Nucleic acid release combination liquid of dried blood spot, and extraction kit and extraction method of genome DNA in dried blood spot

Abstract

The invention discloses a nucleic acid release combination solution for dried blood spots, an extraction kit for genome DNA in the dried blood spots and an extraction method. The nucleic acid release combination solution comprises a cell dispersion solution and a cell lysis solution. The nucleic acid release combination liquid provided by the invention is applied to extraction of genomic DNA in dried blood spots, and the extraction amount of the genomic DNA can be effectively increased.

Description

Technical Field

[0001] The present application relates to the field of biochemical analysis technology, and in particular to a nucleic acid release combination liquid for dried blood spots, a kit for extracting genomic DNA from dried blood spots, and an extraction method. Background Art

[0002] Dried blood spots are usually loaded on a carrier to prepare dried blood spot sheets as samples, which have the advantages of convenient transportation and storage and low cost. Dried blood spots refer to whole blood dripped on filter paper and dried. Compared with the storage of whole blood samples, the storage method is simple and the storage state is stable. Therefore, dried blood paper sheets have been widely used in disease screening samples.

[0003] At present, nanomagnetic bead extraction is one of the main methods for separating and extracting DNA from dried blood spots. However, there are problems such as low extraction yield, low purity and poor extraction stability in DNA extraction from dried blood spots, which affect the application effect of DNA samples in actual clinical practice. Summary of the invention

[0004] In view of the problems in the prior art, the present application provides a nucleic acid release combination liquid for dried blood spots, a kit for extracting genomic DNA from dried blood spots, and an extraction method to increase the amount of genomic DNA extracted from dried blood spots.

[0005] The nucleic acid release composition of dried blood spots in the present application comprises a cell dispersion and a cell lysis solution, wherein the cell dispersion is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and a solvent, wherein the concentration of tris(hydroxymethyl)aminomethane hydrochloride in the cell dispersion is 80 to 120 mM, the concentration of ethylenediaminetetraacetic acid is 10 to 12 mM, and the concentration (m / v) of sodium dodecyl sulfate is 0.6 to 1.2%;

[0006] The cell lysate consists of guanidine hydrochloride, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride), Triton X-100, isopropanol and a solvent. In the cell lysate, the concentration of guanidine hydrochloride is 3-5M, the concentration of sodium chloride is 50-150mM, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 80-120mM, the volume fraction of Triton X-100 is 1-3%, and the volume fraction of isopropanol is 30-50%.

[0007] Several optional methods are also provided below, but they are not intended to be additional limitations on the above-mentioned overall solution, but are merely further supplements or preferences. Under the premise that there are no technical or logical contradictions, each optional method can be combined with the above-mentioned overall solution separately, and multiple optional methods can also be combined.

[0008] Optionally, the cell dispersion contains proteinase K at a concentration of 0.1-1 mg / mL.

[0009] Optionally, the cell lysis solution is prepared by: preparing guanidine hydrochloride solution, sodium chloride solution, tris(hydroxymethylaminomethane) hydrochloride solution, and Triton X-100 solution respectively using solvents in advance, mixing the solutions, and then mixing with the isopropanol.

[0010] The present application also provides a kit for extracting genomic DNA from dried blood spots, comprising the nucleic acid releasing combination solution.

[0011] Optionally, the kit includes a washing solution, the washing solution includes washing solution A and washing solution B, the washing solution A is composed of guanidine isothiocyanate, β-mercaptoethanol and ethanol, the concentration of guanidine isothiocyanate in the washing solution A is 4 to 7 M guanidine isothiocyanate, the volume fraction of the β-mercaptoethanol is 0.08 to 0.12%, and the volume fraction of the ethanol is 50 to 60%;

[0012] The detergent B consists of sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride) and ethanol. The concentration of sodium chloride in the detergent B is 15-23 mM, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 1-3 mM, and the volume fraction of ethanol is 75-90%.

[0013] The present application also provides a method for extracting genomic DNA from a dried blood smear, wherein the dried blood smear is treated with the release combination liquid.

