Streptavidin polymer as well as preparation method and application thereof

Abstract

The invention relates to a streptavidin polymer as well as a preparation method and application thereof, and belongs to the technical field of immunochromatography. The prepared streptavidin polymer has strong affinity with a biotin / fluorescein nucleic acid probe, nucleic acid test paper prepared from the prepared streptavidin polymer can realize rapid and high-sensitivity detection on a detected sample, a result can be obtained within 5 minutes, the false positive rate can be reduced, the cost of the nucleic acid probe is reduced, and the method is suitable for popularization and application. And non-professional technicians can conveniently, easily and accurately judge the result.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochromatography, and relates to streptavidin polymers, a preparation method thereof and applications thereof. Background Art

[0002] The isothermal amplification combined with CRISPR technology is a highly promising molecular POCT detection platform. Rapid amplification combined with CRISPR specific cleavage enables sample-free extraction, rapid, highly sensitive and specific detection. However, compared with nucleic acid test strip detection, isothermal amplification-CRISPR fluorescence detection requires supporting instruments, while nucleic acid test strip detection does not require equipment, and is simple to operate and convenient for grass-roots personnel to use, which can better meet the needs of clinical and on-site rapid detection. The isothermal amplification-CRISPR nucleic acid test strip can be integrally designed in a closed combination with a nucleic acid amplification device, so that amplification and detection are completed in the same device, and contamination caused by opening the lid is avoided during the detection process from sample input to result output.

[0003] The nucleic acid test strip detects the change of a biotin-fluorescein-labeled nucleic acid reporter probe. The isothermal amplification product activates the cleavage activity of the CRISPR effector protein Cas12 / Cas13, which bypasses and cleaves the nucleic acid probe to release biotin and fluorescein. A latex microsphere or colloidal gold labeled with a fluorescein antibody is used. Streptavidin SA at the T line captures biotin and aggregates to show a signal. After the separation of biotin / fluorescein, the latex microsphere / colloidal gold cannot be captured by the T line SA and does not show color. At present, in the isothermal amplification-CRISPR nucleic acid test strip line display method, the T line is placed behind the C line. A positive sample makes the T line strip show color, while a negative sample does not make the T line strip show color. However, in actual detection, the strip of a weakly positive sample will be unclear, thus interfering with the result judgment. In the nucleic acid test strip line disappearance method, the T line is at the front end. For a negative sample, the biotin / fluorescein nucleic acid probe is intact and the T line shows a strip; for a positive sample, the nucleic acid probe is cleaved, biotin and fluorescein are separated, and the free biotin competes with the biotin / fluorescein nucleic acid probe for binding to the T line SA, and the T line does not show a strip, and the positive result interpretation is clear.

[0004] Streptavidin is a protein derived from Streptomyces avidinii, which is composed of four identical subunits. Each subunit can bind a biotin molecule, with a molecular weight of 65Kd and an isoelectric point pI of 6.0. The binding force between streptavidin and biotin is extremely strong, and its dissociation constant is about 10-15M. This characteristic makes the streptavidin-biotin system widely used in traditional biological technologies such as fluorescence microscopy, immunoelectron microscopy, flow cytometry analysis, Western blotting, ELISA detection, etc. and modern frontier research fields such as biochips and nanobiology. Streptavidin does not contain glycosylation modification, and its isoelectric point is close to the physiological pH value, so its non-specific adsorption is significantly lower than the former, and a higher detection signal-to-noise ratio can be provided.

[0005] The isothermal amplification system contains a high concentration of crowding agent, and the solution is viscous. There are differences in the amount of amplification end products of single-copy targets. The biotin / fluorescein nucleic acid probe is cleaved by Cas12 / 13 at 1 pM to 1 nM for 15 minutes. The sensitivity of the conventional SA / fluorescein antibody to detect the biotin / fluorescein nucleic acid probe is around 1 nM, resulting in unstable detection of some weak-positive samples and false negatives. Therefore, it is necessary to improve the affinity between SA and biotin to make the negative samples clearly show the T line, make the weak-positive samples completely disappear the line, and enable non-professional technicians to easily and correctly judge the results to meet the use requirements of nucleic acid test strips. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a streptavidin multimer and its preparation method and application.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A streptavidin multimer, the monomer of the multimer has the following structure:

[0009] Z1(-Z2-Z3) n , wherein,

[0010] Z1 is a carrier and is the core of the multimer;

[0011] Z2 is biotin;

[0012] Z3 is streptavidin;

[0013] "-" is a bond, and n represents a number.

