Gold immunochromatography reagent strip for detecting MRV / LMBV and its preparation and application

Abstract

This invention relates to the field of virus detection technology, and more particularly to MRV / LMBV colloidal gold immunochromatographic reagent strips and their preparation and application. The invention uses monoclonal antibody 2E8 as both the colloidal gold-labeled antibody and the detection line capture antibody; the amino acid sequence of the heavy chain variable region of 2E8 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2, and it specifically recognizes the viral protein MRV-pORF077L. This invention provides an MRV / LMBV colloidal gold immunochromatographic reagent strip that allows for observation and interpretation of detection results without the need for specialized instruments, and can complete the detection within 10-15 minutes. This test strip is suitable for on-site homogenization detection of virus-infected tissues such as the liver, spleen, and kidney of mandarin fish.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to MRV / LMBV colloidal gold immunochromatographic reagent strips and their preparation and application. Background Technology

[0002] Currently, the detection methods for mandarin frog virus (MRV) / largemouth bass iridovirus (LMBV) mainly include: (1) molecular detection technology: using conventional PCR, real-time quantitative PCR (qPCR) and other technologies to detect the MCP gene of the virus; (2) traditional virological methods: virus isolation and culture, transmission electron microscopy (TEM) observation and enzyme-linked immunosorbent assay (ELISA); (3) immunohistochemistry: IHC and IF technology are used for tissue localization detection. The above methods still have shortcomings, such as poor on-site applicability and complicated operation: relying on thermal cycling / constant temperature equipment and clean partitions, cumbersome pretreatment and nucleic acid extraction, long overall turnaround time, which is not conducive to rapid screening on the breeding site; high requirements for operators and quality system: requiring trained personnel and strict partition operation, and relying on complete quality control (positive / negative control, standard curve, sample addition / blank control, retesting rules and external quality assessment); slight deviation may lead to inconsistent results or false negative / false positive, making it difficult to promote on-site.

[0003] Existing technology (CN116840463A) discloses a colloidal gold test strip for detecting antibodies against MRV / LMBV iridovirus in largemouth bass. However, this test strip detects specific antibodies produced by the fish rather than directly detecting viral antigens. Because the production of specific antibodies after infection is somewhat delayed, and antibody levels are easily affected by factors such as fish size, immune status, and stage of infection, this method may not be able to detect infected individuals in the early stages of infection or with weak immune responses in a timely manner. Furthermore, specific antibodies may persist even after viral load decreases or infection recovery, so a positive antibody result cannot directly reflect whether the fish is currently infected. In addition, this method requires the collection of approximately 200 μL of fresh blood for immediate testing, which is not suitable for large-scale screening of small-sized fry or in aquaculture sites; its sensitivity is only expressed by the dilution factor of positive serum, and there is a lack of evaluation of the limit of detection based on viral titer or viral copy number. Therefore, it is still necessary to develop a colloidal gold immunochromatographic reagent strip that can directly detect MRV / LMBV viral antigens, is easy to operate, and is suitable for rapid screening in aquaculture sites. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a new MRV / LMBV colloidal gold immunochromatographic reagent strip, in which the observation and interpretation of the test results do not require special instruments and the test can be completed within 10-15 minutes.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an MRV / LMBV colloidal gold immunochromatographic reagent strip, wherein the test strip comprises a sample pad, a gold label pad, a nitrocellulose NC membrane, and an absorbent pad; The nitrocellulose NC membrane is equipped with a detection line (T line) and a quality control line (C line). The detection line T is coated with monoclonal antibody 2E8; The gold-labeled pad is loaded with colloidal gold-labeled monoclonal antibody 2E8; The quality control line C is coated with goat anti-mouse IgG; The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E8 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0006] Preferably, the monoclonal antibody 2E8 specifically recognizes the viral protein MRV-pORF077L; the amino acid sequence of the viral protein MRV-pORF077L is shown in SEQ ID NO:3.

[0007] The monoclonal antibody 2E8 specifically recognizes the viral protein MRV-pORF077L. The GenBank accession number for MRV-pORF077L is XPZ21335.1; its homologous protein in LMBV is a hypothetical protein (GenBank accession number: UUY86260.1), whose amino acid sequence is completely identical to that shown in SEQ ID NO: 3.

