CN112662663A - RNA extraction kit suitable for normal-temperature transportation and extraction method - Google Patents

Abstract

The invention relates to an RNA extraction kit suitable for normal-temperature transportation, which comprises a lysis binding solution, wherein the lysis binding solution comprises the following components: 1-5M of guanidinium isothiocyanate, 0.2-3M of sodium thiocyanate, 0.01-0.1M of trihydroxymethyl aminomethane, 0.001-0.05M of tetrasodium iminodisuccinate, 0.05-1% of sodium ascorbate, 0.1-2% of betaine, 1-6% of Tween 20, 0.05-0.5M of sodium acetate and 25-55% of isopropanol. The split binding solution does not contain toxic organic solvents such as phenol and the like, and does not contain reagents such as proteinase K and the like which need cold chain transportation, so the RNA extraction kit can be transported at normal temperature. The lysis binding solution simultaneously has the effects of lysing cells/viruses and promoting the binding of nucleic acid and magnetic beads, can complete lysis and binding in one step, and is more suitable for an automatic nucleic acid extractor. The invention combines two protein denaturants, and can enhance the removal capacity of complex chemical property protein and other impurities compared with the single use of one protein denaturant.

Description

RNA extraction kit suitable for normal-temperature transportation and extraction method

Technical Field

The invention relates to the technical field of nucleic acid treatment, in particular to an RNA extraction kit and an extraction method suitable for normal-temperature transportation.

Background

With the development of detection technology, pathogen nucleic acid detection has become the mainstream detection means for respiratory tract pathogen diagnosis due to its high sensitivity and high specific detection performance. By detecting pathogen nucleic acid in respiratory tract specimens such as nasopharyngeal swabs, airway extracts or sputum of patients, common respiratory tract system infection and malignant pathogen infection are accurately and quickly detected and distinguished, pathogen infection is quickly diagnosed, and pathogen types and subtypes/lines can be quickly distinguished, so that basis and guidance are provided for subsequent targeted prevention and control and treatment measures.

Nucleic acid extraction is the first step of pathogenic nucleic acid detection, and the accuracy and reliability of subsequent detection results are directly influenced by the yield and quality of an extraction product. High purity and high concentration of nucleic acid samples are one of the important conditions for obtaining good experimental results in molecular biology experiments. The most commonly used methods for nucleic acid extraction at present are Trizol method, column membrane method and magnetic bead adsorption method. The Trizol method mainly uses phenol as a reagent, the phenol is harmful to human bodies, and the extraction process needs to be carried out by blowing with a gun or violent shaking, centrifuging for many times and the like, so that the Trizol method is not beneficial to automation; although the column membrane method is quick, simple and convenient, the obtained nucleic acid has high purity, does not use toxic solvent, but needs multiple centrifugal operations, has low RNA extraction efficiency, is not beneficial to an automatic extraction process and has low flux. The magnetic bead method takes superparamagnetic nano microspheres as a carrier, can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface, can quickly separate and purify DNA or RNA, does not need centrifugation or filtration in the extraction process, can be manually operated, can also be finished in an automatic working platform mode, has high extraction flux and good automation degree, and is suitable for the requirement of large-sample-quantity extraction. The magnetic bead method for extracting nucleic acid mainly comprises the following operation steps: (1) cracking; (2) combining; (3) washing; (4) and separating the four major parts. Specifically, RNA is released under the action of lysis solution, superparamagnetic silica nano magnetic beads with modified surfaces are specifically combined with the released RNA to form a nucleic acid-magnetic bead compound, washing liquid is added into the nucleic acid-magnetic bead compound subsequently to wash away impurities such as non-specifically adsorbed proteins, the nucleic acid-magnetic bead compound with the impurities removed is separated and enriched with magnetic beads under the condition of a magnetic field, and finally the nucleic acid substance to be extracted is obtained.

