Line elimination immunochromatography test paper and application thereof in CRISPR (clustered regularly interspaced short palindromic repeats) detection and typing

Abstract

The invention relates to a line elimination method immunochromatography test paper, which is formed by bonding a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad on a bottom plate in a mutual overlapping manner in sequence, and the nitrocellulose membrane is provided with a C line for contrast and a T line for detection, a colloidal gold compound marked by streptavidin and a colloidal gold compound marked by rabbit IgG (immunoglobulin G) are sprayed on the combination pad; a T line on the nitrocellulose membrane is coated with an FITC labeled antibody, and a C line on the nitrocellulose membrane is coated with anti-IgG. The line elimination method immunochromatography test paper can eliminate lines when the cutting proportion of the probe reaches 50%, so that the detection sensitivity is greatly improved, and the line elimination method immunochromatography test paper can be applied to target gene detection products in a CRISPR (clustered regularly interspaced short palindromic repeats) mode, including genotyping of pathogenic microorganisms, SNP (Single Nucleotide Polymorphism), insertion and deletion, structural variation, methylation modification site detection or drug resistance gene identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and in particular relates to a high-sensitivity line elimination immunochromatographic test paper and an application thereof in CRISPR detection typing. Background Art

[0002] CRISPR detection technology integrates the rapidity and simplicity of constant temperature amplification technology with the sensitivity and accuracy of gene editing. It can be used for molecular diagnostic projects such as pathogen detection and typing, drug-resistant gene identification, methylation status assessment and precision medicine. In particular, this technology can be well connected with immunochromatographic test strips and can be applied to non-professional scenarios such as home self-testing, battlefield medical treatment and field surveys. Therefore, it is also known as the next generation of molecular diagnostic technology.

[0003] However, to date, immunochromatographic test strips suitable for CRISPR detection still have problems such as: 1. Competition between C line and T line; 2. False positives caused by weak coloration of T line of negative samples; 3. Too high threshold of interpretation of CRISPR detection system. These three drawbacks are related to the interpretation logic of immunochromatographic test strips, and more directly related to the principle of immunochromatographic test strips suitable for CRISPR detection. Taking the most well-known Milenia HybriDetect-1 immunochromatographic test strip from TwistDx as an example, its reaction principle is as follows: when the target nucleic acid to be tested exists in the reaction system, the trans-cleavage activity of the CRISPR detection system is activated, and after cutting off the target nucleic acid to be tested, the CRISPR reporter probe will be further cut off; the two ends of the CRISPR reporter probe are respectively labeled with FAM and Biotin. When the reporter probe is intact, the Biotin at one end of the probe binds to the SA on the test line, and the FAM at the other end binds to the anti-FAM-colloidal gold on the sample pad to form a "sandwich" complex that displays color on the test line; when the reporter probe, FAM and Biotin are no longer connected through the probe, and the test line does not display color.

[0004] In response to the first two drawbacks, domestic and foreign CRISPR detection technology research teams and product development teams have tried to improve them. For example, Zhang Feng et al. in their application literature "Wearable materials with embedded synthetic biology sensors for biomolecule detection" and Li Hao et al. in their invention patent "CN111621598A" clearly defined the method of not showing color in the detection line to represent the presence of the target sequence to be tested, that is, the "line elimination method" interpretation method was used to solve the first two drawbacks. However, this method still has the problem of too high a threshold for interpretation, which is caused by the drawbacks in its detection principle. Specifically, the molecular weight of SA on the test line is 65,000, and it is composed of 4 peptide chains with the same sequence. Each SA peptide chain can bind to 1 biotin molecule, and each SA molecule has 4 binding sites for biotin molecules. Its binding constant is 1015 mol / L, which is about 10,000 times the Ka (105-1011 mol / L) between the probe and the FAM antibody on the sample pad. Therefore, the binding affinity between SA and Biotin on the test line is very strong. As long as a trace amount of complete probe exists, it will show color. Only when the reporter probe in the reaction system is 100% cut can the line be completely eliminated. This means that if the concentration of the target nucleic acid to be tested in the reaction system is not high, the trans-cutting activity of the CRISPR detection system is difficult to be fully activated, resulting in the inability to 100% cut the reporter probe, causing problems such as missed detection of positive samples or too long detection time. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a high-sensitivity line elimination method immunochromatographic test paper to achieve high-sensitivity interpretation of CRISPR molecular detection technology on immunochromatographic test strips.

[0006] The first aspect of the present invention is to provide an immunochromatographic test paper for line elimination method, wherein the immunochromatographic test paper is composed of a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad which are sequentially overlapped and bonded to a bottom plate, the nitrocellulose membrane is provided with a C line for control and a T line for detection, the conjugation pad is sprayed with a colloidal gold complex labeled with streptavidin and a colloidal gold complex labeled with rabbit IgG; the T line on the nitrocellulose membrane is coated with an antibody labeled with FITC, and the C line is coated with anti-IgG.

[0007] The second aspect of the present invention is to provide the use of the above-mentioned elimination line immunochromatographic test paper in the preparation of a target gene detection product.

[0008] The target gene detection includes pathogenic microorganism typing, SNP, insertion and deletion, structural variation, and methylation modification site detection.

[0009] The third aspect of the present invention is to provide a kit for detecting a target gene, comprising the above-mentioned elimination immunochromatographic test paper and a CRISPR cutting system.

