Method and kit for detecting rotavirus based on immunofluorescence method and application of kit

Abstract

The invention relates to the technical field of immunofluorescence detection, and provides a method for detecting rotavirus in vitro based on an immunofluorescence method, and a fixing and permeation integrated composition, a weak acid region anti-quenching sealing liquid and a detection kit which are matched with the method. According to the method, paraformaldehyde and a nonionic surfactant are compounded, so that fixation and permeation synergism is realized, and cell morphology maintenance and antigen epitope accessibility are both considered; and a phosphoric acid-citric acid buffer system is combined with glycerol, and the pH is regulated to a sealing liquid in a weak acid region, so that fluorescence quenching is delayed, and the signal-to-noise ratio and repeatability are improved. According to the invention, the non-specific background is reduced, the observable time is prolonged, and the method is suitable for in-vitro rotavirus immunofluorescence detection.

Description

Technical Field

[0001] This invention relates to the field of immunological detection and bioanalysis, specifically to an in vitro method for detecting rotavirus based on immunofluorescence, and to a matching integrated immobilization and permeation composition, a weakly acidic region anti-quenching mounting solution, and a detection kit. Background Technology

[0002] Rotavirus is a major pathogen causing acute gastroenteritis. In vitro immunofluorescence assays are widely used for viral antigen detection and methodological evaluation due to their advantages of intuitive localization and rapid interpretation. Current procedures typically involve a stepwise process of fixation followed by permeabilization, then incubation with primary / secondary antibodies and neutral mounting. However, problems such as insufficient image signal-to-noise ratio, high background noise, and poor reproducibility are still common across different laboratories, batches, and sample types, affecting the stability and comparability of results.

[0003] The parameters of step-by-step fixation and permeation are highly coupled. Over-fixation can easily cause antigen epitope masking, while insufficient permeation restricts antibody entry. Multiple steps, narrow windows, and operator differences can easily amplify intra-batch / inter-batch variability. Even with the same antibody and microscopic platform, weakened positive signals and non-specific background elevation may still occur, resulting in large data dispersion between fields of view and between wells, making it difficult to establish a unified statistical standard.

[0004] The mounting process directly determines fluorescence fidelity and observation time. Traditional neutral glycerol-PBS mounting solutions are not optimized for the pH sensitivity of fluorophores, which can easily lead to rapid photobleaching and signal attenuation. Although commercially available anti-quenching agents can delay quenching to some extent, their cost, compatibility, and batch stability vary, and the wide pH control range of the mounting solution can further amplify batch-to-batch differences, affecting the consistency of positive determination.

[0005] Given the aforementioned pain points, there is an urgent need for a complete solution that, without replacing existing antibodies and microsurgical platforms, synergistically improves epitope accessibility through an integrated fixation and permeation composition, delays fluorescence quenching and reduces background using a weakly acidic anti-quenching mounting solution, and introduces objective indicators such as SNR and fluorescence half-life as a unified judgment criterion. This solution should also provide clear parameter boundaries and kit-based forms to reduce operational complexity and human-to-human differences, thereby improving the image quality, stability, and reproducibility of rotavirus immunofluorescence detection. Summary of the Invention

[0006] To address the technical deficiencies of existing technologies, this invention provides an in vitro method for detecting rotavirus based on immunofluorescence, comprising:

[0007] a) The sample to be tested is subjected to an integrated fixation and permeation process, and the fixation and permeation system used contains paraformaldehyde and nonionic surfactant;

[0008] b) Immunoincubation and washing with a specific primary antibody against rotavirus antigen and a fluorescently labeled secondary antibody;

[0009] c) Seal the film using a weakly acidic anti-quenching sealing solution;

[0010] d) Acquire fluorescence images and perform signal determination under preset threshold conditions;

[0011] Steps a) and c) respectively enhance epitope accessibility and fluorescence stability through the synergistic effect of the integrated fixation and permeability system and the weak acid region sealing system.

[0012] Preferably, in the fixed and permeable system described in step a), the mass-volume fraction of paraformaldehyde is 1.5%-3.0%, and the volume fraction of nonionic surfactant is 0.10%-0.30%.

[0013] Preferably, the nonionic surfactant is Triton X-100.

[0014] Preferably, the fixation and permeation system uses isotonic phosphate buffer as the medium, with a pH of 6.8-7.6.

[0015] Preferably, the mounting solution in step c) comprises a glycerol and phosphate-citric acid buffer system, and the pH of the mounting solution is 5.2-5.8.

[0016] Preferably, the mounting solution has a pH of about 5.5 and a total buffer concentration of 0.05-0.30 mol / L.

[0017] Preferably, the criteria for determining the fluorescence image include at least one of signal-to-noise ratio (SNR) and fluorescence intensity half-life.

