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Hepatitis B covalently closed circular dna magnetic capture technique

A technology of DNA molecules and magnetic nanometers, which is applied in the direction of recombinant DNA technology, DNA preparation, DNA / RNA fragments, etc., can solve the problems of less cccDNA and the difficulty of cccDNA, and achieve simple separation methods, good application prospects, and high-efficiency and rapid enrichment separation effect

Active Publication Date: 2021-07-09
THE AFFILIATED HOSPITAL OF TRADITIONAL CHINESE MEDICAL TO SOUTHWEST MEDICAL UNIV
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AI Technical Summary

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 capture technique
  • Hepatitis B covalently closed circular dna magnetic capture technique
  • Hepatitis B covalently closed circular dna magnetic capture technique

Examples

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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 capture technology. The present invention prepares the method for HBV cccDNA-specific DNA molecular probe magnetic nano microsphere, and the steps are as follows: (1) adopt chemical co-precipitation method to prepare Fe 3 o 4 Nanoparticles; (2) preparing silicon-shell magnetic nanoparticles by the inverse emulsion method; (3) avidin-modifying the silicon-shell magnetic nanoparticles; (4) coupling ssDNA probes for specific sequences of cccDNA. The invention also discloses the probe magnetic nanometer microsphere prepared by the aforementioned method, and a method for enriching HBV cccDNA by using the microsphere. The HBV cccDNA-specific DNA molecular probe magnetic nano-microspheres prepared by the method of the invention can effectively and specifically capture HBV cccDNA, can realize high-efficiency and rapid enrichment and separation of HBV cccDNA, the separation method is simple, and the application prospect is good.

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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Patent Type & Authority Patents(China)
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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