Bursaphelenchus xylophilus disease detection test strip as well as preparation method and application thereof

Abstract

The invention discloses a pine wood nematode disease detection test strip as well as a preparation method and application thereof, and relates to the technical field of molecular biological detection. The invention discloses a preparation method of a pine wood nematode disease detection test strip. The preparation method comprises the following steps: preparation of a hapten, preparation of an immunogen and a coating antigen, preparation of a monoclonal antibody, preparation of a gold-labeled antibody and preparation of a colloidal gold test strip. The amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO. 1. Results of the embodiment of the invention show that the monoclonal antibody used for preparing the test strip is high in sensitivity; according to the test strip prepared from the colloidal gold labeled monoclonal antibody, the detection time of the pine wood nematode disease is shortened, the whole detection process is optimized, and a good technical support is provided for further promoting early detection of the pine wood nematode disease.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology detection, and more specifically, relates to a pine wood nematode disease detection test strip and a preparation method and application thereof. Background Art

[0002] After being infected by pine wood nematodes, the needles of pine trees lose their green color and turn yellow-brown or reddish-brown, some branches die, and the whole plant wilts. The disease has been studied for many years at home and abroad, and the main focus is on the exploration of scientific issues related to pathogens, such as pathogen types, pathogenic mechanisms, ecological characteristics of pathogenic nematodes, transmission vectors, disease diagnosis and prevention, etc. Due to the complexity of the occurrence and development of the disease, the pathogenic mechanism of the pathogen is not very clear, and there is still a lack of simple and effective prevention and control measures. Once the disease occurs, the entire pine forest will suffer devastating damage. Therefore, if the susceptible pine trees can be detected and diagnosed early when the pine trees do not show obvious symptoms of the disease, it is one of the key technical problems that need to be solved urgently in the current prevention and control of pine wood nematode disease.

[0003] In the early stage of the study, a set of automated molecular detection system for pine wood nematodes was developed by using specific fragments of pine wood nematodes and designing specific primer sequences based on the fragments. Pine wood nematodes were separated by drilling pine debris using the Baermann funnel method, nematode DNA was extracted, and the nematode DNA was tested on the machine. After PCR amplification, the test results were interpreted to determine whether pine wood nematodes were present in the pine tissues. This method requires the isolation of pine wood nematodes and the extraction of nematode DNA from suspected diseased pine trees, at which time the pine trees are relatively in the middle stage of infection, and the step of isolating the pine wood nematode body alone takes at least 3 to 6 hours. Therefore, if pine wood nematodes can be detected at an earlier stage of pine disease and the overall detection time can be shortened, the window period for the prevention and control of pine wood nematode disease can undoubtedly be further grasped.

[0004] Effectors are a type of exogenous protein secreted by pathogens in the early stages of infection, which can change the structure and metabolic pathways of host plant cells, thereby promoting successful infection of the host. Our team has previously identified a number of specific effector proteins that play a key role in the infection stage of pine wood nematodes. Specific effector proteins are highly conserved in pine wood nematodes and have low homology with other homologous species. Therefore, immunological methods can be used to detect effector proteins in diseased trees to determine whether pine wood nematodes exist in the tree.

[0005] The double antibody sandwich method is based on the high affinity between specific antibodies and target molecules and is the most commonly used method for detecting antigens. Antiserum is adsorbed into the small holes of the microtiter plate, and the antigen to be tested is added. If the two are specific, they will bind. Then, an enzyme-linked antibody that reacts specifically with the antigen to be tested is added, so that one unit of antigen binds to two units of antibody at the same time to form a "sandwich"; finally, the substrate of the enzyme is added, and the content of the antigen to be tested is judged according to the depth of the color of the colored product produced. Test strips prepared using this principle are widely used in medicine, environment, food and other fields. For example, pregnancy test strips use double antibody sandwich technology and colloidal gold as an indicator marker to confirm whether a woman is pregnant by detecting the glycoprotein (HCG, i.e. human chorionic gonadotropin) produced by pregnant women during pregnancy. However, this technology is rarely reported in the study of forest diseases. Summary of the invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for preparing a pine wood nematode disease detection test strip. Another technical problem to be solved by the present invention is to provide a pine wood nematode disease detection test strip for early rapid detection of pine wood nematode disease. Another technical problem to be solved by the present invention is to provide an application of a pine wood nematode disease detection test strip in early rapid detection of pine wood nematode disease.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a pine wood nematode disease detection test strip comprises the following steps:

[0009] Preparation of hapten: The pine wood nematode effector protein is hydrolyzed and added with acetic acid to prepare the hapten;

[0010] Preparation of immunogen and coating agent: the hapten is coupled to protein by activated ester method to prepare immunogen and coating agent respectively;

[0011] Preparation of monoclonal antibodies: using the immunogen to prepare monoclonal antibodies, using the coating source to detect the sensitivity and specificity of the monoclonal antibodies through ELISA, and obtaining monoclonal antibodies of pine wood nematode effector proteins;

[0012] Preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to obtain a gold-labeled antibody;

[0013] Preparation of colloidal gold test strips: Use the coating source of pine wood nematode effector protein to make the detection line on the nitrocellulose membrane, use the secondary antibody to make the quality control line on the nitrocellulose membrane, and then assemble it into a test strip.

[0014] The specific steps of preparing the hapten are:

[0015] The effector protein was dissolved in acetic acid and water, heated to reflux, and then an aqueous sodium hydroxide solution was added to reach a pH of 7.5, and pure water was added to dissolve the precipitate. The aqueous solution was washed with n-hexane, and the aqueous layer solution was acidified by separation to a pH of 6, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and passed through a silica gel column to obtain a white solid, which was the hapten.

[0016] The preparation of the immunogen and coating agent is specifically as follows:

[0017] Preparation of immunogen: bovine serum albumin (BSA) is fully dissolved in carbonate buffer (CBS) to obtain a BSA solution, and the activated hapten solution is added dropwise to the BSA solution under magnetic stirring, and the mixture is stirred to react to obtain an immunogen;

[0018] Preparation of coating source: fully dissolve the chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA solution, add the activated hapten solution dropwise into the OVA solution under magnetic stirring, stir the mixture to react, and obtain coating source.

[0019] The preparation of the gold-labeled antibody is specifically as follows: pure water is heated to boiling, a chloroauric acid solution is added under stirring, and then a trisodium citrate solution is added to obtain a colloidal gold solution; a boric acid buffer solution is added to the solution, and after being fully mixed, a monoclonal antibody solution is added dropwise to react, positive branch and negative branch samples are respectively ground, and the samples are respectively diluted with PBS buffer, and then 10% BSA is added dropwise to block to obtain a mixed solution; the mixed solution is centrifuged, the supernatant is discarded, a precipitate is obtained, and the precipitate is dissolved in a heavy suspension to obtain a gold-labeled antibody.

[0020] Pure water is heated to boiling, and a chloroauric acid solution is added under stirring, and then a trisodium citrate solution is added to obtain a colloidal gold solution; a boric acid buffer is added to the solution, and after being fully mixed, a monoclonal antibody solution is added dropwise to react, positive branch and negative branch samples are respectively ground, and the samples are respectively diluted with PBS buffer, and then 10% BSA is added dropwise to block to obtain a mixed solution; the mixed solution is centrifuged, and the supernatant is discarded to obtain a precipitate, and the precipitate is dissolved in a heavy suspension to obtain a gold-labeled antibody.

[0021] The preparation of the colloidal gold test strip is specifically as follows:

[0022] The sample pad is immersed in a PBS solution containing Tween-20, sucrose and BSA and then dried; the coating source is sprayed on the nitrocellulose membrane as a detection line, and the secondary antibody is sprayed on the nitrocellulose membrane as a quality control line; the sample pad, nitrocellulose membrane, absorbent pad and bottom plate are assembled to obtain a finished test strip.

[0023] A pine wood nematode disease detection test strip prepared by any of the preparation methods.

[0024] The test strip is used in detecting pine wood nematode disease.

[0025] The application described, the steps are as follows:

[0026] 1) Grind the pine stem tissue to be tested, dilute it 10 times with lysis buffer, and drop 1 mL of the diluted liquid into the test hole of the test strip; let the test strip stand at room temperature for 10 minutes, observe the color of the quality control line and the test line on the membrane, and judge the result;

[0027] 2) If the quality control line does not show color, replace the test strip and retest until the quality control line shows color; if both the quality control line and the test line show color, the test result of the sample to be tested is positive; if the quality control line shows color and the test line does not, concentrate the sample solution 10 times or prepare a concentrated sample again and test again. If the quality control line still shows color and the test line does not, it is judged as negative.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The present invention has developed a test strip for the pine wood nematode-specific effector protein Bx-FAR-1, and the monoclonal antibody used has high sensitivity and strong specificity, which is beneficial to avoiding false positives in the detection process.

