CN112415202A - Test strip for detecting feline coronavirus, preparation method thereof, kit and detection method - Google Patents
Test strip for detecting feline coronavirus, preparation method thereof, kit and detection method Download PDFInfo
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Abstract
The invention relates to a test strip for detecting feline coronavirus and a kit prepared from the test strip, wherein the test strip comprises a bottom plate, a sample cushion layer, a colloidal gold cushion layer, a nitrocellulose membrane layer and a water absorbing layer are sequentially overlapped on the bottom plate from left to right, and the colloidal gold cushion layer is coated with a feline coronavirus monoclonal antibody marked by colloidal gold particles and a feline infectious peritonitis virus polyclonal antibody; the nitrocellulose membrane layer is coated with a feline coronavirus monoclonal antibody as a T1 detection line, and is also coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line, and is coated with goat anti-chicken IgG as a quality control line. The test strip and the kit are used for detecting the feline coronavirus antigen, the operation is simple, the real POCT detection is achieved, and the virus transmission is effectively controlled. And the test paper strip has the form of multi-antibody markers and multi-antibody coatings, so that the sensitivity of the reagent is improved to the maximum extent, and the detection rate is increased.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a test strip for detecting feline coronavirus, a preparation method thereof, a kit and a detection method.
Background
Feline Infectious Peritonitis (FIP) is caused by infection with feline coronavirus, which was first discovered in the 60's of the 20 th century, and FIP is classified into exudative type and non-exudative type, and non-exudative type FIP is converted into exudative type FIP as the disease progresses. The exudative FIP is mainly clinically characterized by fibrinous peritonitis, pleuritis and pericarditis accompanied by abdominal cavity, thoracic cavity or pericardial effusion; non-exudative FIP has no obvious effusion, and is clinically characterized by granulomatous lesions in different organs, including eyes and central nervous system. Both exudative and non-exudative FIPs are associated with severe systemic disease and produce varying degrees of pleural or peritoneal fluid accumulation.
Feline coronavirus (FCoV) is a member of the genus coronavirus of the family coronaviridae and is the causative agent of the lethal disease FIP in domestic and wild cats. FCoV is divided into two biotypes based on clinical symptoms and the resulting pathological changes, Feline Infectious Peritonitis Virus (FIPV) and Feline Enteric Coronaviruses (FECV). FECV is ubiquitous in the intestinal tract of cats, and cats infected with FECV have mild intestinal symptoms, can heal themselves and have low mortality. The feline infectious disease caused by FIPV infection is a chronic progressive lethal infectious disease, which is mainly characterized by peritonitis and large accumulation of ascites, cats of different ages can be infected, but the incidence of old cats and cats under two years old is high, the incidence of pure cats is higher than that of domestic cats of general species, and the incidence of infection is very high.
FCoV is ubiquitous in cat groups around the world, the positive rate of FCoV infected by cats in different areas, breeding environments and species is greatly different, the virus propagation path is mainly fecal oral transmission, a few cats can be propagated through mechanical paths such as clothes, dishes, bedding, people or insects, the latency period can reach several months, and some cats can be continuously infected with FCoV for 10 years. A serological survey in california in the united states showed 20.0% FCoV positive rates in pet cats kept alone and 87.0% FCoV positive rates in all breeds kept in a herd, so FCoV infections were generally associated with cat numbers and breeding densities. Although group rearing is not the primary reason for pandas who wander to become FCoV, the group rearing environment increases the risk of cats becoming FCoV, and in particular, the environment at a wandering cat rescue station has a higher viral load, making cats more susceptible to virus infection. The results of a study conducted on 226720 cats over 10 years at 24 veterinary medical academy, USA, showed that FIP was one in every 200 new cases. The risk of suffering from FIP of male cats and young cats is high, and the cats at the age of 6-24 months are more likely to suffer from the FIP in the confirmed diagnosis of the American veterinary teaching hospital. By optimizing population density and feeding environment, the incidence rate of FIP development of the group-bred cats infected with FCoV can be greatly reduced.
Laboratory examination methods for FCoV infection mainly comprise serum antibody screening, an RT-PCR method, histopathological staining sections and the like, the detection methods all need to take blood for cats, and blood taking for animals has the problems of difficulty in blood taking and small sample amount. There is a need to find a rapid and convenient diagnostic reagent which can diagnose whether cats are infected with FCoV or not in time and effectively, and avoid the development of FIP which can cause lethal diseases after FCoV infection.
