CN109100518A - Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law - Google Patents
Bird flu, the test strips of human influenza Susceptible population and test card are quickly detected using competition law Download PDFInfo
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- CN109100518A CN109100518A CN201811211047.7A CN201811211047A CN109100518A CN 109100518 A CN109100518 A CN 109100518A CN 201811211047 A CN201811211047 A CN 201811211047A CN 109100518 A CN109100518 A CN 109100518A
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- 238000012360 testing method Methods 0.000 title claims abstract description 146
- 206010022000 influenza Diseases 0.000 title claims abstract description 30
- 206010064097 avian influenza Diseases 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 119
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 83
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 238000010521 absorption reaction Methods 0.000 claims abstract description 19
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- 102000004856 Lectins Human genes 0.000 claims description 63
- 108090001090 Lectins Proteins 0.000 claims description 63
- 239000002523 lectin Substances 0.000 claims description 63
- 239000012528 membrane Substances 0.000 claims description 58
- 238000003908 quality control method Methods 0.000 claims description 58
- 108060002885 fetuin Proteins 0.000 claims description 32
- 102000013361 fetuin Human genes 0.000 claims description 32
- 239000000020 Nitrocellulose Substances 0.000 claims description 30
- 238000000576 coating method Methods 0.000 claims description 30
- 229920001220 nitrocellulos Polymers 0.000 claims description 30
- 210000003296 saliva Anatomy 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 29
- 239000011248 coating agent Substances 0.000 claims description 26
- 238000001035 drying Methods 0.000 claims description 20
- 238000002372 labelling Methods 0.000 claims description 19
- 239000003365 glass fiber Substances 0.000 claims description 18
- 208000002979 Influenza in Birds Diseases 0.000 claims description 17
- 239000006228 supernatant Substances 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 238000001723 curing Methods 0.000 claims description 4
- 125000005630 sialyl group Chemical group 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 101710186708 Agglutinin Proteins 0.000 claims description 3
- 101710146024 Horcolin Proteins 0.000 claims description 3
- 101710189395 Lectin Proteins 0.000 claims description 3
- 101710179758 Mannose-specific lectin Proteins 0.000 claims description 3
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 claims description 3
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 claims description 3
- 239000000910 agglutinin Substances 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 230000028327 secretion Effects 0.000 claims description 2
- 230000002860 competitive effect Effects 0.000 claims 1
- 239000010931 gold Substances 0.000 abstract description 19
- 229910052737 gold Inorganic materials 0.000 abstract description 19
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 239000000084 colloidal system Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 19
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 16
- 241000712461 unidentified influenza virus Species 0.000 description 14
- 238000007885 magnetic separation Methods 0.000 description 8
- 101710154606 Hemagglutinin Proteins 0.000 description 7
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 7
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 7
- 101710176177 Protein A56 Proteins 0.000 description 7
- 239000000185 hemagglutinin Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 239000002253 acid Substances 0.000 description 6
- 238000007605 air drying Methods 0.000 description 6
- 239000006249 magnetic particle Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 5
- 238000009739 binding Methods 0.000 description 5
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
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- 210000004027 cell Anatomy 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000007365 Sialoglycoproteins Human genes 0.000 description 2
- 108010032838 Sialoglycoproteins Proteins 0.000 description 2
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
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- 239000002274 desiccant Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 101001089022 Axinella polypoides Lectin-2 Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101001089088 Cymbosema roseum Lactose-binding lectin-2 Proteins 0.000 description 1
- 241001521402 Maackia <angiosperm> Species 0.000 description 1
- 240000006028 Sambucus nigra Species 0.000 description 1
- 235000003142 Sambucus nigra Nutrition 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 235000008995 european elder Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4724—Lectins
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of quickly to detect bird flu, the test strips of human influenza Susceptible population and test card using competition law.The test strips (card) include the I type test strips (card) for detecting bird flu Susceptible population and/or the II type test strips (card) for detecting human influenza Susceptible population, and test strips (card) include sample pad, colloid gold label pad, detection reaction zone and the water absorption pad set gradually along chromatography direction.Test strips (card) detection sensitivity of the present invention is high, and false positive and false negative rate are low, easy to use, is easy observation difference, is suitable for large-scale application in clinical quick diagnosis and base's epidemiological survey etc..
Description
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to a test strip (card) for rapidly detecting susceptible people of avian influenza and human influenza viruses based on a colloidal gold immunochromatographic technique.
Background
influenza virus HAs an important structural protein on the surface of the influenza virus, exists in the form of an H-polymer and is called Hemagglutinin (HA), the influenza virus invades a human body mainly by the way that the Hemagglutinin (HA) structure on the surface of the influenza virus is combined with a host cell surface specific sugar chain receptor to infect the host, the sialic acid molecule mainly recognized by the HA is combined with α 2-3 and Gal through a terminal C atom to form a SA alpha 2-3Gal structure or form the SA alpha 2-6Gal structure, the host cell surface structure HAs the specificity when the influenza virus is combined with the HA, the influenza virus is combined with the host cell surface sugar chain terminal structure of different types, wherein the different types of the influenza virus specifically recognized SA terminal structure is SA 2-6Gal, and the influenza virus can be used for judging the content of the sialic acid molecule in the human body surface sugar chain structure because the influenza virus HAs the terminal sialic acid chain structure of Gal 2-6 Gal.
