1. An indirect ELISA detection kit for botrytis cinerea which is a pathogenic bacterium of botrytis cinerea is characterized by comprising: an ELISA plate, 10 multiplied by sample coating liquid, 10 multiplied by PBST, skimmed milk powder, an antigen standard solution, 10 multiplied by botrytis cinerea monoclonal antibody, 10 multiplied by enzyme-labeled secondary antibody, TMB substrate liquid, stop solution, a grinding rod and a centrifuge tube; the botrytis cinerea monoclonal antibody is formed by secretion of a hybridoma cell strain, the hybridoma cell strain is named as BcE4, is preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 13 days, and the preservation number is as follows: CCTCC No: C2021116.

2. the indirect ELISA detection kit for Botrytis cinerea which is a pathogen of Botrytis cinerea as claimed in claim 1, wherein the 10 Xsample coating solution is preferably carbonate coating buffer solution with a concentration of 0.5mol/L, pH value of 9.6.

3. The indirect ELISA detection kit for botrytis cinerea as a pathogen of gray mold of claim 1, wherein the 10 XPBST is prepared by weighing 80.00g NaCl and KH₂PO₄ 2.40g、Na₂HPO₄·12H₂O14.4.0 g and KCl 2.00g, adding 800mL of ultrapure water, fully stirring and dissolving, then adding concentrated hydrochloric acid to adjust the pH value to 7.2, adding Tween-205.00mL, and fixing the volume to 1000 mL.

4. The indirect ELISA detection kit for Botrytis cinerea as a pathogen of Botrytis cinerea as claimed in claim 1, wherein the preparation process of the antigen standard solution specifically comprises: selecting a botrytis cinerea strain, inoculating the botrytis cinerea strain to a PDA culture medium, carrying out shake culture at 25 ℃ for 4-5 days, centrifuging at 6000rpm for 20min to collect hypha and spores, washing twice with PBS, carrying out ultrasonic crushing under an ice bath condition until no obvious precipitate exists, centrifuging at 6000rpm for 20min, collecting supernatant containing the antigen, measuring the content of the antigen protein by using a multifunctional microplate reader, and adjusting the concentration of the protein to be 1mg/mL to obtain an antigen standard solution.

Background

The fungus Botrytis cinerea of Ascomycota is one of the pathogenic fungi of Botrytis cinerea of numerous crops. The germs live through the winter by being left in the soil with hypha along with the tissues of the sick and disabled, and infect the fritillaria again in the next year, thus causing serious loss to the agricultural production every year. The sclerotinia fungi are various in species, and the colony, hypha and spore forms of the related species are similar and are difficult to distinguish by depending on the morphological characteristics of pathogenic bacteria. In order to solve the problems of great harm and high identification difficulty of botrytis cinerea, the invention establishes an indirect enzyme-linked immunosorbent (ELISA) method which is rapid, convenient, high in sensitivity and strong in specificity and is suitable for detecting a large number of samples in the field, realizes early prevention and treatment through early identification, realizes symptomatic medicine administration through accurate identification, and provides a practical and reliable basis for prevention and treatment of field botrytis.

Disclosure of Invention

The invention provides an indirect ELISA detection kit for botrytis cinerea, which aims to provide a basis for early diagnosis and accurate diagnosis of botrytis cinerea and prevention and control of the botrytis cinerea.

In order to achieve the purpose, the invention adopts the following technical scheme: an indirect ELISA detection kit for botrytis cinerea which is a pathogenic bacterium of botrytis cinerea, comprising: an ELISA plate, 10 multiplied by sample coating liquid, 10 multiplied by PBST, skimmed milk powder, an antigen standard solution, 10 multiplied by botrytis cinerea monoclonal antibody, 10 multiplied by enzyme-labeled secondary antibody, TMB substrate liquid, stop solution, a grinding rod and a centrifuge tube; the botrytis cinerea monoclonal antibody is formed by secretion of a hybridoma cell strain, the hybridoma cell strain is named as BcE4, is preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 13 days, and the preservation number is as follows: CCTCC No: C2021116.

further, the sample coating solution is preferably carbonate coating buffer solution with the concentration of 0.5 mol/L.

