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WO2020078410A1 - Dispositif et procédé de traitement d'échantillon, et système de pcr numérique comprenant un dispositif de traitement - Google Patents

Dispositif et procédé de traitement d'échantillon, et système de pcr numérique comprenant un dispositif de traitement Download PDF

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Publication number
WO2020078410A1
WO2020078410A1 PCT/CN2019/111626 CN2019111626W WO2020078410A1 WO 2020078410 A1 WO2020078410 A1 WO 2020078410A1 CN 2019111626 W CN2019111626 W CN 2019111626W WO 2020078410 A1 WO2020078410 A1 WO 2020078410A1
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Prior art keywords
sample
extraction
sample processing
cavity
chamber
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PCT/CN2019/111626
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English (en)
Chinese (zh)
Inventor
李昂
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Beijing Zhiyu Biotechnology Ltd
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Beijing Zhiyu Biotechnology Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • 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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the invention relates to the technical field of molecular detection, in particular to a sample processing device and method suitable for digital PCR detection, and a digital PCR system including the sample processing device, especially a droplet-type digital PCR system.
  • molecular detection technologies mainly include nucleic acid molecule hybridization, polymerase chain reaction (PCR) and biochip technology.
  • Molecular testing products are mainly used in the detection of tumors, infections, genetics, prenatal screening and other clinical departments, as well as physical examination centers, technical service centers, third-party testing institutions and microbiological rapid testing markets.
  • PCR technology can qualitatively and quantitatively detect target nucleic acid molecules.
  • digital PCR is an absolute quantitative technology of nucleic acid molecules. It distributes a fluorescent quantitative PCR reaction system to a large number of tiny reactors, each microreactor contains one or more copies of the target nucleic acid molecule, and performs "single molecule template PCR amplification". By counting the number of positive reaction units and statistical methods to calculate the copy number of the target gene in the original sample, digital PCR can perform accurate absolute quantitative detection without relying on the control and standard curve.
  • One of the objects of the present invention is to provide a compact, modular, automated sample processing device and an efficient and convenient sample processing method.
  • the invention also provides a digital PCR system integrating sample processing, droplet generation, amplification and detection.
  • the system has a compact structure, small equipment volume, simple setting and convenient use.
  • the invention also provides an efficient and convenient digital PCR detection method.
  • a sample processing consumable includes a substrate, one or more processing units provided on the substrate, each processing unit includes a plurality of extraction chambers, a sample chamber provided on the substrate, and an extraction chamber provided on the sample chamber
  • a transfer channel for connecting the two, a reagent consumable with a reagent storage cavity, a plurality of extraction chambers are connected through a micro channel at the bottom, and a reagent storage cavity and a sample cavity are connected through a micro channel.
  • each microchannel is preferably set as a channel where liquid does not move from one cavity to another cavity when there is no external force driving or when the driving force is lower than a set value.
  • the pore diameter of the microchannel is, for example, about 60 ⁇ m to 100 ⁇ m, preferably 70 to 100 ⁇ m.
  • the openings of the extraction cavity, the sample cavity and the reagent storage cavity are respectively arranged upward.
  • the sample processing consumable further includes a valve provided at the opening of the extraction chamber, the valve is used to close the extraction chamber or to connect the extraction chamber to the atmosphere or external components other than the extraction chamber.
  • the valve may specifically be a two-way valve, for example.
  • a heat-conducting element and / or a magnetic-permeable element are provided on the substrate corresponding to one or more extraction chambers.
  • the heat conduction element and the magnetic conduction element are provided at the same time.
  • the above-mentioned heat-conducting element can be matched with a heating coil and the like to improve the heating effect and rate of the liquid in the extraction chamber.
  • the heat-conducting element is preferably of elongated shape, which is located at least partially in the extraction chamber and transverse to the axis of the extraction chamber.
  • the heat conduction element provided can also have the effect of turbulent flow, which is beneficial to the mixing of the liquid in the extraction chamber.
  • the above-mentioned magnetic conductive element can be in contact with the magnet for conduction.
  • magnetic beads can be used in combination to achieve nucleic acid separation.
  • the magnetically permeable element has an elongated shape, which is at least partially located in the extraction cavity and transverse to the axis of the extraction cavity.
