Elimination method immunochromatography test strip and its application in CRISPR detection typing

Abstract

The present application relates to a kind of line-elimination method immunochromatography test paper, it is sequentially bonded on base plate by sample pad, binding pad, nitrocellulose membrane, water absorption pad mutual lapping, C line for control and T line for detection are equipped on the nitrocellulose membrane, streptavidin labeled colloidal gold compound and rabbit IgG labeled colloidal gold compound are sprayed on the binding pad;T line on the nitrocellulose membrane is coated with FITC labeled antibody, and C line is coated with anti-IgG.The line-elimination method immunochromatography test paper can eliminate line when probe cutting ratio reaches 50%, thus greatly improve the detection sensitivity, can be applied to the application in CRISPR mode target gene detection product, including pathogenic microorganism genotyping, SNP, insertion and deletion, structural variation, methylation modification site detection, or drug resistance gene identification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular diagnosis, and particularly relates to a high-sensitivity line-eliminating immunochromatographic test paper and application thereof in CRISPR detection typing. BACKGROUND

[0002] The CRISPR detection technology integrates the rapidness and simplicity of the isothermal amplification technology and the sensitivity and accuracy of the gene editing level, and can be used for pathogen detection and typing, drug resistance gene identification, methylation state evaluation and precision medicine and other molecular diagnosis projects; in particular, the technology can be well connected with the immunochromatographic test strip, and can be applied to non-professional scenes such as home self-testing, field medical treatment and field exploration, and is therefore also known as the next generation of molecular diagnosis technology.

[0003] However, up to now, the immunochromatographic test strip suitable for CRISPR detection still has problems such as: 1, competition between C line and T line; 2, false positive problem caused by weak color development of T line of negative sample; 3, high threshold problem of CRISPR detection system. The three drawbacks are related to the reading logic of the immunochromatographic test strip, and more directly related to the principle of the immunochromatographic test strip suitable for CRISPR detection. Taking the most well-known Milenia HybriDetect-1 immunochromatographic test strip of TwistDx company as an example, the reaction principle is as follows: when the target nucleic acid to be detected exists in the reaction system, the transcleavage activity of the CRISPR detection system is activated, and after cutting the target nucleic acid to be detected, the CRISPR reporter probe is further cut; the CRISPR reporter probe is labeled with FAM and Biotin at both ends, when the reporter probe is complete, the Biotin at one end of the probe is combined with SA on the detection line, and the FAM at the other end is combined with anti-FAM-colloidal gold on the sample pad, to form a "sandwich" complex on the detection line to develop color; and when the reporter probe, FAM and Biotin are no longer connected through the probe, the detection line does not develop color.

[0004] In view of the first two disadvantages, CRISPR detection technology research teams and product development teams at home and abroad have tried to improve, such as Zhang Feng et al. in their application document “Wearable materials with embedded synthetic biology sensors for biomolecule detection” and Li Hao et al. in their invention patent “CN111621598A” have clearly stated that the detection line is not colorless to represent the presence of the target sequence to be detected, that is, the “line elimination method” is used to solve the first two disadvantages. However, this method still has the problem of high threshold for judgment, which is caused by the disadvantages of its detection principle. Specifically, the molecular weight of SA on the detection line is 65000, which is composed of 4 sequence identical peptide chains, each SA peptide chain can bind 1 biotin molecule, each SA molecule has 4 biotin molecule binding sites, and 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, so the binding affinity of SA and Biotin on the detection line is very strong. As long as a trace amount of intact probe exists, it will develop color, and only when the reporter probe in the reaction system is 100% cut can the line be completely eliminated. This means that, for example, if the concentration of the target nucleic acid to be detected in the reaction system is not high, the trans-cleavage activity of the CRISPR detection system is difficult to be completely activated, resulting in that the reporter probe cannot be completely cut, causing false negative samples or long detection time. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a high-sensitivity line-elimination method immunochromatography test paper to realize high-sensitivity judgment of CRISPR molecular detection technology on the immunochromatography test paper.

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

[0007] The second aspect of the present application is to provide the use of the above-mentioned line-elimination method immunochromatography 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 application provides a kit for detecting a target gene, comprising the above-mentioned line-elimination immunochromatography test paper and a CRISPR cutting system.