Further, the preparation process of the 10 xPBST is to accurately weigh 80.00g of NaCl, KH₂PO₄ 2.40g，Na₂HPO₄·12H₂14.40g of O, 2.00g of KCl and 800mL of ultrapure water are added and fully stirred to be dissolved, then concentrated hydrochloric acid is added to adjust the pH value to 7.2, Tween-205.00mL is added to be fixedThe volume is 1000 mL.

Further, the preparation process of the antigen standard solution specifically comprises the following steps: selecting a botrytis cinerea strain, inoculating the botrytis cinerea strain to a PDA culture medium, carrying out shake culture at 25 ℃ for 4-5 days, centrifuging at 6000rpm for 20min to collect hypha and spores, washing twice with PBS, carrying out ultrasonic crushing under an ice bath condition until no obvious precipitate exists, centrifuging at 6000rpm for 20min, collecting supernatant containing the antigen, measuring the content of the antigen protein by using a multifunctional microplate reader, and adjusting the concentration of the protein to be 1mg/mL to obtain an antigen standard solution.

Compared with the existing Botrytis cinerea detection technology, the method has the following beneficial effects:

(1) the ELISA kit has strong specificity: the ELISA kit has strong reaction with an antigen of botrytis cinerea, does not react with antigens of alternaria alternata, alternaria tenuissima, fusarium oxysporum, fusarium solani, fusarium equiseti, fusarium semitectum, phoma and phomopsis, and can be used for detecting botrytis cinerea and gray mold caused by the botrytis cinerea.

(2) The ELISA kit has high detection sensitivity: the detection result shows that the detection sensitivity of the monoclonal antibody of the kit on the antigen prepared from the botrytis cinerea hyphae and spores is 312.5ng/mL (namely 31.25ng per hole), and the kit has good detection effect when being applied to an indirect ELISA detection kit.

(3) The monoclonal antibody types in the ELISA kit are Ig kappa and IgG 2A.

(4) Stabilizing the target protein bound by the monoclonal antibody in the ELISA kit: the monoclonal antibody is combined with antigen proteins of about 35kDa and 47kDa of botrytis cinerea.

Drawings

FIG. 1 is a potency detection chart of the monoclonal antibody of the kit;

FIG. 2 is a reaction diagram of monoclonal antibody and different fungal antigens in the kit;

FIG. 3 is a diagram showing the detection sensitivity of the monoclonal antibody in the kit;

FIG. 4 is a diagram showing the identification of the types and subclasses of monoclonal antibodies in the kit;

FIG. 5 is a diagram of a monoclonal antibody-bound target protein of Botrytis cinerea;

Detailed Description

The following description will further describe the embodiments and technical effects of the present invention with reference to the drawings and examples, but the present invention is not limited thereto.

The preparation method of the main solution in the ELISA kit comprises the following steps:

(1)10 × sample coating solution (i.e. concentration 0.5mol/L, 10 × carbonate coating buffer, pH 9.6): accurately weighing Na₂CO₃15.90 g，NaHCO₃29.3g, dissolving with ultrapure water, and metering to 1000 mL;

(2)10 × PBST: accurately weighing 80.00g of NaCl and KH₂PO₄ 2.40g，Na₂HPO₄·12H₂14.40g of O, 2.00g of KCl and 800mL of ultrapure water are added, the mixture is fully stirred and dissolved, then concentrated hydrochloric acid is added to adjust the pH value to 7.2, Tween-205.00mL is added, and the volume is adjusted to 1000 mL;

(3) antigen standard solution: selecting a botrytis cinerea strain, inoculating the botrytis cinerea strain to a PDA culture medium, carrying out shake culture at 25 ℃ for 4-5 days, centrifuging at 6000rpm for 20min to collect hyphae and spores, washing twice with PBS, carrying out ultrasonic crushing (power 200W, crushing for 2s and interval for 2s) under an ice bath condition until no obvious precipitate exists, centrifuging at 6000rpm for 20min, collecting supernatant containing the antigen, measuring the content of the antigen protein by using a multifunctional microplate reader, and adjusting the concentration of the protein to be 1mg/mL to serve as an antigen standard solution.

