CN114839378B - Kit for joint detection of feline herpes and feline calicivirus - Google Patents
Kit for joint detection of feline herpes and feline calicivirus Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a feline herpes and feline calicivirus combined detection kit which comprises a detection card, sample diluent and a sterile cotton swab, wherein a test strip, a test strip nitrocellulose membrane, a water absorption pad, a gold-labeled antibody reaction pad and a sample pad are arranged inside the detection card, a feline herpes virus monoclonal antibody-colloidal gold compound coating and a feline calicivirus monoclonal antibody-colloidal gold compound coating are arranged on the gold-labeled antibody reaction pad, the feline herpes virus monoclonal antibody-colloidal gold compound coating is positioned above the feline herpes virus monoclonal antibody-colloidal gold compound coating, and an isolation pad is arranged between the feline herpes virus monoclonal antibody-colloidal gold compound coating and the feline herpes virus monoclonal antibody-colloidal gold compound coating. The kit is simple to operate, short in detection time, greatly improves the detection efficiency, does not need expensive instruments and equipment for detection, and reduces the detection cost.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for joint detection of feline herpes and feline calicivirus.
Background
The feline upper respiratory disease is a common feline disease, and the common feline upper respiratory disease comprises feline herpes virus (FHV-1), feline leukemia virus (FeLV), Feline Calicivirus (FCV) and the like. Feline herpesvirus (FHV-1), also known as feline rhinotracheitis virus (felinerhinotrachitis virus), belongs to the family a-herpesviridae and is a double-stranded DNA virus with an envelope. The virus mainly attacks kittens, is spread by direct contact, is clinically characterized by keratoconjunctivitis, upper respiratory tract infection and abortion, but has the main respiratory symptoms, the morbidity reaches 100 percent, and the mortality reaches 50 percent. Feline Calicivirus (FCV) is one member of the genus Calicivirus (Calicivirus) belonging to the family Caliciviridae (Caliciviridae), and is a single-stranded positive-sense RNA virus. The virus is highly infectious and can cause Upper Respiratory Disease (URD) and canker sores in cats. FCV infection is a frequently encountered disease in cats, with high morbidity and low mortality. Is distributed worldwide, not only infects cats, but also infects all felines, such as tigers, lions, leopards and the like, and poses certain threat to the health of the wild animals.
FCV is infected very commonly in cat groups, the virus mainly attacks young cats at the age of 6-84 days, the number of the young cats is more than 56-84 days, cats under the age of 1 year are most susceptible, and the incubation period is 2-3 days. Cats that are younger in day are more susceptible to post-infection mortality. The diseased cat and the recessive infected cat are the main infection sources of FCV, and the virus can spread to the outside along with saliva, tears and nasal secretion of the cat, pollute cages, cat beds, padding and surrounding environment, and can also be directly infected to susceptible cats. FCV often causes acute upper respiratory diseases such as mouth ulcers, increased nasal and ocular secretions, keratitis and chronic gastroenteritis, and some strains may also cause claudication. FHV often causes symptoms of the upper respiratory tract and the eye, with corneal dendritic ulceration being a characteristic symptom of this infection. Cats with upper respiratory disease are at a very high risk of contracting FCV or FHV, with respiratory symptoms having a total incidence of FCV and/or FHV infection of 60.2%, and thus for cats with respiratory symptoms, screening for FHV and FCV is a more effective means of identifying respiratory pathogenic microorganisms.
At present, methods for detecting FCV and FHV include serological detection and virological detection. Serological tests include serum neutralization assays, immunofluorescence or enzyme-linked immunosorbent assays, and the like, which are relatively time-consuming; the virus detection method comprises electron microscopy, RT-PCR, fluorescent quantitative RT-PCR and the like, but the detection methods are long in time consumption, expensive instruments and equipment and professional technicians are required for operation, and the detection requirements under basic field conditions such as non-laboratories cannot be met. Therefore, in order to solve the problems, a kit for detecting the feline herpes and the feline calicivirus in a combined manner needs to be developed.
