Nucleic Acid Separation Device and Method Thereof

Abstract

The present invention relates to a nucleic acid separation device and a method thereof. The nucleic acid separation device includes a main body formed such that a biological sample passes from the upper part to the lower part, and an absorption layer provided inside the main body and absorbing an inhibitor contained in the sample and inhibiting polymerase chain reaction (PCR). When the sample passes through the main body, the inhibitor is absorbed by the absorption layer, and the nucleic acid is discharged to the lower part of the main body. 【Representative drawing】Figure 1

Description

Technical Field

[0001] The present invention relates to a nucleic acid separation apparatus and method thereof, and more particularly to a nucleic acid separation apparatus and method capable of removing foreign substances from a biological sample and quickly and easily separating out nucleic acids.

Background Art

[0002] Polymerase chain reaction (PCR) is a method for amplifying a specific target genetic material desired for detection, and is a technique for amplifying a large amount of genetic material having the same base sequence from a small amount of genetic material. The polymerase chain reaction (PCR) is used to amplify human DNA and diagnose various genetic diseases, or is applied to DNA of bacteria, viruses, and fungi for diagnosis of infectious diseases.

[0003] Generally, PCR is repeatedly performed in the following three steps. The three steps are (1) A denaturing step of heating a sample solution containing double-stranded DNA to a specific temperature, for example, about 95° C. to separate the double-stranded DNA into single-stranded DNA, (2) After the denaturing step, an oligonucleotide primer having a sequence complementary to the specific base sequence to be amplified is provided to the sample solution, and the mixture is cooled to a specific temperature, for example, 55° C. together with the separated single-stranded DNA to bind the primer to the specific base sequence of the single-stranded DNA to form a partial DNA·primer complex, which is an annealing step, and (3) After the annealing step, the sample solution is maintained at the active temperature of DNA polymerase, for example, 72° C., and an extension (or amplification) step of forming double-stranded DNA based on the primer of the partial DNA·primer complex by DNA polymerase is included. By repeating the three steps several times, the target nucleic acid having a specific base sequence can be amplified geometrically.

[0004] In order to perform such PCR, nucleic acids must be extracted from a biological sample.

[0005] Conventionally, as an example of nucleic acid extraction technology, there has been a method in which a sample containing cells is solubilized by treatment with SDS or proteinase K, and then proteins are denatured and removed with phenol, and nucleic acids are purified. However, the phenol extraction method requires many processing steps, not only taking a lot of time, but also having the problem that the nucleic acid extraction efficiency depends greatly on the experience and proficiency of the researcher, and the reliability drops significantly. Recently, in order to solve such problems, kits using silica or glass fibers that specifically bind to nucleic acids have also been used. Since the aforementioned silica and glass fibers have a low binding ratio to proteins and cell metabolites, relatively high concentrations of nucleic acids can be obtained. Such a method has the advantage of being simple when compared with the phenol method, but since chaotropic reagents and ethanol that strongly inhibit enzymatic reactions such as PCR are used, these substances must be completely removed, and for this reason, there is the disadvantage that the operation is very complicated and time-consuming.

[0006] Also, in Korean Registered Patent No. 10-0454869, DNA extraction using a cell lysis buffer was performed by the following method: (1) After culturing animal cells, the turbid liquid containing the cells was centrifuged to recover the cells. (2) 300 μL of the cell lysis buffer was added to the recovered cells. (3) After adding the cell lysis buffer, it was left at 70°C for 5 minutes. (4) The entanglement of proteins and the like denatured in the lysis stage was loosened, and the lysed cells were pipetted 2 or 3 times in order to preferably pass through the filter. (5) The lysed cells were transferred to a filter made of a silica membrane, then centrifuged at 13,000 rpm for 1 minute, the solution was discarded, and centrifuged one more time. (6) 500 μL of washing buffer was added to the filter and centrifuged. To enhance efficiency, the solution was discarded, centrifuged one more time, and the washing buffer was completely removed. (7) 200 μL of elution buffer was added to the filter and centrifuged. To obtain a larger amount of genomic DNA, the eluted solution was centrifuged through the filter one more time. (8) The extracted DNA was electrophoresed on a 12% agarose gel, stained with EtBr, and then observed.

[0007] Thus, the conventional nucleic acid extraction method (1) a step of adding cell lysis buffer to cells to lyse the cells; (2) a step of transferring the lysed cells in step 1 to a filter to immobilize nucleic acids; (3) a step of washing the filter in step 2; and (4) a step of recovering nucleic acids from the filter, which had the advantage of being able to extract nucleic acids with high reproducibility in a short number of steps and time. However, when transferring the lysed cells to the filter, washing the filter, or recovering nucleic acids from the filter, a centrifuge was used each time. The use of such a centrifuge had the effect of shortening the time, but was inferior in portability and mobility, and had the problem of complicating the nucleic acid extraction process at the site.

