Amplification-free ultra-sensitive nucleic acid detection system and detection method on basis of digital chromatography and crispr system

Abstract

Provided are an amplification-free ultra-sensitive nucleic acid detection system and detection method on the basis of digital chromatography and a CRISPR system. The detection system comprises a CRISPR reaction system, a chromatography reaction system, and a digital chromatography detection system, wherein the CRISPR reaction system comprises a Cas nuclease, a crRNA, and a nucleic acid probe. The amplification-free ultra-sensitive nucleic acid detection system solves the problems of complicated steps, long term consumption, and high requirements for detection laboratory conditions in an existing nucleic acid detection method, and achieves the amplification-free and ultra-sensitive detection of a target nucleic acid sequence.

Description

A non-amplification super-sensitive nucleic acid detection system and method based on digital tomography and CRISPR system TECHNICAL FIELD

[0001] The present application relates to the field of biological detection system detection, and particularly relates to a non-amplification super-sensitive nucleic acid detection system and method based on digital tomography and CRISPR system. BACKGROUND

[0002] The method of nucleic acid detection analysis has broad application prospects in the fields of clinical disease detection, food safety, microbial inspection and quarantine, and veterinary medicine. At present, most nucleic acid detection technologies require amplification of nucleic acid molecules, i.e., the commonly used polymerase chain reaction (PCR), which amplifies specific target fragment nucleic acids through three steps of melting, annealing and extension. The amplified nucleic acid molecules are detected by fluorescence, colorimetry or turbidity methods. However, traditional PCR reaction steps are complicated, require professional nucleic acid amplification laboratories, and have long detection turnaround time, thus there is an urgent need to develop nucleic acid detection technologies suitable for on-site and rapid detection.

[0003] CRISPR-based nucleic acid detection methods, such as DETECTR and SHERLOCK, amplify target nucleic acid sequences through isothermal amplification, activate the transcleavage activity of crRNA and Cas enzyme, and realize the detection of target nucleic acid through fluorescence or colorimetric methods. However, the current CRISPR detection system has low sensitivity (Anal. Chem. 2024, 96, 20, 8091-8108), and needs to be combined with isothermal nucleic acid amplification technologies such as LAMP, RPA, and RAA. The nucleic acid amplification process not only increases the detection time, but also may cause false negative or false positive results due to amplification errors (ACS Infect. Dis. 2023, 9, 8, 1534-1545). The problem of aerosol pollution in the nucleic acid amplification process also brings great trouble.

[0004] Chinese invention patent application 202310307297.5 discloses a non-amplification gene detection method based on CRISPR-Cas13a system. The invention droplets the nucleic acid probe, detects the digital nucleic acid probe signal of the droplets through a fluorescence microscope, and realizes non-amplification detection of the target nucleic acid sequence. Hajime Shinoda et al. (Commun Biol, 2021 4, 476) reported that the nucleic acid probe cut by CRISPR-Cas13 was dispersed in a microcavity chip, and the non-amplification nucleic acid detection was realized by the method of digital nucleic acid probe detection. Weihua Guan et al. (ACS Sens. 2020, 5, 5, 1273-1280) reported that the CRISPR-Cas12a system was combined with a nanopore, the change of the nucleic acid probe was detected through the nanopore, and non-amplification detection of HIV was realized. Although the above-mentioned CRISPR-based nucleic acid detection system can realize non-amplification nucleic acid detection, the detection steps of the nucleic acid probe signal are complicated, time-consuming, and high in detection cost, which is difficult to realize on-site rapid detection. SUMMARY

[0005] In view of the above-mentioned defects of the prior art, the purpose of the present application is to provide a non-amplification ultra-sensitive nucleic acid detection system based on digital chromatography and CRISPR system, which overcomes the defects of the prior art, such as complicated steps, long time-consuming, and difficulty in standardizing the detection system. The non-amplification nucleic acid detection method provided by the present application has the advantages of simple detection steps, short detection time, and easy standardization of the detection system, which realizes non-amplification, ultra-sensitive and on-site rapid detection of the target nucleic acid sequence. In addition, the present application also provides a method for detecting the above-mentioned non-amplification ultra-sensitive nucleic acid detection system.