[0010] The inventors of the present invention have found that the combination of "SA and Biotin" with strong affinity is used for colloidal gold labeling, and the combination of "FAM and anti-FAM" with relatively weak antigen-antibody interaction is used for T-line interception. The technical idea is likely to solve the problem that the current "line elimination method" nucleic acid immunochromatography test strip can only eliminate the line when the reporter probe is 100% cut, and the judgment efficiency is low. However, only by screening the types of anti-FAM antibodies on the T line, screening the antibody spraying concentration and SA-gold concentration, and further adjusting the colloidal gold test paper buffer system, can a better affinity asymmetric paradigm of "SA and Biotin" and "FAM and anti-FAM" be achieved, so that the reporter probe has a much stronger binding strength with SA-gold particles than the anti-FAM antibody on the T line during the lateral immunochromatography migration process, thereby truly and cleverly lowering the threshold for T-line color elimination, and achieving line elimination when the probe cutting ratio reaches 50%, thereby greatly improving the detection sensitivity and shortening the detection time.

[0011] The line elimination immunochromatographic test strip of the present invention has high sensitivity and can expand the application of nucleic acid immunochromatographic test strips to fields requiring a highly sensitive reading system, such as pathogen detection and typing, and drug-resistant gene identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the high-sensitivity line elimination method immunochromatographic test paper of the present invention.

[0013] Figure 2 The reading efficiency of foreign competitor test strips, test strips made according to domestic competitor methods, and high-sensitivity line elimination method test strips at different cutting ratios.

[0014] Figure 3 Interpretation of low-concentration sample CRISPR fluorescence, foreign competitor test strips, test strips made according to domestic competitor methods, and high-sensitivity line elimination method test strips.

[0015] Figure 4 Schematic diagram of the sensitivity performance comparison of the COVID-19 nucleic acid detection reagents produced by a domestic competitor manufacturer.

[0016] Figure 5 Schematic diagram of the verification results of clinical samples positive for the new coronavirus.

[0017] Figure 6 Sensitivity verification of the novel coronavirus Delta strain typing detection kit.

[0018] Figure 7Specificity verification of the novel coronavirus Delta strain typing detection kit.

[0019] Figure 8 Clinical sample validation of the novel coronavirus Delta strain typing detection kit, wherein 1, 2, 6, 16, 17, 18, 20, and 21 in A and B correspond to sample numbers, and the numbers in C and D correspond to sample numbers. A and C are the experimental results of foreign competitors, and B and D are the experimental results of the kit of the present invention. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0023] Biotin: Biotin.

[0024] SA: Streptavidin.

[0025] FAM: fluorescein.

[0026] FITC: fluorescein isothiocyanate.

[0027] In some embodiments of the present invention, a high-sensitivity line elimination method immunochromatographic test paper is provided, wherein the immunochromatographic test paper is composed of a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad which are sequentially overlapped and bonded to a bottom plate; a C line for control and a T line for detection are provided on the nitrocellulose membrane; a colloidal gold complex labeled with streptavidin and a colloidal gold complex labeled with rabbit IgG are sprayed on the conjugation pad; the T line on the nitrocellulose membrane is coated with an antibody labeled with FITC, and the C line is coated with anti-IgG.

[0028] The conjugate pad is prepared as follows:

[0029] 1) The preparation method of colloidal gold is as follows: 400 mL of double distilled water, 4 mL of 1% (mass volume ratio g:mL) gold chloride, and 4 mL of 1% (mass volume ratio g:mL) trisodium citrate aqueous solution are heated to boiling under stirring, and then cooled to room temperature to obtain a nanogold solution.

[0030] 2) Colloidal gold-labeled streptavidin: For every 10 mL of the above colloidal gold solution, add K2CO3 solution at a ratio of 8-12 μL / mL, mix well, then add 10-20 μg / mL streptavidin, mix well, react for 5-15 minutes to obtain a colloidal gold-labeled streptavidin conjugate, centrifuge and remove the supernatant, take PBS to resuspend the precipitate and make up to 1 mL.

[0031] 3) Colloidal gold labeled rabbit IgG conjugate: The colloidal gold labeled rabbit IgG conjugate was prepared using the same steps and concentrations as above. Herein, the colloidal gold complex labeled with streptavidin, colloidal gold labeled streptavidin, and streptavidin gold are the same substance and can be prepared according to the above method.

[0032] 4) The colloidal gold-labeled streptavidin conjugate, the colloidal gold-labeled rabbit IgG conjugate and PBS were prepared at a ratio of 2:1:17 and dried on glass fiber paper to obtain a colloidal gold conjugate pad.

[0033] In some of the embodiments, in order to achieve a better elimination effect, the volume percentage concentration of colloidal gold-labeled streptavidin used to prepare the conjugate pad in the working solution is 5% to 10%.

[0034] The nitrocellulose membrane includes T line and C line. Prepare T line coating solution: use 10mM phosphate buffer solution as coating diluent, dilute FITC-labeled antibody to a working solution with a concentration of 0.8-1.4mg / mL. Prepare C line coating solution: use 10mM phosphate buffer solution as coating diluent, dilute goat anti-rabbit IgG antibody to a working solution with a concentration of 0.4-0.6mg / mL. Then spray the coating working solution of T and C lines on the nitrocellulose membrane attached to the bottom plate at a speed of 0.1μL / mm, dry, and obtain a nitrocellulose membrane coated with the target material.

[0035] In some of the embodiments, the working concentration of FITC-labeled antibody is 0.9-1.1 mg / mL.

[0036] In some of the embodiments, the antibody is Meridian's K86503M mouse monoclonal antibody.

[0037] In some of these embodiments, the C-line on the nitrocellulose membrane is coated with anti-IgG, preferably goat anti-rabbit IgG.

[0038] The absorbent pad is absorbent filter paper.

