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Extraction reagent and extraction method of hepatitis B virus DNA

A hepatitis B virus and extraction method technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve the problems of cross-infection, high cost, time-consuming and laborious, and achieve the effect of not easy to contaminate

Inactive Publication Date: 2011-06-15
上海裕隆医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the HBV-DNA extraction in the process of sample processing is the commonly used phenol-chloroform method, which is cumbersome, time-consuming and labor-intensive, and easily causes cross-infection, which makes PCR amplification fail.
There are many kits for extracting HBV-DNA on the market. Although the operation is simple and labor-saving, the cost is too high and it is not easy to be extended to general laboratories.

Method used

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  • Extraction reagent and extraction method of hepatitis B virus DNA
  • Extraction reagent and extraction method of hepatitis B virus DNA
  • Extraction reagent and extraction method of hepatitis B virus DNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the preparation of hepatitis B virus DNA extraction reagent

[0027] 1. Preparation of Lysis Buffer 1:

[0028] Using DEPC water, prepare the following solution:

[0029] 10-100mM Tris-HCl pH7.5-8.5

[0030] 0.5-0.9% SDS

[0031] 10-200mM NaOH

[0032] 40-300mM NaCl

[0033] 1-5mM EDTA pH8.0;

[0034] 2. Preparation of Lysis Buffer 1:

[0035] Using DEPC water, prepare the following solution:

[0036] 1-2mg Proteinase K

[0037] 50mM Tris-HCl pH7.6

[0038] 10mM CaCl 2 .

[0039] Note: The above two solutions in the kit cannot be directly mixed for sample DNA extraction.

Embodiment 2

[0040] Embodiment 2: the use of hepatitis B virus DNA extraction reagent

[0041] 1. Collection of Serum and Plasma Samples

[0042] Collect the whole blood sample containing the HBV virus sample into a blood collection tube without any anticoagulant, let it stand at room temperature for 30 minutes, centrifuge at 4000rpm for 5 minutes in a desktop centrifuge, collect the upper serum sample, mix it, and let it stand at room temperature for 30 minutes. Collect upper plasma samples.

[0043] When using the reagent of the present invention to extract HBV DNA, if the extracted DNA is to be further processed by PCR, the serum sample cannot come from EDTA anticoagulated blood.

[0044] 2. Sample Preparation

[0045] Draw 50ul serum sample into the sterilized EP tube, add 15ul Lysis Buffer 1, and mix well. Then add 5ul of Lysis Buffer 2, shake and mix on the shaker, and centrifuge at 3000rpm for 1 minute. The mixture was heated in a 56°C water bath for 30 minutes. Take out the tu...

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Abstract

The invention discloses an extraction method of hepatitis B virus deoxyribonucleic acid (DNA) and belongs to the field of nucleic acid extraction in molecular biology. An alkaline lysis technology is adopted according to particular characteristics of blood and viruses; impurities such as proteins, polysaccharides, lipids and the like are not required to be pre-removed; and the virus DNA is directly extracted through lysis buffer 1 and lysis buffer 2. The method has the characteristics of high efficiency, high speed, simplicity and no pollution, and the whole operation process only lasts for 50 minutes. The obtained hepatitis B virus DNA can be applied to molecular biology experiments such as the common polymerase chain reaction (PCR), fluorescence quantitative PCR and the like.

Description

technical field [0001] The invention relates to a DNA extraction reagent method, in particular to a DNA extraction reagent and method related to hepatitis B virus. Background technique [0002] Viral hepatitis B is a worldwide disease caused by the hepatitis B virus (HBV). The incidence rate is high in developing countries. According to statistics, there are more than 280 million asymptomatic hepatitis B virus carriers (HBsAg carriers) in the world, and my country accounts for about 93 million. Hepatitis B virus (HBV) is a specific hepatotropic virus. Due to the development of nucleic acid molecular hybridization technology in recent years, HBV-DNA can also be detected in extrahepatic organ cells. Positive HBV-DNA is the most reliable indicator of HBV replication. In recent years, the in vitro DNA amplification technology of polymerase chain reaction (PCR) has increased the sensitivity by more than 100 times (10FGfg / ml), and a very small amount of virus can be detected. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 上海裕隆医学检验所股份有限公司
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