KR102160553B1 - Nucleic acid extraction method and cartridge - Google Patents

Abstract

A nucleic acid extraction method according to an embodiment of the present invention includes the steps of: (A) accommodating a solid collector in which infectious bacteria are collected in an inner space of a first chamber; (B) injecting a first solution into the inner space of the first chamber; (C) mixing the collector and the first solution in the inner space of the first chamber to eliminate the infectious bacteria from the collector to form an infectious bacteria solution; (D) adsorbing the infected bacteria to a solid surface while passing the infected bacteria solution through a bead chamber containing a plurality of beads; it provides a nucleic acid extraction method comprising a.

Description

Nucleic acid extraction method and cartridge {NUCLEIC ACID EXTRACTION METHOD AND CARTRIDGE}

The nucleic acid extraction method and cartridge according to the present invention relates to a method for extracting nucleic acid from infectious bacteria and a cartridge used to extract nucleic acid from infectious bacteria through the method.

In general, a method of collecting infectious bacteria present on the surface of an object includes a method of using an adhesive tape or collecting a sample of the surface of the object, but there is a problem that it is difficult to purify impurities other than the infectious bacteria to be collected.

In addition, there is a method of extracting nucleic acids by sweeping the surface by sweeping the surface with a cotton swab and injecting it into a solution, or culturing it in a medium to detect infectious bacteria. There is a problem.

In order to increase the sensitivity of quantitative measurement and measurement of infectious bacteria existing on a large surface, pure nucleic acids must be taken by transferring the infected bacteria to a liquid phase and pre-treating the solution, but the infectious bacteria present in the collection area used when collecting a large surface are liquid. It has to be moved, and there is a problem in that the infectious bacteria transferred to a large volume of liquid must be concentrated.

Republic of Korea Patent Publication 10-2016-0035945 (published on April 1, 2016)

In order to solve the above-described problem, the present invention is to provide a method for automatically extracting nucleic acids of infectious bacteria by accommodating a collection of infectious bacteria collected from a target object having a large surface in a nucleic acid extraction cartridge.

A nucleic acid extraction method according to an embodiment of the present invention includes the steps of: (A) accommodating a solid collector in which infectious bacteria are collected in an inner space of a first chamber; (B) injecting a first solution into the inner space of the first chamber; (C) mixing the collector and the first solution in the inner space of the first chamber to eliminate the infectious bacteria from the collector to form an infectious bacteria solution; (D) adsorbing the infected bacteria to the solid surface while passing the infectious bacteria solution through a bead chamber containing a plurality of beads; it provides a nucleic acid extraction method comprising a.

In this embodiment, the step (B) includes the steps of reducing the inner space of the first chamber by moving the piston; Communicating the first chamber and the second chamber containing the first solution; And injecting the first solution into the first chamber while expanding the inner space of the first chamber in communication with the second chamber by moving the piston.

In this embodiment, step (C) may include mixing the collector and the first solution by moving the magnet accommodated in the inner space of the first chamber.

In this embodiment, (C') after step (C), the first chamber is communicated with the third chamber containing the second solution, and the infected bacteria solution contained in the first chamber is injected into the third chamber, and the second solution and Mixing step; may further include.

In this embodiment, step (D) includes the steps of communicating with each other the third chamber and the first chamber in which the mixed solution of the infectious bacteria solution and the second solution are accommodated via the bead chamber; And adsorbing the infectious bacteria to the solid surface while the mixed solution is passed through the bead chamber.

In the present embodiment, (E) communicating the bead chamber containing the bead and the fourth chamber containing the third solution, and injecting the third solution into the bead chamber; may be further included.

In this embodiment, (F) beating a bead in the bead chamber to extract nucleic acid to form a nucleic acid extract, and injecting the gas contained in the fourth chamber into the bead chamber to eject the nucleic acid extract to the outside using atmospheric pressure. It may further include;

A nucleic acid extraction cartridge according to another embodiment of the present invention includes a first chamber in which a solid collector in which infectious bacteria are collected is accommodated; And a third chamber in which the second solution is accommodated; And a bead chamber in which a plurality of beads are accommodated. A first channel capable of communicating the bead chamber and the first chamber; And a connection hole capable of communicating the bead chamber and the third chamber; a lower layer including; wherein the bead chamber communicates with the first chamber through the first channel and at the same time, according to the relative positions of the upper and lower layers. It provides a nucleic acid extraction cartridge, which is communicated with the third chamber through.