[0018] Preferably, the SNR is calculated as I_signal / I_background, and the threshold and background area selection are performed according to preset quality control standards.

[0019] Preferably, the detection is performed in vitro, and the sample is a cell, tissue section, or clinically derived material that has been preserved.

[0020] The present invention also provides an integrated fixation and permeation composition for immunofluorescence detection, comprising, by mass / volume fraction: 1.5%-3.0% paraformaldehyde; 0.10%-0.30% nonionic surfactant, preferably Triton X-100; and the balance isotonic phosphate buffer, with a pH of 6.8-7.6.

[0021] Preferably, the paraformaldehyde content is 1.8%-2.4%, the Triton X-100 content is 0.15-0.25%, and the pH is 7.0-7.4.

[0022] The present invention also provides an anti-fluorescence quenching mounting solution, characterized in that it comprises a glycerol and phosphoric acid-citric acid buffer system, wherein the pH of the mounting solution is 5.2-5.8 and the total buffer concentration is 0.05-0.30 mol / L.

[0023] The present invention also provides a detection kit for rotavirus immunofluorescence detection, comprising: an integrated fixation and permeabilization composition for immunofluorescence detection; an anti-fluorescence quenching mounting solution; a specific primary antibody against rotavirus antigen and a corresponding fluorescently labeled secondary antibody; a nuclear staining reagent; a diluent and a washing solution.

[0024] The present invention also provides the use of a detection kit in the in vitro immunofluorescence detection of rotavirus.

[0025] The present invention also provides the use of a composition or sealing solution in improving epitope accessibility, reducing nonspecific background and / or delaying fluorescence quenching.

[0026] This invention proposes a systematic solution combining a fixation and permeation composition with a sealing solution for weakly acidic regions to resist quenching: the fixation and permeation are integrated by compounding paraformaldehyde with nonionic surfactants, taking into account both morphological preservation and epitope accessibility; the sealing solution binds glycerol through a phosphate-citric acid buffer and converges the pH to the weakly acidic region, delaying fluorescence quenching and improving the signal-to-noise ratio and repeatability.

[0027] This approach can significantly improve the image quality and stability of in vitro rotavirus immunofluorescence detection without changing the antibody and microscopic equipment. It is highly applicable and cost-effective. Attached Figure Description

[0028] The accompanying drawings are provided to more clearly illustrate the technical solutions in the embodiments of this application. The drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Schematic diagram of the effect of different fixation and permeability systems on fluorescence signal-to-noise ratio.

[0030] Figure 2 Schematic diagram of the effect of different pH mounting solutions on fluorescence intensity half-life. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0032] Viral fluid, rotavirus SA11 strain (ATCCVR-1565), was obtained by amplification in MA104 cells at 37°C and 5% CO2. The harvested supernatant was clarified by centrifugation at 3000 rpm for 10 minutes, filtered through a 0.22 μm membrane, aliquoted, and stored at -80°C with no more than one freeze-thaw cycle to obtain the viral fluid. The virus was then subjected to TCID... 50 The titer was determined by quantitative analysis and was 4.0 × 10⁻⁶. 7 TCID 50 / mL.

[0033] MEM culture medium, commercially available product (Corning, 10-010-CV, 1×, liquid, containing Earle's salts), should be used according to the instructions. Store at 4-8℃ protected from light. Preheat to 37℃ before use.

[0034] Example 1: Immunofluorescence detection method for rotavirus

[0035] 1. Preparation of experimental solutions

[0036] 2.5 mg / mL trypsin solution: 2.5% trypsin (Gibco product number 15090046) diluted 10-fold with 0.01 mol / L PBS buffer;

[0037] Cell growth medium: 10 mL newborn calf serum + 90 mL MEM medium;

[0038] Tissue fixative: 0.2 mL 10% Triton X-100 + 9.8 mL 2% paraformaldehyde;

[0039] Virus maintenance solution: 4 μL 2.5 mg / mL trypsin + 10 mL MEM medium (final concentration 1 μg / mL).

[0040] Mounting solution: 22.2 mL 0.2 mol / L disodium hydrogen phosphate + 27.8 mL 0.1 mol / L citric acid + 50 mL glycerol, adjust pH to 5.5, store at 2-8℃.

[0041] 2. Experimental Procedure

[0042] Cell plating: MA104 cells were digested and diluted to 1.5 × 10⁻⁶. 5 / mL, 4mL per well in a 6-well plate, incubated at 37℃ and 5% CO2 until 85% density;

[0043] Virus activation: Add 8 μL of 2.5 mg / mL trypsin solution to 1 mL of virus solution and incubate at 37°C for 60 min to obtain virus inoculation solution;

[0044] Inoculation and culture: Discard the cell culture medium, add 100 μL of virus inoculation medium to each well, then add 1.5 mL of MEM medium and incubate at 37°C for 16 h;

[0045] Immunofluorescence assay:

[0046] (1) Fixation and permeability

[0047] After discarding the culture medium, add pre-cooled tissue fixation solution and incubate at 4°C for 30 min to complete fixation.