[0014] 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 from 4 to 6.

[0015] To achieve the above purpose, the present invention also provides the following technical solutions:

[0016] A preparation method of a streptavidin multimer, comprising the following steps:

[0017] Step S1: Biotinylation of the carrier protein

[0018] Mix and incubate the carrier Z1 with biotin Z2 to obtain a "carrier-biotin" solution Z1(-Z2)n;

[0019] Step S2: Preparation of the streptavidin multimer polySA

[0020] Add streptavidin Z3 to the "carrier - biotin" solution prepared in step S1 and incubate to obtain the "polySA - carrier" solution Z1(-Z2 - Z3). n .

[0021] Furthermore, the biotin Z2 is NHS - LC - biotin, and step S1 further includes a step of adding an ethanolamine solution for reaction after incubation.

[0022] Furthermore, the mixing molar ratio of the carrier Z1 to biotin Z2 is 1:2 - 1:10.

[0023] Furthermore, the coupling ratio of the carrier - biotin to streptavidin Z3 is 1:4 to 1:6.

[0024] Furthermore, 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.

[0025] Furthermore, the ethanolamine solution in step S1 is 10 μL of 3 M ethanolamine solution.

[0026] Furthermore, the reaction time after adding the ethanolamine solution in step S1 is 30 min.

[0027] Furthermore, the incubation method in step S2 is rotary mixing incubation, and the rotary mixing incubation conditions are as follows: 25°C, 50 rpm, and incubate for 3 h.

[0028] To achieve the above - mentioned purpose, the present invention also provides the following technical solution: the application of streptavidin polymer in the preparation of nucleic acid chromatography detection products.

[0029] Furthermore, the T - line of the chromatography detection product is coated with streptavidin polymer.

[0030] Furthermore, the chromatography detection product is a CRISPR line - disappearing nucleic acid test strip, and the CRSIPR system contains a nucleic acid reporter probe labeled with biotin as the sample loading solution.

[0031] Furthermore, the nucleic acid reporter probe is also labeled with a fluorescent group.

[0032] Furthermore, the preparation method of the CRISPR line - disappearing nucleic acid test strip is as follows:

[0033] Step A: Fluorescent antibody - labeled microspheres

[0034] Label latex microspheres with an antibody that can bind to the fluorescent group of the nucleic acid reporter probe;

[0035] Step B: Sample pad treatment

[0036] Step C: Conjugate pad treatment

[0037] Spray the fluorescent antibody-labeled microspheres prepared in Step A onto the conjugate pad;

[0038] Step D: NC membrane coating

[0039] Use a membrane scribing instrument to spray the C line and T line on the NC membrane, and coat the T line with streptavidin polymer;

[0040] Step E: Nucleic acid test strip assembly

[0041] Paste the sample pad, conjugate pad, NC membrane, and absorbent pad processed in the previous steps onto the bottom plate in sequence to obtain the CRISPR line-erasing nucleic acid test strip.

[0042] Furthermore, the method for the chromatographic detection product to detect nucleic acid is as follows:

[0043] 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 the T line, and the T line will 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 the T line, and the latex microspheres will settle and the T line will show color, reporting negative.

[0044] To achieve the above object, the present invention also provides a CRISPR line-erasing nucleic acid test strip.

[0045] The beneficial effects of the present invention are as follows: The streptavidin polymer prepared by the present invention has a strong affinity for biotin / fluorescein nucleic acid probes. The nucleic acid test strip prepared by using the streptavidin polymer prepared by the present invention can achieve rapid and highly sensitive detection of the sample to be detected, and the result can be obtained in 5 minutes. It can reduce the false positive rate, reduce the cost of nucleic acid probes, and facilitate non-professional technicians to easily and accurately judge the result. Description of the drawings

[0046] In order to make the objects, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail and preferably below in conjunction with the drawings, wherein:

[0047] Figure 1 It is the non-denaturing electrophoresis pattern of poly SA in the embodiment of the present invention;

[0048] Figure 2 It is the schematic diagram of the "line-erasing method" detection principle in the embodiment of the present invention;

