CN106053801A - ELISA kit for detecting IgG antibody of feline calicivirus and detecting method thereof - Google Patents
ELISA kit for detecting IgG antibody of feline calicivirus and detecting method thereof Download PDFInfo
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Abstract
The invention relates to a kit for detecting feline calicivirus and a detecting method thereof, in particular to an ELISA kit for detecting an IgG antibody of feline calicivirus and a detecting method thereof. The ELISA kit and the detecting method solve the problem that current feline calicivirus diagnosis and detection results are not ideal. The kit comprises an ELISA microwell plate, RaMIgG-HRP and substrate developing liquid. The method comprises the steps that firstly, serum is diluted and added into the ELISA microwell plate to be incubated; secondly, the RaMIgG-HRP is added for incubation; thirdly, the substrate developing liquid is added, and reaction is carried out in a shading mode at the room temperature; fourthly, a stop solution is added to terminate the reaction, an ELIASA is used for detecting 450 nm light absorption value, the 450 nm light absorption value and a Cut Off value of the ELISA kit are compared, and then a detection result is obtained. The ELISA kit detection result is high in repeatability and stability, and the speed is high.
Description
Technical field
The present invention relates to a kind of test kit for detecting feline calicivirus and detection method thereof.
Background technology
Feline calicivirus (Feline calicivirus, FCV) is Caliciviridae, chickenpox Tobamovirus, is a kind of single
Strand rna virus.FCV is that cat disease toxicity respiratory infectious disease mainly shows as oral ulcer, eye nasal secretions, sialorrhea, knot
Film inflammation, photophobia, stomatitis, tracheitis, bronchitis, with diphasic fever.Being the frequently-occurring disease of cat, sickness rate is high, and virulent strain infects
Pneumonia, dyspnea, even death can occur.Major source of infection is disease cat and band poison cat, and sick cat can be with secretions in acute stage
A large amount of virus, the susceptible cat of direct contagion is discharged with Excreta；Band poison cat is through treatment, and symptom can disappear, the most long-term after clinical rehabilitation
Toxin expelling, is the dangerous source of infection.
Carrying out diagnosing usually mistaken diagnosis according to medical history, clinical symptoms and epidemic characteristic, delay treatment also causes viral diffusion
And propagation.And the detection of feline calicivirus antibody at present uses the method such as serum neutralization test and indirect immunofluorescence, inspection
Survey time-consuming, poor repeatability and mostly be laboratory internal application, lack unified standard.
Summary of the invention
The invention aims to solve the diagnosis of current feline calicivirus and testing result is undesirable, and the one provided
For detecting ELISA kit and the detection method thereof of feline calicivirus IgG antibody.
The present invention includes ELISA microwell plate, ELIAS secondary antibody for the ELISA kit detecting feline calicivirus IgG antibody
With substrate nitrite ion；Wherein, the feline calicivirus ORF2 recombiant protein of the most coated solubility expression is comprised in ELISA microwell plate
F。
Wherein, the preparation method of the FCV gene ORF2 recombiant protein F of solubility expression:
Feline calicivirus (FCV) VP1 gene order (Genbank:X99445.1) design according to logging in Genbank is expanded
Increase forward primer Sac I: the 5 '-ATT of the F district RNA fragment of feline calicivirus ORF2GAG CTC CTG GGT TAC ACA GGA
ATT GGT-3 ', and downstream primer Xhol I: 5 '-ATACTC GAG TCA TAA TTT TGT CAT ACT ACT-3 ', and adopt
Expand by PCR method, then by the gene fragment clone of amplification to construction recombination plasmid in carrier pET-32a, sequence verification
Proceeding to after errorless use IPTG abduction delivering in expressive host bacterium E.coli BL21 (DE3), condition is: bacterium solution is shaken in 37 DEG C of shaking tables
Adding IPTG to final concentration 0.5mM to exponential phase (OD590nm value is 0.4～0.6), inducing temperature is 20 DEG C, during induction
Between be 7h, it is thus achieved that the feline calicivirus ORF2 recombiant protein F of solubility expression, the amino of feline calicivirus ORF2 recombiant protein F
Acid sequence is VTLALLGYTGIGEQAIGSDRDRVVRISVLPETGARGGNHPIFYKNTIKLGYVIRSI DVFNS
QILHTSRQLSLNHYLLPPDSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSLPTLEFPLSASYMGIQLAKIRLASNIR
SSMTKL。
The preparation method of ELISA microwell plate:
With the carbonate buffer solution that the concentration that pH value is 9.6 is 0.05mol/L as being coated buffer, by cat after purification
After Calicivirus ORF2 recombiant protein F dilution, add microwell plate by 100 μ l/ holes, it is ensured that in every hole, the content of recombiant protein F is
0.2μg；4 DEG C are coated overnight, discard next day and are coated liquid, then by 100 μ l/ holes add concentration be 5% skimmed milk confining liquid, 37 DEG C
Standing 2h, load packaging bag, add desiccant after being washed out drying, drying at room temperature, vacuum preserves.