Sambucus Nigra Agglutinin (SNA) and Maackia Aimrensis Lectin-II (MAL-II) have similarities with regions specifically recognized by influenza virus HA, wherein the SNA specifically recognizes sugar chain receptor structures ending with SA α 2-6Gal, which have similarities with sites specifically bound by human influenza virus, and the MAL-II specifically recognizes sugar chain receptor structures ending with SA α 2-3Gal, which have similarities with sites specifically bound by avian influenza virus.
Chinese patent 'method for rapidly detecting susceptible population of avian influenza and human influenza and test strip' (publication number: CN106771120A) discloses a test strip for rapidly detecting susceptible population of avian influenza and human influenza virus based on colloidal gold chromatography, and the reaction principle is a double lectin sandwich method. However, since lectins do not have immune activity similar to that of antibodies, the binding specificity of lectins to sialoglycoproteins cannot be compared with that of antigen-antibody binding reactions. For detecting substances with antigenic properties such as sialoglycoprotein, a competition method is more reasonable, the specificity of specific binding of lectin is reflected, the detection principle is clearer, and factors influencing the detection result are fewer, so that the false positive and false negative of the detection result are reduced. However, the processing of the detection sample, the concentration of the lectin on the gold-labeled pad, the coating on the detection line and the concentration screening thereof have higher difficulty, and can be determined only by technical clearance, so that the accuracy and reliability of the detection result are ensured. At present, no report of colloidal gold test paper strips (cards) for detecting avian influenza and human influenza susceptible people by using a competition method principle is seen.
Disclosure of Invention
The invention aims to provide a test strip and a test card for rapidly detecting avian influenza and human influenza susceptible population by using a competition method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a test paper for quickly detecting influenza susceptible population by using a competition method comprises a type I test paper for detecting avian influenza susceptible population and/or a type II test paper for detecting human influenza susceptible population, the type I test paper and the type II test paper respectively comprise a sample pad, a colloidal gold labeling pad, a detection reaction area and a water absorption pad which are sequentially arranged along the chromatography direction, the detection reaction area comprises a detection line and a quality control line which are arranged in sequence, the colloidal gold labeling pad of the I-type test paper comprises lectin MAL-II (MAL-II-colloidal gold labeling substance) for labeling colloidal gold, the colloidal gold labeling pad of the II-type test paper comprises lectin SNA (SNA-colloidal gold labeling substance) for labeling colloidal gold, the quality control line of the I-type test paper comprises lectin ConA, the quality control line of the II-type test paper comprises lectin STL, and the detection lines of the I-type test paper and the II-type test paper comprise fetuin.
Preferably, the colloidal gold labeled pad is prepared by coating and drying the colloidal gold labeled lectin MAL-II or the colloidal gold labeled lectin SNA on a glass fiber membrane or a nitrocellulose membrane; the detection line is prepared by coating and drying and solidifying fetuin on a nitrocellulose membrane; the quality control line is made by coating and drying and solidifying the lectin ConA or STL on the nitrocellulose membrane on which the detection line is positioned.
Preferably, the coating of the colloidal gold-labeled lectin MAL-II or the colloidal gold-labeled lectin SNA specifically means that the colloidal gold-labeled lectin MAL-II or the colloidal gold-labeled lectin SNA with the concentration of 30-60 mug/mL is sprayed on a glass fiber membrane or a nitrocellulose membrane; the coating of the fetuin specifically refers to scribing or spraying the fetuin with the concentration of 1-4 mg/mL at the detection line position of the nitrocellulose membrane, and the coating of the lectin ConA or STL specifically refers to scribing or spraying the lectin ConA or STL with the concentration of 1-4 mg/mL at the quality control line position of the nitrocellulose membrane.
Preferably, the test strip is fixed to a substrate or disposed within a card housing having a window.
A preparation method of the test paper comprises the following steps:
preparation of I-type test paper
1.1) coating the lectin MAL-II for marking the colloidal gold on a glass fiber membrane or a nitrocellulose membrane, and then drying (drying at 37-40 ℃ for 18-24 hours) to obtain a colloidal gold marking pad;
1.2) coating fetuin and lectin ConA on a nitrocellulose membrane, and drying (20-30 ℃) to obtain a detection line and a quality control line;
1.3) assembling a sample pad, the colloidal gold labeled pad obtained in the step 1.1), the nitrocellulose membrane containing the detection line and the quality control line obtained in the step 1.2) and a water absorption pad into I-type test paper;
preparation of II-type test paper
2.1) coating the lectin SNA for marking the colloidal gold on a glass fiber membrane or a nitrocellulose membrane, and then drying (drying at 37-40 ℃ for 18-24 hours) to obtain a colloidal gold marking pad;
2.2) coating fetuin and lectin STL on a nitrocellulose membrane, and drying (20-30 ℃) to obtain a detection line and a quality control line;
and 2.3) assembling the sample pad, the colloidal gold labeled pad obtained in the step 2.1), the nitrocellulose membrane containing the detection line and the quality control line obtained in the step 2.2) and the water absorption pad into II-type test paper.
