JP2008005781A - Specimen-treating method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a specimen-treating method by which the treatment of a specimen for extracting nucleic acids from the specimen containing cells can efficiently and quickly be performed with a simple and small device constitution and in simple operations without limiting a performance environment and without using a solvent and a reagent, and especially the treatment can suitably be performed, when a clinical specimen such as blood is targeted. <P>SOLUTION: This specimen-treating method of the present invention comprises setting a specimen containing cells to a cell-adsorbing region of a substrate on whose surface the cell-adsorbing region including regions surface-treated with a cell-adsorbing substance is disposed, removing the specimen from the cell-adsorbing region so as to leave the cells adsorbed to the cell-adsorbing substance, breaking the cells, and then drying the cell-adsorbing region until nucleic acids contained in the cells are exposed, thus extracting the nucleic acid contained in the cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  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.

  In recent years, gene diagnosis such as gene deletion and drug-sensitive SNP (Single Nucleotide Polymorphism) is spreading, and a method for extracting nucleic acid as a gene from a clinical specimen such as blood by an efficient and simple operation, There is a need for a method for simply analyzing nucleic acids, such as a method for simply amplifying nucleic acids.

  Examples of a method for separating or extracting nucleic acid from a sample containing nucleic acid include, for example, a phenol / chloroform extraction method, a method in which a nucleic acid-containing solution is precipitated with an ethanol solution (hereinafter referred to as an ethanol separation method). In some cases), a method disclosed in Patent Document 1, a method in which cells are lysed using an enzyme to separate nucleic acids (hereinafter sometimes referred to as an enzyme separation method), and a blood cell separation method using a centrifugation method There are a nucleic acid extraction method using magnetic beads, a nucleic acid extraction method using a filter, a method disclosed in Patent Document 2, and the like.

  In the phenol / chloroform extraction method, a sample component (protein, lipid, etc.) hardly soluble in water is denatured using phenol / chloroform to dissolve or precipitate the sample component. In the method disclosed in Patent Document 1 (commonly referred to as “Boom method”), contaminants such as proteins and lipids are solubilized in water using a chaotropic solution, and nucleic acid is adsorbed on silica beads as a solid phase carrier and recovered. The collected nucleic acid is washed, and the washed nucleic acid is dissolved again in the aqueous phase. In Patent Document 2, blood components are separated using a micro flow path.

Japanese Patent No. 2680462 JP 2005-224787 A

  However, according to the phenol / chloroform extraction method and the ethanol separation method, there is a problem that manpower is required and the method cannot be easily performed. Furthermore, according to the phenol / chloroform extraction method, since phenol / chloroform is used, there is a problem that the working environment is limited.

  Further, according to the method disclosed in Patent Document 1, operations such as recovery of the solid phase carrier and transfer of the container are required in the process until the nucleic acid is extracted, which makes the operation complicated. There was a problem.

  In addition, according to the enzyme separation method, a method similar to the method described above is used to separate nucleic acids, and thus there are the same problems as described above.

  Moreover, according to the blood cell separation method using the centrifugation 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 nucleic acid extraction method using magnetic beads is used. However, since a mechanism for collecting the beads by magnetism is required, there is a problem that it is difficult to downsize the entire apparatus. In addition, according to the nucleic acid extraction method using magnetic beads, the supernatant liquid is discharged in a state where the magnetic beads are pelleted by collecting the magnetic beads in the process until the nucleic acid is extracted. The liquid to be left may remain in the gaps between the beads, and as a result, the cleaning efficiency may be lowered. Therefore, according to the nucleic acid extraction method using magnetic beads, there is a problem that the operation becomes complicated because the washing must be repeated when the beads are sufficiently washed. In addition, according to the nucleic acid extraction method using magnetic beads, operations such as recovery of the solid-phase carrier made of magnetic beads and transfer of the container are required in the process until nucleic acid is extracted, which makes the work complicated. There was a problem that it was.

  Further, according to the method disclosed in Patent Document 2, the separation element itself for separating blood components can be reduced in size, but a connecting member, a pump for injecting and discharging a sample, Since a valve is required, there is a problem that it is difficult to downsize the entire apparatus.

  That is, according to the above-described method, when separating and extracting nucleic acid from a sample, simplicity, rapidity, degree of automation, and suitability for miniaturization are not always sufficient, and particularly when a clinical specimen such as blood is targeted. Has a problem that it is not always practical.

  Further, according to the above-described method, after nucleic acid is extracted from a sample, the container is transferred when nucleic acid analysis is performed such as nucleic acid amplification, hybridization between a nucleic acid and a probe, or detection of a hybridization reaction. The process from nucleic acid extraction to nucleic acid analysis can be performed with a simple and small device configuration and simple operation. There is a problem that it is difficult to carry out a series of flows without complications.

The present invention has been made in view of the above problems, and it is easy to process a sample such as extracting a nucleic acid from a sample containing cells without restricting the implementation environment or using a solvent or a reagent. An object of the present invention is to provide a sample processing method that can be performed efficiently and quickly with a small apparatus configuration and simple operation, and that can be suitably used particularly for clinical specimens such as blood. To do.
In addition, the present invention enables nucleic acid analysis such as nucleic acid amplification, hybridization between a nucleic acid and a probe, and detection of a hybridization reaction after extraction of the nucleic acid from a sample with a simple and small apparatus configuration and simple operation. An object of the present invention is to provide a sample processing method that can be performed in a series of flows without complications.

