Fluorescence lateral chromatography detection method for canine coronavirus antigen

Abstract

The invention relates to the technical field of medical detection, and discloses a canine coronavirus antigen fluorescence lateral chromatography detection method which comprises the following steps: A, coupling a canine coronavirus paired antibody with carboxyl fluorescent microspheres to form fluorescently-labeled antibody microspheres; b, spraying the fluorescently-labeled antibody microspheres on a conjugate pad, and drying the conjugate pad; c, respectively coating the nitrocellulose membrane with a canine coronavirus antibody and goat anti-canine IgG to form a test line and a control line; and D, assembling the sample pad, the microsphere combination pad, the nitrocellulose membrane and the water absorption pad in sequence, and cutting into a test strip with the width of 4mm for detection. According to the detection method, the influence of interfering substances in the sample can be reduced, good specificity and stability are achieved, meanwhile, the detection design enables the detection method to have a simple and convenient operation process, and complex experimental equipment is not needed. The test strip is relatively simple and convenient to assemble and use, so that veterinarians or related workers can quickly carry out on-site detection.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, in particular to a method for detecting canine coronavirus antigen by fluorescent lateral chromatography. Background Art

[0002] Canine coronavirus disease is an infectious disease of dogs that can cause acute enteritis in dogs. The clinical symptoms of dogs infected with CCoV are mainly diarrhea, which can change from mild loose and soft feces to severe watery diarrhea. When dogs are infected with CCoV alone, the morbidity is high and the mortality rate is low. Clinically, canine coronavirus is often co-infected with pathogens of other intestinal infectious diseases. For example, when CCoV is co-infected with other pathogens such as canine adenovirus (CAV-1), canine parvovirus (CPV-2) and canine distemper virus (CDV), severe clinical symptoms and even death may occur. Studies have shown that CCoV can increase the severity of CPV-2 infection. With the genetic variation of CCoV, a highly virulent pan-tropic CCoV variant has emerged, which can cause fatal and systemic diseases in dogs. This variant can infect dogs of different ages with high morbidity and mortality. It brings great harm to pet dogs.

[0003] At present, the detection method for canine coronavirus includes virus separation, but the nutrient requirements for CCoV isolation and culture are strict, the material cost is high, the culture and passage process is complex, time-consuming and requires professional test personnel. However, for those medical staff who do clinical diagnosis in pet hospitals, it is not suitable for clinical application. Moreover, CCoV is easy to mutate and difficult to separate, so it is not suitable for clinical diagnosis of diseases. There are also literature reports on the establishment of ELISA double antibody sandwich method and neutralization experiment to detect CCoV. Although these methods have good specificity, they are time-consuming and only suitable for laboratory testing, not for clinical testing. In the subsequent medical field, PCR has been used by more people as a simple and fast diagnostic tool that can accurately identify viral genotypes and serotypes, but this method requires sophisticated instruments, which limits its clinical use. In recent years, pet hospitals have widely used colloidal gold test strips for clinical diagnosis of canine coronavirus disease. This technology can make qualitative diagnosis of diarrhea materials suspected of canine coronavirus disease, and the diagnosis results can be detected in 10 minutes. It has the characteristics of convenient operation and fast diagnosis. However, this diagnostic method still has certain disadvantages. It cannot make quantitative diagnosis of CCoV, and viruses with low nucleic acid concentrations are difficult to detect.

[0004] Therefore, those skilled in the art have proposed a canine coronavirus antigen fluorescence lateral flow chromatography detection method to solve the above problems. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for detecting canine coronavirus antigen by fluorescent lateral flow chromatography, which solves the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for detecting canine coronavirus antigen by fluorescent lateral flow chromatography, comprising the following steps:

[0007] A. Use canine coronavirus paired antibodies to couple with carboxyl fluorescent microspheres to form fluorescently labeled antibody microspheres;

[0008] B. spraying the fluorescently labeled antibody microspheres onto the conjugate pad and performing a drying process;

[0009] C. Coating canine coronavirus antibody and goat anti-canine IgG on the nitrocellulose membrane to form a test line and a control line;

[0010] D. Assemble the sample pad, microsphere binding pad, nitrocellulose membrane and absorbent pad in order, and cut them into 4 mm wide test strips for testing.

