Nucleic acid eluent and method for eluting nucleic acids using said nucleic acid eluent

Abstract

The present invention improves the recovery rate of nucleic acid bonded to a nucleic acid capturing carrier, and recovers the nucleic acid at a high concentration. In the present invention, nucleic acid bonded to a nucleic acid capturing carrier is recovered by using a nucleic acid elution liquid containing: a polar solvent for dissolving the nucleic acid; and a hydrophobic solution that undergoes liquid-liquid layer separation with respect to the polar solvent.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid elution solution for eluting nucleic acids from a carrier to which the nucleic acids have been adsorbed, and a method for eluting nucleic acids using the nucleic acid elution solution. [Background technology]

[0002] In recent years, information obtained through nucleic acid analysis, such as cancer genome testing using next-generation sequencing (NGS) systems, has been utilized in various fields, including medicine, clinical testing, the pharmaceutical industry, and the food industry. For this nucleic acid analysis, nucleic acid extraction from various biological samples, such as blood, tissue, and cultured cells, is an essential pretreatment step.

[0003] Nucleic acid extraction methods generally do not use harmful organic solvents such as phenol or chloroform, but rather rely on the property of nucleic acids to bind to silica in the presence of a chaotropic agent or on the property of nucleic acids to bind to silica in the presence of an organic solvent. Using these methods, nucleic acid extraction methods have been reported, such as using a nucleic acid capture chip that incorporates a silica-containing solid phase as a nucleic acid capture carrier, or using magnetic beads (nucleic acid capture carriers) whose surfaces are coated with silica. These methods include a step of binding nucleic acids to the nucleic acid capture carrier and an elution step of eluting the nucleic acids from the nucleic acid capture carrier using an eluent.

[0004] In the method using magnetic beads, after the elution step, the magnetic beads are recovered from the eluent using a magnet. One example is a method in which the eluent containing the magnetic beads is drawn into a dispensing tip, the magnetic beads are retained in the dispensing tip using a magnet, and only the eluent is discharged from the dispensing tip. Another example is a method in which a rod-shaped magnet (which may be covered) is inserted into the eluent containing the magnetic beads, and the magnetic beads are recovered from the eluent.

[0005] Furthermore, Non-Patent Document 1 discloses a method for recovering nucleic acids from a silica membrane by passing an eluent through a silica membrane to which nucleic acids have been adsorbed. In the method disclosed in Non-Patent Document 1, the eluent is passed through the silica membrane by a method such as centrifugation or aspiration. At this time, in order to recover the eluent remaining on the silica membrane, mineral oil is passed through the silica membrane by the same method such as centrifugation or aspiration. By passing the mineral oil through the silica membrane, the eluent remaining on the silica membrane is reliably recovered.

[0006] Furthermore, Patent Documents 1 to 3 disclose systems for eluting nucleic acids captured on magnetic beads using magnetic beads such as those disclosed in Patent Document 4. The systems disclosed in Patent Documents 1 to 3 place aqueous solutions such as washing solutions and eluents in a column via an oil layer, and pass the magnetic beads through the column to wash the magnetic beads and elute the nucleic acids from the magnetic beads. In other words, the provision of an oil layer in the column prevents aqueous solutions such as washing solutions and eluents from mixing. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2017-184626 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-067274 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-093988 [Patent Document 4] Japanese Patent Application Publication No. 2017-176023 [Non-patent literature]

[0008] [Non-Patent Document 1] npj Precision Oncology volume 4, Article number: 3 (2020) Summary of the Invention [Problem to be solved by the invention]

[0009] When nucleic acids are recovered using nucleic acid capture carriers such as magnetic beads as described above, it is preferable to use a system with excellent nucleic acid recovery efficiency. In particular, when analyzing trace amounts of nucleic acids contained in biological samples, low nucleic acid recovery efficiency can lead to the inconvenience of inaccurate analysis using the recovered nucleic acids. On the other hand, if the volume of eluent is increased to efficiently recover the nucleic acids captured on the nucleic acid capture carrier, the nucleic acid concentration in the eluent decreases, which can lead to the inconvenience of requiring complicated operations such as concentrating the nucleic acids prior to subsequent analysis.

[0010] However, in the above-mentioned conventional systems, the only way to increase the efficiency of nucleic acid recovery from nucleic acid capture carriers is to increase the volume of the eluent, which has the problem that high concentrations of nucleic acids cannot be recovered in the eluent. In view of the above-mentioned circumstances, the present invention aims to provide a nucleic acid eluent that can achieve excellent nucleic acid recovery efficiency and recover high concentrations of nucleic acids in the eluent when recovering nucleic acids using nucleic acid capture carriers, and a method for eluting nucleic acids using the nucleic acid eluent. [Means for solving the problem]

[0011] In order to achieve the above-mentioned object, the inventors conducted extensive research and found that by using a nucleic acid elution solution for eluting nucleic acids from a nucleic acid capture carrier as a mixed solvent containing a polar solvent capable of dissolving nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation from the polar solvent, the nucleic acids captured on the nucleic acid capture carrier can be recovered in high concentrations in the polar solvent, thereby completing the present invention.