Preferably, in the preparation of the gold-labeled pad, the gold-labeled lectin MAL-II or gold-labeled lectin SNA is added at a concentration of 30-60. mu.g/mL in an amount of 50-200. mu.L/cm2Coating on a glass fiber membrane or a nitrocellulose membrane; in the preparation of the detection line and the quality control line, fetuin and agglutinin ConA or STL with the concentration of 1-4 mg/mL are coated on the nitrocellulose membrane.
A method for rapidly detecting a sialylated sugar chain by a competition method, comprising the steps of:
1) pretreating the collected detection sample to prevent the degradation of protein in the detection sample to obtain a detection sample;
2) adding a detection sample or a diluted detection sample to the prepared I-type and/or II-type test paper;
3) the fetuin on the type I and/or type II test paper and the sialyl sugar chain with the SA α 2-3Gal structure at the end or the sialyl sugar chain with the SA α 2-6Gal structure at the end in the test sample are used for competitively binding with the lectin MAL-II or SNA of the labeled colloidal gold on the corresponding test paper, so as to form a test result, and the determination of the test result is as follows:
if the detection line and the quality control line are both colored, judging that the sialylated sugar chain of which the tail end has the SA α 2-3Gal structure in the detection sample is negative or has lower content for the I-type test paper, and judging that the sialylated sugar chain of which the tail end has the SA α 2-6Gal structure in the detection sample is negative or has lower content for the II-type test paper;
if only the quality control line is developed, judging that the sialylated sugar chain of which the tail end has the SA α 2-3Gal structure is positive or has higher content in the test sample for the I-type test paper, and judging that the sialylated sugar chain of which the tail end has the SA α 2-6Gal structure is positive or has higher content in the test sample for the II-type test paper;
and if the quality control line does not develop color, judging that the corresponding test paper is invalid.
Preferably, the test sample is selected from saliva or nasal secretions; the test sample is diluted by 1-5 times by using deionized water, injection water, physiological saline (0.9% sodium chloride solution), phosphate buffer solution or borate buffer solution.
Preferably, the pretreatment comprises the following steps: adding a protease inhibitor into the collected detection sample at 0-4 ℃, centrifuging, and adding the protease inhibitor into the supernatant obtained by centrifuging again.
The type I and/or type II test paper is applied to preparation of reagents (such as test paper strips, test paper cards and the like) for rapidly detecting influenza susceptible population (wherein the type I test paper can be used for detecting avian influenza susceptible population, and the type II test paper can be used for detecting human influenza susceptible population).
The invention has the beneficial effects that:
the invention uses fetuin as a detection line, and forms chromatography test paper with lectin MAL-II or SNA marked by colloidal gold respectively, thereby realizing rapid detection of sialylated sugar chain structure (for example, with SA α 2-3Gal or SA α 2-6Gal terminal) based on competition method, the test paper has high detection sensitivity, repetition rate, strong specificity and good stability, the false positive rate and the false negative rate can be controlled below 1 percent and higher than the level of the existing detection reagent based on sandwich method, the detection result is easy to observe and distinguish, the use is convenient, and the invention is suitable for clinical rapid diagnosis of influenza susceptibility and large-scale application of basic level and rural epidemiological investigation, and can also be used for qualitative and semi-quantitative detection of sialylated sugar chain structure.
Drawings
FIG. 1 is a schematic structural diagram of a test strip;
in the figure: 1 is a PVC plate, 2 is a detection line, 3 is a quality control line, 4 is a water absorption pad, 5 is an NC film, 6 is a gold label pad, and 7 is a sample pad.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Preparation of I-type test strip for detecting susceptible population of avian influenza
(1) Preparing colloidal gold: mixing chloroauric acid (HAuCl)4) Preparing a 1% chloroauric acid solution, adding 1mL of the chloroauric acid solution into 99mL of deionized water, heating to boil, immediately adding 2mL of the 1% sodium citrate solution, rapidly and uniformly stirring, continuously heating for 10 minutes, cooling to room temperature, and supplementing deionized water to 100mL to obtain a colloidal gold solution.
(2) Preparing MAL-II-colloidal gold marker: taking 10mL of the prepared colloidal gold solution, adjusting the pH value to 4.9-5.3 (for example, 5.0) by using 0.1mol/L potassium carbonate solution, adding 120 mu g of lectin MAL-II, uniformly mixing, reacting at room temperature for 10 minutes, centrifuging at 4 ℃ and 2000rpm for 20 minutes, and discarding the precipitate. The supernatant was centrifuged at 12000rpm at 4 ℃ for 60 minutes, the supernatant was discarded, and the precipitate was dissolved in 1mL of 0.01mol/L PBS (containing 0.2% BSA) having pH8.0 to obtain a solution of MAL-II-colloidal gold-labeled substance.