[0008] In addition, there are also nucleic acid extraction methods using magnetic beads, nucleic acid extraction methods using a syringe and a filter, nucleic acid extraction methods using DLB (direct lysis buffer), nucleic acid extraction methods using TRIzol (registered trademark), etc. However, for the nucleic acid extraction method using magnetic beads, a magnet is required to fix the nucleic acid to the wall of the tube, and for removing the solution, the use of a pump and valves (automated equipment), or a large number of tips and pipettes (manual) is required. Further, in the nucleic acid extraction method using a syringe and a filter, there is a problem that when a force of a certain intensity or more is applied in the process of transferring the nucleic acid using the syringe, the filter is damaged, making it difficult to extract the nucleic acid. In the nucleic acid extraction method using DLB (direct lysis buffer), since PCR inhibitors are present in the DLB itself, it must be diluted to 1 / 10, so there is a problem that the sensitivity drops significantly due to the dilution. Also, the nucleic acid extraction method using TRIzol (registered trademark) has a problem of using harmful organic solvents such as phenol and chloroform.

[0009] Therefore, there is a need for a device that can rapidly and simply isolate nucleic acids from a biological sample without using additional equipment.

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a nucleic acid separation device and a method thereof that can effectively remove foreign substances from a biological sample and rapidly and easily isolate nucleic acids without using additional equipment.

Means for Solving the Problems

[0011] The nucleic acid separation device for polymerase chain reaction (PCR) testing according to an embodiment of the present invention includes a main body portion formed such that a biological sample passes from top to bottom, and an absorption layer provided inside the main body portion that absorbs inhibitors contained in the sample and inhibits PCR. When the sample passes through the main body portion, the inhibitor is absorbed by the absorption layer, and the nucleic acid is discharged to the lower portion of the main body portion.

[0012] Further, it is desirable that the absorption layer includes at least one resin layer selected from a cation exchange resin layer having a positive charge, an anion exchange resin layer having a negative charge, or a chelate resin layer.

[0013] Further, it is desirable that the absorption layer has an anion exchange resin layer, a chelate resin layer, and a cation exchange resin layer sequentially arranged from the upper part to the lower part of the main body portion.

[0014] Further, the absorption layer includes an anion exchange resin layer, a chelate resin layer, and a cation exchange resin layer, and it is desirable that the volume ratio of the anion exchange resin layer, the chelate resin layer, and the cation exchange resin layer is 1:1:1.

[0015] Further, it is desirable that the flow rate at which the sample passes through the inside of the main body portion is 15 μL / second or less.

[0016] Further, it is desirable to include an input chamber formed with a space for inputting the sample above the main body portion and a collection chamber formed with a space for collecting the nucleic acid discharged from the lower portion of the main body portion.

[0017] Further, it is desirable that the main body portion includes a housing portion that houses the input sample, an adsorption portion provided with the absorption layer, and a discharge portion formed below the adsorption portion.

[0018] Further, it is desirable that an upper filter and a lower filter having a mesh structure for filtering foreign substances contained in the biological sample are respectively provided above and below the absorption layer.

[0019] In addition, the nucleic acid separation method for PCR testing according to another aspect of the present invention includes: injecting a lysis buffer that destroys the cell wall and allows nucleic acids to leak into a biological sample to prepare the sample; introducing the biological sample with exposed nucleic acids into the upper part of the main body; separating and absorbing inhibitors from the biological sample by an absorption layer provided inside the main body to absorb inhibitors that inhibit PCR; and collecting the nucleic acids discharged to the lower part of the main body after the inhibitors are separated from the biological sample.

[0020] Here, it is desirable that the absorption layer includes at least one resin layer selected from a cation exchange resin layer having a positive charge, an anion exchange resin layer having a negative charge, or a chelate resin layer.

[0021] Here, it is desirable that the anion exchange resin layer, the chelate resin layer, and the cation exchange resin layer are sequentially arranged from the upper part to the lower part of the main body.

[0022] Here, the absorption layer includes an anion exchange resin layer, a chelate resin layer, and a cation exchange resin layer, and it is desirable that the volume ratio of the anion exchange resin layer, the chelate resin layer, and the cation exchange resin layer is 1:1:1.

[0023] Here, it is desirable that the flow rate of the sample passing through the inside of the main body is 15 μL / second or less.

