阅读说明：本技术 利用提取盒的核酸提取方法 (Nucleic acid extraction method using extraction cassette ) 是由 赵荣植 李孝根 朴海准 李善永 林冠勋 金芢爱 金宰永 朴婋林 金彤宪 于 2018-12-20 设计创作，主要内容包括：本发明提供一种核酸提取方法,上述核酸提取方法包括：步骤(a),使得核酸提取装置的驱动部连接到配置在活塞上部主体内部空间的控制棒模块及与活塞下部主体相结合的旋转调节模块；步骤(b),驱动旋转调节模块及控制棒模块,从提取盒的相互分离的多个腔室向上述内部空间依次吸入样本及多个试剂,向提取盒的腔室排出上述内部空间的混合液；以及步骤(c),驱动旋转调节模块及控制棒模块,在向活塞上部主体内部空间吸入提取盒的主混合珠腔室内部的试剂之后,向核酸扩增模块排出经混合的试剂。(The invention provides a nucleic acid extraction method, which comprises the following steps: a step (a) of connecting a drive section of the nucleic acid isolation apparatus to a control rod module disposed in an inner space of the upper piston body and a rotation adjustment module coupled to the lower piston body; a step (b) of driving the rotation regulating module and the control rod module, sequentially sucking a sample and a plurality of reagents into the internal space from a plurality of chambers separated from each other of the extraction box, and discharging a mixed liquid in the internal space into the chambers of the extraction box; and (c) driving the rotation control module and the control rod module to suck the reagent in the main mixing bead chamber of the extraction cartridge into the inner space of the upper body of the piston and then discharge the mixed reagent to the nucleic acid amplification module.)

1. A method for extracting nucleic acid using an extraction cassette, comprising:

a step (a) of connecting a drive section of the nucleic acid isolation apparatus to a control rod module disposed in an inner space of the upper piston body and a rotation adjustment module coupled to the lower piston body;

a step (b) of driving the rotation regulating module and the control rod module, sequentially sucking a sample and a plurality of reagents into the internal space from a plurality of chambers separated from each other of the extraction box, and discharging a mixed liquid in the internal space into the chambers of the extraction box; and

and (c) driving the rotation regulating module and the control rod module to suck the reagent in the main mixing bead chamber of the extraction cartridge into the inner space of the upper body of the piston, and then discharging the mixed reagent to the nucleic acid amplification module.

2. The method for extracting nucleic acid using an extraction cassette according to claim 1, wherein in the step (b), the mixed solution is discharged through a nucleic acid trapping filter.

3. The method for extracting nucleic acid using an extraction cassette according to claim 2, wherein the step (b) comprises the step of driving the rotation control module to overlap the liquid port of the lower piston body with the sample chamber port of the extraction cassette, and sucking the sample in the sample chamber into the inner space of the upper body by driving the control rod module.

4. The method for extracting nucleic acid using an extraction cassette of claim 3, further comprising the step of overlapping the liquid port of the lower piston body with the first reagent chamber port of the extraction cassette by driving the rotation control module, and mixing the sample after sucking the first reagent into the inner space of the upper body by driving the control rod module.

5. The method for extracting nucleic acid using an extraction cassette of claim 4, further comprising the step of overlapping the liquid port of the lower piston body with the second reagent chamber port of the extraction cassette by driving the rotation control module, and mixing the mixture after sucking the second reagent into the inner space of the upper body by driving the control rod module.

6. The method for extracting nucleic acid using an extraction cassette of claim 5, comprising the step of driving the rotation control module to overlap the filter port of the lower piston body with the second reagent chamber port of the extraction cassette, and discharging the mixed solution to the second reagent chamber port by driving the control rod module.

7. The method for extracting nucleic acid using an extraction cassette according to claim 6, wherein the step (b) further comprises a plurality of steps of sequentially sucking, mixing, and discharging the plurality of reagents and the other types of reagents.

8. The method for extracting nucleic acid using an extraction cassette of claim 7, wherein the last aspirated reagent is discharged to the main mixing bead chamber of the extraction cassette.

9. The method of extracting nucleic acid using an extraction cassette of claim 8, wherein the step (c) comprises the step of driving the rotation control module to overlap the filter port of the lower piston body with the main mixing bead chamber port of the extraction cassette, and driving the control rod module to discharge the last aspirated reagent to the main mixing bead chamber port.