(3) Preparing a gold label pad: the MAL-II-colloidal gold-labeled solution is diluted 1-2 times (e.g., by 1-fold dilution, the MAL-II concentration is about 60. mu.g/mL) with 0.01mol/L PBS (containing 0.2% BSA) of pH8.0 at 100. mu.L/cm2Uniformly spraying the mixture on a glass fiber membrane, and drying the glass fiber membrane for 24 hours at 40 ℃ under the humidity of 30% to obtain the gold label pad. The gold label pad is sealed by a tin foil bag, is filled with a drying agent and is stored at normal temperature.
(4) Coating of detection line and quality control line: fetuin was diluted to a concentration of 2mg/mL with 0.01mol/L PBS (pH7.2), streaked on an NC membrane (or sprayed on an NC membrane as a coating method instead of streaking), naturally dried at room temperature for 4 hours, and then placed in a 30 ℃ air-drying oven for 1 hour to be air-dried, so that fetuin was fixed on the NC membrane as a test line. At 6 mm from the test line, the line was drawn again with 2mg/mL of the lectin ConA (or sprayed on an NC film as a coating method instead of drawing), and the line was naturally dried at room temperature for 4 hours, and then placed in a 30 ℃ air-drying oven for air-drying for 1 hour to solidify the lectin ConA to obtain a quality control line. The coating, drying and curing of the detection line and the quality control line can be carried out simultaneously.
(5) Assembling the test strip: the test strip consists of a sample pad 7, a gold label pad 6, a nitrocellulose membrane (NC membrane 5) with a detection line 2 and a quality control line 3, a water absorption pad 4 and a substrate (for example, a PVC plate 1); wherein, the water absorption pad 4 can adopt water absorption glass fiber to absorb redundant liquid in the detection sample; the sample pad 7 may be a glass fiber membrane, and contacts the sample to be detected, and the specific structure is shown in fig. 1.
On the PVC plate 1, each test paper part of the test paper strip is sequentially arranged along the chromatography direction, wherein the downstream end is a water absorption pad 4, the upstream end is a sample pad 7, the middle section takes an NC membrane 5 solidified with a detection line 2 and a quality control line 3 as a detection reaction area, and a gold label pad 6 adsorbed with a colloidal gold labeled lectin MAL-II is arranged between the NC membrane 5 and the sample pad 7.
The test paper is attached to the PVC plate 1 and cut into strip-shaped bands with the width of 4 mm; the method comprises the following steps of firstly attaching the NC film 5 to the PVC board 1, then attaching the water absorption pad 4 and the gold label pad 6 to the PVC board 1, respectively partially overlapping with two ends of the NC film 5, and finally attaching the sample pad 7 to the PVC board 1, and partially overlapping with the gold label pad 6. In addition, if the test paper is assembled in the card shell, a test paper card can be formed. The card shell comprises an upper plate and a lower plate, the upper plate is provided with a detection window and a sample adding hole, the sample pad 7 is opposite to the sample adding hole, and the detection reaction area is opposite to the detection window. The detection line 2 solidified on the NC film 5 is closer to the gold label pad 6, and the surface of the test strip (card) is provided with letters 'T' and 'C' as symbols for identifying the detection line and the quality control line, so that the detection line and the quality control line are convenient to distinguish; the test paper strip (card) is stored under a sealed and dry condition for later use.
(II) preparation of II-type test strip for detecting human influenza susceptible population
(1) Preparing colloidal gold: mixing chloroauric acid (HAuCl)4) Preparing a 1% chloroauric acid solution, adding 1mL of the chloroauric acid solution into 99mL of deionized water, heating to boil, immediately adding 2mL of the 1% sodium citrate solution, rapidly and uniformly stirring, continuously heating for 10 minutes, cooling to room temperature, and supplementing deionized water to 100mL to obtain a colloidal gold solution.
(2) Preparation of SNA-colloidal gold label: taking 10mL of the prepared colloidal gold solution, adjusting the pH value to 5.8-6.2 (for example, 6.0) by using 0.1mol/L potassium carbonate solution, adding 120 mu g of lectin SNA, uniformly mixing, reacting at room temperature for 10 minutes, centrifuging at 4 ℃ and 2000rpm for 20 minutes, and discarding the precipitate. The supernatant was centrifuged at 12000rpm at 4 ℃ for 60 minutes, the supernatant was discarded, and the precipitate was dissolved in 1mL of 0.01mol/L PBS (containing 0.2% BSA) having pH8.0 to obtain a solution of SNA-colloidal gold-labeled substance.
(3) Preparing a gold label pad: the SNA-colloidal gold-labeled solution is diluted 1-2 times (for example, by 1-fold dilution, the SNA concentration is about 60. mu.g/mL) with 0.01mol/L PBS (containing 0.2% BSA) having a pH of 8.0, and the concentration is adjusted to 100. mu.L/cm2Uniformly spraying the mixture on a glass fiber membrane, and drying the glass fiber membrane for 24 hours at 40 ℃ under the humidity of 30% to obtain the gold label pad. The gold label pad is sealed by a tin foil bag, is filled with a drying agent and is stored at normal temperature.