  In order to solve the above-described problems and achieve the object, the sample processing method according to claim 1 according to the present invention is a sample processing method for processing a sample containing cells, wherein the cells are substances that adsorb the cells. Using a substrate having a cell adsorption region including a region surface-treated with an adsorbent on its surface, a sample placing step for placing the sample in the cell adsorption region, and after performing the sample placing step, the cell adsorption The nucleic acid contained in the cell by performing the sample removal step of removing the sample from the cell adsorption region and the sample removal step so that the cell adsorbed on the substance remains, and then the cell being 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 cell is exposed.

  The sample processing method according to claim 2 of the present invention is the sample processing method according to claim 1, wherein the sample for selectively destroying the predetermined cells after performing the sample removal step. After performing the destructive substance installation step of placing the destructive substance as a substance in the cell adsorption region and the destructive substance installation step, the destructive substance and the cells destroyed by the destructive substance are removed from the cell adsorption region A destructive substance removal step is further performed, and the nucleic acid exposure drying step is characterized by drying the cell adsorption region after the destructive substance removal step.

  In addition, the sample processing method according to claim 3 according to the present invention is the sample processing method according to claim 1 or 2, wherein the substance for amplifying the nucleic acid after the nucleic acid exposure drying step is performed. Amplifying substance placing step for placing a certain amplifying substance in the cell adsorption region, and after performing the amplifying substance placing step, to prevent evaporation of the amplifying substance so as to cover the extracted nucleic acid and the amplifying substance The anti-transpiration material placement step of placing the anti-transpiration material, which is the substance, in the cell adsorption region, and the temperature exposure step of exposing the cell adsorption region to a predetermined temperature condition after performing the anti-transpiration material placement step. Further, the nucleic acid is amplified by executing the method.

  Moreover, the sample processing method according to claim 4 according to the present invention is the sample processing method according to claim 3, wherein the cell adsorption region includes one or a plurality of types including a sequence complementary to a target nucleic acid sequence. After the execution of the temperature exposure step, after performing the temperature exposure step, after performing the anti-transpiration material removal step of removing the anti-transpiration material from the cell adsorption region, and after performing the anti-transpiration material removal step An adsorption region drying step for drying the cell adsorption region; and a reaction substance that is the substance for performing a hybridization reaction between the nucleic acid and the probe after the adsorption region drying step is performed. The reactant amplification step, and the temperature holding step of maintaining the cell adsorption region at a predetermined temperature after executing the reactant placement step, are amplified. And performing the hybridization reaction serial with the nucleic acid and the probe.

  The sample processing method according to claim 5 according to the present invention is the sample processing method according to claim 4, wherein after the temperature holding step is performed, the reactant removal that removes the reactant from the cell adsorption region. And a reaction detection step of detecting the presence or absence of the hybridization reaction with respect to the amplified nucleic acid after performing the reaction substance removal step.

  The sample processing method according to claim 6 of the present invention is the sample processing method according to any one of claims 1 to 5, wherein the substrate has a hydrophobic region that is the hydrophobic region. In addition, the cell adsorption region is hydrophilic and is surrounded by the hydrophobic region.

  The sample processing method according to claim 7 of the present invention is the sample processing method according to any one of claims 1 to 6, wherein the substrate is provided with a plurality of the cell adsorption regions on the surface thereof. It is characterized by being.

  The sample processing method according to claim 8 according to the present invention is the sample processing method according to claim 7, wherein, in each of the cell adsorption regions, the area of the region surface-treated with the cell adsorption material is It is characterized by being the same.

  The sample processing method according to claim 9 according to the present invention is the sample processing method according to any one of claims 1 to 8, wherein the cell adsorption region has a circular shape and a diameter of 20 μm. It is above and 10,000 micrometers or less, It is characterized by the above-mentioned.

  The sample processing method according to claim 10 according to the present invention is the sample processing method according to any one of claims 1 to 9, wherein the cells are leukocytes and the sample is blood. Features.

  According to the present invention, 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. The sample is removed from the cell adsorption area so that the adsorbed cells remain, and the nucleic acid contained in the cells is extracted by drying the cell adsorption area until the nucleic acids contained in the cells are exposed by the destruction of the cells. Therefore, sample processing, such as extraction of nucleic acids from samples containing cells, can be performed efficiently and quickly with a simple and compact device configuration and simple operation, without limiting the working environment or using solvents or reagents. In particular, there is an effect that it can be suitably used when a clinical specimen such as blood is targeted. In addition, according to the present invention, nucleic acid analysis such as nucleic acid amplification, hybridization between a nucleic acid and a probe, and detection of a hybridization reaction can be performed after extraction of the nucleic acid from a sample. There is an effect that the operation can be performed in a series of flows without complications.

  Embodiments of a sample processing method according to the present invention will be described below in detail with reference to the drawings. In addition, this invention is not limited by this Embodiment.

