CN116183912A - Potato X virus nanometer mimic enzyme test strip and application thereof - Google Patents
Potato X virus nanometer mimic enzyme test strip and application thereof Download PDFInfo
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Abstract
The invention discloses a potato X virus nanometer simulated enzyme test strip and application thereof, wherein the test strip comprises a backing plate, and a sample pad, a bonding pad, an NC film and absorbent paper which are sequentially attached to the backing plate; the sample pad pressure combining pad is combined with a nano enzyme marked monoclonal antibody I, the NC film is provided with a quality control line and a detection line, and the detection line is combined with a monoclonal antibody II paired with the monoclonal antibody I; the specificity is improved by selecting a unidirectional pairing antibody, and the test strip prepared by detection shows that the test strip only reacts with PVX infected plants and does not react with other viruses and healthy plants; the sensitivity detection result shows that the PVX nanometer mimic enzyme test strip is 1:10 3 The virus can still be detected after dilution (w/v, g/mL), and no false positive appears, so that the virus can be used for potato X virus detection.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a potato X virus nanometer mimic enzyme test strip and application of the test strip.
Background
Potato (Solanum tuberosum l.) originates from the andes mountain in south america, the solanaceae, annual herbs, asexual reproduction, tuber-grain crops, one of the most important high-yield crops worldwide, next to wheat, rice and maize. China is the first country of potato production, the potato planting area and the total yield are all the first world, but the single yield level is far lower than that of developed countries such as Europe and America.
In China, after the potato staple food strategy is implemented in 2015, the potato strategic position is further improved, and the quality and the popularity of the detoxified seed potato are important guarantees for the industrial healthy development. The potato is used as a vegetative propagation crop, and after the potato is infected with the virus, the virus is transmitted through tuber generation due to annual planting, so that the incidence rate is increased year by year, and the yield is rapidly reduced. In the field, potato viruses can enable non-toxic potato viruses to infect in a mode of juice friction, aphid virus transmission and the like, so that yield reduction and degradation of potatoes are caused. The potato will take poison for life after being infected by virus, the disease condition aggravates year by year along with the planting algebra, and finally the planting value is lost.
For a long time, virus diseases have plagued scientific workers, and more than 40 virus types are currently found on potato crops, and one virus type is also included. PVX is a member of Potexvirus, also called potato common mosaic virus or potato light mosaic virus, has a wide host range, mainly infects solanaceae crops, and is one of key indexes for judging whether the quality of potato seeds is qualified or not as one of main pests affecting the quality of potato seeds.
After PVX single infects plants, the symptoms of the plants are slightly floral or hidden, and sometimes the leaves are slightly shrunken and the edges of the leaves are wavy, so that a small amount of yield reduction can be caused. However, in field planting, compound infection often occurs, and when the compound infection is carried out on potatoes with PVY, symptoms are aggravated, so that the yield of the potatoes is greatly reduced, and 80% of yield reduction can be caused when the compound infection is serious, so that serious economic loss is caused.
Therefore, a simple, quick, sensitive, economic and accurate potato virus disease detection technology is established, the requirements of potato seed field quality detection service are met, and technical support is provided for comprehensive promotion of potato seed quality detection and authentication work.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide a potato virus X nano-mimic enzyme test strip; the second purpose of the invention is to provide the application of the potato virus X nanometer mimic enzyme test strip in detecting potato virus X.
In order to achieve the above purpose, the present invention provides the following technical solutions:
1. the potato X virus nanometer simulated enzyme test strip comprises a backing plate, and a sample pad, a bonding pad, an NC film and absorbent paper which are sequentially attached to the backing plate; the sample pad pressure combining pad is combined with a nano enzyme marked monoclonal antibody I, the NC film is provided with a quality control line and a detection line, and the detection line is combined with a monoclonal antibody II paired with the monoclonal antibody I;
the monoclonal antibody I is PVX-6, the PVX-6 comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1; the light chain amino acid sequence is shown as SEQ ID NO. 3; the monoclonal antibody II is PVX-2, the PVX-2 comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 5; the light chain amino acid sequence is shown as SEQ ID NO. 7.
Preferably, the monoclonal antibody I consists of a polypeptide with a preservation number of CCTCC NO: hybridoma cell secretion of C2022279; the monoclonal antibody II is prepared from the following components with the preservation number of CCTCC NO: hybridoma cell secretion of C2022280.