[0049] Figure 3 It is the comparison result of the sensitivity of the nucleic acid test strip in the embodiment of the present invention for detecting TB;

[0050] Figure 4 andFigure 5 These are schematic structural diagrams of streptavidin polymers of embodiments of the present invention. Detailed implementation manners

[0051] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0052] Among them, the drawings are only for illustrative purposes, showing only schematic diagrams, not physical diagrams, and should not be construed as a limitation of the present invention; in order to better illustrate the embodiments of the present invention, some components in the drawings will be omitted, enlarged or reduced, which does not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0053] In the drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "rear", etc. indicating the orientation or positional relationship, they are based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the positional relationship in the drawings are only for illustrative purposes and should not be construed as a limitation of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0054] In this embodiment, carrier protein biotinylation is first carried out. Bovine serum albumin BSA is dissolved in PBS buffer to 2 mg / mL, and BSA and NHS-LC-biotin (Thermo) are mixed at a molar ratio of 1:5, 1:10, 1:20, incubated at 25 °C for 1 h, and then 10 μL of 3 M ethanolamine solution is added to react for 30 minutes. The BSA-biotin conjugate is dialyzed into PBS solution through a 10 kD dialysis bag, and the concentration of the conjugate is measured by OD 280 to determine the concentration of the conjugate.

[0055] The purity of the Fc protein (Luoyang Bio-Tech) in this example is above 95%. Dilute the Fc protein to 1 mg / ml with PBS pH 7.4 buffer. Mix Fc and NHS-LC-biotin (Thermo) at molar ratios of 1:5, 1:10, and 1:20, incubate at 25 °C for 1 h, and add 10 μL of 3 M ethanolamine solution and react for 30 minutes. The Fc-biotin conjugate is dialyzed against PBS solution through a 10 kD dialysis bag, and the concentration of the conjugate is determined by OD 280 measurement.

[0056] Subsequently, dissolve streptavidin SA in PBS buffer to 2 mg / mL. According to the ratios of BSA-biotin:SA of 1:2, 1:4, 1:6, and 1:10, add the SA solution to the previously prepared BSA-biotin solution, mix well by rotation at 50 rpm at 25 °C and incubate for 3 h to obtain the streptavidin polymer polySA-BSA. Take 5 μg of polySA-BSA conjugated at a ratio of 1:4 and perform 8% native PAGE electrophoresis. See attached Figure 1 , the purity of polySA-BSA reaches 90%, there is no free BSA protein in the electrophoresis band, and the molecular weight is mainly around 300 kD. Calculated according to the molar ratio, the polymerization ratio of polySA-BSA is close to 1:4, that is, each BSA carrier protein is conjugated with 4 SA molecules.

[0057] Next, prepare polySA-Fc. The method for preparing polySA-Fc using the IgG-Fc fragment is the same as that for preparing polySA-BSA. Among them, the coupling ratio of Fc-biotin to SA is 1:2, 1:4, 1:6, 1:10, and finally obtain the polySA-Fc polymer protein. Take 5 μg of polySA-mFc conjugated at a ratio of 1:4 and perform 8% native PAGE electrophoresis. See attached Figure 1 , the purity of polySA-Fc reaches 90%, there is no free Fc protein in the electrophoresis band, and the molecular weight is mainly around 300 kD. Calculated according to the molar ratio, the polymerization ratio of polySA-Fc is close to 1:4, that is, each Fc protein can be conjugated with 4 SA molecules.

[0058] The amino acid sequence of SA used in this example is as follows: MAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS.

[0059] The amino acid sequence of BSA used in this example is as follows: MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA。

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

[0061] The streptavidin multimer prepared in this example has the following structure: Z1(-Z2-Z3)n, where Z1 is a carrier and the core of the multimer; Z2 is biotin; Z3 is streptavidin; "-" is a bond, and n represents a number.