[0008] This invention screened and discovered that the monoclonal antibody 2E8 can be used both for colloidal gold labeling and as a capture antibody on the detection line. After the MRV / LMBV antigen in the sample binds to the colloidal gold-labeled 2E8, it can be further captured by the 2E8 on the detection line and form a visible band, thus constituting an immunochromatographic system where the same monoclonal antibody completes both recognition and capture. This invention demonstrates the feasibility of this configuration with actual immunochromatographic results, without being limited by specific antigen spatial conformations or epitope presentation mechanisms. This design reduces the types of antibody raw materials required in the production process and is suitable for rapid detection of mandarin fish tissue homogenate supernatant. Utilizing a single, highly efficient monoclonal antibody (2E8) as both a gold-labeled antibody and a membrane-capture antibody reduces production complexity and cost while maintaining good detection specificity. It provides an MRV / LMBV colloidal gold immunochromatographic strip where the observation and interpretation of detection results can be completed within 10-15 minutes without the need for specialized instruments. The test strip features an optimized sample processing procedure, making it suitable for on-site homogenization testing of virus-infected tissues such as the liver, spleen, and kidneys of mandarin fish.

[0009] Preferably, the heavy chain subtype of the monoclonal antibody 2E8 is IgG2b, and the light chain subtype is kappa.

[0010] Preferably, the concentration of the colloidal gold-labeled monoclonal antibody 2E8 is 8–16 μg / mL.

[0011] More preferably, the concentration of the colloidal gold-labeled monoclonal antibody 2E8 is 12 μg / mL.

[0012] Preferably, the concentration of the monoclonal antibody 2E8 coated on the detection line is 0.5–2.0 mg / mL.

[0013] More preferably, the concentration of the monoclonal antibody 2E8 coated on the detection line is 1.0 mg / mL.

[0014] Western blot and LC-MS / MS identified the MRV target protein identified by 2E8 as pORF077L; this protein can be detected in the mid-to-late stages of MRV infection and is present in viral particle-associated components.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned MRV / LMBV colloidal gold immunochromatographic reagent strip, comprising the following steps: (1) Preparation of gold-labeled antibody: Take colloidal gold solution, adjust pH, add diluted monoclonal antibody 2E8, stir, block, centrifuge and resuspend the precipitate to obtain gold-labeled antibody, and spray it onto gold-labeled pad; (2) Membrane application: Spray monoclonal antibody 2E8 onto the NC membrane to form a T line, and spray goat anti-mouse IgG onto the NC membrane to form a C line, and then dry; (3) Test strip assembly: The absorbent pad, NC film, gold label pad and sample pad are sequentially overlapped and pasted on the PET backing, and then cut into strips to obtain the test strip.

[0016] Preferably, the colloidal gold solution contains colloidal gold particles.

[0017] Preferably, the colloidal gold particles have a diameter of 20 nm.

[0018] Thirdly, the present invention provides the application of the above-mentioned MRV / LMBV colloidal gold immunochromatographic reagent strip in the preparation of products for detecting MRV or LMBV antigens.

[0019] The samples tested were liver, spleen, kidney, or pyloric caeca tissue samples from mandarin fish.

[0020] Fourthly, the present invention provides a method for detecting MRV or LMBV viral antigens using the above-described test strip, comprising the following steps: (1) Take a mandarin fish tissue sample, add sample processing solution to homogenize, centrifuge and take the supernatant as the test sample; (2) Add the test sample to the sample pad of the test strip and let it stand for 10-15 minutes; (3) Observation results: If both the test line and the control line show color, the result is positive; if only the control line shows color, the result is negative; if the control line does not show color, the test strip is invalid.

[0021] Fifthly, the present invention provides a detection system for MRV or LMBV viruses, including the above-mentioned MRV / LMBV colloidal gold immunochromatographic reagent strip.

[0022] The beneficial effects of this invention are as follows: The MRV / LMBV colloidal gold immunochromatographic reagent strips provided by this invention have the following advantages: (1) Good detection specificity: 2E8 monoclonal antibody only reacts with MRV and LMBV, and does not cross-react with ISKNV, SDDV, TFV and SCRV, ensuring the accuracy of diagnosis.

[0023] (2) Reliable sensitivity: detection limit can reach 10 4 TCID 50 / mL.

