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WO2007055165A1 - Procede de separation d'acide nucleique, microreacteur et systeme d'essai d'acide nucleique - Google Patents

Procede de separation d'acide nucleique, microreacteur et systeme d'essai d'acide nucleique Download PDF

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Publication number
WO2007055165A1
WO2007055165A1 PCT/JP2006/322086 JP2006322086W WO2007055165A1 WO 2007055165 A1 WO2007055165 A1 WO 2007055165A1 JP 2006322086 W JP2006322086 W JP 2006322086W WO 2007055165 A1 WO2007055165 A1 WO 2007055165A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
microreactor
cells
flow path
liquid
Prior art date
Application number
PCT/JP2006/322086
Other languages
English (en)
Japanese (ja)
Inventor
Akihisa Nakajima
Koji Miyazaki
Yasuhiro Sando
Kusunoki Higashino
Youichi Aoki
Original Assignee
Konica Minolta Medical & Graphic, Inc.
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 Konica Minolta Medical & Graphic, Inc. filed Critical Konica Minolta Medical & Graphic, Inc.
Priority to JP2007544122A priority Critical patent/JPWO2007055165A1/ja
Publication of WO2007055165A1 publication Critical patent/WO2007055165A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • 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
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • 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
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

Definitions

  • nucleic acid separation method Nucleic acid detection microreactor, nucleic acid detection system
  • the present invention relates to a nucleic acid separation method, a nucleic acid detection microreactor, and a nucleic acid detection system including the microreactor. More specifically, the present invention relates to a nucleic acid detection microreactor having a pretreatment means to which a nucleic acid separation method is applied, and a nucleic acid test system including the microreactor.
  • ⁇ -TAS Micro total Analysis System
  • nanoreactor Nanoreactor
  • Lab_on-chips and biochip
  • its application is expected in the medical examination / diagnosis field, environmental measurement field, and agricultural production field. Yes.
  • skilled procedures, and equipment operations as seen in genetic testing, automation, acceleration, and simplification of microanalysis systems are costly and require samples.
  • the benefits of enabling analysis not only in volume and time but also in any time and place are enormous.
  • Patent Document 1 Japanese Patent Laid-Open No. 2001-322099
  • Patent Document 2 JP 2004-108285 A
  • Patent Document 3 Japanese Translation of Special Publication 2003-522521
  • Patent Document 4 Japanese Patent Laid-Open No. 2004-217
  • Patent Document 5 WO02 / 23180
  • the present invention made in view of the above circumstances is a microreactor for nucleic acid detection that enables high-sensitivity analysis using a chip having a pretreatment means and a waste liquid reservoir even for a sample with a small amount or a low concentration. Propose.
  • the microreactor is of the disposal type in order to make the analysis tool less susceptible to infection and contamination.
  • the present invention aims to provide a nucleic acid test system that incorporates a simple configuration and a high-accuracy liquid feeding system, and enables high-precision analysis. Means for solving the problem
  • the method for separating nucleic acids of the present invention is a method for extracting and separating nucleic acids from cells
  • a liquid containing cells is sequentially passed through a filter A having a pore size larger than the cells and a filter B having a pore size smaller than the cells to hold the cells on the supply port side of the filter B
  • the cells are taken out into the washing solution by flowing a washing solution from the outlet of the filter B opposite to the side holding the cells.
  • the nucleic acid extract is then obtained by destroying the cells by passing the washing solution containing the cells through a cell disruption section holding vibrating beads.
  • the nucleic acid extract is introduced from the cell disruption section through the flow path into the nucleic acid capture section where the electric field is applied in the direction crossing the flow path, and the nucleic acid is collected on the force sword side of the nucleic acid capture section.
  • the beads are vibrated by the action of an external actuator attached to the upper surface of the cell destruction part that holds them.
  • This external actuator is preferably a piezoelectric.
  • the electric field is attached to an upper surface of the nucleic acid capturing unit or the contaminant capturing unit having a comb structure.
  • the cell filtration device of the present invention is a cell filtration device used for carrying out the step G) and the step (ii) in the nucleic acid separation method, wherein the filter A and the filter B are used.
  • the partition walls are arranged on the inlet side and the outlet side of the apparatus, respectively, and a space that can be sealed is formed by these partition walls and the side wall of the apparatus, and a cleaning liquid discharge pipe is passed through the side wall surface. Open and close each hole in the mouth and discharge pipe It is characterized by having means to be.
  • the microreactor for nucleic acid testing of the present invention is destroyed in the specimen receiving part that receives a liquid containing cells, the cell destruction part that destroys cells delivered from the specimen receiving part, and the cell destruction part
  • a specimen pretreatment section having a nucleic acid capture section for capturing nucleic acid released from cells, wherein the sample receiving section, the cell destruction section and the nucleic acid capture section are connected in a fine flow path. I'm going
  • the microreactor for nucleic acid detection of the present invention has a reagent storage section for storing a reagent, a waste liquid storage section, a micropump connection section, and a microchannel that communicates each section,
  • the cell disruption section has beads that are vibrated by an external actuator disposed in the cell disruption section to destroy the cells
  • the nucleic acid capturing unit captures a nucleic acid released from a cell destroyed by the cell destructing unit by an electric field applied to an external electrode disposed in the nucleic acid capturing unit.
  • the micropump connection portion includes:
  • a second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than that of the first flow path; a pressure chamber connected to the first flow path and the second flow path;
  • the waste liquid storage section is provided at the bottom of the microreactor, and is characterized in that it is a hollow chamber communicating with at least the end of the specimen pretreatment section and the detection section.
  • microreactor for nucleic acid detection of the present invention comprises:
  • the pressure If the pressure is lower than the preset pressure, the pressure will block the passage of the liquid, and the pressure will be higher than the preset pressure. Then, the liquid feed control unit that allows the passage of liquid,
  • a backflow prevention unit for preventing backflow of liquid in the flow path
  • It is characterized by having a liquid feeding dividing means for controlling the liquid feeding and the quantity of the liquid feeding in the branched flow path.