Preferably, the preparation method of the sample pad pressure comprises the following steps: the nano enzyme marked monoclonal antibody I is sprayed onto a bonding pad by using a spraying speed of 5 mu L/cm after ultrasonic treatment, and is dried.
Preferably, the ultrasonic wave is ultrasonic wave for 5-10 times under the power of 53Khz, and each time is 10-20 seconds.
Preferably, the quality control line is combined with goat anti-mouse IgG.
Preferably, the nano enzyme-labeled monoclonal antibody I is prepared by the following method: and (3) activating carboxyl groups of the cleaned nano enzyme by using NHS and EDC, and then adding the monoclonal antibody I to couple the antibody with the nano enzyme to obtain the nano enzyme marked monoclonal antibody I.
Preferably, the specific conditions for activating the carboxyl group are as follows: mixing the washed nano-enzyme with NHS solution and EDC solution in a volume ratio of 1:1 to ensure that the final concentration of the nano-enzyme is 0.5mg/mL; the concentration of the NHS solution is 10mg/mL, and the concentration of the EDC solution is 10mg/mL; and (3) carrying out ultrasonic mixing, reacting for 30-40 min, and cleaning to obtain the nano-enzyme for activating carboxyl.
Preferably, the coupling is to add the nano-enzyme with activated carboxyl into the antibody with the concentration of 0.1mg/mL to ensure that the final concentration of the nano-enzyme with activated carboxyl is 1mg/mL, uniformly mix the nano-enzyme with ultrasound, react for 14 to 18 hours at the temperature of 2 to 8 ℃, collect the nano-enzyme with activated carboxyl under the action of magnetic force after the reaction, and finally store the nano-enzyme with activated carboxyl by using Tris-buffer with the pH of 7.4 and 50 mM.
Preferably, the quality control line on the NC film is obtained by scribing a sheep anti-mouse IgG solution with the concentration of 1mg/mL according to the scribing speed of 1 mu L/cm, cleaning and drying; the detection line on NC membrane is diluted by monoclonal antibody II to a concentration of 1.5mg/mL, then streaked at a streaking speed of 1 mu L/cm, washed and dried.
2. The potato virus X nano-mimic enzyme test strip is applied to detection of potato virus X.
The invention has the beneficial effects that: the invention discloses a potato virus X nanometer mimic enzyme test strip, which can specifically react with PVX CP protein (PVX-CP) and PVX virus particles by the pairing action of potato virus X monoclonal antibodies PVX-2 and PVX-6, and does not react with PVY virus particles or healthy plants. Further detecting pairing condition by DAS-ELISA method, and if PVX-2 is coated antibody, it can be successfully paired with other antibodies, such as PVX-1, PVX-3 and PVX-6; the sensitivity detection result shows that PVX monoclonal antibodies can be diluted by 1:10240 (w/v, g/mL) times, and can be used as a potato X virus antibody detection kit.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a schematic diagram of the assembly of a nano-scale simulated enzyme test strip;
FIG. 2 shows the interpretation results of a monovalent nano-mimic enzyme test strip;
FIG. 3 shows the PVX nanoenzyme test strip screening results;
FIG. 4 shows PVX nano-mimic enzyme test strip specific detection;
FIG. 5 shows the sensitivity detection of PVX nano-mimic enzyme test strips.
Biological preservation:
2 hybridoma cell lines secreting monoclonal antibodies are sent to China center for preservation, and the preservation addresses are 3D6D9B5 and 4C10F4F5 respectively; the 3D6D9B5 preservation date is 2022, 9 months and 1 day, and the preservation number is CCTCC NO: c2022279, designated as hybridoma cell line 3D6D9B5; the preservation date of 4C10F4F5 is 2022, 9 and 1, and the preservation number is CCTCC NO: c2022280, classified as hybridoma cell line 4C10F4F5.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
The virus CP protein is used as antigen to immunize mice, SP2/0 is fused with spleen cells or lymph node cells of immunized BALB/c mice by using a hybridoma technology, hybridoma cell lines which are high in stability and titer and resistant to potato X virus protein are screened, monoclonal antibody ascites is prepared, hybridoma cells which can stably produce PVX monoclonal antibodies are screened, hybridoma cells which are successfully fused are selected, and only microscopic monoclonal and monoclonal antibodies are selected for detection by a 2-time cell subcloning test. Through detection, the obtained positive monoclonal hybridoma cell strain is subjected to cell expansion culture, the cells are taken for ascites production, and the cells are frozen, and the specific details are shown in table 1.