Disclosure of Invention
The invention aims to: aiming at the defects in the prior art, the kit for jointly detecting the feline herpes and the feline calicivirus is provided, the kit can realize the joint detection of two viruses by one-time sampling, the sampling of pets is reduced, the use is convenient, the operation is simple, and the interference competition existing in the reaction process is reduced.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
a cat herpes and cat calicivirus joint detection kit comprises a detection card, a sample diluent bottle and an aseptic cotton swab, wherein a test strip is arranged inside the detection card and comprises a bottom plate, a nitrocellulose membrane is arranged on the bottom plate, a water absorption pad is arranged above the nitrocellulose membrane, a gold-labeled antibody reaction pad is arranged below the nitrocellulose membrane, a sample pad is arranged below the gold-labeled antibody reaction pad, a cat herpes virus monoclonal antibody-colloidal gold compound coating and a cat calicivirus monoclonal antibody-colloidal gold compound coating are arranged on the gold-labeled antibody reaction pad, the cat calicivirus monoclonal antibody-colloidal gold compound coating is located above the cat herpes virus monoclonal antibody-colloidal gold compound coating, and the cat calicivirus monoclonal antibody-colloidal gold compound coating and the cat herpes virus monoclonal antibody-colloidal gold compound coating are respectively arranged on the bottom plate of the detection kit Isolation pads are arranged between the layers.
As an improved technical scheme, the particle size of the colloidal gold adopted in the feline calicivirus monoclonal antibody-colloidal gold compound coating is 15 nm; the particle size of the colloidal gold adopted in the coat of the feline herpesvirus monoclonal antibody-colloidal gold compound is 40 nm.
As an improved technical scheme, the formation of the coat of the feline calicivirus monoclonal antibody-colloidal gold complex refers to: adding 1mg of feline calicivirus monoclonal antibody into 100mL of colloidal gold solution with the particle size of 15nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline calicivirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:4, spraying the feline calicivirus monoclonal antibody-colloidal gold compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline calicivirus monoclonal antibody-colloidal gold compound; the formation of the coat of the feline herpesvirus monoclonal antibody-colloidal gold compound refers to the following steps: adding 2mg of feline herpesvirus monoclonal antibody into 100mL of colloidal gold solution with the particle size of 40nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline herpesvirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:6, spraying the compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline herpesvirus monoclonal antibody-colloidal gold compound.
As an improved technical scheme, the isolation pad is treated by an isolation pad treatment solution, the isolation pad treatment solution is prepared by 0.2-0.4 wt% of Triton X-100 and 0.3-0.5 wt% of BSA per 1000mL of PBS buffer solution, the concentration of the PBS buffer solution is 25mM, and the pH value is 7.2-7.6.
As an improved technical scheme, a detection line T1 coated with a feline calicivirus monoclonal antibody, a detection line T2 coated with a feline herpesvirus monoclonal antibody and a quality control line coated with a goat anti-cat IgG antibody are arranged on the nitrocellulose membrane.
As an improved technical scheme, the detection line T1, the detection line T2 and the quality control line are obtained by respectively diluting a feline calicivirus monoclonal antibody to 0.8-1.2mg/mL, a feline herpesvirus monoclonal antibody to 0.7-1.0mg/mL and a goat anti-feline IgG to 1.5-2.0mg/mL by using diluents, sequentially spraying the diluted feline calicivirus monoclonal antibody, the feline herpesvirus monoclonal antibody and the goat anti-feline IgG on a nitrocellulose membrane, and drying the nitrocellulose membrane.
As an improved technical scheme, the diluent is prepared by adding 2wt% -4wt% of sucrose and 1wt% -3wt% of trehalose into 1L of 20-60mmol PBS buffer solution, and the pH value of the PBS buffer solution is 7.2-7.6.
As an improved technical scheme, the sample pad is treated by sample treatment liquid in a sample diluent bottle, and the sample treatment liquid is prepared by 0.5wt% of PVA, 0.1wt% to 0.2wt% of S9, 0.01wt% to 0.03wt% of triton X-100, 0.3wt% to 0.5wt% of trehalose and 0.2wt% of casein in each 1000ml of PB buffer solution with the pH value of 8.0 to 8.5.