10. The method for extracting nucleic acid using an extraction cassette according to claim 9, further comprising:

a step of overlapping a liquid port of the lower piston body with a main mixing bead chamber port of the extraction box by driving the rotation adjustment module, and mixing the reagent sucked into the main mixing bead chamber into the inner space of the upper body by driving the control rod module; and

and moving the reagent in the inner space of the piston upper body to the amplification module.

Technical Field

The present invention relates to a nucleic acid extraction method, and more particularly, to a nucleic acid extraction method using an extraction cartridge that can isolate and purify nucleic acids from a sample in order to amplify the nucleic acids.

Background

With the development of modern life engineering techniques, the causes of diseases can be explained at the genetic level. Accordingly, there is an increasing demand for the manipulation and biochemical analysis of biological samples for the cure or prevention of human diseases.

Meanwhile, in addition to diagnosis of diseases, a technique for extracting and analyzing nucleic acids from a sample containing biological samples or cells is required in various fields of new drug development, preliminary examination of whether or not viruses or bacteria are infected, forensic medicine, and the like.

In the conventional nucleic acid extracting apparatus, a separate apparatus is required for each pretreatment step (concentration and purification), and it takes a long time to move to another apparatus after one pretreatment step is finished. Further, as described above, the nucleic acid extraction method using the conventional nucleic acid extraction apparatus has a problem that a large amount of waste liquid, which has been mixed and reacted, is discharged into the separated waste liquid chamber in the extraction step, and thus space utilization efficiency is low.

Therefore, in order to solve such problems, studies are being made on more efficient methods for nucleic acid extraction.

Disclosure of Invention

(problem of the technology)

The present invention aims to provide a more efficient method for extracting nucleic acid.

(means for solving the problems)

In order to achieve the above-described object, a nucleic acid extraction method using an extraction cassette according to an embodiment of the present invention includes: a step (a) of connecting a drive section of the nucleic acid isolation apparatus to a control rod module disposed in an inner space of the upper piston body and a rotation adjustment module coupled to the lower piston body; a step (b) of driving the rotation regulating module and the control rod module, sequentially sucking a sample and a plurality of reagents into the internal space from a plurality of chambers separated from each other of the extraction box, and discharging a mixed liquid in the internal space into the chambers of the extraction box; and (c) driving the rotation control module and the control rod module to suck the reagent in the main mixing bead chamber of the extraction cartridge into the inner space of the upper body of the piston and then discharge the mixed reagent to the nucleic acid amplification module.

According to an embodiment of the present invention, in the step (b), the mixed solution is discharged through a nucleic acid trapping filter.

According to an embodiment of the present invention, the step (b) includes the step of driving the rotation control module to overlap the fluid port of the lower piston body with the sample chamber port of the extraction box, and drawing the sample in the sample chamber into the upper body inner space by driving the control rod module.

According to an embodiment of the present invention, the method includes the step of overlapping the liquid port of the piston lower body with the first reagent chamber port of the extraction cartridge by driving the rotation adjustment module, and mixing the sample after sucking the first reagent into the upper body inner space by driving the control rod module.

According to an embodiment of the present invention, the method further includes the step of overlapping the liquid port of the piston lower body with the second reagent chamber port of the extraction box by driving the rotation adjustment module, and mixing the reagent after sucking the second reagent into the upper body inner space by driving the control rod module.

According to an embodiment of the present invention, the method includes the step of driving the rotation adjustment module to overlap the filter port of the lower piston body with the second reagent chamber port of the extraction box, and driving the control rod module to discharge the mixed liquid to the second reagent chamber port.

According to an embodiment of the present invention, the step (b) further includes a plurality of steps of sequentially sucking, mixing and discharging the plurality of reagents and the other kinds of reagents.

According to an embodiment of the present invention, the last aspirated reagent is expelled towards the main mixing bead chamber of the extraction cartridge.

According to an embodiment of the present invention, the step (c) includes the step of driving the rotation adjustment module to overlap the filter port of the piston lower body with the main mixing bead chamber port of the extraction cartridge, and driving the control rod module to discharge the last sucked reagent to the main mixing bead chamber port.