1000mL is coated buffer by the Na of 1.5g2CO3, the NaHCO of 2.9g3Add ddH2O water dissolution, regulation pH value is to 9.6
After add the water of surplus and make.
Method with above-mentioned ELISA kit detection feline calicivirus:
One, test serum antibody diluent is pressed the dilution of serum endpoint titre, then add ELISA by every hole 100 μ l
Microwell plate, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Two, every hole adds the ELIAS secondary antibody 100 μ l of dilution, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Three, every hole adds substrate nitrite ion 50 μ l, room temperature lucifuge reaction 10～15min；
Four, every hole adds 50 μ l stop buffers termination reactions, microplate reader detection 450nm light absorption value and the Cut of ELISA kit
Off value compares, and i.e. draws testing result.
Cut Off value is the positive marginal value with negative findings.
The Cut Off value establishment method of above-mentioned ELISA kit: with the feline calicivirus positive serum of separate sources step by step
Dilute as one anti-, using PBS as negative control, each positive serum Cigarette dilution detection n times；Then will be right with feminine gender
According to the maximum dilution gradient in significant difference as serum endpoint titre, the serum of the feline calicivirus positive serum of separate sources
End point titres OD450nmAverage+3 × SD is the Cut Off value of ELISA kit.
FCV genome is single-stranded positive RNA, and size is about about 7.8kb, and its RNA5 ' end is covalently bonded with VPg albumen,
FCV genome comprises three ORF (Open Reading Frame), respectively ORF1, ORF2, ORF3.ORF2 main code FCV
Capsid protein, its translation can obtain the capsid precursor albumen of 75KD, and precursor protein is through the protease cutting of virus
Fall the leader protein (LC) of 14kd, obtain the ripe capsid protein VP1 of about the 62KD of maturation.The capsid protein of FCV is not according to
Similarity between homophyletic can be divided into six regions, is broadly divided into A, B, C, D, E, F district.
Selected by the present invention, feline calicivirus detection antigen is the F district recombiant protein F of ORF2, and F district is positioned at the height of capsid protein
The carboxyl terminal that degree is conservative, is positioned at virus surface, for nonneutralizing antibody binding site；Therefore ORF2 recombiant protein F is cat cup-shaped
One of structural protein of virus, have high antigenic, and conservation of amino acids reaches 89.0%～100%, is antigen immunodominant proteins,
Its specificity is good, repetition stability is high.
The present invention uses indirect ElISA method to detect feline calicivirus, and ORF2 recombiant protein F is coated in ELISA microwell plate
In, then with the skimmed milk of 5%, ELISA Plate is closed, add testing sample.Feline calicivirus IgG antibody energy in testing sample
Coated ORF2 recombiant protein F reacts with ELISA Plate, forms F antigen-antibody complex, and enzyme labelled antibody is tied after adding therewith
Closing, then developed the color by substrate nitrite ion, stop buffer terminates reaction.Microplate reader is utilized to measure absorbance, OD under 450nm wavelength
The size of value is directly proportional to the content of feline calicivirus IgG antibody in testing sample.
Owing to FCV infection rate is high, it is difficult to collect enough negative serum samples, so negative serum sample cannot be used
OD450nmLight absorption value demarcates ELISA criterion.The present invention use serum endpoint titre method establish the inventive method
Cut Off value and criterion, have advantage accurate, reliable.
It is short that the present invention has operation short time consumption, quick advantage.Immunofluorescence test is for the strong and weak subjective meaning of fluorescence
Know overweight, and the non-subjective judgment of the inventive method testing result, test result is the clearest and the most definite clear.
Accompanying drawing explanation
Fig. 1 is the indirect ELISA analysis result figure of ORF2 recombinant protein A, B, CD, E, F.
Fig. 2 is No. 1 cat serum sample indirect immunofluorescence assay result figure in embodiment 3.
