Streptavidin multimer, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of immunochromatography. Provided are a streptavidin multimer, and a preparation method therefor and the use thereof. The prepared streptavidin multimer has a strong affinity for a biotin / fluorescein nucleic acid probe. A nucleic acid test strip prepared with the prepared streptavidin multimer can realize rapid and high-sensitivity detection of a sample to be detected. Results can be obtained in 5 minutes, the false positive rate can be reduced, and the cost of the nucleic acid probe is reduced, which is convenient for non-professional technicians to easily and accurately judge the results.

Description

Streptavidin polymer and its preparation method and application Technical Field

[0001] The invention belongs to the technical field of immunochromatography and relates to a streptavidin polymer and a preparation method and application thereof. Background Art

[0002] Constant-temperature amplification combined with CRISPR technology is a highly promising molecular POCT testing platform. Rapid amplification combined with CRISPR-specific cleavage enables rapid, highly sensitive, and specific detection without sample extraction. However, compared with nucleic acid test strips, constant-temperature amplification-CRISPR fluorescence detection requires supporting instruments, while nucleic acid test strips do not require equipment and are easy to operate and convenient for grassroots personnel to use, better meeting the needs of clinical and on-site rapid testing. Constant-temperature amplification-CRISPR nucleic acid test strips can be integrated with the nucleic acid amplification device in a closed combination design, allowing amplification and detection to be completed in the same device, avoiding contamination caused by opening the lid during the sample input and result output process.

[0003] Nucleic acid test strips detect changes in biotin-fluorescein-labeled nucleic acid reporter probes. The isothermal amplification product activates the cleavage activity of the CRISPR effector proteins Cas12 / Cas13, bypassing the nucleic acid probe and releasing biotin and fluorescein. Fluorescent antibodies are then labeled with latex microspheres or colloidal gold. Streptavidin (SA) captures biotin at the T-line, causing it to aggregate and display a signal. After the biotin / fluorescein separates, the latex microspheres / colloidal gold cannot be captured by the T-line (SA), resulting in no color development. Currently, the isothermal amplification-CRISPR nucleic acid test strip development method places the T-line behind the C-line. Positive samples will display the T-line band, while negative samples will not. However, in practice, weakly positive samples will have unclear bands, thus interfering with result interpretation. In contrast, the nucleic acid test strip development method places the T-line at the front. In negative samples, the biotin / fluorescein nucleic acid probe is intact, resulting in a band on the T-line. In positive samples, the nucleic acid probe is cleaved, separating the biotin and fluorescein. Free biotin competes with the biotin / fluorescein nucleic acid probe for binding to the T-line (SA), eliminating the T-line band and ensuring a clear interpretation of a positive result.

[0004] Streptavidin is a protein derived from Streptomyces avidinii. It consists of four identical subunits, each capable of binding to one biotin molecule. It has a molecular weight of 65 kDa and an isoelectric point (pI) of 6.0. Streptavidin binds strongly to biotin, with a dissociation constant of approximately 10-15 M. This property has made the streptavidin-biotin system widely used in traditional biological techniques such as fluorescence microscopy, immunoelectron microscopy, flow cytometry, Western blotting, and ELISA, as well as in cutting-edge research fields such as biochips and nanobiology. Streptavidin lacks sugar modifications and has an isoelectric point close to physiological pH, resulting in significantly lower nonspecific adsorption and a higher signal-to-noise ratio.

[0005] The isothermal amplification system contains a high concentration of crowding agents, resulting in a viscous solution. The final product amount of single-copy target amplification varies, and Cas12 / 13 cleaves 1pM to 1nM of the biotin / fluorescein nucleic acid probe in 15 minutes. Conventional SA / fluorescein antibody detection of biotin / fluorescein nucleic acid probes has a sensitivity of around 1nM, leading to unstable detection of some weakly positive samples and false negatives. Therefore, it is necessary to increase the affinity of SA for biotin, so that negative samples clearly display T-lines, while weakly positive samples completely eliminate them. This also makes it easy for non-professional technicians to correctly interpret the results, thus meeting the needs of nucleic acid test strips.

