Nucleic acid extraction method and extraction agent thereof

An extraction method and reagent technology, applied in the field of biological detection methods and reagents, can solve the problems of easy cross-contamination between samples, cumbersome nucleic acid extraction steps, and contamination between samples, so as to avoid sample contamination, reduce steps and difficulties, and reduce reagents. cost effect

Inactive Publication Date: 2017-08-18
BEIJING KINGHAWK PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing nucleic acid extraction reagents and methods mainly include phenol-chloroform extraction method, alkali lysis method, CTAB extraction method, magnetic bead method, silica matrix method, etc. Lifting, transferring tubes, washing, centrifuging and other steps require instruments and equipment with a rotary door, and it takes about 40 minutes to 2 hours at the same time, and it is easy to cause contamination between samples
[0004] In order to solve the problem

Method used

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  • Nucleic acid extraction method and extraction agent thereof
  • Nucleic acid extraction method and extraction agent thereof
  • Nucleic acid extraction method and extraction agent thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Nucleic acid rapid extraction reagent preparation:

[0039] First dissolve Tris and EDTA with DEPC water, the concentration of Tris is 5-10mM; the concentration of EDTA is 5-10mM; adjust the pH to 6-9, add Tris and EDTA solution, add in 3-[(3-cholesteryl aminopropyl )Dimethylamino]-1-propanesulfonic acid, the volume percentage concentration is 0.005%-0.02%; adding NP-40, the percentage content concentration is 0.01%-0.2%; adding Tween-20, the volume percentage content The concentration is 0.01-0.2%; adding Chelex-100, the volume content is 1-20%.

Embodiment 2

[0041] Nucleic acid extraction and fluorescence quantitative PCR amplification of hand, foot and mouth virus throat swab

[0042] The step of nucleic acid extraction: get 12 routine pharyngeal swab samples to extract respectively with domestic magnetic bead method (CZ), imported silicon substrate membrane (GJ) and extraction reagent (KT) of the present invention,

[0043] Quick (KT) reagent extraction nucleic acid step of the present invention:

[0044] 1) Take 30 μl swab sample and add it to 50 μl extraction solution, lyse at 90°C for 4 minutes, and beat once evenly during the lysis period. Take it out and leave it at room temperature until the particles settle, and the supernatant is the obtained nucleic acid.

[0045] Steps for nucleic acid extraction with domestic magnetic beads (CZ) reagents:

[0046] 1) Take 200 μl of virus liquid into a 1.5ml centrifuge tube (prepared by yourself).

[0047]2) Add 15 μl of magnetic bead suspension G to the centrifuge tube.

[0048] 3...

Embodiment 3

[0077] Serum nucleic acid extraction and HBV fluorescence quantitative PCR amplification

[0078] The steps of nucleic acid extraction: 10 cases of serum samples were extracted by domestic magnetic bead method (CZ), imported silicon matrix membrane (GJ) and extraction reagent (KT) of the present invention.

[0079] Quick (KT) reagent extraction nucleic acid step of the present invention:

[0080] 1) Take 10 μl serum sample and add it to 50 μl extract solution, lyse at 90°C for 4 minutes, and beat evenly once during the lysis period. Take it out and leave it at room temperature until the particles settle, and the supernatant is the obtained nucleic acid.

[0081] The imported silicon matrix membrane (GJ) and the extraction reagent (KT) of the present invention are extracted according to the steps of Example 2.

[0082] HBV nucleic acid amplification was amplified with the "Hepatitis B Virus Nucleic Acid Detection Kit (Fluorescence PCR)" kit produced by Beijing Jinhao Pharmace...

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Abstract

The invention relates to a nucleic acid extraction method. The method includes steps: well mixing a sample with an extraction agent proportionally, performing splitting decomposition for 3-5min at 85-95 DEG C, and uniformizing for 1-2 times during splitting decomposition; after particle settling, taking supernatant which is released nucleic acid. The extraction agent comprises Tris, EDTA, 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid, NP-40, Tween-20, Chelex-100 and water, wherein the concentration of Tris is 5-10mM; the concentration of EDTA is 5-10mM; the volume concentration percentage of 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid is 0.005%-0.02%; the concentration percentage of NP-40 is 0.01%-0.2%; the volume concentration percentage of Tween-20 is 0.01-0.2%; the volume content of Chelex-100 is 1-20%; PH of the extraction agent is 6-9.

Description

[0001] Technical field: [0002] The invention relates to a biological detection method and its reagent, in particular to a nucleic acid extraction method and its extraction reagent. Background technique [0003] Existing nucleic acid extraction reagents and methods mainly include phenol-chloroform extraction method, alkali lysis method, CTAB extraction method, magnetic bead method, silica matrix method, etc. Lifting, transferring tubes, washing, centrifuging and other steps require instruments and equipment with a revolving door, and it takes about 40 minutes to 2 hours at the same time, and it is easy to cause contamination between samples. [0004] In order to solve the problems of cumbersome nucleic acid extraction steps, time-consuming, and easy cross-contamination between samples, and provide simple and rapid nucleic acid extraction reagents and methods for on-site or laboratories with incomplete equipment, the present invention provides a nucleic acid extraction method ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 王月华张誌
Owner BEIJING KINGHAWK PHARMA
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