Fig. 3 is No. 18 cat serum sample indirect immunofluorescence assay result figures in embodiment 3.
Fig. 4 is No. 2 cat serum sample indirect immunofluorescence assay result figures in embodiment 3.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes between each detailed description of the invention
Combination in any.
Detailed description of the invention one: present embodiment includes for the ELISA kit detecting feline calicivirus IgG antibody
ELISA microwell plate, ELIAS secondary antibody and substrate nitrite ion；Wherein, the cat of the most coated solubility expression is comprised in ELISA microwell plate
Calicivirus ORF2 recombiant protein F.
The preparation method of the FCV gene ORF2 recombiant protein F of solubility expression in present embodiment:
Feline calicivirus (FCV) VP1 gene order (Genbank:X99445.1) design according to logging in Genbank is expanded
Increase the primer of the F district RNA fragment of feline calicivirus ORF2, and use PCR method to expand, then the genetic fragment gram by amplification
Grand to construction recombination plasmid in carrier pET-32a, proceed to after sequence verification is errorless expressive host E.coli BL21 (DE3) is used
IPTG abduction delivering；Then the most how the inductive condition that optimization truncated protein is expressed operates, parameter is how many, it is thus achieved that can
The feline calicivirus ORF2 recombiant protein F that dissolubility is expressed.
The preparation method of ELISA microwell plate in present embodiment:
With the carbonate buffer solution that the concentration that pH value is 9.6 is 0.05mol/L as being coated buffer, by cat after purification
After Calicivirus ORF2 recombiant protein F dilution, add microwell plate by 100 μ l/ holes, it is ensured that in every hole, the content of recombiant protein F is
0.2μg；4 DEG C are coated overnight, discard next day and are coated liquid, then by 100 μ l/ holes add concentration be 5% skimmed milk confining liquid, 37 DEG C
Standing 2h, load packaging bag, add desiccant after being washed out drying, drying at room temperature, vacuum preserves.
1000mL is coated buffer by the Na of 1.5g2CO3, the NaHCO of 2.9g3Add ddH2O water dissolution, regulation pH value is to 9.6
After add the water of surplus and make.
Detailed description of the invention two: the present embodiment difference from detailed description of the invention one is: be used for detecting cat cup-shaped sick
The ELISA kit of poison IgG antibody also includes stop buffer, and stop buffer is sulfuric acid solution.Other step and parameter and embodiment
One is identical.
In present embodiment stop buffer be concentration be the sulfuric acid solution of 2mol/L.
Detailed description of the invention three: the present embodiment difference from detailed description of the invention one or two is: substrate nitrite ion is
TMB nitrite ion.Other step and parameter are identical with embodiment one or two.
Detailed description of the invention four: the present embodiment difference from one of detailed description of the invention one to three is: ELIAS secondary antibody
Goat anti-cat lgG for horseradish peroxidase-labeled.Other step and parameter are identical with one of embodiment one to three.
Detailed description of the invention five: the present embodiment difference from one of detailed description of the invention one to four is: be used for detecting
The ELISA kit of feline calicivirus IgG antibody also includes antibody diluent.Other step and parameter and embodiment one to four
One of identical.
In present embodiment, antibody diluent is the PBST of pH value 7.4, and concrete configuration is as follows: containing liquid in 1000ml solution
The KH of 0.27g2PO4, the Na of 3.58g2PO4·12H2The Tween-20 of KCL, 0.5ml of NaCI, 0.2g of O, 8.0g and surplus
Distilled water is made.
Detailed description of the invention six: the present embodiment method of ELISA kit of the present invention detection feline calicivirus:
One, test serum antibody diluent is pressed the dilution of serum endpoint titre, then add ELISA by every hole 100 μ l
Microwell plate, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Two, every hole adds the ELIAS secondary antibody 100 μ l of dilution, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Three, every hole adds substrate nitrite ion 50 μ l, room temperature lucifuge reaction 10～15min；
Four, every hole adds 50 μ l stop buffers termination reactions, microplate reader detection 450nm light absorption value and the Cut of ELISA kit
Off value compares, and i.e. draws testing result.
Present embodiment step one sets repeating hole.ELIAS secondary antibody is the goat anti-cat lgG of horseradish peroxidase-labeled, enzyme
Marking two anti-extension rates is 5000 times.Stop buffer be concentration be the sulfuric acid solution of 2mol/L.Substrate nitrite ion is TMB nitrite ion.
