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Hepatitis B covalently closed circular DNA magnetic trapping technique

A technology of DNA molecules and magnetic nanometers, applied in recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of less cccDNA and greater difficulty of cccDNA

Active Publication Date: 2018-02-16
THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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Problems solved by technology

(2) The content of ccc DNA in cells is less, and it is more difficult to obtain high-purity ccc DNA

Method used

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  • Hepatitis B covalently closed circular DNA magnetic trapping technique
  • Hepatitis B covalently closed circular DNA magnetic trapping technique
  • Hepatitis B covalently closed circular DNA magnetic trapping technique

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experiment example 1

[0027] Experimental example 1 The method of the present invention for enriching and separating covalently closed circular DNA of hepatitis B

[0028] 1. Preparation method

[0029] Preparation process such as figure 1 Shown:

[0030] 1. Preparation of HBV cccDNA-specific DNA molecular probe magnetic nanospheres

[0031] 1.1 Preparation of Fe by chemical coprecipitation method 3 o 4 Nanoparticles:

[0032] 20ml FeCl 3 (1.0mol / L) with 5ml FeCl 2 (2.0mol / L, prepared in 2.0mol / L hydrochloric acid solution) the solution is mixed evenly and added to 250ml 0.7mol / L ammonia solution, and the dark brown precipitate obtained after centrifugation is mixed with 150ml 2.0mol / L perchloric acid The solution was dispersed, washed with ultrapure water until neutral, and dried to obtain Fe 3 o 4 Nanoparticles.

[0033] 1.2 Preparation of silicon-shell magnetic nanoparticles by inverse emulsion method:

[0034] Mix Triton X-100, n-hexanol and cyclohexane evenly at a volume ratio of 1:...

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Abstract

The invention discloses a hepatitis B covalently closed circular DNA magnetic trapping technique. A method for preparing HBV cccDNA specific DNA molecular probe magnetic nano-microspheres comprises the following steps: (1) preparing Fe3O4 nanoparticles by virtue of a chemical coprecipitation method; (2) preparing silicon-shell magnetic nanoparticles by virtue of a reverse emulsion method; (3) conducting avidin modification on the silicon-shell magnetic nanoparticles; and (4) coupling an ssDNA probe for a cccDNA specific sequence. The invention also discloses the probe magnetic nano-microspheres prepared by the method, and a method for enriching HBV cccDNA by virtue of the microspheres. With the application of the HBV cccDNA specific DNA molecular probe magnetic nano-microspheres prepared by the method provided by the invention, the effective specific trapping of HBV cccDNA can be achieved, and the effective and rapid enriching and separation of the HBV cccDNA can be implemented; and the separating method is simple and has a good application prospect.

Description

technical field [0001] The invention relates to a method for enriching and separating covalently closed circular DNA of hepatitis B. Background technique [0002] Chronic hepatitis B is a disease caused by infection with hepatitis B virus (HBV), and there is currently no specific drug that can cure it. HBV cccDNA is the key factor for the persistence of hepatitis B virus infection and is difficult to cure. By detecting HBV cccDNA, we can understand the status and infectivity of HBV infection, evaluate the efficacy of drug treatment of HBV, help understand the degree of HBV clearance from the body, and judge when to stop for clinicians Whether HBV will actively re-replicate and lead to hepatitis B recurrence after antiviral treatment and cessation of treatment provides an objective indicator, and it is also an indicator to evaluate whether extrahepatic tissues are infected by HBV and whether the transplanted liver is reinfected in liver transplantation. [0003] Scholars at ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10B82Y5/00
CPCB82Y5/00C12N15/1013C12Q1/6806C12Q2563/143C12Q2563/155
Inventor 郭永灿涂植光赵朝辉王友强
Owner THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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