TABLE 1 subcloning 6 Positive monoclonal hybridoma cell lines
Example 1 preparation of PVX nanomimetic enzyme test strip
A. PVX monoclonal antibody nanometer mimic enzyme mark screening
(1) Adding purified water into the nano enzyme solution with the required dosage to prepare the concentration of 0.5mg/mL, and performing ultrasonic treatment for 1-2 min (53 kHz);
(2) 13000rpm, centrifuging at room temperature for 5-10 min;
(3) Sucking the supernatant, adding purified water to prepare the solution with the concentration of 0.5mg/mL, and carrying out ultrasonic treatment for 1-2 min after resuspension;
(4) 13000rpm, centrifuging at room temperature for 5-10 min, and sucking the supernatant;
(5) Weighing N-hydroxysuccinimide (NHS) with the mass of 10 times of nano enzyme, adding MES solution (50 mM, pH 6.0), and uniformly mixing to prepare 10mg/mL NHS solution; weighing 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) with the mass of 10 times of nano enzyme, adding MES solution (50 mM, pH 6.0), mixing uniformly, preparing 10mg/mL EDC solution, and taking care of light shielding;
(6) Adding the two solutions prepared in the step (5) into the nano-enzyme washed in the step (4) by equal volume, preparing into the concentration of 0.5mg/mL, re-suspending uniformly, performing ultrasonic treatment for 30-60 s, performing ultrasonic treatment for 3-5 times, and performing room temperature reaction for 30-40 min (taking care of light shielding) on a mixer;
(7) 13000rpm, centrifuging at room temperature for 5-10 min, and sucking the supernatant;
(8) Adding MES solution (50 mM, pH 6.0) to prepare the solution with the concentration of 0.5mg/mL, shaking and mixing uniformly, and then carrying out ultrasonic treatment for 1-2 min;13000rpm, centrifuging at room temperature for 5-10 min, and sucking the supernatant;
(9) 100. Mu.g of PVX monoclonal antibody is added into MES solution (50 mM, pH 8.0) and mixed evenly to prepare antibody solution with concentration of 0.1 mg/mL;
(10) Absorbing the antibody prepared in the step (9), adding the antibody into the nano-enzyme washed in the step (8), preparing a nano-enzyme solution with the concentration of 1mg/mL, re-suspending the solution uniformly, performing ultrasonic treatment for 10-20 s, performing ultrasonic treatment for 5-10 times, and performing reaction on the solution on a mixer at the temperature of 2-8 ℃ for 14-18 h;
(11) And (3) placing the reacted solution on a magnetic rack, sucking the clarified liquid into a new centrifuge tube, and detecting the antibody marking efficiency of the clarified liquid by using an ultramicro protein detector. Adding Tris-buffer (50 mM, pH 7.4) into a centrifuge tube, preparing a nano enzyme solution of 0.5mg/mL, re-suspending uniformly, performing ultrasonic treatment for 10-20 s, performing reaction for 5-10 times at room temperature for 30-40 min;
(12) Placing the reacted solution on a magnetic frame, sucking the supernatant, adding 5% BSA-PBS solution, preparing 1mg/mL nano enzyme solution, re-suspending, performing ultrasonic treatment for 10-20 s, 5-10 times, and controlling the temperature of an ultrasonic cleaner at 2-8 ℃. After re-suspending, placing the mixture on a mixer with the temperature of 2-8 ℃ for sealing for 2-4 hours;
(13) Magnetically adsorbing, discarding supernatant, adding 1% BSA-PBS treatment solution, preparing 1mg/mL nano enzyme solution, re-suspending, performing ultrasonic treatment for 10-20 s, 5-10 times, and controlling the temperature of an ultrasonic cleaner at 2-8 ℃; and (5) preserving at 2-8 ℃ after re-suspending, and sticking a label for later use.
