Tomato spotted wilt virus test strips for tobacco plants

Abstract

The present application discloses a test strip for detecting tomato spotted wilt virus in tobacco plants, comprising: a tomato spotted wilt virus monoclonal antibody-colloidal gold label, wherein the tomato spotted wilt virus monoclonal antibody-colloidal gold label comprises a tomato spotted wilt virus monoclonal antibody; the tomato spotted wilt virus monoclonal antibody comprises: an amino acid sequence and a nucleotide sequence; the amino acid sequence comprises: a heavy chain amino acid sequence as shown in SEQ ID NO:3 and a light chain amino acid sequence as shown in SEQ ID NO:4; the nucleotide sequence comprises: a heavy chain nucleotide sequence as shown in SEQ ID NO:1 and a light chain nucleotide sequence as shown in SEQ ID NO:2. The test strip has a low detection limit of only 1.0 μg / kg, making it suitable for detecting samples with low virus content and in the early stages of infection when the virus has not yet replicated significantly, thereby improving the accuracy of the test results. The test strip is particularly suitable for detecting the virus in tobacco leaves.

Description

Technical Field

[0001] The present application relates to the technical field of tomato spotted wilt virus detection, and in particular to a tomato spotted wilt virus detection test strip for tobacco plants. Background Art

[0002] Tomato spotted wilt virus (TSWV), first discovered in Australia in 1919, is a significant agricultural pest. It infects a variety of plant species, including tomatoes and tobacco. Due to its wide host range and high severity, it is classified as a Category A2 quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO) and is also a quarantine pest for plants entering China.

[0003] There are many types of detection methods for this virus, especially test strips containing antibodies that have a specific reaction to the TSWN virus, which can better detect infected plants.

[0004] The hybridoma cell line disclosed in CN202011163297.5, "A Hybridoma Cell Line and Its Application," secretes monoclonal antibodies against tomato spotted wilt virus. This monoclonal antibody, when formulated into an immunocolloidal gold-labeled rapid test card, enables rapid and large-scale testing of plants. The rapid test card has a detection limit of 0.25 μg / ml for a solution of tomato spotted wilt virus recombinant protein. Furthermore, due to the high plant fiber content in tobacco samples, which adsorbs the virus, accurate detection of samples with low virus loads using this test paper is difficult.

[0005] Tobacco plants do not have any symptoms in the early stages of infection, and it is difficult to detect infected plants by observing their appearance alone. The virus load is low in the early stages of infection. However, the test paper has a high detection limit and cannot accurately detect plants in the early stages of infection, resulting in a high rate of missed detection, which reduces the accuracy of detection of tomato spotted wilt virus infection results in tobacco plants. Summary of the Invention

[0006] In response to the above technical problems, the present application provides a test strip for detecting tomato spotted wilt virus for tobacco plants. After the antibody is made into the test strip, the detection limit is 0.001 μg / ml, and the detection sensitivity is 250 times that of the existing TSWN virus test strip. It is particularly suitable for detecting plants in the early stages of infection. The test strip has high detection accuracy, short detection time, simple operation and low detection cost.

[0007] The present application provides a tomato spotted wilt virus detection test strip for tobacco plants, comprising: a conjugate release pad, a reaction membrane, a sample absorption pad, a water absorbent pad, and a PVC base plate; a tomato spotted wilt virus monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; the conjugate release pad, the reaction membrane, the sample absorption pad, and the water absorbent pad are stacked in sequence on the PVC base plate;

[0008] The tomato spotted wilt virus monoclonal antibody-colloidal gold label includes: a tomato spotted wilt virus monoclonal antibody; the tomato spotted wilt virus monoclonal antibody includes: an amino acid sequence and a nucleotide sequence; the amino acid sequence includes: a heavy chain amino acid sequence as shown in SEQ ID NO: 3 and a light chain amino acid sequence as shown in SEQ ID NO: 4; the nucleotide sequence includes: a heavy chain nucleotide sequence as shown in SEQ ID NO: 1 and a light chain nucleotide sequence as shown in SEQ ID NO: 2.

[0009] The test paper is assembled according to the existing colloidal gold test paper assembly structure and method, which will not be repeated here. Please refer to the embodiments of this application for details. The test strip containing the above-mentioned monoclonal antibody can accurately detect tobacco samples with a virus content of only 1.0 μg / kg, and the test results have a high accuracy rate. The test strip is particularly suitable for detecting the diseased condition of tobacco leaves. In the detection of 20 sample amounts, the result accuracy rate is 100%. The plant fiber content in tobacco samples is relatively high, which causes adsorption of viruses. The low detection limit of the monoclonal antibody provided by this application can effectively achieve accurate detection of such samples.

[0010] Preferably, the method for using the test strip comprises the following steps: taking a sample of the tobacco leaf to be tested, vertically dripping 3 drops of the sampling liquid into the sample addition hole; and reading the test result after standing for 5 to 10 minutes.

