Potato virus X nano-mimic enzyme test strip and its application

Abstract

The present invention discloses a potato virus X nano-mimetic enzyme test strip and its application. The test strip comprises a backing plate and a sample pad, a conjugation pad, an NC membrane and a blotting paper sequentially attached to the backing plate; the sample pad is pressed against the conjugation pad and is conjugated with a nano-enzyme-labeled monoclonal antibody I; a quality control line and a detection line are provided on the NC membrane; the detection line is conjugated with a monoclonal antibody II paired with the monoclonal antibody I; the specificity is improved by selecting a unidirectional paired antibody; the test results show that the prepared test strip reacts only with PVX-infected plants and does not react with other viruses or healthy plants; the sensitivity test results show that the PVX nano-mimetic enzyme test strip has a sensitivity of 1:10 3 The virus can still be detected after dilution (w / v, g / mL) without false positives, so it can be used for potato virus X detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, in particular to a potato virus X nano-mimic enzyme test strip and also to the application of the test strip. Background Art

[0002] Potatoes, as a vegetative crop, are susceptible to viruses. Repeated plantings of potatoes will pass the virus through generations of tubers, leading to increasing disease incidence and a rapid decline in yield. In the field, potato viruses can infect healthy potatoes through sap abrasion and aphid transmission, resulting in reduced yields and degradation. Once infected, potatoes remain infected for life, with the disease becoming increasingly severe with each successive planting generation, ultimately rendering them unsuitable for cultivation.

[0003] Viral diseases have long plagued researchers. Currently, over 40 virus species, including a viroid, have been found in potato crops. Among them, PVX, a member of the genus Potexvirus, also known as potato common mosaic virus or potato mild mosaic virus, has a wide host range, primarily infecting Solanaceae crops. As one of the major pests affecting seed potato quality, PVX is a key indicator for determining seed potato quality.

[0004] When PVX infects a plant alone, symptoms may be mild, such as mosaic or latent leaves, sometimes with slightly wrinkled or wavy leaf margins, resulting in a small yield reduction. However, combined infection with PVY in field crops often exacerbates symptoms, significantly reducing potato yields. In severe cases, yield reductions can reach 80%, leading to significant economic losses.

[0005] Therefore, it is necessary to establish a simple, rapid, sensitive, economical and accurate potato virus disease detection technology to meet the needs of field quality testing services for potato seed potatoes and provide technical support for the comprehensive promotion of potato seed potato quality testing and certification work. Summary of the Invention

[0006] In view of this, one object of the present invention is to provide a potato virus X nano-mimetic enzyme test strip; a second object of the present invention is to provide a use of the potato virus X nano-mimetic enzyme test strip in detecting potato virus X.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A potato virus X nano-mimetic enzyme test strip, comprising a backing plate and a sample pad, a conjugation pad, a NC membrane, and absorbent paper sequentially attached to the backing plate; the sample pad and conjugation pad are conjugated to a nanoenzyme-labeled monoclonal antibody I; the NC membrane is provided with a quality control line and a test line; the test line is conjugated to a monoclonal antibody II paired with the monoclonal antibody I;

[0009] The monoclonal antibody I is PVX-6, and the PVX-6 includes a heavy chain and a light chain, and the heavy chain amino acid sequence is shown in SEQ ID NO.1; the light chain amino acid sequence is shown in SEQ ID NO.3; the monoclonal antibody II is PVX-2, and the PVX-2 includes a heavy chain and a light chain, and the heavy chain amino acid sequence is shown in SEQ ID NO.5; the light chain amino acid sequence is shown in SEQ ID NO.7.

[0010] Preferably, the monoclonal antibody I is secreted by the hybridoma cell with a deposit number of CCTCC NO: C2022280; and the monoclonal antibody II is secreted by the hybridoma cell with a deposit number of CCTCC NO: C2022270.

[0011] Preferably, the conjugate pad is prepared by ultrasonically treating the nanozyme-labeled monoclonal antibody I and spraying it onto the conjugate pad at a spraying speed of 5 μL / cm, followed by drying.