[0024] (3) Simplified antibody raw materials and quality control: The same monoclonal antibody is used for labeling and capture, which simplifies the antibody quality control process. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the structure and principle of a colloidal gold immunochromatographic test strip for mandarin frog virus (MRV); it shows the internal components of a typical colloidal gold test strip, including a sample pad, a gold label pad, a nitrocellulose membrane (NC membrane), and an absorbent pad.

[0026] Figure 2 Figure A shows the morphology of MRV virus particles and the reaction characteristics of the 2E8 monoclonal antibody. Figure B shows the transmission electron microscopy (TEM) image of MRV virus particles, scale bar: 200 nm. Figure C shows the Western blot results of 2E8 on MRV, LMBV, GIV, TFV, ISKNV, and SCRV samples. Figure D shows the Western blot results of pORF077L expression time sequence in samples at 0, 2, 6, 12, 24, 36, 48, and 72 h post-MRV infection, with β-actin as an internal control. Figure D shows the immunofluorescence detection results of 2E8 on MRV-infected MFF-1 cells, scale bar: 10 μm.

[0027] Figure 3The figures show the sensitivity detection results of the MRV / LMBV colloidal gold immunochromatographic test strip of the present invention; in Figure A, the detection results of the MRV virus sample are shown, from left to right as 10. 6 10 5 10 4 10 3 10 2 TCID 50 / mL MRV virus sample and PBS negative control; Figure B shows the detection results of LMBV virus sample, from left to right: 10 6 10 5 10 4 10 3 10 2 TCID 50 / mL LMBV virus sample and PBS negative control.

[0028] Figure 4 The results show the specificity detection of the MRV / LMBV colloidal gold immunochromatographic test strip of the present invention; from left to right in the figure are the negative controls of MRV, LMBV, ISKNV, SDDV, TFV, SCRV and MFF-1 cell culture.

[0029] Figure 5 The image shows the detection results of the 2E8 monoclonal antibody immunochromatographic test strip on clinically infected mandarin fish samples. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0032] The amino acid sequence involved in this invention is as follows: SEQ ID NO:1: EVLLQQSGPELVKPGTSVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYGGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCATAYYRYGYWYFDVWGAGTTVTVSS.

[0033] SEQ ID NO:2: EIVLTQSPTTMAASPGEKITITCRASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGASYSLTIGTMEAEDVATYYCQQGSSILTFGAGTKLELK.

[0034] SEQ ID NO:3: 。

[0035] Example 1: Virus preparation and preparation The MRV virus strain used in this embodiment was a strain preserved in our laboratory, initially isolated from tissue samples of naturally diseased mandarin fish. MRV was inoculated into MFF-1 cells, and after the cells showed obvious cytopathic effects, the cells were repeatedly frozen and thawed three times, and the cell culture supernatant and cell lysate were collected. The samples were centrifuged at 4°C, 8000 ×g for 30 min to remove cell debris, and the supernatant was collected. Subsequently, the samples were ultracentrifuged at 4°C, 80000 ×g for 90 min, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain a crude virus extract. The crude virus extract was spread on a 35% (w / v) sucrose pad and centrifuged at 4°C, 150000 ×g for 1 h. The virus precipitate was collected and further purified by centrifugation using a 30%-60% (w / v) sucrose density gradient, collecting the central milky white viral band, resuspending it in PBS, and storing it at -80°C for later use.

[0036] Purified virus particles were observed by negative staining transmission electron microscopy. Figure 2 A) A typical icosahedral structure of iridovirus, with a diameter of about 150 nm, is visible; SDS-PAGE analysis reveals multiple viral structural protein bands.

[0037] Example 2: Acquisition and Specificity Verification of 2E8 Monoclonal Antibody 1. Using the purified MRV virus particles obtained in Example 1 as an immunogen, BALB / c mice were immunized. The purified viral antigen and mouse monoclonal antibody adjuvant were mixed at a volume ratio of 1:1, with an immunization dose of 50 μg per mouse. Three immunizations were administered: a second immunization 21 days after the first, and a booster immunization 14 days after the second. After the booster immunization, mouse spleen cells were harvested and fused with SP2 / 0 myeloma cells. The fused cells were screened in HAT medium, and positive clones recognizing the MRV antigen were selected using indirect ELISA. Subcloning yielded a hybridoma cell line, 2E8, that stably secretes anti-MRV monoclonal antibodies. Identification showed that its heavy chain subtype was IgG2b, and its light chain was kappa type.

