CN113447653A - Immunochromatography reagent strip for detecting canine circovirus antigen and preparation method thereof - Google Patents
Immunochromatography reagent strip for detecting canine circovirus antigen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an immunochromatographic reagent strip for detecting canine circovirus antigen and a preparation method thereof, belonging to the technical field of in vitro immunodiagnosis detection tools, comprising a PVC bottom plate, and characterized in that: the device also comprises a water absorption pad, a nitrocellulose membrane, a marking pad and a sample pad which are sequentially stuck on the PVC bottom plate; the CanineCV antibody-colloidal gold compound is sprayed on the labeling pad, and a quality control line C and a detection line T are coated on the nitrocellulose membrane; the quality control line C is coated with CanineCV recombinant antigen; the detection line T is coated with CanineCV antibodies; and through (1) preparing colloidal gold solution; (2) preparing a marking pad; (3) coating a quality control line C and a detection line T; (4) pretreating a sample pad; (5) cutting and assembling; and obtaining a finished product. The reagent strip can carry out on-site rapid detection on CanineCV, fills the blank that no CanineCV antigen rapid detection reagent exists in China, and has high detection rate.
Description
Technical Field
The invention relates to the technical field of in-vitro immunodiagnosis detection tools, and particularly belongs to an immunochromatography reagent strip for detecting canine circovirus antigens and a preparation method thereof.
Background
Canine Circovirus (Canine Circovirus, CanineCV) is a single-stranded circular, non-enveloped DNA virus belonging to a newly discovered mammalian Circovirus, first discovered in US Canine serum in 2012. The canine circovirus belongs to the circovirus family, and the circovirus genus also comprises porcine circovirus type 2, duck circovirus, goose circovirus, canary circovirus, avian leukemia virus and the like. The size of the canine circovirus genome is about 2063nt, which is 276-277 nt more than that of the circovirus 2 gene. The canine circovirus has two open reading frames, ORF1 open reading frames are relatively conservative, and the variation degree of ORF2 is relatively large. The ORF1 open reading frame has 912 bases and codes the replicase of canine circovirus, and is related to the replication of the virus; the second open reading frame ORF2 encodes primarily capsid proteins. At present, canine circovirus is detected in Italy, Germany and China mainland, and hosts comprise wild animals of canidae such as wolfs, foxes and badgers besides domestic dogs.
Clinically, CanineCV infection is associated with canine vasculitis, hemorrhagic diarrhea, and gastroenteritis. The virus is mainly present in the intestinal tract, and some viruses are also present in tissues such as liver, spleen and lymph. The clinical symptoms of canine circovirus are very similar to those of porcine dermatitis and nephrotic syndrome caused by circovirus type 2 in both clinical symptoms and pathological changes. Specific primers are designed according to the gene sequence of the canine circovirus and detected by a PCR method, the detection rate of the canine circovirus in dogs with diarrhea reaches 20.1 percent, and the detection rate of the canine circovirus in dogs of a healthy group is only 7.3 percent. Thus, the detection of the CanineCV antigen is time-consuming, energy-consuming and high in cost, and the detection rate is low, so that the timely diagnosis and research of the disease are not facilitated, and even the precious treatment time is delayed. Therefore, a detection means with high detection rate and high speed is needed to detect the CanineCV antigen in time.
Although canine circovirus has received increasing attention, there are few studies on CanineCV and there has been no successful test strip for rapid detection of CanineCV antigen. In 2016, Chinese scholars detect canine circovirus from canine serum in continental land for the first time, and the canine circovirus discovered in China and the strain discovered in Europe and America belong to different evolutionary branches through the analysis of the evolutionary tree. Therefore, the virus is extremely difficult to research, and the factors to be considered in the test are too many, for example, an effective labeling method and a complex solution cannot be developed at present, a small condition can also influence the redissolving effect of the colloidal gold, and further influence the final detection performance, and even if countless tests are carried out, the detection repeatability cannot be realized.
