CN112159806A

CN112159806A - Nucleic acid dissociation liquid and application thereof in nucleic acid extraction

Info

Publication number: CN112159806A
Application number: CN202011378567.4A
Authority: CN
Inventors: 李小锋; 李晨阳; 梁伟伟
Original assignee: Guangdong Hexin Health Technology Co ltd
Current assignee: Guangdong Hexin Health Technology Co ltd
Priority date: 2020-12-01
Filing date: 2020-12-01
Publication date: 2021-01-01
Anticipated expiration: 2040-12-01
Also published as: CN112159806B

Abstract

The invention discloses a nucleic acid dissociation liquid and application thereof in nucleic acid extraction. The nucleic acid dissociation solution provided by the invention consists of guanidinium isothiocyanate, water saturated phenol, an alcohol reducing agent and 8-hydroxyquinoline, wherein the concentration of the guanidinium isothiocyanate is 0.4-2 mol/L, the concentration of the water saturated phenol is 10-50%, the concentration of the alcohol reducing agent is 0.07-0.35%, the concentration of the 8-hydroxyquinoline is 0.02-0.1%, and the pH value of the dissociation solution is 5.0-10.0. The invention also provides the application of the nucleic acid dissociation liquid in nucleic acid extraction, and the treatment by the glucan gel adsorption column has no influence on nucleic acid. The invention can realize the extraction and purification of nucleic acid within 1 minute, the nucleic acid extracted by dissociation can be directly used for PCR reaction, and the integrity of the dissociated nucleic acid can be maintained within 3 minutes to 1 week. This method of nucleic acid extraction requires little equipment.

Description

Nucleic acid dissociation liquid and application thereof in nucleic acid extraction

Technical Field

The invention belongs to the field of molecular biology detection, and particularly relates to a nucleic acid dissociation solution and application thereof in nucleic acid extraction.

Background

The realization of the rapid extraction of nucleic acid is one of the key steps for shortening the clinical nucleic acid diagnosis time, and particularly during epidemic outbreak, the rapid diagnosis of nucleic acid is very important for the prevention and control of epidemic situations. The nucleic acid sample extraction is divided into two parts of protein and nucleic acid dissociation and nucleic acid extraction.

The dissociation of protein and nucleic acid is generally performed by chemical dissociation, enzymatic dissociation, or the like. Chemical dissociation is often used for nucleic acid extraction from cells or viruses. The classical dissociation liquid for dissociation of protein and nucleic acid is Trizol, which can rapidly crack cells, dissociate protein and nucleic acid, and maintain the integrity of RNA; the components contain a large amount of salt ions, so that a precipitation centrifugation method is generally adopted in the RNA extraction process, and the extraction of nucleic acid of a single sample takes about 30 min.

The extraction method of nucleic acid generally adopts precipitation, chromatography, density gradient centrifugation, electrophoresis, physical adsorption and other methods, and for example, the extraction method adopts precipitation centrifugation method mostly when Trizol is used for extraction. At present, the nucleic acid extraction mostly adopts an adsorption method, such as a resin adsorption column method or a magnetic bead adsorption method, the extraction steps are divided into two parts of washing and elution, and the total time is 15-30 min.

Sephadex is a commonly used desalting material, and is commonly used in the field of protein purification.

The dextran gel is an uncharged substance with three-dimensional porous network structure and bead-shaped particles, the fine structure and the mesh of each particle are uniform in diameter, small molecules can enter the mesh of the gel like a sieve, and large molecules are excluded from the particles. When a protein mixture sample containing different molecular sizes is added to a chromatographic column filled with the gel particles, the molecules of which the particles are close to or larger than the meshes of the gel cannot enter the meshes of the gel as eluent passes through the gel column, and move along the gaps of the gel particles along with the eluent under the action of gravity, so that the protein mixture sample is washed out of the chromatographic column firstly as the retardation effect is small and the moving speed is high; molecules smaller than the gel meshes can permeate into the interior of the particles through the gel meshes, and when the molecules are eluted, the molecules continuously penetrate from one mesh to another mesh and diffuse layer by layer, so that the molecules have a large retardation effect and a slow moving speed, and then the molecules are discharged out of the chromatographic column. If the molecule is between the substances that completely exclude or completely penetrate the gel, it will flow out of the column between the two. The different components of the mixture are separated according to the time at which each molecule flows out of the column. Gel columns are convenient devices for their use in the purification of small amounts of protein (30-100 ul).

