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Method for directly counting H9N2 avian influenza virus by naked eyes

An avian influenza virus, direct technology, applied in the field of pathogen detection, can solve the problems of limited sensitivity, high labor intensity, easy to be affected by subjective factors, etc., to achieve the effect of high sensitivity and simple operation

Pending Publication Date: 2019-08-30
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the virus transmission and isolation process is not only time-consuming, but also labor-intensive, which limits its rapid clinical diagnosis
In addition, the hemagglutination test (HA) used to determine positive results is not an identification test, and the judgment results are largely susceptible to subjective factors. It is used for antigen immunodetection, although its specificity is high, but its sensitivity is limited
Detection technologies related to PCR and fluorescent quantitative PCR require relatively expensive equipment, well-trained technicians, clean environment, and high sensitivity, but their use is limited in underdeveloped areas

Method used

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  • Method for directly counting H9N2 avian influenza virus by naked eyes
  • Method for directly counting H9N2 avian influenza virus by naked eyes
  • Method for directly counting H9N2 avian influenza virus by naked eyes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of M13@GNP probe and magnetic probe

[0039] 1. Cleaning of gold nanoparticles

[0040] Add 100 μL of commercially available gold nanoparticles with a diameter of 15 nm coated with protein G into 500 μL of PBS containing 1% BSA in a 1.5 mL centrifuge tube, mix well, put into an Eppendorf 5424 centrifuge, centrifuge at 10,000 rpm for 10 min, and discard the supernatant ; Then add 500 μL of PBS containing 1% BSA to the centrifuge tube, repeat the operation three times; add 100 μL of PBS containing 1% BSA again, oscillate ultrasonically, mix well, and set aside;

[0041] 2. Washing of Magnetic Beads

[0042] Add 10 μL of commercially available protein A-coated magnetic beads with a diameter of 200 nm into 500 μL of PBS containing 1% BSA in a 1.5 mL centrifuge tube, oscillate ultrasonically for 5 min, mix well, and put them on a magnetic separator until the magnetic particles are completely adsorbed. Discard the supernatant; add 500 μL of PBS contain...

Embodiment 2

[0049] Example 2 Capture of H9N2 Avian Influenza Virus by Magnetic Probe

[0050] Take 20 μL of mouse anti-HA antibody-coated magnetic probes and four different virus titers of 100 μL samples to be tested (concentrations are 5PFU / mL, 50PFU / mL, 500PFU / mL, 5000PFU / mL, named as sample 1, Sample 2, sample 3, sample 4), at the same time, the H1N1 virus with a concentration of 5000PFU / mL was used as a negative control, and PBS was used as a blank control to evaluate the specificity of the magnetic probe, and incubated for 1 hour in a Mixer with rotation and shaking at room temperature; after the completion of the binding reaction , put it into a magnetic separator, after the adsorption is complete, add 500 μL of PBS to wash the probe, repeat the operation three times, add 100 μL of PBS to resuspend the magnetic probe to obtain a solution. Such as Figure 5 As shown, the results of the transmission electron microscope showed that the magnetic probes captured the H9N2 avian influenza...

Embodiment 3

[0051] Example 3 Binding count of H9N2 avian influenza virus captured by M13@GNP probe and magnetic probe

[0052] Take 100 μL of the M13@GNP probe in Example 1 and mix it with 20 μL of the four samples, the negative control and the blank control in Example 2, incubate at room temperature for 1 hour, wash with PBS solution containing 1% BSA and separate on the magnetic frame Resuspend in PBS, take 10 μL of the resuspension and 10 μL of M13 host bacteria ER2738 and spread it on an LB agar plate containing IPTG / X-Gal, incubate at 37°C for 8 hours to observe and count the number of blue spots. Such as Figure 6 As shown, the mixture formed blue spots on the LB agar plate containing IPTG / X-Gal. The result is as Figure 6 , by counting, when the virus concentration of sample 1 was 5PFU / mL, the number of coeruleus was 13, when the virus concentration of sample 2 was 50PFU / mL, the number of coeruleus was 27, and when the virus concentration of sample 3 was 500PFU / mL The number was...

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Abstract

The invention discloses an M13@GNP indicator probe and a preparation method thereof, wherein the M13@GNP indicator probe is a protein G-coated gold nanoparticle combined with an M13 monoclonal antibody and a H9N2 monoclonal antibody, and then combined with the M13 phage. The invention also discloses a method for directly counting H9N2 avian influenza virus by naked eyes. The object of the invention is to establish a detection method for directly counting H9N2 influenza virus based on phage@gold nanoprobe, which can detect and quantify H9N2 influenza virus in a sample, shorten the detection period, simplify the detection process, improve the universal applicability of detection methods and is conducive to on-site inspection, so that the detection of H9N2 influenza virus is accurate, sensitive and simple, and timely prevention and control is of great significance.

Description

technical field [0001] The invention belongs to the field of pathogen detection, and relates to a method for directly counting H9N2 avian influenza viruses with naked eyes, in particular to a method for directly counting H9N2 avian influenza viruses with naked eyes using a bacteriophage@gold nanoprobe. Background technique [0002] H9N2 virus is a subtype of avian influenza virus, which spreads widely among chickens in my country and has caused huge economic losses to the poultry industry. Although the H9N2 bird flu virus only occasionally infects humans. But its spread still poses a serious threat to human health. For example, the 1997 outbreak of the deadly human H5N1 influenza virus in Hong Kong was traced to the internal genes of a quail H9N2 virus. In addition, the H7N9 virus, a novel recombinant virus that obtained internal gene segments from the H9N2 virus, caused several human influenza epidemics in China. Similarly, analysis of the gene sequence of the new avian ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/543
CPCG01N33/577G01N33/56983G01N33/54346G01N33/54326G01N2333/11
Inventor 周昕许海旭
Owner YANGZHOU UNIV
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