As a modified technical scheme, the sample diluent contains 0.5wt% BSA, 0.2wt% Tween 20, 0.9wt% NaCl and 0.5w/v% Proclin-950 in every 1000mL of Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 25mM, and the pH value is 8.5-9.0.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
(1) the collection of the samples of the affected cat eye, the nose and the throat swab is more complicated, and the secretion amount is less, so that the kit can reduce the sampling of pets and realize the combined detection of two viruses by one-time sampling; the reaction pad of the test strip is respectively provided with a feline calicivirus monoclonal antibody-colloidal gold compound coating (the particle size of colloidal gold is 15 nm) and a feline herpesvirus monoclonal antibody-colloidal gold compound coating (the particle size of colloidal gold is 40 nm), the feline calicivirus monoclonal antibody-colloidal gold compound coating is positioned above the feline herpesvirus monoclonal antibody-colloidal gold compound coating, and an isolation pad is arranged between the feline calicivirus monoclonal antibody-colloidal gold compound coating and the feline herpesvirus monoclonal antibody-colloidal gold compound coating. The isolation pad is arranged to separate the colloidal gold compounds of different gold particles, the small-particle colloidal gold compound is arranged above the small-particle colloidal gold compound, the large-particle colloidal gold compound is arranged below the small-particle colloidal gold compound, and when the cat calicivirus monoclonal antibody-colloidal gold compound particles are smaller and faster to perform chromatography in the upward chromatography process of a sample, the large-particle colloidal gold compound particles and the large-particle colloidal gold compound particles do not influence each other and are in interference-free competition with the detection line, the isolation pad layer plays a buffering role, the interference competition existing in the reaction process is greatly reduced, and the specificity of the product is ensured.
(2) In practical application, the secretion of conjunctiva, nasal cavity, oral cavity and other parts of eyes of an animal to be detected is collected by adopting an aseptic cotton swab, the sampling swab is inserted into a sample tube filled with 0.5mL of sample diluent, supernatant in the sample tube is taken as detection liquid, the detection liquid is dripped into an injection port of a detection card, and the result can be observed within 5-8 min.
(3) The sample pad can filter impurities in a sample to be detected after being treated by the sample treatment liquid, has good permeability, improves the water absorption speed, accelerates the chromatography speed on the NC membrane, and improves the specificity of the product compared with the performance index of the product. When a quality control line, a detection line T1 and a detection line T2 on the nitrocellulose membrane are coated, a dilution solution containing trehalose and sucrose is used for diluting the feline calicivirus monoclonal antibody, the feline herpesvirus monoclonal antibody and the goat anti-feline IgG, so that the stability of the antibody to be diluted can be improved;
in conclusion, the kit is adopted, only one sampling and one sample processing are needed, one sample buffer solution is used for simultaneously processing two viruses, the simultaneous detection of the two viruses is completed, and the operation is convenient. In a family use scene, pet cat secretion is easier to collect, and the family self-checking use of the pet cat is really realized.
Drawings
FIG. 1 is a schematic structural diagram of a kit according to the present invention;
FIG. 2 is a schematic diagram of a top view of a test strip of the test card of the present invention;
FIG. 3 is a schematic diagram of a side view of a test strip of the test card of the present invention;
the kit comprises a packaging box 1, a detection card 2, a test strip 3, a bottom plate 30, a nitrocellulose membrane 31, a water absorption pad 32, a gold-labeled antibody reaction pad 33, a feline herpesvirus monoclonal antibody-colloidal gold compound coating 330, a feline calicivirus monoclonal antibody-colloidal gold compound coating 331, a sample pad 34, a separation pad 35, a sample diluent bottle 4 and a sterile cotton swab 5.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A feline herpes and feline calicivirus combined detection kit is shown in figures 1 to 3 and comprises a detection card 2, a sample diluent bottle 4 (filled with a sample treatment solution) and a sterile cotton swab 5 (packaged by a packaging box 1), wherein a test strip 3 is arranged inside the detection card 2, the test strip 3 comprises a bottom plate 30, a nitrocellulose membrane 31 is arranged on the bottom plate 30, a water absorption pad 32 is arranged above the nitrocellulose membrane 31, a gold-labeled antibody reaction pad 33 is arranged below the nitrocellulose membrane 31, a sample pad 34 is arranged below the gold-labeled antibody reaction pad 33, a feline herpes virus monoclonal antibody-colloidal gold compound coating and a feline herpes virus monoclonal antibody-colloidal gold compound coating are arranged on the gold-labeled antibody reaction pad 33, the feline herpes virus monoclonal antibody-colloidal gold compound coating 331 is positioned above the feline herpes virus monoclonal antibody-colloidal gold compound coating 330, an isolation pad 35 is arranged between the feline calicivirus monoclonal antibody-colloidal gold compound coating and the feline herpesvirus monoclonal antibody-colloidal gold compound coating.
