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Method for detecting H5N1 influenza A virus hemagglutinin

An influenza A virus and hemagglutinin technology, which is applied in the field of biosensing, can solve the problems of low biosafety, complex reaction system, complicated operation steps, etc., and achieves improved detection sensitivity, wide fluorescence absorption range, and reduced background signal. Effect

Active Publication Date: 2021-01-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Isolation and cultivation of influenza virus is a traditional etiological diagnosis technique. This technique is considered to be the most classic and rigorous method for identifying influenza virus. This process usually takes 2 to 14 days. The disadvantages are cumbersome operation steps, long cycle time and biosafety. lower sex
The serological detection method has a low antibody titer in the early stage of the disease, and the antibody titer can be significantly increased after two weeks. This method can only be applied to epidemiological investigation and research, and cannot be used as a method for early diagnosis
Enzyme-linked immunosorbent assay is a detection technology that uses enzyme-labeled antigens or antibodies to detect antibodies or antigens through chromogenic reaction. The detection results are easy to analyze, safe and efficient, and low in cost, but false positive results may occur, and the serum cannot be distinguished Subtype
With the development of science and technology, molecular biology methods based on isothermal amplification technology can be applied to rapid on-site detection and screening of influenza virus. Problems such as complex system and strict reagent storage conditions

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  • Method for detecting H5N1 influenza A virus hemagglutinin
  • Method for detecting H5N1 influenza A virus hemagglutinin
  • Method for detecting H5N1 influenza A virus hemagglutinin

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Embodiment 1

[0032] The method for detecting H5N1 influenza A virus hemagglutinin may further comprise the steps:

[0033] (1) Using the surface ligand exchange method, polyacrylic acid is used for water-soluble modification of rare earth-doped upconversion luminescent nanoparticles:

[0034] a Preparation of rare earth-doped upconversion luminescent nanoparticles:

[0035] Weigh 2mmol CF 3 COONa and 1.56mmolY(CF 3 COO) 3 , 0.4mmolYb (CF 3 COO) 3 , 0.04mmolEr(CF 3 COO) 3 Added to a three-necked flask, added 10mmol oleic acid, 10mmol oleylamine, 20mmol octadecene, heated to 100°C, stirred for 30min under vacuum; heated to 300°C, kept in nitrogen for 1 hour; cooled to room temperature, added 25ml of ethanol, centrifuged at 8000rpm for 10 minutes to obtain a precipitate of rare earth-doped upconversion luminescent nanoparticles (particle diameter: 17-20nm) (upconversion luminescent nanoparticles before modification), and disperse the precipitate in 15ml of cyclohexane;

[0036] b Wat...

Embodiment 2

[0048] The method for detecting H5N1 influenza A virus hemagglutinin may further comprise the steps:

[0049] Step (1) and (2) are with the step (1) and (2) of embodiment 1;

[0050] (3) Take 100 μg up-converting fluorescent probe and disperse it in 100 μl, 10 mM borate buffer solution with pH=7.8; obtain the dispersion, prepare 10 dispersions in total; add different concentrations of H5N1 HA standard (0, 1 . Graphene (the sheet diameter of graphene oxide is 0.5nm), incubate at 20°C for 20 minutes, set the volume to 300 μl with borate buffer solution of pH=7.8, measure the fluorescence value F of 542nm emission wavelength when 980nm excitation wavelength respectively, simultaneously Determination of blank fluorescence value F 0 , make the relative fluorescence intensity [(F-F 0 ) / F 0 ] Standard curve corresponding to H5N1 HA concentration;

[0051] (4) Take 100 μg up-converting fluorescent probe and disperse it in 100 μl, 10 mM borate buffer solution with pH=7.8, add 20 μ...

Embodiment 3

[0053] The method for detecting H5N1 influenza A virus hemagglutinin may further comprise the steps:

[0054] Step (1) and (2) are with the step (1) and (2) of embodiment 1;

[0055] (3) Disperse 200 μg of the up-converting fluorescent probe in 200 μl, 10 mM borate buffer solution with pH=7.8; obtain the dispersion, and prepare 10 dispersions; add different concentrations of H5N1 HA standard (0, 1 . Graphene (diameter of graphene oxide is 2 μm), incubate at 40°C for 20 minutes, dilute to 300 μl with borate buffer solution with pH = 7.8, respectively measure the fluorescence value F of 542nm emission wavelength at 980nm excitation wavelength, and simultaneously measure Blank fluorescence value F 0 , make the relative fluorescence intensity [(F-F 0 ) / F 0 ] Standard curve corresponding to H5N1 HA concentration;

[0056] (4) Take 200 μg up-converting fluorescent probe and disperse it in 200 μl, 10 mM borate buffer solution with pH=7.8, add 20 μl of human serum sample containi...

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Abstract

The invention discloses a method for detecting H5N1 influenza A virus hemagglutinin. The method comprises the following steps: carrying out water-soluble modification on rare earth doped up-conversionluminescence nanoparticles; coupling an H5N1 HA aptamer on the surfaces of the nanoparticles to obtain an up-conversion fluorescent probe; drawing a standard curve; preparing a detection system of ato-be-detected sample, and determining the relative fluorescence intensity of the to-be-detected sample; and acquiring the concentration of H5N1 HA in the to-be-detected sample according to the standard curve. The detection method is rapid, simple, convenient and stable in performance, the space structure of the original nucleic acid aptamer is changed through the specific recognition effect, andrecovery of fluorescence signals of a detection system is achieved. The fluorescence receptor graphene oxide is wide in fluorescence absorption range, and green up-conversion fluorescence signal quenching can be realized. The detection system can greatly reduce background signals and improve detection sensitivity. The homogeneous wash-free detection system can realize efficient detection of the H5N1 influenza A virus.

Description

technical field [0001] The invention belongs to the technical field of biosensing, and in particular relates to a method for detecting H5N1 influenza A virus hemagglutinin. Background technique [0002] Influenza A virus is an important pathogen that causes seasonal influenza, and its frequent outbreaks pose a huge challenge to global public health. Influenza viruses usually acquire new antigenicity, that is, antigenic shift, which leads to the emergence of new virus strains, and the general lack of corresponding immunity in the population, resulting in influenza pandemics; among them, the hemagglutinin HA of influenza viruses Binding to specific sialosaccharide receptors on the host surface plays a key role in the pathogenicity of the virus, so HA is suitable as a target for detection. Rapid and accurate diagnosis of influenza virus infection is crucial to effectively control the epidemic and reduce morbidity and mortality. [0003] In order to achieve effective preventio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486G01N21/6428
Inventor 王涛赵秋子孙聆东王晓勇杜平王志云
Owner TIANJIN UNIV
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