Method for detecting Tilletia foetida spores by combining ICP-MS with nanogold

Abstract

The invention discloses a method for detecting Tilletia foetida spores by combining ICP-MS with nanogold, and belongs to the technical field of biological detection.The method comprises the steps that a biotin solution reacts with a Tilletia foetida spore polyclonal antibody to obtain a biotin-labeled polyclonal antibody, streptavidin magnetic beads are added, incubation is conducted to obtain immunomagnetic beads, chloroauric acid is added into deionized water, and the immunomagnetic beads are added into the deionized water; the method comprises the following steps: adding sodium citrate after boiling to obtain a nano-gold solution, uniformly mixing the nano-gold solution with a Tilletia foetida spore monoclonal antibody, adding BSA, centrifuging to obtain a nano-gold probe, closing immunomagnetic beads, adding a sample solution containing Tilletia foetida spores, and performing magnetic separation to obtain a spore-magnetic bead compound; the method comprises the following steps: sealing a nanogold probe with a skim milk powder solution, adding a spore-magnetic bead compound, performing magnetic separation to obtain a magnetic bead-spore-nanogold compound, adding a citric acid solution into the magnetic bead-spore-nanogold compound, performing desorption to obtain a desorption solution, and introducing the desorption solution into ICP-MS for detection, so that the sensitivity is high, and the detection is rapid.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a method for detecting Tilletia foetida spores by combining ICP-MS with nano-gold. Background Art

[0002] As one of the main food grains, wheat plays a crucial role in ensuring food security. According to statistics, the sown area of wheat accounts for 20.4% of the total sown area of grains, with a yield of approximately 140 million tons, accounting for 20.1% of the total grain output. The annual purchase, sales, and inventory of wheat all account for about one-third of the total grain volume, making it a crop variety related to food security. Wheat bunt, also known as black smut, black wheat, and fetid black wheat, is one of the world's major diseases of wheat. Once it spreads, the consequences are extremely serious. Generally, it can cause a 10% - 30% reduction in production, and in severe cases, the reduction can reach 50%. Moreover, the fungal spores contain toxic substances and emit stinky trimethylamine-like substances. If humans and livestock consume the flour and wheat grains contaminated with this disease, it will cause poisoning and death, seriously threatening the physical and mental health of the people.

[0003] Research shows that the spores of Tilletia foetida have extremely strong viability. In the natural environment, they can survive in the soil for 2 - 3 years, and some can even survive for up to 20 years. When the number of teliospores reaches a certain level and suitable environmental and host conditions are available, they can reinfect and cause plant diseases. It should be noted that the fungus still has activity after being ingested and digested by livestock and excreted. Moreover, the teliospores have strong heat resistance and can only be inactivated under the conditions of dry heat at 130°C for 30 minutes or wet heat at 80°C for 20 minutes. The existing detection methods for Tilletia foetida spores mainly include morphological identification and laboratory detection methods of molecular biology represented by polymerase chain reaction (PCR). However, due to their cumbersome operation and long detection cycle, they are not suitable for on-site rapid detection of agricultural products. In addition, the relevant standard technologies focus on the detection of Tilletia indica and Tilletia controversa, and there is still a lack of detection methods for common bunt diseases represented by Tilletia foetida and Tilletia caries.

[0004] Therefore, this application proposes to introduce a new amplification strategy into the detection of Tilletia foetida spores based on inductively coupled plasma mass spectrometry (ICP-MS), to construct a new method for fungal spore detection that is rapid, accurate, sensitive, and interference-resistant, and at the same time to broaden the application scope of ICP-MS in the field of food fungal contamination. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned existing technologies, the present application provides a method for detecting Tilletia foetida spores by ICP-MS combined with nanogold, which solves the problems existing in the prior art that the detection of Tilletia tritici spores mainly relies on morphological identification and laboratory detection methods of molecular biology represented by polymerase chain reaction (PCR). However, due to its cumbersome operation and long detection cycle, it is not suitable for on-site rapid detection of agricultural products. Moreover, the relevant standard technologies focus on the detection of Tilletia indica and Tilletia controversa, and there are still deficiencies in the detection methods for common bunt diseases represented by Tilletia foetida and Tilletia caries.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for detecting Tilletia foetida spores by ICP-MS combined with nanogold, comprising the following steps:

[0008] Step 1: React 10 μL of biotin solution with 300 μL of polyclonal antibody against Tilletia foetida spores according to the volume ratio to obtain biotin-labeled polyclonal antibody;

[0009] Step 2: Wash 100 μL of streptavidin magnetic beads 3 times with 100 μL of PBS solution according to the volume ratio, then add 100 μL of biotin-labeled polyclonal antibody, incubate and react, and then wash with PBST solution to obtain immunomagnetic beads;

[0010] Step 3: Add 3 mL of 2% (w / v) HAuCl 4 to 180 mL of deionized water, heat to 140 °C under stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue to heat for 15 min until the solution color turns wine red and no longer changes, stop heating and continue to stir and cool to room temperature to obtain AuNPs nanogold solution;

[0011] Step 4: Take 1.0 mL of AuNPs nanogold solution according to the volume ratio, first adjust the pH to 8.0 - 9.0 with 0.1 mol / L K 2 CO 3 mix it with 15 - 30 μL of 1 mg / mL monoclonal antibody against Tilletia foetida spores, and react overnight at 4 °C. Then add 100 μL of 5% BSA and oscillate and block at 37 °C for 1 h to stabilize the nanogold; centrifuge at 12000 rpm for 10 min to remove the free antibody to obtain the nanogold probe;

