CN113025686B

CN113025686B - Virus RNA releasing agent, kit and application thereof

Info

Publication number: CN113025686B
Application number: CN202110269093.8A
Authority: CN
Inventors: 贾春蕾; 曹振龙; 刘少卿; 宋晓东
Original assignee: Jiangsu Genesmile Precision Medical Technology Co ltd
Current assignee: Jiangsu Genesmile Precision Medical Technology Co ltd
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2023-07-28
Anticipated expiration: 2041-03-12
Also published as: CN113025686A

Abstract

The invention discloses a viral RNA release agent, a kit and application thereof, wherein the release agent comprises 0.01-0.05mmol/L of multi-fruit extract, 0.05-0.5% (v/v) of triton, 5-15mmol/L of potassium chloride and 0.1-0.5% (v/v) of sodium hydroxide. Compared with the prior art, the reagent used in the formula of the release agent has low price, the preparation method is simple, and the contained reagent has no chemical hazard. The reagent can effectively crack viruses, safely and rapidly release nucleic acid, and is simple to operate and short in time consumption. The released product has no inhibition effect on downstream experiments, and the amplification efficiency of subsequent PCR and other experiments is not affected.

Description

Virus RNA releasing agent, kit and application thereof

Technical Field

The invention belongs to the technical field of RNA extraction, and particularly relates to a viral RNA release agent, a kit and application thereof.

Background

Nucleic acid is a generic name of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biomacromolecule compounds polymerized from a number of nucleotide monomers, and are one of the most basic substances for life. Nucleic acid is a kind of biopolymer, is an indispensable constituent substance of all known life forms, is the most important substance in all biomolecules, and is widely existing in all animal and plant cells and microorganisms. The nucleic acid consists of nucleotides, while the nucleotide monomers consist of five carbon sugars, phosphate groups and nitrogen-containing bases. If the five carbon sugar is ribose, then the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.

Nucleic acid extraction is an essential basic technology for modern molecular biology research, and the preparation of high-quality nucleic acid is the key to the success or failure of subsequent research. For example, when detecting nucleic acid against novel coronaviruses in the world, the precondition is that the nucleic acid in the novel coronavirus particles is released or extracted, so that the nucleic acid detection can be smoothly performed, and the requirements of epidemic prevention and control are further met. The principle of nucleic acid extraction firstly ensures the integrity of the primary structure of the nucleic acid and secondly eliminates the contamination of other molecules. Methods for extracting nucleic acids have a long history and various extraction methods are formed. The method for extracting deoxyribonucleic acid (DNA) comprises a classical method of firstly cleaving and releasing DNA and then purifying by chromatography, such as an SDS method, a CTAB method, an enzyme cleavage method and the like, and various commercial kits formed by extending and optimizing DNA extraction methods based on the basis, wherein the most main methods include a column extraction method, a magnetic bead method and the like. For ribonucleic acid (RNA) extraction, for example, extraction of RNA of novel coronaviruses, the classical methods include Trizol method, and commercial kits such as column extraction method and magnetic bead method.

However, the above-mentioned nucleic acid extraction techniques or kits, although widely used, have highlighted their disadvantages in coping with the present novel coronavirus epidemic: long nucleic acid extraction time, complex process, easy occurrence of cross contamination and the like. Under the above circumstances, there is a need and demand for a nucleic acid preparation technique which is convenient and rapid. The novel nucleic acid releasing agent for the China has the advantages that the novel nucleic acid releasing agent for the Santa Classification of Hunan is produced in advance, and the market demand is greatly met.

Even so, existing nucleic acid releasing agents have a low efficiency of viral nucleic acid release, resulting in a reduced sensitivity for nucleic acid detection applications. Actual experimental results prove that the SDS proportion in the existing nucleic acid releasing agent formula is slightly insufficient, so that the subsequent PCR experiment is severely inhibited.

