JP6607846B2 - Nucleic acid amplification automation apparatus and nucleic acid amplification analysis automation apparatus - Google Patents

Description

  The present invention relates to an apparatus for automating nucleic acid amplification, and more particularly to an apparatus for automating DNA amplification by emulsion PCR (emPCR).

  In recent years, next-generation sequencers that perform high-speed sequencing have been developed, and the number of DNAs that can be processed has increased dramatically.

  454 (Roche) using emulsion PCR, SOLiD (Applied Biosystems), etc. are known as main next-generation sequencers currently on the market. The emPCR is a reaction in a micro droplet (emulsion) in which a large number of beads bound to nucleic acids are formed in oil. Miniaturized high-throughput nucleic acid analysis using emulsion PCR (Patent Document 1), multiple sequences A decision reaction (Patent Document 2) and the like have been proposed.

  Emulsion PCR is a technology that can amplify and analyze DNA or RNA molecules derived from a single molecule in the reaction field of individual microfluids, and is also represented by RainDance, Biorad, etc. for applications other than next-generation sequencers. It is adapted to Digital PCR technology. These amplification products are analyzed by Real-time PCR method, end-point PCR method, Flow cytometry method and so on.

However, in order to carry out emulsion PCR, it is necessary to place beads in micro or pico scale wells such as microtiter plates and picotiter plates, so it is difficult to recover samples from individual emulsions. It takes time and effort.
For this reason, automation has been required in order to eliminate the complexity of work.

JP 2010-193484 A JP-T-2007-523627

  An object of the present invention is to provide an apparatus capable of improving the efficiency from preparation of a nucleic acid sample (emulsion generation) to amplification and purification of a nucleic acid, preferably an apparatus that can be automated. Another object of the present invention is to provide an apparatus that can efficiently perform, preferably automatize, from preparation of a nucleic acid sample to amplification and purification of the nucleic acid and measurement (detection or quantification) of the amplified nucleic acid. To do.

  As a result of intensive studies to solve the above problems, the present inventor has provided an emulsion unit, a nucleic acid amplification unit, and a purification concentration unit, and can automatically move the solution between these units. And the automation from the emulsion after nucleic acid amplification to the separation and concentration of nucleic acid succeeded in completing the present invention.

That is, the present invention is as follows.
(Aspect 1) An emulsion generation step of encapsulating nucleic acid-bound beads in a water-in-oil emulsion, a nucleic acid amplification step of amplifying the nucleic acid in the emulsion, an emulsion destruction step of breaking the emulsion, and the amplified A nucleic acid amplification automation apparatus for performing a purification / concentration step for purifying and concentrating nucleic acid, a dispenser unit including a dispenser for dispensing a solution and / or a reagent used in each step, the emulsion generation step, and the Using the emulsion unit that performs the emulsion breaking step, the nucleic acid amplification unit that performs the nucleic acid amplification step using the emulsion generated by the emulsion generation step in the emulsion unit, and the emulsion unit obtained in the emulsion breaking step Purification using a solution containing the amplified nucleic acid And a purified concentration unit to perform condensation step, the nucleic acid amplification automated device.

  (Aspect 2) In the nucleic acid amplification automation apparatus according to Aspect 1, the emulsion unit includes an emulsion container that contains the emulsion, and an agitator that agitates the emulsion to produce and / or break the emulsion. Nucleic acid amplification automation equipment. (Aspect 3) The nucleic acid amplification automation apparatus according to aspect 2, wherein the emulsion container has a thin space for accommodating the emulsion. (Aspect 4) The nucleic acid amplification automation apparatus according to aspect 2 or 3, wherein the emulsion container includes a V-shaped bottom surface protruding downward in a V shape. (Aspect 5) The nucleic acid amplification automation apparatus according to any one of aspects 2 to 4, wherein the agitator is a vibrating body that vibrates the emulsion container.

  (Aspect 6) In the nucleic acid amplification automation apparatus according to any one of aspects 1 to 5, the nucleic acid amplification unit includes a PCR container that houses the emulsion, and a heating and cooling element that heats and cools the PCR container. A nucleic acid amplification automation apparatus. (Aspect 7) The nucleic acid amplification automation apparatus according to aspect 6, wherein the PCR container has a thin space for containing the emulsion. (Aspect 8) The nucleic acid amplification automation apparatus according to Aspect 7, wherein the PCR container has a wide surface and a narrow surface, and the heating and cooling element heats and cools the wide surface. (Aspect 9) In the nucleic acid amplification automation apparatus according to Aspect 8, the wide surfaces are a pair of wide surfaces, and the heating / cooling elements are a pair of heating / cooling elements in contact with each of the pair of wide surfaces. Nucleic acid amplification automation equipment.

