KR20200069180A - Method for eluting a nucleic acid from a nucleic acid-bound particle - Google Patents

Abstract

The present invention relates to a method of eluting nucleic acids from nucleic acid-bound particles in a nucleic acid extraction process. The elution method of the present invention is characterized in that an elution process is performed at a predetermined temperature or higher, thereby increasing efficiency of target detection using eluted nucleic acids.

Description

Method for eluting a nucleic acid from a nucleic acid-bound particle

The present invention relates to a method for eluting nucleic acids from nucleic acid-coupled particles in a nucleic acid extraction process.

As fields requiring nucleic acid analysis, such as disease diagnosis, development of new bio drugs, and criminal investigations, are expanding, there is a need to develop technologies capable of rapidly and efficiently separating nucleic acids from samples in the field of extracting nucleic acids in samples. Current methods for extracting nucleic acids include phenol/chloroform, salting out, chaotropic salts and silica resins, affinity resins, ion exchange chromatography and magnetic beads. And how to do it. These methods are described in US5057426, US4923978, EP0512767, EP0515484, WO95/13368, WO97/10331, and WO96/18731 and the like. These patents and patent applications describe a method for separating nucleic acids after adsorbing them on a solid support.

In particular, the method of extracting nucleic acids using magnetic beads is a method suitable for an automated process, and thus is widely used in screening centers that need to rapidly process a large amount of samples. In screening centers that need to process a large amount of samples within a specified time, it is a very important factor in the time taken to determine whether there is a target pathogen by separating nucleic acids from one sample. This is because the shorter the time required to process one sample, the more samples can be processed. In addition, there is a problem in that the number of diagnostic errors or re-examination increases if each step of the test is not efficiently performed, such as insufficient extraction of nucleic acid in the nucleic acid extraction step or low amplification efficiency in the amplification step.

Therefore, there is a need to improve the nucleic acid extraction process to efficiently and quickly collect nucleic acids in a sample.

Throughout this specification, a number of citations and patent documents are referenced and their citations are indicated. The disclosures of the cited documents and patents are incorporated by reference herein in their entirety and the level of the technical field to which the invention pertains and the contents of the invention are more clearly described.

We have tried to improve the nucleic acid extraction process. In particular, efforts have been made to develop a method for reducing the working time and improving the extraction efficiency, which can be applied to a nucleic acid extraction process suitable for magnetic bead-based automated equipment. As a result, the present inventors perform the step of eluting the nucleic acid from the particles to which the nucleic acid is bound, and extract the nucleic acid by performing at a predetermined temperature or higher, in particular, when the combination of the mixing process is efficiently performed, improved nucleic acid detection results are improved. The present invention was completed by confirming that it can be provided.

Accordingly, an object of the present invention is a nucleic acid-bound particle comprising elution of a nucleic acid by contacting the nucleic acid-bound particle with a elution solution at a temperature of 60 degrees or higher for a predetermined duration. It is to provide a method for eluting nucleic acids from.

Other objects and advantages of the present invention will become more apparent by the following examples, claims and drawings.

I. Method of eluting nucleic acid from particles bound with nucleic acid

According to an aspect of the present invention, the present invention is a nucleic acid from a nucleic acid-bound particle comprising the step of elution of the nucleic acid by contacting the nucleic acid-bound particle with the elution solution at a temperature of 60° C. or higher for a predetermined elution period. It provides a way to elute.

We have tried to improve the nucleic acid extraction process. In particular, efforts have been made to develop a method for reducing the working time and improving the extraction efficiency, which can be applied to a nucleic acid extraction process suitable for magnetic bead-based automated equipment. As a result, the present inventors perform the step of eluting the nucleic acid from the particles to which the nucleic acid is bound to extract the nucleic acid by performing at a predetermined temperature or higher, in particular, when the mixing process is efficiently performed in combination, improved nucleic acid detection results are improved. The present invention was completed by confirming that it can be provided.

Elution means obtaining a target material by dissolving the target material in a liquid phase from a solid material. The term "elution" in the present invention means to obtain by separating the nucleic acid from the nucleic acid-coupled particles.

The elution of the nucleic acid bound to the particle may vary depending on the binding method of the particle and the nucleic acid, and generally may be performed by a method that weakens the binding between the nucleic acid and the particle rather than the binding between the nucleic acid and the solvent. Specifically, when a nucleic acid and a particle capable of binding the nucleic acid are placed in an aqueous solution containing a chaotropic salt in an appropriate concentration, the chaotropic salt binds to a water molecule, thereby weakening the binding of the nucleic acid to the water molecule, and nucleic acid. It is bound to the particles. Then, the nucleic acid may be eluted by obtaining a particle to which the nucleic acid is bound and turbidizing it in an elution solution from which the chaotropic salt has been removed.

The term “nucleic acid” in the present invention is a deoxyribonucleotide or ribonucleotide polymer in single or double chain form (including known analogues of natural nucleotides). The term nucleic acid is meant to include all DNA, cDNA, RNA, unless otherwise specified, and can be used interchangeably with the term. The DNA includes both single-stranded DNA and double-stranded DNA. The RNA includes both single-stranded RNA and double-stranded RNA. According to an embodiment of the present invention, the nucleic acid of the present invention may be double-stranded ribonucleic acid (dsRNA).