In this embodiment, the upper layer further includes a second chamber in which the first solution is accommodated, and the lower layer further includes a second channel capable of selectively communicating the second chamber or the third chamber to the first chamber. can do.

In this embodiment, as the lower portion of the second channel rotates relative to the upper portion, the second or third chambers may be selectively communicated with the first chamber.

In this embodiment, the upper layer further includes a fourth chamber in which the third solution is accommodated, and the connection hole may selectively communicate the third chamber or the fourth chamber to the bead chamber.

In this embodiment, the connection hole may selectively communicate the third chamber or the fourth chamber to the bead chamber as the lower layer portion is rotated relative to the upper layer portion.

In this embodiment, the upper portion further includes a jet hole, and the first channel may selectively communicate the first chamber or the jet hole to the bead chamber.

In this embodiment, as the lower portion of the first channel is rotated relative to the upper portion, the first chamber or the ejection hole may be selectively communicated to the bead chamber.

In this embodiment, the bead chamber may communicate with the ejection hole through the first channel and communicate with the fourth chamber through the connection hole, depending on the relative positions of the upper layer and the lower layer.

In this embodiment, the bead chamber may have at least one protrusion formed on the inner wall.

An object of the present invention is to provide a method of automatically extracting a nucleic acid of an infectious bacteria in a state in which a collection of infectious bacteria is collected from an object having a large surface in a nucleic acid extraction cartridge.

1 is a perspective view showing a nucleic acid extraction cartridge according to an embodiment of the present invention,
2 is a schematic plan view showing an upper portion of the nucleic acid extraction cartridge;
3 is a schematic plan view showing the lower layer of the nucleic acid extraction cartridge,
4 is a schematic side view showing a first chamber of the nucleic acid extraction cartridge;
5 is a schematic side view showing a fourth chamber of the nucleic acid extraction cartridge;
6 is a flow chart showing a nucleic acid extraction method according to another embodiment of the present invention,
FIG. 7 is a graph measuring the degree of dropout of infectious bacteria from the collector by varying the acidity (pH) of the first solution in the nucleic acid extraction method.
8 is a graph measuring the degree of elimination of infectious bacteria from the collector by varying the ethanol concentration of the first solution in the nucleic acid extraction method;
9 is a graph measuring the degree of adsorbing of infectious bacteria to beads by varying the acidity of the second solution in the nucleic acid extraction method;
FIG. 10 is a graph measuring the degree of adsorbing of infectious bacteria to the beads by varying the type of beads and the flow rate of the mixed solution injected into the bead chamber in the nucleic acid extraction method;
11 is a graph measuring the degree of nucleic acid extraction of infectious bacteria by varying the bead beating time in the nucleic acid extraction method;
12 is a graph measuring the degree of nucleic acid extraction of infectious bacteria by varying the length of the protrusion of the bead chamber in the nucleic acid extraction method.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become apparent with reference to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms different from each other, and only these embodiments make the disclosure of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims. Meanwhile, terms used in the present specification are for explaining embodiments and are not intended to limit the present invention. In this specification, the singular form also includes the plural form unless specifically stated in the phrase. As used in the specification, "comprises" and/or "comprising" refers to the presence of one or more other components, steps, actions and/or elements, and/or elements, steps, actions and/or elements mentioned. Or does not exclude additions. Terms such as first and second may be used to describe various elements, but the elements should not be limited by terms. The terms are only used for the purpose of distinguishing one component from another component.

The present invention is a method for collecting infectious bacteria present on the surface by wiping the surface of a target object with a collector, and extracting nucleic acids from infectious bacteria while holding the collector itself in the nucleic acid extraction cartridge 1, and a nucleic acid extraction cartridge using the same It is about (1).

An embodiment of the present invention relates to a nucleic acid extraction cartridge 1 for extracting nucleic acids from infectious bacteria. The nucleic acid extraction cartridge 1 is mounted on a nucleic acid extraction device (not shown) and driven, and relates to a cartridge for extracting nucleic acids from infectious bacteria through a nucleic acid extraction method to be described later.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a perspective view showing a nucleic acid extraction cartridge 1 according to an embodiment of the present invention, FIG. 2 is a schematic plan view showing an upper portion 10 of the nucleic acid extraction cartridge 1, and FIG. 3 is a nucleic acid extraction cartridge 1 ) Is a plan schematic diagram showing the lower layer 11 of the), FIG. 4 is a side schematic view showing the first chamber 101 of the nucleic acid extraction cartridge 1, and FIG. 5 is a fourth chamber 104 of the nucleic acid extraction cartridge 1 It is a schematic side view showing.