The FCV gene ORF2 recombiant protein F of the most coated solubility expression is comprised in ELISA microwell plate.
In present embodiment, the Cut Off value of ELISA kit is 0.295.OD value≤0.295 of test serum sample,
Then this sample is that feline calicivirus IgG antibody is negative；The OD value of test serum sample > 0.295, then this sample is feline calicivirus
IgG antibody is positive.
Detailed description of the invention seven: the present embodiment difference from detailed description of the invention six is: with the cat cup of separate sources
Shape virus-positive serum stepwise dilution as one anti-, using PBS as negative control, each positive serum Cigarette dilution detection
N times；Then using the maximum dilution gradient that with negative control is significant difference as the serum endpoint titre of step one.Other step
And parameter is identical with embodiment six.
N >=3 in present embodiment.
Detailed description of the invention eight: the present embodiment difference from detailed description of the invention seven is: feline calicivirus positive blood
Clear stepwise dilution ratio is 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:21600
And 1:51200.Other step and parameter are identical with embodiment seven.
Detailed description of the invention nine: the Cut Off value establishment method of present embodiment ELISA kit: with separate sources
Feline calicivirus positive serum stepwise dilution as one anti-, using PBS as negative control, each positive serum dilution factor
Detection n times；Then using with negative control be significant difference maximum dilution gradient as serum endpoint titre, the cat of separate sources
Serum endpoint titre OD of Calicivirus positive serum450nmAverage+3 × SD is the Cut Off value of ELISA kit.
N >=3 in present embodiment.Present embodiment selects 20 parts of different feline calicivirus positive serums of originating to try
Test.
Embodiment 1
Utilize molecular biology software to FCV 2280 strain (ATCC numbering VR-2057) VP1 albumen (GenBank:
KC835209.1) A, B, CD, E, F subregion carries out antigenicity, accessibility, hydrophilic, major key pliability, the location of epitope
Etc. the analysis of aspect.
Software DNAstar analysis result shows the B district (125aa～397aa) of VP1 albumen, E district (426aa～524aa), F
(524aa～668aa) is respectively provided with higher antigenicity in district.
Software Megalingn analysis result show amino acid conservative be respectively B district be 90.2%～100%, E district be
67.7%～100%, F district is 89.0%～100%.
Feline calicivirus (FCV) VP1 gene order (Genbank:X99445.1) design according to logging in Genbank is expanded
Increase the primer of A, B, CD, E, F district RNA fragment of feline calicivirus ORF2, and use PCR method to expand, then by amplification
Gene fragment clone, to construction recombination plasmid in carrier pET-32a, proceeds to expressive host E.coli BL21 after sequence verification is errorless
(DE3) IPTG abduction delivering is used in；Then the inductive condition that truncated protein is expressed is optimized.Feline calicivirus ORF2 recombinant protein A,
CD inductive condition: bacterium solution is shaken to exponential phase in 37 DEG C of shaking tables, i.e. OD590nm value is between 0.4～0.6, adds IPTG eventually
Concentration is 0.1mM, and inducing temperature is 16 DEG C, and induction time is 9h.Feline calicivirus ORF2 recombiant protein B, E, F inductive condition:
Bacterium solution is shaken to exponential phase in 37 DEG C of shaking tables, i.e. OD590nm value is between 0.4～0.6, adds IPTG to final concentration of
0.5mM, inducing temperature is 20 DEG C, and induction time is 7h.The final point feline calicivirus ORF2 blocked obtaining solubility expression
Recombiant protein.
Utilize Ni-NTA HIS Bind Resin that the recombiant protein expressed is purified, and use BCA method to measure albumen
Concentration.The ORF2 recombiant protein equivalent loading (every hole 11.7ug) of purification, makees with feline calicivirus Quality Control positive serum (1:500)
Be one resist, the goat anti-cat IgG (1:5000) of HRP labelling as two resist, carry out Western blot analysis, ORF2 recombiant protein
B, E, F acquisition specific protein band at corresponding albumen size respectively, and ORF2 recombinant protein A, CD egg at expection size
The colour developing of informal voucher band is shallower, test result indicate that ORF2 recombiant protein B, E, F have good antigenicity.Measure with ImageJ software
ORF2 recombinant protein A, the immunoblotting analysis gray value of B, CD, E, F be respectively 0,17123,0,18366,19471.Statistics is soft
Part analysis result shows ORF2 recombiant protein B, E, F and recombinant protein A, CD significant difference (P < 0.05).Result above and molecule
Biological software antigenicity predicts the outcome and is consistent, and can primarily determine that recombiant protein B, E, F are antigen immunodominant proteins.