Disclosure of Invention
The invention aims to provide a test strip for detecting feline coronavirus, which mainly aims at solving the problem of how to quickly and accurately detect the feline coronavirus.
In order to achieve the purpose, the application provides a test strip for detecting feline coronavirus, which comprises a bottom plate, wherein a sample cushion layer, a colloidal gold cushion layer, a nitrocellulose membrane layer and a water absorbing layer are sequentially overlapped on the bottom plate from left to right, and the colloidal gold cushion layer is coated with a feline coronavirus monoclonal antibody marked by colloidal gold particles and a feline infectious peritonitis virus polyclonal antibody; the nitrocellulose membrane layer is coated with a feline coronavirus monoclonal antibody as a T1 detection line, and is further coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line, and is coated with goat anti-chicken IgG as a quality control line. In the application, the feline coronavirus monoclonal antibody is mainly used for detecting feline enterocoronavirus, and the feline infectious peritonitis virus polyclonal antibody is mainly used for detecting feline infectious peritonitis virus.
As a further improvement of the application, the concentration of the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody coated on the colloidal gold cushion layer is 15 to 25 mu g/mL; the concentration of the feline coronavirus monoclonal antibody on a T1 detection line is 0.6 mg/mL-1.2 mg/mL; the concentration of the polyclonal antibody of the feline infectious peritonitis virus on the T2 detection line is 0.6 mg/mL-1.2 mg/mL; the concentration of the goat anti-chicken IgG on the quality control line is 1.0 mg/mL-1.5 mg/mL.
As a further improvement of the application, the ratio of the feline coronavirus monoclonal antibody to the feline infectious peritonitis virus polyclonal antibody is 2: 1.
As a further improvement of the application, the colloidal gold cushion layer is also coated with chicken anti-cat IgY antibodies marked by colloidal gold particles.
As a further improvement of the application, the concentration of the chicken anti-cat IgY antibody is 10-15 mu g/mL.
As the further improvement of this application, the cellulose nitrate membrane layer is pasted the intermediate position of bottom plate on the bottom plate the layer that absorbs water is pasted to one side of cellulose nitrate membrane layer, colloidal gold bed course and sample bed course are pasted to the opposite side of cellulose nitrate membrane layer, and colloidal gold bed course one end is pushed down to sample bed course one end 1.0mm ~ 1.5mm, and the cellulose nitrate membrane layer one end 0.5mm ~ 1mm is pushed down to the colloidal gold bed course other end, and the cellulose nitrate membrane layer other end 1.0mm ~ 2.0mm is pushed down to the one end on the layer that absorbs water.
In order to achieve the above object, the present application further provides a preparation method of the test strip for detecting feline coronavirus, which comprises the following steps: s1, preparing a colloidal gold cushion layer: coating the colloidal gold pad with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody marked by colloidal gold particles; s2, preparing a nitrocellulose membrane: coating a cat coronavirus monoclonal antibody on a nitrocellulose membrane layer as a T1 detection line, further coating a cat infectious peritonitis virus polyclonal antibody as a T2 detection line, and coating goat anti-chicken IgG as a quality control line; s3, preparation of a sample cushion layer: firstly, preparing a treating fluid, namely 100mmol/L Tris-HCL buffer solution with pH9.0, which contains 0.5 percent of PEG20000, 0.05 to 0.2 percent of Tween-20, 0.01 to 0.05 percent of surfactant and 0.3 to 1.0 percent of blocking agent; secondly, coating the treatment solution on glass fiber to obtain a sample cushion layer; s4, preparation of the test strip: and (3) sequentially overlapping the sample cushion layer in the step S3, the colloidal gold cushion layer in the step S1, the cellulose nitrate film layer and the water absorbing layer in the step S2 on the bottom plate 1 from left to right to obtain the test strip for detecting the feline coronavirus.
As a further improvement of the application, chicken anti-cat IgY antibodies are coated on the colloidal gold pad.
In order to achieve the above object, the present application further provides a kit for detecting feline coronavirus, which comprises the test strip for detecting feline coronavirus, a sampling swab and a sample diluent.
