Antibody secreting cell strain for detecting ChiVMV-TMV virus, antibody secreted by antibody secreting cell strain and application of antibody secreting cell strain

Abstract

The invention discloses four antibody secreting cell strains for detecting ChiVMV-TMV viruses, antibodies secreted by the four antibody secreting cell strains and application of the four antibody secreting cell strains and belongs to the technical field of microorganisms, the four cell strains are hybridoma cell strains ChiVMV-a, ChiVMV-b, TMV-1 and TMV-2 respectively and are all preserved in the China Center for Type Culture Collection, and the preservation numbers of the four cell strains are CCTCC NO.C202563, CCTCC NO.C202564, CCTCC NO.C2024407 and CCTCC NO.C2024408 respectively. The specific monoclonal antibodies secreted by the four cell strains are utilized, based on the double-antibody sandwich immunochromatography principle, and through the specific antibody combination and double detection line design, the ChiVMV and TMV virus infections can be distinguished at the same time with high precision and high accuracy in single detection, the tedious process of step-by-step detection in a traditional method is avoided, and the detection efficiency is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to an antibody-secreting cell line for ChiVMV-TMV virus detection, an antibody secreted by the cell line, and applications thereof. Background Art

[0002] Chili vein mottle virus (ChiVMV) is a species of the genus Potyvirus in the family Potyviridae. ChiVMV's natural hosts primarily include Solanaceae crops such as peppers, tomatoes, and tobacco, with tobacco and peppers being the most severely affected. In recent years, the virus has spread rapidly in my country, with reports of ChiVMV damage occurring in Hainan, Yunnan, Hunan, Fujian, Sichuan, Shaanxi, and Guizhou, causing severe economic losses to Solanaceae crops. Furthermore, ChiVMV often co-infects with other viruses, leading to leaf mottle, dwarfing, and a sharp drop in yield, resulting in significant economic losses. Given its severity, ChiVMV is listed as a key virus for port quarantine in my country.

[0003] Tobacco mosaic disease (TMV) is a common disease worldwide caused by the tobacco mosaic virus (TMV). The virus's hosts include over 400 plant species, including those in the Solanaceae, Cruciferae, and Cucurbitaceae families. It is particularly devastating to tobacco crops. TMV occurs in both northern and southern tobacco-growing areas of my country, with the southern region being particularly severely affected. The disease can occur from the seedling stage to the harvest, with a field incidence rate generally ranging from 5% to 20% and reaching as high as 90% to 100% in some fields. Early onset can lead to losses of 50% to 70%, or even total crop failure. Furthermore, diseased leaves exhibit uneven color after sun-drying, with a poor flavor and significantly reduced quality. TMV is a single-stranded RNA virus with rod-shaped virions measuring 300 nm x 18 nm. Tobacco mosaic virus has strong resistance to adversity, with high inactivation temperature (up to 90-93°C, 10 minutes), large dilution limit (up to 1,000,000 times), long in vitro preservation period (up to 72 to 96 hours, and even pathogenicity for several years under sterile conditions), and long survival time in dry diseased tissues (up to more than 30 years). This is also an important reason for the continued prevalence of the virus.

[0004] ChiVMV and TMV are two serious pathogenic viruses that harm Solanaceae crops. It's widely acknowledged that rapid and accurate virus detection is crucial for preventing and controlling the spread of viral diseases and minimizing losses in production. Currently, virus detection relies primarily on biological observation, electron microscopy, enzyme-linked immunosorbent assay (ELISA), and PCR. However, these methods have significant limitations: biological observation can only empirically diagnose whether a host plant is infected with a viral disease; electron microscopy requires expensive transmission electron microscope equipment and specialized operators; ELISA involves a multi-step, specialized procedure that can take hours; and PCR is costly, complex, and requires specialized equipment. Consequently, these methods struggle to meet the demands of rapid field testing. While colloidal gold test strips are low-cost and simple to use, they are often designed to detect a single virus. For two pathogens, such as ChiVMV and TMV, separate tests must be performed for both ChiVMV and TMV, leading to duplicate sample processing and low detection efficiency. This makes them unsuitable for simultaneous initial screening of large numbers of samples. Although there are colloidal gold test strips that can deal with two or more pathogens at the same time, it is necessary to find specific antibodies for specific viruses, otherwise it is easy to cross-react, affecting the sensitivity and accuracy of detection.

