Preparation method of tobacco black shank pathogen fluorescence rapid quantitative detection test strip

Abstract

The invention discloses a tobacco black shank pathogen fluorescence rapid quantitative detection test strip preparation method, which comprises: S1, taking 0.5 mL of an activating solution and 30 [mu] L of fluorescent microspheres, activating for 15 min at a room temperature, respectively weighing 5.21 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and using the activating solution to prepare a solution with a concentration of 1 mg / mL, respectively adding 20 microliters and 40 microliters of activated microspheres, and reacting at room temperature for 15 minutes; the method comprises the following steps: diluting 6 [mu] L of a black shank pathogen antibody with 60 [mu] L of deionized water, uniformly mixing, reacting at room temperature for 8 h, adding 200 [mu] L of a confining liquid, uniformly mixing, reacting at room temperature for 30 min, spraying metal on a machine with a metal spraying amount of 2 [mu] L / cm, and directly developing the tobacco black shank fluorescence detection test strip by directly purchasing existing black shank pathogen raw materials (antigen and antibody) on the market. By optimizing a product process system and a sample pretreatment method, the product detection sensitivity is greatly improved, parallelism and stability evaluation is performed on the product prepared by the optimized process, and the product quality meets the internal quality requirement.

Description

Technical Field

[0001] The invention relates to the technical field of testing, and in particular to a method for preparing a fluorescent rapid quantitative testing paper strip for tobacco black shank pathogens. Background Art

[0002] The detection methods of plant viruses (including black leg disease pathogens) mainly include biological detection methods (symptom type identification, host identification, etc.), electron microscopy technology, serological detection methods (enzyme linked immunosorbent assay (ELISA)), and molecular biological detection methods (including PCR technology, nucleic acid probe technology, etc.). Although electron microscopy of isolated viruses is an effective means of diagnosing viruses with strong specificity, it is very time-consuming and labor-intensive, requires professional technicians, and is costly, making it unsuitable for promotion and use. Serological detection methods are relatively time-consuming and labor-intensive. Although immunofluorescence, ELLSA and other methods have the advantages of trace, specific, rapid and accurate, they require relatively complete test instruments and experienced technicians to operate and judge the results. The entire process of testing a batch of samples takes 1 to 2 hours, which is difficult to do at the grassroots level. PCR and nucleic acid probe diagnosis require special instruments and drugs, and have high technical content. They are generally only suitable for laboratory diagnosis or research applications, and are difficult to promote at the grassroots level. There are also related colloidal gold test strips and fluorescent test strips rapid test products on the current market. Test strip rapid test products are simple to operate, short in detection time, low in personnel requirements, and low in detection cost. However, the detection limits of existing colloidal gold rapid test products and fluorescent test strips are basically 100ppb / g or lower, which cannot meet the tobacco industry's demand for black shank disease monitoring. There is an urgent need to establish a black shank disease diagnostic product that is simple, fast, more sensitive, cheap and suitable for grassroots application. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogens, so as to solve the problems raised in the above-mentioned background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen, comprising the following steps:

[0005] S1. Take 0.5mL activation solution + 30μL fluorescent microspheres and activate at room temperature for 15min. Weigh 5.21mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) respectively and prepare 1mg / mL concentration with activation solution. Add 20μL and 40μL activated microspheres respectively and react at room temperature for 15min. Take 60uL deionized water to dilute 6μL black leg pathogen antibody, add all of them and mix well. React at room temperature for 8h, then add 200μL blocking solution and mix well. React at room temperature for 30 minutes, and spray gold on the machine. The spraying amount is 2μL / cm.

[0006] S2. Dilute the antibody with PBS to 1:5, 1:10, 1:20, 1:50, 1:100 as T-line coating solution, and dilute the secondary antibody with PBS + 10% BSA at pH 7.2, 0.02 mol / L as C-line coating solution. Strike the plate at 1 uL / cm, and bake at 37 degrees Celsius for 12 hours.

[0007] S3, cut the ordinary glass fiber into 30*1cm size, treat each piece with 1.5mL of PBS+5%BSA+0.1% preservative buffer solution with pH7.2, 0.02mol / L, and dry at 37 degrees Celsius overnight;

[0008] S4, non-woven fabrics were cut into 30*1.7cm size and treated with 2mL of PBS+0.5% Tween-20+1% trehalose buffer at pH 7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight;

[0009] S5. Assemble the relevant items, cut them into strips with a width of 4 mm, and put them into plastic test cards for testing.

