Fluorescence immunochromatography detection kit for rapidly detecting quality of RNA (Ribonucleic Acid) product, detection method and application

Abstract

The invention belongs to the technical field of biology, and discloses a fluorescence immunochromatography detection kit for rapidly detecting RNA product quality, a detection method and application. The fluorescence immunochromatography detection kit provided by the invention specifically comprises a sample treatment solution and detection test paper, wherein the sample treatment solution comprises 0.1 to 0.5 mg / mL of biotin-poly (dT) 25, 0.1 to 0.5 wt% of polyethylene glycol octyl phenyl ether and 1 to 5 wt% of sodium chloride; the detection test paper comprises a sample pad, a combination pad and a reaction pad which are sequentially contacted; the combination pad is coated with an antibody with a fluorescent nanoparticles-anti-5 '-cap structure; the reaction pad is provided with a detection line, and the detection line is coated with streptavidin. The fluorescence immunochromatography detection kit can be used for detecting a plurality of indexes such as capping rate, integrity and packaging efficiency of RNA products, can realize data acquisition only by adopting a fluorescence signal instrument, is low in instrument requirement and simple to operate, can be well suitable for rapid quality control test in large-scale production of the RNA products, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fluorescent immunochromatography detection kit, a detection method and an application thereof for rapidly detecting the quality of RNA products. Background Art

[0002] In order to ensure the safety and reliability of RNA products such as mRNA vaccines and mRNA drugs, it is necessary to conduct quality control tests on RNA products during the manufacturing process. In the quality control test, the main focus is on multiple indicators such as the capping rate and integrity of mRNA and the encapsulation efficiency of LNP drug preparations.

[0003] At present, the most commonly used test method for RNA capping rate is to obtain oligonucleotide sequences with or without capping at the 5' end by enzyme digestion, and then separate oligonucleotide fragments of different sizes by urea denaturing polyacrylamide gel electrophoresis (PAGE), high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to quantitatively evaluate the capping rate of mRNA. The draft guidance on analytical procedures for mRNA (messenger ribonucleic acid) vaccine quality control issued by the United States Pharmacopeia (USP) recommends the use of capillary electrophoresis (CE) to perform quality control tests on the integrity of mRNA vaccines. The encapsulation efficiency of LNP drug preparations is mainly tested by using the property of RiboGreen to emit green fluorescence after specific insertion into nucleic acids, so as to achieve quantitative detection of RNA in the solution before and after demulsification.

[0004] However, existing methods for quality control testing of RNA products can only detect one or two of multiple indicators such as the uncapping rate, integrity and encapsulation efficiency of RNA products. In addition, the above methods need to be applied to large-scale equipment, and the kits used are expensive, cumbersome and time-consuming to operate. They cannot meet the requirements for rapid quality control testing in large-scale production of RNA products and have great limitations. Summary of the invention

[0005] The first purpose of the present invention is to solve the defects of incomplete test indicators, high equipment requirements, high cost and complicated steps in the existing RNA product quality control test, and provide a fluorescent immunochromatographic detection kit. The fluorescent immunochromatographic detection kit can detect multiple indicators such as the capping rate, integrity and encapsulation efficiency of RNA products, and only needs to use a fluorescent signal instrument to realize data collection. It has low instrument requirements and simple operation, and can be well applied to rapid quality control tests in large-scale production of RNA products, and has good application prospects.

[0006] The second purpose of the present invention is to provide a method for quickly detecting the quality of RNA products.

[0007] The third purpose of the present invention is to provide the use of the above-mentioned fluorescent immunochromatography detection kit in the preparation of RNA products.

[0008] Specifically, the fluorescent immunochromatographic detection kit provided by the present invention comprises: a sample processing solution, wherein the sample processing solution comprises 0.1 to 0.5 mg / mL of biotin-poly (dT) 25 , 0.1-0.5wt% of polyethylene glycol octylphenyl ether and 1-5wt% of sodium chloride; a test strip, the test strip comprising a sample pad, a conjugation pad and a reaction pad in contact with each other in sequence; the conjugation pad is coated with fluorescent nanoparticles-anti-5' cap structure antibodies; the reaction pad is provided with a detection line, and the detection line is coated with streptavidin.