[0006] To achieve the above-mentioned purpose and other related purposes,

[0007] The first aspect of the present application provides a non-amplification ultra-sensitive nucleic acid detection system based on digital chromatography and CRISPR system, comprising: a CRISPR reaction system, a chromatography reaction system and a digital chromatography detection system.

[0008] The CRISPR reaction system comprises a Cas nuclease, a crRNA and a nucleic acid probe.

[0009] The chromatography reaction system comprises a reaction membrane and a tracer nanoparticle, and the detection area of the reaction membrane is fixed with a capture ligand for capturing the nucleic acid probe; the tracer nanoparticle labeled with a labeled ligand can specifically recognize the nucleic acid probe enriched in the detection area.

[0010] The digital chromatography detection system comprises an optical imaging system and an image processing system; the optical imaging system is used for displaying a microscopic image of a reaction membrane of the chromatography reaction system; the image processing system comprises an identification module and a counting module, the identification module is used for identifying a tracer nanoparticle in the microscopic image, and the counting module is used for counting the number of the tracer nanoparticle in the microscopic image.

[0011] The detection area on the reaction membrane of the chromatography reaction system is fixed with a capture ligand, the nucleic acid probe is specifically captured and enriched by the capture ligand, and the tracer nanoparticle specifically recognizes the nucleic acid probe enriched in the detection area.

[0012] The optical imaging system is a fluorescence microscopic amplification or dark field microscopic amplification optical system, and can distinguish a single tracer nanoparticle specifically combined on the reaction membrane of the chromatography reaction system; the image processing system comprises a detection area identification module and a counting module of the specifically combined tracer nanoparticle, and the number of the tracer nanoparticle specifically combined on the detection area of the reaction membrane of the chromatography reaction system is proportional to the concentration of the analyte to be detected.

[0013] In the embodiments of the present application, the chromatography reaction system is selected from a lateral chromatography reaction system or a longitudinal chromatography reaction system.

[0014] The lateral chromatography reaction system comprises a sample pad, a binding pad, a reaction membrane and a water absorption pad; the lateral chromatography reaction system can be the structure in Figure 4 provided by Chinese patent (2020108603286).

[0015] The longitudinal chromatography reaction system comprises a reaction membrane, a water absorption pad and an assembled shell; the longitudinal chromatography reaction system can be the structure in Figure 7 provided by Chinese patent (2020108603286).

[0016] In the embodiments of the present application, the Cas enzyme comprises a Cas9 nuclease, a Cas12a nuclease, a Cas13 nuclease, a Cas14 nuclease and the like.

[0017] The crRNA can specifically bind to the nucleic acid sequence of the sample to be detected, and activate the nucleic acid probe cutting activity of the Cas enzyme.

[0018] In the embodiments of the present application, the nucleic acid probe comprises a single-stranded RNA, a single-stranded DNA and a double-stranded DNA, and the 3' and 5' ends of the nucleic acid probe have a labeling molecule.

[0019] In the embodiments of the present application, the tracer nanoparticle comprises a fluorescent nanoparticle or a plasmonic nanoparticle.

[0020] The fluorescent nanoparticles include at least one of time-resolved fluorescent nanoparticles, organic fluorescent dye nanoparticles, fluorescent quantum dot nanoparticles, and aggregation-induced fluorescent nanoparticles.

[0021] The plasmonic nanoparticles include at least one of gold plasmonic nanoparticles, platinum plasmonic nanoparticles, silver plasmonic nanoparticles, and palladium plasmonic nanoparticles.

[0022] The labeling molecules of the nucleic acid probes include biotin, fluorescein, digoxin, etc.

[0023] In the embodiments of the present application, the concentration of the nucleic acid probe is 1-100 pM.

[0024] The concentration of the tracer nanoparticles is 0.3-0.5 μg / mL, and the particle size of the tracer nanoparticles is 10-500 nm.

[0025] In the embodiments of the present application, the capture ligand of the capture nucleic acid probe is fixed on the detection area of the chromatographic reaction system, and the tracer nanoparticles are labeled with a labeling ligand for identifying the nucleic acid probe.

[0026] In the embodiments of the present application, one end of the nucleic acid probe can specifically bind to the capture ligand, and the other end of the nucleic acid probe can specifically bind to the labeling ligand.

