Quantum dot luminescence technology detection method for animal epidemic diseases

Abstract

The invention discloses a quantum dot luminescence technology detection method for animal epidemic diseases, and the method comprises the following steps: collecting an epidemic disease sample, processing the sample, and obtaining an absorption peak range of the sample; selecting quantum dots by avoiding the absorption peak range of the sample, and selecting two antibodies at the same time; establishing a core-shell-shell structure for the quantum dots, and establishing a model of the quantum dots and the antibody through historical data to obtain an optimal molar ratio of the antibody to the quantum dots; preparing quantum dots with corresponding mass according to a molar ratio, and coupling the quantum dots with an antibody; coating the surface of the solid-phase carrier with another antibody, adding a sample, incubating, removing impurities, adding the quantum dot labeled antibody, and finally exciting the quantum dots and analyzing the content of pathogens in the sample. By detecting the absorption peak value of the sample and selecting the quantum dots, background interference can be effectively reduced, and the detection signal-to-noise ratio and sensitivity are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal epidemic detection, in particular to a quantum dot luminescence technology detection method for animal epidemic. BACKGROUND

[0002] Animal epidemic detection is of great significance to the healthy development of animal husbandry. Traditional detection methods have many drawbacks in practical application. During detection, background interference factors are complex, such as environmental stray light and impurities in the sample, which seriously reduce the signal-to-noise ratio and sensitivity of detection, making it difficult to accurately capture weak epidemic signals. Moreover, the spectra of different epidemic markers overlap with each other, causing frequent spectral cross phenomena, which easily leads to false positive or false negative results, greatly affecting the reliability of the detection results.

[0003] In terms of key detection materials quantum dots, the existing quantum dots have poor fluorescence quantum yield and luminescence intensity, resulting in insufficient detection signal strength and being easily disturbed. At the same time, the coupling process of antibodies and quantum dots lacks precise control, and the coupling rate is not ideal. In addition, the experimental process is complicated, and there is no effective model to determine the number of quantum dots and antibodies, making it difficult to improve the detection efficiency and accuracy, which cannot meet the current demand for rapid and accurate detection of animal epidemics.

[0004] At present, there is no effective solution to the problems in the related art. SUMMARY

[0005] In view of the problems in the related art, the present application proposes a quantum dot luminescence technology detection method for animal epidemic, to overcome the above technical problems existing in the prior art.

[0006] To this end, the specific technical solutions adopted by the present application are as follows:

[0007] A quantum dot luminescence technology detection method for animal epidemic, the method comprising the following steps:

[0008] S1, collecting epidemic samples and processing the samples, and obtaining the absorption peak range of the samples;

[0009] S2, selecting quantum dots away from the absorption peak range of the samples, and selecting two antibodies;

[0010] S3, establishing a core-shell-shell structure for the quantum dots, and establishing a model of quantum dots and antibodies through historical data to obtain the optimal molar ratio of antibodies and quantum dots;

[0011] S4, preparing quantum dots of corresponding mass according to the molar ratio, and coupling the quantum dots with one antibody;

[0012] S5, coat another antibody on the surface of the solid carrier, add the sample, incubate, remove the impurities, add the quantum dot labeled antibody, finally excite the quantum dot and analyze the content of the pathogen in the sample.

[0013] As a preferred embodiment, the collection of epidemic disease samples and the processing of the samples, while obtaining the absorption peak range of the sample, comprises the following steps:

[0014] S11, selecting appropriate samples according to the type of epidemic disease, including blood, tissue, saliva, and feces;

[0015] S12, pretreating the collected samples;

[0016] S13, detecting the absorption peak of the sample using an ultraviolet-visible spectrophotometer to obtain the absorption peak value of the sample.

[0017] All sample processing needs to pay attention to biological safety protection. High pathogenicity pathogens need to be operated in a BSL-3 laboratory, and are stored separately before and after inactivation to avoid cross contamination.

[0018] As a preferred embodiment, the detection of the absorption peak of the sample using an ultraviolet-visible spectrophotometer to obtain the absorption peak value of the sample comprises the following steps:

[0019] S131, setting a blank control group, and using the instrument to perform full-waveband scanning on the sample and the blank control group with a step of 1 nm, and recording the absorbance curve, identifying the characteristic absorption peak, and using the blank control to correct and deduct the scattering baseline;

[0020] S132, obtaining the final sample absorbance curve, marking the highest cap of the absorbance, and analyzing the sample absorbance curve to obtain the range of the sample absorption peak value.