[0012] The present invention encompasses the following.

[0013] [1] A nucleic acid elution solution for eluting nucleic acids bound to a nucleic acid capture carrier, comprising: A nucleic acid elution solution comprising a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation from the polar solvent.

[0014] [2] The hydrophobic solution has a kinematic viscosity of 17 mm at 40 ° C. 2 The nucleic acid eluent according to [1], characterized in that the elution rate is 1 / s or less.

[0015] [3] The hydrophobic solution has a kinematic viscosity of 10 mm at 25 ° C. 2 The nucleic acid eluent according to [1], characterized in that the elution rate is 1 / s or less.

[0016] [4] The nucleic acid eluent according to [1], wherein the hydrophobic solution contains at least one component selected from the group consisting of silicone oil, fluorine-based oil, and liquid paraffin.

[0017] [5] A method for eluting nucleic acid, comprising contacting a nucleic acid capture carrier on which nucleic acid has been captured with a nucleic acid elution solution to elute the nucleic acid from the nucleic acid capture carrier, A method for eluting nucleic acids, wherein the nucleic acid elution solution contains a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation relative to the polar solvent.

[0018] [6] The hydrophobic solution contained in the nucleic acid elution solution has a kinematic viscosity of 17 mm at 40°C. 2 The method for eluting nucleic acids according to [5], characterized in that the elution rate is 1 / s or less.

[0019] [7] The hydrophobic solution contained in the nucleic acid elution solution has a kinematic viscosity of 10 mm at 25°C. 2 The method for eluting nucleic acids according to [5], characterized in that the elution rate is 1 / s or less.

[0020] [8] The method for eluting nucleic acids according to [5], wherein the hydrophobic solution contained in the nucleic acid elution solution contains at least one component selected from the group consisting of silicone oil, fluorine-based oil, and liquid paraffin.

[0021] [9] The method for eluting nucleic acids according to [5], characterized in that after the step of contacting the nucleic acid capture carrier with the nucleic acid elution solution, the nucleic acid capture carrier is separated from the nucleic acid elution solution, and the nucleic acid is recovered in a polar solvent in the nucleic acid elution solution.

[0022]

[10] A method for eluting nucleic acids according to [5], characterized in that it comprises a step of contacting a biological sample prepared from a test organism with a nucleic acid capture carrier, and capturing nucleic acids derived from the biological sample on the nucleic acid capture carrier.

[0023]

[11] A nucleic acid processing device comprising a reaction vessel for bringing a nucleic acid capture carrier having captured nucleic acid into contact with a nucleic acid elution solution, and a separation device for separating either the nucleic acid capture carrier or the nucleic acid elution solution from the reaction vessel, The nucleic acid processing device is characterized in that the nucleic acid elution solution contains a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation relative to the polar solvent.

[0024]

[12] The nucleic acid processing device according to

[11] , characterized in that the nucleic acid capture carriers are magnetic beads, the separation device has a magnetic part containing a magnetic material, and the separation device attracts the nucleic acid capture carriers to the magnetic part and separates the nucleic acid capture carriers from the nucleic acid elution solution in the container. [Effects of the Invention]

[0025] According to the nucleic acid elution solution of the present invention, the amount of liquid can be increased by using a hydrophobic solution when eluting nucleic acids from a nucleic acid capture carrier, and the nucleic acids captured on the nucleic acid capture carrier can be recovered in a polar solvent at a high concentration.

[0026] Furthermore, the nucleic acid elution method of the present invention uses a nucleic acid elution solution containing a polar solvent that dissolves nucleic acids and a hydrophobic solution, so that the amount of liquid can be increased by the hydrophobic solution when eluting nucleic acids from the nucleic acid capture carrier, and the nucleic acids captured on the nucleic acid capture carrier can be recovered in high concentration in the polar solvent.

