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WO2008001691A1 - Procédé de traitement d'échantillon - Google Patents

Procédé de traitement d'échantillon Download PDF

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Publication number
WO2008001691A1
WO2008001691A1 PCT/JP2007/062601 JP2007062601W WO2008001691A1 WO 2008001691 A1 WO2008001691 A1 WO 2008001691A1 JP 2007062601 W JP2007062601 W JP 2007062601W WO 2008001691 A1 WO2008001691 A1 WO 2008001691A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
cell
substance
sample
region
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2007/062601
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English (en)
Japanese (ja)
Inventor
Jun Funazaki
Makoto Bannai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Olympus Corp
Original Assignee
Olympus Corp
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Filing date
Publication date
Application filed by Olympus Corp filed Critical Olympus Corp
Publication of WO2008001691A1 publication Critical patent/WO2008001691A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms

Definitions

  • the present invention relates to a sample processing method for processing a sample such as extracting a nucleic acid contained in the cell from a sample containing the cell.
  • Examples of a method for separating or extracting nucleic acid from a sample containing nucleic acid include, for example, phenol 'chloroform extraction method, a method of separating a nucleic acid by precipitating a nucleic acid-containing solution with an ethanol solution ( Hereinafter, it may be referred to as an ethanol separation method), a method disclosed in Patent Document 1, a method for lysing cells using an enzyme to separate nucleic acids (hereinafter, sometimes referred to as an enzyme separation method), centrifugation Blood cell separation method using a method, nucleic acid extraction method using magnetic beads, nucleic acid extraction method using a filter, and the method disclosed in Patent Document 2
  • a sample component protein, lipid, etc.
  • phenol 'black mouth form' to dissolve or precipitate the sample component.
  • impurities such as proteins and lipids are dissolved in water using a chaotropic solution, and nucleic acids are adsorbed on silica beads as a solid phase carrier.
  • the collected nucleic acid is washed, the collected nucleic acid is washed, and the washed nucleic acid is dissolved again in the aqueous phase.
  • blood components are separated using a micro flow channel.
  • Patent Document 1 Patent No. 2680462
  • Patent Document 2 JP-A-2005-224787
  • the phenol / chloroform extraction method and the ethanol separation method have a problem in that they are manual and cannot be performed easily. Furthermore, according to the phenol “black mouth form” extraction method, since phenol “black mouth form” is used, there is a problem that the working environment is restricted.
  • the blood cell separation method using the centrifugal method a centrifugal separation mechanism is required, and according to the nucleic acid extraction method using a filter, an apparatus for vacuum suction is required, and the magnetic beads are removed.
  • the nucleic acid extraction method used requires a mechanism for collecting the beads by magnetism, which makes it difficult to downsize the entire apparatus.
  • the nucleic acid extraction method using magnetic beads the supernatant liquid is discharged in a state where the magnetic beads are collected by collecting the magnetic beads in the process until the nucleic acid is extracted. There is a possibility that the remaining liquid force and the gap between the nozzles will result in lower cleaning efficiency.
  • nucleic acid extraction method using magnetic beads there is a problem that if the beads are sufficiently washed, the washing must be repeated, so that the operation becomes complicated.
  • operations such as recovery of the solid phase support made of magnetic beads and transfer of the container are required in the process until the nucleic acid is extracted. The problem was that
  • nucleic acid analysis such as nucleic acid amplification, hybridization between a nucleic acid and a probe, and detection of a hybridization reaction is performed.
  • nucleic acid analysis such as nucleic acid amplification, hybridization between a nucleic acid and a probe, and detection of a hybridization reaction is performed.
  • the present invention has been made in view of the above problems.
  • the present invention provides a simple, small-sized apparatus configuration and simple operation, without limiting the working environment or using solvents or reagents, when nucleic acid is extracted from a sample containing cells. It is an object of the present invention to provide a sample processing method that can be performed appropriately and quickly, and that can be suitably used particularly when a clinical specimen such as blood is used as a target.
  • the present invention enables simple and small analysis of nucleic acids such as amplification of nucleic acids, hybridization of nucleic acids and probes, and detection of hybridization reactions after extraction of nucleic acids. It is an object of the present invention to provide a sample processing method that can be carried out in a series of flows without complications with an apparatus configuration and simple operation.
  • the sample processing method according to claim 1 is a sample processing method for processing a sample containing cells, wherein the cells are adsorbed.
  • a sample placing step for placing the sample in the cell adsorption region using a substrate having a cell adsorption region including a region surface-treated with a cell adsorbing material, which is a substance to be treated, and the sample placing step;
  • a sample removal step for removing the sample from the cell adsorption region and a sample removal step so that the cells adsorbed to the cell adsorption substance remain, and then the cells are destroyed.
  • the nucleic acid contained in the cell is extracted by performing a nucleic acid exposure drying step of drying the cell adsorption region until the nucleic acid contained in the cell is exposed.