According to an embodiment of the present invention, the method further comprises: a step of overlapping a liquid port of the lower piston body with a main mixing bead chamber port of the extraction box by driving the rotation adjustment module, and mixing the reagent sucked into the main mixing bead chamber into the inner space of the upper body by driving the control rod module; and moving the reagent in the inner space of the piston upper body to the amplification module.

(effect of the invention)

According to the nucleic acid extraction method using an extraction cartridge according to an embodiment of the present invention, a plurality of samples can be sequentially processed within one extraction cartridge by using an extraction cartridge different from an existing structure. The nucleic acid extraction time can thereby be reduced and the structure of the apparatus can be simplified.

Further, according to the method for extracting nucleic acid using the extraction cartridge according to the embodiment of the present invention, since the used waste liquid can be discharged to the reagent chamber in the extraction cartridge, the nucleic acid extraction step can be efficiently performed.

Drawings

FIG. 1 is a flow chart of a method for extracting nucleic acid according to an embodiment of the present invention;

FIG. 2 is a perspective view of an extraction cartridge for use in the present invention;

FIG. 3 is a bottom perspective view of an extraction cartridge for use with the present invention;

fig. 4 is an exploded view of the extraction cartridge shown in fig. 2.

Detailed Description

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. In this specification, the same or similar reference numerals are given to the same or similar structures even in embodiments different from each other, and the description is replaced with that of the first time. As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The suffixes "module" and "section" of the constituent elements used in the following description are given or mixed for convenience of writing the present specification, and do not have meanings or functions different from each other.

FIG. 1 is a flow chart of a method for extracting nucleic acid according to an embodiment of the present invention.

Referring to fig. 1, the nucleic acid extraction method includes: step S110, sucking a sample into the inner space of the piston; step S210 to step S280, inhaling, mixing, and discharging a plurality of reagents; and step S310 to step S320, so that the extracted nucleic acid moves toward the amplification module.

The method for extracting nucleic acid using the extraction cassette according to an embodiment of the present invention is performed through the following steps.

In the step S110 of drawing the sample into the piston inner space, the sample disposed in the chamber of the extraction box is drawn into the piston upper body 310 inner space by the driving of the piston control rod module.

Before the step of sucking the sample into the inner space of the piston, the step of mounting the extraction cartridge in the nucleic acid extraction apparatus after injecting the sample and the plurality of reagents into each chamber of the extraction cartridge may be further included.

After the extraction cartridge is mounted to the nucleic acid extraction device, the control rod module and the rotation adjustment module are connected to a drive section of the nucleic acid extraction device.

In step S210, in which the first reagent and the second reagent are sucked into the piston inner space and then mixed, the piston is rotated by a predetermined angle by the rotation adjustment module, so that the liquid port of the piston lower body overlaps the first reagent chamber port, and the control rod module is moved to the upper portion of the piston to suck the first reagent. Then, the piston is rotated again by a predetermined angle by rotating the adjustment module, so that the liquid port of the lower piston body overlaps with the second reagent chamber port, and the control rod module is moved to the upper portion of the piston to suck the second reagent.

Then, the control rod module is moved in the vertical direction, and the sample and the plurality of reagents in the internal space are mixed.

Next, the control rod module is moved to the lower portion of the piston by rotating the piston by a predetermined angle again by the rotation adjustment module so that the filter port of the lower piston main body overlaps the second reagent chamber port, and the mixed liquid of the sample and the reagent is discharged into the second reagent chamber (step S220).

The nucleic acid trapping filter may be attached to the filter interface of the lower piston main body to trap nucleic acid discharged when the mixture of the sample and the reagent is discharged.

The piston is rotated again by a predetermined angle by the rotation adjustment module, so that the liquid port of the piston lower body overlaps the third reagent chamber port, and the control rod module is moved to the upper portion of the piston to suck a third reagent (step S230).

Then, the control rod module moves in the vertical direction, and the reagents in the internal space are mixed.

The control rod module is moved to the lower portion of the piston and discharges the reagent into the third reagent chamber by rotating the piston again by a predetermined angle by the rotation adjustment module such that the filter port of the piston lower body overlaps the third reagent chamber port (step S240).

The piston is rotated again by a predetermined angle by the rotation adjustment module, so that the liquid port of the piston lower body overlaps with the fourth reagent chamber port, and the control rod module is moved to the upper portion of the piston to suck a fourth reagent (step S250).