However, the extraction by the magnetic bead method also has some problems at present. Mainly comprises the following aspects: (1) most of nucleic acid extraction kits by a magnetic bead method contain proteinase K, and the proteinase K needs cold chain transportation and is easy to inactivate if transported at normal temperature. The cold chain conveying condition is high in requirement, the cost is high (especially in summer), and the quick and emergency transportation is not facilitated. (2) At present, the extraction process of commercial extraction kits based on the paramagnetic particle method in the market generally needs a heating step for lysis incubation and nucleic acid elution, the heating can damage nucleic acid to a certain extent, especially RNA in a sample, and if the heating at 50-60 ℃ is adopted in the extraction process, RNase (RNase) introduced in the sample, reagent consumables or environment can reach the optimal temperature condition, and further the degradation loss of the RNA sample is caused. Such a loss is not acceptable even in the case of a small amount of a sample to be detected and a small amount of virus in the sample, and is likely to cause serious misjudgment of a result. (3) Most of the magnetic bead method for extracting nucleic acid is to complete sample cracking, combination, washing and separation step by step, is easy to introduce a plurality of containers and consumables, increases the risk of introducing exogenous RNA or RNase, and is not beneficial to automation.

Disclosure of Invention

Technical problem to be solved

In view of the defects and shortcomings of the prior art, the RNA extraction kit suitable for normal-temperature transportation is provided, the problem that the RNA extraction kit based on proteinase K cannot be transported at normal temperature is solved, the RNA extraction kit does not contain toxic organic solvents, heating treatment is not needed in the process of extracting sample RNA, cracking and combination are completed simultaneously by adopting one-step treatment, cracking and combination are not needed to be separated, the automatic nucleic acid extractor is more suitable, the operation process is simple, convenient and quick, the kit is also suitable for pre-packaging reagents into deep-pore plates, and the automatic operation is simpler, more convenient and quicker.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the present invention provides an RNA extraction kit suitable for normal temperature transportation, comprising a lysis binding solution, wherein the lysis binding solution comprises the following components:

1-5M of guanidinium isothiocyanate, 0.2-3M of sodium thiocyanate, 0.01-0.1M of trihydroxymethyl aminomethane, 0.001-0.05M of tetrasodium iminodisuccinate, 0.05-1% of sodium ascorbate, 0.1-2% of betaine, 1-6% of Tween 20, 0.05-0.5M of sodium acetate and 25-55% of isopropanol.

According to a preferred embodiment of the present invention, the composition of the lysis binding solution is:

guanidine isothiocyanate with the concentration of 2M, sodium thiocyanate with the concentration of 1M, trihydroxymethyl aminomethane with the concentration of 0.05M, tetrasodium iminodisuccinate with the concentration of 0.01M, sodium ascorbate with the mass fraction of 0.10%, betaine with the mass fraction of 0.5%, Tween 20 with the mass fraction of 5%, sodium acetate with the concentration of 0.2M and isopropanol with the mass fraction of 45%.

According to a preferred embodiment of the present invention, the RNA extraction kit further comprises a suspension of magnetic beads, a first washing solution, a second washing solution, and an eluent;

preferably, the suspension of magnetic beads consists of: the superparamagnetic silica nano magnetic bead with the solid content of 20-40mg/mL has the particle size of 200-500nm, is dispersed in nuclease-free water, and has the pH value of 4.0-6.0.

Preferably, the first wash liquor consists of: 20-200mM of trihydroxymethyl aminomethane, 1-50mM of imino disuccinic acid tetrasodium, 100-500mM of sodium acetate, 0.1-2% of Tween 20 and 40-60% of ethanol in volume fraction.

Preferably, the second wash solution is 60-80% ethanol by volume fraction.

Preferably, the eluent consists of the following components: 1-10mM tetrasodium iminodisuccinate, 1-100mM tris-HCl buffer, 0.01-0.2% DEPC (diethyl pyrocarbonate).

