JP2016214202A - Nucleic acid amplification apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten a creation time of a PCR product.SOLUTION: In a nucleic acid amplification device 50 of the present invention, a control part 80, after giving heat to a nucleic acid covering body covering template nucleic acid for a predetermined period at temperature which is set in order to break the body, controls a moving mechanism 60 so as to repeat a cycle having a denaturation step of allowing a droplet 20 to stay in a first region 36A which is set to denaturation temperature of target nucleic acid and a synthesis step of allowing the droplet 20 to stay in a second region 36B which is set to synthesis temperature of the target nucleic acid for a plurality of times.SELECTED DRAWING: Figure 7

Description

  The present invention relates to an amplification nucleic acid device.

  The PCR (polymerase chain reaction) method is based on the fact that nucleic acid denaturation and annealing differences occur due to differences in the chain length of nucleic acids such as DNA (deoxyribonucleic acid), etc. It is a technique to amplify nucleic acid by giving.

  As such a PCR method is used, the applicant of the present invention has proposed a PCR apparatus disclosed in Patent Document 1 below. In the biochip attached to the PCR device of Patent Document 1 below, a flow path through which a reaction solution containing a template nucleic acid and the like moves is formed. However, it is filled with a liquid having a small specific gravity and immiscible with the reaction liquid.

  In the PCR device of Patent Document 1 below, when a biochip is mounted on the mounting portion, a heating unit that heats the first region of the flow path formed in the biochip and a temperature different from that of the first region. A heating unit for heating the two regions is provided. In addition, the PCR device disclosed in Patent Document 1 includes a drive mechanism that switches the placement of the mounting portion and the heating portion between the first placement and the second placement. By this drive mechanism, the reaction liquid of the biochip attached to the attachment unit is moved between the first region and the second region that are heated to different temperatures. According to such a PCR apparatus of Patent Document 1 described below, the amplification reaction period can be shortened as compared with the case where the temperature of the entire biochip is switched to different temperatures.

JP 2012-115208 A

  By the way, the template nucleic acid contained in the reaction solution of the biochip described above may be encapsulated by a nucleic acid coating such as a cell wall or an envelope. In this case, a pretreatment is necessary to destroy the nucleic acid coating and extract the template nucleic acid. In this pretreatment, for example, a cationic surfactant is added, and a nucleic acid-surfactant complex that is preferentially dissolved in the organic layer is precipitated with an inorganic salt solution, and then recovered by centrifugation (patent) No. 2,791,367). As another example, there is a method (Japanese Patent Laid-Open No. 2001-352979) in which a nucleic acid is captured with a cationic solid phase carrier having magnetism, and after magnetic separation / washing, the nucleic acid is separated with an anionic substance.

  Since these pretreatments require complicated operations such as precipitation and separation, it is assumed that the PCR product generation time can be shortened if the pretreatment is simplified.

  Therefore, an object of the present invention is to shorten the generation time of a PCR product.

  In order to achieve the above object, the present inventors paid attention to the possibility of simplifying the pretreatment by incorporating it into the PCR apparatus, and as a result of intensive studies, they have reached the nucleic acid amplification apparatus of the present invention.

  The nucleic acid amplifying device of the present invention includes a mounting part capable of mounting a container having a flow path through which a droplet containing a specimen containing a template nucleic acid moves, and when the container is mounted on the mounting part, A movement mechanism for changing the posture to move the droplet from the first region of the container to the second region of the container different from the first region, and from the second region to the first region, a heater, A controller that controls the heater and the moving mechanism, and the controller applies heat for a predetermined period at a temperature set to destroy the nucleic acid coating that coats the template nucleic acid, A plurality of cycles through a denaturation step that keeps the droplet in the first region set at the denaturation temperature of the target nucleic acid and a synthesis step that keeps the droplet in the second region set at the integrity temperature of the target nucleic acid. Repeat the move to repeat Controlling the structure, characterized in that.

It is a figure which shows the cross section of the cartridge of 1st Embodiment. It is a figure which shows a mode that the sample solution is introduce | transduced in the container of a cartridge. It is a figure which shows a mode when the reagent of a freeze-dried state returns to the original state with the template nucleic acid solution. It is a block diagram of a nucleic acid amplifier. It is a figure which shows the mode of a rotation mechanism. It is a figure which shows a mode that the cartridge was mounted | worn with the mounting part. It is a figure which shows the mode of a heat cycle process. It is a figure which shows the cross section of the cartridge of 2nd Embodiment. It is a figure which shows a mode that the sample is introduce | transduced into a tank. It is a figure which shows a mode that the cartridge of 2nd Embodiment was mounted | worn. It is a figure which shows the mode of the rotation mechanism with which the cartridge of 2nd Embodiment was mounted | worn.

  Hereinafter, embodiments for carrying out the present invention will be illustrated with reference to the accompanying drawings. The embodiments and examples illustrated below are for facilitating the understanding of the present invention, and are not intended to limit the present invention. The present invention can be changed and improved without departing from the spirit of the present invention.

(1) Embodiment As an embodiment, after describing a cartridge attached to a nucleic acid amplification device that amplifies nucleic acid, the nucleic acid amplification device will be described.

(1-1) First Embodiment === Cartridge ===
FIG. 1 is a view showing a cross section of the cartridge 1. As shown in FIG. 1, the cartridge 1 includes a container 10 in which a reagent 11 and oil 12 are stored. In the present embodiment, the side wall 10A of the container 10 is cylindrical, and the bottom wall 10B of the container 10 is hollow hemispherical.

  An end of the container 10 facing the bottom wall 10B is open, and a cap 10C is attached to the opening. The cap 10 </ b> C is a lid member that closes the opening of the container 10, and is detachable from the container 10 in this embodiment. Moreover, in this embodiment, the seal | sticker site | part SP of the cap 10C accommodated in the container 10 is made into a column shape.

  A specimen solution is introduced into such a container 10. When the sample solution is introduced into the container 10, the cap 10C is removed from the container 10, and the cap 10C is attached to the container 10 again after the introduction.

  The sample solution introduced into the container 10 is obtained, for example, as follows. That is, specimens such as cells or virus particles derived from organisms such as humans and bacteria are collected by a collection tool such as a cotton swab. Thereafter, the sample solution is purified by placing it in a test tube or the like containing a buffer solution.

