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

[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.

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[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...

[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...