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WO2020093824A1 - 自动化核酸萃取的方法及装置 - Google Patents

自动化核酸萃取的方法及装置 Download PDF

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Publication number
WO2020093824A1
WO2020093824A1 PCT/CN2019/109890 CN2019109890W WO2020093824A1 WO 2020093824 A1 WO2020093824 A1 WO 2020093824A1 CN 2019109890 W CN2019109890 W CN 2019109890W WO 2020093824 A1 WO2020093824 A1 WO 2020093824A1
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Prior art keywords
nucleic acid
column
acid extraction
syringe
liquid tank
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Application number
PCT/CN2019/109890
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English (en)
French (fr)
Inventor
李德政
Original Assignee
开启基因股份有限公司
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 开启基因股份有限公司 filed Critical 开启基因股份有限公司
Priority to US17/290,231 priority Critical patent/US12258555B2/en
Priority to EP19882601.8A priority patent/EP3878939B1/en
Priority to JP2021523988A priority patent/JP7141530B2/ja
Publication of WO2020093824A1 publication Critical patent/WO2020093824A1/zh

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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
    • 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
    • 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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • 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
    • C12M47/12—Purification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1009—Characterised by arrangements for controlling the aspiration or dispense of liquids
    • G01N35/1011—Control of the position or alignment of the transfer device
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
    • G01N35/1083—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane with one horizontal degree of freedom
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0289—Apparatus for withdrawing or distributing predetermined quantities of fluid
    • B01L3/0293—Apparatus for withdrawing or distributing predetermined quantities of fluid for liquids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/103—General features of the devices using disposable tips

Definitions

  • the invention relates to a device and a method, in particular to an automatic nucleic acid extraction device and an automatic nucleic acid extraction method.
  • Precision medicine is a customized medical model. It is based on human genome information and combines proteome, metabolome and other related internal environment information to formulate the best treatment plan for patients in order to maximize the treatment effect and minimize the side effects. .
  • Blood is a red opaque viscous liquid flowing in the blood vessels and heart of the human body.
  • the main components are plasma, blood cells, and genetic material (chromosomes and genes).
  • cell free DNA cfDNA
  • circulating tumor DNA circulating tumor DNA, ctDNA fragment refers to a DNA fragment released from tumor in cfDNA.
  • cfDNA free DNA fragments
  • cfDNA free DNA fragments
  • They may be derived from apoptosis or necrosis of cells, or they may enter the blood through active release (violent exercise).
  • the content of cfDNA in blood is very low. In 1 mL of plasma, the content of cfDNA is about 1 ng to 100 ng, and the content of ctDNA is lower, only accounting for 0.1% to 5% of cfDNA.
  • CN101684463A provides a method for quickly extracting nucleic acids from various micro clinical samples, which includes the following steps: (a) Add the clinical sample to the lysate and mix it; (b) Aspirate the clinical sample and flow through the filter membrane The nucleic acid components in the sample are adsorbed on the filter membrane due to the specific adsorption of the membrane.
  • the filtrate is a waste liquid with cell debris and protein;
  • Aspirate the cleaning solution flow through the filter membrane, and wash the residual residue on the filter membrane For protein or other components, discard the filtrate;
  • TWM477925U provides a sample extraction device including a suction part including a first upper part, an intermediate part communicating below the first upper part and having an inner diameter smaller than the inner diameter of the first upper part, and communicating with the middle A suction portion below the portion and having an inner diameter smaller than the inner diameter of the intermediate portion, whereby the first upper portion or the intermediate portion abuts against the outer periphery of the string member, and the specific liquid is sucked to the
  • the column component; the sample extraction device further includes: a column component; and a connecting component, thereby extracting the expected material from the sample.
  • TWM536238U provides a machine for automatically extracting nucleic acids, including: a machine bottom plate, a tray fixing frame on the plate, the tray fixing frame can move along a horizontal track; a bracket is vertically arranged above the machine bottom plate , And there is a vertical track; a vertical motion unit, the vertical motion unit includes a base plate, a base plate track is provided above the base plate, a motion block is disposed along the base plate track, and a position is below the motion block
  • a syringe fixing unit fixed to the base plate is provided for a syringe to be erected on it; a piston thruster fixing unit is provided for a piston pusher to be erected, so that the moving block is driven up and down along the substrate track
  • the piston pusher generates positive or negative pressure on the syringe
  • the base plate is arranged along the vertical track to drive the moving block and the syringe fixing unit to move up and down corresponding to the bracket.
  • TW201412981A provides a method and device for nucleic acid extraction, which uses a pneumatic extraction method for nucleic acids, and is connected to a device that can apply positive or negative gas pressure at the upper end of the purification tube, so that the specimen can be sucked / discharged from the lower tip of the purification tube ⁇ Cleaning solution and eluent can achieve the effect of extracting nucleic acid easily without centrifuge.
  • the object of the present invention is to provide an automated nucleic acid extraction device and automated nucleic acid extraction method. Compared with the prior art, the automated nucleic acid extraction device and method of the present invention can quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen.
  • nucleic acids such as cfDNA and ctDNA
  • the present invention provides an automated nucleic acid extraction device.
  • the automated nucleic acid extraction device includes a base, a cassette, a driving unit, a moving frame, and a syringe.
  • the seat body has a sample accommodating area, a column accommodating area, a cartridge accommodating area and a collection tube, and the sample accommodating area, the column accommodating area, the cartridge accommodating area and the collection tube are along a Arranged in a linear direction.
  • the cassette is arranged in the cassette accommodating area.
  • the cassette includes two parallel walls and at least two vertical walls.
  • the parallel walls and the vertical walls together form a dissolution liquid tank, at least one washing liquid tank and an elution liquid tank, in which the dissolving liquid is formed
  • the vertical walls of the tank, the washing liquid tank and the eluting liquid tank are respectively provided with an abutting portion, and the dissolving liquid tank, the washing liquid tank and the eluting liquid tank are also arranged in a linear direction, wherein the abutting portion has an arc-shaped wall Or a polygonal wall.
  • the driving unit is disposed on the base.
  • the moving frame is vertically arranged on the seat body, and driven by the driving unit to reciprocate in a linear direction.
  • the syringe is set on the moving frame and moves with the moving frame.
  • the sample containing area has a sample containing space and a binding buffer containing space.
  • the column accommodation area further includes a sample row pipette accommodation space and a column row pipette accommodation space.
  • the automated nucleic acid extraction device further includes a sample discharge pipette, which is movably disposed in the sample discharge pipette receiving space.
  • the automated nucleic acid extraction device further includes a pipe column pipette, which is movably disposed in the pipe column pipette receiving space.
  • the syringe is detachably connected to the sample discharge pipette or the column discharge pipette.
  • the sample accommodating space is used to accommodate a biological sample and a dissolution buffer to perform a dissolution reaction
  • the binding buffer accommodating space is used to accommodate a binding buffer and a dissolution product to perform a binding reaction and dissolve
  • the liquid tank is used to contain the dissolution buffer, defoamer and reaction residue.
  • These washing liquid tanks are used to contain the washing buffer.
  • These elution liquid tanks are used to contain the elution buffer.
  • the accommodating space is used for accommodating the sample row suction pipe, and the column row pipetting accommodating space is used for accommodating the column row pipette.
  • the arc angle of the arc-shaped wall is greater than or equal to 90 degrees.
  • the vertical wall between the washing liquid tank and the eluting liquid tank has a complete hollow cylindrical structure, forming a filter material accommodating space for accommodating a filter material.
  • the polygonal wall includes at least two support walls, and the support walls form an angle of less than 180 degrees with each other.
  • a recess is formed at the bottom of the eluent bath.
  • the cassette further includes a snap fastener for detachably fastening the cassette to the cassette receiving area.
  • the present invention also provides an automated nucleic acid extraction method using the automated nucleic acid extraction device as described above, which includes the following steps: a syringe is used with a column row pipette to draw a combined buffer solution to be mixed in the space After the reaction, the nucleic acid in the reaction is bound to a membrane in the pipette pipette, and the reaction residue is discharged to the cassette, so that the nucleic acid in the reaction residue is bound to the membrane; the syringe is used to match the tube The column suction tube sucks the washing buffer in the at least one washing liquid tank through the diaphragm, and then discharges the washing buffer through the diaphragm with a syringe and a column exhaust tube; and uses the syringe with the column exhaust tube to suck and wash through the diaphragm The elution buffer in the elution tank, and then the syringe and the column exhaust pipette are used to discharge the elution
  • the column in the step of discharging the reaction residue to the cassette, and the step of discharging the washing buffer with a syringe and a column exhaust pipette through the membrane, the column The abutment portion of the suction pipe abuts against the abutment portion of the cassette, and then discharges the reaction residue or the washing buffer.
  • the step The method includes the following steps: abutting the abutting portion of the column row suction tube against the abutting portion of the cassette, and moving the syringe up and down in a direction perpendicular to the linear direction by less than or equal to 5 mm.
  • the effect of the present invention is that the dissolution liquid tank, the washing liquid tank and the elution liquid tank formed by the cartridge can accommodate a large volume of solution through the arrangement of the parallel wall and the vertical wall; and through the washing liquid
  • the tank and the eluent tank are respectively provided with abutting parts. After the syringe discharges a large volume of solution, the sample discharge pipette or the column discharge pipette can be prevented from falling off from the syringe and separating from the syringe.
  • the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen.
  • the automated nucleic acid extraction device of the present invention has a smaller volume, which can save the user's use space.
  • FIG. 1 is a schematic assembly view of a preferred embodiment of an automated nucleic acid extraction device of the present invention.
  • FIG 2 is a side view of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • 3A is a partial schematic diagram of a preferred embodiment of an automated nucleic acid extraction device of the present invention.
  • 3B is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • 3C is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • FIG. 4 is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • 5A is a partial schematic diagram of a preferred embodiment of an automated nucleic acid extraction device of the present invention.
  • 5B is a partial schematic view of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • 5C is a partial schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • 6A is a top view of a preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention.
  • 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention.
  • 6C is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention.
  • 6D to 6H are schematic diagrams of different embodiments of the abutment portion of the cassette of the automated nucleic acid extraction device of the present invention.