Then, the control rod module moves in the vertical direction, and the reagents in the internal space are mixed.

The control rod module is moved to the lower portion of the piston and discharges the reagent into the fourth reagent chamber by rotating the piston again by a predetermined angle by the rotation adjustment module such that the filter port of the lower piston main body overlaps the fourth reagent chamber port (step S260).

The piston is rotated again by a predetermined angle by the rotation adjustment module, so that the liquid port of the piston lower body overlaps the fifth reagent chamber port, and the control rod module is moved to the upper portion of the piston to suck a fifth reagent (step S270).

Then, the control rod module moves in the vertical direction, and the reagents in the internal space are mixed.

The fifth reagent may be an elution reagent that elutes the nucleic acid trapped in the nucleic acid trapping filter.

The control rod module is moved to the lower portion of the piston and discharges the reagent into the main mixing bead chamber by rotating the piston again by a predetermined angle by the rotation adjustment module such that the filter port of the piston lower body overlaps the main mixing bead chamber port (step S280).

When the fifth reagent is discharged, the nucleic acid trapped in the nucleic acid trapping filter is moved into the main mixed bead chamber.

The control rod module moves upward of the piston by rotating the piston by a predetermined angle again by the rotation adjustment module so that the liquid port of the piston lower body overlaps the main mixing bead chamber port, and after the reagent in the main mixing bead chamber is sucked, the control rod module moves in the vertical direction and mixes the reagent in the inner space (step S310).

The control rod module is moved to the lower part of the piston and the reagent is moved to the amplification module by rotating the piston again by a predetermined angle by the rotation adjustment module so that the liquid port of the piston lower body overlaps the amplification module liquid port (step S320).

Hereinafter, the structure of the extraction cartridge will be specifically described with reference to fig. 2 to 4.

Fig. 2 is a perspective view of an extraction cassette used in the present invention, fig. 3 is a bottom perspective view of the extraction cassette used in the present invention, and fig. 4 is an exploded view of the extraction cassette shown in fig. 2.

Referring to fig. 2 to 4, the nucleic acid extraction cartridge may generally include a first body 100, a second body 200, a piston 300, a nucleic acid amplification module 400, and the like.

The first body 100 may be used for storing a plurality of reagents.

As shown, a plurality of chambers 110 may be formed in the first body 100, and the plurality of chambers 110 form compartments separated from each other. Reagents or samples different from each other are disposed in each chamber 110, and the chambers 110 form independent spaces so that the plurality of reagents are not mixed with each other.

The second body 200 includes a path for moving the reagent or the sample stored in the first body 100.

According to an embodiment of the present invention, the second body 200 may include a liquid flow path for moving liquid and an air flow path for moving air, and a pad 220 disposed on an upper surface thereof in order to prevent leakage of liquid when the second body 200 is coupled to the first body 100. If the pad plate 220 is coupled to the second body 200 of the extraction cartridge, the liquid flow path and the air flow path of the second body 200 of the extraction cartridge are blocked by the pad plate 220 to form a space, thereby forming the complete flow path 210.

By connecting the liquid channel to the first body 100, a space in which the sample and the reagent can move or mix is provided.

The air flow path serves to connect the amplification module to a vacuum control portion of the piston 300, to control a vacuum that may be generated when the extracted nucleic acid moves to the amplification module, and to prevent contamination of the nucleic acid amplification product.

A plurality of holes may be formed in the pad plate 220 to penetrate the pad plate 220 in the up-down direction. The liquid flow path and the air flow path in the lower part of the cartridge are connected to the plurality of reagent chambers 110 in the first body 100 of the cartridge through a plurality of holes.

The center portion of the pad plate 220 is combined with the bottom surface of the piston lower body 320 in a snug manner.

As the piston rotates, a plurality of holes formed at the central portion of the pad plate 220 overlap with the filter port or the liquid port of the piston lower body 320.

More specifically, a plurality of flow paths 210 may be formed at an upper portion of the second body 200. Each flow path 210 is formed to extend from the central portion to the peripheral portion of the second body 200 without crossing each other. As shown, one end of a part of the flow paths may be arranged on the same circumference, and the other end may be arranged on the same circumference.

An upper portion of the second body 200 may be combined with the pad 220.