(4)10 × Botrytis cinerea monoclonal antibody: self-made monoclonal antibody aiming at botrytis cinerea. Secreted by a self-made hybridoma cell strain which is named as BcE4, and the hybridoma cell is preserved in China Center for Type Culture Collection (CCTCC) at 13 months of 2021, Wuhan university in Wuhan City, with the preservation number as follows: CCTCC No: C2021116.

(5)10 × enzyme-labeled secondary antibody: horse radish peroxidase-labeled goat anti-mouse IgG (Biosharp);

(6) TMB substrate solution: the substrate solution of TMB produced by Shanghai Biyuntian Biotechnology Co., Ltd is preferable, but not limited thereto.

(7) Stopping liquid: 100mL of concentrated sulfuric acid is accurately measured and slowly added to 800mL of ultrapure water in a dropwise manner.

The storage temperature of the kit is 4 ℃, and the effective period is 6 months.

The method for detecting the botrytis cinerea antigen in the plant tissue grinding fluid based on the ELISA kit specifically comprises the following steps:

(1) preparing a solution: the ELISA kit is firstly placed at room temperature for balancing for 30min, 2mL of 10 multiplied sample coating solution is taken out and purified water is added to 20mL for standby, 60mL of 10 multiplied PBST is taken out and purified water is added to 600mL to prepare 1 multiplied PBST for standby, 0.6g of skimmed milk powder is taken out and 20mL of 1 multiplied PBST is added to prepare 3% skimmed milk solution for standby, 1mL of 10 multiplied Botrytis cinerea monoclonal antibody is taken out and 1 multiplied PBST is added to dilute to 10mL for standby, 1mL of 10 multiplied enzyme-labeled secondary antibody is taken out and 1 multiplied PBST is added to dilute to 10mL for standby.

(2) Sampling and processing: collecting diseased plant tissue, rapidly returning to laboratory, and collecting 1cm tissue for each sample²1mL of the sample coating solution was added to the centrifuge tube and sufficiently ground with a grinding rod.

(3) Sample adding and coating: A1-H1 of a 96-hole enzyme label plate is used as a quality control hole, and a positive antigen standard solution for quality control detection is added into the quality control hole; adding an antigen standard solution and leaving blank holes, wherein each hole is 100 mu L; adding the sample grinding fluid in the step (2) into detection holes B2-H12, wherein each hole is 100 mu L; the microplate was allowed to coat overnight at 4 ℃.

(4) Washing the plate: adding 250 mu L of 1 XPBST into each hole, standing for 3-5 min, then patting dry, and repeatedly washing for three times.

(5) Blocking of non-specific binding: and (4) adding 150 mu L of 3% skimmed milk solution into each well of the overnight coated enzyme label plate, sealing at 37 ℃ for 30min, and repeating the plate washing step (4).

(6) Incubating botrytis cinerea monoclonal antibody: add diluted Botrytis cinerea monoclonal antibody solution (diluted by 10 XBotrytis cinerea monoclonal antibody) 100. mu.L and 3% skimmed milk diluted with 1 XPBST 100. mu.L per well, incubate at 37 ℃ for 1.5h, repeat step (4) wash plate.