[0027] Furthermore, since the nucleic acid processing device of the present invention uses a nucleic acid elution solution containing a polar solvent that dissolves nucleic acids and a hydrophobic solution, the amount of nucleic acid elution solution in the reaction vessel can be increased when eluting nucleic acids from the nucleic acid capture carrier, and the nucleic acids captured on the nucleic acid capture carrier can be recovered in high concentration in the polar solvent. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a flowchart showing an example of a nucleic acid extraction method to which the present invention is applied. [Figure 2] FIG. 1 is a schematic diagram showing an example of a system for extracting nucleic acids using magnetic particles. [Figure 3] FIG. 10 is a schematic diagram showing another example of a system for extracting nucleic acids using magnetic particles. [Figure 4] FIG. 1 is a characteristic diagram showing the relationship between the volume of nucleic acid eluent and the DNA recovery rate. [Figure 5] FIG. 10 is a characteristic diagram showing the relationship between the volume of a nucleic acid elution solution and the recovery rate of the nucleic acid elution solution. [Figure 6] FIG. 1 is a characteristic diagram showing the relationship between the composition conditions of a nucleic acid eluent and the DNA recovery rate. [Figure 7] FIG. 1 is a characteristic diagram showing the relationship between the amount of hydrophobic solution added to a nucleic acid elution solution and the DNA recovery rate. [Figure 8] FIG. 10 is a characteristic diagram showing the relationship between the amount of hydrophobic solution added to the nucleic acid elution solution and the recovery rate of the nucleic acid elution solution. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[0030] The nucleic acid elution solution according to the present invention contains a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation from the polar solvent. The nucleic acid elution solution can be brought into contact with a nucleic acid capture carrier that has captured nucleic acids, thereby eluting the nucleic acids from the nucleic acid capture carrier. Use of the nucleic acid elution solution according to the present invention can significantly improve the recovery rate of nucleic acids from the nucleic acid capture carrier (nucleic acid recovery rate).

[0031] A polar solvent is a solvent that has a polarity capable of dissolving hydrophilic nucleic acids and is also called a highly polar solvent. Typical examples of polar solvents include low-salt aqueous solutions and pure water. For example, a solvent consisting of 10 mmol / L Tris (pH 8.5) and 0.1 mmol / L EDTA can be used.

[0032] A hydrophobic solution is a solution primarily composed of nonpolar molecules and exhibits the property of liquid-liquid phase separation from the polar solvent. Therefore, a nucleic acid elution solution containing a hydrophobic solution and a polar solvent is separated into a layer containing the hydrophobic solution and a layer containing the polar solvent. The composition of the hydrophobic solution is not particularly limited, but it is preferable that the hydrophobic solution contains an oil, i.e., a hydrocarbon compound, as its primary component. Examples of such hydrophobic solutions include liquids containing an oil selected from the group consisting of silicone oil, fluorine-based oil, and liquid paraffin. The hydrophobic solution is not limited to a solution consisting of a single component, but may also be a mixed solution consisting of multiple components. Examples of silicone oils that can be used include dimethylsilicone oil, methylhydrogensilicone oil, methylphenylsilicone oil, and cyclic dimethylsilicone oil.

[0033] The hydrophobic solution has a kinematic viscosity of 17 mm at 40°C. 2 / s or less is preferable, and 14 mm 2 / s or less, and the kinematic viscosity at 25°C is preferably 10 mm 2 It is most preferable that the kinematic viscosity of the hydrophobic solution at 40°C is in the range of 17 mm / s or less. 2 / s or less, 14mm 2 / s or less, or a kinematic viscosity of 10mm at 25°C 2When the kinetic viscosity is in the range of 1 / s or less, the nucleic acid recovery rate can be further improved. The kinetic viscosity of a hydrophobic solution is defined as a value at a specific temperature, but the kinetic viscosity tends to decrease as the temperature increases. Therefore, by setting the temperature when using the nucleic acid eluent at 40°C or higher, the kinetic viscosity at 40°C can be reduced to 17 mm 2 Excellent nucleic acid recovery rates can be achieved even when using hydrophobic solutions exceeding 1 / s.

[0034] The nucleic acid eluent having the above-described structure can be used to elute nucleic acids from nucleic acid capture carriers to which nucleic acids are bound. The nucleic acid eluent according to the present invention can be applied, for example, to the nucleic acid extraction method shown in the flowchart of FIG. 1. The nucleic acid eluent according to the present invention is used in step 4: elution from nucleic acid capture carriers in the flowchart of FIG. 1. The application of the nucleic acid eluent according to the present invention is not limited to the flowchart shown in FIG. 1.

[0035] In this nucleic acid extraction method, first, in step 1, a lysis reagent for preparing a sample containing nucleic acids and a binding reagent for binding nucleic acids to a nucleic acid-binding carrier are prepared, and then the lysis reagent and binding reagent are added to a biological sample such as animal cells. Alternatively, the lysis reagent and binding reagent may be prepared as a single liquid and added to the biological sample. According to step 1, nucleic acids contained in the biological sample are dissolved in a solution, thereby preparing a sample containing nucleic acids. Here, nucleic acids include deoxyribonucleic acids and ribonucleic acids that are double-stranded, single-stranded, or partially double-stranded or single-stranded. In this nucleic acid extraction method, biological samples are not limited to animal cells; microbial cells such as bacteria and fungi, plant cells, or viruses may also be used. However, biological samples such as whole blood, plasma, serum, sputum, urine, cultured cells, and cultured bacteria are particularly desirable.