  • FIG. 7A is a partial enlarged view of the automated nucleic acid extraction device of FIG.
  • FIG. 7B is a schematic diagram of the operation of the automated nucleic acid extraction device of FIG. 7A.
  • FIG. 7C is another schematic diagram of the automated nucleic acid extraction device of FIG. 7A.
  • FIG. 8A is a partial schematic diagram of the automated nucleic acid extraction device of FIG. 1A.
  • FIG. 8B is an exploded schematic view of the automated nucleic acid extraction device of FIG. 8A.
  • FIG. 9 is a flowchart of a preferred embodiment of the automated nucleic acid extraction method of the present invention.
  • the automated nucleic acid extraction device of the present invention can quickly and conveniently extract nucleic acids of higher yield and higher concentration, such as cfDNA and ctDNA, from the specimen.
  • the specimen includes, but is not limited to: blood, plasma, urine, saliva, tissue fluid, and tissue. The structure and features of the automated nucleic acid extraction device will be described below with examples.
  • FIG. 1 is a combined schematic diagram of a preferred embodiment of the automated nucleic acid extraction device of the present invention
  • FIG. 2 is a side view of a preferred embodiment of the automated nucleic acid extraction device of the present invention.
  • the automated nucleic acid extraction device includes a base B, a cassette 31, a driving unit M, a moving frame 4 and a syringe 5.
  • the seat body B has a sample accommodating area 1, a column accommodating area 2, and a cartridge accommodating area 3.
  • the sample accommodating area 1 has a sample accommodating space 11 and a binding buffer accommodating space 12, And the sample accommodating area 1, the column accommodating area 2 and the cassette accommodating area 3 are arranged along a linear direction L1.
  • the order in which the sample accommodating area 1, the column accommodating area 2 and the cassette accommodating area 3 are arranged along the linear direction L1 can be, for example, but not limited to, the first sample content Placement area 1, column accommodation area 2 and then cartridge accommodation area 3; first column accommodation area 2, cartridge accommodation area 3 and then sample accommodation area 1; first cartridge accommodation area 3 and sample accommodation Zone 1 is followed by column accommodating zone 2; or cartridge accommodating zone 3 first, column accommodating zone 2 and then sample accommodating zone 1.
  • the cassette 31 is disposed in the cassette accommodating area 3.
  • the cassette 31 includes two parallel walls 31a and at least two vertical walls 31b.
  • the parallel walls 31a and the vertical walls 31b together form a solution
  • the liquid tank 311, the washing liquid tank 312, and the elution liquid tank 313 are also arranged along the linear direction L1.
  • the number of vertical walls 31b is nine, and together with the parallel walls 31a, two dissolution liquid tanks 311, four washing liquid tanks 312, and two elution liquid tanks 313 are formed.
  • the vertical walls 31b, dissolving The number of the liquid tank 311, the washing liquid tank 312 and the elution liquid tank 313 can be adjusted according to the actual needs of the user (for example, if the sample is a tissue that is difficult to extract, the dissolving liquid tank 311 and the elution liquid tank 313 Can be increased), the present invention is not limited.
  • the dissolution liquid tank 311, the washing liquid tank 312, and the elution liquid tank 313 formed therein can accommodate a large volume of buffer solution (dissolution buffer, washing buffer Liquid and / or elution buffer), such as but not limited to more than 2 ml, more than 5 ml, or more than 10 ml.
  • buffer solution dissolution buffer, washing buffer Liquid and / or elution buffer
  • the driving unit M is disposed on the base B, and the moving frame 4 is vertically disposed on the base B, and is driven by the driving unit M to reciprocate along the linear direction L1.
  • the syringe 5 is provided on the moving frame 4 and moves with the moving frame 4.
  • the driving unit M is shown as a transmission roller belt, the driving unit M may also be other driving devices, such as a linear module or other device that can drive the linear movement of the moving frame 4, and the present invention is not limited.
  • the column receiving area 2 further includes a sample discharge pipe receiving space 21 and a column discharge pipe receiving space 22.
  • the automated nucleic acid extraction device further includes a sample discharge pipette 6, which is movably disposed in the sample discharge pipette accommodating space 21.
  • the automated nucleic acid extraction device further includes a column row pipette 7, which is movably disposed in the column row pipette receiving space 22.
  • the syringe 5 is detachably connected to the sample discharge pipette 6 or the column discharge pipette 7.
  • the automated nucleic acid extraction device further includes a collection tube 8, which is arranged along the linear direction L1 with the cassette 31 and the receiving areas.
  • the collection tube 8 may be disposed adjacent to the cassette 31 (but the invention is not limited to this), because the automated nucleic acid extraction method is to extract along the linear direction L1, and then the eluate is discharged in the last step Into the collection tube 8, the design of the collection tube 8 arranged adjacent to the cassette 31 and arranged in the linear direction L1 is consistent with the moving direction of the mobile rack 4, which can avoid the mobile rack 4 in the process of automated nucleic acid extraction, reciprocating through the collection Above the tube 8, causing cross contamination.
  • the sample accommodating space 11 is used for accommodating a biological sample and a lysis buffer to perform a lysis reaction.
  • the biological sample includes, but is not limited to: blood, plasma, urine, saliva, tissue fluid, and tissue.
  • the binding buffer accommodating space 12 is used to accommodate a binding buffer and a lysate for binding reaction.
  • the dissolving solution tank 311 is used to contain a dissolving buffer (lysis buffer), an antifoaming agent (defoaming agent), and reaction residues.
  • These washing liquid tanks 312 are used to contain washing buffers.
  • These elution liquid tanks 313 are used for accommodating elution buffer.
  • the sample discharge pipette accommodating space 21 is used for accommodating the sample discharge pipette 6.
  • the pipe row suction pipe accommodating space 22 is used to house the pipe row suction pipe 7.
  • the sample accommodating space 11, the combined buffer accommodating space 12, the dissolution liquid tank 311, and the washing liquid tank 312 can contain, for example, but not limited to, 30 mL of specimen or buffer, so compared to the prior art (Usually 2mL) has a higher sample capacity.
  • the increase in the volume of the dissolution buffer can improve the dissolution reaction of the sample to increase the concentration of nucleic acid; the increase in the volume of the washing buffer can wash the solution remaining in the pipette 7 In order to increase the concentration and purity of nucleic acids extracted in the subsequent steps.
  • the number of the dissolving liquid tank, the washing liquid tank, and the eluting liquid tank can be adjusted according to the actual needs of the user, and the present invention is not limited.
  • a diaphragm 74 is disposed inside the column row suction pipe 7.
  • the material of the membrane 74 is, for example, but not limited to, a silicone membrane, which is positively charged, and the nucleic acid contained in the specimen may be negatively charged during the nucleic acid extraction process.
  • the sucking property allows the nucleic acid to attach to the membrane 74, and then extracts the nucleic acid attached to the membrane 74 through a subsequent procedure.
  • the seat body B may further include at least one heating element H, which is disposed below the sample accommodating space 11 and / or the column exhaust pipette accommodating space 22.
  • heating element H when the sample accommodating space 11 is provided with When heating element H, it can promote the dissolution reaction between the biological sample and the dissolution buffer, so that the biological sample can be dissolved more completely, and then increase the concentration of nucleic acid extracted by the subsequent procedure; when the column row suction tube accommodating space 22 is provided with When the heating element H is heated, it can promote the volatilization of the residual solvent on the membrane 74 of the column exhaust pipette 7, thereby increasing the concentration and purity of the nucleic acid extracted by the subsequent procedure.
  • the heating element H below the sample accommodating space 11 may not be turned on (that is, it is not heated during the dissolution reaction) to avoid the ribonucleic acid being decomposed and affected The concentration of ribonucleic acid after extraction.
  • a concave portion f is formed at the bottom of these elution liquid tanks 313.
  • a smaller amount of elution buffer can be used when the specimen is eluted, thereby increasing the concentration of nucleic acid extraction.
  • the figure shows that only the eluent tank 313 adjacent to the collecting tube 8 has a recess f, however, other eluent tanks 313 may also have a recess f, and the present invention is not limited.
  • the volume of the elution buffer is, for example but not limited to, 1 ml, 500 ⁇ l, 200 ⁇ l, 100 ⁇ l, 50 ⁇ l, 30 ⁇ l, etc., which can be adjusted according to the user's needs, and the invention is not limited .
  • the cassette 31 further includes an elastic fastener 316 for detachably fastening the cassette 31 to the cassette accommodating area 3.
  • FIGS. 8A and 8B are an exploded views of FIG. 8A.
  • the automated nucleic acid extraction device of this embodiment may further include an iron frame I, which is detachably disposed on the base B.
  • the plurality of cassettes 31 and the collection tube 8 can be set on the iron frame I by the user first, and then set on the base B together with the iron frame I, so as to facilitate the user to set up the plurality of cassettes 31 and the collection tube 8.
  • the user can also set the iron frame I on the seat body B, and then set the cassette 31 and the collection tube 8.
  • the present invention is not limited.
  • the iron frame I can be removed from the seat body B to facilitate the user to take out multiple cassettes 31 and collection tubes 8 at a time.
  • the iron frame I may include two handles I1 to facilitate the user to take out the iron frame I from the seat body B or put the iron frame I into the seat body B.
  • the sample discharge pipette 6 includes an assembly portion 61 and a tip portion 62 connected to the assembly portion 61.
  • the pipe column suction pipe 7 includes an assembling portion 71, an abutting portion 72, and a tip portion 73 connected to the abutting portion 72, wherein the length D2 of the tip portion 73 is less than the depth D1 of the collection tube 8 (as shown in FIG. 3C) Show).
  • the tip portion 73 equipped with nucleic acid enters the collection tube 8
  • the force generated by the nucleic acid-containing specimen after being discharged from the tip portion 73 is too large to cause the specimen to splash out of the collection tube 8 To increase the yield of nucleic acid extraction.
  • the assembling portion 61 and the tip portion 62 are integrally formed; the assembling portion 71, the abutting portion 72 and the tip portion 73 are integrally formed.