1 to 5, a nucleic acid extraction cartridge 1 according to an embodiment of the present invention may include an upper layer portion 10 and a lower layer portion 11.

Specifically, referring to FIG. 2, the upper layer portion 10 includes a first chamber 101, a second chamber 102, a third chamber 103, and a fourth chamber 104 formed in the body 10a. , It may include a jet hole 105 and pistons (P1, P2).

The first chamber 101 may be a chamber in which a solid collector in which infectious bacteria are collected is accommodated. The first chamber 101 may be separated from the nucleic acid extraction cartridge 1. Collectors are for collecting infectious bacteria, and those having a porous and flexible material such as sponge, tissue paper, and cleaning cloth may be used. In this specification, the term'solid phase' is a state of a solid object, a state having substantially uniform properties and shapes physically and chemically no matter which part of the object is taken at room temperature, and is defined as including a semi-solid state in a soft state. Can be. In addition, the term'infectious bacteria' may be a microorganism including bacteria (Bacteria) and virus (Virus).

A magnet may be accommodated in the inner space of the first chamber 101. The magnet is for removing infectious bacteria from the collector, and for example, a neodymium magnet may be used. The piston P1 may be press-fit into the first chamber 101. The first chamber 101 may be coupled to the nucleic acid extraction cartridge 1 after the piston P1 is press-fitted with the collector and the magnet accommodated in the inner space thereof. The first chamber 101 may be a member formed in a cylindrical shape and provided with an internal space. However, the shape of the first chamber 101 is not limited to a cylindrical shape, and will not be limited as long as it can accommodate the collector. A first hole H1, which is a hole for connecting the first channel 112 and the second channel 113 to be described later, may be formed under the first chamber 101.

The second chamber 102 may be a chamber in which the first solution is accommodated. The second chamber 102 may be partitioned adjacent to the left side of the first chamber 101. A second hole H2 as a hole for connecting the second channel 113 may be formed under the second chamber 102.

The third chamber 103 may be a chamber in which the second solution is accommodated. The third chamber 103 may be partitioned adjacent to the right side of the first chamber 101. A third hole H3 that is a hole for connecting the first channel 112 and the second channel 113 may be formed under the third chamber 103.

The fourth chamber 104 may be a chamber in which the third solution is accommodated. The fourth chamber 104 may be disposed adjacent to the left side of the second chamber 102. The fourth chamber 104 may be separable from the nucleic acid extraction cartridge 1. The piston P2 may be press-fitted into the fourth chamber 104 while the third solution and gas are accommodated. After that, the fourth chamber 104 can be coupled to the nucleic acid extraction cartridge 1. A fourth hole H4, which is a hole for connecting the connection hole 114 to be described later, may be formed under the fourth chamber 104.

The ejection hole 105 may be a hole for ejecting the nucleic acid extract to the outside. The ejection hole 105 may be disposed adjacent to the right side of the third chamber 103. The ejection hole 105 may be connected to the first channel 112.

Specifically, the ejection hole 105 may be formed in an open shape in a portion of the side of the upper layer. The ejection hole 105 may be connected to a fifth hole H5, which is a hole formed under the upper layer portion 10 adjacent thereto. Further, the fifth hole H5 may be connected to the first channel 112.

The first to fifth holes H1 to H5 may be disposed to form the same distance with respect to the holes adjacent to each other. For example, the arrangement of the first to fifth holes H1 to H5 may be arranged at each vertex of a regular pentagon.

The lower layer 11 may include a bead chamber 111, a first channel 112, a second channel 113, and a connection hole 114 in the body 11a. The lower layer 11 may be coupled to the upper layer 10 so that the body 11a can be rotated around the central axis 10b on the lower central axis 10b line.
In this case, the body 11a may be coupled to the lower part of the body 10a of the upper layer 10 through the lower case 11b.