[0012] Step 5: Take 50 μg of immunomagnetic beads according to the mass-to-volume ratio. After washing twice with PBS, add 100 μL of 1% skim milk powder solution and incubate for 30 min. Remove the skim milk powder solution by magnetic separation. Add 100 μL of the sample solution containing Tilletia foetida spores, mix well, and place it on a constant temperature shaker set at 37 °C with a rotation speed of 120 rpm for reaction for 15 - 90 min. Use magnetic separation to remove the supernatant from the obtained complex, and wash it 3 times with PBS to obtain the spore-magnetic bead complex;

[0013] Step 6: Take 200 μL of 1% skim milk powder solution according to the volume ratio to block 10 μL of nanogold probe for 1 h. Then add it to the spore-magnetic bead complex, mix well, and place it on a constant temperature shaker at 37 °C with a rotation speed of 120 rpm for labeling for 15 - 90 min; After the labeling is completed, remove the excess nanogold by magnetic separation, wash it twice with 200 μL of PBST, and then wash it once with 200 μL of PBS. Remove the supernatant by magnetic separation to obtain the magnetic bead-spore-nanogold complex for subsequent desorption;

[0014] Step 7: Take 100 μL of 0.25 - 2 mol / L citric acid solution and add it to the magnetic bead-spore-nanogold complex. After vortex mixing, desorb it at room temperature for 10 min to obtain the desorbed solution. After magnetic separation, take 100 μL of the desorbed solution and introduce it into ICP-MS to measure the response signal intensity of Au, and calculate the concentration of Tilletia foetida spores after substituting it into the standard curve.

[0015] Preferably, in the first step, the biotin solution is conjugated biotin; the polyclonal antibody against Tilletia foetida spores is a polyclonal antibody against Tilletia foetida spores prepared by immunizing 13-week-old male New Zealand white rabbits.

[0016] Preferably, the preparation steps of the polyclonal antibody against Tilletia foetida spores are as follows: Inject 1 mL of the complete suspension of Tilletia foetida spores subcutaneously into the back of the neck of male New Zealand white rabbits 3 times at intervals of 7 days for immunization; Take carotid artery blood 14 days after the last immunization; Incubate the plasma at 37 °C for 1 h and centrifuge at 500 g for 30 min; Collect the serum, package it, and store it at -80 °C for later use.

[0017] Preferably, the preparation method of the suspension of Tilletia foetida spores is as follows: Take 1 g of wheat infected with Tilletia foetida, place it in a 50 mL centrifuge tube, add 20 mL of sterile water, shake it for 12 h and then break it, filter it through a 150-mesh sieve, centrifuge it, and discard the supernatant. Add 0.25% sodium hypochlorite aqueous solution and let it stand at room temperature for 1 min for surface disinfection, and then wash it 3 times with sterile water to obtain the suspension of Tilletia foetida spores. Count it using a hemocytometer and store it at 4 °C for later use.

[0018] Preferably, in the first step and the second step, the reaction temperature is 37 °C and the oscillating reaction is carried out for 45 min.

[0019] Preferably, the monoclonal antibody against Tilletia foetida spores in the fourth step is a monoclonal antibody against Tilletia foetida spores prepared by immunizing 6-8-week-old Blbc mice.

[0020] Preferably, the specific preparation steps of the monoclonal antibody against Tilletia foetida spores in the fourth step are as follows: after breaking Tilletia foetida spores, immunize mice to produce antibodies against antigens, then perform fusion screening to obtain stable monoclonal cell lines. After culturing the cell lines, inject them into the abdominal cavity of mice, purify and collect the antibodies from the ascites, and store them at -80 °C for later use.

[0021] Preferably, in the fourth step, after centrifuging at 12,000 rpm for 10 min to remove free antibodies, wash three times with an equal volume of 0.01 mol / L salt-free PBS with pH = 8.0 - 9.0 and centrifuge to remove excess antibodies. Finally, disperse it in salt-free PBS with the same volume as the AuNPs nanogold solution for reconstitution to obtain nanogold probes.

[0022] Preferably, the preparation method of the sample solution containing Tilletia foetida spores in the fifth step is to dilute the concentration of Tilletia foetida spores to 200 - 50,000 / mL with PBS.

[0023] Preferably, the preparation method of the 1% skim milk powder solution in the fifth step is to dilute skim milk powder and pure water according to a volume ratio of 1:100.

[0024] Principle explanation: The analytical strategy based on elemental labeling and inductively coupled plasma-mass spectrometry (ICP-MS) detection has been widely applied to the field of biological detection and analysis, including protein quantitative analysis, nucleic acid analysis, cell counting analysis, pathogen analysis such as bacteria and virus particles, single-cell analysis, and mass spectrometry imaging analysis of cells and tissues, etc.; in these biological analysis applications, ICP-MS exhibits high sensitivity, good resistance to matrix interference, and excellent multi-target simultaneous analysis ability; in this invention, immunomagnetic beads are used for the biological separation of spores in complex wheat samples, nanogold is used to label the captured spores, and ICP-MS is used for the highly sensitive detection of nanogold, so as to realize the analysis of target spores.