[0010] Preferably, the pH value of the activation solution in S1 is 6.0, the activation solution is 0.01moL / L citric acid-sodium citrate buffer, and the activation solution formula is: 0.01moL / L citric acid: 0.01moL / L sodium citrate volume ratio (3.8:16.2).

[0011] Preferably, the fluorescent microspheres in S1 are fluorescent microspheres with a diameter of 200 nm, and the blocking solution is a 10% BSA aqueous solution.

[0012] Preferably, the overnight drying time of S3 and S4 is more than 12 hours.

[0013] Preferably, the pH of PBS in S2 is 7.2 and its concentration is 0.02 mol / L.

[0014] Preferably, the method further includes a potency test, a sensitivity test and a detection limit test for fresh tobacco leaf samples, wherein the potency test is performed by diluting the black leg pathogen antigen concentration to 0 ng / mL (PBS buffer) and 50 ng / mL with a PBS buffer having a pH of 7.2 and 0.02 mol / L, and taking 100 uL with a pipette and adding them to the sample wells of the fluorescent test strips, reacting at room temperature for 10 minutes, and analyzing the results with a fluorescent reader.

[0015] Preferably, the sensitivity test selects 1:10 and 1:20 times coating concentrations for sensitivity test according to the potency test results, and the sensitivity test method is: dilute the black leg pathogen standard substance concentration to 0 ng / mL (PBS buffer), 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL with PBS at pH 7.2 and 0.02 mol / L, and take 100 uL with a pipette and add it to the fluorescent test strip sample wells, react at room temperature for 10 minutes, and analyze the results with a fluorescence reader.

[0016] Preferably, the detection limit test of fresh tobacco leaf samples is performed by adding black shank pathogen antigen to fresh tobacco leaf samples according to the sensitivity test results to evaluate the detection limit concentration of the samples, and the added concentrations are 0ng / g, 10ng / g, 50ng / g, 100ng / g, and 500ng / g.

[0017] Preferably, the pretreatment method of the fresh tobacco leaf sample is: weigh 0.1g of fresh tobacco leaf sample and add it to a 2mL polystyrene centrifuge tube, add 1mL of sample extract, repeatedly crush the tobacco leaf sample with a small glass rod, take 100uL of supernatant with a pipette and add it to the sample well of the fluorescent test strip, react at room temperature for 10 minutes, and analyze the results with a fluorescence reader, the pH of the extract is 7.2, and the concentration is 0.02mol / L PBS+5% methanol solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are: by directly purchasing the existing black shank pathogenic bacteria raw materials (antigens, antibodies) on the market, directly developing black shank pathogenic bacteria fluorescent detection test strips, and by optimizing the product process system and sample pretreatment methods, achieving a significant improvement in product detection sensitivity, and conducting parallelism and stability evaluation on the products prepared by the optimized process, the product quality meets internal quality requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the structure of the fluorescent product test strip of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] See also Figure 1 The present invention provides a technical solution: a method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogens, comprising the following steps:

[0022] S1, Antibody-coupled Microspheres

[0023] Take 0.5mL activation solution (pH 6.0 0.01moL / L citric acid-sodium citrate buffer) + 30μL fluorescent microspheres (200nm diameter fluorescent microspheres purchased from Jiangsu Weidu Biotechnology Co., Ltd.) and activate at room temperature for 15min. Weigh 5.21mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) and prepare 1mg / mL concentration with activation solution. Add 20μL and 40μL activated microspheres respectively and react at room temperature for 15min. Take 60uL deionized water to dilute 6μL black leg pathogen antibody (purchased by Qingdao Tobacco Research Institute), add all and mix well, react at room temperature for 8h, then add 200μL blocking solution (10% BSA aqueous solution) and mix well and react at room temperature for 30 minutes, then spray gold on the machine, with a spraying amount of about 2μL / cm.

[0024] Activation solution formula: 0.01moL / L citric acid: 0.01moL / L sodium citrate volume ratio (3.8:16.2).