  • the sample processing method according to claim 2 is the sample processing method according to claim 1, wherein after the sample removal step is executed, the predetermined cells are selectively used. After performing the destructive substance installation step of placing the destructive substance, which is the substance to be destroyed, in the cell adsorption region, and the destructive substance installation step, the destructive substance and the destructive substance are destroyed from the cell adsorption region. And a step of removing the destructive substance and the like, which removes the cells, and the nucleic acid exposure drying step dries the cell adsorption region after the destructive substance etc. removal step.
  • the sample processing method according to claim 3 is the sample processing method according to claim 1 or 2, wherein the nucleic acid is amplified after the nucleic acid exposure drying step is performed.
  • the amplification substance installation step of placing the amplification substance as the substance in the cell adsorption region and the amplification substance installation step are performed, the amplification substance is evaporated so as to cover the extracted nucleic acid and the amplification substance.
  • the anti-fusible material installation step of placing the anti-fusible material, which is the substance for preventing the anti-evaporation, in the cell adsorption region and the anti-evaporation material installation step the cell adsorption region is exposed to a predetermined temperature condition.
  • the nucleic acid is amplified by further performing a temperature exposure step.
  • the sample processing method according to claim 4 is the sample processing method according to claim 3, wherein the cell adsorption region includes a sequence complementary to a target nucleic acid sequence.
  • the cell adsorption region includes a sequence complementary to a target nucleic acid sequence.
  • One or a plurality of types of probes are provided in separate sections, and after performing the temperature exposure step, the anti-transpiration material removal step of removing the anti-transpiration material from the cell adsorption region, and the anti-transpiration material removal step And the substance for performing a hybridization reaction between the nucleic acid and the probe after the adsorption region drying step is performed and the adsorption region drying step is performed.
  • the sample processing method according to claim 5 is the sample processing method according to claim 4, wherein after the temperature holding step is performed, the reactant is removed from the cell adsorption region. After performing the reactant removal step to be removed and the reactant removal step, the reaction for detecting the presence or absence of the hybridization reaction for the amplified nucleic acid is performed. And a response detecting step.
  • sample processing method according to claim 6 according to the present invention is the sample processing method according to any one of claims 1 to 5, wherein the substrate is the hydrophobic region.
  • a hydrophobic region is further provided, and the cell adsorption region is hydrophilic and is surrounded by the hydrophobic region.
  • the sample processing method according to claim 7 according to the present invention is the sample processing method according to any one of claims 1 to 6, wherein the substrate has the cell adsorption region on its surface. It is characterized by having multiple areas.
  • sample processing method according to claim 8 is the sample processing method according to claim 7, wherein each of the cell adsorption regions is surface-treated with the cell adsorption substance.
  • the area of the region is the same.
  • the shape of the cell adsorption region is circular, Its diameter is 20 ⁇ m or more and 10,000 ⁇ m or less.
  • the cell in the sample processing method according to any one of claims 1 to 9, the cell is a white blood cell, and the sample Is characterized by blood.
  • a sample is placed in a cell adsorption region using a substrate provided with a cell adsorption region including a region surface-treated with a cell adsorption material, which is a substance that adsorbs cells.
  • a cell adsorption material which is a substance that adsorbs cells.
  • the method can be performed quickly, and in particular, it can be suitably used when a clinical sample such as blood is used as a target.
  • nucleic acid amplification after nucleic acid is extracted from the sample cartridge, nucleic acid amplification, hybridization between the nucleic acid and the probe, and hybridization are performed.
  • Nucleic acid analysis such as reaction detection, can be performed in a series of flows with simple and small apparatus configuration and simple operation without any complexity.
  • FIG. 1 is a diagram showing an example of the configuration of a sample processing apparatus 100 that works on the present embodiment.
  • FIG. 2 is a perspective view showing an example of a substrate 102 according to the present embodiment.
  • FIG. 3 is a perspective view showing an example of a substrate 102 according to the present embodiment.
  • FIG. 4 is a perspective view showing an example of a substrate 102 according to the present embodiment.
  • FIG. 5 is a perspective view showing an example of a substrate 102 according to the present embodiment.
  • FIG. 6 is a diagram showing an example of a sample processing method executed by the sample processing apparatus 100.
  • FIG. 7 is a diagram showing an example of a sample processing method executed by the sample processing apparatus 100. Explanation of symbols
  • FIG. 1 is a diagram showing an example of the configuration of a sample processing apparatus 100 according to the present embodiment.
  • the sample processing apparatus 100 includes a substrate 102, a microtiter plate 104, a dispensing device 106, a thermal cycler 108, a discharge device 110, a discharge head cleaning device 112, and a fluorescence scanner. 114 and a control device 116.
