can realize large-scale, core-shell DNA sphere structures through oligonucleotide thermally induced phase separation and hybridization-driven assembly. It has advantages such as simple design, low cost, high loading capacity and modularity.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] The present invention aims to provide a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor, comprising a DNA signal amplification reactor, a Cas13a recognition module, and a Cas12a signal output module. The DNA signal amplification reactor contains a fluorescently labeled probe. The Cas13a recognition module is used to identify the target RNA and trigger the cleavage of the linker strand, releasing the DNA signal amplification reactor. The Cas12a signal output module is used to identify the released DNA signal amplification reactor and generate a fluorescent signal. After identifying the released DNA signal amplification reactor, the Cas12a signal output module activates trans-cleavage activity, cleaving the fluorescent reporter probe to achieve two-stage signal amplification.

[0008] Furthermore, the DNA signal amplification reactor includes a core sequence and a shell sequence. The core sequence contains a polyadenine sequence and functional module X (Cas12a activation substrate), and the shell sequence contains a polythymine sequence and functional module J (hybridization link of the strand coupled to the magnetic bead). The core-shell structure is formed by supramolecular single-stranded DNA through thermally induced phase separation and hybridization self-assembly.

[0009] Nuclear layer sequence SEQ ID NO.1: 5'-TTAGGATAGATATACGGGTTCAAAAAAAAAAAAAAAAAAAA-3'.

[0010] The sequence SEQ ID NO.2 of functional template X is: 5'-TTAGGATAGATATACGGGTTC-3'.

[0011] The sequence of polyadenine (A20) is SEQ ID NO.3: AAAAAAAAAAAAAAAAAAAA.

[0012] Shell sequence SEQ ID NO. 4: 5'-TTTTAGAGGATCGTGTGGTTTTTTTTTTTTTTTTTTTT-3'.

[0013] The sequence SEQ ID NO.5 of functional module J is: 5'-TTTTAGAGGATCGTGTGGTTTT-3'.

[0014] The sequence of polythymine (T20) is SEQ ID NO.6: TTTTTTTTTTTTTTTTTTTTTTTTT.

[0015] The sequence of the fluorescently labeled probe, SEQ ID NO.7, is: 5'-GAACCCGTATATCTATCCTAA-3'.

[0016] Furthermore, the Cas13a recognition module includes the Cas13a protein and its crRNA, which are used to specifically recognize pathogen RNA and cleave the linker strand to release the DNA signal amplification reactor; the Cas12a signal output module includes the Cas12a protein, its crRNA, and a fluorescent reporter probe, which are used to recognize the released DNA reactor and generate a fluorescent signal.

[0017] The sequence of the crRNA of the Cas13a protein, SEQ ID NO.8, is: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUCAACAUCAGUCUGAUAAGCUA-3'.

[0018] The sequence of the crRNA of the Cas12a protein, SEQ ID NO.9, is: 5'-UAAUUUCUACUAAGUGUAGAUGAACCCGUAUAUCUAUCCUAA-3'.

[0019] The sequence of the fluorescent reporter probe, SEQ ID NO.10, is: 5'6-FAM-UUUUU-3'BHQ1.

[0020] A method for preparing a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor includes the following steps:

[0021] Step 1) Synthesis of DNA signal amplification reactor: The core sequence and shell sequence are mixed, separated by adding Mg²⁺ induction phase, heated and then cooled to form a core-shell structured DNA signal amplification reactor, and fluorescently labeled probes are loaded in the DNA signal amplification reactor;

[0022] Step 2) Construction of nanoprobes: After incubating streptavidin magnetic beads with biotinylated RNA, they were added to a DNA signal amplification reactor for incubation. The nanoprobes were formed by binding through the RNA linker strand.

[0023] Step 3) Assembly of the CRISPR / Cas13a-Cas12a cascade system: Add Cas13a protein, Cas13a protein crRNA and target RNA to the nanoprobe, incubate in the DNA signal amplification reactor, and after magnetic separation, add Cas12a protein, Cas12a protein crRNA and fluorescent reporter probe to monitor the fluorescence signal.

[0024] Furthermore, in step 1), the mass ratio of the core sequence to the shell sequence is 4-8:1, and the concentration of Mg²⁺ is 40-60 mM. Preferably, the mass ratio of the core sequence to the shell sequence is 6:1, and the concentration of Mg²⁺ is 50 mM.

[0025] Furthermore, in step 1), the heating conditions are 90-100℃ for 10-15 min and the cooling rate is 0.01-0.05℃ / s. Preferably, the heating conditions are 95℃ for 12 min and the cooling rate is 0.01℃ / s.