Disclosure of Invention

The invention aims to: aiming at the defects in the prior art, the invention provides a viral RNA releasing agent and application thereof, the releasing agent has low cost, convenient and quick use, can improve the release efficiency of nucleic acid, and the extracted product has no inhibition effect on the subsequent PCR reaction and has no special requirement on temperature.

The technical scheme is as follows: in order to achieve the aim of the invention, the invention adopts the following technical scheme:

a viral RNA delivery agent comprising 0.01-0.04mmol/L of dodine (dodecylguanidine acetate), 0.05% -0.5% (v/v) Triton (Triton X-100), 5-15mmol/L potassium chloride (KCl), 0.1% -0.5% (v/v) sodium hydroxide.

Preferably, the sodium hydroxide is a 50% strength aqueous sodium hydroxide solution.

In the release agent, the use of the polyvidone instead of the traditional guanidine salt can reduce the inhibition effect on downstream experiments, and can effectively crack a sample to release nucleic acid. The triton is a widely used nonionic surfactant, and can crack viral coat proteins and separate nucleic acids from proteins. The potassium chloride can provide salt ions, damage the stability of protein and increase the release efficiency. The sodium hydroxide is capable of denaturing proteins.

Preferably, the release agent comprises 0.02-0.03mmol/L of multi-fruit, 0.05% -0.1% (v/v) of triton, 5-10mmol/L of potassium chloride and 0.1% -0.3% (v/v) of sodium hydroxide.

The concentration of the multocarry is controlled to be 0.02-0.03mmol/L, and the multocarry is used as a reagent component to obviously reduce the inhibition effect on the PCR reaction. The addition amount of the triton is controlled within the range of 0.05% -0.1% (v/v) and the best effect is achieved. The sodium hydroxide added in the reagent is 0.1% -0.3% (v/v), and can exist more stably with other reagents.

The Triton (Triton X-100) reagent is sigma brand, proclin300 is Solarbio brand, and the reagent is directly used after purchase.

Preferably, the release agent further comprises 0.05% -0.1% (v/v) proclin300, which enables a substantial increase in the stabilizing effect of the agent.

Preferably, the solvent of the release agent is selected from deionized water.

The invention finally provides application of the viral RNA releasing agent in extracting viral RNA.

The releasing agent provided by the invention avoids the requirement on temperature in the extraction and release process, and the reaction can be extracted at normal temperature, so that the operation is simple and convenient. The releasing agent can complete cracking in the reaction process only for 5-10min, thus greatly saving the reaction time and improving the efficiency

The beneficial effects are that: compared with the prior art, the reagent used in the formula of the release agent has low price, the preparation method is simple, and the contained reagent has no chemical hazard. The reagent can effectively crack viruses, safely and rapidly release nucleic acid, and is simple to operate and short in time consumption. The released product has no inhibition effect on downstream experiments, and the amplification efficiency of subsequent PCR and other experiments is not affected.

Drawings

FIG. 1 is a graph showing PCR amplification after releasing pseudoviral nucleic acid by the nucleic acid releasing agent of the present invention.

FIG. 2 is a graph of PCR amplification after release of pseudoviral nucleic acid by the St.Hunan organism nucleic acid releasing agent.

FIG. 3 is a combined PCR amplification curve of pseudoviral nucleic acid released by the nucleic acid releasing agent of the present invention.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the examples described below, unless otherwise specified, are all conventional; the materials, reagents, instruments and the like used, unless otherwise specified, are commercially available.

EXAMPLE 1 nucleic acid Release agent preparation procedure of the invention

1. Reagent preparation: preparing multocaryum, potassium chloride, triton and sodium hydroxide.

Wherein, the multi-fruit order is from sigma company, the name: dodin, cat No.: 45466, dry powder; triton (Triton-X100) was purchased from sigma company under the name: triton-X100, cat: t8787, liquid (100%); sodium hydroxide was purchased from sigma company under the name: sodium hydroxidesolution, cargo number: STBJ3989, liquid (content 50% in H ₂ O); the potassium chloride is dry powder or granule, and is obtained from common commercial brands in China and is chemically pure. proclin300 is a Solarbio brand, 100% liquid, and is used directly after purchase.