Wherein the particle size of the colloidal gold adopted in the feline calicivirus monoclonal antibody-colloidal gold compound coating is 15 nm; the particle size of the colloidal gold adopted in the coat of the feline herpesvirus monoclonal antibody-colloidal gold compound is 40 nm.
Wherein the formation of the coat of the feline calicivirus monoclonal antibody-colloidal gold complex refers to: adding 1mg of feline calicivirus monoclonal antibody into 100mL of colloidal gold solution (0.2 g/L) with the particle size of 15nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline calicivirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:4, spraying the compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline calicivirus monoclonal antibody-colloidal gold compound; the formation of the coat of the feline herpesvirus monoclonal antibody-colloidal gold complex refers to: adding 2mg of feline herpesvirus monoclonal antibody into 100mL of colloidal gold solution (0.2 g/L) with the particle size of 40nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline herpesvirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:6, spraying the compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline herpesvirus monoclonal antibody-colloidal gold compound.
Wherein the isolation pad is prepared by coating the isolation pad treatment solution on glass fiber and drying at 37 deg.C for 8 hr; the treatment solution for spacer was prepared to contain 0.2wt% Triton X-100 and 0.3wt% BSA per 1000mL of PBS buffer at 25mM and pH 7.2.
Wherein the nitrocellulose membrane is provided with a detection line T1 coated with the feline calicivirus monoclonal antibody, a detection line T2 coated with the feline herpesvirus monoclonal antibody and a quality control line C coated with the goat anti-feline IgG antibody. The detection line T1, the detection line T2 and the quality control line are obtained by diluting the feline calicivirus monoclonal antibody to 0.8mg/mL, diluting the feline herpesvirus monoclonal antibody to 0.7mg/mL and diluting the goat anti-feline IgG to 1.5mg/mL respectively by using a diluent, sequentially spraying the diluted feline calicivirus monoclonal antibody, the feline herpesvirus monoclonal antibody and the goat anti-feline IgG on a nitrocellulose membrane, and drying the nitrocellulose membrane.
Wherein the diluent is prepared by adding 2wt% of sucrose and 1wt% of trehalose into 1L of 20mmol of PBS buffer solution, and the pH value of the PBS buffer solution is 7.2.
Wherein the sample pad is obtained by coating the sample treatment solution on glass fiber and drying; the sample treatment solution was prepared by adding 0.5wt% PVA, 0.1wt% S9, 0.01wt% Triton X-100, 0.3wt% trehalose and 0.2wt% casein to 1000ml of PB buffer solution having a pH of 8.0 to 8.5.
Wherein the sample diluent contains 0.5wt% BSA, 0.2wt% Tween 20, 0.9wt% NaCl and 0.5w/v% Proclin-950 per 1000mL of Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 25mM, and the pH value is 8.5.
Example 2
The difference from example 1 is:
wherein the isolation pad is prepared by coating the isolation pad treatment solution on glass fiber and drying at 37 deg.C for 8 hr; the treatment solution for spacer was prepared to contain 0.3wt% Triton X-100 and 0.4wt% BSA per 1000mL of PBS buffer at 25mM and pH 7.4.
Wherein the nitrocellulose membrane is provided with a detection line T1 coated with the feline calicivirus monoclonal antibody, a detection line T2 coated with the feline herpesvirus monoclonal antibody and a quality control line coated with the goat anti-feline IgG antibody. The detection lines T1, T2 and quality control line refer to that the feline calicivirus monoclonal antibody, the feline herpesvirus monoclonal antibody and the goat anti-feline IgG are respectively diluted to 1.0mg/mL, 0.85mg/mL and 1.8mg/mL by using diluent, and the test lines T1, T2 and quality control line are sequentially sprayed on a nitrocellulose membrane and dried.
The diluent is prepared by adding 3wt% of sucrose and 2wt% of trehalose into 1L of 40mmol of PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.4.
Wherein the sample pad is treated by a sample treatment solution, and the sample treatment solution is prepared by adding 0.5wt% of PVA, 0.15wt% of S9, 0.02wt% of Triton X-100, 0.4wt% of trehalose and 0.2wt% of casein into 1000ml of PB buffer solution with the pH of 8.0-8.5.