[0062] See the appendix Figure 4 and the appendix Figure 5 , which is a schematic structural diagram of the streptavidin multimer prepared in this example. In the appendix Figure 5 , Protein represents the carrier Z1 (BSA or Fc). It should be noted that the streptavidin multimer prepared in this example is not the chain structure shown in the appendix Figure 5 , but only a part of the chemical formula of the multimer prepared in this example is shown to those skilled in the art. The multimer prepared in this example is a branched structure centered on the carrier, as shown in the appendix Figure 5 . Figure 4 Shown

[0063] Next, this example uses the above polySA-BSA and polySA-Fc to prepare a nucleic acid test strip for isothermal amplification-CRISPR line elimination method. The preparation method is as follows:

[0064] Step A: Fluorescent antibody-labeled microspheres

[0065] Label 200nm red latex microspheres with rabbit anti-FITC antibody at a ratio of 1:10;

[0066] Step B: Sample pad treatment

[0067] Soak glass fiber in a solution with a pH of 8.6 containing 10 mM Tris, 0.1% Casein, 0.1% Tween-20, 0.1% PVP, and 5% sucrose for 2 hours, and then dry at 50°C for 4 hours;

[0068] Step C: Conjugate pad treatment

[0069] Adjust the concentration of the fluorescent antibody-labeled microspheres prepared in step A to 1 mg / ml, and then spray them on the conjugate pad with a gold-spraying and membrane-drawing instrument at a spraying parameter of 10 μL / cm, and then dry at 45°C for 3 hours;

[0070] Step D: NC membrane coating

[0071] Take a 2.5 cm long CN120 NC membrane, coat the NC membrane with a PBS solution containing 2% trehalose, and spray the C line and T line on the NC membrane with a membrane-drawing instrument at a speed of 0.8 μL / cm. The C line is coated with 2 mg / mL goat anti-mouse IgG, and the T line is coated with polySA-BSA or 1.5 mg / mL SA solution or polySA-mFc solution. Among them, polySA-BSA and polySA-Fc are calculated according to the SA molar concentration, and then dry at 37°C for 4 hours;

[0072] Step E: Assembly of nucleic acid test strip

[0073] After pasting the sample pad, conjugation pad, NC membrane, and absorbent pad processed in the previous steps onto the base plate in sequence, and then cutting them according to a width of 3 mm, a constant-temperature amplification-CRISPR nucleic acid test strip is obtained. The schematic diagram of the test strip settings and the detection principle are shown in the appendix. Figure 2 .

[0074] In this example, an evaluation experiment on the affinity of the prepared constant-temperature amplification-CRISPR nucleic acid test strip was carried out. The main process is as follows.

[0075] Take 50 μL of the nucleic acid reporter probe standard to an Ep tube of 1.5 mL, put it into the nucleic acid test strip for detection, and record the results after 5 minutes. As shown in Table 1, when the SA biotin / fluorescein nucleic acid probe is 2.7 nM, a positive color band appears. When polySA-BSA is coupled at a ratio of 1:4, a positive band 2+ appears for the 100 pM nucleic acid probe, and the coupling activity of 1:4 > 1:6 > 1:2 coupling effect. When polySA-Fc is coupled at a ratio of 1:4, a positive band 3+ appears for the 100 pM nucleic acid probe, and the coupling activities of 1:4 and 1:6 are close, and the affinity is higher than that of the 1:2 coupling effect. Complexes are prepared at molar ratios of 1:1 / 1:2 / 1:4 / 1:6, 1:10. When the coupling ratio is 1:4 - 1:6, the optimal detection results are achieved. When polySA is coupled at a ratio of 1:2, due to insufficient SA concentration, the number of SA molecules coupled to the carrier protein is insufficient; when coupled at a ratio of 1:10, SA is in excess, resulting in the presence of free SA, interfering with polySA coating, and the affinity is slightly reduced.

[0076] Table 1 Comparison of the affinity of polySA-nucleic acid probes

[0077]

[0078] "-" indicates negative, and there is no band on the T line. "+" indicates positive, and a band appears on the T line, and the number indicates the relative brightness of the band.

[0079] Based on the results above and cost considerations, in this example, the carrier proteins streptavidin SA at ratios of 1:4 and 1:6 are selected to prepare polySA-BSA and polySA-Fc to prepare nucleic acid test strips and used to detect samples.

[0080] In this example, Mycobacterium tuberculosis IS6110 was used as the target nucleic acid, and the target concentrations were set to 0, 1, 10, and 100 copies. The upstream primer was: TCGGAAGCTCCTATGACAATGCACTAGCCG; the downstream primer was: GGCCAACTCGACATCCTCGATGGACCGCCA. The RAA amplification reagent was produced by Shanghai Liangrun Biomedical Technology Co., Ltd. The components were added according to the system shown in Table 2, and the reaction was carried out at 42 °C for 20 min; then 4 μL of the amplification product was taken and diluted 375 times with sterile water, and 5 μL of the isothermal amplification product was added to the CRISPR system (Table 3) and reacted at 37 °C for 15 min.