  • the provided magnetic conductive element can also play a turbulent effect, which is conducive to the mixing of the liquid in the extraction chamber.
  • the sample processing consumable further comprises a spoiler rod at least partially located in the sample cavity and disposed transverse to the axis of the sample cavity.
  • a plurality of extraction chambers are distributed at intervals, the axis lines of the extraction chamber and the sample chamber respectively extend along the height direction of the substrate, and the axis line of the transfer channel is transverse to the axis of the extraction chamber and the sample chamber Heart line.
  • a plurality of extraction chambers of each processing unit are sequentially arranged along the length direction of the substrate, and both ends of the transfer channel communicate with the sample chamber and the extraction chamber closest to the sample chamber, respectively.
  • the transfer channel includes a first channel and a second channel communicating at one end, wherein the inner diameter of the first channel is larger than the second channel, the other end of the first channel communicates with the extraction chamber, and the other end of the second channel The part communicates with the sample chamber.
  • the base material includes a first base material portion formed with a plurality of extraction chambers, a second base material portion formed with a sample chamber, and both ends are respectively connected to the first base material portion and the second base material portion
  • the transfer channel includes a first channel extending inside the elongated connecting portion along the length of the elongated connecting portion.
  • the second channel is formed on the second substrate portion.
  • the reagent consumable and the substrate are detachably connected.
  • the substrate is provided with an interface
  • the reagent consumable includes a housing, a U-shaped reagent tube provided in the housing, and a pair of mating plugs provided on the housing and the interface of the substrate A plug, a reagent storage cavity formed by the inner cavity of the reagent tube, two ends of the reagent tube are respectively connected with micro tubes, and the two micro tubes are respectively arranged through a pair of plugs.
  • the two micro tubes The inner cavity of one constitutes a microchannel that connects the inner cavity of the reagent tube with the bottom of the sample cavity, and the other is used to connect the driving module or the atmosphere, and the driving module is a device capable of driving liquid flow.
  • the sample processing consumable further includes one or more reagents required for sample processing, and the reagents are encapsulated in a cavity selected from a plurality of extraction chambers and reagent storage chambers.
  • the sample processing consumables are PCR detection sample processing consumables.
  • Each processing unit includes more than 6 extraction chambers.
  • the sample processing consumables further include a valve provided at the opening of the extraction chamber. The valve is used to close the extraction chamber Or, the extraction chamber is communicated with external components other than the atmosphere or the extraction chamber.
  • the base material corresponding to one or more extraction chambers is provided with a heat conduction element and a selective magnetic conduction element.
  • a magnetically conductive element is provided.
  • a heat-conducting element is provided on the substrate corresponding to one of the extraction chambers, and a magnetic-conducting element is provided on the substrate corresponding to the other adjacent extraction chamber.
  • the magnetic elements are arranged transversely to the axis of the extraction cavity and each have exposed contacts.
  • the sample processing consumables further include substances required for nucleic acid extraction.
  • the substances required for nucleic acid extraction include washing solution, cell lysate, enzymes, eluent, and magnetic beads.
  • the liquid and the magnetic beads are respectively encapsulated in different extraction chambers, and the cell lysate and the enzyme are encapsulated in the same extraction chamber or in different extraction chambers.
  • a sample processing device including one or more of the above-mentioned sample processing consumables of the present invention, a driving module connected to the sample processing consumables, and a control module, the driving module is used to drive the liquid
  • the sample processing consumables flow in each cavity or channel, and the drive module is connected to and controlled by the control module.
  • the sample processing device further includes a heating module, the heating module includes a heating coil, and the heating module is connected to and controlled by the control module.
  • the heating module is preferably movably arranged.
  • the sample processing device further includes a magnet provided outside the extraction chamber. Using magnets and magnetic beads, nucleic acid separation can be achieved.
  • the magnet is preferably movably arranged.
  • the sample processing apparatus further includes a sliding base that is slidably disposed relative to the base material, and the heating coil and / or the magnet are disposed on the sliding base. Further, the sliding direction of the sliding base may be an up-down direction.
  • the arrangement of the driving module is not particularly limited, and the pneumatic driving method is preferably used.
  • the drive module includes a valve, an air pump, a pressure sensor, and a gas line that are sealingly connected to the opening of each cavity.