[0039] As a general rule, the filter paper is overlapped with the C line side of the nitrocellulose membrane, that is, the C line is close to the absorbent pad, the filter paper is attached to the top of the membrane, the conjugate pad is overlapped with the T line side of the nitrocellulose membrane, the conjugate pad is attached to the top of the membrane, the sample pad is overlapped with the conjugate pad, and the sample pad is attached to the top of the conjugate pad.

[0040] Conventionally, the sample pad contains Tris buffer, BSA, NaCl, Tween20 and Proclin 300. 20 mL of 1M Tris (pH 7.5) solution, 5-15 g of BSA powder, 5-15 g of NaCl powder, 5-15 mL of Tween20 solution and 5-15 g of Proclin 300 should be weighed in a reagent bottle, fixed to 1 L with ddH2O, fully mixed, applied on glass fiber paper, and dried for more than 4 hours to obtain a sample pad.

[0041] See also Figure 1 .

[0042] The material of the sample pad is a glass fiber membrane, and the sample to be tested is dropped onto the sample pad.

[0043] The immunochromatographic test paper of the present invention has high sensitivity and can be used for different types of typing detection, including pathogenic microorganism typing, SNP, insertion and deletion, structural variation, methylation modification sites, etc.

[0044] The detection method of the present invention comprises an amplification system, a cutting system, and a high-sensitivity elimination immunochromatographic test paper.

[0045] Suitable nucleic acid amplification systems include PCR, RPA, LAMP, etc.

[0046] The compatible cutting reaction systems are Cas13a system, Cas12a system, Cas12b system, Ago system, etc.

[0047] The adapted reporter probe is 8 to 12 bases, and is labeled with biotin and other markers at both ends, respectively. The other markers may be FAM, TAMRA, DIGOXIN, and the like.

[0048] The target nucleic acid detection kit composed of the line-elimination method immunochromatographic test paper of the present invention comprises the line-elimination method immunochromatographic test paper and also comprises a CRISPR cutting system.

[0049] In some of the embodiments, a target nucleic acid amplification system is also included.

[0050] In some of the embodiments, according to the convention, the main components of the amplification system are amplification enzyme, reaction buffer, activator, primer, sample and ddH2O. The amplification enzyme, reaction buffer and activator are obtained by purchasing. The primers need to be designed according to the sequence information of the target gene and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sample is a nucleic acid sample, which can be a nucleic acid sample extracted and purified by magnetic beads / column method, or a nucleic acid sample obtained by a method of rapid sample release. The method for using the amplification system is: prepare 50 μL amplification system, containing one person's amplification enzyme lyophilized powder, 20-30 μL reaction buffer, 5 μM 2-5 μL primer, 10 μL nucleic acid sample, 2.5 μL activator, and ddH2O to a total volume of 50 μL. The reaction conditions are: after mixing, place in a constant temperature metal bath or water bath at 37°C to 42°C for 10 to 30 minutes.

[0051] In some of the embodiments, according to the routine, the main components of the cutting system are buffer, MgCl2, rNTP, RNase inhibitor, T7 RNA polymerase, LwaCas13a enzyme, crRNA and reporter probe. The method of using the cutting system is as follows: prepare a 50 μL cutting system, containing 5 μL buffer, 1M 1 μL MgCl2 solution, 25mM 4 μL rNTP solution, 40U / μL 1 μL RNase inhibitor, 50U / μL1.5 μL T7 RNA polymerase, 5μM 0.4μL LwaCas13a enzyme, 10μM0.2μL crRNA, 10μM0.2μL reporter probe, 10μL amplification system reaction product, ddH2O to a total volume of 50μL. The reaction conditions are, after mixing, place in a 37°C to 39°C constant temperature metal bath or water bath for 10 to 30 minutes.

[0052] Take 50 μL of the above reaction solution for immunochromatography, wait for 5 minutes to observe the experimental results, and read the results within 5 to 15 minutes. The quality control line C should be constantly colored, and if it cannot be colored, it should be retested; the detection line T is used to capture the complete reporter probe and the cut probe Biotin modified end in the cleavage reaction. If it is positive, the T line will not be colored, and if it is negative, the T line will be colored.

[0053] To address the pain point that conventional line-erasing immunochromatographic test strips can only be erased when the reporter probe is 100% cut and have low interpretation efficiency, we adopted the method of "coating and labeling marker interchange" based on the principle that the binding force between SA and Biotin is much stronger than the binding force between FAM and anti-FAM. This has solved the problem of high interpretation threshold of conventional line-erasing immunochromatographic test strips and greatly improved the detection sensitivity. We gave an example of its application in the typing of the new coronavirus Delta strain (constant temperature amplification-CRISPR-immunochromatography).

[0054] The high-sensitivity immunochromatographic test paper of the present invention can be applied to different types of typing tests, including pathogen detection and typing, drug-resistant gene identification, gene variation and methylation modification site detection, etc.

[0055] The present invention is further described in detail below with reference to specific embodiments.

[0056] Example 1: Detection of the efficiency of reporter probe reading by high-sensitivity immunochromatographic test strips

[0057] At present, the linac immunochromatographic test strips used for CRISPR at home and abroad are all judged by whether the reporter probe is cut or not.

[0058] When the target nucleic acid to be tested exists in the reaction system, the reporter probe is cut, the T line does not show color, and the result is positive; if there is no target nucleic acid to be tested, the reporter probe remains intact, the T line shows color, and the result is negative. When the concentration of the target nucleic acid to be tested is not high, the trans-cutting activity of the CRISPR detection system is difficult to be fully activated, resulting in the inability of the reporter probe to be 100% cut. Therefore, this embodiment simulates a CRISPR reaction system with different reporter probe cutting ratios, that is, in a reaction system without a target nucleic acid to be tested, a simulated complete probe and a simulated single-end probe are added to simulate the cutting state of the reporter probe, so that the amount of the reporter probe to be added to the system = the amount of the simulated complete probe + (the amount of the simulated single-end probe F + the amount of the simulated single-end probe R) / 2, and the specific amount added is shown in Table 2. Thereby, the reaction system with different cutting degrees can be simulated more quantitatively; and the interpretation effectiveness of the domestic and foreign competitive line elimination immunochromatographic test strips and high-sensitivity line elimination immunochromatographic test strips is observed under the condition of low reporter probe cutting (i.e., simulating weak positive samples).

