CN111948381A - Canine coronavirus fluorescence aptamer test strip - Google Patents
Canine coronavirus fluorescence aptamer test strip Download PDFInfo
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- CN111948381A CN111948381A CN202010846532.2A CN202010846532A CN111948381A CN 111948381 A CN111948381 A CN 111948381A CN 202010846532 A CN202010846532 A CN 202010846532A CN 111948381 A CN111948381 A CN 111948381A
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 55
- 241000711506 Canine coronavirus Species 0.000 title claims abstract description 51
- 241000282465 Canis Species 0.000 claims abstract description 36
- 239000002299 complementary DNA Substances 0.000 claims abstract description 22
- 108020004635 Complementary DNA Proteins 0.000 claims abstract description 21
- 238000010804 cDNA synthesis Methods 0.000 claims abstract description 21
- 238000010791 quenching Methods 0.000 claims abstract description 12
- 230000000171 quenching effect Effects 0.000 claims abstract description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 14
- 239000000020 Nitrocellulose Substances 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 12
- 229920001220 nitrocellulos Polymers 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002715 modification method Methods 0.000 claims description 6
- 238000005086 pumping Methods 0.000 claims description 6
- 238000006748 scratching Methods 0.000 claims description 6
- 230000002393 scratching effect Effects 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 6
- 230000002745 absorbent Effects 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 238000007098 aminolysis reaction Methods 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 241000711573 Coronaviridae Species 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 5
- 239000007850 fluorescent dye Substances 0.000 abstract description 3
- 230000000295 complement effect Effects 0.000 abstract description 2
- 238000001215 fluorescent labelling Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 229920000915 polyvinyl chloride Polymers 0.000 description 6
- 239000004800 polyvinyl chloride Substances 0.000 description 6
- 239000000523 sample Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091028026 C-DNA Proteins 0.000 description 1
- 241000701931 Canine parvovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241001292005 Nidovirales Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 206010067470 Rotavirus infection Diseases 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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Abstract
The invention discloses a canine coronavirus fluorescent aptamer test strip, and relates to the technical field of test strips. The invention carries out fluorescence labeling on canine coronavirus-aptamer and labels a quenching group on the 5' end of a DNA single chain which is complementary with the aptamer; when the canine coronavirus is absent from the system, the canine coronavirus-aptamer binds to the aptamer complementary DNA, such that fluorescence on the aptamer is quenched; when the canine coronavirus is added to be combined with the canine coronavirus-aptamer to form a canine coronavirus fluorescent aptamer complex, the structure of the aptamer is changed, the aptamer complementary DNA single strand is released, and the quenched fluorescence is recovered again. By the change of fluorescence, the concentration of the canine coronavirus can be quantitatively detected by using a fluorescence quantitative instrument.
Description
Technical Field
The invention belongs to the technical field of test strips, and particularly relates to a canine coronavirus fluorescent aptamer test strip.
Background
Canine Coronavirus (CCV) belongs to Nidovirales, Coronaviridae, Coronavirales, and Coronavirales group I. The CCV virus particle has a diameter of 60-200 nm, is spherical and has an envelope. The nucleocapsid is filamentous, helically symmetric, like other enveloped viruses. The disease is frequently encountered in cold winter, is rapidly spread and is frequently nest outbreak within a few days; although the disease occurs regardless of breed, age, and sex, it is common for puppies to first develop the disease and then spread to dogs of other ages when they are prevalent in a canine population.
Both the incidence and mortality of puppies is higher than that of adult dogs. Symptoms worsen in puppies, vomiting and diarrhea are the main symptoms of the disease, and symptoms are mild in adult dogs. The clinical symptoms and epidemiology of the disease are similar to rotavirus infection, and the disease is often infected with rotavirus, canine parvovirus and the like in a mixed way, so that the diagnosis is difficult.
The test strip for the fluorescent aptamer of the canine coronavirus is provided to solve the problems.