Again using ORF2 recombinant protein A, B, CD, E, F as envelope antigen, same to concentration (2 μ g/ml) wrapper sheet, every hole 100 μ l,
Using FCV positive quality control serum (1:12800 dilution) as one resist, using 1:5000 dilution HRP labelling goat anti-cat IgG as
Two resist, and set negative quality controlled serum comparison, blank.Every hole is all provided with two parallel controls, measures OD450nmValue.According to S/N value
Size judges the antigenicity power of recombinant protein A, B, CD, E, F, carries out statistical test after 8 repetitive operations.Result shows
The OD of ORF2 recombiant protein F450S/N value is the highest, notable (P < 0.05) (as shown in Figure 1) with other four recombiant protein diversityes.
In conjunction with bioinformatics software DNAstar and Western Blot antigenicity analysis result, determine ORF2 restructuring egg
White F is FCV VP1 dominant antigen fragment.
Positive quality control serum: cat serum is again through 56 DEG C, 30min inactivation of complement process after vaccine immunity.
Negative quality controlled serum: the most ill and non-immunized cat serum again through 56 DEG C, 30min inactivation of complement processes.
Embodiment 2
Take feline panleucopenia virus, feline herpetovirus, cat infectiousness rhino-bronchitis virus, feline infectious peritonitis virus and cat
Calicivirus totally five parts of virus-positive serum, the ELISA kit detecting feline calicivirus IgG antibody by the present invention is examined
Survey.Only feline calicivirus positive serum testing result is positive, and remaining four parts of serum sample is illustrated as feminine gender, and experiment proves
The specificity of the ELISA kit that the present invention detects feline calicivirus IgG antibody is good.
Using different batches coated ELISA microwell plate to operate, the coefficient of variation is 0.05%～0.26%, is less than
10%, it was demonstrated that the present invention detect the ELISA kit of feline calicivirus IgG antibody batch between repetition stability high.
Using same batch coated ELISA microwell plate to operate, the coefficient of variation is 0.01%～0.06%, is less than
10%, it was demonstrated that batch interior repetition stability that the present invention detects the ELISA kit of feline calicivirus IgG antibody is high.
Embodiment 3
By 20 parts source different cat serum do respectively indirect ELISA experiment (using test kit of the present invention to detect),
Neutralization test and indirect immunofluorescence experiment.
Experimental result is as shown in table 1, and the Cut Off value of indirect ELISA experiment is 0.295.
Table 1
| Serum is numbered | Neutralization test titer | IFA result (1:50) | ELISA result (1:100 dilution) |
| 1 | 595 | + | 1.685 |
| 2 | 354 | + | 1.586 |
| 3 | 149 | + | 1.322 |
| 4 | 44 | + | 1.444 |
| 5 | 8 | + | 1.055 |
| 6 | 178 | + | 1.719 |
| 7 | 18 | + | 1.48 |
| 8 | 31 | + | 1.522 |
| 9 | 45 | + | 1.488 |
| 10 | 13 | + | 1.418 |
| 11 | 354 | + | 1.725 |
| 12 | 354 | + | 2.091 |
| 13 | 354 | + | 2.084 |
| 14 | 0 | + | 0.81 |
| 15 | 0 | - | 0.231 |
| 16 | 0 | - | 0.188 |
| 17 | 0 | - | 0.23 |
| 18 | 0 | - | 0.128 |
| 19 | 0 | - | 0.239 |
| 20 | 0 | - | 0.237 |
No. 1, No. 18 and No. 2 cat serum sample indirect immunofluorescence assay results are the most as shown in Figure 2, Figure 3 and Figure 4.
Using the testing result of test kit of the present invention and the coincidence rate of neutralization test testing result is 95%；With immunity indirectly
The coincidence rate of fluorescent test testing result is 100%.
Claims (9)
1. for detecting the ELISA kit of feline calicivirus IgG antibody, it is characterised in that be used for detecting feline calicivirus IgG
The ELISA kit of antibody includes ELISA microwell plate, ELIAS secondary antibody and substrate nitrite ion；Wherein, comprise in ELISA microwell plate
The feline calicivirus ORF2 recombiant protein F of the most coated solubility expression.
