CN113588949A - Preparation and use method of canine coronavirus chemiluminescence detection reagent - Google Patents
Preparation and use method of canine coronavirus chemiluminescence detection reagent Download PDFInfo
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Abstract
The invention discloses a preparation method of a canine coronavirus chemiluminescence detection reagent, belonging to the technical field of biological detection preparations, and the specific preparation process comprises the following steps: preparation of magnetic microsphere-G19 conjugate: activating magnetic microspheres by using EDC and NHS, adding a canine coronavirus antibody G19, performing oscillation coupling, and storing in a magnetic microsphere buffer solution for later use; preparation of G15-acridinium ester conjugate: mixing G15 and acridinium ester, labeling, coupling, vibrating, sealing, purifying, and storing in luminous reagent buffer solution; and the method is used to obtain the luminosity RLU of the detection sample. The preparation and use methods of the chemiluminescence detection reagent fill the blank of the domestic canine coronavirus chemiluminescence detection reagent, and the qualitative, quantitative and rapid detection of the canine coronavirus antigen is really realized.
Description
Technical Field
The invention relates to the technical field of biological detection preparations, and particularly belongs to a preparation method and application of a detection reagent for canine coronavirus enteritis.
Background
Canine coronavirus enteritis is a viral infectious disease source caused by Canine Coronavirus (CCV), and can cause dogs to have gastroenteritis symptoms of different degrees, and is characterized by symptoms such as frequent vomiting, diarrhea, depression, anorexia and the like. The disease can occur all the year round, and is frequently seen in winter, the disease dog is the main infectious agent, and the dog can be infected by respiratory tract, digestive tract, excrement and pollutants. Once the disease occurs, the infection can be caused by the difficulty in controlling the same-house dogs of the same litter dogs, and the death rate of puppies is higher. Canine coronavirus enteritis is one of the most common viral infectious diseases in dogs. Aiming at the serious harm of canine coronavirus enteritis to dogs and people, the early and accurate screening of diseases is a key link for preventing infection and transmission of the diseases and ensuring the health of dogs. Currently, there are 3 immunological detection methods commonly used in clinical practice: enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, and immunofluorescence microsphere method. The ELISA method is a classic immunological method which is developed and applied earlier, has higher sensitivity and lower difficulty of vector standardization, but has slow detection speed, easy pollution and more complicated steps. The colloidal gold method is the most widely applied pet disease rapid diagnosis method in the current market, breaks through the limitation of the existing detection technology on personnel and places, shortens the detection time, is convenient and rapid to operate, but is easy to generate false positive, and has the problems of omission and the like. The immunofluorescence method can realize quantitative detection, but the false positive rate is high, so that the wide application of the method is influenced. A new detection technique, chemiluminescence, may be ideal for addressing the shortcomings of the detection methods described above.
The chemiluminescence immunoassay combines a high-sensitivity chemiluminescence assay technology with a high-specificity immunoreaction and is used for detecting various antigens, antibodies, hormones and the like. The method has the characteristics of higher sensitivity than ELISA, strong specificity, wide linear range, stable result, simplified operation and the like, is widely applied to the detection of clinical specimens, and is developed rapidly in recent years. Is a latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and time-resolved fluoroimmunoassay. The pet disease diagnosis is different from the human disease diagnosis, the scale of the pet hospital is far smaller than that of the human hospital, the conventional chemiluminescence detector has too large volume and complicated operation and is not suitable for the pet hospital, and along with the development of portability, rapidity and easy operation of the chemiluminescence detector, the chemiluminescence detection technology is inevitably applied to the rapid screening market of pet infectious diseases.
However, no practical and specific technical scheme is really researched, and the chemiluminescence immunoassay method is really used for early screening and detecting the canine coronavirus enteritis.
Disclosure of Invention
Aiming at the defects and shortcomings in the background technology, the invention provides a preparation method and a use method of a canine coronavirus chemiluminescence detection reagent, fills the blank of the canine coronavirus chemiluminescence detection reagent in China, and truly realizes qualitative, quantitative and rapid detection of canine coronavirus antigens.