Disclosure of Invention
The invention aims to provide a test strip for a canine coronavirus fluorescent aptamer, which is characterized in that a canine coronavirus-aptamer is subjected to fluorescent labeling, and the 5' end of a DNA single chain which is complementary with the aptamer is subjected to labeling of a quenching group; when the canine coronavirus is absent from the system, the canine coronavirus-aptamer binds to the aptamer complementary DNA, such that fluorescence on the aptamer is quenched; when the canine coronavirus is added to be combined with the canine coronavirus-aptamer to form a canine coronavirus fluorescent aptamer complex, the structure of the aptamer is changed, the aptamer complementary DNA single strand is released, and the quenched fluorescence is recovered again. By the change of fluorescence, the concentration of the canine coronavirus can be quantitatively detected by using a fluorescence quantitative instrument.
In order to solve the technical problems, the invention is realized by the following technical scheme:
the invention relates to a canine coronavirus fluorescent aptamer test strip, which comprises water absorption paper, a sample pad, a PVC (polyvinyl chloride) bottom plate, a nitrocellulose membrane and a canine coronavirus-aptamer, wherein the canine coronavirus-aptamer has the following sequence: 5 '-. x × -3'; an aptamer complementary DNA single strand, the aptamer complementary DNA single strand sequence being: 5 '— 3'; the 3' end of the canine coronavirus-aptamer sequence is modified by a fluorescent group FAM; the 5' end of the aptamer complementary DNA single strand is marked with a quenching group; marking fluorescein on a canine coronavirus-aptamer, and then scratching the fluorescein on the nitrocellulose membrane to serve as a detection line T of the test strip; scratching a goat anti-mouse antibody on the nitrocellulose membrane to form a quality control line C of the test strip; when the fluorescein-labeled canine coronavirus-aptamer is hybridized with a quencher-labeled aptamer complementary DNA single strand (cDNA), the fluorescein is quenched by the proximity of the aptamer to the quencher.
Further, the 3' end of the canine coronavirus-aptamer sequence is modified with a fluorescent group FAM; the modification method of the fluorescent group comprises the following steps: directly synthesizing a DNA sequence by using 3' -end fluorescent hydroxyl polystyrene, then aminolysis, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
Further, the aptamer complements a 5' end modified quencher group (TAMRA) of the DNA single strand; the modification method of the quenching group comprises the following steps: directly synthesizing a DNA sequence by using a 5' end fluorescence quenching group, then aminolyzing, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
Further, the method for detecting the canine coronavirus by using the test strip comprises the following steps:
when the canine coronavirus is absent in the system, the canine coronavirus-aptamer binds to the aptamer complementary DNA single strand, so that the fluorescence on the canine coronavirus-aptamer is quenched;
when the canine coronavirus is added, the canine coronavirus is combined with the canine coronavirus-aptamer to form a canine coronavirus fluorescent aptamer complex, the structure of the canine coronavirus-aptamer is changed, an aptamer complementary DNA single strand is released, and quenched fluorescence is recovered again;
the concentration of canine coronavirus was quantitatively determined by the change in fluorescence using a fluorescence quantitative analyzer.
Further, the use method of the test strip is as follows: after the absorbent paper, the sample pad, the PVC base plate and the nitrocellulose membrane were assembled, the change in fluorescence was observed with an ultraviolet lamp when detecting canine coronavirus.
The invention has the following beneficial effects:
1. the invention has more accurate detection and can detect the virus concentration;
2. the detection result of the invention is more convenient for veterinarians to make accurate diagnosis.
Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the test strip for canine coronavirus fluorescent aptamer of the present invention when the test strip is positive;
FIG. 2 is a schematic diagram of the test strip for canine coronavirus fluorescence aptamer of the invention when the test strip is negative.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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 invention.