[0024] Here, in the step of separating and absorbing inhibitors from the biological sample, a first sub-body provided with an anion exchange resin layer, a second sub-body provided with a chelate resin layer, and a third sub-body provided with a cation exchange resin layer are separately provided, and it is desirable that the sample sequentially passes through the first sub-body, the second sub-body, and the third sub-body.

[0025] Here, it is desirable to use Lysis Buffer as the lysis buffer.

[0026] Here, the absorption layer is polarized, and the main body is filled with a storage buffer that maintains the polarity of the absorption layer. Before introducing the sample into the main body, it is desirable to include a washing step of discharging the storage buffer from the main body.

[0027] Here, the nucleic acid is preferably used for PCR.

Advantages of the Invention

[0028] The nucleic acid separation device and method according to an embodiment of the present invention effectively remove foreign substances from a biological sample and provide the effect of rapidly and easily separating nucleic acids.

[0029] In addition, the present invention eliminates the use of additional equipment such as a centrifuge for nucleic acid separation, facilitating portability and movement to the site and improving user convenience.

[0030] In addition, when the biological sample is moved vertically inside the main body, inside the main body, foreign substances are adsorbed and removed, and nucleic acids are discharged downward and collected. Therefore, the amount of the introduced sample and the extracted nucleic acids are substantially the same, and the effect of ensuring a large amount of nucleic acids from which inhibitors have been removed in one operation is provided.

Brief Description of the Drawings

[0031]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0032] Hereinafter, various embodiments of the present invention will be described in relation to the accompanying drawings. The various embodiments of the present invention can be subject to various modifications and can have various embodiments, but specific embodiments are illustrated in the drawings and related detailed descriptions are provided. However, they are not intended to limit the various embodiments of the present invention to the specific embodiments, and it should be understood that they include all modifications and / or equivalents or alternatives included in the spirit and technical scope of the various embodiments of the present invention. Regarding the description of the drawings, similar reference numerals are used for similar components.

[0033] Expressions such as "comprising" or "also being a thing that comprises" that can be used in various embodiments of the present invention indicate the existence of the disclosed corresponding function, operation, or component, and do not limit one or more further functions, operations, or components. Also, in various embodiments of the present invention, terms such as "comprising" or "having" are used to specify that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, and it should not be understood that they preclude the possibility of the existence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof in advance.

[0034] When it is mentioned that a certain component is "connected to" another component, it should be understood that the aforementioned certain component may be directly connected to the aforementioned other component, but there may be further other components between the aforementioned certain component and the aforementioned other component. On the other hand, when it is mentioned that a certain component is "directly connected to" or "directly coupled to" another component, it should be understood that there are no further other components between the aforementioned certain component and the aforementioned other component.

[0035] The terms used in various embodiments of the present invention are used only for the purpose of describing specific embodiments and are not intended to limit the various embodiments of the present invention. Singular expressions include plural expressions unless clearly stated otherwise in the context.

[0036] Unless otherwise defined, technical or scientific terms are included. All terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the various embodiments of the present invention belong.

[0037] Commonly used terms that are pre-defined should be construed to have a meaning consistent with the meaning in the context of the related art, and should not be construed to have an ideal or overly formal meaning unless clearly defined in the various embodiments of the present invention.

[0038] The present invention relates to a nucleic acid separation device and a method thereof, and is related to a device for removing inhibitors from a biological sample and separating and obtaining nucleic acids in order to perform a polymerase chain reaction (PCR) on the biological sample. Here, the nucleic acid separation device according to the present invention can be utilized not only when nucleic acid extraction is required for the purpose of disease diagnosis, treatment, or prevention, but also when nucleic acid extraction from a sample is required in various fields such as new drug development and detection of environmental hormones.

[0039] First, the term "PCR (polymerase chain reaction) or polymerase chain reaction (PCR)" used in this specification means a reaction that utilizes a thermostable DNA polymerase to amplify a specific target nucleic acid molecule. In addition to the DNA polymerase, the PCR may use a reaction mixture containing primers (forward primers, reverse primers), which are oligonucleotides that can hybridize specifically to the target nucleic acid, a deoxynucleotide triphosphate mixture (dNTP mixture), and divalent ions such as Mg2+.

[0040] "Primer" is used for initiating the PCR reaction and refers to an oligonucleotide or polynucleotide that hybridizes complementarily to template DNA. The primers for the PCR reaction can be a pair consisting of a forward primer (or sense primer) selected from the same sense strand as the gene coding progression direction of the nucleic acid molecule to be amplified, and a reverse primer (or antisense primer) selected from the antisense strand complementary to the sense strand.