So far, epidemiology, genotyping, pathogenicity, mechanism research and the like of the virus are still blank, no reagent for rapidly detecting the canine circovirus exists in China, and the control on CanineCV epidemic disease and epidemiological investigation thereof are hindered and are not in advance.
Disclosure of Invention
Aiming at the defects and shortcomings in the background technology, the invention provides the immunochromatography reagent strip for detecting the canine circovirus antigen, which can carry out on-site rapid detection on CanineCV, fills the blank that no CanineCV antigen rapid detection reagent exists in China, and has high detection rate.
The invention also aims to provide a preparation method of the immunochromatography reagent strip for detecting the canine circovirus antigen, the preparation method can effectively mark the compound, ensure the redissolution effect of the colloidal gold, enable the prepared reagent strip to carry out on-site rapid detection on the CanineCV antigen, and has high detectable rate and strong repeatability.
In order to realize the purpose, the invention adopts the following technical scheme to realize the purpose:
the immunochromatography reagent strip for detecting the canine circovirus antigen comprises a PVC (polyvinyl chloride) base plate, and further comprises a water absorption pad, a nitrocellulose membrane, a marking pad and a sample pad which are sequentially stuck on the PVC base plate;
the sample pad is arranged on one end of the PVC bottom plate; the sample pad is provided with a sample dripping area; one end of the sample pad is erected on the marking pad, the CanineCV antibody-colloidal gold compound is sprayed on the marking pad, and one end of the marking pad far away from the sample pad is erected on the nitrocellulose membrane; the nitrocellulose membrane is fixedly connected to the PVC bottom plate, a quality control line C and a detection line T are coated on the nitrocellulose membrane, and the quality control line C and the detection line T are parallel to each other and are arranged perpendicular to the extension direction of the test strip; the quality control line C is coated with CanineCV recombinant antigen; the detection line T is coated with CanineCV antibodies; and one end of the nitrocellulose membrane far away from the sample pad is erected below absorbent paper.
Further measures taken are: the spraying amount of the CanineCV antibody-colloidal gold compound on the labeling pad is 2-4 mul/cm, the ratio of the colloidal gold labeling CanineCV antibody is 5-10 mug per ml of labeling amount, the concentration multiple of the CanineCV antibody-colloidal gold compound is 20-30 times, namely, the colloidal gold is concentrated to 1ml per 20-30 ml, and the spraying amount is 2-4 mul/cm.
Further measures taken are: the concentration of CanineCV antibodies coated by the detection line T on the nitrocellulose membrane is 0.5-1.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm; the concentration of the CanineCV antigen coated by the quality control line C is 1.0-2.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm.
Further measures taken are: the grain size of the colloidal gold is 20-50 nm.
The preparation method of the immunochromatography reagent strip for detecting the canine circovirus antigen comprises the following steps:
(1) preparing a colloidal gold solution;
(2) preparing a labeling pad, including preparing a Canine CV antibody-colloidal gold compound, namely adding 2-10 mu l of 0.1mol/l K2CO3 solution into 1ml of colloidal gold solution, uniformly mixing, adding 5-10 mu g of Canine CV antibody, uniformly mixing for 15min, adding 50-100 mu l of 10% BSA solution, sealing for 10min, placing the mixture in a refrigerated centrifuge, centrifuging for 30min at 10000rpm, removing supernatant, re-dissolving the precipitate to 30-50 mu l with the colloidal gold re-solution, and storing for later use at 2-8 ℃;
(3) coating a quality control line C and a detection line T;
(4) pretreating a sample pad;
(5) cutting and assembling; and obtaining a finished product.
Further measures taken are: the colloidal gold redissolution contains 0.02mol/l Tris buffer solution with the pH value of 8.2, 0.1 percent of Casein, 0.3 to 0.5 percent of PVP40 and 2 to 5 percent of trehalose mixed solution.
Further measures taken are: the preparation process in the step (1) comprises the steps of weighing 100ml of ultrapure water into a conical flask, adding 1.0ml of 1% chloroauric acid solution into the conical flask, uniformly mixing, heating to boiling, rapidly adding a proper amount of 1% trisodium citrate, observing the color change of the solution, continuing heating for 5 minutes after the color is stable, stopping adding liquid, cooling the solution to room temperature, and storing at 2-8 ℃.