[0048] After fixation, the liquid was discarded, pre-cooled methanol was added, and the mixture was incubated at 4°C for 10 min for permeation.

[0049] Wash twice with PBS for 5 minutes each time to remove residual fixation / permeabilization reagents.

[0050] The integrated fixation and permeation system uses isotonic PBS as the medium, with the pH controlled between 6.8 and 7.6.

[0051] (2) Blocking and antibody incubation

[0052] Add 2% BSA solution to each well and incubate at 37°C for 30 minutes to complete the sealing.

[0053] After discarding the blocking solution, add the primary antibody working solution diluted 1:1000 and incubate at 37°C for 90 min.

[0054] Wash three times with PBS, 5 minutes each time.

[0055] Add the secondary antibody working solution diluted 1:200 and incubate at 37°C for 60 minutes, avoiding light throughout.

[0056] Wash three more times with PBS for 5 minutes each time to remove unbound secondary antibody.

[0057] (3) Nucleation and sealing

[0058] Add DAPI working solution prepared at a ratio of 1:1000 and incubate at room temperature in the dark for 5 minutes.

[0059] Rinse once with PBS to remove free dye.

[0060] Add mounting solution and cover with a coverslip, then let stand at room temperature for 10 minutes to stabilize the fluorescence signal.

[0061] The mounting solution was a combination of glycerol and phosphate-citric acid buffer system, pH 5.5; the total buffer concentration was controlled at 0.15 mol / L.

[0062] (4) Results observation

[0063] Under a fluorescence microscope, blue fluorescence of the cell nucleus was observed in the ultraviolet excitation channel, and green fluorescence of the viral antigen was observed in the blue light excitation channel. When a clear colocalization signal of blue cell nucleus and green fluorescence is visible within the same cell, the cell is considered positive.

[0064] The negative control only shows blue nuclear fluorescence; no specific green signal should be observed.

[0065] 3. Image Judgment and Quality Control Standards

[0066] Image acquisition was performed under uniform exposure, gain, and threshold conditions. The signal-to-noise ratio (SNR) was defined as SNR = I_signal / I_background, where I_signal is the average fluorescence intensity of the positive cell target region, and I_background is the average fluorescence intensity of the extracellular background region in the same field of view. Background region selection was performed according to preset quality control standards (avoiding cells and edge artifacts). The fluorescence intensity half-life T... 1 / 2 This refers to the time it takes for the fluorescence intensity in the same field of view to decrease to 50% of its initial value under isochronous acquisition conditions.

[0067] Test Example 1: Sensitivity and Specificity Verification

[0068] The traditional stepwise fixation + neutral mounting (old method) and the "fixation-permeability integration + weak acid mounting" method of Example 1 were compared under the same antibody and microscopic parameters. Results showed that the SNR increased from 3.4±0.5 to 6.4±0.6 (n=3, P<0.01); T 1 / 2 The time increased from 8.2±1.0 min to 15.2±1.4 min (n=3, P<0.001); composite index SNR×T 1 / 2 The improvement is approximately 252%. Accordingly, the immobilization permeability and weak acid sealing system of the present invention has significant advantages in improving epitope accessibility, reducing nonspecific background, and delaying fluorescence quenching.

[0069] Test Example 2: Positive Rate / Background Rate and Repeatability

[0070] Counting ≥400 cells in 5 random fields of view per well. Results of Example 1: Positive rate 64.0±4.2% (old method 48.3±5.3%, P<0.05); background positive rate 2.2±0.6% (old method 4.9±1.0%, P<0.05); intra-well and intra-batch CV% ≤20%. The above indicators meet the preset quality control thresholds.

[0071] The method of this invention is applicable to in vitro samples, including but not limited to cell samples, tissue section samples, and clinically derived materials that have undergone preservation treatments such as cryopreservation or fixation. For different sample types, only equivalent adjustments need to be made according to the parameter ranges of the fixation-permeability and mounting system in this specification.

[0072] like Figure 1 As shown, under the same antibody and imaging parameters, the experimental group using the integrated fixation and permeabilization composition had a significantly higher SNR than the control group using stepwise fixation, stepwise permeabilization, and neutral mounting (mean ± SD, n ≥ 3, P < 0.05), indicating that epitope accessibility and low background were improved simultaneously.

