JP2020042051A - Sample pretreatment cartridge - Google Patents

Abstract

To provide a sample pretreatment cartridge capable of reducing time required for pretreatment and handling a large or small amount of blood plasma.SOLUTION: A sample pretreatment cartridge 10 provided herein is used for pretreatment for extracting nucleic acids from blood plasma samples. The sample pretreatment cartridge 10 is integrally formed with multiple reaction containers 11 to allow a blood plasma sample to be dispensed in accordance with the amount of the blood plasma sample. The blood plasma sample is dispensed to a number of reaction containers 11 in accordance with the amount of the blood plasma sample.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a sample pretreatment cartridge.

  When analyzing nucleic acids contained in plasma, pretreatment for extracting nucleic acids from plasma is performed. For example, the pretreatment includes a step of isolating the nucleic acid by decomposing the protein bound to the nucleic acid, a step of adsorbing the isolated nucleic acid to the magnetic particles, and a step of washing impurities adsorbed on the magnetic particles. Releasing the nucleic acid from the magnetic particles to extract the nucleic acid. Patent Document 1 discloses that a nucleic acid is extracted from a specimen using a single reaction container having a capacity of about 1 mL, and nucleic acids derived from a target bacterium or virus among the extracted nucleic acids are amplified by PCR to obtain a target nucleic acid. An analyzer for performing an analysis is described.

JP-T-2015-514223 A

  In the analyzer of Patent Literature 1, a single reaction container having a capacity of about 1 mL is used to extract nucleic acids from a small amount of plasma of about 25 μL to 75 μL. However, when nucleic acids are extracted from a large amount of plasma of 1 mL or more, in a reaction vessel having a volume of about 1 mL, the process of extracting nucleic acids from a small amount of plasma must be performed in a plurality of times. Take it. In particular, when extracting and analyzing nucleic acids derived from cancer cells, since the amount of nucleic acids derived from cancer cells that can be extracted when the cancer progresses in the initial state is very small, in the pretreatment, a large amount of plasma is used to extract the nucleic acids. Need to be extracted. When pretreatment is performed on such a large amount of plasma, the time required for pretreatment is further increased.

  In the configuration described in Patent Literature 1, a large amount of plasma cannot be processed at one time because one reaction container having a capacity of about 1 mL is used. In that case, it is conceivable to process a large amount of plasma using a reaction vessel having a large capacity. However, in the case of infants, infants or children, it is difficult to collect a large amount of plasma, so it is necessary to extract nucleic acids from a small amount of plasma. There is a possibility that a small amount of plasma in the reaction container may not be completely aspirated as compared with the case where a small reaction container is used. Therefore, there is a demand for a method that can reduce the time required for the pretreatment and that can handle both large and small amounts of plasma.

  A main aspect of the present invention relates to a sample pretreatment cartridge used for pretreatment for extracting nucleic acids from a plasma sample. In the cartridge according to this aspect, a plurality of reaction vessels are integrally formed such that the plasma sample is dispensed according to the amount of the plasma sample.

  ADVANTAGE OF THE INVENTION According to this invention, the time required for pre-processing can be shortened and it can respond to a large amount of plasma and a small amount of plasma.

FIG. 1 is a schematic diagram when the internal configuration of the sample pretreatment device according to the embodiment is viewed from above. FIG. 2A is a perspective view illustrating a configuration of the sample pretreatment cartridge according to the embodiment. FIG. 2B is a perspective view illustrating a configuration of the reagent cartridge according to the embodiment. FIGS. 3A to 3F are diagrams for explaining the flow of processing by the sample pretreatment device according to the embodiment. FIG. 4 is a schematic diagram when the configuration of the dispensing unit according to the embodiment is viewed from above. FIG. 5 is a diagram illustrating a configuration of a reaction unit, a washing unit, and an elution unit according to the embodiment. FIG. 6 is a block diagram illustrating a configuration of the sample pretreatment device according to the embodiment. FIG. 7 is a flowchart illustrating a process of the sample pretreatment device for storing information on the amount of the plasma sample and starting the nucleic acid extraction process according to the embodiment. FIG. 8 is a diagram illustrating a configuration of a setting screen according to the embodiment. FIG. 9 is a flowchart illustrating a nucleic acid extraction process according to the embodiment. FIGS. 10A to 10F are diagrams for explaining that DNA is extracted from a plasma sample by the first to third processes according to the embodiment. FIG. 11 is a flowchart illustrating a process of a first step according to the embodiment. FIG. 12 is a flowchart illustrating the processing of the second step according to the embodiment. FIG. 13 is a flowchart illustrating the processing of the second step according to the embodiment. FIGS. 14A to 14H are views for explaining sample dispensing in the first half of the second step according to the embodiment. FIG. 15 is a flowchart illustrating the processing of the second step according to the embodiment. FIG. 16 is a flowchart illustrating the processing of the second step according to the embodiment. FIG. 17 is a flowchart illustrating the processing of the third step according to the embodiment. FIGS. 18A to 18E are schematic diagrams illustrating a modified example of the sample pretreatment cartridge according to the embodiment.

  In the following embodiments, the present invention is applied to a sample pretreatment device for extracting nucleic acids from a plasma sample. The sample pretreatment device of the embodiment particularly extracts DNA. After the DNA is extracted by the sample pretreatment device of the embodiment, the gene is then detected based on, for example, the BEAMing (Bead, Emulsion, Amplification, and Magnetics) method. That is, the sample pretreatment device of the embodiment performs pretreatment for extracting DNA before gene detection. The sample pretreatment cartridge of the embodiment is used for pretreatment for extracting DNA before gene detection.

  As shown in FIG. 1, the sample pretreatment device 100 includes plate members 110, 120, 130, holding members 140, 150, 160, a dispensing unit 200, six reaction units 300, and six cleaning units 400. , Six elution units 500, six cooling units 600, a control unit 701, and a storage unit 702. In FIG. 1, the XYZ axes are orthogonal to each other. The X-axis positive direction indicates the left direction, the Y-axis positive direction indicates the rear, and the Z-axis positive direction indicates the vertically downward direction. In the following drawings, the XYZ axes are the same as the XYZ axes shown in FIG.

  Holes are formed in the plate members 110, 120, and 130 as described below. Each of the holding members 140, 150, and 160 has a hole-shaped holding portion that is depressed with respect to the upper surface. These holes and holding portions are arranged along rows 101 to 106 extending in the Y-axis direction. The columns 101 to 106 are arranged in order in the X-axis positive direction in the sample pretreatment device 100. The holes and the holding units arranged along the rows 101 to 106 respectively correspond to the processing area of one sample. Therefore, the sample pretreatment device 100 has six plasma sample processing regions.

  Five holes 111, one hole 112, and one hole 113 are formed in the plate member 110 along the rows 101 to 106, respectively. The holes 111 to 113 penetrate the plate member 110. When one sample pretreatment cartridge 10 is installed, the five reaction containers 11 of the sample pretreatment cartridge 10 are respectively inserted into five holes 111, one hole 112, and one hole 113 arranged in the Y-axis direction. One washing container 12 and one elution container 13 are passed through. The five reaction vessels 11 are supported by the reaction section 300 having a hole-shaped holding section below the plate member 110, and the elution vessel 13 is provided with an elution having a hole-shaped holding section below the plate member 110. Supported by the unit 500. Thereby, one sample pretreatment cartridge 10 is installed on the plate member 110. Six sample pretreatment cartridges 10 can be installed on the plate member 110 along the rows 101 to 106.

  When the processing of the plasma sample is started, a new sample pretreatment cartridge 10 is previously installed at the position of the row where the sample container 41 is installed among the rows 101 to 106. The configuration of the sample pretreatment cartridge 10 will be described later with reference to FIG.

  Instead of the reaction vessel 11 and the elution vessel 13 being supported by the reaction section 300 and the elution section 500, the flat section 10a of the sample pretreatment cartridge 10 shown in FIG. Thus, the sample pretreatment cartridge 10 may be installed on the plate member 110.

  Eight reagent container holding portions 121 are formed on the plate member 120 along the rows 101 to 106, respectively. The reagent container holding section 121 is a hole provided in the plate member 120. The reagent container holding part 121 penetrates the plate member 120. When one reagent cartridge 20 is installed, the reagent containers 21 to 28 of the reagent cartridge 20 are passed through eight reagent container holding parts 121 arranged in the Y-axis direction. Then, by supporting the flat portion 20 a of the reagent cartridge 20 shown in FIG. 2B on the upper surface of the plate member 120, one reagent cartridge 20 is installed on the plate member 120. Six reagent cartridges 20 can be installed on the plate member 120 along the rows 101 to 106.

  When the processing of the plasma sample is started, the reagent cartridge 20 containing the reagent is installed in advance at the position of the row where the sample container 41 is installed among the rows 101 to 106. The reagent containers 21 to 28 of the reagent cartridge 20 contain a solubilizing solution, a preparation solution, an extract solution, a first reagent, a first washing solution, a second washing solution, a third washing solution, and a second reagent, respectively. The configuration of the reagent cartridge 20 will be described later with reference to FIG.