In order to realize the purpose, the invention adopts the following technical scheme to realize the purpose:
a preparation method of a canine coronavirus chemiluminescence detection reagent comprises a magnetic microsphere-G19 conjugate and a G15-acridinium ester conjugate, and specifically comprises the following steps:
preparation of magnetic microsphere-G19 conjugate: activating magnetic microspheres by using EDC and NHS, adding a canine coronavirus antibody G19, performing oscillation coupling, sealing the magnetic microspheres by using a sealing solution, cleaning the magnetic microspheres in a magnetic frame, and storing the magnetic microspheres in a magnetic microsphere buffer solution for later use to obtain a magnetic microsphere-G19 conjugate;
preparation of G15-acridinium ester conjugate: centrifuging and concentrating a canine coronavirus antibody G15 and acridinium ester to remove impurities, then fully and uniformly mixing G15 and the acridinium ester for labeling and coupling, slightly shaking for labeling at room temperature, adding a luminescent confining liquid for sealing, and dialyzing to remove unbound acridinium ester; purifying by a Sephadex G50 gel chromatography column, and storing in a luminescent reagent buffer solution for later use to obtain a G15-acridinium ester conjugate;
the canine coronavirus chemiluminescence detection reagent comprises a magnetic microsphere-G19 conjugate and a G15-acridinium ester conjugate.
Further measures taken are: in the step of preparing the magnetic microsphere-G19 conjugate, a canine coronavirus antibody G19 is coupled for 4 hours in a shaking way, a magnetic microsphere confining liquid is sealed for 2 hours, and the magnetic microsphere is cleaned in a magnetic frame for 2 times;
in the step of preparing the G15-acridinium ester conjugate: according to the following steps of 4: and (3) fully and uniformly mixing G15 and acridinium ester according to the mass ratio of 1, carrying out label coupling, slightly shaking the label for 2 hours at room temperature, and adding a luminescent confining liquid for sealing for 1 hour.
Further measures taken are: in the step of preparing the magnetic microsphere-G19 conjugate, the carboxyl magnetic beads are activated by a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC/N-hydroxysuccinimide (NHS) method and then coupled with G19 to prepare antibody magnetic beads.
Further measures taken are: in the step of preparing the magnetic microsphere-G19 conjugate, EDC and NHS are added, oscillation activation is carried out for 30min in a 37 ℃ thermostat, canine coronavirus antibody G19 is added, oscillation reaction is carried out for 4h in the 37 ℃ thermostat, a magnetic microsphere confining liquid is sealed for 2h, the magnetic microspheres are washed in a magnetic frame for 2 times, and the magnetic microsphere buffer solution is diluted and then placed in a 4 ℃ refrigerator for storage for later use.
Further measures taken are: the magnetic microsphere confining liquid is prepared by mixing 0.05M Tris, 0.05% casein, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.8.
Further measures taken are: the magnetic microsphere buffer solution is prepared by mixing 0.05M Tris, 1.5% trehalose, 1% bovine serum albumin, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.4.
Further measures taken are: the luminous confining liquid is prepared by mixing 0.01M PBS, 0.15% lysine and 0.1% Proclin300, and the pH value of the confining liquid is 7.5.
Further measures taken are: the luminescence reagent buffer solution is prepared by mixing PBS, 1% trehalose, 0.5% TrionX-100, 0.1% Proclin300 and 0.5% casein, and the pH value is 6.3.
The application method of the dog coronavirus chemiluminescence detection reagent comprises the following steps of taking a dog vomit or excrement sample, properly diluting the dog vomit or excrement sample with a sample diluent, sequentially adding the diluted sample or standard or quality control product, a magnetic microsphere-G19 conjugate and a G15-acridine ester conjugate into a transparent reaction tube, oscillating and incubating the sample or standard or quality control product, the magnetic microsphere-G19 conjugate and the G15-acridine ester conjugate at room temperature for reaction, then cleaning the reaction tube under the action of a magnetic field, enabling the reaction tube to enter a detection darkroom, pumping a substrate excitation liquid A and a bottom excitation liquid B into the reaction tube by using a full-automatic luminometer, and finally reading relative luminosity RLU of each hole on the chemiluminescence meter.