[0014] Optionally, the extraction method comprises:

[0015] placing the dried blood smear in the cell dispersion and performing a first incubation treatment, and after the incubation treatment is completed, centrifuging the dried blood smear to place it at the bottom;

[0016] The mixed solution obtained from the first incubation treatment is taken, and the cell lysate is added thereto for a second incubation treatment.

[0017] Optionally, the extraction method includes: directly adding magnetic beads to the system after the second incubation treatment.

[0018] Optionally, the temperature of the first incubation treatment is 50-60° C. and the time is 40-50 min.

[0019] Optionally, the temperature of the second incubation treatment is 55-65° C., and the time is 8-15 min.

[0020] Optionally, each incubation treatment is performed under shaking conditions.

[0021] Compared with the prior art, the nucleic acid releasing combination solution provided in the present application is applied to extract genomic DNA from dried blood smears, which can effectively increase the amount of genomic DNA extracted. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of an extraction method in one embodiment of the present application. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0025] Whole blood refers to blood collected directly from the human body, including all components of blood cells and plasma. Whole blood is dripped onto a filter paper and dried to obtain a dried blood film. Generally, blood cell groups in dried blood form clumps after drying, and these clumps are surrounded by a fibrin network. The internal tissue morphology of dried blood spots formed by blood from different sources will be different, mainly manifested in changes in the fibrin network and blood cell clumps. In the prior art, genomic DNA in dried blood films is separated and extracted using a nanomagnetic bead extraction method, and blood cells in dried blood films are lysed using a relatively high concentration of lysing solution, and genomic DNA is extracted by nanomagnetic bead adsorption separation. Although this method can quickly obtain genomic DNA samples, there are problems such as genomic DNA loss and low impurity removal rate during the processing process, which affect the extraction amount and purity of genomic DNA samples.

[0026] In order to solve the above technical problems, the present application provides a nucleic acid release combination liquid for dried blood spots, including a cell dispersion liquid and a cell lysis liquid, wherein the cell dispersion liquid is composed of tris-HCl, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS) and a solvent, wherein the concentration of tris-HCl is 80-120 mM, the concentration of EDTA is 10-12 mM, and the concentration (m / v) of sodium dodecyl sulfate is 0.6-1.2%. The cell lysis liquid is composed of guanidine hydrochloride, sodium chloride, tris-HCl, Triton X-100, isopropanol and a solvent, wherein the concentration of guanidine hydrochloride in the cell lysis liquid is 3-5 M, the concentration of sodium chloride is 50-150 mM, the concentration of tris-HCl is 80-120 mM, the volume fraction of Triton X-100 is 1-3%, and the volume fraction of isopropanol is 30-50%.

[0027] Among them, the cell dispersion solution is used to expose and disperse the cells and DNA in the dry blood tissue clumps, and separate the cells, DNA and other matrices. Among its components, tris(hydroxymethylaminomethane) hydrochloride plays a buffering role, and the pH of its solution is 7.5; sodium dodecyl sulfate mainly plays the role of dissolving the fibrin network and dispersing blood cell clumps, so that the blood cells and DNA are fully released, exposed and dispersed; ethylenediaminetetraacetic acid in the cell dispersion solution is an important metal chelating agent that can inhibit enzyme activity, and the pH of its solution is 7.5.

[0028] Tris(hydroxymethyl)aminomethane hydrochloride) in the cell lysis solution plays a buffering role, and the pH of the solution is 7.5. Guanidine hydrochloride and Triton X-100 play the role of lysing cell membranes, destroying nuclear membranes, inhibiting the activity of nucleases, and promoting protein denaturation, so that cells can release a large amount of nucleic acids. For fully exposed and dispersed blood cells, only lower concentrations of guanidine hydrochloride and Triton X-100 are needed to achieve effects such as cell lysis, and lower concentrations effectively reduce damage to genomic DNA, such as breakage or digestion, which is beneficial to increase the amount of genomic DNA extracted. In addition, sodium chloride and isopropanol in the cell lysis solution play a role in precipitating DNA.