[0005] Therefore, there is an urgent need to find dual-virus bispecific monoclonal antibodies that can avoid cross-reactions so that ChiVMV and TMV can be detected simultaneously at low cost using colloidal gold test strips, thereby realizing a simple, rapid, sensitive, inexpensive and grassroots-suitable viral diagnostic method for ChiVMV and TMV. Summary of the Invention

[0006] The purpose of the present invention is to provide four secretory cell lines that respectively secrete antibodies for ChiVMV-TMV virus detection. Through these four secretory cell lines, dual-virus bispecific monoclonal antibodies corresponding to colloidal gold test strips that avoid cross-reaction can be obtained, thereby solving the technical problem that the existing ChiVMV and TMV colloidal gold test strips cannot cope with the simultaneous initial screening of large quantities of samples due to repeated sample processing and low detection efficiency.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] Antibody-secreting cell lines for ChiVMV-TMV virus detection include hybridoma cell line ChiVMV-a, hybridoma cell line ChiVMV-b, hybridoma cell line TMV-1, and hybridoma cell line TMV-2. The deposit number of the hybridoma cell line ChiVMV-a is CCTCCNO.C202563, its Latin name is Hybridoma cell line ChiVMV-a, the deposit time is March 11, 2025, and the deposit unit is China Center for Type Culture Collection; the deposit number of the hybridoma cell line ChiVMV-b is CCTCCNO.C202564, its Latin name is Hybridoma cell line ChiVMV-b, the deposit time is March 11, 2025, and the deposit unit is China Center for Type Culture Collection; the deposit number of the hybridoma cell line TMV-1 is CCTCCNO.C2024407, its Latin name is Hybridoma cell line TMV-1, the preservation date is December 25, 2024, and the preservation unit is the China Center for Type Culture Collection; the preservation number of the hybridoma cell line TMV-2 is CCTCCNO.C2024408, its Latin name is Hybridoma cell line TMV-2, the preservation date is December 25, 2024, and the preservation unit is the China Center for Type Culture Collection.

[0009] Antibodies for detecting ChiVMV-TMV virus include specific monoclonal antibodies ChiVMV-a, ChiVMV-b, TMV-1, and TMV-2. The specific monoclonal antibody ChiVMV-a is secreted and produced by the hybridoma cell line ChiVMV-a according to claim 1; the specific monoclonal antibody ChiVMV-b is secreted and produced by the hybridoma cell line ChiVMV-b according to claim 1; the specific monoclonal antibody TMV-1 is secreted and produced by the hybridoma cell line TMV-1 according to claim 1; and the specific monoclonal antibody TMV-2 is secreted and produced by the hybridoma cell line TMV-2 according to claim 1.

[0010] Application of the ChiVMV-TMV virus detection antibody in the preparation of ChiVMV and / or TMV virus detection products.

[0011] Furthermore, the ChiVMV and / or TMV virus detection product is a ChiVMV-TMV dual virus colloidal gold detection test strip.

[0012] A ChiVMV-TMV dual virus colloidal gold detection test strip comprises a base plate, and a sample pad, a gold label pad, a NC membrane, and a water-absorbing filter paper fixed to the base plate in a partially overlapping manner from top to bottom along the extension direction of the base plate, wherein the gold label pad is coated with the colloidal gold-labeled ChiVMV-a antibody and TMV-1 antibody according to claim 2, and the NC membrane is provided with a T1 line, a T2 line, and a C line, wherein the T1 line is coated with the ChiVMV-b antibody according to claim 2, the T2 line is coated with the TMV-2 antibody according to claim 2, and the C line is coated with a goat anti-mouse IgG secondary antibody.