[0010] The inventor of the present application found that the technical idea of using the combination of "SA and Biotin" with strong affinity for colloidal gold labeling and the combination of "FAM and anti-FAM" with relatively weak antigen-antibody interaction for intercepting the T line can solve the problem that the current "line-elimination" nucleic acid immunochromatography test strip can only eliminate the line when the reporter probe is 100% cut, and the interpretation efficiency is low. However, only by screening the type of anti-FAM antibody on the T line, screening the concentration of antibody spray film, screening the concentration of SA-gold, and further adjusting the colloidal gold test paper buffer system, the asymmetric affinity of "SA and Biotin" and "FAM and anti-FAM" can be optimized, so that the binding strength of the reporter probe to the SA-gold particles during lateral flow immunochromatography is much higher than that to the anti-FAM antibody on the T line, thereby truly and ingeniously lowering the threshold for eliminating the color of the T line, 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 immunochromatography test paper has high sensitivity, and can expand the application of nucleic acid immunochromatography test strips to the fields of pathogen detection and typing, drug resistance gene identification, and other fields requiring high sensitivity interpretation systems. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The schematic diagram of the high-sensitivity line-elimination immunochromatography test paper.

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

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

[0015] Figure 4 The schematic diagram of the comparison results of the sensitivity performance of the new crown nucleic acid detection reagent of a domestic competitor.

[0016] Figure 5 The schematic diagram of the verification results of the new crown virus positive clinical sample.

[0017] Figure 6 Sensitivity verification of the new crown virus Delta strain typing test kit.

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

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

[0020] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application 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 application more thorough and comprehensive.

[0021] The experimental methods in the following examples not specified in the specific conditions, generally in accordance with the conventional conditions, for example, Green and Sambrook, editor of the fourth edition of "molecular cloning laboratory manual" (Molecular Cloning: A Laboratory Manual) has been published in 2013, or in accordance with the manufacturer's recommended conditions. The various common chemical reagents used in the examples are commercially available products.

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

[0023] Biotin: biotin.

[0024] SA: streptavidin.

[0025] FAM: fluorescein.

[0026] FITC: fluorescein isothiocyanate.

[0027] In some embodiments of the present application, a high-sensitivity line-eliminating immunochromatographic test paper is provided, which is sequentially and mutually bonded on a bottom plate by a sample pad, a binding pad, a nitrocellulose membrane, and a water absorption pad. The nitrocellulose membrane is provided with a C line for control and a T line for detection. The binding pad is sprayed with streptavidin-labeled colloidal gold complex and rabbit IgG-labeled colloidal gold complex. The T line on the nitrocellulose membrane is coated with FITC-labeled antibody, and the C line is coated with anti-IgG.

[0028] The preparation of the binding pad is 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, 4 mL of 1% (mass volume ratio g:mL) aqueous solution of trisodium citrate, heated to boiling under stirring, and then cooled to room temperature to obtain a gold nanoparticle solution.

[0030] 2) Colloidal gold labeled streptavidin: for every 10 mL of the above colloidal gold solution, 8-12 μL / mL of K2CO3 solution is added, mixed, and then 10-20 μg / mL of streptavidin is added, mixed, and reacted for 5-15 min to obtain a colloidal gold labeled streptavidin complex. After centrifugation, the supernatant is removed, and the precipitate is resuspended in PBS to a volume of 1 mL.

[0031] 3) Colloidal gold labeled rabbit IgG complex: the colloidal gold labeled rabbit IgG complex is prepared by the same method and concentration as described above. In this paper, the colloidal gold labeled streptavidin complex, colloidal gold labeled streptavidin, and streptavidin gold are the same substance. They can be prepared according to the above method.

[0032] 4) The colloidal gold labeled streptavidin complex, colloidal gold labeled rabbit IgG complex, and PBS are mixed in a ratio of 2:1:17, dried on glass fiber paper, and a colloidal gold binding pad is obtained.

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

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

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

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

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

[0038] The water-absorbing pad is water-absorbing filter paper.

[0039] According to the convention, the filter paper is overlapped with the C-line side of the nitrocellulose membrane, that is, the C-line is close to the water-absorbing pad, the filter paper is pasted on the upper side of the membrane, the binding pad is overlapped with the T-line side of the nitrocellulose membrane, the binding pad is pasted on the upper side of the membrane, and the sample pad is overlapped with the binding pad, and the sample pad is pasted on the upper side of the binding pad.

[0040] According to the convention, the sample pad contains Tris buffer, BSA, NaCl, Tween20 and Proclin300. 20mL of 1M Tris (pH7.5) solution, 5-15g of BSA powder, 5-15g of NaCl powder, 5-15mL of Tween20 solution and 5-15g of Proclin300 are weighed in a reagent bottle, ddH2O is added to 1L, and it is mixed thoroughly and coated on glass fiber paper and dried for more than 4h to obtain the sample pad.

[0041] Referring to Figure 1 .

[0042] The material of the sample pad is glass fiber membrane, and the sample to be tested is added dropwise on the sample pad.

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

[0044] The detection method comprises an amplification system, a cleavage system and a high-sensitivity line-elimination immunochromatographic test paper.

[0045] The suitable nucleic acid amplification system is PCR, RPA, LAMP, etc.

