CN106754890B

CN106754890B - Extraction kit and extraction method of virus RNA

Info

Publication number: CN106754890B
Application number: CN201710052461.7A
Authority: CN
Inventors: 吴英松; 刘天才; 杨学习; 陈瑶
Original assignee: Southern Medical University
Current assignee: GUANGZHOU BDS BIOLOGICAL TECHNOLOGY Co.,Ltd.
Priority date: 2017-01-24
Filing date: 2017-01-24
Publication date: 2020-05-08
Anticipated expiration: 2037-01-24
Also published as: CN106754890A

Abstract

The invention provides a virus RNA extraction kit, which comprises a cracking binding solution A, a washing solution B, an eluent C and a digestion solution D, wherein the cracking binding solution A contains 20-50 mM of sucrose, 20-50 mM of ammonium sulfate, 10-50 mM of Tris-HCl, 1-5% (V/V) of Brij-58 and 0.2-0.5 mg/mL of magnetic beads; the pH value is 4.5-5.5, the washing liquid B contains 1-5% (V/V) Brij-58, 5-25 mM Tris-HCl and 0.5-1 mg/mL proteinase K, and the pH value is 6-7; the kit is used for extracting RNA, the method does not depend on the traditional Chaotropic salt, RNA can be cracked, adsorbed and washed under the low-salt ion environment, and meanwhile, the washing liquid does not contain alcohol substances, so that related salt and impurities can be removed. The method is simple and feasible, has low cost and more practical application value.

Description

Extraction kit and extraction method of virus RNA

Technical Field

The invention relates to the technical field of molecular biology detection, and particularly relates to a kit and a method for extracting viral RNA.

Background

Nucleic acid extraction is the first step of the whole technical process of nucleic acid detection, and is also one of the most critical methods in molecular biology. It is the basis for downstream nucleic acid detection and scientific research, and the quality and integrity of extracted nucleic acids can directly affect the results of clinical research or diagnosis. The general nucleic acid extraction process comprises two major steps: and (4) cracking and purifying the sample. Lysis means releasing nucleic acid in a sample into a reaction system, and purification means completely separating nucleic acid from other components in the reaction system, such as proteins, salts, and other impurities. Common RNA extraction methods include TRIZOL method, guanidinium isothiocyanate method, CTAB-LiCl method, and total RNA extraction kits of various biological companies at home and abroad. The classical method for extracting RNA is a TRIZOL method, which is a method based on the extraction principle of isothiocyanate muscle/phenol/chloroform, the method has wide application range, is suitable for both animals and plants, and the extracted RNA basically has no pollution of genome DNA, but the method has complicated operation steps, uses toxic reagents such as phenol, chloroform and the like in an extraction reagent, and is not suitable for extracting the RNA of a material rich in polysaccharide and polyphenol. The CTAB-LiCl method is commonly used for extracting RNA from plant tissues, but since CTAB itself is a reagent having a function similar to SDS, although it can form an insoluble complex with RNA and DNA, the CTAB method usually involves other operations such as LiCl precipitation, which makes the method cumbersome to operate, and chloroform, a toxic reagent, is used for extraction. The traditional extraction reagent has relatively low extraction yield and complex steps, which bring certain influence on extraction work, so that qualified RNA samples are difficult to obtain.

The magnetic bead method is a nucleic acid extraction method developed in recent years. The magnetic beads have a diameter of several tens of nanometers to several micrometers, exhibit magnetism in a magnetic field, and are spherical composite particles composed of inorganic/organic or inorganic/inorganic materials. The magnetic bead method for extracting nucleic acid does not need organic solvents with high toxicity, such as phenol, chloroform and the like, and the extracted nucleic acid has high purity and high yield. Patent numbers 201010281633.6, 201510023368.4, etc. disclose methods for extracting viral DNA or RNA, but these methods are based on the cleavage and adsorption of DNA or RNA under Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, sodium perchlorate, etc.), followed by washing with a proportion of ethanol, and finally releasing the nucleic acids into the eluate. The Chaotropic salt is a salt which destroys the stability of molecular force (hydrogen bond), can make hydrophobic protein more soluble in water, can not interfere the interaction formed by non-covalent bond between molecules in low salt, the magnetic bead in the Chaotropic salt can enhance the capability of adsorbing nucleic acid, and the RNA extraction in the current domestic published patent is basically derived on the basis. Few methods for extracting RNA that are not based on Chaotropic salts exist in the prior art.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a kit for extracting viral RNA.