[0013] The preparation method of the ChiVMV-TMV dual virus colloidal gold detection test strip comprises the following steps:

[0014] (a) Preparation of NC membrane: T1 and T2 lines were drawn using ChiVMV-b and TMV-2 antibodies, and C line was drawn using goat anti-mouse IgG secondary antibody. The membrane was then dried to obtain the NC membrane.

[0015] (b) preparing a gold-labeled pad: ChiVMV-a antibody and TMV-1 antibody were mixed with colloidal gold solution, respectively, and the mixture was purified by centrifugation after pH adjustment and BSA blocking to obtain a colloidal gold-labeled antibody solution. The colloidal gold-labeled antibody solution was then sprayed onto a gold-labeled pad test strip to obtain a gold-labeled pad;

[0016] (c) Assembling the test strip: The sample pad, gold label pad, NC membrane, and absorbent filter paper are fixed to the bottom plate in sequence with partial overlap, and the ends of the gold label pad and the NC membrane overlap by 1-2 mm.

[0017] Furthermore, the process of the colloidal gold solution in step (b) is: reacting the chloroauric acid solution with the trisodium citrate solution under boiling conditions to form colloidal gold particles with a particle size of 40 nm.

[0018] Furthermore, the concentration of the colloidal gold-labeled antibody solution obtained in step (b) is 0.4 mg / mL.

[0019] The ChiVMV-TMV dual virus colloidal gold detection test strip is used as a detection test strip for TMV and / or ChiVMV virus.

[0020] The method for simultaneously detecting ChiVMV and TMV viruses in tobacco samples using the ChiVMV-TMV dual virus colloidal gold test strip comprises the following steps:

[0021] (i) adding the sample extract to be tested to the sample pad, and allowing the liquid to flow along the test strip;

[0022] (ii) Observe the test results after 8-10 minutes:

[0023] -If the C line is colored and both the T1 and T2 lines are colored, then both ChiVMV and TMV are considered positive;

[0024] -If the C line is colored and only the T1 line is colored, it is considered ChiVMV positive and TMV negative;

[0025] -If the C line is colored and only the T2 line is colored, then the test is considered TMV positive and ChiVMV negative;

[0026] - If only the C line is colored, both ChiVMV and TMV are considered negative;

[0027] - If the C line does not show color, the test is considered invalid.

[0028] Compared with the existing technology, the present invention solves the bottleneck problems of low efficiency, high cost and complex operation of traditional detection methods through innovative antibody combination, process optimization and structural design, and provides an efficient, economical and easy-to-promote solution for plant virus diagnosis. It has significant practical value and application prospects. The specific effects are as follows:

[0029] 1. Efficient simultaneous detection: The test strip prepared by the present invention is based on the principle of double-antibody sandwich immunochromatography. Through the combination of specific antibodies (ChiVMV-a / ChiVMV-b and TMV-1 / TMV-2) and dual detection lines (T1, T2), it can simultaneously identify ChiVMV and TMV virus infections in a single test, avoiding the cumbersome step-by-step testing process required by traditional methods, significantly improving detection efficiency, and is particularly suitable for rapid initial screening of large quantities of samples in the field.

[0030] 2. Rapid and sensitive: The test strip prepared by the present invention takes only 8-10 minutes to detect, which is much shorter than the time requirements of ELISA (4-8 hours) and PCR technology. In addition, the test strip has a detection sensitivity of up to 1 ppm for viral particles, which can effectively detect low-concentration infected samples and meet the needs of early disease diagnosis.