[0036] In step 1, techniques such as heating and stirring can be appropriately applied to promote dissolution of the biological sample. After dissolving the biological sample with the lysis reagent, solids may be removed by centrifugation.

[0037] Substances contained in the above-mentioned binding reagent that promote the binding of nucleic acids to the nucleic acid-binding solid phase include chaotropic agents such as NaI (sodium iodide), KI (potassium iodide), NaClO4 (sodium perchlorate), NaSCN (sodium thiocyanate), GuSCN (guanidine thiocyanate), GuHCl (guanidine hydrochloride), etc. The binding reagent is not limited to these, and any substance that promotes the binding of nucleic acids to the nucleic acid-binding solid phase may be used.

[0038] Next, in step 2 of this nucleic acid extraction method, a nucleic acid capture carrier is contacted with the nucleic acid-containing sample prepared in step 1, and nucleic acid is bound to the nucleic acid-binding carrier. Examples of nucleic acid capture carriers include glass particles, silica particles, silica-coated magnetic particles, quartz filter paper, quartz wool or crushed products thereof, and diatomaceous earth. In other words, any substance containing silicon oxide or an organic polymer having hydroxyl groups on its surface can be used as a nucleic acid capture carrier. The nucleic acid capture carrier is not limited to these, and any substance that can capture nucleic acid can be used.

[0039] In particular, the present invention can be applied to a method for extracting nucleic acids using silica-coated magnetic particles (also referred to as magnetic beads or magnetic beads). Specifically, as shown in FIG. 2, first, a plurality of magnetic particles 2 are mixed in a reaction vessel 3 with a sample 1 containing nucleic acids prepared in step 1. This allows the nucleic acids contained in the sample 1 to bind to the magnetic particles 2. At this time, by using a dispensing tip 4 to aspirate and discharge the magnetic particles 2 together with the sample 1, the sample 1 can be effectively brought into contact with the magnetic particles 2. At this time, a pipetter, syringe, pump, or the like is directly or indirectly attached to the dispensing tip 4 in an airtight state, and the sample 1 can be aspirated or discharged by vacuum decompression, pressurization, centrifugation, or the like using these.

[0040] 2, while the magnetic particles 2 are attracted to the dispensing tip 4 together with the sample 1, a magnetic body 5 is brought close to the side of the dispensing tip 4. As a result, the magnetic particles 2 in the dispensing tip 4 are captured on the inner wall of the dispensing tip 4. In this state, the sample 1 in the dispensing tip 4 is discharged into a reaction vessel 3, thereby separating the magnetic particles 2 to which the nucleic acid is bound from the sample 1.

[0041] 3, first, a plurality of magnetic particles 2 are mixed in a reaction vessel 3 with the sample 1 containing nucleic acid prepared in step 1. This allows the nucleic acid contained in the sample 1 to bind to the magnetic particles 2. At this time, the sample 1 containing the magnetic particles 2 is stirred, thereby allowing the sample 1 to be brought into effective contact with the magnetic particles 2.

[0042] 3, the cylindrical cover 6 with the magnetic rod 7 inserted therein is immersed in the reaction vessel 3, thereby capturing the magnetic particles 2 in the sample 1 on the bottom and sides of the cover 6. Then, the cylindrical cover 6 is removed from the reaction vessel 3 with the magnetic rod 7 still inserted therein, thereby separating the magnetic particles 2 with the nucleic acid bound thereto from the sample 1.

[0043] Furthermore, by removing the magnetic rod 7 from the cover 6 while the magnetic particles 2 in the sample 1 are trapped on the bottom and side surfaces of the cover 6, the magnetic particles 2 trapped on the bottom and side surfaces of the cover 6 can be returned to the sample 1. In this way, by repeating the operation of trapping the magnetic particles 2 in the sample 1 on the bottom and side surfaces of the cover 6 and then returning the magnetic particles 2 trapped on the bottom and side surfaces of the cover 6 to the sample 1, the sample 1 can be brought into effective contact with the magnetic particles 2.

[0044] Although not shown, the nucleic acid capture carrier may be disposed inside a dispensing tip, and may bind nucleic acids in a solution containing nucleic acids by aspirating the solution into the dispensing tip. In this case, repeated aspirating and discharging of the solution can effectively bring the solution into contact with the nucleic acid capture carrier disposed in the dispensing tip. Furthermore, although not shown, a nucleic acid capture column packed with nucleic acid capture carriers can also be used. In this case, a sample containing nucleic acids can be added to the top of the column, and the sample can be passed through by pressurization, centrifugation, vacuum decompression, etc., to bring the sample into contact with the nucleic acid capture carrier.