[0038] 2. Target protein identification: Purified MRV virus particles were separated by SDS-PAGE and transferred to a membrane. Western blot analysis was performed using the 2E8 monoclonal antibody. The results showed that the 2E8 monoclonal antibody specifically recognized a protein band of approximately 97 kDa in the MRV virus particles, and the band was uniform with no obvious non-specific bands, indicating that 2E8 has good recognition specificity for MRV virus particle-related proteins.

[0039] The approximately 97 kDa band was further excised and analyzed by LC-MS / MS. Mass spectrometry identification results (Table 1) showed that the band corresponds to the protein encoded by MRV-pORF077L, predicted to be a putative 2-cysteine ​​adaptor domain protein, with a sequence coverage of 68%, 57 uniquely matched peptides, and an identification score of 605.44. These results indicate that the target protein recognized by 2E8 is MRV-pORF077L.

[0040] Western blot was used to detect the reactivity of 2E8 to different fish virus samples. The results showed that ( Figure 2 (B) 2E8 can recognize protein bands with similar molecular weights in MRV and LMBV samples, but no obvious reaction signal was observed in ISKNV, GIV, TFV and SCRV samples, indicating that 2E8 can recognize MRV / LMBV related antigens and has no obvious cross-reaction with the above non-target fish viruses.

[0041] To analyze the expression characteristics of pORF077L during MRV infection, Western blot analysis was performed on samples from cells at different time points after MRV infection. The results showed that ( Figure 2 (C) The pORF077L signal identified by 2E8 began to appear 24 h post-infection, significantly increased at 36 h, and remained detectable at 48 h and 72 h; β-actin as an internal control showed that the protein loading amounts were basically consistent across samples. These results indicate that pORF077L is a virus-associated protein expressed in the mid-to-late stages of MRV infection and can be present in virus particle-associated components, making it suitable as a target protein for MRV / LMBV colloidal gold immunochromatographic detection.

[0042] Table 1: LC-MS / MS identification results of the target protein of the 2E8 monoclonal antibody 3. Antibody reactivity at the cellular level: Immunofluorescence assay ( Figure 2 D) Further confirmation indicates that the 2E8 monoclonal antibody can induce a specific immune response in MRV-infected MFF-1 cells, with the fluorescence signal mainly concentrated at the viral assembly site in the cytoplasm. These results fully demonstrate that the 2E8 monoclonal antibody can serve as a high-quality core ingredient for constructing colloidal gold immunochromatographic test strips.

[0043] Example 3: Preparation, assembly, and use of 2E8 monoclonal antibody colloidal gold immunochromatographic test strips 1. Preparation of colloidal gold-labeled 2E8 monoclonal antibody Take a colloidal gold solution with a particle size of 20 nm and adjust its pH to 8.8 with K2CO3 solution. Add purified 2E8 monoclonal antibody to the colloidal gold solution to a final concentration of 12 μg / mL, mix well, and react at room temperature for 30 min. Then add BSA to a final concentration of 1%, and block at room temperature for 30 min. After blocking, centrifuge at 4 ℃ and 12000 rpm for 30 min, discard the supernatant, and resuspend the precipitate with a gold-labeled reconstitution solution containing 1% BSA to obtain the colloidal gold-labeled 2E8 monoclonal antibody solution for later use.

[0044] 2. Sample pad treatment The sample pad was thoroughly immersed in the sample pad treatment solution. The sample pad treatment solution consisted of borate buffer, pH 7.4-7.6, containing 0.1% Tween-20, 1% BSA, and 0.1 M NaCl. After immersion for 120 min, the sample pad was removed and dried at 37 °C for 2 h. The dried sample pad was then sealed and stored for later use.

[0045] 3. Preparation of gold-labeled conjugate pads The colloidal gold-labeled 2E8 monoclonal antibody solution obtained in step 1 was uniformly sprayed onto the gold-labeled conjugate pad and dried at 37°C for 2 h to obtain the gold-labeled conjugate pad containing the colloidal gold-labeled 2E8 monoclonal antibody. The dried gold-labeled conjugate pad was sealed and stored for later use.