The bead chamber 111 may be a portion in which a plurality of beads are accommodated. A magnet for bead beating may be accommodated in the inner space of the bead chamber 111. At least one protrusion 111a may be formed on the inner wall of the bead chamber 111. Preferably, four protrusions 111a may be provided along the inner wall of the bead chamber 111. The shape of the protrusion 111a may be a rectangular parallelepiped shape. The protrusions 111a may be arranged at equal intervals on the inner surface of the bead chamber 111. Preferably, the protrusion 111a may protrude from the inner surface of the bead chamber 111 toward the center of the bead chamber 111 to a length of 2 mm. While the beads inside the bead chamber 111 are beating, they are also beating to the protrusion 111a, the movement of the beads is prevented, and the number of beatings increases, thereby increasing the efficiency of extracting nucleic acids from infectious bacteria.

The bead chamber 111 communicates with the first chamber 101 through the first channel 112 and the third chamber through the connection hole 114 according to the relative position of the upper layer portion 10 and the lower layer portion 11. Can communicate with (103).

The first channel 112 may be a channel capable of communicating the bead chamber 111 and the first chamber 101. In addition, the first channel 112 may selectively communicate the first chamber 101 or the ejection hole 105 to the bead chamber 111. The first channel 112 may selectively communicate the first chamber 101 or the ejection hole 105 to the bead chamber 111 as the lower layer 11 is rotated relative to the upper layer 10.
One end of the first channel 112 is connected to the bead chamber 111 and the other end is connected to the first hole H1 of the first chamber 101 to communicate the bead chamber 111 and the first chamber 101 I can make it. In addition, one end of the first channel 112 is connected to the bead chamber 111 and the other end of the first channel 112 is connected to the fifth hole H5 to communicate the bead chamber 111 and the ejection hole 105.

The second channel 113 may be a portion capable of selectively communicating the second chamber 102 or the third chamber 103 to the first chamber 101. The second channel 113 selectively selects the second chamber 102 or the third chamber 103 to the first chamber 101 as the lower layer 11 is rotated relative to the upper layer 10. You can communicate with.
Specifically, one end of the second channel 113 is connected to the first hole H1 of the first chamber 101 and the other end is connected to the second hole H2 of the second chamber 102, 101 and the second chamber 102 can be communicated.
In addition, one end of the second channel 113 is connected to the first hole H1 of the first chamber 101 and the other end of the second channel 113 is connected to the third hole H3 of the third chamber 103. ) And the third chamber 103 can be communicated.

Preferably, the second channel 113 is rotated by 10 degrees in a counterclockwise direction with respect to the upper layer 10 while the upper layer portion 10 and the lower layer portion 11 of the nucleic acid extraction cartridge 1 are aligned. The first chamber 101 and the second chamber 102 may be communicated through In this state, the first solution accommodated in the second chamber 102 may be injected into the inner space of the first chamber 101 while discharging the piston P1 pressed into the first chamber 101.
Then, the infectious bacteria are eliminated from the collecting body to form an infectious bacteria solution, and the lower layer 11 is rotated 72 degrees clockwise with respect to the upper layer 10 so that the first chamber 101 is formed through the second channel 113. And the third chamber 103 can be communicated with each other.
Then, by pressing the piston P1 into the inner space of the first chamber 101, the infected bacteria solution contained in the first chamber 101 may be injected into the third chamber 103 containing the second solution.

The connection hole 114 may be a hole capable of communicating the bead chamber 111 and the third chamber 103. The connection hole 114 may be formed on the bead chamber 111. The connection hole 114 may selectively communicate the third chamber 103 or the fourth chamber 104 to the bead chamber 111. The connection hole 114 may selectively communicate the third chamber 103 or the fourth chamber 104 to the bead chamber 111 as the lower layer 11 is rotated relative to the upper layer 10. Specifically, the connection hole 114 can communicate with the bead chamber 111 and the third chamber 103 while being connected to the third hole H3, and the bead chamber 111 while being connected to the fourth hole H4 And the fourth chamber 104 can be communicated with each other.