[0030] 2) The present invention shortens the detection time of pine wood nematode disease by using a test strip prepared by using colloidal gold-labeled monoclonal antibodies, optimizes the overall detection process, and provides technical support for further promoting the early detection of pine wood nematode disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the immunopotency diagram of the pine wood nematode effector protein Bx-FAR-1;

[0032] Figure 2 Flow cytometry quantitative assessment diagram for cell bank construction;

[0033] Figure 3 This is a test strip for pine wood nematode effector protein detection (the left is a positive result, and the right is a negative result);

[0034] Figure 4 This is a commercial pine wood nematode effect protein test strip diagram;

[0035] Figure 5 This is a graph showing the results of testing pine samples using pine wood nematode effector protein test strips. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0037] The pine stem segments infected with pine wood nematodes and the pine stem segments not inoculated with pine wood nematodes used in the following examples were all stored in the Forest Pathology Laboratory of Nanjing Forestry University.

[0038] Example 1

[0039] 1. Purification and extraction of pine wood nematode effector protein Bx-FAR-1

[0040] Referring to the article previously published by our laboratory (DOI: 10.3969 / j.issn.2095-1191.2020.11.016), the main steps include: cloning of the target gene (Bx-FAR-1 gene of pine wood nematode (BXY_1685000.1)), construction and identification of the prokaryotic expression vector, protein expression and purification, and finally obtaining a high-purity Bx-FAR-1 protein (artificial antigen) that meets the use requirements.

[0041] 2. Pine wood nematode effector protein Bx-FAR-1 is concentrated in fluorescent markers

[0042] The protein solution obtained above is placed on a cellulose membrane, and the water is filtered out by high pressure. At this time, the protein remains on the membrane, and then it is placed in a high-pressure filter, and it is continuously stirred and filtered to make the liquid in the bag seep out, and the negative pressure is pumped to obtain highly concentrated protein. Then the GFP green fluorescent marker is coupled to the protein so that the protein carries a green marker to ensure the subsequent single B cell screening. The purity of the induced protein is detected by western blot.

[0043] The results are as follows Figure 1 As shown, the induced Bx-FAR-1 protein band is single and the width of the protein band is deepened. The results show that the Bx-FAR-1 protein after enrichment and concentration has high purity and concentration and can be used for the preparation of monoclonal antibodies.

[0044] Example 2

[0045] 1. Preparation of hapten

[0046] The effector protein was dissolved in acetic acid and water, heated to reflux, and then an aqueous sodium hydroxide solution was added to reach a pH of 7.5, and pure water was added to dissolve the precipitate. The aqueous solution was washed with n-hexane, and the aqueous layer solution was acidified by separation to a pH of 6, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and passed through a silica gel column to obtain a white solid, which was the hapten.

[0047] 2. Preparation of immunogen and coating

[0048] Preparation of immunogen: bovine serum albumin (BSA) is fully dissolved in carbonate buffer (CBS) to obtain a BSA solution, and the activated hapten solution is added dropwise to the BSA solution under magnetic stirring, and the mixture is stirred to react to obtain an immunogen;

[0049] Preparation of coating source: fully dissolve the chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA solution, add the activated hapten solution dropwise into the OVA solution under magnetic stirring, stir the mixture to react, and obtain coating source.

[0050] 3. Immunity titer of pine wood nematode effector protein Bx-FAR-1

[0051] The obtained pure protein was diluted with PBS at different times (10 times, 100 times, 1000 times and 10000 times), and the diluted Bx-FAR-1 protein solution was dripped on the solid phase carrier to form the coated protein; the coated protein was used as an antigen to perform abdominal immunization on mice (purchased from Sigma-Aldrich (Wuxi) Biochemical Technology Co., Ltd. (Sigma-Aldrich)). The specific steps are as follows:

[0052] 1) Antigen emulsification and immunization

[0053] Each mouse was given 100 μg of each immune protein, mixed with Freund's adjuvant in equal volumes of 1:1, and ultrasonic emulsification was performed with a power of 40%, ultrasonic for 1 second and 4 seconds, and ultrasonic time of 5-10 minutes. 300 μL of the emulsified antigen was taken for subcutaneous immunization. The interval between the first, second, and third immunizations was 10 to 14 days.