Further measures taken are: the step (2) further comprises S2, spraying gold, and spraying the CanineCV antibody-colloidal gold compound onto a marking pad by using a three-dimensional gold spraying film scratching instrument, wherein the spraying amount is 2-4 mu l/cm; and (3) drying the sprayed marking pad in a drying chamber (the humidity is less than 30%) for 12h, adding a drying agent, and sealing for later use.
Further measures taken are: the treatment process of the step (4) comprises the steps of soaking the sample pad in sample pad pretreatment liquid for 30min, draining, drying in a drying chamber (the humidity is less than 30%) for 12h, adding a drying agent, and sealing for later use;
the sample pad pretreatment solution contains a mixed solution of a Tris-HCl buffer solution, 0.5% Tween 20 and 0.5% trehalose, and the pH value of the mixed solution is 7-9.
Further measures taken are: the specific steps of the step (3) comprise S1, quality control line C and detection line T coating liquid preparation: diluting CanineCV into a quality control line C solution of 1.0-2.0 mg/ml by using 0.01mol/L PBS buffer solution with the pH value of 7.4; taking CanineCV antibodies, and diluting the CanineCV antibodies into 0.5-1.0 mg/ml detection line T solution by using 0.01mol/L PBS buffer solution with pH7.4;
s1, scribing: coating the coating liquid of the quality control line C and the detection line T to the positions of the quality control line and the detection line of the nitrocellulose membrane respectively by using a three-dimensional metal spraying and membrane scratching instrument, wherein the interval between C, T lines is 5-8 mm, and the coating amount is 1.0-2.0 mu l/cm; and (3) drying the scratched film in a drying chamber (the humidity is less than 30%) for 12 hours, adding a drying agent, and sealing for later use.
Further measures taken are: the process of the step (5) comprises the steps of taking the water absorption pad, the processed sample pad and the gold label pad, cutting according to the size of (15-20mm) × 300mm, and respectively adding a drying agent into the cut water absorption pad, the cut sample pad and the cut gold label pad and sealing for later use;
taking the cut water absorption pad, the sample pad, the gold mark pad and the PVC bottom plate with the membrane, pasting the gold mark pad to enable the gold mark pad to be pressed on the nitrocellulose membrane by 1-2 mm, pasting the sample pad to enable the sample pad to be pressed on the gold mark pad by 3-5 mm, and finally pasting the water absorption pad to be pressed on the nitrocellulose membrane by 1-2 mm; and cutting the assembled PVC base plate into 3-4 mm colloidal gold immunochromatographic reagent strips for detecting CanineCV antigens to obtain finished products.
In order to rapidly detect CanineCV, facilitate disease diagnosis, avoid delaying valuable treatment opportunity and reduce energy and cost consumption, the inventor group successfully develops an immunochromatography reagent strip for detecting canine circovirus antigens through deep research and research, the CanineCV antigens are coated on a quality control line C of the test strip, CanineCV monoclonal antibodies are coated on a detection line T, and meanwhile, CanineCV antibody-colloidal gold compounds are sprayed on a marking pad; the CanineCV antigen and the CanineCV antibody can be specifically combined, so that the sensitivity and the accuracy of detection can be improved.
According to the invention, colloidal gold with the particle size of 20-50nm is utilized, the proportion of the CanineCV antibody marked by the colloidal gold is strictly controlled to be 5-10 mu g per ml of marked quantity, the optimal control condition for uniformly mixing the colloidal gold solution and the CanineCV antibody is found, the concentration of each substance is controlled to be proper, and the like, so that the CanineCV antibody-colloidal gold compound is successfully and fully marked, the sensitivity and the accuracy of final detection are ensured, the detection rate is high, the repeatability is strong, the use quantity of the required antibody is low, the production cost of a reagent can be reduced, and the effect is remarkable.