[0073] like Figure 2 As shown, while keeping fixation / permeabilization and immunoincubation constant, simply adjusting the mounting medium to a weakly acidic range (pH 5.2-5.8) significantly prolonged T. 1 / 2 (Mean ± SD, n ≥ 3, P < 0.05), suggesting that the weak acid buffer system makes a stable contribution to fluorescence quenching resistance.

[0074] Product Example A: Preparation and Storage of an Integrated Immobilization and Permeability Composition

[0075] Dissolve 2.0% paraformaldehyde and 0.20% Triton X-100 in isotonic PBS by mass / volume fraction, adjust pH to 7.2, sterilize by 0.22μm filtration, and store at 2-8℃ protected from light. Shelf life is 1-3 months. Permissible range: paraformaldehyde 1.5-3.0%, nonionic surfactant 0.10-0.30%, pH 6.8-7.6.

[0076] Product Example B: Preparation and Storage of Anti-fluorescence Quenching Sealing Solution

[0077] Glycerol was mixed with phosphate-citrate buffer (total buffer concentration 0.10 mol / L), and the pH was adjusted to 5.5. The mixture was then sterilized by 0.22 μm filtration and stored at 2-8°C protected from light. Permissible range: pH 5.2-5.8, total buffer concentration 0.05-0.30 mol / L. Stability assessment showed that the SNR function retention rate was ≥90% after 3 months, and the appearance and pH remained essentially stable.

[0078] Kit Examples and Usage Instructions

[0079] The kit contains: a fixation and permeabilization integrated composition (Product Example A), an anti-fluorescence quenching mounting solution (Product Example B), a specific primary antibody against rotavirus antigen and a corresponding fluorescently labeled secondary antibody, nuclear staining reagent (DAPI), diluent, and washing buffer. Each component should be stored separately at 2–8°C protected from light. Instructions for use are included. This kit is suitable for in vitro immunofluorescence detection of rotavirus.

[0080] Compared with traditional step-by-step fixation and neutral sealing, the integrated fixation and permeability composition and the weakly acidic anti-quenching sealing solution provided by this invention can significantly improve epitope accessibility and signal-to-noise ratio, reduce non-specific background and delay fluorescence quenching when used for rotavirus immunofluorescence detection, thereby improving detection sensitivity, interpretation stability and repeatability.

[0081] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting rotavirus based on immunofluorescence, characterized in that, include: a) The sample to be tested is subjected to an integrated fixation and permeation process, and the fixation and permeation system used contains paraformaldehyde and nonionic surfactant; b) Immunoincubation and washing with a specific primary antibody against rotavirus antigen and a fluorescently labeled secondary antibody; c) Seal the film using a weakly acidic anti-quenching sealing solution; d) Acquire fluorescence images and perform signal determination under preset threshold conditions; Steps a) and c) respectively enhance epitope accessibility and fluorescence stability through the synergistic effect of the integrated fixation and permeability system and the weak acid region sealing system.

2. The method for detecting rotavirus based on immunofluorescence assay according to claim 1, characterized in that, In the fixed and permeable system described in step a), the mass-volume fraction of paraformaldehyde is 1.5%-3.0%, and the volume fraction of nonionic surfactant is 0.10%-0.30%.

3. The method for detecting rotavirus based on immunofluorescence as described in claim 2, characterized in that, The nonionic surfactant is Triton X-100.

4. The method for detecting rotavirus based on immunofluorescence assay according to any one of claims 1-3, characterized in that, The fixation and permeation system uses isotonic phosphate buffer as the medium, with a pH of 6.8-7.

6.

5. The method for detecting rotavirus based on immunofluorescence as described in claim 1, characterized in that, The mounting solution in step c) comprises a glycerol and phosphate-citric acid buffer system, and the pH of the mounting solution is 5.2-5.

8.

6. The method for detecting rotavirus based on immunofluorescence assay according to any one of claims 1-3, characterized in that, The criteria for determining fluorescence images include at least one of signal-to-noise ratio (SNR) and fluorescence intensity half-life.

7. The method for detecting rotavirus based on immunofluorescence as described in claim 6, characterized in that, SNR is calculated as I_signal / I_background, and the threshold and background area selection are performed according to preset quality control standards.

8. The method for detecting rotavirus based on immunofluorescence assay according to claim 6, characterized in that, The test is performed in vitro, and the sample is a cell, tissue section, or clinically derived material that has been preserved.

9. A detection kit for rotavirus immunofluorescence detection, characterized in that, It contains: a fixed and permeable integrated composition; an anti-fluorescence quenching mounting solution; a specific primary antibody against rotavirus antigen and a corresponding fluorescently labeled secondary antibody; nuclear staining reagent; diluent and washing solution.

10. Use of the detection kit of claim 9 in in vitro immunofluorescence detection of rotavirus.

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