[0027] In the embodiments of the present application, the tracer nanoparticles specifically combined with the nucleic acid probe in the detection area can be distinguished by single particle under the optical imaging system, and the counting module of the specifically combined tracer nanoparticles can count the number of the specifically combined tracer nanoparticles in the detection area.

[0028] A method for detecting by using the above-mentioned non-amplification super-sensitive nucleic acid detection system, comprising the following steps:

[0029] Step one, adding the detection sample into the CRISPR reaction system for 5-30 minutes;

[0030] Step two, adding the sample after the CRISPR reaction to the chromatographic reaction system for 5-15 minutes of chromatographic reaction;

[0031] Step three, obtaining the microscopic image of the detection area on the reaction membrane of the chromatographic reaction system under the optical imaging system, and counting the tracer nanoparticles in the detection area by the image processing system;

[0032] Step four, calculating the concentration of the nucleic acid to be detected in the sample by the fitting relationship curve between the concentration of the calibration sample and the number of the tracer nanoparticles.

[0033] For the present application, the detection sample is added to the CRISPR reaction system, and after the crRNA binds to the nucleic acid sequence of the sample to be detected, the nucleic acid probe cutting activity of the Cas enzyme can be activated, thereby cutting the nucleic acid probe. After the CRISPR reaction, the sample is added dropwise to the chromatographic reaction system, one end of the uncut nucleic acid probe can be specifically combined with the capture ligand, and the other end of the nucleic acid probe can be specifically combined with the labeled ligand on the surface of the tracer nanoparticle, thereby enriching the tracer nanoparticle in the detection area; the cut nucleic acid probe will not be enriched in the detection area. The micrograph of the detection area on the reaction film of the chromatographic reaction system is obtained on the optical imaging system, thereby obtaining the micrograph of the tracer nanoparticle specifically combined with all uncut nucleic acid probes, and the number of uncut nucleic acid probes is obtained by counting the tracer nanoparticles in the detection area through the image processing system, thereby calculating the number of cut nucleic acid probes and the number of nucleic acid sequences in the sample to be detected.

[0034] As described above, the present application has the following beneficial effects:

[0035] The present application provides a non-amplification super-sensitive nucleic acid detection system and method based on digital chromatography and CRISPR system, wherein the digital chromatography detection system can significantly improve the detection and analysis sensitivity of the chromatography detection method by combining microscopic signal amplification and single nanoparticle counting.

[0036] The present application creatively adds a CRISPR system, which can be specifically activated by the target nucleic acid sequence in the sample. Due to the super-sensitive detection capability of digital chromatography, the concentration of the nucleic acid probe in the CRISPR detection system can be greatly reduced, thereby amplifying the signal of the nucleic acid probe cut by CRISPR enzyme, and realizing non-amplification super-sensitive detection of the target nucleic acid sequence.

[0037] The non-amplification super-sensitive nucleic acid detection system of the present application overcomes the problems of existing nucleic acid detection methods, such as complicated steps, long time consumption, and high requirements for laboratory conditions. The present application uses the signal amplification of the CRISPR detection system and the signal amplification of the digital chromatography detection system to realize non-amplification super-sensitive detection of the target nucleic acid sequence. At the same time, the detection steps of the system are simple, the time consumption is short, the detection system is easy to standardize, and the system has the advantages of rapidness, convenience, low cost, and easy industrialization of chromatography test paper. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of the composition structure of the non-amplification super-sensitive nucleic acid detection system based on digital chromatography and CRISPR system in the embodiment 1 of the present application.

[0039] Figure 2 is the optimization of the concentration of fluorescently labeled particles in Example 3 of the present application.

[0040] Figure 3 is the optimization of the concentration of nucleic acid probes in Example 3 of the present application.

[0041] Figure 4 is the fluorescence photos and fluorescence micrographs of the detection zone of the CRISPR nucleic acid test paper without amplification for detecting HIV-1 RNA in Example 4 of the present application.