The second purpose of the invention is to provide a method for extracting nucleic acid from virus RNA by using the RNA extraction kit.

The purpose of the invention is realized by the following technical scheme:

an extraction kit of virus RNA comprises a cleavage binding solution A, a washing solution B, an eluent C and a digestion solution D, wherein the cleavage binding solution A contains 20-50 mM of sucrose, 20-50 mM of ammonium sulfate, 10-50 mM of Tris-HCl, 1-5% (V/V) of Brij-58 and 0.2-0.5 mg/mL of magnetic beads; the pH value is 4.5-5.5, the washing liquid B contains 1-5% (V/V) Brij-58, 5-25 mM Tris-HCl and 0.5-1 mg/mL proteinase K, and the pH value is 6-7.

The working principle of the kit is as follows: under the action of the digestion solution D, the shell of the virus is damaged, the RNA of the virus nucleic acid is released, meanwhile, under the action of the lysis binding solution A, the RNA can be bound to the magnetic beads, then the RNA on the magnetic beads is washed by the washing solution B, redundant salt and protein molecules are removed, and finally the RNA on the magnetic beads is eluted by the eluent C.

The pH value of the cracking binding solution A is ensured to be 4.5-5.5, RNA can be ensured not to be dissolved, meanwhile, the environment with low pH value is beneficial to magnetic beads to adsorb nucleic acid, and the nucleic acid can be bound on the magnetic beads to the maximum extent.

Preferably, the eluent C contains 10-20 mM Tris-HCl and 0.5mM EDTA, and the pH value is 8.5.

Preferably, the digestion solution D contains 20mg/mL proteinase K.

Preferably, the particle size of the magnetic beads is 0.5-1 μm.

The invention also provides a method for extracting nucleic acid by using the kit, which comprises the following steps:

s1, adding the digestion solution D and the lysis binding solution A into the sample, mixing uniformly, centrifuging, and absorbing and removing supernatant;

s2, adding the washing liquid B, uniformly mixing for 10min, centrifuging, and then absorbing and removing the supernatant again;

s3, adding the washing liquid B, uniformly mixing for 1min, centrifuging, and then absorbing and removing the supernatant again;

and S4, adding the eluent C, carrying out warm bath for a period of time, shaking and uniformly mixing during the warm bath, centrifuging, and absorbing the supernatant into a new centrifugal tube to obtain the nucleic acid.

Preferably, the mixing volume ratio of the sample and the lysis binding solution A in S1 is 1: 3.

preferably, the temperature bath condition of S4 is 56-75 ℃, and the adding volume of the eluent C is 50-100 mu L.

Preferably, the addition volume of the washing solution B of S2 and S3 is 300-600 muL.

As a specific embodiment, the method for extracting nucleic acid according to the present invention comprises the steps of:

s1, adding 200 mu L of fresh plasma or serum sample into a 1.5ml centrifugal tube, adding 20 mu 1 of digestion solution D and 600 mu 1 of lysis binding solution A into the centrifugal tube, uniformly mixing for 15 minutes at room temperature, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 1min, and sucking and removing supernatant;

s2, adding 500 mu 1 of washing solution B into the centrifuge tube, uniformly mixing at room temperature for 10 minutes, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 1min, and sucking and removing the supernatant;

s3, adding 500 mu 1 of washing solution B into the centrifuge tube, uniformly mixing at room temperature for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 1 minute, and sucking and removing the supernatant;