[0021] As a preferred embodiment, the selection of quantum dots avoiding the absorption peak range of the sample, and the selection of two antibodies comprises the following steps:

[0022] S21, selecting quantum dots with an emission wavelength in a low interference waveband according to the avoidance interval of the sample absorption peak;

[0023] S22, selecting two antibodies, one of which is fixed on a solid carrier through thiol modification, and the other of which is coupled with quantum dots labeled with streptavidin;

[0024] S23, confirming that the two antibodies do not interfere with each other through competitive ELISA to ensure the stability of the sandwich structure.

[0025] The non-interference antibody binding ensures that only the target pathogen can be accurately combined with the two antibodies to form a sandwich structure, effectively excluding the interference of other non-target substances, greatly reducing the probability of false positive results, and making the detection results more truly reflect the presence of the target pathogen in the sample.

[0026] As a preferred embodiment, the core-shell-shell structure of the quantum dots is established, and a model of the quantum dots and the antibodies is established based on historical data to obtain the optimal molar ratio of the antibodies to the quantum dots, including the following steps:

[0027] S31, selecting materials of the core, the inner shell and the outer shell based on the target emission wavelength;

[0028] S32, layer-by-layer coating by thermal injection method, adjusting reaction temperature and time to optimize shell thickness, and verifying structure integrity and luminous efficiency by transmission electron microscopy and fluorescence spectrum;

[0029] S33, collecting historical experimental data including different quantum dot surface modification groups, antibody types, coupling conditions, molar ratios and corresponding fluorescence intensities, and establishing a formula of the number of core-shell-shell quantum dots to the number of antibodies based on the data to obtain the optimal molar ratio of the antibodies to the quantum dots.

[0030] As a preferred embodiment, the core-shell-shell structure of the quantum dots is established, and a model of the quantum dots and the antibodies is established based on historical data to obtain the optimal molar ratio of the antibodies to the quantum dots, including the following steps:

[0031] S331, collecting quantum dots, antibodies, coupling conditions and fluorescence intensity parameters, and determining core variables affecting the molar ratio;

[0032] S332, establishing a formula of the quantum dots and the antibodies, and the specific formula is:

[0033]

[0034] wherein N1 is the total number of antibodies successfully coupled with the quantum dots, N2 is the number of quantum dots, k d is a dissociation constant, indicating the affinity of the antibody to the quantum dots, k max is the maximum number of antibodies that can be combined with a single quantum dot;

[0035] Inputting the number of quantum dots and obtaining the number of antibodies, and finally obtaining the molar ratio of the antibodies to the quantum dots.

[0036] As a preferred embodiment, the core-shell-shell structure of the quantum dots is established, and a model of the quantum dots and the antibodies is established based on historical data to obtain the optimal molar ratio of the antibodies to the quantum dots, including the following steps:

[0037] S41, activate the quantum dots, and mix the quantum dots with the antibody according to a molar ratio after activation, and add a PBS buffer with a pH of 7.4;

[0038] S42, react for 2 hours at room temperature in the dark, add 1% BSA or 5% skimmed milk powder to block unbound sites, and incubate for 30 minutes at room temperature;

[0039] S43, remove free antibodies by centrifugation with an ultrafiltration tube, and further purify the coupling complex by using a Sephadex G-25 column after centrifugation.

[0040] As a preferred embodiment, the method of coating another antibody on the surface of a solid-phase carrier, adding a sample for incubation, removing impurities, adding quantum dot-labeled antibodies, exciting the quantum dots, and analyzing the content of the pathogen in the sample comprises the following steps:

[0041] S51, dilute the capture antibody to 1-10 μg / mL, add 100 μL per well, incubate overnight at 4°C or for 2 hours at 37°C, discard the coating solution, add 200 μL of blocking solution per well, incubate for 1 hour at 37°C, and finally wash 3 times with PBST;

[0042] S52, add 100 μL of the sample to the antibody-coated well, incubate for 1 hour at 37°C, and remove unbound impurities by washing 3 times with PBST

[0043] S53, add quantum dot-antibody conjugates, incubate for 1 hour at 37°C in the dark, and completely remove free quantum dots by washing 5 times with PBST;

[0044] S54, excite the quantum dots using a fluorescence instrument, and detect the emission wavelength;

[0045] S55, read the fluorescence intensity of each well using a fluorescence microplate reader, and calculate the concentration of the pathogen according to a standard curve;

[0046] S56, dilute the known concentration of the pathogen antigen by gradient, repeat the above steps, and fit to establish a quantitative relationship curve between the fluorescence intensity and the concentration of the target.