[0042] Figure 5 is a comparison of the detection results of the fluorescence test paper for detecting HIV-1 RNA without amplification by CRISPR nucleic acid test paper in Example 4 of the present application using fluorescence analog signals and fluorescent particle digital signals. DETAILED DESCRIPTION

[0043] The following describes the embodiments of the present application by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0044] Example 1

[0045] As shown in Figure 1, a non-amplification ultra-sensitive nucleic acid detection system based on digital chromatography and CRISPR system, the detection system comprises: a CRISPR reaction system, a chromatography reaction system, a digital chromatography detection system;

[0046] The CRISPR reaction system comprises Cas nuclease, crRNA and nucleic acid probes;

[0047] The chromatography reaction system is a lateral flow chromatography reaction system or a vertical flow chromatography reaction system, wherein the lateral flow chromatography reaction system comprises a sample pad, a conjugate pad, a reaction membrane, and a water absorption pad; the vertical flow chromatography reaction system comprises a reaction membrane, a water absorption paper, and an assembled card shell; the capture ligand is fixed on the detection zone of the reaction membrane of the chromatography reaction system, the capture ligand is used for specific capture and enrichment of nucleic acid probes, and the labeled ligand marked by tracer nanoparticles is used for specific recognition of the enriched nucleic acid probes in the detection zone;

[0048] The digital chromatography detection system comprises an optical imaging system and an image processing system, the optical imaging system is a fluorescence microscopic magnification optical system or a dark field microscopic magnification optical system, and the optical imaging system can distinguish a single tracer nanoparticle specifically bound on the reaction membrane of the chromatography reaction system; the image processing system comprises a detection zone recognition module and a counting module of specifically bound tracer nanoparticles, and the number of specifically bound tracer nanoparticles in the detection zone is proportional to the concentration of the analyte to be detected.

[0049] Example 2

[0050] A method for non-amplification ultra-sensitive nucleic acid detection using the above system, comprising the following steps:

[0051] Step 1, 0.5 μl of sample (10 μM Target RNA) is added to the CRISPR reaction system, and the reaction is carried out at 37°C for 5-30 minutes;

[0052] Step 2, after the CRISPR reaction, the sample is added dropwise to the chromatographic reaction system, and after the chromatographic reaction for 5-15 minutes, the microscopic image of the detection area on the reaction film of the chromatographic reaction system is obtained on the optical imaging system, and the tracer nanoparticles in the detection area are counted by the image processing system;

[0053] Step 3, the concentration of the nucleic acid to be detected in the sample is calculated by fitting the concentration of the calibrator and the number of tracer nanoparticles.

[0054] Example 3

[0055] A non-amplification gene detection method based on CRISPR system mainly includes two parts: one is the construction of CRISPR reaction system, and the other is the detection of nucleic acid probe by immunochromatography and the reading and counting of single particles in the detection area.

[0056] 1. Optimization of tracer nanoparticle concentration

[0057] In order to achieve ultra-sensitive detection, the concentration of tracer nanoparticles in the chromatographic test paper is first optimized, and the concentration of fluorescent particles in the detection area is also reduced accordingly.

[0058] As shown in Figure 2, after dilution of the tracer nanoparticle concentration, the fluorescence signal on the detection line gradually weakens, and when the tracer nanoparticle concentration is 0.31 μg / mL, the fluorescence signal on the detection line is weak and can be distinguished by the naked eye.

[0059] 2. Optimization of nucleic acid probe concentration

[0060] In order to achieve ultra-sensitive detection, the concentration of nucleic acid probe in the CRISPR reaction system is further optimized, and the concentration of nucleic acid probe is as low as possible, and the concentration of target probe required to start CRISPR cutting can also be reduced accordingly.

[0061] As shown in Figure 3, the nucleic acid probe is diluted successively from the highest concentration of 10 nM to 0.01 nM, and when the nucleic acid probe concentration is 10 pM (0.01 nM), the signal of the detection line begins to decrease, indicating that the concentration ratio of nucleic acid probe and tracer nanoparticle at this concentration is relatively optimal.

[0062] Example 4

[0063] Based on CRISPR-Cas13 and digital chromatographic test paper, the ultra-sensitive detection of HIV-1 RNA includes the following steps:

[0064] 1. Quantum dot nanosphere labeled anti-fluorescein (anti-FAM) antibody

[0065] 1.1) Take 100 microliters of quantum dot nanospheres with carboxyl groups on the surface (emission wavelength 620 nanometers, red), dilute to 300 microliters with a phosphate buffer at pH 6.0, add 0.3 mg of the activator 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), place in a rotary mixer, and react at room temperature for 0.5 hours.