[0046] The suitable cleavage reaction system is Cas13a system, Cas12a system, Cas12b system, Ago system, etc.

[0047] The suitable reporter probe is 8-12 bases, and the two ends are respectively labeled with biotin and other markers, and the other markers can be FAM, TAMRA, DIGOXIN, etc.

[0048] The target nucleic acid detection kit composed of the line-elimination immunochromatographic test paper comprises the line-elimination immunochromatographic test paper and further comprises a CRISPR cleavage system.

[0049] In some embodiments thereof, the target nucleic acid amplification system is further included.

[0050] In some embodiments, the amplification system mainly consists of amplification enzyme, reaction buffer, activator, primer, sample and ddH2O. The amplification enzyme, reaction buffer and activator are obtained by outsourcing. The primer needs to be designed according to the sequence information of the target gene and synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. The sample is a nucleic acid sample, which can be a nucleic acid sample extracted and purified by magnetic bead method / column method, or a nucleic acid sample obtained by a sample rapid release method. The preparation method of the amplification system is as follows: 50 μL of the amplification system is prepared, which contains one serving of amplification enzyme freeze-dried powder, 20-30 μL of reaction buffer, 5 μM 2-5 μL of primer, 10 μL of nucleic acid sample, 2.5 μL of activator, and ddH2O is added to a total volume of 50 μL. The reaction conditions are as follows: after mixing, incubate in a constant temperature metal bath or water bath at 37-42°C for 10-30 min.

[0051] In some embodiments, the cutting system mainly consists of buffer, MgCl2, rNTP, RNAase inhibitor, T7 RNA polymerase, LwaCas13a enzyme, crRNA and reporter probe. The preparation method of the cutting system is as follows: 50 μL of the cutting system is prepared, which contains 5 μL of buffer, 1 μL of 1M MgCl2 solution, 4 μL of 25mM rNTP solution, 1 μL of 40U / μL RNAase inhibitor, 1.5 μL of 50U / μL T7 RNA polymerase, 0.4 μL of 5 μM LwaCas13a enzyme, 0.2 μL of 10 μM crRNA, 0.2 μL of 10 μM reporter probe, 10 μL of amplification system reaction product, and ddH2O is added to a total volume of 50 μL. The reaction conditions are as follows: after mixing, react in a constant temperature metal bath or water bath at 37-39°C for 10-30 min.

[0052] Take 50 μL of the above-mentioned reaction solution for immunochromatography, and observe the experimental results after waiting for 5 min. The results are read within 5-15 min. The quality control line C should be constant in color development. If it cannot be developed, it should be retested. The detection line T is used to capture the intact reporter probe and the cut probe Biotin modified end in the cutting reaction. If it is positive, the T line does not develop color. If it is negative, the T line develops color.

[0053] We solve the problem of high detection threshold of the conventional line-elimination immunochromatography test strip, which can only eliminate lines when the reporter probe is 100% cut, and has low reading efficiency, based on the principle that the binding force of SA and Biotin is much stronger than that of FAM and anti-FAM. The method of "coating and labeling marker exchange" greatly improves the detection sensitivity and exemplifies the application in the new crown Delta strain typing (isothermal amplification-CRISPR-immunochromatography method).

[0054] The high-sensitivity line-elimination method immunochromatographic test paper provided by the application can be applied to different types of detection, including pathogen detection and typing, drug resistance gene identification, gene variation and methylation modification site detection, etc.

[0055] The application will be further described in detail in combination with specific examples.

[0056] Example 1: High-sensitivity line-elimination method immunochromatographic test paper for report probe interpretation efficiency detection

[0057] At present, the line-elimination method immunochromatographic test paper applied to CRISPR at home and abroad is all judged by whether the report probe is cut or not.

[0058] When the target nucleic acid to be detected exists in the reaction system, the report probe is cut, the T line does not develop color, and the result is positive; if there is no target nucleic acid to be detected, the report probe remains intact, the T line develops color, and the result is negative. When the concentration of the target nucleic acid to be detected is not high, the trans-cleavage activity of the CRISPR detection system is difficult to be completely activated, which leads to the report probe being unable to be cut by 100%. Therefore, this embodiment simulates the CRISPR reaction system with different report probe cutting ratios, that is, in the reaction system without the target nucleic acid to be detected, the amount of the report probe that should be added = 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. Thus, the reaction system with different cutting degrees can be quantitatively simulated; and the interpretation efficiency of the line-elimination method immunochromatographic test paper at home and abroad and the high-sensitivity line-elimination method immunochromatographic test paper in the case of low report probe cutting (i.e. simulating a weak positive sample) is observed.