[0041] "Sample" refers to a genetic material such as a nucleic acid to be amplified or a biological solution containing such a genetic material. And "reaction reagent" is for detecting the aforementioned target genetic material and includes fluorescent dyes, primers, etc. The primer can also consist of a pair of primers 15 - 30 bp in length that can bind to both ends of a specific site of the target gene. Also, a DNA polymerase that does not lose its activity even at a high temperature of 90°C or higher is used.

[0042] Figure 1 schematically illustrates a nucleic acid separation device according to an embodiment of the present invention, Figure 2 illustrates the configuration of an absorption layer according to another embodiment of the present invention, and Figure 3 illustrates a nucleic acid separation device according to another embodiment of the present invention. Figure 4 illustrates a flowchart of a nucleic acid separation method according to an embodiment of the present invention, and Figure 5 is a diagram showing the sample injection process.

[0043] Hereinafter, preferred embodiments according to the present invention will be described in detail with reference to the accompanying drawings.

[0044] A nucleic acid separation device according to an embodiment of the present invention includes a main body 10 and an absorption layer 124 provided inside the main body 10. When a biological sample passes through the main body 10, an inhibitor that inhibits PCR is absorbed by the absorption layer 124, and the nucleic acid is discharged to the lower part of the main body 10, thereby separating the nucleic acid from the sample.

[0045] The main body 10 is formed such that a biological sample passes through it from top to bottom. The main body 10 can be provided in a tubular or tube shape that is open in the vertical direction. The main body 10 can also be made of materials such as glass, PC, PMMA, PP, PE, metal, and synthetic resin. The main body 10 is generally cylindrical in shape.

[0046] According to this embodiment, the main body 10 includes a storage part 11, an adsorption part 12, and a discharge part 13.

[0047] The storage part 11 provides a space for storing the introduced biological sample. When the biological sample is introduced, since it gradually moves downward from the inside of the main body 10, the storage part 11 provides a space where the introduced sample temporarily stays. The size of the space is sufficient as long as it can accommodate the volume of a single sample introduced to isolate the required nucleic acid capacity, and specifically, there are no restrictions on the size and shape.

[0048] The adsorption part 12 is a part provided inside the main body 10 where an absorption layer 124 for absorbing and removing inhibitors contained in the sample and inhibiting PCR is provided. The inhibitors include salts, proteins, and other intracellular chemical substances contained in the sample. During the implementation of PCR, these substances act as factors interfering with nucleic acid amplification and are removed by the absorption layer 124 of the adsorption part 12. With the absorption layer 124, the inhibitors contained in the sample are adsorbed and removed, and only nucleic acids are discharged downward and can be collected, so that nucleic acids can be separated simply and quickly. The present invention provides a new concept of a separation device and its method for separating nucleic acids without going through the process of fixing nucleic acids using a predetermined adsorption filter or the like and then re-separating nucleic acids from the adsorption filter to separate nucleic acids from a sample.

[0049] The absorption layer 124 also includes at least one resin layer selected from the negatively charged anion exchange resin layer 121, the positively charged cation exchange resin layer 123, and the chelate resin layer 122. The chelate resin layer 122 has a weak positive charge and has the property of capturing divalent metal ions. Further, each resin layer contains spherical resin, and an inhibitor is adsorbed on the spherical resin. The diameter of the spherical resin can be formed to be approximately 0.18 mm to 0.3 mm.

[0050] The absorption layer 124 is polar, and an inhibitor having a polarity in a biological sample can be absorbed and removed by the absorption layer 124. The absorption layer 124 can be formed by any one layer or a combination of a plurality of layers among the anion exchange resin layer 121, the cation exchange resin layer 123, and the chelate resin layer 122.

[0051] According to the present embodiment, the adsorption unit 12 is composed of a plurality of layers. As shown in FIG. 1, the absorption layer 124 includes an anion exchange resin layer 121, a chelating resin layer 122, and a cation exchange resin layer 123. According to the present embodiment, the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123 are arranged in that order from the upper part to the lower part. It is desirable that the volumes of the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123 are substantially provided in a ratio of 1:1:1. Here, it goes without saying that the volume ratio of the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123 is not limited to 1:1:1. The substances contained in the biological sample are polar, and while passing through the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123, they are absorbed by each layer, and only the nucleic acid can flow downward. The anion exchange resin layer 121 also contains at least one of TMA (trimethylamine), DMEA (dimethylethanolamine), tertiary amine, etc., and the chelating resin layer 122 also contains at least one of a carboxyl group (-COOH), iminodiacetic acid chelate, etc. And the cation exchange resin layer 123 also contains at least one of a sulfate group (-SO3H), a carboxyl group (-COOH), a sulfonic acid group (-SO3H), etc.