[0029] Furthermore, the cell dispersion solution also contains proteinase K, which can accelerate the dispersion rate of cells and DNA. However, in the process of treating dried blood spots, the amount of proteinase K used is relatively low, and it is sufficient to degrade the fibrin network to disperse the blood cell clusters. Its concentration is, for example, 0.1 to 1 mg / mL, preferably 0.5 to 1 mg / mL. Specifically, proteinase K uses Hanhaixin enzyme proteinase K.

[0030] In one embodiment, the concentration (m / v) of sodium dodecyl sulfate in the cell dispersion solution is 0.6-1%, preferably 1%, which can meet the requirements of fibrin network dissolution and dispersion of blood cell clumps. If the concentration is too high, most cells will be lysed prematurely, resulting in premature release of nucleic acids (genomic DNA). The released nucleic acids have the probability of being damaged by the cell lysis solution and adsorbed by the filter paper of the dried blood film, affecting the final amount of genomic DNA extracted.

[0031] Furthermore, the concentration of tris(hydroxymethyl)aminomethane hydrochloride in the cell dispersion is preferably 90-100 mM, more preferably 100 mM. The concentration of ethylenediaminetetraacetic acid in the cell dispersion is preferably 10-11 mM, preferably 10 mM.

[0032] In one embodiment, the concentration of guanidine hydrochloride in the cell lysate is preferably 3-4 M, more preferably 4 M; the volume fraction of Triton X-100 is preferably 2-3%, more preferably 2%.

[0033] Furthermore, the concentration of sodium chloride in the cell lysate is preferably 80-100 mM, more preferably 100 mM. The concentration of tris(hydroxymethyl)aminomethane hydrochloride in the cell lysate is preferably 80-100 mM, more preferably 100 mM.

[0034] The solvents used in the cell dispersion and the cell lysate are all pure water. The preparation method of the cell dispersion is to prepare the solutions of each component by using solvent distribution, and then mix the solutions of each component in a certain proportion. The preparation method of the cell lysate is: prepare guanidine hydrochloride solution, sodium chloride solution, tris(hydroxymethyl)aminomethane hydrochloride solution, and Triton X-100 solution by using solvents in advance, mix the solutions, and then mix them with isopropanol.

[0035] One embodiment of the present application provides a kit for extracting genomic DNA from a dried blood smear, comprising the above-mentioned nucleic acid releasing composition solution.

[0036] As for the nanomagnetic bead extraction method, the extraction kit also includes a detergent and an eluent, wherein the eluent is a tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0) having a concentration of 15 to 30 mM, preferably 18 to 23 mM, and more preferably 20 mM.

[0037] For the solution obtained by treating the dried blood smear with the nucleic acid releasing combination liquid of the present application, the configured detergents include detergent A and detergent B, wherein detergent A is composed of guanidine isothiocyanate, β-mercaptoethanol and ethanol, and the concentration of guanidine isothiocyanate is 4-7M guanidine isothiocyanate, preferably 4-5M, and more preferably 5M; the volume fraction of β-mercaptoethanol in detergent A is 0.08-0.12%, preferably 0.09-0.1%, and more preferably 0.1%; the volume fraction of ethanol is 50-60%, preferably 55-60%, and more preferably 56%.

[0038] Detergent B is composed of sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and ethanol. The concentration of sodium chloride in detergent B is 15-23 mM, preferably 18-20 mM, more preferably 20 mM, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 1-3 mM, preferably 1-2 mM, more preferably 2 mM; the volume fraction of ethanol is 75-90%, preferably 75-80%, more preferably 80%.

[0039] See also Figure 1 In one embodiment of the present application, a method for extracting genomic DNA from a dried blood smear is provided, wherein the dried blood smear is treated with the above-mentioned release combination liquid. Specifically, the method comprises the following steps:

[0040] Step S100, placing the dried blood smear in a cell dispersion and performing a first incubation treatment, and after the incubation treatment is completed, centrifuging the dried blood smear to place it at the bottom;

[0041] Step S200: Take the mixed solution obtained by the first incubation treatment, add the cell lysis solution thereto, and perform the second incubation treatment.