  The reagent 11 is a reagent used for the amplification reaction of the target nucleic acid, and is freeze-dried and fixed to the bottom wall portion 10B of the container 10. The reagent 11 contains at least a DNA (deoxyribonucleic acid) polymerase, a primer, and dNTP (deoxyribonucleotide triphosphate). The reagent 11 may contain a buffer solution. However, when the reagent 11 is freeze-dried, the water disappears, so ions such as magnesium ions and potassium contained in the buffer solution are contained in the container 10. It is fixed to the bottom wall 10B.

  Note that the nucleic acid of the sample in the sample solution introduced into the container 10 is a template nucleic acid, and all or a part of the nucleic acid to be amplified in the template nucleic acid is the target nucleic acid. This target nucleic acid is, for example, a DNA fragment, a cDNA (complementary DNA) fragment or a PNA (peptide nucleic acid).

  When the template nucleic acid is RNA (ribonucleic acid), reverse transcriptase, a reverse transcriptase primer, and the like are also included as the reagent 11 used for the amplification reaction of the target nucleic acid in order to obtain cDNA of the RNA.

  In addition, when quantifying the amplification degree of a target nucleic acid using a real-time PCR method, a fluorescence for quantification such as a probe to which a fluorescent dye such as TaqMan probe, Molecular Beacon or cycling probe binds, or a fluorescent dye for intercalator such as SYBR Green is used. A labeled probe is also contained as the reagent 11 used for the amplification reaction of the target nucleic acid.

  The oil 12 has a specific gravity smaller than that of the sample solution introduced into the container 10 and is phase-separated from the sample solution, for example, 2CS silicone oil or mineral oil.

  FIG. 2 is a diagram illustrating a state in which the sample solution is introduced into the container of the cartridge, and FIG. 3 is a diagram illustrating a state in which the lyophilized reagent is returned to the original state by the sample solution.

  As shown in FIG. 2, when the sample solution is introduced into the container 10 of the cartridge 1, the sample solution has a function of reducing the surface area of the interface, so that the sample solution is compatible with the oil 12 in the container 10. Separated into droplets 20. Since the specific gravity of the droplet 20 is larger than the specific gravity of the oil 12, the droplet 20 settles along the side wall portion 10 </ b> A in the container 10. That is, the cylindrical side wall portion 10 </ b> A in the container 10 becomes a flow path for the droplet 20.

  As shown in FIG. 3, when the droplet 20 settles down to the bottom wall portion 10 </ b> B of the container 10, the lyophilized reagent 11 is restored to its original state by the moisture in the droplet 20. The reagent 11 is taken into the droplet 20.

  When the reagent 11 changed from the freeze-dried state to the original state is taken into the droplet 20, the sample and the reagent 11 used for amplification of the target nucleic acid in the sample are included in the droplet 20, The droplet 20 serves as a place where the nucleic acid amplification reaction proceeds.

=== Nucleic Acid Amplifier ===
FIG. 4 is a block diagram of the nucleic acid amplification device. As shown in FIG. 4, the nucleic acid amplification device 50 includes a rotation mechanism 60, a fluorescence measuring device 70, and a control unit 80.

<Rotation mechanism>
FIG. 5 is a diagram illustrating a state of the rotation mechanism. In the following description of the nucleic acid amplification device 50, as shown in the figure, the top, bottom, front, back, left and right are defined. That is, the vertical direction when the base 51 of the nucleic acid amplification device 50 is installed horizontally is defined as “vertical direction”, and “upper” and “lower” are defined according to the direction of gravity. Further, the axial direction of the rotation axis AX of the cartridge 1 is defined as “left-right direction”, and the vertical direction and the direction perpendicular to the left-right direction are defined as “front-rear direction”.

  As shown in FIG. 5, the rotating mechanism 60 includes a rotating body 61 and a rotating motor 66 (FIG. 4) that rotates the rotating body 61. The rotating body 61 is provided with a heater 65 having an insertion hole 64 from which the cartridge 1 can be attached and detached. The rotating body 61 rotates around the rotation axis AX supported by the support base 52 fixed to the base 51 without changing the relative position between the heater 65 and the cartridge 1 mounted in the insertion hole 64 of the heater 65. To do.

  Note that the insertion hole 64 of the heater 65 in this embodiment also serves as a hole through which the cartridge 1 can be taken in and out and a mounting portion for mounting the cartridge 1 inserted into the hole. However, the hole and the mounting portion are separate. The nucleic acid amplification device 50 may be provided. Further, the number of holes through which the cartridge 1 can be taken in and out may be plural so that the nucleic acid amplification device 50 can mount a plurality of cartridges 1.

  The rotating motor 66 (FIG. 4) rotates the rotating body 61 according to an instruction from the control unit 80 so that the cartridge 1 mounted in the insertion hole 64 of the heater 65 is turned upside down.

  FIG. 6 is a diagram illustrating a state where the cartridge is mounted. As shown in FIG. 6, the heater 65 includes a first heater 65B and a second heater 65C.

  The first heater 65B surrounds the first region 36A corresponding to one end of the side wall portion 10A serving as the flow path of the droplet 20 in the container 10 of the cartridge 1 mounted in the insertion hole 64 of the heater 65, and the first region 36A is surrounded by the first region 36A. Heat. The heating temperature can be switched to a temperature at which the denaturation reaction of the target nucleic acid proceeds or a temperature at which the nucleic acid coating covering the nucleic acid, such as a bacterial cell wall or a virus envelope, is destroyed. The temperature at which the target nucleic acid denaturation reaction proceeds is 95-105 ° C., and the temperature at which the nucleic acid coating is destroyed is 50-110 ° C.

  The second heater 65 </ b> C surrounds the second region 36 </ b> B corresponding to the other end side of the side wall portion 10 </ b> A serving as the flow path of the droplet 20 in the container 10 of the cartridge 1 mounted in the insertion hole 64 of the heater 65. Heat. The heating temperature can be switched to a temperature at which a target nucleic acid synthesis reaction proceeds or a temperature at which a nucleic acid coating such as bacteria or virus is destroyed. The temperature at which the target nucleic acid synthesis reaction proceeds is 50 to 75 ° C.

  A spacer 65D that suppresses heat conduction between the first heater 65B and the second heater 65C is disposed between the first heater 65B and the second heater 65C. The spacer 65D is formed with a through hole at a position along the longitudinal direction of the insertion hole 64 of the first heater 65B and the second heater 65C, which prevents the insertion of the container 10 of the cartridge 1 into the insertion hole 64. Is prevented.