[0011] Preferably, the tobacco leaves to be tested are harvested tobacco leaves to be flue-cured or newly flue-cured tobacco leaves; and the test strips are stored at 4-30° C. for 12 months.

[0012] Preferably, the detection limit of the test strip is 1.0 μg / kg.

[0013] This test strip has high accuracy for the aforementioned tobacco leaves, with a detection limit as low as 1.0 μg / kg, which translates to 0.001 μg / ml. It effectively detects early-stage diseased tobacco leaves, avoiding missed detections and improving comprehensiveness of tomato spotted wilt virus testing. The test strip has a zero false-positive rate and a zero false-negative rate, demonstrating high accuracy. It effectively prevents the impact of false positives or negatives on test results.

[0014] Preferably, the preparation method of the tomato spotted wilt virus monoclonal antibody-colloidal gold marker comprises the following steps:

[0015] 1) adding 20-50 μg of tomato spotted wilt virus monoclonal antibody per ml of colloidal gold solution, and stirring to adjust the final concentration of tomato spotted wilt virus monoclonal antibody in the colloidal gold solution to 1% by volume to obtain a mixed solution;

[0016] 2) The mixed solution was allowed to stand, centrifuged, and the supernatant was discarded. The precipitate was washed twice with a reconstitution buffer, and the precipitate was resuspended to obtain a tomato spotted wilt virus monoclonal antibody-colloidal gold label.

[0017] When the marker prepared by the above method is used in a test strip, accurate and effective detection of diseased tobacco leaves can be achieved.

[0018] Preferably, step 1) comprises: adjusting the pH value of the colloidal gold solution to 7.0.

[0019] Preferably, the pH is adjusted using a potassium carbonate solution with a concentration of 0.2 mol / L; and the standing time is 10 min.

[0020] Preferably, the centrifugation conditions are: 12000 r / min, 4° C., 40 min.

[0021] Specifically, the preparation method comprises adjusting the pH of colloidal gold to 7.0 with a 0.2 mol / L potassium carbonate solution under magnetic stirring, adding 20 to 50 μg of the above-mentioned tomato spotted wilt virus monoclonal antibody per milliliter of colloidal gold solution, continuing to stir and mix for 30 minutes, adding 10% BSA to a final concentration of 1% (volume fraction) in the colloidal gold solution, and allowing to stand for 10 minutes. Centrifuging at 12,000 rpm and 4°C for 40 minutes, discarding the supernatant, and washing the precipitate twice with reconstitution buffer. The precipitate is then resuspended in a reconstitution buffer with a volume that is 1 / 10 of the initial colloidal gold volume and stored at 4°C for later use.

[0022] Preferably, the reconstitution buffer used is a 0.02 mol / L phosphate buffer with a pH of 7.2, to which are added: 0.02% to 0.1% by mass of casein and 0.05% to 0.2% by mass of Tween-80.

[0023] Preferably, the preparation of the conjugate release pad comprises the following steps:

[0024] The conjugate release pad was soaked in a phosphate buffer solution containing bovine serum albumin (0.5% bovine serum albumin in the buffer) at a pH of 7.2 and 0.5 mol / L, and allowed to soak evenly for 1 hour. The pad was then dried at 37°C for 3 hours for later use. The prepared tomato spotted wilt virus monoclonal antibody-colloidal gold label was evenly sprayed onto the conjugate release pad using an Isoflow sprayer, with 0.01 ml of the tomato spotted wilt virus monoclonal antibody-colloidal gold label applied per 1 cm of the conjugate release pad. The pad was then placed in an environment at 37°C (humidity <20%) for 60 minutes, removed, and stored in a dry environment (humidity <20%) for later use.

[0025] Preferably, the preparation of the sample absorption pad comprises the following steps: soaking the sample absorption pad in a 0.1 mol / L phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH 7.2 for 2 hours, and baking at 37° C. for 2 hours for later use.

[0026] The beneficial effects of this application include:

[0027] 1) The present application provides a tomato spotted wilt virus detection test strip for tobacco plants. After the tomato spotted wilt virus monoclonal antibody is made into a colloidal gold test strip, the liquid sample to be tested combines with the tomato spotted wilt virus monoclonal antibody-colloidal gold marker during the flow process to form a drug-antibody-colloidal gold marker. The test result is obtained based on the presence or color depth of the red strip on the test line. The detection efficiency is high and the detection cost is low. The test strip has a low detection limit of only 1.0 μg / kg, which is suitable for detecting samples with low virus content and where the virus has not replicated in large quantities in the early stage of infection, which is conducive to improving the accuracy of the test results. The test strip is particularly suitable for detecting the virus in tobacco leaves.

[0028] 2) The test strips for detecting tomato spotted wilt virus in tobacco plants provided herein have the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for use by various units, simple storage, and long shelf life. The method for detecting tomato spotted wilt virus residues using the test strips of the present invention is simple, rapid, intuitive, accurate, widely applicable, low cost, and easily promoted for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the fused cells obtained in Example 1 of the present application;

[0030] Figure 2 This is a schematic diagram of a single cell after cloning obtained in Example 1 of the present application;

[0031] Figure 3 This is a schematic diagram of the expanded culture state of cells obtained in Example 1 of the present application; DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0033] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0034] The technical means that are not described in detail in this application and are not used to solve the technical problems of this application are all set according to the common knowledge in this field, and can be implemented in a variety of common knowledge settings.