  • the assembling portion 61 and the tip portion 62 can also be detached from each other, and the assembling portion 71, the abutting portion 72 and the tip portion 73 can also be detached from each other, and the present invention is not limited.
  • the sample discharge pipette 6 may have the same configuration as the column discharge pipette 7, the only difference between the two is that the column discharge pipette 7 has a diaphragm 74, while the sample discharge pipette 6 does not have a membrane 74 (i.e.,
  • the sample discharge pipette 6 can also have an assembly part, an abutment part and a tip part, and only a single mold can be used to manufacture the sample discharge pipette 6 and the column discharge pipette 7, and the sample is separately prepared by taking out or putting in the diaphragm 74 Suction pipe 6 and pipe column suction pipe 7).
  • the bottom of the syringe 5 further includes a connector 51, wherein the syringe 5 is connected to the assembly parts 61 and 71 through the connector 51.
  • the joint 51 cooperates with the structure of the fitting portion 61 of the sample discharge pipette 6 or the fitting portion 71 of the column discharge pipette 7 to tightly connect the syringe 5 and the fitting portions 61 and 71.
  • the syringe 5 and the connector 51 may be integrally formed.
  • FIG. 7A is an enlarged view of the area A in FIG. 1.
  • the moving frame 4 includes a ejection plate 41, and a spring mechanism 42 is provided on both sides of the moving frame 4 respectively.
  • the spring mechanism 42 drives the ejection plate 41 to control the assembling parts 61 and 71 to be detached from the bottom of the syringe 5.
  • the spring mechanism 42 moves in the L3 direction and drives the ejector plate 41 to move in the L4 direction to separate the sample discharge pipette 6 or the column discharge pipette 7 from the syringe 5.
  • the mobile rack 4 may further include at least one syringe fixing member 43 to fix the syringe 5 on the mobile rack 4.
  • the syringe fixing member 43 may further include a syringe locking groove 431 and at least one concave hole 432, and an elastic member R may be disposed in the concave hole 432.
  • the syringe 5 is locked in the syringe locking groove 431 of the two syringe fixing members 43, and the syringe 5 is better fixed through the elastic member R provided in the concave hole 432 At the syringe locking groove 431.
  • the number of the syringe fixing member 43, the concave hole 432 and the elastic member R can be adjusted according to the needs of the user, as long as the syringe 5 can be firmly fixed in the syringe clamping groove 431, the present invention does not No restrictions.
  • FIG. 5A, FIG. 5B and FIG. 6A are partial schematic diagrams of a preferred embodiment of the automated nucleic acid extraction device of the present invention
  • FIG. 6A is a cassette of the automated nucleic acid extraction device of the present invention.
  • the abutting portion 314 has an arc-shaped wall 3141 and the arc angle ⁇ of the arc-shaped wall 3141 is greater than or equal to 90 degrees.
  • the arc angle ⁇ may be 90 degrees; preferably, the arc angle ⁇ may be 120 degrees; preferably, the arc angle ⁇ may be 180 degrees; preferably, the arc angle ⁇ may be 270 degrees, as long as it can
  • the suction pipe 7 for supplying the pipe column only needs to abut.
  • the vertical wall 31b has an arc-shaped wall 3141, and the vertical wall 31b is connected to the parallel wall 31a, that is, both ends of the arc-shaped wall 3141 are not directly connected to the parallel wall 31a.
  • FIGS. 1, 5C and 6C wherein FIG. 5C is a partial schematic view of a preferred embodiment of the automated nucleic acid extraction device of the present invention, and FIG. 6C is another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention.
  • the abutment portion 314 has a polygonal wall.
  • the polygonal wall may include at least two support walls.
  • a structure including a first support wall 3142 and a second support wall 3143 will be used for explanation.
  • the first supporting wall 3142 and the second supporting wall 3143 have an included angle e smaller than 180 degrees.
  • the angle e can be 150 degrees; preferably, the angle e can be 120 degrees; preferably, the angle e can be 90 degrees; preferably, the angle e can be 45 degrees, as long as It is sufficient for the pipe column suction pipe 7 to abut and stand.
  • FIGS. 6D to 6H illustrate different embodiments of the abutment portion of the cassette of the automated nucleic acid extraction device of the present invention.
  • the abutment portion 314 is, for example but not limited to, an arc-shaped wall 3141 (as shown in FIG. 6E) or a polygonal wall (as shown in FIGS.
  • the polygonal wall has, for example but not limited to, two support walls and three supports Walls, four support walls, five support walls, six support walls or a structure larger than six support walls, each support wall has an angle, the number of support walls and the angle of the angle are not limited, as long as the pipe can be supplied
  • the column row suction pipe 7 only needs to abut and stand.
  • both ends of the polygonal wall of the abutment portion 314 may directly contact the parallel wall 31a, however, as shown in FIGS. 6F to 6H, the polygonal wall of the abutment portion 314 of the vertical wall 31b The two ends are not directly connected to the parallel wall 31a, but are connected by the vertical wall 31b and the parallel wall 31a, which is not limited herein.
  • the abutment portion 72 of the column discharge pipette 7 abuts on the arc-shaped wall 3141 of the abutment portion 314 or the abutment portion 72 of the column discharge pipette 7 during the discharge of liquid (eg, reactants, washing buffer, etc.) It is between the first support wall 3142 and the second support wall 3143, so that when the syringe 5 discharges liquid, the column suction pipe 7 will not fall off from the syringe 5 due to excessive pressure and separate.
  • liquid eg, reactants, washing buffer, etc.
  • the abutment portion 72 of the column discharge pipe 7 can be made to stand against the stand by the design of the abutment portion 314 On the part 314 without being completely separated from the syringe 5, and then the syringe 5 can be closely matched with the column row suction tube 7 again by a subsequent step of moving the syringe 5 up and down in a direction L2 perpendicular to the linear direction L1, In order to facilitate the subsequent extraction step.
  • FIG. 6B is a top view of another preferred embodiment of the cassette of the automated nucleic acid extraction device of the present invention.
  • the vertical wall 31b between the washing liquid tank 312 and the eluting liquid tank 313 has a complete hollow cylindrical structure, forming a filter material accommodating space 315 for accommodating a filter material.
  • the filter material is used to filter the dissolved lysate to fix the tissue fragments or impurities on the outside of the filter material and clarify (no tissue fragments or impurities)
  • the lysate can be sucked into the sample discharge pipette 6 to prevent the tissue fragments or impurities from plugging the tip 62 of the sample discharge pipette 6.
  • the step of lysing the tissue sample can be completed on the automated nucleic acid extraction device of the present invention, so that the tissue sample can also achieve the effect of fully automatic nucleic acid purification.
  • the filter material accommodating space 315 can also accommodate enzymes required in the middle of the extraction process, for example, deoxyribonuclease (DNase), in the extraction process.
  • DNase deoxyribonuclease
  • the DNA in the sample is removed from the sample, and the DNA decomposing enzyme (DNase) can also be washed away by subsequent washing steps, so that after the extraction is completed, ribonucleic acid without DNA can be obtained (RNA).
  • the moving frame 4 and the syringe 5 move linearly L1 (that is, the syringe moves relative to the desktop and the cassette does not move), so the depth of the machine is only the base B ’s
  • the length D3 is added to the thickness D4 of the moving frame 4 (D3 + D4).
  • the seat body B is moved linearly.
  • the depth of the machine must be at least twice the length D3 of the seat body B plus the moving frame 4 The thickness of D4 (D3 ⁇ 2 + D4).
  • the length D3 of the base B of the automated nucleic acid extraction device of this experimental example is, for example, but not limited to 30 cm
  • the thickness D4 of the moving frame 4 is, for example, but not limited to 20 cm
  • the depth of the machine for example, but not limited to about 50 cm
  • FIG. 9 is a preferred embodiment of the automated nucleic acid extraction method of the present invention.
  • the automated nucleic acid extraction method of the present invention is performed using the automated nucleic acid extraction device as described above.
  • the bottom of the is provided with a driving roller belt, which will drive the moving frame 4 to move in the linear direction L1.
  • the automated nucleic acid extraction method includes the following steps: Step S05 uses the syringe 5 and the column pipette 7 to suck the mixed reactant in the binding buffer storage space, so that the nucleic acid in the reactant is bound to The column discharges a membrane 74 in the pipette 7, and discharges the reaction residue to the cassette 31, so that the nucleic acid in the reaction residue is bound to the membrane 74. Then, proceed to step S06, use the syringe 5 and the column row suction pipe 7 to suck the washing buffer in at least one washing liquid tank through the membrane 74, and then use the syringe 5 and the column row suction pipe 7 to pass the membrane 74 through the membrane 74 discharge.
  • step S08 proceed to step S08 to draw the elution buffer in the eluent bath through the syringe 74 with the syringe 5 and the column exhaust pipette 7, and then use the syringe 5 with the column exhaust pipette 7 to pass the nucleic acid
  • the elution buffer is drained to the collection tube 8.
  • a step S07 may be arranged between steps S06 and S08 to move the column row suction pipe 7 to the column row suction pipe accommodating space 22 for heating to minimize the residual amount of the washing buffer on the membrane 74.
  • the above steps S05 to S08 are performed by the automated nucleic acid extraction device. The dotted line shown in FIG.
  • steps S01 to S04 can be performed manually by the user outside the automated nucleic acid extraction device, or all by the automated nucleic acid Extraction device.
  • the user can first dissolve and centrifuge the sample, then mix the supernatant with the binding buffer, and then add the binding buffer to the storage space, and the automated nucleic acid extraction device can perform the subsequent steps; or, the user can first After the sample is dissolved and centrifuged, the supernatant is added to the binding buffer storage space and mixed with the binding buffer, and the automated nucleic acid extraction device performs the next steps; or, the user can first dissolve the sample and centrifuge, then add the dissolved product to the dissolving solution In the tank, subsequent steps are performed by an automated nucleic acid extraction device, and the invention is not limited.
  • steps S01 to S08 are performed by an automated nucleic acid extraction device.
  • the automated nucleic acid extraction device and the moving frame 4 move to a specific direction along the linear direction L1
  • the sample discharge pipette 6 placed in the sample discharge pipette storage space 21 is connected to take out the sample discharge pipette 6 from the sample discharge pipette storage space 21.