[0009] Furthermore, the sample treatment solution is a Tris-HCl buffer, and the concentration of the Tris-HCl buffer is 5-15 mM and the pH is 7.5-8.

[0010] Furthermore, the coating amount of the fluorescent nanoparticle-anti-5' cap structure antibody on the conjugate pad is 0.01-0.2 μg / cm 2 .

[0011] Furthermore, in the fluorescent nanoparticle-anti-5' cap structure antibody, the particle size of the fluorescent nanoparticle is 100-300 nm.

[0012] Furthermore, on the detection line, the coating amount of streptavidin is 10-15 μg / cm 2 .

[0013] Furthermore, a quality control line is provided on the detection pad, and the quality control line is coated with an antibody against the 5' cap structure.

[0014] Furthermore, on the quality control line, the coating amount of the anti-5' cap structure antibody is 0.01-2 μg / cm 2 .

[0015] The method for rapidly detecting the quality of RNA products provided by the present invention uses the above-mentioned fluorescent immunochromatography detection kit to detect the RNA product to be detected.

[0016] Furthermore, the method comprises: S1, mixing the RNA product to be tested with the sample treatment solution to obtain a loading solution; S2, performing chromatography on the loading solution on the test paper; S3, detecting the fluorescence signal of the test line, and calculating and obtaining the RNA product quality index.

[0017] Furthermore, in step S1, based on the total volume of the sample treatment solution, the added amount of the RNA product to be tested is 0.01-0.05 pmol / μL.

[0018] Furthermore, in step S2, the loading volume of the loading solution is 0.1 to 5 μL.

[0019] The present invention also provides the use of the fluorescent immunochromatography detection kit in the preparation of RNA products.

[0020] Beneficial effects:

[0021] The fluorescent immunochromatographic detection kit provided by the present invention includes a sample processing solution and a detection test paper, wherein the sample processing solution specifically includes a specific concentration of biotin-poly (dT) 25 , polyethylene glycol octylphenyl ether, sodium chloride and Tris-HCl buffer, which contains biotin-poly (dT) 25 The test strip is used to detect RNA products treated with a sample treatment solution. The fluorescent nanoparticles-anti-5' cap structure antibodies on the binding pad can bind to RNA molecules with 5' cap structures in RNA products, and the synergistic effect of polyethylene glycol octylphenyl ether and sodium chloride can effectively change the biotin-poly (dT) complex bound to the polyA tail structure. 25 The three-dimensional spatial structure of the RNA allows the biotin on it to be completely released and exposed, thereby combining with the streptavidin on the detection line to achieve the enrichment of the fluorescent signal. The enriched fluorescent signal represents the RNA molecule with both a 5' cap structure and a polyA tail. By detecting and calculating the fluorescent signal, multiple quality control indicators such as the capping rate, integrity and encapsulation efficiency of the RNA product can be detected. It has the advantages of fast detection speed, simple operation and low price, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the structure of the test paper provided in Preparation Example 1 of the present invention;

[0023] Figure 2 This is one of the experimental result diagrams of the specific recognition ability test of the fluorescent immunochromatographic detection kit provided in Example 7 of the present invention;

[0024] Figure 3 FIG2 is the experimental result of the specific recognition ability test of the fluorescent immunochromatographic detection kit provided in Example 7 of the present invention;

[0025] Figure 4This is one of the experimental result graphs of the detection resolution test of the fluorescent immunochromatographic detection kit provided in Example 8 of the present invention;

[0026] Figure 5 FIG2 is a second experimental result of the detection resolution test of the fluorescent immunochromatographic detection kit provided in Example 8 of the present invention;

[0027] Figure 6 Figure 3 is the experimental result of the detection resolution test of the fluorescent immunochromatographic detection kit provided in Example 8 of the present invention;

[0028] Figure 7 FIG4 is an experimental result of the detection resolution test of the fluorescent immunochromatographic detection kit provided in Example 8 of the present invention;

[0029] Figure 8 This is a graph showing the experimental results of the fluorescent immunochromatographic detection kit provided in Example 9 of the present invention for detecting the relevant performance test of RNA integrity (-80°C);

[0030] Fig. 9 This is a graph showing the experimental results of the fluorescent immunochromatographic detection kit provided in Example 9 of the present invention for detecting the relevant performance test of RNA integrity (37° C.);

[0031] Fig.10 This is a graph showing the experimental results of the relevant performance test of the fluorescent immunochromatography detection kit provided in Example 10 of the present invention for detecting the encapsulation efficiency of LNP drug preparations.