[0052] The discharge unit 13 is formed below the adsorption unit 12, and the nucleic acid remaining after the inhibitor is removed from the sample is discharged downward. According to the present embodiment, the sample moves inside the main body unit 10 at a speed of 15 μL / second or less, and as a result, the flow rate per unit time of the nucleic acid discharged through the discharge unit 13 is 15 μL or less per second. The flow rate per unit time of the nucleic acid discharged through the discharge unit 13 can be adjusted by appropriately forming the cross-sectional area of the discharge unit 13 through which the nucleic acid passes.

[0053] The larger the cross-sectional area of the discharge section 13 is, the larger the amount of nucleic acid discharged becomes. However, the speed at which the sample passes through the inside of the main body section 10 becomes relatively fast, and it becomes impossible to ensure the time for the inhibitor to be sufficiently adsorbed on the absorption layer 124. Therefore, it was confirmed that when the discharge flow rate of the nucleic acid is adjusted to 15 μL or less per second, the absorption layer 124 can sufficiently remove the inhibitor.

[0054] According to another embodiment of the present invention, the main body section 10 may also be composed of a sub-main body section having a single layer of the absorption layer 124. The sub-main body sections can be provided in plurality. For example, as shown in FIG. 2, the main body section 10 may be provided such that the first sub-main body section 16, the second sub-main body section 17, and the third sub-main body section 18 are separated from each other. An anion exchange resin layer 121 may be provided in the first sub-main body section 16. A chelating resin layer 122 may be provided in the second sub-main body section 17. A cation exchange resin layer 123 may be provided in the third sub-main body section 18. Further, according to the present embodiment, as shown in FIG. 2, the sample is made to primarily pass through the first sub-main body section 16 provided with the anion exchange resin layer 121, and then pass through the second sub-main body section 17 provided with the chelating resin layer 122 and the third sub-main body section 18 provided with the cation exchange resin layer 123 in that order. Pumping means may be used to transfer the sample to the adjacent sub-main body sections. When the first sub-main body section 16, the second sub-main body section 17, and the third sub-main body section 18 are arranged at a distance from each other, the flow rate of the nucleic acid discharged from the third sub-main body section 18 is adjusted to 15 μL or less per second so that the inhibitor is sufficiently adsorbed and removed in each sub-main body section. When using sub-main body sections separated from each other, the discharge flow rate of the nucleic acid can be adjusted by the pumping means. As described above, when the main body section 10 is constituted by the first sub-main body section 16, the second sub-main body section 17, and the third sub-main body section 18, it is possible to form only a single layer of the absorption layer 124 in the sub-main body section without the need to laminate the absorption layer 124, thereby improving the manufacturing convenience.

[0055] According to the present embodiment, it further includes an input chamber 20, a collection chamber 30, an upper filter 14, and a lower filter 15.

[0056] The loading chamber 20 is disposed above the main body 10 and provides a space for loading the sample. That is, the sample can be loaded into the loading chamber 20. As shown in FIG. 1, the loading chamber 20 is provided above the main body 10, and the loading chamber 20 and the main body 10 are also connected by a connecting pipe. Referring to FIG. 1, the loading chamber 20 is formed such that the width becomes narrower from the upper side to the lower side, facilitating the loading of the sample from the upper side and allowing the sample to be quickly discharged to the lower side. However, the shape of the loading chamber 20 is not limited thereto and can be variously changed.

[0057] The collection chamber 30 provides a space for collecting the nucleic acid discharged from the lower part of the main body 10. As shown in FIG. 1, the nucleic acid that has fallen through the discharge part 13 of the main body 10 is collected in the internal space of the collection chamber 30. The collection chamber 30 is in a form that provides a space for collecting the nucleic acid, and its form can be variously changed. For example, as shown in FIG. 3, the collection chamber 30 can be embodied in a form coupled to the main body 10. Referring to FIG. 3, the collection chamber 30 has a cylindrical structure with an open upper part, and the main body 10 can be provided with locking claws that hang on the edge of the collection chamber 30 in the circumferential direction of the outer peripheral surface. The upper part of the locking claw hangs around the upper part of the collection chamber 30, and the lower part of the locking claw can be coupled in a form inserted into the inside of the collection chamber 30. In such a case, the accommodation part 11 at the upper part of the main body 10 can perform the function of the loading chamber 20.