According to the preferred embodiment of the present invention, the solid content of the magnetic bead suspension is 25 mg/mL; the first wash solution consists of the following components: 100mM of tris (hydroxymethyl) aminomethane, 10mM of tetrasodium iminodisuccinate, 140mM of sodium acetate, 0.50% of Tween 20 and 55% of ethanol; the second washing solution is 70% absolute ethyl alcohol; the eluent consists of the following components: 1mM tetrasodium iminodisuccinate, 10mM tris-HCl buffer, 0.010% DEPC (diethyl pyrocarbonate).

The remaining parts of the cleavage-bound solution, the first washing solution, the second washing solution, and the eluent, which are not specifically described, are RNase (nuclease) -free water.

In a second aspect, the present invention also provides a method for extracting viral nucleic acid from a sample using the RNA extraction kit described above, the method comprising the steps of:

(1) sample preparation: soaking the sampling swab sample in physiological saline, fully shaking and uniformly mixing, standing for 2-5min, and centrifuging at the speed of 800 plus 12,000rpm to prepare a virus sample preservation solution;

(2) sample lysis and nucleic acid adsorption: taking 300-500 mu L of virus sample preservation solution for detection; adding 1-2 times of the volume of the lysis binding solution into the virus sample preservation solution obtained in the step (1), simultaneously adding 15-40uL of magnetic bead suspension, uniformly mixing, placing on a magnetic frame, standing, and removing liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;

(3) rinsing: adding 550-; adding 550-;

(4) sample elution: and (4) adding 550-650 mu L of eluent into the magnetic beads obtained in the step (3), vibrating and uniformly mixing, placing on a magnetic frame, collecting the eluent after the magnetic beads are adsorbed, namely the obtained nucleic acid solution, and storing at low temperature (-80 ℃).

According to a preferred embodiment of the method of the present invention, the extraction process is performed using a 32-channel automatic nucleic acid extractor.

According to the preferred embodiment of the method of the present invention, the method of extracting using a 32-channel automatic nucleic acid extractor comprises: after obtaining the virus sample preservation solution, the following steps are carried out:

step 1: adding 400-500uL lysis binding solution, 300-500uL virus sample preservation solution and 15-40uL magnetic bead suspension into the deep pore plate 1/7; the extractor is set as follows: mixing for 15min at high speed for 60s without heating;

step 2: 600uL of the first washing solution 550-550 was added in the column of the deep-hole plate 2/8; the extractor is set as follows: mixing for 2min at medium speed for 60s without heating;

and step 3: 600uL of the second washing solution 550-550 was added in the column of the deep-hole plate 3/9; the extractor is set as follows: mixing for 2min at medium speed for 60s without heating;

and 4, step 4: adding eluent 55-60uL into a deep hole plate 4/10 column; the extractor is set as follows: after waiting for 6min, mixing for 10min, wherein the mixing speed is low, the magnetic attraction time is 90s, and heating is not started.

And finally, the magnetic beads are recycled, namely the magnetic beads in the eluent are transferred to the washing liquid in front, so that the magnetic beads are prevented from being adhered to the magnetic rod sleeve to pollute the detection environment.

(III) advantageous effects

(1) The lysis binding solution simultaneously comprises a lysis component and a binding accelerator, wherein guanidinium isothiocyanate and sodium thiocyanate are protein denaturants and have the function of cell/virus structure lysis; sodium acetate and isopropanol are used as binding promoters to promote the binding of nucleic acid and magnetic beads; wherein, the trihydroxymethyl aminomethane, the iminodisuccinic acid tetrasodium and the sodium ascorbate are stable components, the trihydroxymethyl aminomethane plays a role of a buffer agent to provide a stable buffer environment, the iminodisuccinic acid tetrasodium is a metal ion chelating agent to play a role in inhibiting RNase (nuclease) to prevent nucleic acid degradation, and the sodium ascorbate is a reducing agent to stabilize an RNA molecular structure; wherein, betaine is zwitterionic surfactant, and Tween 20 is nonionic surfactant, and has effects of removing impurities such as protein and esters, and reducing impurities adsorbed by magnetic beads. The iminodisuccinic acid tetrasodium salt is a biodegradable green metal ion chelating agent, so that the problems of water body pollution and difficult decomposition caused by EDAT are avoided.