  • the substrate 102 is provided with a cell adsorption region including a region surface-treated with a cell adsorbent, which is a substance that adsorbs cells specifically or non-specifically, specifically, a slide glass. is there.
  • a cell adsorbent which is a substance that adsorbs cells specifically or non-specifically, specifically, a slide glass. is there.
  • FIGS. 2 and 3 and FIGS. 4 and 5 are perspective views showing an example of the substrate 102 that is useful for the present embodiment.
  • the substrate 102 shown in FIG. 2 has a total of 48 (4 rows and 12 columns) circular hydrophilic capillar regions 102a having a diameter of 1.6 mm surrounded by a hydrophobic region 102b arranged on the surface at equal intervals. ing.
  • the hydrophilic capsule region 102a corresponds to the cell adsorption region according to the present invention, and is a lectin corresponding to the cell adsorption material according to the present invention (lectin is a substance that adsorbs cells non-specifically). It is a hydrophilic region that has been partially surface-treated.
  • the hydrophobic region 102b is a hydrophobic region surrounding the hydrophilic capture region 102a provided in an annular shape (ring shape) as shown in FIG.
  • the hydrophilic cap region 102a is formed by treating the surface of the slide glass as the substrate 102 with, for example, a silane coupling agent.
  • the hydrophobic region 102b is formed on the surface of the slide glass so as to avoid the hydrophilic cap region 102a and to surround the hydrophilic cap region 102 by, for example, a method of printing fluorine resin or the like.
  • the hydrophilic cap region 102a is partially provided with a non-specific cell adsorption coat layer 102d surface-treated with lectin.
  • Non-specific cell adsorption coat layer 1 02d is formed in the same area (for example, 300 m 2 to 78 mm 2 ) between the hydrophilic cap region 102a by a method such as screen printing.
  • the hydrophilic cap region 102a includes a detection probe spot 102c in which one or more types of detection probes including a sequence complementary to a target nucleic acid sequence are arranged via an appropriate linker as shown in FIG.
  • the non-specific cell-adsorbing coat layer 102d is partitioned into spots and arranged in spots at regular intervals.
  • a plurality of detection probes a plurality of target nucleic acids can be detected and analyzed at the same time, and the multi-analysis is excellent.
  • the substrate 102 shown in Fig. 4 has a total of 48 (4 rows and 12 columns) circular hydrophilic capillar regions 102a with a diameter of 1.6 mm surrounded by a hydrophobic region 102b arranged at equal intervals on the surface. ing.
  • the hydrophilic capsule region 102a is an antibody used for beads or the like that specifically binds to leukocytes, and is a hydrophilic region partially surface-treated with a leukocyte-specific antibody corresponding to the cell-adsorbing substance according to the present invention. is there.
  • the hydrophobic region 102b is a hydrophobic region that surrounds the hydrophilic cap region 102a that is provided on the entire surface of the substrate 102 as shown in FIG.
  • the hydrophilic cap region 102a is formed by treating the surface of the slide glass as the substrate 102 with, for example, a silane coupling agent. Further, the hydrophobic region 102b is formed on the surface of the slide glass so as to avoid the hydrophilic capture region 102a and surround the hydrophilic capture region 102 by, for example, a method of printing fluorine resin.
  • the hydrophilic cap region 102a is partially provided with a leukocyte-specific cell adsorption coat layer 102e surface-treated with a leukocyte-specific antibody as shown in FIG.
  • the leukocyte-specific cell adsorption coating layer 102e is formed with the same area (for example, 300 ⁇ m 2 to 78 mm 2 ) between the hydrophilic hydrophilic cap regions 102a by a method such as screen printing.
  • the hydrophilic cap region 102a is a test in which one or a plurality of types of detection probes including a sequence complementary to a target nucleic acid sequence are arranged through an appropriate linker. As shown in FIG. 5, the outgoing probe spots 102c are divided into spots within the region of the leukocyte-specific cell adsorption coat layer 102e and arranged in spots at regular intervals.
  • the substrate according to the present invention is not limited to the substrate 102 shown in FIGS. 2 to 5 described above that works on the present embodiment.
  • the substrate 102 is not limited to a slide glass.
  • the arrangement state and the number of the hydrophilic cap region 102a are not limited to those shown in FIGS.
  • the shape of the hydrophilic cap region 102a is not limited to the above-mentioned circular shape with a diameter of 1.6 mm, for example, a substantially circular shape with a diameter of 20 / zm to 10,000 / zm (including an ellipse, for example). Or other than these.
  • the shape of the hydrophilic cap region 102a is a substantially circular shape with a diameter of 20 ⁇ m and a force of 10,000 ⁇ m
  • the hydrophilic cap region 102a depends on the shape of the sample, the destructive substance and the amplification according to the present invention. Liquids such as substances, anti-transpiration materials, and reactants can be held stably and reliably.