(7) Incubation of enzyme-labeled secondary antibody: mu.L of diluted 1 × enzyme-labeled secondary antibody solution (diluted with 10 × enzyme-labeled secondary antibody), i.e., 100. mu.L of HRP-labeled goat-anti-mouse secondary antibody per well, was added to each well, incubated at 37 ℃ for 1h, and the plate washing in step (4) was repeated.

(8) And (3) color development and termination judgment: adding 100 mu L of TMB substrate liquid into each hole, developing for 15-20min under the condition of keeping out of the sun at 37 ℃ until the quality control color development is obvious, then adding 50 mu L of stop solution into each hole to stop the color development reaction, and considering the hole with obvious color reaction (showing bright yellow color different from the blank hole) as a positive hole due to the principle of antigen-antibody specific binding and enzyme label amplification, namely proving that the tested sample contains botrytis cinerea antigen with the content of more than or equal to 31.25ng, otherwise, the tested sample is negative reaction.

TABLE 1 kit contents

Material	Number of		Material	Number of
					Enzyme label plate	12 holes by 8 strips		10 × enzyme-labeled secondary antibody	1mL
10X sample coating liquid	2mL		TMB substrate solution	10mL
					10×PBST	60mL		Stopping liquid	6mL
Defatted milk powder	0.6g		Grinding rod	1 root of Chinese thorowax
					Antigen standard solution	1mL		1.5mL centrifuge tube	96 pieces of
10X Botrytis cinerea monoclonal antibody	1mL

Note: 10 × sample coating solution and 10 × PBST were diluted with purified water, and skim milk powder and 10 × mAb were diluted with 1 × PBST.

Example 1: preparation of hybridoma cell line

(1) Preparation of antigen: selecting single colonies of botrytis cinerea, respectively inoculating the single colonies into a potato glucose liquid culture medium, carrying out shake culture at 25 ℃ for 4-5 days, collecting spores and hyphae by using a 50mL centrifuge tube, centrifuging at 6000r/min for 20min, washing for 2 times by using PBS, carrying out ultrasonic crushing (power 200W, crushing for 2s and interval for 2s), centrifuging the crushed liquid at 6000r/min for 20min, collecting supernatant, measuring the content of albumin by using a Coomassie brilliant blue method, adjusting the protein concentration to 1000 mu g/mL, serving as an initial antigen for immune antigen and later detection, subpackaging a small amount of antigen liquid, and freezing and storing at-80 ℃. A small amount of the extract is stored at-20 deg.C before use.

(2) 3 healthy BaL b/c mice of 6-8 weeks are selected, and an intraperitoneal injection method is adopted, wherein 200 mu L of antigen emulsified by equivalent Freund's complete adjuvant is injected into each mouse. The immunization was carried out 3 weeks later with 200. mu.L of antigen emulsified with an equal amount of Freund's incomplete adjuvant. After another 3 weeks, immunization was performed with antigen without adjuvant. 3 days after the last immunization, antisera (polyclonal antibodies) were collected and mouse spleen lymphocytes were taken under sterile conditions for cell fusion.

(3) Polyethylene glycol (PEG-4000) was used as the fusogenic agent, and the mouse myeloma cell line was SP 2/0. The whole process is operated under aseptic conditions. The mouse spleen cells and myeloma cells are put into a 50mL centrifuge tube, mixed and centrifuged (1200r/min, 2min), and then supernatant liquid is sucked up and the cells are homogenized by fingers. The centrifuge tube was placed in a water-containing beaker pre-warmed in a 37 ℃ water bath, and 0.7mL of a cell fusion agent (50% PEG, pH adjusted to 9.0) was slowly added over 1 min. After standing for 1min, 40mL of RMPI-1640 medium, which had been pre-warmed to 37 ℃, was gradually added to dilute the PEG and lose its effect. After centrifugation (1000r/min, 2min), the supernatant was discarded. The precipitated cells were suspended in 40mL of HAT medium, and then distributed in 96-well cell culture plates previously supplemented with feeder cells, and subjected to 5% (V/V) CO₂Incubate at 37 ℃ in an incubator. Half of HAT culture medium is changed after 3 days, half of HAT culture medium is changed after 5 days, and HT culture medium is used for continuous culture after 5 days, at the moment, parents die, and if cells grow, the hybridoma cells are obtained.