s4, adding 100 mu 1 of eluent C into the centrifuge tube, carrying out warm bath for 6 minutes (shaking and mixing uniformly without stopping), placing the centrifuge tube on a magnetic frame after instantaneous centrifugation, carrying out magnetic separation for 1min, and absorbing the supernatant into a new centrifuge tube.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a virus RNA extraction kit, which comprises a cracking binding solution A, a washing solution B, an eluent C and a digestion solution D, wherein the cracking binding solution A contains 20-50 mM of sucrose, 20-50 mM of ammonium sulfate, 10-50 mM of Tris-HCl, 1-5% (V/V) of Brij-58 and 0.2-0.5 mg/mL of magnetic beads; the pH value is 4.5-5.5, the washing liquid B contains 1-5% (V/V) Brij-58, 5-25 mM Tris-HCl and 0.5-1 mg/mL proteinase K, and the pH value is 6-7; the kit is used for extracting RNA, the method does not depend on the traditional Chaotropic salt, RNA can be cracked, adsorbed and washed under the low-salt ion environment, and meanwhile, the washing liquid does not contain alcohol substances (ethanol) and can remove related salt and impurities. The method of the invention is simple and feasible, has low cost and does not contain any component polluting the environment.

Detailed Description

The invention is further illustrated by the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures, in which specific conditions are not indicated in the examples below, are generally carried out according to conditions conventional in the art or as recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.

Example 1

A kit for extracting viral RNA comprises a lysis binding solution A, a washing solution B, an eluent C and a digestion solution D (20 mg/mL proteinase K).

The cracking binding liquid A comprises the following components: 20mM sucrose, 50mM ammonium sulfate, 10mM Tris-HCl (pH 4.0), 1% (V/V) Brij-58, 0.5mg/ml magnetic beads, and autoclaved water as a solvent, and the final pH of the cleavage conjugate A was 4.5.

The composition of washing solution B was: 1% (V/V) Brij-58, 5mM Tris-HCl (pH 6.6), 1mg/ml proteinase K, autoclaved water as a solvent, and washing solution B at a final pH of 6.6.

The composition of eluent C was: 10mM Tris-HCl, 0.5mM EDTA in water, final pH 8.5 for eluent C.

The method for extracting HIV-1 plasma or serum sample RNA by adopting the kit comprises the following steps:

(1) adding 200 mu L of fresh HIV-1 plasma or serum sample into a 1.5ml centrifugal tube, adding 20 mu L of digestion solution D and 600 mu L of cleavage binding solution A into the centrifugal tube, uniformly mixing for 15 minutes at room temperature, placing the centrifugal tube on a magnetic frame, performing magnetic separation for 1min, and sucking and removing supernatant;

(2) adding 500 mu L of washing liquid B into the centrifuge tube, uniformly mixing for 10 minutes at room temperature, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 1min, and sucking and removing the supernatant;

(3) adding 500 mu L of washing liquid B into the centrifuge tube, uniformly mixing at room temperature for 1 minute, placing the centrifuge tube on a magnetic frame, performing magnetic separation for 1min, and sucking and removing the supernatant;

(4) adding 100 mu 1 of eluent C into the centrifuge tube, carrying out warm bath for 6 minutes (shaking and mixing uniformly without stop), placing the centrifuge tube on a magnetic frame after instantaneous centrifugation, carrying out magnetic separation for 1min, and absorbing the supernatant into a new centrifuge tube.

The sample amount extracted by using a QIAamp Viral RNA Mini Kit as a control reagent is 200 mul, and the elution volume is 100 mul.

After extraction is finished, 20 mul of the nucleic acid RNA extracting solution is taken as a template, detection is carried out by applying a human immunodeficiency syndrome virus type 1 nucleic acid detection kit (PCR fluorescence probe method) of the Daan gene, and the detection result is shown in the following table 1.

TABLE 1 comparison of CT values for different samples

Sample numbering	Inventive test results (CT value)	Qiagen reagent test results (CT value)
			1	24.21	24.35
2	35.98	35.32
			3	30.47	31.05
4	27.12	27.31
			5	32.34	32.67
6	36.21.	36.01

The result shows that the efficiency of extracting nucleic acid by using the kit of the embodiment 1 of the invention is in the same level with the extraction efficiency of the existing nucleic acid extraction reagent, so the invention has obvious advantages in the aspect of HIV-1 RNA nucleic acid extraction application.

Example 2

The cracking binding liquid A comprises the following components: 30mM sucrose, 30mM ammonium sulfate, 30mM Tris-HCl (pH 4.0), 3% (V/V) Brij-58, 0.4mg/ml magnetic beads, and autoclaved water as a solvent, and the final pH of the cleavage conjugate A was 5.0.