[0058] The foregoing upper filter 14 and lower filter 15 are provided for filtering foreign substances contained in a biological sample, such as sections or fragments of cells. The upper filter 14 is provided above the absorption layer 124, and the lower filter 15 is provided below the absorption layer 124. As shown in FIG. 1, the upper filter 14 is bonded above the anion exchange resin layer 121, and the lower filter 15 is bonded below the cation exchange resin layer 123. By the upper filter 14, foreign substances contained in the sample are primarily filtered and moved to the absorption layer 124. By the lower filter 15, before falling into the collection chamber 30 through the absorption layer 124, foreign substances are secondarily filtered, and only nucleic acids can be collected in the collection chamber 30.

[0059] Hereinafter, the operation and effects of the nucleic acid separation device according to the configuration will be described based on experimental results.

[0060] First, when the sample does not contain an inhibitor, after passing the nucleic acid through the polar resin layer and then performing PCR, as a result, conventionally, when performing PCR after passing the direct lysis buffer (DLB), which is provided using a lysis buffer, through at least one absorption layer 124 during PCR, it was confirmed that the influence on the number of repetitions of the PCR cycle for detecting a specific base sequence is negligible. The PCR cycle is defined as one cycle consisting of the following three steps. The three steps are: (1) a denaturing step in which a sample solution containing double-stranded DNA is heated to a specific temperature, for example, about 95°C, to separate the double-stranded DNA into single-stranded DNA; (2) after the denaturing step, an oligonucleotide primer having a sequence complementary to the specific base sequence to be amplified is provided to the sample solution, and together with the separated single-stranded DNA, it is cooled to a specific temperature, for example, 55°C, to bind the primer to the specific base sequence of the single-stranded DNA and form a partial DNA·primer complex, which is an annealing step; and (3) after the annealing step, the sample solution is maintained at the active temperature of the DNA polymerase, for example, 72°C, and the DNA polymerase forms double-stranded DNA based on the primer of the partial DNA·primer complex, which is an extension (or amplification) step. The PCR repeats the three steps several times until the detection of the specific base sequence is possible, and geometrically amplifies the target nucleic acid having the specific base sequence. In Table 1 below, a Ct value of 29.56 means that the number of repetitions is on average 29.56. From such experimental results, when performing PCR after passing the nucleic acid through the anion exchange resin layer 121, the chelate layer, and the cation exchange resin layer 123, respectively, each having a weak negative charge, and when passing through the absorption layer 124 composed of these absorption layers 124 in three stages, it was confirmed that the number of repetitions of the PCR cycle that enables the detection of a specific gene is negligibly different from the conventional method, and that the nucleic acid is not affected by the resin layer.

[0061] [Table 1]

[0062] And when the sample contains an inhibitor, as shown in Table 2 below, conventionally, the sample prepared using a lysis buffer does not amplify a specific base sequence due to the inhibitor even when the three steps for PCR are performed. However, as shown in Table 2, after passing the sample through the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123, and then repeatedly performing the three steps for PCR, it was confirmed that the specific base sequence was amplified to a detectable level. In particular, as shown in FIG. 1, when the anion exchange resin layer 121, the chelating resin layer 122, and the cation exchange resin layer 123 are laminated from the upper side to the lower side to form the absorption layer 124, it was confirmed that the number of repetitions of the PCR cycle for detecting the specific base sequence is the shortest. That is, when the three-layer resin layer is laminated to form the absorption layer 124, the amplification of the specific base sequence can be completed rapidly.

[0063]

Table 2

[0064] In addition, according to another aspect of the present invention, a nucleic acid separation method is provided.

[0065] The nucleic acid separation method according to the present embodiment, as shown in FIG. 4, includes a step (S1) of injecting a lysis buffer that destroys the cell wall and leaks nucleic acid into a biological sample to prepare a sample, a step (S2) of introducing the biological sample with exposed nucleic acid into the upper part of the main body 10, a step (S3) of separating and absorbing an inhibitor from the biological sample by an absorption layer 124 provided to absorb an inhibitor that inhibits PCR inside the main body 10, and a step (S4) of collecting the nucleic acid discharged to the lower part of the main body 10 after the inhibitor is separated from the biological sample.

[0066] The step of preparing the sample (S1) is a step of injecting a predetermined lysis buffer, for example, Lysis Buffer, into a biological sample to break the cell wall and leak nucleic acids. Lysis Buffer is a lysis buffer used to break the cell wall. That is, in the step of preparing the sample (S1), the cells are lysed using the lysis buffer to elute intracellular nucleic acids.

[0067] The step of loading the sample (S2) is a step of loading the sample with exposed nucleic acids onto the upper part of the main body 10, and it is carried out by loading the sample into the aforementioned loading chamber 20. Here, as shown in FIG. 3, when the accommodating part 11 of the main body 10 substitutes the function of the loading chamber 20, it goes without saying that the sample can be loaded into the accommodating part 11.