[0045] Next, in step 3 of this nucleic acid extraction method, the nucleic acid-bound nucleic acid capture carrier is washed to remove non-specifically bound substances from the nucleic acid capture carrier. The washing reagent is not particularly limited, but may be, for example, any reagent that can remove the reagent added in step 1 and impurities from the nucleic acid capture carrier while maintaining the binding of nucleic acid to the nucleic acid capture carrier. Organic compounds such as lower alcohols and low-molecular-weight ketones can be used as the washing reagent. For example, ethanol and isopropanol can be used as the washing reagent, and it is particularly preferable to use ethanol with a concentration of 70% or higher.

[0046] The technique for contacting the cleaning reagent with the nucleic acid capture carrier is not particularly limited, but the technique used when contacting the above-mentioned nucleic acid-containing sample with the nucleic acid capture carrier (for example, the method shown in Figures 2 and 3) can be applied as appropriate.

[0047] Next, in step 4 of the nucleic acid extraction method, the nucleic acid bound to the nucleic acid capture carrier is eluted from the nucleic acid capture carrier. Specifically, the nucleic acid is recovered in the nucleic acid elution solution by contacting the nucleic acid-bound nucleic acid capture carrier with the nucleic acid elution solution. The technique for contacting the nucleic acid elution solution with the nucleic acid capture carrier is not particularly limited, but the technique used when contacting the nucleic acid-containing sample with the nucleic acid capture carrier (e.g., the method shown in Figures 2 and 3) can be used as appropriate.

[0048] The nucleic acid elution solution according to the present invention contains a polar solvent that dissolves nucleic acids and a hydrophobic solution, and nucleic acids are dissolved in the polar solvent. Therefore, by using the nucleic acid elution solution according to the present invention, nucleic acids can be eluted in a high concentration into the polar solvent.

[0049] For example, when contacting a nucleic acid capture carrier with a nucleic acid elution solution according to the methods shown in Figures 2 and 3, it is difficult to elute all or most of the nucleic acids from the nucleic acid capture carrier unless the entire nucleic acid capture carrier, such as the magnetic particles 2, is brought into contact with the nucleic acid elution solution. Furthermore, even in methods other than those shown in Figures 2 and 3, it is important to ensure that the nucleic acid elution solution is brought into contact with the nucleic acid capture carrier in order to elute all or most of the nucleic acids from the nucleic acid capture carrier.

[0050] In the method shown in Figure 2, when the amount of nucleic acid elution liquid is small, it is preferable to reduce the inner diameter of the dispensing tip according to the amount of liquid that can be aspirated. However, if the inner diameter is too small, the nucleic acid capture carrier will clog the dispensing tip, so it is preferable to ensure a certain size. Therefore, when a certain inner diameter is maintained, it is preferable to limit the amount of nucleic acid elution liquid that can be aspirated into the dispensing tip.

[0051] The same applies to the method shown in Figure 3, where it is preferable to fill the nucleic acid elution solution up to the height where the nucleic acid capture carrier is covered on the magnetic rod, it is preferable to ensure a certain level of liquid volume, and it is also preferable to limit the amount of nucleic acid elution solution accordingly.

[0052] The nucleic acid elution solution according to the present invention is stirred including the hydrophobic solution, so that the amount of polar solvent can be kept small even when a liquid volume that ensures contact with the nucleic acid capture carrier is used. In other words, by using the nucleic acid elution solution according to the present invention, it is possible to ensure that the nucleic acid elution solution is brought into contact with the nucleic acid capture carrier, thereby eluting all or most of the nucleic acids from the nucleic acid capture carrier, and also to dissolve high concentrations of nucleic acids in the polar solvent.

[0053] Thus, by using the nucleic acid eluent according to the present invention, it is possible to increase the recovery rate of nucleic acids from nucleic acid-bound nucleic acid capture carriers and obtain a highly concentrated nucleic acid solution. In the obtained nucleic acid solution, the hydrophobic solution layer and the polar solvent layer containing nucleic acids can be separated and the polar solvent layer can be used for subsequent analysis, or the two layers of the hydrophobic solution layer and the polar solvent layer containing nucleic acids can be used for subsequent analysis as they are.

[0054] The analysis using the obtained nucleic acid solution is not particularly limited, but examples include sequence analysis using a next-generation sequencer or the like, electrophoresis, high-performance liquid chromatography mass spectrometry, etc. When subjected to these specific analyses, the two layers, the hydrophobic solution layer and the polar solvent layer containing nucleic acids, can be used as they are. [Example]

[0055] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0056] Example 1 1. Purpose In this example, we investigated the relationship between nucleic acid concentration, nucleic acid eluent volume, and extraction efficiency, and then investigated a concentration method for extracting nucleic acids at high concentrations in small volumes. Specifically, a polar solvent (main component: water) to which a hydrophobic solution (oil) was added was used as the nucleic acid eluent. DNA can be dissolved in polar solvents, but is insoluble in hydrophobic solutions (oil).