[0006] Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a streptavidin polymer and a preparation method and application thereof.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] Streptavidin polymer, wherein the polymer monomer has the following structure:

[0010] Z1(-Z2-Z3) n ,in,

[0011] Z1 is the carrier and the polymer core;

[0012] Z2 is biotin;

[0013] Z3 is streptavidin;

[0014] “-” is a key and n represents a number.

[0015] Furthermore, the carrier is any one of BSA and Fc protein. When the carrier is BSA, the multimer is polySA-BSA; when the carrier is Fc, the multimer is polySA-mFc, and n is a positive integer of 4-6.

[0016] In order to achieve the above object, the present invention also provides the following technical solutions:

[0017] The preparation method of streptavidin polymer comprises the following steps:

[0018] Step S1: Biotin labeling of carrier protein

[0019] The carrier Z1 is mixed with biotin Z2 and incubated to obtain a "carrier-biotin" solution Z1(-Z2)n;

[0020] Step S2: Preparation of streptavidin polymer polySA

[0021] Add streptavidin Z3 to the "carrier-biotin" solution prepared in step S1 and incubate to obtain "polySA-carrier" solution Z1(-Z2-Z3) n .

[0022] Furthermore, the biotin Z2 is NHS-LC-biotin, and step S1 further includes the step of adding ethanolamine solution to react after incubation.

[0023] Furthermore, the molar ratio of the carrier Z1 to the biotin Z2 is 1:2-1:10.

[0024] Furthermore, the coupling ratio of the carrier-biotin and streptavidin Z3 is 1:4 to 1:6.

[0025] Furthermore, in step S1, the incubation temperature of the carrier Z1 and NHS-LC-biotin was 25° C., and the incubation time was 1 h.

[0026] Furthermore, the ethanolamine solution in step S1 is 10 μL of 3 M ethanolamine solution.

[0027] Furthermore, the reaction time after adding the ethanolamine solution in step S1 is 30 minutes.

[0028] Furthermore, the incubation method in step S2 is rotation mixing incubation, and the rotation mixing incubation conditions are as follows: 25° C. 50 rpm, incubation for 3 hours.

[0029] To achieve the above-mentioned object, the present invention further provides the following technical solution: application of streptavidin polymers in the preparation of nucleic acid chromatography detection products.

[0030] Furthermore, the T-line of the chromatographic detection product is coated with a streptavidin polymer.

[0031] Furthermore, the chromatography detection product is a CRISPR elimination method nucleic acid test paper, and the CRISPR system contains a nucleic acid reporter probe labeled with biotin as a sample solution.

[0032] Furthermore, the nucleic acid reporter probe is also labeled with a fluorescent group.

[0033] Furthermore, the preparation method of the CRISPR elimination method nucleic acid test paper is as follows:

[0034] Step A: Fluorescent antibody labeling of microspheres

[0035] labeling the latex microspheres with antibodies that can bind to the fluorescent group of the nucleic acid reporter probe;

[0036] Step B: Sample pad treatment

[0037] Step C: Conjugate Pad Treatment

[0038] Spray the fluorescent antibody-labeled microspheres prepared in step A onto the conjugate pad;

[0039] Step D: NC membrane coating

[0040] Use a membrane streaking device to spray C lines and T lines on the NC membrane, and coat the T lines with streptavidin polymers;

[0041] Step E: Nucleic acid test strip assembly

[0042] The sample pad, conjugate pad, NC membrane and absorbent pad processed in the previous steps are sequentially pasted on the bottom plate to obtain the CRISPR elimination method nucleic acid test strip.

[0043] Furthermore, the method for detecting nucleic acid by the chromatography detection product is as follows:

[0044] If the sample to be tested contains the target nucleic acid, the nucleic acid reporter probe will be cleaved in the CRISPR system, and will not bind to the streptavidin polymer when passing through the T line, the T line will not show color, and the report is positive; if the sample to be tested does not contain the target nucleic acid, the nucleic acid reporter probe will not be cleaved in the CRISPR system, and will bind to the streptavidin polymer when passing through the T line, the latex microspheres will precipitate the T line and show color, and the report is negative.