[0032] Figure numerals: 1, PVC base plate; 2, sample pad; 3, binding pad; 4, reaction pad; 5, water-absorbing pad; 6, detection line; 7, quality control line. DETAILED DESCRIPTION

[0033] The fluorescent immunochromatographic detection kit provided by the present invention specifically includes a sample treatment solution and a test strip, and the RNA product is treated by the sample treatment solution so that it can be better detected by the test strip. The sample treatment solution includes 0.1 to 0.5 mg / mL of biotin-poly (dT) 25, such as 0.1mg / mL, 0.15mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL or any value therebetween; 0.1-0.5wt% of polyethylene glycol octylphenyl ether, such as 0.1wt%, 0.18wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt% or any value therebetween; 1-5wt% of sodium chloride, such as 1wt%, 1.3wt%, 1.5wt%, 2wt%, 3wt%, 4wt%, 5wt% or any value therebetween. The test paper comprises a sample pad, a conjugation pad and a reaction pad in contact with each other in sequence; the conjugation pad is coated with fluorescent nanoparticles-anti-5' cap structure antibodies; the reaction pad is provided with a detection line, and the detection line is coated with streptavidin.

[0034] In the present invention, the polyethylene glycol octylphenyl ether and sodium chloride in the sample treatment solution can cooperate to optimize the three-dimensional spatial structure of RNA so that the biotin-poly (dT) bound thereto is 25 The biotin on the RNA can be fully released and exposed, so that it can be recognized and bound by the streptavidin on the detection line, so as to finally achieve the quantitative determination of multiple indicators such as the capping rate, integrity and encapsulation efficiency of the RNA product.

[0035] In the present invention, the sample treatment solution is preferably a Tris-HCl buffer. In some specific embodiments, the concentration of the Tris-HCl buffer is preferably 5 to 15 mM, such as 5 mM, 7.5 mM, 10 mM, 12.5 mM, 13 mM, 15 mM, or any value therebetween; the pH is preferably 7.5 to 8, such as 7.5, 7.6, 7.7, 7.8, 8, or any value therebetween. At this time, RNA can have a more stretched three-dimensional structure in the sample treatment solution, which is beneficial for its binding to biotin-poly (dT) 25 The combination with the antibody against the 5' cap structure can achieve better detection effect.

[0036] In the present invention, the coating amount of the fluorescent nanoparticle-anti-5' cap structure antibody on the conjugate pad is preferably 0.01-0.2 μg / cm 2 For example, 0.04 μg / cm 2 , 0.042μg / cm 2 , 0.048μg / cm 2 , 0.06μg / cm 2 , 0.12μg / cm 2 , 0.2μg / cm 2 or any value in between.

[0037] In the invention, in the fluorescent nanoparticle-anti-5' cap structure antibody, the fluorescent nanoparticle is a type of raw material commonly used in the production of existing immunochromatographic test strips, and is not particularly limited. In some specific embodiments, the fluorescent nanoparticle can be but is not limited to: modified polystyrene microspheres modified with fluorescent complexes of the rare earth element europium.

[0038] In some specific embodiments, the particle size of the fluorescent nanoparticles is preferably 100-300 nm, such as 100 nm, 105 nm, 110 nm, 120 nm, 150 nm, 200 nm, 240 nm, 270 nm, 300 nm or any value therebetween.

[0039] In the present invention, the coating amount of streptavidin on the detection line is preferably 10-15 μg / cm 2 , such as 10μg / cm 2 , 11μg / cm 2 , 12.5μg / cm 2 , 13.8μg / cm 2 , 14μg / cm 2 , 15μg / cm 2 or any value in between.