The “contact” means to make two or more independent substances into a state capable of causing chemical and physical interactions. For example, in the present invention, contacting a nucleic acid-bound particle with an elution solution means making the nucleic acid bound to the particle be separated from the particle by the elution solution.

The predetermined elution period means the time at which the elution reaction occurs. The predetermined elution period may be, for example, 1 minute, 2 minutes, 3 minutes, 5 minutes or more. According to one embodiment of the present invention, the elution period may be 5 minutes or more. In addition, the predetermined elution period may be 30 minutes, 20 minutes, 15 minutes, 10 minutes or less. If the elution period is too long, the effect may be inferior to the increased time. In addition, when the elution period is long, there is a problem that the time required for the entire process increases, so the elution period is preferably shortened. However, if the elution period is too short, there may be a problem that the elution is not sufficient. According to one embodiment of the present invention, the predetermined elution period may be 3 minutes to 15 minutes. According to one embodiment of the present invention, the predetermined elution period may be 5 minutes to 10 minutes.

The method of the present invention is characterized in that the nucleic acid is eluted at a temperature of 60°C or higher. The nucleic acid obtained by performing the elution step at high temperature then shows improved detection results in the nucleic acid detection process. The constant temperature may be, for example, 60°C, 70°C, 80°C, 90°C or 95°C or higher. The elution method of the present invention is characterized in that the nucleic acid-bound particles are contacted with the elution solution at a temperature of 60° C. or higher to elute the nucleic acid.

According to the method of the present invention, it is possible to shorten the time required for the nucleic acid analysis process by performing the elution step while heating to a predetermined temperature or more. Performing the elution reaction at high temperature may denature the nucleic acid eluted by the elution reaction. If the process following the elution step requires a denaturation process, such as a reverse transcription process, if it elutes by applying the elution method of the present invention, there is no need to add a separate modification process. This can significantly shorten the time for the entire nucleic acid analysis process. The temperature at which denaturation may occur may differ depending on the type of nucleic acid. Therefore, the experimenter can determine the temperature of the elution step in consideration of the type of nucleic acid to be extracted. The elution may be performed at a temperature of 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C. or higher, and preferably 80° C. or higher. .

Therefore, according to one embodiment of the present invention, the elution step may be performed at a minimum temperature or more at which the nucleic acid is denatured.

According to one embodiment of the invention, the elution step may be carried out at a temperature of 70 ℃ or more. According to another embodiment of the invention, the elution step may be performed at a temperature of 80 ℃ or more.

The elution solution can be any solution that can remove or weaken the binding force between the particle and the nucleic acid to separate the nucleic acid from the particle. Specifically, the elution solution of the present invention has a low salt concentration and may be a solution without a hydroxyl donor. The elution solution of the present invention may be, for example, water, or TRIS-HCl, TRIS-EDTA, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), potassium phosphate or sodium in water It may be a solution containing a buffering agent such as phosphate. The concentration of the buffering agent in the elution solution is, for example, 0.1 mM-1000 mM, 0.1 mM-800 mM, 0.1 mM-600 mM, 0.1 mM-300 mM, 0.1 mM-100 mM, 0.1 mM-90 mM, 0.1 mM-80 mM, 0.1 mM- It can be 70mM, 0.1mM-50mM, 0.1mM-40mM, 0.1mM-30mM, 0.1mM-20mM, 0.1mM-10mM, 0.5mM-10mM, 1mM-10mM or 1mM-5mM. The elution solution of the present invention may include one or more buffering agents. In addition, the elution solution of the present invention may further include ethanol, a chaotropic salt, and the like, if necessary.

The pH of the elution solution may be 4 to 12, for example, 7 to 10, 8 to 10, and 8 to 9. According to one embodiment of the invention, the pH of the elution solution may be 8 to 9.

Meanwhile, according to an embodiment of the present invention, the elution step of the present invention may include a process of mixing the particles and the elution solution.

Since the particles are generally insoluble solid particles, the mixture in the present invention means a suspension in which the particles are suspended in a solvent. Mixing in the present invention means that the particles are suspended in a solvent.

Mixing in the present invention may be to maintain the suspended state of the particles by applying a physical force from the outside. Preferably, the mixing may be mixing involving heat loss that lowers the temperature of the mixture of particles and elution solution.

For example, a mixing method that involves changing the contact area between the mixture and the reaction vessel or a method of repeating the operation of inserting and removing objects into the mixture may continuously discharge heat of the mixture through the reaction vessel. Such mixing may prevent the mixture from being maintained at a predetermined temperature or higher, thereby lowering the elution efficiency or lowering the denaturation rate of the nucleic acid molecule, and consequently prevents the nucleic acid detection reaction from occurring effectively.

According to one embodiment of the present invention, the mixing may be to mix the particles and the elution solution by up and down reciprocating motion. The up-and-down reciprocating motion means that the elution solution reciprocates downward and upward.

The up-and-down reciprocating motion repeats the operation of inserting and removing an object such as a rod-type mixing means into the elution solution, or suctioning the elution solution using a device such as, for example, a pipette ( It can be implemented by repeating aspiration and dispensing.