The method solves the problem of poor redissolution effect of the CanineCV antibody and the colloidal gold through the most economical, simplified and rigorous preparation method. According to the invention, the colloidal gold redissolving solution is prepared into 0.02mol/l Tris buffer solution with the pH value of 8.2, 0.1% Casein, 0.3% -0.5% PVP40 and 2-5% trehalose, the sample pad pretreatment solution adopts a mixed solution of pH 7-9, Tris-HCl buffer solution, 0.5% Tween 20 and 0.5% trehalose, unexpected outstanding effects are shown, the pH value and the content of trehalose and other substances are also important, and the redissolving effect, the repeatability of detection and the final detection performance of the colloidal gold can be finally ensured.
The invention fills the blank state of the CanineCV rapid detection reagent in China at present, fills the blank that the CanineCV rapid detection reagent does not exist in China, clears obstacles for the prevention and control of CanineCV epidemic diseases and the epidemiological investigation thereof, and has important significance for the prevention and control of CanineCV epidemic diseases, and the guarantee of the safety of pet dogs/canids and the epidemiological investigation thereof.
Through the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. compared with the PCR method detection in the prior art, the immunochromatographic reagent strip does not need special instruments and equipment and technicians, can realize on-site rapid detection, can obtain results within 5-10 minutes, and is convenient for diagnosing CanineCV diseases so as to provide treatment in time.
2. The immunochromatography reagent strip has the advantages of high detection rate, high accuracy, strong repeatability, good stability and accelerated stability of at least more than 20 days.
3. The immunochromatography reagent strip is simple and convenient to use and operate, relatively low in manufacturing cost and convenient to popularize and apply, fills up the blank that no CanineCV virus antigen rapid detection reagent exists in China, and has important significance for preventing and controlling CanineCV epidemic diseases and guaranteeing the safety of pet dogs/canidae animals and epidemiological investigation.
Drawings
FIG. 1 is a schematic structural diagram of an embodiment of the present invention;
FIG. 2 is a diagram illustrating a part of the detection results of a sample under accuracy investigation;
FIG. 3 is a diagram showing the results of detecting a part of samples in a specific examination.
In the figure: 1. a sample pad; 2. a marking pad; 3. a nitrocellulose membrane; 4. a water absorbent pad; 5. A sample dropping area; 6. detecting a line T; 7. a quality control line C; 8. a PVC base plate;
in fig. 2, the 1 st test strip arranged from left to right is a recombinant antigen detection result, the 2 nd test strip is a positive sample detection result, and the 3 rd test strip is a negative sample detection result;
in fig. 3, the 1 st test strip arranged from left to right is a positive sample test result, the 2 nd test strip is a CDV test result, the 3 rd test strip is a CPV test result, the 4 th test strip is a PCV2 test result, and the 5 th test strip is a DuCV test result.
In the figure: 1. a water absorbent pad; 2. a nitrocellulose membrane; 3. a marking pad; 4. a sample pad; 5. A sample dropping area; 6. a quality control line C; 7. and detecting a line T.
Detailed Description
In order to clearly understand the technical solutions adopted by the present invention, the following description of the preferred embodiments of the present invention is provided, and it should be understood that the embodiments described herein are only used for illustrating and explaining the present invention, and not for limiting the present invention, and the terms such as "upper", "inner", "middle", "left", "right", and "a" and the like in the present specification are also used for convenience of description, and are not used to limit the scope of the present invention, and the changes or modifications of the relative relationships thereof are also regarded as the scope of the present invention without substantial technical changes.
Example (b): the immunochromatography reagent strip for detecting the canine circovirus antigen comprises a PVC (polyvinyl chloride) base plate, and further comprises a water absorption pad 1, a nitrocellulose membrane 2, a marking pad 3 and a sample pad 4 which are sequentially adhered to the PVC base plate;
the sample pad 4 is arranged on one end of the PVC bottom plate; the sample pad 4 is provided with a sample dripping area 5; one end of the sample pad 4 is erected on the labeling pad 3, the CanineCV antibody-colloidal gold compound is sprayed on the labeling pad 3, and one end of the labeling pad 3, which is far away from the sample pad 4, is erected on the nitrocellulose membrane 2; the nitrocellulose membrane 2 is fixedly connected to a PVC bottom plate, a quality control line C6 and a detection line T7 are coated on the nitrocellulose membrane 2, and the quality control line C6 and the detection line T7 are parallel to each other and are perpendicular to the extension direction of the test strip; the quality control line C6 is coated with CanineCV recombinant antigen; the detection line T7 is coated with a CanineCV antibody; one end of the nitrocellulose membrane 2, which is far away from the sample pad 4, is erected under absorbent paper.