[0031] 3. Simple operation and low cost: No complex instruments or specialized operators are required; testing can be completed by simply adding 100μL of sample extract to the test strip, significantly reducing equipment dependence and technical barriers. Simultaneously, the test strip preparation process is simplified (e.g., batch spraying of colloidal gold-labeled antibodies and optimized NC membrane coating), significantly reducing the cost of a single test, making it suitable for grassroots promotion.

[0032] 4. High specificity and anti-interference ability: The present invention uses specific monoclonal antibodies secreted by hybridoma cell lines with deposit numbers CCTCCNO.C202563 / C202564 and CCTCCNO.C2024407 / C2024408. Through antibody pairing design (the gold label pad and the NC membrane detection line antibody belong to different epitopes), the cross-reaction between ChiVMV and TMV is completely avoided, and it has strong anti-interference ability against impurities in tobacco samples (such as plant proteins and pigments).

[0033] 5. Intuitive and reliable result interpretation: The test strip's validity is verified by the color development of the control line (C line), while the presence of the test lines (T1 and T2) is directly reflected in the presence of the virus. Interpretation of the results requires no specialized expertise, thus avoiding human error. Experimental data demonstrate that the test strips provided by this invention provide consistent results with theoretical expectations for ChiVMV / TMV single or mixed infection samples, demonstrating excellent reproducibility.

[0034] 6. Wide applicability: The test strips can be adapted to sample extracts from tobacco leaves, peppers and other Solanaceae crops, and the detection conditions (room temperature, pH 7.4 PBS buffer system) are highly universal, suitable for on-site rapid detection in different environments, providing timely data support for disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the structure of the dual virus colloidal gold test strip prepared in Example 2, wherein A is a main view, B is a top view, and C is a schematic diagram. 1 is a sample pad; 2 is a marking pad; 3 is an NC membrane (result observation area); 4 is an absorbent pad; 5 is a control area (line C); 6 is a detection area (lines T1 and T2); and 7 is a bottom plate.

[0036] Figure 2 Graph showing the detection results of the ChiVMV / TMV dual virus colloidal gold test strip in Example 3 for blank sample buffer, ChiVMV / TMV virus particle mixed solution, ChiVMV virus particle solution, and TMV virus particle solution.

[0037] Figure 3 This is a graph showing the test results of the ChiVMV / TMV dual virus colloidal gold test strip in Example 3 for ChiVMV and TMV double-negative tobacco sample extracts, ChiVMV and TMV double-positive tobacco sample extracts, ChiVMV-positive sample extracts, and TMV-positive sample extracts. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below in conjunction with specific embodiments, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than limiting the present invention.

[0039] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0040] The reagents, antibodies, and instruments involved in the embodiments of the present invention are shown in Table 1:

[0041] Table 1

[0042]

[0043]

[0044]

[0045] Example 1

[0046] In this example, ChiVMV and TMV monoclonal antibodies were prepared using secretion from four cell lines. The specific experimental steps are as follows:

[0047] 1. Cell recovery: ChiVMV monoclonal antibody hybridoma cell lines (hybridoma cell line ChiVMV-a, hybridoma cell line ChiVMV-b) with deposit numbers CCTCC NO. C202563 and CCTCC NO. C202564 and TMV monoclonal antibody hybridoma cell lines (hybridoma cell line TMV-1, hybridoma cell line TMV-2) with deposit numbers CCTCC NO. C2024407 and CCTCC NO. C2024408 were cultured in cell culture flasks for subsequent preparation of ChiVMV antibody a, ChiVMV antibody b, TMV antibody 1, and TMV antibody 2, respectively.

[0048] 2. Ascites Preparation and Purification

[0049] (1) Preparation of ascites: One week in advance, mineral oil was injected into the peritoneal cavity of mice. A certain number of resuscitated cells (hybridoma cell line ChiVMV-a, hybridoma cell line ChiVMV-b, hybridoma cell line TMV-1, hybridoma cell line TMV-2) were injected into the peritoneal cavity of mice. The ascites were collected about 10 days later and centrifuged at 4000 rpm. The supernatant was the monoclonal antibody ascites.