In particular, according to one embodiment of the present invention, the mixing may be to mix the particles and the elution solution by up and down reciprocating motion of the rod-type mixing means.

The rod-shaped mixing means is not particularly limited, and may be, for example, a columnar or polygonal columnar shape. The rod-shaped mixing means may have a constant diameter, or the diameter may be different for each position (height) of the mixing means whose diameter is measured in order to mix the solution well. The rod-shaped mixing means forms a columnar empty space therein and may be in the form of a tube with an open top. According to one embodiment of the present invention, the rod-type mixing means may be a tube guiding a rod including a magnetic force generating material of a liquid transfer device.

The tube can be made of a metal or alloy or non-metallic material. The metal or alloy may be aluminum, iron, stainless steel, or an alloy containing them. Preferably, the tube is magnetically permeable, and may be made of a material that is unlikely to undergo a chemical reaction upon direct contact with a reaction solution containing a specific component. The tube may preferably be a plastic material or a mixture of plastic and other materials, the plastic material being, for example, polyethylene, polyethylene terephthalate, polypropylene, polystyrene, polyvinyl chloride, polycarbonate, melamine resin, Phenol resin or a mixture thereof, but is not limited thereto.

According to the bead transfer method, which is one of nucleic acid extraction methods using magnetic beads, which are frequently used in automated processes, the beads bound with nucleic acids are attached to tubes using a rod-shaped tube in which a magnetic bar is accommodated. After transferring the attached tube to the reaction vessel with the elution solution, the magnetic bar inside is removed to transfer the beads to the elution solution. Subsequently, the tube from which the magnetic bar has been removed is repeatedly moved up and down in the elution solution to mix the magnetic beads with the elution solution and the nucleic acid. This mixing method does not maintain a high reaction temperature and does not have a special effect on the process of performing the elution step. However, in order to shorten the elution time or to simultaneously proceed with the elution and nucleic acid denaturation process, when introducing a mixing method accompanying the effect of lowering the temperature of the mixture as described above in the process of performing the elution step in a high temperature environment, the The mixing method keeps the temperature of the elution solution lower than the set temperature, which negatively affects the nucleic acid detection reaction.

This problem can be solved by further increasing the reaction temperature to prevent the heat loss generated. Therefore, according to one embodiment of the present invention, the elution step may be performed at least 80 ℃ or more. In this case, the mixing can be carried out continuously during the elution period.

On the other hand, according to the method of the present invention, it is possible to solve the above problems by adjusting the proportion of the mixing process in the elution step. The present inventors have confirmed that, rather than continuously mixing the mixture of particles and the elution solution in order to maintain the suspension, it is better to limit the mixing period to a certain ratio over the entire elution period.

According to one embodiment of the present invention, the mixing may be continuously performed during the elution period, may be performed in a certain section of the elution period, or may be performed at a specific time interval during the elution period.

If the temperature during the elution step is sufficiently high, satisfactory dissolution results can be obtained even if a mixing process is performed throughout the elution period. However, preferably, the period for performing the mixing process may be performed in a certain section of the elution period. The predetermined section may be a start section including a time point at which the elution period starts or an end section including a time point at which the elution period ends, and preferably a start section.

For example, but not limited to, the period for performing the mixing process is 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30 of the elution period %, 40%, 50%, 60%, 80% or 100% or less. Optionally, the mixing process may not be performed during the elution period.

According to an embodiment of the present invention, the period for performing the mixing process may be 100% or less of the elution period.

According to an embodiment of the present invention, the period for performing the mixing process may be 30% or less of the elution period.

According to an embodiment of the present invention, the period for performing the mixing process may be 20% or less of the elution period.

Meanwhile, the duration of the mixing process of the present invention may be limited in time. The period for performing the mixing process of the present invention is 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 in a range not exceeding the elution period. It can be no more than minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes.

During the elution step, the period during which the mixing process is not performed may preferably set the mixture of the particles and the elution solution at a temperature of 60° C. or higher. In addition, an additional mixing process may be performed during a period in which the mixing process is not performed during the elution step.

In the present invention, in order to distinguish the aforementioned mixing process from the additional mixing process, the above-described mixing process is referred to as a “basic mixing process” or “main mixing process”, and the additional mixing process is “additional mixing process” or “ Secondary mixing process”.

In the present invention, the additional mixing process is distinguished from the mixing process which is limited in proportion or time in the above-mentioned elution period, in particular, the mixing process by up and down motion.

In the present invention, the additional mixing process is distinguished from the above-described relatively high heat loss mixing process, in particular, the mixing process by vertical motion.

The additional mixing process may be performed by a method that does not involve heat loss, such as shaking. In particular, the shaking may be an orbital shaking method.

The elution is preferably performed to such an extent that the nucleic acid can be separated from the particles, and may be, for example, 1 minute, 2 minutes, 3 minutes, or 5 minutes or more. On the other hand, if the elution time is too long, the entire nucleic acid extraction process may be delayed, and further, the effect of extending the elution time may be insufficient compared to the delay. Therefore, the elution period of the present invention may be 30 minutes, 20 minutes, 10 minutes or 5 minutes or less.