[0057] In this example, we aimed to elute highly concentrated DNA using a smaller volume (less than 50 μL) of nucleic acid eluent containing a hydrophobic solution. We also considered that the addition of a hydrophobic solution would result in a more concentrated DNA in the polar solvent than in the conventional method, provided that the DNA recovery rate remained the same.

[0058] 2. Target In this example, as an example, a nucleic acid preparation system compatible with liquid biopsy was used, and cell-free DNA (hereinafter referred to as cfDNA) in blood was targeted.

[0059] 3.Material 3-1.Reagents Table 1 shows the kinematic viscosity and flash point (values ​​listed in the catalog and SDS) of the hydrophobic solutions (oils) used in the nucleic acid elution solution. Hydrophobic solutions (oils) A, B, and D are silicone oils with a dimethylpolysiloxane structure. They are extremely chemically inert and feature excellent heat and cold resistance and viscosity stability, and are available in a wide range of viscosities. Hydrophobic solutions (oils) C and E (mineral oils) are hydrophobic, low-viscosity oils that are added to PCR reaction solutions to prevent evaporation during PCR, but do not inhibit the PCR reaction after nucleic acid extraction.

[0060] [Table 1]

[0061] 3-2.Mock plasma sample A 140-bp PCR product was added to cfDNA and DNA-free human plasma as a simulated cfDNA (hereafter referred to as simulated cfDNA). The concentration of the simulated cfDNA was adjusted according to each experiment.

[0062] 4. Method 4-1.cfDNA extraction method 4-1-1.cfDNA extraction method 1 We performed extraction of simulated cfDNA. An example of the extraction method used in this example is shown in Figure 3. First, a reagent that solubilizes blood proteins is added, binding the DNA in the sample to the magnetic beads. Agitation is performed by the up-and-down movement of a plastic rod-shaped comb. A magnet is placed inside the comb, and the comb is immersed in the solution, attracting the magnetic beads with DNA bound to the tip of the comb. Next, with the magnet still attached inside the comb, the comb is transferred to a washing solution with the magnetic beads still attached. The comb is left in the washing solution, and the magnet is removed, releasing the magnetic beads into the washing solution. This process of agitation, magnetic bead adsorption, and magnetic bead transfer is repeated, and the sample is washed with multiple washing solutions. Next, the magnetic beads are transferred to a nucleic acid elution solution, and the DNA bound to the magnetic beads is eluted into the nucleic acid elution solution. After the DNA is released, the magnetic beads are attracted to the magnet via the comb and separated from the nucleic acid elution solution.

[0063] The specific procedure for cfDNA extraction is as follows: First, two 24-deep-well plates were prepared, and each reagent was dispensed into the designated locations on the plates according to Table 2.

[0064] [Table 2]

[0065] Next, 5 mL of the simulated plasma sample was added to columns A and B of Plate 1 and gently mixed by pipetting. Nucleic acid extraction was then performed. After nucleic acid extraction was completed, the DNA extracted from column A of Plate 2 was immediately collected in a 500 μL tube for storage.

[0066] 4-1-2.cfDNA extraction method 2 cfDNA extraction was also performed using a different method, the specific procedures of which are shown below.

[0067] 1 mL of simulated plasma sample was mixed with the reagents shown in Table 3. Multiple samples were treated in batches.

[0068] [Table 3]

[0069] Next, the mixture was incubated at 60°C for 20 minutes. Every 5 minutes, the mixture was removed from the incubator and stirred for 1 minute. The mixture was then placed on ice for 5 minutes. Finally, the sample was mixed with the magnetic bead-containing buffer as shown in Table 4. Multiple samples were processed in batches.

[0070] [Table 4]

[0071] After thoroughly mixing the solution in the tube, place it on a magnet for 5 minutes. The supernatant was then completely removed. The tube was removed from the magnet, and 1 mL of lysis and binding reagent was added per 1 mL of sample to the magnetic bead-containing tube. The tube was then vortexed and transferred to a new 1.5 mL tube. The tube was then placed back on the magnet, and the collected supernatant was returned to the original tube. Any remaining magnetic beads were then further collected and added to the 1.5 mL tube. The supernatant was then completely removed while the tube was still attached to the magnet. The tube was then removed from the magnet, and 1 mL of lysis and binding reagent was added and vortexed for 30 seconds. The vortexed sample was then placed on the magnet for 2 minutes or until the supernatant was clear. The supernatant was then completely removed while the tube was still attached to the magnet. The tube was then removed from the magnet, and 1 mL of 80% ethanol was added and vortexed for 30 seconds. The vortexed sample was then placed on the magnet for 2 minutes or until the supernatant was clear. The supernatant was then completely removed while the tube was still attached to the magnet.