  First, the configuration of a sample processing apparatus 100 that is an apparatus for carrying out the sample processing method according to the present embodiment will be described with reference to FIG. FIG. 1 is a diagram illustrating an example of a configuration of a sample processing apparatus 100 according to the present embodiment. As shown in FIG. 1, 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, a fluorescent scanner 114, and the like. , And a control device 116.

  The substrate 102 is provided with a cell adsorption region including a region surface-treated with a cell adsorption material, which is a material that specifically or non-specifically adsorbs cells, and is specifically a slide glass. Here, an example of the board | substrate 102 concerning this Embodiment is demonstrated with reference to FIG.2 and FIG.3 and FIG.4 and FIG.5. 2 to 5 are perspective views showing an example of the substrate 102 according to the present embodiment.

  The substrate 102 shown in FIG. 2 has a total of 48 (4 rows and 12 columns) circular hydrophilic capture regions 102a having a diameter of 1.6 mm surrounded by a hydrophobic region 102b arranged at equal intervals on the surface thereof. The hydrophilic capture 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 a ring shape as shown in FIG. The hydrophilic capture 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 surround the hydrophilic capture region by avoiding the hydrophilic capture region 102a by, for example, a method of printing a fluororesin or the like.

Here, the details of the hydrophilic capture region 102a will be described with reference to FIG. The hydrophilic capture region 102a is partially provided with a non-specific cell adsorption coat layer 102d surface-treated with lectin as shown in FIG. The non-specific cell adsorption coat layer 102d is formed with the same area (for example, 300 μm ² to 78 mm ² ) between the hydrophilic capture regions 102a by, for example, a method such as screen printing. In addition, as shown in FIG. 3, the hydrophilic capture region 102a has a detection probe spot 102c in which one or a plurality of types of detection probes including a sequence complementary to a target nucleic acid sequence is arranged via an appropriate linker. Partitions are divided in the region of the non-specific cell adsorption coat layer 102d and arranged in spots at regular intervals. In addition, by arranging a plurality of detection probes, a plurality of target nucleic acids can be detected and analyzed simultaneously, and the multi-item analytical performance is excellent.

  In the substrate 102 shown in FIG. 4, a total of 48 circular capture regions 102a having a diameter of 1.6 mm surrounded by a hydrophobic region 102b are arranged on the surface at equal intervals (4 rows and 12 columns). The hydrophilic capture 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. . Examples of the leukocyte-specific antibody include “Dynabeads HLA Class I 2 ml” (model number: DB21001) manufactured by Invitrogen, and “Lymph T / B Quick 50tests” (model number: LK-50) manufactured by Wan Lambda. -TB) and other antibodies used for beads. The hydrophobic region 102b is a hydrophobic region that surrounds the hydrophilic capture region 102a provided on the entire surface of the substrate 102 as shown in FIG. The hydrophilic capture 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 surround the hydrophilic capture region by avoiding the hydrophilic capture region 102a by, for example, a method of printing a fluororesin or the like.

Here, the details of the hydrophilic capture region 102a will be described with reference to FIG. The hydrophilic capture 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 coat layer 102e is formed with the same area (for example, 300 μm ² to 78 mm ² ) between the hydrophilic capture regions 102a by a method such as screen printing. As shown in FIG. 5, the hydrophilic capture region 102a has a detection probe spot 102c in which one or a plurality of types of detection probes including a sequence complementary to a target nucleic acid sequence is arranged via an appropriate linker. The white blood cell-specific cell-adsorbing coat layer 102e is divided into spots and arranged in spots at regular intervals.

  In addition, the board | substrate concerning this invention is not limited to the board | substrate 102 shown to FIGS. 2-5 mentioned above concerning this Embodiment. For example, the substrate 102 is not limited to a slide glass. Further, the arrangement state and the number of the hydrophilic capture regions 102a are not limited to those shown in FIGS.

  Further, the shape of the hydrophilic capture region 102a is not limited to the above-described circular shape having a diameter of 1.6 mm, and may be, for example, a substantially circular shape (for example, including an ellipse) having a diameter of 20 μm to 10,000 μm, or other shapes. . In addition, when the shape of the hydrophilic capture region 102a is a substantially circular shape having a diameter of 20 μm to 10,000 μm, the hydrophilic capture region 102a depends on the shape of the sample, the destructive substance, the amplification substance, and the transpiration substance according to the present invention. And a liquid such as a reaction substance can be stably and reliably held, and a small amount of droplets of about 2 pl (picoliters) to 260 μl (microliters) is held in a substantially hemispherical shape depending on the diameter. be able to. As a result, the reagent used for the sample and various substances can be effectively reduced.

  Further, 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 capture region 102a, and may be provided in the entire hydrophilic capture region 102a. You may provide in the whole hydrophilic capture area | region 102a except the probe spot 102c.

  The hydrophobic region 102b may be provided so as to surround the hydrophilic capture region 102a, and may be provided in an annular shape as shown in FIGS. 2 and 3, or on the surface of the substrate 102 as shown in FIGS. In addition to providing the entire region, for example, it may be partially provided on the surface of the substrate 102 excluding the hydrophilic capture region 102a. Further, the arrangement state and the number of detection probe spots 102c are not limited to those shown in FIGS.

