A nucleic acid POCT detection kit and a detection method

Abstract

The application belongs to the technical field of nucleic acid detection, and particularly relates to a nucleic acid POCT detection kit. The detection system provided by the application is constructed based on the trans-cleavage activity and strand displacement / hybridization chain reaction of Cas12a / crRNA based on Toehold regulation. The detection method based on the detection system is high in sensitivity, good in specificity, simple in operation, and does not need complex instruments.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a nucleic acid POCT detection kit and detection method. Background Technology

[0002] Nucleic acids, as one of the most fundamental substances of life, are widely present in the cells of all animals, plants, and microorganisms. The discovery of the DNA double helix structure and base pairing rules by Watson and Crick marked the formal entry of life science research into the era of molecular biology. With the continuous development of nucleic acid synthesis and sequencing technologies, nucleic acid detection has been widely applied in infectious diseases, tumors, genetic diseases, parasitic diseases, and forensic medicine. Therefore, an increasing number of nucleic acid detection methods have been developed and applied. These methods mainly include nucleic acid electrophoresis, nucleic acid hybridization, gene chips, gene sequencing, polymerase chain reaction (PCR), and related technologies such as multiplex PCR and digital real-time polymerase chain reaction. However, these methods are not conducive to large-scale application due to their cumbersome operation, high cost, long processing time, and the need for complex equipment, demanding experimental environments, and specialized operators. They cannot meet the clinical demand for early and immediate diagnosis of diseases, leading to prolonged diagnosis time for patients, missed optimal treatment opportunities, and even endangering their lives.

[0003] Point-of-care testing (POCT) is a testing method that uses portable analytical instruments and matching reagents to quickly obtain test results at the sampling site. Due to its advantages such as portability, rapid testing, ease of operation, high applicability to testing conditions and personnel, ability to be conducted near patients, and ability to detect environmental pathogens outdoors or in resource-poor areas, it is widely used in many fields such as cardiovascular diseases, diabetes, oncology, and infectious diseases.

[0004] The CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-Associated Protein) is an adaptive immune system in bacteria and archaea, protecting them from invasion by bacteriophages, viruses, and plasmids. Cas9 is a widely used hallmark nuclease in type II CRISPR / Cas systems, widely applied for targeted recognition and gene editing due to its specific cleavage of target double-stranded DNA. Further research into the CRISPR-Cas system has revealed that Cas12 and Cas13 possess similar properties; after binding to target DNA, they can not only specifically cleave the target gene but also non-specifically cleave associated ssDNA / ssRNA in the system, demonstrating great promise for high-specificity and rapid nucleic acid detection. However, current reports on CRISPR-Cas systems primarily utilize fluorescence and electrical signals for signal output, requiring complex instruments for signal collection, which cannot meet the needs of point-of-care testing (POCT). Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a nucleic acid POCT detection kit and detection method.

[0006] The solution adopted in this invention is as follows: A nucleic acid POCT detection kit, comprising:

[0007] The Cas12a / crRNA digestion system includes hybrid double-stranded DNA Toehold, Cas12a, and crRNA;

[0008] Chain displacement / HCR amplification system, including avidin magnetic beads, G4-H1 and G4-H2;

[0009] The TMB colorimetric system includes a potassium-containing buffer solution, heme chloride, and TMB.

[0010] The hybrid double-stranded DNA Toehold was formed by co-incubation of biotin-H0, G4-CT and G4-S.

[0011] The biotin-H0 sequence is shown in SEQ ID NO.1;

[0012] The G4-CT sequence is shown in SEQ ID NO.2;

[0013] The G4-S sequence is shown in SEQ ID NO.3;

[0014] The G4-H1 sequence is shown in SEQ ID NO.4;

[0015] The G4-H2 sequence is shown in SEQ ID NO.5.

[0016] Preferably, the molar ratio of biotin-H0, G4-CT, and G4-S is 10:1:10; biotin-H0, G4-CT, and G4-S are incubated in a metal bath at 90-100°C for 3-8 minutes, and then cooled to 35-40°C for incubation to form hybrid double-stranded DNA Toehold.