[0040] In the present invention, the detection pad is preferably further provided with a quality control line, on which an antibody against the anti-5' cap structure is coated, wherein the antibody against the anti-5' cap structure is used to recognize and bind to the free fluorescent nanoparticle-anti-5' cap structure.

[0041] In some specific embodiments, the coating amount of the anti-5' cap structure antibody is 0.01-2 μg / cm 2 , such as 0.01 μg / cm 2 , 0.05μg / cm 2 , 0.08μg / cm 2 , 0.12μg / cm 2 , 0.15μg / cm 2 , 0.2μg / cm 2 , 0.8μg / cm 2 , 1μg / cm 2 , 1.5μg / cm 2 , 2μg / cm 2 or any value in between.

[0042] In the present invention, the sample pad, conjugate pad and reaction pad included in the test paper are materials conventionally used in the art, and are limited to being able to detect RNA. The present invention does not impose any particular limitation on their specific materials, preparation processes and sources.

[0043] The present invention also provides a method for rapidly detecting the quality of RNA products, which uses the above-mentioned fluorescent immunochromatographic detection kit to detect the RNA product to be detected. More specifically, the method for rapidly detecting the quality of RNA products specifically includes: S1, taking the RNA product to be detected and mixing it with the sample treatment solution to obtain a loading solution; S2, taking the loading solution and performing chromatography on the detection test paper; S3, detecting the fluorescent signal of the detection test paper, and calculating and obtaining the RNA product quality index.

[0044] In the present invention, in step S1, based on the total volume of the sample processing solution, the added amount of the RNA product to be tested is preferably 0.01-0.05 pmol / μL, such as 0.01 pmol / μL, 0.02 pmol / μL, 0.03 pmol / μL, 0.04 pmol / μL, 0.05 pmol / μL or any value therebetween.

[0045] In the present invention, in step S2, the loading volume of the loading solution is preferably 0.1 to 5 μL, such as 0.1 μL, 0.3 μL, 0.75 μL, 1 μL, 1.38 μL, 2 μL, 3 μL, 5 μL or any value therebetween.

[0046] In the present invention, in step S2, the chromatography is a type of technical means conventionally used in fluorescent immunochromatography detection. Those skilled in the art can make adaptive choices on the specific conditions to be adopted according to the actual sample solution and the test paper, and the present invention does not impose any particular limitation on it.

[0047] In some specific embodiments, in step S2, the chromatography time is preferably 10 to 20 min, such as 10 min, 13 min, 15 min, 18 min, 20 min or any value therebetween.

[0048] In the present invention, the method for detecting the fluorescent signal of the test line and / or the quality control line on the test strip and the method for calculating the capping rate, integrity, sealing efficiency and other related indicators in step S3 are a type of method conventionally used in the art, and are limited to being able to realize the detection of the fluorescent signal and the quantitative calculation of the related indicators, and the present invention does not impose any special limitation on them.

[0049] The present invention also provides the use of the above-mentioned fluorescent immunochromatography detection kit in the preparation of RNA products. The kit can be used to quickly and accurately detect relevant indicators such as RNA capping rate, integrity, and sealing efficiency, thereby realizing rapid quality control testing of large-scale production of RNA products, and has good application prospects.

[0050] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0051] The reagents and sources of the following preparation examples and examples are specifically included:

[0052] Antibody against 5' cap structure (Abcam, catalog number ab300741, rabbit IgG antibody);

[0053] Activated fluorescent microspheres (Meiniu Biotechnology Co., Ltd., catalog number MF-02);

[0054] Biotin-poly(dT) 25 (Qingke Biotechnology, catalog number NS-050400-001);

[0055] Streptavidin (Solyb, Catalog No. S9171);

[0056] Anti-rabbit IgG antibody (Jingda Biotechnology, catalog number W4011);

[0057] Cap1 capping kit (Novozymes, catalog number DD4109-01);

[0058] Firefly Luciferase-mRNA (Yisheng Biotechnology, catalog number 17101ES);

[0059] mRNA COVID-19 vaccine Spikevax (Moderna).