  Holes 131 are formed in the plate member 130 at the positions of the rows 101 to 106, respectively. The hole 131 penetrates the plate member 130. When the reagent container 31 is installed, the reagent container 31 is passed through the hole 131. Then, the reagent container 31 is installed on the plate member 130 by being supported by the cooling section 600 having a hole-shaped holding portion below the plate member 130. Six reagent containers 31 can be installed on the plate member 130 at the positions of the rows 101 to 106. When the processing of the plasma sample is started, the reagent container 31 containing the reagent is installed in advance at the position of the row where the sample container 41 is installed among the rows 101 to 106. The reagent container 31 contains the proteinase K. The reagent container 31 containing the proteinase K is cooled by the cooling unit 600 until the dispensing of the proteinase K is performed.

  Hole-shaped sample container holding portions 141 are formed in the holding member 140 at the positions of the rows 101 to 106, respectively. The sample container holding unit 141 holds the sample container 41. The holding member 140 can hold six sample containers 41 at the positions of the rows 101 to 106. When the processing of the plasma sample is started, the sample container 41 containing the plasma sample is set in the sample container holding unit 141 in advance.

  Eight holding portions 151 are formed on the holding member 150 along the rows 101 to 106, respectively. The holding portion 151 has a hole shape that is recessed downward from the upper surface of the holding member 150. Of the eight holding parts 151 arranged in the Y-axis direction, the six holding parts 151 on the Y-axis positive side hold the chip 51, and the two holding parts 151 on the Y-axis negative side hold the chip 52. The holding member 150 can hold 36 chips 51 and 12 chips 52 at the positions of the rows 101 to 106. When the processing of the plasma sample is started, new chips 51 and 52 are installed in advance in the holders 151 of the rows of the rows 101 to 106 where the sample containers 41 are installed.

  In the holding member 160, hole-shaped holding portions 161 are formed at the positions of the rows 101 to 106, respectively. The holding unit 161 holds the container 61. The holding member 160 can hold six containers 61 at the positions of the rows 101 to 106. When the processing of the plasma sample is started, a new container 61 is installed in advance in the holding unit 161 of the row in which the sample containers 41 are installed among the rows 101 to 106.

  The dispensing unit 200 includes a pair of nozzles 201 and 202 at the positions of the rows 101 to 106, respectively. The dispensing section 200 has a configuration for moving the nozzles 201 and 202 in the Y-axis direction and the Z-axis direction. The lower ends of the nozzles 201 and 202 have a cylindrical shape. When the nozzle 201 is positioned just above the chip 51 held by the holding member 150 and is lowered, the chip 51 is attached to the lower end of the nozzle 201. Similarly, when the nozzle 202 is positioned just above the chip 52 held by the holding member 150 and is lowered, the chip 52 is attached to the lower end of the nozzle 202. Further, the sample pretreatment device 100 includes a discard unit (not shown) for discarding the chips 51 and 52 mounted on the nozzles 201 and 202. When the nozzles 201 and 202 are moved in the Y-axis direction with the nozzles 201 and 202 inserted in the holes of the discarding portions, the chips 51 and 52 are detached from the lower ends of the nozzles 201 and 202, and the detached chips 51 and 52 are collected in the discarding portion. You.

  The dispensing section 200 is configured to be able to dispense a plasma sample and a reagent via the chips 51 and 52. The dispensing unit 200 performs suction while lowering the lower ends of the chips 51 and 52 below the liquid surface in a state where the chips 51 and 52 are mounted on the nozzles 201 and 202, respectively. After performing the suction, the dispensing unit 200 moves the lower ends of the chips 51 and 52 into the container to be discharged, and discharges the liquid stored in the chips 51 and 52 to the container to be discharged by suction. When the aspirated liquid is discarded, the dispensing unit 200 discharges the liquid stored in the chips 51 and 52 to a discarding unit (not shown). The configuration of the dispensing section 200 will be described later with reference to FIG.

  The six reaction units 300 are provided below the plate member 110, and are arranged along the rows 101 to 106, respectively. Five reaction vessels 11 of one sample pretreatment cartridge 10 are arranged in one reaction section 300. The reaction unit 300 heats the placed reaction container 11 to promote the reaction between the reagent and the plasma sample contained in the reaction container 11.

  The six cleaning units 400 are installed below the plate member 110 and the holding members 140 and 150, and are arranged along the rows 101 to 106, respectively. In one cleaning unit 400, the cleaning container 12 of one sample pretreatment cartridge 10 is arranged. The cleaning unit 400 performs a process for removing impurities adsorbed on the magnetic particles in the disposed cleaning container 12.

  The six elution portions 500 are provided below the plate members 110 and 120, and are arranged along the rows 101 to 106, respectively. In one elution section 500, an elution container 13 of one sample pretreatment cartridge 10 is arranged. The elution section 500 performs a process for extracting DNA by separating magnetic particles and DNA in the disposed elution container 13.

  The six cooling units 600 are provided below the plate member 130, and are arranged in rows 101 to 106, respectively. One cooling unit 600 is provided with one reagent container 31. The cooling unit 600 cools the placed reagent container 31 to a predetermined temperature.

  The control unit 701 receives a signal from each unit of the sample pretreatment device 100 and controls each unit based on the program stored in the storage unit 702.

  As shown in FIG. 2A, the sample pretreatment cartridge 10 includes a flat portion 10a extending in the Y-axis direction, five reaction containers 11, one washing container 12, and one elution container 13. Prepare. The five reaction vessels 11, one washing vessel 12, one elution vessel 13, and the plane portion 10a are integrally formed. Specifically, each of the five reaction vessels 11, one washing vessel 12, one elution vessel 13, and the flat portion 10a is a separate part, and the respective parts are adhered by an adhesive or the like, so that the sample is obtained. The pre-processing cartridge 10 is formed integrally. In addition to this, for example, by injection molding a resin material such as plastic, the five reaction vessels 11, one washing vessel 12, one elution vessel 13, and the plane portion 10a are integrally molded of the same material. As a result, the sample pretreatment cartridge 10 may be integrally formed. The reaction container 11, the washing container 12, and the elution container 13 are formed on the lower surface side of the flat portion 10a. Openings 11a, 12a, and 13a are formed in the upper portions of the reaction vessel 11, the washing vessel 12, and the elution vessel 13, respectively. Chips 51 and 52 are inserted into the reaction vessel 11, the washing vessel 12, and the elution vessel 13 from above through the openings 11a, 12a, and 13a, respectively.

  The inner surface of the reaction vessel 11 has the same diameter as the diameter of the opening 11a from the opening 11a to a predetermined depth position. The inner surface of the reaction vessel 11 gradually decreases in diameter from this depth position toward the positive direction of the Z-axis, and is subsequently connected to a bottom surface which is concave in a spherical shape. That is, the reaction vessel 11 has a cylindrical inner surface, a conical inner surface, and a spherically concave bottom surface. Similarly to the reaction container 11, the washing container 12 and the elution container 13 also have a cylindrical inner surface, a conical inner surface, and a spherically concave bottom surface. The diameter and depth of the washing container 12 and the elution container 13 are different from those of the reaction container 11. The capacity of the washing vessel 12 is larger than the capacity of the elution vessel 13, and the capacity of the reaction vessel 11 is larger than the capacity of the washing vessel 12.

  In the reaction container 11, a plasma sample and a reagent for allowing nucleic acids in the plasma sample to be adsorbed to the magnetic particles are dispensed. A sample after the reaction in the reaction container 11 is dispensed into the washing container 12 in order to remove impurities adsorbed on the magnetic particles. In the elution container 13, a sample that has been processed in the washing container 12 is dispensed in order to separate magnetic particles (described later) from DNA in the plasma sample.

  Since the five reaction vessels 11, one washing vessel 12, and one elution vessel 13 are integrally formed in the sample pretreatment cartridge 10, these vessels can be easily used for the sample pretreatment apparatus 100. It can be attached and detached at the same time.

  The five reaction vessels 11, one washing vessel 12, and one elution vessel 13 may be provided in the sample pretreatment device 100 in advance. In this case, each container is washed and used for each plasma sample. However, in this case, if the plasma sample remains in the container due to insufficient washing, there is a possibility that another plasma sample is mixed with the plasma sample to be processed. Therefore, in the embodiment, as described above, when the processing of the plasma sample is started, a new sample pretreatment cartridge 10 is installed. When the sample pretreatment cartridge 10 is exchanged for each plasma sample in this manner, other plasma samples are mixed with the plasma sample as compared with the case where the container provided in the sample pretreatment device 100 is washed and used each time. Such contamination can be prevented.

  The five reaction vessels 11 have the same shape as each other. Thereby, the reaction can be similarly advanced in the five reaction vessels 11. The five reaction vessels 11 are linearly arranged in a line along the longitudinal direction of the plane portion 10a. This makes it easier to arrange a plurality of sample pretreatment cartridges 10 side by side. Specifically, if the sample pretreatment cartridges 10 are arranged in a direction intersecting with the direction in which the reaction vessels 11 are arranged, the plurality of sample pretreatment cartridges 10 can be easily installed compactly in the sample pretreatment device 100.