<Fluorescence measuring instrument>
The fluorescence measuring instrument 70 is a measuring instrument that measures the fluorescence intensity of the droplet 20 accommodated in the container 10 in the cartridge 1, and as shown in FIG. 5, the end of the cartridge 1 mounted in the insertion hole 64 of the heater 65. Are arranged in a state of facing each other with a predetermined distance.

  The fluorescence measuring device 70 irradiates excitation light corresponding to the fluorescent dye contained in the droplet 20 in accordance with a measurement instruction from the control unit 80 and measures the fluorescence intensity emitted from the droplet 20. Further, the fluorescence measuring instrument 70 gives data indicating the fluorescence intensity obtained as a measurement result to the control unit 80. The fluorescence measuring instrument 70 may measure the fluorescence intensity corresponding to one fluorescent dye, or may measure the fluorescence intensity corresponding to a plurality of fluorescent dyes.

<Control unit>
As shown in FIG. 4, the control unit 80 includes a storage unit 91, and an input unit 92 and a display unit 93 are connected to the control unit 80. The storage unit 91 includes an area for storing a program, an area for storing various data such as setting data input from the input unit 92 and data obtained by nucleic acid amplification processing, and an area for developing the program and data. Is included.

  The control unit 80 appropriately controls the rotation mechanism 60 and the fluorescence measuring device 70 based on the program and setting data stored in the storage unit 91, and appropriately executes nucleic acid elution processing, thermal cycle processing, or amplification analysis processing.

≪Nucleic acid elution treatment≫
When the control unit 80 receives an instruction to execute the nucleic acid elution process from the input unit 92, the control unit 80 sets a temperature at which the first heater 65B and the second heater 65C destroy the nucleic acid covering, and the first heater 65B. The second heater 65C is driven for a predetermined period. Here, the predetermined time refers to a period defined as necessary and sufficient to destroy the nucleic acid coating. As a result, the oil 12 filled in the container 10 of the cartridge 1 is heated, and heat is applied to the nucleic acid coating body of the specimen in the droplet 20 surrounded by the oil 12. As a result, the nucleic acid coating is destroyed by heat, and the nucleic acid of the specimen (template nucleic acid) is eluted in the droplet 20. The driving time (heating time) of the first heater 65B and the second heater 65C is preferably 2 seconds to 30 seconds.

  In the present embodiment, as part of the input unit 92, process start buttons 92A to 92C are provided in predetermined portions of the nucleic acid amplifying apparatus 50 for each type of specimen, and the temperature at which the nucleic acid covering of the specimen is destroyed is the temperature of the specimen. Each type is stored in the storage unit 91.

  For example, a first process start button 92A for gram-positive bacteria, a second process start button 92B for gram-negative bacteria, and a third process start button 92C for viruses are provided. The temperature for destroying Gram-positive bacteria is 100 ° C to 110 ° C, the temperature for destroying Gram-negative bacteria is 90 ° C to 100 ° C, and the temperature for destroying virus capsids or envelopes is 50 ° C to 90 ° C. Is done. The cell walls of Gram-positive bacteria tend to be thicker or harder than the cell walls of Gram-negative bacteria, and the cell walls of Gram-negative bacteria tend to be thicker or harder than viral capsids and envelopes.

  Based on the command given from the processing start button and the temperature stored in the storage unit 91, the control unit 80, for example, a temperature at which the cell wall of a Gram-positive bacterium, the cell wall of a Gram-negative bacterium, or a virus capsid or envelope is destroyed. Recognize This recognized temperature is set for the first heater 65B and the second heater 65C. That is, the control unit 80 switches the temperature to be set for the first heater 65B and the second heater 65C according to the type of specimen.

  As a result, even if the thickness or hardness of the nucleic acid coating differs depending on the type of specimen, the nucleic acid coating is destroyed while the damage to the nucleic acid wrapped in the nucleic acid coating is suppressed.

≪Thermal cycle treatment≫
FIG. 7 is a diagram showing a state of the heat cycle process. Specifically, FIGS. 7A and 7B show the state of the target nucleic acid synthesis stage, and FIGS. 7C and 7D show the state of the target nucleic acid denaturation stage.

  When the controller 80 finishes performing the nucleic acid elution process, the controller 80 changes the temperature set in the first heater 65B from the temperature at which the nucleic acid coating is destroyed to the temperature at which the target nucleic acid denaturation reaction proceeds, The heating temperature to be heated for the first region 36A of the container 10 is switched. In addition, the control unit 80 changes the temperature set in the second heater 65C to a temperature at which the target nucleic acid synthesis reaction proceeds, and switches the heating temperature to be heated for the second region 36B of the container 10. As a result, a temperature gradient is formed in the oil 12 filled in the container 10 of the cartridge 1.

  After the first heater 65B and the second heater 65C are driven, a predetermined period is required until the oil 12 in the first region 36A reaches, for example, 98 ° C. and the oil 12 in the second region 36B reaches, for example, 54 ° C. . Since the amplification reaction of the target nucleic acid does not proceed appropriately during this period, the control unit 80 waits with this period as a standby period.

  At this time, as shown in FIG. 7A, the cap 10C side of the container 10 mounted in the insertion hole 64 of the heater 65 is disposed on the upper side, and the bottom wall 10B side of the container 10 is disposed on the lower side. The rotating body 61 is located at the reference position. When the rotating body 61 is located at the reference position, as shown in FIG. 7B, the droplet 20 settles down by its own weight and remains in the second region 36B. Therefore, the target nucleic acid contained in the droplet 20 does not move to the first denaturation stage.

  When the above-described standby period has elapsed, the control unit 80 rotates the rotating body 61 by 180 degrees. In this case, as shown in FIG. 7C, the cap 10C side of the container 10 mounted in the insertion hole 64 of the heater 65 is disposed on the lower side, and the bottom wall portion 10B side of the container 10 is disposed on the upper side. The rotating body 61 is positioned at the reverse position. When the rotating body 61 is located at the reversal position, as shown in FIG. 7D, the droplet 20 settles due to its own weight and moves to the first region 36A. Accordingly, the target nucleic acid contained in the droplet 20 moves to the denaturation stage.