[0012] Preferably, the ultrasound is performed at a power of 53 Khz for 5 to 10 times, each time for 10 to 20 seconds.

[0013] Preferably, the quality control line is combined with goat anti-mouse IgG.

[0014] Preferably, the nanozyme-labeled monoclonal antibody I is prepared by the following method: the carboxyl group of the washed nanozyme is activated with NHS and EDC, and then the monoclonal antibody I is added to couple the antibody with the nanozyme to obtain the nanozyme-labeled monoclonal antibody I.

[0015] Preferably, the specific conditions for activating the carboxyl group are as follows: the cleaned nanozyme is mixed with an NHS solution and an EDC solution in a volume ratio of 1:1 to make the final concentration of the nanozyme 0.5 mg / mL; the concentration of the NHS solution is 10 mg / mL, and the concentration of the EDC solution is 10 mg / mL; after ultrasonic mixing, the reaction is carried out for 30 to 40 minutes, and the nanozyme with activated carboxyl group is obtained by washing.

[0016] Preferably, the coupling is to add the activated carboxyl nanozyme to the antibody at a concentration of 0.1 mg / mL to make the final concentration of the activated carboxyl nanozyme 1 mg / mL, and then react at 2~8°C for 14~18 hours after ultrasonic mixing. After the reaction, it is collected under the action of a magnetic force and finally stored in Tris-buffer with pH 7.4 and 50 mM.

[0017] Preferably, the quality control line on the NC membrane is obtained by streaking a 1 mg / mL goat anti-mouse IgG solution at a streaking speed of 1 μL / cm, washing, and drying; the detection line on the NC membrane is obtained by diluting monoclonal antibody II to a concentration of 1.5 mg / mL, then streaking at a streaking speed of 1 μL / cm, washing, and drying.

[0018] 2. Application of the potato virus X nano-mimic enzyme test strip in detecting potato virus X.

[0019] The present invention provides a beneficial effect: a potato virus X nano-mimetic enzyme test strip, utilizing the paired action of potato virus X monoclonal antibodies PVX-2 and PVX-6, specifically reacts with the CP protein (PVX-CP) of PVX and PVX virus particles, while not reacting with PVY virus particles or healthy plants. Further DAS-ELISA testing revealed that when PVX-2 was the coated antibody, it could successfully pair with other antibodies, such as PVX-1, PVX-3, and PVX-6. Sensitivity testing demonstrated that the PVX monoclonal antibody had a sensitivity of up to 1:10240 (w / v, g / mL), making it suitable for use as a test kit for detecting potato virus X antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0021] Figure 1 This is the assembly diagram of the nano-mimetic enzyme test strip;

[0022] Figure 2 Interpretation of results for monovalent nano-mimetic enzyme test strips;

[0023] Figure 3 Screening results for PVX nanozyme test strips;

[0024] Figure 4 It is a specific detection method for PVX nano-mimetic enzyme test strips;

[0025] Figure 5 This is the sensitivity test of PVX nano-mimetic enzyme test strips.

[0026] Biological Deposits:

[0027] Two hybridoma cell lines secreting monoclonal antibodies were deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and named 3D6D9B5 and 4C10F4F5 respectively; the deposit date of 3D6D9B5 was September 1, 2022, the deposit number was CCTCC NO: C2022279, and the classification name was hybridoma cell line 3D6D9B5; the deposit date of 4C10F4F5 was September 1, 2022, the deposit number was CCTCC NO: C2022280, and the classification name was hybridoma cell line 4C10F4F5. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0029] Mice were immunized with the viral CP protein as an antigen. Using hybridoma technology, SP2 / 0 cells were fused with spleen cells or lymph node cells from immunized BALB / c mice. Stable, high-titer hybridoma cell lines expressing anti-potato virus X protein were screened for ascites production, and monoclonal antibody production was performed. Hybridomas that stably produced PVX monoclonal antibodies were then screened for successful fusion. Successful hybridomas were selected and subjected to two cell subcloning experiments, with only microscopically detected monoclonal and secondary clones selected for testing. Positive monoclonal hybridoma cell lines were then expanded for culture, harvested for ascites production, and cryopreserved, as shown in Table 1.