Preferably, in a state in which the infectious bacteria solution is injected into the third chamber 103 and mixed with the second solution to form a mixed solution, the lower layer 11 is rotated 108 degrees with respect to the upper layer 10 to make the third chamber 103 The bead chamber 111 may be disposed under the bead chamber 111, and the third hole H3 of the third chamber 103 may be connected to the connection hole 114 of the bead chamber 111. In this state, the bead chamber 111 and the first chamber 101 may communicate with each other through the first channel 112.
Then, while discharging the piston (P1) pressed into the first chamber (101), the mixed solution accommodated in the inner space of the third chamber (103) passes through the bead chamber (111), and the infectious bacteria are adsorbed to the beads. It can be injected into the inner space of 101.
Then, the lower layer 11 is rotated 144 degrees with respect to the upper layer 10 to connect the fourth hole H4 of the fourth chamber 104 and the connection hole 114 of the bead chamber 111 to connect the fourth chamber. The bead chamber 111 and 104 can be communicated.
Thereafter, the inner space in which the third solution is accommodated in the fourth chamber 104 is pressed into the piston P2 to inject the third solution into the bead chamber 111.
Thereafter, bead beating is performed to extract nucleic acids of infectious bacteria adsorbed on the beads.

In addition, the bead chamber 111 communicates with the ejection hole 105 through the first channel 112 and the fourth chamber through the connection hole 114 according to the relative position of the upper layer portion 10 and the lower layer portion 11. Can communicate with (104).
Specifically, the first channel 112 is connected to the fifth hole H5 to communicate the bead chamber 111 and the ejection hole 105, and the connection hole 114 is connected to the fourth hole H4 to The chamber 111 and the fourth chamber 104 may be communicated with each other. Therefore, by pressing the piston P2 into the inner space of the fourth chamber 104, the gas contained in the inner space of the fourth chamber 104 is injected into the bead chamber 111, and the nucleic acid extract contained in the bead chamber 111 Can be ejected to the outside through the ejection hole 105.

Hereinafter, a nucleic acid extraction method according to another embodiment of the present invention will be described. 6 is a flow chart showing a nucleic acid extraction method according to another embodiment of the present invention.

6, in the nucleic acid extraction method according to an embodiment of the present invention, first (A) a step (S1) of accommodating a solid trap in which infectious bacteria are collected in the inner space of the first chamber 101 is performed. I can. Infectious bacteria can be collected by wiping the surface of the target object with a collector.

After the (A) step, (B) the step (S2) of injecting the first solution into the inner space of the first chamber 101 may be performed. In addition, step (B) may be performed before step (A).

In the step (B), the step of reducing the internal space of the first chamber 101 by moving the piston P1, communicating the first chamber 101 and the second chamber 102 containing the first solution Steps and steps of injecting the first solution into the first chamber 101 while expanding the inner space of the first chamber 101 communicated with the second chamber 102 by moving the piston P1 may be performed in sequence. have. The first chamber 101 may be coupled to the nucleic acid extraction cartridge 1 in a state in which the volume of the internal space is reduced by pressing the piston P1 into the internal space. Here, while the volume of the inner space of the first chamber 101 is reduced, the volume of the collector may be reduced. For this purpose, it is preferable that the collector has a porous and flexible property.

Then, the second channel 113 is connected to the hole of the first chamber 101 and the hole of the second chamber 102 by rotating the lower layer 11 with respect to the upper layer 10, and the first chamber 101 ) And the second chamber 102 can be communicated.

Then, the piston P1 is extruded to transfer the first solution accommodated in the inner space of the second chamber 102 to the inner space of the first chamber 101. The first solution may be a solution for eliminating infectious bacteria from the collector. The first solution may be a basic solution. Preferably, a solution having a pH (acidity) of 9 may be used.

7 is a graph of measuring pathogen desorption of infectious bacteria from a collector by varying the acidity (pH) of the first solution in the nucleic acid extraction method.

Referring to FIG. 7, compared to the first solution having a pH of 5 and the first solution having a pH of 9, it can be seen that the first solution having a pH of 9 has a high degree of dropout from the trapping body in both bacteria and viruses.

FIG. 8 is a graph measuring pathogen desorption of infectious bacteria from a collection by varying the ethanol concentration of the first solution in the nucleic acid extraction method.

Referring to Figure 8, the first solution may contain ethanol. It can be seen that pathogen desorption of the infectious bacteria is increased when ethanol is included in 5% or more than ethanol is not included in the first solution.

After the step (B), (C) a step (S3) of forming an infectious bacteria solution by mixing the collector and the first solution in the inner space of the first chamber 101 to eliminate the infectious bacteria from the collector may be performed. have.