Wherein the spraying amount of the CanineCV antibody-colloidal gold compound on the labeling pad 3 is 2-4 mul/cm, the ratio of the colloidal gold labeled CanineCV antibody is 5-10 mug per ml of labeling amount, the concentration multiple of the CanineCV antibody-colloidal gold compound is 20-30 times, namely, each 20-30 ml of colloidal gold is concentrated to 1ml, and the spraying amount is 2-4 mul/cm.
The concentration of CanineCV antibodies coated by a detection line T7 on the nitrocellulose membrane 2 is 0.5-1.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm; the concentration of CanineCV antigen coated by the quality control line C6 is 1.0-2.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm.
The grain size of the colloidal gold is 20-50 nm.
The preparation method comprises the following steps:
(1) preparing a colloidal gold solution:
weighing 100ml of ultrapure water into a conical flask, adding 1.0ml of 1% chloroauric acid solution into the conical flask, uniformly mixing, heating to boil, quickly adding a proper amount of 1% trisodium citrate, observing the color change of the solution, continuing heating for 5 minutes after the color is stable, stopping adding liquid, cooling the solution to room temperature, and storing at 2-8 ℃;
(2) preparation of marking pad:
s1 CanineCV antibody-colloidal gold complexThe preparation method comprises the steps of taking 1ml of colloidal gold solution, and adding 2-10 mu l of 0.1mol/l K2CO3Uniformly mixing the solution, adding 5-10 mu g of CanineCV antibody, uniformly mixing for 15min, adding 50-100 mu l of 10% BSA solution, sealing for 10min, placing in a refrigerated centrifuge, centrifuging for 30min at 4 ℃ and 10000rpm, removing supernatant, redissolving the precipitate with colloidal gold redissolution to 30-50 mu l, and storing at 2-8 ℃ for later use; the colloidal gold redissolution contains 0.02mol/l Tris buffer solution with the pH value of 8.2, 0.1 percent of Casein, 0.3 to 0.5 percent of PVP40 and 2 to 5 percent of trehalose mixed solution;
s2, spraying the CanineCV antibody-colloidal gold compound onto a marking pad by using a three-dimensional gold spraying film scratching instrument, wherein the spraying amount is 2-4 mu l/cm; placing the sprayed marking pad in a drying chamber (the humidity is less than 30%) for drying for 12h, adding a drying agent, and sealing for later use;
(3) coating a quality control line C and a detection line T:
s1, preparing a quality control line C and a detection line T coating solution, namely taking CanineCV, and diluting the CanineCV into a quality control line C solution of 1.0-2.0 mg/ml by using 0.01mol/L PBS buffer solution with the pH value of 7.4; taking CanineCV antibodies, and diluting the CanineCV antibodies into 0.5-1.0 mg/ml detection line T solution by using 0.01mol/L PBS buffer solution with pH7.4;
s1, scribing a film, namely respectively coating liquids of a quality control line C and a detection line T to the positions of the quality control line and the detection line of the nitrocellulose film by using a three-dimensional metal spraying and film scribing instrument, wherein the interval between C, T lines is 5-8 mm, and the coating amount is 1.0-2.0 mu l/cm; placing the marked film in a drying chamber (the humidity is less than 30%) to dry for 12h, adding a drying agent and sealing for later use;
(4) pretreatment of the sample pad:
soaking the sample pad in the sample pad pretreatment solution for 30min, draining, drying in a drying chamber (humidity less than 30%), drying for 12h, adding a drying agent, and sealing for later use;
the sample pad pretreatment solution contains a mixed solution of a Tris-HCl buffer solution, 0.5%% Tween 20 and 0.5% trehalose, wherein the pH value of the mixed solution is 7-9;
(5) cutting and assembling:
cutting the water absorption pad, the processed sample pad and the gold mark pad according to the size of (15-20mm) × 300mm, and respectively adding a drying agent into the cut water absorption pad, the cut sample pad and the cut gold mark pad and sealing the mixture for later use;
taking the cut water absorption pad, the sample pad, the gold mark pad and the PVC bottom plate with the membrane, pasting the gold mark pad to enable the gold mark pad to be pressed on the nitrocellulose membrane by 1-2 mm, pasting the sample pad to enable the sample pad to be pressed on the gold mark pad by 3-5 mm, and finally pasting the water absorption pad to be pressed on the nitrocellulose membrane by 1-2 mm; and cutting the assembled PVC base plate into 3-4 mm colloidal gold immunochromatographic reagent strips for detecting CanineCV antigens to obtain finished products.