The glucan gels are classified into 10, 15, 25, 50, 75, 100, etc. according to their swelling coefficients; of these, a swelling factor of 25 is more common, indicating that 10 grams of dry powder has the ability to absorb 25ml of water.

Generally, most of the current nucleic acid extraction and purification methods adopt a resin adsorption method or a magnetic bead adsorption method, a high-speed centrifuge or an automatic nucleic acid extractor is required, and the total time consumption is long, namely about 15-30 minutes. Nucleic acid extraction and purification are not performed using a gel column, and nucleic acid extraction and purification are completed in a short time.

Disclosure of Invention

In order to solve the problems, the invention provides a nucleic acid dissociation solution and details the application thereof in nucleic acid extraction, and the dissociation solution can effectively complete the extraction of nucleic acid within one minute by combining with the extraction method of the invention and can ensure the normal operation of the subsequent PCR reaction.

The solution of the invention is as follows:

the invention provides a nucleic acid dissociation solution, which consists of guanidinium isothiocyanate, water-saturated phenol, an alcohol reducing agent and 8-hydroxyquinoline, and comprises the following components in concentration and pH value: guanidine isothiocyanate: the molar concentration is 0.4mol/L-2 mol/L; water-saturated phenol: the mass percentage concentration is 10% -50%; alcohol reducing agent: the mass percentage concentration is 0.07-0.35%; 8-hydroxyquinoline: the mass percentage concentration is 0.02% -0.1%; the pH value of the dissociation liquid is 5.0-10.0.

Preferably, the alcohol reducing agent is 2-mercaptoethanol or dithiothreitol.

The invention also provides application of the nucleic acid dissociation solution in nucleic acid extraction.

Preferably, the nucleic acid extraction is performed by using a gel column filled with sephadex. The gel adsorption column adsorbs various anions and cations in the dissociation solution and inhibitors of PCR reaction, and the sample treated by the nucleic acid dissociation solution can be directly used for PCR reaction after the gel column adsorbs the ions and the inhibitors of PCR reaction.

More preferably, the sephadex has a swelling factor of 25.

The application method of the invention comprises the following steps:

(1) collecting a throat swab, soaking the collected throat swab in the dissociation liquid, and fully mixing to obtain a dissociated sample;

(2) and centrifuging the gel column, adding the dissociated sample into the gel column, and centrifuging to obtain filtrate, namely the extracted nucleic acid.

The invention has the beneficial effects that: the dissociation liquid and the gel column are combined for application, and the dissociation liquid has the following beneficial effects:

(1) the cell is rapidly cracked, nucleic acid combined with protein in a throat swab and a sputum sample which are clinically collected can be effectively dissociated, nucleic acid extraction can be completed within 1 minute, the dissociated and extracted nucleic acid can be directly used for PCR reaction, and the whole operation time of PCR nucleic acid diagnosis is greatly shortened;

(2) the integrity of nucleic acids can be maintained for a long time: the dissociation liquid can also protect dissociated nucleic acid, and can keep the integrity of the nucleic acid within three minutes and a week after the nucleic acid is extracted from a sample and transported to a laboratory;

(3) expensive equipment is not required to be equipped, and only one palm centrifugal machine is needed.

Drawings

FIG. 1 is a graph showing the comparative amplification of a target gene in a test sample 1 using a dissociation solution, an extraction method and a Trizol conventional nucleic acid extraction method as described in example 2;

FIG. 2 is a graph showing the amplification of a target gene in a comparative test sample 1 using the dissociation solution, the extraction method and the Trizol combined gel column nucleic acid extraction method in example 2;

FIG. 3 is a graph showing the amplification of target genes in Trizol conventional nucleic acid extraction method and Trizol conventional nucleic acid extraction method in comparison with detection sample 1.

Detailed Description

The invention will be further illustrated by the following detailed description of specific embodiments, which are not to be construed as limiting the invention but are intended to be exemplary only. The reagents used therein are only those specifically selected in this particular example, and it is understood that one skilled in the art can select other corresponding reagents as needed to achieve the objectives of the present invention.

Example 1: a preferred embodiment of the invention is the use of the formula composition of the dissociation liquid in nucleic acid extraction

The nucleic acid dissociation solution of the embodiment is composed of guanidinium isothiocyanate, hydrocortisone, 2-mercaptoethanol and 8-hydroxyquinoline, and the components in the dissociation solution, the concentrations thereof and the PH values are shown in table 1:

TABLE 1 formulation composition of dissociation liquid in example 1

The main materials required for the nucleic acid extraction method are as follows: gel column, 7.4mM filter, Sephadex G25, 2mM Tris pH9.0 buffer.

