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
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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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