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WO2022088511A1 - Réactif pour la lyse d'un échantillon de virus - Google Patents

Réactif pour la lyse d'un échantillon de virus Download PDF

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Publication number
WO2022088511A1
WO2022088511A1 PCT/CN2021/071117 CN2021071117W WO2022088511A1 WO 2022088511 A1 WO2022088511 A1 WO 2022088511A1 CN 2021071117 W CN2021071117 W CN 2021071117W WO 2022088511 A1 WO2022088511 A1 WO 2022088511A1
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WO
WIPO (PCT)
Prior art keywords
reagent
virus
sample
titanium dioxide
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/071117
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English (en)
Chinese (zh)
Inventor
张敏
杨蓉
张权峰
宋学文
叶凯璐
周康
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vivachek Biotech (hangzhou) Co Ltd
Original Assignee
Vivachek Biotech (hangzhou) Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vivachek Biotech (hangzhou) Co Ltd filed Critical Vivachek Biotech (hangzhou) Co Ltd
Publication of WO2022088511A1 publication Critical patent/WO2022088511A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of disease diagnosis, and relates to a reagent for splitting virus samples, in particular to a reagent for splitting novel coronavirus samples.
  • the general immunochromatographic detection test paper includes a support layer, a sample pad, a marker pad, a reaction area, and an absorbent paper.
  • the sample pad, the marker pad, the reaction area, and the absorbent paper are overlapped on the support layer in turn, and the reaction area includes the quality control line (C line ) and the detection line (T line), when a certain amount of the sample to be tested is applied to the sample pad, due to capillary action, after being evenly dispersed through the sample pad, after the marking of the marking pad, in the reaction area, it is respectively connected with the C line and the C line of the reaction area.
  • the antigen or antibody on the T line reacts, and the remaining liquid after the reaction is absorbed by the absorbent paper.
  • the C line signal value tends to be stable, the result of the reaction with the antigen or antibody shows the strength of the T line signal value.
  • lysis When conducting virus detection, such as new coronavirus detection, it is necessary to lyse the collected samples on the nasal swab or throat swab to obtain the sample to be tested, and then apply it to the fluorescent immunochromatography test paper for reaction, lysis
  • the lysis effect of the solution on the component to be tested (virus) in the sample is directly reflected in the strength of the T-line signal value, so the formulation of the lysis solution is particularly important. Due to the poor lysis effect of the existing virus lysate, the detection sensitivity of the virus is affected, especially when the virus content is particularly low, it is very easy to cause false detection and missed detection.
  • the present invention provides a new lysis solution formulation, which contains nano-titanium dioxide, which can significantly improve the lysis effect of the components to be tested in the sample, thereby greatly improving the virus detection sensitivity, especially for novel The lysis of the coronavirus.
  • the present invention provides a virus-splitting reagent, comprising titanium dioxide, buffer, surfactant and preservative.
  • titanium dioxide is nano titanium dioxide.
  • the content of the nano titanium dioxide is 0.01%-0.1%.
  • the buffer solution includes sodium chloride, phosphate and sodium caseinate.
  • the buffer solution includes sodium chloride 0.15-0.45 mol/L, phosphate 1.15-2.9 g/L, and sodium caseinate content of 0.5%.
  • the surfactant is Triton X-100.
  • the preservative is Proclin 300.
  • the present invention also provides a fluorescence immunochromatography detection kit, which is characterized in that it includes the virus splitting reagent according to any one of claims 1 to 7, and an immune layer for detecting virus antigens Analysis test paper.
  • the present invention provides the use of nano-titanium dioxide for preparing a reagent for splitting virus samples.
  • virus sample is a novel coronavirus sample.
  • the beneficial effect of the present invention is that the virus can be better split through the splitting of the present invention, and more antigens or antigenic sites can be exposed, thereby improving the detection sensitivity.