[0068] The step of absorbing and separating the inhibitor (S3) is a step in which the inhibitor is absorbed and removed by the absorption layer 124 provided inside the main body 10. The main body 10 and the absorption layer 124 provided inside it can be configured as shown in FIG. 1.

[0069] According to the present embodiment, the absorption layer 124 also includes at least one resin layer selected from among the cation exchange resin layer 123 having a positive electrode, the anion exchange resin layer 121 having a negative electrode, or the chelate resin layer 122 as described above. That is, the absorption layer 124 is made by selecting one layer among the resin layers, or by a combination of two or three layers.

[0070] According to this embodiment, in the absorption layer 124, an anion exchange resin layer 121, a chelating resin layer 122, and a cation exchange resin layer 123 are arranged in this order from the upper part to the lower part of the main body 10. That is, it is composed of three resin layers, and the volume ratio of the anion exchange resin layer 121, the chelating resin layer, and the cation exchange resin layer 123 is substantially 1:1:1. Further, the flow rate at which the sample passes through the inside of the main body 10 is adjusted to 15 μL / second or less. By adjusting the flow rate at which the sample passes through the inside of the main body 10 to 15 μL / second or less, and adjusting the flow rate of the nucleic acid discharged to the lower part of the main body 10 to 15 μL or less per second, the inhibitor can be sufficiently absorbed while the sample passes through the absorption layer 124. When the discharge flow rate of the nucleic acid exceeds 15 μL per second, the moving speed of the sample becomes fast and the inhibitor cannot be sufficiently absorbed.

[0071] Further, according to another embodiment of the present invention, in the inhibitor absorption and separation step (S3), a first sub main body 16 provided with an anion exchange resin layer 121, a second sub main body 17 provided with a chelating resin layer 122, and a third sub main body 18 provided with a cation exchange resin layer 123 are provided separately from each other, and the sample can be passed through the first sub main body 16, the second sub main body 17, and the third sub main body 18 in this order. The above-mentioned first sub main body 16, second sub main body 17, and third sub main body 18 are arranged separately from each other, the sample that has passed through the first sub main body 16 is transferred to the second sub main body 17 and passed through it, and then passed through the third sub main body 18. In such a case, in the nucleic acid collection step (S4), the nucleic acid can be collected at the rear end of the third sub main body 18.

[0072] The above-mentioned step (S4) of collecting the nucleic acid is a step of discharging the nucleic acid to the lower part of the main body 10 and collecting the discharged nucleic acid if the inhibitor is absorbed and removed by the absorption layer 124. According to the embodiment of the present invention, when 500 μL of a biological sample is injected, the inhibitor contained in the sample is removed, and the flow rate discharged to the lower part of the main body 10 is substantially maintained at 500 μL, and inside it, the inhibitor is removed and only the nucleic acid is obtained in a contained state. This greatly improves the ensured flow rate of the sample containing only the nucleic acid compared to the prior art.

[0073] Note that according to the embodiment of the present invention, before introducing a biological sample into the main body 10, a washing step of the main body 10 is included.

[0074] The left side of FIG. 5 illustrates a step of primarily washing a storage buffer that maintains the polarity of the absorption layer 124. According to the embodiment of the present invention, the absorption layer 124 is formed of a resin layer having a polarity, and the polarity of the absorption layer 124 must be maintained until the main body 10 is used. Therefore, the inside of the main body 10 is filled with a storage buffer for maintaining the polarity of the absorption layer 124.

[0075] When using the nucleic acid separation device according to the present invention, the storage buffer is washed. As shown on the left side of FIG. 5, a washing buffer (e.g., water) is introduced into the introduction chamber 20 to wash the storage buffer inside the main body 10. Subsequently, the right side of FIG. 5 illustrates a state in which a sample is introduced after washing the storage buffer, and while the sample is being introduced, the washing buffer filled in the main body 10 is discharged first, and the lower collection chamber 30 is filled. After all the washing buffer is pushed out and discharged by the introduced sample, the nucleic acid to be discharged is collected below the main body 10.

[0076] As described above, the nucleic acid separation device and method according to the embodiment of the present invention can effectively remove inhibitors from a biological sample and quickly and easily secure nucleic acids. The rapid securing of nucleic acids provides an effect of significantly shortening the PCR implementation time. In addition, the present invention eliminates the use of additional equipment such as a centrifuge for nucleic acid separation, making it convenient to carry and move to the site and improving the convenience for users.

[0077] As described above, the present invention has been described in detail with reference to the preferred embodiments. However, the present invention is not limited to the embodiments, and various modifications can be provided within the scope not departing from the scope of the present invention.