  • a nucleic acid test system characterized in that a nucleic acid is measured by attaching the microreactor to the apparatus main body.
  • the nucleic acid detection system of the present invention separates the chip 'component, which is a microreactor for each specimen, and the control' detection component, which is the main body of the device, equipped with elements for reagents and liquid delivery systems. System configuration. Therefore, cross-contamination and carry-over are difficult for microanalysis and amplification reactions.
  • the microreactor chip has a very limited volume into which a sample can be introduced. Therefore, it is difficult to capture the target nucleic acid or bacteria when the concentration is low.
  • the microreactor for nucleic acid detection of the present invention it has a specimen pretreatment unit capable of extracting nucleic acids and genes as analysis target substances from cells, separating them and concentrating them. Therefore, the substance can be concentrated even in a dilute sample, and at the same time, harmful substances that interfere with the reaction and contaminants that clog the flow path can be excluded.
  • the microreactor for nucleic acid test of the present invention has a flow channel configuration that can simultaneously analyze positive control and negative control in order to eliminate the influence of reaction inhibition, contamination, and increase in backround. Such a microreactor realizes highly reliable and highly accurate analysis even with a very small amount of sample.
  • nucleic acid testing system and nucleic acid testing microreactor of the present invention are based on the configuration and analysis principle of the apparatus, and the conventional nucleic acid sequence analysis, restriction enzyme analysis, and nucleic acid hybridization. Much smaller sample volume compared to analysis It is clear that highly accurate results can be obtained with effort and a simple device.
  • the nucleic acid detection system of the present invention can be used for various gene analyzes, clinical tests, diagnosis, pharmaceutical screening, safety of drugs, agricultural chemicals or various chemicals, examination of toxicity, environmental analysis, food It can be used for inspection, forensic medicine, chemistry, brewing, fishery, livestock, agricultural production, agriculture and forestry. Brief Description of Drawings
  • FIG. 1 is a schematic diagram of a nucleic acid test system comprising a nucleic acid test microreactor and an apparatus main body.
  • FIG. 2 is a schematic view of a microreactor for nucleic acid testing in one embodiment of the present invention.
  • the micropump belongs to a separate device from the nucleic acid testing microreactor.
  • FIG. 3 is a schematic view of the cell filtration device of the present invention, with the upper view showing a front view and the lower view showing a cross-sectional view.
  • FIG. 4 is a diagram showing a procedure for recovering cells using a cell filtration device.
  • FIG. 5 is a cross-sectional view of a pretreatment unit attached to the nucleic acid detection microreactor of the present invention.
  • the left compartment represents a cell disruption part
  • the right part represents a nucleic acid capture part.
  • FIG. 6 shows a piezo pump
  • FIG. 3 (a) is a cross-sectional view showing an example of this pump.
  • FIG. 3 (b) is a top view thereof.
  • FIG. 3 (c) is a cross-sectional view showing another example of a piezo pump.
  • FIG. 7 is a diagram showing a configuration around a pump connection part of a microreactor for nucleic acid detection when the piezo pump is separated from the microreactor. Explanation of symbols
  • the nucleic acid detection microreactor of the present invention (hereinafter sometimes simply referred to as a microreactor) and a nucleic acid detection system including the microreactor, a micropump, various control devices, and a detection device will be described below.
  • the “specimen” is a fluid containing the nucleic acid to be measured.
  • “Gene” may also refer to DNA or RNA, which is simply a chemical entity, that refers to DNA or RNA that carries genetic information that expresses some function. These and the polynucleotide are collectively referred to as “nucleic acid”.
  • “Element” means a functional component installed in the microphone port reactor.
  • the “fine channel” is a channel formed in the microreactor of the present invention.
  • the method for separating nucleic acids of the present invention is a method for extracting and separating nucleic acids from cellular force
  • a liquid containing cells is sequentially passed through a filter A having a pore size larger than the cells and a filter B having a pore size smaller than the cells to hold the cells on the supply port side of the filter B
  • the cells are taken out into the washing solution by flowing a washing solution from the outlet of the filter B opposite to the side holding the cells.
  • the nucleic acid extract is introduced from the cell disruption section through the flow path into the nucleic acid capture section where the electric field is applied in the direction crossing the flow path, and the nucleic acid is collected on the force sword side of the nucleic acid capture section.
  • the filter mesh used in the method for separating nucleic acids of the present invention takes into consideration the size of cells, blood cells, winoles, bacteria, and the like.
  • the form of the filter and the thickness of the filter layer should be set appropriately according to the purpose. For example, insoluble materials and dust are first filtered out, and then two types of filters with different sizes are used in combination to carry out the prescribed treatment.
  • As the shape of the filter there is a force S such as a layered configuration, a resin layer, a hollow fiber aggregate configuration, etc., but it can be selected arbitrarily.
  • the filter A is a filter containing a target nucleic acid and having a pore size larger than that of the cells in the sample.
  • the filter A is a so-called “prefilter” for removing insoluble contaminants, dust, and the like by filtration.
  • the filter B is a “sterile filter” having a pore size smaller than that of the cell, and the cell cannot pass through the filter B.
  • the filter A having a large hole size filters coarse particles such as salt crystals, cell debris, hair, and tissue. Therefore, the average hole size should be small enough to filter the sample fluid force such coarse particles and large enough to pass the target cells and viruses containing nucleic acids. Selected. In general, the hole size should be in the range of about 2-25 ⁇ m, and preferably about 5 ⁇ m.
  • the filter B having a small hole size captures cells and viruses in the sample fluid.
  • the average hole size is selected according to the average size of the cell or virus containing the nucleic acid of interest. For example, 0.1 to 5 zm, preferably 0.5 to 2 zm.