[0025] Beneficial effects:

[0026] 1. This application first uses inductively coupled plasma-mass spectrometry combined with nanogold to detect Tilletia foetida spores. Based on the polyclonal antibody / monoclonal antibody against Tilletia foetida spores, capture and enrich the spores through immunomagnetic beads, label them with nanogold tags, and then introduce ICP-MS for detection;

[0027] 2. The method for detecting Tilletia foetida spores by combining ICP-MS with nanogold in this application has the advantages of high sensitivity, simple pretreatment, rapid detection, etc. It can be used for the detection of trace Tilletia foetida spores in a large number of wheat samples, and can be used for the preliminary screening and prevention and control of bunt-infected wheat in various production and processing links such as harvesting, storage, and circulation, improving the safety supervision of grain production and processing;

[0028] 3. The method for detecting Tilletia foetida spores by combining ICP-MS with nanogold in this application can be applied to the early detection and application of various grain fungal contaminations, providing technical support for the supervision and prevention and control of grain safety;

[0029] 4. Compared with other methods, this method has the advantages of simplicity and rapidity; compared with other immunological and PCR techniques, the sample pretreatment of the new method is much simpler, shortening the time required for spore enrichment, separation, and detection from 24 hours to within 2 hours, greatly improving the detection efficiency; this method can detect as low as 200 spores / mL, and the sensitivity is much higher than that of sensory detection and other detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the principle for detecting Tilletia foetida spores by combining ICP-MS with nanogold in this application;

[0031] Figure 2 It is an electron micrograph of the immunomagnetic beads in this application;

[0032] Figure 3 It is an electron micrograph of the nanogold in this application;

[0033] Figure 4 It is an electron micrograph of the immunomagnetic beads capturing spores in this application;

[0034] Figure 5 It is a diagram showing the influence of immunomagnetic beads with different particle sizes on the detection signal in this application;

[0035] Figure 6 It is a diagram showing the influence of different capture times on the detection signal in this application;

[0036] Figure 7 It is a diagram showing the influence of different labeling times on the detection signal in this application;

[0037] Figure 8 It is a diagram showing the influence of different desorbents on the detection signal in this application;

[0038] Figure 9 It is a diagram showing the influence of different analyte concentrations on the detection signal in this application;

[0039] Figure 10 It is a diagram showing the influence of the number of desorption times on the detection signal in this application;

[0040] Figure 11 This is the standard curve graph of this application. Specific embodiments

[0041] The present invention will be further described in detail below in conjunction with embodiments. It should be understood that the following embodiments are only for the explanation and illustration of the present invention, but do not limit the present invention to these specific embodiments, and do not limit the scope of the present invention in any way.

[0042] The biotinylated conjugate used in this application was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: D601048-0200.

[0043] Male New Zealand white rabbits and 6-8-week-old Blbc mice were purchased from Absin (Shanghai) Biotechnology Co., Ltd.

[0044] Streptavidin magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd., product number: MB1003.

[0045] PBST solution was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: B640011-0010.

[0046] The sample solution of Tilletia foetida spores was taken from the wheat sample of Zhangjiagang Xizhang Grain and Oil Purchase and Sale Co., Ltd., and the concentration of Tilletia foetida spores was diluted to 200-50,000 / mL with PBS.

[0047] Skim milk powder solution was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: A600669-0250.

[0048] Example 1

[0049] A method for detecting Tilletia foetida spores by ICP-MS combined with gold nanoparticles, the preparation of immunomagnetic beads and the optimization of capture conditions, including the following steps:

[0050] Step 1: React 10 μL of biotin-conjugated with 300 μL of polyclonal antibody against Tilletia foetida spores according to the volume ratio to obtain biotin-labeled polyclonal antibody; the polyclonal antibody against Tilletia foetida spores is a polyclonal antibody against Tilletia foetida spores prepared by immunizing 13-week-old male New Zealand white rabbits. The preparation steps are as follows: Subcutaneously inject 1 mL of a complete suspension of Tilletia foetida spores into the back of the neck of male New Zealand white rabbits three times at intervals of 7 days for immunization; draw carotid artery blood 14 days after the last immunization; incubate the plasma at 37 °C for 1 h and centrifuge at 500 g for 30 min; collect the serum, package it, and store it at -80 °C for later use; the preparation method of the Tilletia foetida spore suspension is as follows: Take 1 g of wheat infected with Tilletia foetida, place it in a 50 mL centrifuge tube, add 20 mL of sterile water, shake it for 12 h and then break it, filter it through a 150-mesh sieve, discard the supernatant after centrifugation, add 0.25% sodium hypochlorite aqueous solution and let it stand at room temperature for 1 min for surface disinfection, and then wash it three times with sterile water to obtain a Tilletia foetida spore suspension, count it using a hemocytometer, and store it at 4 °C for later use;

[0051] Step 2: Select streptavidin magnetic beads with particle sizes of 300 nm, 500 nm, and 1 μm respectively. Dilute the 10 mg / mL streptavidin magnetic beads to 0.5 mg / mL. Wash 100 μL of streptavidin magnetic beads three times with 100 μL of PBS solution according to the volume ratio, then add 100 μL of biotin-labeled polyclonal antibody, react at 37 °C for 45 min, wash it with PBST solution after incubation reaction, and obtain immunomagnetic beads, and perform electron microscopy characterization on them. As Figure 2 shown, it is streptavidin magnetic beads with a particle size of 1 μm;

[0052] Step 3: Add 3 mL of 2% (w / v) HAuCl 4 to 180 mL of deionized water, heat it to 140 °C under stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue to heat for 15 min until the solution color turns wine red and no longer changes, stop heating and continue to stir and cool to room temperature to obtain AuNPs gold nanoparticle solution;