[0017] A nucleic acid POCT detection method, wherein the method is for non-therapeutic or diagnostic purposes, and the method uses the nucleic acid POCT detection kit described above;

[0018] The method includes the following steps:

[0019] (1) Pre-incubate Cas12a and crRNA, then add DNA Toehold and test solution, and perform CRISPR / Cas12a digestion reaction;

[0020] (2) Add the CRISPR / Cas12a digestion solution to avidin magnetic beads, shake to carry out the coupling reaction, and then collect the coupled avidin magnetic beads by magnetic separation;

[0021] (3) HCR amplification was performed on the coupled avidin magnetic beads using G4-H1 and G4-H2 to obtain HCR amplified avidin magnetic beads;

[0022] (4) Incubate potassium-containing buffer, heme chloride and HCR-amplified avidin magnetic beads in the dark, then collect the magnetic beads by magnetic separation, add TMB and incubate in the dark, and terminate the reaction with concentrated sulfuric acid.

[0023] Preferably, in step (1), the molar ratio of Cas12a, crRNA, and DNA Toehold is 2:5:2.

[0024] Preferably, in step (2), the avidin magnetic beads are first washed with B&W buffer and then coupled; in step (3), the CRISPR / Cas12a enzyme digestion solution and the avidin magnetic beads are shaken vigorously at room temperature for 25-40 minutes to complete the coupling reaction.

[0025] Preferably, in step (3), G4-H1 and G4-H2 are incubated in a metal bath at 90-100℃ for 5-15 min, and then cooled to room temperature and incubated with DNA Toehold-coupled avidin magnetic beads and HCR hybridization buffer at 35-40℃ with shaking for 0.8-1.2 h.

[0026] Preferably, in step (4), Tris-Hcl-KCl, heme chloride solution, and avidin magnetic beads amplified by HCR are incubated at 35-40℃ in the dark for 25-40 min; the magnetic beads collected by magnetic separation are incubated with TMB solution at room temperature in the dark for 10-20 min.

[0027] Preferably, the presence of the target nucleic acid is qualitatively determined by visually observing the solution after the TMB incubation reaction. When the system does not contain the target nucleic acid, TMB will show color, and when the system contains the target nucleic acid, TMB will not show color.

[0028] Preferably, the target pathogen can be quantitatively detected by scanning the absorbance of the solution at 360 nm–520 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0029] The beneficial effects of the present invention are as follows: The detection system provided by the present invention is constructed based on the trans-cleavage activity of Cas12a / crRNA regulated by Toehold and the strand substitution / hybridization chain reaction. The detection method based on this detection system has high sensitivity, good specificity, and is easy to operate, without the need for complex instruments. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0031] Figure 1 A schematic diagram illustrating the principle of ultrasensitive nucleic acid colorimetric detection based on the trans-cleavage activity and strand substitution / hybridization chain reaction of CRISPR-Cas12a regulated by Toehold;

[0032] Figure 2 12% gel electrophoresis pattern to demonstrate the feasibility of HCR amplification on magnetic beads;

[0033] Figure 3 The G4 conjugate on the magnetic beads catalyzes the TMB-H2O2 reaction;

[0034] Figure 4 Validation of the Cas12a / crRNA cleavage system;

[0035] Figure 5 To assess the feasibility of a CRISPR / Cas12a-SD-HCR-based nucleic acid detection protocol;

[0036] Figure 6Based on the CRI-SDHCR nucleic acid detection performance verification, a represents the fluorescence change of the detection system; b represents the linear relationship between the change in absorbance at 50 nm and the logarithm of DNA concentration from 10 fM to 1 nM.

[0037] Figure 7 For the specificity validation results of the CRISPR / Cas12a-SD-HCR nucleic acid detection protocol, a represents the fluorescence change of the detection system; b represents the fluorescence value of the detection system at 522 nm. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0039] This invention provides a nucleic acid colorimetric detection biosensor based on Toehold-regulated trans-cleavage activity of Cas12a / crRNA and SD / HCR amplification reaction, which is named CRI-SDHCR. The detection process mainly includes the following three parts: Cas12a / crRNA digestion, SD / HCR amplification, and TMB color development. The detection principle diagram is shown below. Figure 1 As shown.