[0035] Example

[0036] Unless otherwise specified, the instruments and reagents used in the following examples were obtained from commercial channels.

[0037] Example 1 Obtaining Monoclonal Antibodies to Tomato Spotted Wilt Virus

[0038] 1.1 Immunization of animals

[0039] Immunize mice according to the general procedure, collect blood, and perform serum testing. Select mice that meet the fusion requirements and receive a booster injection of antigen three days before fusion. The antigen used is a prokaryotic protein, the protein (NP) of Tomato Swollen Weed Virus (TSWV). The immunization time and frequency are as follows:

[0040] Table 1 Immunization process

[0041] Immunization and blood collection time operate 2021 / 12 / 1 First Immunity 2021 / 12 / 16 Second Immunity 2021 / 12 / 31 The third immune 2022 / 1 / 7 First blood draw 2022 / 1 / 14 Fourth Immunity 2022 / 1 / 21 Second blood draw 2022 / 1 / 28 Fifth Immunity 2022 / 2 / 4 The third blood collection

[0042] 1.2 Mouse polyclonal antibody detection

[0043] 1.2.1 Package board

[0044] Prepare antigen coating plate, dilute TSWV-NP protein to 1ug / ml with CB solution, apply 100ul to each well of ELISA plate, and coat at 4℃ overnight; after coating, wash the plate once with PBST solution, add 150ul blocking solution to each well, block at 37℃ for 2h, pat dry, and store at 4℃ for later use.

[0045] 1.2.2 Mouse polyclonal antibody serum positive test

[0046] Mouse serum was diluted 1000-fold with antibody diluent and then serially diluted threefold six times. Seven serum samples of varying concentrations were added sequentially, starting from the top and ending at 100 μl per well, to an ELISA plate coated with antigen. The plate was sealed with a cover film and incubated at 37°C for 30 minutes. The plate was washed three times and patted dry. Then, 100 μl of goat anti-mouse IgG enzyme-linked secondary antibody was added to each well and incubated at 37°C for 30 minutes. The plate was washed four times and patted dry. 100 μl of substrate solution (AB mixture) was added to each well and incubated at room temperature for 15 minutes. Stop solution (50 μL / well) was added and the OD values were measured at dual wavelengths of 450 nm and 630 nm using an enzyme-linked immunosorbent assay (ELISA). Fusion mice were selected based on the OD values. Wells with a color development value of approximately 1.0 (≥1.0) corresponded to the dilution factor indicating the antibody titer, and negative sera had an OD value of less than 0.4. ELISA test data are shown in Table 2, Serum Test Data.

[0047] One week after the fifth immunization, blood was collected from the eyeball vein of the mice to separate the serum, and the serum titer was detected by ELISA. Table 2 shows the data of the last blood collection (three collections).

[0048] Table 2 Mouse serum test results

[0049]

[0050] As shown in Table 2, according to the ELISA test of the third blood collection, the serum titer of mice 2#, 3#, and 4# was 24.3×104, which was higher than that of mice 1# and 5#. Among them, the 0D value of mouse 3# was the highest, indicating that the serum antibody content of mouse 3# was the highest. Therefore, mouse 3# was selected for subsequent cell fusion operations.

[0051] 1.3 Cell fusion

[0052] 1.3.1 Preparation

[0053] The fusion agent was divided into 1 ml portions and placed in a 37°C CO2 incubator before fusion.

[0054] HAT medium: Prepare before fusion (500 ml HAT medium contains 5 mg HAT, 20% FBS, 100 U / ml penicillin, and 0.1 mg / ml streptomycin), and place in a 37°C incubator to warm up.

[0055] HT culture medium: Prepare before the first cloning (500 ml DMEM containing 5 mg HT, 20% FBS, 100 U / ml penicillin, and 0.1 mg / ml streptomycin), and place in a 37°C incubator to warm up.

[0056] Cloning medium: except for the first clone, other clones were used (500ml DMEM containing 20% FBS, penicillin 100U / ml, streptomycin 0.1mg / ml

[0057] Cell expansion medium: used for expansion culture after cell establishment (500ml DMEM containing 15% FBS, penicillin 100U / ml, streptomycin 0.1mg / ml)

[0058] 1.3.2.Resuscitation and cultivation of SP2 / 0

[0059] Thaw SP2 / 0 cells 2 weeks before fusion. Remove cryogenically stored SP2 / 0 and quickly warm in a 37°C water bath (within 1-2 minutes). Once thawed, add to a centrifuge tube containing 5-10 ml of DMEM. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the pellet in expanded culture medium, and transfer to a cell flask for culture. Observe the cells and perform culture medium changes as needed. Handle the SP2 / 0 cells as needed one day before fusion. Replace with fresh culture medium approximately 8 hours before fusion.