[0054] 2) Potency test

[0055] ① Antigen coating: Take the ELISA plate, add 100 μL / well of 2 μg / mL antigen protein (diluted with coating solution), and incubate at 4°C overnight or at 37°C for 2 hours;

[0056] ② Blocking: discard the coating solution, spin dry, wash three times with PBST (200 μL / well), 3 min / time, add 1% BSA to block, 37°C, 2h

[0057] ③ Serum to be tested: discard the blocking solution, spin dry, wash three times with PBST (200 μL / well), 3min / time, add 100 μL / well of gradient diluted serum (PBST from 1:400 dilution, 8 gradients of continuous dilution), 37°C, 60min. Blank control is negative serum.

[0058] ④ Secondary antibody: discard the liquid, spin dry, wash three times with PBST (200 μL / well), 3 min / time, add HRP-labeled goat anti-mouse IgG secondary antibody 100 μ / well, 37°C, 45 min;

[0059] ⑤ Color development: discard the liquid, spin dry, wash three times with PBST (200 μL / well), 3 min / time, add TMB 100 μ / well, protect from light for 10 min, and add 100 μL of stop solution to each well after staining to terminate the reaction.

[0060] ⑥ Interpretation of results: The value is read by the ELISA reader at 450nm (A450), and the titer is above 1:10000.

[0061] The results are as follows Figure 2 As shown, among the four mice tested, the antibody titer in three mice reached more than 200,000. The results showed that the antibody titer produced by mice immunized with Bx-FAR-1 pure protein as antigen was good.

[0062] 3) Intraperitoneal impact immunization

[0063] After the mice are immunized and tested to show a meaningful serum titer (>1:10000), the animals rest for 3 to 6 weeks. This period of time allows the enhanced secondary response to decay, so that new B cells can be activated and expanded after the last contact with the antigen. The "rested" mice are given a final pre-fusion shock immunization, with the antigen being pyrogen-free saline or phosphate buffered saline without adjuvant. The most effective route for shock immunization is intravenous injection, or direct delivery of the antigen to the spleen, where newly activated B cells can be harvested for fusion. 3 days after intravenous shock immunization (4 days after intraperitoneal shock immunization), harvest immune spleen cells or lymphocytes for fusion.

[0064] 4. Obtaining antigen target cells

[0065] 1) Enrichment of lymphocytes

[0066] ①Pinch the skin at the back of the mouse's neck and fix the head. Use your other hand to reach back to the tail, pinch the middle of the tail, straighten the mouse, lift it up 30 degrees diagonally, and instantly break the neck.

[0067] ② Immerse it in 75% ethanol for 5 minutes, and use tweezers to hold it in 75% ethanol and stir it from time to time to fully disinfect it, then take it out, drain the alcohol, fix it on a fixing plate, and take out the mouse spleen in the clean bench;

[0068] ③Put 4-5mL of mouse lymphocyte separation solution in a 35mm culture dish, put the spleen on the cell sieve, and the grinding time of each spleen should be controlled within 5mins; suck the liquid obtained by grinding into a 15mL centrifuge tube, add 500-1mL of 1640 incomplete culture medium to 6mL, and centrifuge at 800g in a horizontal rotor for 30mins;

[0069] ④After centrifugation, the bottom tube contains red blood cells and debris, the upper tube contains separation solution, the top layer is 1640, and below 1640 is a thinner, cloud-like white film, i.e., lymphocyte layer. Carefully pipette this layer into a new centrifuge tube, add 1640 to 10 mL, 250 g, and centrifuge for 10 minutes;

[0070] ⑤After centrifugation, remove the supernatant, add 3 to 5 times the volume of red blood cells lysis buffer, lyse for 2 minutes, 400g, centrifuge for 5 minutes, remove the supernatant after centrifugation, wash twice at 1640; then resuspend with 5 mL of 1640.