[0040] The CRISPR / Cas system (Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-Associated Protein) is a prokaryotic immune defense system used to resist the invasion of foreign genetic material, such as bacteriophages and viruses. Simultaneously, it provides bacteria with acquired immunity (similar to secondary immunity in mammals), developing a corresponding "memory" when bacteria are invaded by viruses. When a virus re-invades, the CRISPR system can recognize the foreign DNA, cut it, and silence the expression of the foreign gene, resisting viral interference. It is precisely because of this precise targeting function that the CRISPR / Cas system has been developed into a highly efficient gene editing tool. CRISPR / Cas can perform precise, targeted gene editing.

[0041] By designing crRNAs, specific recognition of target nucleic acids of different pathogens can be achieved. The specific crRNA design process is as follows: A 35 bp dsDNA segment containing a PAM sequence (TTTN) is randomly designed as the target nucleic acid. The scaffold sequence of Cas12a (UAA UUU CUA CUA AGU GUA GAU) is then added to the 20 bases following the PAM sequence of the target nucleic acid. Taking Vibrio vulnificus as an example, the designed Cas12a sequence is 5'—UAA UUU CUA CUA AGU GUA GAUGGG CCU AGA UAA AGA AGA AC—3'.

[0042] Chain displacement reactions are competitive binding reactions, primarily consisting of three elements: the substrate strand, the initiator strand, and the output strand. The substrate and output strands are partially complementary DNA double strands, with a single strand remaining on the substrate strand—the toehold region. The toehold acts as a cleavage point, guiding the initiator strand to competitively bind with the partially complementary DNA double strand to replace the output strand, thus releasing the free output strand. Toehold-mediated chain displacement is a method that ensures high specificity detection, overcoming common problems of biosensors such as complex operation, susceptibility to matrix interference, and the need for sophisticated instruments and demanding experimental environments. Introducing toehold-mediated chain displacement can significantly improve the detection specificity of sensors, reduce operational steps, and enable rapid detection of analytes.

[0043] The detection system provided by this invention innovatively incorporates a 5' biotin-modified DNA toehold, connecting the recognition and colorimetric portions of the detection system. On one hand, the DNA toehold can enhance the trans-cleavage activity of Cas12a / crRNA by regulating the length of the DNA single-strand portion. On the other hand, it can also act as the HO in the HCR reaction, forming a G-rich DNA strand through strand substitution and hybridization chain reactions. Under the action of heme chloride and potassium ions, a G4 conjugate is formed, acting as a DNase to catalyze the TMB-H2O2 reaction. Qualitative analysis is achieved by visually observing color changes. After terminating the reaction with concentrated sulfuric acid, quantitative detection of pathogenic bacteria can be achieved using an ELISA reader. When the target gene is present in the system, Cas12a / crRNA can specifically recognize and cleave the target gene. Simultaneously, the trans-cleavage activity of Cas12a / crRNA is activated, non-specifically cleaving ssDNA (i.e., DNA Toehold). Only the 5' portion of the double strand of the cleaved DNA Toehold can couple with avidin magnetic beads, performing strand displacement. The missing DNA strand prevents strand displacement and HCR reactions, failing to form G4 conjugates, and TMB does not develop color. When the system does not contain the target gene, the DNA Toehold cannot be non-specifically cleaved by Cas12a / crRNA. The 5' end of the biotin-modified DNA Toehold couples to the magnetic beads via avidin-biotin interaction. Subsequently, through strand displacement and HCR reactions, densely packed DNA double strands form on the magnetic beads. Then, under the action of heme chloride and potassium ions, G4 conjugates catalyze the TMB-H2O2 reaction, changing the solution from colorless to blue, achieving qualitative detection. Finally, the reaction was terminated by adding concentrated sulfuric acid, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to achieve quantitative detection of pathogenic bacteria.

[0044] The following are the sources of the reagents used in the various embodiments of this invention: Avidin magnetic beads were purchased from Suzhou Beaver Biotechnology Co., Ltd. LbCas12a was purchased from NEB Corporation, USA. TMB and heme chloride were purchased from Shanghai Aladdin Biotechnology Co., Ltd. DNA Ladder (20 bp) was purchased from Beijing Baori Biotechnology Co., Ltd. Ultra GelRed staining solution was purchased from Beijing Novizan Biotechnology Co., Ltd. DNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. All experimental water was ultrapure water with a conductivity of 18.2 MΩ cm.

[0045] Table 1 Major DNA Sequences

[0046]

[0047] Note: Biotin is a biotin-modified substance.