(4) Coating of detection line and quality control line: fetuin was diluted to a concentration of 2mg/mL with 0.01mol/L PBS (pH7.2), streaked on an NC membrane (or sprayed on an NC membrane as a coating method instead of streaking), naturally dried at room temperature for 4 hours, and then placed in a 30 ℃ air-drying oven for 1 hour to cure fetuin on the NC membrane as a detection line. At 6 mm from the test line, a line was drawn again with 2mg/mL lectin STL (or sprayed on an NC film as a coating method instead of drawing), dried naturally at room temperature for 4 hours, and then placed in a 30 ℃ air-drying oven for air-drying for 1 hour to solidify the lectin STL, thereby obtaining a quality control line. The spraying, drying and curing of the detection line and the quality control line can be carried out simultaneously.
(5) Assembling the test strip: the test strip consists of a sample pad 7, a gold label pad 6, a nitrocellulose membrane (NC membrane 5) with a detection line 2 and a quality control line 3, a water absorption pad 4 and a substrate (for example, a PVC plate 1); wherein, the water absorption pad 4 can adopt water absorption glass fiber to absorb redundant liquid in the detection sample; the sample pad 7 may be a glass fiber membrane, and contacts the sample to be detected, and the specific structure is shown in fig. 1.
On the PVC plate 1, each test paper part of the test paper strip is sequentially arranged along the chromatography direction, wherein the downstream end is a water absorption pad 4, the upstream end is a sample pad 7, the middle section takes an NC membrane 5 solidified with a detection line 2 and a quality control line 3 as a detection reaction area, and a gold-labeled pad 6 adsorbed with the colloidal gold-labeled lectin SNA is arranged between the NC membrane 5 and the sample pad 7.
The test paper is attached to the PVC plate 1 and cut into strip-shaped bands with the width of 4 mm; the method comprises the following steps of firstly attaching the NC film 5 to the PVC board 1, then attaching the water absorption pad 4 and the gold label pad 6 to the PVC board 1, respectively partially overlapping with two ends of the NC film 5, and finally attaching the sample pad 7 to the PVC board 1, and partially overlapping with the gold label pad 6. In addition, if the test paper is assembled in the card shell, a test paper card can be formed. The card shell comprises an upper plate and a lower plate, the upper plate is provided with a detection window and a sample adding hole, the sample pad 7 is opposite to the sample adding hole, and the detection reaction area is opposite to the detection window. The detection line 2 solidified on the NC film 5 is closer to the gold label pad 6, and the surface of the test strip (card) is provided with letters 'T' and 'C' as symbols for identifying the detection line and the quality control line, so that the detection line and the quality control line are convenient to distinguish; the test paper strip (card) is stored under a sealed and dry condition for later use.
(III) principle of detection
A test paper strip (card) for detecting avian influenza susceptible population, wherein a colloidal gold labeled pad (i.e., gold labeled pad) adsorbs a MAL-II-colloidal gold marker, a detection line is coated with fetuin, a quality control line is coated with lectin ConA capable of binding to lectin MAL-II, neither the detection line nor the quality control line shows color before any sample to be detected is provided, when the sample is added to the sample pad, SA α 2-3Gal sugar chains in the sample are chromatographically migrated to a detection reaction zone together with the MAL-II-colloidal gold marker, since SA α 2-3Gal sugar chains in the sample and SA α 2-3Gal sugar chains in fetuin are competitively bound to the MAL-II-colloidal gold marker, when the SA α 2-3Gal sugar chain concentration in the sample exceeds a certain amount, the MAL-II-gold marker cannot bind to fetuin in the detection line, no mared line appears in the position, or when SA α 2-3Gal sugar chains in the sample exceed a certain amount, the MAL-II-gold marker does not bind to the detection line, and whether SA α 2-3Gal sugar chains in the detection line are bound to the detection line is lower than a certain amount, and the SA-Gal sugar chains in the detection line shows that the detection line is effectively bound to the detection line.
A test strip (card) for detecting a human influenza susceptible population, wherein a colloidal gold labeled pad (i.e., gold labeled pad) adsorbs an SNA-colloidal gold label, a detection line is coated with fetuin, a quality control line is coated with lectin STL capable of binding to lectin SNA, neither the detection line nor the quality control line shows color before any sample is supplied, SA α 2-6Gal sugar chains in the sample are chromatographically migrated to a detection reaction region together with the SNA-colloidal gold label when the sample is added to the sample pad, no purplish red line appears at a position where SA α 2-6Gal sugar chains in the sample and SA α 2-6Gal sugar chains in fetuin compete with the SNA-colloidal gold label, and no purplish red line appears when the SA α 2-6Gal sugar chains in the sample exceed a certain amount, SA α 2-6Gal sugar chains in the sample are not bound to fetuin in the detection line, and no matter if SA α 2-6Gal sugar chains in the sample are bound to the detection line, a red line appears at a position where SA α 2-6Gal sugar chains in the detection line is less than a certain amount, the detection line, and a red line shows effective detection result that no purplish red line appears in the detection line.
the fetuin is bovine fetuin which comprises 12 cysteine residues and mainly comprises 74% of polypeptide, 8.3% of hexose, 5.5% of hexosamine, and 8.7% of sialic acid, wherein the content of sialic acid linked to SA α 2-3Gal sugar chain is 4.8% and the content of sialic acid linked to SA α 2-6Gal sugar chain is 2.8%, and the fetuin can be used for preparing a detection line in a test strip (card) designed because it contains a quantitative sugar chain.