In consideration of shortening the time required for the nucleic acid extraction process, the predetermined elution period in the method of the present invention is 3 minutes to 15 minutes, and the period for performing the mixing process may be 80% or less of the predetermined elution period.

Preferably, in the method of the present invention, the predetermined elution period is 5 minutes to 10 minutes, and the period for performing the mixing process may be 20% or less of the predetermined elution period.

In the present invention, the particle may be composed of any material such as silica, glass, latex, plastic, polymer, cellulose, and refers to a small mass that may exist in any size and shape. The particles of the present invention include any shape, and may be, for example, a random shape, a needle shape, a fibrous shape, a spherical shape, an elliptical shape, a plate shape, and generally a spherical shape or a shape close to a spherical shape.

The size of the particles of the present invention is not particularly limited, for example, the diameter may be 1000um or less, 100um or less, 50um or less, 10um or less, 5um or less. In addition, the diameter of the particles of the present invention may be 1um or more, 500nm or more, 100nm or more, 50nm or more, 10nm or more, 1nm or more. Preferably, the diameter of the particles of the present invention may be 1 nm to 1000 um, 100 nm to 100 um, 500 nm to 10 um, or 500 nm to 5 um.

According to one embodiment of the present invention, the particles of the present invention may be selected from the group consisting of silica beads, polymer beads and cellulose beads. According to the method of the present invention, only one kind of beads of the silica beads, polymer beads, and cellulose beads may be used, or two or more of the silica beads, polymer beads, and cellulose beads may be mixed and used.

In the present invention, the term "bead" is not essential, but mostly refers to a particle or a collection of particles having a spherical or nearly spherical shape.

The particles of the present invention may include a magnetic, paramagnetic or superparamagnetic material to interact with a magnetic field or magnet.

According to one embodiment of the invention, the particles of the invention may be magnetic beads. The magnetic beads of the present invention refer to particles comprising a magnetic, paramagnetic or superparamagnetic material. Preferably, the particles of the present invention may be paramagnetic beads or superparamagnetic beads. Paramagnetic beads refer to particles comprising paramagnetic materials. Superparamagnetic beads refer to particles comprising superparamagnetic materials. Paramagnetic materials or superparamagnetic materials exhibit magnetic properties when there is an external magnetic field, but lose the magnetic properties when the external magnetic field disappears.

According to one embodiment of the present invention, the particle may include a binding site that binds to a nucleic acid. The binding site refers to a site containing a functional group capable of binding a nucleic acid. When the substance constituting the particle itself can bind to the nucleic acid, the particle itself may be a binding site that binds to the nucleic acid. Alternatively, when a functional group capable of binding a nucleic acid is bound to the surface of the particle, the functional group may be a binding site of the present invention. The binding site may be any structure capable of binding nucleic acids, and preferably may be a structure including a hydroxyl donor. The binding of the particle to the nucleic acid is a reversible bond.

According to one embodiment of the present invention, the surface of the particle may include a functional group capable of binding a nucleic acid. Since the nucleic acid has a negative charge on the phosphoric acid group of the backbone, it can bind to a functional group which is a hydroxyl donor, for example.

Accordingly, according to one embodiment of the present invention, the particles may be hydroxyl group donors (hydroxyl donor) or may include functional groups that act as hydroxyl group donors. The functional group serving as the hydroxyl group donor may be, for example, a hydroxyl group, an amine group or a carboxyl group.

According to an embodiment of the present invention, the particle containing the hydroxyl group donor functional group may be a particle containing at least one functional group selected from the group consisting of hydroxyl group, amine group and carboxyl group.

The method of the present invention can be mainly applied to a method for extracting nucleic acids from cells or viruses. Accordingly, the method of the present invention may further include the step of binding the nucleic acid from the cell or virus to the particle.

According to one embodiment of the present invention, the method of the present invention comprises the steps of: (a) lysing a virus or cell in a sample prior to the elution step; (b) a nucleic acid binding step in which the nucleic acid is bound to the particle by contacting the particle with the lysate; And (c) a washing step of removing a substance not bound to the particles from the result of the nucleic acid binding step.

In step (a), the virus or cell in the sample is lysed.

The nucleic acid is exposed to the outside through the lysis, and is in a state capable of separation.

As used herein, the term “sample” includes biological samples (eg, cells, tissues, and body fluids) and non-biological samples (eg, food, water, and soil), and the biological samples include, for example, viruses, bacteria, tissues, cells, Blood (including whole blood, plasma and serum), lymph, bone marrow, saliva, sputum, swab, aspiration, milk, urine, feces, ocular fluid, semen, brain extract, spinal fluid, joint fluid , Thymus fluid, bronchial washing fluid, ascites and amniotic fluid.

The lysis means rupturing the cell membrane, viral protein envelope, and the like. The dissolution may be performed through physical, bio, and chemical methods. The biochemical dissolution method may be a method of treating a solvent such as enzymatic dygestion, pH adjustment, an alkali solution, or a water-soluble anionic detergent. The physical method may be a sonication method or a heat treatment method.

In step (b), the nucleic acid is bound to the particle by contacting the particle with the lysate. The step (b) is referred to as a nucleic acid binding step.