[0059] 1) Reagent preparation

[0060] (1) In this embodiment, the main components of the cutting system are buffer, MgCl2, rNTP, RNAase inhibitor, T7 RNA polymerase, LwaCas13a enzyme and crRNA. The use method of the cutting system is as follows: 1500 μL of the cutting system is prepared, containing 150 μL of buffer, 1M 30 μL of MgCl2 solution, 25mM 120 μL of rNTP solution, 40U / μL 30 μL of RNAase inhibitor, 50U / μL 45 μL of T7 RNA polymerase, 5 μM 12 μL of LwaCas13a enzyme, 10 μM 6 μL of crRNA, and ddH2O is added to a total volume of 1500 μL.

[0061] (2) In this embodiment, the simulation probe is designed, and the cutting system prepared in (1) is used to dilute the simulation complete probe and the simulation single-end probe in two tubes respectively, 700 μL per tube. Tube 1 contains only simulation complete probe, and the final dilution concentration is 40 nM; tube 2 contains simulation single-end probe F and simulation single-end probe B, and the final dilution concentration is 40 nM. The complete double-end probe, and the simulation single-end probe sequence and modification information are shown in Table 3:

[0062] Table 3 Sequence and Label Information

[0063] Mock probe 5' FAM-UUUUUUUU-Biotin 3' Mock single-end probe F 5' FAM-UUUUUUUU Mock single-end probe B 5' Biotin-UUUUUUUU

[0064] (3) In the present embodiment, the high-sensitivity line-elimination method immunochromatographic test paper mainly comprises 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, Tween 20, 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 Tween 20 solution, and 10 g of Proclin 300 are added to a reagent bottle, and ddH2O is added to a volume of 1 L, followed by thorough mixing, spreading on glass fiber paper, and drying for more than 4 h to obtain the 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) sodium citrate are heated to boiling under stirring, and then cooled to room temperature to obtain a nano-gold solution. For every 10 mL of the above colloidal gold solution, 8 μL / mL of K2CO3 solution is added, followed by mixing, adding 20 μg / mL of streptavidin, mixing, and reacting for 8 min to obtain a conjugate of colloidal gold-labeled streptavidin. After centrifugation, the supernatant is removed, and the precipitate is resuspended in PBS to a volume of 1 mL. A conjugate of colloidal gold-labeled rabbit IgG is prepared by the same procedure and concentration. The conjugate of colloidal gold-labeled streptavidin, the conjugate of colloidal gold-labeled rabbit IgG, and PBS buffer are mixed at a ratio of 2:1:17 (volume percentage concentration of colloidal gold-labeled streptavidin complex is 10%), and dried on glass fiber paper to obtain a colloidal gold conjugate pad. The nitrocellulose membrane comprises a T line and a C line. A T line coating solution is prepared by diluting FITC antibody to a concentration of 1 mg / mL in a 10 mM phosphate buffer solution as a coating diluent. A C line coating solution is prepared by diluting goat anti-rabbit IgG antibody to a concentration of 0.5 mg / mL in a 10 mM phosphate buffer solution as a coating diluent. Then, the coating working solutions of the T and C lines are sprayed on the nitrocellulose membrane attached to the base plate at a speed of 0.1 μL / mm by a quantitative membrane sprayer, and dried to obtain a nitrocellulose membrane coated with target materials. The water-absorbing pad is a thick water-absorbing filter paper. The filter paper is overlapped with the C line side of the nitrocellulose membrane, the filter paper is attached above the membrane, the conjugate pad is overlapped with the T line side of the nitrocellulose membrane, the conjugate pad is attached above the membrane, and the sample pad is overlapped with the conjugate pad, and the sample pad is attached above the conjugate pad.

[0065] 2) Test method

[0066] The analog probe prepared in this embodiment (2) is prepared in different proportions, wherein the analog complete probe tube 1 and the tube 2 containing the analog single-ended probe are prepared in different proportions, and the reading effect of the domestic and foreign competitors (in this embodiment, the foreign competitor is Milenia HybriDetect 1 of TwistDx Company in the United Kingdom, and the domestic competitor is RT-RAA-nfo nucleic acid amplification reagent (test strip type) of Zhongce Biology) is tested. The reading effect of the line-elimination method test strip and the high-sensitivity line-elimination method immunochromatography test strip in the system containing the analog probe when the cutting proportion of the probe is different. After preparation in different proportions, the final concentration of the probe is calculated in Table 4:

[0067] Table 4: Preparation of analog probes with different cutting proportions

[0068]

[0069]

