WO2020071715A1 - Procédé d'extraction d'acide nucléique et cartouche l'utilisant - Google Patents

Procédé d'extraction d'acide nucléique et cartouche l'utilisant

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Publication number
WO2020071715A1
WO2020071715A1 PCT/KR2019/012767 KR2019012767W WO2020071715A1 WO 2020071715 A1 WO2020071715 A1 WO 2020071715A1 KR 2019012767 W KR2019012767 W KR 2019012767W WO 2020071715 A1 WO2020071715 A1 WO 2020071715A1
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WO
WIPO (PCT)
Prior art keywords
chamber
nucleic acid
solution
bead
acid extraction
Prior art date
Application number
PCT/KR2019/012767
Other languages
English (en)
Korean (ko)
Inventor
민준홍
백창윤
유현진
이원녕
정원석
Original Assignee
중앙대학교 산학협력단
재단법인 바이오나노헬스가드연구단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 중앙대학교 산학협력단, 재단법인 바이오나노헬스가드연구단 filed Critical 중앙대학교 산학협력단
Publication of WO2020071715A1 publication Critical patent/WO2020071715A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the nucleic acid extracting method and cartridge according to the present invention relates to a method for extracting the nucleic acid of an infectious bacterium and a cartridge used to extract the nucleic acid of an infectious bacterium through it.
  • the method of collecting the infectious bacteria present on the surface of the object has a method of using an adhesive tape or a method of collecting a surface sample of the object, but there is a problem that it is difficult to purify impurities other than the infecting bacterium to be collected.
  • the present invention is to provide a method capable of automatically extracting nucleic acids of infectious bacteria by receiving a collection of infectious bacteria from a target object having a large surface in a nucleic acid extraction cartridge.
  • a nucleic acid extraction method comprises the steps of: (A) receiving a solid-phase collection body in which infectious bacteria have been collected into an interior space of a first chamber; (B) injecting a first solution into the interior space of the first chamber; (C) mixing the collection body and the first solution in the interior space of the first chamber to remove the infecting bacteria from the collecting body to form an infecting bacteria solution; (D) the step of re-adsorbing the infecting bacteria to the solid surface while passing the infecting bacteria solution through a bead chamber containing a plurality of beads; provides a nucleic acid extraction method comprising a.
  • step (B) includes moving the piston to shrink the inner space of the first chamber; Communicating the first chamber and the second chamber containing the first solution; And moving the piston to expand the inner space of the first chamber in communication with the second chamber and injecting the first solution into the first chamber.
  • step (C) may include a step of mixing the collector and the first solution by moving the magnet accommodated in the inner space of the first chamber.
  • the first chamber is communicated with the third chamber in which the second solution is accommodated, and the infectious bacteria solution accommodated in the first chamber is injected into the third chamber, and the second solution and Mixing; may further include.
  • step (D) comprises the step of communicating the first chamber and the third chamber containing the mixed solution of the infectious bacteria solution and the second solution through the bead chamber; And re-adsorbing the infecting bacteria on the solid surface while the mixed solution passes through the bead chamber.
  • Nucleic acid extraction cartridge includes a first chamber in which a solid-phase collection of infectious bacteria is received; 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 lower layer part including a connecting hole capable of communicating the bead chamber and the third chamber.
  • the bead chamber is connected to the first chamber through the first channel at the same time according to the relative positions of the upper and lower layers. It provides a nucleic acid extraction cartridge, which communicates with the third chamber.
  • the upper layer portion further includes a second chamber in which the first solution is received
  • the lower layer portion further includes a second channel capable of selectively communicating the second chamber or the third chamber to the first chamber. can do.
  • the upper layer portion further includes a fourth chamber in which the third solution is accommodated, and the connection hole can selectively communicate the third chamber or the fourth chamber to the bead chamber.
  • connection hole may selectively communicate the third chamber or the fourth chamber to the bead chamber.
  • the upper layer portion further includes a jet hole, and the first channel may selectively communicate the first chamber or jet hole to the bead chamber.
  • the first channel may selectively communicate the first chamber or the ejection hole to the bead chamber.
  • the bead chamber may communicate with the ejection hole through the first channel and communicate with the fourth chamber through the connection hole according to the relative positions of the upper and lower layers.
  • the bead chamber may be formed with at least one protrusion on the inner wall.