The composition of washing solution B was: 3% (V/V) Brij-58, 18 mM Tris-HCl (pH 6.6), 0.8mg/ml proteinase K, autoclaved water as the solvent, and washing solution B at a final pH of 6.0.

The composition of eluent C was: 15mM Tris-HCl, 0.5mM EDTA in water, final pH 8.5 for eluent C.

Example 3

The cracking binding liquid A comprises the following components: 50mM sucrose, 20mM ammonium sulfate, 50mM Tris-HCl (pH 4.0), 5% (V/V) Brij-58, 0.5mg/ml magnetic beads, and autoclaved water as a solvent, and the final pH of the cleavage conjugate A was 5.5.

The composition of washing solution B was: 5% (V/V) Brij-58, 25mM Tris-HCl (pH 6.6), 0.5mg/ml proteinase K, autoclaved water as the solvent, and Wash B at a final pH of 7.0.

The composition of eluent C was: 20mM Tris-HCl, 0.5mM EDTA in water, final pH 8.5 for eluent C.

Comparative example 1

The only difference between the RNA kit used in this comparative example and the RNA kit described in example 1 was that the pH of the lysis buffer a was 7.0, and the results of the detection using the method described in example 1 were: the results of comparative example 1 are significantly inferior to those of example 1, and the low concentration sample has no test result. The results are shown in Table 2.

TABLE 2 comparison of CT values for different samples

Sample numbering	Example 1 results (CT value)	Comparative example 1 test results (CT value)
			1	24.58	28.98
2	35.12	-
			3	30.06	38.05
4	27.32	32.14
			5	32.54	38.37
6	36.83	-

Comparative example 2

The RNA kit used in this comparative example was identical to the RNA kit described in example one, except that the concentration of Brij-58 in the lysate was 10% (V/V), and the results were shown by performing the assay as described in example 1: the results of comparative example 2 are significantly worse than example 1, demonstrating that the extraction efficiency is not optimal. The results are shown in Table 3.

TABLE 3 comparison of CT values for different samples

Sample numbering	Example 1 results (CT value)	Comparative example 2 test results (CT value)
			1	24.58	27.17
2	35.12	38.72
			3	30.06	32.15
4	27.32	29.52
			5	32.54	34.37
6	36.83	38.85

Claims

1. The extraction kit of the virus RNA is characterized by comprising a cleavage binding solution A, a washing solution B, an eluent C and a digestion solution D, wherein the cleavage binding solution A contains 20-50 mM of sucrose, 20-50 mM of ammonium sulfate, 10-50 mM of Tris-HCl, 1-5% of V/V Brij-58 and 0.2-0.5 mg/mL of magnetic beads, and the pH value is 4.5-5.5; the washing solution B contains 1-5% of V/VBrij-58, 5-25 mM Tris-HCl and 0.5-1 mg/mL proteinase K, and the pH value is 6-7; the eluent C contains 10-20 mM Tris-HCl and 0.5mM EDTA, and the pH value is 8.5; the digestion solution D contains 20mg/mL proteinase K.

2. The kit for extracting viral RNA according to claim 1, wherein the magnetic beads have a particle size of 0.5 to 1 μm.

3. A method for extracting nucleic acid using the kit of claim 1 or 2, comprising the steps of:

s1, taking a virus sample, adding a digestion solution D and a lysis binding solution A, mixing uniformly, centrifuging, and then sucking and removing supernatant;

s2, adding the washing liquid B, uniformly mixing for 10min, centrifuging, and then, absorbing and removing the supernatant again;

s4, adding the eluent C, carrying out warm bath for a period of time, shaking and uniformly mixing during the warm bath, centrifuging, and then absorbing the supernatant into a new centrifugal tube to obtain the virus RNA.

4. The method of claim 3, wherein the mixing volume ratio of the sample and lysis binding solution A of S1 is 1: 3.

5. the method according to claim 3, wherein the temperature bath condition of S4 is 56-75 ℃, and the volume of eluent C added is 50-100 μ L.

6. The method according to claim 3, wherein the washing solution B is added in a volume of 300 to 600. mu.L in S2 and S3.