  The washing container 12 and the elution container 13 are formed at the end of the sample pretreatment cartridge 10. This facilitates installation of the washing unit 400 in the sample pretreatment apparatus 100 while being separated from the reaction container 11 and the elution container 13. Similarly, it becomes easy to dispose the elution unit 500 from the reaction container 11 and the washing container 12 and install it in the sample pretreatment device 100. Further, the washing container 12 and the elution container 13 are formed at positions not adjacent to each other. Accordingly, it is easy to separately install the washing unit 400 and the elution unit 500 in the sample pretreatment device 100.

  As shown in FIG. 2B, the reagent cartridge 20 includes a flat portion 20a extending in the Y-axis direction, and reagent containers 21 to 28. The reagent containers 21 to 28 and the plane portion 20a are formed integrally. Specifically, each of the reagent containers 21 to 28 and the flat portion 20a is a separate component, and the reagent cartridge 20 is integrally formed by bonding each component with an adhesive or the like. In addition, for example, the resin container such as plastic is injection-molded, and the reagent containers 21 to 28 and the flat portion 20a are integrally formed of the same material, so that the reagent cartridge 20 is integrally formed. Is also good. The reagent containers 21 to 28 are formed on the lower surface side of the plane portion 20a, and have the same shape as each other. Each of the reagent containers 21 to 28 has a cylindrical inner surface, a conical inner surface, and a bottom surface depressed into a spherical shape, similarly to the reaction container 11. The reagent containers 21 to 28 may have different shapes depending on the volume of the reagent to be stored. Openings 21a to 28a are formed in the upper portions of the reagent containers 21 to 28, respectively. Chips 51 and 52 are inserted into reagent containers 21 to 28 from above through openings 21a to 28a, respectively.

  As described above, the solubilizing solution, the preparation solution, the extraction solution, the first reagent, the first washing solution, the second washing solution, the third washing solution, and the second reagent are stored in the reagent containers 21 to 28 of the reagent cartridge 20, respectively. Is accommodated. The preparation liquid, the extract, and the first reagent are reagents for adsorbing DNA in the plasma sample to the magnetic particles. The second reagent is a reagent for releasing DNA adsorbed on the magnetic particles.

  For example, lysates include Tris-HCl, EDTA-2Na, Guanidine Thiocyanate, and Tween®20. The extract contains Tris-HCl, EDTA-2Na, Guanidine Thiocyanate, and Tween®20. The preparation solution contains Isopropanol. The first reagent includes sodium azide and magnetic particles for adsorbing DNA in a plasma sample. The magnetic particles are magnetic particles whose surfaces are covered with silica. The particles constituting the magnetic particles are, for example, iron oxide. The first washing solution contains EDTA-2Na, Guanidine Hydrochloride, Sodium Azide, and Ethanol. The second washing solution contains Sodium Azide and Ethanol. The third washing solution contains Ethanol. The second reagent contains Tris-HCl, EDTA-2Na, and Sodium Azide.

  As described above, when processing of a plasma sample is started, a new reagent cartridge 20 containing a reagent is placed in the row where the plasma sample is placed. This eliminates the need for the dispensing section 200 to include a configuration for moving the nozzles 201 and 202 in the X-axis direction in order to dispense the reagent.

  The eight reagents contained in the reagent containers 21 to 28 and the reagent contained in the reagent container 31 may be contained in nine containers provided in the sample pretreatment device 100 in advance. In this case, for example, the nine containers are installed on the X-axis negative side of the holding member 150, and the reagents contained in each container are commonly used for the processing of the rows 101 to 106. In this case, the dispensing section 200 needs to have a mechanism for moving the nozzles 201 and 202 in the X-axis direction. Further, the installation area of the sample pretreatment device 100 in the X-axis direction increases. On the other hand, since the containers accommodating the nine reagents are commonly used for each plasma sample, the area for disposing the six reagent cartridges 20 and the six reagent containers 31 can be reduced, and the sample pretreatment device 100 Can be reduced in the Y-axis direction.

  Next, with reference to FIG. 1 and FIGS. 3A to 3F, an outline of a processing flow by the sample pretreatment device 100 will be described.

  The operator places the sample container 41 containing the plasma sample in the sample container holding unit 141. The operator places the sample container 41 in the sample container holding unit 141 according to the number of plasma samples to be processed. FIG. 1 illustrates an example in which, among the six sample container holding units 141, the sample containers 41 are installed in the three sample container holding units 141 on the right side corresponding to the rows 101 to 103, and processing is performed on three plasma samples. It is shown.

  The operator inputs the amount of the plasma sample to be processed for each sample container 41 installed in the sample container holding unit 141 via an input unit 704 shown in FIG. Specifically, the operator inputs one of 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL via the input unit 704 for each plasma sample. The control unit 701 obtains the input value as information on the amount of the plasma sample, and stores the obtained information on the amount of the plasma sample in the storage unit 702. In addition, the operator places the sample container 41 in advance in the sample container 41 by storing the plasma sample in an amount equal to or larger than the input amount of the sample container 41 in the sample container 41 in advance. Thereafter, the operator inputs an instruction to start the processing via the input unit 704.

  Note that the information regarding the amount of the plasma sample is not limited to being obtained via the input unit 704. For example, by aspirating the entire amount of the plasma sample in the sample container 41, the measurement unit provided in the dispensing unit 200 measures the amount aspirated, and the control unit 701 determines the amount of the plasma sample based on the detection signal of the measurement unit. May be obtained. In this case, for example, a value obtained by truncating the decimal part of the amount of the plasma sample obtained by the dispensing unit 200 is obtained. At this time, if the value whose decimal part is truncated is 6 or more, the value is set to 5. The value thus obtained is used as information on the amount of the plasma sample.

  However, in this example, the information on the plasma sample is automatically determined according to the amount of the plasma sample contained in the sample container 41. Therefore, the operator determines that the amount of the plasma sample is the desired amount to be processed. Therefore, the amount of the plasma sample to be stored in the sample container 41 must be finely adjusted in advance. Therefore, as in the embodiment, regardless of the amount of the plasma sample contained in the sample container 41, the information on the amount of the plasma sample is determined based on the value input by the operator, thereby simplifying the operation of the operator. .

  The information on the amount of the plasma sample is not limited to being obtained via the input unit 704, and may be read from a barcode or RFID attached to the sample container 41.

  Subsequently, when a start instruction is input, the control unit 701 performs processing of the plasma samples contained in each sample container 41 in parallel. For the processing of the plasma sample, the sample pretreatment cartridge 10, the reagent cartridge 20, the reagent container 31, the chip 51, and the 52 and a container 61 are used.

  The control unit 701 reads the information on the amount of the plasma sample stored in the storage unit 702, and stores the plasma sample contained in the sample container 41 and the reagent cartridge in the number of reaction containers 11 corresponding to the information on the amount of the plasma sample. The dispensing section 200 is controlled so as to dispense the reagent contained in the reagent container 20 and the reagent contained in the reagent container 31. Specifically, when 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL is input as the amount of the plasma sample to be processed, the plasma sample is stored in one, two, three, four, or five reaction vessels 11. And reagents are dispensed.

  For example, in the case of a plasma sample in which 5 mL is input as the amount to be processed, the plasma sample and the reagent are dispensed into five reaction containers 11 arranged in the same row as the plasma sample. In the case of a plasma sample in which 3 mL has been input as the amount to be processed, the plasma sample and the reagent are dispensed into three reaction containers 11 arranged in the same row as the plasma sample. When three reaction vessels 11 are used, of the five reaction vessels 11 arranged in the row direction, three reaction vessels 11 on the Y axis negative side are used, and two reaction vessels 11 on the Y axis positive side are not used. .

  The capacity of each of the five reaction vessels 11 is set to a capacity suitable for efficiently reacting the dispensed plasma sample with the reagent in a short time. That is, the reagent can be dispensed at a predetermined ratio with respect to the plasma sample dispensed into the reaction container 11, and the plasma sample and the reagent accommodated in the reaction container 11 are quickly heated to a predetermined temperature, The capacity of each reaction vessel 11 is set so that the reaction can be promoted.

  Hereinafter, for the sake of convenience, the processing of a plasma sample in which 5 mL is input as the processing amount will be described. The following dispensing process is performed by the control unit 701 controlling the dispensing unit 200.

  First, the proteinase K contained in the reagent container 31 is dispensed into five reaction containers 11. An equal amount of proteinase K is dispensed into each reaction vessel 11. Subsequently, as shown in FIG. 3A, the plasma sample contained in the sample container 41 is dispensed into five reaction containers 11. An equal amount of a plasma sample is dispensed into each reaction container 11. Specifically, 1 mL of the plasma sample is dispensed into each of the five reaction vessels 11.