In order to achieve the above object, the present application provides a method for detecting feline coronavirus by using the above-mentioned kit, comprising the following steps: s1, placing the sample in a sample diluent, fully mixing and standing, and taking the supernatant as a detection liquid sample; and S2, dripping the detection liquid sample into a sample hole of the kit, standing for a period of time, and judging the result.
The reagent strip comprises a bottom plate, a sample cushion layer, a colloidal gold cushion layer, a cellulose nitrate film layer and a water absorbing layer are sequentially overlapped on the bottom plate from left to right, and the colloidal gold cushion layer is coated with a feline coronavirus monoclonal antibody marked by colloidal gold particles and a feline infectious peritonitis virus polyclonal antibody; the nitrocellulose membrane layer is coated with a feline coronavirus monoclonal antibody as a T1 detection line, and is further coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line, and is coated with goat anti-chicken IgG as a quality control line. The test strip and the kit are used for detecting the feline coronavirus antigen, are simple to operate, can be applied to pet hospitals, families and other places, achieve real POCT detection, and can effectively control the virus propagation.
The test strip adopts the form of multi-antibody marking and multi-antibody coating, the colloidal gold cushion layer is prepared in a mode of marking two types of antibodies, and the two types of monoclonal antibodies are respectively coated on the nitrocellulose membrane, so that compared with a single-antibody-marked monoclonal antibody and a single-antibody coating technology, the reagent sensitivity is improved to the maximum extent, and the detection rate is increased. The detection method of the kit provided by the invention has the advantages of simple steps and strong operability, and can be used for simultaneously detecting the feline coronavirus antigens in different media of excrement, secretion and ascites of the cats, so as to realize the detection of the feline coronavirus antigens in different media. The pet cat secretion and excrement in the family are easier to collect, and the self-checking use of the pet cat family is realized.
Drawings
FIG. 1 is a schematic structural diagram of a test strip for detecting feline coronavirus;
FIG. 2 is a schematic diagram of the test strip showing that the test strip has a negative determination result;
FIG. 3 is a schematic diagram of the test strip showing that the test strip shows a positive result;
FIG. 4 is a schematic diagram of the test strip showing the test strip being invalid;
FIG. 5 is a schematic diagram showing the test results of the specificity test paper for different virus samples;
in the figure: 1. a base plate; 2. a sample cushion layer; 3. a colloidal gold cushion layer; 4. a cellulose nitrate film layer; 5. a water-absorbing layer; 41. t1 detection line; 42. t2 detection line; 43. and (4) quality control line.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be understood that the described embodiments are only a few embodiments of the present application, not all embodiments, and are not intended to limit the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In order to rapidly and accurately detect the feline coronavirus, the test strip for detecting the feline coronavirus comprises a bottom plate 1, wherein a sample cushion layer 2, a colloidal gold cushion layer 3, a nitrocellulose membrane layer 4 and a water absorbing layer 5 are sequentially overlapped on the bottom plate 1 from left to right, and the colloidal gold cushion layer 3 is coated with a feline coronavirus monoclonal antibody marked by colloidal gold particles and a feline infectious peritonitis virus polyclonal antibody; the nitrocellulose membrane layer 4 is coated with a feline coronavirus monoclonal antibody as a T1 detection line 41, and is further coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line 42, and is coated with goat anti-chicken IgG as a quality control line 43. In the application, the cat coronavirus monoclonal antibody and the cat infectious peritonitis virus polyclonal antibody are mixed according to a certain proportion, and 2 biological cat coronavirus antibodies are mixed on the colloidal gold cushion layer 3, so that two biological types of the cat coronavirus are covered, the missing detection is reduced, and the detection rate of the cat coronavirus is favorably improved.
In the present application, as a preferred embodiment, the concentration of the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody coated on the colloidal gold backing layer 3 is 15 μ g/mL to 25 μ g/mL; the concentration of the feline coronavirus monoclonal antibody on the T1 detection line 41 is 0.6 mg/mL-1.2 mg/mL; the concentration of the polyclonal antibody of the feline infectious peritonitis virus on the T2 detection line 42 is 0.6 mg/mL-1.2 mg/mL; the concentration of the goat anti-chicken IgG on the quality control line 43 is 1.0 mg/mL-1.5 mg/mL. As a further preferred embodiment, the ratio of said feline coronavirus monoclonal antibody and said feline infectious peritonitis virus polyclonal antibody is 2: 1.