[0047] The present application has the following beneficial effects:

[0048] 1, the present application can effectively reduce the background interference during quantum detection by collecting epidemic samples and processing the samples, obtaining the absorption peak range of the samples, and selecting quantum dots according to the absorption peak range of the samples, can make the specific luminescent signal of the quantum dot-labeled antibody and the background form high contrast, thereby improving the detection signal-to-noise ratio and sensitivity, and avoiding false positive or false negative problems caused by spectral cross, thereby improving the reliability and stability of the detection results.

[0049] 2、The present application can improve the fluorescence quantum yield and the light intensity of quantum dots by establishing a core-shell-shell structure for quantum dots; in addition, the coupling rate of antibodies and quantum dots will be affected due to the establishment of the core-shell-shell structure for quantum dots, therefore, by establishing a model formula of the number of quantum dots and the number of antibodies based on historical data, not only can the number of quantum dots and antibodies be more accurately obtained, the coupling rate of quantum dots and antibodies can be improved, but also the experimental steps can be simplified to assist in accelerating the progress of the experiment. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0051] Figure 1 is a flow chart of a quantum dot luminescence technology detection method for animal epidemic diseases according to an embodiment of the present application. DETAILED DESCRIPTION

[0052] In order to further illustrate the embodiments, the present application provides drawings which are part of the disclosure of the present application, mainly used to illustrate the embodiments, and can be explained in combination with the related description of the specification to explain the operating principle of the embodiments. Those skilled in the art should understand other possible embodiments and advantages of the present application by referring to these contents. The components in the drawings are not drawn to scale, and similar component symbols are usually used to represent similar components.

[0053] According to an embodiment of the present application, a quantum dot luminescence technology detection method for animal epidemic diseases is provided.

[0054] The present application will be further described in combination with the drawings and specific embodiments, as shown in Figure 1 A quantum dot luminescence technology detection method for animal epidemic diseases according to an embodiment of the present application, the method comprises the following steps:

[0055] S1, collecting epidemic samples and processing the samples, and obtaining the absorption peak range of the samples;

[0056] Further, collecting epidemic samples and processing the samples, and obtaining the absorption peak range of the samples comprises the following steps:

[0057] S11, selecting appropriate samples according to the type of epidemic, including blood, tissue, saliva, and feces;

[0058] S12, pretreating the collected samples;

[0059] S13, using a UV-visible spectrophotometer to detect the absorption peak of the sample, and obtaining the absorption peak value of the sample.

[0060] It should be noted that different epidemic samples have different processing methods. Blood samples are usually collected using an anticoagulant tube, and then the serum or plasma is separated by centrifugation. It should be noted that high pathogenicity pathogens need to be inactivated by heating at 56°C for 30 minutes or adding 0.1% Triton X-100, and then extracting nucleic acids with a commercial kit or directly for antibody detection; tissue samples need to be quickly frozen and stored in the lesion area, homogenized and added with lysis buffer to break the cells, centrifuged to take the supernatant, and for bacterial infection, lysozyme can be added to digest the cell wall, and for prions, strong denaturing buffer is used for inactivation; saliva samples need to be centrifuged to remove mucus and cell fragments, and a stabilizer is added to preserve the integrity of nucleic acids, and for rabies virus, mouse neuroblastoma cells are used for separation and culture, and for Mycobacterium tuberculosis, NALC-NaOH is used to digest the mucus and then centrifuged to enrich; fecal samples need to be suspended in PBS to remove impurities, and polyvinylpyrrolidone is used to adsorb polyphenolic inhibitors, and for viral diarrhea samples, nucleic acids are extracted after chloroform inactivation, and for parasite eggs, saturated brine is used for enrichment, and for bacterial pathogens, selective enrichment culture is required.

[0061] Further, using a UV-visible spectrophotometer to detect the absorption peak of the sample, and obtaining the absorption peak value of the sample includes the following steps:

[0062] S131, setting a blank control group, and using the instrument to scan the sample and the blank control group in the full wave band, with a step of 1 nm, and recording the absorbance curve, identifying the characteristic absorption peak, and using the blank control to correct and deduct the scattering baseline;

[0063] S132, obtaining the final sample absorbance curve, marking the highest cap of absorbance, and analyzing the sample absorbance curve to obtain the range of sample absorption peak value.