  Further, the control unit 80 moves the rotator 61 only during the denaturation reaction period set as the period of the denaturation stage required for the denaturation reaction of the target nucleic acid from the time when the rotator 61 has been rotated 180 degrees (the rotator 61 is stopped). Stop. Thereby, the denaturation reaction of the target nucleic acid contained in the droplet 20 proceeds. The denaturation reaction period is at least between the first region 36A corresponding to one end side of the side wall portion 10A serving as the flow path of the droplet 20 in the container 10 and the second region 36B corresponding to the other end side of the side wall portion 10A. The period is longer than the period during which the droplet 20 moves.

  Next, when the denaturation reaction period has elapsed, the control unit 80 rotates the rotator 61 by 180 degrees to switch the rotator 61 from the reverse position to the reference position, and as shown in FIG. The droplet 20 is moved to 36B. Thereby, the target nucleic acid contained in the droplet 20 moves to the synthesis stage.

  The control unit 80 also rotates the rotator 61 only during the synthesis reaction period set as the period of the synthesis stage required for the target nucleic acid synthesis reaction from the time when the rotator 61 has been rotated 180 degrees (the rotator 61 is stopped). Stop. Thereby, the annealing reaction and extension reaction of the target nucleic acid contained in the droplet 20 proceed. Note that the synthesis reaction period is a period longer than the period during which the droplet 20 moves between at least the first region 36A and the second region 36B, as in the above-described modification period.

  In this way, the control unit 80 switches the rotating body 61 alternately between the reverse position and the reference position, moves the droplet 20 to the first region 36A, and moves the droplet 20 to the second region 36B. The cycle that goes through the synthesis step is repeated several times. The number of cycles to be repeated is set in the control unit 80, for example, 50 times.

≪Amplification analysis process≫
The amplification analysis process is executed in parallel with the thermal cycle process. That is, the control unit 80 gives a measurement instruction to the fluorescence measuring device 70 for each synthesis reaction period, and stores data indicating the fluorescence intensity given from the fluorescence measuring device 70 as the measurement instruction result in the storage unit 91.

  As shown in FIGS. 7A and 7B, since the rotating body 61 is in the reference position during the synthesis reaction period, the droplet 20 in the container 10 settles toward the bottom wall portion 10B. Go. However, immediately after the rotating body 61 reaches the reference position, the droplet 20 may not reach the bottom wall portion 10B. Therefore, it is desirable that the control unit 80 give the measurement instruction to the fluorescence measuring instrument 70 after a predetermined time has elapsed since the rotation body 61 has been rotated from the reverse position to the reference position. In particular, it is desirable to be immediately before the rotation from the reference position to the reverse position.

  In addition, the control unit 80 reads data indicating the fluorescence intensity for the number of times set as the number of cycles to be repeated from the storage unit 91 according to the command input from the input unit 92, and based on the data, the fluorescence corresponding to the number of cycles An amplification curve showing the intensity transition is generated. Then, when generating the amplification curve, the control unit 80 determines whether the reference amplification efficiency is acceptable based on the amplification curve, and causes the display unit 93 to appropriately display both or one of the determination result and the amplification curve. .

<Summary>
As described above, in the nucleic acid amplification device 50 of the present embodiment, the sample 20 and the reagent 11 used for amplification of the target nucleic acid in the sample are contained in the droplet 20 in the oil 12 accommodated in the container 10. The control unit 80 of the nucleic acid amplification device 50 applies heat to the nucleic acid coating body that coats the nucleic acid of the sample for a predetermined period.

  In the case of the present embodiment, the control unit 80 includes a first heater 65B that heats the first region 36A of the container 10 mounted in the insertion hole 64 of the heater 65, and a second heater 10 that is different from the first region 36A. A temperature at which the nucleic acid coating is destroyed is set for both of the second heaters 65C for heating the region 36B, and heat is applied to the nucleic acid coating for a predetermined period.

  As a result, heat is applied to the droplet 20 surrounded by the oil 12 filled in the container 10 from the entire periphery, and the nucleic acid coating body of the sample in the droplet 20 is destroyed by the heat, and the nucleic acid (template nucleic acid) of the sample is destroyed. ) Is eluted in the droplet 20.

  Thereafter, the control unit 80 sets a denaturation temperature for the first heater 65B and sets a synthesis temperature for the second heater 65C, controls the rotating mechanism 60 to execute a thermal cycle process, and performs the droplet 20 The nucleic acid eluted in is amplified.

  As described above, the nucleic acid amplification device 50 of the present embodiment can shorten the generation time of the PCR product by allowing the amplification reaction to proceed after the nucleic acid film body is destroyed regardless of the procedure.

  By the way, the specimen is included in the double of the droplet 20 and the oil 12 smaller than the specific heat of the droplet 20. For this reason, the oil 12 is easier to warm than water, and the oil 12 gives heat to the droplet 20 from the entire periphery. Therefore, it is possible to destroy the nucleic acid coating and elution of the nucleic acid of the specimen (template nucleic acid) in a shorter time than when heat is applied to the specimen solution in which the specimen is placed in the buffer solution. Further, since the droplet 20 is contained in the oil 12, it is difficult to evaporate. Therefore, the size of the droplet 20 can be further reduced, and the nucleic acid coating can be destroyed in a short time by the amount of the size reduction, and the nucleic acid (template nucleic acid) of the specimen can be eluted.

(1-2) Second Embodiment Next, a second embodiment will be described. However, among the components of the cartridge and the nucleic acid amplification device according to the second embodiment, the same or equivalent components as those of the first embodiment are denoted by the same reference numerals, and redundant descriptions are omitted as appropriate.

=== Cartridge ===
FIG. 8 is a view showing a cross section of the cartridge 1 of the second embodiment. As shown in FIG. 8, the cartridge 1 of the present embodiment is configured by connecting a droplet introduction container 90 to the container 10 of the first embodiment.

  The droplet introducing container 90 is for introducing the droplet 20 of the solution containing the template nucleic acid and the reagent 11 into the container 10, and has a tank 100, a plunger 110, and a tube 120.

  The tank 100 is a part into which the sample solution is placed, and is detachable from the opening end of the plunger 110. When the sample is put into the tank 100, as shown in FIG. 9, it is removed from the plunger 110, and the sample solution 140 is put through the opening 101 of the tank 100 communicating with the opening end of the plunger 110. Thereafter, the tank 100 is attached to the open end of the plunger 110. Note that the specimen 141 contained in the specimen solution 140 is, for example, bacteria or viruses collected from a human or the like.