  • the filter may be a commercially available filter such as cellulose acetate, Membrane filters such as cellulose nitrate mixed ester, polytetrafluoroethylene, polyethersulfone, nylon, polyvinylidene difluoride, and glass fiber are shown. Polycarbonate, polyethylene, polypropylene, and glass are used as filter housing materials. Etc.
  • the cells can be taken out into the washing solution by pouring the washing solution into the space from the take-out port on the side opposite to the cell holding side of the filter B.
  • the washing liquid in which the cells are dispersed is passed through the flow path to the cell destruction section that holds the vibrating beads.
  • the nucleic acid extract containing the released nucleic acid and the like is further passed through a nucleic acid capturing part to which an electric field is applied in a direction crossing the flow path.
  • Nucleic acids which are negatively charged linear molecules, tend to migrate and collect on the anode side.
  • Nucleic acids collected on the force sword side of the nucleic acid capture unit can be separated from the nucleic acid capture unit with a nucleic acid recovery solution.
  • the cleaning liquid, the extraction liquid, and the recovery liquid are not particularly limited, but are various salt solutions, buffer solutions, physiological saline, and aqueous solutions.
  • nucleic acid separation method of the present invention is easily carried out in the following sample filtration apparatus and specimen pretreatment unit installed on a microreactor.
  • the cell filtration device of the present invention is a cell filtration device used for carrying out the steps G) and (i) in the nucleic acid separation method of the present invention, and has the following configuration. Yes.
  • the filter A and the filter B are arranged as partition walls on the inlet side 81 and the outlet side 82 of the apparatus, respectively, and a space (filtration space) 86 that can be sealed is formed by these partition walls and the apparatus side wall.
  • the side wall surface has a cleaning liquid discharge pipe 83, and each hole in the inlet, the outlet and the discharge pipe has means that can be opened and closed arbitrarily (FIG. 3).
  • the space 86 is a filtrate reservoir, but preferably has a cylindrical shape, such as a syringe type.
  • the device is convenient.
  • Transparent plastics such as polyethylene, polypropylene, polycarbonate and the like are suitable materials because they are easy to handle and readily manufactured, but are not particularly limited thereto.
  • the opening for introducing the sample liquid containing cells, the outlet for discarding the filtrate, and the hole in the cleaning liquid discharge pipe for collecting the cleaning liquid have means that can be opened and closed. Open and close those holes as needed at each stage.
  • a preferable example of such means may be a detachable screw cap, a plug stopper or a three-way cock.
  • the size of the filtration device of the present invention is appropriately set depending on the type and concentration of the sample. In general:! ⁇ 50ml capacity is easy to handle and operate.
  • steps (i) and (ii) in the method for separating nucleic acids of the present invention can be carried out simply as follows (FIG. 4).
  • the inlet side 81 of the cell filtration device of the present invention is placed in a sample solution in a sample container (such as a blood collection tube, a urine cup, a vial, or a sample bottle) containing the collected sample liquid.
  • a sample container such as a blood collection tube, a urine cup, a vial, or a sample bottle
  • Aspirate from the outlet 82 on the opposite side and introduce the sample liquid into the filtration space (filtrate reservoir) 86 through the filter A84.
  • the portion remaining in the filtration space includes the particles and cells that cannot pass through the pores of the filter B85.
  • the suction may be performed by an appropriate pressure reducing means such as a water pump, but when there is no need to increase the amount of the specimen, a disposable syringe, a vacuum blood collection tube, or the like is used. Install it in the hole on the outlet side and suck it. In the case of a vacuum blood collection tube, it is automatically sucked after installation.
  • the filtrate that has passed through filter B is convenient because it is accommodated in a disposable syringe or vacuum blood collection tube.
  • a washing liquid is fed into the space from the take-out port side 82, and the liquid containing cells is washed. Unnecessary cleaning solution is removed by suction from the outlet side 82. Repeat if necessary.
  • the hole on the inlet side 81 is closed, the hole of the cleaning liquid discharge pipe 83 that has been closed until then is opened, air is supplied from the outlet side 82, and the air is pressurized, so that the inlet side is supplied from the outlet side. Remove the cleaning fluid from the open cleaning fluid drain.
  • the washing solution contains cells.
  • the washing solution containing the cells is directly injected into the sample receiving portion of the microreactor for nucleic acid analysis of the present invention. For this reason, it is preferable that the cleaning liquid discharge tube and the specimen receiving portion are joined in a liquid tight manner.
  • the position, size, etc., of the washing liquid discharge pipe provided on the side wall of the cell filtration device are designed to be optimal in the above relationship with the microreactor as a chip.
  • a needle tip may be attached to the washing liquid discharge tube and injected into the sample insertion port made of silicon rubber in the sample receiving part of the microreactor.
  • the configuration, size, etc. of the cell filtration device according to various circumstances of the specimen can be designed separately from the microreactor by using a separate device.
  • the cell filtration device of the present invention has a very simple configuration.
  • Insoluble contaminants can be removed in advance, and the cells can be easily separated from the sample solution.
  • a washing solution containing only the target cells can be injected into the sample receiving part of the microreactor in an appropriate amount.
  • the number of cells in the washing liquid in which the cells are suspended can be easily adjusted.
  • the nucleic acid to be detected especially the genes of microorganisms such as viruses or fungi (bacteria, fungi)
  • viruses or fungi are present at very low concentrations.
  • Gram-negative bacteria are present in less than 10 copies per milliliter of blood
  • Taliptosporidium is usually only a few copies per gallon of drinking water
  • anthrax is less than 100 copies per milliliter of water.
  • the microreactor chip has a very limited capacity for introducing the sample, so the concentration of the target bacteria in the sample solution is low. It is virtually impossible to capture this. Therefore, it is extremely useful that a large amount of sample solution can be passed through filter A and concentrated in filter B.
  • the cell filtration device of the present invention By using the cell filtration device of the present invention, it is possible to separate cells easily and cleanly without being contaminated by bacteria, viruses, etc., and to transfer the specimen to the nucleic acid detection microreactor of the present invention. .