Wherein the sample diluent contains 0.5wt% BSA, 0.2wt% Tween 20, 0.9wt% NaCl and preservative in each 1000mL of Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 25mM, and the pH value is 8.8.
Example 3
The difference from example 1 is:
wherein the isolation pad is prepared by coating the isolation pad treatment solution on glass fiber and drying at 37 deg.C for 8 hr; the treatment solution for spacer was prepared to contain 0.4wt% Triton X-100 and 0.5wt% BSA per 1000mL of PBS buffer at 25mM and pH 7.6.
Wherein the nitrocellulose membrane is provided with a detection line T1 coated with the feline calicivirus monoclonal antibody, a detection line T2 coated with the feline herpesvirus monoclonal antibody and a quality control line coated with the goat anti-feline IgG antibody. The detection line T1, the detection line T2 and the quality control line refer to that the feline calicivirus monoclonal antibody, the feline herpesvirus monoclonal antibody and the goat anti-feline IgG are respectively diluted to 1.2mg/mL, 1.0mg/mL and 2.0mg/mL by using diluent, and the detection lines are sequentially sprayed on a nitrocellulose membrane and dried.
Wherein the diluent is prepared by adding 4wt% of sucrose and 3wt% of trehalose into 1L of 60mmol PBS buffer solution, and the pH value of the PBS buffer solution is 7.6.
Wherein the sample pad is treated with a sample treatment solution prepared by adding 0.5wt% PVA, 0.2wt% S9, 0.03wt% Triton X-100, 0.5wt% trehalose and 0.2wt% casein to 1000ml of PB buffer solution with pH of 8.0-8.5.
Wherein the sample diluent contains 0.5wt% BSA, 0.2wt% Tween 20, 0.9wt% NaCl and preservative in each 1000mL of Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 25mM, and the pH value is 9.0.
Example 4
The use method of the combined detection kit for the feline herpes and feline calicivirus comprises the following steps:
(1) sample processing
a. The detection sample is the pharyngeal secretion, the conjunctival secretion or the nasal secretion of cats;
b. and collecting the secretion of the animal to be detected by using a sterile cotton swab. Collecting secretion samples from conjunctiva, nasal cavity, oral cavity, etc. of animal eye with a sampling swab, preferably the swab is fully wetted.
c. Inserting the sampling swab into a sample tube filled with 0.5mL of sample diluent, squeezing the swab from the outer wall of the sample processing tube, squeezing the processing tube and rotating the swab 15-20 times to leave the processed mixed solution in the tube as much as possible, and discarding the swab. And standing for 1min after fully and uniformly mixing, wherein the supernatant is the detection liquid (sample to be detected).
(2) Detection of
a. Taking out the detection card from the kit and placing the detection card on a table top;
b. dropping 3 drops (about 100 muL) of samples to be detected to the sample adding holes of the detection cards;
c. the results were interpreted within 5-8 minutes and after 10 minutes the results were not valid.
(3) Result judgment
a. Positive (+): one red line appears on each of the control line (C) and the detection lines (T1 or/and T2).
b. Negative (-): only one red line appears on the quality control line (C), and no red line appears on the detection line (T).
c. And (4) invalidation: the appearance of a wireless strip at the position of the quality control line (C) indicates misoperation or reagent failure.
Example 5
Examination of sensitivity, specificity and stability of the kit of the invention:
1. sensitivity study
The sensitivity studies were carried out by diluting the recombinant antigens of the feline calicivirus and the feline herpesvirus in a gradient of 1000, 500, 100, 50, 10 and 5pg/mL, respectively, and the results are shown in the following Table 1:
remarking: "+" represents positive, "+/-" represents weak positive, and "-" represents negative.
The lowest detection limits of the kit (kit in example 2) for detecting the feline calicivirus recombinant antigen and the feline herpesvirus recombinant antigen are 50pg/mL and 10pg/mL respectively.
2. Investigation of specificity
The kit in the embodiment 2 is adopted to verify the cross-reaction pathogenic microorganisms with the concentrations in the table below which are easy to cause the same or similar symptoms, and the specific detection results are shown in the table 2;
the results shown in table 2 show that: the cross-reactive substances at the following concentrations had no effect on the detection of the kit of the present invention, indicating that the kit of the present invention has good specificity.