In this application, as a preferred embodiment, the colloidal gold cushion layer 3 is further coated with a chicken anti-cat IgY antibody labeled with colloidal gold particles, and the chicken anti-cat IgY antibody and the mammal immunoglobulin (such as rheumatoid factor and autoantibody) will not undergo a serum immune cross reaction, thereby avoiding the generation of false positive. As a preferable example, the concentration of the chicken anti-cat IgY antibody is 10 to 15 mu g/mL.
In the application, a preparation method of the test strip for detecting the feline coronavirus is also provided for quickly and accurately detecting the feline coronavirus, and S1 and the preparation of the colloidal gold cushion layer 3 are as follows: coating the colloidal gold pad with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody marked by colloidal gold particles; s2, preparation of nitrocellulose membrane layer 4: coating a feline coronavirus monoclonal antibody on the nitrocellulose membrane layer 4 as a T1 detection line 41, further coating a feline infectious peritonitis virus polyclonal antibody as a T2 detection line 42, and coating goat anti-chicken IgG as a quality control line 43; s3, preparation of sample pad layer 2: firstly, preparing a treating fluid, namely 100mmol/L Tris-HCL buffer solution with pH9.0, which contains 0.5 percent of PEG20000, 0.05 to 0.2 percent of Tween-20, 0.01 to 0.05 percent of surfactant and 0.3 to 1.0 percent of blocking agent; secondly, coating the treatment fluid on glass fiber to obtain a sample pad; s4, preparation of the test strip: and sequentially overlapping the sample cushion layer 2 in the step S3, the colloidal gold cushion layer 3 in the step S1, the nitrocellulose membrane layer 4 and the water absorbing layer 5 in the step S2 on the bottom plate 1 from left to right to obtain the test strip for detecting the feline coronavirus. Further, the commonly used blocking agent is BSA, skimmed milk powder, casein, serum and the like, and the surfactant can be triton X-100. In the application, the mass percentage of the Tween-20 is further limited to 0.05-0.1%.
In the present application, as a preferred embodiment, in the preparation of the gold colloid pad layer 3: the preparation method comprises the following steps of coating a colloidal gold pad with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody marked by colloidal gold particles: the colloidal gold is prepared by a trisodium citrate reduction method, the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody are slowly added into the colloidal gold according to the proportion of 15 mu g/mL-25 mu g/mL, and further preference is given, slowly adding the cat coronavirus monoclonal antibody and the cat infectious peritonitis virus polyclonal antibody into the colloidal gold according to the proportion of 20 mu g/mL, fully reacting to obtain a first colloidal gold mixture, centrifuging the first colloidal gold mixture at the centrifugal speed of 7000 rpm-10000 rpm for 30 minutes, discarding supernatant, concentrating, standing for reacting for 20 min-30 min to obtain the cat coronavirus monoclonal antibody and the cat infectious peritonitis virus polyclonal antibody marked by the colloidal gold particles, namely a first colloidal gold compound, and spraying the first colloidal gold compound on the glass fiber to obtain a colloidal gold cushion layer 3; as a further preferred example, the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody are mixed in a ratio of 2:1, the concentration of the first colloidal gold complex prepared from the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody is preferably 15% by a factor of 20 for the concentration of the colloidal gold mixture. In the present application, as a preferred embodiment, the specific spraying process of the first colloidal gold compound is as follows: putting glass fiber into a stainless steel disc, measuring 45mL of a first colloidal gold compound, pouring the first colloidal gold compound containing 4.5mL of the feline coronavirus monoclonal antibody marked by the colloidal gold particles and 2.25mL of the feline infectious peritonitis virus polyclonal antibody marked by the colloidal gold particles into the stainless steel disc to ensure that the first colloidal gold compound is completely and uniformly absorbed, and then putting the first colloidal gold compound into an electrothermal blowing dry box at 37 ℃ for drying for 12 hours to obtain a colloidal gold cushion layer 3 for storage and later use.