[0042] As for the nanomagnetic bead extraction method, the extraction method also includes: step S300, directly adding magnetic beads to the system after the second incubation treatment, and performing a third incubation treatment, without the need to pre-centrifuge the system obtained in step S200. In step S100, the cells have been separated from other matrices, and the cell membrane layer may also be partially degraded, just like eggs after peeling off the eggshell. These cells are lysed after the incubation treatment in step S200, and a large amount of nucleic acids are quickly released. Extraction and purification are performed by adsorption by magnetic beads to meet the extraction purity requirements. Among them, the nanomagnetic beads use MA200H magnetic beads.

[0043] Specifically, in step S100, the temperature of the first incubation treatment is 50-60°C, the time is 40-50 minutes, preferably, the temperature is 56°C, and the time is 45 minutes; instant centrifugation refers to centrifugation at 1000rpm for 1 second. In step S200, the temperature of the second incubation treatment is 55-65°C, the time is 8-15 minutes, preferably, the temperature is 60°C, and the time is 10 minutes. In step S300, the temperature of the third incubation treatment is room temperature, preferably 25°C, and the time is 5-15 minutes, preferably 10 minutes.

[0044] Further, each incubation treatment is carried out under shaking conditions, wherein the rotation speed of the first incubation treatment is 1000-1500 rpm, preferably 1200 rpm. The rotation speed of the second incubation treatment is 1200-1500 rpm, preferably 1400 rpm. The third incubation treatment is carried out in a vertical mixer at a rotation speed of 10-30 rpm, preferably 20 rpm.

[0045] See also Figure 1 , the extraction method of the present application also includes:

[0046] Step 400, transferring the system of step S300 to a magnetic rack, washing the magnetic beads with a washing solution, and drying the magnetic beads after washing;

[0047] Step S500: using an elution solution to elute the genomic DNA from the dried magnetic beads.

[0048] Specifically, step 400 includes:

[0049] Step S410: After the system of step S300 is allowed to stand, the supernatant is discarded, and the magnetic beads are washed for the first time using washing solution A. After washing, the beads are centrifuged and the supernatant is discarded;

[0050] Step S410: washing the magnetic beads for a second time with washing solution B, centrifuging and discarding the supernatant after washing; wherein the second washing is performed multiple times, for example, twice.

[0051] Step S500 includes: placing the dried magnetic beads in an eluent for a fourth incubation treatment at a temperature of 50 to 60°C, preferably 56°C, for 8 to 15 minutes, preferably 10 minutes. The fourth incubation treatment is performed under shaking conditions at a speed of 1300 to 1600 rpm, preferably 1400 rpm. After the incubation is completed, centrifuge and place on a magnetic rack to extract the supernatant, thereby obtaining a sample containing genomic DNA. The eluent is Tris-HCl (pH 8.0), and its concentration is 8 to 10 mM, preferably 10 mM.

[0052] The extraction kit and extraction method of the present application can be used to extract genomic DNA from dried blood samples of newborns and adults, have good applicability, and the amount and purity of extracted genomic DNA are high.

[0053] The following provides specific application examples in combination with specific parameters to further illustrate the present application.

[0054] Application Example 1

[0055] The reagents and materials used are shown in Table 1:

[0056] Table 1 Reagents and materials for Application Example 1

[0057]

[0058]

[0059] Steps:

[0060] (1) Cut each of the dried blood samples 1 to 6 into three pieces with a diameter of 3.5 mm and place them in a container;

[0061] (2) Add 350 μL of cell dispersion and 10 μL of Hanhaixin enzyme proteinase K to each container, and place each container on a constant temperature mixer for the first incubation at a temperature of 56°C and a rotation speed of 1200 rpm for 45 min. After the incubation, centrifuge briefly to allow the dried blood to remain at the bottom;