The method for dissociating and extracting the clinical throat swab by the nucleic acid dissociation liquid comprises the following steps:

the throat swab collected clinically is immersed in the dissociation liquid in the embodiment, and the dissociation sample is obtained after the throat swab is fully and uniformly mixed. And centrifuging the gel column for 3 seconds by using a palm centrifuge (the rotating speed is 4000-.

Example 2: second preferred embodiment of the invention dissociation liquid formula composition applied in nucleic acid extraction

The nucleic acid dissociation solution of the embodiment is composed of guanidinium isothiocyanate, hydrocortisone, 2-mercaptoethanol and 8-hydroxyquinoline, and the components in the dissociation solution, the concentrations thereof and the PH values are shown in table 2:

TABLE 2 formulation composition of dissociation liquid in example 2

The main materials required for the nucleic acid extraction method are as follows: gel column, 7.4mM filter disc, Sephadex G25, 2mM TrisPh9.0 buffer.

Example 3: third preferred embodiment of the invention dissociation liquid formula composition applied in nucleic acid extraction

The nucleic acid dissociation solution of the embodiment is composed of guanidinium isothiocyanate, hydrocortisone, 2-mercaptoethanol and 8-hydroxyquinoline, and the components in the dissociation solution, the concentrations thereof and the PH values are shown in table 3:

TABLE 3 composition of dissociation liquid formulation in example 3

Example 4: comparative test between nucleic acid dissociation extraction method and magnetic bead nucleic acid extraction method

3 clinical throat swabs were stored in physiological saline and used as test samples for comparative experiments and this example. Labeled as sample 1, sample 2, sample 3, respectively. The

samples

1, 2 and 3 were subjected to nucleic acid extraction by the comparative test method and the method of the present invention, respectively. The specific operation is as follows:

method of comparative experiment: nucleic acid extraction is carried out by combining a nucleic acid extraction reagent HY-112A of the research and development science and technology Limited company with a full-automatic nucleic acid automatic extraction instrument of the company. The operation steps are as follows: (1) taking out a 96-deep-hole plate, adding 400 mu l of a first-hole reagent into the first hole, and sealing 250 mu l of a cover agent; (2) adding 600 mul of reagent into the second hole; (3) adding 620 mu l of a third hole reagent into the third hole; (4) adding 620 mu l of reagent in the fourth well; (5) adding 500 mu l of reagent into the sixth well; (6) penetrating the first hole through a capping agent, adding 100 mu l of throat swab sample and 20ul of HY-112A binding solution, and uniformly mixing for 5-8 times; (7) placing the 96 deep-hole plate on a reagent tray of the automatic nucleic acid extraction instrument, installing a magnetic rod sleeve, and covering an instrument cover; (8) clicking the program "HY-112A" to perform full-automatic nucleic acid extraction; (9) and after the program operation is finished, taking out the 96 deep-hole plate, wherein the liquid in the sixth hole is the extracted nucleic acid.

The method of the embodiment comprises the following steps: and taking 10 mul of a throat swab sample, adding 40 mul of dissociation liquid in the embodiment 1, and vibrating and uniformly mixing to obtain the dissociated sample. The dissociated sample was subjected to nucleic acid extraction by the gel column extraction method in example 1.

For two target genes: the target genes 1 and 2 were subjected to amplification detection using the same PCR reaction solution. The results of comparing the amplified CT values of the nucleic acids extracted by the two methods are shown in table 4:

TABLE 4 CT values obtained from comparative experiments using the dissociation solution and extraction method of example 1 and the magnetic bead nucleic acid extraction method

From the results shown in Table 4, it was found that when the nucleic acid extracted by the method of extracting nucleic acid and the dissociation solution in example 1 of the present invention and the nucleic acid extracted by the magnetic bead extraction method were subjected to PCR nucleic acid amplification reaction detection, the CT values of the detection of both were almost the same.

Thus, it was demonstrated that the dissociation solution and the nucleic acid extraction method in example 1 of the present invention are equivalent to the effect of the extracted nucleic acid obtained by the conventional magnetic nucleic acid bead extraction method; the nucleic acid extraction method can be completed within 1 minute, and only one centrifuge is needed to be carried out on the instrument without other large-scale equipment; however, the conventional method for extracting nucleic acid magnetic beads takes 30 minutes.