In the present application, as another preferred example, in the preparation of the gold colloidal pad 3: the preparation method comprises the following steps of coating a colloidal gold pad with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody marked by colloidal gold particles, and coating a chicken anti-feline IgY antibody on the colloidal gold pad at the same time: the first colloidal gold complex is prepared as described above, and the second colloidal gold complex is prepared as follows: slowly adding the chicken anti-cat IgY antibody into the colloidal gold according to the proportion of 10-15 mu g/mL, fully reacting to obtain a second colloidal gold mixture, centrifuging the second colloidal gold mixture at the centrifugal speed of 8000-10000 rpm for 30 minutes, discarding supernatant, and concentrating to obtain the chicken anti-cat IgY antibody marked by the colloidal gold particles, namely a second colloidal gold compound. As another preferred embodiment, the gold colloid cushion layer 3 is prepared by spraying the first gold colloid compound on the glass fiber at a concentration of 10% to 15% and the second gold colloid compound at a concentration of 3% to 4%, and drying to obtain the gold colloid cushion layer 3.
In the present application, as a preferred example, the preparation of the nitrocellulose membrane layer 4: the cellulose nitrate membrane layer 4 is coated with a feline coronavirus monoclonal antibody as a T1 detection line 41, a feline infectious peritonitis virus polyclonal antibody as a T2 detection line 42 and a sheep anti-chicken IgG as a quality control line 43, and the preparation method comprises the following steps: diluting the feline coronavirus monoclonal antibody with the concentration of 0.6-1.2 mg/mL by using 50mmol/L PBS buffer solution, adding a sucrose solution with the mass percentage of 2-4%, coating the solution on a nitrocellulose membrane, and drying the solution for 2 hours at the temperature of 60 ℃ to obtain a T1 detection line 41 after the feline coronavirus monoclonal antibody is coated on a nitrocellulose membrane layer 4; diluting the feline infectious peritonitis virus polyclonal antibody with the concentration of 0.6-1.2 mg/mL by using 50mmol/L PBS buffer solution, adding a sucrose solution with the mass percentage of 2-4%, coating the solution on a nitrocellulose membrane, and drying the cellulose nitrate membrane for 2 hours at the temperature of 60 ℃ to obtain a T2 detection line 42 coated with the feline infectious peritonitis virus polyclonal antibody on the nitrocellulose membrane layer 4; diluting goat anti-chicken IgG with the concentration of 1.0-1.5 mg/mL by using 50mmol/L PBS buffer solution, adding a sucrose solution with the mass percentage of 2-4%, coating the solution on a nitrocellulose membrane, and drying the solution for 2 hours at the temperature of 60 ℃ to obtain a quality control line 43 for coating the goat anti-chicken IgG on the nitrocellulose membrane layer 4.
In this application, as a preferred embodiment, the preparation of the sample pad: firstly, preparing a treatment solution, namely a Tris-HCL buffer solution with pH9.0, wherein the Tris-HCL buffer solution contains 0.5 percent of PEG20000 macromolecular polymer, 0.05 to 0.2 percent of Tween-20, 0.01 to 0.05 percent of triton X-100, 0.5 percent of BSA, and 0.3 percent of casein in 100 mmol/L; secondly, coating the treatment solution on glass fiber, and drying to obtain a sample pad; as another preferred embodiment, preparation of the sample pad: firstly, preparing a treatment solution, which comprises 0.5 percent of PEG20000 macromolecular polymer, 0.05 to 0.2 percent of Tween-20, 0.01 to 0.05 percent of Triton X-100, 0.5 percent of 100mmol/L of casein sodium and Tris-HCL buffer solution with pH9.0; the detailed process of the treatment solution preparation is as follows: weighing 12.114g Tris (Tris (hydroxymethyl aminomethane)) and dissolving in 900mL ultrapure water, sequentially adding 5g PEG20000, 5g sodium caseinate, 2mL Tween-20 and 0.2mL Triton X-100, fully dissolving and mixing, adjusting pH to 9.0 with HCL, and adding ultrapure water to a constant volume of 1L; next, the treatment liquid is applied to glass fibers and dried, and it is more preferable to use: cutting glass fiber into 30cm × 25cm cushion, uniformly coating the cushion with 50ml of treatment solution, drying in an electrothermal blowing drying oven at 37 deg.C for 16 hr to obtain sample cushion, and storing for use.