Claims

1. A main body 10 formed to allow a biological sample to pass from top to bottom; An absorption layer 124 provided inside the main body 10 and absorbing an inhibitor that inhibits polymerase chain reaction (PCR) contained in the biological sample. A nucleic acid separation device, characterized in that when the biological sample passes through the main body 10, the inhibitor is absorbed by the absorption layer 124 and nucleic acid is discharged from the lower part of the main body 10.

2. The nucleic acid separation device according to claim 1, characterized in that the absorption layer 124 includes at least one resin layer selected from a cation exchange resin layer 123 having a positive charge, an anion exchange resin layer 121 having a negative charge, and a chelate resin layer 122.

3. The nucleic acid separation device according to claim 1, characterized in that the anion exchange resin layer 121, the chelate resin layer 122, and the cation exchange resin layer 123 are arranged in that order from the upper part to the lower part of the main body 10.

4. The absorption layer 124 includes an anion exchange resin layer 121, a chelate resin layer 122, and a cation exchange resin layer 123. The nucleic acid separation device according to claim 1, characterized in that the volume ratio of the anion exchange resin layer 121, the chelate resin layer 122, and the cation exchange resin layer 123 is 1:1:

1.

5. The nucleic acid separation device according to claim 1, characterized in that the flow rate at which the biological sample passes through the inside of the main body 10 is 15 μL / second or less.

6. An injection chamber 20 having a space for injecting the biological sample into the upper part of the main body 10; The nucleic acid separation device according to claim 1, further comprising a collection chamber 30 having a space for collecting the nucleic acid discharged from the lower part of the main body 10.

7. The main body 10 A housing part 11 for housing the biological sample introduced into the main body; An adsorption part 12 where the absorption layer 124 is provided; The nucleic acid separation device according to claim 1, characterized in that it includes a discharge part 13 formed below the adsorption part 12.

8. The nucleic acid separation device according to claim 1, characterized in that an upper filter 14 and a lower filter 15 having a mesh structure for filtering foreign substances contained in the biological sample are respectively provided above and below the absorption layer 124.

9. Injecting a lysis buffer that breaks the cell wall and leaks nucleic acid into the biological sample to prepare the sample; The step of introducing a biological sample with exposed nucleic acid into the upper part of the main body 10; The step of absorbing and separating the inhibitor from the biological sample by an absorption layer 124 provided inside the main body 10 and absorbing an inhibitor that inhibits polymerase chain reaction (PCR); The step of collecting the nucleic acid discharged to the lower part of the main body 10 after the inhibitor is separated from the biological sample, characterized in that it comprises a nucleic acid separation method.

10. The absorption layer 124 includes at least one resin layer selected from a cation exchange resin layer 123 having a positive electrode, an anion exchange resin layer 121 having a negative electrode, and a chelate resin layer 122, according to claim 9. Nucleic acid separation method described.

11. The absorption layer 124 is characterized in that the anion exchange resin layer 121, the chelate resin layer 122, and the cation exchange resin layer 123 are arranged in that order from the upper part to the lower part of the main body 10, according to claim 9. Nucleic acid separation method described.

12. The absorption layer 124 includes an anion exchange resin layer 121, a chelate resin layer 122, and a cation exchange resin layer 123. The volume ratio of the anion exchange resin layer 121, the chelate resin layer 122, and the cation exchange resin layer 123 is 1:1:1, according to claim 9. Nucleic acid separation method described.

13. The flow rate at which the biological sample passes through the inside of the main body 10 is 15 μL / second or less, according to claim 9. Nucleic acid separation method described.

14. The main body 10 further includes a first sub-main body 16 in which an anion exchange resin layer 121 is arranged, a second sub-main body 17 in which a chelate resin layer 122 is arranged, and a third sub-main body 18 in which a cation exchange resin layer 123 is arranged, which are separated from each other. The step of absorbing and separating the inhibitor from the biological sample is: The nucleic acid separation method according to claim 9, characterized in that the biological sample is passed through the first sub-main body 16, the second sub-main body 17, and the third sub-main body 18 in that order.

15. The lysis buffer contains Lysis Buffer, according to claim 9. Nucleic acid separation method described.

16. The absorption layer 124 has a polarity. The main body 10 is filled with a storage buffer that maintains the polarity of the absorption layer 124. The nucleic acid separation method according to claim 9, further comprising a washing step of discharging the storage buffer from the main body 10 before introducing the biological sample into the main body 10.

17. The nucleic acid separation method according to claim 9, wherein the nucleic acid is used for a polymerase chain reaction (PCR) test.

Images