  The plunger 110 is a pusher that pushes a predetermined amount of liquid in the tube 120 functioning as a syringe from the end on the tube 120 side to the container 10, and has a cylindrical portion 111 and a rod-like portion 112.

  The cylindrical part 111 is a part that communicates with the opening 101 of the tank 100 via the adapter 113. Around the opening on the tank 100 side of the tubular portion 111, a flange-shaped mounting base 111A that protrudes outward from the tubular portion 111 toward the outside of the opening is formed. By fitting the adapter 113 attached to the mounting base 111 </ b> A into the opening of the tubular portion 111, the opening of the tubular portion 111 and the opening of the tank 100 are communicated. Note that the mounting base 111 </ b> A of the cylindrical portion 111 is a pressing portion that is pressed by the nucleic acid amplification device 50.

  The end of the tubular portion 111 on the tube 120 side is fitted to the inner wall of the upper syringe 121 of the tube 120 and is slidable along the upper syringe 121 while being in contact with the inner wall of the upper syringe 121. The interval between the mounting base 111A of the plunger 110 and the upper edge of the tube 120 is the slide length of the plunger 110.

  The rod-shaped part 112 is supported by a rib 114 protruding from the inner wall of the tubular part 111 on the tube 120 side end. The end of the rod-like portion 112 on the tube 120 side is located inside the upper syringe 121 and away from the lower syringe 122 in the initial state where the plunger 110 is not pushed ((A) in FIG. 8). On the other hand, the end of the rod-shaped portion 112 on the tube 120 side is inserted into the lower syringe 122 of the tube 120 when the plunger 110 is pushed, and along the lower syringe 122 while being in contact with the inner wall of the lower syringe 122. Slide ((B) of FIG. 8).

  A seal 112A is formed at the tip of the rod-shaped portion 112 on the tube 120 side, and the liquid in the tube 120 is prevented from flowing back to the plunger 110 by the seal 112A. Note that the liquid in the tube 120 is pushed downstream by an amount corresponding to the volume of the seal 112A slid in the lower syringe 122.

  A buffer solution 130 is accommodated inside the plunger 110. When the cartridge 1 is set up with the mounting base 111A of the plunger 110 facing up, the sample 141 contained in the sample solution 140 in the tank 100 contains the buffer solution 130 as shown in FIG. Settling.

≪Tube≫
The tube 120 has an upper syringe 121, a lower syringe 122, and a capillary 123. The inner diameters of the upper syringe 121, the lower syringe 122, and the capillary 123 are gradually reduced from the plunger 110 toward the container 10, and each part is filled with the buffer solution 130.

  The upper syringe 121 is a cylindrical portion that functions as a syringe for the cylindrical portion 111 of the plunger 110, and the cylindrical portion 111 of the plunger 110 is slidably in contact with the inner wall of the upper syringe 121 as described above. ing.

  The lower syringe 122 is a cylindrical portion that functions as a syringe for the rod-shaped portion 112 of the plunger 110, and the seal 112A of the rod-shaped portion 112 of the plunger 110 is slidable on the inner wall of the lower syringe 122 as described above. Mated.

  The capillary 123 is a narrow tube part having a plurality of types of liquids. The end of the capillary 123 on the container 10 side is inserted into the container 10, and the tip of the insertion portion is tapered. That is, the inner diameter of the end of the capillary 123 (opening diameter of the capillary 123) is smaller than the inner diameter of the capillary 123 other than the end.

  A reaction solution plug 150 is disposed in the capillary 123. The “plug” means a liquid that occupies a specific section in the capillary 123, and is held in a columnar shape in the example of FIG. Preferably there are no bubbles in the plug.

  The reaction solution plug 150 contains the reagent 11 used for the amplification reaction of the target nucleic acid. In the case of the first embodiment, the reagent 11 is placed on the bottom wall portion 10B of the container 10 in a lyophilized state. On the other hand, in the present embodiment, the reagent 11 is not arranged in the container 10 but is arranged in a plug state on the capillary 123 of the droplet introduction container 90 connected to the container 10. The reagent 11 is mixed with the sample 141 in the sample solution 140 that settles from the tank 100 via the buffer solution 130 and remains in the reaction solution plug 150.

  On such an outer wall surface of the tube 120, a fixing claw 124 and a guide plate 125 for mounting the cartridge 1 on a predetermined part of the nucleic acid amplification device are formed.

≪Container≫
The cap 10 </ b> C of the container 10 in the present embodiment is configured by an oil receiving portion 131 and a step portion 132. The oil receiving part 131 is a cylindrical part and functions as a reservoir for receiving the oil 12 filled in the container 10 located on the downstream side of the oil receiving part 131. There is a gap between the inner wall of the oil receiving portion 131 and the outer wall of the capillary 123 of the tube 120, and this gap becomes an oil receiving space 131 </ b> A for receiving the oil 12 overflowing from the container 10. The volume of the oil receiving space 131A is larger than the volume in which the seal 112A of the plunger 110 slides with the lower syringe 122 of the tube 120.

  The inner wall on the upstream side of the oil receiving portion 131 is sealed by contacting the annular convex portion of the tube 120. The seal portion is formed with a vent so that the oil does not leak due to the surface tension of the oil.

  The step portion 132 is a portion having a step provided on the downstream side of the oil receiving portion 131. The inner diameter of the downstream part of the step part 132 is smaller than the inner diameter of the oil receiving part 131. The inner wall of the stepped portion 132 is in contact with the outer wall on the downstream side of the capillary 123 of the tube 120. Sealing occurs when the inner wall of the stepped portion 132 and the outer wall of the tube 120 come into contact with each other. The seal portion resists the flow while allowing the oil 12 of the container 10 to flow into the oil receiving space 131A.

  As described above, in the initial state where the plunger 110 is not pushed, the rod-like portion 112 of the plunger 110 is located inside the upper syringe 121 of the tube 120 (see FIG. 8A). For this reason, the liquid in the tube 120 is not pushed out into the container 10. In this initial state, the interface of the oil 12 is located relatively downstream of the oil receiving space 131A (see FIG. 8A).