[0081] Put the nucleic acid test strip into the reaction solution and observe the result after 5 minutes.

[0082] 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; when the obtained reaction system passes through the sample pad of the "line elimination method" immunochromatographic test strip, it binds to the colloidal gold-labeled rabbit anti-FITC antibody, and a system containing the colloidal gold-labeled rabbit anti-FITC antibody-FAM group is obtained. When passing through the T line, it does not bind to streptavidin and the T line does not show color. 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. When the obtained reaction system passes through the sample pad of the "line elimination method" immunochromatographic test strip, it binds to the colloidal gold-labeled rabbit anti-FITC antibody, and a system containing the colloidal gold-labeled rabbit anti-FITC antibody-FAM group-biotin is obtained. 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 settle and the T line shows color. The principle is as Figure 2 shown.

[0083] The results are shown in Table 4 and Appendix Figure 3 , the detection sensitivity of the polySA-BSA (coupling ratio 1:4) nucleic acid test strip is 1 copy, while the detection sensitivity of the SA nucleic acid test strip is 10 copies, and the detection sensitivity difference is 10 times; the detection sensitivity of the PolySA-Fc (coupling ratio 1:4) nucleic acid test strip reaches 1 copy. It can be seen that the detection sensitivities of the polySA-BSA and polySA-Fc nucleic acid test strips are better than that of SA.

[0084] Table 2 RAA amplification system

[0085] 1×RAA Mix / Polyethylene glycol (PEG) 29.7 μL Forward primer F (100 μM) 0.125 μL Reverse primer R (100 μM) 0.125 μL IS6110 5 μL buffer 2.5ul <![CDATA[DEPC-H2O]]> Make up to 50 μL

[0086] Table 3 Cas12a reaction system

[0087]

[0088] Table 4 Detection performance of nucleic acid test strips

[0089]

[0090] Line elimination method: "-" indicates negative, and there is a band on the T line. "+" indicates positive, and there is no band on the T line.

[0091] In this embodiment, the above nucleic acid test strip was also used to detect sputum samples. Among them, 10 TB-positive sputum samples and 10 TB-negative control samples were compared with a fluorescence PCR kit. The Jiangsu Weizhen Biological TB isothermal amplification-CRISPR nucleic acid test strip device was used, and the isothermal amplification system was the same as the aforementioned sample amplification system. As shown in Table 5, the results showed that the polySA-BSA / Fc nucleic acid test strips prepared with a coupling ratio of 1:4 or 1:6 had a positive coincidence rate of 100% (10 / 10) and a negative coincidence rate of 100% (30 / 30) for detecting TB isothermal amplification CRISPR samples, which was consistent with the performance of qPCR.

[0092] Table 5 Comparison of the results of isothermal amplification-CRISPR nucleic acid test strips and PCR

[0093]

[0094] Line elimination method: "-" indicates negative, and there is a band on the T line. "+" indicates positive, and there is no band on the T line.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by 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 , where, Z1 is a carrier and is the polymer core; Z2 is biotin; Z3 is streptavidin; "-" represents a bond, and n represents a number.

2. The streptavidin multimer according to claim 1, wherein 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 further includes the step of adding an ethanolamine solution for reaction after incubation.

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

10.

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

6.

7. The preparation method according to claim 4, wherein 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, characterized in that, The chromatography detection product is a CRISPR line-eliminating nucleic acid test strip, and the CRSIPR system is used as the sample loading solution containing 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, wherein The preparation method of the CRISPR line-eliminating nucleic acid test strip is as follows: Step A: Labeling of fluorescent antibody microspheres 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 on the conjugate pad; Step D: NC membrane coating Use a membrane drawing instrument to spray C line and T line on the NC membrane, and the T line 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 on the bottom plate in sequence to obtain the CRISPR line-eliminating nucleic acid test strip.

16. The application according to claim 15, characterized in that, 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 it will not bind to the streptavidin polymer when passing through the T line, so the T line 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 it will bind to the streptavidin polymer when passing through the T line, and the latex microspheres will settle and the T line shows color, reporting negative.

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

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