  • FIG. 1 is a perspective view showing a nucleic acid extraction cartridge according to an embodiment of the present invention
  • Figure 2 is an exploded perspective view of Figure 1
  • Figure 3 is a plan view showing the upper portion of the nucleic acid extraction cartridge
  • Figure 4 is a plan view showing the lower layer of the nucleic acid extraction cartridge
  • Figure 5 is a side view showing the first chamber of the nucleic acid extraction cartridge
  • Figure 6 is a side view showing the fourth chamber of the nucleic acid extraction cartridge
  • FIG. 7 is a sectional view taken along line I-I 'in FIG. 4,
  • FIGS 8 to 11 are schematic plan views showing various arrangement states of the upper and lower layers using the first and second channels of the present invention.
  • FIG. 12 is a flowchart showing a nucleic acid extraction method according to another embodiment of the present invention.
  • 13 is a graph measuring the degree to which infectious bacteria have been removed from a collector by varying the acidity (pH) of the first solution in the nucleic acid extraction method
  • 15 is a graph measuring the degree to which infectious bacteria are adsorbed on beads by varying the acidity of the second solution in the nucleic acid extraction method
  • Figure 16 is a graph measuring the degree of infection of the bacteria in 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
  • Figure 17 is a graph measuring the degree of nucleic acid extraction of infectious bacteria by varying the bead beat time in the nucleic acid extraction method
  • 18 is a graph measuring the degree of nucleic acid extraction of infectious bacteria by varying the length of the projections of the bead chamber in the nucleic acid extraction method.
  • first channel 113 second channel
  • connection hole P1, P2 piston
  • the present invention is a method of extracting a nucleic acid of an infectious bacterium while collecting the infectious bacteria present on the surface by wiping the surface of the target object with the collector, and receiving the collector itself in the nucleic acid extraction cartridge 1, and a nucleic acid extraction cartridge using the same (1).
  • nucleic acid extraction cartridge 1 for extracting nucleic acids of infectious bacteria.
  • the nucleic acid extraction cartridge 1 is mounted and driven in a nucleic acid extraction device (not shown), and relates to a cartridge for extracting nucleic acids of infectious bacteria through a nucleic acid extraction method described later.
  • FIG. 1 is a perspective view showing a nucleic acid extraction cartridge according to an embodiment of the present invention
  • FIG. 2 is an exploded perspective view of FIG. 1.
  • a nucleic acid extraction cartridge 1 may include an upper layer portion 10 and a lower layer portion 11.
  • the upper layer 10 includes a first chamber 101, a second chamber 102, a third chamber 103, a fourth chamber 104, and a blowout hole 105 ) And pistons P1 and P2.
  • the first chamber 101 may be a chamber in which a solid-phase collector in which infectious bacteria are collected is accommodated.
  • the first chamber 101 may be separated from the nucleic acid extraction cartridge 1.
  • the collecting body is for collecting infectious bacteria, and those having a porous flexible material such as sponge or tissue paper and cleaning cloth may be used.
  • the term 'solid phase' is a state of a solid object, which is a state having physically and chemically substantially uniform properties and shapes even when taking any part of the object at room temperature, and is defined to include a semi-solid phase in a soft state. Can be.
  • the term 'infectious bacteria' may be a microorganism including bacteria and viruses.
  • a magnet may be accommodated in the inner space of the first chamber 101.
  • the magnet is intended to eliminate infectious bacteria from the collector, and for example, a neodymium magnet may be used.
  • the piston P1 may be pressed into the first chamber 101.
  • the first chamber 101 may be coupled to the nucleic acid extraction cartridge 1 after the piston P1 is pressurized while the collector and the magnet are accommodated in the inner space.
  • the first chamber 101 may be a member having a cylindrical shape and an inner 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 below 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 disposed adjacent to the sinus side of the first chamber 101 in the drawing.
  • a second hole H2 which is 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 disposed adjacent to the right side of the first chamber 101 in the drawing.
  • a third hole H3, which 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 removable from the nucleic acid extraction cartridge 1.
  • the piston P2 may be pressurized while the third solution and gas are accommodated in the fourth chamber 104. Thereafter, the fourth chamber 104 may 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.
  • the ejection hole 105 may be formed in an open shape on a part of the side surface of the upper layer.
  • the ejection hole 105 may be connected to the fifth hole H5, which is a hole formed in the lower portion of the upper layer 10 adjacent thereto.
  • the fifth hole H5 may be connected to the first channel 112 (see FIG. 6).
  • the first to fifth holes H1 to H5 may be disposed to form the same distance with respect to mutually adjacent holes.
