Cell lysis reagent for extracting and purifying nucleic acids in biological samples

A technology for lysing reagents and biological samples, applied in the field of biochemistry, can solve the problems of affecting the effect of cell lysis, reducing the yield of nucleic acid extraction, heating and dissolving reagents, and inconvenient suction operations, etc., and achieves the effect of low cost and easy operation

Active Publication Date: 2012-07-04
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In actual operation, due to the effect of guanidinium salt on reducing the pH value of the solution, the guanidinium salt in these cell lysates is prone to precipitate when the pH value is close to neutral or slightly acidic, which n...

Method used

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  • Cell lysis reagent for extracting and purifying nucleic acids in biological samples
  • Cell lysis reagent for extracting and purifying nucleic acids in biological samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the preparation of cell lysis reagent

[0031] According to the content of the invention, at first prepare basic reagent 100mM Tris-HCl (pH7.0) and 50mM EDTA, obtain following five kinds of cell lysis reagents after adding other components:

[0032] (1) Cell Lysis Reagent A

[0033] 50mM Tris-HCl, 25mM EDTA, guanidine hydrochloride 6M, SDS 1% (w / v), Triton X-100 10% (v / v), add ammonia water to make NH 4 + The concentration is 0.5M, and the reagent pH is about 9.0.

[0034] (2) Cell Lysis Reagent B

[0035] 50 mM Tris-HCl, 25 mM EDTA, 6M guanidine hydrochloride, SDS 1% (w / v), Triton X-100 10% (v / v), the pH of the reagent was adjusted to about 9.0 with 10% NaOH.

[0036] (3) Cell Lysis Reagent C

[0037] 50mM Tris-HCl, 25mM EDTA, 2M guanidine isothiocyanate, Triton X-100 20% (v / v), add ammonia to make NH 4 + The concentration is 1M, and the pH of the reagent is about 13.0.

[0038](4) Cell Lysis Reagent D

[0039] 50mM Tris-HCl, 25mM EDTA,...

Embodiment 2

[0044] Example 2: Application of Cell Lysis Reagent in Serum Hepatitis B Virus DNA Extraction

[0045] (1) Extraction of HBV DNA from serum samples

[0046] Three concentrations of HBV DNA-positive serum calibrated by the China National Institute for the Control of Pharmaceutical and Biological Products’ hepatitis B virus (HBV) nucleic acid quantitative standards were used as samples to be determined (each concentration was 5.0×10 2 IU / ml, 5.0×10 4 IU / ml, 5.0×10 6 IU / ml), five kinds of cell lysis reagents in the measurement embodiment 1 extract the effect of serum HBVDNA.

[0047] In the experiment, Roche Highpure Total Nucleic Acid Kit (silica gel membrane method) was used as the nucleic acid extraction control reagent, and the solid phase material used in the prepared cell lysis reagent was the silica gel membrane column in the Roche control reagent. The control nucleic acid extraction reagent was operated according to the kit instructions provided by Roche. The sam...

Embodiment 3

[0062] Example 3: Application of Cell Lysis Reagent in Nasopharyngeal Swab Type A H1N1 Influenza Virus RNA Extraction

[0063] (1) Sample processing

[0064] Take 3 nasopharyngeal swabs collected clinically and identified as positive for Influenza A H1N1 virus, add 1.5ml of normal saline to each swab collection tube, oscillate fully with a vortex mixer, and collect the rinse solution for nucleic acid extraction. Then take Formosa Plastics DNA / RNA extraction reagent (magnetic bead method) as control nucleic acid extraction reagent, compare the extraction effect of 5 kinds of cell lysis reagents prepared in Example 1 to influenza A virus RNA in nasopharyngeal swab.

[0065] The Formosa Plastics magnetic bead method nucleic acid extraction reagent was operated according to its instructions. The three swabs each absorbed 200 μl of rinsing solution, and after extraction, each used 100 μl of elution buffer to separate the nucleic acid from the magnetic beads. The solid phase mate...

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Abstract

The invention discloses a cell lysis reagent for extracting and purifying nucleic acids in biological samples. The solution of the reagent is alkaline and contains the following components: guanidinium, ammonium ions and a surface active agent, thereby, cells in the biological samples can be more fully cracked in an alkali solution-phase environment, and nucleic acids [deoxyribonucleic acid/ ribonucleic acid (DNA/RNA)] released out of the cells can be promoted to be combined and absorbed with a solid material, and finally, is separated from the solid material through an elution step. The cell lysis reagent disclosed by the invention has the advantages of simplness and convenience in operation and high extraction yield of the nucleic acids, and can be used for extracting and purifying the nucleic acids in the biological samples with various types, such as whole blood, serum, blood plasma, saliva, urine, cerebrospinal fluid, cell and tissue culture solution or human nasopharynx and urogenital canal swabs and the like.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to cell lysis and nucleic acid extraction and purification in biological samples. Background technique [0002] The extraction and purification of nucleic acid (including DNA and RNA) is a basic operation in molecular biology. In cells, nucleic acid is always combined with various proteins. Nucleic acid extraction and separation mainly refers to releasing nucleic acid from biological sample cells to the outside of the cell and separating it from biological macromolecules such as proteins, polysaccharides, and fats. The extracted nucleic acid should be Only by ensuring the integrity of its molecular primary structure and excluding other impurity molecules, can it better meet the quality requirements of downstream nucleic acid hybridization, amplification, sequencing and other molecular biology gene operations or clinical gene detection for nucleic acid templates. ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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