[0050] (2) Monoclonal antibody purification: Ascites was centrifuged for 15 min (4000 rpm, room temperature), the supernatant was collected, saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4); saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, stirring was continued for 30 min, centrifuged for 30 min (13000 rpm, 4°C), and the supernatant was discarded; the precipitate was dissolved in an appropriate amount of PBS (0.01 M, pH 7.4), dialyzed at 4°C overnight, the antibody content was determined, and the precipitate was frozen at -20°C for later use. After ammonium sulfate precipitation, continue purification using a Protein G column. First, pass the new column with 5 mL of ultrapure water, and then equilibrate the purification column with 5 mL of 0.4 M PB buffer (pH 7.0); the antibody is passed through the column slowly during the process to ensure better binding of the antibody protein to the binding site; continue to equilibrate the purification column with 10 mL of 0.4 M PB buffer (pH 7.0); elute the antibody from the binding site with 5 mL of 0.1 M glycine-HCl buffer (pH 2.7), and add 1 M Tris-HCl (pH 8.0) to neutralize the glycine to keep the pH neutral, which is suitable for antibody storage.

[0051] 3. Potency testing

[0052] The purified antibodies were tested for titer using the indirect ELISA method, and those with qualified titers (titer not less than 1:10^5) were used for subsequent assays.

[0053] Example 2

[0054] In this example, ChiVMV antibody a, ChiVMV antibody b, TMV antibody 1, and TMV antibody 2 prepared in Example 1 were used to prepare TMV dual virus colloidal gold test strips. The preparation steps were as follows:

[0055] 1. Preparation of nitrocellulose membrane containing test lines T1, T2 and quality control line C

[0056] 1) Preparation of coating antibodies

[0057] ChiVMV antibody b was diluted to 1 mg / mL with PBS buffer solution with a concentration of 10 mM and a pH value of 7.4 to obtain ChiVMV-b antibody solution, which was used as the coating antibody for the detection line T1.

[0058] TMV antibody 2 was diluted to 1 mg / mL with a PBS buffer solution having a concentration of 10 mM and a pH value of 7.4 to obtain a TMV antibody 1 solution, which was used as the coating antibody for the detection line T2.

[0059] The goat anti-mouse IgG secondary antibody (purchased from Hangzhou Longji) was diluted to 1 mg / mL with a PBS buffer solution with a concentration of 10 mM and a pH value of 7.4 and used as the coating antibody for line C.

[0060] 2) Coating

[0061] A PALL170 nitrocellulose membrane (NC membrane) was selected and a gold-sprayed membrane marker was used to draw a T1 line at 1.0 μL / cm with a ChiVMV-b antibody solution at a concentration of 1.0 mg / mL as the detection line; a TMV-2 antibody solution at a concentration of 1.0 mg / mL was drawn at 1.0 μL / cm with a T2 line as another detection line; a goat anti-mouse IgG secondary antibody solution at a concentration of 1 mg / mL was drawn at 1.0 μL / cm with a C line as the quality control line; the membrane was dried at 37°C for 24 hours and set aside for use; the antibody-coated NC membrane was obtained for later use.

[0062] 2. Preparation of gold label pad for immobilization of specific monoclonal antibodies labeled with colloidal gold

[0063] 1) Preparation of colloidal gold

[0064] a. Preparation: Clean a 500mL beaker, a 20mL small beaker, a rotor, a brown bottle, and a glass rod, then place them in an acid tank (potassium dichromate: concentrated sulfuric acid: ultrapure water = 120g:200mL:1000mL) and soak for 24 hours. Remove and rinse with tap water 3-4 times, then with ultrapure water 3-4 times, and dry in a 37°C oven until ready to use.