[0030] Table 1. Six positive monoclonal hybridoma cell lines were screened out through subcloning

[0031] Hybridoma cell number Antibody No. Antibody type Antibody subclass Chain 1F3B2A10 PVX-1 MAbs IgG1 Kappa 3D6D9B5 PVX-2 MAbs IgG1 Kappa 4F9G10A7 PVX-3 MAbs IgG1 Kappa 2G5F11D4 PVX-4 MAbs IgG1 Kappa 4A2E5B4 PVX-5 MAbs IgG1 Kappa 4C10F4F5 PVX-6 MAbs IgG1 Kappa

[0032] Example 1: Preparation of PVX Nano-Mimetic Enzyme Test Strips

[0033] A. PVX monoclonal antibody nanomimetic enzyme labeling screening

[0034] (1) Take the required amount of nanozyme solution, add purified water to a concentration of 0.5 mg / mL, and sonicate for 1-2 min (53 kHz);

[0035] (2) Centrifuge at 13,000 rpm at room temperature for 5–10 min.

[0036] (3) Remove the supernatant and add purified water to a concentration of 0.5 mg / mL. Resuspend and sonicate for 1–2 min.

[0037] (4) Centrifuge at 13,000 rpm at room temperature for 5–10 min and remove the supernatant.

[0038] (5) Weigh 10 times the mass of the nanozyme N-hydroxysuccinimide (NHS), add MES solution (50 mM, pH 6.0) and mix well to prepare a 10 mg / mL NHS solution; weigh 10 times the mass of the nanozyme 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), add MES solution (50 mM, pH 6.0) and mix well to prepare a 10 mg / mL EDC solution. Keep away from light;

[0039] (6) Pipette equal volumes of the two solutions prepared in step (5) and add them to the washed nanozyme in step (4) to a concentration of 0.5 mg / mL. Resuspend and mix thoroughly. Ultrasonicate for 30-60 s, 3-5 times in total, and react on a mixer at room temperature for 30-40 min (keep away from light).

[0040] (7) Centrifuge at 13,000 rpm at room temperature for 5–10 min and remove the supernatant;

[0041] (8) Add MES solution (50 mM, pH 6.0) to a concentration of 0.5 mg / mL, shake to mix, and then sonicate for 1-2 minutes; centrifuge at 13,000 rpm at room temperature for 5-10 minutes, and remove the supernatant;

[0042] (9) Take 100 μg of PVX monoclonal antibody and add it to MES solution (50 mM, pH 8.0) and mix well to prepare an antibody solution with a concentration of 0.1 mg / mL;

[0043] (10) Pipette the antibody prepared in step (9) and add it to the nanozyme washed in step (8) to prepare a nanozyme solution with a concentration of 1 mg / mL. Resuspend and mix, sonicate for 10-20 seconds, sonicate 5-10 times in total, and react on a mixer at 2-8°C for 14-18 hours.

[0044] (11) Place the reaction solution on a magnetic rack, draw the clarified liquid into a new centrifuge tube, and use an ultra-micro protein detector to detect the antibody labeling efficiency of the clarified liquid. Add Tris-buffer (50 mM, pH 7.4) to the centrifuge tube to prepare a 0.5 mg / mL nanozyme solution, resuspend and mix, sonicate for 10-20 s, 5-10 times, and react at room temperature for 30-40 min.

[0045] (12) Place the reaction solution on a magnetic rack, remove the supernatant, add 5% BSA-PBS solution to prepare a 1 mg / mL nanozyme solution, resuspend, and ultrasonicate for 10-20 s, 5-10 times. The temperature of the ultrasonic cleaning instrument should be controlled at 2-8 °C. After resuspension, place it on a 2-8 °C mixer and seal it for 2-4 h.