A fluorescence analysis method for detecting the canine coronavirus can be established by utilizing the aptamer fluorescent probe based on the fluorescence resonance energy transfer principle; the canine coronavirus is excited at the wavelength of 375nm and can fluoresce at the position of 430nm, after the canine coronavirus is combined with the aptamer marked with fluorescein, fluorescence resonance energy transfer can occur between the canine coronavirus and the aptamer, and the fluorescence intensity of the fluorescein at the position of 518nm is enhanced; the detection of the canine coronavirus can be realized according to the change of the fluorescence intensity at 518 nm.
The invention relates to a canine coronavirus fluorescent aptamer test strip, which comprises water absorption paper, a sample pad, a PVC (polyvinyl chloride) bottom plate, a nitrocellulose membrane and a canine coronavirus-aptamer, wherein the sequence of the canine coronavirus-aptamer is as follows: 5 '-. x × -3'; the aptamer complementary DNA single strand has the following sequence: 5 '— 3'; the 3' end of the canine coronavirus-aptamer sequence is modified by a fluorescent group FAM; the 5' end of the aptamer complementary DNA single strand is marked with a quenching group; marking fluorescein on a canine coronavirus-aptamer, and then scratching the fluorescein on a nitrocellulose membrane to serve as a detection line T of the test strip; scratching a goat anti-mouse antibody on a nitrocellulose membrane to form a quality control line C of the test strip; when the fluorescein-labeled canine coronavirus-aptamer is hybridized with a quencher-labeled aptamer-complementary single-stranded DNA (c-DNA), the fluorescein is quenched by the proximity of the aptamer to the quencher.
Wherein, the 3' end of the canine coronavirus-aptamer sequence is modified with a fluorescent group FAM; the modification method of the fluorescent group comprises the following steps: directly synthesizing a DNA sequence by using 3' -end fluorescent hydroxyl polystyrene, then aminolysis, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
Wherein, the aptamer complements 5' end of the DNA single strand to modify a quenching group (TAMRA); the modification method of the quenching group comprises the following steps: directly synthesizing a DNA sequence by using a 5' end fluorescence quenching group, then aminolyzing, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
The method for detecting the canine coronavirus through the test strip comprises the following steps:
as shown in fig. 2: when the canine coronavirus is absent in the system, the canine coronavirus-aptamer binds to the aptamer complementary DNA single strand, so that the fluorescence on the canine coronavirus-aptamer is quenched;
as shown in fig. 1: when the canine coronavirus is added, the canine coronavirus is combined with the canine coronavirus-aptamer to form a canine coronavirus fluorescent aptamer complex, the structure of the canine coronavirus-aptamer is changed, an aptamer complementary DNA single strand is released, and quenched fluorescence is recovered again;
the concentration of canine coronavirus was quantitatively determined by the change in fluorescence using a fluorescence quantitative analyzer.
The use method of the test strip comprises the following steps: after the absorbent paper, the sample pad, the PVC base plate and the nitrocellulose membrane are assembled, the change of fluorescence is observed by using an ultraviolet lamp when the canine coronavirus is detected, the detection is more accurate, and the virus concentration can be detected; the detection result of the invention is more convenient for veterinarians to make accurate diagnosis.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (5)
1. The utility model provides a dog coronavirus fluorescence aptamer test paper strip, includes absorbent paper, sample pad, PVC bottom plate and nitrocellulose membrane, its characterized in that still includes:
a canine coronavirus-aptamer having the sequence: 5 '-. x × -3';
an aptamer complementary DNA single strand, the aptamer complementary DNA single strand sequence being: 5 '— 3';
the 3' end of the canine coronavirus-aptamer sequence is modified by a fluorescent group FAM;
the 5' end of the aptamer complementary DNA single strand is marked with a quenching group;
marking fluorescein on a canine coronavirus-aptamer and then scratching the fluorescein on the nitrocellulose membrane to serve as a detection line of the test strip;
scratching a goat anti-mouse antibody on the nitrocellulose membrane to form a quality control line of the test strip;
upon hybridization of the fluorescein-labeled canine coronavirus-aptamer to the quencher-labeled aptamer complementary DNA single strand, the fluorescein is quenched by the proximity of the aptamer to the quencher.