In this application, as a preferred embodiment, the test strip is prepared by: sequentially overlapping the sample cushion layer 2 in the step S3, the colloidal gold cushion layer 3 in the step S1, the nitrocellulose membrane layer 4 in the step S2 and the water absorbing layer 5 on the bottom plate 1 from left to right, wherein the bottom plate 1 is a PVC (polyvinyl chloride) plate, the nitrocellulose membrane layer 4 is prepared at the middle position of the bottom plate 1 in an adhering manner, and the water absorbing layer 5 is adhered to one side of the bottom plate 1 at which the nitrocellulose membrane is fixed; sticking a prepared colloidal gold cushion layer 3 and a prepared sample cushion layer 2 on the other side of the position of the bottom plate 1, wherein one end of the sample cushion layer 2 presses one end of the colloidal gold cushion layer 3 for 1.0-1.5 mm, the other end of the colloidal gold cushion layer 3 presses one end of the nitrocellulose membrane layer 4 for 0.5-1 mm, one end of the water absorption layer 5 presses the other end of the nitrocellulose membrane layer 4 for 1.0-2.0 mm, and cutting the product obtained in the step into test strips with the width of 3.0mm after the product is pressed to be flat, so as to obtain the test strips for detecting the feline coronavirus,
the application also provides a kit for detecting feline coronavirus antigens, which comprises the test strip for detecting feline coronavirus, a swab for sampling and a sample diluent; as a preferred embodiment, the sample diluent is 10mmol/L Tris-HCl buffer containing bovine serum albumin BSA, tween-80 and a preservative.
The application also provides a method for detecting the feline coronavirus antigen, which comprises the following steps: s1, placing the sample in a sample diluent, fully mixing and standing, and taking the supernatant as a detection liquid sample; and S2, dripping the detection liquid sample into a sample hole of the kit, standing for a period of time, and judging the result. As a preferred embodiment, the detailed detection steps are as follows: s1, wherein the sample is obtained from cat excrement, secretion and ascites. During sampling, the stool sample is not required to be collected in too large amount, and the swabs 1/3-2/3 are preferably covered; the swab is preferably fully wetted for secretion samples, such as secretion samples of conjunctiva, nasal cavity, oral cavity, anus and other parts of eyes of an animal to be detected; aiming at ascites, an ascites sample is extracted from the abdominal cavity of the sick cat by using a syringe, and the sampling amount is generally 2 mL-3 mL. The sample was placed in the sample dilution and the detailed procedure was as follows: if the sample is a fecal sample or a secretion sample, directly inserting a sampling swab into a sample tube filled with a buffer solution; if the sample is ascites, 0.2mL of the ascites sample is added to the sample tube containing the buffer. Placing the sample in a sample diluent, fully mixing uniformly, standing for 1min, taking the supernatant as a detection liquid sample, sucking the detection liquid sample to be detected by using a disposable plastic pipette, dripping 2-3 drops (80-100 mu L) into a sample hole of the kit, standing for 5-8 min to judge the result, and judging the result ineffectively after the standing time is more than 10 min. The criteria for the result judgment are as follows:
negative, as shown in fig. 2: only one red strip appears at the position of a quality control line 43C, and no red strip appears at the detection lines T1 and T2 of the feline coronavirus antigen test strip, which indicates that the feline coronavirus antigen is a negative result, and the sample does not contain feline coronavirus (FECV) and feline infectious peritonitis virus (FCoV);
positive, as shown in fig. 3:
1. three red bands appear. Two red bands appear at the T1 detection line and the T2 detection line, respectively, and the other red band appears at the quality control line 43C. The positive result shows that: the sample contains both feline coronavirus (FECV) and feline infectious peritonitis virus (FCoV);
2. two red bands appear. One red band appears at the T1 detection line and the other red band appears at the quality control line 43C, respectively. The positive result shows that: the sample contains feline coronavirus (FECV);
3. two red bands appear. One red band appears at the T2 detection line and the other red band appears at the quality control line 43C, respectively. The positive result shows that: the sample contains feline infectious peritonitis virus (FCoV);
invalid, as shown in fig. 4: no red band appeared at the control line 43C, indicating a malfunction or reagent failure. If the result is invalid, repeated experiments are required.