  Subsequently, as shown in FIG. 3B, the solubilizing solution, the preparation solution, the extract solution, and the first reagent contained in the reagent cartridge 20 are dispensed into five reaction containers 11. Equal amounts of reagents are dispensed into each reaction vessel 11, and DNA in the plasma sample is adsorbed on the magnetic particles.

  Subsequently, as shown in FIG. 3C, a predetermined amount of the sample is sequentially dispensed into the washing container 12 from each reaction container 11, and impurities contained in the sample are removed as a liquid component. Further, after all the samples in the reaction containers 11 are dispensed into the washing container 12, the first to third washing liquids contained in the reagent cartridge 20 are dispensed into the washing container 12, as shown in FIG. The impurities contained in the sample in the washing container 12 are removed as a liquid component.

  Subsequently, as shown in FIG. 3E, the sample in the washing container 12 and the second reagent contained in the reagent cartridge 20 are dispensed into the elution container 13, and the magnetic particles and DNA are separated. You. Subsequently, with the magnetic force applied to the elution container 13, the supernatant in the elution container 13 is dispensed into the container 61 as shown in FIG. The sample in the container 61 contains the extracted DNA. Thus, the processing for one plasma sample is completed. Processing for other plasma samples is performed in parallel in the same manner.

  When an amount other than 5 mL is input as the amount of the plasma sample to be processed, the number of reaction vessels 11 used for the process is changed according to the input amount of the plasma sample. For example, when 3 mL is input as the amount of the plasma sample to be processed, the plasma sample is dispensed into each of the three reaction vessels 11 by 1 mL. The reagent is dispensed into these three reaction vessels 11.

  As described above, the plasma sample and the reagent are dispensed into the number of reaction containers 11 corresponding to the amount of the plasma sample, and the reaction on the plasma sample is performed in parallel in each reaction container 11 in which the plasma sample and the reagent are dispensed. Done. That is, when the amount of the plasma sample is large, for example, 5 mL, five reaction containers 11 are used, and when the amount of the plasma sample is small, for example, 1 mL, one reaction container 11 is used.

  Therefore, the time required for the reaction between the plasma sample and the reagent can be reduced as compared with the case where the plasma sample to be processed and the reagent are collectively dispensed into one large-capacity reaction container. Further, when the reaction is performed in a large-capacity reaction container, the degree of progress of the reaction may vary depending on the amount of the plasma sample, so that it is difficult to stably extract DNA. However, when the reaction is performed in the number of reaction vessels 11 corresponding to the amount of the plasma sample as described above, DNA can be stably extracted regardless of the amount of the plasma sample.

  Further, the number of reaction vessels 11 into which the plasma sample is dispensed is determined according to the amount of the plasma sample. For this reason, the plasma sample can be processed at a time in the total volume that can be accommodated in all the reaction vessels 11, that is, in the range up to 5 mL in the embodiment. Therefore, according to the sample pretreatment device 100 of the embodiment, the time required for pretreatment for extracting DNA can be reduced, and a large amount of plasma sample and a small amount of plasma sample can be handled.

  As described above, the plasma sample is evenly distributed from the sample container 41 to each reaction container 11 by 1 mL. That is, an equal amount of the plasma sample is dispensed into each reaction container 11. This makes it possible to shorten the time required until the reaction is completed in all the reaction vessels 11 as compared with the case where the plasma sample to be processed is dispensed unequally into each reaction vessel 11. In addition, the reaction proceeds in each reaction vessel 11 almost equally.

  Note that the control unit 701 may receive a value other than an integer value as the information on the amount of the plasma sample to be processed. In this case, the number of the reaction containers 11 into which the plasma sample is dispensed and the amount of the plasma sample to be dispensed may be determined so that the amount of the plasma sample to be dispensed into the reaction container 11 is close to 1 mL. . For example, when 3.6 mL is input, 0.9 mL of the plasma sample may be dispensed to each of the four reaction vessels 11. When 4.4 mL is input, 1.1 mL of the plasma sample may be dispensed into each of the four reaction vessels 11. If there is no large difference between the reactions in the respective reaction vessels 11, different amounts of the plasma sample may be dispensed into the respective reaction vessels 11. For example, when 3.6 mL is input, 1 mL, 1 mL, 0.8 mL, and 0.8 mL of the plasma sample may be dispensed into the four reaction vessels 11 respectively.

  The control unit 701 may receive an amount of less than 1 mL as the information on the amount of the plasma sample to be processed. In this case, the received amount of the plasma sample is dispensed to only one reaction container 11. This makes it possible to deal with even smaller amounts of plasma samples.

  In the embodiment, the sample pretreatment cartridge 10 includes the five reaction containers 11, but is not limited thereto, and may include 2 to 4 or 6 or more reaction containers 11.

  As shown in FIG. 4, the dispensing unit 200 includes nozzles 201 and 202, a pressure applying unit 210, six vertical transfer units 220, and a front and rear transfer unit 230.

  The pressure application unit 210 includes six valves 211, six valves 212, six pressure sensors 213, and six pumps 214. When the pump 214 is driven when the valve 211 is opened and the valve 212 is closed, the liquid can be dispensed through the tip 51 mounted on the nozzle 201. When the pump 214 is driven when the valve 211 is closed and the valve 212 is opened, liquid can be dispensed through the tip 52 attached to the nozzle 202. The pressure sensor 213 detects the pressure of the flow path connecting the pump 214 and the valves 211 and 212.

  The six vertical transfer units 220 are installed on the lower surface side of the support unit 231 corresponding to the nozzles 201 and 202 provided along the rows 101 to 106. The vertical transfer unit 220 includes a motor (not shown), and drives the pair of nozzles 201 and 202 arranged in the row direction in the vertical direction by driving the motor. The six upper and lower transfer units 220 can be individually driven. This makes it possible to individually dispense the plasma samples and the reagents arranged in the rows 101 to 106. The front-rear transfer unit 230 includes a support unit 231 and two rails 232. The front-rear transfer unit 230 includes a motor (not shown), and drives the support unit 231 in the Y-axis direction along the rail 232 by driving the motor.

  When sucking the liquid, the control unit 701 controls the dispensing unit 200 and lowers the chips 51 and 52 from above the liquid level. When the lower ends of the chips 51 and 52 come into contact with the liquid surface, the pressure of the flow path connecting the pump 214 and the valves 211 and 212 changes. The control unit 701 detects that the lower ends of the chips 51 and 52 have contacted the liquid surface based on a change in the detection signal of the pressure sensor 213. Then, the control unit 701 controls the dispensing unit 200 to move the nozzles 201 and 202 downward according to the dispensed amount, and sucks a predetermined amount of liquid via the chips 51 and 52. When discharging the liquid, the control unit 701 controls the dispensing unit 200 to position the lower ends of the chips 51 and 52 in the container to be discharged. Then, the control unit 701 controls the dispensing unit 200 to discharge the liquid positioned in the chips 51 and 52.

  With reference to FIG. 5, the configuration of the reaction unit 300, the cleaning unit 400, and the elution unit 500 will be described. In FIG. 5, when the sample pretreatment cartridge 10, the plate member 110, and the conductive members 312 and 512 are cut by a plane parallel to a YZ plane passing through the center position of the sample pretreatment cartridge 10 in the X axis direction. Is shown. For other configurations, the appearance when viewed in the negative direction of the X axis is shown for convenience.

  The reaction section 300 includes a heating section 310 for heating the reaction vessel 11. The heating unit 310 includes two heaters 311, a conduction member 312, and two heat radiation members 313. The two heaters 311 are provided on the lower surface of the conductive member 312, and heat the five reaction vessels 11 by heating the conductive member 312. A Peltier element may be used instead of the heater 311.

  The conductive member 312 is made of a metal having high thermal conductivity, and conducts the heat of the heater 311 to the reaction vessel 11. The two heat radiating members 313 are provided on the lower surfaces of the two heaters 311 respectively, and after the heating by the heater 311 is completed, the heat of the heater 311 and the conductive member 312 is efficiently radiated. The conductive member 312 includes five reaction container holding portions 312a for holding the five reaction containers 11, respectively. The five reaction container holders 312a are formed in circular holes depressed from the upper surface of the conductive member 312. The diameter of the reaction container holding part 312a is substantially the same as the diameter of the reaction container 11. The inner surface of the reaction container holding portion 312a has a shape to which the outer surface of the reaction container 11 fits.

  The cleaning unit 400 includes a magnetic force application unit 410 that applies a magnetic force to the cleaning container 12. The magnetic force applying unit 410 includes a magnet 411 and a magnet driving unit 412 that moves the magnet 411. The magnet drive unit 412 includes a rail 412a, a moving member 412b, a motor 412c, and a drive shaft 412d. The moving member 412b is configured to be movable along the rail 412a. The magnet 411 is installed on the moving member 412b via a rod-shaped member. The motor 412c is fixed inside the sample pretreatment device 100. One end of the drive shaft 412d is connected to the shaft of the motor 412c. A screw groove is formed on the drive shaft 412d. The screw groove of the drive shaft 412d is connected to a screw hole formed in the moving member 412b.