[0059] 1) Reagent preparation

[0060] (1) In this embodiment, the main components of the cutting system are buffer, MgCl2, rNTP, RNase inhibitor, T7 RNA polymerase, LwaCas13a enzyme and crRNA. The method of using the cutting system is as follows: prepare 1500μL cutting system, containing 150μL buffer, 1M 30μL MgCl2 solution, 25mM120μL rNTP solution, 40U / μL 30μL RNase inhibitor, 50U / μL 45μL T7 RNA polymerase, 5μM 12μL LwaCas13a enzyme, 10μM 6μL crRNA, and ddH2O to a total volume of 1500μL.

[0061] (2) In this embodiment, a simulated probe was designed, and the cleavage system prepared in (1) was used to dilute the simulated complete probe and the simulated single-ended probe in two tubes, 700 μL in each tube. Tube 1 contained only the simulated complete probe, and the final dilution concentration was 40 nM; Tube 2 contained the simulated single-ended probe F and the simulated single-ended probe B, and the final dilution concentration was 40 nM. The complete double-ended probe and the simulated single-ended probe sequence and modification information are shown in Table 3:

[0062] Table 3 Sequence and marker information

[0063] Simulate a complete probe 5'FAM-UUUUUUUU-Biotin3' Analog single-ended probe F 5'FAM-UUUUUUUU Analog single-ended probe B 5'Biotin-UUUUUUUU

[0064] (3) In this embodiment, the main components of the high-sensitivity line elimination immunochromatographic test paper are a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad. The sample pad contains Tris buffer, BSA, NaCl, Tween20, and Proclin 300. 20 mL of 1M Tris (pH 7.5) solution, 10 g of BSA powder, 10 g of NaCl powder, 10 mL of Tween20 solution, and 10 g of Proclin 300 should be measured in a reagent bottle, and ddH2O is adjusted to 1 L, mixed thoroughly, applied on glass fiber paper, and dried for more than 4 hours to obtain a sample pad. The conjugate pad contains gold chloride, sodium citrate, potassium carbonate, streptavidin, and rabbit IgG. 400 mL of double distilled water, 4 mL of 1% (mass volume ratio g: mL) gold chloride, and 4 mL of 1% (mass volume ratio g: mL) trisodium citrate aqueous solution are heated to boiling under stirring, and then cooled to room temperature to obtain a nanogold solution. For every 10mL of the above colloidal gold solution, add K2CO3 solution at a ratio of 8μL / mL, mix well, then add 20μg / mL streptavidin, mix well, react for 8min, and obtain a colloidal gold-labeled streptavidin conjugate. After centrifugation, remove the supernatant, take PBS to resuspend the precipitate and make up to 1mL. Use the same steps and concentrations to prepare a colloidal gold-labeled rabbit IgG conjugate. The above streptavidin conjugate, rabbit IgG conjugate and PBS buffer are configured at a ratio of 2:1:17 (the volume percentage concentration of the streptavidin-labeled colloidal gold complex is 10%), and dried on glass fiber paper to obtain a colloidal gold conjugate pad. The nitrocellulose membrane includes a T line and a C line. Prepare the T line coating solution: Use 10mM phosphate buffer solution as the coating diluent and dilute the FITC antibody to a working solution with a concentration of 1mg / mL. Prepare C-line coating solution: Use 10mM phosphate buffer solution as the coating diluent, and dilute the goat anti-rabbit IgG antibody to a working solution with a concentration of 0.5mg / mL. Then spray the T and C-line coating working solutions onto the nitrocellulose membrane attached to the bottom plate at a speed of 0.1μL / mm using a quantitative membrane sprayer, and dry to obtain a nitrocellulose membrane coated with the target material. The absorbent pad is a thickened absorbent filter paper. Overlap the filter paper with the C-line side of the nitrocellulose membrane, attach the filter paper to the top of the membrane, overlap the conjugate pad with the T-line side of the nitrocellulose membrane, attach the conjugate pad to the top of the membrane, overlap the sample pad with the conjugate pad, and attach the sample pad to the top of the conjugate pad.

[0065] 2) Test methods

[0066] The simulated probe was prepared using (2) in this embodiment, wherein the simulated complete probe tube 1 and the simulated single-ended probe tube 2 were prepared in different proportions, and the domestic and foreign competing products (in the present invention, the foreign competing product is Milenia HybriDetect 1 of TwistDx Company of the United Kingdom, and the domestic competing product is RT-RAA-nfo nucleic acid amplification reagent (test strip type) of Zhongce Biology) were tested for the interpretation effect of the line elimination method test paper and the high-sensitivity line elimination method immunochromatographic test paper in the system containing the simulated probe at different probe cutting ratios. After preparation in different proportions, the final concentration of the probe is calculated as shown in Table 4:

[0067] Table 4 Probe ratios for simulating different cutting ratios

[0068]

[0069]

[0070] After testing, according to the method of this embodiment, the foreign competitor test strips need to reach a CRISPR probe cutting ratio of up to 100% to completely eliminate the T line, and there is no obvious color difference from the negative when the cutting efficiency reaches 95%; the test strips made by the domestic competitor method need to reach a CRISPR probe cutting ratio of 100% to completely eliminate the line, and only a certain color difference can be seen when the CRISPR probe cutting ratio is 95%. According to the interpretation logic, the result when the T line does not show color is positive, and the result when the T line shows color is negative. The foreign competitor test strips and the test strips made according to the domestic competitor method both require a very high cutting ratio to eliminate the line and be interpreted as positive. Therefore, when the sample concentration is not high and the CRISPR probe is not completely cut, false negative results are easily generated. The present invention designs a "line elimination method" with a wider interpretation limit. The "line elimination method" can eliminate the line when the probe cutting ratio is 50%. This design reduces the limitations of false negatives caused by the upper limit of the reaction system performance and improves the interpretation efficiency of the line elimination method. Results are shown Figure 2 .