Further measures taken are: sequentially adding 50 mu L of diluted sample or standard or quality control product, 30 mu L of magnetic microsphere-G19 conjugate and 40 mu L of LG 15-acridinium ester conjugate into a transparent reaction tube, performing shake incubation reaction for 10min at room temperature, and then washing for 3 times under the action of a magnetic field;
the substrate exciting liquid A is a mixture of 0.1M concentrated nitric acid, 0.15% hydrogen peroxide and 0.1% TrionX-100;
the substrate excitation solution B is a mixture of 0.35M sodium hydroxide and 2.5% TrionX-100.
Chemiluminescence immunoassay has been studied for a long time in the technical field of detection, and although researchers in the field have also realized that the method has great prospect in pet infectious disease detection, the research depth and application of chemiluminescence immunoassay in pet infectious disease detection are shallow, and no practical and practicable technical scheme is developed after countless experimental researches and tests, so that the research is once stopped, domestic chemiluminescence detection reagents for canine coronavirus are still blank, and chemiluminescence immunoassay is still guessed and conceived when the chemiluminescence immunoassay is applied to pet infectious disease, particularly to detection of canine new coronavirus.
The magnetic microsphere-G19 conjugate and the G15-acridine ester conjugate are successfully prepared through specific steps, so that the detection of the canine coronavirus antigen by using a chemiluminescence immunoassay method is really realized, the blank that no canine coronavirus chemiluminescence detection reagent exists in China is filled, a technical foundation is laid for further research on a detection technology, and a technical support is provided; the method provides convenience and creates opportunity for discovering and treating canine coronavirus enteritis as soon as possible.
The detection reagent prepared by the method has the characteristics of rapid detection, high sensitivity and good accuracy, and can realize qualitative and quantitative detection of the canine coronavirus antigen, so that an experienced doctor can judge the disease duration and the following disease trend according to the content of the canine coronavirus, and more accurate treatment measures and schemes can be provided conveniently and then.
Through continuous and intensive research, the invention discovers that the weight ratio of the organic silicon compound is 4: mixing G15 and acridinium ester fully and uniformly in a mass ratio of 1, and performing label coupling; the magnetic microsphere confining liquid is prepared by mixing 0.05M Tris, 0.05% casein, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.8; the magnetic microsphere buffer solution is prepared by mixing 0.05M Tris, 1.5 percent of trehalose, 1 percent of bovine serum albumin, 0.05 percent of Triton X-100 and 0.01 percent of Proclin300, and the pH value is 7.4; and a luminescent reagent buffer solution and the like are controlled by special components and proportion, so that the preparation of the high-sensitivity magnetic microsphere-G19 conjugate and the G15-acridinium ester conjugate can be better ensured, the accuracy and the sensitivity of a detection result are ensured, and the qualitative and quantitative analysis of the canine coronavirus is carried out.
The invention also provides a using method of the dog coronavirus detection reagent, and the method comprises the specific steps and adopts a full-automatic luminometer to pump a specific substrate excitation liquid A and a specific bottom excitation liquid B in sequence, so that the relative luminosities RLU of all holes can be read on a chemiluminescence meter, the luminosities RLU of the samples can be obtained, and the method can be used for detecting the luminosities RLU of the standard products.
Preparing a standard substance antigen concentration standard curve by detecting the obtained standard substance luminosity RLU value, then utilizing the luminosity RLU value to be in positive correlation with the antigen concentration in the sample, substituting the luminosity RLU value into the sample, then carrying out quantitative calculation on the canine coronavirus antigen concentration according to the standard curve, and judging whether to infect the canine coronavirus according to a qualitative standard, thereby realizing the qualitative and quantitative analysis of the canine coronavirus antigen.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the detection reagent for the canine coronavirus is successfully prepared by the invention, so that the detection of the canine coronavirus antigen by using a chemiluminescence immunoassay method is really realized, the blank that no canine coronavirus chemiluminescence detection reagent exists in China is filled, and a technical basis is laid for further researching the detection of the pet infectious disease; the method provides convenience and creates opportunity for discovering and treating canine coronavirus enteritis as soon as possible.