  • the arrangement form of the first to fifth holes H1 to H5 may be arranged at each vertex portion of the 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 (see FIG. 2) in the body 11a.
  • the lower layer 11 may be rotatably coupled around the central axis on the central axis line below the upper layer 10. In this case, the lower layer 11 may be rotated through a handle (not shown) provided on the bottom surface of the body 11a.
  • the body 11a may be fitted to the lower portion of the upper layer 10 via the lower case 11b.
  • the lower case 11b is formed with a center through which the body 11a can be rotated.
  • the bead chamber 111 may be a portion in which a plurality of beads are accommodated.
  • a magnet (not shown) 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 protrusion 111a may be arranged at the same interval on the inner surface of the bead chamber 111.
  • the protrusion 111a may protrude to a length of 2 mm from the inner surface of the bead chamber 111 toward the center of the bead chamber 111. While the beads inside the bead chamber 111 are bent, they are also bent in the protrusion 111a, the movement of the beads is prevented, and the efficiency of extracting nucleic acids from the infecting bacteria may increase as the number of beats increases.
  • the bead chamber 111 communicates with the first chamber 101 through the first channel 112 at the same time according to the relative positions of the upper layer 10 and the lower layer 11, and the connection hole It can be communicated with the third chamber 103 through (114).
  • first channel 112 may be a channel capable of communicating the bead chamber 111 and the first chamber 101.
  • 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 so that the bead chamber 111 and the first chamber 101 ).
  • one end of the first channel 112 is connected to the bead chamber 111 and the other end is connected to the fifth hole H5 to connect the bead chamber 111 and the ejection hole 105. I can communicate.
  • the second channel 113 may be a portion capable of selectively communicating the second chamber 102 or the third chamber 103 with the first chamber 101.
  • the second channel 113 selectively selects the second chamber 102 or the third chamber 103 in the first chamber 101 as the lower layer 11 is rotated relative to the upper layer 10. Can communicate with.
  • 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 H2 of the second chamber 102 It is connected to the first chamber 101 and the second chamber 102 to communicate.
  • 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.
  • the first chamber 101 and the third chamber 103 can communicate with each other.
  • the second channel 113 is rotated 10 degrees counterclockwise with respect to the upper portion 10 while the upper portion 10 and the lower portion 11 of the nucleic acid extraction cartridge 1 are aligned.
  • the first chamber 101 and the second chamber 102 may communicate with each other (see FIG. 10). 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.
  • the infectious bacteria are removed 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 to rotate the first chamber 101 through the second channel 113. And the third chamber 103 can be communicated (see FIG. 11).
  • the infectious bacteria solution accommodated in the first chamber 101 can be injected into the third chamber 103 containing the second solution.
  • the connection hole 114 may be a hole through which the bead chamber 111 and the third chamber 103 can communicate.
  • 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 portion 11 is rotated relative to the upper layer portion 10.
  • the connection hole 114 may be connected to the third hole (H3) while communicating the bead chamber 111 and the third chamber 103, and connected to the fourth hole (H4), the bead chamber 111 And the fourth chamber 104 may communicate.
  • the lower portion 11 is rotated 108 degrees relative to the upper portion 10 to turn the third chamber 103
  • the bead chamber 111 may be disposed at the lower portion of the bead, and the third hole H3 of the third chamber 103 may be connected to the connection hole 114 of the bead chamber 111.
  • the bead chamber 111 and the first chamber 101 may be communicated through the first channel 112 (see FIG. 8).
  • the mixed liquid accommodated in the inner space of the third chamber 103 passes through the bead chamber 111, and the infecting bacteria are adsorbed on the bead. It may be injected into the interior space of (101).
  • the lower layer 11 is rotated 144 degrees relative 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 may be communicated with the 104.
  • the inner space in which the third solution of the fourth chamber 104 is accommodated may be pressurized with the piston P2 to inject the third solution into the bead chamber 111 (see FIG. 9).
  • bead beading can be performed to extract the nucleic acid of the infecting bacteria adsorbed on the beads.
  • the bead chamber 111 communicates with the ejection hole 105 through the first channel 112 according to the relative positions of the upper layer portion 10 and the lower layer portion 11, and at the same time, the fourth chamber through the connection hole 114. It can be communicated with 104 (see Fig. 9).
  • the first channel 112 is connected to the fifth hole (H5) to communicate the bead chamber 111 and the ejection hole 105
  • the connection hole 114 is connected to the fourth hole (H4) beads
  • the chamber 111 and the fourth chamber 104 may communicate. Therefore, the piston P2 is pressed into the inner space of the fourth chamber 104 to inject the gas contained in the inner space of the fourth chamber 104 into the bead chamber 111, and the nucleic acid extract solution contained in the bead chamber 111 It can be ejected to the outside through the ejection hole (105).