[0062] (3) Pipette 300 μL of the mixed solution in step (2) into a container, add 600 μL of cell lysis solution, and place the container on a constant temperature mixer for a second incubation at 60°C, a rotation speed of 1400 rpm, and a time of 10 min;

[0063] (4) After the second incubation, 100 μL of magnetic beads MA2OOH were directly added to the system of step (3), and all were placed on a vertical mixer for a third incubation at a temperature of 25°C, a rotation speed of 20 rpm, and a time of 10 min;

[0064] (5) Place the system after incubation in step (4) on a magnetic stand and let it stand for 1 min, discard the supernatant, add 750 μL of detergent A and wash for 15 s. After washing, immediately discard the supernatant;

[0065] (6) Add 750 μL of detergent B and wash for 15 seconds. After washing, discard the supernatant. Wash again with detergent B. After washing, discard the supernatant and dry the magnetic beads.

[0066] (7) Add 50 μL of elution buffer and place in a thermostatic mixer for the fourth incubation at 56°C, 1400 rpm, and 10 min. After the incubation, centrifuge and collect the supernatant to obtain a sample containing genomic DNA.

[0067] In this application example, dried blood samples 1, 2, and 3 are from different newborns, and dried blood samples 4, 5, and 6 are from different adults.

[0068] Comparative Example 1

[0069] Take samples from the same batch as the samples used in the application example and use a commercially available nucleic acid extraction kit. The specific components are shown in Table 2:

[0070] Table 2 Reagents and materials for Comparative Example 1

[0071]

[0072]

[0073] Extract nucleic acid from samples 1 to 6 according to the kit extraction instructions. The specific steps are as follows:

[0074] (1) Cut each of the dried blood samples 1 to 6 into three pieces with a diameter of 3.5 mm and place them in a container;

[0075] (2) Add 200 μL of lysis buffer MDA and 20 μL of Proteinase K to the container, vortex for 10 seconds, and place in a constant temperature oscillator preheated to 65°C for 30 minutes;

[0076] (3) During the lysis of the dried blood spot sample, add 30 μL of 300 nm silicon hydroxy magnetic beads and 600 μL of magnetic bead binding solution Buffer DLTP to a new container and mix by whipping or oscillating for 10 seconds to obtain a mixture of magnetic beads and magnetic bead binding solution. After the dried blood spot sample is lysed, the mixture of magnetic beads and magnetic bead binding solution is briefly centrifuged and placed at room temperature for 2 minutes. The supernatant of the lysate is transferred to the mixture of magnetic beads and magnetic bead binding solution, which is then placed in a constant temperature oscillator and shaken at 900 rpm for 30 minutes at room temperature.

[0077] (4) Place the container on the magnetic stand and let it stand for 1 min. When the magnetic beads are completely adsorbed, carefully remove the liquid, add 800 μL of the first rinse buffer DW to the container, vortex and oscillate for 30 seconds to resuspend the magnetic beads, place the container on the magnetic stand and let it stand for 30 seconds. When the magnetic beads are completely adsorbed, carefully remove the liquid;

[0078] (5) Repeat step (4) once;

[0079] (6) Remove the container from the magnetic stand, add 800 μL of the second rinse buffer Buffer MWP (MWE2 + 4 times the volume of ethanol), vortex for 30 seconds to resuspend the magnetic beads, place the container on the magnetic stand and let it stand for 30 seconds. When the magnetic beads are completely adsorbed, carefully remove the liquid.

[0080] (7) Remove the container from the magnetic stand, centrifuge briefly, and place the container on the magnetic stand for 30 seconds. When the magnetic beads are completely adsorbed, carefully remove the liquid, place the container on the magnetic stand, open the tube cover, and dry it at room temperature for 5 minutes.

[0081] (8) Remove the container from the magnetic rack, add 60-100 μL of elution buffer EB, place it in a thermostatic oscillator preheated to 65°C, and incubate at 1600 rpm for 10 min; after a brief centrifugation, place the container on the magnetic rack and let it stand for 1 min until the magnetic beads are completely adsorbed. After the magnetic beads are completely adsorbed, a genomic DNA solution is obtained. The genomic DNA solution is transferred to a new container and stored.