[0025] S2. Film cutting

[0026] Use PBS with pH 7.2 and 0.02 mol / L to dilute the antibody to 1:5, 1:10, 1:20, 1:50, and 1:100 as T-line coating solution, and use PBS with pH 7.2 and 0.02 mol / L + 10% BSA to dilute the secondary antibody as C-line coating solution. Striking was performed at 1uL / cm and then baked at 37 degrees Celsius for 12 hours (the back plate was Shanghai Jieyi 30*6cm2 line PVC board, the absorbent paper was Shanghai Jie Ning Spec30*20cm, and the NC membrane was Shanghai Jie Ning JN-90).

[0027] Preparation method of PBS buffer solution at pH 7.2, 0.02 mol / L

[0028]

[0029]

[0030] S3, Gold Label Pad Processing

[0031] Shanghai Jieyi ordinary glass fiber was cut into 30*1cm size and treated with 1.5mL of PBS+5%BSA+0.1% preservative (P300) buffer solution at pH 7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight (more than 12 hours);

[0032] S4. Sample pad processing

[0033] Shanghai Jie Ning SF-06 non-woven fabric was cut into 30*1.7cm size and treated with 2mL of PBS+0.5% Tween-20+1% trehalose buffer at pH 7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight (more than 12 hours);

[0034] S5. Product Assembly

[0035] according to Figure Ⅰ The structural standard shown assembles the relevant items, cuts them into strips with a width of 4mm, and puts them into a plastic test card for testing. The figure includes:

[0036] PVC sheet: Shanghai Jieyi 30*6cm2 PVC sheet, used as the carrier of raw materials for fluorescent test strips;

[0037] Chromatographic membrane: Shanghai Jie Ning JN-90, used as a chromatographic carrier for marking quality control lines, test lines and sample testing;

[0038] Sample pad: Shanghai Jie Ning SF-06 non-woven fabric was cut into 30*1.7cm size and treated with 2mL of PBS+0.5% Tween-20+1% trehalose buffer at pH 7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight (more than 12 hours). Its main function is sample buffering during sample detection.

[0039] Gold label pad: (for labeling antibody fluorescent microspheres): Shanghai Jieyi ordinary glass fiber is cut into 30*1cm size and treated with 1.5mL of PBS+5%BSA+0.1% preservative (P300) buffer at pH7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight (more than 12 hours). It mainly serves as a storage carrier after the antibody is labeled on the fluorescent microspheres;

[0040] Water-absorbing pad: Shanghai Jie Ning Spec30*20cm, used for chromatographic attraction during sample testing;

[0041] Quality control line (C line): labeled secondary antibody (sheep anti-mouse antibody), used to verify the effectiveness of the test strip product;

[0042] Test line (T line): Labeled black shank disease pathogen antibody, which can combine with the analyte (black shank disease pathogen antigen) and the black shank disease pathogen antibody-labeled fluorescent microsphere conjugate in the sample (double antibody sandwich) to produce color depth changes to achieve the purpose of detection;

[0043] S6. Potency test

[0044] Dilute the black leg disease pathogen antigen (provided by Yunnan Tobacco Science Research Institute, the concentration was 78.6ug / mL after third-party protein concentration determination) with PBS buffer at pH 7.2 and 0.02mol / L to 0ng / mL (PBS buffer) and 50ng / mL. Use a pipette to take 100uL and add it to the sample well of the fluorescent test strip. After reacting at room temperature for 10 minutes, use a fluorescence reader to analyze the results. The test results are as follows:

[0045]

[0046] Table 1 Potency test data

[0047] S7, sensitivity test

[0048] According to the results of 6. Potency test, 1:10 and 1:20 times coating concentration were selected for sensitivity test. The test method was: dilute the black shank pathogen standard substance (provided by Yunnan Tobacco Science Research Institute, the concentration was 78.6ug / mL after third-party protein concentration determination) with PBS at pH 7.2 and 0.02mol / L to the concentrations of 0ng / mL (PBS buffer), 1ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, and 50ng / mL, and use a pipette to take 100uL respectively and add it to the fluorescent test strip sample well. After reacting at room temperature for 10 minutes, use a fluorescent reader to analyze the results. The test results are as follows:

[0049]

[0050] Table 2 Sensitivity test data

[0051] Conclusion of the sensitivity test experiment: It can be seen from the data that the potency is 1:10 to 1:20 times that of the conventional system process; the sensitivity is approximately 10ng / mL.