[0070] According to the method of this embodiment, the foreign competitor test strip needs to reach a CRISPR probe cutting proportion of 100% to completely eliminate the T line, and the cutting efficiency is 95% when there is no obvious color difference with the negative; the test strip prepared according to the domestic competitor method needs a CRISPR probe cutting proportion of 100% to completely eliminate the line, and a CRISPR probe cutting proportion of 95% can only see a certain color difference. According to the reading logic, the T line is positive when it does not color, and the T line is negative when it colors. The foreign competitor test strip and the test strip prepared according to the domestic competitor method both need a very high cutting proportion to eliminate the line and judge as positive, so in the case of low sample concentration and incomplete CRISPR probe cutting, false negative results are easily produced. The present application designs a "line-elimination method" with a wider reading limit. The "line-elimination method" can eliminate the line when the probe cutting proportion is 50%. This design reduces the limitations of false negatives caused by the upper limit of the performance of the reaction system, and improves the reading efficiency of the line-elimination method. The results are shown in Figure 2 .

[0071] Example 2: Sensitivity test of high-sensitivity line-elimination method immunochromatography test strip in new coronavirus nucleic acid detection kit

[0072] The present embodiment includes the preparation and application of a new coronavirus nucleic acid detection kit (CRISPR-immunochromatography method). The new coronavirus nucleic acid detection kit (CRISPR-immunochromatography method) includes a new coronavirus N gene isothermal amplification reagent, a CRISPR reaction reagent, and a high-sensitivity line-elimination method immunochromatography test strip. Another fluorescent signal comparison reagent is prepared to directly visualize and compare the cutting proportion of the probe.

[0073] 1) Reagent composition

[0074] (1) New coronavirus N gene isothermal amplification reagent: The primers in the reagent are self-designed and synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd. The primers are selected for amplification of the sequence of the new coronavirus (2019-nCoV) N gene. The primer sequence information is shown in Table 5. The synthesized primer probe and the RNA isothermal rapid amplification mixed solution (basic type) purchased from Ampure Future Biotechnology Co., Ltd. are freeze-dried together to produce, as shown in Table 5.

[0075] Table 5 primer sequence information

[0076]

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

[0079] Table 6 crRNA and reporter probe sequence information

[0080]

[0081] (3) High-sensitivity line-elimination immunochromatography test paper: The high-sensitivity line-elimination immunochromatography test paper used in this example is made in the same way as in Example 1.

[0082] 2) Method for use

[0083] (1) Sample preparation: Take the new coronavirus (2019-nCoV) genomic RNA standard substance (GBW091099) purchased from China Institute of Metrology, dilute the sample to 20 copies / μL and 2 copies / μL.(2) Isothermal amplification reaction: 10 μL of diluted sample is mixed with 40 μL of DNase / RNase free water, then added to the new crown N gene isothermal amplification freeze-dried reagent, mixed up and down, then centrifuged with a low-speed centrifuge, and the isothermal amplification reaction was carried out in a constant temperature metal bath (reaction conditions: 42℃, 30min).(3) CRISPR reaction: Take 40 μL of CRISPR reaction reagent, add 10 μL of isothermal amplification reaction product to the system, mix up and down, centrifuge to the bottom of the tube, and react in a 37℃ constant temperature metal bath or water bath for 30min.

[0084] (4) CRISPR fluorescence: Take 40 μL of CRISPR fluorescence reagent, add 10 μL of isothermal amplification reaction product to the system, mix up and down, centrifuge to the bottom of the tube, and react in a 37℃ constant temperature metal bath or water bath for 30min.

[0085] (5) Result interpretation: Take 50 μL of all the reaction liquid in the above (3) for immunochromatography, observe the experimental results after 5 minutes, and interpret the results within 5-15 minutes. The quality control line C should be constant color development, if it cannot be developed, the test strip should be replaced for retesting; 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 positive, the T line does not develop color, if negative, the T line develops color. The results are shown in Figure 3 .

[0086] The results are shown in Figure 3As shown, when the results are interpreted using the CRISPR fluorescence method (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 fluorescence signal produced is not saturated, and the relative value is low. When the results are interpreted using the CRISPR line-elimination immunochromatography test paper (right 1-3), for example, when detecting 200 copies of the novel coronavirus (2019-nCoV) genomic RNA, since the CRISPR probe is completely cut, the foreign and domestic competitors and the line-elimination immunochromatography test paper of the present application are all interpreted as positive; for example, when detecting 20 copies of the novel coronavirus (2019-nCoV) genomic RNA, since the CRISPR probe is not completely cut, the estimated proportion of cut probes is less than 50%, the foreign and domestic competitors cannot be interpreted as positive by the line-elimination method, while the high-sensitivity line-elimination immunochromatography test paper of the present application can be detected. This shows that the product of the present application has higher sensitivity and can realize the detection of low-abundance samples.