[0065] b. Preparation of Solution A: Use a plastic measuring spoon to weigh 1 g of chloroauric acid powder (purchased from Sigma) into a brown bottle, add 99 mL of ultrapure water to fully dissolve, and store at 4°C in the dark.

[0066] c. Preparation of Solution B: Weigh 1 g of trisodium citrate (purchased from Sigma) and dissolve it in 99 mL of ultrapure water and mix well.

[0067] d. Preparation of gold: Measure 99 mL of ultrapure water into a beaker, add 1 mL of burnt gold solution A, place on a constant temperature magnetic stirrer and stir to mix, turn on the heat until the solution boils, quickly add 2 mL of freshly prepared burnt gold solution B, continue stirring and heating, the solution gradually turns blue-black, then purple-black, and then turns red when heated. Continue boiling to turn a transparent orange-red color, continue boiling for 10 minutes, cool naturally to room temperature, add ultrapure water to 100 mL. Pour into a brown bottle and store at 4°C in the dark to obtain a colloidal gold solution (wherein the colloidal gold particle size is 40 nm and the concentration is 1 / 10,000).

[0068] 2) Antibody labeling

[0069] a. Labeling: Take 1.5 mL of the colloidal gold solution prepared in step 1), adjust the pH with 0.1 M K2CO3, add 20 μg of ChiVMV-a antibody and 20 μg of TMV-1 antibody, and react at room temperature for 40 minutes. Stop the reaction by adding 10% BSA to each solution and let it stand for 30 minutes.

[0070] c. Antibody purification: The above-mentioned static product was first centrifuged at low speed (1500 r / min), the precipitate formed by the agglomerated gold colloid particles was discarded, and the supernatant was collected; then the mixture was centrifuged at high speed (8500 r / min) for 30 minutes, the supernatant was carefully aspirated, the precipitate was collected, and the precipitate was re-dissolved in 50 μL of 0.1 M PBS (pH 7.4) containing 1% (mass percentage) BSA and stored at 4°C; a colloidal gold-labeled ChiVMV-a solution (concentration of 0.4 mg / mL) and a colloidal gold-labeled TMV-1 antibody solution (concentration of 0.4 mg / mL) were obtained.

[0071] 3) Gold spraying and film scratching

[0072] The colloidal gold labeled ChiVMV antibody 2 solution (0.4 mg / mL) and the colloidal gold labeled TMV antibody 2 solution (0.4 mg / mL) prepared in 2) above were sprayed onto the pretreated gold label pad test strip at 1.0 μL / cm, dried and set aside to obtain a gold label pad immobilized with colloidal gold-labeled specific monoclonal antibodies.

[0073] 3. Assemble the test strip: partially overlap the sample pad 1, gold label pad 2, NC membrane 3 and absorbent filter paper 4 in sequence and fix them on the bottom plate 7, wherein the gold label pad 2 overlaps the end of the NC membrane by 1-2 mm. The obtained ChiVMV / TMV dual virus colloidal gold test strip has the following structure: Figure 1 As shown, it includes a bottom plate 7, a sample pad 1, a gold label pad 2, an NC membrane 3 and a water-absorbing filter paper 4 according to Figure 1 The sequence from left to right in A partially overlaps the bottom plate 7, Figure 1 As can be seen, the NC membrane 3 is tightly attached to the base plate 7, the gold label pad 2 and the absorbent filter paper 4 partially overlap at each end of the NC membrane 3, and the sample pad 1 partially overlaps the end of the gold label pad 2 away from the NC membrane 3. The NC membrane 3 is provided with a control area marked by line C and a detection area marked by two detection lines T1 and T2.

[0074] Example 3

[0075] This example tests the ChiVMV / TMV dual virus colloidal gold test strip prepared in Example 2 for detection effectiveness. The test process and results are as follows:

[0076] (1) The test strip was inserted into 100 μL of blank sample solution (pH 7.4, 0.1 mol / L PBS) and the color development was observed after 8 minutes of reaction at room temperature. Figure 2 ), the control line C showed obvious wine red, and the T1 and T2 lines showed no color.