[0052] S8, Detection limit test of fresh tobacco leaf samples

[0053] According to the sensitivity test sensitivity test results, the black leg pathogen antigen addition test of fresh tobacco leaf samples was carried out to evaluate the sample detection limit concentration, and the addition concentration was 0ng / g, 10ng / g, 50ng / g, 100ng / g, and 500ng / g. The sample pretreatment method is: weigh 0.1g of fresh tobacco leaf sample and add it to a 2mL polystyrene centrifuge tube, add 1mL of sample extract (pH7.2, 0.02mol / L PBS+5% methanol solution), repeatedly smash the tobacco leaf sample with a glass rod, take 100uL of supernatant with a pipette and add it to the fluorescent test strip sample well, react at room temperature for 10 minutes, and analyze the results with a fluorescence reader. The test results are as follows:

[0054]

[0055]

[0056] Table 3 Detection limit test data of fresh tobacco leaf samples

[0057] Conclusion of the fresh tobacco leaf sensitivity test experiment: It can be seen from the data that the potency is 1:10 and 1:20 times using the conventional system process; the sensitivity is approximately 100ng / g.

[0058] Process system optimization plan

[0059] Option 1: Optimizing T-line coating solution

[0060] Coating standard: The antibody dilution ratio is 1:10 and the plate is streaked at 1uL / cm. After streaking, it is placed at 37 degrees Celsius and baked for 12 hours (the back plate is Shanghai Jieyi 30*6cm2 line PVC board, the absorbent paper is Shanghai Jie Ning Spec30*20cm, and the NC membrane is Shanghai Jie Ning JN-90). The rest of the process is the same as described in the above method and does not change for the time being.

[0061] Control solution CK: PBS + 10% BSA, pH 7.2, 0.02 mol / L;

[0062] Optimization scheme 1: pH 9.5 0.05 mol / L CB;

[0063] Optimization scheme 2: pH 7.2 0.02 mol / L PB + 5% BSA;

[0064] Optimization scheme 3: pH 7.2 0.02 mol / L PBS + 5% sucrose;

[0065] Optimization plan 4: Optimize reagents.

[0066] The optimized reagent formula is as follows:

[0067]

[0068] The test data of different coating schemes are as follows:

[0069]

[0070] Table 4 Test data of different coating schemes

[0071] Experimental conclusion: Optimization Scheme 1 is slightly worse than the control scheme and has a lower sensitivity; Optimization Scheme 2 is slightly better than the control scheme and has a slightly higher sensitivity; Optimization Scheme 3 has similar effects to Optimization Scheme 1; Optimization Scheme 4 is darker in color than all the schemes and has a higher sensitivity, so Optimization Scheme 4 can be selected as an alternative.

[0072] Option 2: Optimize the marking process

[0073] Experimental plan: The control plan CK and other process plans are the same as those described in the above method and remain unchanged for the time being.

[0074] Optimization scheme 1: Take 0.5mL activation solution + 30μL microspheres, weigh 5.21mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) respectively, prepare a concentration of 1mg / ml with activation solution, add 20μL and 40μL respectively, activate at 35℃ constant temperature oscillator at 200rpm for 15min, take 60uL deionized water to dilute 6μL black leg disease virus antibody (purchased from Qingdao Tobacco Research Institute), add all to mix, react at 35℃ constant temperature oscillator at 200rpm for 4h, add 200μL blocking solution (formula see the table below) into the reaction bottle, at 35℃ constant temperature oscillator at 200rpm for 30min, spray gold on the machine, and the gold spraying amount is about 2μL / cm.

[0075] Blocking solution formula table

[0076]

[0077]

[0078] Optimization scheme 2: Take 0.5mL activation solution + 30μL microspheres, weigh 5.21mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) respectively, prepare 1mg / mL concentration with activation solution, add 20μL and 40μL respectively, activate in 40℃ water bath with ultrasonic oscillation for 15min, take 60uL deionized water to dilute 6μL black leg virus antibody (purchased from Qingdao Tobacco Research Institute), add all to mix, stir at 4℃ constant temperature (the stirrer is placed in a 4℃ refrigerator) for reaction for 12h, add 200μL blocking solution (the formula is shown in the blocking solution formula table) into the reaction bottle, oscillator at 35℃ constant temperature at 200 rpm for 30 minutes, then add 100uL 20% trehalose aqueous solution to react for 30 minutes, spray gold on the machine, and the gold spraying amount is about 2μL / cm.