[0072] The tube was fixed to the magnet and left to stand for 3-5 minutes, then dried, and the remaining liquid was completely removed. The tube was then removed from the magnet, and a polar solvent, a dissolving and binding reagent, was added. Depending on the experimental conditions, a hydrophobic solution (oil) was also added. The amount of liquid varied depending on the experimental conditions.

[0073] The mixture was then stirred for 5 minutes, and the resulting sample was placed on a magnet for 2 minutes or until the supernatant became transparent, after which the supernatant was collected.

[0074] 4-1-3. Evaluation of nucleic acid eluent The volume of nucleic acid eluate and DNA concentration were determined, and the DNA recovery rate, which is synonymous with nucleic acid extraction efficiency, was calculated.

[0075] The volume of nucleic acid elution solution was calculated by measuring the weight of the empty tube and the tube after nucleic acid elution solution recovery, and then calculating the volume from the difference. The volume of simulated cfDNA added to the simulated plasma sample was the volume of the solution dispensed with the pipette. The recovery rate of the nucleic acid elution solution was calculated according to the following formula:

[0076]

number

[0077] The DNA concentration in the nucleic acid eluent and the DNA concentration in the experimental sample were measured using a fluorometer. The same experiment was performed on a simulated plasma sample without the addition of simulated cfDNA, and the measured values ​​were subtracted as background values.

[0078] The DNA recovery rate was calculated by multiplying the liquid volume by the DNA concentration. Then, the DNA recovery rate was calculated according to the following formula:

[0079]

number

[0080] 5. Effect of the volume of nucleic acid eluent Simulated plasma samples spiked with simulated cfDNA at 15 ng / 5 mL of plasma were processed in a nucleic acid elution device, and DNA was eluted using three nucleic acid eluent volumes: 50, 100, and 200 μL. Figure 4 shows the DNA recovery rate for each volume of nucleic acid eluent. The measured DNA recovery rates for 50 μL and 100 μL were 63.8% and 58.9%, respectively, with no significant difference observed. On the other hand, the DNA recovery rate for 200 μL was improved to 77.7%. Figure 5 shows the recovery rate for each volume of nucleic acid eluent. When using 50, 100, and 200 μL of nucleic acid eluent in the method shown in Figure 3, the recovery rate was 80.4–91.5%, lower than the expected volume. Furthermore, the recovery rate of nucleic acid eluent increased with increasing volume of nucleic acid eluent. This indicates that a smaller volume of nucleic acid eluent increases the percentage of nucleic acid eluent remaining on the magnetic beads.

[0081] 6. Effect of hydrophobic solutions on nucleic acid elution 6-1. Comparison of dynamic viscosity of hydrophobic solutions (oils) Nucleic acids were manually eluted from 1 mL of simulated plasma sample to which simulated cfDNA was added so as to be 50 to 120 ng / 1 mL of plasma. The nucleic acid eluent used consisted of 50 μL or 100 μL of polar solvent, and 50 μL of polar solvent to which 50 μL of hydrophobic solution (oil) was added. Four types of hydrophobic solution (oil) with different kinetic viscosities, excluding C in Table 1, were compared. Figure 6 shows the DNA recovery rate. As shown in Figure 6, the DNA recovery rate for 50 μL of nucleic acid eluent to which no hydrophobic solution (oil) was added, was an average of 62.7%. In contrast, the DNA recovery rate for 50 μL of nucleic acid eluent to which no hydrophobic solution (oil) was added was 62.7%. 2 When hydrophobic solutions (oils: A, B, and D) with a kinematic viscosity of 14 to 17 mm / s (25°C) were added, the DNA recovery rate was 72.6 to 77.6%, which was either unchanged or improved compared to the samples without hydrophobic solutions (oils). 2 When E was added at 1 / s (38°C), the DNA recovery rate was 32.8% on average, which clearly decreased the DNA recovery rate.

[0082] The experimental conditions under which the DNA recovery rate did not improve or decreased may have been due to insufficient mixing conditions between the hydrophobic solution (oil) and the polar solvent, and it is believed that the DNA recovery rate can be improved by optimizing these.

[0083] 6-2. Comparison of the amount of hydrophobic solution (oil) added Nucleic acid extraction was performed using 4-1-2.cfDNA extraction method 2 from 1 mL of human plasma sample to which simulated cfDNA had been added to give a concentration of approximately 0.75 ng / 1 mL of plasma. The nucleic acid eluent used was 50 μL of polar solvent alone, to which 50 or 150 μL of hydrophobic solution (oil) was added, or 15 μL of polar solvent alone, to which 35, 85, or 185 μL of hydrophobic solution (oil) was added. All hydrophobic solutions (oils) had a kinematic viscosity of 5 mm. 2 / s (C does not have a measurement temperature listed in the SDS, B is 25°C) C and B were used. From the considerations in the previous section, B was selected because of its low viscosity and relatively high flash point (102°C).