[0046] (13) Magnetic adsorption, discard the supernatant, add 1% BSA-PBS treatment solution to prepare 1 mg / mL nanozyme solution, resuspend, and ultrasonicate for 10-20 s, 5-10 times. The temperature of the ultrasonic cleaning instrument should be controlled at 2-8°C. After resuspension, store at 2-8°C and label for later use.

[0047] Pretreatment of conjugate pads, sample pads, and absorbent pads

[0048] Conjugate pad pretreatment:

[0049] (1) Cut the glass fiber membrane into bonding pads with a width of 7 mm using an instrument;

[0050] (2) Prepare the conjugate pad pretreatment solution (1% Triton X-100, 50 mM sodium borate, pH 8.0);

[0051] (3) Take an appropriate amount of cut conjugate pad and place it in the conjugate pad pretreatment box. Use a pipette to absorb the conjugate pad pretreatment solution and drop it on the conjugate pad to soak it completely. Soak for 25-35 minutes.

[0052] (4) Use tweezers to clamp the soaked bonding pads onto a drying net, arrange them neatly, and dry them in an oven at 40°C for 2 h until completely dry.

[0053] (5) Place the dried bonding pad in a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier for later use.

[0054] Sample pad pretreatment:

[0055] (1) Cut the glass fiber membrane into sample pads with a width of 11 mm using an instrument;

[0056] (2) Prepare sample pad pretreatment solution (10 mM PBS, 1% Tween 20, 0.1 g / L PVP K30, pH 7.4);

[0057] (3) Take an appropriate amount of cut sample pad and place it in the sample pad pretreatment box. Use a pipette to absorb the sample pad pretreatment liquid droplets and add them to the sample pad to soak it completely. Soak for 25-35 minutes.

[0058] (4) Use tweezers to clamp the soaked sample pads onto the drying net, arrange them neatly, and dry them in an oven at 40°C for 2.5 h until they are completely dry.

[0059] (5) Place the dried sample pad in a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier for later use.

[0060] Absorbent Pad Treatment:

[0061] (1) Cut the absorbent paper into absorbent pads with a width of 22 mm using an instrument;

[0062] (2) Place the cut absorbent pads on a drying net, arrange them neatly, and dry them in an oven at 40°C for 2.5 hours;

[0063] (3) Place the dried absorbent pad in a sealed bag, add an appropriate amount of desiccant, write a label, and store it in a dehumidifier for later use.

[0064] C. NC film scribing operation

[0065] (1) Take a certain amount of PVX antibody, add coating buffer solution, and dilute to a coating antibody solution with a concentration of 1.5 mg / mL;

[0066] (2) Take a certain amount of goat anti-mouse IgG, add coating buffer solution, and dilute to a goat anti-mouse IgG solution with a concentration of 1 mg / mL;

[0067] (3) Turn on the streak sprayer and perform the cleaning procedure. After cleaning, place the catheter of pump 1 in the diluted PVX (test line) antibody solution, and place the catheter of pump 3 in the diluted goat anti-mouse IgG (quality control line) solution;

[0068] (4) Place the PVC backing plate with the NC film on the correct position of the marking sprayer and execute the marking procedure at a marking speed of 1 μL / cm;

[0069] (5) After marking, perform the cleaning procedure and turn off the machine. Mark the PVC backing board and place it in an oven to dry for 1 hour at 37°C.

[0070] D. Bonding pad gold spraying operation

[0071] (1) Take the nanozyme labeled with PVX antibody, put it into a centrifuge tube, adsorb it with a magnetic stand, discard the solution, add a certain amount of nanozyme labeled antibody diluent (50 mM / L Tris, 10% trehalose (w / v), 5% BSA (w / v), 1% Triton X-100 (w / v), 1% Tween 20 (w / v), 0.05% proclin (w / v), 1% PVP K30 (w / v), pH 8.5), and dilute it to a nanozyme concentration of 1 mg / mL;

[0072] (2) Place the diluted nanozyme-labeled antibody solution in an ultrasonic instrument and sonicate 5 to 10 times, each time for 10 to 20 seconds;

[0073] (3) Turn on the line sprayer and perform the cleaning procedure. After cleaning, place the catheter of pump 2 into the nanozyme-labeled antibody solution after ultrasound.