  (Aspect 10) The nucleic acid amplification automation apparatus according to any one of aspects 6 to 9, wherein the PCR container includes a V-shaped bottom surface protruding downward in a V shape. (Aspect 11) In the nucleic acid amplification automation apparatus according to any one of aspects 1 to 10, the purification and concentration unit includes a filter unit including one or a plurality of filter tubes for purifying the amplified nucleic acid, and the amplified nucleic acid. A nucleic acid amplification automation apparatus comprising a purification concentration container for concentration. (Aspect 12) The nucleic acid amplification automation apparatus according to aspect 11, wherein the filter unit includes a heating member that heats the one or more filter tubes.

  (Aspect 13) In the nucleic acid amplification automation apparatus according to Aspect 11 or 12, the one or more filter tubes include a filter of a size that does not allow the beads to pass through, and filtering is performed by reducing pressure from below the filter. A nucleic acid amplification automation apparatus. (Aspect 14) In the nucleic acid amplification automation apparatus according to any one of Aspects 11 to 13, the purification and concentration container includes a magnet movable outside the purification and concentration container, and the magnet is attached to the purification and concentration container. A nucleic acid amplification automation apparatus that adsorbs the magnetic beads bound to the beads to the inner surface of the purification and concentration container by approaching the outer surface.

  (Aspect 15) The nucleic acid amplification automation apparatus according to any one of aspects 1 to 14, comprising a stirrer that stirs the filter tube. (Aspect 16) The nucleic acid amplification automation apparatus according to Aspect 15, wherein the stirring body is a vibrating body that vibrates the filter unit. (Aspect 17) The nucleic acid amplification automation apparatus according to Aspect 4 or 10, wherein the dispenser has an elongated capillary tube that is attached to suck a solution from the V-shaped bottom surface. (Aspect 18) The nucleic acid amplification automation device according to any one of aspects 1 to 17, further comprising a stage on which the emulsion unit, the nucleic acid amplification unit, and the purification and concentration unit are mounted, and the dispenser unit includes the dispenser A nucleic acid amplification automation apparatus comprising a moving mechanism that can move three-dimensionally on a stage.

  (Aspect 19) The nucleic acid amplification automation apparatus according to any one of aspects 1 to 18, wherein the dispenser moves the emulsion from the emulsion unit to the nucleic acid amplification unit for the nucleic acid amplification step, The emulsion containing amplified nucleic acid is transferred from the nucleic acid amplification unit to the emulsion unit for the emulsion breaking step, and obtained in the emulsion breaking step from the emulsion unit to the purification concentration unit for the purification concentration step. A nucleic acid amplification automation device that moves a solution containing amplified nucleic acid.

  (Aspect 20) In order to analyze the nucleic acid in the nucleic acid analysis automation apparatus comprising the nucleic acid amplification automation apparatus according to any one of aspects 1 to 19, and analyzing the nucleic acid obtained from the nucleic acid amplification automation apparatus, A nucleic acid analysis automation apparatus comprising a DNA chip or a sequencer. (Aspect 21) In a nucleic acid analysis automation apparatus for analyzing a nucleic acid obtained from the nucleic acid amplification automation apparatus according to any one of aspects 1 to 19, the bead is a fluorescent bead, and the amplified nucleic acid attached to the fluorescent bead A nucleic acid analysis automation device comprising: a DNA chip that binds to the fluorescent beads; a light source that irradiates the fluorescent beads with excitation light; and a fluorescence measurement device that measures fluorescence generated by the excitation light.

  The present invention provides a nucleic acid amplification automation apparatus and a nucleic acid amplification analysis automation apparatus. The apparatus of the present invention can automatically perform nucleic acid preparation, emulsion generation, nucleic acid amplification, emulsion breakage, and nucleic acid purification and concentration, thereby avoiding the complexity of user work and improving work efficiency. Can do.

1 is a perspective view showing a nucleic acid amplification automation apparatus according to a first embodiment of the present invention. It is a top view which shows the stage of the nucleic acid amplification automation apparatus of FIG. It is a conceptual diagram which shows the emulsion unit of FIG. FIG. 3 is a perspective view showing the PCR unit of FIG. 2. It is a conceptual diagram which shows the PCR container used for the PCR unit of FIG. It is a conceptual diagram which shows the filter part of the refinement | purification concentration unit of FIG. It is a conceptual diagram which shows the enrichment tube of the 2 refinement | purification concentration unit of a figure. FIG. 2 is a control block diagram of the nucleic acid amplification automation device according to the first embodiment of the present invention. It is a flowchart which shows emPCR operation | movement by the nucleic acid amplification automation apparatus which concerns on the 1st Embodiment of this invention. It is a schematic diagram which shows operation | movement of the nucleic acid amplification detection automation apparatus which concerns on the 3rd Embodiment of this invention. It is a schematic diagram which shows operation | movement of the digital PCR apparatus which concerns on the 4th Embodiment of this invention.