[0053] Step 4: Take 1.0 mL of the AuNPs gold nanoparticle solution according to the volume ratio and first use 0.1 mol / L K 2 CO 3Adjust the pH to 8.0 - 9.0, mix it with 15 - 30 μL of 1 mg / mL monoclonal antibody against Tilletia foetida spores, and react overnight at 4°C. Then add 100 μL of 5% BSA and incubate with shaking at 37°C for 1 h to stabilize the nanogold. After centrifuging at 12,000 rpm for 10 min to remove the free antibody, wash it three times with an equal volume of 0.01 mol / L salt-free PBS with pH = 8.0 - 9.0 and centrifuge to remove the excess antibody. Finally, redissolve it in salt-free PBS with the same volume as the AuNPs nanogold solution to obtain the nanogold probe. The monoclonal antibody against Tilletia foetida spores is a monoclonal antibody against Tilletia foetida spores prepared by immunizing 6 - 8-week-old Blbc mice. The specific preparation steps of the monoclonal antibody against Tilletia foetida spores are as follows: after breaking Tilletia foetida spores, immunize mice to produce antibodies against the antigen, then perform fusion screening to obtain a stable monoclonal cell line. Culture the cell line and inject it into the abdominal cavity of mice, and purify and collect the antibody from the ascites for storage at -80°C for later use;

[0054] Step 5: Take 50 μg of immunomagnetic beads according to the mass-volume ratio. After washing twice with PBS, add 100 μL of 1% skim milk powder solution and incubate for 30 min. Remove the skim milk powder solution by magnetic separation. Add 100 μL of the sample solution containing Tilletia foetida spores, mix well, and react on a constant temperature shaker set at 37°C with a rotation speed of 120 rpm for 15 - 90 min. Use magnetic separation to remove the supernatant of the obtained complex, wash it 3 times with PBS to obtain the spore-magnetic bead complex, and perform electron microscopy characterization on it, as Figure 4 shown; The preparation method of the sample solution containing Tilletia foetida spores is to dilute the concentration of Tilletia foetida spores to 200 - 50,000 / mL with PBS; The preparation method of the 1% skim milk powder solution is to dilute skim milk powder and pure water at a volume ratio of 1:100;

[0055] Step 6: Take 200 μL of 1% skim milk powder solution according to the volume ratio to block 10 μL of the nanogold probe for 1 h, then add it to the spore-magnetic bead complex, mix well, and incubate on a constant temperature shaker at 37°C with a rotation speed of 120 rpm for 15 - 90 min; The presence of milk powder can reduce the non-specific adsorption of magnetic beads to nanogold and reduce the blank of the method. After the labeling is completed, use magnetic separation to remove the excess nanogold, wash it twice with 200 μL of PBST, and then wash it once with 200 μL of PBS. Use magnetic separation to remove the supernatant to obtain the magnetic bead-spore-nanogold complex for subsequent desorption;

[0056] Step 7: Add 100 μL of 1 mol / L citric acid solution to the magnetic bead-spore-nanogold complex. After vortex mixing, desorb at room temperature for 10 min to obtain the desorbed solution. After magnetic separation, take 100 μL of the desorbed solution and introduce it into ICP-MS for detection. Measure the response signal intensity of Au. As Figure 5 and Figure 6 shown, after substituting into the standard curve, the concentration of Tilletia foetida spores is calculated.

[0057] Figure 5 The results show that the response signal value of streptavidin magnetic beads with a particle size of 1 μm is significantly higher than that of 300 nm and 500 nm, indicating that the capture effect on spores is better under this condition. Therefore, immunomagnetic beads with a particle size of 1 μm are selected for the experiment.

[0058] Figure 6 It shows that with the increase of the capture time, the detection signal intensity also decreases. A reaction time of 15 minutes is sufficient to complete the capture of the target spores. Therefore, 15 minutes is selected as the capture time for this experiment.

[0059] Example 2

[0060] A method for detecting Tilletia foetida spores by ICP-MS combined with nanogold, and the preparation of nanogold probes and the optimization of labeling conditions, including the following steps:

[0061] Step 1: React 10 μL of biotin-coupled with 300 μL of polyclonal antibody against Tilletia foetida spores of wheat by volume ratio to obtain biotin-labeled polyclonal antibody. The polyclonal antibody against Tilletia foetida spores of wheat is a polyclonal antibody against Tilletia foetida spores of wheat prepared by immunizing 13-week-old male New Zealand white rabbits. The preparation steps are as follows: Inject 1 mL of a complete suspension of Tilletia foetida spores of wheat subcutaneously into the back of the neck of male New Zealand white rabbits 3 times at intervals of 7 days for immunization; Take carotid artery blood 14 days after the last immunization; Incubate the plasma at 37 °C for 1 h and centrifuge at 500 g for 30 min; Collect the serum, package it, and store it at -80 °C for later use; The preparation method of the suspension of Tilletia foetida spores of wheat is as follows: Take 1 g of wheat infected with Tilletia foetida, place it in a 50 mL centrifuge tube, add 20 mL of sterile water, shake it for 12 h and then break it, filter it through a 150-mesh sieve, discard the supernatant after centrifugation, add 0.25% sodium hypochlorite aqueous solution and let it stand at room temperature for 1 min for surface disinfection, and then wash it 3 times with sterile water to obtain the suspension of Tilletia foetida spores of wheat. Count with a hemocytometer and store it at 4 °C for later use;

[0062] Step 2: Respectively select streptavidin magnetic beads with particle sizes of 300 nm, 500 nm, and 1 μm. Dilute the 10 mg / mL streptavidin magnetic beads to 0.5 mg / mL. Wash 100 μL of streptavidin magnetic beads 3 times with 100 μL of PBS solution according to the volume ratio. Then add 100 μL of biotin-labeled polyclonal antibody and react at 37 °C for 45 min. After the incubation reaction, wash with PBST solution to obtain immunomagnetic beads, and perform electron microscopy characterization on them. As Figure 2 shown, it is streptavidin magnetic beads with a particle size of 1 μm;

[0063] Step 3: Add 3 mL of 2% (w / v) HAuCl 4 to 180 mL of deionized water, heat to 140 °C under stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue to heat for 15 min until the solution color turns wine red and no longer changes, stop heating and continue stirring to cool to room temperature to obtain AuNPs nanogold solution;