[0071] 2) Flow sorting

[0072] ① After centrifugation of the cells, discard the supernatant and follow the procedure of 1 mL: 10 7 Add the suspension in a ratio of 1×10 5 Single fluorescent staining was performed for compensation, and the remaining cells were mixed fluorescently stained and incubated at 4 degrees Celsius for 30 minutes;

[0073] ② Centrifuge and discard the supernatant, suspend the precipitate, wash again, and then resuspend according to the cell ratio to a concentration of about 1×10 7 / mL;

[0074] ③ Add the resuspended cell solution to the flow cytometry tube, place it on ice, and wait for the flow cytometry;

[0075] ④First use a single staining tube to adjust compensation, sorting logic: remove adhesion, then negatively select dead cells and T cells, then positively select IgG, CD19 and antigen target cells labeled with fluorescent antibodies, and collect the target cells.

[0076] The results are as follows Figure 3 As shown in the figure, since the green fluorescent marker is added when the antigen protein is concentrated, it is possible to see with the naked eye whether each B cell has a fluorescent marker. The results show that the number of labeled B cells is large and the activity is good, which can be used to extract antibodies.

[0077] 5. Antibody Expression

[0078] 1) Cell passaging

[0079] ① Inoculate the cells into fresh SMM-293TII medium to a final density of 0.3-0.4×10 6 cells / mL;

[0080] ② Place the culture bottle in an incubator (37℃160rpm5% CO2) and loosen the bottle cap 1 / 4 turn to allow gas exchange.

[0081] ③ After 3-4 days of subculture, the density of viable cells can generally reach 3×10 6 cells / mL and the cell viability was greater than 90%.

[0082] 2) Cell cryopreservation

[0083] ① Count cells and calculate the required freezing volume based on the number of cells;

[0084] ② Mix 45% fresh culture medium SMM293-TII and 10% DMSO and place in a 4°C refrigerator for use;

[0085] ③ Centrifuge an appropriate amount of cells (100×g for 5 min), retain 45% of the supernatant, discard the excess supernatant, resuspend the cells, and then add the prepared freezing solution drop by drop;

[0086] ④ Gently mix the cells to make a cell mixture and dispense it into cryopreservation tubes. 1mL to 1.5mL per tube is recommended.

[0087] ⑤ Place the cryovial in a cell freezing box with isopropanol added, place it in a -80°C refrigerator overnight, and then place it in a liquid nitrogen tank for long-term storage.

[0088] 3) Cell transfection

[0089] On the day of transfection, take samples to count cell density and viability. The cell density should be between 3-5×10 6 cells / mL, with a viability of more than 90%. Adjust the cell density to 3×10 6 cells / mL, the volume of each bottle of cell fluid is 20mL;

[0090] Detect the expression of the target gene 48-72 hours after transfection. If long-term expression and production of recombinant proteins and antibodies is required, add 0.7mL SMS293-SUPI feed solution, 60μL enhancer1, and 200μL enhancer2 24 hours after transfection, and then add feed solution (0.7mL) and enhancer every 48 hours. Samples can be collected 6-10 days after transfection at the longest, depending on the expression level required.

[0091] 4) Antibody purification

[0092] ① The collected cell supernatant was centrifuged at 1000g for 10 min to remove cells and other impurities, and the supernatant after centrifugation was centrifuged at 5000g for 20 min to remove cell debris. The supernatant after centrifugation was filtered through a 0.45μm filter.

[0093] ② Equilibrate the protein A column with 5 times column volume of PBS. After equilibration, slowly add the sample to be purified into the column.

[0094] ③ Elute nonspecifically adsorbed proteins with 10 column volumes of PBS and collect the effluent.

[0095] ④ Elute the antibody with 5 to 10 column volumes of glycine (pH 3.0), and collect the effluent in separate tubes, 500 μL to 1 mL per tube. Immediately adjust the eluted target protein to neutrality with Tris-Hcl (pH 9.0) to prevent the low pH from denaturing and inactivating the purified protein.

[0096] The amino acid sequence of the monoclonal antibody that meets the requirements for test strip development is as follows: KFHQVRPELRDLLPKNMLKFMDSFTPEDEKLFEETDDSGGLSEYLKRVGAKN PKLAQRGSVIVKDIVTRMAALDQTTSDFVKTVMGALDRKLGKLNDQEVIETA KVLLAGFDKLTLGRRSGLKAMFPSIHDTLLSDKFKELAKGKL.