[0046] 4. Preparation of testing lines and quality control lines A nitrocellulose membrane with a pore size of 15 μm and a capillary flow rate of 90–120 s / 4 cm was selected as the reaction membrane. 2E8 monoclonal antibody was diluted to 1 mg / mL with coating buffer and used as the detection line coating antibody; goat anti-mouse IgG was diluted to 0.5 mg / mL with coating buffer and used as the control line coating antibody. The 2E8 monoclonal antibody solution and goat anti-mouse IgG solution were respectively sprayed onto the nitrocellulose membrane using a coating apparatus to form parallel detection lines (T-lines) and control lines (C-lines), with the T-line located closer to the gold-labeled binding pad and the C-line closer to the absorbent pad. After coating, the nitrocellulose membrane was dried at 37 ℃ for 2 h, sealed, and stored for later use.

[0047] 5. Assembly of test strips ( Figure 1 ) A nitrocellulose membrane with a test line (T-line) and a control line (C-line) marked on it is adhered to the center of a PET backing. A gold-labeled conjugate pad and a sample pad are sequentially attached to the end of the nitrocellulose membrane near the test line (T-line), and an absorbent pad is attached to the other end near the control line (C-line). The gold-labeled conjugate pad, the sample pad, and the absorbent pad are overlapped by 1 mm each to ensure continuous capillary chromatography of the test solution. The absorbent pad has an absorption capacity of 180–200 μL / cm. After assembly, the membrane is cut into 6 mm wide test strips using a strip cutter. The test strips are inserted into the test cartridge, with the sample pad corresponding to the sample application hole and the test line (T-line) and control line (C-line) corresponding to the observation window. The packaged test strips and desiccant are sealed in an aluminum foil bag and stored at 4°C.

[0048] 6. Use of test strips and interpretation of results Take 60 μL of the processed sample and add it to the sample well of the test strip. Allow it to stand at room temperature for 15 min and then observe the results. The result interpretation criteria are as follows: If both the test line (T line) and the control line (C line) show visible red bands, it indicates that the sample contains MRV and / or LMBV antigens; if only the control line (C line) shows a visible red band and the test line (T line) does not show color, it indicates that MRV or LMBV antigens are not detected in the sample, or that the antigen content is below the minimum detection limit of this test strip; if the control line (C line) does not show a red band, regardless of whether the test line (T line) shows color, the test is considered invalid, and a new test strip should be used for retesting.

[0049] Example 4: Pairing screening of MRV monoclonal antibodies To screen antibody combinations suitable for MRV colloidal gold immunochromatographic test strips, seven monoclonal antibodies against MRV—1C4, 1G1, 2E8, 3G7, 3B11, 4B2, and 4F3 (all disclosed in the literature *Production and characterization of monoclonal antibodies against mandarinfish ranavirus and first identification of pyloric caecum as the major target tissue*, obtained using the same method as in Example 2)—were selected for pairing and screening. Following the aforementioned colloidal gold labeling and test strip preparation methods, the antibody combinations listed in Table 2 were used for screening (T line), with the control line (C line) coated with goat anti-mouse IgG. Except for the antibody combinations, the preparation conditions for each test strip were identical. The same batch of MRV virus solution (titer 10) was used. 7.5 TCID 50 / mL), add 60 μL of sample to each test strip, react at room temperature for 15 min, and then interpret the results. The test strip is valid when the C line develops color; according to the visual color intensity of the T line, the result is recorded as strong signal (++), weak signal (+), or no signal (-). The detection results of each antibody combination are shown in Table 2. In the table, each row is the colloidal gold-labeled antibody, and each column is the T-line coated antibody.

[0050] The results showed that only when 2E8 was paired with itself did a clear, strong signal appear in the T-line; a weak signal appeared when 2E8 was used as the colloidal gold-labeled antibody and 4B2 was used as the T-line coating antibody; no visible T-line was observed for the other antibody combinations tested. These results indicate that 2E8 can simultaneously serve as both the colloidal gold-labeled antibody and the T-line coating antibody to form an effective 2E8 self-pairing sandwich immunochromatographic system. Based on a comprehensive comparison of the color intensity and readability of each combination, 2E8 paired with itself was determined to be the preferred antibody combination for the subsequent preparation of MRV colloidal gold immunochromatographic test strips.

[0051] Table 2: Results of MRV monoclonal antibody pairing screening Note: +++ indicates a strong signal; + indicates a weak signal; − indicates no signal; this table is displayed by antibody combination, rows are colloidal gold-labeled antibodies, columns are T-line coated antibodies, and blank indicates that no reverse detection was performed.