[0060] Preparation Example 1

[0061] This preparation example is used to illustrate a test paper and its preparation. Figure 1The test paper includes a PVC base plate 1, a sample pad 2, a conjugation pad 3, a reaction pad 4 and a water-absorbing pad 5. The sample pad 2, the conjugation pad 3, the reaction pad 4 and the water-absorbing pad 5 are fixed on the PVC base plate 6, and one side of the sample pad 2 is overlapped on the conjugation pad 3, the side of the conjugation pad 3 away from the sample pad 2 is overlapped on the reaction pad 4, and one side of the water-absorbing pad 5 is overlapped on the side of the reaction pad 4 away from the conjugation pad 3. The reaction pad 4 is provided with a detection line 6 and a quality control line 7, and the detection line 6 is provided on the side of the reaction pad 4 close to the conjugation pad 3. Among them, the conjugation pad 3 is coated with fluorescent nanoparticles-anti-mRNA 5' end capping structure antibody, and the coating amount of the fluorescent nanoparticles-anti-5' capping structure antibody is 0.12μg / cm 2 Detection line 6 is coated with streptavidin, and the coating amount is 12.5 μg / cm 2 The quality control line 7 is coated with anti-rabbit IgG antibody, and the coating amount is 1μg / cm 2 , and the distance between the quality control line and the detection line is 5mm.

[0062] The preparation method of the test paper provided in this preparation example specifically comprises the following steps:

[0063] (1) Preparation of conjugate pad 3: S1. Preparation of fluorescent nanoparticle-anti-5' cap structure antibody: 30 μg of anti-5' cap structure antibody was mixed with 3 μg of activated fluorescent microspheres (particle size of 200 nm), mixed at room temperature for 1 hour, centrifuged at 15000 rpm for 30 minutes, the supernatant was removed, and the precipitate was blocked with phosphate buffer (pH=7.4) solution containing 5% BSA (bovine serum albumin) for 1 hour, stirred and mixed; centrifuged at 15000 rpm for 10 minutes, the supernatant was removed, and the fluorescent nanoparticle-anti-5' cap structure antibody was obtained.

[0064] S2. Spray the fluorescent nanoparticles-anti-5' end cap structure antibody onto the glass cellulose membrane according to the above coating amount, and dry it at 37° C. to obtain a conjugate pad 3.

[0065] (2) Preparation of reaction pad 4: Streptavidin and anti-rabbit IgG antibody were streaked at different positions on the glass cellulose membrane using a streaking instrument according to the above coating amount to form a detection line 6 and a quality control line 7, and then dried at 37° C. to obtain a reaction pad 4.

[0066] (3) Assembly of fluorescent immunochromatographic test strips: Figure 1 The structure shown is that the PVC base plate 1, the sample pad 2, the conjugation pad 3, the reaction pad 4 and the water absorbent pad 5 are assembled to obtain the test paper.

[0067] Preparation Example 2

[0068] This preparation example adopts the method provided in Preparation Example 1 to prepare the test strip, except that the coating amount of the fluorescent nanoparticle-anti-5' end cap structure antibody on the conjugate pad is 0.1 μg / cm 2 The coating amount of streptavidin on the detection line is 10μg / cm 2 The coating amount on the anti-rabbit IgG antibody was 0.1 μg / cm 2 , other conditions are the same, and the test paper is obtained.

[0069] Preparation Example 3

[0070] This preparation example adopts the method provided in Preparation Example 1 to prepare the test strip, except that the coating amount of the fluorescent nanoparticle-anti-5' end cap structure antibody on the conjugate pad is 0.2 μg / cm 2 The coating amount of streptavidin on the detection line is 15μg / cm 2 The coating amount on the anti-rabbit IgG antibody was 2 μg / cm 2 , other conditions are the same, and the test paper is obtained.

[0071] Example 1

[0072] This example is used to illustrate a fluorescent immunochromatographic detection kit, which specifically includes a sample treatment solution and a test strip provided in Preparation Example 1. The sample treatment solution includes 0.2 mg / mL of biotin-poly(dT) 25 , 0.2 wt % of polyethylene glycol octylphenyl ether, 2 wt % of sodium chloride and 10 mM Tris-HCl buffer (pH = 7.5).