[0079] The test data of different marking schemes are as follows:

[0080]

[0081] Table 5 Test data of different marking schemes

[0082] Experimental conclusion: Optimization Scheme 1 has better sensitivity than the control scheme; Optimization Scheme 2 has the best sensitivity among the three schemes compared with the control scheme, so Optimization Scheme 2 can be selected as an alternative.

[0083] Scheme 3: Combine and compare optimized scheme 4 in scheme 1 and optimized scheme 2 in scheme 2. The process is the same as described in the above method.

[0084] The test data is as follows:

[0085]

[0086] Table 6 Optimization scheme combination comparison test data

[0087] Experimental conclusion: The combination of Scheme 1 Optimization Scheme 4 and Scheme 2 Optimization Scheme 2 can further improve the detection sensitivity; therefore, the optimized combination scheme can be selected as the system scheme.

[0088] Sample processing method optimization plan (final optimization plan for process system)

[0089] Control scheme CK: The CK scheme is the optimized combination scheme in Table 6.

[0090] Solution 1: Physically reduce the dilution factor to improve sensitivity

[0091] Weigh 10g of fresh tobacco leaf sample and put it into a sterilized sampling bag. Rub the tobacco leaf with both hands to crush it, collect the relevant juice and mix it evenly. Then take 100uL and add 400uL of sample extract ((PH7.2, 0.02mol / L PBS+5% methanol solution) and mix it for testing.

[0092] Solution 2: Optimize sample extraction solution

[0093] Weigh 0.1g of fresh tobacco leaf sample into a 2mL polystyrene centrifuge tube, add 11mL of sample extract, crush thoroughly with a glass rod, and take 100uL for testing;

[0094] The formula of sample extract 1 is as follows:

[0095]

[0096]

[0097] Solution 3: Optimize sample extraction solution

[0098] Weigh 0.1g of fresh tobacco leaf sample into a 2mL polystyrene centrifuge tube, add 21mL of sample extract, crush thoroughly with a glass rod, and take 100uL for testing;

[0099] The formula of sample extract 2 is as follows:

[0100]

[0101]

[0102] Table 7 Sample processing method test data

[0103] Experimental conclusion: Schemes 1, 2, and 3 can all improve the detection sensitivity from the experimental data, but the detection limit of 1ng / g is not obvious. Scheme 1 directly reduces the sample dilution multiple and directly improves the detection sensitivity. Schemes 2 and 3 mainly use optimized sample extracts (adding cell lysis reagent components to fully improve sample extraction efficiency). Scheme 3 is better than Scheme 2.

[0104] Combination of sample processing methods (combination of Scheme 1 and Scheme 3)

[0105] Control scheme CK: The CK scheme is the optimized combination scheme in Table 6.

[0106] Combination scheme: Weigh 10g of fresh tobacco leaf sample and put it into a sterilized sampling bag. Rub the tobacco leaves with both hands to crush them, collect the relevant juice and mix them evenly. Then take 100uL and add 400uL of sample extract 2 and extract to mix for inspection.

[0107] The test data is as follows:

[0108]

[0109] Table 8 Test data of sample treatment method combination scheme

[0110] Experimental conclusion: The combined solution can better improve the sample detection sensitivity, and the color development is higher than that of the CK solution. The detection sensitivity of fresh samples can reach 1ng / g, which can greatly improve the product detection sensitivity; therefore, it can be used as the final solution.

[0111] 3. Final product parameter verification

[0112] 1. Parallelism verification

[0113]

[0114]

[0115] Table 9 Product fresh tobacco leaf parallelism test data

[0116] Experimental conclusion: The parallel verification of the products is relatively good, and the parallelism meets the internal quality requirements (qualified standard CV%<15%).

[0117] 2. Stability verification

[0118]

[0119] Table 10 Product stability test data

[0120] Experimental conclusion: The product stability was verified according to the internal quality requirements at 4°C and 37°C. The data showed that the product was relatively stable and met the internal quality requirements (the qualified standard was that the T / C value of the product stored at 37°C for 12 days did not change by more than 20% compared with that at 4°C).