2. The detection reagent prepared by the invention has the characteristics of rapid detection, high sensitivity and good accuracy, and can realize qualitative and quantitative detection of the canine coronavirus antigen, so that an experienced doctor can judge the disease duration and the following disease trend according to the content of the canine coronavirus, and more accurate treatment measures and methods can be conveniently provided.
2. The invention also provides a using method of the dog coronavirus detection reagent, the relative luminosity RLU of each hole can be read on a chemiluminescence apparatus by the method, so that the luminosity RLU of the sample can be obtained, and the luminosity RLU of the standard can be detected by the method; by obtaining the luminosity RLU of the sample and the standard substance, the standard curve of the canine coronavirus can be conveniently prepared after subsequent data processing, and the concentration of the canine coronavirus antigen can be quantitatively calculated according to the standard curve after the standard curve is substituted into the luminosity RLU of the sample.
Drawings
FIG. 1 is a standard graph of a canine coronavirus chemiluminescent reagent.
In the figure: the abscissa represents the concentration of circovirus and the ordinate represents the luminescence value RLU.
Detailed Description
In order to clearly understand the technical solutions adopted by the present invention, the following description is made on the preferred embodiments of the present invention, and it should be understood that the embodiments described herein are only used for illustrating and explaining the present invention, and are not used to limit the present invention.
Example 1: a preparation method of a canine coronavirus chemiluminescence detection reagent comprises a magnetic microsphere-G19 conjugate and a G15-acridinium ester conjugate, and specifically comprises the following steps:
preparation of magnetic microsphere-G19 conjugate: activating magnetic microspheres by using EDC and NHS, adding a canine coronavirus antibody G19, performing oscillation coupling for 4h, sealing the magnetic microspheres in a sealing solution for 2h, cleaning the magnetic microspheres in a magnetic frame for 2 times, and then storing the magnetic microspheres in a magnetic microsphere buffer solution for later use to obtain a magnetic microsphere-G19 conjugate;
preparation of G15-acridinium ester conjugate: the canine coronavirus antibody G15 and acridinium ester were concentrated by centrifugation to remove impurities, and the ratio of the antibody to the acridinium ester was adjusted according to 4: mixing G15 and acridinium ester uniformly by mass ratio of 1, performing label coupling, slightly shaking the label at room temperature for 2h, adding luminescent blocking liquid, sealing for 1h, and dialyzing to remove unbound acridinium ester; purifying by a Sephadex G50 gel chromatography column, and storing in a luminescent reagent buffer solution for later use to obtain a G15-acridinium ester conjugate;
and obtaining a finished product of the chemiluminescence detection reagent by obtaining a magnetic microsphere-G19 conjugate and a G15-acridinium ester conjugate.
In the step of preparing the magnetic microsphere-G19 conjugate, activating carboxyl magnetic beads by adopting a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC/N-hydroxysuccinimide (NHS) method, then coupling the activated carboxyl magnetic beads with G19 to prepare antibody magnetic beads, and specifically, adding EDC and NHS, oscillating and activating for 30min in a 37 ℃ constant temperature box, adding canine coronavirus antibody G19, oscillating and reacting for 4h in the 37 ℃ constant temperature box, sealing a magnetic microsphere sealing solution for 2h, cleaning the magnetic microspheres for 2 times in a magnetic frame, diluting by using a magnetic microsphere buffer solution, and storing in a 4 ℃ refrigerator for later use.