  • FIG. 12 is a flow chart showing a nucleic acid extraction method according to another embodiment of the present invention.
  • a step (S1) of accommodating the solid collection body of the infected bacteria into the inner space of the first chamber 101 is performed.
  • Collection of infectious bacteria can be performed by wiping the surface of the target object with a collection body.
  • step (B) of injecting the first solution into the inner space of the first chamber 101 may be performed.
  • step (B) may be performed before step (A).
  • step (B) moving the piston P1 to reduce the internal space of the first chamber 101, the first chamber 101 and the second chamber 102 containing the first solution to communicate
  • the step and the step of injecting the first solution into the first chamber 101 while moving the piston P1 to expand the inner space of the first chamber 101 in communication with the second chamber 102 may be performed in order.
  • the first chamber 101 may be coupled to the nucleic acid extraction cartridge 1 in a state in which the volume of the inner space is reduced by pressing the piston P1 into the inner space.
  • the volume of the collector can also be reduced.
  • the collector has a porous flexible property.
  • 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 portion 11 with respect to the upper layer portion 10, and the first chamber 101 ) And the second chamber 102 can be communicated.
  • the first solution may be a solution for removing infectious bacteria from the collector.
  • a basic solution may be used as the first solution.
  • a solution having a pH (acidity) of 9 may be used.
  • FIG. 13 is a graph of measuring the degree of pathogen desorption of infectious bacteria from the collection by varying the acidity (pH) of the first solution in the nucleic acid extraction method.
  • FIG. 14 is a graph measuring the degree of pathogen desorption of infectious bacteria from a collector by varying the ethanol concentration of the first solution in the nucleic acid extraction method.
  • the first solution may include ethanol. It can be seen that the pathogen desorption of the infecting bacteria increases when the ethanol is contained in 5% or more than the ethanol is not included in the first solution.
  • step (B) step (C) step (S3) of mixing the collector and the first solution in the inner space of the first chamber 101 to remove the infecting bacteria from the collector and forming the infecting bacteria solution may be performed.
  • step (S3) of mixing the collector and the first solution in the inner space of the first chamber 101 to remove the infecting bacteria from the collector and forming the infecting bacteria solution may be performed.
  • the 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.
  • the device in which the nucleic acid extraction cartridge 1 is mounted may include an electromagnet, and the electromagnet may magnetically move the neodymium magnet accommodated in the inner space of the first chamber 101. 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 infecting bacteria may be eliminated from the collector as the first solution.
  • step (C) the first chamber 101 is communicated with the third chamber 103 in which the second solution is accommodated, and the infectious bacteria solution accommodated in the first chamber 101 is in the third chamber 103 ) To mix with the second solution to form a mixed solution (S4) may be performed.
  • the second channel 113 is connected to the holes of the first chamber 101 and the holes of the third chamber 103,
  • the first chamber 101 and the third chamber 103 can be communicated (see FIG. 11).
  • the piston P1 may be injected into the inner space of the first chamber 101 to inject the infectious bacteria contained in the first chamber 101 into the third chamber 103.
  • the absorbed infectious liquid may also be injected into the third chamber 103, and the porous collector may act as a filter to remove impurities. Therefore, the infecting bacteria solution having a high concentration of infecting bacteria may be injected into the third chamber 103.
  • the 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 the infecting bacteria to the solid surface.
  • the second solution may be an acidic solution.
  • the solid surface may mean a surface of another solid object such as a bead surface and / or a filter.
  • 15 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.
  • an experiment performed by varying the pH of the second solution 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) were injected into 20 mL of the first solution to generate an infectious bacteria solution containing infectious bacteria, which was passed through beads at a rate of 1 mL / min and remained. The amount of bacteria present was checked. As a result, it was confirmed that the adsorption rate (adsorption) of the infectious bacteria was high under the condition that the pH of the second solution was low.
  • a solution having a pH of 5 may be used as the second solution.
  • the step (S5) of re-adsorbing the infecting bacteria on the solid surface may be performed while passing the infecting bacteria solution through the bead chamber 111 containing a plurality of beads.
  • the solid surface may mean a surface of another solid object such as a bead surface and / or a filter.
  • a step of re-adsorbing the infecting bacteria on the solid surface may be performed.