[0073] Comparative Example 1

[0074] The fluorescent immunochromatographic detection kit provided in this comparative example is basically the same as that in Example 1, except that, in the fluorescent immunochromatographic detection kit, an equal volume of 10 mM Tris-HCl buffer (pH = 7.5) is used to replace the sample treatment solution, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0075] Comparative Example 2

[0076] The fluorescent immunochromatographic detection kit provided in this comparative example is basically the same as that in Example 1, except that, in the fluorescent immunochromatographic detection kit, the sample processing liquid does not include polyethylene glycol octylphenyl ether, that is, the concentration of polyethylene glycol octylphenyl ether is 0wt%, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0077] Comparative Example 3

[0078] The fluorescent immunochromatographic detection kit provided in this comparative example is basically the same as that in Example 1, except that in the fluorescent immunochromatographic detection kit, the sample processing liquid does not include sodium chloride, that is, the concentration of sodium chloride is 0wt%. Other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0079] Example 2

[0080] The fluorescent immunochromatographic detection kit provided in this example is basically the same as that in Example 1, except that the detection paper provided in Preparation Example 2 is used instead of the detection paper provided in Preparation Example 1, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0081] Example 3

[0082] The fluorescent immunochromatographic detection kit provided in this example is basically the same as that in Example 1, except that the test paper provided in Preparation Example 3 is used instead of the test paper provided in Preparation Example 1, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0083] Example 4

[0084] The fluorescent immunochromatographic detection kit provided in this embodiment is basically the same as that in embodiment 1, except that the biotin-poly(dT) in the sample treatment solution 25 The concentration of is 0.5 mg / mL, the concentration of polyethylene glycol octylphenyl ether is 0.5 wt %, the concentration of sodium chloride is 3 wt %, the concentration of Tris-HCl buffer is 15 mM, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0085] Example 5

[0086] The fluorescent immunochromatographic detection kit provided in this embodiment is basically the same as that in embodiment 1, except that the biotin-poly(dT) in the sample treatment solution 25 The concentration of is 0.1 mg / mL, the concentration of sodium chloride is 5 wt %, the concentration of Tris-HCl buffer is 5 mM, and the pH is 8, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0087] Example 6

[0088] The fluorescent immunochromatographic detection kit provided in this embodiment is basically the same as that in Example 1, except that the pH of the Tris-HCl buffer in the sample treatment solution is 6.5, and other conditions are the same to obtain a fluorescent immunochromatographic detection kit.

[0089] Example 7

[0090] This example is used to illustrate the specific recognition ability of the fluorescent immunochromatographic detection kit provided in the above examples and comparative examples for RNA with a complete structure (5' cap and polyA tail), specifically including:

[0091] (1) 132 ng of fully capped mRNA-GFP with a polyA tail, fully uncapped mRNA-GFP with a polyA tail, fully capped mRNA-GFP without a polyA tail, and fully uncapped mRNA-GFP without a polyA tail were respectively mixed with 50 μL of the sample treatment solution provided in each embodiment and comparative example to obtain a loading solution.

[0092] The nucleotide sequences of mRNA-GFP and polyA are shown in Table 1.

[0093] Table 1.

[0094]

[0095]

[0096] (2) According to the RNA loading amount of 0.5 pmol, the loading solution was added dropwise to the sample pad 2 of the test paper corresponding to each sample treatment solution. After chromatography for 15 minutes, the fluorescence intensity of the test line 6 and the quality control line 7 was measured by a fluorescence detector, and the T / C ratio was calculated according to the following formula. The results are as follows: Figure 2 and 3 shown.

[0097] T / C ratio = (fluorescence intensity of the test line / fluorescence intensity of the quality control line) × 2000

[0098] Depend on Figure 2 and 3 It can be seen from the test results shown that, compared with the comparative example, the fluorescent immunochromatographic detection kit provided in Examples 1 to 6 of the present invention has excellent specific recognition ability for RNA with a complete structure (5' cap and polyA tail).