[0011] Preferably, the steps of preparing fluorescent microspheres in step A are as follows:

[0012] A1. Add 20 μl of fluorescent microspheres into 500 μL of 0.05 mol / L PBS buffer, mix, and then add 50 μl of LEDC and 50 μl of NHS activator for activation;

[0013] A2, after oscillation activation and centrifugation, the supernatant was discarded, and 800 μL of 0.05 mol / L PBS buffer was added to redissolve the precipitate;

[0014] A3. Add 10 μg of canine coronavirus antibody for coupling, followed by centrifugation and precipitation.

[0015] Preferably, the components of the sample pad in step D include: PBS buffer containing 1% BSA, 0.9% NAC1, 0.1% sucrose, 1% PEG4000, 0.5% TritonX-100 and 0.3‰ Procline300, wherein the pH of the buffer is 8.0.

[0016] Preferably, the microsphere protective solution in steps A to D is composed of 0.2 M boric acid-borax buffer, 2% sucrose, 0.5% BSA and 0.3‰ Proc lin to improve the stability of the fluorescent microspheres.

[0017] Preferably, a specific test is also included, wherein the specific test is performed by using the above-mentioned test strips to detect positive samples of canine parvovirus, canine adenovirus, canine distemper virus, feline calicivirus, feline coronavirus and feline herpesvirus, and setting up negative samples as controls.

[0018] Preferably, a sensitivity experiment is also included, wherein the sensitivity experiment is performed by diluting the cytotoxicity of canine coronavirus in four gradients to determine the minimum detection limit of the method.

[0019] Preferably, the four gradient dilutions in the sensitivity test are: 1:10, 1:100, 1:1000, 1:10000.

[0020] Preferably, a repeatability test is also included, in which dilutions of 1:10 and 1:100 are selected as quality control products, and test strips from the same batch and different batches are tested respectively to calculate the intra-batch and inter-batch coefficients of variation, and the test results are read and analyzed by a fluorescent imaging system.

[0021] The present invention provides a method for detecting canine coronavirus antigen by fluorescent lateral flow chromatography. It has the following beneficial effects:

[0022] 1. The present invention can reduce the influence of interfering substances in the sample through the detection method, and has good specificity and stability. At the same time, the prepared immunofluorescence chromatography test strips are used to compare with the fluorescence quantitative PCR results to detect 20 clinical samples. The detection results of the two methods are completely consistent, with a positive compliance rate of 100%, a negative compliance rate of 100%, and a total compliance rate of 100%. The detection results are accurate and reliable, the detection range is wider, the operation is simple, fast, and the cost is low.

[0023] 2. The design of the detection method of the present invention enables it to have a simple operation process without the need for complex experimental equipment. The assembly and use of the test strips are relatively simple, allowing veterinarians or related staff to quickly conduct on-site testing. This convenience not only improves the efficiency of the test, but also allows the results to be obtained quickly under on-site conditions, supporting timely decision-making and treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example:

[0027] Please see attached Figure 1 , a method for detecting canine coronavirus antigen by fluorescent lateral flow chromatography, comprising the following steps:

[0028] A. Use canine coronavirus paired antibodies to couple with carboxyl fluorescent microspheres to form fluorescently labeled antibody microspheres;

[0029] The microsphere protection solution in step A to step D is composed of 0.2M boric acid-borax buffer, 2% sucrose, 0.5% BSA and 0.3‰ Proc lin to improve the stability of the fluorescent microspheres.

[0030] The steps for the fluorescent microspheres in step A are as follows:

[0031] A1. Add 20 μl of fluorescent microspheres into 500 μL of 0.05 mol / L PBS buffer, mix, and then add 50 μl of LEDC and 50 μl of NHS activator for activation;

[0032] A2, after oscillation activation and centrifugation, the supernatant was discarded, and 800 μL of 0.05 mol / L PBS buffer was added to redissolve the precipitate;

[0033] B, A3, add 10 μg canine coronavirus antibody for coupling, followed by centrifugal precipitation.

[0034] B. Spray the fluorescently labeled antibody microspheres onto the conjugate pad and dry them;

[0035] C. Coating canine coronavirus antibody and goat anti-canine IgG on the nitrocellulose membrane to form a test line and a control line;

[0036] D. Assemble the sample pad, microsphere binding pad, nitrocellulose membrane and absorbent pad in order, and cut them into 4 mm wide test strips for testing.

[0037] The components of the sample pad in step D include: PBS buffer containing 1% BSA, 0.9% NACl, 0.1% sucrose, 1% PEG4000, 0.5% TritonX-100 and 0.3‰ Procline300, wherein the pH of the buffer is 8.0.