B. Conjugate pad, sample pad and absorbent pad pretreatment
Pretreatment of the bonding pad:
(1) Cutting the glass fiber film into a bonding pad with the width of 7mm by an instrument;
(2) Preparing a binding pad pretreatment solution (1% Triton X-100, 50mM sodium borate, pH 8.0);
(3) Placing a proper amount of cut bonding pad in a bonding pad pretreatment box, sucking the bonding pad pretreatment liquid by a pipetting gun, dripping the bonding pad pretreatment liquid on the bonding pad, soaking the bonding pad in the bonding pad pretreatment box for 25-35 min;
(4) Clamping the soaked bonding pad on a drying net by using tweezers, neatly arranging, and drying in a drying oven at 40 ℃ for 2 hours until the bonding pad is completely dried;
(5) And (3) placing the dried combination pad into a sealing bag, placing a proper amount of drying agent, writing a label, and storing in a dehumidification cabinet for standby.
Sample pad pretreatment:
(1) Cutting the glass fiber film into sample pads with the width of 11mm by an instrument;
(2) Preparing sample pad pretreatment liquid (10mM PBS,1%Tween 20,0.1g/L PVP K30, pH 7.4);
(3) Placing a proper amount of cut sample pad into a sample pad pretreatment box, sucking sample pad pretreatment liquid drops by a liquid-transferring gun, adding the sample pad pretreatment liquid drops on the sample pad, soaking the sample pad in the sample pad pretreatment liquid drops for 25-35 min;
(4) Clamping the soaked sample pad on a drying net by using tweezers, neatly arranging, and drying in a drying oven at 40 ℃ for 2.5 hours until the sample pad is completely dried;
(5) And (3) placing the dried sample pad into a sealing bag, placing a proper amount of drying agent, writing a label, and storing in a dehumidification cabinet for standby.
And (3) water absorption pad treatment:
(1) Cutting the absorbent paper into absorbent pads with the width of 22mm by an instrument;
(2) Placing the cut water absorption pad on a drying net, arranging the water absorption pad in order, and drying the water absorption pad in a drying oven at 40 ℃ for 2.5 hours;
(3) And (3) placing the dried water absorption pad into a sealing bag, placing a proper amount of drying agent, writing a label, and storing in a dehumidification cabinet for standby.
C. NC film scribing operation
(1) Taking a certain amount of PVX antibody, adding a coating buffer solution, and diluting to a coating antibody solution with the concentration of 1.5 mg/mL;
(2) Taking a certain amount of goat anti-mouse IgG, adding a coating buffer solution, and diluting to a concentration of 1mg/mL;
(3) Opening a scribing film spraying instrument, executing a cleaning program, putting a guide pipe of a pump 1 into diluted PVX (detection line) antibody solution after cleaning, and putting a guide pipe of a pump 3 into diluted goat anti-mouse IgG (quality control line) solution;
(4) Placing the PVC backing plate with the NC film on the correct position of a scribing film spraying instrument, and executing a scribing program according to a scribing speed of 1 mu L/cm;
(5) After scribing, a cleaning procedure is carried out and the machine is turned off, a PVC backing plate is marked, and the PVC backing plate is put into an oven for drying for 1h at 37 ℃.
D. Bond pad metal spraying operation
(1) The nanometer enzyme marked with PVX antibody is put into a centrifuge tube, adsorbed by a magnetic frame, the solution is discarded, a certain amount of nanometer enzyme marked 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) and pH 8.5) is added, and the nanometer enzyme is diluted to the concentration of 1mg/mL;
(2) Placing the diluted nano enzyme marked antibody solution in an ultrasonic instrument, and performing ultrasonic treatment for 5-10 times for 10-20 seconds each time;
(3) Opening a scribing film spraying instrument, executing a cleaning program, and placing a catheter of the pump 2 into the ultrasonic nano enzyme marked antibody solution after cleaning;
(4) Placing the pretreated bonding pad at the correct position of a scribing and film spraying instrument, and executing a metal spraying procedure according to the film spraying speed of 5 mu L/cm;
(5) After the metal spraying procedure is completed, the cleaning procedure is executed and the machine is turned off, after the bonding pad is marked, the bonding pad is put into an oven for drying for 1h, and the temperature is 37 ℃.