[0099] Example 8

[0100] This example is used to illustrate the resolution of the fluorescent immunochromatography detection kit provided in the above examples and comparative examples for detecting RNA capping rate, specifically including:

[0101] (1) mRNA-GFP completely capped and having a polyA tail and mRNA-GFP completely uncapped and having a polyA tail were mixed in different proportions to obtain RNA products with different capping rates; wherein the nucleotide sequences of mRNA-GFP and polyA are shown in Table 1.

[0102] (2) 132 ng of the RNA product was taken and mixed evenly with 50 μL of the sample treatment solution provided in each embodiment and comparative example to obtain a loading solution.

[0103] (3) According to the mRNA loading amount of 0.5 pmol, the loading solution was added dropwise to the sample pad 2 of the test paper corresponding to each sample treatment solution. After chromatography for 15 minutes, the fluorescence intensity of the test line 6 and the quality control line 7 was measured by a fluorescence detector, and the normalized T / C ratio and S / N ratio were calculated according to the following formula. The results are as follows: Figures 4 to 7 shown.

[0104] Normalized T / C ratio = (T / C ratio) / 2000

[0105] S / N ratio = fluorescence intensity of the RNA to be tested / fluorescence intensity of RNA without 5' cap structure and with polyA tail

[0106] Depend on Figures 4 to 7 From the test results shown, it can be seen that compared with comparative examples 1 to 3, the fluorescent immunochromatographic detection kit provided by Examples 1 to 6 of the present invention detects RNA products with different capping rates, and there is a good linear relationship between the normalized T / C ratio and the capping rate, and has a better signal-to-noise ratio S / N value. The capping rate discrimination of RNA products is as low as 5%, and it has excellent discrimination ability for RNA products with different capping rates.

[0107] Example 9

[0108] This example is used to illustrate the performance of the fluorescent immunochromatographic detection kit provided in Example 1 and Comparative Examples 1 to 3 for detecting RNA integrity, specifically including:

[0109] (1) The commercially available Firefly Luciferase-mRNA was stored at -80°C and 37°C for 14 days, respectively. On the 0th, 3rd, 7th and 14th day of storage, 132 ng of Firefly Luciferase-mRNA was mixed with 50 μL of the sample treatment solution provided in Example 1 and the comparative example to obtain a loading solution.

[0110] (2) According to the mRNA loading amount of 0.5 pmol, the loading solution was added dropwise to the sample pad 2 of the test paper corresponding to each sample treatment solution. After chromatography for 15 minutes, the fluorescence intensity of the test line 6 and the quality control line 7 was measured by a fluorescence detector to obtain the normalized T / C ratio, and the intact RNA ratio was calculated according to the following formula.

[0111] RNA integrity ratio = (normalized T / C ratio at storage day X) / (normalized T / C ratio at storage day 0) × 100%

[0112] (3) At the same time, the results of the above Firefly Luciferase-mRNA detection by capillary electrophoresis (CE) were used as a control. Figure 8 and 9 shown.

[0113] Depend on Figure 8 and 9 It can be seen from the test results shown that, compared with comparative examples 1 to 3, the RNA integrity ratio tested by the fluorescent immunochromatography detection kit provided by the present invention is similar to the result measured by the existing known capillary electrophoresis (CE) method. The test results have good reliability and can be used for real-time detection of RNA integrity.

[0114] Example 10

[0115] This example is used to illustrate the performance of the fluorescent immunochromatographic detection kit provided in the above examples and comparative examples for detecting the encapsulation efficiency of LNP drug preparations, specifically including:

[0116] (1) The commercially available mRNA COVID-19 vaccine Spikevax was used as the LNP experimental model, and centrifuged at 4°C and 1000 rpm to collect the precipitate and supernatant, respectively. The precipitate was washed 5 times with deionized water, and the washing solution and the supernatant were combined to obtain the RNA sample solution;

[0117] (2) 132 ng of the precipitate was mixed evenly with 50 μL of the sample treatment solution provided in Example 1 and the comparative example to obtain a loading solution I;

[0118] 1 μL of RNA sample solution was mixed evenly with 50 μL of sample treatment solution provided in Example 1 and the comparative example to obtain a loading solution II;

[0119] (3) Take 1 μL of sample solution I and II respectively and add them to the sample pad 2 of the test paper corresponding to each sample treatment solution. After chromatography for 15 minutes, use a fluorescence detector to measure the fluorescence intensity of the test line 6 and the quality control line 7 to calculate the normalized T / C ratio, and calculate the encapsulation efficiency according to the following formula.