In the step (C), a step of mixing the collector and the first solution by moving the magnet accommodated in the inner space of the first chamber 101 may be performed. The magnet may be a neodymium magnet. A device in which the nucleic acid extraction cartridge 1 is mounted may include an electromagnet, and the electromagnet may move a neodymium magnet accommodated in the inner space of the first chamber 101 by magnetic. As the magnet accommodated in the inner space of the first chamber 101 is continuously moved, the collector and the first solution may be mixed, and in this process, the infectious bacteria may be eliminated from the collector into the first solution.

After the step (C), (C') the first chamber 101 is communicated with the third chamber 103 containing the second solution, and the infected bacteria solution contained in the first chamber 101 is transferred to the third chamber 103 ) And mixing with the second solution to form a mixed solution (S4) may be performed.

By rotating the lower part 11 with respect to the upper part 10 of the nucleic acid extraction cartridge 1, the second channel 113 is connected to the hole of the first chamber 101 and the hole of the third chamber 103, The first chamber 101 and the third chamber 103 can be communicated with each other. By pressing the piston P1 into the inner space of the first chamber 101, the infected bacteria solution accommodated in the first chamber 101 may be injected into the third chamber 103. In this process, as the volume of the porous collector decreases, the absorbed infected bacteria solution may be injected into the third chamber 103, and the porous collector serves as a filter to remove impurities. Accordingly, an infectious bacteria solution having a high concentration of infectious bacteria may be injected into the third chamber 103. Infectious bacteria solution may be mixed with the second solution accommodated in the third chamber 103. The second solution may be a solution for adsorbing infectious bacteria to the solid surface. The second solution may be an acidic solution. Here, the solid surface may mean the surface of a bead and/or a surface of another solid object such as a filter.

9 is a graph measuring the degree of adsorption of infectious bacteria to beads by varying the acidity of the second solution in the nucleic acid extraction method.

Referring to FIG. 9, an experiment performed by varying the pH of the second solution in order to optimize the conditions of the second solution capable of adsorbing bacteria and viruses to the beads can be seen. Bacteria (10 ³ CFU/mL) and virus (10 ³ PFU/mL) are injected into 20 mL of the first solution to create an infectious bacteria solution, which is passed through beads at a rate of 1 mL/min, and then remains. The amount of bacteria present was checked. As a result, it was confirmed that the infectious bacteria adsorption rate (Adsorption) was high under the condition that the pH of the second solution was low. Preferably, as the second solution, a solution having a pH of 5 may be used.

After the step (C'), (D) a step (S5) of adsorbing the infected bacteria to the solid surface while passing the infected bacteria solution through the bead chamber 111 in which a plurality of beads are accommodated may be performed. Here, the solid surface may mean a surface of a bead and/or a surface of another solid object such as a filter.

As the bead, a functional bead capable of adsorbing infectious bacteria may be used. Functional beads may have a size of about 70 to 100 micrometers, and graphene oxide may be coated on beads made of a glass material. The total weight of the plurality of functional beads accommodated in the bead chamber 111 may be approximately 0.4 g. Functional beads not only can easily and quickly adsorb infectious bacteria, but also have excellent functions of extracting nucleic acids by destroying the adsorbed infectious bacteria with bead beating.

In the step (D), the step of communicating the third chamber 103 and the first chamber 101 containing the mixed solution of the infected bacteria solution and the second solution through the bead chamber 111 to each other, and the mixed solution While passing through the chamber 111, the step of adsorbing the infectious bacteria to the solid surface may be performed. Here, the solid surface may mean a surface of a bead and/or a surface of another solid object such as a filter.

FIG. 10 is a graph measuring the degree of adsorption of infectious bacteria to the beads by varying the type of beads and the flow rate of the mixed solution injected into the bead chamber 111 in the nucleic acid extraction method.

Referring to FIG. 10, it can be seen that the flow rate of passing the mixed solution through the bead chamber 111 affects the degree of adsorbing to the beads. The adsorption rate according to the rate of flowing the solution containing the desorbed infectious bacteria into the bead chamber 111 containing the functional beads (GO bead) was confirmed. 20 mL (10 ³ CFU/mL) of the bacterial solution was injected into the bead chamber 111, and the amount of bacteria in the discharged solution was compared and analyzed by a polymerase chain reaction (PCR). As a result, in the case of the functional beads, even when the solution was passed through the solution at a relatively high rate of 5 mL/min and 10 mL/min, the infectious bacteria adsorption rate was higher than about 80%. In addition, it can be seen that, compared to general glass beads, functional beads have higher infectious bacteria adsorption rate than glass beads, regardless of the injection rate of the bacterial solution.