[0064] It should be noted that different epidemic samples have different absorbance curves, and different epidemic samples have different absorbance curves. The absorption peak data obtained according to the absorbance curves of different epidemic samples are shown in Table 1 as follows:

[0065] Table 1: Sample Absorption Peak Data Table

[0066]

[0067] S2, selecting quantum dots away from the absorption peak range of the sample, and selecting two antibodies;

[0068] Further, selecting quantum dots away from the absorption peak range of the sample, and selecting two antibodies includes the following steps:

[0069] S21, selecting quantum dots with a low-interference wavelength band according to a sample absorption peak avoidance interval;

[0070] S22, selecting two antibodies, one of which is fixed on a solid carrier through thiol modification, and the other of which is coupled with streptavidin-labeled quantum dots;

[0071] S23, confirming that the two antibodies do not interfere with each other through competitive ELISA to ensure the stability of the sandwich structure.

[0072] S3, establishing a core-shell-shell structure of quantum dots, and establishing a model of quantum dots and antibodies through historical data to obtain an optimal molar ratio of antibodies and quantum dots;

[0073] Further, the establishment of a core-shell-shell structure of quantum dots and the establishment of a model of quantum dots and antibodies through historical data to obtain an optimal molar ratio of antibodies and quantum dots include the following steps:

[0074] S31, selecting the materials of the core, inner shell and outer shell based on the target emission wavelength;

[0075] S32, adjusting the reaction temperature and time to optimize the shell thickness through a thermal injection method, and verifying the structural integrity and luminescent efficiency through transmission electron microscopy and fluorescence spectroscopy;

[0076] S33, collecting historical experimental data, including different quantum dot surface modification groups, antibody types, coupling conditions, molar ratios and corresponding fluorescence intensities, and establishing a formula of the number of core-shell-shell quantum dots and the number of antibodies according to these data to obtain an optimal molar ratio of antibodies and quantum dots.

[0077] It should be noted that the establishment of a core-shell-shell structure of quantum dots can significantly improve the fluorescence quantum yield of quantum dots, enhance the luminescent intensity, and also optimize the emission spectrum to make it narrower and more symmetrical, thereby improving the purity and quality of luminescence;

[0078] Further, the collection of historical experimental data, including different quantum dot surface modification groups, antibody types, coupling conditions, molar ratios and corresponding fluorescence intensities, and the establishment of a formula of the number of core-shell-shell quantum dots and the number of antibodies according to these data to obtain an optimal molar ratio of antibodies and quantum dots include the following steps:

[0079] S331, collecting quantum dots, antibodies, coupling conditions, and fluorescence intensity parameters, and determining core variables that affect the molar ratio;

[0080] S332, establishing a formula of quantum dots and antibodies, and the specific formula is:

[0081]

[0082] Wherein, N1 is the total number of antibodies successfully coupled with quantum dots, N2 is the number of quantum dots, k d is the dissociation constant, indicating the affinity of the antibody to the quantum dot, k max is the maximum number of antibodies that can be bound by a single quantum dot;

[0083] Input the number of quantum dots and obtain the number of antibodies, and finally obtain the molar ratio of antibodies to quantum dots.

[0084] It should be noted that the model formula established is simulated by the following historical experimental data, as shown in Table Two:

[0085] Table Two: Quantum Dot and Antibody Quantity Table

[0086]

[0087]

[0088] S4, prepare the corresponding mass of quantum dots according to the molar ratio, and couple the quantum dots with an antibody;

[0089] Further, preparing the corresponding mass of quantum dots according to the molar ratio and coupling the quantum dots with an antibody includes the following steps:

[0090] S41, activate the quantum dots, and mix the quantum dots with the antibody according to the molar ratio after activation, and add PBS buffer solution with pH 7.4;

[0091] S42, react at room temperature for 2 hours, add 1% BSA or 5% skimmed milk powder to block the unbound sites, and incubate at room temperature for 30 minutes;

[0092] S43, remove the free antibody by centrifugation with an ultrafiltration tube, and further purify the coupled complex using a Sephadex G-25 column after centrifugation is completed

[0093] S5, coat another antibody on the surface of a solid carrier, add the sample for incubation, remove impurities, add quantum dot labeled antibody, finally excite the quantum dots and analyze the pathogen content in the sample.