[0052] Example 5: Sensitivity detection of MRV / LMBV colloidal gold immunochromatographic test strip 1. To evaluate the detection sensitivity of the MRV / LMBV colloidal gold immunochromatographic test strip of the present invention for MRV viral antigen, samples were collected after TCID45 testing. 50 The MRV virus solution used for testing was serially diluted 10-fold with the sample processing solution to prepare 10 samples. 6 10 5 10 4 10 3 and 10 2 TCID 50 / mL of MRV virus sample, with PBS as a negative control.

[0053] During testing, 60 μL of MRV virus samples at different concentrations were added to the sample pad of the test strip, and the results were observed after being left at room temperature for 15 minutes. The lowest viral concentration at which the control line (C line) showed normal color development and a positive band was visible to the naked eye on the test line (T line) was used as the limit of detection.

[0054] Test results are shown Figure 3 A. The results showed that 10 6 10 5 10 4 TCID 50MRV virus samples at / mL all showed a detection line (T line) and a control line (C line); when the virus concentration dropped to 10... 3 TCID 50 At a concentration of 10 mL / mL, the T-line of the test strip was either indistinct or unreadable; the PBS negative control only showed the C-line (control line) and not the T-line. These results indicate that the limit of detection for MRV / LMBV colloidal gold immunochromatographic test strip of this invention is 10⁻⁶ m / mL. 4 TCID 50 / mL.

[0055] 2. The same method was used to perform sensitivity testing on LMBV virus samples. The samples were then subjected to TCID... 50 The LMBV virus solution was serially diluted 10-fold to prepare 10 concentrations. 6 10 5 10 4 10 3 and 10 2 TCID 50 LMBV virus samples of various concentrations were collected at a concentration of / mL, with PBS used as a negative control. 60 μL of each concentration was added to the sample area of ​​the test strip, and the results were observed after incubation at room temperature for 15 min.

[0056] Test results are shown Figure 3 B. The results showed a concentration of 10. 6 10 5 and 10 4 TCID 50 All LMBV virus samples at / mL showed both the control line (C line) and the detection line (T line), of which 10 4 TCID 50 The T line for samples of / mL is lighter in color but still readable to the naked eye; when the virus concentration decreases to 10... 3 and 10 2 TCID 50 At a concentration of 10 / mL, only the control line (C line) appeared, and the test line (T line) was not visible to the naked eye; the PBS negative control also only showed the control line (C line). Therefore, the limit of detection for LMBV viral antigen of this invention is 10. 4 TCID 50 / mL.

[0057] Example 6: Specific detection of MRV / LMBV colloidal gold immunochromatographic test strip To evaluate the detection specificity of the MRV / LMBV colloidal gold immunochromatographic test strip of the present invention, MRV virus solution, LMBV virus solution, infectious spleen and kidney necrosis virus (ISKNV) virus solution, desquamation disease virus (SDDV) virus solution, tiger frog virus (TFV) virus solution, mandarin fish rhabdovirus (SCRV) virus solution, and MFF-1 cell culture negative control were tested respectively.

[0058] All viral samples were strains preserved in our laboratory. Viral culture media were collected after amplification in corresponding sensitive cells. Before testing, the viral culture media were centrifuged at 4℃ and 8000 rpm for 10 min to remove cell debris, and the supernatant was used as the test sample. The MFF-1 cell culture negative control was processed using the same method. During testing, 60 μL of the test sample was added to the sample pad of the test strip, and the results were observed after incubation at room temperature for 15 min.

[0059] Test results are shown Figure 4 The figure shows, from left to right, negative controls for MRV, LMBV, ISKNV, SDDV, TFV, SCRV, and MFF-1 cell cultures. The results show that both the MRV and LMBV virus samples showed a T-line (detection line) and a C-line (control line), indicating a positive result. The negative controls for ISKNV, SDDV, TFV, SCRV, and MFF-1 cell cultures only showed a C-line (control line) and no T-line (detection line), indicating a negative result.