  • step S01 the sample discharge pipette 6 connected to the syringe 5 moves to the cassette 31 of the cassette accommodating area 3 in the linear direction L1.
  • step S01 the dissolution buffer solution is sucked from the dissolution solution tank 311 of the cassette 31, and then moved to the sample storage area 1.
  • step S02 is performed to discharge the dissolution buffer to the sample accommodating space 11 of the sample accommodating area 1 and perform mixing.
  • step S03 is performed, the mixed dissolution product in the sample accommodation space 11 is sucked, and moved to the binding buffer accommodation space 12 of the sample accommodation area 1.
  • step S04 is performed, and the dissolved product is discharged to the binding buffer storage space 12 and mixed with the binding buffer to perform a binding reaction.
  • the syringe 5 and the sample discharge pipette 6 are moved to the sample discharge pipette accommodating space 21, and the ejection plate 41 is driven by the spring mechanism 42 to disassemble the sample discharge pipette 6 from the syringe 5 to the sample
  • the pipe column suction pipe 7 is connected to take out the pipe column suction pipe 7 provided with the diaphragm 74 from the pipe column accommodating area 2.
  • step S05 is performed, and the formed reactant is sucked by the syringe 5 and the column exhaust pipette 7 so that the reactant passes through the membrane 74 and the nucleic acid contained in the reactant is attached to the membrane 74.
  • the syringe 5 moves to the cassette 31 in the linear direction L1.
  • step S06 the column discharge pipette 7 with nucleic acid moves along the syringe 5 and the moving rack 4 in the linear direction L1 to the washing liquid tank 312 containing the washing buffer, and then the washing buffer is sucked and discharged for washing , The washing buffer is passed through the membrane 74 twice to wash the reaction residues remaining in the membrane 74 clean.
  • the column discharge pipette 7 with nucleic acid can be moved to the column discharge pipette accommodating space 22 along the linear direction L1 along with the syringe 5 and the moving frame 4 to stay the column discharge pipette 7 in the column row
  • the pipette accommodating space 22 is heated by the heating element H to volatilize the liquid (for example, washing buffer) remaining on the membrane 74 of the column row pipette 7 to fully dry it (ie, step S07).
  • the column discharge pipette 7 with nucleic acid is moved along with the syringe 5 and the moving frame 4 in the linear direction L1 to the elution liquid tank 313 containing elution buffer solution and proceeds to step S08, by sucking and discharging the elution buffer The solution is eluted to obtain an eluate (elution buffer with nucleic acid).
  • the column row suction pipe 7 sucks the obtained eluate, and then moves to the collection tube 8 in the linear direction L1, and then discharges the eluate to the collection tube 8.
  • the heating time of the heating element H is, for example but not limited to, 30 seconds, 1 minute, 3 minutes, and 5 minutes, as long as the liquid remaining on the membrane 74 of the column exhaust pipette 7 is volatilized and sufficiently dried, the present invention does not No restrictions.
  • step S05 in the step of discharging the reaction residue to the cassette 31 (step S05), and the step of discharging the washing buffer with the syringe 5 and the column exhaust pipette 7 through the diaphragm 74 (step S06), the abutment portion 72 of the column discharge pipette 7 is abutted on the abutment portion 314 of the cassette 31, and then the reaction residue or the washing buffer is discharged.
  • the pipe column suction pipe 7 is abutted on the abutment portion 314, so that the pipe column suction pipe 7 will not be affected by pressure during the process of discharging the liquid If it is too large, it falls off from the syringe 5 and separates.
  • the abutment section 72 of the column discharge pipe 7 can be made to stand against the abutment section 314 by the design of the abutment section 314 Without completely separating from the syringe 5, the syringe 5 and the column row suction tube 7 can be tightly matched again through subsequent steps.
  • the abutment portion 72 of the column discharge pipette 7 is abutted on the abutment portion 314 of the cassette 31, and then the reaction residue or the washing buffer is discharged (step S05, After S06), it further includes the following steps: abutting the abutting portion 72 of the column row suction tube 7 against the abutting portion 314 of the cassette 31, and moving the syringe 5 up and down in a direction L2 perpendicular to the linear direction L1 by less than Or equal to 5 mm. Through this step, the pipe column suction pipe 7 and the syringe 5 can be tightly connected to prevent the pipe column suction pipe 7 from falling off.
  • the syringe 5 can move up and down along the direction L2 by 5 mm; preferably, the syringe 5 can move up and down along the direction L2 by 4.5 mm; preferably, the syringe 5 can move up and down along the direction L2 by 3 mm to move
  • the column row suction pipe 7 and the syringe 5 are tightly connected again.
  • the number of washing liquid tanks 312 can be adjusted according to the actual needs of users, and the invention is not limited. In detail, if the number of washing liquid tanks 312 is greater than one, before moving the column row suction pipe 7 to the column row suction pipe accommodating space 22 for heating, the syringe 5 and the column row suction pipe 7 may be repeated through the membrane at least once The step of sucking the washing buffer solution in the at least one washing solution tank by the sheet 74, and then discharging the washing buffer solution through the membrane 74 with the syringe 5 and the column exhaust pipette 7.
  • the column discharge pipette 7 with nucleic acid moves along the syringe 5 and the moving rack 4 in the linear direction L1 to the washing liquid tank 312 containing the washing buffer and repeats the steps of sucking and discharging the washing buffer to perform washing It can be performed in different washing liquid tanks 312, and the number of washing steps depends on the number of washing liquid tanks 312.
  • the steps of discharging the dissolution buffer to the sample accommodation space 11 and mixing (step S02), and the step of sucking the mixed dissolution product in the sample accommodation space 11 (step S03) are It also includes the steps of moving to the filter material accommodating space 315, combining the sample discharge pipe 6 matched with the syringe 5 with the filter material in the filter material accommodating space 315; and after mixing in the suction sample accommodating space 11
  • the method further includes the steps of moving the sample drain pipe 6 and the filter material to the dissolving liquid tank 311 and reciprocating in the linear direction in the dissolving liquid tank 311.
  • the filter material is used to filter the dissolved lysate to fix the tissue fragments or impurities on the outside of the filter material, and then proceed to step S03 to clarify (not The lysate containing tissue fragments or impurities) is drawn into the syringe 5. Then, move the sample suction pipe 6 and the filter material to the dissolution liquid tank 311 and reciprocate in the linear direction in the dissolution liquid tank 311 to clean the impurities stuck on the outside of the filter material, and then dissolve the syringe 5 The product is discharged to the binding buffer accommodating space 12 to perform the binding reaction, so as to prevent tissue fragments or impurities from affecting the effect of the binding reaction and affecting the extraction efficiency. That is, when the biological sample is a tissue or other sample with impurities, the foregoing steps may be added.
  • the sample accommodating space 11, the combined buffer accommodating space 12, the sample exhaust pipette accommodating space 21, the column exhaust pipette accommodating space 22, the dissolution liquid tank 311, the washing liquid tank 312, the washing The number of the liquid extraction tank 313, the abutment portion 314, the syringe 5, the sample discharge pipette 6, the column discharge pipette 7, the collection pipe 8 and the joint 51 can be adjusted according to the actual needs of the user, and the invention is not limited.
  • the cassette 31, the sample accommodation space 11, the combined buffer accommodation space 12, the sample discharge pipette accommodation space 21, the column discharge pipette accommodation space 22, the syringe 5 and the collection tube 8 are along the linear direction L1 Arrangement, the order of arrangement is not limited here.
  • the automatic nucleic acid extraction device of the present invention has the design of the abutment portion 314 through the dissolution liquid tank 311, the washing liquid tank 312, and the adjacent elution liquid tank 313, so that the user can perform automatic nucleic acid extraction It can prevent the reactants from splashing out of the dissolving liquid tank 311, the washing liquid tank 312 or the eluting liquid tank 313, and can also prevent the column row suction pipe 7 from loosening and falling off.
  • the automated nucleic acid extraction device of the present invention can indeed quickly and conveniently extract higher yields and higher concentrations of nucleic acids, such as cfDNA and ctDNA, from the specimen.