2. The canine coronavirus fluorescent aptamer test strip of claim 1, wherein the 3' end of the canine coronavirus-aptamer sequence is modified with a Fluorophore (FAM);
the modification method of the fluorescent group comprises the following steps: directly synthesizing a DNA sequence by using 3' -end fluorescent hydroxyl polystyrene, then aminolysis, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
3. The canine coronavirus fluorescent aptamer test strip of claim 1, wherein the aptamer is a complementary DNA single strand modified at the 5' end with a quencher;
the modification method of the quenching group comprises the following steps: directly synthesizing a DNA sequence by using a 5' end fluorescence quenching group, then aminolyzing, purifying by a high performance liquid chromatograph, quantifying, and obtaining the sequence after pumping.
4. The canine coronavirus fluorescent aptamer test strip of claim 1, wherein the test strip is used for detecting canine coronavirus, and the method comprises the following steps:
when the canine coronavirus is absent in the system, the canine coronavirus-aptamer binds to the aptamer complementary DNA single strand, so that the fluorescence on the canine coronavirus-aptamer is quenched;
when the canine coronavirus is added, the canine coronavirus is combined with the canine coronavirus-aptamer to form a canine coronavirus fluorescent aptamer complex, the structure of the canine coronavirus-aptamer is changed, an aptamer complementary DNA single strand is released, and quenched fluorescence is recovered again;
the concentration of canine coronavirus was quantitatively determined by the change in fluorescence using a fluorescence quantitative analyzer.
5. The canine coronavirus fluorescent aptamer test strip of claim 4, wherein the test strip is used in a method comprising: after the absorbent paper, the sample pad, the PVC base plate and the nitrocellulose membrane were assembled, the change in fluorescence was observed with an ultraviolet lamp when detecting canine coronavirus.
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| CN202010846532.2A CN111948381A (en) | 2020-08-21 | 2020-08-21 | Canine coronavirus fluorescence aptamer test strip |
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| CN202010846532.2A CN111948381A (en) | 2020-08-21 | 2020-08-21 | Canine coronavirus fluorescence aptamer test strip |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738619A (en) * | 2014-12-11 | 2016-07-06 | 杨挥 | Test paper strip and method for detecting canine coronavirus through immunofluorescence chromatography technique |
| CN107831317A (en) * | 2017-11-01 | 2018-03-23 | 杭州微瑞科技有限公司 | Canine coronavirus antibody Quantitative detection card and application method |
| CN208672650U (en) * | 2018-05-04 | 2019-03-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card |
| CN109765384A (en) * | 2019-01-29 | 2019-05-17 | 北京勤邦生物技术有限公司 | A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application |
| CN109856406A (en) * | 2018-12-24 | 2019-06-07 | 北京勤邦生物技术有限公司 | A kind of canine parvovirus antibody fluorescence test strip and its preparation method and application |
-
2020
- 2020-08-21 CN CN202010846532.2A patent/CN111948381A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105738619A (en) * | 2014-12-11 | 2016-07-06 | 杨挥 | Test paper strip and method for detecting canine coronavirus through immunofluorescence chromatography technique |
| CN107831317A (en) * | 2017-11-01 | 2018-03-23 | 杭州微瑞科技有限公司 | Canine coronavirus antibody Quantitative detection card and application method |
| CN208672650U (en) * | 2018-05-04 | 2019-03-29 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card |
| CN109856406A (en) * | 2018-12-24 | 2019-06-07 | 北京勤邦生物技术有限公司 | A kind of canine parvovirus antibody fluorescence test strip and its preparation method and application |
| CN109765384A (en) * | 2019-01-29 | 2019-05-17 | 北京勤邦生物技术有限公司 | A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application |
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