[0097] The nucleotide sequence encoding the monoclonal antibody is shown below. The nucleotide sequence is: AAGTTTCATCAGGTCCGGCCAGAACTTCGAGACCTCTTGCCAAAGAATATGCTGAAGTTCATGGATAGTTTCACCCCCGAAGATGAAAAACTTTTTGAGGAAACTGATGATTCAGGTGGACTTTCCGAGTACCTGAAGAGGGTCGGTGCCAAAAACCCCAAGCTGGCTCAGCGTGGAAGCGTTATTGTAAAGGATATTGTGACGAGAA TGGCTGCCTTGGATCAGACCACATCTGATTTTGTTAAAACAGTTATGGGAGCGCTAGACCGTAAGCTTGGAAAGCTCAATGACCAAGAAGTTATCGAGACGGCCAAGGTTCTACTTGCCGGATTCGACAAGCTCACCCTTGGGCGGAGGAGCGGTCTGAAGGCGATGTTTCCAAGTATTCACGACACCTTATTATCTGACAAATTCAAGGAGTTAGCTAAAGGCAAACTG(SEQ ID NO.1).

[0098] Example 3

[0099] 1. Preparation of gold-labeled antibodies

[0100] The stem segments of pine trees infected with pine wood nematodes were cut as positive treatments, and the stem segments of pine trees not inoculated with pine wood nematodes were used as negative controls. The critical threshold of the binding between the effector protein and the antibody was tested on a nitrocellulose membrane, and the appropriate antibody dilution multiple was screened to determine the optimal range of colloidal gold color development. The specific steps are as follows:

[0101] Pure water is heated to boiling, chloroauric acid solution is added under stirring, and then trisodium citrate solution is added to obtain colloidal gold solution; boric acid buffer is added to the solution, and monoclonal antibody solution is added dropwise to react after being fully mixed, positive branch and negative branch samples are ground respectively, and they are diluted with PBS buffer respectively, and then 10% BSA is added dropwise to block to obtain a mixed solution; the mixed solution is centrifuged, the supernatant is discarded, a precipitate is obtained, and the precipitate is dissolved in a resuspension to obtain a gold-labeled antibody. The optimal color development range of colloidal gold is 200-800 mg / μL.

[0102] 2. Preparation of pine wood nematode effector protein test strips

[0103] The sample pad is immersed in a PBS solution containing Tween-20, sucrose and BSA and then dried; the coating source is sprayed on the nitrocellulose membrane as a detection line, and the secondary antibody is sprayed on the nitrocellulose membrane as a quality control line; the sample pad, nitrocellulose membrane, absorbent pad and bottom plate are assembled to obtain a finished test strip.

[0104] The results are as follows Figure 4 The test strip color development results show that positive tissues show two lines on the membrane, while negative tissues show one line. This result shows that the appropriate range of colloidal gold binding to antibodies has been verified, and the test strip has achieved success in the experimental stage.

[0105] 3. Use test strips to detect pine wood nematodes in samples

[0106] After grinding the suspected infected pine stem tissue, dilute it 10 times with lysis solution, take 1mL of the diluted liquid and drop it into the test hole of the test strip. Let the test strip stand at room temperature for 10 minutes, wait for the quality control line on the membrane to show color, indicating that the sample extract and the test strip have no quality and technical problems, and observe whether there is a red line on the test line.

[0107] The results are as follows Figure 5As shown, 78 suspected infected pine samples were tested, of which 70 samples detected positive (quality control line, detection line are all colored), 2 test strips quality control lines did not develop color, and 6 samples did not match the actual situation (quality control line color, detection line did not develop color). Among them, the samples of 2 test strips quality control lines that did not develop color were re-tested, and positive (quality control line, detection line are all colored), and the reason for the non-color development of the test strip quality control line was that the antigen adsorption rate of the nitrocellulose membrane of the two test strips was poor, and the problem can be effectively solved by controlling the raw material quality prepared by the test strips. 6 samples to be tested were analyzed for not matching the actual situation, and the reason was that the sample protein concentration was lower than 0.1ng / uL, so that the correct result was not shown. By re-extracting the sample (or concentrating the prepared sample), and controlling its concentration to be within the specified range of this application (greater than 0.1ng / uL), it was detected again (re-tested), and the result was positive.

[0108] In summary, combined with the test results of the samples to be tested, the re-test results and the cause analysis, it is shown that the pine wood nematode disease detection test strip is successfully prepared, and the test results are accurate, reliable and highly sensitive, and can be used for the rapid detection of pine wood nematode disease.