The lysate means the result of dissolving the sample. The lysate may include nucleic acids of cells or viruses in the sample. The nucleic acid in the lysate is bound to the particles. The binding is a reversible binding, and can be adjusted according to specific conditions, such as salt, pH, and temperature. The binding can preferably be carried out in the presence of chaotropic salts, such as guanidine salts.

In the washing step (c), a substance not bound to the particles is removed from the result of the nucleic acid binding step. The washing step refers to a step of removing substances derived from samples other than the nucleic acid bound to the particles. In the washing step, a nucleic acid bound to the particles is not separated, but a solution that thoroughly washes unwanted substances is used. The washing step is preferably performed by incubating the washing solution with the particles and then removing the washing solution. When using a magnetic bead as the particles of the present invention, a magnetic field may be applied in the process of removing the washing solution to prevent the nucleic acid-bound particles from being removed together with the washing solution. The washing step may be performed once or more than once. For example, the washing step may be carried out repeatedly 1 to 10 times, 1 to 5 times, 2 to 5 times, or 2 to 4 times.

The nucleic acid extracted from the crushed cells and bound to the beads can be eluted according to the elution method of the present invention. The dissolution method is as described above.

The method of mixing the magnetic beads with a solution suitable for each step may be by a reaction solution transfer method or a bead transfer method.

The reaction solution transfer method is performed by fixing the magnetic beads to the reaction container with a magnet or the like, and removing the reaction solution after introducing the solution necessary for extraction into the container. Specifically, after the sample and the lysate are reacted by introducing a sample and a lysate into a reaction vessel in which the magnetic beads are fixed, the sample lysate is removed, and the rinse solution is washed to remove the rinse solution, and the eluent is introduced to elute. It is a method of obtaining the elution solution after.

In the bead transfer method, the sample and the dissolution solution, the washing solution, and the elution solution are respectively pre-dispensed in a plurality of containers, and the magnetic beads are magnetic rods in the container with the sample and dissolution solution, the container with the cleaning solution, and the container with the elution solution Nucleic acid is extracted by sequentially moving using a magnetic rod. In particular, the cover in which the magnetic rod is inserted is used together.

The container for nucleic acid elution by the method of the present invention includes a multi-well plate.

Meanwhile, according to an embodiment of the present invention, the method of the present invention may include detecting a target nucleic acid from the eluted nucleic acid.

The terms “target nucleic acid”, “target molecule” or “target nucleic acid molecule” mean a target nucleic acid to be finally amplified or detected, and may be used interchangeably.

Target nucleic acid molecules can be any naturally occurring prokaryotic, eukaryotic (e.g., protozoa and parasites, fungi, yeast, higher plants, lowland and higher animals including mammals and humans), viruses (e.g., herpes Virus, HIV, influenza virus, hepatitis virus, polio virus, etc.), or a nucleic acid molecule of a viroid. The nucleic acid molecule can also be any nucleic acid molecule that can be recombinantly produced or chemically synthesized. Thus, nucleic acid sequences may or may not be found in nature.

The target nucleic acid molecule includes not only the double strand but also the single strand, and the nucleic acid sequence newly generated in the reaction process can be the target nucleic acid molecule as well as when it exists from the beginning in the sample to be analyzed. The present invention does not require that the target nucleic acid molecule to be detected has any specific sequence or length, and the target nucleic acid molecule of the present invention may include any DNA (gDNA and cDNA) and RNA molecules.

The detection method can be by a known target nucleic acid detection method. Various methods are known using the signal-generating means to generate a signal indicative of the presence of a target nucleic acid. Representative examples include: TaqMan probe method (US Patent No. 5,210,015), molecular beacon method (Tyagi et al., Nature Biotechnology v.14 MARCH 1996), Scorpion method (Whitcombe et al., Nature Biotechnology 17:804- 807 (1999)), Sunrise or Amplifluor method (Nazarenko et al., 2516-2521 Nucleic Acids Research, 25(12):2516-2521 (1997), and U.S. Patent No. 6,117,635), Lux method (US Patent No. 7,537,886), CPT (Duck P, et al. Biotechniques, 9:142-148 (1990)), LNA method (US Patent No. 6,977,295), Flexor method (Sherrill CB, et al., Journal of the American Chemical Society, 126:4550-4556 (2004)), Hybeacons (DJ French, et al., Molecular and Cellular Probes (2001) 13, 363-374 and U.S. Patent No. 7,348,141), double-labeled self- Dual-labeled, self-quenched probe (US Pat. No. 5,876,930), hybridization probe (Bernard PS, et al., Clin Chem 2000, 46, 147-148), PTOCE (PTO cleavage and extension) method ( WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (PCT/KR2013/012312) And CER method (WO 2011/0373 06).

Detection of the target nucleic acid may proceed with amplification of the nucleic acid. Accordingly, according to one embodiment of the present invention, the method of the present invention may further include a step of amplifying the nucleic acid by dispensing the eluted nucleic acid in the nucleic acid amplifying composition.