  On the other hand, when the plunger 110 is pushed, the rod-like portion 112 of the plunger 110 slides into the lower syringe 122 of the tube 120 (see FIG. 8B), so that the liquid in the tube 120 is pushed out to the container 10. It is.

  Specifically, first, the reaction solution plug 150 of the tube 120 flows from the tube 120 into the container 10. Since the inner diameter of the side wall 10 </ b> A of the container 10 is larger than the inner diameter of the capillary 123, the columnar reaction liquid plug 150 in the tube 120 becomes a droplet 20 in the oil of the container 10. Since the droplet 20 has a specific gravity greater than that of the oil 12, the droplet 20 settles on the side wall 10 </ b> A of the container 10 that becomes the flow path of the droplet 20.

=== Nucleic Acid Amplifier ===
FIG. 10 is a diagram illustrating a state in which the cartridge according to the second embodiment is mounted. FIG. 11 is a diagram illustrating a state of the rotation mechanism to which the cartridge according to the second embodiment is mounted. As shown in FIG. 10 and FIG. 11, in the nucleic acid amplification device 50 of the present embodiment, a fixing portion 160 is newly provided opposite to the opening side of the insertion hole 64. The fixed portion 160 is provided with a guide rail 161 for guiding the cartridge 1 to the insertion hole 64 while restraining the guide plate 125 in the cartridge 1 of the present embodiment in the front-rear direction.

  When the container 10 of the cartridge 1 guided by the guide rail 161 is inserted into the insertion hole 64, the fixing claw 124 (FIG. 8) of the cartridge 1 is caught by the notch portion of the fixing portion 160, so that the cartridge 1 is mounted. Is done.

  When the cartridge 1 is thus mounted, the mounting base 111A of the plunger 110 in the cartridge 1 is pressed by the rod 171. The rod 171 is controlled by the control unit 80 via the rod driving unit 172. Note that the direction in which the rod 171 pushes the mounting base 111A is inclined 45 degrees with respect to the vertical direction, not the vertical direction. Therefore, when the plunger 110 is pushed by the rod 171, the rotating body 61 is rotated 45 degrees, and the longitudinal direction of the cartridge 1 is matched with the moving direction of the rod 82.

  Further, in the nucleic acid amplification device 50 of the present embodiment, when the cartridge 1 is mounted on the fixing portion 160, the third heater 170 surrounding the reaction solution plug 150 of the cartridge 1 is newly provided in the heater 65.

  The third heater 170 heats the region including the reaction solution plug 150. The heating temperature is set to a temperature at which the nucleic acid coating is destroyed, and is set to 50 to 110 ° C. by the control unit 80 as in the first embodiment. That is, in the first embodiment, the first heater 65B and the second heater 65 are used not only as a heater for proceeding with the denaturation reaction and synthesis reaction of the template nucleic acid but also as a heater for destroying the nucleic acid coating. . On the other hand, in the present embodiment, the first heater 65B and the second heater 65 are dedicated heaters that allow the template nucleic acid denaturation reaction and the synthesis reaction to proceed. The third heater 170 is a dedicated heater for destroying the nucleic acid coating.

  For example, after detecting that the cartridge 1 is mounted based on a sensor (not shown) provided at a predetermined part of the fixing unit 160, the control unit 80 sets the temperature at which the third heater 170 destroys the nucleic acid covering. Set and heat the nucleic acid coating for a predetermined period. As a result, the nucleic acid coating of the sample 141 that settles from the tank 100 via the buffer solution 130 and remains in the reaction solution plug 150 is destroyed, and the nucleic acid (template nucleic acid) of the sample 141 is eluted into the reaction solution plug 150. . For this reason, the reaction liquid plug 150 contains the template nucleic acid and the reagent 11.

  Next, the control unit 80 rotates the rotating body 61 by 45 degrees so that the longitudinal direction of the cartridge 1 matches the moving direction of the rod 82. Thereafter, the control unit 80 controls the rod 171 via the rod driving unit 172 and presses the mounting base 111 </ b> A of the plunger 110 of the cartridge 1 fixed to the fixing unit 160 with the rod 171. As a result, the reaction solution plug 150 is pushed out into the container 10, and the droplet 20 of the solution containing the template nucleic acid and the reagent 11 is introduced into the container 10.

  Next, the control unit 80 sets the denaturation temperature for the first heater 65B and sets the synthesis temperature for the second heater 65C, controls the rotation mechanism 60 to execute the thermal cycle process, and performs the droplet 20 The nucleic acid eluted in is amplified. Note that the timing for setting the denaturation temperature for the first heater 65B and the synthesis temperature for the second heater 65C was before the temperature for destroying the nucleic acid coating was set for the third heater 170. May be.

<Summary>
As described above, in the nucleic acid amplification device 50 of the present embodiment, the droplet introduction container 90 for introducing the droplet 20 of the solution containing the template nucleic acid and the reagent 11 is connected to the container 10. A third heater 170 is provided for heating the predetermined part. Then, the controller 80 controls the rotation mechanism 60 after driving the third heater 170 to apply heat to the nucleic acid coating body for a predetermined period.

  In this way, even when the nucleic acid coating is destroyed by the third heater 170 provided separately from the first heater 65B and the second heater 65, the nucleic acid film can be used regardless of the procedure as in the first embodiment. Since the amplification reaction can proceed after destroying the body, the generation time of the PCR product can be shortened.

  In addition, since the third heater 170 is a dedicated heater for destroying the nucleic acid coating, switching of the temperatures to be set for the first heater 65B and the second heater 65 is avoided. Accordingly, there is no time until the heater reaches the target temperature after the heater is switched, and the PCR product generation time can be shortened accordingly.

(2) Modifications In the above embodiment, the specific gravity of the droplets 20 is greater than the specific gravity of the oil 12. However, the specific gravity of the droplet 20 may be smaller than the specific gravity of the oil 12. Even if it does in this way, there exists an effect similar to the said embodiment.

  In the above embodiment, the side wall 10A of the container 10 is cylindrical, and the bottom wall 10B of the container 10 is hollow hemispherical. However, the shape of the container 10 can be various shapes.

  In the first embodiment, the cap 10 </ b> C is detachable from the container 10, and the template nucleic acid solution is introduced with the cap 10 </ b> C removed from the container 10. However, the cap 10C may be fixed to the container 10, and the template nucleic acid solution may be introduced through a needle that penetrates the cap 10C. Note that the cap 10 </ b> C may be formed integrally with the container 10.