[0026] Furthermore, in step 3), the incubation conditions are 35-40℃ and the incubation time is 10-20 min. Preferably, the incubation conditions are 37℃ and the incubation time is 15 min.

[0027] Furthermore, in step 3), the concentration ratio of Cas13a protein to Cas12a protein is 1:1.

[0028] Application of a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor or a method for preparing a CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor in the preparation of pathogen RNA detection kits.

[0029] Furthermore, pathogen RNAs include sepsis bacteria RNA, SARS-CoV-2 RNA, or Mycobacterium tuberculosis RNA.

[0030] The preparation method of the CRISPR cascade nucleic acid detection system based on the DNA signal amplification reactor includes the following steps:

[0031] Step 1) Synthesis of the DNA signal amplification reactor

[0032] The core sequence p(A20-X) and shell sequence P(T20-J) were designed and synthesized. The core sequence contains the polyadenine sequence A20 and functional module X, while the shell sequence contains the polythymine sequence T20 and functional module J. p(A20-X) and P(T20-J) were mixed at a mass ratio of 4-8:1, and separated by adding a 40-60 mM Mg²⁺ induction phase. The mixture was heated to 90-100℃ and held for 10-15 min, then cooled at a rate of 0.01-0.05℃ / s to form a core-shell DNA reactor. Fluorescently labeled probes were added to the system, and hybridization was used to load them into the DNA reactor.

[0033] Step 2) Construction of nanoprobes

[0034] Streptavidin magnetic beads were incubated with biotinylated RNA at 35-40°C to form a solid-phase carrier. A DNA signal amplification reactor was then incubated with this carrier at 35-40°C, allowing binding via the RNA linker strand to form a nanoprobe.

[0035] Step 3) Assembly of the CRISPR / Cas13a-Cas12a cascade system

[0036] Cas13a protein, its crRNA, and target RNA were added to a nanoprobe and incubated at 35-40°C for 10-20 min to cleave the biotinylated RNA, releasing a DNA signal amplification reactor. The released DNA signal amplification reactor was collected by magnetic separation. Cas12a protein, its crRNA, and a fluorescent reporter probe were then added, and the fluorescence signal was monitored. The concentration ratio of Cas13a protein to Cas12a protein was 1:1.

[0037] This invention optimizes the particle size, shell density, and loading efficiency of the DNA reactor by adjusting the ratio of p(A20-X) to P(T20-J) (mass ratio 4-8:1), thermal pyrolysis time (90-100℃ for 10-15 min), annealing rate (cooling rate 0.01-0.05℃ / s), and Mg²⁺ concentration (40-60 mM).

[0038] This invention improves the signal amplification efficiency and detection sensitivity of the cascade system by optimizing the concentration ratio of Cas13a protein to Cas12a protein (1:1), the crRNA sequences of Cas13a protein and Cas12a protein (SEQ ID NO.8 and SEQ ID NO.9), the reaction time (incubation conditions: 35-40℃, incubation time: 10-20 min), and the Mg²⁺ ion strength (concentration: 40-60 mM).

[0039] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0040] (1) This invention is the first to combine a DNA signal amplification reactor with a CRISPR / Cas13a-Cas12a dual-enzyme cascade system, achieving amplification-free and highly sensitive detection of pathogen RNA. The DNA signal amplification reactor has a high signal loading capacity, which can significantly enhance the detection signal and make up for the shortcomings of low signal-to-noise ratio and insufficient modularity of existing CRISPR systems.

[0041] (2) Through the design of “structural response + functional coupling”, the present invention triggers the release of DNA signal amplification reactor after Cas13a recognizes the target RNA, and Cas12a further cuts the fluorescent probe to achieve dual-stage signal amplification, which significantly improves the detection sensitivity and can meet the detection needs of low-abundance pathogen RNA.

[0042] (3) The system of the present invention has good biocompatibility and modular design. It can be adapted to a variety of pathogen targets by changing the crRNA sequence. It has versatility and rapid deployment capability and is suitable for clinical point diagnosis scenarios.

[0043] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the detection principle of the CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor according to the present invention.

[0045] Figure 2 This is a confocal laser scanning microscope image of the DNA signal amplification reactor of the present invention, where A is the p(A20-M) core layer; B is the p(T20-N) shell layer; and C is the "core-shell structure" DNA signal amplification reactor.

[0046] Figure 3 This is a diagram showing the fluorescence signal output of the present invention.

[0047] Figure 4 This is a diagram showing the sensitivity fluorescence signal output of the present invention. Detailed Implementation

[0048] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0049] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0050] Example 1

[0051] Preparation method of CRISPR cascade nucleic acid detection system based on DNA signal amplification reactor.

