阅读说明：本技术 一种rna病毒核酸提取液及提取方法 (RNA virus nucleic acid extracting solution and extracting method ) 是由 孙子奎 王展 朱月艳 徐福桥 于 2020-08-18 设计创作，主要内容包括：本发明公开了一种RNA病毒核酸提取液,其特征在于,包括：200μl～400ul裂解液；400ul～600ul漂洗液A；400ul～600ul漂洗液B；50ul～100ul洗脱液；20ul～40ul磁珠；5～6μlCarrierRNA；所述裂解液中,以1升的含量计算,包括如下组分：50mM～100mMTris；10mM～50mMEDTA；0.1M～0.2MNACL；4.5M～5MGITC；0.1％～0.2％SDS；5％～8％Tween-20；3％～5％TritonX-100；0.05％～0.2％β-ME。本发明还公开了提取方法。本发明通过在裂解液中加入β-ME,抑制了Rnase酶活性,从而有效地防止了RNA的降解,通过控制上样比例在保证得率的同时,降低了提取时样本间的污染。另外本发明的方法20min即可实现核酸的提取,提取效率高。(The invention discloses an RNA virus nucleic acid extracting solution which is characterized by comprising the following components: 200 mul to 400ul of lysate; 400 ul-600 ul of rinsing liquid A; 400 ul-600 ul of rinsing liquid B; 50ul to 100ul of eluent; 20 ul-40 ul of magnetic beads; 5-6 μ l of Carrier rRNA; the lysis solution comprises the following components in 1 liter: 50 mM-100 mM Tris; 10 mM-50 mM EDTA; 0.1M-0.2 MNACL; 4.5M to 5 MGITC; 0.1 to 0.2 percent SDS; 5 to 8 percent of Tween-20; 3 to 5 percent of TritonX-100; 0.05 to 0.2 percent of beta-ME. The invention also discloses an extraction method. The invention inhibits the RNase activity by adding beta-ME into the lysis solution, thereby effectively preventing the degradation of RNA, ensuring the yield by controlling the sample loading proportion and reducing the pollution among samples during extraction. In addition, the method can realize the extraction of nucleic acid within 20min, and has high extraction efficiency.)

1. An RNA virus nucleic acid extraction solution, comprising:

200 mul to 400ul of lysate;

400 ul-600 ul of rinsing liquid A;

400 ul-600 ul of rinsing liquid B;

50ul to 100ul of eluent;

20 ul-40 ul of magnetic beads;

5～6μl CarrierRNA

the lysis solution comprises the following components in 1 liter:

50mM～100mMTris；

10mM～50mMEDTA；

0.1M～0.2MNACL；

4.5M～5MGITC；

0.1％～0.2％SDS；

5％～8％Tween-20；

3％～5％TritonX-100；

0.05％～0.2％β-ME。

2. the RNA virus nucleic acid extraction solution of claim 1, wherein the rinsing solution A comprises the following components in an amount of 1 liter:

5mM～15mMTris；

0.5M～1.0MNaOAc；

1.5％～2％Tween-20；

50％～60％EOH。

3. the RNA virus nucleic acid extraction solution of claim 1, wherein the rinsing solution B comprises the following components in an amount of 1 liter:

5mM～20mMTris；

70％～80％EOH。

4. the RNA viral nucleic acid extract according to claim 1, wherein the eluate contains, in an amount of 1 liter, the following components:

2.5mM～5mMTris。

5. the RNA virus nucleic acid extraction solution according to claim 1, wherein the magnetic beads are commercially available magnetic beads and have a concentration of 50 mg/ml; the CarrierrRNA is 1ug/ul of CarrierrRNA.

6. The RNA virus nucleic acid extract according to claim 1, wherein the pH of the lysate, the rinse A, the rinse B and the eluate is 8.0.

7. The method for extracting nucleic acid from RNA virus of any one of claims 1 to 6, comprising the steps of:

adding the lysis solution, the CarrierRNA and the magnetic beads into a test tube filled with a sample to be detected, performing magnetic mixing on a magnetic rack, standing until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

taking the test tube off the magnetic rack, adding the rinsing liquid A, uniformly mixing, standing, placing on the magnetic rack again for magnetic adsorption until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

taking the test tube off the magnetic rack, adding the rinsing liquid B, uniformly mixing, standing, placing on the magnetic rack again for magnetic adsorption until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

and taking the test tube off the magnetic frame, adding the eluent to elute the nucleic acid on the magnetic beads, wherein the eluted nucleic acid is RNA solution.