[0064] Step 4: Take 1.0 mL of the AuNPs nanogold solution according to the volume ratio. First, adjust the pH to 8.0 - 9.0 with 0.1 mol / L K 2 CO 3 . Mix it with 15 - 30 μL of 1 mg / mL monoclonal antibody against Tilletia foetida spores and react overnight at 4 °C. Then add 100 μL of 5% BSA and oscillate and block at 37 °C for 1 h to stabilize the nanogold; centrifuge at 12000 rpm for 10 min to remove the free antibody, then wash three times with an equal volume of 0.01 mol / L salt-free PBS with pH = 8.0 - 9.0 and centrifuge to remove the excess antibody. Finally, redissolve it in salt-free PBS with the same volume as the AuNPs nanogold solution to obtain a nanogold probe, and perform electron microscopy characterization on it. As Figure 3 shown; the monoclonal antibody against Tilletia foetida spores is a monoclonal antibody against Tilletia foetida spores prepared by immunizing 6 - 8-week-old Blbc mice. The specific preparation steps of the monoclonal antibody against Tilletia foetida spores are as follows: After breaking Tilletia foetida spores, immunize mice to produce antibodies against the antigen, then perform fusion screening to obtain a stable monoclonal cell line. Cultivate the cell line and inject it into the abdominal cavity of mice, and purify and collect the antibody from the ascites for storage at -80 °C for later use;

[0065] Step 5: Take 50 μg of immunomagnetic beads according to the mass-to-volume ratio. After washing twice with PBS, add 100 μL of 1% skim milk powder solution and incubate for 30 min. Remove the skim milk powder solution by magnetic separation. Add 100 μL of the sample solution containing Tilletia foetida spores, mix well, and place on a constant temperature shaker set at 37 °C with a rotation speed of 120 rpm for reaction for 45 min. The resulting complex is subjected to magnetic separation to remove the supernatant, and washed 3 times with PBS to obtain the spore-magnetic bead complex. The preparation method of the sample solution containing Tilletia foetida spores is to dilute the concentration of Tilletia foetida spores to 200 - 50,000 / mL with PBS. The preparation method of the 1% skim milk powder solution is to dilute skim milk powder and pure water at a volume ratio of 1:100.

[0066] Step 6: Take 200 μL of 1% skim milk powder solution according to the volume ratio to block 10 μL of the nanogold probe for 1 h, and then add it to the spore-magnetic bead complex. After mixing well, place it on a constant temperature shaker at 37 °C with a rotation speed of 120 rpm for labeling for 15, 30, 45, 60, 75, 90 min. The presence of milk powder can reduce the non-specific adsorption of magnetic beads to nanogold and reduce the blank of the method. After the labeling is completed, the excess nanogold is removed by magnetic separation, washed 2 times with 200 μL of PBST, and then washed once with 200 μL of PBS. The supernatant is removed by magnetic separation to obtain the magnetic bead-spore-nanogold complex for subsequent desorption.

[0067] Step 7: Take 100 μL of 1 mol / L citric acid solution and add it to the magnetic bead-spore-nanogold complex. After vortex mixing, desorb at room temperature for 10 min to obtain the desorbed solution. After magnetic separation, take 100 μL of the desorbed solution and introduce it into ICP-MS for detection. Measure the response signal intensity of Au, and calculate the concentration of Tilletia foetida spores after substituting into the standard curve.

[0068] Figure 7 It is shown that as the labeling time increases, the detection signal shows a trend of first increasing and then decreasing, indicating that the non-specific adsorption of nanogold will increase with the prolongation of the labeling time. Therefore, this experiment selects 45 min with a higher detection signal to carry out the experiment.

[0069] Example 3

[0070] A method for detecting Tilletia foetida spores by ICP-MS combined with nanogold, optimization of desorption conditions, includes the following steps:

[0071] Step 1: React 10 μL of biotin-conjugated with 300 μL of polyclonal antibody against Tilletia foetida spores according to the volume ratio to obtain biotin-labeled polyclonal antibody; the polyclonal antibody against Tilletia foetida spores is a polyclonal antibody against Tilletia foetida spores prepared by immunizing 13-week-old male New Zealand white rabbits. The preparation steps are as follows: Inject 1 mL of a complete suspension of Tilletia foetida spores subcutaneously into the back of the neck of male New Zealand white rabbits three times at 7-day intervals; draw carotid artery blood 14 days after the last immunization; incubate the plasma at 37 °C for 1 h and centrifuge at 500 g for 30 min; collect the serum, package it, and store it at -80 °C for later use; the preparation method of the Tilletia foetida spore suspension is as follows: Take 1 g of wheat infected with Tilletia foetida, place it in a 50 mL centrifuge tube, add 20 mL of sterile water, shake it for 12 h and then break it, filter it through a 150-mesh sieve, discard the supernatant after centrifugation, add 0.25% sodium hypochlorite aqueous solution and let it stand at room temperature for 1 min for surface disinfection, and then wash it three times with sterile water to obtain a Tilletia foetida spore suspension, count it using a hemocytometer, and store it at 4 °C for later use;

[0072] Step 2: Select streptavidin magnetic beads with particle sizes of 300 nm, 500 nm, and 1 μm respectively. Dilute 10 mg / mL streptavidin magnetic beads to 0.5 mg / mL. Wash 100 μL of streptavidin magnetic beads three times with 100 μL of PBS solution according to the volume ratio, then add 100 μL of biotin-labeled polyclonal antibody, react at 37 °C for 45 min, and wash with PBST solution after incubation to obtain immunomagnetic beads;