[0066] 1.2) After activation of the carboxyl quantum dot microspheres, centrifuge at 12000 rpm for 10 minutes, remove the supernatant, and disperse the activated quantum dot nanospheres in 300 microliters of phosphate buffer, add about 100 micrograms of anti-FAM labeled antibody, place in a rotary mixer, and react at room temperature for 1 hour.

[0067] 1.3) After the antibody and quantum dot nanosphere coupling reaction, centrifuge at 12000 rpm for 10 minutes, remove the supernatant, disperse the antibody-quantum dot nanosphere in 300 microliters of phosphate buffer, add 10 mg of BSA, place in a rotary mixer, and block at room temperature for 2 hours to obtain the anti-FAM-quantum dot nanosphere conjugate.

[0068] 2. Assembly of digital immunochromatographic test strips

[0069] 2.1) Select quantum dot nanospheres (emission wavelength 520 nanometers, green) as detection zone indicator particles, mix 100 microliters of green quantum dot nanospheres with 10 mg of BSA, add 0.3 mg of EDC, place in a rotary mixer, and react at room temperature for 2 hours to obtain the detection zone tracer particles.

[0070] 2.2) The chromatographic reaction membrane is selected as CN 140 nitrocellulose membrane (NC membrane) from Sartorius, with streptavidin at a concentration of 1 mg per milliliter as the detection zone, and goat anti-mouse IgG polyclonal antibody at a concentration of 1 mg per milliliter as the quality control zone. Then, with a spraying amount of 1 microliter per centimeter, evenly draw the membrane on the NC membrane to form the detection zone and the quality control zone with a separation of 4 millimeters.

[0071] 2.3) Stack the sample pad, reaction membrane, and water absorption pad in sequence on the PVC adhesive backing plate, assemble into a large plate, and cut it into 4 millimeter wide test strip strips with a paper cutter, which is a lateral chromatographic reaction system.

[0072] 3. CRISPR-Cas13 reaction

[0073] The crRNA is designed for a conserved region sequence of HIV-1, and the CRISPR reaction system comprises 10 μM crRNA 0.5 μl, 10 μM Cas13 enzyme 0.5 μl, enzyme-free water 15.5 μl, HOLMES Buffer 22 μl, 10 pM CRISPR-LFA ssRNA reporter 1 μl, and reaction at room temperature for 30 minutes.

[0074] 4. Chromatographic test paper detection

[0075] 20 μl of the solution after the CRISPR reaction is mixed with 50 μl of anti-FAM quantum dot nanosphere complex, added to the sample pad of the chromatographic test paper, after reaction for 15 minutes, the fluorescence signal intensity of the test band and the quality control band of the test paper is detected by the fluorescence immunochromatographic analyzer; at the same time, a fluorescence microscope is used to take a picture to obtain a fluorescence microscopic image of the test band.

[0076] The fluorescence photos (upper) and the fluorescence microscopic photos (lower) of the test paper strip are shown in FIG. 4, the red quantum dot nanomicrosphere in the detection area is counted by using ImageJ software. The detection results of the fluorescence immunochromatographic analyzer (FIG. 5 left), the microscopic counting detection results (FIG. 5 middle) and the comparison of the two detection results (FIG. 5 right) are obtained. The concentration of HIV-1 RNA in the sample to be detected is calculated by the fitting equation between the series of known concentrations of HIV-1 RNA calibrators and the tracer particle microscopic counting.

[0077] In summary, the non-amplification super-sensitive nucleic acid detection system of the present application overcomes the problems of the prior art nucleic acid detection method, such as complicated steps, long time consumption, and high requirements for laboratory conditions. The present application can realize non-amplification and super-sensitive detection of target nucleic acid sequences through signal amplification of the CRISPR detection system and signal amplification of the digital chromatographic detection system. At the same time, the detection steps of the system are simple, the time consumption is short, the detection system is easy to standardize, and the system has the advantages of rapidness, convenience, low cost and easy industrialization of the chromatographic test paper. Therefore, the present application effectively overcomes the various shortcomings in the prior art and has high industrial utilization value.