  • the gas line connects the air pump, the pressure sensor, the valve, and the extraction cavity, and the pressure sensor and the control module signal connection.
  • the invention also provides a digital PCR system, which includes a base, a moving mechanism provided on the base, an operating platform provided on the base, a droplet generating device with a sampling needle, a nucleic acid amplification temperature control device, and a product
  • the signal collection device and the control device, the moving mechanism, the droplet generation device, the nucleic acid amplification temperature control device, and the product signal collection device are respectively connected to the control device and controlled by the control device.
  • the digital PCR system also includes the aforementioned sample processing device of the present invention. The sample processing consumables of the sample processing device are set on the operating platform.
  • the driving module of the sample processing device is connected to the moving mechanism and moves under the driving of the moving mechanism.
  • multiple extraction chambers in each processing unit are arranged side by side along the length direction of the digital PCR system, and the driving modules of the droplet generation device and the sample processing device are arranged along the width direction of the digital PCR system cloth.
  • the moving mechanism has an upright fixed block
  • the driving module further includes a connecting seat for installing the gas sensor and the valve
  • the connecting seat can be set to slide up and down
  • the digital PCR system further includes The first longitudinal moving device that slides up and down on the connection base.
  • the present invention also provides a method for processing a sample for digital PCR detection, including a nucleic acid extraction step of extracting nucleic acid from a sample to be tested, and a mixing step of mixing the nucleic acid solution obtained by the extraction step with other reagents to prepare the test sample
  • a nucleic acid extraction step of extracting nucleic acid from a sample to be tested
  • a mixing step of mixing the nucleic acid solution obtained by the extraction step with other reagents to prepare the test sample
  • the aforementioned sample processing device of the present invention is used to perform the extraction step and the mixing step, specifically including the following steps:
  • Materials required for nucleic acid extraction include cleaning solution, cell lysate, enzymes, eluent, magnetic beads, cleaning solution, washing The deliquoring and magnetic beads are located in different extraction chambers respectively, and the cell lysate and enzymes are in the same extraction chamber or different extraction chambers, where the eluent is stored in the extraction chamber connected to the transfer channel;
  • Nucleic acid elution The elution solution is sent to the extraction chamber where the magnetic beads are located to elute the nucleic acid. After elution, the eluted nucleic acid solution is returned to the original extraction chamber;
  • the delivery of various liquids is driven by the positive or negative pressure realized by the drive module.
  • a sample for digital PCR detection that is, an aqueous solution for nucleic acid analysis
  • a nucleic acid amplification reaction solution which constitutes the aqueous phase of digital PCR droplets.
  • Nucleic acid amplification reaction solution can be of different types, such as nucleic acid amplification reaction solution using deoxyribonucleic acid (DNA) as a template (may be referred to as DNA amplification reaction solution) or ribonucleic acid (RNA) as a template
  • the reverse transcription nucleic acid amplification reaction solution (which may be referred to as RNA reverse transcription reaction solution) may also be other nucleic acid amplification reaction solutions, such as a loop-mediated isothermal amplification (LAMP) reaction solution.
  • LAMP loop-mediated isothermal amplification
  • a typical DNA amplification reaction solution contains the nucleic acid (template) to be detected, a buffered aqueous solution, deoxyribonucleoside triphosphate (dNTP), primers, polymerase, and product labeling substance such as fluorescent dye or fluorescent probe.
  • template nucleic acid
  • dNTP deoxyribonucleoside triphosphate
  • primers primers
  • polymerase polymerase
  • product labeling substance such as fluorescent dye or fluorescent probe.
  • the substances required for nucleic acid extraction vary according to different nucleic acid extraction methods, and those skilled in the art can select appropriate components according to actual needs, such as cleaning solution, cell lysate, Enzymes, eluents, magnetic beads, etc. These substances are known, and common enzymes such as lysozyme, proteinase K and so on.
  • step (1) there are two or more other reagents in the reagent storage cavity.
  • step (7) after the mixing is completed, the solution in the sample chamber is layered, the upper layer is the sample solution, and the lower layer is the isolation oil.
  • the present invention also relates to a digital PCR detection method, which uses the above-mentioned digital PCR system of the present invention, and the detection method includes sequential steps of sample processing, droplet generation, amplification reaction, and signal collection and processing, wherein the sample processing takes The above-mentioned sample processing method of the present invention; after obtaining the sample, the sampling needle of the droplet generating device is inserted into the sample cavity to suck the sample.