8. The method for extracting nucleic acid from RNA virus of any one of claims 1 to 6, comprising the steps of:

step one, adding the following reagents into an extraction plate:

sequentially adding the lysis solution, the sample to be detected, CarrierRNA and the magnetic beads into the first hole;

adding the rinsing liquid A into the second hole;

adding the rinsing liquid B into the third hole;

adding the eluent into the fifth hole

And step two, setting the extraction steps according to the table 1, and then extracting, wherein the solution in the fifth hole is the RNA virus genome nucleic acid.

9. The method for extracting nucleic acid from RNA viruses according to claim 7, wherein the volume ratio of the lysate to the sample to be tested is 1: 1.

10. the method for extracting nucleic acid from RNA viruses according to claim 8, wherein the volume ratio of the lysate to the sample to be tested is 1: 1.

Technical Field

The invention relates to the field of nucleic acid extraction, and particularly relates to an RNA virus nucleic acid extracting solution and an extraction method.

Background

RNA virus is one of important pathogens of infectious diseases, and the detection of the RNA virus has important significance for clinical treatment. In clinical practice, virus nucleic acid detection can be used as an important reference basis for virus infection, and virus nucleic acid extraction is the primary work. However, the RNase enzyme is widely existed, the RNA virus nucleic acid is extremely easy to degrade in the extraction process, so that the false negative phenomenon can occur in the detection process.

At present, most of nucleic acid extraction of RNA viruses adopts a silicon matrix membrane adsorption method, RNA is adsorbed by using a centrifugal adsorption column, and then impurities such as protein, polysaccharide and the like are washed away by using a specific rinsing liquid so as to achieve the purposes of separation and purification.

The silicon substrate membrane adsorption method has the defects that repeated centrifugation is needed, and waste liquid is poured; the operation is complicated, the time consumption is long, and cross contamination is easy to occur when a plurality of samples are processed; because the sample is independently processed by a single tube, the automatic operation is difficult to form, and the clinical requirement of high-throughput detection cannot be met.

In addition, the redistilled phenol used in the TRIzol process is toxic; the magnetic bead method has higher cost and low extraction rate; the common water boiling method cannot be directly used for extracting RNA, mainly because endogenous RNA enzymes exist in a sample, and the RNA enzymes degrade the RNA. Therefore, there is a need in the art for a method that can be easily and conveniently performed and that can extract RNA with high efficiency.

Disclosure of Invention

In order to overcome the above defects of the prior art, the present invention aims to provide an RNA virus nucleic acid extraction solution and an extraction method.

In order to realize the purpose of the invention, the adopted technical scheme is as follows:

an RNA virus nucleic acid extraction solution comprising:

200 mul to 400ul of lysate;

400 ul-600 ul of rinsing liquid A;

400 ul-600 ul of rinsing liquid B;

50ul to 100ul of eluent;

20 ul-40 ul of magnetic beads;

5～6μl CarrierRNA

the lysis solution comprises the following components in 1 liter:

50mM～100mMTris；

10mM～50mMEDTA；

0.1M～0.2MNACL；

4.5M～5MGITC；

0.1％～0.2％SDS；

5％～8％Tween-20；

3％～5％TritonX-100；

0.05％～0.2％β-ME。

in a preferred embodiment of the invention, the rinsing liquid A comprises the following components in 1 liter:

5mM～15mMTris；

0.5M～1.0MNaOAc；

1.5％～2％Tween-20；

50％～60％EOH。

in a preferred embodiment of the present invention, the rinsing liquid B comprises the following components in an amount of 1 liter:

5mM～20mMTris；

70％～80％EOH。

in a preferred embodiment of the present invention, the eluent comprises the following components in an amount of 1 liter:

2.5mM～5mMTris。

in a preferred embodiment of the present invention, the magnetic beads are commercially available magnetic beads with a concentration of 50 mg/ml.