[0084] Figure 7 shows the DNA recovery rate under each condition. When 50 μL of polar solvent alone was used as the nucleic acid eluent, the average was 77.7%. When a hydrophobic solution (oil) was further added, the DNA recovery rate improved to 93.7-97.4%. No difference was observed depending on the type of hydrophobic solution (oil). When 15 μL of polar solvent alone was used as the nucleic acid eluent, the DNA recovery rate averaged 70.1%. Furthermore, when the amount of hydrophobic solution (oil) added was 85 or 185 μL, the DNA recovery rates were 82.8-87.7% and 77.8-94.2%, respectively, demonstrating an improvement over the DNA recovery rate when no hydrophobic solution (oil) was added. Oil type C had a higher recovery rate than oil type B.

[0085] Figure 8 shows the recovery rate of the nucleic acid eluate recovered when eluted under each condition. In all cases, the recovery rate for the nucleic acid eluate amount was 69.3 to 93.5%, which was lower than the expected nucleic acid eluate amount of 15 μL or 50 μL. When the nucleic acid eluate amount was 15 μL, the recovery rate of the nucleic acid eluate tended to increase by adding hydrophobic solution C. Correspondingly, the DNA recovery rate also tended to increase. This indicates that the amount of nucleic acid eluate remaining on the magnetic beads, etc., was reduced.

[0086] 7. Review of Results From the results of this example, it was revealed that by using a nucleic acid elution solution containing a polar solvent that dissolves nucleic acids and a hydrophobic solution, nucleic acids can be eluted at a high concentration even if the polar solvent is used in a small volume (for example, less than 50 μL). The dynamic viscosity of the hydrophobic solution was 10 mm 2 It was revealed that a range of 1 / s or less is preferable, but it was suggested that nucleic acids can be eluted at high concentrations even outside this range by considering the mixing conditions with polar solvents.

[0087] Regarding the amount of hydrophobic solution added, the DNA recovery rate tended to increase with the amount of hydrophobic solution added. However, even if the amount of hydrophobic solution was small, it was suggested that the nucleic acid could be eluted at a high concentration by reducing evaporation during operation and residue when separating the magnetic beads and nucleic acid eluent.

Claims

1. A nucleic acid elution solution for eluting nucleic acids bound to a nucleic acid capture carrier after stirring, comprising: A nucleic acid elution solution characterized in that it is a mixed solvent containing a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation from the polar solvent, wherein the hydrophobic solution is selected from the group consisting of silicone oil, fluorine-based oil and liquid paraffin, each having a dynamic viscosity of 10 mm2 / s or less at 25°C, and the nucleic acid capture carrier is a magnetic bead.

2. A method for eluting nucleic acid, comprising contacting a nucleic acid capture carrier on which nucleic acid has been captured with a nucleic acid elution solution while stirring, and eluting the nucleic acid from the nucleic acid capture carrier, comprising: The nucleic acid elution method is characterized in that the nucleic acid elution solution is a mixed solvent containing a polar solvent that dissolves nucleic acids and a hydrophobic solution that undergoes liquid-liquid phase separation in the polar solvent, the hydrophobic solution being selected from the group consisting of silicone oil, fluorine-based oil and liquid paraffin, each having a dynamic viscosity of 10 mm2 / s or less at 25°C, and the nucleic acid capture carrier is a magnetic bead.

3. The nucleic acid elution method according to claim 2, characterized in that after the step of contacting the nucleic acid capture carrier with the nucleic acid elution solution while stirring, the nucleic acid capture carrier is separated from the nucleic acid elution solution, and the nucleic acid is recovered in a polar solvent in the nucleic acid elution solution.

4. 3. The method for eluting nucleic acids according to claim 2, characterized in that it comprises a step of contacting a biological sample prepared from an organism to be tested with a nucleic acid capture carrier, and capturing nucleic acids derived from the biological sample on the nucleic acid capture carrier.

5. A nucleic acid processing device comprising a reaction vessel in which a nucleic acid capture carrier having captured nucleic acid is brought into contact with a nucleic acid elution solution while stirring, and a separation device that separates either the nucleic acid capture carrier or the nucleic acid elution solution from the reaction vessel, The nucleic acid elution solution is a mixed solvent containing a polar solvent that dissolves nucleic acids and a hydrophobic solution that causes liquid-liquid phase separation in the polar solvent, the hydrophobic solution being selected from the group consisting of silicone oil, fluorine-based oil and liquid paraffin, each having a dynamic viscosity of 10 mm2 / s or less at 25°C, and the nucleic acid capture carrier is a magnetic bead.

6. The nucleic acid processing device described in Claim 5, characterized in that the separation device has a magnetic part containing a magnetic material, and the separation device attracts the nucleic acid capture carrier to the magnetic part and separates the nucleic acid capture carrier from the nucleic acid elution solution in the container.

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