E. PVX nanometer simulated enzyme test strip assembly
(1) Opening the plate pasting machine, pasting the PVC plate inwards to be tightly attached to the working plate position of the plate pasting machine, fixing the PVC plate according to starting suction, and placing the PVC plate in the middle position as far as possible so as to avoid being in a movable state when lifting the release paper;
(2) Assembling the test strip according to the selected combination;
(3) Pressing a start key, assembling the dried NC film, the bonding pad, the absorbent paper and the pretreated sample pad according to the specification shown in figure 1, pressing the NC film by the absorbent paper by 2mm, pressing the NC film by 2mm, pressing the bonding pad by 2mm, setting the bonding pad by the sample pad, and attaching the sample pad on a backing plate;
(4) After the paste plate procedure is finished, the paste plate machine is closed. The large reagent strip plate is placed in a light-shielding environment with humidity less than or equal to 30% and stored for standby;
(5) Placing the stuck PVC plate on a chopper, turning on a power supply and a switch, lifting the roll-over stand, adjusting the position of the object placing plate, clamping the finished product large plate, aligning the left end of the finished product large plate with the blade, and putting down the roll-over stand;
(6) Setting the chopping width of the test strip to be 0.4cm, and starting chopping by clicking;
(7) Loading the cut test paper strips into a reagent card shell, opening a power supply of an automatic shell pressing machine, placing the reagent paper strips loaded with the shells on a shell pressing machine conveyor belt, clicking an operation button, operating the machine until all reagent card shells are pressed, and closing the shell pressing machine;
(8) The test strip is put into a heat-sealing bag with a drying agent for heat sealing;
(9) After the heat sealing is finished, marking is carried out, and the test strip is stored in a dehumidification cabinet with humidity not higher than 30%.
Judging test strip results: when PVX is detected by using a monovalent nanometer simulated enzyme Test strip, when PVX is contained in a sample to be detected, brown strips appear at a detection line (T line) and a quality Control line (C line) of the nanometer simulated enzyme Test strip respectively, which indicates that the detection result is positive; when the sample to be detected does not contain PVX, no brown strip appears at the T line, and a brown strip appears at the C line, which indicates that the test strip is negative; if only the T line showed a brown band, but the C line did not show a brown band, or neither the T line nor the C line showed a brown band after the sample was added dropwise (FIG. 2).
Example 2, PVX nanoscopic enzyme test strip Performance detection
A. Screening by PVX nanometer simulated enzyme test paper
(1) Grinding PVX infected tissue culture seedlings into powder by liquid nitrogen, adding an extraction buffer solution, mixing uniformly by vortex, and centrifuging at room temperature for 4000rpm for 2min;
(2) Respectively dripping positive control and blank control according to the assembled test strip;
(3) Dropping the sample into the sample hole, 80 mu L/hole, and timing for 15min at room temperature;
(4) And observing the test strip to detect whether false positive occurs, and recording the serial number of the test strip without the false positive.
The antibodies for preparing the nanometer mimic enzyme test strip are unidirectional paired antibodies and cannot be mutual paired antibodies, so that the paired antibodies are PVX-5 and PVX-1, PVX-2 and PVX-3, PVX-2 and PVX-6 and PVX-5 and PVX-3 respectively, the nanometer mimic enzyme test strips prepared by the pairs of antibodies are screened, wherein the PVX-1 and PVX-5 paired antibodies, whether PVX-5 or PVX-1 is used as a labeled antibody, and the nanometer mimic enzyme test strip prepared by using PVX-1 or PVX-5 as a coated antibody is dripped with a negative sample, and only a quality control line (C line) is shown although false positive does not appear, but a detection line (T line) does not appear after a positive sample is added, and a positive result cannot be detected; after a nanometer mimic enzyme test strip prepared by taking PVX-6 as a coating antibody, PVX-3 or PVX-2 as a binding antibody and PVX-3 as a coating antibody and PVX-6 as a detection antibody is added into a positive sample, the C line and the T line are simultaneously present, a positive result can be detected, but after a negative control is added, the C line and the T line are simultaneously present, and false positive is shown; only PVX-6 is used as a binding antibody, the C line and the T line are simultaneously appeared after a positive sample is added into a nanometer mimic enzyme test strip prepared by PVX-2 as a coating antibody, the result is positive, only the C line is not the T line after a negative control is added, and the result is negative (figure 3).