Three comparative examples were prepared for testing in the present invention.
Comparative example 1: the same immunochromatographic reagent strip for detecting canine circovirus antigens as that used in the above examples was prepared, except that comparative example 1 used 1ml of colloidal gold solution, 15. mu.l of 0.1mol/l K2CO3 solution was added and mixed well, and then 15. mu.g of CanineCV antibody was added and mixed well for 15min for labeling, i.e., 1ml of colloidal gold solution and 15. mu.g of antibody were labeled.
Comparative example 2: the same immunochromatographic reagent strip for detecting canine circovirus antigen as that used in the above example was prepared except that comparative example 2 used a colloidal gold complex solution having a pH of 8.5 and a buffer of 0.02mol/l Tris containing 0.1% Casein, 0.3% to 0.5% PVP40, and 5.5% trehalose.
Comparative example 3: the same immunochromatographic reagent strip for detecting canine circovirus antigen as that used in the above example was prepared, except that in comparative example 3, a mixture of Tris-HCl buffer, 0.5% Tween 20, and 0.6% trehalose was added to the pretreatment solution for sample pad
The immunochromatographic reagent strips for detecting canine circovirus antigens obtained in the above examples, and comparative examples 1 to 3 were subjected to performance tests.
First, accuracy investigation
28 CanineCV positive fecal samples (nucleic acid-determined CanineCV positive samples) and 36 healthy dog fecal samples were collected at the canine site and tested using the reagent.
The detection result shows that: the immunochromatographic reagent strip for detecting canine circovirus antigen obtained in the example is positive in detection results of 28 porcine CanineCV positive samples and negative in detection results of 36 healthy control samples. The accuracy rates of the positive sample and the negative sample reach 100 percent, the detection rate is high, and the kit meets the requirements of the kit.
The test results of the test strips obtained in comparative examples 1-3 were negative for 28 porcine CanineCV positive samples and 36 healthy control samples. The coincidence rate of the positive reference substance reaches 0%, so that the detection cannot be carried out, and the examination of specificity, sensitivity and the like of the reagent strips obtained by the comparison ratios 1-3 is not needed.
(II) examination of specificity
The reagent strips obtained in the present example were used to detect Canine Distemper (CDV), Canine Parvo (CPV), porcine circovirus type 2 (PCV2), and duck circovirus (DuCV) samples (positive samples confirmed by the nucleic acid method), respectively, and the detection results are shown in table 1.
TABLE 1 results of specificity test
As can be seen from the test results in the table above, the test strips obtained in this example were negative to the test results of CDV, CPV, PCV2 and DuCV, indicating that the colloidal gold reagent of the present invention has good specificity.
(III) repeatability examination
The test was repeated 5 times using 28 CanineCV positive fecal samples (CanineCV positive samples determined by the nucleic acid method) and 36 healthy dog fecal samples obtained in this example.
And (3) detection results: the results of 5 repeated detections are consistent, which shows that the reagent has good repeatability and strong practicability.