[0045] To achieve the above-mentioned purpose, the present invention also provides a CRISPR elimination method nucleic acid test paper.

[0046] The beneficial effects of the present invention are as follows: the streptavidin polymer prepared by the present invention has a strong affinity with the biotin / fluorescein nucleic acid probe, and the nucleic acid test paper prepared using the streptavidin polymer prepared by the present invention can achieve rapid and high-sensitivity detection of the sample to be tested, and the result can be obtained in 5 minutes, which can reduce the false positive rate and the cost of the nucleic acid probe, making it convenient for non-professional technicians to easily and accurately judge the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0048] FIG1 is a non-denaturing electrophoresis diagram of poly-SA according to an embodiment of the present invention;

[0049] FIG2 is a diagram showing the principle of detection of the “line elimination method” according to an embodiment of the present invention;

[0050] FIG3 is a comparison of the sensitivity of nucleic acid test strips for detecting TB according to an embodiment of the present invention;

[0051] FIG4 and FIG5 are both schematic diagrams of the structures of streptavidin polymers according to embodiments of the present invention. DETAILED DESCRIPTION

[0052] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0053] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0054] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0055] In this example, the carrier protein was first biotin-labeled. Bovine serum albumin (BSA) was dissolved in PBS buffer to 2 mg / mL. BSA and NHS-LC-biotin (Thermo) were mixed at a molar ratio of 1:5, 1:10, and 1:20, and incubated at 25°C for 1 hour. Then, 10 μL of 3M ethanolamine solution was added and reacted for 30 minutes. The BSA-biotin marker was dialyzed into PBS solution using a 10 kD dialysis bag and the OD value was calculated. 280 Determine marker concentration.

[0056] The purity of the Fc protein (Luoyang Bioton) in this example was above 95%. The Fc protein was diluted to 1 mg / ml with PBS pH 7.4 buffer, and Fc and NHS-LC-biotin (Thermo) were mixed at a molar ratio of 1:5, 1:10, and 1:20, incubated at 25°C for 1 hour, and 10 μL of 3M ethanolamine solution was added to react for 30 minutes. The Fc-biotin marker was dialyzed into PBS solution through a 10 kD dialysis bag and the OD 280 Determine marker concentration.

[0057] Streptavidin SA was then dissolved in PBS buffer to 2 mg / mL. The SA solution was added to the previously prepared BSA-biotin solution at BSA-biotin:SA ratios of 1:2, 1:4, 1:6, and 1:10, and the mixture was mixed and incubated at 25°C and 50 rpm for 3 hours to obtain the streptavidin polymer polySA-BSA. 5 μg of the 1:4 ratio-coupled polySA-BSA was subjected to 8% gel non-denaturing PAGE electrophoresis. As shown in Figure 1, the purity of the polySA-BSA reached 90%, with no free BSA protein in the electrophoretic band. The molecular weight was mainly around 300 kd. Calculated by molar ratio, the polymerization ratio of polySA-BSA was close to 1:4, that is, each BSA carrier protein was coupled to 4 SA molecules.

[0058] Next, polySA-Fc was prepared using an IgG-Fc fragment, using the same method as polySA-BSA. The conjugation ratios of Fc-biotin to SA were 1:2, 1:4, 1:6, and 1:10, ultimately yielding a polySA-Fc polymer protein. 5 μg of the 1:4 conjugated polySA-mFc was subjected to 8% native polyacrylamide gel electrophoresis (Figure 1). The purity of the polySA-Fc reached 90%, with no free Fc protein present in the electrophoretic band. The molecular weight was primarily around 300 kd. Calculated on a molar basis, the polymerization ratio of the polySA-Fc polymer was close to 1:4, meaning each Fc protein could be conjugated to four SA molecules.

[0059] The amino acid sequence of SA used in this example is as follows: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS.

[0060] The amino acid sequence of BSA used in this example is as follows:

[0061] The amino acid sequence of Fc used in this example is as follows: KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0062] The streptavidin polymer prepared in this example has the following structure: Z1(-Z2-Z3)n, wherein Z1 is a carrier and the core of the polymer; Z2 is biotin; Z3 is streptavidin; "-" is a bond, and n represents a number.