[0048] The following are the sources of the instruments and equipment used in the various embodiments of the present invention: multifunctional microplate reader (SpectraMax iD3, Meigu Molecular Instruments Co., Ltd., Shanghai), and heat-collecting constant temperature magnetic stirrer (DF-101S type, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.).

[0049] The following are the experimental procedures used in the various embodiments of the present invention:

[0050] (1) CRISPR / Cas12a trans-cutting

[0051] First, 1 μL of biotin-H0 (100 μM), 10 μL of G4-CT (1 μM), and 10 μL of LG4-S (10 μM) were incubated in a 95°C metal bath for 5 minutes, then slowly cooled to 37°C and held for 1 hour to form a hybrid double-stranded DNA toehold. 10 µL of 200 nM Cas12a and 10 µL of 200 nM crRNA were pre-incubated at 37°C for 20 minutes, then 1 µL of 5 µM DNA toehold, 2 µL of target gene solution, and 2 µL of 10×2.1 NEB buffer were added, and the mixture was incubated at 37°C for 60 minutes.

[0052] (2) Coupling of avidin magnetic beads with biotin-modified DNA Toehold

[0053] Wash avidin beads (15 μg) three times with 2×B&W buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl). Then add the CRISPR / Cas12a digested solution to the avidin beads and shake vigorously at room temperature for 30 min. Collect the conjugated avidin beads magnetically and discard the supernatant.

[0054] (3) SD-HCR reaction

[0055] First, incubate 100 μM G4-H1 and 100 μM G4-H2 in a metal bath at 95 °C for 10 min, then slowly cool to room temperature. Add 2 μL of 100 μM G4-H1 and 100 μM G4-H2 to avidin magnetic beads coupled with DNA Toehold, respectively. Make up the volume to 20 μL with HCR hybridization buffer (50 mM Na2HO4·H2O, 500 mM NaCl, pH 6.8), and incubate with shaking at 37 °C for 1 h. Collect the HCR-amplified avidin magnetic beads magnetically, discarding the remaining reaction solution. Finally, wash the avidin magnetic beads three times with 1×B&W buffer.

[0056] (4) TMB color development

[0057] Add 10 μL of 500 mM Tris-HCl-KCl and 5 μL of 50 μM heme chloride solution to the avidin magnetic beads amplified by HCR and incubate at 37°C in the dark for 30 min. Collect the magnetic beads magnetically and wash them three times with 1×B&W buffer. Then, add 35 μL of TMB solution to a PCR tube and incubate at room temperature in the dark for 15 min. Collect the incubated TMB solution magnetically, add 8 μL of 2.0 M H2SO4 to terminate the reaction, and transfer the reaction solution to a 384-well plate. Scan the absorbance of the solution using a microplate reader from 360 nm to 520 nm.

[0058] Example 1: Feasibility Analysis of HCR Amplification on Magnetic Beads

[0059] The feasibility of HCR amplification on magnetic beads was verified by 12% polyacrylamide gel electrophoresis, and the results are as follows: Figure 2 As shown in the diagram. Lane 1 contains the 5' biotin-modified DNA toehold, which is H0 acting as the initiator in the HCR amplification system; lanes 2 and 3 contain two hybridizable complementary DNA strands, H1 and H2, with sticky ends, respectively; lane 4 contains H1 plus H2; lane 5 contains the magnetic beads after HCR amplification for 1 h, heated at 100°C for 10 min, eluted with hybridization buffer, and electrophoretically analyzed. The results show a new, larger band, confirming successful HCR amplification on the magnetic beads. This is because the H0 coupled to the magnetic beads acts as the initiating strand, activating and opening the hairpin probe H1 in the solution. The opened H1 hybridizes with the hairpin probe H2, opening the neck loop structure of H2. The exposed single-stranded DNA then partially complements H1, thus opening a new H1 strand. This cycle repeats, forming a long double-stranded DNA strand on the magnetic beads.