  In the first embodiment, the seal part SP of the cap 10C has a cylindrical shape. However, a hemispherical or conical depression may be formed in a portion facing the bottom wall portion 10B of the container 10 in the seal portion SP. When this dent tapers away from the side wall 10A of the container 10, the droplet 20 can be stopped at a fixed position, so that the droplet 20 can be heated under the same conditions. In addition, the bottom wall part 10B of the container 10 may be tapered as it separates from the side wall part 10A of the container 10.

In the first embodiment, the first heater 65B and the second heater are used during the nucleic acid elution process from when the first heater 65B and the second heater 65C set to a temperature at which the nucleic acid coating film is broken are driven to stop. Both 65Cs were driven. However, only one of the first heater 65B or the second heater 65C may be driven.
However, the rotator 61 during the nucleic acid elution process from when the first heater 65B and the second heater 65C set to a temperature at which the nucleic acid coating film is destroyed is stopped after being driven is positioned at the reference position, and the droplet 20 is It settles by its own weight and remains in the second region 36B ((B) of FIG. 7). For this reason, it is preferable to drive the second heater 65C from the viewpoint of heating the oil 12 faster during the nucleic acid elution process.

In the first embodiment, the nucleic acid coating film is subjected to the nucleic acid elution process from when the first heater 65B and the second heater 65C set to a temperature at which the nucleic acid coating film is broken to when the nucleic acid coating process is stopped. Heat was given for a predetermined period. In the second embodiment, heat is applied to the nucleic acid coating film for a predetermined period at the time of nucleic acid elution processing from when the third heater 170 set to a temperature at which the nucleic acid coating film is broken is driven to when it is stopped. It was. However, vibration may be applied to the nucleic acid coating film along with heat.
When vibration is applied to the nucleic acid coating film, for example, the control unit 80 controls the rotation mechanism 60 so that the droplet 20 alternately moves between the reference position and the reverse position, and vibrates the nucleic acid coating film. give. As another example, the control unit 80 controls the rotation mechanism 60 so that the droplets 20 are alternately moved to a reference position and a position shifted from the reference position in the direction of the reversal position by a predetermined angle. Give vibration to it.
As another example, one or two or more vibration sources that generate mechanical vibrations or sonic vibrations are provided in the rotating body 61 or the heater 65, and the vibration source is driven by the control unit 80 during the nucleic acid elution process to the nucleic acid coating film. Give vibration.
As described above, when vibration is applied to the nucleic acid coating film together with heat during the nucleic acid elution process, the number of template nucleic acids eluted from the nucleic acid film per unit sample is larger than when vibration is not applied. As a result, amplification efficiency can be improved.
When the control unit 80 controls the rotation mechanism 60 so that the droplet 20 moves during a predetermined period in which heat is applied to the nucleic acid coating, a case where a vibration source for moving the droplet 20 is separately provided. Compared to the above, it is possible to reduce the size as much as the vibration source can be omitted.
By the way, the amount of vibration to be applied to the nucleic acid coating film may be switched according to the type of specimen. The amount of vibration is the magnitude or frequency of vibration per unit time.
For example, when the control unit 80 receives a command from the second process start button 92B for gram-negative bacteria, the control unit 80 compares with a command received from the first process start button 92A for gram-positive bacteria. Reduce the magnitude or frequency of vibration per unit time. In addition, the control unit 80 receives a command from the second process start button 92B for gram-negative bacteria, as compared to a command received from the third process start button 92C for virus. Increase the magnitude or frequency of vibration per unit time.
Further, heat and vibration to be applied to the nucleic acid coating film may be selectively switched according to the type of specimen.
For example, when receiving a command from the third process start button 92C for virus, the control unit 80 gives heat to the droplet 20. On the other hand, when the control unit 80 receives a command from the second processing start button 92B for gram-negative bacteria, the control unit 80 vibrates the droplet 20. On the other hand, when receiving a command from the first process start button 92A for gram positive bacteria, the control unit 80 gives heat and vibration to the droplet 20.
Furthermore, the period of heat or vibration to be applied to the nucleic acid coating film may be switched according to the type of specimen.
For example, when the control unit 80 receives a command from the second process start button 92B for gram-negative bacteria, the control unit 80 compares with a command received from the first process start button 92A for gram-positive bacteria. Thus, the period of heat or vibration to be applied to the droplet 20 is shortened. In addition, the control unit 80 receives a command from the second process start button 92B for gram-negative bacteria, as compared to a command received from the third process start button 92C for virus. The period of heat or vibration to be applied to the droplet 20 is increased.
In this way, when switching according to the type of specimen is applied, even if the thickness or hardness of the nucleic acid coating varies depending on the type of specimen, damage to the nucleic acid wrapped in the nucleic acid coating is suppressed. However, the nucleic acid coating can be destroyed.

In the above embodiment, the surfactant is not included in the reagent 11. However, a surfactant may be included in the reagent 11 for the purpose of assisting the destruction of the nucleic acid coating. When the surfactant is included in the reagent 11, for example, the surfactant is fixed in the container 10 in a freeze-dried state together with the reagent 11, or the surfactant is contained in the reaction solution plug 150 together with the reagent 11. This can be realized.
When the surfactant is included in the reagent 11, the ratio of the number of template nucleic acids eluted from the nucleic acid film per unit sample can be increased compared to the case where the surfactant is not given. Amplification efficiency can be improved.
The surfactant is not particularly limited as long as it is generally used for elution of nucleic acid from cells or the like. Specific examples include Triton-based surfactants such as Triton-X, nonionic surfactants such as Tween 20, and anionic surfactants such as sodium N-lauroyl sarcosine (SDS).