  • the solid surface may mean a surface of another solid object such as a bead surface and / or a filter.
  • FIG. 16 is a graph measuring the adsorption rate of infected bacteria on 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.
  • the flow rate of the infectious bacteria passing through the mixed solution through the bead chamber 111 affects the degree to which the beads are adsorbed. Adsorption rate according to the rate at which the solution containing the detached infectious bacteria flows into the bead chamber 111 containing the functional beads (GO bead) was confirmed. Bacterial solution was injected into 20 mL (10 3 CFU / mL) of the bead chamber 111, and the amount of bacteria in the discharged solution was compared and analyzed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the functional beads even when the solution was passed through at a relatively fast rate of 5 mL / min and 10 mL / min, the adsorption rate of infectious bacteria was high at about 80% or more.
  • the functional beads have a higher rate of adsorption of infectious bacteria than the glass beads, regardless of the injection rate of the bacterial solution.
  • Step S6 of injecting into 111) may be performed.
  • the bead chamber 111 and the fourth chamber 104 may be communicated by rotating the lower layer portion 11 of the nucleic acid extraction cartridge 1 relative to the upper layer portion 10.
  • a 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.
  • step (E) the beads in the bead chamber 111 and / or the beads adsorbed by the infecting bacteria are beaten to extract nucleic acids to form a nucleic acid extract, and the gas contained in the fourth chamber 104 is beaded.
  • step (S7) of injecting the nucleic acid extract to the outside by injecting it into the chamber 111 using air pressure may be performed.
  • Bead beading may be performed by moving the beads accommodated inside the bead chamber 111 by using the electromagnet provided in the nucleic acid extraction device to move the beads to collide with each other. In addition, it can be performed by rotating the bead chamber 111 to move the beads.
  • 17 is a graph measuring the degree of nucleic acid extraction (Ct value) of infectious bacteria by varying the beading time in the nucleic acid extraction method.
  • the bead beating efficiency was checked by time. Gram-positive bacteria and virus samples having difficulty in nucleic acid extraction were used to compare nucleic acid extraction rates. As a result, it was confirmed that performing the bead beating for 1 minute at a frequency of 50 Hz showed a nucleic acid extraction rate equivalent to that of the bead beating that is generally performed.
  • 18 is a graph measuring the degree of nucleic acid extraction of infectious bacteria by varying the length of the protrusion 111a of the bead chamber 111 in the nucleic acid extraction method.
  • the experiment was performed by adjusting the size and / or number of the protrusions 111a formed on the inner wall of the bead chamber 111.
  • the 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
  • the nucleic acid of the infecting bacteria was extracted to confirm the bead beating efficiency.
  • Gram-positive bacteria having difficulty in nucleic acid extraction were used to adjust the size and number of protrusions 111a, and compared with conventional bead-biting.
  • four projections 111a protruding 2 mm from the inner wall of the bead chamber 111 were used, it was confirmed that they exhibited the same efficiency as the existing bead beating.
  • nucleic acid extraction cartridge 1 By extracting the nucleic acid from the infecting bacteria through the above-described nucleic acid extraction method, it is possible to automatically extract the nucleic acid of the infecting bacteria in a state in which the collection of the infecting bacteria from the target object having a large surface is accommodated in the nucleic acid extraction cartridge 1.
  • the nucleic acid of the infecting bacteria is automatically extracted from the collecting body.
  • the nucleic acid of the infecting bacteria can be efficiently extracted.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Un procédé d'extraction d'acide nucléique selon un mode de réalisation de la présente invention comporte les étapes consistant à : (A) recevoir un collecteur en phase solide ayant des agents pathogènes infectieux collectés à l'intérieur de celui-ci dans l'espace interne d'une première chambre ; (B) injecter une première solution dans l'espace interne de la première chambre ; (C) mélanger le collecteur et la première solution pour séparer les agents pathogènes infectieux du collecteur, formant ainsi une solution d'agents pathogènes infectieux, dans l'espace interne de la première chambre ; et (D) faire passer la solution d'agents pathogènes infectieux à travers une chambre de billes recevant une pluralité de billes pour permettre aux agents pathogènes infectieux d'être réadsorbés sur des surfaces solides.
PCT/KR2019/012767 2018-10-01 2019-09-30 Procédé d'extraction d'acide nucléique et cartouche l'utilisant WO2020071715A1 (fr)

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CN114032173B (zh) * 2022-01-11 2022-03-15 至美时代生物智能科技(北京)有限公司 一种密闭式空气采样瓶

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