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Reagent for removing non-nuclear red blood cells in biological sample and application of reagent to DNA extraction

A technology for biological samples and nucleated red blood cells, applied in DNA extraction, and in the field of lysing reagents for removing anucleated red blood cells from biological samples, can solve the problems of adverse effects of extraction or culture, incomplete lysis of red blood cells, and low purity of nucleated cells , to achieve the effects of safe and environmentally friendly reagent preparation and use, convenient downstream genetic manipulation detection, and chemical stability of reagent components

Pending Publication Date: 2019-06-07
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used nucleated cell separation methods are: (1) Ficoll and Percoll density gradient centrifugation. Although it is generally recognized that red blood cells are removed and monocytes can be purely separated, the lymphocyte separation liquid reagent used is relatively large in volume and cumbersome to operate. Time-consuming; (2) The ammonium chloride cracking red blood cell method is convenient to use, but the effect of removing red blood cells is not good, and repeated washing is required. At the same time, the thermal stability of the ammonium chloride solution is poor, and it needs to be prepared immediately or stored / transported in low-temperature refrigeration
For example, the invention patent "A red blood cell lysate and its lysing method" (CN104178244A) discloses that the red blood cell lysate used contains ammonium chloride, potassium bicarbonate, disodium edetate and other components. It needs to be mixed for 15-30 minutes, the cracking effect is not good, and it is very time-consuming. At the same time, the ammonium chloride and potassium bicarbonate components in the cracking solution are thermally unstable components, which can only be filtered and sterilized and stored in cold storage. Preheating, its preparation, use and storage are inconvenient
The invention patent "A Method and Kit for Separating Nucleated Cells in Vitro and Removing Red Blood Cells" (CN102747035A) also discloses that the erythrocyte lysate is composed of ammonium chloride and Tris-HCl, while the invention patent "Reagents for extracting DNA and using the The method for extracting mammalian DNA with reagents" (CN1635134A) discloses that the erythrocyte lysate contains Tris-HCl, magnesium chloride and sodium chloride, and has the disadvantages of incomplete lysis of erythrocytes, low purity of nucleated cells, and long operation time.
The disadvantages of these disclosed erythrocyte lysates will adversely affect subsequent nucleated cell DNA extraction or cultivation, etc.

Method used

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  • Reagent for removing non-nuclear red blood cells in biological sample and application of reagent to DNA extraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation of erythrocyte lysing reagent

[0032] Prepare four kinds of erythrocyte lysis reagents A to D according to the content of the invention, and prepare the cell lysis reagent E (ACK ammonium chloride erythrocyte lysate) as a control, as follows:

[0033] (1) Erythrocyte Lysis Reagent A

[0034] EDTA 5 mmol / L, NaCl 15 mmol / L, Tween 20 0.4% (v / v), Tris-HCl 5 mmol / L, pH value about 8.0.

[0035] (2) Erythrocyte Lysis Reagent B

[0036] NTA 3mmol / L, NaCl 50 mmol / L, Triton X-100 0.8% (v / v), Tris-HCl 15 mmol / L, pH value about 7.6.

[0037] (3) Erythrocyte Lysis Reagent C

[0038] EDTA 1mmol / L, KCl 40 mmol / L, Triton X-100 0.3% (v / v), Tris-HCl 20 mmol / L, pH value about 7.5.

[0039] (4) Erythrocyte Lysis Reagent D

[0040] EDTA 2mmol / L, NaCl 35 mmol / L, Triton X-100 0.5% (v / v), Tris-HCl 10 mmol / L, the reagent pH is about 7.8.

[0041] (5) Erythrocyte Lysis Reagent E

[0042] NH 4 Cl 155 mmol / L, KHCO 3 12mmol / L, EDTA 0.15mmol / L, pH7.2-7.4.

[...

Embodiment 2

[0044] Example 2: Comparison of the lysing and removing effects of different formulations of cell lysing reagents on peripheral blood erythrocytes

[0045] (1) Sample collection: Collect 1 part of EDTA anticoagulated whole blood with a volume of 5ml, mix well and distribute 400μl / tube into 2ml centrifuge tubes, and divide into 10 tubes of samples.

[0046] (2) Take the 5 kinds of erythrocyte lysis reagents prepared in Example 1, according to the ratio of whole blood: red blood cell lysate volume of 1:3, absorb 1.2ml of each erythrocyte lysis reagent and add it to the whole blood, each red blood cell lysate Lyse 2 tubes of samples, a total of 5 groups of 10 tubes of samples, and mix well for 1 min.

[0047] (3) Centrifuge 5 groups of 10 tubes of solution at 10,000 rpm for 1 min, discard the supernatant and retain the enriched cell pellet.

[0048] (4) Add 1ml of red blood cell lysis reagent to the enriched cell pellet, mix well and resuspend the pellet.

[0049] (5) Centrifug...

Embodiment 3

[0054] Example 3: DNA extraction application of cell lysis reagent in Epstein-Barr virus positive whole blood sample

[0055] (1) DNA extraction solution: prepared according to the formula of 10mmol / L NaOH, 0.1% SDS, for DNA extraction.

[0056] (2) Sample collection: human infection with Epstein-Barr virus causes a variety of related diseases. First, it proliferates in the epithelial cells of the oropharynx, then infects B lymphocytes, enters the blood circulation and causes systemic infection, and can remain latent in human lymphoid tissues and white blood cells for a long time . The present invention collects 5 cases of EDTA anticoagulated human whole blood samples identified as Epstein-barr virus (Epstein-barr virus) positive from the clinic, each case is divided into 400 μl / tube and placed in a 1.5ml centrifuge tube, and divided into 2 groups with a total of 10 Tube.

[0057] (3) Take the erythrocyte lysis reagent D and the control erythrocyte lysis reagent E in Example...

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Abstract

The invention relates to a splitting reagent for removing non-nuclear red blood cells in a biological sample and an application of the reagent to DNA extraction. The splitting reagent for removing non-nuclear red blood cells comprises a metal ion chelating agent, inorganic salts, a surfactant and a solution buffer system. The invention further provides an application method of the splitting reagent to DNA extraction. The splitting reagent for removing non-nuclear red blood cells provided by the invention overcomes defects that a conventional product is poor in stability, needs frozen preservation and needs to be preheated for use after being taken out, the red blood cells in blood samples or tissue cell samples of people, mice and the like can be effectively removed through splitting, andnucleated cells in the samples are separated and enriched. The splitting reagent is used for extracting nucleic acid(DNA) of the nucleated cells, and has the advantages of being stable in components,safe, environmental-friendly, high in splitting efficacy of the red blood cells, and quick and simple to operate. The reagent can be stored at normal temperature for a long term, cold chain transportation is not needed, and the preparation and use cost is low.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a lysing reagent for removing anucleated red blood cells in biological samples and its application in DNA extraction. Background technique [0002] Red blood cells or erythrocytes (Red Blood Cells, RBCs) are the most numerous blood cells in the blood, and are also the main medium for transporting oxygen through the blood in vertebrates, and have immune functions. The mature red blood cells in the circulating blood of mammalian vertebrates (except camels and alpacas) have no nucleus and DNA; while the red blood cells of avian vertebrates have nucleus and DNA, and the total number of normal adult red blood cells is (3.5-5.5) x 10 9 pieces / ml. Leukocytes or white blood cells (Leukocyte, White Blood Cell, WBC) are colorless, spherical, blood cells with nuclei and DNA in the blood. They mainly play a defensive role in mammals and participate in the body's immune defe...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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