Using method for extracting virus DNA by using micro-nucleic acid releasing agent

A nucleic acid release agent and release agent technology, applied in the field of molecular biology, can solve the problems of cumbersome steps, repeated extraction, multiple tube changes, complicated operations, etc., to avoid false negatives and ensure the efficiency of nucleic acid cleavage.

Inactive Publication Date: 2015-10-07
宝瑞源生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although the nucleic acid separation technology is developing rapidly at present, there are some shortcomings. For example, although the phenol-chloroform extraction method has high nucleic acid purity, its main components are harmful to the human body and are likely to cause environmental pollution. At the same time, the extraction process requires repeated extractions. Change the tube, the steps are cumbersome, which will cause the loss and contamination of the sample; alkaline lysis method: the nucleic acid extracted by this method is not easy to preserve; the concentration of nucleic acid DNA extracted by the silica gel membrane adsorption column method is low and the operation is complicated; the magnetic bead method is expensive due to the high extraction cost , which limits its wide application; this reagent is used for the extraction of virus DNA from biological samples, with less sample demand, simple operation, and no cumbersome steps such as centrifugation and elution. The functions of various factors such as proteins, drugs, and hemolysis that interfere with PCR amplification can avoid pollution and nucleic acid loss caused by repeated centrifugation, elution and uncapping; realize the "one-step method" and "one-chamber method" of PCR extraction "Operation"

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Examples

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Effect test

Embodiment 1

[0016] Embodiment 1: use the method of the present invention to extract the gradient dilution of hepatitis B virus nucleic acid plasma sample (HBV-DNA); 1) take known concentration (2 * 10 7 IU / ml) HBV-positive samples were diluted tenfold with matrix serum to 200IU / ml for later use; 2) Prepare a corresponding number of 200μl nuclease-free PCR reaction tubes, add 5μl trace nucleic acid release agent to each tube, and add 5μl HBV samples to be tested ( 2×10 7 IU / ml~200IU / ml six concentrations), pipette repeatedly for 10 times;

[0017] 3) Add 30 μl blocking agent;

[0018] 4) Cover the tube cap and place it in an ordinary PCR instrument for reaction. The specific parameters are as follows: Step 1: 95°C, 10 minutes; Step 2: 4°C, 2 minutes;

[0019] 5) Take out the lysed PCR reaction tube from the ordinary PCR machine, carefully open the tube cap, add 2.5 μl of release-promoting agent to each tube and repeatedly blow and mix with a pipette for 10 times;

[0020] 6) Add the HBV...

Embodiment 2

[0021] Embodiment 2: Extract the precision of hepatitis B virus nucleic acid plasma sample (HBV-DNA) by the method of the present invention;

[0022] 1) Take the known concentration (2×10 7 IU / ml) HBV-positive samples were diluted ten-fold with matrix serum to 2×10 3 IU / ml spare;

[0023] 2) Prepare a corresponding number of 200 μl nuclease-free PCR reaction tubes, add 5 μl trace nucleic acid release agent to each tube, add 5 μl HBV samples to be tested (2×10 5 IU / ml and 2×10 3 Two concentrations of IU / ml, 8 replicate wells for each concentration sample), repeated pipetting 10 times with a pipette;

[0024] 3) Add 30 μl blocking agent;

[0025] 4) Cover the tube cap and place it in an ordinary PCR instrument for reaction. The specific parameters are as follows: Step 1: 95°C, 10 minutes; Step 2: 4°C, 2 minutes;

[0026] 5) Take out the lysed PCR reaction tube from the ordinary PCR machine, carefully open the tube cap, add 2.5 μl of release-promoting agent to each tube and ...

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Abstract

The invention discloses a method for extracting virus DNA contained in a biological sample by utilizing a micro-nucleic acid releasing agent, belongs to the technical field of molecular biology, and particularly relates to a reagent for extracting virus DNA by utilizing the micro-nucleic acid releasing reagent and a using method thereof. The micro-nucleic acid releasing agent cracks the virus DNA contained in the biological sample (a serum or a plasma) and can more effectively ensure the cracking and release efficiency through the assistance of a release promoting agent; the micro-nucleic acid releasing agent completely releases nucleic acid and also has the function of closing various factor substances, namely protein, drugs, hemolyzed blood and the like which are contained in a sample and have interference effects on PCR amplification, so that false negative in a detection process is prevented. The method disclosed by the invention is simple, convenient, flexible, fast and accurate in sample extraction, prevents the pollution and nucleic acid loss which are caused by repeated centrifugation and elution uncapping, and realizes the 'one-step' and 'one-room' operation of PCR extraction.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a reagent for extracting viral nucleic acid (DNA) by using a trace nucleic acid releasing agent and a method for using the same. Background technique [0002] Nucleic acid extraction is the starting point of downstream nucleic acid detection, research or product development. The quality and integrity of the isolated nucleic acid directly affect the research or diagnostic results. Therefore, nucleic acid extraction is one of the most critical methods in molecular biology. Usually successful nucleic acid isolation Purification requires four important steps: disruption and lysis of tissues or cells, denaturation of nucleoprotein complexes, inactivation of nucleases, and removal of contaminants. An ideal extraction method can obtain high-quality target nucleic acids without protein, Glycolipids and other nucleic acid contamination, there are many specialized nucleic acid ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 郭冬冬迟磊吕翔张美娜杨海侠
Owner 宝瑞源生物技术(北京)有限公司
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