  When the sample pretreatment cartridge 10 is installed, as shown in FIG. 5, the washing container 12 is positioned below the hole 111 shown in FIG. 1 via the flat portion 10a. When the motor 412c is driven in this state, the magnet 411 is moved between the position near the bottom of the cleaning container 12 and the position far from the bottom of the cleaning container 12 via the moving member 412b and the drive shaft 412d. You. The rail 412a is arranged such that the positive side of the Y axis is lifted in the negative direction of the Z axis with respect to a direction parallel to the Y axis. Thus, the magnet 411 moves obliquely with the driving of the motor 412c.

  The dissolution unit 500 includes a heating unit 510 for heating the dissolution container 13, and a magnetic force application unit 520 for applying a magnetic force to the dissolution container 13. The heating unit 510 includes a heater 511, a conduction member 512, and a heat radiation member 513. The heater 511 is provided on the lower surface of the conductive member 512 and heats the conductive member 512. Instead of the heater 511, a Peltier element may be used.

  The conductive member 512 is made of a metal having high thermal conductivity, and conducts the heat of the heater 511 to the elution container 13. The heat dissipating member 513 is provided on the lower surface of the heater 511, and efficiently dissipates heat of the heater 511 and the conductive member 512 after the heating by the heater 511 is completed. The conductive member 512 includes an elution container holding portion 512a for holding the elution container 13, and a hole 512b connected to the elution container holding portion 512a. The elution container holding portion 512a is formed in a circular hole shape depressed from the upper surface of the conductive member 512. The diameter of the elution container holding portion 512a is substantially the same as the diameter of the elution container 13. The inner surface of the elution container holding portion 512a has a shape to which the outer surface of the elution container 13 fits. The hole 512b is connected to the elution container holding part 512a from the side surface of the conductive member 512.

  The magnetic force applying unit 520 includes a magnet 521 and a magnet driving unit 522 that moves the magnet 521. The magnet driving unit 522 includes a rail 522a, a moving member 522b, a motor 522c, and a belt 522d. The moving member 522b is configured to be movable along the rail 522a. The magnet 521 is installed on the moving member 522b via a rod-shaped member. The motor 522c is fixed in the sample pretreatment device 100. The belt 522d is hung on two pulleys. The pulley on the Y axis positive side is connected to the shaft of the motor 522c. The moving member 522b is connected to the belt 522d by a fastener.

  When the sample pretreatment cartridge 10 is installed, as shown in FIG. 5, the elution container 13 is held by the elution container holder 512a. When the motor 522c is driven in this state, the magnet 521 moves through the moving member 522b and the belt 522d along the hole 512b to a position near the bottom of the elution container 13 and a position far from the bottom of the elution container 13. Moved between. The rail 522a is arranged such that the negative side of the Y axis is lifted in the negative direction of the Z axis with respect to a direction parallel to the Y axis. The hole 512b extends parallel to the rail 522a. Accordingly, the magnet 521 moves obliquely with the driving of the motor 522c.

  When the sample pretreatment cartridge 10 is installed, the five reaction vessels 11 passed through the holes 111 of the plate member 110 shown in FIG. 1 are inserted into the reaction vessel holding section 312a and held by the reaction vessel holding section 312a. Is done. One elution container 13 passed through the hole 113 of the plate member 110 shown in FIG. 1 is inserted into the elution container holding portion 512a, and is held by the elution container holding portion 512a. Thereby, the five reaction vessels 11 are arranged in the reaction section 300, the washing vessel 12 is arranged in the washing section 400, and the elution vessel 13 is arranged in the elution section 500.

  When the sample pretreatment cartridge 10 is installed, the outer surface of the reaction container 11 is held without gap by the reaction container holding unit 312a, and the outer surface of the elution container 13 is held without gap by the elution container holding unit 512a. Is desirable. Thus, the heat generated from the heater 311 is efficiently transmitted to the reaction container 11 via the conductive member 312, and the heat generated from the heater 511 is efficiently transmitted to the elution container 13 via the conductive member 512.

  In the embodiment, the reaction unit 300, the washing unit 400, and the elution unit 500 are provided corresponding to the sample pretreatment cartridges 10 arranged for each plasma sample, that is, corresponding to the rows 101 to 106. . Thus, the processing by the reaction unit 300, the washing unit 400, and the elution unit 500 can be independently performed for each plasma sample. In the reaction section 300, a heater may be individually provided for each of the five reaction vessels 11.

  When the processing of the plasma samples in the columns 101 to 106 is performed in synchronization with each other, the reaction unit 300, the washing unit 400, and the elution unit 500 are provided one by one in common for the columns 101 to 106. Is also good. In this case, the heaters 311, 511, the conductive members 312, 512, and the heat radiating members 313, 513 are extended in the X-axis direction so as to correspond to the six sample pretreatment cartridges 10 arranged in the rows 101 to 106. . Further, six magnets 411 corresponding to each washing container 12 are driven by one magnet driving unit 412, and six magnets 521 corresponding to each elution container 13 are driven by one magnet driving unit 522.

  As shown in FIG. 6, the sample pretreatment device 100 includes a control unit 701, a storage unit 702, a display unit 703, an input unit 704, a dispensing unit 200, a reaction unit 300, a cleaning unit 400, An elution unit 500 and a cooling unit 600 are provided.

  The control unit 701 is configured by a CPU or a microcomputer. The control unit 701 receives a signal from each unit of the sample pretreatment device 100 and controls each unit. The storage unit 702 includes a RAM, a ROM, a hard disk, and the like. The control unit 701 may be configured by a CPU and a microcomputer. In this case, for example, a microcomputer may control each unit of the sample pretreatment device 100, and a CPU communicably connected to the microcomputer may transmit an instruction signal to the microcomputer. That is, the control unit 701 may be configured by a plurality of control units.

  The display unit 703 includes a display. The input unit 704 includes a mouse and a keyboard. The sample pretreatment device 100 may include a display input unit configured by a touch panel display instead of the display unit 703 and the input unit 704.

  Next, processing performed by the sample pre-processing apparatus 100 will be described with reference to a flowchart.

  As shown in FIG. 7, in step S1, the control unit 701 determines whether an operator has accepted a setting start instruction via the input unit 704. Upon receiving the setting start instruction, the control unit 701 displays the setting screen 800 on the display unit 703 in step S2.

  As shown in FIG. 8, the setting screen 800 includes setting display areas 801 to 806, a start button 811 and a cancel button 812. Each of the setting display areas 801 to 806 includes a value input unit that can set the amount of the plasma sample to be processed in the columns 101 to 106, respectively. In the example shown in FIG. 8, the value input section is configured by six radio buttons. The six radio buttons correspond to none, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL, respectively. In the initial state, the radio button corresponding to “none” is selected. The value input units of the setting display areas 801 to 806 are not limited to the radio buttons as described above, and may be configured by a list box, a text box, or the like in which the amount of the plasma sample can be set.

  The operator selects the radio button via the input unit 704 for the amount of the plasma sample to be processed among the plasma samples contained in the sample container 41 in the setting display area corresponding to the row in which the sample containers 41 are installed. Set by Then, the operator presses the start button 811 via the input unit 704 to start the nucleic acid extraction processing by the sample pretreatment device 100. At this time, the control unit 701 does not start the processing for the column in which “none” is set. When the operator discards the settings in the setting display areas 801 to 806 and closes the setting screen 800, the operator presses a cancel button 812 via the input unit 704.

  When the setting screen 800 is displayed during the process of nucleic acid extraction, the setting display area corresponding to the column where the process is being performed is in a state where input is not accepted. For example, when the processing of the plasma sample corresponding to the column 103 has already been performed, the setting display area 803 does not accept an input as indicated by a broken line display as in the example shown in FIG. "Processing" indicating that the processing is being performed is displayed below the. In this way, even if there is a column that is being processed, if there is a column that has not been processed, an input for a column that has not been processed is ready to be accepted. When the operator selects the amount of the plasma sample in the setting display area where input can be received via the input unit 704 and presses the start button 811, the nucleic acid extraction processing corresponding to the set row is started. The processing started later is performed in parallel with the nucleic acid extraction processing already performed.

  Returning to FIG. 7, in step S3, the control unit 701 determines whether or not the operator has pressed the start button 811 via the input unit 704. When determining that the start button 811 has been pressed, the control unit 701 stores information on the amount of the plasma sample in the storage unit 702 based on the setting display areas 801 to 806 in step S4. In step S4, when any value of 1 mL to 5 mL is set in the setting display area corresponding to the column where the processing is not performed, information on the amount of the plasma sample is set based on the setting display area. It is memorized. Then, in step S5, the control unit 701 starts the nucleic acid extraction processing on the plasma sample in which information on the amount of the plasma sample is stored.

  As shown in FIG. 9, in the nucleic acid extraction process, the control unit 701 executes a first step of step S11, a second step of step S12, and a third step of step S13 in order. The processing of steps S11 to S13 is performed for each plasma sample. Details of each step will be described later with reference to FIGS. Here, before the detailed description of each step, how the DNA is extracted from the plasma sample by each step will be described.