3. Stability survey
The inventive kit (example 2) was subjected to a destructive test at a temperature of 45 ℃ for a period of 6 months, and the stability of the kit was examined at day 1, day 3, day 7, day 10, day 15, month 1, day 45, month 2, month 3, month 4, month 5 and month 6, respectively, according to the following criteria:
(1) physical state of sample dilution
Appearance: colorless, transparent and clear liquid, no particles, floccules, precipitates and no volatility.
(2) Performance index
1) The negative quality control product coincidence rate is as follows: and (3) detecting by using 10 cat calicivirus negative quality control substances, wherein all detection results are negative, and the accuracy of the kit meets the requirement.
2) The positive quality control product conformity rate: and (3) detecting by using 10 positive (including strong, medium and weak positives) quality control substances of the feline calicivirus, wherein all detection results are positive, and the accuracy of the kit meets the requirement.
Minimum detection limit: and (4) detecting by using the reference substance S with the lowest detection limit, wherein the result is positive.
Repeatability: the result variation coefficients of the negative reference substance, the positive reference substance and the lowest detection limit from 1 day to 6 months are not more than 15%.
The test result of the stability of the kit is as follows:
the sample diluent physical state stability results are shown in tables 3 and 4;
the results of the 45 ℃ accelerated destruction test of the kit are shown in Table 4;
remarking: "+" represents positive; "+/-" indicates weak positivity; "-" represents negative;
the test shows that the product can be stabilized for at least 60 days at 45 ℃, and according to the stability experiment principle, the Arrhenius formula: d (Ink)/dT = Ea/RT2 Ea, is preserved for 24 months at normal temperature, is equivalent to 60 days of destruction at 45 ℃, can meet the clinical requirements of clinics and health quarantine departments of pet hospitals, and can also be used for diagnosis and research of pet diseases of colleges and universities and scientific research institutions.
According to the operation, detecting the performance index of the kit by using 10 parts of the feline herpesvirus negative quality control and 10 parts of the feline herpesvirus positive quality control; the test result of the stability of the kit is as follows:
the sample diluent physical state stability results are shown in table 5;
the results of the 45 ℃ accelerated destruction test of the kit are shown in Table 6;
remarking: "+" represents positive; "+/-" indicates weak positivity; "-" represents negative;
the test shows that the product can be stabilized for at least 60 days at 45 ℃, and according to the stability experiment principle, the Arrhenius formula: d (Ink)/dT = Ea/RT2 Ea, is preserved for 24 months at normal temperature, is equivalent to 60 days of destruction at 45 ℃, can meet the clinical requirements of clinics and health quarantine departments of pet hospitals, and can also be used for diagnosis and research of pet diseases of colleges and universities and scientific research institutions.
Example 6
The kit of the invention is used for clinical trial investigation:
1. the kit and the commercially available kit are adopted to simultaneously detect 83 samples, wherein 18 positive samples (infected by feline calicivirus) and 67 negative samples have detection results shown in the following table 7:
the results show that: the joint detection kit has the same detection effect as a commercially available kit for separately detecting the feline calicivirus.
2. The kit and the commercially available kit are adopted to simultaneously detect 60 samples, wherein 11 positive samples (infected by feline herpesvirus) and 49 negative samples have detection results shown in the following table 8:
the results show that: the joint detection kit and the commercially available kit for separately detecting the feline herpesvirus have the same detection effect.
The present invention is not limited to the above-described embodiments, and various modifications made by those skilled in the art without inventive skill from the above-described conception fall within the scope of the present invention.
Claims (7)
1. A cat herpes and cat calicivirus combined detection kit is characterized in that: the kit comprises a detection card, a sample diluent bottle and a sterile cotton swab, wherein a test strip is arranged in the detection card, the test strip comprises a bottom plate, a nitrocellulose membrane is arranged on the bottom plate, a water absorption pad is arranged above the nitrocellulose membrane, a gold-labeled antibody reaction pad is arranged below the nitrocellulose membrane, a sample pad is arranged below the gold-labeled antibody reaction pad, the gold-labeled antibody reaction pad is provided with a feline herpesvirus monoclonal antibody-colloidal gold compound coating and a feline calicivirus monoclonal antibody-colloidal gold compound coating, the feline calicivirus monoclonal antibody-colloidal gold compound coating is positioned above the feline herpesvirus monoclonal antibody-colloidal gold compound coating, an isolation pad is arranged between the feline calicivirus monoclonal antibody-colloidal gold compound coating and the feline herpesvirus monoclonal antibody-colloidal gold compound coating;
wherein the particle size of the colloidal gold adopted in the feline calicivirus monoclonal antibody-colloidal gold compound coating is 15 nm; the particle size of the colloidal gold adopted in the feline herpesvirus monoclonal antibody-colloidal gold compound coating is 40 nm;
the isolation pad is treated by an isolation pad treatment solution, the isolation pad treatment solution is prepared by 0.2-0.4 wt% of Triton X-100 and 0.3-0.5 wt% of BSA in each 1000mL of PBS buffer solution, the concentration of the PBS buffer solution is 25mM, and the pH value is 7.2-7.6.