(IV) stability examination
The test strips obtained in this example were left at 37 ℃ for 5 days, 10 days, 15 days, 20 days, and 30 days, respectively, to perform accelerated destructive tests, and then 5 positive samples were detected, respectively, with the test results shown in table 2.
Table 2 stability test results
As can be seen from the test results in the above table, the test results of the test strip of this example which was left for 15 days were all positive, and the positive rate began to decrease after 30 days. It can be seen that the test strip of this example has an accelerated stability of at least 20 days, which is comparable.
(V) comparison with nucleic acid detection method
The samples to be detected collected in 28 cases of canine fields are respectively detected by a nucleic acid detection kit (RT-PCR method) and the reagent of the invention. The SPSS software performs the chi-square test, the results of which are shown in table 3.
TABLE 3 chi-square test results of colloidal gold method and RT-PCR method of the present invention
As is clear from the results in Table 3 above, the P value of the paired Chi Square test (McNemar test) of the colloidal gold method of this example is 1.000, and the Kappa value of the consistency test (Kappa test) is 0.984. The chi-square test result shows that the colloidal gold method and the RT-PCR method have no significant difference in clinical sample detection and high accuracy.
The above results show that the test strip prepared by the embodiment can realize on-site rapid detection without special instruments and technical personnel, facilitates diagnosis of CanineCV diseases, provides treatment in time, has high accuracy and strong repeatability of detection results, and is suitable for popularization and application.
The test strip provided by the invention fills the blank that no CanineCV virus antigen rapid detection reagent exists in China, and has important significance for preventing and controlling CanineCV epidemic diseases and guaranteeing the safety of pet dogs/canids and epidemiological investigation thereof.
The above description is only for the purpose of illustrating the present invention and not for the purpose of limiting the same, and equivalent modifications and variations of the present invention will be apparent to those skilled in the art without departing from the overall spirit of the present invention.
Claims (10)
1. An immunochromatography reagent strip for detecting canine circovirus antigens, which comprises a PVC bottom plate and is characterized in that: the device also comprises a water absorption pad, a nitrocellulose membrane, a marking pad and a sample pad which are sequentially stuck on the PVC bottom plate;
the sample pad is arranged on one end of the PVC bottom plate; the sample pad is provided with a sample dripping area; one end of the sample pad is erected on the marking pad, the CanineCV antibody-colloidal gold compound is sprayed on the marking pad, and one end of the marking pad far away from the sample pad is erected on the nitrocellulose membrane; the nitrocellulose membrane is fixedly connected to the PVC bottom plate, a quality control line C and a detection line T are coated on the nitrocellulose membrane, and the quality control line C and the detection line T are parallel to each other and are arranged perpendicular to the extension direction of the test strip; the quality control line C is coated with CanineCV recombinant antigen; the detection line T is coated with CanineCV antibodies; and one end of the nitrocellulose membrane far away from the sample pad is erected below absorbent paper.
2. The immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 1, which is characterized in that: the spraying amount of the CanineCV antibody-colloidal gold compound on the labeling pad is 2-4 mul/cm, the ratio of the colloidal gold labeling CanineCV antibody is 5-10 mug per ml of labeling amount, the concentration multiple of the CanineCV antibody-colloidal gold compound is 20-30 times, namely, the colloidal gold is concentrated to 1ml per 20-30 ml, and the spraying amount is 2-4 mul/cm.
3. The immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 1, which is characterized in that: the concentration of CanineCV antibodies coated by the detection line T on the nitrocellulose membrane is 0.5-1.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm; the concentration of the CanineCV antigen coated by the quality control line C is 1.0-2.0 mg/ml, and the scribing amount is 1.0-2.0 mu l/cm.
4. The immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 1, which is characterized in that: the grain size of the colloidal gold is 20-50 nm.
5. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen as recited in any one of claims 1 to 4, which is characterized in that: comprises the following steps:
(1) preparing a colloidal gold solution;
(2) preparing a labeling pad, including preparing CanineCV antibody-colloidal gold compound, taking 1ml of colloidal gold solution, and adding 2-10 mu l of 0.1mol/l K2CO3Uniformly mixing the solution, adding 5-10 mu g of CanineCV antibody, uniformly mixing for 15min, adding 50-100 mu l of 10% BSA solution, sealing for 10min, placing in a refrigerated centrifuge, centrifuging for 30min at 4 ℃ and 10000rpm, removing supernatant, redissolving the precipitate with colloidal gold redissolution to 30-50 mu l, and storing at 2-8 ℃ for later use;
(3) coating a quality control line C and a detection line T;
(4) pretreating a sample pad;
(5) cutting and assembling; and obtaining a finished product.
6. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 5, which is characterized in that: the colloidal gold redissolution contains 0.02mol/l Tris buffer solution with the pH value of 8.2, 0.1 percent of Casein, 0.3 to 0.5 percent of PVP40 and 2 to 5 percent of trehalose mixed solution.
7. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 5, which is characterized in that: the preparation process in the step (1) comprises the steps of weighing 100ml of ultrapure water into a conical flask, adding 1.0ml of 1% chloroauric acid solution into the conical flask, uniformly mixing, heating to boiling, rapidly adding a proper amount of 1% trisodium citrate, observing the color change of the solution, continuing heating for 5 minutes after the color is stable, stopping adding liquid, cooling the solution to room temperature, and storing at 2-8 ℃.
8. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 5, which is characterized in that: the step (2) further comprises S2, spraying gold, and spraying the CanineCV antibody-colloidal gold compound onto a marking pad by using a three-dimensional gold spraying film scratching instrument, wherein the spraying amount is 2-4 mu l/cm; and (3) drying the sprayed marking pad in a drying chamber (the humidity is less than 30%) for 12h, adding a drying agent, and sealing for later use.
9. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 5, which is characterized in that: the treatment process of the step (4) comprises the steps of soaking the sample pad in sample pad pretreatment liquid for 30min, draining, drying in a drying chamber (the humidity is less than 30%) for 12h, adding a drying agent, and sealing for later use;
the sample pad pretreatment solution contains a mixed solution of a Tris-HCl buffer solution, 0.5% Tween 20 and 0.5% trehalose, and the pH value of the mixed solution is 7-9.
10. The method for preparing an immunochromatographic reagent strip for detecting canine circovirus antigen according to claim 5, which is characterized in that: the specific steps of the step (3) comprise S1, quality control line C and detection line T coating liquid preparation: diluting CanineCV into a quality control line C solution of 1.0-2.0 mg/ml by using 0.01mol/L PBS buffer solution with the pH value of 7.4; taking CanineCV antibodies, and diluting the CanineCV antibodies into 0.5-1.0 mg/ml detection line T solution by using 0.01mol/L PBS buffer solution with pH7.4;
s1, scribing: coating the coating liquid of the quality control line C and the detection line T to the positions of the quality control line and the detection line of the nitrocellulose membrane respectively by using a three-dimensional metal spraying and membrane scratching instrument, wherein the interval between C, T lines is 5-8 mm, and the coating amount is 1.0-2.0 mu l/cm; placing the marked film in a drying chamber (the humidity is less than 30%) to dry for 12h, adding a drying agent and sealing for later use;
the process of the step (5) comprises the steps of taking the water absorption pad, the processed sample pad and the gold label pad, cutting according to the size of (15-20mm) × 300mm, and respectively adding a drying agent into the cut water absorption pad, the cut sample pad and the cut gold label pad and sealing for later use;
taking the cut water absorption pad, the sample pad, the gold mark pad and the PVC bottom plate with the membrane, pasting the gold mark pad to enable the gold mark pad to be pressed on the nitrocellulose membrane by 1-2 mm, pasting the sample pad to enable the sample pad to be pressed on the gold mark pad by 3-5 mm, and finally pasting the water absorption pad to be pressed on the nitrocellulose membrane by 1-2 mm; and cutting the assembled PVC base plate into 3-4 mm colloidal gold immunochromatographic reagent strips for detecting CanineCV antigens to obtain finished products.
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Application publication date: 20210928 |