[0060] 1.3.3 Preparation of feeder cells

[0061] Peritoneal macrophages were used as feeder cells. BALB / c mice were killed by cervical dislocation and then immersed in 75% alcohol for disinfection for 5 minutes. The mice were taken out and stood upright for half a minute until no more alcohol was dripping from them. Then they were transferred to a clean bench with the abdomen facing upwards. The abdominal skin was cut open to expose the endothelium of the peritoneal cavity. An appropriate amount of DMEM was drawn up with a syringe and injected into the mouse's abdominal cavity. The internal organs should not be punctured. The mice were shaken several times, and then the DMEM was aspirated and transferred to a 50 ml centrifuge tube. Repeat this 3-4 times. The centrifuge tube containing the feeder cells was placed in a centrifuge at 1000 r / min and centrifuged for 5 minutes. The supernatant was discarded, and an appropriate amount of HAT culture medium was added. The feeder cells in the tube were blown evenly for use.

[0062] 1.3.4 Preparation of SP2 / 0 cells

[0063] Take SP2 / 0 cells in good growth condition, pour out the culture medium, add appropriate amount of DMEM culture medium, blow off the cells with a curved pipe, and make a suspension for later use.

[0064] 1.3.5 Preparation of splenocytes

[0065] Immunized BALB / c mice were sacrificed by cervical dislocation and then disinfected by soaking in 75% alcohol for 5 minutes. The spleen was removed, excess tissue removed, and the spleen was rinsed with DMEM before being placed in a sterile culture dish. DMEM culture medium was drawn up with a syringe and injected into the spleen to dissipate splenocytes. Repeat this process 3-5 times to dissipate as many splenocytes as possible. The spleen cell suspension was then transferred to a centrifuge tube.

[0066] 1.3.6 Cell fusion

[0067] Add the SP2 / 0 suspension to the spleen cell suspension, mix thoroughly, and centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant, keeping the tube facing downward. Use filter paper to remove any remaining liquid from the tube walls. Tap the cells at the bottom of the tube to disperse them evenly, as close to a single layer as possible. Add 1 ml of fusion agent uniformly over 1 minute along the tube walls, rotating the tube as you add. After addition, let the tube lie flat and allow fusion to proceed for 1 minute. Terminate fusion with 15 ml of DMEM medium, adding 1 ml uniformly over the first minute, 2 ml over the second minute, and so on.

[0068] After adding, centrifuge at 1000r / min at room temperature for 5 minutes. Discard the supernatant and mix the precipitate with an appropriate amount of HAT culture medium. Add the corresponding HAT working solution as needed, and spread the cells on a 96-well cell plate at 100-150ul / well. Then spread the prepared feeder cells on a 96-well cell plate containing fused cells at 100-150ul / well, and culture in a 5% CO2 37℃ constant temperature cell culture incubator for 7-10 days. Observe the cell morphology and determine the detection time according to the number and size of the cells. The photo of the cells obtained after fusion is as follows: Figure 1 shown.

[0069] 1.4 Screening and cloning of hybridoma cells

[0070] 1.4.1 Fusion Plate Detection

[0071] Observe the cell status 4-5 days after fusion and prepare the antigen coating plate. The steps are the same as 1.2.1.

[0072] After 7 days of fusion, take 50 μl of sample from each well and add it to the ELISA plate. Seal the plate with a cover film. Follow the same steps as in 1.2.2.

[0073] 1.4.2 Cloning and Detection

[0074] Based on the test results, cells were selected for cloning and cultured in HT medium.

[0075] Prepare the culture medium and a new cell plate in advance for cloning. Pipet the selected cell wells to evenly distribute the cells. Aspirate a portion of the cell suspension to dilute the cells and count them on a cell counter. Dilute the cells to 1-5 cells per microliter based on the count results. Calculate the amount of suspension required for 1-2 cells per well based on the amount of plated cells. Aspirate the suspension into HT culture medium, mix well, plate into a new 96-well cell plate, and plate the feeder cells.

[0076] After 6-8 days of cloning, the cells can be tested. The single cell photos obtained after cloning are as follows: Figure 2As shown. Wrap the plate 1-2 days in advance. The wrapping and testing steps are the same as 1.2.1. The testing steps are the same as 1.2.2. If the test results are qualified and there are single cells in the cell plate wells, transfer the cells to 24-well cell plates for culture. After testing the titer and cross-culture, determine the strain and expand the culture. The expanded culture photo is shown in Figure 3 If there is no single cell, perform the second and third cloning until a single cell appears.

[0077] Through fusion and cloning screening, 6 cell lines were finally identified. The number of clones and fusion detection data of each cell line are shown in the table below.

[0078] Table 3 Cell clone details

[0079]

[0080] As shown in Table 3, 6 cell lines were screened for secondary cloning to obtain single cells with high OD values. After expansion culture, the single cells can secrete antibodies, i.e., monoclonal antibodies. The titers are shown in the expansion culture table.