[0073] Step 3: Add 3 mL of 2% (w / v) HAuCl 4 to 180 mL of deionized water, heat it to 140 °C with stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue to heat for 15 min until the solution color turns wine red and no longer changes, stop heating and continue to stir and cool to room temperature to obtain AuNPs gold nanoparticle solution;

[0074] Step 4: Take 1.0 mL of AuNPs gold nanoparticle solution according to the volume ratio and first use 0.1 mol / L K 2 CO 3Adjust the pH to 8.0 - 9.0, mix it with 15 - 30 μL of 1 mg / mL monoclonal antibody against Tilletia foetida spores of wheat, and react overnight at 4°C. Then add 100 μL of 5% BSA and incubate with shaking at 37°C for 1 h to stabilize the gold nanoparticles. Centrifuge at 12,000 rpm for 10 min to remove the free antibody, wash three times with an equal volume of 0.01 mol / L salt-free PBS with pH = 8.0 - 9.0 and centrifuge to remove the excess antibody. Finally, resuspend it in salt-free PBS with the same volume as the AuNPs gold nanoparticle solution to obtain the gold nanoprobe; the monoclonal antibody against Tilletia foetida spores of wheat is a monoclonal antibody against Tilletia foetida spores of wheat prepared by immunizing 6 - 8-week-old Blbc mice. The specific preparation steps of the monoclonal antibody against Tilletia foetida spores of wheat are as follows: after breaking Tilletia caries spores of wheat, immunize mice to produce antibodies against the antigen, then perform fusion screening to obtain a stable monoclonal cell line. Culture the cell line and inject it into the abdominal cavity of mice, purify and collect the antibody from the ascites, and store it at -80°C for later use;

[0075] Step 5: Take 50 μg of immunomagnetic beads according to the mass-volume ratio. After washing twice with PBS, add 100 μL of 1% skim milk powder solution and incubate for 30 min. Remove the skim milk powder solution by magnetic separation, add 100 μL of the sample solution containing Tilletia foetida spores of wheat, mix well, and react on a constant temperature shaker set at 37°C with a rotation speed of 120 rpm for 45 min. Use magnetic separation to remove the supernatant of the obtained complex, wash 3 times with PBS to obtain the spore-magnetic bead complex; the preparation method of the sample solution containing Tilletia foetida spores of wheat is to dilute the concentration of Tilletia foetida spores of wheat to 200 - 50,000 / mL with PBS; the preparation method of the 1% skim milk powder solution is to dilute skim milk powder and pure water according to the volume ratio of 1:100;

[0076] Step 6: Take 200 μL of 1% skim milk powder solution according to the volume ratio to block 10 μL of the gold nanoprobe for 1 h, then add it to the spore-magnetic bead complex, mix well, and incubate on a constant temperature shaker at 37°C with a rotation speed of 120 rpm for 15 - 90 min; the presence of milk powder can reduce the non-specific adsorption of magnetic beads to gold nanoparticles and reduce the blank of the method. After the labeling is completed, use magnetic separation to remove the excess gold nanoparticles, wash twice with 200 μL of PBST, then wash once with 200 μL of PBS, and use magnetic separation to remove the supernatant to obtain the magnetic bead-spore-gold nanoparticle complex for subsequent desorption;

[0077] Step 7: Respectively take 100 μL of formic acid, citric acid, acetic acid or nitric acid solution with a concentration of 0.25, 0.5, 1, 1.5 or 2 mol / L and add it to the magnetic bead-spore-nano gold complex. After vortex mixing, desorb at room temperature for 10 min to obtain the desorbed solution. After magnetic separation, take 100 μL of the desorbed solution and introduce it into ICP-MS for detection. Measure the response signal intensity of Au, and calculate the concentration of Tilletia foetida spores after substituting into the standard curve.

[0078] Figure 8 It shows that at the same concentration, the detection signal after desorption with citric acid is higher, and the blank value is also within the acceptable range. Therefore, citric acid is selected as the analytical agent for this experiment.

[0079] Figure 9 It shows that with the increase of the concentration of citric acid, the signal of the experimental group also increases, and it basically remains unchanged after reaching 1 mol / L. Therefore, 1 mol / L of citric acid is selected as the concentration of the analytical agent for this experiment.

[0080] Repeat the desorption 3 times with 1 mol / L of citric acid to compare the influence of the desorption times on the detection signal. Figure 10 It shows that with the increase of the desorption times, the detection signal decreases significantly. In order to improve the detection sensitivity, 1 desorption time is selected to carry out this experiment.

[0081] Example 4

[0082] A method for detecting Tilletia foetida spores by ICP-MS combined with nano gold, comprising the following steps:

[0083] Step 1: React 10 μL of biotinylated conjugate with 300 μL of polyclonal antibody against Tilletia foetida spores of wheat according to the volume ratio to obtain biotin-labeled polyclonal antibody; the polyclonal antibody against Tilletia foetida spores of wheat is a polyclonal antibody against Tilletia foetida spores of wheat prepared by immunizing 13-week-old male New Zealand white rabbits. The preparation steps are as follows: inject 1 mL of the complete suspension of Tilletia foetida spores of wheat subcutaneously into the back of the neck of male New Zealand white rabbits 3 times at intervals of 7 days for immunization; take carotid artery blood 14 days after the last immunization; incubate the plasma at 37 °C for 1 h and centrifuge at 500 g for 30 min; collect the serum, package it and store it at -80 °C for later use; the preparation method of the suspension of Tilletia foetida spores of wheat is as follows: take 1 g of wheat infected with Tilletia foetida, place it in a 50 mL centrifuge tube, add 20 mL of sterile water, oscillate for 12 h and then break it, filter it through a 150-mesh sieve, discard the supernatant after centrifugation, add 0.25% sodium hypochlorite aqueous solution and let it stand at room temperature for 1 min for surface disinfection, and then wash it 3 times with sterile water to obtain the suspension of Tilletia foetida spores of wheat. Count with a hemocytometer and store it at 4 °C for later use.