[0074] (4) Place the pretreated conjugate pad in the correct position on the line sprayer and perform the gold spraying procedure at a spraying speed of 5 μL / cm;

[0075] (5) After the gold spraying process is completed, perform the cleaning process and shut down the machine. After marking the bonding pad, place it in an oven and dry it for 1 hour at 37°C.

[0076] E. PVX nano-mimetic enzyme test strip assembly

[0077] (1) Turn on the laminating machine, place the PVC board close to the working plate of the laminating machine, press the start suction to fix the PVC board, and try to place the PVC board in the middle position to prevent it from being in an active state when the release paper is opened;

[0078] (2) Assemble the test strips according to the selected combination;

[0079] (3) Press the start button and place the dried NC membrane, conjugate pad, absorbent paper, and pre-treated sample pad in the same manner as ( Figure 1 ) were assembled as shown, with absorbent paper pressing 2 mm against the NC membrane, the binding pad pressing 2 mm against the NC membrane, and the sample pad pressing 2 mm against the binding pad, and then attached to the backing plate;

[0080] (4) After the plate attaching process is completed, turn off the plate attaching machine. Place the reagent strip plate in a dark environment with a humidity of ≤30% for storage;

[0081] (5) Place the pasted PVC board on the cutting machine, turn on the power and switch, lift the turning frame, adjust the position of the storage plate, clamp the finished large board, align the left end of the finished large board with the blade, and lower the turning frame;

[0082] (6) Set the test strip cutting width to 0.4 cm and click Start cutting;

[0083] (7) Place the cut test strips into the reagent cartridge, turn on the power of the automatic cartridge press, place the cartridge-loaded test strips on the cartridge press conveyor, click the "Run" button, and the machine will run until all reagent cartridges are cartridge-pressed, then turn off the cartridge press;

[0084] (8) Place the test strips into a heat-sealed bag containing desiccant and heat-seal it;

[0085] (9) After heat sealing, mark the test strips and store them in a dehumidifier with a humidity not exceeding 30%.

[0086] Interpretation of test strip results: When using a monovalent nano-mimetic enzyme test strip to detect PVX, if the sample to be tested contains PVX, a brown band will appear at the test line (T line) and the control line (C line) of the nano-mimetic enzyme test strip, indicating a positive test result; if the sample to be tested does not contain PVX, no brown band will appear at the T line, but a brown band will appear at the C line, indicating a negative test result; if a brown band only appears at the T line after the sample is added, but no brown band appears at the C line, or no brown band appears at both the T line and the C line, it indicates an invalid result ( Figure 2 ).

[0087] Example 2: Performance testing of PVX nano-mimetic enzyme test strips

[0088] A. PVX nano-simulated enzyme test strips can be used for screening

[0089] (1) Grind the PVX-infected tissue culture seedlings into powder using liquid nitrogen, add extraction buffer, vortex mix, and centrifuge at room temperature at 4000 rpm for 2 min;

[0090] (2) Add the positive control and blank control to the assembled test strips respectively;

[0091] (3) Add the sample dropwise to the sample wells, 80 μL / well, and time at room temperature for 15 min;

[0092] (4) Observe the test strips to see if there are any false positives, and record the test strip numbers if there are no false positives.