(2) The cracking binding solution does not contain toxic organic solvents such as phenol and the like, and does not contain reagents such as proteinase K and the like which need cold chain transportation, so that the RNA extraction kit can be transported at normal temperature.

(3) The lysis binding solution simultaneously has the functions of cell/virus lysis and nucleic acid and magnetic bead combination promotion, so that the lysis and combination can be realized by adopting a one-step method without separate operation, the RNA loss or interference introduced by exogenous RNA is reduced, the lysis binding solution is more suitable for an automatic nucleic acid extractor, and the operation process is simple and convenient.

(4) The cracking binding solution combines the two protein denaturants with different action mechanisms, sodium thiocyanate is a chaotropic agent, has the characteristic of destroying the structural stability of water molecules, acts on hydrophobic molecules, can increase the water solubility of the molecules, is matched with a surfactant for use, and is mainly used for accelerating the digestion speed of protein and lipid substances and improving the treatment efficiency. Guanidine isothiocyanate belongs to a decoupling agent, is a strong protein denaturant, is mainly used for dissolving protein and leading the secondary structure of the protein to disappear, leads the cellular structure to be degraded, and quickly separates nucleoprotein from nucleic acid. Compared with the single protein denaturant, the removal capability of impurities such as complex chemical protein can be enhanced.

The invention uses different types of surfactants (betaine and Tween 20) in combination for playing a role in cracking decontamination by matching with sodium thiocyanate. Protein is a complex biological macromolecule containing non-polar, polar and charged groups, different types of surfactants interact differently with protein, betaine belongs to zwitterionic surfactants, tween 20 belongs to nonionic surfactants, and the addition of both types of surfactants can enhance detergency for the complexity of protein chemistry.

According to the invention, two binding promoters, namely sodium acetate and isopropanol, are used in a combined manner, the sodium acetate can change the charge distribution on the surface of nucleic acid, sodium ions can serve as an electric bridge between hydroxyl groups on the surface of magnetic beads and negatively charged groups on the surface of nucleic acid, so that the nucleic acid is electrostatically adsorbed onto the magnetic beads, and the isopropanol can damage hydration layers around RNA to promote the RNA to be dehydrated, aggregated and bound onto the magnetic beads. If one of the binding promoters is used alone, the use concentration needs to be increased, and a high concentration of sodium acetate causes difficulty in washing, and the extracted nucleic acid carries a large amount of salt, which inhibits the subsequent PCR reaction. And too high concentration of isopropanol is not favorable for removing other impurities in the sample.

The invention also adds stable components such as tris (hydroxymethyl) aminomethane, a metal chelating agent, a reducing agent sodium ascorbate and the like which have a pH buffering effect, is favorable for ensuring the stability of the extracted RNA and reducing loss, and is particularly favorable for extracting the virus RNA in trace test samples such as throat swabs and the like.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail below with reference to specific embodiments.

The invention provides an RNA extraction kit which does not contain proteinase K, phenol and other toxic organic matters, and comprises a lysis binding solution, a magnetic bead suspension, a first washing solution, a second washing solution and an eluent. Wherein the lysis binding solution comprises the following components:

1-5M of guanidinium isothiocyanate, 0.2-3M of sodium thiocyanate, 0.01-0.1M of trihydroxymethyl aminomethane, 0.001-0.05M of tetrasodium iminodisuccinate, 0.05-1% of sodium ascorbate, 0.1-2% of betaine, 1-6% of Tween 20, 0.05-0.5M of sodium acetate and 25-55% of isopropanol.

The magnetic bead suspension is obtained by dispersing superparamagnetic silica nano magnetic beads with the particle size of 200-500nm in nuclease-free water, wherein the solid content is 20-40mg/mL, and the pH value is 4.0-6.0.