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Abstract

一种自动化核酸萃取的方法及装置。该自动化核酸萃取装置包含座体、卡匣、驱动单元、移动架及针筒。座体具有样品容置区、管柱容置区、卡匣容置区以及收集管，且沿线性方向排列。卡匣设置于卡匣容置区，包含二平行壁及至少二垂直壁，平行壁与垂直壁共同形成溶解液槽、洗涤液槽及洗提液槽，其中形成溶解液槽、洗涤液槽及洗提液槽的垂直壁上分别设置有抵靠部，且溶解液槽、洗涤液槽及洗提液槽沿线性方向排列。抵靠部具有圆弧状壁或多边形壁。驱动单元及移动架设置于座体。针筒设置于移动架，且受到驱动单元的驱动而随移动架沿线性方向往复移动。该自动化核酸萃取装置能快速且方便地由检体中萃取出较高产量及较高浓度的核酸。

Description

自动化核酸萃取的方法及装置 技术领域
本发明涉及一种装置及方法，特别涉及一种自动化核酸萃取装置及自动化核酸萃取方法。
背景技术
随着人类基因组测序技术的革新、生物医学分析技术的进步以及大数据分析工具的出现，精准医疗(Precision Medicine)的时代已经到来。精准医疗是一种定制医疗模式，它以人体基因组信息为基础，结合蛋白质组、代谢组等相关内环境信息，为患者量身制定出最佳治疗方案，以期达到治疗效果最大化和副作用最小化。
血液是流动在人体的血管和心脏中的一种红色不透明的粘稠液体，主要成分为血浆、血细胞、遗传物质(染色体和基因)。其中，无细胞DNA(cell free DNA，cfDNA)就是在血浆中发现的游离DNA片段，而循环肿瘤DNA(circulating tumor DNA，ctDNA)片段是指cfDNA中来自肿瘤释放的DNA片段。
几乎每个人的血液中都含有游离的DNA片段(亦即cfDNA)，它们可能来源于细胞的凋亡、坏死，也可能是通过主动释放的方式(剧烈运动)进入血液。然而，血液中的cfDNA含量很低，在1mL的血浆中，cfDNA的含量约为1ng至100ng之间，ctDNA的含量更低，仅占cfDNA的0.1％至5％之间。
有研究已经证实，肿瘤患者外周血中的cfDNA总量高于健康人，通过这一点，cfDNA的含量若增多，可以起到一个很好的提示作用，由此作为肿瘤初筛的手段。因此，由液态生物检体(Liquid Biopsy)中纯化出极微量的cfDNA(核酸的一种)乃是精准医疗的第一步。
目前本领域的研究人员已致力于开发萃取例如cfDNA或ctDNA等核酸的装置及方法。例如，CN101684463A提供了一种从各种微量临床样本中快速提取核酸的方法，其包含以下步骤：(a)将临床样本加入裂解液，混匀；(b)抽吸临床样本，流经过滤膜，样本中的核酸成分因膜的特异性吸附而吸附在过滤膜上，滤液为有细胞碎屑和蛋白质的废液；(c)抽吸清洗液，流经过滤膜，清洗过滤膜上残留的蛋白质或其它成分，弃滤液；(d)抽吸洗脱液，流经过滤膜，将吸附在膜上的核酸成分洗脱，而得到不含有其他杂质的核酸水溶液，用于核酸扩增。
TWM477925U提供了一种样品萃取装置，包含吸取部件，所述吸取部件包含第一上部、连通于所述第一上部下方且内径小于所述第一上部的内径的中间部、及连通于所述中间部下方且内径小于所述中间部的内径的吸取部，由此所述第一上部或所述中间部配合抵接所述管柱部件的外周，且经由所述吸取部将特定液体吸至所述管柱部件；所述样品萃取装置还包含：管柱部件；及应接部件，由此自样品中萃取出预期材料。
TWM536238U提供了一种自动化萃取核酸的机台，包含：一机台底板，其上一托盘固 定架，所述托盘固定架可沿一水平轨道移动；一支架其垂直设置于所述机台底板上方，且其上设有一垂直轨道；一垂直运动单元，所述垂直运动单元包含一基板，所述基板上方设有一基板轨道，一运动块沿所述基板轨道设置，及一位于所述运动块下方并固锁于所述基板的一针筒固定单元供一针筒架设于其上；一活塞推进器固定单元供一活塞推进器架设，使所述运动块沿所述基板轨道上下移动时带动该活塞推进器而使所述针筒产生正压或负压，且所述基板沿所述垂直轨道设置而可带动所述运动块及所述针筒固定单元对应所述支架上下运动。
TW201412981A提供了一种抽取核酸的方法及装置，其利用气压式萃取核酸的方法，在纯化管上端与一可施以正或负气体压力装置接合，即可由纯化管下尖部吸取/排出检体、清洗液及冲提液，达到不需要离心机即可简便抽取核酸的效果。
虽已存在上述现有技术，但这些现有技术仍有无法大量萃取出cfDNA或ctDNA的缺点。因此，若能开发出一种能够大量萃取cfDNA或ctDNA以提高产量的自动化核酸萃取装置，将会对精准医疗带来突破。
发明内容
本发明的目的为提供一种自动化核酸萃取装置及自动化核酸萃取方法。相较于现有技术，本发明的自动化核酸萃取装置及方法可快速且方便地由检体中萃取出较高产量及较高浓度的核酸，例如cfDNA及ctDNA。
于是本发明提供一种自动化核酸萃取装置，该自动化核酸萃取装置包含一座体、一卡匣、一驱动单元、一移动架以及一针筒。座体具有一样品容置区、一管柱容置区、一卡匣容置区以及一收集管，且样品容置区、管柱容置区、卡匣容置区以及收集管是沿一线性方向排列。卡匣设置于卡匣容置区，卡匣包含二平行壁及至少二垂直壁，平行壁与垂直壁共同形成一溶解液槽、至少一洗涤液槽以及一洗提液槽，其中形成溶解液槽、洗涤液槽以及洗提液槽的垂直壁上分别设置有一抵靠部，且溶解液槽、洗涤液槽以及洗提液槽亦沿线性方向排列，其中抵靠部具有一圆弧状壁或一多边形壁。驱动单元设置于座体。移动架垂直设置于座体，且受到驱动单元的驱动而沿线性方向往复移动。针筒设置于移动架并随移动架移动。
在一实施例中，样品容置区具有一样品容置空间及一结合缓冲液容置空间。
在一实施例中，管柱容置区还包含一样品排吸管容置空间及一管柱排吸管容置空间。
在一实施例中，自动化核酸萃取装置还包含一样品排吸管，移动地设置于样品排吸管容置空间。
在一实施例中，自动化核酸萃取装置还包含一管柱排吸管，移动地设置于管柱排吸管容置空间。
在一实施例中，针筒与样品排吸管或管柱排吸管可拆卸地相接。
在一实施例中，样品容置空间是用于容置一生物样品以及溶解缓冲液以进行溶解反应，结合缓冲液容置空间是用于容置结合缓冲液以及溶解产物以进行结合反应，溶解液槽是用 于容置溶解缓冲液、消泡剂以及反应残余物，这些洗涤液槽是用于容置洗涤缓冲液，这些洗提液槽是用于容置洗提缓冲液，样品排吸管容置空间是用于容置样品排吸管，以及管柱排吸管容置空间是用于容置管柱排吸管。
在一实施例中，圆弧状壁的弧角大于或等于90度。
在一实施例中，在洗涤液槽与洗提液槽之间的垂直壁具有一完整的空心圆柱结构，形成一滤材容置空间，用以容置一滤材。
在一实施例中，多边形壁包含至少二支撑壁，这些支撑壁互相夹有一小于180度的夹角。
在一实施例中，洗提液槽的底部形成一凹部。
在一实施例中，卡匣还包含一弹扣件，用以将卡匣可拆卸地扣合于卡匣容置区。
本发明还提供一种自动化核酸萃取方法，该自动化核酸萃取方法应用如上所述的自动化核酸萃取装置，其包含下列步骤：以针筒搭配一管柱排吸管吸取一结合缓冲液容置空间中混合后的反应物，使反应物中的核酸结合至管柱排吸管中的一膜片，并将反应残余物排出至卡匣，使反应残余物中的核酸结合至膜片；以针筒搭配管柱排吸管通过膜片吸取至少一洗涤液槽中的洗涤缓冲液，再将洗涤缓冲液以针筒搭配管柱排吸管通过膜片排出；以及以针筒搭配管柱排吸管通过膜片吸取洗提液槽中的洗提缓冲液，再以针筒搭配管柱排吸管通过膜片将带有核酸的洗提缓冲液排至收集管。
在一实施例中，在自动化核酸萃取方法中，在将反应残余物排出至卡匣的步骤、以及将洗涤缓冲液以针筒搭配管柱排吸管通过膜片排出的步骤中，是将管柱排吸管的抵靠部抵靠在卡匣的抵靠部上，再将反应残余物、或洗涤缓冲液排出。
在一实施例中，在自动化核酸萃取方法中，在将管柱排吸管的抵靠部抵靠在卡匣的抵靠部上，再将反应残余物、或洗涤缓冲液排出的步骤之后，更包含下列步骤：将管柱排吸管的抵靠部抵靠在卡匣的抵靠部上，并将针筒依与线性方向垂直的方向上下移动小于或等于5毫米。
综上所述，本发明的功效在于：通过卡匣的平行壁及垂直壁的设置，使其形成的溶解液槽、洗涤液槽以及洗提液槽可容纳大体积的溶液；且通过洗涤液槽以及洗提液槽分别设置有抵靠部的设计，在针筒排出大体积的溶液之后，可避免样品排吸管或管柱排吸管从针筒脱落而与针筒分离。另外，通过卡匣以及容置空间沿线性方向排列以及移动架与针筒沿线性方向往复移动的设计，可达到在一线性方向自动进行核酸萃取的功效而避免检体污染且提高萃取效率，因此，本发明自动化核酸萃取装置确实能快速且方便地由检体中萃取出较高产量及较高浓度的核酸，例如cfDNA及ctDNA。再者，相较于现有技术，其是由卡匣以及容置空间进行线性移动(亦即相对于桌面，卡匣移动而针筒不动)，而本发明则是由移动架与针筒进行线性移动(亦即相对于桌面，针筒移动而卡匣不动)，因此本发明的自动化核酸萃取装置的作动空间较小，约为卡匣以及容置空间的长度的一倍，而现有技术由卡匣以及容置空间进行线性移动的作动空间则约为卡匣以及容置空间的长度的2至3倍。 因此，本发明自动化核酸萃取装置的体积较小，可节省使用者的使用空间。
附图说明
图1是本发明自动化核酸萃取装置的一较佳实施例的组合示意图。
图2是本发明自动化核酸萃取装置的一较佳实施例的侧视图。