The amplification refers to a template-dependent process that increases the amount of target nucleic acid compared to the initial amount. The amplification repetitively complementary nucleic acid strands using both the nucleic acid present in the sample as a template, and constructing a complementary nucleic acid strand, and using both the reconstituted complementary nucleic acid strand and the nucleic acid present in the initial sample as a template. It may be a method of exponentially increasing the amount of the target nucleic acid in a manner to restructure.

For amplification of the nucleic acid, the nucleic acid isolated from the sample is reacted with the nucleic acid amplification composition. The nucleic acid amplification composition may include, for example, polymerase, dNTP, primer and buffer.

In addition, in the case of amplification of the present invention, when the nucleic acid present in the sample is RNA, a reverse transcription step is essential before the annealing step is performed. For details, see Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. Press, Cold Spring Harbor, NY (2001); And Noonan, K. F. et al., Nucleic Acids Res. 16:10366 (1988). The reverse transcription process refers to a process of preparing a DNA strand complementary thereto using RNA as a template. For reverse transcription reactions, oligonucleotide dT primers, random primers or target-specific primers can be used.

According to an embodiment of the present invention, the method of the present invention may be a method of extracting nucleic acid from a nucleic acid-coupled particle in a nucleic acid extraction method using a nucleic acid extraction device.

The nucleic acid extraction device may be a liquid transfer device including a liquid transfer means. Alternatively, the nucleic acid extraction apparatus may be a bead transfer method apparatus including particle collection and transport means.

Specifically, the bead transfer-based nucleic acid extraction device includes a rod containing a magnetic force generating material, a tube (cover or strip) into which the rod is inserted, and a module for moving the magnetic rod and tube. According to an embodiment of the present invention, in the nucleic acid extraction apparatus, the magnetic rod and the tube are independently movable in the vertical direction. According to an embodiment of the present invention, in the nucleic acid extraction device, the magnetic rod and the tube are movable in independent or dependent left and right directions. In the nucleic acid extracting device, the magnetic rod and the tube are movable in independent or dependent front-rear directions.

The tube may serve to cover or guide the magnetic rod. The magnetic rod is inserted into the tube, and when the magnet rod moves up and down, the tube can move up and down together. Alternatively, the tube may serve to mix the solution in a process for nucleic acid extraction (eg, dissolution, nucleic acid binding, washing or elution).

According to another aspect of the present invention, the present invention provides a nucleic acid extraction apparatus for performing the method of the present invention for eluting nucleic acids from particles to which nucleic acids are bound.

II. Record carriers, devices and computer programs

The recording medium, apparatus and computer program of the present invention are for carrying out the above-described method of the present invention, and contents common to them are omitted to avoid excessive complexity of the present specification by repeated description.

According to another aspect of the present invention, the present invention provides a computer readable recording medium comprising instructions for implementing a processor for executing a method for eluting nucleic acids from nucleic acid bound particles, the method comprising: Includes:

Elution of the nucleic acid by bringing the nucleic acid-bound particle into contact with the elution solution at a temperature of 60° C. or higher for a predetermined duration.

According to another aspect of the present invention, the present invention provides a computer program stored in a computer-readable recording medium, embodying a processor for executing a method for eluting nucleic acid from a nucleic acid-coupled particle, the method comprising: The steps include:

Elution of the nucleic acid by bringing the nucleic acid-bound particle into contact with the elution solution at a temperature of 60° C. or higher for a predetermined duration.

The program instructions, when executed by a processor, cause the processor to execute the method of the present invention described above. Program instructions for executing a method for eluting nucleic acids from nucleic acid-bound particles are to elution nucleic acids by bringing the nucleic acid-bound particles into contact with the elution solution for a predetermined duration at a temperature of 60° C. or higher. Instructions may include.

The method of the present invention is executed in a processor, and the processor may be a stand-alone computer connected to a nucleic acid extraction device, a network attached computer, or a processor embedded in the nucleic acid extraction device.

Computer readable recording media are various storage media known in the art, such as CD-R, CD-ROM, DVD, flash memory, floppy disk, hard drive, portable HDD, USB, magnetic tape, MINIDISC, non-volatile memory card , EEPROM, optical disk, optical storage medium, RAM, ROM, system memory and web server.

Instructions for implementing a processor implementing the invention may be included in a logic system. The instructions may be provided on a software recording medium (eg, portable HDD, USB, floppy disk, CD and DVD), but are downloadable and other memory such as a hard drive or local or attached RAM or ROM. ). Computer code for implementing the present invention can be executed in various coding languages such as C, C++, Java, Visual Basic, VBScript, JavaScript, Perl and XML. In addition, various languages and protocols can be used for external and internal storage and delivery of instructions according to the present invention.

According to another aspect of the invention, the invention elutes a nucleic acid from a nucleic acid bound particle comprising (a) a computer processor, and (b) the computer readable recording medium of the invention coupled to the computer processor. It provides a device for doing.

According to an embodiment of the present invention, the device of the present invention is a reaction vessel that can accommodate a mixture of nucleic acid-bound particles and an elution solution, a nucleic acid-bound particle or nucleic acid bound from a mixture in the reaction vessel Means for separating the solution excluding particles and/or temperature control means may be additionally included.

