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A method for detecting h5n1 influenza A virus hemagglutinin

A technology of influenza A virus and hemagglutinin, which is applied in the field of biosensing, can solve the problems of complex reaction system, low biological safety, and low antibody titer, and achieve a wide range of fluorescence absorption, improved detection sensitivity, and high specificity The effect of sex detection

Active Publication Date: 2022-08-05
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

Method used

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  • A method for detecting h5n1 influenza A virus hemagglutinin
  • A method for detecting h5n1 influenza A virus hemagglutinin
  • A method for detecting h5n1 influenza A virus hemagglutinin

Examples

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Embodiment 1

[0032] A method for detecting H5N1 influenza A virus hemagglutinin, comprising the following steps:

[0033] (1) Water-soluble modification of rare earth-doped upconversion luminescent nanoparticles using polyacrylic acid by surface ligand exchange method:

[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 Put it into a three-necked flask, add 10mmol oleic acid, 10mmol oleylamine, 20mmol octadecene, heat to 100°C, stir under vacuum for 30min; heat to 300°C, keep under nitrogen for 1 hour; cool to room temperature, add 25 ml of ethanol, centrifuged at 8000 rpm for 10 minutes to obtain a precipitate of rare earth-doped upconversion luminescent nanoparticles (particle diameter of 17-20 nm) (upconversion luminescent nanoparticles before modification), and the precipitate was dispersed in 15 ml of cyclohexane;

[0036] b Water-soluble m...

Embodiment 2

[0048] A method for detecting H5N1 influenza A virus hemagglutinin, comprising the following steps:

[0049] Steps (1) and (2) are the same as steps (1) and (2) of Example 1;

[0050] (3) Disperse 100 μg of the up-conversion fluorescent probe in 100 μl of borate buffer at 10 mM pH=7.8; obtain a dispersion, and prepare 10 dispersions in total; add different concentrations of H5N1 HA standard (0, 1 , 2, 5, 10, 20, 50, 80, 100, 150ng / ml) standard solution (solvent is 10mM, pH=7.4PBS), incubate at 20°C for 60 minutes, add the final concentration of 50μg / ml of oxidized Graphene (the sheet diameter of graphene oxide is 0.5 nm), incubate at 20°C for 20 minutes, dilute the volume to 300 μl with borate buffer with pH=7.8, and measure the fluorescence value F at the emission wavelength of 542 nm at the excitation wavelength of 980 nm. Determination of blank fluorescence value F 0 , the relative fluorescence intensity [(F-F 0 ) / F 0 ] standard curve corresponding to H5N1 HA concentrat...

Embodiment 3

[0053] A method for detecting H5N1 influenza A virus hemagglutinin, comprising the following steps:

[0054] Steps (1) and (2) are the same as steps (1) and (2) of Example 1;

[0055] (3) Disperse 200 μg of the up-conversion fluorescent probe in 200 μl of borate buffer at 10 mM pH=7.8; obtain a dispersion, and prepare 10 dispersions in total; add H5N1 HA standard products of different concentrations (0, 1 , 2, 5, 10, 20, 50, 80, 100, 150ng / ml) standard solution (solvent is 10mM, pH=7.4PBS), incubate at 40°C for 20 minutes, add the final concentration of 300μg / ml of oxidative Graphene (the sheet diameter of graphene oxide is 2 μm), incubate at 40°C for 20 minutes, dilute the volume to 300 μl with borate buffer with pH=7.8, and measure the fluorescence value F at the emission wavelength of 542 nm at the excitation wavelength of 980 nm, and simultaneously measure Blank fluorescence value F 0 , the relative fluorescence intensity [(F-F 0 ) / F 0 ] standard curve corresponding to...

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Abstract

The invention discloses a method for detecting H5N1 influenza A virus hemagglutinin. The steps include: water-soluble modification of rare earth-doped up-conversion luminescent nanoparticles; completing the coupling of H5N1 HA nucleic acid aptamer on the surface of the nanoparticles to obtain up-conversion Fluorescent probe; making a standard curve; preparing a detection system for the sample to be tested, and measuring the relative fluorescence intensity of the sample to be tested; obtaining the concentration of H5N1 HA in the sample to be tested according to the standard curve; the detection method of the present invention is fast, simple and stable in performance , Recognizing the original nucleic acid aptamer changes its own spatial structure through specific recognition, and realizes the recovery of the fluorescence signal of the detection system. The fluorescence acceptor graphene oxide has a wide fluorescence absorption range and can achieve green up-conversion fluorescence signal quenching. The detection system can greatly reduce the background signal and improve the detection sensitivity. The homogeneous wash-free detection system can realize the efficient detection of H5N1 influenza A virus.

Description

technical field [0001] The invention belongs to the technical field of biosensing, in particular to a method for detecting H5N1 influenza A virus hemagglutinin. Background technique [0002] Influenza A virus is an important pathogen causing seasonal influenza, and its frequent outbreaks have brought enormous challenges to global public health. Influenza viruses usually acquire new antigenicity, that is, antigenic transfer, which leads to the emergence of new virus strains, which generally lack corresponding immunity in the population, resulting in influenza pandemics; among them, the hemagglutinin HA of influenza viruses Binding to specific sialic sugar 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 essential to effectively control the epidemic and reduce morbidity and mortality. [0003] In order to achieve effective prevention and ...

Claims

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Application Information

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Patent Type & Authority Patents(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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