[0082] The concentration and purity of genomic DNA in each sample were detected. The higher the concentration, the larger the extraction amount. 260 / 280 It indicates the degree of interfering protein components in the extracted genomic DNA, and the value is generally required to be between 1.8 and 2.

[0083] Referring to Table 3, the results show that, compared with Comparative Example 1, the extraction reagent and extraction method of the present application can improve the extraction amount and purity of genomic DNA in the dried blood sample.

[0084] Table 3 Test results

[0085]

[0086] The nucleic acid releasing combination solution provided in the present application is used to extract genomic DNA from dried blood smears, which can effectively improve the extraction amount and purity of genomic DNA.

[0087] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A nucleic acid release composition for dried blood spots, characterized in that: The invention comprises a cell dispersion solution and a cell lysis solution, wherein the cell dispersion solution is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, sodium dodecyl sulfate and a solvent, wherein the concentration of tris(hydroxymethyl)aminomethane hydrochloride in the cell dispersion solution is 80-120 mM, the concentration of ethylenediaminetetraacetic acid is 10-12 mM, and the concentration (m / v) of sodium dodecyl sulfate is 0.6-1.2%; The cell lysate consists of guanidine hydrochloride, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride), Triton X-100, isopropanol and a solvent. In the cell lysate, the concentration of guanidine hydrochloride is 3-5M, the concentration of sodium chloride is 50-150mM, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 80-120mM, the volume fraction of Triton X-100 is 1-3%, and the volume fraction of isopropanol is 30-50%.

2. The nucleic acid release composition according to claim 1, characterized in that: The cell dispersion contains proteinase K at a concentration of 0.1-1 mg / mL.

3. The nucleic acid release composition according to claim 1, characterized in that: The preparation method of the cell lysis solution is: guanidine hydrochloride solution, sodium chloride solution, tris(hydroxymethylaminomethane) hydrochloride solution and Triton X-100 solution are prepared in advance by using solvents respectively, and the solutions are mixed and then mixed with the isopropanol.

4. A kit for extracting genomic DNA from dried blood smears, characterized in that: The invention comprises the nucleic acid releasing composition liquid according to any one of claims 1 to 3.

5. The extraction kit according to claim 4, characterized in that It comprises a washing liquid, wherein the washing liquid comprises washing liquid A and washing liquid B, the washing liquid A is composed of guanidine isothiocyanate, β-mercaptoethanol and ethanol, the concentration of guanidine isothiocyanate in the washing liquid A is 4 to 7 M guanidine isothiocyanate, the volume fraction of β-mercaptoethanol is 0.08 to 0.12%, and the volume fraction of ethanol is 50 to 60%; The detergent B consists of sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride) and ethanol. The concentration of sodium chloride in the detergent B is 15-23 mM, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 1-3 mM, and the volume fraction of ethanol is 75-90%.

6. A method for extracting genomic DNA from a dried blood smear, characterized in that: The dried blood piece is treated with the release composition according to any one of claims 1 to 3.

7. The extraction method according to claim 6, characterized in that include: placing the dried blood smear in the cell dispersion and performing a first incubation treatment, and after the incubation treatment is completed, centrifuging the dried blood smear to place it at the bottom; The mixed solution obtained from the first incubation treatment is taken, and the cell lysate is added thereto for a second incubation treatment.

8. The extraction method according to claim 7, characterized in that include: Magnetic beads were directly added to the system after the second incubation treatment for the third incubation treatment.

9. The extraction method according to claim 7, characterized in that The first incubation treatment is performed at a temperature of 50 to 60° C. for 40 to 50 minutes. The temperature of the second incubation treatment is 55-65° C. and the time is 8-15 minutes.

10. The extraction method according to claim 7, characterized in that Each incubation treatment was carried out under shaking conditions.

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