  • the present invention also relates to another digital PCR detection method, which uses the above-mentioned digital PCR system of the present invention, and the detection method includes sequential steps of sample processing, droplet generation, amplification reaction, and signal collection and processing, wherein the sample processing
  • the above sample processing method of the present invention is adopted; after obtaining the sample, the sampling needle of the droplet generating device is inserted into the isolation oil through the sample solution, and suction is performed until the sample is completely removed or continues to be drawn after the sample solution is drawn Partially isolate oil.
  • This sampling method can avoid sample residue and loss.
  • a component when a component is described to be transverse to another component, it means that the two components are intersecting, and a typical situation includes the two components being perpendicular.
  • the present invention has the following advantages compared with the prior art:
  • the sample processing device of the present invention can have various functions such as nucleic acid extraction and reagent mixing, and is suitable for obtaining samples directly used for digital PCR detection.
  • the device is simple, compact, small in size, modular in design and suitable for Build an integrated droplet digital PCR system.
  • the sample processing method of the present invention reduces the complexity of user operations, improves work efficiency, shortens the processing cycle, and reduces operating errors.
  • the digital PCR system of the present invention integrates sample processing, droplet formation, amplification, and detection, and realizes automatic control from sample processing to droplet generation to PCR reaction to result detection.
  • the structure design is reasonable and the overall volume of the system is reduced. At the same time, it reduces the complexity of user operations, improves work efficiency, shortens the detection cycle, and reduces operating errors.
  • FIG. 1 is a perspective schematic view of the sample processing device of Embodiment 1 (the pumps and sensors in the drawings are only schematic illustrations);
  • Example 2 is a schematic top view of the sample processing device of Example 1;
  • FIG. 3 is a schematic cross-sectional view at A-A in FIG. 2;
  • Example 4 is a perspective schematic view of the reagent consumables of Example 1;
  • FIG. 5 is a perspective schematic view of the sample processing device of Embodiment 2.
  • Example 6 is a schematic perspective view of the valve base of Example 2.
  • Example 7 is a schematic perspective view of the digital PCR system of Example 3.
  • Drop generation mechanism 300, sampling needle; 310, drop container; 700, first longitudinal movement device; 710, second longitudinal movement device; 8, product signal acquisition mechanism; 91, X direction Guide rail; 92, mobile base frame; 93, Y-direction guide rail; 94, movable seat; 95, fixed block; 951, first slide rail; 952, second Rail.
  • the invention provides a new sample processing device especially suitable for digital PCR sample processing, and organically combines the new sample processing device with other modules of the digital PCR system to construct an integrated digital PCR system with small volume, convenient operation and simple setting.
  • the new sample processing device mainly includes a sample processing consumable, a driving module connected to the sample processing consumable, a heating module, and a control module.
  • the driving module is used to drive the liquid in each of the sample processing consumables. Flow in the cavity or channel, the drive module is connected to the control module and controlled by the control module.
  • the design of the driving module, the heating module and the control module itself can be set by those skilled in the art according to the introduction of this document and the well-known knowledge in the art without any creative labor.
  • a sample processing apparatus includes a sample processing consumable 200, a driving module 100 connected to the sample processing consumable 200, and a control module (not shown in the figure).
  • the driving module 100 is used for driving In the device for liquid flow, the driving module 100 is connected to and controlled by the control module.
  • the sample processing consumable 200 includes a base material 210 and a reagent consumable 230, which are detachably connected. Further, the sample processing consumable 200 includes a first base portion 211, a second base portion 212, and elongated connecting portions 270 connected to the first base portion 211 and the second base portion 212 at both ends.
  • the first base portion 211 is provided with a plurality of extraction chambers 260 spaced along the longitudinal direction thereof, and the second base portion 212 is formed with a sample chamber 250.
  • the bottoms of the plurality of extraction chambers 260 communicate with each other through microchannels 280 opened at the bottom of the first base portion 211.
  • the extraction chamber 261 closest to the sample chamber 250 among the plurality of extraction chambers 260 communicates with the sample chamber 250 through a transfer channel.