[0087] Example 3: Comparison of the sensitivity performance of the novel coronavirus nucleic acid detection reagent with a domestic competitor

[0088] 1) Novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography method): In this example, the novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography method) of a domestic competitor (RT-RAA-nfo nucleic acid amplification reagent (test strip type) of Zhongce Biology) and the novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography method) based on the high-sensitivity line-elimination method (strong and weak binding force interconversion) described in Example 2 were used.

[0089] 2) Detection method:

[0090] (1) Sample preparation: Take the novel coronavirus (2019-nCoV) genomic RNA standard material (GBW091099) purchased from the China Institute of Metrology, and sequentially dilute the sample to 100 copies / μL, 10 copies / μL, 5 copies / μL and 1 copies / μL.

[0091] (2) The constant temperature amplification reaction, the CRISPR reaction and the result interpretation part are the same as in Example 2. The negative control, the reagent kit of the competitor manufacturer and the reagent kit in Example 2 all normally color; when the sample concentration is 1000 copies per reaction, the reagent kit of the competitor manufacturer and the reagent kit in Example 2 can all be detected; when the sample concentration is 100 copies per reaction, the detection rate of the competitor manufacturer is 1 / 3, and the reagent kit in Example 2 can all be detected; when the sample concentration is 50 copies per reaction, the competitor manufacturer cannot be detected, and the reagent kit in Example 2 can all be detected; when the sample concentration is 10 copies per reaction, the detection rate of the competitor manufacturer is 1 / 3, and the reagent kit in Example 2 can all be detected. The results are shown in Figure 4 .

[0092] Example 4: Detection of clinical samples

[0093] 1) Novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography method): the novel coronavirus nucleic acid detection kit (CRISPR-immunochromatography method) described in Example 2 is used in this embodiment.

[0094] 2) Detection method:

[0095] (1) Sample preparation: 24 clinical samples of novel coronavirus nucleic acid were collected, and the sample CT value information is shown in Figure 5 . After the sample was taken out and thawed, nucleic acid extraction and purification were performed with a nucleic acid extraction and purification reagent.

[0096] (2) The constant temperature amplification reaction, the CRISPR reaction and the result interpretation part are the same as in Example 2. There are 7 samples with CT values less than 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. Among them, the sample with a CT value of 38 has a T line color of non-detection, and the rest of the samples have no color and are detected. The results are shown in Figure 5 : In nucleic acid detection, especially in new crown detection, the CT value ≤ 35 is used as the positive judgment value standard, and the sample with a CT value of 38 is an extremely weak sample, which is not judged as positive in clinical practice. The results show that the detection kit has high sensitivity, which meets the traditional positive judgment standard of nucleic acid detection.

[0097] Example 5: Sensitivity verification of novel coronavirus Delta strain typing detection kit

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

[0099] The kit is used for distinguishing the EFR156-158G mutation site in the S gene of the new coronavirus Delta strain. The mutation site only occurs in the Delta mutant strain as of the filing of the present patent, does not cross-react with the original strain and other mutant strains, but does not rule out the possibility of future new mutant strains mutating at this site, causing the kit to fail.(1) New coronavirus Delta strain typing isothermal amplification freeze-dried ball reagent: The primers in the reagent are independently designed, and sequences around the mutation site specific to the Delta strain in the S gene of the new coronavirus (2019-nCoV) are selected for amplification. The primer sequence information is shown in Table, and the synthesized primer probe and the RNA isothermal rapid amplification mixed solution (basic type) purchased from Ampfu Biotech Co., Ltd. are freeze-dried together for production, as shown in 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-BufferA-O: 1.31x buffer, 26.24mM MgCl2, 2.62mM rNTP solution, 1.05U / μL RNAase inhibitor, 52.49nM crRNA, 52.49nM reporter probe. The crRNA and reporter probe are independently designed, and 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 sequence is shown in Table 8:

[0103] Table 8 New coronavirus S gene wild type sequence crRNA and reporter probe sequence information

[0104]

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

[0106] Table 9 New coronavirus S gene DELTA mutant sequence crRNA and reporter probe sequence information

[0107]

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

[0109] (5) High-sensitivity line-elimination immunochromatographic test paper: The high-sensitivity line-elimination immunochromatographic test paper used in this example was made in the same way as in Example 1 above.

[0110] 3) Detection method

[0111] (1) Sample preparation: The new coronavirus (2019-nCoV) Delta B.1.617 variant genomic RNA standard material (GBW09316) purchased from the China Institute of Metrology was taken, and the sample was diluted to 10 copies / µL, 5 copies / µL, 2 copies / µL, 1 copies / µL, 0.5 copies / µL, 0.3 copies / µL, and 0.1 copies / µL.