The magnetic microsphere confining liquid is prepared by mixing 0.05M Tris, 0.05% casein, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.8; the magnetic microsphere buffer solution is prepared by mixing 0.05M Tris, 1.5 percent of trehalose, 1 percent of bovine serum albumin, 0.05 percent of Triton X-100 and 0.01 percent of Proclin300, and the pH value is 7.4; the luminous confining liquid is prepared by mixing 0.01M PBS, 0.15% lysine and 0.1% Proclin300, and the pH value of the confining liquid is 7.5; the luminescence reagent buffer solution is prepared by mixing PBS, 1% trehalose, 0.5% TrionX-100, 0.1% Proclin300 and 0.5% casein, and the pH value is 6.3.
The application method of the prepared dog coronavirus chemiluminescence detection reagent comprises the following steps of taking a dog vomit or excrement sample, appropriately diluting the dog vomit or excrement sample with a sample diluent, sequentially adding 50 mu L of diluted sample, 30 mu L of magnetic microsphere-G19 conjugate and 40 mu L of LG 15-acridinium ester conjugate into a transparent reaction tube, performing shake incubation reaction for 10min at room temperature, then cleaning for 3 times under the action of a magnetic field, allowing the reaction tube to enter a detection darkroom, pumping a substrate excitation liquid A and a bottom excitation liquid B into the reaction tube by using a full-automatic luminometer, and finally reading relative luminosity RLU of each hole on the chemiluminescence meter.
Wherein the substrate exciting liquid A is a mixture of 0.1M concentrated nitric acid, 0.15% hydrogen peroxide and 0.1% TrionX-100; the substrate exciting solution B is a mixture of 0.35M sodium hydroxide and 2.5% TrionX-100.
Example 2: the same preparation method as that of the above example 1 was adopted to obtain a canine coronavirus chemiluminescence detection reagent.
Then diluting a series of standard products with different concentrations, and detecting the standard products with different concentrations of the canine coronavirus one by one through the following using method of the canine coronavirus chemiluminescence detection reagent, wherein the specific operation steps are as follows:
taking a dog vomit or excrement sample, properly diluting the dog vomit or excrement sample by using a sample diluent, sequentially adding 50 mu L of diluted standard substances with different concentrations, 30 mu L of magnetic microsphere-G19 conjugate and 40 mu L of LG 15-acridinium ester conjugate into a transparent reaction tube, carrying out oscillation incubation reaction for 10min at room temperature, then cleaning the reaction tube for 3 times under the action of a magnetic field, feeding the reaction tube into a detection darkroom, pumping a substrate excitation liquid A and a bottom excitation liquid B by using a full-automatic luminometer, and finally reading relative luminosity RLU of each hole on a chemiluminescence meter.
Wherein the substrate exciting liquid A is a mixture of 0.1M concentrated nitric acid, 0.15% hydrogen peroxide and 0.1% TrionX-100; the substrate exciting solution B is a mixture of 0.35M sodium hydroxide and 2.5% TrionX-100.
The present invention also carried out the following validation tests.
(I) preparing standard curve of canine coronavirus chemiluminescence reagent
The results of the standard curves prepared by summarizing the RLU values of the luminosity obtained by detecting a series of canine coronavirus standards with different concentrations in example 2 are shown in fig. 1.
And the linear formula of the standard curve is Y35001X +37327, the correlation coefficient of the standard curve is R2 0.9907, which shows that the linear correlation is good, the detection sensitivity is 6.3pg/mL, and the detection sensitivity is high.
(II) standard recovery test
The detection reagent and the using method obtained in example 1 were used to measure the recovery rate of the added standard by adding high, medium and low concentrations of CCV recombinant antigen to vomit or excrement of healthy dogs, and the measurement was repeated 3 times for each sample, and the results were summarized as shown in Table 1 below.
TABLE 1 recovery test results
As can be seen from the statistics in table 1, the normalized recovery of CCV recombinant antigen in canine clinical samples was: 95.03% -105.35%, indicating good recovery.
(III) specific assay
The method comprises the steps of selecting common canine viral infectious disease recombinant antigens including canine distemper, canine parvovirus, canine influenza and canine adenovirus, carrying out specificity analysis tests on the common canine viral infectious disease recombinant antigens and the new canine coronavirus antigen (CCV) by using the detection reagent and the method in the embodiment 1, and summarizing detection results as shown in the following table 2.