Example 5: the invention relates to a nucleic acid extraction method by combining a nucleic acid dissociation solution with a gel column, a Trizol traditional nucleic acid extraction method, and a comparison experiment of four methods of nucleic acid extracted by the Trizol traditional nucleic acid extraction method through the gel column and the Trizol combined gel column nucleic acid extraction method

samples

method one of the comparative experiments: trizol traditional nucleic acid extraction method

The operation steps are as follows: taking 100 mul of throat swab sample, adding 400 mul of Trizol, and vibrating and mixing uniformly; then adding 100 mul of chloroform, shaking and mixing uniformly; centrifuge at 12000rpm for 5 min. Taking the supernatant to a new EP tube, adding isopropanol with the same volume, shaking and mixing evenly, and centrifuging at 12000rpm for 10 min. The supernatant was removed, 1ml of 75% ethanol was added, inverted several times, and centrifuged at 12000rpm for 5 min. The supernatant was removed and 500. mu.l of DEPC water was added to dissolve the nucleic acid precipitate.

Method two of the comparative experiment: gel column is crossed to nucleic acid that Trizol tradition nucleic acid extraction method extracted

The operation steps are as follows: 50ul of nucleic acid extracted in the first comparative experiment of this example was purified using the gel column of example 2.

Method three of the comparative experiment: trizol combined gel column nucleic acid extraction method

The operation steps are as follows: taking 10 mul of a throat swab sample, adding Trizol 40 mul, and vibrating and mixing uniformly to obtain a dissociated sample. The sample after Trizol dissociation was subjected to nucleic acid extraction using the gel column extraction method in example 2.

The method of the embodiment comprises the following steps: and taking 10 mul of a throat swab sample, adding 40 mul of dissociation liquid in the embodiment 2, and vibrating and uniformly mixing to obtain the dissociated sample. The dissociated sample was subjected to nucleic acid extraction using the gel column extraction method of example 2.

And carrying out amplification detection on the same target gene by using the same PCR reaction solution. The results of comparing the CT values after amplification of the nucleic acids extracted and purified by the four methods are shown in table 5:

TABLE 5 CT values obtained from comparative experiments using the dissociation liquid, extraction method, Trizol conventional nucleic acid extraction method-extracted nucleic acid gel column and Trizol combination gel column nucleic acid extraction method of example 2

FIG. 1 is a graph showing the comparative amplification of a target gene in a test sample 1 using a dissociation solution, an extraction method and a Trizol conventional nucleic acid extraction method as described in example 2; FIG. 2 is a graph showing the amplification of a target gene in a comparative test sample 1 using the dissociation solution, the extraction method and the Trizol combined gel column nucleic acid extraction method in example 2; FIG. 3 is a graph showing the amplification of target genes in Trizol conventional nucleic acid extraction method and Trizol conventional nucleic acid extraction method in comparison with detection sample 1.

As is clear from the results shown in Table 5 and FIG. 1, the nucleic acids obtained by the method of extracting nucleic acids and the dissociation solution in example 2 of the present invention were detected by PCR nucleic acid amplification reaction using the nucleic acids obtained by Trizol conventional nucleic acid extraction method, and the CT values obtained by amplification were almost the same, but the fluorescence values obtained by the former amplification were slightly lower because the nucleic acids obtained by the method contained a certain amount of protein and had some influence on the fluorescence values of PCR reaction, but did not affect the CT values, and thus had no influence on the interpretation of the clinical nucleic acid detection results.

From the results shown in Table 5 and FIG. 2, it is understood that the PCR nucleic acid amplification reaction was carried out using the nucleic acid obtained by the dissociation liquid and the nucleic acid extraction method of example 2 of the present invention and the nucleic acid obtained by the Trizol combination gel column, and the CT value of the nucleic acid amplification obtained by the latter was 2 to 3 greater than that of the nucleic acid obtained by the former because the PCR amplification reaction was inhibited because the gel column purified by removing all the salt ions because of a large amount of salt ions contained in Trizol, and a large amount of salt ions remained in the filtrate obtained by centrifugation. The Trizol dissociated sample is not suitable for one-time purification using a gel column, and multiple purifications may be required to remove salt ions from the dissociated sample.