[0060] Example 7: Detection and verification of MRV infection in different tissue samples of mandarin fish To verify the applicability of the MRV / LMBV colloidal gold immunochromatographic test strip of the present invention to different tissue samples of MRV-infected mandarin fish, suspected MRV-infected mandarin fish with clinical symptoms such as ascites and hepatosplenomegaly were collected. After being confirmed as MRV positive by PCR testing, liver, spleen, kidney, pyloric caeca, intestine and gill tissues were taken for testing.

[0061] The sample preparation method is as follows: Weigh 50 mg of the above tissue sample and add 500 μL of sample preparation solution to thoroughly homogenize. The sample preparation solution is 20 mM PBS, pH 7.4, containing 0.1% Triton X-100, 1% BSA, and 0.05% Tween-20. After homogenization, centrifuge at 8000 rpm for 10 min at 4℃, and take the supernatant as the sample to be tested. During detection, add 60 μL of the sample to the sample pad of the test strip, and observe the results after incubating at room temperature for 10–15 min.

[0062] Test results are shown Figure 5The results showed that liver, spleen, kidney, and pyloric caeca tissue samples all showed a test line (T line) and a control line (C line), indicating MRV positivity. Intestinal and gill tissue samples only showed a control line (C line) and no obvious test line (T line), indicating negative results or detection below the limit of detection of this test strip. These results demonstrate that the test strip of this invention can be used for rapid detection of MRV antigen in MRV-infected mandarin fish tissue samples such as liver, spleen, kidney, and pyloric caeca.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An MRV / LMBV colloidal gold immunochromatographic reagent strip, characterized in that, The test strip includes a sample pad, a gold label pad, a nitrocellulose NC membrane, and an absorbent pad; The nitrocellulose NC membrane is equipped with a detection line (T line) and a quality control line (C line). The detection line T is coated with monoclonal antibody 2E8; The gold-labeled pad is loaded with colloidal gold-labeled monoclonal antibody 2E8; The quality control line C is coated with goat anti-mouse IgG; The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2E8 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The MRV / LMBV colloidal gold immunochromatographic reagent strip as described in claim 1, characterized in that, The monoclonal antibody 2E8 specifically recognizes the viral protein MRV-pORF077L; the amino acid sequence of MRV-pORF077L is shown in SEQ ID NO:

3.

3. The MRV / LMBV colloidal gold immunochromatographic reagent strip as described in claim 1, characterized in that, The heavy chain subtype of the monoclonal antibody 2E8 is IgG2b, and the light chain subtype is kappa.

4. The MRV / LMBV colloidal gold immunochromatographic reagent strip as described in claim 1, characterized in that, The concentration of the colloidal gold-labeled monoclonal antibody 2E8 is 8–16 μg / mL.

5. The MRV / LMBV colloidal gold immunochromatographic reagent strip as described in claim 1, characterized in that, The concentration of the monoclonal antibody 2E8 coated on the detection line is 0.5–2.0 mg / mL.

6. A method for preparing an MRV / LMBV colloidal gold immunochromatographic reagent strip as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of gold-labeled antibody: Take colloidal gold solution, adjust pH, add diluted monoclonal antibody 2E8, stir, block, centrifuge and resuspend the precipitate to obtain gold-labeled antibody, and spray it onto gold-labeled pad; (2) Membrane application: Spray monoclonal antibody 2E8 onto the NC membrane to form a T line, and spray goat anti-mouse IgG onto the NC membrane to form a C line, and then dry; (3) Test strip assembly: The absorbent pad, NC film, gold label pad and sample pad are sequentially overlapped and pasted on the PET backing, and then cut into strips to obtain the test strip.

7. The use of the MRV / LMBV colloidal gold immunochromatographic reagent strip as described in any one of claims 1-5 in the preparation of products for detecting MRV or LMBV antigens.

8. The application as described in claim 7, characterized in that, The samples tested were liver, spleen, kidney, or pyloric caeca tissue samples from mandarin fish.

9. A method for detecting MRV or LMBV viruses using the test strip according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Take a mandarin fish tissue sample, add sample processing solution to homogenize, centrifuge and take the supernatant as the test sample; (2) Add the test sample to the sample pad of the test strip and let it stand for 10-15 minutes; (3) Observation results: If both the test line and the control line show color, the result is positive; if only the control line shows color, the result is negative; if the control line does not show color, the test strip is invalid.

10. A detection system for MRV or LMBV viruses, characterized in that, Includes the MRV / LMBV colloidal gold immunochromatographic reagent strip as described in any one of claims 1-5.

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