After the step (D), (E) the bead chamber 111 in which the beads and/or the infectious bacteria are adsorbed is in communication with the fourth chamber 104 in which the third solution is accommodated, and the third solution is connected to the bead chamber ( Injecting to 111) (S6) may be performed.

The lower layer 11 of the nucleic acid extraction cartridge 1 is rotated with respect to the upper 10 to allow the bead chamber 111 and the fourth chamber 104 to communicate. The third solution may be accommodated in the inner space of the fourth chamber 104. The third solution may be a solution for extracting nucleic acids. The third solution may be injected into the bead chamber 111 by pressing the piston P2 into the inner space of the fourth chamber 104.

After the step (E), (F) the beads in the bead chamber 111 and/or the beads adsorbed with infectious bacteria are beaten to extract nucleic acids to form a nucleic acid extract, and the gas contained in the fourth chamber 104 is beaded. Injecting into the chamber 111 and ejecting the nucleic acid extract to the outside using atmospheric pressure (S7) may be performed. The bead beating may be performed by moving the magnets accommodated in the bead chamber 111 using an electromagnet provided in the nucleic acid extraction apparatus, while moving the beads to collide with each other. In addition, it may be performed by rotating the bead chamber 111 to move the beads.

FIG. 11 is a graph measuring the nucleic acid extraction degree (Ct value) of an infected bacteria by varying the bead beating time in the nucleic acid extraction method.

Referring to FIG. 11, in order to confirm the nucleic acid extraction rate (Ct value) of the infected bacteria adsorbed on the functional beads, the bead beating efficiency was confirmed by time. Nucleic acid extraction rates were compared and analyzed using Gram-positive bacteria and virus samples, respectively, having difficulty in nucleic acid extraction. As a result, it was confirmed that performing a bead beating for 1 minute at a frequency of 50 Hz exhibits a nucleic acid extraction rate equivalent to that of a generally performed bead beating.

12 is a graph in which the degree of nucleic acid extraction of an infected bacteria is measured by varying the length of the protrusion 111a of the bead chamber 111 in the nucleic acid extraction method.

Referring to FIG. 12, it can be seen that the experiment was performed by adjusting the size and/or number of protrusions 111a formed on the inner wall of the bead chamber 111. Infectious bacteria inside the bead chamber 111 using a neodymium magnet accommodated in the bead chamber 111 and an electromagnet provided in the nucleic acid extraction device to check the nucleic acid extraction rate of the infectious bacteria adsorbed on the functional beads present in the bead chamber 111 The adsorbed functional beads were moved to extract nucleic acids of infectious bacteria, and the bead beating efficiency was confirmed. The size and number of protrusions 111a were adjusted using Gram-positive bacteria, which are difficult to extract nucleic acids, and compared with conventional bead betting. When four protrusions 111a protruding 2 mm from the inner wall of the bead chamber 111 were used, it was confirmed that the same efficiency as the existing bead beating was used.

By extracting the nucleic acid from the infectious bacteria through the above-described nucleic acid extraction method, the nucleic acid of the infectious bacteria can be automatically extracted in a state in which the collection body obtained by collecting the infectious bacteria from a target object having a large surface is accommodated in the nucleic acid extraction cartridge 1.

The nucleic acid extraction method and the user of the cartridge substantially wipe the surface of the target object with the collector, accommodate it in the first chamber 101, and press-in the piston P1, and the nucleic acid of the infectious bacteria is automatically extracted from the collector. Nucleic acids of infectious bacteria can be efficiently extracted.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the art to which the present invention pertains will be able to make various modifications and variations without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the claims below, and all technical ideas within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

1: nucleic acid extraction cartridge 10: upper portion
10a: main body 11: lower layer
11a: body 101: first chamber
102: second chamber 103: third chamber
104: fourth chamber 105: ejection hole
111: bead chamber 111a: protrusion
112: first channel 113: second channel
114: connection hole P1, P2: piston

Claims (16)

