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Influenza A virus nucleic acid labeling method based on rare earth up-conversion nanoprobe

An influenza A virus and rare earth up-conversion technology, which is applied in the fields of biotechnology and materials, can solve the problems of difficult real-time fluorescence monitoring for a long time, poor photostability, and wide emission spectrum of organic fluorescent dyes, and achieves the effect of high scientific research application prospects.

Pending Publication Date: 2022-02-25
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Organic fluorescent dyes have a wide emission spectrum and poor photostability. It is relatively difficult to monitor real-time fluorescence for a long time, and has a certain impact on virus activity.
There are also studies that use the stem-loop structure to connect quantum dots to label viral nucleic acids through the principle of complementary base pairing. However, whether it is organic dyes or quantum dots, it is more suitable for in vitro virus tracing after virus labeling. Due to the existence of biological tissues and light Poor penetration, limiting their use in vivo

Method used

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  • Influenza A virus nucleic acid labeling method based on rare earth up-conversion nanoprobe
  • Influenza A virus nucleic acid labeling method based on rare earth up-conversion nanoprobe

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Take the surface carboxylated rare earth up-conversion nanoparticle solution, add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxyl succinimide) was activated by rotating overnight, centrifuged, discarded the supernatant, and added ddH 2 O resuspended with streptavidin:ddH 2 O volume ratio is 1:1000, add streptavidin, rotate and mix well, centrifuge and add ddH 2 O resuspended to obtain streptavidinized rare earth up-converting nanoparticles; the volume ratio of stem-loop solution: streptavidinized rare earth upconverting nanoparticles solution was 1:100 to streptavidinized rare earth upconverting nanoparticles The stem-loop DNA is added to the solution of the rare earth upconversion nanoparticle, and the rare earth upconversion nanoprobe for labeling the viral nucleic acid is obtained after the rotation reaction. After labeling influenza A virus nucleic acid with rare-earth up-conversion nanoprobes in the cells, add mouse anti-HA a...

Embodiment 2

[0028] Take the surface carboxylated rare earth up-conversion nanoparticle solution, add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxyl succinimide) was activated by rotating overnight, centrifuged, discarded the supernatant, and added ddH 2 O resuspended with streptavidin:ddH 2 Add streptavidin at a volume ratio of 1:500, rotate and mix well, then centrifuge and add ddH 2 O resuspended to obtain streptavidinized rare earth up-converting nanoparticles; the volume ratio of stem-loop solution: streptavidinized rare earth upconverting nanoparticles solution was 1:100 to streptavidinized rare earth upconverting nanoparticles The stem-loop DNA is added to the solution of the rare earth upconversion nanoparticle, and the rare earth upconversion nanoprobe for labeling viral nucleic acid is obtained after the rotation reaction. After labeling influenza A virus nucleic acid with rare-earth up-conversion nanoprobes in the cells, add mouse anti-HA...

Embodiment 3

[0030] Take the surface carboxylated rare earth up-conversion nanoparticle solution, add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxyl succinimide) was activated by rotating overnight, centrifuged, and the supernatant was discarded, and ddH was added 2 O resuspended with streptavidin:ddH 2The O volume ratio is 1:200, add streptavidin, rotate and mix, centrifuge and add ddH 2 O resuspended to obtain streptavidinized rare earth up-converting nanoparticles; the volume ratio of stem-loop solution: streptavidinized rare earth upconverting nanoparticles solution was 1:100 to streptavidinized rare earth upconverting nanoparticles The stem-loop DNA is added to the solution of the rare earth upconversion nanoparticle, and the rare earth upconversion nanoprobe for labeling the viral nucleic acid is obtained after the rotation reaction. After labeling influenza A virus nucleic acid with rare-earth up-conversion nanoprobes in the cells, add mouse ...

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Abstract

The invention relates to a viral nucleic acid labeling method based on a rare earth up-conversion nanoprobe. The method comprises the following steps: combining the rare earth up-conversion nanoprobe with a DNA stem-loop structure by utilizing a characteristic reaction of streptavidin and biotin; by means of the self-assembly process in the influenza A virus breeding process, combining the rare earth up-conversion nanoprobe-DNA stem loop to the influenza A virus RNA, and labeling the influenza A virus nucleic acid by the rare earth up-conversion nanoprobe. The co-localization result of the fluorescence signal of the fluorescently-labeled influenza A virus specific antibody and the fluorescence signal of the rare earth up-conversion nanoprobe is utilized to verify the efficiency of labeling the viral nucleic acid by the rare earth up-conversion nanoprobe, and the efficiency of labeling the viral nucleic acid by the rare earth up-conversion nanoprobe can reach more than 80%. The DNA stem-loop-rare earth up-conversion nano probe can be used for labeling influenza A virus nucleic acid by utilizing a base complementary pairing principle, and has a relatively high scientific research and application prospect in the fields of biotechnologies such as virology and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and material technology, and in particular relates to a method for labeling influenza A virus nucleic acid based on a rare earth upconversion nano-probe. Background technique [0002] Since the genome of the virus is encapsulated in a complete virus particle, it cannot be marked by commonly used dyes. At present, intercalating dyes are mainly used to mark viral nucleic acids. Organic fluorescent dyes have a wide emission spectrum and poor photostability, making real-time fluorescence monitoring for a long time relatively difficult, and have a certain impact on virus activity. There are also studies that use the stem-loop structure to connect quantum dots to label viral nucleic acids through the principle of complementary base pairing. However, whether it is organic dyes or quantum dots, it is more suitable for in vitro virus tracing after virus labeling. Due to the existence of biological tissues and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6816C12R1/93
CPCC12Q1/701C12Q1/6816C12Q2563/107C12Q2525/301C12Q2565/601
Inventor 常津庞高举王汉杰张英英潘惠卓
Owner TIANJIN UNIV
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