[0071] Example 2: Sensitivity test of high-sensitivity line elimination immunochromatographic test paper in the COVID-19 nucleic acid detection kit

[0072] This embodiment includes the preparation and application of a novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography). The novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography) includes a novel coronavirus N gene constant temperature amplification reagent, a CRISPR reaction reagent, and a high-sensitivity line elimination immunochromatography test paper. A fluorescent signal comparison reagent is also prepared to visualize and directly compare the cutting ratio of the probe.

[0073] 1) Reagent composition

[0074] (1) Novel coronavirus N gene isothermal amplification reagent: The primers in the reagent are independently designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and are selected to amplify the novel coronavirus (2019-nCoV) N gene sequence. The primer sequence information is shown in Table 5. The synthesized primer probe and the commercially available RNA isothermal rapid amplification mixture (basic type) of Ampo Biotech Co., Ltd. are freeze-dried together for production, as shown in Table 5.

[0075] Table 5 Primer sequence information

[0076]

[0077] (2) CRISPR reaction reagents: 1.25× buffer, 25mM MgCl2, 2.5mM rNTP solution, 1U / μL RNase inhibitor, 1.875U / μL T7 RNA polymerase, 50nM LwaCas13a enzyme, 50nM crRNA, 50nM reporter probe 1. The crRNA is designed at the conserved sequence position between the primers selected for isothermal amplification, and the reporter probe has FAM and Biotin labels at both ends. See Table 6.

[0078] (3) CRISPR fluorescent reagent: 1.25× buffer, 25mM MgCl2, 2.5mM rNTP solution, 1U / μL RNase inhibitor, 1.875U / μL T7 RNA polymerase, 50nM LwaCas13a enzyme, 50nM crRNA, 50nM reporter probe 2. The crRNA was designed at a conserved sequence position between the primers selected for isothermal amplification, and the reporter probe had FAM and BHQ labels at both ends.

[0079] Table 6 crRNA and reporter probe sequence information

[0080]

[0081] (3) High-sensitivity line-elimination immunochromatographic test paper: The preparation method of the high-sensitivity line-elimination immunochromatographic test paper used in this example is the same as that in Example 1.

[0082] 2) Usage

[0083] (1) Sample preparation: Take the novel coronavirus (2019-nCoV) genome RNA standard material (GBW091099) purchased from the National Institute of Metrology, China, and dilute the sample to 20 copies / μL and 2 copies / μL. (2) Constant temperature amplification reaction: Take 10μL of the diluted sample and mix it with 40μL DNase / RNase free water, then add it to the SARS-CoV-2 N gene constant temperature amplification freeze-dried reagent, mix it upside down, centrifuge it instantly with a low-speed centrifuge, and perform constant temperature amplification reaction in a constant temperature metal bath (reaction conditions are 42°C, 30min). (3) CRISPR reaction: Take 40μL of CRISPR reaction reagent, add 10μL of constant temperature amplification reaction product to the system, mix it upside down, centrifuge it instantly to the bottom of the tube, and place it in a 37°C constant temperature metal bath or water bath for reaction for 30min.

[0084] (4) CRISPR fluorescence: Take 40 μL of CRISPR fluorescence reagent, add 10 μL of constant temperature amplification reaction product to the system, mix by inversion, centrifuge to the bottom of the tube, place in fluorescent quantitative PCR ABI 7500 at 37°C for 30 min, and collect fluorescence every 1 min.

[0085] (5) Interpretation of results: Take 50 μL of the total reaction solution in (3) above for immunochromatography, wait for 5 minutes and then observe the experimental results, and interpret the results within 5 to 15 minutes. The quality control line C should be constantly colored. If it cannot be colored, the test strip should be replaced and retested; the detection line T is used to capture the complete reporter probe and the cut-off probe Biotin-modified end in the CRISPR reaction. If it is positive, the T line will not be colored, and if it is negative, the T line will be colored. See the results. Figure 3 .

[0086] The results are as follows Figure 3As shown, when the CRISPR fluorescence method is used for result interpretation (left 1), 200 copies of the novel coronavirus (2019-nCoV) genomic RNA can activate a sufficient amount of Cas enzyme to produce a saturated fluorescence signal, proving that the CRISPR probe is completely cut; while 20 copies of the novel coronavirus (2019-nCoV) genomic RNA cannot activate a sufficient amount of Cas enzyme to complete the cutting of the probe, so the generated fluorescence signal is unsaturated and the relative value is low. When the CRISPR elimination line immunochromatographic test paper is used for result interpretation (right 1-3), if 200 copies of the novel coronavirus (2019-nCoV) genomic RNA are detected, since the CRISPR probe is completely cut, foreign competitors, domestic competitors and this elimination line immunochromatographic test paper are all judged as positive; if 20 copies of the novel coronavirus (2019-nCoV) genomic RNA are detected, since the CRISPR probe is not completely cut, the estimated cut probe ratio is less than 50%, and foreign competitors and domestic competitors cannot be judged as positive by the elimination line method, while the high-sensitivity elimination line immunochromatographic test paper of the present invention can detect it. This shows that the product of the present invention has higher sensitivity and can detect low-abundance samples.