TABLE 2 results of specificity test
The results in the table 2 show that when the kit is used for determining high-concentration canine distemper, canine parvovirus, canine influenza and canine adenovirus recombinant antigens, the determination concentrations are far lower than the theoretical concentrations, and the determination concentrations of the canine new coronavirus antigens (CCV) are quite close to the original concentrations, so that the canine coronavirus detection reagent obtained by the preparation method has remarkable specificity and accuracy, and the occurrence of false positive detection results can be avoided.
(IV) precision test (Interbatch and Interbatch)
The detection kit obtained in this example 1 was used to measure the concentrations of three accurately quantified standards, high, medium, and low, each of which was set with 10 duplicate wells, and the results of the measurements are summarized in table 3.
TABLE 3 results of precision test
As can be seen from the test results in Table 3 above, both the intra-batch variation coefficient and the inter-batch variation coefficient of the kit are lower than 10%, and the kit meets the specified requirements of the kit.
(V) stability test
The stability of the chemiluminescence kit is determined by adopting a thermal stability acceleration test, the detection reagent prepared in the embodiment 1 is sealed and placed in an electrothermal blowing drying oven at 37 ℃, and CCV recombinant antigens with the concentrations of 10pg/mL, 100pg/mL and 1000pg/mL are prepared for detection, and the detection is carried out once a week for 4 weeks; accelerated 4 weeks at 37 ℃ is equivalent to storage at 4 ℃ for more than two years, and the accelerated stability of the method is examined, and the detection results are summarized as shown in the following table 4.
TABLE 4 results of accelerated thermal stability test
From the detection results in table 4 above, it can be seen that the detection results of the detection reagent prepared in this embodiment 1 have variation coefficients less than 5% within 4 weeks, which indicates that the chemiluminescence detection reagent of the present invention has good stability and can be popularized and applied.
(VI) establishing qualitative standard
Collecting feces of healthy dogs in pet clinics and pet stores more than 100 parts, quantitatively detecting with the chemiluminescence reagent prepared in the embodiment 1 and the using method, analyzing normal distribution, determining 95% reference interval, and adopting a formula
Qualitative criteria were determined.
And (3) displaying a detection result: fecal measurements from 100 healthy dogs were in accordance with a normal distribution,
namely when the concentration of the fecal CCV is more than or equal to 27.28pg/mLDogs have a greater probability of infecting coronavirus; when the fecal CCV concentration is less than or equal to 27.28pg/mL, the possibility of infecting the dog with coronavirus is small.
In conclusion, the chemiluminescent reagent obtained by the invention has strong specificity, high sensitivity and stable property, can realize qualitative, quantitative, rapid and accurate detection of the canine coronavirus antigen by matching with the using method of the invention, and is suitable for popularization and application.
The above description is only for the purpose of illustrating the embodiments of the present invention and not for the purpose of limiting the same, and equivalent modifications and variations of the embodiments of the present invention will be apparent to those skilled in the art without departing from the overall spirit of the invention.
Claims (10)
1. A preparation method of a canine coronavirus chemiluminescence detection reagent is characterized by comprising the following steps: the dog coronavirus chemiluminescence detection reagent comprises a magnetic microsphere-G19 conjugate and a G15-acridinium ester conjugate, and the specific preparation process comprises the following steps:
preparation of magnetic microsphere-G19 conjugate: EDC and NHS are adopted to activate magnetic microspheres, dog coronavirus antibody G19 is added to carry out oscillation coupling, the magnetic microspheres are sealed by sealing liquid, the magnetic microspheres are washed in a magnetic frame and then stored in magnetic microsphere buffer solution for later use, and the magnetic microsphere-G19 conjugate is obtained.