  Returning to FIG. 1, the microtiter plate 104 is a sample according to the present invention placed on the hydrophilic capture region 102 a and various substances according to the present invention (for example, a destructive substance, an amplification substance, a transpiration substance, a reactive substance according to the present invention, Furthermore, a buffer solution or the like) is held separately.

  Here, the sample includes at least nucleated cells, such as blood composed of whole blood anticoagulated with heparin or the like, or a buffy coat composed mainly of white blood cells from which blood components have been separated. . The destructive substance is a substance for selectively destroying a predetermined cell, and is, for example, a lysate with 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 a nucleic acid, and is, for example, an amplification reaction solution containing a PCR (Polymerase Chain Reaction) reagent. The anti-transpiration material is a material for preventing evaporation such as an amplification material and a reaction material, and is, for example, a sealing oil such as mineral oil or silicone oil. As mineral oil, there is a mineral oil “M8662” manufactured by Sigma-Aldrich, and as a silicone oil, there is a silicone oil for PCR “10890-010” manufactured by Invitrogen. The reaction substance is a substance for performing a hybridization reaction between the nucleic acid and the detection probe, for example, a hybridization reaction solution.

  The dispensing device 106 includes a multi-pipter 106 a, and the multi-pipetter 106 a sucks a sample and various substances (for example, a destructive substance, an amplification substance, a vapor-proofing substance, a reactive substance, and a buffer solution) from the microtiter plate 104. Or a sample or various substances sucked are dispensed into the hydrophilic capture region 102a. The thermal cycler 108 applies a thermal cycle related to a temperature condition necessary for the PCR reaction to the substrate 102 or each hydrophilic capture region 102a, or maintains the substrate 102 or each hydrophilic capture region 102a at room temperature or a predetermined temperature. Or

  The ejection device 110 includes an ejection head 110a. The ejection head 110a sucks a sample and various substances placed in each hydrophilic capture area 102a, and the sucked sample and various substances are collected in another hydrophilic capture area. Discharge to 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. Specifically, the control device 116 is a commercially available personal computer, and is connected to the dispensing device 106, the thermal cycler 108, the ejection device 110, the ejection head cleaning device 112, and the fluorescent scanner 114 so as to be communicable. 106, the thermal cycler 108, the ejection device 110, the ejection head cleaning device 112, and the fluorescent scanner 114 are controlled. The control device 116 has a function of receiving the 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.

  Next, a sample processing method executed by the sample processing apparatus 100 described above will be described with reference to FIGS. 6 and 7 are diagrams illustrating an example of a sample processing method executed by the sample processing apparatus 100. FIG.

  A blood B consisting of whole blood anti-coagulated with heparin containing white blood cells W and red blood cells R, and a lysis solution So with a surfactant adjusted in salt concentration so as to selectively dissolve red blood cells; An amplification reaction solution P containing a PCR reagent containing a fluorescently labeled primer, a sealing oil S, a hybridization reaction solution H, and a buffer solution are placed in a predetermined section of the microtiter plate 104 and shown in FIGS. When the substrate 102 is placed at a predetermined position, various conditions are set via an input device such as a keyboard and a mouse on the start menu screen displayed on the monitor of the control device 116, and the start button on the start menu screen is pressed. The sample processing apparatus 100 executes the sample processing method shown in FIG.

  First, the control device 116 issues a command to the dispensing device 106. When the dispensing device 106 receives the command, the multipipetter 106a sucks the blood B from the microtiter plate 104, and the sucked blood B is sent to the respective dispensing devices 106a. About 1 μl (microliter) is dropped on the hydrophilic capture region 102a (step 1: sample placement step). Thereby, the blood B is held as a hemispherical droplet in the hydrophilic capture region 102a, and when a predetermined time has elapsed from the dropping, a part of the blood cell contained in the blood B is simply deposited on the leukocyte-specific cell adsorption coat layer 102e by sedimentation or the like. Of the blood cells in contact with each other in the form of a single layer, only white blood cells W are adsorbed to the leukocyte-specific cell adsorption coat layer 102e.

  Next, the control device 116 issues a command to the dispensing device 106 when a predetermined time elapses after completing Step 1, and the dispensing device 106 adsorbs to the leukocyte-specific cell adsorption coat layer 102e upon receiving the command. The blood B is removed from each hydrophilic capture region 102a by the multi-pipettor 106a so that the white blood cells W are left (step 2: sample removal step). Since the leukocytes are adsorbed on the leukocyte-specific cell adsorption coat layer 102e, the blood B can be removed from the hydrophilic capture region 102a by being sucked by the multipipetter 106a of the dispensing device 106. When the blood B is sucked by the multi-pipetter 106a, the tip of the nozzle and the leukocyte-specific cell adsorption coat are not destroyed at the tip of the nozzle of the multi-pipetter 106a so as to prevent destruction of the leukocytes W adsorbed on the leukocyte-specific cell adsorption coat layer 102e. It is desirable to provide a gap about the thickness of white blood cells (about several tens of μm) between the layer 102e.