[0084] Step 2: Select streptavidin magnetic beads with particle sizes of 300 nm, 500 nm, and 1 μm respectively. Dilute the 10 mg / mL streptavidin magnetic beads to 0.5 mg / mL. Wash 100 μL of streptavidin magnetic beads 3 times with 100 μL of PBS solution according to the volume ratio. Then add 100 μL of biotin-labeled polyclonal antibody, and react at 37 °C for 45 min. After the incubation reaction, wash with PBST solution to obtain immunomagnetic beads;

[0085] Step 3: Add 3 mL of 2% (w / v) HAuCl 4 to 180 mL of deionized water, heat to 140 °C under stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue to heat for 15 min until the solution color turns wine red and no longer changes, stop heating and continue to stir and cool to room temperature to obtain the AuNPs nanogold solution;

[0086] Step 4: Take 1.0 mL of the AuNPs nanogold solution according to the volume ratio. First, adjust the pH to 8.0 - 9.0 with 0.1 mol / L K 2 CO 3 Mix it with 15 - 30 μL of 1 mg / mL monoclonal antibody against Tilletia foetida spores and react overnight at 4 °C. Then add 100 μL of 5% BSA and oscillate and block at 37 °C for 1 h to stabilize the nanogold; centrifuge at 12000 rpm for 10 min to remove the free antibody, and then wash three times with an equal volume of 0.01 mol / L salt-free PBS with pH = 8.0 - 9.0 and centrifuge to remove the excess antibody. Finally, redissolve it in salt-free PBS with the same volume as the AuNPs nanogold solution to obtain the nanogold probe; the monoclonal antibody against Tilletia foetida spores is a monoclonal antibody against Tilletia foetida spores prepared by immunizing 6 - 8-week-old Blbc mice. The specific preparation steps of the monoclonal antibody against Tilletia foetida spores are as follows: after breaking Tilletia foetida spores, immunize mice to produce antibodies against the antigen, then perform fusion screening to obtain a stable monoclonal cell line. After culturing the cell line, inject it into the abdominal cavity of mice, and purify and collect the antibody from the ascites for storage at -80 °C for later use;

[0087] Step 5: Take 50 μg of immunomagnetic beads according to the mass-to-volume ratio. After washing twice with PBS, add 100 μL of 1% skim milk powder solution and incubate for 30 min. Remove the skim milk powder solution by magnetic separation. Add 100 μL of the sample solution containing Tilletia foetida spores, mix well, and place on a constant temperature shaker set at 37°C with a rotation speed of 120 rpm for reaction for 15 - 90 min. Use magnetic separation to remove the supernatant from the resulting complex, and wash 3 times with PBS to obtain the spore-magnetic bead complex. The preparation method of the sample solution containing Tilletia foetida spores is to dilute the concentration of Tilletia foetida spores to 200 - 50,000 spores / mL with PBS; the preparation method of the 1% skim milk powder solution is to dilute skim milk powder and pure water at a volume ratio of 1:100;

[0088] Step 6: Take 200 μL of 1% skim milk powder solution according to the volume ratio to block 10 μL of the nanogold probe for 1 h, then add it to the spore-magnetic bead complex, mix well, and place on a constant temperature shaker at 37°C with a rotation speed of 120 rpm for labeling for 15 - 90 min; the presence of milk powder can reduce the non-specific adsorption of magnetic beads to nanogold and reduce the blank of the method. After the labeling is completed, use magnetic separation to remove the excess nanogold, wash 2 times with 200 μL of PBST, then wash once with 200 μL of PBS, and use magnetic separation to remove the supernatant to obtain the magnetic bead-spore-nanogold complex for subsequent desorption;

[0089] Step 7: Take 100 μL of 0.25 - 2 mol / L citric acid solution and add it to the magnetic bead-spore-nanogold complex. After vortexing and mixing, desorb at room temperature for 10 min to obtain the desorbed solution. After magnetic separation, take 100 μL of the desorbed solution and introduce it into ICP-MS for detection. Measure the response signal intensity of Au, and calculate the concentration of Tilletia foetida spores after substituting into the standard curve.

[0090] According to the detection results, with the spore concentration as the abscissa and the detection signal value as the ordinate, draw a standard curve, as Figure 11 shown. The linear range for detecting Tilletia foetida spores based on inductively coupled plasma - ICP-MS combined with nanogold is 200 - 50,000 spores / mL, the linear correlation coefficient R 2 = 0.9922, and the lowest detection limit is 200 spores / mL.