[0038] It also includes a specific test, which uses the above-mentioned test strips to detect positive samples of canine parvovirus, canine adenovirus, canine distemper virus, feline calicivirus, feline coronavirus and feline herpes virus, and sets up negative samples as controls.

[0039] Specificity test:

[0040] The prepared canine coronavirus antigen fluorescent immunochromatographic test strips were used to specifically detect positive samples of canine parvovirus, canine adenovirus, canine distemper virus, feline calicivirus, feline coronavirus, feline herpes virus and canine coronavirus, and negative samples were set as negative controls.

[0041] Specifically, sample preparation:

[0042] Collect and label all positive samples and negative control samples.

[0043] Ensure that each sample is handled following safe procedures to avoid cross contamination.

[0044] Testing process:

[0045] Add each positive sample drop by drop onto the sample pad of the canine coronavirus antigen test strip.

[0046] At the same time, under the same conditions, a negative control sample was added to the test strip to ensure the accuracy of the test results.

[0047] Result interpretation:

[0048] The test results are read within an appropriate time and interpreted based on the color development of the test line and the control line.

[0049] Record the test results for each sample and confirm that only the canine coronavirus sample develops color on the test line, and other samples should only develop color on the control line.

[0050] Result analysis:

[0051] By comparing the results of positive samples and negative control samples, the specificity of the test strip was evaluated to ensure that it can effectively distinguish canine coronavirus from other canine and feline viruses.

[0052] It also includes a sensitivity experiment, in which the cytotoxic agent of canine coronavirus is diluted in four gradients. The four gradient dilutions in the sensitivity experiment are: 1:10, 1:100, 1:1000, and 1:10000.

[0053] Sensitivity test:

[0054] The prepared canine coronavirus antigen immunofluorescence chromatography test strips were used for sensitivity test, and the cytotoxicity of canine coronavirus was diluted into 4 gradients (1:10, 1:100, 1:1000, 1:10000).

[0055] It also includes repeatability tests. The repeatability tests select dilutions of 1:10 and 1:100 as quality control products, and test the test strips from the same batch and different batches to calculate the intra-batch and inter-batch coefficients of variation. The test results are read and analyzed by a fluorescent imaging system.

[0056] Specifically, the experimental steps are:

[0057] Sample dilution:

[0058] Dilute the canine coronavirus cytotoxin in the following four gradients: 1:10 1:100 1:1000 1:10000

[0063] Each gradient should be mixed thoroughly to ensure uniform concentration.

[0064] Testing process:

[0065] Each dilution gradient sample was dropped onto the sample pad of the canine coronavirus antigen test strip.

[0066] Make sure each test is performed under the same environmental conditions to ensure consistent results.

[0067] Result interpretation:

[0068] Read the test strip result within the specified time.

[0069] Observe the color development of the test line and record the test results at each dilution gradient.

[0070] Repeatability test:

[0071] The dilutions of 1:10 and 1:100 were selected as quality control products, and the canine coronavirus antigen immunofluorescence chromatography test strips of the same batch were tested, and the intra-batch coefficient of variation was calculated. The canine coronavirus antigen immunofluorescence chromatography test strips of 3 different batches were tested, and the inter-batch coefficient of variation was calculated.

[0072] Clinical sample testing:

[0073] The 20 collected clinical samples were tested using the prepared canine coronavirus antigen immunofluorescence chromatography test strips and fluorescent quantitative PCR.

[0074] result:

[0075] Specific results:

[0076] From the results in Table 1, it can be seen that the negative samples of CDV, CAV, FCoV, CPV, FCV, and FHV were all negative, and only the canine coronavirus positive sample was positive, indicating that the prepared canine coronavirus antigen immunofluorescence chromatography test strip has good specificity and has no cross reaction with other related pathogens.

[0077] Table 1 Specificity test results of canine coronavirus antigen immunofluorescence chromatography test strip

[0078]

[0079]

[0080] Sensitivity results:

[0081] From the results in Table 2, it can be seen that the minimum detection amount of the prepared canine coronavirus antigen immunofluorescence chromatography test strip for canine coronavirus cytotoxicity is 1:100.

[0082] Table 2 Sensitivity test results of canine coronavirus antigen immunofluorescence chromatography test strips

[0083] Cytotoxic dilution Fluorescence value (TU / mL) Result judgment 1:10 126.37 Positive 1:100 14.25 Weak Yang 1:100 <10 Negative 1:1000 <10 Negative

[0084] Reproducible results

[0085] As can be seen from Table 3, the intra-batch coefficients of variation were 2.87% and 7.01%, respectively; the inter-batch coefficients of variation were 4.14% and 9.59%, respectively. The intra-batch and inter-batch coefficients of variation were all below 10%, proving that the prepared test strips had good repeatability.