图3A是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图3B是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图3C是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图4是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图5A是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图5B是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图5C是本发明自动化核酸萃取装置的一较佳实施例的局部示意图。
图6A是本发明自动化核酸萃取装置的卡匣的一较佳实施例的俯视图。
图6B是本发明自动化核酸萃取装置的卡匣的另一较佳实施例的俯视图。
图6C是本发明自动化核酸萃取装置的卡匣的又一较佳实施例的俯视图。
图6D至图6H是本发明自动化核酸萃取装置的卡匣的抵靠部的不同实施例的示意图。
图7A为图1的自动化核酸萃取装置的局部放大图。
图7B为图7A的自动化核酸萃取装置的动作示意图。
图7C为图7A的自动化核酸萃取装置的另一示意图。
图8A为图1A的自动化核酸萃取装置的部分示意图。
图8B为图8A的自动化核酸萃取装置的分解示意图。
图9是本发明自动化核酸萃取方法的一较佳实施例的流程图。
具体实施方式
以下将参照相关附图图式，说明依据本发明自动化核酸萃取装置的较佳实施例，其中相同的组件将以相同的参照符号加以说明。
本发明的自动化核酸萃取装置能快速且方便地由检体中萃取出较高产量及较高浓度的核酸，例如cfDNA及ctDNA。特别地，检体包含，但不限于：血液、血浆、尿液、唾液、组织液及组织。以下将以实施例来说明自动化核酸萃取装置的结构与特征。
请参照图1及图2，其中图1是本发明自动化核酸萃取装置的一较佳实施例的组合示意图，以及图2是本发明自动化核酸萃取装置的一较佳实施例的侧视图。
如图1及图2所示，自动化核酸萃取装置包含一座体B、一卡匣31、一驱动单元M、一移动架4以及一针筒5。
座体B具有一样品容置区1、一管柱容置区2，以及一卡匣容置区3，样品容置区1具有一样品容置空间11及一结合缓冲液容置空间12，且样品容置区1、管柱容置区2以及卡匣容置区3是沿一线性方向L1排列。特别地，样品容置区1、管柱容置区2以及卡匣容置区3沿线性方向L1(沿图面方向由右至左，下同)排列的顺序可以例如但不限于先样品容 置区1、管柱容置区2再卡匣容置区3；先管柱容置区2、卡匣容置区3再样品容置区1；先卡匣容置区3、样品容置区1再管柱容置区2；或者先卡匣容置区3、管柱容置区2再样品容置区1。
请参照图2并搭配图6A，卡匣31设置于卡匣容置区3，卡匣31包含二平行壁31a及至少二垂直壁31b，这些平行壁31a与这些垂直壁31b共同形成一溶解液槽311、至少一洗涤液槽312以及一洗提液槽313，其中形成溶解液槽311、洗涤液槽312以及洗提液槽313的这些垂直壁31b上分别设置有一抵靠部314，且溶解液槽311、洗涤液槽312以及洗提液槽313亦沿线性方向L1排列。在本实施例中，垂直壁31b的数目为9个，并与平行壁31a共同形成2个溶解液槽311、4个洗涤液槽312及2个洗提液槽313，然而垂直壁31b、溶解液槽311、洗涤液槽312及洗提液槽313的数目可依据使用者的实际需求而自行调整(举例而言，若样品为较难萃取的组织，溶解液槽311以及洗提液槽313可以增加)，本发明并无限制。另外，通过卡匣31的平行壁31a及垂直壁31b的设置，使其形成的溶解液槽311、洗涤液槽312以及洗提液槽313可容纳大体积的缓冲液(溶解缓冲液、洗涤缓冲液及/或洗提缓冲液)，例如但不限于2毫升以上、5毫升以上、或10毫升以上。
请再参照图1及图2，在本实施例中，驱动单元M设置于座体B，移动架4垂直设置于座体B，且受到驱动单元M的驱动而沿线性方向L1往复移动。针筒5设置于移动架4并随移动架4移动。特别地，图中虽显示驱动单元M为传动滚带，然而，驱动单元M亦可为其他驱动装置，例如直线模块或其他可驱动移动架4线性移动的装置，本发明并无限制。
请参照图1、2及图4，在本实施例中，管柱容置区2还包含一样品排吸管容置空间21及一管柱排吸管容置空间22。
请参照图4，在本实施例中，自动化核酸萃取装置还包含一样品排吸管6，移动地设置于样品排吸管容置空间21。在本实施例中，自动化核酸萃取装置还包含一管柱排吸管7，移动地设置于管柱排吸管容置空间22。特别地，针筒5与样品排吸管6或管柱排吸管7可拆卸地相接。
请参照图1至图3A，自动化核酸萃取装置还包含一收集管8，与卡匣31及这些容置区沿线性方向L1排列。在较佳的情况下收集管8可以是邻近于卡匣31设置(但本发明不以此为限)，因自动化核酸萃取方法是沿线性方向L1进行萃取，于最后步骤再将洗出物排出至收集管8中，故收集管8设置于邻近卡匣31且沿线性方向L1排列的设计，与移动架4移动的方向一致，可避免移动架4于自动化核酸萃取的过程中，往复通过收集管8的上方，而造成交叉污染的情形。
在本实施例中，样品容置空间11是用于容置生物样品(biological sample)以及溶解缓冲液(lysis buffer)以进行溶解反应(lysis reaction)。在本实施例中，生物样品包含，但不限于：血液、血浆、尿液、唾液、组织液及组织。结合缓冲液容置空间12是用于容置结合缓冲液(binding buffer)以及溶解产物(lysate)以进行结合反应(binding reaction)。溶解液槽311是用于容置溶解缓冲液(lysis buffer)、消泡剂(defoaming agent)以及反应残余 物。这些洗涤液槽312是用于容置洗涤缓冲液(wash buffer)。这些洗提液槽313是用于容置洗提缓冲液(elution buffer)。样品排吸管容置空间21是用于容置样品排吸管6。管柱排吸管容置空间22是用于容置管柱排吸管7。在本实施例中，样品容置空间11、结合缓冲液容置空间12、溶解液槽311以及洗涤液槽312可容纳例如但不限于30mL的检体或缓冲液，因此相较于现有技术(通常是2mL)具有较高的检体容量，溶解缓冲液体积增加可改善样品的溶解反应，以提高核酸的浓度；洗涤缓冲液的体积增加可将残留于管柱排吸管7溶液洗涤干净，以提高后续步骤萃取的核酸的浓度及纯度。在本实施例中，溶解液槽、洗涤液槽、洗提液槽的数目可依据使用者的实际需求而自行调整，本发明并无限制。
请参照图3C，管柱排吸管7的内部设置有一膜片74。膜片74的材质例如，但不限于硅胶膜片(silica membrane)，并带有正电，而检体在进行核酸萃取的过程中，其内含有的核酸可带有负电，利用正负电相吸的特性可使核酸附着于膜片74上，再透过后续的程序将附着于膜片74上的核酸萃取出来。
在本实施例中，座体B更可包含至少一加热件H，设置于样品容置空间11和/或管柱排吸管容置空间22下方，于此，当样品容置空间11下方设置有加热件H时，可促进生物样品以及溶解缓冲液之间的溶解反应，使生物样品能溶解的更完全，进而增加后续程序萃取出来的核酸浓度；当管柱排吸管容置空间22下方设置有加热件H时，可促进管柱排吸管7膜片74上残余溶剂的挥发，进而增加后续程序萃取出来的核酸浓度及纯度。特别地，当欲萃取的核酸为核糖核酸(RNA)时，样品容置空间11下方的加热件H可以不开启(亦即在进行溶解反应时不加热)，以避免核糖核酸被分解，而影响萃取后核糖核酸的浓度。
请参照图3A及图3B，这些洗提液槽313的底部形成一凹部f。由此设计，检体在进行洗提时可以使用更小量的洗提缓冲液，进而提高核酸萃取的浓度。特别地，图中虽显示仅邻近于收集管8的洗提液槽313具有凹部f，然而，其他洗提液槽313亦可具有凹部f，本发明并无限制。另外，洗提缓冲液的体积例如但不限于1毫升、500微升、200微升、100微升、50微升、30微升等，视使用者的需求可进行调整，本发明并无限制。
请参照图6A至图6C，卡匣31还包含一弹扣件316，用以将卡匣31可拆卸地扣合于卡匣容置区3。特别地，请参考图8A及8B，为了便于了解，图8A仅显示图1自动化核酸萃取装置中的部分结构，图8B为图8A的分解图。本实施例的自动化核酸萃取装置还可以包含一铁架I，铁架I可拆卸的设置于座体B上。多个卡匣31及收集管8可先由使用者设置到铁架I上，再连同铁架I一并设置到座体B上，以方便使用者设置多个卡匣31及收集管8。另外，使用者亦可以先将铁架I设置到座体B上，再设置卡匣31及收集管8，本发明并无限制。再者，使用自动化核酸萃取装置萃取核酸之后，可以将铁架I从座体B上取出，以方便使用者一次取出多个卡匣31及收集管8。特别地，铁架I可包含两把手I1，方便使用者将铁架I从座体B上取出或将铁架I放入座体B中。
请参照图4，样品排吸管6包含一装配部61以及一与装配部61相接的尖端部62。管柱排吸管7包含一装配部71、一抵靠部72以及一与抵靠部72相接的尖端部73，其中尖端 部73的长度D2是小于收集管8的深度D1(如图3C所示)。由此设计，当装配有核酸的尖端部73进入收集管8时，可防止带有核酸的检体在从尖端部73排出后所产生的力道太大而使检体从收集管8中飞溅出，以提高核酸萃取的产量。在本较佳实施例中，装配部61及尖端部62是一体成型；装配部71、抵靠部72以及尖端部73是一体成型。另择地，装配部61及尖端部62亦可彼此拆卸，装配部71、抵靠部72以及尖端部73亦可彼此拆卸，本发明并无限制。特别地，样品排吸管6可以与管柱排吸管7具有相同的构形，两者的差异仅在于管柱排吸管7具有膜片74，而样品排吸管6不具有膜片74(亦即，样品排吸管6亦可具有装配部、抵靠部以及尖端部，仅需使用一种模具即可制造样品排吸管6与管柱排吸管7，另通过取出或放入膜片74各自制成样品排吸管6与管柱排吸管7)。