[0081] 1.4.3 Expanded cultivation

[0082] Culture cells in a 24-well plate and observe. Perform assays when cells have spread to over 80% of the wells. Dilute the supernatant 30-fold, then serially dilute 3-fold 6-7 times. Follow the same procedures as described in Section 1.2.2 of the polyclonal antibody inhibition assay. See Table 4 for cell supernatant assay data. After assay, transfer the cells to a flask for expansion.

[0083] After testing, a total of 17 positive cell lines were screened out, and their names and titers are shown in the following table.

[0084] Table 4 Cell supernatant detection results

[0085] code name Original cell line number Cell supernatant dilution multiple OD value 101 2B7-C10-F8 1350 1.291 102 2B7-C10-D9 1350 2.733 103 2B7-C10-E9 1350 2.865 104 2B7-C10-F9 1350 0.570 105 2B7-C10-H9 1350 2.284 201 5D8-B7-G1 450 1.106 202 5D8-B7-A2 450 0.973 203 5D8-B7-C3 450 1.425 204 5D8-B7-G9 450 1.286 205 5D8-B7-D10 450 1.084 301 3E6-D10-D5 1350 1.643 401 2E6-F1-D2 450 0.531 501 5D10-D1-D2 450 1.338 502 5D10-D1-B3 450 1.062 503 5D10-D1-B6 450 1.160 601 1A5-E6-E3 270 1.047 602 1A5-E6-D5 270 1.155

[0086] As shown in the table above, the antibody titer in the supernatant of cells cloned from 2B7 and 3E6 (101 / 102 / 103 / 104 / 105 / 301) was the highest, reaching over 103 (1350); the antibody titer in the supernatant of cells cloned from 5D8, 2E6, and 5D10 reached over 102 (450), and that of 1A5 was around 1:270. Ascites was prepared from all of the above cells.

[0087] 1.5 Cryopreservation

[0088] Before freezing, ensure that the cells are plump and in good condition (round, translucent, and abundant), occupying 80% of the total volume. Use a curved pipette to blow off the cells, transfer them to a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add freezing solution to mix the cells, transfer them to a cryovial, label them, and place them in a cryovial. Place them in a -80°C freezer overnight. Short-term storage is possible at -80°C, but long-term storage requires transfer to liquid nitrogen.

[0089] 1.6 Preparation and detection of monoclonal antibody ascites

[0090] Induce ascites in animals. Prepare a certain number of BALB / c mice. Before injecting cells, inject 0.5 ml of sterile paraffin oil into the mouse's peritoneal cavity. Wait at least 4 days before injecting cells. When the cells are plump and in good condition, use a curved pipe to blow the cells out of the bottle and transfer them to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add physiological saline and mix thoroughly. Use a syringe to draw up the mixed cell suspension and inject it into the mouse's peritoneal cavity. Each mouse can be injected with 0.5 ml of liquid. Depending on the number of cells, each mouse can be injected with approximately 1*10e6 cells.

[0091] Ascites will develop 7-10 days after cell inoculation. Closely monitor the mouse's health and signs of ascites. When the mouse's abdomen becomes distended, remove the ascites with a syringe. Repeat this process until the mouse dies. Centrifuge the ascites at 8000 rpm for 10 minutes in a refrigerated centrifuge, aliquot, and freeze at -20°C until ready for use. ELISA plate coating procedures are the same as in 1.2.1. Testing procedures are the same as in 1.2.2. Dilutions can be performed starting at 1000 and using a 3-fold serial dilution step of 6-7. Test data are shown in Table 5, Ascites Titer Test Table.

[0092] The obtained ascites was diluted with PBS and the OD values were detected and the results are shown in the following table.

[0093] Table 5 Ascites detection data

[0094]

[0095] As shown in Table 5, the antibody titers of ascites numbered MC-2 / MC-3 / MC-4 / MC-6 / MC-7 / MC-8 / MC-10 / MC-11 / MC-12 / MC-13 / MC-14 / MC-15 / MC-16 / MC-17 / MC-20 / MC-21 / MC-22 were relatively high, reaching 104. After paired screening, MC-1 and MC-4 were derived from the same cell line 201 and had basically the same properties. MC-6 and MC-3 were derived from the same cell line 201 and had basically the same properties. Therefore, MC-4 (MC-1) and MC-6 (MC-3) were selected for subsequent experiments.

[0096] 1.7 Antibody Purification

[0097] 1.7.1 Antibody Purification

[0098] Ammonium sulfate precipitation plus affinity chromatography was used. The mouse ascites prepared from the above-mentioned MC-4 (MC-1) and MC-6 (MC-3) cell lines was centrifuged to remove impurities, transferred to a new centrifuge tube, and an equal volume of PB buffer was added and mixed. A saturated ammonium sulfate solution of the same volume as the buffered solution was added dropwise and the solution was mixed evenly. The final ammonium sulfate concentration was 50%. The solution was allowed to precipitate overnight at 4°C. After precipitation, the solution was centrifuged and the supernatant was discarded. PB buffer (60% of the volume of the ascites) was added to redissolve the precipitate. After redissolution, the solution was transferred to a dialysis bag for dialysis. After dialysis, the precipitate was centrifuged to obtain a preliminarily purified antibody. The protein solution was purified using a Protein G affinity column. After obtaining a high-purity antibody protein solution, the concentration was measured, a preservative was added, and the solution was aliquoted and frozen for later use.