Finally, PVX-2 is selected as a coating antibody, PVX-6 is selected as a labeling antibody to prepare a nanometer mimic enzyme test strip, and specificity and sensitivity are detected.
Biological preservation of hybridoma cells 3D6D9B5 and 4C10F4F5 producing PVX-2 and PVX-6 antibodies; the 3D6D9B5 preservation date is 2022, 9 months and 1 day, and the preservation number is CCTCC NO: c2022279, designated as hybridoma cell line 3D6D9B5; the preservation date of 4C10F4F5 is 2022, 9 and 1, and the preservation number is CCTCC NO: c2022280, classified as hybridoma cell line 4C10F4F5.
Antibodies produced by 3D6D9B5 and 4C10F4F5 are named PVX-2 and PVX-6 respectively, PVX-2 and PVX-6 are sequenced, the heavy chain amino acid sequence of PVX-6 is shown as SEQ ID NO.1, the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO.2, the light chain amino acid sequence of PVX-6 is shown as SEQ ID NO.3, and the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO. 4. The heavy chain amino acid sequence of PVX-2 is shown as SEQ ID NO.5, the nucleotide sequence for encoding the amino acid is shown as SEQ ID NO.6, the light chain amino acid sequence of PVX-2 is shown as SEQ ID NO.7, and the nucleotide sequence for encoding the amino acid is shown as SEQ ID N O.8.
B. PVX nanometer mimic enzyme test strip specificity detection
In order to detect the specificity of the prepared PVX nanometer simulated enzyme test strip, the test strip prepared in advance is taken out for standby, PVA, PVM, PVS, PVY, PLRV and PVX plants and healthy plants (H) are ground into powder by liquid nitrogen, extraction buffer solution is added according to 1:10 (w/v, g/mL) for vortex mixing, after centrifugation at 4000rpm for 2min at room temperature, supernatant is dripped into a sample hole, and the sample hole is placed for 15min, and the observation result is shown in figure 4.
The result shows that PVA, PVM, PVS, PVY, PLRV and healthy plant (H) sap drop into the test strip, the test strip only presents a C line, and is a negative result, which indicates that the test strip does not immunoreact with PVA, PVM, PVS, PVY, PLRV and healthy plants, PVX plant sap drop into the test strip, C line and T line both appear, and the test strip presents a positive result, which indicates that the test strip immunoreacts with PVX, and the test strip has better specificity.
C. PVX nanometer simulated enzyme test strip sensitivity detection
In order to detect the sensitivity of the prepared PVX nanometer simulated enzyme test strip, the test strip prepared in advance is taken out for standby, the infected PVX plant is ground into powder by liquid nitrogen, the extraction buffer solution is added according to 1:10 (w/v, g/mL), vortex mixing is carried out, after centrifugation at 4000rpm for 2min at room temperature, the supernatant is respectively added according to 1:10 1 ～1:10 5 (w/v, g/mL) was diluted, and after mixing, the sample was added dropwise to the sample well and left at room temperature for 15min, and the observation results were shown in FIG. 5.
The results show that PVX nanometer simulated enzyme test strips can be arranged in a ratio of 1:10 to 1:10 2 、1:10 3 After dilution (w/v, g/mL), C line and T line appear simultaneously, the detection result is positive, PVX can be detected, and when the dilution multiple is 1:10 4 、1:10 5 After dilution (w/v, g/mL), only line C appeared, no line T appeared, the detection result was negative, and PVX could not be detected.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. The potato X virus nanometer mimic enzyme test strip is characterized in that: the test strip comprises a backing plate, and a sample pad, a bonding pad, an NC film and absorbent paper which are sequentially attached to the backing plate; the sample pad pressure combining pad is combined with a nano enzyme marked monoclonal antibody I, the NC film is provided with a quality control line and a detection line, and the detection line is combined with a monoclonal antibody II paired with the monoclonal antibody I;
the monoclonal antibody I is PVX-6, the PVX-6 comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1; the light chain amino acid sequence is shown as SEQ ID NO. 3; the monoclonal antibody II is PVX-2, the PVX-2 comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 5; the light chain amino acid sequence is shown as SEQ ID NO. 7.