[0063] See Figures 4 and 5, which are schematic diagrams of the structure of the streptavidin multimer prepared in this example. Protein in Figure 5 represents the carrier Z1 (BSA or Fc). It should be noted that the streptavidin multimer prepared in this example does not have a chain structure as shown in Figure 5. Figure 5 is only used to show a portion of the chemical formula of the multimer prepared in this example to those skilled in the art. The multimer prepared in this example has a branched structure centered on the carrier, as shown in Figure 4.

[0064] Next, this example uses the above-mentioned polySA-BSA and polySA-Fc to prepare a constant temperature amplification-CRISPR elimination method nucleic acid test paper. The preparation method is as follows:

[0065] Step A: Fluorescent antibody labeling of microspheres

[0066] 200 nm red latex microspheres were labeled with rabbit anti-FITC antibody at a ratio of 1:10;

[0067] Step B: Sample pad treatment

[0068] The glass fiber was soaked in a solution containing 10 mM Tris, 0.1% Casein, 0.1% Tween-20, 0.1% PVP, and 5% sucrose at pH 8.6 for 2 hours and then dried at 50°C for 4 hours;

[0069] Step C: Conjugate Pad Treatment

[0070] Adjust the concentration of the fluorescent antibody-labeled microspheres prepared in step A to 1 mg / ml and spray them onto the conjugate pad using a gold sprayer at a spraying parameter of 10 μL / cm. Then dry them at 45°C for 3 hours.

[0071] Step D: NC membrane coating

[0072] A 2.5-cm-long CN120 NC membrane was coated with a PBS solution containing 2% trehalose. A film streaker was used to spray C and T lines onto the membrane at a rate of 0.8 μL / cm. The C line was coated with 2 mg / mL goat anti-mouse IgG, and the T line was coated with polySA-BSA, 1.5 mg / mL SA solution, or polySA-mFc solution. The concentrations of polySA-BSA and polySA-Fc were calculated based on the SA molar concentration. The membrane was then dried at 37°C for 4 hours.

[0073] Step E: Nucleic acid test strip assembly

[0074] After the sample pad, conjugate pad, NC membrane, and absorbent pad processed in the previous steps are sequentially attached to the base plate, they are cut into 3 mm widths to obtain a constant temperature amplification-CRISPR nucleic acid test strip. The schematic diagram of the test strip setup and the detection principle are shown in Figure 2.

[0075] This example conducted an affinity evaluation experiment on the isothermal amplification-CRISPR nucleic acid test strip prepared above. The main process is described below.

[0076] 50 μL of the nucleic acid reporter probe standard was diluted to 1.5 mL in an Eppendorf tube, placed on a nucleic acid test strip, and the results were recorded after 5 minutes. As shown in Table 1, the results show that the SA biotin / fluorescein nucleic acid probe exhibited a positive color band at 2.7 nM. When conjugated with polySA-BSA in a 1:4 ratio, 100 pM of the nucleic acid probe exhibited a positive band of 2+, and the 1:4 conjugation had greater activity than the 1:6 conjugation, which was greater than the 1:2 conjugation. When conjugated with polySA-Fc in a 1:4 ratio, 100 pM of the nucleic acid probe exhibited a positive band of 3+, and the 1:4 and 1:6 conjugation ratios were similar, with higher affinity than the 1:2 conjugation. Complexes were prepared at molar ratios of 1:1 / 1:2 / 1:4 / 1:6, and 1:10, with optimal detection results achieved at a conjugation ratio of 1:4 to 1:6. When polySA is coupled at a ratio of 1:2, the number of SA molecules coupled to the carrier protein is insufficient due to insufficient SA concentration; when the coupling ratio is 1:10, SA is excessive, resulting in the presence of free SA, which interferes with polySA coating and slightly reduces affinity.

[0077] Table 1 Comparison of polySA-nucleic acid probe affinities "-" indicates negative, with no bands on the T line. "+" indicates positive, with bands on the T line. The numbers indicate the relative brightness of the bands.