[0087] Example 3: Comparison of sensitivity performance of COVID-19 nucleic acid detection reagents with a domestic competitor

[0088] 1) Novel Coronavirus Nucleic Acid Detection Kit (CRISPR-Immunochromatography): This example uses a novel coronavirus nucleic acid detection kit (CRISPR-Immunochromatography) from a domestic competitor manufacturer (RT-RAA-nfo nucleic acid amplification reagent (test strip type) from Zhongce Biological) and a novel coronavirus nucleic acid detection kit (CRISPR-Immunochromatography) based on the high-sensitivity elimination method (interchange of strong and weak binding forces) described in Example 2.

[0089] 2) Detection method:

[0090] (1) Sample preparation: The novel coronavirus (2019-nCoV) genome RNA standard material (GBW091099) purchased from the National Institute of Metrology was used and the samples were diluted to 100 copies / μL, 10 copies / μL, 5 copies / μL, and 1 copy / μL, respectively.

[0091] (2) The isothermal amplification reaction, CRISPR reaction and result interpretation are the same as in Example 2. For the negative control, the competitor's product and the kit in Example 2 both showed normal color development; when the sample concentration was 1000 copies / reaction, the competitor's product and the kit in Example 2 were able to detect all of them; when the sample concentration was 100 copies / reaction, the competitor's product had a detection rate of 1 / 3, while the kit in Example 2 could detect all of them; when the sample concentration was 50 copies / reaction, the competitor's product was unable to detect, while the kit in Example 2 could detect all of them; when the sample concentration was 10 copies / reaction, the competitor's product had a detection rate of 1 / 3, while the kit in Example 2 could detect all of them. See the results. Figure 4 .

[0092] Example 4 Clinical sample detection

[0093] 1) Novel Coronavirus Nucleic Acid Detection Kit (CRISPR-Immunochromatography): This example uses the Novel Coronavirus Nucleic Acid Detection Kit (CRISPR-Immunochromatography) described in Example 2.

[0094] 2) Detection method:

[0095] (1) Sample preparation: A total of 24 clinical samples of novel coronavirus nucleic acid were collected. The sample CT value information is available at Figure 5 After the samples were taken out and thawed, nucleic acid extraction and purification reagents were used to extract and purify nucleic acids.

[0096] (2) The isothermal amplification reaction, CRISPR reaction and result interpretation were the same as in Example 2. There were 7 samples with CT values ​​below 20, 4 samples with CT values ​​between 20 and 25, 5 samples with CT values ​​between 26 and 30, 7 samples with CT values ​​between 31 and 35, and 1 sample with a CT value greater than 35. The sample with a CT value of 38 had a T line coloring result of not detected, and the rest of the samples had no coloring result, which was detected. See the results. Figure 5 : In nucleic acid testing, especially COVID-19 testing, a CT value of ≤35 is used as the standard for positive judgment. Here, a sample with a CT value of 38 is an extremely weak sample and is not clinically judged to be positive. The results show that the detection kit of the present invention has a high sensitivity and meets the traditional positive judgment standard for nucleic acid testing.

[0097] Example 5: Verification of the sensitivity of the novel coronavirus Delta strain typing detection kit

[0098] 1) Novel coronavirus Delta strain typing kit (constant temperature amplification-CRISPR-immunochromatography):

[0099] This kit is used to distinguish the EFR156-158G mutation site of the S gene in the Delta strain of the novel coronavirus. As of the submission of this patent, this mutation site only appears in the Delta mutant strain and does not cross-react with the original strain and other mutant strains. However, it does not rule out the possibility that new mutant strains will mutate at this site in the future, causing the kit to become ineffective. (1) Constant-temperature amplification freeze-dried ball reagent for typing the Delta strain of the novel coronavirus: The primers in the reagent are independently designed, and the sequences around the Delta strain-specific mutation site in the S gene of the novel coronavirus (2019-nCoV) are selected for amplification. The primer sequence information is shown in the table. The synthesized primer probe and the commercially available RNA constant-temperature rapid amplification mixture (basic type) of Anpu Future Biotechnology Co., Ltd. are freeze-dried together for production, see Table 7.

[0100] Table 7 Primer sequence information

[0101] Upstream primer 5'-CGCTACTAATGTTGTTATTAAAGTCTGTGAATTTC-3'(SEQ ID NO:4) Downstream primer 5'-GAAAAGGCTGAGAGACATATTCAAAAGTGCAA-3'(SEQ ID NO:5)

[0102] (4) CRISPR-Buffer A-O: 1.31× buffer, 26.24mM MgCl2, 2.62mM rNTP solution, 1.05U / μL RNase inhibitor, 52.49nM crRNA, 52.49nM reporter probe. The crRNA and reporter probe are independently designed. The crRNA specifically recognizes the EFR156-158 site and its adjacent sequence of other subtypes of strains except the Delta strain, and does not cross-react with the Delta strain. The crRNA and reporter probe sequences are shown in Table 8:

[0103] Table 8 SARS-CoV-2 S gene wild-type sequence crRNA and reporter probe sequence information

[0104]

[0105] (5) CRISPR-Buffer A-DELTA: 1.31× buffer, 26.24mM MgCl2, 2.62mM rNTP solution, 1.05U / μL RNase inhibitor, 52.49nM crRNA, 52.49nM reporter probe. The crRNA and reporter probe are independently designed. The crRNA specifically recognizes the Delta strain EFR156-158G mutation site and its adjacent sequence, and does not cross-react with other subtypes. The crRNA and reporter probe sequences are shown in Table 9:

[0106] Table 9 SARS-CoV-2 S gene DELTA mutation sequence crRNA and reporter probe sequence information

[0107]

[0108] (4) CRISPR-Buffer B: 1.05 pmol / μL Cas13a enzyme, 39.47 U / μL T7 RNA polymerase.