[0091] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification are only preferred examples of the present invention and do not limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting spores of Tilletia sutchuenensis using ICP-MS combined with nanogold, characterized in that: The following steps are involved: Step 1: react 10 μL of biotin solution with 300 μL of polyclonal antibody against spores of wheat smut fungus according to the volume ratio to obtain biotin-labeled polyclonal antibody; Step 2: Wash 100 μL of streptavidin magnetic beads with 100 μL of PBS solution three times according to the volume ratio, then add 100 μL of biotin-labeled polyclonal antibody, incubate the reaction, and then wash with PBST solution to obtain immunomagnetic beads; Step 3: Add 3 mL of 2% (w / v) HAuCl4 to 180 mL of deionized water by volume, heat to 140°C with stirring, and react for 15 min; then add 10 mL of 1% (w / v) sodium citrate, continue heating for 15 min until the solution color turns to wine red and does not change, stop heating and continue stirring to cool to room temperature to obtain AuNPs nanogold solution; Step 4: Take 1.0 mL of AuNPs nanogold solution by volume, adjust the pH to 8.0-9.0 with 0.1 mol / L K2CO3, mix it with 15-30 μL of 1 mg / mL wheat light smut fungus spore monoclonal antibody and react overnight at 4°C, then add 100 μL of 5% BSA and shake at 37°C for 1 hour to stabilize the nanogold; centrifuge at 12000 rpm for 10 minutes to remove free antibodies, and obtain the nanogold probe; Step 5: Take 50 μg of immunomagnetic beads by mass volume ratio, wash twice with PBS, add 100 μL of 1% skimmed milk powder solution, block for 30 minutes, remove the skimmed milk powder solution by magnetic separation, add 100 μL of sample solution containing wheat smut fungus spores, mix well and place on a constant temperature shaker set at 37°C with a speed of 120 rpm to react for 15-90 minutes, remove the supernatant of the obtained complex by magnetic separation, wash three times with PBS to obtain a spore-magnetic bead complex; Step 6: Take 200 μL of 1% skim milk powder solution by volume to block 10 μL of nanogold probe for 1 hour, then add it to the spore-magnetic bead complex, mix well and place on a constant temperature shaker at 37°C and 120 rpm for labeling for 15-90 minutes; after labeling, remove excess nanogold by magnetic separation, wash twice with 200 μL PBST, wash once with 200 μL PBS, remove supernatant by magnetic separation, and obtain the magnetic bead-spore-nanogold complex for subsequent desorption; Step 7: Take 100 μL of 0.25-2 mol / L citric acid solution and add it to the magnetic bead-spore-nanogold complex. After vortex mixing, desorb at room temperature for 10 min to obtain the desorption solution. After magnetic separation, take 100 μL of the desorption solution and introduce it into ICP-MS to determine the response signal intensity of Au. After entering the standard curve, the concentration of Ustilago farfara spores is calculated.

2. The method for detecting spores of Tilletia glabra using ICP-MS combined with nanogold according to claim 1, characterized in that: The biotin solution in the first step is conjugated biotin; the polyclonal antibody against spores of wheat stinking spores is prepared by immunizing 13-week-old male New Zealand white rabbits.

3. The method for detecting spores of Tilletia sutchuenensis by combining ICP-MS with nanogold according to claim 2, characterized in that: The preparation steps of the polyclonal antibody to wheat spores are as follows: 1 mL of complete wheat spore suspension is subcutaneously injected into the back of the neck of male New Zealand white rabbits for 3 times, with an interval of 7 days; carotid artery blood is collected 14 days after the last immunization; plasma is incubated at 37°C for 1 hour, and centrifuged at 500g for 30 minutes; serum is collected, packaged, and stored at -80°C for use.

4. The method for detecting spores of Tilletia sutchuenensis by combining ICP-MS with nanogold according to claim 3, characterized in that: The method for preparing the wheat spore suspension of light stinking smut fungus is as follows: 1 g of wheat infected with light stinking smut fungus is taken, placed in a 50 mL centrifuge tube, 20 mL of sterile water is added, the mixture is broken after oscillation for 12 hours, filtered through a 150-mesh sieve, the supernatant is discarded after centrifugation, a 0.25% sodium hypochlorite aqueous solution is added, and the mixture is allowed to stand at room temperature for 1 minute for surface disinfection, and then washed with sterile water for 3 times to obtain the wheat spore suspension of light stinking smut fungus, which is counted using a hemocytometer, and stored at 4°C for future use.

5. The method for detecting spores of Tilletia sutchuenensis by combining ICP-MS with nanogold according to claim 1, characterized in that: The reaction temperature in the first step and the second step is 37° C., and the shaking reaction is performed for 45 minutes.

6. The method for detecting spores of Tilletia glabra using ICP-MS combined with nanogold according to claim 1, characterized in that: The monoclonal antibody against spores of wheat tinderica spores in the fourth step is prepared by immunizing 6-8 week old Blbc mice.

7. The method for detecting spores of Tilletia glabra using ICP-MS combined with nanogold according to claim 6, characterized in that: The specific preparation steps of the monoclonal antibody against spores of wheat smut fungus in the fourth step are as follows: crushing the spores of wheat smut fungus, immunizing mice to produce antibodies against the antigen, performing fusion screening to obtain a stable monoclonal cell line, culturing the cell line and injecting it into the peritoneal cavity of mice, purifying and collecting antibodies from the ascites, and storing them at -80°C for use.

8. The method for detecting spores of Tilletia glabra using ICP-MS combined with nanogold according to claim 1, characterized in that: In the fourth step, the free antibodies were removed by centrifugation at 12000 rpm for 10 min, and then washed three times with an equal volume of 0.01 mol / L salt-free PBS with a pH of 8.0-9.0 and centrifuged to remove excess antibodies. Finally, it was dispersed in an equal volume of salt-free PBS as the AuNPs nanogold solution to obtain a nanogold probe.

9. The method for detecting spores of Tilletia sutchuenensis by combining ICP-MS with nanogold according to claim 1, characterized in that: The method for preparing the sample solution containing wheat spores in the fifth step is to dilute the wheat spores to a concentration of 200-50,000 spores / mL with PBS.

10. The method for detecting spores of Tilletia glabra using ICP-MS combined with nanogold according to claim 1, characterized in that: The 1% skimmed milk powder solution in the fifth step is prepared by diluting skimmed milk powder and pure water in a volume ratio of 1:100.

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