(4) When the cells in the small holes grow to cover one fourth of the area of the bottom of the holes, the supernatant can be sucked up and the antibody can be detected by indirect ELISA. Selecting strong positive cell strains which do not react with antigens of fusarium equiseti, fusarium semitectum, alternaria alternata, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma phomopsis and phomopsis, performing multiple cloning culture by using a limiting dilution method until all the holes are positive to obtain a cell strain BcE4 secreting the monoclonal antibody, and further performing expanded culture on a cell strain BcE4 for preparing monoclonal antibody ascites and freezing and storing liquid nitrogen. The hybridoma cells are preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 13 days, and the preservation numbers of Wuhan university in Wuhan City are as follows: CCTCC No: C2021116.

example 2: generation of monoclonal antibodies

Taking about 8-week-old Balb/c mice, performing intraperitoneal injection of 0.2mL of pristanane, and performing intraperitoneal injection of 0.2mL of pristanane containing 5-10 × 10 after 7 days⁵And (3) carrying out cell suspension on the hybridoma cell BcE4, wherein the abdominal cavity of the mouse obviously swells 7-10 days after injection, taking ascites, centrifuging at 2000r/min for 3min, and collecting supernatant, namely the ascites monoclonal antibody. Purifying the monoclonal antibody by Protein A column chromatography to obtain the monoclonal antibody capable of specifically identifying the botrytis cinerea, and storing at-80 ℃.

Example 3: monoclonal antibody working concentration determination

The titer of the antibody was determined by indirect ELISA. The 1000 mug/mL botrytis cinerea antigen is diluted by 500 times by using a sample coating solution, then a whole enzyme label plate (namely 2 mug/mL) is coated, the temperature is kept overnight at 4 ℃, PBST is washed for three times, and then the whole enzyme label plate is sealed by using skimmed milk for 60 min. Diluting the monoclonal antibody BcE4 times, adding into the coated well, adding 100 μ L of monoclonal antibody into each well, washing at 37 deg.C for 1h, adding 100 μ L of enzyme-labeled secondary antibody after washing PBST for three times, washing at 37 deg.C for 1h, adding OPD substrate color development solution after washing PBST for color development, terminating the reaction with 50 μ L of stop solution, reading OD with enzyme-labeling instrument_490nmAnd taking a sample with the ratio of the sample to the negative control being more than 2 as a positive test to determine the titer of the monoclonal antibody ascites.

The detection proves that the potency of BcE4 is 2.56 multiplied by 10⁴Double, BcE4 dilution was determined 1000-fold for other detection experiments (see fig. 1).

Example 4: kit specificity detection

The detection objects are respectively botrytis cinerea, fusarium equiseti, fusarium semitectum, alternaria tenuis, fusarium oxysporum, fusarium solani, phoma and phomopsis, and the antigens (1000 mug/mL) are diluted to 2 mug/mL by using a sample coating solution and then coated on an enzyme label plate. And (3) detecting the specificity of the monoclonal antibody by using an indirect ELISA method by using the sample coating solution as a blank control.

Through the detection, the antibody and the antigen of the fungus do not have cross reaction, but the antibody has strong reaction with the botrytis cinerea antigen, and whether the botrytis cinerea antigen exists can be obviously judged (figure 2).

Example 5: kit detection sensitivity determination

Diluting the 1000 mug/mL botrytis cinerea antigen with the sample coating liquid by multiple times to be 1: 100X 2⁰～1:100×2¹⁴And the enzyme is coated on an enzyme label plate, and the concentration is from top to bottom from high to low. Repeat 8 times, column 12 is blank, coated with sample coating solution only.