请参照图3B至图5B，针筒5的底部还包含一接头51，其中针筒5通过接头51与装配部61、71连接。详细而言，接头51与样品排吸管6的装配部61或管柱排吸管7的装配部71的结构配合，用以将针筒5与装配部61、71紧密地相接。特别的，针筒5与接头51可以是一体成型的。
请参照图1及图7A至图7B，图7A为图1中A区域的局放大图。移动架4包含一退料板41，且移动架4两侧分别设置有一弹簧机构42，弹簧机构42带动退料板41作动以控制装配部61、71从针筒5的底部拆卸。详细而言，如图7B所示，弹簧机构42沿L3方向移动，带动退料板41往L4方向移动，以将样品排吸管6或管柱排吸管7与针筒5分离。
请参照图7C并搭配图7A，移动架4更可包含至少一针筒固定件43，以将针筒5固定在移动架4上。针筒固定件43更可包含一针筒卡固槽431以及至少一凹孔432，凹孔432中可设置有弹性件R。如图7A所示，针筒5卡固在两个针筒固定件43的针筒卡固槽431中，并透过设置于凹孔432中的弹性件R，将针筒5更好的固定在针筒卡固槽431。特别地，针筒固定件43、凹孔432及弹性件R的数目可以依据使用者的需求进行调整，只要能将针筒5稳固的固定在针筒卡固槽431中即可，本发明并无限制。
请参照图1、图5A、图5B及图6A，其中图5A及图5B是本发明自动化核酸萃取装置的一较佳实施例的局部示意图，图6A是本发明自动化核酸萃取装置的卡匣的一较佳实施例的俯视图。如图5A、图5B及图6A所示，抵靠部314具有一圆弧状壁3141且圆弧状壁3141的弧角θ大于或等于90度。较佳的，弧角θ可以为90度；较佳的，弧角θ可以为120度；较佳的，弧角θ可以为180度；较佳的，弧角θ可以为270度，只要能供管柱排吸管7抵靠即可。特别地，如图6A所示，垂直壁31b具有圆弧状壁3141，由垂直壁31b与平行壁31a相接，亦即，圆弧状壁3141的两端不直接与平行壁31a相接。
请参照图1、图5C及图6C，其中图5C是本发明自动化核酸萃取装置的一较佳实施例的局部示意图，图6C是本发明自动化核酸萃取装置的卡匣的又一较佳实施例的俯视图。如图5C、图6C所示，抵靠部314具有多边形壁，多边形壁可包含至少两个支撑壁，于此先以包含一第一支撑壁3142及一第二支撑壁3143的结构用于说明，第一支撑壁3142与第二支撑壁3143夹有一小于180度的夹角e。较佳的，夹角e可以为150度；较佳的，夹角e 可以为120度；较佳的，夹角e可以为90度；较佳的，夹角e可以为45度，只要能供管柱排吸管7抵靠并站立即可。特别地，请参照图6D至图6H，用以说明本发明自动化核酸萃取装置的卡匣的抵靠部的不同实施态样。抵靠部314例如但不限于圆弧状壁3141(如图6E所示)或多边形壁(如图6D、图6F至图6H)，多边形壁例如但不限于具有两个支撑壁、三个支撑壁、四个支撑壁、五个支撑壁、六个支撑壁或大于六个支撑壁的结构，各个支撑壁之间具有夹角，支撑壁的数目以及夹角的角度不限制，只要能供管柱排吸管7抵靠及站立即可。特别地，如图6D所示，抵靠部314的多边形壁的两端可直接与平行壁31a相接，然而，如图6F至6H所示，垂直壁31b的抵靠部314的多边形壁的两端不直接与平行壁31a相接，而是由垂直壁31b与平行壁31a相接，于此不作限制。
通过上述抵靠部314的设计，在排出液体(例如反应物、洗涤缓冲液等)的过程中，管柱排吸管7的抵靠部72抵靠在抵靠部314的圆弧状壁3141或是第一支撑壁3142及第二支撑壁3143之间，以使得针筒5在排出液体时，管柱排吸管7不会因压力过大而从针筒5脱落而分离。即使管柱排吸管7于针筒5在排出液体后于针筒5上升时松开，可通过抵靠部314的设计，使管柱排吸管7的抵靠部72抵靠并站立于抵靠部314上，而不会与针筒5完全分离，接着可通过后续使针筒5依与线性方向L1垂直的方向L2上下移动的步骤来将针筒5与管柱排吸管7再次紧密配合，以利后续萃取步骤的进行。
请参照图1、图6A及图6B，其中图6B是本发明自动化核酸萃取装置的卡匣的另一较佳实施例的俯视图。图6B的卡匣，在洗涤液槽312与洗提液槽313之间的垂直壁31b具有一完整的空心圆柱结构，形成一滤材容置空间315，用以容置一滤材。详细而言，当生物样品为组织或其他具有杂质的样品时，滤材用以过滤溶解后的溶解产物，以将组织碎片或杂质卡固在滤材外侧，澄清(不含组织碎片或杂质)的溶解产物则可吸入样品排吸管6中，避免组织碎片或杂质塞住样品排吸管6的尖端部62。由此设计，组织样品可直接上机，不需在机器外先利用离心机离心，取得澄清的溶解产物再上机。故组织样品裂解的步骤可于本发明的自动化核酸萃取装置上完成，让组织样品也可达到全自动核酸纯化的功效。特别地，欲萃取检体中的核糖核酸(RNA)时，滤材容置空间315更可容置萃取流程中间所需的酶，例如是脱氧核糖核酸分解酶(DNase)，以在萃取的过程中去除样品中的脱氧核糖核酸(DNA)，而该脱氧核糖核酸分解酶(DNase)亦可被后续的洗涤步骤冲洗去除，故萃取完成后即可得到不含脱氧核糖核酸(DNA)的核糖核酸(RNA)。
请参照图1及图2，本实验例由移动架4与针筒5进行线性移动L1(亦即相对于桌面，针筒移动而卡匣不动)，因此机器的深度仅为座体B的长度D3再加上移动架4的厚度D4(D3+D4)。然而，现有技术是由座体B进行线性移动，为了使针筒能够吸取到各个容置空间中的溶液，机器的深度必须至少为座体B的长度D3的2倍再加上移动架4的厚度D4(D3×2+D4)。举例而言，本实验例的自动化核酸萃取装置座体B的长度D3例如但不限于30公分、移动架4的厚度D4例如但不限于20公分，机器的深度例如但不限于约为50公分，因此能够节省使用者的使用空间。
请参照图9并搭配图1至3C，其为本发明自动化核酸萃取方法的较佳实施例，本发明的自动化核酸萃取方法是应用如前所述的自动化核酸萃取装置进行的，自动化核酸萃取装置的底部设置有传动滚带，其会带动移动架4沿线性方向L1移动。在本较佳实施例中，自动化核酸萃取方法包含下列步骤：步骤S05以针筒5搭配管柱排吸管7吸取结合缓冲液容置空间中混合后的反应物，使反应物中的核酸结合至管柱排吸管7中的一膜片74，并将反应残余物排出至卡匣31，使反应残余物中的核酸结合至膜片74。继而进行步骤S06，以针筒5搭配管柱排吸管7通过膜片74吸取至少一洗涤液槽中的洗涤缓冲液，再将洗涤缓冲液以针筒5搭配管柱排吸管7通过膜片74排出。接着进行步骤S08以针筒5搭配管柱排吸管7通过膜片74吸取洗提液槽中的洗提缓冲液，再以针筒5搭配管柱排吸管7通过膜片74将带有核酸的洗提缓冲液排至收集管8。其中，步骤S06与S08之间可以安排一个步骤S07，将管柱排吸管7移动至管柱排吸管容置空间22加热，以尽量降低洗涤缓冲液在膜片74上的残留量。上述步骤S05至步骤S08是由自动化核酸萃取装置进行的，图9中所示的虚线部分(步骤S01至步骤S04)，可以由使用者在自动化核酸萃取装置外手动进行，或是全部由自动化核酸萃取装置进行。举例而言，使用者可以先将样品溶解离心后，再将上清液与结合缓冲液混合后再加入结合缓冲液容置空间，由自动化核酸萃取装置进行后续步骤；或者，使用者可以先将样品溶解离心后，再将上清液加入结合缓冲液容置空间与结合缓冲液混合，由自动化核酸萃取装置进行后续步骤；或者，使用者可以先将样品溶解离心后，将溶解产物加入溶解液槽中，由自动化核酸萃取装置进行后续步骤，本发明并无限制。
本实施例用以说明步骤S01至步骤S08皆由自动化核酸萃取装置进行的情况，在步骤S01至步骤S08中，自动化核酸萃取装置与移动架4(带动针筒5)沿线性方向L1移动至特定位置进行前述步骤，详细描述如下：在步骤S01之前将设置于移动架4的针筒5依线性方向L1移动至管柱容置区2的样品排吸管容置空间21，使针筒5与容置于样品排吸管容置空间21中的样品排吸管6相接而将样品排吸管6由样品排吸管容置空间21取出。接着，与针筒5相接的样品排吸管6沿线性方向L1移动至卡匣容置区3的卡匣31。然后进行步骤S01，从卡匣31的溶解液槽311中吸取溶解缓冲液，接着移动至样品容置区1。接下来进行步骤S02将溶解缓冲液排出至样品容置区1的样品容置空间11并进行混合。之后进行步骤S03，吸取样品容置空间11中混合后的溶解产物，并移动至样品容置区1的结合缓冲液容置空间12。然后进行步骤S04，将溶解产物排出至结合缓冲液容置空间12并与结合缓冲液混合以进行结合反应。接着在进行步骤S05之前，将针筒5及样品排吸管6移动至样品排吸管容置空间21，通过弹簧机构42带动退料板41作动以使样品排吸管6从针筒5拆卸至样品排吸管容置空间21，再将针筒5沿线性方向L1移动至管柱容置区2的管柱排吸管容置空间22，使针筒5与设置于管柱排吸管容置空间22的管柱排吸管7相接而将内部设置有膜片74的管柱排吸管7由管柱容置区2取出。之后，与针筒5相接的管柱排吸管7沿线性方向L1移动至结合缓冲液容置空间12。接着进行步骤S05，以针筒5搭配管柱排吸管7吸取所形成的反应物，以使得反应物通过膜片74而反应物所含有的核酸附着于膜片74。接着， 针筒5沿线性方向L1移动至卡匣31。然后使反应物沿重力方向再次通过膜片74而使内含的核酸附着于膜片74，至于反应残余物会沿重力方向排出至卡匣31，使反应残余物通过膜片两次以确保反应物中的核酸能附着于膜片74上。接着，进行步骤S06，带有核酸的管柱排吸管7随针筒5及移动架4沿线性方向L1移动至装有洗涤缓冲液的洗涤液槽312继而通过吸取及排出洗涤缓冲液以进行洗涤，使洗涤缓冲液通过膜片74两次，以将残留在膜片74的反应残余物洗涤干净。在洗涤之后，可先将带有核酸的管柱排吸管7随针筒5及移动架4沿线性方向L1移动至管柱排吸管容置空间22，将管柱排吸管7停留在管柱排吸管容置空间22中，通过加热件H加热，以使管柱排吸管7的膜片74上残留的液体(例如洗涤缓冲液)挥发，使其充分干燥(即步骤S07)。最后再将带有核酸的管柱排吸管7随针筒5及移动架4沿线性方向L1移动至装有洗提缓冲液的洗提液槽313并进行步骤S08，通过吸取及排出洗提缓冲液以进行洗提，由此而得到洗出物(eluate，带有核酸的洗提缓冲液)。之后，管柱排吸管7吸取所得到的洗出物，接而沿线性方向L1移动至收集管8，然后将洗出物排至收集管8。特别地，加热件H加热的时间例如但不限于30秒、1分钟、3分钟、5分钟，只要使管柱排吸管7的膜片74上残留的液体挥发并充分干燥即可，本发明并无限制。