[0109] (5) High-sensitivity line-elimination immunochromatographic test paper: The preparation method of the high-sensitivity line-elimination immunochromatographic test paper used in this example is the same as that of the above-mentioned Example 1.

[0110] 3) Detection method

[0111] (1) Sample preparation: The 2019-nCoV Delta B.1.617 variant genomic RNA standard material (GBW09316) purchased from the National Institute of Metrology was used and the samples were diluted to 10 copies / μL, 5 copies / μL, 2 copies / μL, 1 copy / μL, 0.5 copies / μL, 0.3 copies / μL, and 0.1 copies / μL, respectively.

[0112] (2) Constant-temperature amplification reaction: Pipette 10 μL of the diluted sample, mix with 40 μL DNase / RNase free water, and add to the constant-temperature amplification freeze-dried ball reagent for the new coronavirus Delta strain typing. Mix by inverting the tube upside down, centrifuge briefly in a low-speed centrifuge, and perform a constant-temperature amplification reaction in a constant-temperature metal bath (reaction conditions are 42°C, 30 min).

[0113] (3) CRISPR reaction: Prepare two tubes of CRISPR reaction reagents for each amplification reaction. CRISPR-O: Add 76.2μL CRISPR-BufferA-O and 3.8μL CRISPR-BufferB to the PCR tube and mix well; CRISPR-DELTA: Add 76.2μL CRISPR-BufferA-DELTA and 3.8μL CRISPR-BufferB to the PCR tube and mix well. Take 20μL of the amplification reaction product and add it to CRISPR-O and CRISPR-DELTA respectively. After mixing by inverting, perform instant centrifugation in a low-speed centrifuge, and perform CRISPR reaction in a constant temperature metal bath (reaction conditions are 37°C, 30min).

[0114] (4) Interpretation of results: Mix the CRISPR-O and CRISPR-DELTA reaction products and centrifuge them. Take 65 μL of each and add it to the sample pad of the high-sensitivity line elimination immunochromatographic test strip. Interpret the results within 5 to 15 minutes. When the C line of the CRISPR-O test strip is colored and the T line is not colored, and the C line of the CRISPR-DELTA test strip is colored and the T line is colored, it is positive for non-DELTA strains of new coronavirus; when the C line of the CRISPR-O test strip is colored and the T line is colored, and the C line of the CRISPR-DELTA test strip is colored and the T line is not colored, it is positive for the DELTA strain of new coronavirus; when the C line of the CRISPR-O test strip is colored and the T line is colored, and the C line of the CRISPR-DELTA test strip is colored and the T line is not colored, it is positive for the DELTA strain of new coronavirus. When the C line of the CRISPR-O test strip is colored and the T line is colored, and the C line of the CRISPR-DELTA test strip is colored and the T line is colored, it is negative for the new coronavirus. See the results. Figure 6 (The upper figure is the result of Milenia HybriDetect 1 of TwistDx, a foreign competitor, and the lower figure is the high-sensitivity immunochromatographic test strip of this embodiment.) The results show that the same reaction product can be detected with a sensitivity as low as 3 copies / reaction using the kit of the present invention, which is better than the 100 copies / reaction of foreign competitors.

[0115] Example 6: Specificity of the COVID-19 typing test kit

[0116] 1) Novel coronavirus Delta strain typing kit (constant temperature amplification-CRISPR-immunochromatography): This example uses the Novel coronavirus Delta strain typing kit (constant temperature amplification-CRISPR-immunochromatography) of Example 3.

[0117] 2) Detection method:

[0118] (1) Sample preparation: The novel coronavirus (2019-nCoV) in vitro transcribed RNA standard material (GBW09299), the novel coronavirus (2019-nCoV) beta B.1.351 variant genomic RNA standard material (GBW09315), the novel coronavirus (2019-nCoV) delta B.1.617 variant genomic RNA standard material (GBW09316), the novel coronavirus (2019-nCoV) gamma P.1 variant genomic RNA standard material (GBW09317), and the novel coronavirus (2019-nCoV) omega BA.1.1 variant genomic RNA standard material (GBW09318) purchased from the National Institute of Metrology were diluted to 10 copies / μL respectively.

[0119] (2) The isothermal amplification reaction, CRISPR reaction and result interpretation were the same as in Example 5. Figure 7The results showed that the high-sensitivity elimination line detection kit of the present invention can specifically distinguish the Delta strain from different variants of the new coronavirus at a concentration as low as 10 copies / μL.

[0120] Example 7: Clinical sample verification of the COVID-19 typing test kit

[0121] 1) Novel coronavirus Delta strain typing kit (constant temperature amplification-CRISPR-immunochromatography): This example uses the Novel coronavirus Delta strain typing kit (constant temperature amplification-CRISPR-immunochromatography) of Example 3.

[0122] (1) Nucleic acid extraction and purification reagents: nucleic acid extraction or purification reagents of Guangzhou Wondfo Biotech Co., Ltd., registration number: Yuesui Xiebei 20210234.

[0123] (2) Fluorescence PCR detection reagent: Daan Gene's 2019-nCoV nucleic acid detection kit (fluorescence PCR method), registration number: National Medical Device Registration No. 20203400063.