The first washing liquid comprises the following components: 20-200mM of trihydroxymethyl aminomethane, 1-50mM of imino disuccinic acid tetrasodium, 100-500mM of sodium acetate, 0.1-2% of Tween 20 and 40-60% of ethanol in volume fraction.

The second washing solution is ethanol with the volume fraction of 60-80%.

The eluent is: 1-10mM tetrasodium iminodisuccinate, 1-100mM tris-HCl buffer, 0.01-0.2% DEPC (diethyl pyrocarbonate). Diethylpyrocarbonate is a potent, but not exhaustive, rnase inhibitor.

The cleavage binding solution, the suspension of magnetic beads, the washing solution, the eluent, and the like, which are not specifically described, are water containing no RNase or a buffer solution for adjusting/maintaining a desired pH.

Examples

RNA pseudovirus nucleic acid extraction is carried out by adopting the RNA virus nucleic acid extraction kit (Cellpro) and the RNA virus nucleic acid extraction kit (CompA) sold in the market, and detection is carried out by a reverse transcription and fluorescence quantitative PCR detection kit.

Sample preparation: RNA pseudovirus concentration is 5000copies/mL, 500copies/mL samples, each concentration of each nucleic acid extraction kit extracts 8 copies in parallel, according to the respective kit instruction extraction program for nucleic acid extraction. And carrying out reverse transcription and qPCR detection after extraction is finished.

The RNA virus nucleic acid extraction kit (Cellpro) comprises the following components:

cleavage of the binding solution, the composition is as follows (table 1):

② magnetic bead suspension, which comprises the following components:

the particle size of the superparamagnetic silicon oxide nano magnetic bead is 200-500nm, and the superparamagnetic silicon oxide nano magnetic bead is dispersed in nuclease-free water to obtain a superparamagnetic silicon oxide nano magnetic bead suspension with the solid content of 25mg/mL, wherein the pH value of the superparamagnetic silicon oxide nano magnetic bead suspension is 5.0.

③ the first washing liquid, the composition is as follows (table 2):

components	Concentration of
		Tris (hydroxymethyl) aminomethane	100mM
Imino disuccinic acid tetrasodium salt	10mM
		Sodium acetate	140mM
Tween 20	0.50％
		Ethanol	55％

Second washing liquid

70% ethanol solution.

Fifthly, eluent (Table 3)

Eluent	Concentration of
		Imino disuccinic acid tetrasodium salt	1mM
tris-HCl	10mM
		DEPC	0.10％

The RNA extraction kit with the composition is extracted by adopting a 32-channel automatic nucleic acid extractor, and the operation steps are as follows:

step 1, adding 400uL of lysis binding solution, 350uL of RNA virus sample preservation solution and 20uL of magnetic bead suspension into a deep-well plate 1/7 column.

The sample includes: the concentration of the RNA pseudovirus is 5000copies/mL and 500 copies/mL;

step 2, adding 600uL of first washing solution into 2/8;

step 3, adding 600uL of second washing solution into 3/9;

step 4, add 60uL of eluent in column 4/10.

The parameters of the 32-channel automatic nucleic acid extractor were set as follows (table 4):

finally, the results of the detection (Ct value of PRC amplification) in RNA pseudovirus experiments are shown in the following table (table 5):

the result shows that the CT value of the RNA extracted by the RNA virus nucleic acid detection kit is about 1 CT in advance of the CT value of the RNA detected by qPCR in the market, and the repeatability (cv) of 8 samples is better. The detection result shows that the kit has better RNA virus nucleic acid extraction effect.

The above examples are described by taking the example of extraction in a 32-channel automatic nucleic acid extractor. It can be understood that manual operation can also be adopted, and the specific process is as follows:

(1) sample lysis and nucleic acid adsorption: preparing sample preservation solution for detection by taking 300-400 mu L pseudovirus; adding 1-2 times of splitting binding solution into pseudovirus sample preservation solution, simultaneously adding 15-40uL of magnetic bead suspension, uniformly mixing, placing on a magnetic frame, standing, and removing liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;

(2) rinsing: adding 550-; adding 550-;

(3) sample elution: adding 550 and 650 mu L of eluent into the rinsed magnetic beads obtained in the step (2), shaking and uniformly mixing, placing on a magnetic frame, and collecting the eluent, namely the obtained nucleic acid solution after the magnetic beads are adsorbed, and storing at-80 ℃.