  • a commercially available disposable syringe and syringe filter can be used to perform the same treatment as described above, although it is somewhat complicated. Specifically, first attach a commercially available syringe 'filter (a small-capacity filtration filter of the type attached to the syringe) equivalent to filter A to a plastic syringe of appropriate volume, and put the sample liquid in the sample liquid. Aspirate into syringe. Next, filter A is removed, and instead a commercially available syringe corresponding to filter B is attached as the second filter, and the sample liquid in the syringe is filtered through the filter by pushing the plunger.
  • a commercially available syringe 'filter a small-capacity filtration filter of the type attached to the syringe
  • the second filter can be removed, and a needle tip is attached instead, and the washing solution containing the cells can be transferred to the mouth reactor.
  • FIG. 1 is a schematic view in one embodiment of a nucleic acid inspection system comprising a microreactor for nucleic acid testing detachable from an apparatus main body and the apparatus main body.
  • FIG. 2 is a schematic view of a microreactor for nucleic acid detection according to an embodiment of the present invention.
  • the present invention can be arbitrarily modified and changed in accordance with the spirit of the present invention, and these are included in the present invention. That is, as long as the structure, configuration, arrangement, shape, dimensions, material, method, method, etc. of the microreactor for nucleic acid detection and the nucleic acid detection system of the present invention are all or part of the present invention, are consistent with the spirit of the present invention, It can be various.
  • the microreactor 2 for nucleic acid detection shown in FIGS. 1 and 2 is a single chip produced by appropriately combining one or more members such as plastic resin, glass, silicon, ceramics and the like.
  • the micro flow path and the housing of the microreactor are formed of a plastic resin that is easy to process and inexpensive, and easy to dispose of by incineration.
  • polystyrene The resin is preferable because it has excellent moldability and has a strong tendency to adsorb streptavidin and the like, as will be described later, so that the detection part can be easily formed on the fine channel.
  • the fine channel is formed with a width of about 100 xm and a depth of about 100 zm, for example.
  • the detection part covering at least the detection part of the fine channel on the surface of the microphone outlet reactor is transparent.
  • Fig. 2 shows an example of a typical flow path configuration of the microreactor for nucleic acid detection of the present invention.
  • the nucleic acid test system of the present invention includes a device body 1 in which a micro pump, a control device that controls the micro pump, a temperature control device that controls temperature, a detection device, and the like are integrated, and the device body 1 is mounted.
  • Possible microreactor 2 When the sample liquid is injected into the sample receiving part of the microreactor 2 in which the reagent is preliminarily sealed and the microreactor 2 is attached to the main body 1 of the nucleic acid detection system, a mechanical connection for operating the liquid feed pump is necessary. For example, electrical connection for control is also made. Therefore, when the apparatus main body 1 and the microreactor 2 are joined, the flow path of the microreactor 2 is also activated.
  • the flow of analytes and reagents, amplification of nucleic acids based on mixing, reactions such as nucleic acid and probe binding, detection of reactants and optical measurement are a series of sequential steps.
  • Measurement data is stored in a file together with necessary conditions and recorded items, and nucleic acid measurement is automatically performed.
  • the detection device is an optical detection means such as a photodiode or a photomultiplier tube, which irradiates measurement light from, for example, an LED on the detection section on the analysis flow path for each inspection item. It is a device that detects transmitted light or reflected light. There are various optical devices with different principles as means of optical detection, but an ultraviolet / visible spectrophotometer is desirable. It may be a device incorporated in the above detection device, or may be connected as a separate device at the time of use.
  • the detection device for optically detecting the nucleic acid contained in the specimen is incorporated in the device main body 1 together with the liquid feeding means including the micropump and the temperature control device, It has an integrated configuration.
  • Control system, optical detection, data collection and processing related to control of liquid feeding, temperature and reaction constitutes the main body of the nucleic acid detection system of the present invention together with the micropump and the optical device.
  • the apparatus main body 1 is commonly used for a specimen sample by mounting a microreactor 2 for nucleic acid detection.
  • the reaction and detection such as amplification described above are performed under the conditions set in advance with respect to the order of delivery, volume, timing, etc., and the nucleic acid detection system as a program along with the micropump, temperature control, and optical detection data processing. It is built into the software installed in the.
  • the control program stored in the main unit 1 can be modified as appropriate.
  • nucleic acid test system of the present invention all components are miniaturized and are in a form that is convenient to carry. Therefore, the nucleic acid test system is excellent in workability and operability without being restricted by the place and time of use. Because it can be measured quickly regardless of location and time, it can be used for emergency medical treatment or for personal use in home medical care. Numerous micropump units used for liquid delivery are built into the main body 1 side, so the microreactor for nucleic acid testing can be used as a disposable type.
  • the microreactor and nucleic acid detection system for nucleic acid testing of the present invention can be suitably used particularly for testing genes or nucleic acids.
  • a mechanism for DNA amplification is installed on the nuclear acid detection microreactor.
  • the basic configuration of nucleic acids other than genes is almost the same.
  • the microreactor for nucleic acid test of the present invention has a sample receiving part, a sample pretreatment part, a reagent storage part, a waste liquid storage part, a micropump connection part, and a fine channel.
  • the cell filtration device has a specimen receiving part that receives a washing liquid containing cells through its washing liquid discharge pipe, and a cell destruction part and a nucleic acid capturing part that are specimen pretreatment parts. It communicates with the channel.
  • a cleaning liquid containing cells and viruses in the specimen receiving section is sent as a specimen liquid to the specimen pretreatment section through a fine flow path, and the specimen liquid is pretreated using the pretreatment means of the specimen pretreatment section.
  • the nucleic acid to be measured is extracted, separated, and concentrated, and the substance is measured by flowing it to the flow path constituting the reaction part and then the flow path constituting the detection part provided downstream of the fine flow path.