According to an embodiment of the present invention, the device of the present invention may be a nucleic acid extraction device. According to an embodiment of the present invention, the apparatus of the present invention may include a rod including a magnetic force generating material and a tube guiding the rod when the rod is inserted to move the rod in the vertical direction.

The computer processor may be constructed such that one processor performs all of the above-described performances. Alternatively, the processor unit may be constructed such that several processors divide and execute the above-described performance.

According to one embodiment of the present invention, the processor may be implemented by installing software in a conventional device (eg, nucleic acid extraction device) used to separate nucleic acids from a sample.

The effects of the present invention are as follows.

(a) The nucleic acid eluate obtained according to the elution method of the present invention provides improved nucleic acid detection results (eg, a small Ct value).

(b) When the time of the mixing process is limited to a certain ratio compared to the elution period, better nucleic acid detection results can be obtained than when the mixing process is continuously performed within the elution period.

(c) When the time of the mixing process is limited to a certain ratio compared to the elution period, heat loss in the mixing process can be reduced, so that the nucleic acid is eluted from the particles at a desired temperature. In addition, when denaturation of a nucleic acid eluting at a desired temperature occurs, it is possible to maintain the temperature at which the denaturation occurs.

(d) When a high temperature elution temperature is used, denaturation of the eluted nucleic acid can be simultaneously performed in the elution process. If the eluted nucleic acid is used directly in the detection step, the time required for a separate denaturation process can be removed or reduced, and the time required for the entire nucleic acid detection process can be shortened. This is particularly useful when the elution target is RNA that requires reverse transcription.

(e) In the case of a device for simultaneously extracting nucleic acids from a plurality of samples, the surrounding environment may be different depending on the position of the container where the elution step is performed. In this case, even if the elution step is performed under the same conditions, the heat loss environments for each container may be different. The method of the invention, in particular the method of adjusting the mixing process, can solve the problems arising from these environmental differences.

1 is a result of confirming the change in nucleic acid detection effect according to the elution protocol change using the Rotavirus standard strain.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, according to the gist of the present invention, the scope of the present invention is not limited by these examples to those skilled in the art to which the present invention pertains. It will be obvious.

Example

Example 1 Protocol comparison of elution step during Rotavirus nucleic acid extraction process

<1-1> Rotavirus nucleic acid extraction according to elution method

The elution step optimization test according to the method of the present invention was performed with a standard Rotavirus strain. The Rotavirus used was the ATCC VR-2018 standard strain. A test sample was prepared by diluting 1000 times with Phosphate-Buffered Saline (PBS).

Nucleic acid extraction was performed with SGprep32 (Seegene, Korea), an extraction automation equipment using STARMag 96 UniPlate/UniTube extraction kit (Seegene, Korea).

The cover of the STARMag 96 UniPlate/UniTube extraction kit was removed, 200ul of the prepared test sample was dispensed into a well containing 150ul of the Lysis buffer containing protenase K, and then left at room temperature for 5 minutes to proceed with Lysis.

After Lysis is completed, 650 μl of Binding buffer containing Sodium perchlorate (20-40%), Ethanol (35-55%) and Magnetic beads is administered to each well, and then mounted on SGprep32 to mount nucleic acids according to Uniprotocol-A. Was extracted.

Nucleic acid-bound magnetic beads generated from the extracted binding buffer were washed with 500ul of washing buffer 1 (Sodium perchlorate (5-20%), ethanol (25-35%)) and 2 of washing buffer (ethanol (75-87%)). Elution was performed after washing by sequentially mixing in 500ul and separating.

In the elution step of the SGprep32 equipment uni-protocol-A step, experiments were conducted under the following four conditions, and extraction of 3 repetitions per condition was performed. The elution buffer used was Tris/HCl buffer (pH 8.5).

(1) Uni-Protocol A-1: Mix in 80ul elution buffer 100ul for 5 minutes

(2) Uni-Protocol A-2: 100ul of elution buffer at 80℃ for 10 minutes

(3) Uni-Protocol A-3: After mixing 100ul of elution buffer for 30 seconds at 80℃, stand for 4 minutes and 30 seconds.

(4) Uni-Protocol A-4: After mixing 100ul of elution buffer for 30 seconds at 80℃, stand for 9 minutes and 30 seconds.

In the above protocol, mixing was performed by moving the disposable cover mounted on the magnetic bar up and down in the solution in each well when extracting with SGprep32.

After the elution was completed, magnetic beads were removed from the elution buffer to obtain a total of 12 nucleic acid extracts, three for each of the four protocols, and a detection test using the same was conducted.

<1-2> Comparison of Rotavirus detection effect according to elution method

Rotavirus detection effect was tested using the results of extracting 12 nucleic acids obtained for each of the elution protocols.

The test was performed using the Allplex™ GI-Virus Assay product (Seegene, Korea). 5 ul of the extracted nucleic acid per test, 5X GI-V MOM 5 ul, RNase-free Water 8 ul, 5X Real-time One-Step Buffer 5 ul, Real-time One-Step Enzyme 2 ul was mixed to prepare a reaction mixture in a total volume of 25 ul.