  • the transfer channel includes a first channel 271 and a second channel 272 having an inner diameter smaller than the first channel 271.
  • the first channel 271 is disposed in the elongated connection portion 270 along the length of the elongated connection portion 270, and the second channel 272 is formed in On the second base material portion 212, the first channel 271 and the second channel 272 communicate at one end, and the other ends of the first channel 271 and the second channel 272 communicate with the extraction chamber 261 and the sample chamber 250, respectively.
  • the adoption of the transfer channel structure is more conducive to controlling the transfer of quantitative liquid accurately and conveniently.
  • the port of the first channel 271 is higher than the bottom of the extraction chamber 261
  • the port of the second channel 272 is higher than the bottom of the sample chamber 250
  • the first channel 271 and the second channel 272 are both arranged horizontally and both The axis lines are at the same level.
  • the reagent consumable 230 includes a housing 231, a U-shaped reagent tube 232 provided in the housing 231, and an interface provided on the housing 231 to cooperate with the interface of the second base part 212 Pair of a pair of plugs 234.
  • the inner cavity of the reagent tube 232 constitutes a reagent storage cavity.
  • the two ends of the reagent tube 232 are respectively connected with micro tubes 233, and the two micro tubes 233 are respectively disposed through a pair of plugs 234.
  • the two micro tubes The inner cavity of one of the tubes 233 constitutes a microchannel that connects the inner cavity of the reagent tube 232 and the bottom of the sample cavity 250, and the other is used to connect the driving module 100 or the atmosphere.
  • the openings of the extraction chamber 260, the sample chamber 250, and the reagent storage chamber are all arranged upward.
  • a heat conducting element 281 is provided at one of the extraction chambers 260, and a magnetic conductive element 282 is provided at the other adjacent extraction chamber 260.
  • the heat-conducting element 281 and the magnetic-permeable element 282 are both elongated shapes, which are respectively transverse to the axis of the corresponding extraction cavity 260 and have exposed contact portions.
  • the heat conducting element 281 can be in contact with an external heating coil to improve the heating effect and rate, and the magnetic conducting element 282 can be in contact with an external magnet to conduct magnetic force.
  • the sample processing device further includes a sliding base 29 that is slidably disposed relative to the base material, and the heating coil and / or the magnet are disposed on the sliding base 29.
  • the sliding direction of the sliding base 29 may be an up-down direction, as shown in FIG. 7.
  • the pore diameters of the microchannel 280 and the transfer channel are relatively small, and the pore diameters of the microchannel and the transfer channel are about 90 microns to 100 microns, respectively.
  • the driving module 100 is connected to the ends of the partial extraction chamber 260 and the microtube 233 respectively.
  • Each driving module 100 includes an air pump 110, a pressure sensor 140, a valve 120, and a gas line 130, respectively.
  • An opening seal 150 is provided at the opening of the extraction chamber 260, and the valve 120 is sealedly connected to the sample processing consumable 200 through the seal 150.
  • control module is not shown in the figure, those skilled in the art can easily understand that after the drive module 100 is connected to the control module, the control module can control the opening and closing of each valve 120 according to a preset program, so that the required Positive or negative pressure is generated in the cavity, so that the liquid in the cavity is transferred from one cavity to another cavity, thereby automatically completing each processing operation that should be manually completed.
  • the sample processing device of this example is very suitable for the processing of digital PCR samples.
  • An exemplary processing step is as follows: (1) Referring to FIG. 3, the auxiliary oil is enclosed in the extraction chamber 268, and absolute ethanol is enclosed in the extraction chamber 267.
  • the cavity 266 encapsulates the cleaning solution 1
  • the extraction cavity 265 encapsulates the cleaning solution 2
  • the extraction cavity 264 encapsulates the cell lysate and the enzyme
  • the extraction cavity 263 adds the sample to be tested
  • the extraction cavity 262 encapsulates the magnetic beads
  • the extraction cavity 261 encapsulates the eluent
  • reagents The inner cavity of the tube 232 encapsulates PCR reaction reagents other than the nucleic acid solution.
  • isolation oil Different PCR reaction reagents such as polymerase, dNTP, and labeling substances are separated by an isolation oil.
  • the isolation oil and the auxiliary oil are the same or different.