  • the hydrophilic capture region 102a can hold a small amount of droplets of about 2 pl (picoliter) to 260 1 (microliter) in a substantially hemispherical shape, depending on its diameter. As a result, the reagents used for the sample and various substances can be effectively reduced.
  • the non-specific cell adsorption coat layer 102d and the leukocyte-specific cell adsorption coat layer 102e are not limited to being partially provided in the hydrophilic cap region 102a, but are provided in the entire hydrophilic cap region 102a. It may be provided over the entire hydrophilic cap region 102a excluding the detection probe spot 102c.
  • the hydrophobic region 102b may be provided so as to surround the hydrophilic cap region 102a.
  • the hydrophobic region 102b is provided in an annular shape as shown in FIGS. 2 and 3, or is provided on the entire surface of the substrate 102 as shown in FIGS. 4 and 5, for example, a hydrophilic cap region. It may be partially provided on the surface of the substrate 102 excluding 102a. Further, the arrangement state and the number of detection probe spots 102c are not limited to those shown in FIGS.
  • the microtiter plate 104 is used for the sample according to the present invention to be placed in the hydrophilic cap region 102a and various substances related to the present invention (for example, the destructive material and the amplification related to the present invention). Substances, anti-transpiration materials, reactants, and buffer solutions) are kept in separate compartments.
  • the sample contains at least nucleated cells.
  • the sample is, for example, blood composed of whole blood anti-coagulated with heparin or the like, or a buffy coat mainly composed of leukocytes by separating blood components from whole blood.
  • the destructive substance is a substance for selectively destroying a predetermined cell.
  • the destructive substance is, for example, a lysate using a surfactant or the like whose salt concentration is adjusted so as to selectively lyse red blood cells.
  • the amplification substance is a substance for amplifying nucleic acid.
  • the amplification substance is an amplification reaction solution containing, for example, a PCR (Polymerase Chain Reaction) reagent.
  • Anti-transpiration material is a substance to prevent transpiration such as amplification material and reaction material.
  • the anti-fusible material is, for example, a sealing oil such as mineral oil or silicon oil. Examples of mineral oil include Sigma Aldrich's mineral oil “M8662”, and examples of silicone oil include Invitrogen's silicone oil for PCR “10890-010”.
  • the reactive substance is a substance for performing a hybridization reaction between the nucleic acid and the detection probe.
  • the reactant is, for example, a noble reaction solution.
  • the dispensing device 106 includes a multi-pipeter 106a, and the multi-pipetter 106a is used to collect samples and various substances (for example, destructive substances, amplification substances, anti-transpiration substances, reactive substances, and buffer liquids).
  • the sample is sucked from the microtiter plate 104, or the sucked sample and various substances are dispensed into the hydrophilic cap region 102a.
  • the thermal cycler 108 applies a thermal cycle related to the temperature condition necessary for the PCR reaction to the substrate 102 or each hydrophilic cap region 102a, or the substrate 102 or each hydrophilic cap region 102a to room temperature or a predetermined temperature. Or hold.
  • the discharge device 110 includes a discharge head 110a, and the discharge head 110a sucks the sample and various substances placed in each hydrophilic cap region 102a, and the sucked sample and various substances are discharged to other materials. Discharge to the hydrophilic cap region 102a.
  • the discharge head cleaning device 112 cleans the discharge head 110a.
  • the fluorescence scanner 114 includes a substrate insertion unit 114a, and scans the substrate 102 inserted into the substrate insertion unit 114a to create fluorescence image data.
  • the control device 116 is a commercially available personal computer.
  • the control device 116 includes a dispensing device 106, a thermal cycler 108, a discharge device 110, a discharge head cleaning device 112, and a fluorescent scanner 114. It is connected so as to be able to communicate, and controls the dispensing device 106, the thermal cycler 108, the discharge device 110, the discharge head cleaning device 112, and the fluorescent scanner 114.
  • the control device 116 has a function of receiving fluorescence image data transferred from the fluorescence scanner 114 and detecting the presence or absence of a hybridization reaction based on the fluorescence image data.
  • FIG. 6 and FIG. 6 and 7 are diagrams showing an example of a sample processing method executed by the sample processing apparatus 100.
  • FIG. 6 and FIG. 6 and 7 are diagrams showing an example of a sample processing method executed by the sample processing apparatus 100.
  • the sample processing apparatus 100 executes the sample processing method shown in FIG.
  • the control device 116 issues a command to the dispensing device 106.
  • the dispensing device 106 sucks blood B from the microtiter plate 104 by the multipipette 106a and sucks the sucked blood.
  • B is dropped about 11 (microliter) into each hydrophilic cap region 102a (step 1: sample placement step).
  • blood B is held in the hydrophilic cap region 102a as a hemispherical droplet, and after a predetermined time, a part of the blood cell contained in blood B is simply applied to the leukocyte-specific cell adsorption coat layer 102e by sedimentation or the like.