[0052] Step 1) Synthesis of the DNA signal amplification reactor:

[0053] Core sequence p(A20-X) and shell sequence P(T20-J) were designed. ssDNA was obtained by rolling circle amplification and characterized by PAGE electrophoresis. p(A20-X) and P(T20-J) were mixed at a 6:1 ratio, 50 mM Mg²⁺ was added, and the mixture was heated to 95 °C and held for 12 min. The mixture was then slowly cooled at a ramp rate of 0.01 °C / s to form a core-shell DNA signal amplification reactor. Fluorescently labeled (FAM and Cy3) barcode oligomeric sequences were added to the system, and the mixture was stirred at room temperature for 3 h. Detection was performed using CLSM.

[0054] Step 2) Construction of nanoprobes:

[0055] Streptavidin magnetic beads (50 μL, 500 μg) were incubated with biotinylated RNA (10 μL, 20 μM) at 37°C for 1 h, followed by washing three times with PBST. 10 μL of the solution was added to a DNA signal amplification reactor, and the mixture was incubated at 37°C for 2 h, followed by washing three times with PBST to obtain the nanoprobe.

[0056] Step 3) Assembly and testing of the CRISPR / Cas13a-Cas12a cascade system:

[0057] Cas13a protein crRNA (2 μL, 1 μM), Cas13a protein (1 μL, 1 μM), reaction buffer (2 μL, 10×), and target RNA (2 μL) were added to the nanoprobe and incubated at 37°C for 15 min to cleave the biotinylated RNA, releasing the DNA signal amplification reactor. The free DNA signal amplification reactor was collected by magnetic separation, and Cas12a protein (1 μL, 1 μM), Cas12a protein crRNA (2 μL, 1 μM), Reporter (2 μL, 10 μM), and r2.1 reaction buffer (2 μL, 10×) were added. Fluorescence values ​​were monitored using a qPCR instrument.

[0058] Example 2

[0059] Preparation method of CRISPR cascade nucleic acid detection system based on DNA signal amplification reactor.

[0060] Step 1) Synthesis of the DNA signal amplification reactor:

[0061] Core sequence p(A20-X) and shell sequence P(T20-J) were designed. ssDNA was obtained by rolling circle amplification and characterized by PAGE electrophoresis. p(A20-X) and P(T20-J) were mixed at an 8:1 ratio, 60 mM Mg²⁺ was added, and the mixture was heated to 100 °C and held for 15 min. The mixture was then slowly cooled at a ramp rate of 0.03 °C / s to form a core-shell DNA signal amplification reactor. Fluorescently labeled (FAM and Cy3) barcode oligomeric sequences were added to the system, and the mixture was stirred at room temperature for 2 h. Detection was performed using CLSM.

[0062] Step 2) Construction of nanoprobes:

[0063] Streptavidin magnetic beads (50 μL, 500 μg) were incubated with biotinylated RNA (10 μL, 20 μM) at 40 °C for 1 h, followed by washing three times with PBST. 10 μL of the solution was added to a DNA signal amplification reactor, and the mixture was incubated at 40 °C for 2 h, followed by washing three times with PBST to obtain the nanoprobe.

[0064] Step 3) Assembly and testing of the CRISPR / Cas13a-Cas12a cascade system:

[0065] Cas13a protein crRNA (2 μL, 1 μM), Cas13a protein (1 μL, 1 μM), reaction buffer (2 μL, 10×), and target RNA (2 μL) were added to the nanoprobe and incubated at 40°C for 20 min to cleave the biotinylated RNA, releasing the DNA signal amplification reactor. The free DNA signal amplification reactor was collected by magnetic separation, and Cas12a protein (1 μL, 1 μM), Cas12a protein crRNA (2 μL, 1 μM), Reporter (2 μL, 10 μM), and r2.1 reaction buffer (2 μL, 10×) were added. Fluorescence values ​​were monitored using a qPCR instrument.

[0066] Example 3

[0067] Preparation method of CRISPR cascade nucleic acid detection system based on DNA signal amplification reactor.

[0068] Step 1) Synthesis of the DNA signal amplification reactor:

[0069] Core sequence p(A20-X) and shell sequence P(T20-J) were designed. ssDNA was obtained by rolling circle amplification and characterized by PAGE electrophoresis. p(A20-X) and P(T20-J) were mixed in a 4:1 ratio, 40 mM Mg²⁺ was added, and the mixture was heated to 90 °C and held for 10 min. The mixture was then slowly cooled at a ramp rate of 0.05 °C / s to form a core-shell DNA signal amplification reactor. Fluorescently labeled (FAM and Cy3) barcode oligomeric sequences were added to the system, and the mixture was stirred at room temperature for 3 h. Detection was performed using CLSM.