A first chamber formed in the main body to accommodate the solid collector in which infectious bacteria are collected, a second chamber partitioned from the first chamber in the main body and receiving a first solution, and a first chamber in the main body, and An upper layer portion formed in a partition with the second chamber and including a third chamber in which a second solution is accommodated; And
The body is rotatably coupled around the central axis on the central axis line of the lower part of the upper layer, and a bead chamber provided to accommodate a plurality of beads in the body, and a bead chamber formed in the body through rotation operation of the body A first channel capable of communicating the bead chamber and the first chamber, a connection hole provided in the bead chamber to communicate the bead chamber and the third chamber, and a rotation of the body formed in the body Includes; a lower layer portion including a second channel capable of selectively communicating the second chamber or the third chamber to the first chamber through manipulation,
The bead chamber,
Depending on the relative position of the upper layer and the lower layer, the nucleic acid extraction cartridge is communicated to the first chamber through the first channel and to the third chamber through the connection hole.
The method of claim 1,
The second channel is a nucleic acid extraction cartridge capable of selectively communicating the second chamber or the third chamber to the first chamber as the lower layer portion is rotated relative to the upper layer portion.
The method of claim 1,
The upper layer further includes a fourth chamber in which a third solution is accommodated,
The connection hole is capable of selectively communicating the third chamber or the fourth chamber to the bead chamber, a nucleic acid extraction cartridge.
The method of claim 3,
The connection hole is a nucleic acid extraction cartridge capable of selectively communicating the third chamber or the fourth chamber to the bead chamber as the lower layer is rotated relative to the upper layer.
The method of claim 3,
The upper layer further includes a jet hole,
The first channel is capable of selectively communicating the first chamber or the ejection hole to the bead chamber, a nucleic acid extraction cartridge.
The method of claim 5,
The first channel is a nucleic acid extraction cartridge capable of selectively communicating the first chamber or the ejection hole to the bead chamber as the lower layer portion is rotated relative to the upper layer portion.
The method of claim 5,
The bead chamber,
Depending on the relative position of the upper layer and the lower layer, the nucleic acid extraction cartridge is communicated to the ejection hole through the first channel and communicates with the fourth chamber through the connection hole.
The method of claim 1,
The bead chamber,
At least one protrusion formed on the inner wall, nucleic acid extraction cartridge.
In the nucleic acid extraction method using the nucleic acid extraction cartridge of any one of claims 1 to 8,
(A) accommodating the solid collector in which the infectious bacteria are collected in the inner space of the first chamber;
(B) injecting a first solution into the inner space of the first chamber;
(C) mixing the collector and the first solution in the inner space of the first chamber to eliminate the infectious bacteria from the collector to form an infectious bacteria solution; And
(D) adsorbing the infected bacteria on the solid surface while passing the infectious bacteria solution through a bead chamber containing a plurality of beads;
Nucleic acid extraction method comprising a.
The method of claim 9,
The (B) step;
Moving a piston to reduce the inner space of the first chamber;
Communicating the first chamber and a second chamber containing the first solution; And
Injecting the first solution into the first chamber while moving the piston to expand the inner space of the first chamber in communication with the second chamber;
Nucleic acid extraction method comprising a.
The method of claim 9,
The (C) step,
Mixing the collector and the first solution by moving a magnet accommodated in the inner space of the first chamber;
Nucleic acid extraction method comprising a.
The method of claim 9,
(C') After the step (C), the first chamber is communicated with the third chamber containing the second solution, and the infectious bacteria solution contained in the first chamber is injected into the third chamber and mixed with the second solution. Thus forming a mixed solution;
Nucleic acid extraction method further comprising a.
The method of claim 12,
The step (D),
Communicating with each other a third chamber in which the mixed solution is accommodated and the first chamber via the bead chamber; And
Adsorbing the infectious bacteria to the solid surface while the mixed solution passes through the bead chamber;
Nucleic acid extraction method comprising a.
The method of claim 9,
(E) communicating the bead chamber containing the bead and the fourth chamber containing the third solution, and injecting the third solution into the bead chamber;
Nucleic acid extraction method further comprising a.
The method of claim 14,
(F) beating the bead in the bead chamber to extract nucleic acid to form a nucleic acid extract, and injecting gas contained in the fourth chamber into the bead chamber to eject the nucleic acid extract to the outside using atmospheric pressure;
Nucleic acid extraction method further comprising a. delete

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