  • the blood cells in contact with each other in the form of a single layer only leukocytes W are adsorbed on the leukocyte-specific cell adsorption coat layer 102e.
  • the control device 116 issues a command to the dispensing device 106 when the force is also passed for a predetermined time after completing the step 1, and when the dispensing device 106 receives the command, the leukocyte-specific cell adsorption codec.
  • Blood B is removed from each hydrophilic cap region 102a by the multi-pipetter 106a so that the leukocytes W remain adsorbed on the layer 102e (step 2: sample removal step). Since leukocytes are adsorbed on the leukocyte-specific cell adsorption coat layer 102e, blood B can be removed from the hydrophilic cap region 102a by sucking with the multipipette 106a of the dispensing apparatus 106.
  • the white blood cell W adsorbed to the leukocyte-specific cell adsorption coat layer 102e by the tip of the multipipeter 106a nozzle is not destroyed. It is desirable to provide a gap of about the thickness of leukocytes (about several tens of ⁇ m) between the leukocyte-specific cell adsorption coat layer 102e.
  • Step 2 when Step 2 is completed, erythrocytes R and the like other than leukocytes W are formed into a single layer on the nonspecific cell adsorption coat layer 102d. Since there is a possibility of adsorption (see step 2 shown in FIG. 7), when the substrate 102 is used, the controller 116 completes step 2 and dispenses the force as shown in FIG.
  • the dispensing device 106 receives the command, the multi-pipetter 106a sucks the lysate So from the microtiter plate 104, and the sucked lysate So for each hydrophilic capillaries.
  • Approximately 1 1 (microliter) is dropped on one area 102a (process 2 ': destructive substance installation process), and after a predetermined time has elapsed, red blood cells R and the like are removed from each hydrophilic cap area 102a by the multipipette 106a.
  • Dissolved solution So may be removed (step 2 ': broken Substances such as removing step).
  • the lysate So is retained in the hydrophilic cap region 102a as hemispherical droplets, and the red blood cells R adsorbed to the non-specific cell adsorption coat layer 102d are lysed by the lysate So to inhibit amplification of nucleic acids such as hemoglobin.
  • the components to be eluted are dissolved in the lysate So, and only the leukocytes W can be adsorbed to the non-specific cell adsorption coat layer 102d. That is, hemoglobin, which inhibits nucleic acid amplification by dissolving erythrocytes R, can be efficiently and effectively removed together with the lysis solution So. As a result, step 4 relating to nucleic acid amplification can be efficiently performed. As a result, accuracy in step 5 relating to the hybridization reaction between the nucleic acid and the detection probe and step 6 relating to the detection of the hybridization reaction is also improved. Can be improved.
  • the control device 116 issues a command to the thermal cycler 108, and when the thermal cycler 108 receives the command, the white blood cell W destroys the nucleus contained in the white blood cell W.
  • the substrate 102 or each hydrophilic cap region 102a By holding the substrate 102 or each hydrophilic cap region 102a at room temperature or an appropriate temperature until the acid is exposed, water on the surface of each hydrophilic cap region 102a is evaporated, and each hydrophilic cap region 102a is evaporated.
  • the region 102a is dried (Step 3: Nucleic acid exposure drying step).
  • nucleic acid N contained in leukocytes W could be extracted easily and easily from a very small amount of blood B. As a result, the physical and mental burden on the subject can be reduced.
  • the control device 116 issues a command to the dispensing device 106.
  • the dispensing device 106 sucks the amplification reaction solution P from the microtiter plate 104 by the multipipette 106a, About 1 ⁇ 1 (microliter) of the amplified amplification reaction solution P is dropped on each hydrophilic cap region 102a (step 4: amplification substance installation step).
  • the amplification reaction liquid is held in the hydrophilic cap region 102a as a hemispherical droplet.
  • the control device 116 continues to issue a command to the dispensing device 106.
  • the dispensing device 106 sucks and sucks the sealing oil S from the microtiter plate 104 by the multipipette 106a.
  • the sealing oil S Approximately 51 (microliters) of sealing oil S in each hydrophilic cap region 102a so as to cover nucleic acid N and amplification reaction solution P (on the upper layer of nucleic acid N and amplification reaction solution P as shown in FIG. 6) Dripping (process 4: anti-transpiration material installation process).
  • the amplification reaction solution P is sealed to prevent evaporation of the amplification reaction solution P in the thermal cycle.
  • the controller 116 issues a command to the thermal cycler 108, and upon receiving the command, the thermal cycler 108 applies the thermal cycle necessary for the PCR reaction to the substrate 102 or each hydrophilic cap region 102a.
  • Process 4 Temperature exposure process
  • the nucleic acid N extracted in the step 3 can be easily and easily amplified on the same substrate 102 and the same hydrophilic cap region 102a by the step 4.