  Here, when the substrate 102 shown in FIGS. 2 and 3 is used, the red blood cells R and the like other than the white blood cells W are adsorbed to the non-specific cell adsorption coating layer 102d in a single layer when Step 2 is completed. Since there is a possibility (see step 2 shown in FIG. 7), when the substrate 102 is used, the controller 116 instructs the dispensing device 106 after completing step 2 as shown in FIG. When the dispenser 106 receives the instruction, the dispensing device 106 sucks the lysate So from the microtiter plate 104 by the multi-pipetter 106a, and about 1 μl (microliter) of the sucked lysate So in each hydrophilic capture region 102a. Liters) (step 2 ′: destructive substance placement step), and after a predetermined time has passed, red blood cells R and the like are removed from each hydrophilic capture region 102a by the multi-pipettor 106a. May be removed lysate So that solution (Step 2 ': depleting substances such as removing step). As a result, the lysate So is retained as a hemispherical droplet in the hydrophilic capture region 102a, 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 eluted in the lysis solution So, and only the white blood cells W can be adsorbed to the non-specific cell adsorption coat layer 102d. That is, hemoglobin that inhibits nucleic acid amplification by dissolving erythrocytes R can be efficiently and effectively removed together with the lysis solution So. As a result, the step 4 relating to the amplification of the nucleic acid can be efficiently performed, and as a result, the accuracy in the step 5 relating to the hybridization reaction between the nucleic acid and the detection probe and the step 6 relating to the detection of the hybridization reaction can be improved. it can.

  Returning to FIG. 6, the control device 116 issues a command to the thermal cycler 108, and upon receipt of the command, the thermal cycler 108 destroys the leukocytes W until the nucleic acids contained in the leukocytes W are exposed, or each of the substrates 102 or each of them. By holding the hydrophilic capture region 102a at room temperature or an appropriate temperature, the water on the surface of each hydrophilic capture region 102a is evaporated, and each hydrophilic capture region 102a is dried (step 3: nucleic acid exposure drying). Process).

  As described above, the sample processing such as extraction of the nucleic acid N from the blood B by the steps 1 to 3 can be performed with a simple and small apparatus configuration and a simple operation without limiting the working environment or using a solvent or a reagent. Therefore, it was possible to carry out efficiently and quickly. In addition, the nucleic acid N contained in the leukocytes W could be easily and easily extracted from a very small amount of blood B. Thereby, a test subject's physical and mental burden can be reduced.

Next, the control device 116 issues a command to the dispensing device 106, and when the dispensing device 106 receives the command, the amplification reaction liquid P is sucked from the microtiter plate 104 by the multipipette 106a, and the sucked amplification reaction. About 1 μl (microliter) of the liquid P is dropped on each hydrophilic capture region 102a (step 4: amplification substance placing step). As a result, the amplification reaction solution P is held in the hydrophilic capture region 102a as a hemispherical droplet.
Then, the control device 116 continues to issue a command to the dispensing device 106. Upon receiving the command, the dispensing device 106 sucks the sealing oil S from the microtiter plate 104 by the multi-pipetter 106a, and sucks the sealing oil S About 5 μl (microliter) is dropped onto each hydrophilic capture region 102a so as to cover the nucleic acid N and the amplification reaction solution P (on the upper layer of the nucleic acid N and the amplification reaction solution P as shown in FIG. 6) (step 4). : Anti-transpiration material installation process). As a result, the amplification reaction solution P is sealed to prevent evaporation of the amplification reaction solution P in the thermal cycle.
Then, the controller 116 issues a command to the thermal cycler 108. Upon receiving the command, the thermal cycler 108 applies a thermal cycle necessary for the PCR reaction to the substrate 102 or each hydrophilic capture region 102a (step 4). : Temperature exposure process).

  As described above, the nucleic acid N extracted in the step 3 can be simply and easily amplified in the same substrate 102 and the same hydrophilic capture region 102a by the step 4. In other words, the process from the extraction of the nucleic acid N to the amplification of the nucleic acid N is carried out with only one substrate 102 without requiring a container transfer operation or a new container as in the prior art between the steps 1 to 4. Was easily and easily performed. Further, in step 4, a fluorescent label could be introduced into the amplified nucleic acid N. As a result, the amount of expensive reagents such as amplification reagents used can be reduced. The nucleic acid amplification method is not limited to the PCR method shown in step 4, and may be, for example, an isothermal amplification method or other various known amplification methods.