Preparation of G15-acridinium ester conjugate: centrifuging and concentrating a canine coronavirus antibody G15 and acridinium ester to remove impurities, then fully and uniformly mixing G15 and the acridinium ester for labeling and coupling, slightly shaking for labeling at room temperature, adding a luminescent confining liquid for sealing, and dialyzing to remove unbound acridinium ester; and purifying by a Sephadex G50 gel chromatography column, and storing in a luminescent reagent buffer solution for later use to obtain the G15-acridinium ester conjugate.
2. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps:
in the step of preparing the magnetic microsphere-G19 conjugate, a canine coronavirus antibody G19 is coupled for 4 hours in a shaking way, a magnetic microsphere confining liquid is sealed for 2 hours, and the magnetic microsphere is cleaned in a magnetic frame for 2 times; in the step of preparing the G15-acridinium ester conjugate: according to the following steps of 4: and (3) fully and uniformly mixing G15 and acridinium ester according to the mass ratio of 1, carrying out label coupling, slightly shaking the label for 2 hours at room temperature, and adding a luminescent confining liquid for sealing for 1 hour.
3. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps: in the step of preparing the magnetic microsphere-G19 conjugate, the carboxyl magnetic beads are activated by a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC/N-hydroxysuccinimide (NHS) method and then coupled with G19 to prepare antibody magnetic beads.
4. The method for preparing the reagent for detecting canine coronavirus chemiluminescence according to claim 3, wherein the reagent comprises: in the step of preparing the magnetic microsphere-G19 conjugate, EDC and NHS are added, oscillation activation is carried out for 30min in a 37 ℃ thermostat, canine coronavirus antibody G19 is added, oscillation reaction is carried out for 4h in the 37 ℃ thermostat, a magnetic microsphere confining liquid is sealed for 2h, the magnetic microspheres are washed in a magnetic frame for 2 times, and the magnetic microsphere buffer solution is diluted and then placed in a 4 ℃ refrigerator for storage for later use.
5. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps: the magnetic microsphere confining liquid is prepared by mixing 0.05M Tris, 0.05% casein, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.8.
6. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps: the magnetic microsphere buffer solution is prepared by mixing 0.05M Tris, 1.5% trehalose, 1% bovine serum albumin, 0.05% Triton X-100 and 0.01% Proclin300, and the pH value is 7.4.
7. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps: the luminous confining liquid is prepared by mixing 0.01M PBS, 0.15% lysine and 0.1% Proclin300, and the pH value of the confining liquid is 7.5.
8. The method for preparing a canine coronavirus chemiluminescent detection reagent according to claim 1, wherein the method comprises the following steps: the luminescence reagent buffer solution is prepared by mixing PBS, 1% trehalose, 0.5% TrionX-100, 0.1% Proclin300 and 0.5% casein, and the pH value is 6.3.
9. The method of claim 1, wherein the reagent for the chemiluminescent detection of canine coronavirus comprises: taking a dog vomit or excrement sample, properly diluting the dog vomit or excrement sample by using a sample diluent, sequentially adding the diluted sample or a standard substance or a quality control substance, a magnetic microsphere-G19 conjugate and a G15-acridine ester conjugate into a transparent reaction tube, oscillating and incubating the mixture at room temperature for reaction, then cleaning the reaction tube under the action of a magnetic field, putting the reaction tube into a detection darkroom, pumping a substrate exciting liquid A and a bottom exciting liquid B into the detection darkroom by using a full-automatic luminometer, and finally reading the relative luminosity RLU of each hole on a chemiluminescence meter.
10. The method of using the reagent for the chemiluminescent detection of canine coronavirus according to claim 9, wherein: adding 50 μ L of diluted sample or standard or quality control, 30 μ L of magnetic microsphere-G19 conjugate, and 40 μ L of LG 15-acridinium ester conjugate in sequence into a transparent reaction tube;
the substrate exciting liquid A is a mixture of 0.1M concentrated nitric acid, 0.15% hydrogen peroxide and 0.1% TrionX-100;
the substrate excitation solution B is a mixture of 0.35M sodium hydroxide and 2.5% TrionX-100.
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