[0077] (2) The test strip was inserted into 100 μL of 1 ppm ChiVMV / TMV virus particle mixture solution (sampled with the same PBS solution as above, the ChiVMV / TMV virus concentration was 1 ppm), and the same operation steps as the blank sample were followed. The results showed that ( Figure 2 ), the control line C and the detection line T1 in the observation hole are colored, and the detection line T2 is also colored.

[0078] (3) The test strip was inserted into 100 μL of 100 ppm and 1 ppm ChiVMV virus particle solution (sampled and diluted with the same PBS solution as above) and the same operation steps as the blank sample were followed. The results showed that ( Figure 2 ), the control line C and the detection line T1 in the observation hole are colored, while the detection line T2 is not colored.

[0079] (4) The test strip was inserted into 100 μL of 10ppm, 1ppm, and 0.5ppm TMV virus particle solutions (sampled and diluted with the same PBS solution as above) and the same operation steps as the blank sample were followed. The results showed that ( Figure 2 ), the control line C and the detection line T1 in the observation hole do not show color, the detection line T2 shows color, and the color changes in a gradient.

[0080] (5) The test strip was inserted into 100 μL of ChiVMV positive sample extract and the same operation steps as the blank sample were followed. The results showed that ( Figure 3 ), the control line C and the detection line T1 in the observation hole are colored, the detection line T2 is not colored, and the color changes in a gradient.

[0081] (6) The test strip was inserted into 100 μL of TMV positive sample extract and the same operation steps as the blank sample were followed. The results showed that ( Figure 3 ), the control line C and the detection line T1 in the observation hole do not show color, the detection line T2 shows color, and the color changes in a gradient.

[0082] The above experimental results show that the ChiVMV / TMV dual virus colloidal gold detection test strip prepared in Example 2 can detect ChiVMV with a virus concentration of 1 ppm and TMV virus with a virus concentration of 0.5 ppm, with high detection accuracy, and can simultaneously detect the ChiVMV / TMV virus particle mixed solution. When the ChiVMV / TMV virus concentration is 1 ppm, the sample can also be clearly detected. This shows that the ChiVMV / TMV dual virus colloidal gold detection test strip prepared in Example 2 completely avoids the cross-reaction of ChiVMV and TMV, and has strong anti-interference properties against impurity components (such as plant proteins and pigments) in tobacco samples.

[0083] The above embodiment is only one of the preferred implementation methods of the present invention and should not be used to limit the scope of protection of the present invention. Any changes or modifications that have no substantive meaning made to the main design concept and spirit of the present invention, as long as the technical problems solved are still consistent with the present invention, should be included in the scope of protection of the present invention.

Claims

1. Antibody-secreting cell line for ChiVMV-TMV virus detection, characterized in that: Including hybridoma cell line ChiVMV-a, hybridoma cell line ChiVMV-b, hybridoma cell line TMV-1, hybridoma cell line TMV-2, among which the deposit number of hybridoma cell line ChiVMV-a is CCTCCNO.C202563, the deposit time is March 11, 2025, and the deposit unit is China Center for Type Culture Collection; the deposit number of hybridoma cell line ChiVMV-b is CCTCCNO.C202564, the deposit time is March 11, 2025, and the deposit unit is China Center for Type Culture Collection; The deposit time is March 11, 2025, and the depository is China Center for Type Culture Collection; the deposit number of hybridoma cell line TMV-1 is CCTCCNO.C2024407, and the deposit time is December 25, 2024, and the depository is China Center for Type Culture Collection; the deposit number of hybridoma cell line TMV-2 is CCTCCNO.C2024408, and the deposit time is December 25, 2024, and the depository is China Center for Type Culture Collection.