(IV) false positive rate and false negative rate test experiment of test paper strip (card) for detecting avian influenza and human influenza susceptible population
4.1, saliva sample collection and pretreatment: in 50 healthy people, two hours after meal, between about 9 and 10 o' clock, after three rinses with physiological saline, naturally secreted whole saliva, at least 1mL each, was collected quickly and immediately placed on ice, and protease inhibitors (10 μ L per mL of saliva) were added to prevent protein degradation. Combining the collected saliva samples (50 cases of mixing), centrifuging at 12000rpm at 4 deg.C for 1h (removing saliva residues and exfoliated cells), and collecting supernatant; the centrifuged saliva sample supernatant can be used as a sample to be assayed (4 ℃) by adding a Protease Inhibitor (1. mu.L per 10 mL), for example, a Protease Inhibitor Cocktail from SIGMA.
4.2, removal of SA α 2-6Gal sugar chains and SA α 2-3Gal sugar chains in the sample:
1. preparation of lectin magnetic microparticle complexes
50 2mL centrifuge tubes were placed on a magnetic separation rack, and 3mg of each epoxidized magnetic microparticle (epoxidized magnet was suspended in an aqueous solution and stored at about 14mg/mL) was pipetted into the centrifuge tubes. And (4) magnetically separating, and discarding the supernatant after the supernatant is clear. Adding boric acid borax coupling buffer solution (5 mL, 0.05mol/L borax solution, 45mL0.2mol/L boric acid solution and 0.15mol/L NaCl) 1mL respectively, adjusting pH to 7.4 with hydrochloric acid or sodium hydroxide, oscillating, mixing, washing magnetic particles on a magnetic separation frame, performing magnetic separation for 3min, and removing supernatant after supernatant is clear. Adding 600 mu L of SNA/MAL-II solution (1mg/mL, solvent is coupling buffer) into the washed epoxidized magnetic particles, uniformly mixing, placing on a constant temperature oscillator, carrying out oscillation reaction for 3h at 25 ℃ and 180rpm, carrying out magnetic separation for 3min after the coupling reaction is finished, and removing supernatant. 1mL of the blocking solution (aqueous solution containing 2% ethanolamine and 0.1% BSA) was added to each of the centrifuge tubes, and the reaction was carried out at 25 ℃ and 180rpm for 1 hour with shaking. And after the sealing is finished, placing the mixture on a magnetic separation frame, and after magnetic separation, removing the supernatant to respectively obtain the SNA magnetic particle compound and the MAL-II magnetic particle compound.
2. removal of alpha 2-6-linked terminal sialylated sugar chain
and (3) adding the saliva sample treated in the step (4.1) into the prepared SNA magnetic particle compound, adding 700 mu L (saliva) into each tube, carrying out oscillation reaction for 3 hours at the temperature of 25 ℃ and the speed of 180rpm, taking out the saliva sample after the reaction is finished, placing the saliva sample on a magnetic separation frame for separation, and collecting supernatant to obtain the saliva sample with SA α 2-6Gal sugar chains removed.
3. removal of alpha 2-3-linked terminal sialylated sugar chain
and adding the saliva sample with the SA α 2-6Gal sugar chains removed into the prepared MAL-II magnetic particle compound, carrying out oscillation reaction for 3 hours at 25 ℃ and 180rpm, taking out the saliva sample after the reaction is finished, placing the saliva sample on a magnetic separation frame for separation, and collecting supernatant to obtain the saliva sample with the SA α 2-6Gal sugar chains and the SA α 2-3Gal sugar chains removed.
4.3 false Positive test
1. I type test paper strip (card) for detecting avian influenza susceptible population
(1) 5mL of the treated saliva sample (excluding SA α 2-6Gal sugar chains and SA α 2-3Gal sugar chains) was taken.
(2) 100 mu L of saliva sample and 100 mu L of deionized water are dropped into a sample pad of a type I test paper strip (card) for detecting the susceptible population of avian influenza, and the result is observed within 50 cases in 5 minutes to judge whether the sample contains SA α 2-3Gal sugar chains.