[0078] The above embodiments only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A non-amplification ultra-sensitive nucleic acid detection system based on digital tomography and CRISPR system, characterized in that, The application relates to a CRISPR reaction system, a chromatographic reaction system and a digital chromatographic detection system. The CRISPR reaction system comprises a Cas nuclease, a crRNA and a nucleic acid probe. The chromatographic reaction system comprises a reaction membrane and a tracer nanoparticle, the detection area of the reaction membrane is fixed with a capture ligand for capturing the nucleic acid probe, and the tracer nanoparticle is marked with a marking ligand which can specifically recognize the nucleic acid probe enriched in the detection area. The digital chromatographic detection system comprises an optical imaging system and an image processing system, the optical imaging system is used for displaying the microscopic image of the reaction membrane of the chromatographic reaction system, and a single tracer nanoparticle in the detection area can be distinguished, the image processing system comprises an identification module and a counting module, the identification module is used for identifying the tracer nanoparticle in the microscopic image, and the counting module is used for counting the number of the tracer nanoparticle in the microscopic image. The chromatographic reaction system is selected from a lateral chromatographic reaction system or a longitudinal chromatographic reaction system. 2.The non-amplification ultra-sensitive nucleic acid detection system based on digital tomography and CRISPR system according to claim 1, wherein: The lateral chromatographic reaction system comprises a sample pad, a binding pad, a reaction membrane and a water absorption pad. The longitudinal chromatographic reaction system comprises a reaction membrane, a water absorption pad and an assembled card shell. The Cas enzyme comprises a Cas9 nuclease, a Cas12a nuclease, a Cas13 nuclease or a Cas14 nuclease. 3.The non-amplification ultra-sensitive nucleic acid detection system based on digital tomography and CRISPR system according to claim 1, wherein: After the crRNA is combined with the nucleic acid sequence of the sample to be detected, the nucleic acid probe cutting activity of the Cas enzyme can be activated. The nucleic acid probe comprises single-stranded RNA, single-stranded DNA and double-stranded DNA, and the 3' and 5' ends of the nucleic acid probe are provided with a marking molecule.

4. The digital tomography and CRISPR system based amplification-free ultrasensitive nucleic acid detection system according to claim 1, characterized in that: The tracer nanoparticle comprises a fluorescent nanoparticle or a plasmonic nanoparticle.

5. The digital tomography and CRISPR system based amplification-free ultrasensitive nucleic acid detection system according to claim 4, characterized in that: The fluorescent nanoparticle comprises at least one of a time-resolved fluorescent nanoparticle, an organic fluorescent dye nanoparticle, a fluorescent quantum dot nanoparticle and an aggregation-induced fluorescent nanoparticle. The plasmonic nanoparticle comprises at least one of a gold plasmonic nanoparticle, a platinum plasmonic nanoparticle, a silver plasmonic nanoparticle and a palladium plasmonic nanoparticle. The marking molecule of the nucleic acid probe comprises at least one of biotin, fluorescein and digoxin. The concentration of the nucleic acid probe is 1-100 pM.

6. The digital tomography and CRISPR system based amplification-free ultrasensitive nucleic acid detection system according to claim 5, characterized in that: The particle size of the tracer nanoparticle is 10-500 nm. The detection area of the chromatographic reaction system is fixed with a capture ligand for capturing the nucleic acid probe, and the surface of the tracer nanoparticle is marked with a marking ligand for identifying the nucleic acid probe.

7. The digital tomography and CRISPR system based amplification-free ultrasensitive nucleic acid detection system according to claim 1, wherein: One end of the nucleic acid probe can be specifically combined with the capture ligand, and the other end of the nucleic acid probe can be specifically combined with the marking ligand.

8. The digital tomography and CRISPR system based amplification-free ultrasensitive nucleic acid detection system according to claim 7, characterized in that: The method comprises the following steps:

9. A method for detection using the non-amplification ultrasensitive nucleic acid detection system according to any one of claims 1-8, characterized in that, Step one, adding the detection sample into the CRISPR reaction system for 5-30 minutes; Step two, adding the sample after the CRISPR reaction into the chromatographic reaction system for 5-15 minutes; Step three, acquiring the microscopic image of the detection area of the reaction membrane of the chromatographic reaction system on the optical imaging system, and counting the tracer nanoparticles in the detection area through the image processing system; Step four, calculating the concentration of the nucleic acid to be detected in the sample through the fitting relationship curve between the concentration of the calibration sample and the number of the tracer nanoparticles. ​

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