Then, the PCR plate containing the reaction mixture was subjected to real-time PCR using a real-time PCR device (CFX96 Real-time cycler, Bio-Rad, USA). The amplification reaction was 50 min at 20°C and 15 min at 95°C. The reaction was repeated 45 times at 95°C for 10 seconds, at 60°C for 60 seconds, and at 72°C for 30 seconds. Fluorescence was measured at 60°C and 72°C every cycle.

The data analysis was conducted by Seegene Viewer S/W (Seegene, Korea), and the Ct value (threshold cycle) and analysis results are described in Table 1 and FIG. 1.

Elution conditions Uni-ProtocolA-1
(80℃, 5 minutes mixing) Uni-ProtocolA-2
(80℃, 10 minutes mixing) Uni-ProtocolA-3
(After mixing at 80℃ for 30 seconds
4 minutes 30 seconds fixed) Uni-ProtocolA-4
(After mixing at 80℃ for 30 seconds
9 minutes 30 seconds fixed) Test 1 (Ct) 33.69 32.72 31.10 31.15 Test 2 (Ct) 34.32 31.47 31.11 30.69 Test 3 (Ct) 35.19 34.25 32.64 31.68 Average 34.40 32.81 31.62 31.17 Standard Deviation 0.75 1.39 0.89 0.50 Max 35.19 34.25 32.64 31.68 Min 33.69 31.47 31.10 30.69 Δ
(Max-Min) 1.50 2.78 1.54 0.99

As shown in Table 1 and Figure 1, comparing the average Ct of uni-protocols A-1 and A-3, the mixing process is limited to 30 seconds rather than elution while continuously mixing at 80°C for 5 minutes, and 4 It can be seen that it is more efficient to stand for 30 minutes. These results can also be confirmed by comparing the average Ct of uni-protocols A-2 and A-4 in which the elution step was performed for 10 minutes. Also, comparing the average Ct of the uni-protocols A-2 and A-3, it can be seen that it is more effective to limit the time of the mixing process than to simply increase the target detection time.

As a result, it can be seen that limiting the duration of the mixing process in the elution step has better target detection effect than elution while continuously mixing.

Claims (22)

A method for eluting nucleic acids from particles bound with nucleic acids comprising:
Elution of the nucleic acid by bringing the nucleic acid-bound particle into contact with the elution solution at a temperature of 60° C. or higher for a predetermined duration. The method of claim 1, wherein the elution step comprises mixing the particles and the elution solution. The method according to claim 2, wherein the mixing is continuously performed during the elution period, is performed at a certain section of the elution period, or is performed at a specific time interval during the elution period. 3. The method of claim 2, wherein the period for performing the mixing process is 30% or less of the elution period. The method of claim 1, wherein the predetermined elution period is 3 to 15 minutes, and the period for performing the mixing process is 80% or less of the predetermined elution period. The method of claim 1, wherein the elution period is 5 minutes or more. The method of claim 2, wherein the mixing (mixing) is characterized in that the particles and the elution solution is mixed by up and down reciprocating motion. The method of claim 1, wherein the elution step is carried out at a temperature above the minimum temperature at which the nucleic acid is denatured. The method of claim 1, wherein the elution step is performed at a temperature of 70° C. or higher. The method of claim 1, wherein the elution step is performed at a temperature of 80°C or higher. The method of claim 1, wherein the particles are selected from the group consisting of silica beads, polymer beads, and cellulose beads. The method of claim 1, wherein the particles are magnetic beads. The method of claim 1, wherein the surface of the particle comprises a functional group capable of binding a nucleic acid. 14. The method of claim 13, wherein the particle is a hydroxyl donor or a method comprising a functional group that acts as a hydroxyl donor. The method according to claim 1, wherein the particle containing a functional group that serves as a hydroxyl donor is a particle comprising at least one functional group selected from the group consisting of hydroxyl group, amine group and carboxyl group. The method of claim 1, wherein the method is prior to the elution step.
(a) lysing the virus or cells in the sample (lysis);
(b) a nucleic acid binding step of binding the nucleic acid to the particle by contacting the particle with the lysate; And
(c) A method comprising the step of removing a substance that is not bound to the particles from the result of the nucleic acid binding step. The method of claim 1, wherein the method comprises detecting a target nucleic acid from the eluted nucleic acid. The method of claim 1, wherein the method further comprises the step of amplifying the nucleic acid by dispensing the eluted nucleic acid in a nucleic acid amplification composition. The method of claim 1, wherein the nucleic acid is a double stranded ribonucleic acid (dsRNA). A computer readable recording medium comprising instructions for implementing a processor to implement a method for eluting nucleic acids from nucleic acid bound particles comprising the following steps:
Elution of the nucleic acid by bringing the nucleic acid-bound particle into contact with the elution solution at a temperature of 60° C. or higher for a predetermined duration. An apparatus for eluting nucleic acids from nucleic acid bound particles comprising (a) a computer processor, and (b) a computer readable recording medium of claim 20 coupled to the computer processor. A computer program stored on a computer readable recording medium embodying a processor for executing a method for eluting nucleic acids from particles to which nucleic acids are bound, comprising:
Elution of the nucleic acid by bringing the nucleic acid-bound particle into contact with the elution solution at a temperature of 60° C. or higher for a predetermined duration.

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