[0078] Based on the above results and cost considerations, this example selected carrier protein: streptavidin SA in a ratio of 1:4 and 1:6 to prepare polySA-BSA and polySA-Fc to prepare nucleic acid test paper, which was used to detect samples.

[0079] In this example, Mycobacterium tuberculosis IS6110 was used as the target nucleic acid, with target concentrations set at 0, 1, 10, and 100 copies. The upstream primer was: TCGGAAGCTCCTATGACAATGCACTAGCCG; the downstream primer was: GGCCAACTCGACATCCTCGATGGACCGCCA. RAA amplification reagent, manufactured by Shanghai Liangrun Biopharmaceutical Technology Co., Ltd., was added according to the system shown in Table 2 and reacted at 42°C for 20 minutes. Then, 4 μL of the amplified product was diluted 375-fold with sterile water. 5 μL of the isothermal amplified product was added to the CRISPR system (Table 3) and reacted at 37°C for 15 minutes.

[0080] Place the nucleic acid test paper into the reaction solution and observe the results after 5 minutes.

[0081] If the test sample contains the target nucleic acid, the nucleic acid reporter probe will be cleaved by the CRISPR system. The resulting reaction system, when passing through the sample pad of the "line elimination method" immunochromatographic test strip, binds to the colloidal gold-labeled rabbit anti-FITC antibody, resulting in a system containing colloidal gold-labeled rabbit anti-FITC antibody-FAM group. When passing through the T line, it does not bind to streptavidin, and the T line does not develop color. If the test sample does not contain the target nucleic acid, the nucleic acid reporter probe will not be cleaved by the CRISPR system. The resulting reaction system, when passing through the sample pad of the "line elimination method" immunochromatographic test strip, binds to the colloidal gold-labeled rabbit anti-FITC antibody, resulting in a system containing colloidal gold-labeled rabbit anti-FITC antibody-FAM group-biotin. When passing through the T line, the biotin on the colloidal gold-labeled rabbit anti-FITC (fluorescein) antibody-FAM group-biotin binds to the streptavidin on the T line, and the latex microspheres sediment the T line to develop color. The principle is shown in Figure 2.

[0082] The results are shown in Table 4 and Figure 3. The detection sensitivity of the polySA-BSA (coupling ratio 1:4) nucleic acid test paper is 1 copy, while the detection sensitivity of the SA nucleic acid test paper is 10 copies, with a difference of 10 times; the detection sensitivity of the PolySA-Fc (coupling ratio 1:4) nucleic acid test paper reaches 1 copy. It can be seen that the detection sensitivity of polySA-BSA and polySA-Fc nucleic acid test papers is better than that of SA.

[0083] Table 2 RAA amplification system

[0084] Table 3 Cas12a reaction system

[0085] Table 4 Detection performance of nucleic acid test paper Elimination method: "-" indicates negative, with a band on the T line. "+" indicates positive, with no band on the T line.

[0086] This example also tested sputum samples using the aforementioned nucleic acid test strips. Ten TB-positive sputum samples and 10 TB-negative control samples were compared with a fluorescent PCR kit. The test strips were used using a Jiangsu Weizhen Biotech TB constant-temperature amplification-CRISPR nucleic acid test strip device, using the same constant-temperature amplification system as the aforementioned sample amplification system. As shown in Table 5, the results show that the polySA-BSA / Fc nucleic acid test strips prepared with a 1:4 or 1:6 coupling ratio achieved a 100% coincidence rate (10 / 10) for positive TB constant-temperature amplified CRISPR samples and a 100% coincidence rate (30 / 30) for negative TB samples, consistent with qPCR performance.

[0087] Table 5 Comparison of constant temperature amplification-CRISPR nucleic acid test paper and PCR test results Elimination method: "-" indicates negative, with a band on the T line. "+" indicates positive, with no band on the T line.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A streptavidin multimer, characterized in that, The polymer monomer has the structure shown below: Z1(-Z2 - Z3) n , wherein, Z1 is a carrier and is the core of the polymer; Z2 is biotin; Z3 is streptavidin; "-" represents a bond, and n represents a number.