Comparative example 1

In this example, the concentration of "guanidinium isothiocyanate" was changed to 3M by removing the protein denaturing agent "sodium thiocyanate" from the "cleavage solution" in example 1.

Comparative example 2

In this example, the protein denaturing agent "guanidine isothiocyanate" was removed from the "cleavage solution" in example 1, and the concentration of "sodium thiocyanate" was changed to 3M.

Comparative example 3

This example was based on the "split binding solution" of example 1, with the sodium acetate removed and the isopropanol added to a volume fraction of 60%.

Comparative example 4

In this example, on the basis of the "lysis binding solution" in example 1, betaine was removed and the amount of Tween-20 was increased to 7.5%.

Comparative example 5

In this example, based on the "lysis binding solution" of example 1, Tween-20 was removed and the amount of betaine was increased to 5.5%.

Using the RNA extraction kit of comparative examples 1 to 5, extraction was performed using a 32-channel automatic nucleic acid extractor, the parameters of which are shown in Table 4.

The results of the detection (Ct value of PRC amplification) of the RNA pseudovirus assay are shown in the following table (Table 6):

therefore, the lysis binding solution of the invention combines two protein denaturants with different action mechanisms, compared with the single protein denaturant, the lysis binding solution of the invention combines sodium thiocyanate and guanidine isothiocyanate, has better amplification effect, and the average CT value of PCR amplification of the nucleic acid extracted in the example 1 is 3-4 CTs ahead of the average CT value of the PCR amplification of the nucleic acid extracted in the comparison with the comparison ratios 1 and 2. The results show that the concentration of the extracted nucleic acid is low and the cracking effect is poor when one protein denaturant is used alone.

The invention combines two binding promoters, namely sodium acetate and isopropanol, and has better nucleic acid binding effect compared with the method of singly using one binding promoter. The nucleic acid amplification result shows that the undetected sample (NA) appears in the comparative example 3, and the CT value of the amplified sample is obviously later. It is difficult to bind nucleic acids to magnetic beads more completely using one binding promoter alone, and nucleic acids are more easily bound to magnetic beads using the synergistic effect of multiple binding promoters.

The invention jointly uses different types of surfactants (betaine and Tween 20) for playing a role in cracking by matching with sodium thiocyanate, and compared with the method of singly using one surfactant, the extracted nucleic acid has a better amplification effect. Comparative examples 4 and 5, which use one type of surfactant alone, cause a decrease in detergency due to the diversity of protein groups, and a decrease in PCR amplification ability, even if not detected at a later stage in CT. Aiming at the complex diversity of the chemical properties of protein groups, various types of surfactants are added for combined use, so that the interaction between the surfactants and the proteins can be enhanced.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. The RNA extraction kit suitable for normal-temperature transportation is characterized by comprising a lysis binding solution, wherein the lysis binding solution contains the following components:

1-5M of guanidinium isothiocyanate, 0.2-3M of sodium thiocyanate, 0.01-0.1M of trihydroxymethyl aminomethane, 0.001-0.05M of tetrasodium iminodisuccinate, 0.05-1% of sodium ascorbate, 0.1-2% of betaine, 1-6% of Tween 20, 0.05-0.5M of sodium acetate and 25-55% of isopropanol.

2. The RNA extraction kit suitable for transportation at room temperature according to claim 1, wherein the lysis binding solution comprises:

guanidine isothiocyanate with the concentration of 2M, sodium thiocyanate with the concentration of 1M, trihydroxymethyl aminomethane with the concentration of 0.05M, tetrasodium iminodisuccinate with the concentration of 0.01M, sodium ascorbate with the mass fraction of 0.10%, betaine with the mass fraction of 0.5%, Tween 20 with the mass fraction of 5%, sodium acetate with the concentration of 0.2M and isopropanol with the mass fraction of 45%.