  • Other substances are miscible, specifically select the formula oil with the same or similar composition as the oil phase of digital PCR droplets;
  • nucleic acid elution The valve 121 is opened to communicate with the atmosphere, so that a negative pressure is generated in the extraction chamber 262. The eluent is drawn into the extraction chamber 262 for nucleic acid elution. After the elution is completed, a positive pressure is generated in the extraction chamber 262 Press the nucleic acid solution back into the extraction chamber 261;
  • the valve 121 opens to communicate with the atmosphere, applying positive pressure to the auxiliary oil in the extraction chamber 268, so that the auxiliary oil enters the extraction chamber 261, so that the liquid level of the nucleic acid solution in the extraction chamber rises to the port of the transfer channel Above, then the valve 121 closes and continues to pressurize the auxiliary oil in the extraction chamber 268 to fill the nucleic acid into the transfer channel.
  • the pressure sensor feedbacks the relevant signal to judge that the filling is completed, and then the valve 121 opens to generate the extraction chamber 268
  • the negative pressure lowers the level of the nucleic acid solution below the port of the transfer channel, then the valve 121 closes, a positive pressure is generated in the extraction chamber 268, and the nucleic acid solution in the transfer channel is pressed into the sample chamber 250 using the gas in the chamber;
  • the sample processing device has a simple structure, can be modularized, has a small size, and is easy to set up, which not only avoids human operation, reduces work time, but also reduces Operating errors improve the accuracy of the results.
  • This embodiment provides a sample processing device, which is basically the same as that in Embodiment 1.
  • the specific design layout of the driving module for driving the liquid flow in each cavity of the sample processing consumable 200 and the driving module of Embodiment 1 It is different.
  • Each driving module includes an air pump (not shown in the figure, the air pump interface and the intake port 131 in the figure are connected by a gas line), a gas sensor (installed inside the connection base 170, not shown in the figure), and a pneumatic valve group 160 and gas lines (not shown in the figure).
  • the pneumatic valve group 160 includes a valve base 180 fixedly connected to the connecting seat 170, and a plurality of valves 182 provided on the valve base 180 (one-to-one correspondence with the extraction chamber and the sample chamber).
  • the valve base 180 is provided with a channel communicating with the mouth of each valve 182.
  • the bottom of the valve base 180 is provided with a sample processing consumables interface 181 corresponding to the position below each valve 182. These interfaces 181 are respectively connected to a plurality of extraction chambers and sample chambers The connection is sealed by the seal 150.
  • the entire pneumatic valve group 160 is provided with a total air source interface 190, which is connected to the pressure sensor and the air pump through a gas pipeline. Compared with Embodiment 1, this embodiment has a more compact structure, is more convenient to set up, and is more suitable for constructing an integrated digital PCR system.
  • the system includes a base 1, a moving mechanism provided on the base 1, an operating platform 2 provided on the base, and a droplet with a sampling needle 300
  • the generating device 3 the nucleic acid amplification temperature control device, the product signal collection device 8, the control device (not shown in the figure), and the sample processing device as shown in Example 2.
  • the moving mechanism, the sample processing device, the droplet generation device 3, the nucleic acid amplification temperature control device, and the product signal collection device 8 are respectively connected to the control device and controlled by the control device.
  • the moving mechanism includes an X-direction guide rail 91 extending along the length of the micro-digital PCR system, a moving base 92 slidingly connected to the X-direction guide rail, provided on the moving base 92 and extending in the width direction of the digital PCR system
  • the Y-direction guide 93 is a movable base 94 that is slidingly connected to the Y-direction guide 93.
  • the moving base 94 further includes an upright fixed block 95, and the fixed block 95 is respectively provided with a first slide rail 951 and a second slide rail 952 extending in the up-down direction.
  • the connection base 170 of the driving module 100 of the sample processing device and the droplet generating device 3 are respectively slidingly connected to the first slide rail 951 and the second slide rail 952.
  • the digital PCR system further includes a first longitudinal movement device 700 for driving the connection base 170 to slide up and down, a second longitudinal movement device 710 for driving the droplet generation device 3 to slide up and down, the first longitudinal movement device 700, and the second longitudinal movement
  • the device 710 is not particularly limited, and may be implemented by a motor-driven screw nut mechanism or a motor-driven rack and pinion structure.