And (3) specific detection: in order to investigate the specificity of the test strip, the cats with the feline panleukosis virus, the feline enterocoronavirus and the feline infectious peritonitis virus are respectively sampled and detected, wherein the sample with the feline panleukosis virus is used as a negative sample, the detection method is adopted for detection, the detection result is shown in fig. 5, wherein the detection result of the feline panleukosis virus corresponds to the detection result shown in fig. 5, the detection result of the feline infectious peritonitis virus corresponds to the detection result shown in fig. 5, the detection result of the feline panleukosis virus corresponds to the detection result shown in fig. 5, and the detection results of the feline panleukosis virus sample and the feline infectious peritonitis virus sample are negative and positive according to the detection results.
And (3) detecting the sensitivity of the feline coronavirus: the concentration is respectively 101TCID50/mL、102TCID50/mL、102.5TCID50/mL、103TCID50/mL、103.5TCID50/mL、104TCID50/mL、104.5TCID50/mL、105TCID50/mL、105.5TCID50The detection method is adopted to detect the/mL feline coronavirus sample, and the detection result shows that the minimum detection limit of the test strip is 104.5TCID50/mL。
And (3) detecting the sensitivity of the feline infectious peritonitis virus: the concentration is respectively 102TCID50/mL、102.5TCID50/mL、103TCID50/mL、103.5TCID50/mL、104TCID50/mL、104.5TCID50/mL、105TCID50/mL、105.5TCID50/mL、106TCID50the/mL feline infectious peritonitis virus sample is detected according to the detection method, and the detection result shows that the minimum detection limit of the test strip is 105TCID50/mL。
And (3) stability test: the stability of different batches of immunochromatographic test strips was tested on days 1, 5, 10, 15, 19, 20, 21 and 22 by using 104.5TCID50Perml feline coronavirus and 105TCID50The detection method is adopted to detect the feline infectious peritonitis virus, and test strips used for detection are respectively stored for 1, 5, 10, 15, 19, 20, 21 and 22 days at 37 ℃. The stability detection results of different batches are the same and are all positive.
In summary, the application provides a test strip for detecting feline coronavirus and a kit prepared from the test strip, the test strip comprises a bottom plate 1, a sample cushion layer 2, a colloidal gold cushion layer 3, a nitrocellulose membrane layer 4 and a water absorbing layer 5 are sequentially overlapped on the bottom plate 1 from left to right, and the colloidal gold cushion layer 3 is coated with a feline coronavirus monoclonal antibody marked by colloidal gold particles and a feline infectious peritonitis virus polyclonal antibody; the nitrocellulose membrane layer 4 is coated with a feline coronavirus monoclonal antibody as a T1 detection line 41, and is further coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line 42, and is coated with goat anti-chicken IgG as a quality control line 43. The test strip and the kit are used for detecting the feline coronavirus antigen, are simple to operate, have good specificity, detection sensitivity and stability, can be applied to pet hospitals, families and other places, achieve real POCT detection, and can effectively control the virus propagation.
The test strip adopts the form of multi-antibody marking and multi-antibody coating, the colloidal gold cushion layer 3 is prepared in a mode of marking two types of antibodies, and the monoclonal antibodies of the two types are coated on the nitrocellulose membrane respectively, so that compared with a single-antibody-marked monoclonal antibody and a single-antibody coating technology, the reagent sensitivity is improved to the maximum extent, and the detection rate is increased. The detection method of the kit provided by the invention has the advantages of simple steps and strong operability, and can be used for simultaneously detecting the feline coronavirus antigens in different media of excrement, secretion and ascites of the cats, so as to realize the detection of the feline coronavirus antigens in different media. The pet cat secretion and excrement in the family are easier to collect, and the self-checking use of the pet cat family is realized.
It is to be emphasized that: the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and all simple modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
Claims (10)
1. The test strip for detecting the feline coronavirus comprises a base plate (1), wherein a sample cushion layer (2), a colloidal gold cushion layer (3), a nitrocellulose membrane layer (4) and a water absorbing layer (5) are sequentially overlapped on the base plate (1) from left to right, and the test strip is characterized in that the colloidal gold cushion layer (3) is coated with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody which are marked by colloidal gold particles; the nitrocellulose membrane layer (4) is coated with a feline coronavirus monoclonal antibody as a T1 detection line (41), and is also coated with a feline infectious peritonitis virus polyclonal antibody as a T2 detection line (42), and is coated with goat anti-chicken IgG as a quality control line (43).