  • a nucleic acid detection microreactor characterized in that waste liquid generated as a result of concentration and measurement of nucleic acid is transferred to and confined in the waste liquid reservoir.
  • each element such as a liquid feed control section, a backflow prevention section, a reagent quantification section, and a mixing section has a microfabrication technique at a functionally appropriate position. It is installed by.
  • RNA it is converted to cDNA using an appropriate reverse transcriptase and then used for analysis.
  • Such a pre-processing operation is an operation different from a method in which a predetermined amount of sample is applied to a predetermined position of a conventional analysis chip at a time and the sample is measured.
  • the present invention is characterized in that it is composed of two stages: a pretreatment before the application to the nucleic acid detection microreactor and a pretreatment performed on the nucleic acid detection microphone reactor thereafter.
  • the specimen pretreatment unit is a part that preliminarily performs extraction, separation, concentration, and the like of the nucleic acid to be analyzed by cell destruction (bacterial or hemolysis).
  • the shape, structure, etc. are arbitrary, and pretreatment means such as vibrating beads and an electric field by an external electrode are applied.
  • an extraction liquid, a cleaning liquid, a recovery liquid, and the like are sealed as necessary.
  • the specimen pretreatment unit includes at least two independent parts in the flow path, a cell destruction unit, and a cell destruction unit. And a nucleic acid trap.
  • the specimen containing cells and viruses is sent from the specimen receiving section to the cell destruction section of the specimen pretreatment section.
  • the cell destruction part nucleic acids are extracted by physically destroying cells and viruses.
  • the cell destruction part provided on the fine flow path downstream from the specimen receiving part is a space that is wider than the flow path before and after it and spreads up and down. Inside the space, the required number of beads are added. And retained ( Figure 5). Therefore, it should be thick enough to provide space for the beads to move or to rupture cells or viruses.
  • the size of the beads needs to be larger than the diameter of the fine flow path so as to remain in the space, and if arranged, it is in the range of 50 to 600 / im, preferably 100 to 400 x m.
  • the average diameter of the beads does not need to be the same as long as it stays in the space inside the cell disruption that can be selected according to the cells, bacteria, or viruses to be ruptured by the beads.
  • the shape of the beads can be either porous or non-porous.
  • the material of the beads is not particularly limited, but suitable beads for rupturing cells or viruses include zirconia, borosilicate glass, lime glass, silica, and polystyrene beads. In particular, materials that do not adsorb nucleic acids are preferred.
  • Beads that remove unwanted substances eg, proteins and peptides
  • chemicals eg, salts, metal ions, surfactants
  • ion-exchange beads for removing proteins are known, and beads having a metal ion chelate H glaze such as iminoniacetic acid remove metal ions from biological samples.
  • the beads are vibrated by an external actuator attached to the cell destruction portion.
  • a piezoelectric PZT (lead zirconate titanate) ceramic resonator is a suitable example of such an actuator.
  • Cells that come into contact with or collide with beads that are vibrated by the PZT vibrator have their membrane structure destroyed, and intracellular substances are released out of the cells.
  • the intracellular substance contains the target nucleic acid.
  • bacterial cells and viruses are lysed very quickly, and as a result, the nucleic acid comes out into the liquid, and the nucleic acid extract becomes can get.
  • the vibration strength of the beads is appropriate for the type and nature of cells in the specimen. Just set it up.
  • a lysis agent for example, a surfactant, an enzyme, or an organic compound
  • a lysis agent for example, a surfactant, an enzyme, or an organic compound
  • the extract must be treated with chaotropic salt to denature contaminating proteins (enzymes used) and potentially interfering proteins.
  • the above chemical agent force suppresses subsequent DNA amplification
  • surfactants denaturants such as ethanol may change the water repellency of the water repellent valve of the fine flow path. This necessitates a step of separating the target nucleic acid from the chemical agent, resulting in a reduction in the amount of nucleic acid as well as complexity.
  • the nucleic acid capture unit may be located on the downstream side of the cell destruction unit and may be substantially adjacent.
  • a pair of external electrodes are attached to the outer surface of the chip that is located above and below the trapping part and the contaminant trapping part facing each other (Fig. 5).
  • An electric field is applied in the crossing direction.
  • electrophoresis is supplementarily performed for nucleic acid separation, an electric field is generated in a direction crossing the flow path and applied to cell components. Since the dimension across the flow path, for example, the width direction or height direction of the flow path, is extremely small compared to the length direction of the flow path, the applied voltage required to generate the desired electric field can be small.
  • the voltage applied between the channel widths is about 40-60V.
  • electrophoresis is performed as a driving force for biomolecules, it can be seen that a very low voltage can be applied compared to the need to apply a voltage of several kV. Note that the application of voltage to the electrodes and the action of the PZT vibrator of the cell destruction unit are electrically controlled from the apparatus body while the microreactor is mounted on the apparatus body.
  • the pair of opposed external electrodes may be formed by depositing an electrode such as platinum on the upper surface of the nucleic acid capturing unit or on the side wall of the channel.
  • these electrodes and the structure having a comb structure can be manufactured by an etching technique as disclosed in, for example, Japanese Patent Application Laid-Open No. 2002-233792.
  • a nucleic acid extract containing a nucleic acid from the cell disruption section is sent to the nucleic acid capturing section through the fine channel.
  • the nucleic acid extract is passed through a nucleic acid capture section where an electric field is applied across the flow path, the nucleic acid having a negative charge moves toward the anode (anode) and hits the anode.
  • the positively charged contaminant is the opposite surface corresponding to the force sword (cathode), that is, the surface of the flat portion facing the concave comb structure in FIG. Is trapped).
  • the liquid containing other cell components passes through the trapping part and flows downstream.