  • steps 1 and 4 there is no need for a container transfer operation or a new container as in the prior art, and only one substrate 102 is used to extract the nucleic acid N from the extraction of the nucleic acid N.
  • a fluorescent label could be introduced into the amplified nucleic acid N.
  • the nucleic acid amplification method is not limited to the PCR method shown in step 4, but may be, for example, an isothermal amplification method or other various known amplification methods.
  • the control device 116 issues a command to the dispensing device 106.
  • the dispensing device 106 removes the sealing oil S from each hydrophilic cap region 102a by the multi-pipette 106a ( Process 5: Process for removing fugitive substances).
  • the control device 116 issues a command to the thermal cycler 108.
  • the thermal cycler 108 holds the substrate 102 or each hydrophilic cap region 102a at room temperature or an appropriate temperature.
  • Each hydrophilic cap region 102a is dried (step 5: adsorption region drying step).
  • the control device 116 issues a command to the dispensing device 106.
  • the dispensing device 106 sucks the hybridization reaction liquid H from the microtiter plate 104 by the multipipette 106a, and sucks the suctioned nozzle.
  • the hybridization reaction liquid is held in the hydrophilic cap region 102a as hemispherical droplets. If the amplification reaction solution P is further mixed with reagents for the hybridization reaction, this step can be omitted.
  • the control device 116 issues a command to the dispensing device 106 following the reactant placing step, and the dispensing device 106 sucks the sealing oil S from the microtiter plate 104 by the multi-pipeter 106a when receiving the command. Then, the aspirated sealing oil S covers the nucleic acid N (containing fluorescently labeled target nucleic acid N1) and the hybridization reaction solution H (as shown in FIG. 6). About 5 ⁇ l (microliter) may be dropped onto each hydrophilic cap region 102a (on the upper layer of the reaction solution H). As a result, the sealing oil S is held as a hemispherical droplet in the hydrophilic cap region 102a.
  • the control device 116 then issues a command to the thermal cycler 108, and the thermal cycler In accordance with the directive, the substrate 108 holds the substrate 102 or each hydrophilic cap region 102a at a room temperature or a temperature suitable for the substrate 102 or each hydrophilic cap region 102a (for example, a temperature of 60 ° C). ) Is applied to maintain the temperature (step 5: temperature holding step).
  • step 5 the hybridization reaction between the target nucleic acid N1 fluorescently labeled in step 4 and the detection probe can be carried out easily and on the same substrate 102 and the same hydrophilic cap region 102a. It was easy to do. In other words, between step 1 and step 5, there is no need for a container transfer operation or a new container as in the prior art.
  • the process from the extraction of nucleic acid N to the hybridization reaction could be carried out simply and easily with 2 alone.
  • the hybridization method is not limited to the hybridization method shown in step 5.
  • a fluorescent label is not introduced, and in step 5, a target nucleic acid is used using an intercalator.
  • a hybridization reaction between N1 and the detection probe may be performed.
  • the control device 116 issues a command to the dispensing device 106.
  • the dispensing device 106 removes the hybridization reaction liquid H by the multipipette 106a (step 6). : Reactive substance removal step).
  • the control device 116 issues a command to the dispensing device 106 following the reactant removal process, and when the dispensing device 106 receives the command, the multipipette 106a sucks the buffer solution from the microtiter plate 104.
  • the hydrophilic cap regions 102a may be dried by holding the substrate 102 or the respective hydrophilic cap regions 102a at room temperature or an appropriate temperature.
  • the control device 116 issues a command to the fluorescent scanner 114.
  • the fluorescent scanner 114 confirms whether the substrate 102 is properly inserted into the substrate insertion portion 114a, and the substrate 102 is properly inserted. If it is confirmed that the substrate is scanned, the substrate 102 is scanned, the fluorescence image data of the scanned substrate 102 is transferred to the control device 116, and the control device 116 receives the fluorescence image data transferred from the fluorescence scanner 114. Then, based on the fluorescence image data, the presence or absence of a hybridization reaction is detected for the fluorescently labeled target nucleic acid N1 (step 6: reaction detection step).
  • the target nucleic acid N1 hybridized with the detection probe in the fluorescently labeled target nucleic acid N1 can be easily and easily detected by the step 6 on the same substrate 102 and the same hydrophilic cap region 102a. I was able to. In other words, between step 1 and step 6, the container transfer operation and a new container are not required as in the prior art, and the nucleic acid N is extracted from the hybridization using only one substrate 102. The process up to the detection of the reaction could be performed easily and easily.
  • the hydrophilic cap region including the leukocyte-specific cell adsorption coat layer 102e surface-treated with the leukocyte-specific antibody.
  • blood B containing white blood cells W such as whole blood is placed in the hydrophilic cap region 102a, so that the white blood cells W adsorbed on the hydrophilic cap region 102a remain.