[0124] 4) Detection method:

[0125] (1) Sample preparation: A total of 15 clinical samples of the new coronavirus were collected, including 2 negative samples and 13 positive samples. Among the positive samples, 7 were Delta strain samples and 6 were Alpha strain samples. The sample information is shown in Table 10. After the samples were taken out and thawed, nucleic acid extraction and purification reagents were used for nucleic acid extraction and purification.

[0126] (2) The extracted nucleic acid was tested using a fluorescent PCR detection reagent. The results are shown in Table 10:

[0127] Table 10 Ct values ​​of fluorescence quantitative PCR detection of COVID-19 Alpha and Delta samples

[0128]

[0129]

[0130] (3) The isothermal amplification reaction, CRISPR reaction and result interpretation were the same as in Example 3. Figure 8 The results show that 15 clinical samples identified by PCR were tested, of which 2 were negative, 6 were Alpha positive, and 7 were Delta positive. The high-sensitivity line elimination test kit described in the present invention can detect 6 / 6 Alpha strains and 7 / 7 Delta strains. The foreign competitor control product (Milenia HybriDetect 1 of TwistDx, UK) can only detect 5 / 6 Alpha strains and 4 / 7 Delta strains.

[0131] Example 8 Comparative Experiment

[0132] In order to achieve a better line elimination effect, we optimized the concentration of streptavidin gold on the conjugate pad, as shown in Table 1. The 1%, 5%, 10%, and 15% in Table 8-1 correspond to the volume percentage concentrations of streptavidin gold of 1%, 5%, 10%, and 15%, respectively. The results shown are the colorimetric obtained by the percentage cutting probe below, where C1 to C8 are the colorimetric from strong to weak. The greater the difference between 0% cutting and other cutting ratios, the easier it is to read. The results show that the optimal range of streptavidin gold concentration is 5% to 10%. The preparation method of the immunochromatographic test paper is as described in Examples 1 and 2. In this embodiment, a 1:1 mixture of a probe labeled with biotin on one end and a probe labeled with FAM on one end is used as a 100% cutting probe, and a probe labeled with biotin and FAM on both ends is used as a 0% cutting probe.

[0133] Table 8-1 Streptavidin gold concentration screening

[0134]

[0135] The nitrocellulose membrane includes a T line and a C line. Prepare the T line coating solution: use 10mM phosphate buffer solution as the coating diluent, and dilute the FITC antibody to a working solution with a concentration of 1mg / mL. Prepare the C line coating solution: use 10mM phosphate buffer solution as the coating diluent, and dilute the goat anti-rabbit IgG antibody to a working solution with a concentration of 0.5mg / mL. Then spray the coating working solution of the T and C lines on the nitrocellulose membrane attached to the bottom plate at a speed of 0.1μL / mm, dry it, and obtain the nitrocellulose membrane coated with the target material.

[0136] In order to achieve a better line elimination effect, we compared the FITC antibodies and concentrations on the conjugate pad. Except for the different T-line antibodies and concentrations, the other preparation parameters are the same as in Example 1 and Example 2. In the table, C-1 (C-1 is the mouse monoclonal antibody Meridian's sold product K45219M), C-2 (Meridian's K86503M), and C-3 (mouse monoclonal antibody Fitzgerald's 10-F50C, clone number M25286) are different types of antibodies. The 0.5 and 1.0 in the table represent the concentrations of the corresponding antibodies of 0.5 mg / mL and 1.0 mg / mL. As shown in Table 8-2, it was found that Meridian's K86503M mouse monoclonal antibody (C-2) had the best line elimination effect, and the optimal concentration was 1 mg / mL.

[0137] Table 8-2 T-line antibody and concentration screening

[0138]

[0139] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An immunochromatographic test paper for line elimination method, which is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad which are sequentially overlapped and bonded to a bottom plate, wherein a C line for control and a T line for detection are provided on the nitrocellulose membrane, characterized in that: The conjugate pad is sprayed with colloidal gold complexes labeled with streptavidin and colloidal gold complexes labeled with rabbit IgG; the T line on the nitrocellulose membrane is coated with FITC labeled antibody, the C line is coated with anti-IgG, and the C line is close to the water absorbent pad.

2. The immunochromatographic test paper according to claim 1, characterized in that: The volume percentage concentration of the colloidal gold complex labeled with streptavidin on the binding pad is 5% to 10%.

3. The immunochromatographic test paper according to claim 2, characterized in that: The dosage ratio of the colloidal gold complex labeled with streptavidin to the colloidal gold complex labeled with rabbit IgG on the conjugate pad is 2:

1.

4. The immunochromatographic test paper according to claim 1, characterized in that: The working concentration of the T-line coated with FITC-labeled antibody is 0.8-1.4 mg / mL, preferably 0.9-1.1 mg / mL.

5. The immunochromatographic test paper according to claim 1, characterized in that: The antibody in the FITC-labeled antibody is K86503M mouse monoclonal antibody.

6. The immunochromatographic test paper according to claim 1, characterized in that: The working concentration of the C-line coated anti-IgG is 0.4-0.6 mg / mL.

7. Use of the line elimination immunochromatographic test paper according to any one of claims 1 to 6 in the preparation of a target gene detection product using the CRISPR method.

8. The use according to claim 7, characterized in that: The target gene detection includes genotyping of pathogenic microorganisms, SNP, insertion and deletion, structural variation, methylation modification site detection, or drug resistance gene identification.

9. A kit for detecting a target gene, characterized in that: It comprises the line elimination immunochromatographic test paper according to any one of claims 1 to 6, and a CRISPR cutting system.

10. The kit according to claim 9, characterized in that It also includes a target nucleic acid amplification system; and / or the CRISPR cleavage system is a Cas13a system, a Cas12a system, a Cas12b system, and an Ago system.

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