The judgment is based on the following:
negative (none): the detection line and the quality control line are purple red;
positive (with): only the quality control line appears purple red;
and (4) invalidation: if the quality control line does not show purple red, the detection result of the test strip (card) is judged to be invalid no matter whether the detection line shows purple red or not;
the judgment result is as follows: the saliva sample is positive in 1 part, negative in 49 parts, and all effective, the false positive rate is 2%, and the false positive rate can be controlled below 1% along with the increase of sample cases.
2. II type test paper strip (card) for detecting human influenza susceptible population
(1) 5mL of the treated saliva sample (excluding SA α 2-6Gal sugar chains and SA α 2-3Gal sugar chains) was taken.
(2) 100 mu L of saliva sample and 100 mu L of deionized water are dropped into a sample pad of a II type test paper strip (card) for detecting people susceptible to human influenza, and the result is observed within 50 cases in 5 minutes to judge whether the sample contains SA α 2-6Gal sugar chain.
The judgment is based on the following:
negative (none): the detection line and the quality control line are purple red;
positive (with): only the quality control line appears purple red;
and (4) invalidation: if the quality control line does not show purple red, the test result of the test strip (card) is judged to be invalid no matter whether the test line shows purple red or not;
the judgment result is as follows: the saliva sample is positive in 2 parts and negative in 48 parts, all the saliva samples are effective, the false positive rate is 4%, and the false positive rate can be controlled to be below 1% along with the increase of sample cases.
4.4 false negative test
1. Sample preparation:
10mL of saliva sample from which SA α 2-6Gal sugar chains and SA α 2-3Gal sugar chains were removed was taken, and 16.4mg of fetuin was added to the saliva sample, and the mixture was dissolved and mixed to prepare a test sample.
2. I type test paper strip (card) for detecting avian influenza susceptible population
(1) 5mL of the prepared test sample was taken.
(2) 100 mu L of test sample and 100 mu L of deionized water are dropped into a sample pad of a type I test paper strip (card) for detecting the susceptible population of avian influenza, and the result is observed within 50 cases in 5 minutes to judge whether the sample contains SA α 2-3Gal sugar chain.
The judgment is based on the following:
negative (none): the detection line and the quality control line are purple red;
positive (with): only the quality control line appears purple red;
and (4) invalidation: if the quality control line does not show purple red, the detection result of the test strip (card) is judged to be invalid no matter whether the detection line shows purple red or not;
the judgment result is as follows: 1 part of saliva samples are negative, 49 parts are positive, all the saliva samples are effective, the false negative rate is 2%, and the false negative rate can be controlled to be below 1% along with the increase of sample cases.
3. II type test paper strip (card) for detecting human influenza susceptible population
(1) 5mL of the prepared test sample was taken.
(2) 100 mu L of test sample and 100 mu L of deionized water are dropped into a sample pad of a II type test strip (card) for detecting the susceptible population of human influenza, and the result is observed within 50 cases in 5 minutes to judge whether the sample contains SA α 2-6Gal sugar chain.
The judgment is based on the following:
negative (none): the detection line and the quality control line are purple red;
positive (with): only the quality control line appears purple red;
and (4) invalidation: if the quality control line does not show purple red, the detection result of the test strip (card) is judged to be invalid no matter whether the detection line shows purple red or not;
the judgment result is as follows: 1 part of saliva samples are negative, 49 parts are positive, all the saliva samples are effective, the false negative rate is 2%, and the false negative rate can be controlled to be below 1% along with the increase of sample cases.
Claims (10)
1. A test paper for rapidly detecting influenza susceptible population by using a competition method is characterized in that: the test paper comprises I-type test paper for detecting avian influenza susceptible population and/or II-type test paper for detecting human influenza susceptible population, wherein the I-type test paper and the II-type test paper comprise a sample pad (7), a colloidal gold labeling pad, a detection reaction zone and a water absorption pad (4) which are sequentially arranged along a chromatography direction, the detection reaction zone comprises a detection line (2) and a quality control line (3), the colloidal gold labeling pad of the I-type test paper comprises lectin MAL-II for labeling colloidal gold, the colloidal gold labeling pad of the II-type test paper comprises lectin SNA for labeling colloidal gold, the quality control line (3) of the I-type test paper comprises lectin ConA, the quality control line (3) of the II-type test paper comprises lectin STL, and the detection line (2) of the I-type test paper and the II-type test paper comprises fetuin.
2. The test paper for rapidly detecting influenza susceptible population by using a competition method according to claim 1, which is characterized in that: the colloidal gold labeled pad is prepared by coating and drying the lectin MAL-II labeled with colloidal gold or the lectin SNA labeled with colloidal gold on a glass fiber membrane or a nitrocellulose membrane; the detection line (2) is prepared by coating and drying and curing fetuin on a nitrocellulose membrane; the control line (3) is made by coating and drying and curing lectin ConA or STL on a nitrocellulose membrane.