[0112] (2) Isothermal amplification reaction: 10 µL of the diluted sample was mixed with 40 µL of DNase / RNase free water, then added to the new crown Delta strain typing isothermal amplification freeze-dried ball reagent, mixed well by inverting up and down, and then centrifuged with a low-speed centrifuge. The isothermal amplification reaction was performed on a constant temperature metal bath (reaction conditions: 42°C, 30 min).

[0113] (3) CRISPR reaction: Two tubes of CRISPR reaction reagent were prepared for each amplification reaction. CRISPR-O: 76.2 µL of CRISPR-BufferA-O and 3.8 µL of CRISPR-BufferB were added to the PCR tube and mixed well; CRISPR-DELTA: 76.2 µL of CRISPR-BufferA-DELTA and 3.8 µL of CRISPR-BufferB were added to the PCR tube and mixed well. 20 µL of the amplification reaction product was added to CRISPR-O and CRISPR-DELTA, respectively, mixed well by inverting up and down, and then centrifuged with a low-speed centrifuge. The CRISPR reaction was performed on a constant temperature metal bath (reaction conditions: 37°C, 30 min).

[0114] (4) Result interpretation: After mixing the CRISPR-O and CRISPR-DELTA reaction products, centrifuge, take 65 μL respectively and add to the sample pad of high sensitivity line-out immunochromatography test paper, and interpret the results within 5-15 minutes. When the CRISPR-O corresponding test paper C line develops color, T line does not develop color, and the CRISPR-DELTA corresponding test paper C line develops color, T line develops color, it is non-DELTA strain new coronavirus positive; when the CRISPR-O corresponding test paper C line develops color, T line develops color, and the CRISPR-DELTA corresponding test paper C line develops color, T line does not develop color, it is DELTA strain new coronavirus positive; when the CRISPR-O corresponding test paper C line develops color, T line develops color, and the CRISPR-DELTA corresponding test paper C line develops color, T line develops color, it is new coronavirus negative. The results are shown in Figure 6 (The upper figure is the result of the foreign competitor - Milenia HybriDetect 1 of the British TwistDx company, and the lower figure is the high sensitivity line-out immunochromatography test paper of the present embodiment). The results show that the same reaction product, the kit described in the present application can detect a sensitivity as low as 3 copies / reaction, which is better than the 100 copies / reaction of the foreign competitor.

[0115] Example 6: Specificity of new coronavirus typing test kit

[0116] 1) New coronavirus Delta strain typing kit (isothermal amplification-CRISPR-immunochromatography method): The new coronavirus Delta strain typing kit (isothermal amplification-CRISPR-immunochromatography method) of Example 3 is used in this embodiment.

[0117] 2) Detection method:

[0118] (1) Sample preparation: Take the commercially available new coronavirus (2019-nCoV) in vitro transcription RNA standard material (GBW09299), new coronavirus (2019-nCoV) beta B.1.351 variant genome RNA standard material (GBW09315), new coronavirus (2019-nCoV) delta B.1.617 variant genome RNA standard material (GBW09316), new coronavirus (2019-nCoV) gamma P.1 variant genome RNA standard material (GBW09317), and new coronavirus (2019-nCoV) omicron BA.1.1 variant genome RNA standard material (GBW09318) from China Institute of Metrology, and dilute them to 10 copies / μL respectively.

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

[0120] Example 7: Verification of new crown typing detection kit for clinical samples

[0121] 1) New crown Delta strain typing kit (isothermal amplification-CRISPR-immunochromatography method): The new crown Delta strain typing kit (isothermal amplification-CRISPR-immunochromatography method) of Example 3 is used in this embodiment.

[0122] (1) Nucleic acid extraction and purification reagent: nucleic acid extraction and purification reagent of Guangzhou Wanfu Biotechnology Co., Ltd., Record No.: Yue Sui Jiansheng Record 20210234.

[0123] (2) Fluorescent PCR detection reagent: 2019-nCoV nucleic acid detection kit (fluorescent PCR method) of Daan Gene, Registration No.: Guojianzhunqing 20203400063.

[0124] 4) Detection method:

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

[0126] (2) The extracted nucleic acid was detected by fluorescent PCR detection reagent, and the results are shown in Table 10:

[0127] Table 10 Ct value of fluorescent quantitative PCR detection of new crown Alpha and Delta samples

[0128]

[0129]

[0130] (3) The isothermal amplification reaction, CRISPR reaction and result interpretation part are the same as Example 3. The results are shown in Figure 8 The results show that among the 15 PCR-identified clinical samples, 2 were negative, 6 were Alpha positive, and 7 were Delta positive. The high-sensitivity line-elimination detection kit of the application can detect 6 / 6 Alpha strains and 7 / 7 Delta strains. The foreign competitor control product (Milenia HybriDetect 1 of British TwistDx Company) can only detect 5 / 6 Alpha strains and 4 / 7 Delta strains.