A placing part 201 depressed deeply toward the lower end may be formed at the upper portion of the second body 200, and the setting plate 220 may be engaged with the placing part 201 at the upper portion of the second body 200 in a meshing manner.

The gasket 220 is closely attached to the upper surface of the second body 200 and can seal the plurality of flow paths 210. The pad plate 220 may be formed of rubber or synthetic resin having elasticity so that the pad plate 220 can be more strongly adhered to the second body 200.

A plurality of holes may be formed in the pad plate 220 to penetrate the pad plate 220 in the up-down direction.

According to an embodiment of the present invention, the plurality of holes are arranged to vertically overlap end portions of the plurality of channels 210. In other words, the plurality of holes formed in the pad plate 220 are paired with each other and connected by the flow path 210.

The pad 220 may include a plurality of holes 221 arranged on the same circumference C1 of the central portion and a plurality of holes 221 arranged on the same circumference C2 of the peripheral portion.

The piston 300 may be composed of a piston upper body 310 and a piston lower body 320.

An internal space in which a reagent and a sample can be mixed is formed in the upper body 310 of the piston 300, and a control rod module of the piston 300 that is movable in the vertical direction is disposed in the internal space.

The piston control rod module may include: a binding part 301 that binds to a drive part of the nucleic acid isolation apparatus; and a contact portion 302 which is in contact with the piston internal space and moves in the vertical direction.

The piston lower main body 320 is formed as one body by being combined with the piston upper main body 310.

The piston lower body 320 may be combined with a rotation regulation module 330.

As shown, the piston upper body 310 is inserted into a hole formed at the central portion of the first body 100, and the shaft 324 of the piston lower body 320 is inserted into a hole formed at the central portion of the second body 200.

The shaft 324 of the piston lower body 320 is engaged with and fixed to a rotation adjusting module 330, and the rotation adjusting module 330 is combined with the lower end of the second body 200.

The nucleic acid amplification module 400 may be combined with the first body 100 or the second body 200.

The internal channel 210 may be formed inside the nucleic acid amplification module 400, and one end of the internal channel 210 may be formed to overlap at least one of the plurality of channels 210 formed in the second body 200.

According to an embodiment of the present invention, the nucleic acid amplification module 400 may be provided with a fixing member 410, and the fixing member 410 may cover the nucleic acid amplification module 400 and be coupled to the first body 100 and the second body 200 so as not to allow the nucleic acid amplification module 400 to be freely separated.

The first body 100 may include a plurality of reagent chambers 110, each of the chambers 110 being formed in a spaced-apart manner from each other.

An interface overlapping one end of the flow path 210 or a plurality of holes formed in the pad plate 220 is formed at the lower end of each chamber 110. The ports may be formed to have different radii from each other according to the use of the chamber 110.

Each reagent chamber 110 includes a single reagent chamber fluid interface. Each reagent chamber liquid port is connected to a liquid flow path in the lower part of the extraction cartridge via a liquid port connection hole of the rubber pad 220. The sample chamber interface and the main mixing bead chamber interface are combined with separate sample and main mixing bead chambers 111, respectively.

The sample chamber ports may be arranged on the same circumference, and the main mixing bead chamber ports may be arranged on other circumferences.

The sample chamber may include a plurality of dry beads required for sample extraction, and the main mixing bead chamber may include a plurality of dry beads required for nucleic acid amplification.

The sample chamber and the main mixing bead chamber are connected to a sample chamber port and a main mixing bead chamber port, respectively, and each port is connected to a hole formed in the pad and a flow path of the second body 200 of the extraction cartridge to form a structure in which liquid can move.

According to the nucleic acid extraction method using the extraction cartridge as described above, a plurality of samples can be sequentially processed within one extraction cartridge by using an extraction cartridge different from the existing structure. The nucleic acid extraction time can thereby be reduced and the structure of the apparatus can be simplified.

Further, according to the method for extracting nucleic acid using the extraction cartridge according to the embodiment of the present invention, since the used waste liquid can be discharged to the reagent chamber in the extraction cartridge, the nucleic acid extraction step can be efficiently performed.

The method for extracting nucleic acid using the extraction cassette described above is not limited to the structure and method in the above-described embodiments, and all or a part of each embodiment may be selectively combined to produce various modifications of the above-described embodiments.