  As shown in FIG. 10A, the plasma sample in the sample container 41 is a DNA 71, a histone 72 that is a DNA binding protein that binds to DNA in the plasma sample, an enzyme 73 that degrades the DNA 71, and a protein 74 in the plasma sample. Etc. are included. In the first step, proteinase K is dispensed into the reaction container 11, the plasma sample in the sample container 41 is dispensed into the reaction container 11, and the lysate is dispensed into the reaction container 11. Proteinase K degrades histone 72 bound to DNA 71 and separates histone 72 from DNA 71. Further, proteinase K degrades the enzyme 73 and suppresses the function of the enzyme 73. In addition, proteinase K degrades protein 74 in the plasma sample. The lysate creates an environment in which proteinase K works.

  Subsequently, the prepared liquid and the extract are dispensed into the reaction vessel 11. Since the DNA 71 has high hydrophilicity, it easily forms a hydrogen bond with a water molecule in a solution. On the other hand, silica covering the surface of the magnetic particles 77 shown in FIG. 10C has high hydrophobicity. Therefore, the DNA 71 in the initial state hardly binds to the silica of the magnetic particles 77. The preparation removes the hydrated molecules of DNA 71 to make DNA 71 hydrophobic. Thereby, the hydrophobic DNA 71 comes to be adsorbed on the silica of the magnetic particles 77. The extract creates an environment for the DNA 71 to be adsorbed on the magnetic particles 77. In the first step, when the reaction proceeds in the reaction vessel 11, the inside of the reaction vessel 11 becomes, for example, a state shown in FIG.

  As shown in FIG. 10B, the sample in the reaction vessel 11 at this time contains DNA 71, enzyme 73, protein 74 in plasma sample, denatured substances 75 and 76, and the like. The denaturing substance 75 is obtained by denaturing and decomposing the histone 72. The denaturing substance 76 is obtained by denaturing and decomposing the enzyme 73 and the protein 74 in the plasma sample. As the reaction proceeds in the reaction vessel 11, the histone 72 is separated from the DNA 71, and the histone 72, the enzyme 73, and the protein 74 in the plasma sample are denatured and decomposed. Further, in the first step, a first reagent containing magnetic particles is dispensed into the reaction vessel 11. As a result, the DNA 71 is adsorbed on the magnetic particles 77, and the inside of the reaction container 11 is brought into a state shown in FIG. 10C, for example. The denatured substances 75 and 76 in the plasma sample also adsorb to the magnetic particles 77.

  In the second step, the sample in the reaction vessel 11 is dispensed into the cleaning vessel 12, and the magnetic particles 77 are adsorbed on the inner wall of the cleaning vessel 12 by the magnet 411 of the cleaning section 400 shown in FIG. Then, the supernatant liquid is removed from the washing container 12. As a result, denatured substances 75 and 76 that are not bound to the magnetic particles 77 in the plasma sample are removed. Subsequently, the first cleaning liquid is dispensed into the cleaning container 12. As a result, the denatured substances 75 and 76 bound to the magnetic particles 77 are separated, and the inside of the cleaning container 12 is in a state shown in FIG. 10D, for example. Then, the supernatant is removed from the washing container 12 using the magnet 411, so that the denatured substances 75 and 76 are removed from the sample. Subsequently, the second cleaning liquid is dispensed into the cleaning container 12. Then, the supernatant is removed from the washing container 12 using the magnet 411, whereby the denaturing substances 75 and 76 are further removed from the sample.

  Subsequently, the third cleaning liquid is dispensed into the cleaning container 12. Then, the sample in the washing container 12 is dispensed into the elution container 13, and the magnetic particles 77 are adsorbed on the inner wall of the elution container 13 by the magnet 521 of the elution unit 500 shown in FIG. Then, by removing the supernatant from the elution container 13, the denaturing substances 75 and 76 are further removed from the sample.

  In the third step, the second reagent is dispensed into the elution container 13. As a result, the DNA 71 is released from the magnetic particles 77, and the inside of the elution container 13 becomes, for example, a state shown in FIG. Subsequently, the magnetic particles 77 are adsorbed on the inner wall of the elution container 13 by the magnet 521 of the elution unit 500 shown in FIG. 5, and the supernatant of the elution container 13 is dispensed into the container 61. At this time, the sample in the container 61 is in a state where unnecessary substances other than the DNA 71 have been removed, as shown in FIG. Thus, the nucleic acid extraction processing for one plasma sample is completed.

  Next, the first to third steps will be described with reference to FIGS.

  In the flowchart shown below, the processing of each step is executed by the control unit 701 controlling the dispensing unit 200, the reaction unit 300, the cleaning unit 400, and the elution unit 500. As described above, since the first to third steps are performed for each plasma sample for which the process has been started, the following description relates to the process performed for one plasma sample.

  As shown in FIG. 11, in step S101, the control unit 701 reads information on the amount of the plasma sample corresponding to the plasma sample to be processed from the storage unit 702, and stores the read plasma sample in the reaction container 11 to be dispensed. Is determined according to the information on the amount of

  Specifically, when the information on the amount of the plasma sample corresponds to 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL, the number of reaction vessels 11 to be dispensed is one, two, three, and four, respectively. And five. The reaction vessels 11 to be dispensed are determined in order from the reaction vessel 11 on the Y axis negative side among the five reaction vessels 11 arranged on the plate member 110. For example, when the information on the amount of the plasma sample corresponds to 3 mL, the three reaction containers 11 on the Y axis negative side are the dispensing targets. Therefore, in the following process, only the reaction container 11 to be dispensed among the five reaction containers 11 is used for the process. That is, the dispensing process is performed only on the reaction container 11 to be dispensed.

  In step S102, the control unit 701 mounts the new chip 51 held by the holding member 150 on the nozzle 201. In step S103, the control unit 701 dispenses a predetermined amount of the proteinase K contained in the reagent container 31 to each reaction container 11. In step S104, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 51 on the nozzle 201. In step S105, the control unit 701 dispenses 1 mL of the plasma sample stored in the sample container 41 to each reaction container 11.

  In step S106, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 51 on the nozzle 201. In step S107, the control unit 701 dispenses a predetermined amount of the solubilizing solution contained in the reagent container 21 to each reaction container 11. In step S108, the control unit 701 heats each reaction vessel 11 by the two heaters 311 of the reaction unit 300, and heats the sample in each reaction vessel 11. As a result, the reaction proceeds in the reaction vessel 11, and as described with reference to FIG. 10B, the histone 72 is separated from the DNA 71, and the histone 72, the enzyme 73, and the protein 74 in the plasma sample are denatured and decomposed. You.

  In step S109, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 51 on the nozzle 201. In step S110, the control unit 701 dispenses a predetermined amount of the preparation liquid stored in the reagent container 22 to each reaction container 11. Further, in step S111, the control unit 701 dispense a predetermined amount of the extract contained in the reagent container 23 into each reaction container 11.

  In step S112, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 52 on the nozzle 202. In step S113, the control unit 701 dispenses the first reagent contained in the reagent container 24 to each reaction container 11 by a predetermined amount. In step S114, the control unit 701 stirs the sample in the reaction container 11 by continuously performing the operation of sucking and discharging the sample in the reaction container 11 in each reaction container 11. Such a stirring operation is hereinafter referred to as “suction / discharge stirring”. In step S115, the control unit 701 heats each reaction vessel 11 by the two heaters 311 to heat the sample in each reaction vessel 11. Thus, the DNA 71 is adsorbed on the magnetic particles 77 as described with reference to FIG. Thus, the first step is completed.

  The amount of the plasma sample and the amount of the reagent to be dispensed into the reaction container 11 are determined so that the reaction can be efficiently performed in the reaction container 11. From such a viewpoint, in the embodiment, the amount of the plasma sample dispensed into the reaction container 11 is determined to be 1 mL, and the amount of the reagent dispensed into the reaction container 11 is each determined to be a predetermined amount. . Thus, at the time when the first step is completed, each of the reaction vessels 11 contains 2.92 mL of the sample.

  Note that the dispensing of proteinase K in step S103, the dispensing of the plasma sample in step S105, the dissolving solution in step S107, and the heating in step S108 are not performed in the order described above. You may. Each process may be performed in any order, and may be performed in parallel.

  As shown in FIG. 12, in step S201, the control unit 701 brings the magnet 411 of the cleaning unit 400 closer to the cleaning container 12. In step S202, the control unit 701 discards the chip 52 mounted on the nozzle 202 and mounts a new chip 51 on the nozzle 201. In step S203, the control unit 701 sets the processing position of the reaction vessel 11 to 1. Thereby, the position of the reaction container 11 located closest to the Y axis negative side among the reaction containers 11 to be dispensed becomes the processing position. The processing position is sequentially moved one by one toward the Y axis positive side by being incremented by one in step S210 described later. The value of the processing position is stored in the storage unit 702.