[0093] The antibodies used to prepare the nano-mimetic enzyme test strips should be unidirectional paired antibodies and cannot be mutually paired antibodies. Therefore, PVX-5 and PVX-1, PVX-2 and PVX-3, PVX-2 and PVX-6, and PVX-5 and PVX-3 were selected as paired antibodies, and the nano-mimetic enzyme test strips prepared with these pairs of antibodies were screened. Among them, the PVX-1 and PVX-5 paired antibodies, regardless of whether PVX-5 or PVX-1 was used as the labeled antibody, and PVX-1 or PVX-5 was used as the coated antibody, when the negative sample was added to the nano-mimetic enzyme test strips, although no false positives occurred and only the quality control line (C line) appeared, after the positive sample was added, the detection line (T line) did not appear, and the positive result could not be detected; When the nano-mimetic enzyme test strip prepared with PVX-3 as the coating antibody, PVX-3 or PVX-2 as the binding antibody, PVX-3 as the coating antibody, and PVX-6 as the detection antibody was added to the positive sample, the C line and the T line appeared at the same time, and a positive result was detected. However, after the negative control was added, the C line and the T line appeared at the same time, indicating a false positive. When the nano-mimetic enzyme test strip prepared with PVX-6 as the binding antibody and PVX-2 as the coating antibody was added to the positive sample, the C line and the T line appeared at the same time, indicating a positive result. After the negative control was added, only the C line appeared without the T line, indicating a negative result ( Figure 3 ).

[0094] Finally, PVX-2 was selected as the coating antibody and PVX-6 as the labeling antibody to prepare the nano-mimetic enzyme test strips, and their specificity and sensitivity were tested.

[0095] The hybridoma cells 3D6D9B5 and 4C10F4F5 that produce PVX-2 and PVX-6 antibodies were biologically deposited; the deposit date of 3D6D9B5 was September 1, 2022, the deposit number was CCTCC NO: C2022279, and the classification name was hybridoma cell line 3D6D9B5; the deposit date of 4C10F4F5 was September 1, 2022, the deposit number was CCTCC NO: C2022280, and the classification name was hybridoma cell line 4C10F4F5.

[0096] The antibodies produced by 3D6D9B5 and 4C10F4F5 were named PVX-2 and PVX-6, respectively. PVX-2 and PVX-6 were sequenced. The heavy chain amino acid sequence of PVX-6 is shown in SEQ ID NO. 1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO. 2. The light chain amino acid sequence of PVX-6 is shown in SEQ ID NO. 3, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO. 4. The heavy chain amino acid sequence of PVX-2 is shown in SEQ ID NO. 5, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO. 6. The light chain amino acid sequence of PVX-2 is shown in SEQ ID NO. 7, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO. 8.

[0097] B. PVX nano-simulated enzyme test strip specific detection

[0098] In order to detect the specificity of the prepared PVX nano-mimetic enzyme test strips, the prepared test strips were taken out for use, and the plants of PVA, PVM, PVS, PVY, PLRV and PVX and the healthy plants (H) were ground into powder with liquid nitrogen, and the extraction buffer was added at a ratio of 1:10 (w / v, g / mL) and vortexed to mix. After centrifugation at 4000 rpm for 2 min at room temperature, the supernatant was added dropwise to the sample well and left for 15 min. The results were observed. Figure 4 shown.

[0099] The results showed that when the juice of PVA, PVM, PVS, PVY, PLRV and healthy plants (H) was dripped into the test strip, only the C line appeared on the test strip, which was a negative result, indicating that the test strip did not have an immune reaction with PVA, PVM, PVS, PVY, PLRV and healthy plants. When the juice of PVX plants was dripped into the test strip, both the C line and the T line appeared, which was a positive result, indicating that the test strip had an immune reaction with PVX and had good specificity.

[0100] C. PVX nano-simulated enzyme test strip sensitivity detection

[0101] In order to detect the sensitivity of the prepared PVX nano-mimetic enzyme test strips, the prepared test strips were taken out for use, the infected PVX plants were ground into powder with liquid nitrogen, and the extraction buffer was added at a ratio of 1:10 (w / v, g / mL), vortexed and mixed, and centrifuged at 4000 rpm for 2 min at room temperature. The supernatant was then diluted with water at a ratio of 1:10. 1 ~1:10 5 (w / v, g / mL) for dilution, mix well and drop the sample into the sample well. Leave at room temperature for 15 min and observe the results. Figure 5 shown.