2. The preparation process of the reagent comprises the following steps:

a, mother liquor configuration: preparing a mother solution with the concentration of 2mmol/L with the multocaryum according to the state or concentration of the original material, and preparing a mother solution with the concentration of 1mol/L with potassium chloride;

b, according to 10ml:1.5ml:10ml:1.5ml: the mother liquor of multocaryum, the triton reagent, the mother liquor of potassium chloride, sodium hydroxide and proclin300 are added in sequence in a volume ratio of 0.5ml, and after all the materials are added, deionized water is used for metering the volume to 1L.

c, mixing the prepared liquids to obtain the nucleic acid releasing agent.

EXAMPLE 2 Effect application of the nucleic acid releasing agent of the present invention

1. The pseudoviruses are prepared for extraction, and the pseudoviruses are inactivated at 56 ℃ for 30min. After inactivation, the pseudoviruses were diluted to 200copies/ul and prepared for RNA release.

2. The consumables used for the experiment were prepared, 10ul of diluted pseudovirus was added to a 1.5ml centrifuge tube, and then the release agent of the invention obtained in example 1 was added in a 1:1 ratio or a 1:2 ratio. The reaction is carried out at room temperature for 5 to 10 minutes. Release of RNA was completed and 3 replicates were made.

3. Taking 5ul of released product, adding 15ul of amplification reagent, and performing PCR reaction on a macrostone SLAN-96P fluorescent quantitative PCR instrument, wherein the reaction program is 55 ℃ for 10min,95 ℃ for 1min, (95 ℃ for 10S,60 ℃ for 1min for 45 cycles).

4. After the PCR amplification is finished, an amplification curve graph 1 is obtained;

example 3 St.Hunan biological nucleic acid Release agent application test

1. The pseudoviruses were prepared for extraction and inactivated at 56℃for 30min under the same conditions as in example 2. After inactivation, the pseudoviruses were diluted to 200copies/ul and prepared for RNA release.

2. The consumables used for the experiment were prepared, 10ul of diluted pseudovirus was added to a 1.5ml centrifuge tube, and then nucleic acid releasing reagent was added in a 1:1 ratio or a 1:2 ratio to the san Hunan organism Co., ltd. The reaction is carried out at room temperature for 5 to 10 minutes. Release of RNA was completed and 3 replicates were made.

3. 5ul of the released product was taken, 15ul of amplification reagent was added, and PCR was performed on a macrostone SLAN-96P fluorescent quantitative PCR apparatus together with example 2, the reaction procedure was 55℃for 10min,95℃for 1min, (95℃for 10S,60℃for 1min45 cycles).

4. After the PCR amplification is finished, an amplification curve graph 2 is obtained;

5. overlapping the PCR amplification curves of example 2 and example 3 to obtain an overlapping amplification curve graph 3; 6. the data show that both example 2 and example 3 achieved better amplification efficiency, and that the results shown in FIG. 3 after the combined analysis found that the amplification efficiency of example 2 was equivalent or due to the experimental results of example 3.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.

Claims

1. A viral RNA releasing agent is characterized in that the releasing agent comprises 0.02-0.03mmol/L of multi-fruit extract, 0.05-0.1% (v/v) Triton X-100, 5-10mmol/L of potassium chloride, 0.1-0.3% (v/v) sodium hydroxide and 0.05-0.1% (v/v) proclin300, and the solvent of the releasing agent is selected from deionized water.

2. A kit for extracting viral RNA, comprising the viral RNA delivery agent of claim 1.

3. Use of the viral RNA delivery agent of claim 1 for extracting viral RNA.

4. Use of the kit of claim 2 for extracting viral RNA.