[0060] The feasibility verification of the TMB-H2O2 reaction catalyzed by G4 conjugates on magnetic beads was conducted in two groups. The experimental group, after HCl amplification, formed densely packed long DNA strands on the magnetic beads. These DNA strands, rich in G bases, could form G4 conjugates, catalyzing the color development of TMB (TMB appears blue, but due to the presence of yellow magnetic beads, it appears green to the naked eye). The control group lacked functionalized H0, and no G4 conjugates formed, thus failing to catalyze the TMB color development. Magnetic separation was performed on both groups, and the reaction was terminated with a stop solution. Absorbance was measured at 450 nm. The absorption spectra showed that the absorbance of the experimental group was significantly higher than that of the control group. These results demonstrate that the TMB-H2O2 reaction catalyzed by G4 conjugates on magnetic beads is successful and can be used as a signal output component for the construction of detection methods.

[0061] Example 2: Feasibility Analysis of the Cas12a / crRNA Cutting System

[0062] The complete Cas12a / crRNA cleavage system includes Cas12a, crRNA, a fluorescent probe, and the target gene. Only when the system is intact, i.e., the target gene is present, can Cas12a / crRNA specifically recognize the target gene, activating its trans-cleavage activity and enabling non-specific cleavage of ssDNA (the fluorescent probe) within the system. Because the probe is modified with fluorescent and fluorescence-quenching groups at both ends, according to the principle of fluorescence resonance transfer, when the probe is intact, the fluorescence of the fluorescent group is quenched by the quenching group; and when the probe is cleaved, the fluorescent and quenching groups at both ends move away, emitting a strong fluorescence signal. The trans-cleavage activity of Cas12a / crRNA can be determined by quantitatively detecting the fluorescence changes using a microplate reader. Results are as follows: Figure 4 As shown, the strong fluorescence value at 522 nm indicates that the Cas12a / crRNA trans-cleavage activity was activated, and the probe was non-specifically cleaved. In contrast, no fluorescence was produced in the control group, indicating that non-specific cleavage did not occur in the absence of target DNA. The strong fluorescence value at 522 nm in the experimental group demonstrates that the Cas12a / crRNA trans-cleavage activity was activated, and the probe was non-specifically cleaved. These results prove the successful establishment of the CRISPR / Cas12a cleavage system.

[0063] Example 3: Feasibility of CRISPR / Cas12a-SD-HCR Detection Solution

[0064] To further verify the reliability of the CRI-SDHCR assay, the Vibrio vulnificus-specific target gene vvhA was selected to validate the entire assay protocol. The results are as follows: Figure 5 As shown, when the initiating strand is single-stranded DNA H0, the trans-cleavage activity of Cas12a is activated in the presence of the target gene, but the cleavage efficiency is low, with only a slight decrease in absorbance at 450 nm compared to the absence of the target genome. When the initiating strand is DNA Toehold, the trans-cleavage activity of Cas12a activated by the target gene is greatly increased, demonstrating that the developed Toehold-based CRI-SDHCR detection system has good detection performance.

[0065] Example 4: Performance Verification of CRI-SDHCR Detection

[0066] Different concentrations of the vvhA gene were detected under optimal conditions. The results are as follows: Figure 6 As shown in figure a, the absorbance at 450 nm gradually decreases as the DNA concentration increases from 0 fM to 1 nM. Figure 6 As shown in b, there is a good linear relationship between the change in absorbance at 450 nm and the logarithm of DNA concentration from 10 fM to 1 nM, with a detection limit of 4.54 fm and a regression equation of ∆Abs = 0.0531. lg[target DNA concentration(fM)]-0.0091, correlation coefficient R2=0.9903.

[0067] Example 5: Application of bacterial detection

[0068] The target bacterium, Vibrio vulnificus, and six other common environmental pathogens—Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter baumannii, and Escherichia coli—were selected to validate the specificity of CRI-SDHCR. The genomes of these seven bacteria were extracted, and the Vibrio vulnificus-specific target gene vvhA was amplified to verify the performance of CRI-SDHCR in bacterial specificity detection. Figure 7 As shown in the figure, the absorbance of Vibrio vulnificus at 450 nm was significantly reduced compared to the other six strains, indicating that this method has high specificity.