3. The RNA extraction kit suitable for transportation at room temperature according to claim 1 or 2, wherein the RNA extraction kit further comprises a suspension of magnetic beads, a first washing solution, a second washing solution and an eluent;

the magnetic bead suspension consists of the following components: superparamagnetic silica nano magnetic beads with solid content of 20-40mg/mL, the particle size of the superparamagnetic silica nano magnetic beads is 200-500nm, the superparamagnetic silica nano magnetic beads are dispersed in nuclease-free water, and the pH value of a magnetic bead suspension is 4.0-6.0;

the first wash solution consists of the following components: 20-200mM of trihydroxymethyl aminomethane, 1-50mM of imino disuccinic acid tetrasodium, 100-500mM of sodium acetate, 0.1-2% of Tween 20 and 40-60% of ethanol in volume fraction;

the second washing solution is ethanol with the volume fraction of 60-80%;

the eluent consists of the following components: 1-10mM of imino disuccinic acid tetrasodium, 1-100mM of tris-HCl buffer solution and 0.01-0.2% of DEPC.

4. The RNA extraction kit suitable for transportation at room temperature according to claim 3, wherein the solid content of the magnetic bead suspension is 25 mg/mL; the first wash solution consists of the following components: 100mM of tris (hydroxymethyl) aminomethane, 10mM of tetrasodium iminodisuccinate, 140mM of sodium acetate, 0.50% of Tween 20 and 55% of ethanol; the second washing solution is 70% absolute ethyl alcohol; the eluent consists of the following components: 1mM tetrasodium iminodisuccinate, 10mM tris-HCl buffer, 0.010% DEPC.

5. A method for extracting viral nucleic acid from a sample using the RNA extraction kit of any one of claims 1 to 4, comprising the steps of:

(1) sample preparation: soaking the sampling swab sample in physiological saline, fully shaking and uniformly mixing, standing for 2-5min, and centrifuging at the speed of 800 plus 12,000rpm to prepare a virus sample preservation solution;

(2) sample lysis and nucleic acid adsorption: taking 300-500 mu L of virus sample preservation solution for detection; adding 1-2 times of the volume of the lysis binding solution into the virus sample preservation solution obtained in the step (1), simultaneously adding 15-40uL of magnetic bead suspension, uniformly mixing, placing on a magnetic frame, standing, and removing liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;

(3) rinsing: adding 550-; adding 550-;

(4) sample elution: and (4) adding 550-650 mu L of eluent into the rinsed magnetic beads obtained in the step (3), vibrating and uniformly mixing the mixture, placing the mixture on a magnetic frame, collecting the eluent after the magnetic beads are adsorbed, namely the obtained nucleic acid solution, and storing the solution at low temperature.

6. The method of claim 5, wherein the extraction is performed using a 32-channel automated nucleic acid extractor.

7. The method of claim 6, wherein the extraction is performed using a 32-channel automated nucleic acid extractor by: after obtaining the virus sample preservation solution, the following steps are carried out:

step 1: adding 400-500uL lysis binding solution, 300-500uL virus sample preservation solution and 15-40uL magnetic bead suspension into the deep pore plate 1/7; the extractor is set as follows: mixing for 15min at high speed for 60s without heating;

step 2: 600uL of the first washing solution 550-550 was added in the column of the deep-hole plate 2/8; the extractor is set as follows:

mixing for 2min at medium speed for 60s without heating;

and step 3: 600uL of the second washing solution 550-550 was added in the column of the deep-hole plate 3/9; the extractor is set as follows:

mixing for 2min at medium speed for 60s without heating;

and 4, step 4: adding eluent 55-60uL into a deep hole plate 4/10 column; the extractor is set as follows: after waiting for 6min, mixing for 10min, wherein the mixing speed is low, the magnetic attraction time is 90s, and heating is not started.