  • Hydrophilic cap region 102a force Extracts nucleic acid contained in leukocyte W by drying hydrophilic cap region 102a so that blood B is removed and leukocyte W is destroyed to expose nucleic acid contained in the leukocyte. To do.
  • nucleic acid N when nucleic acid N is extracted from blood B containing leukocytes W, the sample can be processed with a simple and small device configuration and simple operation without restricting the working environment or using solvents or reagents. Can be done efficiently and quickly. In other words, “nuclear nucleic acid extraction” related to the analysis of nucleic acid can be performed easily and easily.
  • a substrate 102 as shown in FIGS. 2 to 5 is used. This effectively reduces the amount of blood and various reagents used in clinical tests for genetic diagnosis such as gene deletion and drug-sensitive SNPs, resulting in the physical burden and mental health of the subject. The burden can be reduced and the inspection cost can be reduced. it can.
  • the substrate 102 shown in FIGS. 2 and 3 the substrate 102 having the nonspecific cell adsorption coating layer 102d
  • leukocytes W such as whole blood are removed.
  • the lysate So The solution So may be removed from each hydrophilic cap region 102a after a predetermined time has elapsed after being placed in the hydrophilic cap region 102a.
  • erythrocytes R having hemoglobin which is unnecessary for nucleic acid analysis and is an inhibitory substance can be selectively and effectively eliminated.
  • the accuracy of nucleic acid analysis can be improved.
  • the amplification reaction solution P is placed in the hydrophilic cap region 102a, and the sealing oil S is applied to the hydrophilic cap region 102a so as to cover the extracted nucleic acid N and the amplification reaction solution P.
  • the nucleic acid N is amplified by exposing the hydrophilic cap region 102a to a predetermined temperature condition.
  • the extracted nucleic acid N can be easily and easily amplified on the same substrate 102 and the same hydrophilic cap region 102a.
  • Japanese Patent No. 3743090 which detects a nucleic acid by amplifying it in a hydrophilic region, has been disclosed.
  • the patent publication discloses that a reaction solution is dropped on a heating resistor coated with a hydrophobic material surrounding a hydrophilic material and a heat cycle is applied to the reaction solution.
  • Techniques for performing nucleic acid amplification are disclosed.
  • a separate element for extracting and detecting nucleic acids is required. For this reason, it is difficult to analyze nuclear acid efficiently and quickly with a simple and small apparatus configuration and simple operation.
  • the process of extracting nucleic acid N and amplification of nucleic acid N can be performed simply and easily with only one substrate 102 with a simple and small apparatus configuration and simple operation. it can.
  • the sealing oil S is removed from the hydrophilic cap region 102a, the hydrophilic cap region 102a is dried, and the hybridization is performed.
  • the reaction solution H By placing the reaction solution H in the hydrophilic cap region 102a and keeping the hydrophilic cap region 102a at a predetermined temperature, a hybridization reaction between the amplified nucleic acid N and the probe is performed.
  • the hybridization reaction between the target nucleic acid N1 and the detection probe can be easily and easily performed on the same substrate 102 and the same hydrophilic cap region 102a.
  • Japanese Patent No. 3386391 discloses a method in which a probe containing a sequence complementary to a target nucleic acid is provided on a flat substrate in divided areas.
  • Patent No. 3393528 discloses a method of detecting the presence or absence of a target nucleic acid by an hybridization reaction with a sample nucleic acid provided with a label or the like.
  • 3625826 discloses a method in which a droplet isolated by surface tension is subjected to a chemical reaction, and in particular, a nucleic acid is detected by hybridization with a probe.
  • these patent publications do not disclose a technique relating to extraction of nucleic acid from a sample or amplification of a small amount of extracted nucleic acid. Therefore, when analyzing nucleic acids, apart from the method for detecting nucleic acids in these patent publications, a method for amplifying a small amount of nucleic acids by extracting samples of nucleic acids is required. Therefore, it is difficult to perform nucleic acid analysis efficiently and quickly with a simple and small apparatus configuration and simple operation.
  • the nucleic acid N extraction process and the hybridization reaction can be performed simply and easily with only one substrate 102 with a simple and small apparatus configuration and simple operation. be able to.
  • the hybridization reaction solution H is removed from the hydrophilic cap region 102a, and the presence or absence of the hybridization reaction is detected by the amplified nucleic acid.
  • the target nucleic acid N1 hybridized with the detection probe in the target nucleic acid N1 can be easily and easily detected on the same substrate 102 and the same hydrophilic cap region 102a.
  • the sample processing apparatus 100 from the extraction of the nucleic acid N from the blood B, the amplification of the nucleic acid N, the hybridization between the nucleic acid N and the probe, and the detection of the hybridization reaction
  • the analysis of nucleic acid N can be performed in a series of flows without complications with a single substrate 102 with a simple and small apparatus configuration and simple operation.