As is apparent from the results of Table 5 and FIG. 3, when the nucleic acid extracted by Trizol conventional nucleic acid extraction was purified using the gel column of example 2 of the present invention, the CT value of the nucleic acid amplified by gel column purification was 1-2 smaller than that of the nucleic acid extracted by Trizol conventional nucleic acid extraction in PCR detection, because the nucleic acid extracted by Trizol conventional nucleic acid extraction still contained some unknown PCR inhibitors, thereby inhibiting the PCR reaction. After the purification by the gel column, certain unknown PCR inhibitors are removed, so that the purity of the nucleic acid is higher, the number of the inhibitors is less, and the PCR amplification reaction is more sufficient. From this, it can be seen that the gel column can further purify the nucleic acid extracted by the trizol conventional nucleic acid extraction method.

Example six: the application of gel column in combination with other lysis solutions.

This example is a one-step alkaline lysis solution.

3 clinical throat swabs were stored in physiological saline and used as test samples for comparative experiments and this example. Labeled as sample 1, sample 2, sample 3, respectively. The comparative experiment was performed by using the comparative experiment method for sample 1, sample 2 and sample 3, and purifying the nucleic acid extracted in the comparative experiment using a gel column. The specific operation is as follows:

method of comparative experiment:

100 mul of throat swab sample is taken, 300 mul of one-step method alkali lysate is added, and the sample nucleic acid is obtained after shaking and mixing.

The method of the embodiment comprises the following steps:

50 μ l of sample nucleic acid of the comparative experiment in this example was taken, and the nucleic acid extracted by the one-step alkaline lysis method was purified by the gel column in example 2.

And carrying out amplification detection on the same target gene by using the same PCR reaction solution. The results of comparing the CT values after amplification of the nucleic acids extracted and purified by the two methods are shown in table 6:

TABLE 6 CT values obtained from comparative experiments on nucleic acids extracted by one-step alkaline lysis and nucleic acids purified by one-step alkaline lysis using gel column

As is clear from the results in Table 6, the CT value of the purified nucleic acid obtained by the one-step alkaline lysis method using the gel column was 3 to 4 smaller than that of the nucleic acid obtained by the one-step alkaline lysis method. Because the gel column removes PCR inhibitors in nucleic acid extracted by a one-step alkaline lysis method, thereby reducing the amplification reaction of the gel column on PCR.

Therefore, the gel column can remove some unknown PCR inhibitors in the nucleic acid extracted by the one-step alkaline lysis method, and further purify the nucleic acid. By analogy, the gel column can further purify nucleic acid extracted by other nucleic acid extraction methods.

In summary, the CT values of the PCR amplification reactions of the nucleic acids obtained by the dissociation solution and the nucleic acid extraction method of the present invention are substantially the same as those obtained by the magnetic bead nucleic acid extraction method or the Trizol traditional nucleic acid extraction method. The nucleic acid extraction method can be completed within 1 minute, while the other two methods require about 30 minutes. In addition, the nucleic acid extraction method only needs one palm centrifuge, and does not need a high-speed centrifuge or a nucleic acid extraction instrument, thereby effectively saving the cost of an experimental field and instruments.

In addition, the gel column can be used for further purifying nucleic acid extracted by other methods, and certain unknown PCR inhibitors can be removed. And other samples lysed by the lysate can also be used for nucleic acid extraction and purification by using a gel column. The number of gel column purifications may vary from lysate to lysate.

Claims

1. The nucleic acid dissociation liquid is characterized by comprising guanidinium isothiocyanate, water-saturated phenol, an alcohol reducing agent and 8-hydroxyquinoline, wherein the components in the dissociation liquid, the concentrations and the pH values of the components are as follows:

guanidine isothiocyanate: the molar concentration is 0.4mol/L-2 mol/L;

water-saturated phenol: the mass percentage concentration is 10% -50%;

alcohol reducing agent: the mass percentage concentration is 0.07-0.35%;

8-hydroxyquinoline: the mass percentage concentration is 0.02% -0.1%;

the pH value of the dissociation liquid is 5.0-10.0.

2. The nucleic acid dissociation solution according to claim 1, wherein the alcohol reducing agent is 2-mercaptoethanol or dithiothreitol.

3. Use of the nucleic acid dissociation solution as set forth in claim 1 in nucleic acid extraction.

4. The use of claim 3, wherein the nucleic acid extraction is performed using a sephadex-packed gel column.

5. Use according to claim 4, wherein the sephadex has a swelling factor of 25.

6. Use according to claim 4, characterized in that it comprises the following steps:

(1) collecting a throat swab, soaking the collected throat swab in the nucleic acid dissociation liquid, and shaking and mixing for 2-5s to obtain a dissociated sample;

(2) and centrifuging the gel column, adding the dissociated sample into the gel column, and centrifuging for 10-20s to obtain filtrate which is the extracted nucleic acid.