  • the amount of virus in the sample is particularly low, it is hoped that a positive result can be obtained, and more antigen fragments or virus fragments are expected to be obtained.
  • the lysis solution provided by the present invention to lyse the sample can significantly increase the virus antigen concentration in the sample. Therefore, the immunofluorescence test strip is used to increase the minimum threshold of detection and prevent missed detection.
  • Fig. 1 is the T-line mean value level comparison diagram obtained for lysate formulations 1-6 in Example 8
  • the positive samples in the following examples are virus cultures with a virus titer of 1.51 ⁇ 10 6 TCID 50 /mL, diluted to 15,000 times, 10,000 times, 5,000 times, and 1,000 times, respectively, as samples to be tested.
  • a virus titer 1.51 ⁇ 10 6 TCID 50 /mL, diluted to 15,000 times, 10,000 times, 5,000 times, and 1,000 times, respectively, as samples to be tested.
  • the T line signal value is similar to the background value, and it is judged as negative.
  • the background value refers to the signal value detected when no sample is injected.
  • the preparation method of the fluorescence immunochromatography detection test paper used in the present invention is as follows, and the fluorescence immunochromatography detection test paper used in the present invention is the test paper produced in the same batch:
  • Step 1 Fluorescent microspheres are labeled with monoclonal antibody against NP protein of novel coronavirus.
  • sample pad treatment solution 0.01M pH 7.4 phosphate buffered saline solution, fixed mass fraction of BSA, surfactant, PVP K30.
  • Step 4 Dilute and spray the antibody marker
  • step 1 The novel coronavirus NP protein monoclonal antibody labeled with fluorescent microspheres in step 1 was sprayed on the glass fiber treated in step 3 at a speed of 3.0uL/cm with a gold sprayer, and placed in a drying oven at 37°C overnight. Dry, take out and place in an aluminum foil bag and seal it for later use;
  • Step 5 Scribing the nitrocellulose membrane
  • Embodiment 2 Lysis solution formula 1
  • Lysis solution 1 contains the following components by mass fraction: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.12g KH2PO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300.
  • the pH of the lysate was 8.0.
  • the new coronavirus positive culture concentration is 1/15000 of the original concentration
  • the T line has a signal
  • the line background signal that is, the T line signal when the culture concentration is When the result is negative.
  • the culture concentration was 1/10000, 1/5000 and 1/5000 of the original concentration, it was judged as a positive result.
  • Lysis solution 2 contains the following components in mass fractions: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300.
  • the pH of the lysate was 8.0.
  • Embodiment 4 Lysis solution formula 3
  • Lysis solution 3 contains the following components in mass fractions: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.12g KH2PO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.01% Nano Titanium Dioxide.
  • the pH of the lysate was 8.0.
  • Embodiment 5 Lysis solution formula 4
  • Lysis solution 4 contains the following components in mass fractions: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.02% nano-titanium dioxide.
  • the pH of the lysate was 8.0.
  • Embodiment 6 Lysis solution formula 5
  • Lysis solution 5 contains the following components in mass fractions: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.03% nano-titanium dioxide.
  • the pH of the lysate was 8.0.
  • Embodiment 7 Lysis solution formula 6
  • Lysis solution 6 contains the following components by mass fraction: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.05% nano-titanium dioxide.
  • the pH of the lysate was 8.0.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un réactif permettant de lyser un échantillon de virus. Le réactif contient du dioxyde de titane nanométrique, de sorte qu'un effet de lyse sur un composant à tester dans l'échantillon peut être manifestement amélioré, améliorant ainsi considérablement la sensibilité du test de virus, en particulier la lyse du SARS-CoV-2.
PCT/CN2021/071117 2020-10-29 2021-01-11 Réactif pour la lyse d'un échantillon de virus Ceased WO2022088511A1 (fr)

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CN202011178357.0A CN113281503B (zh) 2020-10-29 2020-10-29 一种用于裂解病毒样本的试剂
CN202011178357.0 2020-10-29

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