  • the sample pretreatment unit receives the sample from the sample receiving unit 20 and the treatment liquid used for the pretreatment from the reagent storage unit 18 (which may be provided separately from the other reagent storage units), and the reagent storage unit. It communicates with the flow path from 18 to the reaction section downstream of the fine flow path. Since there is a waste liquid reservoir (waste liquid reservoir) on the bottom surface of the chip, for example, a valve disposed at the boundary with the pretreatment section is opened, and unnecessary sample pretreatment waste liquid and cleaning liquid are discarded there. be able to.
  • the nucleic acid capturing part preferably has a comb structure as a microstructure.
  • the nucleic acid is attracted by electrophoresis under an applied electric field to gather the nucleic acid at a high density away from the fluid flow.
  • the fluid is pumped out, but the nucleic acid is taken into the comb-shaped partial region so that it does not flow along with the fluid.
  • the comb-shaped structure does not occupy the entire cross-section of the flow path, and nothing is arranged on the other side of the flow path, so it does not obstruct the flow of fluid and is continuously efficient.
  • there is an advantage that the nucleic acid is separated from other components and recovered.
  • the nucleic acid collected in the concave nucleic acid capture unit on the anode side can be separated from the nucleic acid capture unit with a nucleic acid recovery solution.
  • the biasing means applies a voltage to the counter electrode so as to generate an electric field in the opposite direction to the nucleic acid
  • the nucleic acid is separated and collected more efficiently.
  • the electric field is turned off, the foreign matter trapped on the flat surface of the force sword (contaminant catching part) also detaches, but such foreign matter tends to flow out before the nucleic acid in the recess. Therefore, when changing the direction of the electric field, it is desirable to make adjustments so that the electric field is weaker than the trapped voltage.
  • Nucleic acids can be concentrated by such treatment.
  • the pre-treated specimen as described above can then be The liquid is divided into two or more micro channels for sample analysis by the following liquid sending and dividing means, and is sent to the downstream analysis flow channel that communicates.
  • the divided specimens enter from the Sampno report into the fine channel through which the reagents flow.
  • the sample receiving part 20 of the microreactor for nucleic acid detection of the present invention has the following configuration.
  • the sample receiving unit 20 communicates with the sample injection unit to temporarily store the sample (that is, the above-described washing liquid containing cells or viruses) and supply the sample to the mixing unit.
  • the cell-containing liquid injected into the sample receiving unit 20 is connected to the micropump 11 and the pump connection unit 12, and is sent to the sample pretreatment unit by these actions.
  • the portion (sample injection portion) for injecting the sample on the upper surface of the sample receiving portion 20 is made of an elastic material such as a rubber-like material to prevent leakage, infection and contamination to the outside and to ensure sealing performance. It is desirable that the plug is covered with a resin or reinforced film such as polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the specimen in the syringe is injected with a needle pierced with the stopper made of the rubber material or a needle through a pore with a lid. In the former case, it is preferable that the needle hole is immediately closed when the needle is pulled out. Alternatively, you can install another specimen injection mechanism.
  • the sample to be measured in the present invention is not particularly limited to a biological sample itself, but most biological samples such as whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum, etc.
  • the sample is applicable.
  • gene, DNA or RNA is the target of analysis as a nucleic acid that is a type of amplification reaction.
  • the specimen may be prepared or isolated from a sample that may contain such nucleic acid. Therefore, in addition to the above samples, cell cultures; nucleic acid-containing samples such as viruses, bacteria, molds, yeasts, plants and animals; samples that may contain or contain microorganisms, and other nucleic acids. Any sample that may be present is considered.
  • the microreactor for nucleic acid testing of the present invention requires a very small amount of sample compared to the case of manual testing. For example, a blood sample of about 2 to 3 zL is simply injected into a chip with a length and width of several centimeters. For example, in the case of genes, 0.001 to 100 ng as DNA It is. For this reason, including the case where only a very small amount of specimen can be obtained, the nucleic acid detection mic mouth reactor of the present invention has few restrictions on the specimen surface, and inevitably requires a small amount of reagents, and the inspection cost is low. Reduction.
  • the waste liquid storage part is a hollow chamber provided at the bottom of the microreactor, and it should be used to store excess specimens, cleaning liquid generated in the specimen separation / concentration process, waste liquid, and waste liquid generated as a result of specimen reaction and measurement. It is a sealed waste liquid reservoir for storing.
  • the specimen pretreatment unit when the analyte concentration step is performed as a pretreatment, it is necessary to treat the waste liquid generated by the concentration and washing. Such waste liquid is less troublesome to store automatically inside than to be discharged outside the microreactor for processing.
  • the waste liquid storage part is provided immediately below the valve part of the specimen pretreatment part of the microreactor, and communicates with at least the specimen pretreatment part, the reaction part of the flow path, the end part of the detection part and the like and has a necessary capacity.
  • Any hollow structure in which a through-hole communicating with each part that generates waste liquid is formed may be used.
  • the waste liquid storage part may be in the form of a multi-compartment cavity divided into a plurality of compartments that may be a single hollow chamber.
  • the volume, shape, etc. are not particularly limited, but it is necessary to consider the strength of the chip itself and the space limitations associated with the arrangement of other elements.
  • the capacity of the waste liquid storage part is several hundreds of square liters when it is provided at the bottom of most chips of several centimeters square.
  • a valve that opens and closes at an appropriate place in the through hole may be provided as necessary so that the waste liquid in the waste liquid reservoir does not enter other parts of the microreactor or leak outside. Good.
  • the installation and structure of the waste liquid reservoir further provide the following advantages to the user. Workers who analyze clinical samples are always at risk of infection from the sample, such as bacteria or viruses. Not only the handling and pre-treatment of specimens, but also the treatment of cleaning liquid during measurement and waste liquid after measurement are not only complicated, but there is still a risk of infection and the possibility of environmental pollution.
  • a waste liquid storage part is provided at the bottom of the microphone mouth reactor, and all of the above unnecessary specimens, waste liquids, etc. are finally disposed in the waste liquid reservoir. Yes.