[0070] Step 2) Construction of nanoprobes:

[0071] Streptavidin magnetic beads (50 μL, 500 μg) were incubated with biotinylated RNA (10 μL, 20 μM) at 35°C for 2 h, followed by washing three times with PBST. 10 μL of the solution was added to a DNA signal amplification reactor, and the mixture was incubated at 35°C for 3 h, followed by washing three times with PBST to obtain the nanoprobe.

[0072] Step 3) Assembly and testing of the CRISPR / Cas13a-Cas12a cascade system:

[0073] Cas13a protein crRNA (2 μL, 1 μM), Cas13a protein (2 μL, 2 μM), reaction buffer (2 μL, 10×), and target RNA (2 μL) were added to the nanoprobe and incubated at 35°C for 10 min to cleave the biotinylated RNA, releasing the DNA signal amplification reactor. The free DNA signal amplification reactor was collected by magnetic separation, and Cas12a protein (2 μL, 2 μM), Cas12a protein crRNA (2 μL, 1 μM), Reporter (2 μL, 10 μM), and r2.1 reaction buffer (2 μL, 10×) were added. Fluorescence values ​​were monitored using a qPCR instrument.

[0074] like Figure 3 The results showed that the system of the present invention can successfully detect target RNA, and the fluorescence signal intensity is positively correlated with the target concentration.

[0075] Detection performance evaluation and clinical sample validation.

[0076] Under optimized conditions, the detection limit was determined through gradient dilution experiments, and RNA mutants were designed to verify specificity. Anti-interference performance was tested in simulated plasma samples, and accuracy was evaluated through spiked recovery experiments. Blood samples were collected from sepsis patients to validate the system's ability to detect low-abundance pathogen RNA, and the results were compared with PCR, blood culture, and other methods.

[0077] The results show that, Figure 4 The system of this invention has high sensitivity and high specificity, with a detection limit at a low abundance level (1pM), and it is stable in complex samples. The clinical sample detection results are in good agreement with traditional methods.

[0078] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0079] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A CRISPR-cascaded nucleic acid detection system based on a DNA signal amplification reactor, characterized in that, The device includes a DNA signal amplification reactor, a Cas13a recognition module, and a Cas12a signal output module. The DNA signal amplification reactor contains a fluorescently labeled probe. The Cas13a recognition module is used to trigger the cleavage of the linker strand after recognizing the target RNA, releasing the DNA signal amplification reactor. The Cas12a signal output module is used to recognize the released DNA signal amplification reactor and generate a fluorescent signal. The DNA signal amplification reactor includes a core sequence and a shell sequence. The core sequence is shown in SEQ ID NO.1, and the shell sequence is shown in SEQ ID NO.

4. The functional module X sequence in the core sequence, SEQ ID NO.2, is the Cas12a activation substrate. The sequence of the fluorescently labeled probe is shown in SEQ ID NO.7; The method for preparing the detection system includes the following steps: Step 1) Synthesis of DNA signal amplification reactor: The core sequence and shell sequence are mixed, separated by adding Mg²⁺ induction phase, heated and then cooled to form a core-shell structured DNA signal amplification reactor, and fluorescently labeled probes are loaded in the DNA signal amplification reactor; Step 2) Construction of nanoprobes: After incubating streptavidin magnetic beads with biotinylated RNA, they were added to a DNA signal amplification reactor for incubation. The nanoprobes were formed by binding through the RNA linker strand.

2. The CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor as described in claim 1, characterized in that: The Cas13a recognition module includes the Cas13a protein and its crRNA; the sequence of the Cas13a protein crRNA is shown in SEQ ID NO. 8; the Cas12a signal output module includes the Cas12a protein, its crRNA, and a fluorescent reporter probe; the sequence of the Cas12a protein crRNA is shown in SEQ ID NO. 9; the sequence of the fluorescent reporter probe is shown in SEQ ID NO.

10.

3. The CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor as described in claim 1, characterized in that: In step 1), the mass ratio of the core sequence to the shell sequence is 4-8:1, and the concentration of Mg²⁺ is 40-60 mM.

4. The CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor as described in claim 3, characterized in that: In step 1), the heating conditions are 90-100℃ for 10-15 min, and the cooling rate is 0.01-0.05℃ / s.

5. The preparation method of the CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor as described in claim 2, characterized in that: The concentration ratio of Cas13a protein to Cas12a protein is 1:

1.

6. The application of the CRISPR cascade nucleic acid detection system based on a DNA signal amplification reactor as described in claim 1 in the preparation of pathogen RNA detection kits.

7. The application as described in claim 6, characterized in that: Pathogen RNAs include sepsis bacteria RNA, SARS-CoV-2 RNA, or Mycobacterium tuberculosis RNA.

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