[0094] Further, coating another antibody on the surface of a solid carrier, adding the sample for incubation, removing impurities, adding quantum dot labeled antibody, and finally exciting the quantum dots and analyzing the pathogen content in the sample include the following steps:

[0095] S51, dilute the capture antibody to 1-10 μg / mL, add 100 μL per well, incubate at 4°C overnight or 37°C for 2 hours, discard the coating solution, add 200 μL of blocking solution per well, incubate at 37°C for 1 hour, and finally wash with PBST for 3 times;

[0096] S52, 100 μL sample is added to the antibody-coated well, incubated at 37℃ for 1 hour, and washed 3 times with PBST to remove unbound impurities

[0097] S53, add quantum dot and antibody conjugate, incubate at 37℃ for 1 hour in the dark, and wash 5 times with PBST to completely remove free quantum dots;

[0098] S54, use a fluorescence instrument to excite quantum dots, and detect the emission wavelength;

[0099] S55, use a fluorescence enzyme-labeled instrument to read the fluorescence intensity of each well, and calculate the pathogen concentration according to the standard curve;

[0100] S56, gradient dilution of known concentration of pathogen antigen, repeat the above steps, and fit to establish the quantitative relationship curve between fluorescence intensity and target concentration.

[0101] It should be noted that by selecting two antibodies, an effective "sandwich" detection structure can be formed, which can more efficiently capture and label the target pathogen, enhance the detection signal, enable the detection method to identify a lower concentration of pathogen in the sample, and improve the lower limit of detection; in terms of repeatability of detection, the stable sandwich structure ensures that the binding mode of the antibody and the pathogen is relatively fixed during each detection process, reducing experimental errors.

[0102] In summary, the present application collects the epidemic sample and processes the sample, simultaneously obtains the absorption peak range of the sample, and selects quantum dots through the absorption peak range of the sample, which can effectively reduce the background interference during quantum detection, and by selecting quantum dots with different absorption peak values from the sample, the specific luminescence signal of the quantum dot labeled antibody can form high contrast with the background, thereby improving the detection signal-to-noise ratio and sensitivity, and avoiding false positive or false negative problems caused by spectral cross, thereby improving the reliability and stability of the detection results; the present application establishes a core-shell-shell structure for quantum dots, which can improve the fluorescence quantum yield of quantum dots and enhance the luminescence intensity; in addition, since the quantum dots establish a core-shell-shell structure, the coupling rate of the antibody and the quantum dots will be affected, therefore, by establishing a model formula of the number of quantum dots and the number of antibodies through historical data, not only the number of quantum dots and the number of antibodies can be more accurately obtained, the coupling rate of quantum dots and antibodies is improved, but also the experimental steps are simplified, and the progress of the experiment is accelerated.

[0103] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A quantum dot luminescence detection method for animal diseases, characterized in that, The method includes the following steps: S1. Collect disease samples and process the samples, while obtaining the absorption peak range of the samples; S2. Select quantum dots while avoiding the absorption peak range of the sample, and select two antibodies at the same time; S3. Establish a core-shell-shell structure for quantum dots and build a model of quantum dots and antibodies using historical data to obtain the optimal molar ratio of antibodies to quantum dots; S4. Prepare quantum dots of the appropriate mass according to the molar ratio, and conjugate the quantum dots with an antibody; S5. Coat another antibody onto the surface of a solid-phase carrier, add the sample for incubation and impurity removal, then add quantum dot-labeled antibody, and finally excite the quantum dots and analyze the pathogen content in the sample.

2. The quantum dot luminescence detection method for animal diseases according to claim 1, characterized in that, The process of collecting and processing disease samples, and obtaining the absorption peak range of the samples, includes the following steps: S11. Select appropriate samples based on the type of disease, including blood, tissue, saliva, and feces; S12. Preprocess the collected samples; S13. Use a UV-Vis spectrophotometer to detect the absorption peak of the sample and obtain the absorption peak value of the sample.

3. The quantum dot luminescence detection method for animal diseases according to claim 2, characterized in that, The process of using a UV-Vis spectrophotometer to detect the absorption peak of the sample and obtain the sample's absorption peak value includes the following steps: S131. Set up a blank control group, and use the instrument to perform full-band scanning of the sample and the blank control group with a step size of 1 nm, and record the absorbance curve, identify the characteristic absorption peak, and use the blank control for correction and subtract the scattering baseline. S132. Obtain the final sample absorbance curve, mark the highest absorbance peak, and analyze the sample absorbance curve to obtain the range of sample absorption peaks.