2. The streptavidin multimer according to claim 1, characterized in that, The carrier is any one of BSA and Fc protein. When the carrier is BSA, the polymer is polySA-BSA. When the carrier is Fc, the polymer is polySA-mFc, and n is a positive integer from 4 to 6.

3. The method for preparing the streptavidin multimer according to any one of claims 1-2, characterized in that, It includes the following steps: Step S1: Biotinylation of the carrier protein Mix the carrier Z1 with biotin Z2 and incubate them to obtain the "carrier-biotin" solution Z1(-Z2). n ; Step S2: Preparation of streptavidin polymer polySA Add streptavidin Z3 to the "carrier-biotin" solution prepared in step S1 and incubate to obtain the "polySA-carrier" solution Z1 (-Z2-Z3). n .

4. The preparation method according to claim 3, characterized in that, The biotin Z2 is NHS-LC-biotin, and step S1 also includes the step of adding an ethanolamine solution for reaction after incubation.

5. The preparation method according to claim 4, characterized in that, The mixing molar ratio of the carrier Z1 and biotin Z2 is 1:2 - 1:

10.

6. The preparation method according to claim 3, wherein The coupling ratio of the carrier-biotin and streptavidin Z3 is 1:4 to 1:

6.

7. The preparation method according to claim 4, characterized in that, In step S1, the incubation temperature of the carrier Z1 and NHS-LC-biotin is 25°C, and the incubation time is 1 h for both.

8. The preparation method according to claim 4, characterized in that, The ethanolamine solution in step S1 is 10 μL of 3 M ethanolamine solution.

9. The preparation method according to claim 4, characterized in that, The reaction time after adding the ethanolamine solution in step S1 is 30 min.

10. The preparation method according to claim 3, characterized in that, The incubation method in step S2 is rotary mixing incubation, and the rotary mixing incubation conditions are as follows: 25°C, 50 rpm, and incubation for 3 h.

11. Use of the streptavidin polymer according to any one of claims 1 - 2 in the preparation of a nucleic acid chromatography detection product.

12. The application according to claim 11, wherein The T line of the chromatography detection product is coated with the streptavidin polymer.

13. The application according to claim 12, wherein The chromatography detection product is a CRISPR line-eliminating nucleic acid test strip, and the CRSIPR system is used as the sample loading solution and contains a nucleic acid reporter probe labeled with biotin.

14. The application according to claim 13, characterized in that, The nucleic acid reporter probe is also labeled with a fluorescent group.

15. The application according to claim 14, characterized in that, The preparation method of the CRISPR line-eliminating nucleic acid test strip is as follows: Step A: Labeling of microspheres with fluorescent antibodies Label latex microspheres with an antibody that can bind to the fluorescent group of the nucleic acid reporter probe; Step B: Sample pad treatment Step C: Conjugate pad treatment Spray the fluorescent antibody-labeled microspheres prepared in step A onto the conjugate pad; Step D: NC membrane coating Use a membrane dispenser to spray line C and line T on the NC membrane, and line T is coated with the streptavidin polymer; Step E: Assembly of the nucleic acid test strip Paste the sample pad, conjugate pad, NC membrane, and absorbent pad treated in the previous steps onto the bottom plate in sequence to obtain the CRISPR line-eliminating nucleic acid test strip.

16. The application according to claim 15, wherein The method for the chromatography detection product to detect nucleic acid is as follows: If the target nucleic acid is contained in the sample to be tested, the nucleic acid reporter probe will be cleaved in the CRISPR system and will not bind to the streptavidin polymer when passing through line T, so line T does not show color, reporting positive; if the target nucleic acid is not contained in the sample to be tested, the nucleic acid reporter probe will not be cleaved in the CRISPR system and will bind to the streptavidin polymer when passing through line T, and the latex microspheres will settle and line T will show color, reporting negative.

17. A CRISPR nucleic acid test strip for eliminating lines, characterized in that, The test strip is coated with the streptavidin polymer according to any one of claims 1 - 2.

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