  • the substrate 102 used in the sample processing apparatus 100 further includes a hydrophobic region 102b in addition to the hydrophilic cap region 102a, and the hydrophilic cap region 102a is surrounded by the hydrophobic region 102b.
  • a hydrophobic region 102b in addition to the hydrophilic cap region 102a, and the hydrophilic cap region 102a is surrounded by the hydrophobic region 102b.
  • the substrate 102 used in the sample processing apparatus 100 is provided with a plurality of hydrophilic cap region 102a on the surface thereof. As a result, a plurality of samples can be analyzed at the same time, and the multi-sample processing ability is excellent.
  • the area of the non-specific cell adsorption coat layer 102d or the leukocyte-specific cell adsorption coat layer 102e is the same between the hydrophilic capture regions 102a.
  • the amount of leukocytes adsorbed on the monolayer can be made constant between the hydrophilic capillaries 102a, and as a result, the amount of nucleic acid to be extracted becomes constant, and the nucleic acid can be extracted with good quantitativeness. Further, it is not necessary to measure the concentration of the extracted nucleic acid, and stable amplification of the nucleic acid is possible. That is, nucleic acid analysis is easy and easy, and detection accuracy is improved.
  • the shape of the hydrophilic cap region 102a is circular and the diameter thereof is 20 ⁇ m or more and 10,000 ⁇ m or less.
  • blood B and various substances can be held stably and surely, and a small amount of liquid droplets of about 260 ⁇ 1 (microliters), with a force of 2 pl (picolitol), can be held in a generally hemispherical shape. Is possible. This can reduce the amount of reagent used for blood clots and various substances.
  • sample used in the sample processing apparatus 100 is blood containing white blood cells, it can be suitably used in clinical examinations such as gene diagnosis such as gene deficiency and drug-sensitive SNP.
  • the sample processing method according to the present invention can be suitably used in various fields such as biopharmaceuticals and medical treatments, and particularly when nucleic acids are extracted from clinical specimens such as blood (for example, , Gene diagnosis such as gene deletion and drug sensitivity SNP) Can do.

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Abstract

La présente invention concerne un procédé simple de traitement d'un échantillon, grâce auquel le traitement d'un échantillon contenant des cellules, destiné à l'extraction d'un acide nucléique, peut être efficacement et rapidement réalisé en utilisant un appareil simple et de petite taille. Le procédé ne limite cependant pas l'environnement de travail, n'utilise pas de solvant ou de réactif, et il est particulièrement approprié à tester un spécimen clinique tel que du sang. Ce procédé de traitement d'échantillon consiste à utiliser un plateau comportant une zone dont la surface a été traitée avec un adsorbant de cellules, lequel est un matériau capable d'adsorber des cellules à sa surface, à placer un échantillon contenant des cellules dans la zone d'adsorption cellulaire, à extraire l'échantillon de la zone d'adsorption cellulaire tout en y laissant les cellules adsorbées par l'adsorbant cellulaire, et à sécher la zone d'adsorption cellulaire jusqu'à ce que les cellules soient disloquées de façon à ce qu'un acide nucléique contenu dans les cellules soit exposé et puisse être ainsi extrait desdites cellules.
PCT/JP2007/062601 2006-06-29 2007-06-22 Procédé de traitement d'échantillon Ceased WO2008001691A1 (fr)

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US9249445B2 (en) * 2008-09-02 2016-02-02 National Institute Of Advanced Industrial Science And Technology Cell detection method, and microarray chip for use in the method
US20140295415A1 (en) * 2011-11-04 2014-10-02 Diagnostics For All, Inc. Low cost, disposable molecular diagnostic devices

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2680462B2 (ja) * 1989-03-23 1997-11-19 アクゾ・エヌ・ヴエー 核酸の単離方法
WO2003085109A2 (fr) * 2002-04-09 2003-10-16 Genesystems Procede et automate d'extraction d'acides nucleiques a partir d'un melange complexe
WO2005066343A1 (fr) * 2003-12-30 2005-07-21 Agency For Science, Technology And Research Puce de purification d'acide nucleique
JP2005224787A (ja) * 2004-02-15 2005-08-25 Eiichi Tamiya 不溶性物質の分離方法及び不溶性物質分離デバイス

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2680462B2 (ja) * 1989-03-23 1997-11-19 アクゾ・エヌ・ヴエー 核酸の単離方法
WO2003085109A2 (fr) * 2002-04-09 2003-10-16 Genesystems Procede et automate d'extraction d'acides nucleiques a partir d'un melange complexe
WO2005066343A1 (fr) * 2003-12-30 2005-07-21 Agency For Science, Technology And Research Puce de purification d'acide nucleique
JP2005224787A (ja) * 2004-02-15 2005-08-25 Eiichi Tamiya 不溶性物質の分離方法及び不溶性物質分離デバイス

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