  • the first longitudinal moving device 700 and the second longitudinal moving device 710 use a motor-driven screw nut mechanism, and the specific setting of the mechanism is conventional, which will not be repeated here.
  • the driving module 100 of the sample processing device and the droplet generating device 3 can move in the three dimensions of front-rear, left-right, and up-down directions.
  • the product signal acquisition device 8 is installed on the moving base 94, and can move in the front-rear and left-right directions under the driving of the moving mechanism.
  • the sample processing apparatus includes a plurality of sample processing consumables 200, and the length direction of each sample processing consumable 200 is consistent with the length direction of the digital PCR system.
  • the driving modules of the droplet generating device 3 and the sample processing device are arranged along the width direction of the digital PCR system. This layout structure is very compact and easy to operate.
  • the droplet generation device uses a micro-pipe vibration to generate droplets, and its specific structural design is not limited, and an installation method known in the art may be used.
  • the operation platform 2 is provided with a droplet container mounting portion 21 through which the droplet container 310 can be detachably mounted for cooperation with the droplet generation device 3 to obtain droplets for subsequent amplification reactions and provide A place for nucleic acid amplification reactions.
  • the product signal acquisition device 8 is known and includes a camera, an optical fiber, an excitation light wheel motor, etc., and can be specifically configured in a conventional manner in the art.
  • the method of processing PCR samples in Example 1 is used to obtain samples and take samples; after sampling, the droplet generation mechanism 3 is driven to the position of the droplet container, and the sampling needle 300 is inserted into the droplet container 310 below the oil phase liquid surface to start Vibration and sample pushing, so that droplets of uniform size are generated in the droplet container 310; after the droplets are generated, the nucleic acid amplification and temperature control device starts heating to perform nucleic acid amplification. After the heating cycle is completed, the product signal collection device 8 moves to Observe and take photos of the location of the droplet container, and send it to the control module for data processing and analysis.

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Abstract

Dispositif et procédé de traitement d'échantillon, et système de PCR numérique comprenant ledit dispositif. Le dispositif de traitement d'échantillon comprend un consommable de traitement d'échantillon, un module d'entraînement connecté audit dispositif, et un module de commande. Le consommable de traitement d'échantillon comprend un substrat, de multiples cavités d'extraction, une cavité d'échantillon, un canal de transfert disposé entre la cavité d'échantillon et la cavité d'extraction et utilisé pour communiquer avec toutes deux, et un consommable d'agent ayant une cavité de stockage d'agent. Les multiples cavités d'extraction communiquent entre elles par le fond au moyen d'un microcanal. La cavité de stockage d'agent communique avec la cavité d'échantillon au moyen du microcanal. Le dispositif de traitement d'échantillon de la présente invention peut avoir de multiples fonctions telles que l'extraction d'un acide nucléique et le mélange d'un agent, et est applicable pour obtenir un échantillon directement utilisé pour la détection de PCR numérique. De plus, le dispositif est simple et compact quant à sa structure, est de petite taille, peut être assemblé de manière modulaire, et peut être utilisé pour construire un système de PCR numérique à gouttelettes intégré.
PCT/CN2019/111626 2018-10-17 2019-10-17 Dispositif et procédé de traitement d'échantillon, et système de pcr numérique comprenant un dispositif de traitement Ceased WO2020078410A1 (fr)

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CN114989940A (zh) * 2022-06-22 2022-09-02 北京金诺美科技股份有限公司 一种基于膜吸附法的核酸提取装置以及提取方法
WO2024141009A1 (fr) * 2022-12-29 2024-07-04 深圳迈瑞生物医疗电子股份有限公司 Analyseur de diagnostic moléculaire et procédé d'extraction d'acide nucléique
CN117210309A (zh) * 2023-11-09 2023-12-12 迈克生物股份有限公司 生物样品处理装置及生物检测系统
CN117210309B (zh) * 2023-11-09 2024-01-26 迈克生物股份有限公司 生物样品处理装置及生物检测系统
WO2025139793A1 (fr) * 2023-12-29 2025-07-03 深圳迈瑞生物医疗电子股份有限公司 Analyseur d'échantillons
CN119086222A (zh) * 2024-11-08 2024-12-06 杭州智灵龙生物科技有限公司 集成核酸样本制备与体积计量的一体化耗材装置及方法

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