[0121] Final conclusion: By optimizing the product process system and sample pretreatment methods, the product detection sensitivity is greatly improved, and the parallelism and stability of the products prepared by the optimized process are evaluated. The product quality meets the internal quality requirements.

[0122] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogens, characterized in that: The following steps are involved: S1. Take 0.5mL activation solution + 30μL fluorescent microspheres and activate at room temperature for 15min. Weigh 5.21mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) respectively and prepare 1mg / mL concentration with activation solution. Add 20μL and 40μL activated microspheres respectively and react at room temperature for 15min. Take 60uL deionized water to dilute 6μL black leg disease pathogen antibody, add all of them and mix well. React at room temperature for 8h, then add 200μL blocking solution and mix well. React at room temperature for 30 minutes, and spray gold on the machine. The spraying amount is 2μL / cm. S2. Dilute the antibody with PBS to 1:5, 1:10, 1:20, 1:50, 1:100 as T-line coating solution, and dilute the secondary antibody with PBS + 10% BSA at pH 7.2, 0.02 mol / L as C-line coating solution. Strike the plate at 1 uL / cm, and bake at 37 degrees Celsius for 12 hours. S3, cut the ordinary glass fiber into 30*1cm size, treat each piece with 1.5mL of PBS+5%BSA+0.1% preservative buffer solution with pH7.2, 0.02mol / L, and dry at 37 degrees Celsius overnight; S4, non-woven fabrics were cut into 30*1.7cm size and treated with 2mL of PBS+0.5% Tween-20+1% trehalose buffer at pH 7.2, 0.02mol / L, and dried at 37 degrees Celsius overnight; S5. Assemble the related items, cut them into strips with a width of 4 mm, and put them into plastic test cards for testing.

2. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 1, characterized in that: The pH value of the activation solution in S1 is 6.0, and the activation solution is 0.01 mol / L citric acid-sodium citrate buffer. The activation solution formula is: 0.01 mol / L citric acid: 0.01 mol / L sodium citrate volume ratio (3.8:16.2).

3. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 1, characterized in that: The fluorescent microspheres in the S1 are fluorescent microspheres with a diameter of 200 nm, and the blocking solution is a 10% BSA aqueous solution.

4. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 1, characterized in that: The overnight drying time of S3 and S4 is more than 12 hours.

5. The method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 1, characterized in that: The pH of PBS in S2 is 7.2, and its concentration is 0.02 mol / L.

6. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 1, characterized in that: It also includes potency testing, sensitivity testing and detection limit testing of fresh tobacco leaf samples. The potency testing method is to dilute the tobacco black shank pathogen antigen concentration to 0 ng / mL (PBS buffer) and 50 ng / mL with a PBS buffer of pH 7.2 and 0.02 mol / L, and use a pipette to take 100uL respectively and add it to the sample well of the fluorescent test strip. After reacting at room temperature for 10 minutes, use a fluorescence reader to analyze the results.

7. A method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 6, characterized in that: The sensitivity test selects 1:10 and 1:20 times coating concentrations for sensitivity testing according to the potency test results. The sensitivity test method is: dilute the black leg disease pathogen standard substance concentration to 0 ng / mL (PBS buffer), 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL with PBS at pH 7.2 and 0.02 mol / L, and take 100 uL with a pipette and add it to the fluorescent test strip sample well, react at room temperature for 10 minutes, and analyze the results with a fluorescence reader.

8. The method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 7, characterized in that: The fresh tobacco leaf sample detection limit test is performed on the fresh tobacco leaf sample for tobacco black shank pathogen based on the sensitivity test result, and the sample detection limit concentration is evaluated, and the added concentrations are 0ng / g, 10ng / g, 50ng / g, 100ng / g, and 500ng / g.

9. The method for preparing a fluorescent rapid quantitative detection test strip for tobacco black shank pathogen according to claim 8, characterized in that: The pretreatment method of the fresh tobacco leaf sample is as follows: weigh 0.1g of the fresh tobacco leaf sample and add it to a 2mL polystyrene centrifuge tube, add 1mL of sample extract, repeatedly smash the tobacco leaf sample with a small glass rod, take 100uL of supernatant with a pipette and add it to the sample well of the fluorescent test paper strip, react at room temperature for 10 minutes, and analyze the results with a fluorescence reader, wherein the pH of the extract is 7.2, and the concentration is 0.02mol / L PBS+5% methanol solution.

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