[0131] Example 8 Comparative Experiment

[0132] To achieve better line-elimination effect, we optimized the concentration of streptavidin gold on the conjugate pad. As shown in Table 1, 1%, 5%, 10%, and 15% in Table 8-1 correspond to the volume percentage concentration of streptavidin gold of 1%, 5%, 10%, and 15%, respectively. The results shown are the color development degrees of the lower percentage cleaved probes, in which C1-C8 are color development degrees from strong to weak. The greater the difference between 0% cleavage and other cleavage percentages, the easier to interpret. It is found that the optimal range of streptavidin gold concentration is 5%-10%. The preparation method of the immunochromatographic test strip is as described in Examples 1 and 2. In this example, the mix of the single-end biotin-labeled probe and the single-end FAM-labeled probe mixed at 1:1 is used as the 100% cleaved probe, and the probe with biotin and FAM labeled at both ends is used as the 0% cleaved probe.

[0133] Table 8-1 Screening of Streptavidin Gold Concentration

[0134]

[0135] The nitrocellulose membrane includes T lines and C lines. The T line coating solution is prepared by diluting the FITC antibody to a concentration of 1 mg / mL in a 10 mM phosphate buffer solution as the coating diluent. The C line coating solution is prepared by diluting the goat anti-rabbit IgG antibody to a concentration of 0.5 mg / mL in a 10 mM phosphate buffer solution as the coating diluent. Then the coating working solution of the T and C lines is sprayed on the nitrocellulose membrane pasted on the bottom plate at a speed of 0.1 μL / mm, and dried to obtain the nitrocellulose membrane coated with the target material.

[0136] To achieve better line-elimination effect, we compared the FITC antibody and concentration on the conjugate pad. Except for the different T line antibodies and concentrations, other preparation parameters are as described in Examples 1 and 2. In the table, C-1 (C-1 is the mouse monoclonal antibody Meridian product K45219M), C-2 (Meridian K86503M), and C-3 (mouse monoclonal antibody Fitzgerald 10-F50C, clone number M25286) are different types of antibodies. In the table, 0.5 and 1.0 represent the concentrations of the corresponding antibodies of 0.5 mg / mL and 1.0 mg / mL, respectively, as shown in Table 8-2. It is found that the Meridian K86503M mouse monoclonal antibody (C-2) has the best line-elimination effect, and the optimal concentration is 1 mg / mL.

[0137] Table 8-2 Screening of T Line Antibody and Concentration

[0138]

[0139] The above embodiments only express several implementation manners of the present application, which are described in a more specific and detailed manner, but cannot be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A line-eliminating immunochromatographic test strip, comprising a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad sequentially overlapped and bonded to a base plate, wherein the nitrocellulose membrane has a C-line for control and a T-line for detection, characterized in that, The conjugation pad is coated with a streptavidin-labeled colloidal gold complex and a rabbit IgG-labeled colloidal gold complex; the T-line of the nitrocellulose membrane is coated with FITC antibody, and the C-line is coated with anti-IgG, with the C-line close to the absorbent pad; the FITC antibody is Meridian's K86503M mouse monoclonal antibody; the working concentration of the FITC antibody coated on the T-line is 0.8~1.4 mg / mL; and the volume percentage concentration of the streptavidin-labeled colloidal gold complex on the conjugation pad is 5%~10%.

2. The immunochromatographic test strip according to claim 1, characterized in that, The ratio of the streptavidin-labeled colloidal gold complex to the rabbit IgG-labeled colloidal gold complex on the conjugate pad is 2:

1.

3. The immunochromatographic test strip using the line-elimination method according to claim 1, characterized in that, The working concentration of the FITC antibody coated on the T line is 0.9-1.1 mg / mL.

4. The immunochromatographic test strip according to claim 1, characterized in that, The working concentration of the FITC antibody coated on the T line is 1 mg / mL.

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

6. The immunochromatographic test strip according to claim 1, characterized in that, The working concentration of the C-line coated anti-IgG is 0.5 mg / mL.

7. The use of the line-eliminating immunochromatographic test strip according to any one of claims 1-6 in the preparation of CRISPR-based target gene detection products.

8. The application according to claim 7, characterized in that, The target gene detection includes pathogen genotyping, SNP, insertion / deletion, structural variation, methylation modification site detection, or drug resistance gene identification.

9. A kit for detecting a target gene, characterized in that, Includes the line-elimination immunochromatographic test strip as described in any one of claims 1-6, and the CRISPR cutting system.

10. The reagent kit according to claim 9, characterized in that, It also includes target nucleic acid amplification systems; and / or CRISPR cutting systems such as Cas13a, Cas12a, Cas12b, and Ago systems.

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