[0099] After purification, the antibody concentration in the solution was tested using a protein concentration detector. The results are shown in the table below.

[0100] Table 6 Antibody purification results

[0101]

[0102]

[0103] As shown in Table 6, the antibodies produced by the cell lines 201 / 203S corresponding to MC-4 (MC-1) and MC-6 (MC-3) have high concentrations and purities after purification, meeting the standards for subsequent experiments and are ready for use.

[0104] The obtained protein solution is a monoclonal antibody to Tomato Wilt Virus. The protein was sequenced using existing methods, and its heavy chain amino acid sequence is shown in SEQ ID NO: 3:

[0105] EVHLQQFGAELVKPGTSLKISCKASGYTFTDYIMDWVKQSHGESLEWIGDIDPKYDSTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARLTTATAYWGQGTLVTVSA

[0106] The light chain amino acid sequence is shown in SEQ ID NO: 4:

[0107] DIVITQDELSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELLTFGAGTKLELK

[0108] Antibodies with higher titers are preferably selected, and the nucleotide sequences determined include: heavy chain nucleotide sequence and light chain nucleotide sequence, wherein the heavy chain nucleotide sequence is as shown in SEQ ID NO: 1:

[0109] gaggtccatctgcaacagtttggagctgagctggtgaagcctgggacttcattgaagatttcctgcaaggcttctggctacacattcactgactacatcatggactgggtg aagcagagccatggagagagccttgagtggattggagatattgatcctaagtatgatagtactaactacaaccagaagttcaagggaaaggccacattgactgtagacaag

[0110] tcctccagcacagcctacatggagctccgcagcctgacatctgaggacactgc

[0111] agtctattactgtgcacgacttactacggctacggcttattggggccaagggactctggtcactgtctctgcgg

[0112] The light chain nucleotide sequence is shown in SEQ ID NO: 2:

[0113] gatatcgtgataacacaagatgaactctccaatccagtcactcttggaacatcagcttccatctcctgcaggtctagtaagagtctcctacatagtaatggcatcacttatttgtattggtatctgcagaagccaggccagtctcctcagctcctgatttatcagat gtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggctgaggatgtgggtgtttattactgtgctcaaaatctagaactgctcacgttcggtgctgggaccaagctggagctgaaac

[0114] Example 2 Preparation of Tomato Spotted Wilt Virus Detection Test Strips

[0115] The preparation method of the test strip comprises the following steps:

[0116] 1) preparing a release pad sprayed with a tomato spotted wilt virus monoclonal antibody-colloidal gold marker conjugate;

[0117] 2) preparing a reaction membrane having a test line coated with TSWV-NP protein and a quality control line coated with goat anti-mouse antibody;

[0118] 3) Assemble the conjugate release pad, reaction membrane, sample absorption pad, water absorption pad and PVC bottom plate prepared in 1) and 2) into a test strip.

[0119] Specifically, the method includes the following steps:

[0120] 2.1 Preparation of goat anti-mouse antibodies

[0121] Sheep are used as immune animals, and mouse antibodies are used as immunogens to immunize pathogen-free sheep to obtain sheep anti-mouse anti-antibodies. The specific operation is carried out according to existing preparation methods and will not be repeated here.

[0122] 1.1 Preparation of Tomato Spotted Wilt Virus Monoclonal Antibody-Colloidal Gold Label

[0123] 1.1.1 Preparation of colloidal gold

[0124] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double-distilled deionized water. Place 100 ml in a conical flask and heat to boiling using a thermostatic electromagnetic stirrer. Add 2.5 ml of 1% trisodium citrate while continuing to stir at high temperature until the solution turns a translucent red. Cool to room temperature, then return to the original volume with deionized water and store at 4°C. The prepared colloidal gold should be pure, translucent, and free of precipitates and floating matter.

[0125] 1.1.2 Preparation of Tomato Spotted Wilt Virus Monoclonal Antibody-Colloidal Gold Label

[0126] Under magnetic stirring, the pH of the colloidal gold solution was adjusted to 7.0 with 0.2 mol / L potassium carbonate solution. 30 μg of the tomato spotted wilt virus monoclonal antibody prepared in Example 1 was added per milliliter of the colloidal gold solution. Stirring and mixing were continued for 30 minutes. 10% BSA was added to a final concentration of 1% (volume fraction) in the colloidal gold solution. The solution was allowed to stand for 10 minutes. The solution was centrifuged at 12,000 rpm and 4°C for 40 minutes. The supernatant was discarded, and the precipitate was washed twice with reconstitution buffer. The precipitate was resuspended in reconstitution buffer at a volume 1 / 10 of the initial colloidal gold volume and stored at 4°C for later use.