[0086] Table 3 Repeatability test results of canine coronavirus antigen immunofluorescence chromatography test strips

[0087]

[0088]

[0089] Clinical sample test results

[0090] As can be seen from Table 4, one canine coronavirus positive sample was detected in 20 clinical samples, and the others were negative. The prepared test strips were compared with the results of fluorescence quantitative PCR technology, and the test results were completely consistent, with a positive coincidence rate of 100%, a negative coincidence rate of 100%, and a total coincidence rate of 100%, indicating that the prepared test strips have a good accuracy rate.

[0091] Table 4 Results of canine coronavirus antigen immunofluorescence chromatography test strips and ELISA kits for clinical samples

[0092]

[0093]

[0094] Note: Fluorescence value > 10TU / mL is positive, and fluorescence value < 10TU / mL is negative.

[0095] In summary: the prepared immunofluorescence chromatography test strip for detecting canine coronavirus antigen has good specificity and stability, reduces the influence of interfering substances in the sample, and the prepared immunofluorescence chromatography test strip is compared with the fluorescence quantitative PCR results to detect 20 clinical samples. The detection results of the two methods are completely consistent, with a positive compliance rate of 100%, a negative compliance rate of 100%, and a total compliance rate of 100%. The test results are accurate and reliable, the detection range is wider, the operation is simple, fast, and the cost is low.

[0096] At the same time, the design of the detection method of the present invention enables it to have a simple operation process without the need for complex experimental equipment. The assembly and use of the test strips are relatively simple, allowing veterinarians or related staff to quickly conduct on-site testing. This convenience not only improves the efficiency of the test, but also allows the results to be obtained quickly under on-site conditions, supporting timely decision-making and treatment plans.

[0097] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting canine coronavirus antigen by fluorescence lateral flow chromatography, characterized in that: The following steps are involved: A. Use canine coronavirus paired antibodies to couple with carboxyl fluorescent microspheres to form fluorescently labeled antibody microspheres; B. spraying the fluorescently labeled antibody microspheres onto the conjugate pad and performing a drying process; C. Coating canine coronavirus antibody and goat anti-canine IgG on the nitrocellulose membrane to form a test line and a control line; D. Assemble the sample pad, microsphere binding pad, nitrocellulose membrane and absorbent pad in order, and cut them into 4 mm wide test strips for testing.

2. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: The steps of fluorescent microspheres in step A are as follows: A1. Add 20 μl of fluorescent microspheres into 500 μL of 0.05 mol / L PBS buffer, mix, and then add 50 μL of LEDC and 50 μL of NHS activator for activation; A2, after oscillation activation and centrifugation, the supernatant was discarded, and 800 μL of 0.05 mol / L PBS buffer was added to redissolve the precipitate; A3. Add 10 μg of canine coronavirus antibody for coupling, followed by centrifugation and precipitation.

3. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: The components of the sample pad in step D include: PBS buffer containing 1% BSA, 0.9% NACl, 0.1% sucrose, 1% PEG4000, 0.5% TritonX-100 and 0.3‰ Proclin300, wherein the pH of the buffer is 8.

0.

4. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: The microsphere protection solution in steps A to D is composed of 0.2M boric acid-borax buffer, 2% sucrose, 0.5% BSA and 0.3‰ Proclin to improve the stability of the fluorescent microspheres.

5. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: It also includes a specific test, which uses the above-mentioned test strips to detect positive samples of canine parvovirus, canine adenovirus, canine distemper virus, feline calicivirus, feline coronavirus and feline herpes virus, and sets up negative samples as controls.

6. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: A sensitivity test was also included, which was performed by making four serial dilutions of the cytotoxic canine coronavirus.

7. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 6, characterized in that: The four gradient dilutions in the sensitivity test were: 1:10, 1:100, 1:1000, and 1:10000.

8. A canine coronavirus antigen fluorescence lateral flow detection method according to claim 1, characterized in that: It also includes a repeatability test, in which dilutions of 1:10 and 1:100 are selected as quality control products, and test strips from the same batch and different batches are tested to calculate the intra-batch and inter-batch coefficients of variation. The test results are read and analyzed by a fluorescent imaging system.

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