[0102] The results showed that PVX nano-simulated enzyme test strips could be used at 1:10 and 1:10 2 , 1:10 3 (w / v, g / mL) After dilution, C line and T line appear at the same time, the test result is positive, and PVX can be detected. When the dilution ratio is 1:10 4 , 1:10 5 After dilution (w / v, g / mL), only the C line appears and no T line appears. The test result is negative and PVX cannot be detected.

[0103] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Potato virus X nano-mimic enzyme test strip, characterized by: The test strip includes a backing plate and a sample pad, a conjugation pad, an NC membrane and absorbent paper sequentially attached to the backing plate; the sample pad presses the conjugation pad, the conjugation pad is conjugated to a nanozyme-labeled monoclonal antibody I, a quality control line and a detection line are provided on the NC membrane, and the detection line is conjugated to a monoclonal antibody II paired with the monoclonal antibody I; the monoclonal antibody I is PVX-6, the PVX-6 includes a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO.1; the light chain amino acid sequence is shown in SEQ ID NO.3; the monoclonal antibody II is PVX-2, the PVX-2 includes a heavy chain and a light chain, the heavy chain amino acid sequence is shown in SEQ ID NO.5; the light chain amino acid sequence is shown in SEQ ID NO.7; the monoclonal antibody I is secreted by the hybridoma cell line 4C10F4F5 with a deposit number of CCTCC NO: C2022280; the monoclonal antibody II is secreted by the hybridoma cell line 4C10F4F5 with a deposit number of CCTCC NO: C2022280 NO: Secretion of hybridoma cell line 3D6D9B5 of C2022279.

2. The potato virus X nano-mimic enzyme test strip according to claim 1, characterized in that: The preparation method of the conjugate pad is as follows: after ultrasonic treatment, the nanozyme-labeled monoclonal antibody I is sprayed onto the conjugate pad at a spraying speed of 5 μL / cm, and dried.

3. The potato virus X nano-mimic enzyme test strip according to claim 2, characterized in that: The ultrasound is performed at a power of 53 Khz for 5 to 10 times, each time for 10 to 20 seconds.

4. The potato virus X nanomimetic enzyme test strip according to claim 1, characterized in that: The quality control line is combined with goat anti-mouse IgG.

5. The potato virus X nanomimetic enzyme test strip according to claim 1, characterized in that: The nanozyme-labeled monoclonal antibody I is prepared by the following method: the carboxyl group of the washed nanozyme is activated with NHS and EDC, and then the monoclonal antibody I is added to couple the antibody with the nanozyme to obtain the nanozyme-labeled monoclonal antibody I.

6. The potato virus X nanomimetic enzyme test strip according to claim 5, characterized in that: The specific conditions for activating the carboxyl group are as follows: the cleaned nanozyme is mixed with an NHS solution and an EDC solution in a volume ratio of 1:1 to obtain a final concentration of the nanozyme of 0.5 mg / mL; the concentration of the NHS solution is 10 mg / mL, and the concentration of the EDC solution is 10 mg / mL; after ultrasonic mixing, the reaction is carried out for 30 to 40 minutes, and the nanozyme with activated carboxyl group is obtained by washing.

7. The potato virus X nano-mimic enzyme test strip according to claim 5, characterized in that: The coupling is to add the activated carboxyl nanozyme to the antibody with a concentration of 0.1 mg / mL to make the final concentration of the activated carboxyl nanozyme 1 mg / mL, and then react at 2~8°C for 14~18 hours after ultrasonic mixing. After the reaction, it is collected under the action of magnetism and finally stored in pH 7.4, 50 mM Tris-buffer.

8. The potato virus X nano-mimic enzyme test strip according to claim 5, characterized in that: The quality control line on the NC membrane was obtained by streaking with 1 mg / mL goat anti-mouse IgG solution at a streaking speed of 1 μL / cm, washing, and drying; The test line was obtained by diluting monoclonal antibody II to a concentration of 1.5 mg / mL, then streaking at a speed of 1 μL / cm, washing, and drying.

9. Use of the potato virus X nanomimetic enzyme test strip according to any one of claims 1 to 8 in detecting potato virus X.

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