Next, the control device 116 issues a command to the dispensing device 106. Upon receiving the command, the dispensing device 106 removes the sealing oil S from each hydrophilic capture region 102a by the multipipette 106a (Step 5: Anti-transpiration material removal process).
Then, the control device 116 issues a command to the thermal cycler 108. Upon receipt of the command, the thermal cycler 108 holds each substrate 102 or each hydrophilic capture region 102a at room temperature or an appropriate temperature, thereby maintaining each hydrophilic property. The property capture region 102a is dried (step 5: adsorption region drying step).
Then, the control device 116 issues a command to the dispensing device 106. Upon receiving the command, the dispensing device 106 sucks the hybridization reaction solution H from the microtiter plate 104 by the multi-pipette 106a and sucks the sucked hybridization. About 1 μl (microliter) of the reaction solution H is dropped on each hydrophilic capture region 102a (step 5: reactant mounting step). As a result, the hybridization reaction solution H is held in the hydrophilic capture region 102a as a hemispherical droplet. When the amplification reaction solution P is further mixed with reagents for the hybridization reaction, this step can be omitted.
Here, the control device 116 issues a command to the dispensing device 106 following the reactant placing step, and when receiving the command, the dispensing device 106 sucks the sealing oil S from the microtiter plate 104 by the multi-pipette 106a. The aspirating sealing oil S is applied to the upper layer of the target nucleic acid N1 and the hybridization reaction solution H so as to cover the nucleic acid N (including the fluorescently labeled target nucleic acid N1) and the hybridization reaction solution H (as shown in FIG. 6). ) About 5 μl (microliter) may be dropped on each hydrophilic capture region 102a. Accordingly, the sealing oil S is held in the hydrophilic capture region 102a as a hemispherical droplet.
Then, the control device 116 issues a command to the thermal cycler 108. Upon receiving the command, the thermal cycler 108 holds the substrate 102 or each hydrophilic capture region 102a at room temperature, or the substrate 102 or each hydrophilic capture region. An appropriate temperature (for example, a temperature of 60 ° C.) is applied to the region 102a to hold the temperature (step 5: temperature holding step).

  As described above, by the step 5, the hybridization reaction between the target nucleic acid N1 fluorescently labeled in the step 4 and the detection probe can be easily and easily performed on the same substrate 102 and the same hydrophilic capture region 102a. In other words, the steps from the extraction of the nucleic acid N to the hybridization reaction are carried out with only one substrate 102 without the need for a container transfer operation or a new container as in the prior art between Step 1 and Step 5. It was easy and easy. The hybridization method is not limited to the hybridization method shown in step 5. For example, in step 4, a fluorescent label is not introduced, and in step 5, the target nucleic acid N1 is detected using an intercalator. A hybridization reaction with a probe may be performed.

Next, the control device 116 issues a command to the dispensing device 106. Upon receiving the command, the dispensing device 106 removes the hybridization reaction solution H with the multipipette 106a (step 6: reactant removal step). When the sealing oil S is dropped on the upper layer of the hybridization reaction solution H in step 5, the dispensing device 106 removes the sealing oil S and the hybridization reaction solution H with the multipipette 106a.
Here, 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 multipipetter 106a sucks the buffer solution from the microtiter plate 104, A suitable amount of the sucked buffer solution is dropped on each hydrophilic capture region 102a to clean each hydrophilic capture region 102a, and the controller 116 issues a command to the thermal cycler 108. The thermal cycler 108 Upon receipt, each hydrophilic capture region 102a may be dried by holding the substrate 102 or each hydrophilic capture region 102a at room temperature or at an appropriate temperature.
Then, the operator inserts the substrate 102 into the substrate insertion portion 114a of the fluorescent scanner 114.
Then, the control device 116 issues a command to the fluorescent scanner 114. Upon receiving the command, the fluorescent scanner 114 confirms whether or not the substrate 102 is properly inserted into the substrate insertion portion 114a, and the substrate 102 is properly inserted. When the substrate is scanned, the fluorescence image data of the scanned substrate 102 is transferred to the control device 116. When the control device 116 receives the fluorescence image data transferred from the fluorescence scanner 114, 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).

  As described above, in Step 6, the target nucleic acid N11 hybridized with the detection probe among the fluorescently labeled target nucleic acids N1 can be detected easily and easily on the same substrate 102 and also on the same hydrophilic capture region 102a. In other words, from step 1 to step 6, from the extraction of the nucleic acid N to the detection of the hybridization reaction with only one substrate 102 without the need for a container transfer operation or a new container as in the prior art. The process was simple and easy.

  As described above in detail, according to the sample processing apparatus 100 according to the present embodiment, the hydrophilic capture region 102a including the leukocyte-specific cell adsorption coat layer 102e surface-treated with the leukocyte-specific antibody is provided on the surface thereof. The hydrophilic capture region 102a is used so that the blood B containing white blood cells W such as whole blood is placed in the hydrophilic capture region 102a and the white blood cells W adsorbed on the hydrophilic capture region 102a remain. Since the hydrophilic capture region 102a is dried so that the nucleic acid contained in the leukocyte is exposed by removing the blood B from the blood B and destroying the leukocyte W, the nucleic acid contained in the leukocyte W is extracted. Sample processing such as extraction of nucleic acid N from blood B containing without restricting the implementation environment or using solvents or reagents , A simple, compact apparatus configuration and simple operation, can be done efficiently and quickly. In other words, “nucleic acid extraction from blood” related to nucleic acid analysis can be performed easily and easily. In addition, according to the sample processing apparatus 100, since the substrate 102 as shown in FIGS. 2 to 5 is used, the amount of blood and the amount of various reagents used in clinical tests in gene diagnosis such as gene deletion and drug-sensitive SNP are determined. As a result, the physical burden and the mental burden on the subject can be reduced, and the examination cost can be reduced.