  • This waste liquid reservoir is completely sealed inside the microreactor, and is a disposable type nucleic acid test microarray of the present invention. After use, the reactor is stored in a hazard box installed in the inspection room and incinerated.
  • the microreactor for nucleic acid testing of the present invention In the microreactor for nucleic acid testing of the present invention, necessary reagents (including a cleaning solution) are sealed in a predetermined amount in a reagent storage unit 18 in the microreactor in advance. Accordingly, the microreactor for nucleic acid testing of the present invention is ready for use without having to fill the required amount of reagent each time it is used.
  • necessary reagents are known, and various reagents for amplification, probes used for detection, color reagents, and the like can be mentioned.
  • a micropump 11 is provided for each of the specimen receiving part 20, the reagent containing part 18 and, if necessary, the control containing part, to feed the contents of these containing parts.
  • the micropump 11 is connected to the upstream side of the reagent storage unit 18, and supplies the driving liquid to the reagent storage unit side by the micropump 11, thereby pushing out the reagent to the flow path and feeding the liquid.
  • the micropump unit is incorporated in an apparatus main body (nucleic acid detection apparatus) separate from the microreactor. By attaching the microreactor to the apparatus main body, the micropump unit is connected to the microreactor from the pump connection portion 12. (Fig. 7).
  • a piezo pump is used as the micro pump. That is,
  • a second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than that of the first flow path; a pressure chamber connected to the first flow path and the second flow path;
  • a medicine container is provided, and a pump connection part is provided on the upstream side of each of these container parts, and the micropump is connected to these pump connection parts, and the driving liquid is supplied from each micropump.
  • the reaction liquid necessary for gene amplification analysis is started by extruding the sample solution and the reagent in each container and joining them together.
  • Mixing of reagent and reagent, and mixing of specimen and reagent may be performed at a desired ratio in a single mixing section, or a plurality of merging sections may be provided by dividing either force or both. They may be mixed so as to have a desired mixing ratio.
  • the form of such a reaction part is not particularly limited, and various forms and modes are conceivable. Basically, at least two kinds of liquids including a reagent and a specimen liquid are fed by a micropump and joined together,
  • a fine channel that is provided first from the merging portion and in which each of the liquids is diffused and mixed, and is provided first from a downstream end of the fine channel, and a space wider than the fine channel, It is preferable to provide a liquid reservoir for storing and reacting the mixed liquid that has been diffusively mixed in the fine channel.
  • the amplification method is not limited.
  • DNA amplification technology can use PCR amplification methods that are widely used in various fields.
  • Various conditions for implementing the amplification technique have been examined in detail and described in various documents including improvements.
  • the present inventors have already proposed a flow channel device that enables temperature control suitable for a microchip ( JP 2004-108285 A).
  • This device system may be applied to the amplification flow path of the microreactor for nucleic acid detection of the present invention.
  • the heat cycle can be switched at high speed, and the microchannel is a micro reaction cell with a small heat capacity. This can be done in a much shorter time than the method.
  • the ICAN (Isothermal chimera primer initiated nucleic acid amplification) method recently developed as an improvement of the PCR amplification method is 50-65. It has a feature that DNA amplification can be carried out in a short time at an arbitrary constant temperature in C (see Japanese Patent No. 3433929). Accordingly, the ICAN method is a suitable amplification technique because the nucleic acid detection microreactor of the present invention requires simple temperature control. In the manual operation, this method, which takes 1 hour, is completed in 10 to 20 minutes, preferably 15 minutes, in the nucleic acid detection microreactor of the present invention.
  • a detection unit for detecting amplified nucleic acid is provided on the downstream side of the reaction unit of the fine channel. At least the detection part is made of a transparent material, preferably a transparent plastic, in order to enable optical measurement. Furthermore, the biotin-affinity protein (avidin, streptavidin) adsorbed on the detection section on the microchannel is either piotin labeled on the probe substance or piotin labeled on the 5 'end of the primer used for gene amplification reaction. Binds specifically. As a result, the probe labeled with biotin or the amplified nucleic acid is trapped in this detection unit.
  • biotin-affinity protein avidin, streptavidin
  • the method for detecting the separated amplified nucleic acid is not particularly limited, but as a preferred embodiment, it is basically carried out in the following steps. That is, using the microreactor for nucleic acid detection of the present invention,
  • a step of amplifying the nucleic acid in the microchannel of the reaction unit a step of mixing the amplification reaction solution containing the amplified nucleic acid and the denaturing solution in the microchannel, and denaturing the amplified DNA into a single strand, A solution obtained by denaturing the amplified DNA into a single strand is sent to the detection section in the microchannel adsorbed with the piotin affinity protein, and the amplified nucleic acid is trapped through the trapping of the amplified gene.
  • a probe DNA whose end is fluorescently labeled with FITC fluorescein isothiocyanate is allowed to flow through the detection section in the fine channel, and this is hybridized to the immobilized gene.
  • FITC fluorescently labeled with fluorescein isothiocyanate
  • a gold colloid solution whose surface is modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow into the microchannel, and the gold colloid is adsorbed to the FITC-modified probe hybridized to DNA.
  • streptavidin When streptavidin is immobilized in a microchannel formed on a polystyrene substrate, it is not necessary to perform a special chemical treatment. It is only necessary to apply the piotino-affinity protein onto the fine flow path downstream of the amplification reaction part and to adsorb the biotin-affinity protein onto the flow path.
  • a probe DNA for genetic testing fluorescently labeled oligonucleotides are preferably used.
  • the DNA base sequence a sequence that is complementary to a part of the gene base sequence to be detected is selected. By appropriately selecting the base sequence of the probe DNA, it is possible to detect with high sensitivity without being affected by the coexisting DNA and background by specifically binding to the target gene.
  • FITC anti-FITC antibodies for example, gold colloid anti-FITC anti-mouse IgG are preferable because they are available.
  • the probe may be labeled with horseradish peroxidase (HRP) instead of the fluorescent dye.