The maximum dilution multiple of the 1000 mug/mL botrytis cinerea antigen obtained by detection is 3200 times, namely the detection sensitivity is as follows: 1000. mu.g/mL 3200.312.5 ng/mL, i.e., 31.5ng per well (FIG. 3).

Example 6: detection of type and subclass of monoclonal antibody

The monoclonal antibody was identified using a mouse monoclonal antibody subclass identification kit (Baiotton center for laboratory materials, lot: 20200809). 1000. mu.g/mL of Botrytis cinerea antigen diluted 1000-fold with sample coating solution was applied to an ELISA plate at 100. mu.L/well, 16 wells were repeated, incubated at 37 ℃ for 2h or placed in a4 ℃ apparatus for 12h, and washed 1 time with PBST after sample coating solution was discarded. Adding 100 μ L of monoclonal antibody diluted 1000 times with PBST into each well of ELISA plate, incubating at 37 deg.C for 30min, washing with PBST for 5 times, taking 100 μ L of each enzyme-labeled antibody of 8 detection antibody Ig types and subclasses in the kit, adding into each well, repeating each enzyme-labeled antibody for 2 times, incubating at 37 deg.C for 30min, washing with PBST for 5 times, adding TMB color developing solution, developing for 15min in dark place, and adding 50 μ L of 2M H₂SO₄The reaction was terminated. Detection OD of enzyme-linked immunosorbent assay (OD)_450nmThe type and subclass of the antibody Ig corresponding to the positive hole are the type and subclass of the antibody of the monoclonal antibody.

The antibody types of the BCA4 were determined to be Ig kappa and IgG2A by detection (FIG. 4).

Example 7: detection of monoclonal antibody binding proteins

The detection of the antibody binding protein by SDS-PAGE and Western blot technique includes the following steps:

(1) 5% (M/V) of the concentrated gel and 10% (M/V) of the separation gel were prepared.

(2) 80 mu L of botrytis cinerea antigen and 20 mu L of 5 multiplied sample buffer solution are mixed in a 1.5mL centrifuge tube, and are immediately placed in an ice bath for 3min after being subjected to metal bath at 99 ℃ for 10min, and then are centrifuged for 5min at 10000 r/min.

(3) Samples were added to the sample wells with a microsyringe, 2 wells for each sample, and protein Marker was added simultaneously.

(4) Electrophoresis: and (3) firstly keeping the constant voltage of 80V, changing the constant voltage of 120V after the bromophenol blue indicator enters the separation gel, and stopping electrophoresis when the bromophenol blue indicator moves to about 1cm of the lower opening of the gel plate.

(5) And (3) transferring the gel for 30min by a semi-dry transfer method at a constant pressure of 25V, and transferring the protein onto the nitrocellulose membrane.

(6) The transferred membrane was washed 4 times with PBST for 5min each.

(7) The membrane was immersed in 3% skim milk and sealed at room temperature for 1 h.

(8) The membrane was immersed in a 2000-fold diluted monoclonal antibody solution in 3% skim milk, shaken at 75r/min for 1h at room temperature, and incubated overnight at 4 ℃. Washing with the step (6).

(9) The membrane was immersed in 8000-fold dilution of horseradish peroxidase-labeled antibody with 3% BSA and shaken at room temperature for 1h at 75 r/min. Washing with the step (6).

(10) The membrane was immersed in 10mL of a freshly prepared DAB color developing solution, slowly shaken away from light until color development was achieved, and the color development reaction was stopped with distilled water. The band developed on the membrane is the specific protein combined by the antibody, and the relative molecular weight of the combined protein is calculated according to the protein Marker.

The antibody was detected to specifically bind to a protein having a molecular weight of about 35kDa and 47kDa from Botrytis cinerea (FIG. 5).

Finally, it should also be noted that the above list is only a specific implementation example of the present invention. It is obvious that the invention is not limited to the above examples of facts, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.