[0127] The reconstitution buffer is a 0.02 mol / L phosphate buffer with a pH of 7.2, and the following components are added: 0.08% by mass of casein and 0.15% by mass of Tween-80.

[0128] 3.1 Preparation of conjugate release pad

[0129] The conjugate release pad was soaked in a phosphate buffer solution containing bovine serum albumin (0.5% bovine serum albumin in the buffer) at a pH of 7.2 and 0.5 mol / L, and allowed to soak evenly for 1 hour. The pad was then dried at 37°C for 3 hours for later use. The prepared tomato spotted wilt virus monoclonal antibody-colloidal gold label was evenly sprayed onto the conjugate release pad using an Isoflow sprayer, with 0.01 ml of the tomato spotted wilt virus monoclonal antibody-colloidal gold label applied per 1 cm of the conjugate release pad. The pad was then placed in an environment at 37°C (humidity <20%) for 60 minutes, removed, and stored in a dry environment (humidity <20%) for later use.

[0130] 3.2 Preparation of reaction membrane

[0131] The tomato spotted wilt virus hapten-ovalbumin conjugate is coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.

[0132] Coating process: Dilute the tomato spotted wilt virus hapten-ovalbumin conjugate to 10 mg / ml in phosphate buffer and coat it onto the test line (T line) on the nitrocellulose membrane using an Isoflow applicator at a coating volume of 0.8 μl / cm. Dilute the goat anti-mouse antibody to 200 μg / ml in 0.01 mol / L, pH 7.4 phosphate buffer and coat it onto the control line (C line) on the nitrocellulose membrane using an Isoflow applicator at a coating volume of 1.0 μl / cm. Dry the coated membrane at 37°C for 2 hours before use.

[0133] 3.3 Preparation of sample absorption pad

[0134] The sample absorption pad was placed in a phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH 7.2, and 0.1 mol / L and soaked for 2 h, and then dried at 37° C. for 2 h for later use.

[0135] 3.4 Assembly of test strips

[0136] A sample absorption pad, a conjugate release pad, a reaction membrane, and a water absorbent pad are sequentially attached to a PVC base plate; one-third of the conjugate release pad from its starting end is covered by the sample absorption pad, the end of the conjugate release pad is connected to the starting end of the reaction membrane, the end of the reaction membrane is connected to the starting end of the water absorbent pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the end of the water absorbent pad is aligned with the ending end of the PVC base plate; a test line (T line) and a quality control line (C line) are formed on the reaction membrane, both of which are strips perpendicular to the length of the test strip; the test line is located on the side closest to the end of the conjugate release pad; and the quality control line is located on the side away from the end of the conjugate release pad. The resulting test strip is cut into 3 mm wide strips by a machine and placed in a specially made plastic card.

[0137] Example 3 Detection of the Tomato Spotted Wilt Virus Colloidal Gold Test Strip Obtained in Example 2

[0138] 1. Test strip detection limit test

[0139] Take blank tobacco leaves (harvested tobacco leaves to be cured, freshly cured tobacco leaves) and prepare samples for testing according to the following steps:

[0140] Place 0.15g of fresh tobacco leaves in a centrifuge tube and rotate and squeeze the tube several times with a grinding rod to ensure that the leaf tissue is fully ground. Add 2mL of 0.02mol / L PB buffer and repeat the grinding to thoroughly mix the sample and buffer. Remove the grinding rod and let it stand for at least 1 minute. The supernatant is the test fluid.

[0141] Tomato spotted wilt virus was added to the obtained samples to a final concentration of 0.1 μg / kg, 0.5 μg / kg, 1.0 μg / kg, 1.5 μg / kg, and 2.0 μg / kg in the solution. The test strips obtained in Example 2 were used for testing, and each sample was measured three times.

[0142] The colloidal gold test strip for tomato spotted wilt virus obtained in Example 2 was used as follows: 3 drops of the sample solution to be tested were vertically dripped into the sample well using a pipette. The timing began when the liquid began to flow, and the reaction lasted for 5 to 10 minutes (the time for reading the test results for each sample was within this range, with an average reading time of 10 minutes). The result was determined. The method for using the test strip in this example was the same.

[0143] Analysis of the test results of the tomato spotted wilt virus colloidal gold test strip obtained in Example 2 (the analysis method of the test strip results in this example is the same):

[0144] Read the results using a colloidal gold analyzer (referred to as the "analyzer"):

[0145] Negative (-): indicates that the concentration of the analyte in the sample is lower than the detection limit;

[0146] Positive (+): indicates that the concentration of the substance to be tested in the sample is equal to or higher than the detection limit;

[0147] Invalid: Indicates that retesting is required.

[0148] When the test strips were used to test tobacco leaf samples (both harvested and newly cured), the analyzer displayed negative results when TSWV was spiked at concentrations of 0.1 μg / kg and 0.5 μg / kg; however, the analyzer displayed positive results when TSWV was spiked at concentrations of 1.0 μg / kg, 1.5 μg / kg, and 2.0 μg / kg. Therefore, the detection limit of the test strips was 1.0 μg / kg.