2. The kit for the combined detection of feline herpes and feline calicivirus according to claim 1, wherein: the formation of the feline calicivirus monoclonal antibody-colloidal gold complex coating refers to the following steps: adding 1mg of feline calicivirus monoclonal antibody into 100mL of colloidal gold solution with the particle size of 15nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline calicivirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:4, spraying the feline calicivirus monoclonal antibody-colloidal gold compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline calicivirus monoclonal antibody-colloidal gold compound; the formation of the coat of the feline herpesvirus monoclonal antibody-colloidal gold compound refers to the following steps: adding 2mg of feline herpesvirus monoclonal antibody into 100mL of colloidal gold solution with the particle size of 40nm, centrifuging after reaction, discarding supernatant, adding 2mL of gold redissolving solution, standing for reaction, diluting the prepared feline herpesvirus monoclonal antibody-colloidal gold compound with the gold redissolving solution according to the volume ratio of 1:6, spraying the compound on a hydrophilic polyester film at the volume ratio of 2-3ul/cm, and drying to obtain the feline herpesvirus monoclonal antibody-colloidal gold compound.
3. The kit for the combined detection of feline herpes and feline calicivirus according to claim 1, wherein: the nitrocellulose membrane is provided with a detection line T1 coated with the feline calicivirus monoclonal antibody, a detection line T2 coated with the feline herpesvirus monoclonal antibody and a quality control line coated with the goat anti-cat IgG antibody.
4. The kit for the combined detection of feline herpes and feline calicivirus according to claim 3, wherein: the detection line T1, the detection line T2 and the quality control line are obtained by diluting a feline calicivirus monoclonal antibody to 0.8-1.2mg/mL, diluting a feline herpesvirus monoclonal antibody to 0.7-1.0mg/mL and diluting goat anti-feline IgG to 1.5-2.0mg/mL respectively with a diluent, sequentially spraying the diluted feline calicivirus monoclonal antibody, the diluted feline herpesvirus monoclonal antibody and the diluted feline anti-feline IgG on a nitrocellulose membrane and drying the nitrocellulose membrane.
5. The kit for the combined detection of feline herpes and feline calicivirus according to claim 4, characterized in that: the diluent is prepared by adding 2wt% -4wt% of sucrose and 1wt% -3wt% of trehalose into 1L of 20-60mmol of PBS buffer solution, and the pH value of the PBS buffer solution is 7.2-7.6.
6. The kit for the combined detection of feline herpes and feline calicivirus according to claim 1, wherein: the sample pad is treated by sample treatment liquid in a sample diluent bottle, and the sample treatment liquid is prepared by 0.5wt% of PVA, 0.1wt% -0.2wt% of S9, 0.01wt% -0.03wt% of Triton X-100, 0.3wt% -0.5wt% of trehalose and 0.2wt% of casein in 1000ml of PB buffer solution with the pH of 8.0-8.5.
7. The kit for the combined detection of feline herpes and feline calicivirus according to claim 1, wherein: the sample diluent contains 0.5wt% BSA, 0.2wt% Tween 20, 0.9wt% NaCl and 0.5w/v% Proclin-950 in every 1000mL of Tris-HCl buffer solution, the concentration of the Tris-HCl buffer solution is 25mM, and the pH value is 8.5-9.0.
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| CN116735866A (en) * | 2023-02-01 | 2023-09-12 | 深圳粒影生物科技有限公司 | Test strip for triple detection of feline panleukopenia virus, feline herpesvirus and feline calicivirus antigen and preparation method thereof |
| CN117825697B (en) * | 2024-03-01 | 2024-05-07 | 中国科学院过程工程研究所 | Hazard factor detection reagent card and detection method |
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