In the above embodiment, the control unit 80 sets the temperature (temperature at which the covering is destroyed) to be set for the first heater 65B and the second heater 65C or the third heater 170 according to the type of the specimen. Switched. However, this temperature (temperature at which the covering is destroyed) may not be switched according to the type of specimen.
For example, when the process start button is omitted and the control unit 80 receives a command to execute the nucleic acid elution process from the input unit 92, the first heater 65B and the second heater 65C or the third heater for each unit period The temperature of the heater 170 (the temperature at which the covering is destroyed) is switched. Even in this manner, as in the above embodiment, even if the type of the specimen is unknown, the nucleic acid covering body of the specimen can be destroyed by heating.
In short, if the control unit 80 switches the temperature at which the nucleic acid coating is destroyed within a predetermined temperature range (50 ° C. to 110 ° C.), the nucleic acid coating of an unknown specimen can be destroyed by heating.
In addition, although the case where the amount of vibration to be applied to the nucleic acid covering is switched according to the type of specimen has been described above, the amount of vibration is also switched for each unit period without switching according to the type of specimen. It is possible.
In addition, the case where the heat and vibration to be applied to the nucleic acid covering body are selectively switched according to the type of the specimen has been described above, but the selection of the heat and vibration is also dependent on the type of specimen. It is possible to switch every unit period without switching.
Furthermore, although the case where the period of heat or vibration to be applied to the nucleic acid coating is switched according to the type of specimen has been described above, the period is also changed for each unit period without switching according to the type of specimen. It is possible to switch to.

  In the above embodiment, the start of the denaturation reaction period and the synthesis reaction period is the time when the rotating body 61 has been rotated 180 degrees (the rotating body 61 is stopped), but the rotating body 61 starts to rotate 180 degrees. It may be a point in time.

  In the above embodiment, the rotating mechanism 60 is employed as a moving mechanism that moves the droplet 20 in the container 10 to the first region 36A and the second region 36B. However, the droplets 20 are alternately placed in the first region 36A, which is set to the denaturation temperature of the target nucleic acid in the container 10, and the second region 36B, which is different from the first region 36A and is set to the synthesis temperature of the target nucleic acid. Various moving mechanisms other than the rotating mechanism 60 can be applied as long as the mechanism is moved to the right.

  In the above-described embodiment, the region to which the droplet 20 is to be moved in the container 10 is a first region 36A that is set to the denaturation temperature of the target nucleic acid, and is a region that is different from the first region 36A and that is the target nucleic acid synthesis A second region 36B to be heated was disposed. However, for example, as described in Japanese Patent Application No. 2014-107844, three regions may be arranged. That is, as the first region 36A, a region that is brought to the denaturation temperature of the target nucleic acid is arranged. In addition, two different regions are arranged as the second region 36B, one region is set to an annealing temperature set as a temperature at which the annealing reaction in the target nucleic acid synthesis reaction proceeds, and the other region is an extension of the target nucleic acid. The elongation temperature is set as the temperature at which the reaction proceeds. As described above, the container is not limited to the above-described embodiment in which the temperature change in the cycle is the two stages of the denaturation stage and the synthesis stage, and even when the three stages of the denaturation stage, the annealing stage, and the extension stage are used. The droplet 20 can be moved in the interior. In addition, even if the temperature change in a cycle is made into three steps, it is possible to apply various moving mechanisms other than a rotating mechanism.

  In the above embodiment, the nucleic acid amplification device 50 including at least the first heater 65B and the second heater 65C is applied. However, a nucleic acid amplification device other than the nucleic acid amplification device 50 of the above embodiment may be applied as long as a temperature gradient can be formed inside the container 10. For example, only the high temperature side heater may be provided, and the second heater 65C may be changed to a cooler. Alternatively, the first heater 65B and the second heater 65C may be provided outside the rotating body 61. Alternatively, the part where the first heater 65B is provided and the part where the second heater 65C is provided may be reversed.

  Moreover, in the said embodiment, although the fluorescence measuring device 70 was provided, you may abbreviate | omit. If the fluorescence measuring instrument 70 is omitted, nucleic acid amplification cannot be quantified, but the nucleic acid can be amplified.

  DESCRIPTION OF SYMBOLS 1 ... Cartridge, 10 ... Container, 10A ... Side wall part, 10B ... Bottom wall part, 10C ... Cap, 11 ... Reagent, 12 ... Oil, 20 ... Liquid Drop, 50 ... nucleic acid amplification device.

Claims (9)

A mounting part capable of mounting a container having a channel through which a droplet containing a template nucleic acid moves; and
When the container is mounted on the mounting portion, the posture of the container is changed so that the liquid droplets are changed from the first region of the container to the second region of the container different from the first region, and the first A moving mechanism for moving from two areas to the first area;
A heater,
A controller for controlling the heater and the moving mechanism;
With
The controller applies heat for a predetermined period at a temperature set to destroy the nucleic acid coating that coats the template nucleic acid, and then drops the droplet into the first region set at the target nucleic acid denaturation temperature. Controlling the transfer mechanism to repeat a plurality of cycles through a denaturation step that retains and a synthesis step that retains the droplet in the second region set at the integrity temperature of the target nucleic acid;
A nucleic acid amplification device. The heater includes a first heater that heats the first region, a second heater that heats the second region,
With
The controller sets a temperature at which at least one of the first heater and the second heater destroys the nucleic acid coating, and applies heat to the nucleic acid coating for a predetermined period of time, and then applies the heat to the first heater. And setting the denaturation temperature for the second heater and setting the synthesis temperature to control the moving mechanism,
The nucleic acid amplification device according to claim 1. A droplet introduction container for introducing the template nucleic acid and the droplet into the container is connected to the container;
The heater includes a first heater that heats the first region to a denaturation temperature of the target nucleic acid, a second heater that heats the second region to a synthesis temperature of the target nucleic acid, and a predetermined portion of the droplet introduction container. A third heater for heating
With
The controller controls the movement mechanism after driving the third heater and applying heat to the nucleic acid coating for a predetermined period.
The nucleic acid amplification device according to claim 1. The control unit can switch the temperature set to destroy the nucleic acid coating within a predetermined temperature range,
The nucleic acid amplification device according to any one of claims 1 to 3, wherein The predetermined temperature range is 50 ° C to 110 ° C.
The nucleic acid amplification device according to claim 4, wherein The control unit switches the temperature at which the nucleic acid coating is destroyed according to the type of specimen,
The nucleic acid amplification apparatus according to claim 4 or 5, wherein The controller switches the temperature at which the nucleic acid coating is destroyed every unit time.
The nucleic acid amplification apparatus according to claim 4 or 5, wherein The control unit controls the moving mechanism so that the droplet moves during the predetermined period in which heat is applied to the nucleic acid coating.
The nucleic acid amplification device according to any one of claims 1 to 7, wherein The droplet contains a surfactant,
The nucleic acid amplification apparatus according to any one of claims 1 to 8, wherein the nucleic acid amplification apparatus is characterized in that

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