  In step S204, the control unit 701 determines whether there is a sample of 1.2 mL or more in the reaction container 11 at the processing position. As described above, when the first step is completed, the sample contained in each reaction container 11 is 2.92 mL, and the control unit 701 determines the amount of the sample sucked from the reaction container 11 in step S206 described later. Is stored in the storage unit 702. In step S204, the control unit 701 determines whether or not there is a sample of 1.2 mL or more in the reaction container 11 at the processing position based on the information. The threshold for the determination in step S204 is set to the maximum amount that can be dispensed at one time via the chip 51.

  If the control unit 701 determines in step S204 that there is a sample of 1.2 mL or more in the reaction container 11 at the processing position, the control unit 701 sucks and discharges the sample contained in the reaction container 11 at the processing position in step S205. In step S206, the control unit 701 dispenses a 1.2 mL sample from the reaction vessel 11 at the processing position to the cleaning vessel 12. The sample dispensing amount in step S206 is set to the maximum amount that can be dispensed at one time via the chip 51. Thereby, the number of times of dispensing from the reaction container 11 to the washing container 12 can be minimized. By the dispensing in step S206, the magnetic particles 77 in the sample are adsorbed on the inner wall of the cleaning container 12. In step S207, the control unit 701 aspirates the supernatant in the cleaning container 12, that is, the supernatant of the sample in the cleaning container 12, and discards the supernatant in the disposal unit. Then, control unit 701 returns the process to step S204.

  When the control unit 701 determines in step S204 that there is no sample of 1.2 mL or more in the reaction vessel 11 at the processing position, it determines in step S208 whether the processing position is the last position. That is, in step S208, the control unit 701 determines whether or not the processing position is the position of the reaction container 11 closest to the Y axis positive side among the reaction containers 11 to be dispensed.

  When the control unit 701 determines in step S208 that the processing position is not the last processing position, in step S209, the control unit 701 divides all the samples contained in the reaction container 11 at the processing position into the reaction container 11 at the processing position + 1. Note. In step S210, the control unit 701 moves the processing position by one to the Y axis positive side by incrementing the value of the processing position by one. Then, control unit 701 returns the process to step S204. On the other hand, if the control unit 701 determines in step S208 that the processing position is the last position, the processing proceeds to step S211 in FIG.

  As shown in FIG. 13, in step S211, the control unit 701 sucks and discharges the sample contained in the reaction container 11 at the processing position. In step S212, the control unit 701 dispenses all the samples contained in the reaction container 11 at the processing position to the cleaning container 12. In step S213, the control unit 701 removes the supernatant in the washing container 12. In step S214, the control unit 701 separates the magnet 411 of the cleaning unit 400 from the cleaning container 12, and advances the processing to step S215 in FIG.

  As described above, in the processing of steps S201 to S214, a predetermined amount of the sample is dispensed from the reaction container 11 in which the reaction is completed into the cleaning container 12, and the magnetic particles are adsorbed on the inner wall of the cleaning container 12, and then the cleaning container The operation of removing the supernatant from is performed several times. Thereby, the supernatant containing unnecessary components can be quickly and reliably removed as compared with a case where the sample in each reaction vessel 11 is collectively transferred to a large-capacity vessel and the operation of removing the supernatant is performed only once in this vessel. Can be removed.

  Here, the dispensing of the sample in steps S203 to S210 in FIG. 12 will be described with reference to FIGS. In the examples shown in FIGS. 14A to 14H, only the two reaction vessels 11 on the Y axis negative side are used for the processing.

  In step S203, the processing position is set to 1. In step S204, it is determined that there is a sample of 1.2 mL or more in the reaction container 11 on the Y axis negative side at the processing position. Therefore, as shown in FIG. 14A, in step S206, a 1.2 mL sample is dispensed from the reaction container 11 on the negative side of the Y axis at the processing position to the cleaning container 12. Thus, as shown in FIG. 14B, the liquid amount at the processing position decreases, and the liquid amount in the cleaning container 12 increases. Then, in step S207, the supernatant in the washing container 12 is removed, and the process returns to step S204.

  In step S204, it is determined that there is still a sample of 1.2 mL or more in the reaction container 11 on the Y axis negative side at the processing position. Therefore, as shown in FIG. 14 (c), the sample is dispensed in step S206, and as shown in FIG. 14 (d), the liquid amount at the processing position decreases, and the liquid amount of the cleaning container 12 becomes To increase. Then, in step S207, the supernatant in the washing container 12 is removed, and the process returns to step S204.

  At this time, as shown in FIG. 14E, a sample less than 1.2 mL, which is the maximum suction amount of the chip 51, is left in the reaction container 11 at the processing position. Here, 2.92−1.2 × 2 = 0.52 mL is left. As described above, a predetermined amount of the plasma sample and the reagent are dispensed into the reaction vessel 11 so that the reaction proceeds efficiently in one reaction vessel 11, and as a result, the reaction vessel 11 has a 2.92 mL sample. Will be accommodated. The amount of the sample sucked from the reaction container 11 is 1.2 mL, which is the maximum amount that can be dispensed at one time via the tip 51. Therefore, in the embodiment, when a sample is dispensed from one reaction container 11, a predetermined amount of the sample is left.

  In step S204, it is determined that there is no sample of 1.2 mL or more in the reaction container 11 on the Y axis negative side at the processing position. Then, in step S208, it is determined that the processing position is not the last position. For this reason, as shown in FIG. 14E, in step S209, all the samples contained in the reaction container 11 on the Y-axis negative side at the processing position are converted into the reaction on the Y-axis positive side at the processing position +1. Dispensed into the container 11. Thereby, as shown in FIG. 14 (f), the liquid volume of the reaction vessel 11 at the processing position becomes 0, and the liquid volume of the reaction vessel 11 at the processing position +1 increases. Then, in step S210, the processing position is incremented by one, and the process returns to step S204.

  In step S204, it is determined that there is a sample of 1.2 mL or more in the reaction container 11 on the Y axis positive side at the processing position. For this reason, as shown in FIG. 14G, in step S206, a 1.2 mL sample is dispensed from the reaction container 11 on the Y axis positive side at the processing position to the cleaning container 12. Thus, as shown in FIG. 14H, the liquid amount at the processing position decreases, and the liquid amount in the cleaning container 12 increases. Similarly, from the reaction container 11 on the Y axis positive side, a plasma sample is dispensed into the washing container 12 in 1.2 mL increments.

  Thereafter, in step S204, it is determined that there is no sample of 1.2 mL or more in the reaction container 11 on the Y axis positive side at the processing position, and in step S208, it is determined that the processing position is the last position. The process proceeds to Step S211. Then, in step S212, all the samples contained in the reaction container 11 on the Y axis positive side at the processing position are dispensed into the cleaning container 12. In this way, a sample is dispensed from each reaction container 11 in which the reaction has been completed to the washing container 12.

  As described above, when the amount of the sample in the reaction container 11 at the processing position is less than a certain amount, the sample remaining in the reaction container 11 at the processing position is dispensed into the reaction container 11 at the processing position +1. Thereby, the number of times of removing the supernatant in the washing container 12 can be reduced, so that the time required for the entire processing can be reduced. After the sample remaining in the reaction vessel 11 at the processing position is dispensed to the reaction vessel 11 at the processing position +1, the dispensing of the sample from the reaction vessel 11 at the processing position +1 to the cleaning vessel 12 is advanced. Accordingly, when there is another reaction container 11 for accommodating a sample other than the reaction container 11 at the processing position +1, the total amount of the chip 51 is smaller than when dispensing a sample from another reaction container 11 is performed. The moving distance can be shortened. Therefore, the time required for the entire process can be reduced.

  As described above, when the second step is started, the amount of the sample contained in each reaction vessel 11 is 2.92 mL. In the embodiment, the reaction vessel 11 containing 2.92 mL of the sample is configured to be able to further contain a fixed amount of the sample. Specifically, the reaction container 11 of the embodiment is configured to be able to accommodate a sample of 2.92 + 1.2 = 4.12 mL. Thereby, when it is determined that the sample to be stored is less than 1.2 mL, the sample is stored in one reaction container 11 at the processing position +1 without being stored in the two reaction containers 11 separately. be able to. Therefore, the time required for dispensing can be reduced.

  If the amount of the sample accommodated in the reaction container 11 and the amount of suction of the chip 51 are set to values other than those of the embodiment, in step S206, all the samples at the processing position are dispensed into the cleaning container 12. It is conceivable that it will be done. In this case, if the processing position is not the last position, the processing position is incremented by 1 after the processing of step S207, and the processing returns to step S204. If the processing position is the last position, after the processing in step S207, the processing proceeds to step S214.

  As illustrated in FIG. 15, in step S215, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 51 on the nozzle 201. In step S216, the control unit 701 dispense the first cleaning liquid stored in the reagent container 25 into the cleaning container 12 by a predetermined amount. In step S217, the control unit 701 sucks and discharges the sample in the cleaning container 12. In step S218, the control unit 701 causes the magnet 411 of the cleaning unit 400 to approach the cleaning container 12. Thereby, the magnetic particles 77 in the sample are adsorbed on the inner wall of the washing container 12. In step S219, the control unit 701 removes the supernatant in the washing container 12. This completes the cleaning with the first cleaning liquid. In step S220, the control unit 701 separates the magnet 411 of the cleaning unit 400 from the cleaning container 12.