2. Antibodies for detecting ChiVMV-TMV viruses, including specific monoclonal antibodies ChiVMV-a, ChiVMV-b, TMV-1, and TMV-2, characterized in that: The specific monoclonal antibody ChiVMV-a is secreted and produced by the hybridoma cell line ChiVMV-a according to claim 1; the specific monoclonal antibody ChiVMV-b is secreted and produced by the hybridoma cell line ChiVMV-b according to claim 1; the specific monoclonal antibody TMV-1 is secreted and produced by the hybridoma cell line TMV-1 according to claim 1; and the specific monoclonal antibody TMV-2 is secreted and produced by the hybridoma cell line TMV-2 according to claim 1.

3. Use of the ChiVMV-TMV virus detection antibody according to claim 2 in the preparation of ChiVMV and / or TMV virus detection products.

4. The use according to claim 3, characterized in that The ChiVMV and / or TMV virus detection product is a ChiVMV-TMV dual virus colloidal gold detection test strip.

5. A ChiVMV-TMV dual virus colloidal gold test strip, characterized in that: The invention comprises a base plate, and a sample pad, a gold label pad, a NC membrane and a water-absorbing filter paper fixed on the base plate in sequence and partially overlapped from top to bottom along the extension direction of the base plate, wherein the gold label pad is coated with the colloidal gold-labeled ChiVMV-a antibody and TMV-1 antibody according to claim 2, and the NC membrane is provided with a T1 line, a T2 line and a C line, wherein the T1 line is coated with the ChiVMV-b antibody according to claim 2, the T2 line is coated with the TMV-2 antibody according to claim 2, and the C line is coated with a goat anti-mouse IgG secondary antibody.

6. The method for preparing the ChiVMV-TMV dual virus colloidal gold detection test strip according to claim 5, wherein: The steps include: (a) Preparation of NC membrane: T1 and T2 lines were drawn using ChiVMV-b and TMV-2 antibodies, and C line was drawn using goat anti-mouse IgG secondary antibody. The membrane was then dried to obtain the NC membrane. (b) preparing a gold-labeled pad: ChiVMV-a antibody and TMV-1 antibody were mixed with colloidal gold solution, respectively, and the mixture was purified by centrifugation after pH adjustment and BSA blocking to obtain a colloidal gold-labeled antibody solution. The colloidal gold-labeled antibody solution was then sprayed onto a gold-labeled pad test strip to obtain a gold-labeled pad; (c) Assembling the test strip: The sample pad, gold label pad, NC membrane, and absorbent filter paper are fixed to the bottom plate in sequence with partial overlap, and the ends of the gold label pad and the NC membrane overlap by 1-2 mm.

7. The preparation method according to claim 6, wherein The process of the colloidal gold solution in step (b) is: reacting the chloroauric acid solution with the trisodium citrate solution under boiling conditions to form colloidal gold particles with a particle size of 40 nm.

8. The preparation method according to claim 7, wherein The concentration of the colloidal gold-labeled antibody solution obtained in step (b) is 0.4 mg / mL.

9. Use of the ChiVMV-TMV dual virus colloidal gold detection test strip as claimed in claim 5 as a detection test strip for TMV and / or ChiVMV virus.

10. A method for simultaneously detecting ChiVMV and TMV viruses in tobacco samples using the ChiVMV-TMV dual virus colloidal gold test strip according to claim 5, characterized in that: The following steps are involved: (i) adding the sample extract to be tested to the sample pad, and allowing the liquid to flow along the test strip; (ii) Observe the test results after 8-10 minutes: -If the C line is colored and both the T1 and T2 lines are colored, then both ChiVMV and TMV are considered positive; -If the C line is colored and only the T1 line is colored, it is considered ChiVMV positive and TMV negative; -If the C line is colored and only the T2 line is colored, then the test is considered TMV positive and ChiVMV negative; - If only the C line is colored, both ChiVMV and TMV are considered negative; - If the C line does not show color, the test is considered invalid.

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