[0149] 2. False positive rate and false negative rate test

[0150] Twenty positive samples of tobacco leaves (harvest tobacco leaves and first-cured tobacco leaves) with known TSV levels greater than 1.0 μg / kg (both pre-cured and first-cured) and 20 negative samples of tobacco leaves (harvest tobacco leaves and first-cured tobacco leaves) with levels less than 1.0 μg / kg were collected and tested using three batches of test strips. The positive and negative rates were calculated. The results are shown in Tables 7 and 8.

[0151] Table 7 Tobacco leaf (harvested tobacco leaves to be cured) test sample results

[0152]

[0153] Table 8 Tobacco leaf (first flue-cured tobacco leaf) test sample results

[0154]

[0155] The results in Tables 7 and 8 show that when the test strips produced in three batches were used to detect positive tobacco leaf samples (harvested tobacco leaves to be cured and newly cured tobacco leaves), all the results were positive. It can be seen that the positive sample compliance rate of the test strips was 100%, the false negative rate was 0, and the positive result detection accuracy was 100%.

[0156] When 20 negative tobacco leaf samples (harvested tobacco leaves to be cured and newly cured tobacco leaves) were tested, all the results were negative. It can be seen that the negative compliance rate was 100%, the false positive rate was 0, and the negative result detection accuracy rate was 100%.

[0157] The test strip for detecting tomato spotted wilt virus provided in this application is particularly suitable for rapid detection of tomato spotted wilt virus residues in tobacco leaves (harvested tobacco leaves to be cured and newly cured tobacco leaves), and the detection results have a high accuracy rate.

[0158] Example 4

[0159] The difference from Example 2 is that the reconstitution buffer is a 0.02 mol / L phosphate buffer with a pH of 7.2, to which 0.02% by mass of casein and 0.05% by mass of Tween-80 are added.

[0160] In 1.1.2, add 20 μg of tomato spotted wilt virus monoclonal antibody per ml of colloidal gold solution.

[0161] Example 5

[0162] The difference from Example 2 is that the reconstitution buffer is a 0.02 mol / L phosphate buffer with a pH of 7.2, and 0.1% by mass of casein and 0.2% by mass of Tween-80 are added thereto.

[0163] In 1.1.2, add 50 μg of tomato spotted wilt virus monoclonal antibody per ml of colloidal gold solution.

[0164] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A test strip for detecting tomato spotted wilt virus for tobacco plants, characterized in that: include: Conjugate release pad, reaction membrane and sample absorption pad, water absorbent pad and PVC bottom plate; Tomato spotted wilt virus monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; The conjugate release pad, reaction membrane, sample absorption pad, and water absorbent pad are stacked on the PVC base in sequence; The tomato spotted wilt virus monoclonal antibody-colloidal gold label includes: a tomato spotted wilt virus monoclonal antibody; the tomato spotted wilt virus monoclonal antibody includes: an amino acid sequence; the amino acid sequence includes: a heavy chain amino acid sequence as shown in SEQ ID NO: 3 and a light chain amino acid sequence as shown in SEQ ID NO:

4.

2. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 1, characterized in that: The method of using the test strips includes the following steps: taking a tobacco leaf sample to be tested, vertically dripping 3 drops of the sampling liquid into the sample well; and reading the test results after standing for 5 to 10 minutes.

3. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 1, wherein: The tobacco leaves to be tested are harvested tobacco leaves to be flue-cured or newly flue-cured tobacco leaves; the test strips are stored at 4-30°C for 12 months.

4. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 1, wherein: The detection limit of the test strip is 1.0μg / kg.

5. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 1, wherein: The preparation method of tomato spotted wilt virus monoclonal antibody-colloidal gold label comprises the following steps: 1) adding 20-50 μg of tomato spotted wilt virus monoclonal antibody per ml of colloidal gold solution, and stirring to adjust the final concentration of tomato spotted wilt virus monoclonal antibody in the colloidal gold solution to 1% by volume to obtain a mixed solution; 2) The mixed solution was allowed to stand, centrifuged, and the supernatant was discarded. The precipitate was washed twice with a reconstitution buffer, and the precipitate was resuspended to obtain a tomato spotted wilt virus monoclonal antibody-colloidal gold label.

6. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 5, characterized in that: Step 1) includes: adjusting the pH value of the colloidal gold solution to 7.

0.

7. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 6, characterized in that: The pH was adjusted using a potassium carbonate solution with a concentration of 0.2 mol / L; the standing time was 10 min.

8. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 6, wherein: The centrifugation conditions were: 12000 r / min, 4°C for 40 min.

9. The tomato spotted wilt virus detection test strip for tobacco plants according to claim 6, wherein: The reconstitution buffer used is a 0.02 mol / L phosphate buffer with a pH of 7.2, and is added with: 0.02% to 0.1% casein by mass and 0.05% to 0.2% Tween-80 by mass.

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