H5N1 type highly pathogenic avian influenza nanometer quantum point detection method

A nano-quantum dot, H5N1 technology, applied in the field of inspection and quarantine, can solve the problems of unfavorable rapid diagnosis and immunization procedures, difficulty in large-scale detection, and high sensitivity, achieve good promotion and application prospects, facilitate high-throughput detection, conditions less demanding effects

Inactive Publication Date: 2013-05-08
BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. Virus isolation and identification is a reliable method for diagnosing AI, but virus isolation and culture takes a long time, at least 7 days, which is not conducive to rapid diagnosis and rational formulation of immunization procedures, and is difficult to meet the needs of rapid diagnosis of highly pathogenic AI
[0006] 2. Agar diffusion (AGP) test is used for virus antigen specificity test. Although this method is simple and easy to implement, it has poor sensitivity and is prone to false positives.
Neutralization test (NT) is a relatively sensitive and specific serological method, but its operation is cumbersome, time-consuming and material-consuming, and it is hardly used in clinical practice.
[0007] 3. ELISA is a relatively mature method developed in recent years, with high sensitivity. The disadvantage is that the sensitivity of the test is greatly affected by the quality of the sample.
[0008] 4. The RT-PCR method is a sensitive and specific method, and its sensitivity is higher than that of ELISA. Due to the high technical requirements, the field samples are complex and changeable, the interference is large, and the sample processing is difficult, so large-scale detection is difficult.

Method used

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  • H5N1 type highly pathogenic avian influenza nanometer quantum point detection method
  • H5N1 type highly pathogenic avian influenza nanometer quantum point detection method
  • H5N1 type highly pathogenic avian influenza nanometer quantum point detection method

Examples

Experimental program
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Effect test

preparation example Construction

[0062] 2. Preparation of Quantum Dots

[0063] Tri-n-octyl phosphorus oxide (TOPO, 90%, Aldrich), tri-n-butyl phosphorus (TBP, 90%, TCI, Japan), octadecylamine (ODA, 90%, ACROS), octadecene (ODE, 90 %, ACROS), elemental sulfur (S, 99.9% Aldrich), oleic acid (OA, 90%, Aldrich), cadmium oxide (CdO, 99.5%, Aldrich) and Se powder (100mesh, 99.99%, Aldrich), toluene, Ethanol, methanol, n-hexane, chloroform, dichloromethane and acetone were all of analytical grade and purchased from Tianjin University Kewei Reagent Company.

[0064] UV-2450 ultraviolet-visible spectrophotometer (Shimadzu Corporation), P JEM-100CX II transmission electron microscope (erkin Elmer Inc.), F-4500 fluorescence spectrophotometer (Hitachi), G2F20 transmission electron microscope (Tecnai), SHJM-1 digital display constant temperature stirring electric heating mantle (Juancheng Fuli Scientific Research Instrument Factory, Shandong Province), Hettich Mikro22R refrigerated centrifuge (JEOL company), electronic ...

Embodiment 1

[0071] Embodiment 1: Propagation, purification and concentration of virus

[0072] 1. Reproduction of the virus

[0073] Take AIV H5N1 virus, make 1×10 4 Double dilution, inoculate 10-day-old chicken embryos in the allantoic cavity, 0.2ml / piece, culture at 37°C, illuminate eggs 3 times a day, eliminate dead embryos that died within 24 hours, and collect chicken embryos that did not die within 24-96 hours. Cool at 4°C and collect the allantoic fluid aseptically.

[0074] 2. Virus inactivation and purification

[0075] (1) Inactivation: Formaldehyde was added to the collected allantoic fluid to make the final concentration 0.1%, and placed in a biochemical incubator at 37°C for inactivation.

[0076] (2) Differential centrifugation at 4000 rpm, 45 minutes at 4°C, and discard the precipitate. The supernatant was ultracentrifuged at 100,000×g at 4°C for 2 hours, the precipitate was collected, the virus was resuspended in PBS (pH 7.4) with 1 / 20 volume of the original allantoic ...

Embodiment 2

[0083] Example 2: Preparation and Purification of H5N1 Type Avian Influenza Monoclonal Antibody

[0084] 1. Mouse Immunization Procedure

[0085] The virus purified and concentrated in Example 1 was used as the immune antigen to immunize 8-week-old Balb / c mice. A total of four immunizations were carried out. The first immunization was fully emulsified with the same amount of Freund's adjuvant, the second and third immunizations were added with the same amount of Freund's incomplete adjuvant, and the fourth was without adjuvant. Each immunization interval was 2 weeks. The virus dose for each immunization was 150 μg per mouse, and the immunization route was multi-point injection under the back of the neck. Two weeks after the fourth immunization, blood was collected from the tail feeder to measure the HA value of the serum. If the serum titer was reasonable, a booster immunization was carried out three days before the fusion, and the virus antigen without adjuvant was injected...

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Abstract

The invention discloses a method for detecting a nano quantum dot of the highly pathogenic bird flu of H5N1, belonging to the disease inspection field. The method comprises the following steps that: (1) a monoclonal antibody or a polyclonal antibody of the bird flu of H5N1 is prepared and purified; (2) a water-solubility carboxylation nano quantum dot is prepared; (3) the nano quantum dot is usedfor marking the monoclonal antibody or the polyclonal antibody of the bird flu of H5N1; and (4) the sample is detected. The method has the advantages that the nano quantum dot is applied to the diagnosis of the highly pathogenic bird flu of H5N1, the method has high speed, stability, convenient detection of high flux, low condition requirement, simple and convenient operation and easy popularization; the method has the characteristics of simple operation, high speed and high sensitivity, etc. The method provides an effective way to detect and diagnose the highly pathogenic bird flu of H5N1 inChina and has good popularization and application potential in the aspect of quarantine inspection in the process of import and export.

Description

technical field [0001] The invention relates to a nano-quantum dot detection method for H5N1 highly pathogenic avian influenza, more specifically, a method for detecting monoclonal or polyclonal antibodies to avian influenza virus labeled with quantum dots prepared by ultrasonic emulsification modification, which belongs to inspection and quarantine field. Background technique [0002] Avian influenza (abbreviated as avian influenza, avian influenza, A I) is a severe infectious disease of poultry caused by type A influenza virus, and is designated as a type A infectious disease by the International Office of Epizootics, of which H5N1 subtype avian influenza is the most severe outbreak in recent years Frequent occurrence of a highly pathogenic avian influenza has brought huge economic losses to the poultry industry and threatened human life and health. As cases of human infection with avian influenza have also been discovered in our country, the prevention and control of hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/533G01N33/569
Inventor 马贵平常津刘旭辉张兵波李冰玲史喜菊李炎鑫刘全国
Owner BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU INSPECTION & QUARANTINE TECH CENT
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