  • HRP horseradish peroxidase
  • Typical coloring substances for this purpose are 3,3 ', 5,5' _tetramethylbenzidine (0 ⁇ 48), 3,3'-diaminobenzidine (D AB), p-phenylenediamine (OPD) Etc. are known.
  • enzyme / chromogenic systems such as alkaline phosphatase and galactosidase can be used.
  • Preferred embodiments of the microreactor for nucleic acid testing of the present invention include: Within one chip,
  • a sample pretreatment unit for performing pretreatment of the sample for performing pretreatment of the sample
  • a reagent container for storing reagents used in probe binding reactions, detection reactions (including gene amplification reactions, etc.);
  • a probe container that houses a probe for example, a probe that is hybridized to a gene to be detected amplified by a gene amplification reaction
  • a pump connection portion connectable to a separate micropump for feeding the liquid in each storage portion and the flow path;
  • a micropump is connected to the chip via a pump connection unit, and the nucleic acid extracted from the sample stored in the sample receiving unit and the reagent stored in the reagent storage unit are fed to the channel, and the microchannel
  • the reaction solution for example, the amplification reaction portion, is mixed and reacted, and then the processing solution obtained by treating the reaction solution and the probe accommodated in the probe accommodating portion are sent to the detection portion in the downstream flow path. Liquid, mix in the flow path and bind (or hybridize) with the probe, detect nucleic acid based on this reaction product,
  • the sample pretreatment unit and the detection unit communicate with each other through a through-hole, and is a hollow chamber provided at the bottom of the microreactor. Measurement of excess sample, cleaning liquid generated in the sample concentration process, waste liquid, and sample As a result, it is configured to have a waste liquid storage portion that is a sealed waste liquid reservoir for storing the generated waste liquid and the like.
  • the liquid feed dividing means includes a branched fine channel, a liquid feeding control unit 13 and a backflow prevention unit.
  • the liquid supply control unit 13 blocks the passage of the liquid until the liquid supply pressure in the forward direction (usually the direction in which the fluid is pushed out by the pump, that is, the downstream direction) reaches a predetermined pressure, and supplies the liquid above the predetermined pressure. Allow fluid to pass by applying hydraulic pressure.
  • the backflow prevention unit that prevents the backflow of the liquid in the flow path is a check valve in which the valve body closes the flow path opening due to the backflow pressure, or the valve body is moved to the flow path opening by the valve body deforming means. It consists of an active valve that presses and closes the opening.
  • the micropump In the microchannel of the microreactor for nucleic acid test of the present invention, the micropump, a liquid feeding control unit capable of controlling the passage of liquid by the pump pressure, and a backflow prevention for preventing the backflow of the liquid in the channel Depending on the part, the liquid feeding, the fixed amount of the liquid feeding amount, and the mixing of each liquid in the branched flow path are controlled. Reagents and specimens are divided at an appropriate ratio by the powerful liquid dividing means and the action of the micro pump 11.
  • Nucleic acid analysis is usually performed in parallel with sample analysis by adding a negative control to the analysis. This is because it is essential for correction of contamination, for example, color development and fluorescence of substances mixed in reagents. Furthermore, in order to increase the reliability of the analysis results, it is also necessary to add positive controls. This is useful for detecting interfering factors in added reagents, verifying the appropriateness of set conditions, and nonspecific interactions. Similarly, it is often necessary to provide internal control, especially for quantitative analysis.
  • Simultaneous positive control and internal control are particularly important for gene amplification by PCR. This is because it is particularly necessary to check that the PCR reaction is occurring correctly. For example, when a problem occurs, it is optimal for verifying whether it originates from set conditions, reagents, operation, analysis system, or sample. The ability of PCR to amplify trace amounts of genes present in specimens several hundred thousand to several million times, and the effects of contamination such as cross-contamination are extremely serious.

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Abstract

L'invention concerne un microréacteur et un système d'essai d'un acide nucléique qui comprennent un dispositif de prétraitement et permettent de parvenir à une certaine efficacité d'un essai extrêmement fiable. Un système d'essai d'acide nucléique comprend un composant de puce pour chaque spécimen doté d'éléments d'acheminement de réactifs et de liquides, et un composant de commande/détection constituant le corps principal dudit appareil d'essai et séparé dudit composant de puce. Comme ce système présente une partie de prétraitement de spécimen conçu pour réaliser un prétraitement par extraction d'un acide nucléique provenant du spécimen et par concentration dudit acide nucléique, la substance cible peut être concentrée, même lors de l'utilisation d'un échantillon dilué, parallèlement à l'élimination simultanée d'une substance nocive interférant avec la réaction et de contaminants provoquant un blocage dans une voie. En outre, ce système présente une telle constitution de voie, qu'il permet l'analyse simultanée d'un témoin positif et d'un témoin négatif. Du fait de sa constitution, ce système ne présente guère de problèmes graves de microanalyse ou d'amplification, par exemple, de contamination croisée et de contamination rémanente.
PCT/JP2006/322086 2005-11-11 2006-11-06 Procede de separation d'acide nucleique, microreacteur et systeme d'essai d'acide nucleique WO2007055165A1 (fr)

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CN110283709A (zh) * 2019-05-09 2019-09-27 广州和实生物技术有限公司 全自动核酸分子杂交仪
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JP2008116318A (ja) * 2006-11-03 2008-05-22 Japan Advanced Institute Of Science & Technology Hokuriku 検体の捕捉方法
CN101538567B (zh) * 2008-03-20 2012-09-19 杭州优思达生物技术有限公司 过滤式微量核酸临床样品快速处理方法
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CN110283709A (zh) * 2019-05-09 2019-09-27 广州和实生物技术有限公司 全自动核酸分子杂交仪
WO2022070662A1 (fr) * 2020-09-29 2022-04-07 富士フイルム株式会社 Récipient d'essai, dispositif d'essai et méthode d'essai
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