  In step S221, the control unit 701 dispenses a predetermined amount of the second cleaning liquid stored in the reagent container 26 into the cleaning container 12. Then, the control unit 701 performs the processing of steps S222 to S225 as in the processing of steps S217 to S220. By removing the supernatant in step S224, the washing with the second washing liquid is completed. By dispensing the first cleaning liquid and the second cleaning liquid, the denatured substances 75 and 76 bound to the magnetic particles 77 are separated as described with reference to FIG. Then, denaturants 75 and 76 are removed from the sample by removing the supernatant in steps S219 and S224.

  As shown in FIG. 16, in step S226, the control unit 701 dispenses a predetermined amount of the third cleaning liquid stored in the reagent container 27 into the cleaning container 12. In step S227, the control unit 701 sucks and discharges the sample in the cleaning container 12. In step S228, the control unit 701 brings the magnet 521 of the elution unit 500 closer to the elution container 13. In step S229, the control unit 701 dispenses the sample contained in the washing container 12 to the elution container 13. That is, the control unit 701 dispenses a sample from the washing container 12 in which washing with the first and second washing liquids has been completed to the elution container 13.

  In step S230, the control unit 701 removes the supernatant in the elution container 13. This completes the cleaning with the third cleaning liquid. By removing the supernatant in step S230, the denaturing substances 75 and 76 are removed from the sample. In step S231, the control unit 701 heats the elution container 13 by the heater 511 of the elution unit 500, and heats the sample in the elution container 13. Thereby, the remaining reagent evaporates from the sample in the elution container 13. In step S232, the control unit 701 separates the magnet 521 from the elution container 13. Thus, the second step is completed.

  The first to third cleaning liquids are dispensed into the cleaning container 12 with the magnet 411 separated from the cleaning container 12. Accordingly, the sample in the cleaning container 12 is stirred by the first to third cleaning liquids, so that the stirring in the cleaning container 12 can be performed more efficiently.

  As shown in FIG. 17, in step S301, the control unit 701 discards the chip 51 mounted on the nozzle 201 and mounts a new chip 52 on the nozzle 202. In step S302, the control unit 701 dispenses a predetermined amount of the second reagent contained in the reagent container 28 to the elution container 13. In step S303, the control unit 701 sucks and discharges the sample in the elution container 13. In step S304, the control unit 701 heats the elution container 13 by the heater 511 of the elution unit 500, and heats the sample in the elution container 13. As a result, the reaction proceeds in the elution container 13 and the DNA 71 is released from the magnetic particles 77 as described with reference to FIG.

  In step S305, the control unit 701 brings the magnet 521 close to the elution container 13. Thereby, the magnetic particles 77 in the sample are adsorbed on the inner wall of the elution container 13. In step S306, the control unit 701 dispenses the supernatant in the elution container 13 into the container 61. Thus, as described with reference to FIG. 10F, the sample in the container 61 is in a state where unnecessary substances other than the DNA 71 have been removed. In step S307, the control unit 701 separates the magnet 521 from the elution container 13. Thus, the third step is completed.

<Example of changing sample pretreatment cartridge>
The sample pretreatment cartridge 10 may be configured as shown in FIGS.

  In the configuration shown in FIG. 18A, six reaction vessels 11 are provided between the washing vessel 12 and the elution vessel 13 in two rows in the Y-axis direction, as compared with the configuration shown in FIG. ing. In the configuration shown in FIG. 18B, the cleaning container 12 and the elution container 13 are formed on the same side end of the flat portion 10a as compared with the configuration shown in FIG. Further, three reaction vessels 11 are provided on the Y axis positive side of the washing vessel 12 and the elution vessel 13, respectively. In the configuration shown in FIG. 18C, the cleaning container 12 is provided between the elution container 13 and the reaction container 11 on the Y axis positive side, as compared with the configuration shown in FIG. In the configuration shown in FIG. 18D, each container is provided along a circle as compared with the configuration shown in FIG. In the example illustrated in FIG. 18E, the flat portion 10a is extended in the Y-axis positive direction, and the reagent containers 21 to 28 are positioned at the extended flat portion 10a as compared with the configuration illustrated in FIG. Is provided.

  In the examples shown in FIGS. 18A, 18B and 18D, since the containers of the sample pretreatment cartridge 10 are not arranged in a line, it is necessary to move the nozzles 201 and 202 also in the X-axis direction. . However, since the width of the sample pretreatment cartridge 10 in the Y axis direction is reduced, the installation area of the sample pretreatment device 100 in the Y axis direction can be reduced.

  In the example shown in FIG. 18C, since the cleaning container 12 is not provided at the end of the sample pretreatment cartridge 10, the cleaning unit 400 moves from the position of the cleaning container 12 to the X axis so as to be separated from other containers. Need to be installed in the direction. Similarly, in the example shown in FIG. 18 (e), since the elution container 13 is not provided at the end of the sample pretreatment cartridge 10, the elution unit 500 is positioned so that it is separated from other containers. From the X-axis direction. As described above, in the examples illustrated in FIGS. 18C and 18E, the installation area of the sample pretreatment device 100 in the X-axis direction increases. Therefore, the washing container 12 and the elution container 13 are desirably provided at the end of the sample pretreatment cartridge 10.

  In the configuration shown in FIG. 18E, since the reagent containers 21 to 28 are provided in the sample pretreatment cartridge 10, it is not necessary to separately prepare the reagent cartridge 20 shown in FIG. Thus, the reaction container 11, the washing container 12, the elution container 13, and the reagent containers 21 to 28 can be set in the sample pretreatment device 100 only by installing one sample pretreatment cartridge 10.

  The sample pretreatment cartridge 10 shown in FIG. 1 was provided with a washing container 12 and an elution container 13 for performing a process for removing impurities contained in a sample and a process for separating DNA from magnetic particles. However, the present invention is not limited to this, and the sample pretreatment cartridge 10 may further include a container in addition to the washing container 12 and the elution container 13 for removing impurities and separating DNA.

Reference Signs List 10 sample pretreatment cartridge 11 reaction container 12 washing container 13 elution container 24, 28 reagent container 41 sample container 51, 52 chip 100 sample pretreatment device 121 reagent container holding unit 141 sample container holding unit 200 dispensing unit 300 reaction unit 311 heater 312a Reaction vessel holding section 400 Cleaning section 410 Magnetic force applying section 500 Elution section 510 Heating section 511 Heater 512 Conductive member 512a Elution vessel holding section 512b Hole 520 Magnetic force applying section 521 Magnet 522 Magnet driving section 701 Control section 704 Input section

Claims (11)

A sample pretreatment cartridge used for pretreatment for extracting nucleic acids from a plasma sample,
A sample pretreatment cartridge, wherein a plurality of reaction vessels are integrally formed such that the plasma sample is dispensed according to the amount of the plasma sample.   The sample pretreatment cartridge according to claim 1, wherein the plurality of reaction vessels have the same shape as each other.   The sample pretreatment cartridge according to claim 1, wherein the plurality of reaction containers are linearly arranged in a line along a longitudinal direction of the sample pretreatment cartridge. A flat portion extending in the longitudinal direction of the sample pretreatment cartridge is further formed,
4. The sample pretreatment cartridge according to claim 1, wherein the flat portion is formed integrally with the plurality of reaction vessels. 5. A first reagent container for containing a first reagent containing magnetic particles for adsorbing the nucleic acid in the plasma sample, and a second reagent container for containing a second reagent for releasing the nucleic acid adsorbed on the magnetic particles. And two reagent containers are further formed,
The sample pretreatment cartridge according to any one of claims 1 to 4, wherein the first reagent container and the second reagent container are formed integrally with the plurality of reaction containers.   The sample pretreatment cartridge according to claim 5, wherein the plurality of reaction containers are configured to dispense the first reagent into a number of the reaction containers according to an amount of the plasma sample.   In order to remove a liquid component in the sample containing the plasma sample and the first reagent, a washing container in which the number of the reaction-completed samples in the reaction container according to the amount of the plasma sample is dispensed, 7. The sample pretreatment cartridge according to claim 5, wherein the sample pretreatment cartridge is formed integrally with the plurality of reaction vessels.   The sample pretreatment cartridge according to claim 7, wherein the cleaning container is configured to apply a magnetic force so as to perform a process for removing impurities adsorbed on the magnetic particles.   9. The method according to claim 5, wherein an elution container into which the second reagent is dispensed for separating the magnetic particles and the nucleic acid is further formed integrally with the plurality of reaction containers. A sample pretreatment cartridge according to the item.   10. The sample pretreatment cartridge according to claim 9, wherein the elution container is configured to be applied with a magnetic force and heated so that a pre-nucleic acid extraction process is performed.   The sample pretreatment cartridge according to claim 9, wherein each of the plurality of reaction containers has a larger capacity than the elution container.

Images