  Further, according to the sample processing apparatus 100, when the substrate 102 (the substrate 102 having the nonspecific cell adsorption coat layer 102d) shown in FIGS. 2 and 3 is used, the blood B containing white blood cells W such as whole blood. Is placed in the hydrophilic capture region 102a and blood B is removed from the hydrophilic capture region 102a so that the white blood cells W adsorbed on the hydrophilic capture region 102a remain, and then the lysate So is added to each hydrophilic capture region 102a. Since the lysate So may be removed from each hydrophilic capture region 102a after a predetermined time has elapsed, the blood cells adsorbed on the hydrophilic capture region 102a are unnecessary and are not necessary for nucleic acid analysis. It is possible to selectively and effectively eliminate red blood cells R having hemoglobin as a substance, thereby improving the accuracy of nucleic acid analysis. Rukoto can.

Further, according to the sample processing apparatus 100, the amplification reaction solution P is placed in the hydrophilic capture region 102a, and the sealing oil S is placed in the hydrophilic capture region 102a so as to cover the extracted nucleic acid N and the amplification reaction solution P. Since the nucleic acid N is amplified by exposing the sexual capture 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 capture region 102a.
So far, as a technique related to nucleic acid amplification, for example, Japanese Patent No. 3743090, which detects nucleic acid in a region having hydrophilicity, has been disclosed. Specifically, in the patent publication, 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. However, in this patent publication, in order to analyze nucleic acids, an additional element for extracting and detecting nucleic acids is required. Therefore, nucleic acid analysis can be performed efficiently and quickly with a simple and small apparatus configuration and simple operation. Difficult to do.
However, according to the sample processing apparatus 100, the steps from the extraction of the nucleic acid N to the amplification of the nucleic acid N can be performed simply and easily with only one substrate 102 with a simple and small apparatus configuration and simple operation.

Further, according to the sample processing apparatus 100, the sealing oil S is removed from the hydrophilic capture region 102a, the hydrophilic capture region 102a is dried, the hybridization reaction solution H is placed in the hydrophilic capture region 102a, and the hydrophilic capture region 102a. Is kept at a predetermined temperature to carry out a hybridization reaction between the amplified nucleic acid N and the probe, so that the hybridization reaction between the target nucleic acid N1 and the detection probe is performed on the same substrate 102 and also on the same hydrophilic capture region 102a. It can be carried out simply and easily.
Until now, as a technique related to the hybridization reaction, a patent publication of Japanese Patent No. 3386391, a patent publication of Japanese Patent No. 3393528, a patent publication of Japanese Patent No. 3625826, and the like have been disclosed. Japanese Patent No. 3386391 discloses a method in which a probe including a sequence complementary to a target nucleic acid is provided on a flat substrate in divided areas. Japanese Patent No. 3393528 discloses a method for detecting the presence or absence of a target nucleic acid by a hybridization reaction with a sample nucleic acid provided with a label or the like. Japanese Patent No. 3625826 discloses a method in which a droplet isolated by surface tension is chemically reacted, and in particular, a nucleic acid is detected by hybridization with a probe. However, these patent publications do not disclose a technique related to the extraction of nucleic acids from a sample or amplification of a small amount of extracted nucleic acids. Therefore, when analyzing nucleic acids, what are the nucleic acid detection methods in these patent publications? In addition, since a method for amplifying a small amount of nucleic acid by extracting a nucleic acid from a sample is required, it is difficult to analyze nucleic acid efficiently and quickly with a simple and small apparatus configuration and simple operation.
However, according to the sample processing apparatus 100, the steps from the extraction of the nucleic acid N to the hybridization reaction can be performed simply and easily with only one substrate 102 with a simple and small apparatus configuration and simple operation.

  Further, according to the sample processing apparatus 100, the hybridization reaction solution H is removed from the hydrophilic capture region 102a, and the presence or absence of a hybridization reaction is detected for the amplified nucleic acid. Therefore, the target hybridized with the detection probe in the target nucleic acid N1. The nucleic acid N11 can be easily and easily detected on the same substrate 102 and the same hydrophilic capture region 102a.

  As described above, according to the sample processing apparatus 100, one substrate is used from the extraction of the nucleic acid N from the blood B to the analysis of the nucleic acid N such as amplification of the nucleic acid N, hybridization of the nucleic acid N and the probe, and detection of the hybridization reaction. With 102, a simple and small device configuration and simple operation can be performed in a series of flows without complications.

  In addition, the substrate 102 used in the sample processing apparatus 100 further includes a hydrophobic region 102b in addition to the hydrophilic capture region 102a, and the hydrophilic capture region 102a is surrounded by the hydrophobic region 102b. The substance can be reliably held in the hydrophilic capture region 102a. In addition, since the substrate 102 used in the sample processing apparatus 100 is provided with a plurality of hydrophilic capture regions 102a on the surface thereof, a plurality of samples can be analyzed at the same time, and the multi-sample processing ability is excellent. Further, in the substrate 102 used in the sample processing apparatus 100, since 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 single layer adsorption is performed. The amount of white blood cells can be made constant between the hydrophilic capture regions 102a. As a result, the amount of nucleic acid to be extracted becomes constant, and nucleic acids 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 simple and easy, and detection accuracy is improved. Further, in the substrate 102 used in the sample processing apparatus 100, the shape of the hydrophilic capture region 102a is circular and the diameter thereof is 20 μm or more and 10,000 μm or less, so that the blood B and various substances can be stably and reliably held. In addition, a small amount of droplets of about 2 pl (picoliter) to 260 μl (microliter) can be held in a substantially hemispherical shape. Thereby, the quantity of the reagent used for the blood B and various substances can be reduced.