In a preferred embodiment of the invention, the CarrierRNA is 1ug/ul of CarrierRNA.

In a preferred embodiment of the present invention, the pH of the lysis solution, the rinsing solution A, the rinsing solution B and the eluent is 8.0.

A method for extracting RNA virus nucleic acid extracting solution comprises the following steps:

adding the lysis solution, the CarrierRNA and the magnetic beads into a test tube filled with a sample to be detected, performing magnetic mixing on a magnetic rack, standing until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

taking the test tube off the magnetic rack, adding the rinsing liquid A, uniformly mixing, standing, placing on the magnetic rack again for magnetic adsorption until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

taking the test tube off the magnetic rack, adding the rinsing liquid B, uniformly mixing, standing, placing on the magnetic rack again for magnetic adsorption until the magnetic beads are completely adsorbed on the tube wall of the test tube, and then discarding the liquid in the test tube;

and taking the test tube off the magnetic frame, adding the eluent to elute the nucleic acid on the magnetic beads, wherein the eluted nucleic acid is RNA solution.

An automatic extraction method of RNA virus nucleic acid comprises the following steps:

step one, adding the following reagents into an extraction plate:

sequentially adding the lysis solution, the sample to be detected, CarrierRNA and the magnetic beads into the first hole;

adding the rinsing liquid A into the second hole;

adding the rinsing liquid B into the third hole;

adding the eluent into the fifth hole.

And step two, setting the extraction steps according to the table 1, and then extracting, wherein the solution in the fifth hole is the RNA virus genome nucleic acid.

In a preferred embodiment of the present invention, the volume ratio of the lysis solution to the sample to be tested is 1: 1.

the invention has the beneficial effects that:

through adding beta-ME into the lysate, the RNase activity is inhibited, so that the degradation of RNA is effectively prevented, and the pollution among samples during extraction is reduced while the yield is ensured by controlling the sample loading proportion. In addition, the method can realize the extraction of nucleic acid within 20min, and has high extraction efficiency.

Drawings

FIG. 1 is a diagram showing the results of HCV quality control extraction.

FIG. 2 is a schematic diagram of the detection result of HIV quality control extraction.

FIG. 3 is a schematic diagram showing the detection result of the extraction of the pseudovirus of the new coronavirus.

FIG. 4 is a graph showing the results of the contamination test between samples.

Detailed Description

The main principle of the invention is as follows: through adding beta-ME into the lysis solution, the RNase enzyme activity is inhibited, thereby effectively preventing the degradation of RNA.

By controlling the sample loading ratio (the volume ratio of the lysate to the sample to be detected), the yield is ensured, and the pollution among samples during extraction is reduced.

The extraction method of the RNA virus nucleic acid extracting solution can realize the extraction of nucleic acid within 20min, and has high extraction efficiency.

The automatic extraction method of RNA virus nucleic acid can meet the requirement of high clinical flux.

Extraction example 1

A method for extracting RNA virus nucleic acid comprises the following steps:

and (3) cracking the sample: adding 200 mul of sample into 1.5ml of EP tube, adding 200 mul of lysis solution and 6 mul of Carrier rRNA, mixing uniformly, standing at room temperature for 10min, placing the EP tube on a magnetic frame, adsorbing for 20s, and absorbing the liquid in the EP tube when the magnetic beads are completely adsorbed on the wall of the EP tube;

washing nucleic acid: taking off the EP tube from the magnetic frame, adding the rinsing liquid A600ul into the EP tube, uniformly mixing, standing at room temperature for 2min, placing the EP tube on the magnetic frame, adsorbing for 15s, and absorbing and discarding the liquid in the EP tube when the magnetic beads are completely adsorbed on the wall of the EP tube;

washing nucleic acid, taking off the EP tube from the magnetic rack, adding a rinsing liquid B600ul into the EP tube, uniformly mixing, standing at room temperature for 1min, placing the EP tube on the magnetic rack, adsorbing for 10s, and when magnetic beads are completely adsorbed on the wall of the EP tube, sucking and discarding the liquid in the EP tube, and repeating once;

elution of nucleic acids: and taking down the EP tube from the magnetic frame, and adding 50 ul-100 ul of eluent to elute the nucleic acid on the magnetic beads to obtain the RNA solution.