在本较佳实施例中，在将反应残余物排出至卡匣31的步骤(步骤S05)、以及将洗涤缓冲液以针筒5搭配管柱排吸管7通过膜片74排出的步骤中(步骤S06)，是将管柱排吸管7的抵靠部72抵靠在卡匣31的抵靠部314上，再将反应残余物、或洗涤缓冲液排出。通过抵靠部314的设计，使得针筒5在排出液体时，管柱排吸管7是抵靠在抵靠部314上，由此使管柱排吸管7于排出液体的过程中不会因压力过大而从针筒5脱落而分离。即使管柱排吸管7于针筒5在排出液体后于上升时松开，可通过抵靠部314的设计，使管柱排吸管7的抵靠部72抵靠并站立于抵靠部314上，而不会与针筒5完全分离，可通过后续步骤将针筒5与管柱排吸管7再次紧密配合。
在本较佳实施例中，在将管柱排吸管7的抵靠部72抵靠在卡匣31的抵靠部314上，再将反应残余物、或洗涤缓冲液排出的步骤(步骤S05、S06)之后，更包含下列步骤：将管柱排吸管7的抵靠部72抵靠在卡匣31的抵靠部314上，并将针筒5依与线性方向L1垂直的方向L2上下移动小于或等于5毫米。通过此步骤，可将管柱排吸管7与针筒5紧密地相接，避免管柱排吸管7松脱掉落。较佳的，针筒5可沿方向L2上下移动5毫米；较佳的，针筒5可沿方向L2上下移动4.5毫米；较佳的，针筒5可沿方向L2上下移动3毫米，以将管柱排吸管7与针筒5再次紧密地相接。
在本较佳实施例中，洗涤液槽312的数目可依据使用者的实际需求而自行调整，本发明并无限制。详细而言，若洗涤液槽312的数目大于一个，在将管柱排吸管7移动至管柱排吸管容置空间22加热之前，可重复至少一次以针筒5搭配管柱排吸管7通过膜片74吸取至少一洗涤液槽中的洗涤缓冲液，再将洗涤缓冲液以针筒5搭配管柱排吸管7通过膜片74排出的步骤。亦即，带有核酸的管柱排吸管7随针筒5及移动架4沿线性方向L1移动至装有洗涤缓冲液的洗涤液槽312并通过反复吸取及排出洗涤缓冲液以进行洗涤的步骤可在 不同的洗涤液槽312中进行，洗涤步骤的次数依据洗涤液槽312的数目决定。
在本较佳实施例中，在将溶解缓冲液排出至样品容置空间11并进行混合的步骤(步骤S02)，以及吸取样品容置空间11中混合后的溶解产物的步骤(步骤S03)之间，更包含移动至滤材容置空间315，将针筒5搭配的样品排吸管6与滤材容置空间315中的滤材组合的步骤；以及在吸取样品容置空间11中混合后的溶解产物的步骤(步骤S03)之后，更包含将样品排吸管6与滤材移动至溶解液槽311并在溶解液槽311中沿线性方向往复移动的步骤。详细而言，当生物样品为组织或其他具有杂质的样品时，滤材用以过滤溶解后的溶解产物，以将组织碎片或杂质卡固在滤材外侧，再进行步骤S03，将澄清(不含组织碎片或杂质)的溶解产物吸取至针筒5中。随后将样品排吸管6与滤材移动至溶解液槽311并在溶解液槽311中沿线性方向往复移动，以将卡固在滤材外侧的杂质清洗干净后，再将针筒5中的溶解产物排出至结合缓冲液容置空间12以进行结合反应，避免组织碎片或杂质影响结合反应的效果进而影响萃取效率。亦即，当生物样品为组织或其他具有杂质的样品时可增设前述步骤。
在本较佳实施例中，样品容置空间11、结合缓冲液容置空间12、样品排吸管容置空间21、管柱排吸管容置空间22、溶解液槽311、洗涤液槽312、洗提液槽313、抵靠部314、针筒5、样品排吸管6、管柱排吸管7、收集管8及接头51的数目可依据使用者的实际需求而自行调整，本发明并无限制。特别地，卡匣31、样品容置空间11、结合缓冲液容置空间12、样品排吸管容置空间21、管柱排吸管容置空间22、针筒5及收集管8是沿线性方向L1排列，排列的顺序于此不作限制。
综上所述，本发明自动化核酸萃取装置通过各个溶解液槽311、洗涤液槽312以及相邻的洗提液槽313分别设置有抵靠部314的设计，使用者在进行自动化核酸萃取时可避免反应物自溶解液槽311、洗涤液槽312或洗提液槽313溅出，亦可避免管柱排吸管7松脱掉落。另外，通过卡匣31以及容置空间沿线性方向L1排列以及移动架4与针筒5沿线性方向L1反复移动的设计，可达到在一线性方向L1自动进行核酸萃取的功效而避免检体污染且提高萃取效率。因此，本发明自动化核酸萃取装置确实能快速且方便地由检体中萃取出较高产量及较高浓度的核酸，例如cfDNA及ctDNA。
以上所述仅为举例性，而非为限制性。任何未脱离本发明的精神与范畴，而对其进行的等效修改或变更，均应包含于权利要求书中。

Claims (10)

  1. 一种自动化核酸萃取装置，其特征在于，所述自动化核酸萃取装置包含：
    一座体，所述座体具有一样品容置区、一管柱容置区、一卡匣容置区，以及一收集管，且所述样品容置区、所述管柱容置区、所述卡匣容置区以及所述收集管是沿一线性方向排列；
    一卡匣，设置于所述卡匣容置区，所述卡匣包含二平行壁及至少二垂直壁，这些平行壁与这些垂直壁共同形成一溶解液槽、至少一洗涤液槽以及一洗提液槽，其中形成所述溶解液槽、所述洗涤液槽以及所述洗提液槽的这些垂直壁上分别设置有一抵靠部，且所述溶解液槽、所述洗涤液槽以及所述洗提液槽亦沿所述线性方向排列，其中所述抵靠部具有一圆弧状壁或一多边形壁；
    一驱动单元，设置于所述座体；
    一移动架，垂直设置于所述座体，且受到所述驱动单元的驱动而沿所述线性方向往复移动；
    以及一针筒，设置于所述移动架并随所述移动架移动。
  2. 如权利要求1所述的自动化核酸萃取装置，其特征在于，所述样品容置区具有一样品容置空间及一结合缓冲液容置空间。
  3. 如权利要求1所述的自动化核酸萃取装置，其特征在于，所述管柱容置区还包含一样品排吸管容置空间及一管柱排吸管容置空间，而所述自动化核酸萃取装置还包含一样品排吸管及一管柱排吸管，且所述样品排吸管及所述管柱排吸管移动地设置于所述样品排吸管容置空间及所述管柱排吸管容置空间。
  4. 如权利要求3所述的自动化核酸萃取装置，其特征在于，所述针筒与所述样品排吸管或所述管柱排吸管可拆卸地相接。
  5. 如权利要求1所述的自动化核酸萃取装置，其特征在于，当所述抵靠部具有所述圆弧状壁时所述圆弧状壁的弧角大于或等于90度；当所述抵靠部具有所述多边形壁时所述多边形壁包含至少二支撑壁，这些支撑壁互相夹有一小于180度的夹角。
  6. 如权利要求1所述的自动化核酸萃取装置，其特征在于，在所述洗涤液槽与所述洗提液槽之间的所述垂直壁具有一完整的空心圆柱结构，形成一滤材容置空间。
  7. 如权利要求1所述的自动化核酸萃取装置，其特征在于，所述洗提液槽的底部形成一凹部，且所述卡匣还包含一弹扣件。
  8. 一种自动化核酸萃取方法，应用于如权利要求1至7中任一项所述的自动化核酸萃取装置，其特征在于，所述自动化核酸萃取方法包含下列步骤：
    以所述针筒搭配一管柱排吸管吸取一结合缓冲液容置空间中混合后的反应物，使反应物中的核酸结合至所述管柱排吸管中的一膜片，并将反应残余物排出至所述卡匣，使反应残余物中的核酸结合至所述膜片；
    以所述针筒搭配所述管柱排吸管通过所述膜片吸取所述至少一洗涤液槽中的洗涤缓冲 液，再将洗涤缓冲液以所述针筒搭配所述管柱排吸管通过所述膜片排出；
    以及以所述针筒搭配所述管柱排吸管通过所述膜片吸取所述洗提液槽中的洗提缓冲液，再以所述针筒搭配所述管柱排吸管通过所述膜片将带有核酸的洗提缓冲液排至所述收集管。
  9. 如权利要求8所述的自动化核酸萃取方法，其特征在于，在将反应残余物排出至所述卡匣的步骤、以及将洗涤缓冲液以所述针筒搭配所述管柱排吸管通过所述膜片排出的步骤中，是将所述管柱排吸管的所述抵靠部抵靠在所述卡匣的所述抵靠部上，再将反应残余物、或洗涤缓冲液排出。
  10. 如权利要求9所述的自动化核酸萃取方法，其特征在于，在将所述管柱排吸管的所述抵靠部抵靠在所述卡匣的抵靠部上，再将反应残余物、或洗涤缓冲液排出的步骤之后，还包含下列步骤：
    将所述管柱排吸管的所述抵靠部抵靠在所述卡匣的所述抵靠部上，并将所述针筒依与所述线性方向垂直的方向上下移动小于或等于5毫米。
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