3. The test paper for rapidly detecting influenza susceptible population by using a competition method according to claim 2, which is characterized in that: the coating of the colloidal gold-labeled lectin MAL-II or the colloidal gold-labeled lectin SNA specifically means that the colloidal gold-labeled lectin MAL-II or the colloidal gold-labeled lectin SNA with the concentration of 30-60 mu g/mL is sprayed on a glass fiber membrane or a nitrocellulose membrane; the coating of the fetuin specifically refers to scribing or spraying the fetuin with the concentration of 1-4 mg/mL at the detection line position of the nitrocellulose membrane, and the coating of the lectin ConA or STL specifically refers to scribing or spraying the lectin ConA or STL with the concentration of 1-4 mg/mL at the quality control line position of the nitrocellulose membrane.
4. The test paper for rapidly detecting influenza susceptible population by using a competition method according to claim 1, which is characterized in that: the test paper is fixed on a substrate or arranged in a card shell with a window.
5. The method for preparing the test paper for rapidly detecting influenza susceptible population by using the competition method according to claim 1, which is characterized in that: the method comprises the following steps:
1) coating the lectin MAL-II for labeling the colloidal gold on a glass fiber membrane or a nitrocellulose membrane and then drying to obtain a colloidal gold labeling pad;
2) coating fetuin and lectin ConA on a nitrocellulose membrane, and drying to obtain a detection line (2) and a quality control line (3);
3) assembling a sample pad (7), a colloidal gold labeled pad, a nitrocellulose membrane containing a detection line (2) and a quality control line (3) and a water absorption pad (4) into I-type test paper for detecting avian influenza susceptible people;
or,
1) coating the lectin SNA for marking the colloidal gold on a glass fiber membrane or a nitrocellulose membrane and then drying to obtain a colloidal gold marking pad;
2) coating fetuin and lectin STL on a nitrocellulose membrane, and drying to obtain a detection line (2) and a quality control line (3);
3) and assembling the sample pad (7), the colloidal gold labeled pad, the nitrocellulose membrane containing the detection line (2) and the quality control line (3) and the water absorption pad (4) into II-type test paper for detecting the susceptible population of human influenza.
6. The method of claim 5, wherein: in the step 1), the lectin MAL-II labeled with colloidal gold or the lectin SNA labeled with colloidal gold with the concentration of 30-60 mu g/mL is mixed according to the concentration of 50-200 mu L/cm2Coating on a glass fiber membrane or a nitrocellulose membrane; in the step 2), fetuin and agglutinin ConA or STL with the concentration of 1-4 mg/mL are coated on the nitrocellulose membrane.
7. A method for rapidly detecting a sialylated sugar chain by a competition method, characterized in that: the method comprises the following steps:
1) pretreating the collected detection sample to prevent the degradation of protein in the detection sample to obtain a detection sample;
2) adding a detection sample or a diluted detection sample to I-type and/or II-type test paper, wherein the I-type test paper and the II-type test paper comprise a sample pad (7), a colloidal gold labeling pad, a detection reaction area and a water absorption pad (4) which are sequentially arranged along a chromatography direction, the detection reaction area comprises a detection line (2) and a quality control line (3), the colloidal gold labeling pad of the I-type test paper comprises lectin MAL-II for labeling colloidal gold, the colloidal gold labeling pad of the II-type test paper comprises lectin SNA for labeling colloidal gold, the quality control line (3) of the I-type test paper comprises lectin ConA, the quality control line (3) of the II-type test paper comprises lectin STL, and the detection line (2) of the I-type and II-type test paper comprises fetuin;
3) the fetuin on the type I and/or type II test paper and the sialyl sugar chain with the SA α 2-3Gal structure at the end or the sialyl sugar chain with the SA α 2-6Gal structure at the end in the test sample are combined with the lectin MAL-II or SNA of the labeled colloidal gold on the corresponding test paper in a competitive manner to form a test result, and the test result is judged as follows:
if the detection line (2) and the quality control line (3) are both colored, determining that the sialylated sugar chain of which the tail end has the SA α 2-3Gal structure is negative or low in content in the detection sample for the type I test paper, and determining that the sialylated sugar chain of which the tail end has the SA α 2-6Gal structure is negative or low in content in the detection sample for the type II test paper;
if only the quality control line (3) is developed, judging that the sialylated sugar chain of which the tail end has the SA α 2-3Gal structure is positive or has higher content in the test sample for the I-type test paper, and judging that the sialylated sugar chain of which the tail end has the SA α 2-6Gal structure is positive or has higher content in the test sample for the II-type test paper;
if the quality control line (3) does not develop color, the corresponding test paper is judged to be invalid.
8. The method for rapidly detecting a sialylated sugar chain by a competition method according to claim 7, wherein: the detection sample is selected from saliva or nasal secretion; the test sample is diluted by deionized water, injection water, normal saline, phosphate buffer solution or borate buffer solution with the dilution multiple of 1-5 times.
9. The method for rapidly detecting a sialylated sugar chain by a competition method according to claim 7, wherein: the pretreatment comprises the following steps: and adding a protease inhibitor into the collected detection sample, centrifuging, and adding the protease inhibitor into the supernatant obtained by centrifuging again to obtain the detection sample.
10. Use of a strip according to claim 1 in the preparation of a reagent for the rapid detection of influenza susceptible populations.
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