2. The test strip for detecting feline coronavirus according to claim 1, wherein the concentration of the feline coronavirus monoclonal antibody and the feline infectious peritonitis virus polyclonal antibody coated on the colloidal gold backing layer (3) is 15 to 25 μ g/mL;
the concentration of the feline coronavirus monoclonal antibody on the T1 detection line (41) is 0.6 mg/mL-1.2 mg/mL;
the concentration of the feline infectious peritonitis virus polyclonal antibody on a T2 detection line (42) is 0.6 mg/mL-1.2 mg/mL;
the concentration of the goat anti-chicken IgG on the quality control line (43) is 1.0 mg/mL-1.5 mg/mL.
3. The test strip for detecting feline coronavirus according to claim 1, wherein the ratio of the feline coronavirus monoclonal antibody to the feline infectious peritonitis virus polyclonal antibody is 2: 1.
4. The test strip for detecting feline coronavirus according to claim 1, wherein the colloidal gold pad layer (3) is further coated with a chicken anti-feline IgY antibody labeled with colloidal gold particles.
5. The test strip for detecting feline coronavirus according to claim 4, wherein the concentration of the chicken anti-feline IgY antibody is 10 μ g/mL-15 μ g/mL.
6. The test strip for detecting feline coronavirus according to claim 1, wherein the nitrocellulose membrane layer (4) is adhered to an intermediate position of the base plate (1), a water-absorbing layer (5) is adhered to one side of the nitrocellulose membrane layer (4) on the base plate (1), a colloidal gold cushion layer (3) and a sample cushion layer (2) are adhered to the other side of the nitrocellulose membrane layer (4), one end of the sample cushion layer (2) presses 1.0mm to 1.5mm of one end of the colloidal gold cushion layer (3), the other end of the colloidal gold cushion layer (3) presses 0.5mm to 1mm of one end of the nitrocellulose membrane layer (4), and one end of the water-absorbing layer (5) presses 1.0mm to 2.0mm of the other end of the nitrocellulose membrane layer (4).
7. The method for preparing the test strip for detecting feline coronavirus according to any one of claims 1-6, comprising the steps of:
s1, preparing a colloidal gold cushion layer (3): coating the colloidal gold pad with a feline coronavirus monoclonal antibody and a feline infectious peritonitis virus polyclonal antibody marked by colloidal gold particles;
s2, preparing a nitrocellulose membrane layer (4): coating a feline coronavirus monoclonal antibody on a nitrocellulose membrane layer (4) as a T1 detection line (41), further coating a feline infectious peritonitis virus polyclonal antibody as a T2 detection line (42), and coating goat anti-chicken IgG as a quality control line (43);
s3, preparation of a sample cushion layer (2): firstly, preparing a treating fluid, namely 100mmol/L Tris-HCL buffer solution with pH9.0, which contains 0.5 percent of PEG20000, 0.05 to 0.2 percent of Tween-20, 0.01 to 0.05 percent of surfactant and 0.3 to 1.0 percent of blocking agent; secondly, coating the treatment solution on glass fiber to obtain a sample cushion layer (2);
s4, preparation of the test strip: and (3) sequentially overlapping the sample cushion layer (2) in the step S3, the colloidal gold cushion layer (3) in the step S1, the nitrocellulose membrane layer (4) and the water absorbing layer (5) in the step S2 on the bottom plate 1 from left to right to obtain the test strip for detecting the feline coronavirus.
8. The method for preparing the test strip for detecting feline coronavirus according to claim 6, wherein the colloidal gold pad is coated with the chicken anti-feline IgY antibody.
9. A kit for detecting feline coronavirus, comprising the test strip for detecting feline coronavirus of any one of claims 1-6, a sampling swab, and a sample diluent.
10. A method for detecting feline coronavirus using the kit of claim 9, comprising the steps of:
s1, placing the sample in a sample diluent, fully mixing and standing, and taking the supernatant as a detection liquid sample;
and S2, dripping the detection liquid sample into a sample hole of the kit, standing for a period of time, and judging the result.
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