[0120] Encapsulation efficiency = (fluorescence intensity of sample solution I) / (fluorescence intensity of sample solution I + fluorescence intensity of sample solution II) × 100%

[0121] (4) At the same time, a 10 mM Tris-HCl buffer (pH = 7.5) containing 0.2 wt% of polyethylene glycol octylphenyl ether was used as a demulsifier, 132 ng of mRNA new crown vaccine Spikevax was mixed evenly with 50 μL of the demulsifier, and the fluorescence signal before and after demulsification was measured by RiboGreen fluorescence method, and the encapsulation efficiency was calculated. The test results are as follows Fig.10 shown.

[0122] Encapsulation efficiency = (RNA concentration after emulsion breaking - RNA concentration before emulsion breaking) / RNA concentration before emulsion breaking × 100%

[0123] Depend on Fig.10 It can be seen from the test results shown that, compared with comparative examples 1 to 3, the encapsulation efficiency tested by the fluorescent immunochromatographic detection kit provided by the present invention is similar to the existing known RiboGreen fluorescence method, and the detection results have good reliability and can be used for real-time detection of LNP products.

[0124] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A fluorescent immunochromatography detection kit, characterized in that: The kit includes: A sample processing solution comprising 0.1 to 0.5 mg / mL of biotin-poly(dT) 25 , 0.1-0.5wt% of polyethylene glycol octylphenyl ether and 1-5wt% of sodium chloride; The test strip comprises a sample pad, a conjugation pad and a reaction pad which are in contact with each other in sequence; the conjugation pad is coated with fluorescent nanoparticles-anti-5' cap structure antibodies; the reaction pad is provided with a detection line which is coated with streptavidin.

2. The fluorescent immunochromatographic detection kit according to claim 1, characterized in that: The sample treatment solution is a Tris-HCl buffer solution, and the concentration of the Tris-HCl buffer solution is 5-15 mM, and the pH value is 7.5-8.

3. The fluorescent immunochromatographic detection kit according to claim 1, characterized in that: On the binding pad, the coating amount of the fluorescent nanoparticle-anti-5' cap structure antibody is 0.01-0.2 μg / cm 2 ; Optionally, in the fluorescent nanoparticle-anti-5' cap structure antibody, the particle size of the fluorescent nanoparticle is 100-300 nm.

4. The fluorescent immunochromatographic detection kit according to claim 1, characterized in that: The coating amount of streptavidin on the detection line is 10-15 μg / cm 2 .

5. The fluorescent immunochromatographic detection kit according to claim 1, characterized in that: The detection pad is also provided with a quality control line, and the quality control line is coated with an antibody against the 5' cap structure; Optionally, on the quality control line, the coating amount of the anti-5' cap structure antibody is 0.01-2 μg / cm 2 .

6. A method for rapidly detecting the quality of RNA products, characterized in that: The method uses the fluorescent immunochromatographic detection kit described in any one of claims 1 to 6 to detect the RNA product to be detected.

7. The method for rapid detection of RNA product quality according to claim 6, characterized in that: The method comprises: S1, mixing the RNA product to be tested with the sample treatment solution to obtain a loading solution; S2, performing chromatography on the loading solution on the test paper; S3, detecting the fluorescent signal of the test paper, and calculating and obtaining the RNA product quality index.

8. The method for rapid detection of RNA product quality according to claim 7, characterized in that: In step S1, based on the total volume of the sample treatment solution, the added amount of the RNA product to be tested is 0.01-0.05 pmol / μL.

9. The method for rapid detection of RNA product quality according to claim 7, characterized in that: In step S2, the loading volume of the loading solution is 0.1 to 5 μL.

10. Use of the fluorescent immunochromatographic detection kit according to any one of claims 1 to 5 in the preparation of RNA products.

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