ELISA kit for detecting feline calicivirus IgG antibody the most according to claim 1, it is characterised in that use
ELISA kit in detection feline calicivirus IgG antibody also includes stop buffer, and stop buffer is sulfuric acid solution.
ELISA kit for detecting feline calicivirus IgG antibody the most according to claim 1 and 2, it is characterised in that
Substrate nitrite ion is TMB nitrite ion.
ELISA kit for detecting feline calicivirus IgG antibody the most according to claim 3, it is characterised in that enzyme
Mark two resists the goat anti-cat lgG for horseradish peroxidase-labeled.
ELISA kit for detecting feline calicivirus IgG antibody the most according to claim 1 and 2, it is characterised in that
Antibody diluent is also included for detecting the ELISA kit of feline calicivirus IgG antibody.
6. by the method for ELISA kit detection feline calicivirus described in claim 1, it is characterised in that detection method is by following
Step is carried out:
One, test serum antibody diluent is pressed the dilution of serum endpoint titre, then add ELISA micropore by every hole 100 μ l
Plate, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Two, every hole adds the ELIAS secondary antibody 100 μ l of dilution, hatches 1h, then cleans 3～5 times with antibody diluent for 37 DEG C；
Three, every hole adds substrate nitrite ion 50 μ l, room temperature lucifuge reaction 10～15min；
Four, every hole adds 50 μ l stop buffers termination reactions, microplate reader detection 450nm light absorption value and the Cut Off of ELISA kit
Value compares, and i.e. draws testing result.
The method of detection feline calicivirus the most according to claim 6, it is characterised in that the cat cup-shaped with separate sources is sick
Poison positive serum stepwise dilution as one anti-, using PBS as negative control, each positive serum Cigarette dilution detection n times；
Then using the maximum dilution gradient that with negative control is significant difference as the serum endpoint titre of step one.
The method of detection feline calicivirus the most according to claim 7, it is characterised in that feline calicivirus positive serum by
Level dilution ratio is 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:21600 and 1:
51200。
9. the Cut Off value establishment method of ELISA kit described in claim 1, it is characterised in that with the cat cup of separate sources
Shape virus-positive serum stepwise dilution as one anti-, using PBS as negative control, each positive serum Cigarette dilution detection
N times；Then using with negative control be significant difference maximum dilution gradient as serum endpoint titre, the cat cup-shaped of separate sources
Serum endpoint titre OD of virus-positive serum450nmAverage+3 × SD is the Cut Off value of ELISA kit.
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| CN108362877A (en) * | 2018-01-24 | 2018-08-03 | 中国农业科学院兰州畜牧与兽药研究所 | Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof |
| CN109991413A (en) * | 2017-12-31 | 2019-07-09 | 江苏雷森生物科技有限公司 | A kind of feline calicivirus fluorogenic quantitative detection card and detection method |
| CN112321722A (en) * | 2020-11-13 | 2021-02-05 | 杭州亿米诺生物科技有限公司 | Cat calicivirus VP1-VP2 recombinant protein and preparation method and application thereof |
| CN113817726A (en) * | 2021-11-22 | 2021-12-21 | 苏州蝌蚪生物技术有限公司 | Amplification primer composition and kit for detecting feline calicivirus |
| CN116183915A (en) * | 2022-12-27 | 2023-05-30 | 深圳粒影生物科技有限公司 | Triple antibody detection test strip for feline panleukopenia virus, feline herpesvirus and feline calicivirus and application thereof |
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| 蒋艳妹等: "猫杯状病毒ＶＰ１衣壳蛋白的截短表达及其抗原性分析", 《中国兽医科学》 * |
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| CN108362877A (en) * | 2018-01-24 | 2018-08-03 | 中国农业科学院兰州畜牧与兽药研究所 | Feline calicivirus fluorescence immune chromatography test strip and preparation method thereof |
| CN112321722A (en) * | 2020-11-13 | 2021-02-05 | 杭州亿米诺生物科技有限公司 | Cat calicivirus VP1-VP2 recombinant protein and preparation method and application thereof |
| CN113817726A (en) * | 2021-11-22 | 2021-12-21 | 苏州蝌蚪生物技术有限公司 | Amplification primer composition and kit for detecting feline calicivirus |
| CN116183915A (en) * | 2022-12-27 | 2023-05-30 | 深圳粒影生物科技有限公司 | Triple antibody detection test strip for feline panleukopenia virus, feline herpesvirus and feline calicivirus and application thereof |
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