2. The potato virus X nano-mimetic enzyme test strip of claim 1, wherein: the monoclonal antibody I is prepared from the following components with the preservation number of CCTCC NO: hybridoma cell secretion of C2022279; the monoclonal antibody II is prepared from the following components with the preservation number of CCTCC NO: hybridoma cell secretion of C2022280.
3. The potato virus X nano-mimetic enzyme test strip of claim 1, wherein: the preparation method of the sample pad pressure comprises the following steps: the nano enzyme marked monoclonal antibody I is sprayed onto a bonding pad by using a spraying speed of 5 mu L/cm after ultrasonic treatment, and is dried.
4. The potato virus X nano-mimetic enzyme test strip of claim 3, wherein: the ultrasonic treatment is carried out for 5-10 times under the power of 53Khz for 10-20 seconds each time.
5. The potato virus X nano-mimetic enzyme test strip of claim 1, wherein: and the quality control line is combined with goat anti-mouse IgG.
6. The potato virus X nano-mimetic enzyme test strip of claim 1, wherein: the nano enzyme marked monoclonal antibody I is prepared by the following method: and (3) activating carboxyl groups of the cleaned nano enzyme by using NHS and EDC, and then adding the monoclonal antibody I to couple the antibody with the nano enzyme to obtain the nano enzyme marked monoclonal antibody I.
7. The potato virus X nano-mimetic enzyme test strip of claim 6, wherein: the specific conditions for activating the carboxyl group are as follows: mixing the washed nano-enzyme with NHS solution and EDC solution in a volume ratio of 1:1 to ensure that the final concentration of the nano-enzyme is 0.5mg/mL; the concentration of the NHS solution is 10mg/mL, and the concentration of the EDC solution is 10mg/mL; and (3) carrying out ultrasonic mixing, reacting for 30-40 min, and cleaning to obtain the nano-enzyme for activating carboxyl.
8. The potato virus X nano-mimetic enzyme test strip of claim 6, wherein: the coupling is to add nano-enzyme with activated carboxyl into an antibody with the concentration of 0.1mg/mL to ensure that the final concentration of the nano-enzyme with activated carboxyl is 1mg/mL, uniformly mix the nano-enzyme with ultrasonic waves, react for 14-18 h at the temperature of 2-8 ℃, collect the nano-enzyme with activated carboxyl under the action of magnetic force after the reaction, and finally store the nano-enzyme with the activated carboxyl by using Tris-buffer with the pH of 7.4 and 50 mM.
9. The potato virus X nano-mimetic enzyme test strip of claim 6, wherein: the quality control line on the NC film is obtained by scribing a sheep anti-mouse IgG solution with the concentration of 1mg/mL according to the scribing speed of 1 mu L/cm, cleaning and drying; the detection line is diluted by monoclonal antibody II to a concentration of 1.5mg/mL, then streaked at a streaking speed of 1 mu L/cm, washed and dried to obtain the antibody.
10. Use of the potexvirus nano-mimic enzyme test strip according to any one of claims 1 to 9 in the detection of potexvirus.
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| CN202211515129.7A CN116183912A (en) | 2022-11-30 | 2022-11-30 | Potato X virus nanometer mimic enzyme test strip and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116535500A (en) * | 2022-11-30 | 2023-08-04 | 西南大学 | Potato X virus monoclonal antibody PVX-6 and application thereof |
| CN116589567A (en) * | 2022-11-30 | 2023-08-15 | 西南大学 | Potato X virus monoclonal antibody PVX-2 and application thereof |
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2022
- 2022-11-30 CN CN202211515129.7A patent/CN116183912A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116535500A (en) * | 2022-11-30 | 2023-08-04 | 西南大学 | Potato X virus monoclonal antibody PVX-6 and application thereof |
| CN116589567A (en) * | 2022-11-30 | 2023-08-15 | 西南大学 | Potato X virus monoclonal antibody PVX-2 and application thereof |
| CN116589567B (en) * | 2022-11-30 | 2024-03-19 | 西南大学 | Potato X virus monoclonal antibody PVX-2 and application thereof |
| CN116535500B (en) * | 2022-11-30 | 2024-03-22 | 西南大学 | Potato X virus monoclonal antibody PVX-6 and application thereof |
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