4. The quantum dot luminescence detection method for animal diseases according to claim 1, characterized in that, The selection of quantum dots, avoiding the absorption peak range of the sample, and the simultaneous selection of two antibodies includes the following steps: S21. Based on the sample absorption peak avoidance range, select quantum dots whose emission wavelength is located in the low interference band; S22. Select two antibodies: one antibody is immobilized on a solid support by thiol modification, and the other antibody is coupled to streptavidin-labeled quantum dots. S23. Confirm that the two antibodies bind independently through competitive ELISA to ensure the stability of the sandwich structure.

5. The quantum dot luminescence detection method for animal diseases according to claim 1, characterized in that, The process of establishing a core-shell-shell structure for quantum dots and creating a model of quantum dots and antibodies using historical data to obtain the optimal molar ratio of antibodies to quantum dots includes the following steps: S31. Select the materials for the core, inner shell, and outer shell based on the target emission wavelength; S32. The shell thickness was optimized by layer-by-layer coating through hot injection, and the reaction temperature and time were controlled. The structural integrity and luminescence efficiency were verified by transmission electron microscopy and fluorescence spectroscopy. S33. Collect historical experimental data, including different quantum dot surface modification groups, antibody types, coupling conditions, molar ratios and their corresponding fluorescence intensities, and establish a formula for the number of core-shell-shell quantum dots and the number of antibodies based on these data, so as to obtain the optimal molar ratio of antibodies to quantum dots.

6. The quantum dot luminescence detection method for animal diseases according to claim 5, characterized in that, The collection of historical experimental data, including different quantum dot surface modification groups, antibody types, coupling conditions, molar ratios, and their corresponding fluorescence intensities, and the establishment of a formula for the number of core-shell-shell quantum dots and the number of antibodies based on this data to obtain the optimal molar ratio of antibodies to quantum dots, includes the following steps: S331. Collect quantum dot, antibody, coupling conditions, and fluorescence intensity parameters, and determine the core variables affecting the molar ratio; S332. Establish the formula for quantum dots and antibodies. The specific formula is as follows: Where N1 is the total number of antibodies successfully coupled with quantum dots, N2 is the number of quantum dots, and k d k is the dissociation constant, representing the affinity between the antibody and the quantum dot. max This represents the maximum number of antibodies that a single quantum dot can bind to. Input the number of quantum dots and obtain the number of antibodies, and finally obtain the molar ratio of antibodies to quantum dots.

7. The quantum dot luminescence detection method for animal diseases according to claim 1, characterized in that, The preparation of quantum dots of appropriate mass according to the molar ratio and the conjugation of quantum dots with an antibody include the following steps: S41. Activate the quantum dots, and after activation, mix the quantum dots with the antibody at a molar ratio and add PBS buffer at pH 7.

4. S42, react at room temperature in the dark for 2 hours, add 1% BSA or 5% skim milk powder to block unbound sites, and incubate at room temperature for 30 minutes; S43. Remove free antibody by centrifugation using an ultrafiltration tube. After centrifugation, further purify using a Sephadex G-25 column and collect the conjugate complex.

8. The quantum dot luminescence detection method for animal diseases according to claim 1, characterized in that, The process of coating another antibody onto the surface of a solid-phase support, adding a sample for incubation and impurity removal, adding a quantum dot-labeled antibody, and finally exciting the quantum dots and analyzing the pathogen content in the sample includes the following steps: S51. Dilute the capture antibody to 1-10 μg / mL, add 100 μL to each well, incubate overnight at 4°C or for 2 hours at 37°C, then discard the coating solution, add 200 μL of blocking solution to each well, incubate at 37°C for 1 hour, and finally wash 3 times with PBST. S52. Add 100 μL of sample to the antibody-coated wells, incubate at 37°C for 1 hour, and wash three times with PBST to remove unbound impurities. S53. Add quantum dots and antibody conjugate, incubate at 37°C in the dark for 1 hour, and wash 5 times with PBST to completely remove free quantum dots; S54. Excite the quantum dots using a fluorescence spectrometer and detect the emission wavelength; S55. Use a fluorescence microplate reader to read the fluorescence intensity of each well and calculate the pathogen concentration based on the standard curve; S56. Serially dilute known concentrations of pathogen antigen, repeat the above steps, and fit to establish a quantitative relationship curve between fluorescence intensity and target concentration.

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