[0109] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a pine wood nematode disease detection test strip, characterized in that: The following steps are involved: Preparation of hapten: The pine wood nematode effector protein is hydrolyzed and added with acetic acid to prepare the hapten; Preparation of immunogen and coating agent: the hapten is coupled to protein by activated ester method to prepare immunogen and coating agent respectively; Preparation of monoclonal antibodies: using the immunogen to prepare monoclonal antibodies, using the coating source to detect the sensitivity and specificity of the monoclonal antibodies through ELISA, and obtaining monoclonal antibodies of pine wood nematode effector proteins; Preparation of gold-labeled antibody: labeling the monoclonal antibody with colloidal gold to obtain a gold-labeled antibody; Preparation of colloidal gold test strips: Use the coating source of pine wood nematode effector protein to make a test line on a nitrocellulose membrane, use the secondary antibody to make a quality control line on the nitrocellulose membrane, and then assemble into a test strip; The amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.

1.

2. The method for preparing the pine wood nematode disease detection test strip according to claim 1, characterized in that: The specific steps of preparing the hapten are: The effector protein was dissolved in acetic acid and water, heated to reflux, and then an aqueous sodium hydroxide solution was added to reach a pH of 7.5, and pure water was added to dissolve the precipitate. The aqueous solution was washed with n-hexane, and the aqueous layer solution was acidified by separation to a pH of 6, and then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and passed through a silica gel column to obtain a white solid, which was the hapten.

3. The method for preparing the pine wood nematode disease detection test strip according to claim 1, characterized in that: The preparation of the immunogen and coating agent is specifically as follows: Preparation of immunogen: bovine serum albumin (BSA) is fully dissolved in carbonate buffer (CBS) to obtain a BSA solution, and the activated hapten solution is added dropwise to the BSA solution under magnetic stirring, and the mixture is stirred to react to obtain an immunogen; Preparation of coating source: fully dissolve the chicken ovalbumin OVA in carbonate buffer CBS to obtain OVA solution, add the activated hapten solution dropwise into the OVA solution under magnetic stirring, stir the mixture to react, and obtain coating source.

4. The method for preparing the pine wood nematode disease detection test strip according to claim 1, characterized in that: The preparation of the gold-labeled antibody is specifically as follows: Pure water is heated to boiling, and a chloroauric acid solution is added under stirring, and then a trisodium citrate solution is added to obtain a colloidal gold solution; a boric acid buffer is added to the solution, and after being fully mixed, a monoclonal antibody solution is added dropwise to react, positive branch and negative branch samples are respectively ground, and the samples are respectively diluted with PBS buffer, and then 10% BSA is added dropwise to block to obtain a mixed solution; the mixed solution is centrifuged, and the supernatant is discarded to obtain a precipitate, and the precipitate is dissolved in a heavy suspension to obtain a gold-labeled antibody.

5. The method for preparing the pine wood nematode disease detection test strip according to claim 1, characterized in that: The preparation of the colloidal gold test strip is specifically as follows: The sample pad is immersed in a PBS solution containing Tween-20, sucrose and BSA and then dried; the coating source is sprayed on the nitrocellulose membrane as a detection line, and the secondary antibody is sprayed on the nitrocellulose membrane as a quality control line; the sample pad, nitrocellulose membrane, absorbent pad and bottom plate are assembled to obtain a finished test strip.

6. A pine wood nematode disease detection test strip prepared by the method for preparing a pine wood nematode disease detection test strip according to any one of claims 1 to 5.

7. Use of the pine wood nematode disease detection test strip according to claim 6 in detecting pine wood nematode disease.

8. The use according to claim 7, characterized in that: Here are the steps: 1) Grind the pine stem tissue to be tested, dilute it 10 times with lysis buffer, and drop 1 mL of the diluted liquid into the test hole of the test strip; let the test strip stand at room temperature for 10 minutes, observe the color of the quality control line and the test line on the membrane, and judge the result; 2) Both the quality control line and the test line are colored, and the test result of the sample to be tested is positive; If the quality control line shows color but the test line does not, the sample solution is concentrated 10 times or the concentrated sample is re-prepared and tested again. If the quality control line still shows color but the test line does not, it is judged as negative; If the quality control line does not show color, replace the test strip and retest.

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