  In addition, since the sample used in the sample processing apparatus 100 is blood containing white blood cells, it can be suitably used in clinical tests such as gene diagnosis such as gene deletion and drug-sensitive SNP.

  As described above, the sample processing method according to the present invention can be suitably used in various fields such as biotechnology, pharmaceuticals, and medicine, and particularly when nucleic acids are extracted from clinical specimens such as blood (for example, gene deletion) And gene diagnosis such as drug-sensitive SNPs).

It is a figure which shows an example of a structure of the sample processing apparatus 100 concerning this Embodiment. It is a perspective view which shows an example of the board | substrate 102 concerning this Embodiment. It is a perspective view which shows an example of the board | substrate 102 concerning this Embodiment. It is a perspective view which shows an example of the board | substrate 102 concerning this Embodiment. It is a perspective view which shows an example of the board | substrate 102 concerning this Embodiment. FIG. 3 is a diagram illustrating an example of a sample processing method executed by the sample processing apparatus 100. FIG. 3 is a diagram illustrating an example of a sample processing method executed by the sample processing apparatus 100.

Explanation of symbols

100 Sample processing equipment
102 substrates
102a Hydrophilic capture region
102b Hydrophobic region
102c Detection probe spot
102d Non-specific cell adsorption coating layer
102e leukocyte specific cell adsorption coat layer
104 Microtiter plate
106 Dispensing device
106a Multi Pipetter
108 thermal cycler
110 Discharge device
110a Discharge head
112 Discharge head cleaning device
114 Fluorescent scanner
116 Control device

Claims (10)

In a sample processing method for processing a sample containing cells,
Using a substrate provided with a cell adsorption region on the surface thereof, including a region surface-treated with a cell adsorption material that is a substance that adsorbs the cells,
A sample placement step of placing the sample in the cell adsorption region;
A sample removal step of removing the sample from the cell adsorption region so that the cells adsorbed to the cell adsorption material remain after the sample placement step;
After performing the sample removal step, the nucleic acid exposure drying step of drying the cell adsorption region until the nucleic acid contained in the cell is exposed by the destruction of the cell,
By running
A sample processing method, wherein the nucleic acid contained in the cell is extracted. After performing the sample removal step, a destructive substance placement step of placing a destructive substance, which is the substance for selectively destroying the predetermined cells, in the cell adsorption region,
After performing the destructive substance placing step, further performing a destructive substance removing step for removing the destructive substance and the cells destroyed by the destructive substance from the cell adsorption region,
The sample processing method according to claim 1, wherein, in the nucleic acid exposure drying step, the cell adsorption region is dried after the destructive substance removal step is executed. After performing the nucleic acid exposure and drying step, an amplification substance placement step of placing the amplification substance, which is the substance for amplifying the nucleic acid, in the cell adsorption region;
After performing the amplification substance installation step, the anti-transpiration substance, which is the substance for preventing evaporation of the amplification substance so as to cover the extracted nucleic acid and the amplification substance, is placed in the cell adsorption region. Substance detention process;
A temperature exposure step of exposing the cell adsorption region to a predetermined temperature condition after performing the anti-fusible material placing step;
By further executing
The sample processing method according to claim 1 or 2, wherein the nucleic acid is amplified. The cell adsorption region is provided with one or a plurality of types of probes including a sequence complementary to a target nucleic acid sequence, divided into sections,
After performing the temperature exposure step, the anti-fusogenic material removing step of removing the anti-fusible material from the cell adsorption region,
An adsorption region drying step of drying the cell adsorption region after performing the anti-transpiration material removing step;
After performing the adsorption region drying step, a reaction material placing step of placing the reaction material, which is the material for performing a hybridization reaction between the nucleic acid and the probe, in the cell adsorption region;
A temperature maintaining step for maintaining the cell adsorption region at a predetermined temperature after performing the reactant placing step;
By running
The sample processing method according to claim 3, wherein the hybridization reaction between the amplified nucleic acid and the probe is performed. A reactant removal step of removing the reactant from the cell adsorption region after performing the temperature holding step;
5. The sample processing method according to claim 4, wherein after performing the reactant removal step, a reaction detection step of detecting the presence or absence of the hybridization reaction is performed on the amplified nucleic acid. The substrate further includes a hydrophobic region that is the hydrophobic region,
The sample processing method according to claim 1, wherein the cell adsorption region is hydrophilic and is surrounded by the hydrophobic region. The sample processing method according to claim 1, wherein a plurality of the cell adsorption regions are provided on a surface of the substrate. 8. The sample processing method according to claim 7, wherein in each of the cell adsorption regions, the area of the region surface-treated with the cell adsorption material is the same. The sample processing method according to any one of claims 1 to 8, wherein the cell adsorption region has a circular shape and has a diameter of 20 µm or more and 10,000 µm or less. The sample processing method according to claim 1, wherein the cells are white blood cells, and the sample is blood.

Images