[0069] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention. sequence list <120> A nucleic acid POCT detection kit and detection method <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 31 <212> DNA <213> Artificial sequence <400> 1 tttttttttt ttagaagaag gtgtttaagt a 31 <210> 2 <211> 12 <212> DNA <213> Artificial sequence <400> 2 ctaaaaaaaa aa 12 <210> 3 <211> 13 <212> DNA <213> Artificial sequence <400> 3 tacttaaaca cct 13 <210> 4 <211> 55 <212> DNA <213> Artificial sequence <400> 4 agggcgggtg ggtgtttaag ttggagaatt gtacttaaac accttcttct tgggt 55 <210> 5 <211> 54 <212> DNA <213> Artificial sequence <400> 5 gggtcaattc tccaacttaa actagaagaa ggtgtttaag ttgggtaggg cggg 54

Claims

1. A nucleic acid POCT detection kit, characterized in that, include: The Cas12a / crRNA digestion system includes hybrid double-stranded DNA Toehold, Cas12a, and crRNA; Chain displacement / HCR amplification system, including avidin magnetic beads, G4-H1 and G4-H2; The TMB colorimetric system includes a potassium-containing buffer solution, heme chloride, and TMB. The hybrid double-stranded DNA Toehold was formed by co-incubation of biotin-H0, G4-CT and G4-S. The biotin-H0 sequence is shown in SEQ ID NO.1; The G4-CT sequence is shown in SEQ ID NO.2; The G4-S sequence is shown in SEQ ID NO.3; The G4-H1 sequence is shown in SEQ ID NO.4; The G4-H2 sequence is shown in SEQ ID NO.5; The crRNA specifically recognizes the nucleic acid to be tested.

2. The nucleic acid POCT detection kit according to claim 1, characterized in that: The molar ratio of biotin-H0, G4-CT, and G4-S is 10:1:10; biotin-H0, G4-CT, and G4-S are incubated in a metal bath at 90-100℃ for 3-8 minutes, and then cooled to 35-40℃ for incubation to form hybrid double-stranded DNA Toehold.

3. A nucleic acid point-of-care testing (POCT) method, wherein the method is for non-therapeutic or non-diagnostic purposes, characterized in that, It uses the nucleic acid POCT detection kit as described in claim 1 or 2; The method includes the following steps: (1) Pre-incubate Cas12a and crRNA, then add DNA Toehold and test solution, and perform CRISPR / Cas12a digestion reaction; (2) Add the CRISPR / Cas12a digestion solution to avidin magnetic beads, shake to carry out the coupling reaction, and then collect the coupled avidin magnetic beads by magnetic separation; (3) HCR amplification was performed on the coupled avidin magnetic beads using G4-H1 and G4-H2 to obtain HCR amplified avidin magnetic beads; (4) Incubate potassium-containing buffer, heme chloride and HCR-amplified avidin magnetic beads in the dark, then collect the magnetic beads by magnetic separation, add TMB and incubate in the dark, and terminate the reaction with concentrated sulfuric acid.

4. The nucleic acid POCT detection method according to claim 3, characterized in that: In step (1), the molar ratio of Cas12a, crRNA, and DNA Toehold is 2:5:

2.

5. The nucleic acid POCT detection method according to claim 3, characterized in that: In step (2), avidin magnetic beads are first washed with B&W buffer and then coupled; the CRISPR / Cas12a digested solution and avidin magnetic beads are shaken vigorously at room temperature for 25-40 minutes to complete the coupling reaction.

6. The nucleic acid POCT detection method according to claim 3, characterized in that: In step (3), G4-H1 and G4-H2 are incubated in a metal bath at 90-100℃ for 5-15 min, and then cooled to room temperature. They are then incubated with DNA Toehold-coupled avidin magnetic beads and HCR hybridization buffer at 35-40℃ with shaking for 0.8-1.2 h.

7. The nucleic acid POCT detection method according to claim 3, characterized in that: In step (4), Tris-Hcl-KCl, heme chloride solution, and avidin magnetic beads amplified by HCR are incubated at 35-40℃ in the dark for 25-40 min; the magnetic beads collected by magnetic separation are incubated with TMB solution at room temperature in the dark for 10-20 min.

8. The nucleic acid POCT detection method according to claim 3, characterized in that: The presence of the target nucleic acid is qualitatively determined by visually observing the solution after the TMB incubation reaction. When the system does not contain the target nucleic acid, TMB will show color; when the system contains the target nucleic acid, TMB will not show color.

9. The nucleic acid POCT detection method according to claim 3, characterized in that: The target pathogen can be quantitatively detected by scanning the absorbance of the solution at 360 nm–520 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

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