  Hereinafter, embodiments of the nucleic acid amplification automation apparatus and nucleic acid amplification analysis automation apparatus of the present invention will be described in detail. In the present invention, "automatic", "automated", and "automatic" do not exclude any manual work, and the apparatus executes at least a part of the processing in each process according to a predetermined program. Also included.

[Outline of First Embodiment]
The nucleic acid amplification automation apparatus according to the first embodiment of the present invention automatically performs an emulsion generation step, an amplification step for amplifying nucleic acids in the emulsion, an emulsion breaking step, and a purification and concentration step for separating, purifying and concentrating the amplified nucleic acids. To process. First, in describing the apparatus of the present invention, a series of steps from the emulsion generation step to the nucleic acid purification and concentration step performed in the present invention will be described.

  In the emulsion generation step, a water-in-oil type emulsion is prepared by mixing and stirring beads, to which a nucleic acid sample is bound, a PCR reagent (polymerase, primer, PCR buffer, etc.), and a hydrophobic solvent in a container. In order to facilitate the preparation of the emulsion, it is preferable to mechanically stir, vibrate, or vortex the emulsion container described below.

  Subsequently, the produced emulsion is subjected to an amplification step by PCR (polymerase chain reaction). Amplification can be performed by a thermal cycler (a PCR unit described later) in a reaction solution of a milliliter level, for example, a solution having a volume of 3 to 10 ml.

  After PCR, the emulsion is broken (called breaking) with a breaking solution such as isopropanol, and the amplification solution is washed. After washing, beads to which the amplified nucleic acid is bound are selected and the target nucleic acid is concentrated (enriched). For this selection, it is preferable to use magnetic beads.

  In the present invention, each unit or container that performs the above-described basic emulsion generation and destruction step, nucleic acid amplification step, and purification concentration step is made independent. Movement of the solution between each unit or container shall be performed automatically using a dispenser.

[Overall structure of nucleic acid amplification automation system]
The nucleic acid amplification automation apparatus 1 according to the first embodiment automates the above steps, and will be described with reference to FIGS. In FIG. 1, the nucleic acid amplification automation apparatus 1 includes a stage 10 that includes the units illustrated in FIG. 2, and a dispenser unit 20 that is provided on the stage 10 and includes a dispenser 21. Under the stage 10, one or both of a waste liquid bottle 11 in which various waste liquids are stored and a waste box 12 in which chips attached to the dispenser 21 are discarded can be optionally provided.

  The movement mechanism of the dispenser unit 20 can move the dispenser 21 three-dimensionally on the stage 10, dispense a solution or a reagent to each unit, and suck the solution from each unit. . This moving mechanism moves the dispenser unit 20 in the X direction and the X-axis direction moving unit 23 for moving the dispenser unit 20 in the X direction and the dispenser unit 20 in the Y direction for three-dimensional movement of the dispenser unit 20. It is comprised from the Y-axis direction moving part 24 and the Z-axis direction moving part 25 which moves the dispensing machine 21 to a Z-axis direction.

  FIG. 2 is a top view of the working stage 10 provided in the nucleic acid amplification automation apparatus 1. The stage 10 includes an emulsion unit 30 that performs an emulsion generation step and an emulsion breaking step, a PCR unit (nucleic acid amplification unit) 40 that performs a nucleic acid amplification step, and a purification concentration unit 50 that performs a purification and concentration step of amplified nucleic acid. .

  Further, the stage 10 stores a chip cassette 13 for storing a plurality of chips mounted on the dispenser 21, a disposal opening 14 for chips and the like connected to the disposal box 12, and a smaller amount of a plurality of reagents. First reagent cassette 15 to be stored, second reagent cassette 16 for storing a larger amount of a plurality of reagents, sample tube 17 for storing a sample such as nucleic acid before amplification, and output tube for storing nucleic acid after purification and concentration 18 may be optionally provided, or any combination of any of the above.

(Emulsion unit)
In the emulsion unit 30, the emulsion is generated and broken (braking). The emulsion unit 30 is demonstrated using the schematic diagram of FIG. The emulsion unit 30 includes a flat emulsion container 31 having a thin internal space, a vibration body (stirring body) 33 that is arranged around the emulsion container 31 and vibrates the container 31, and a vibration body driving unit (stirring) that vibrates the vibration body 33. Body drive unit) 35. The emulsion container (emulsion vial) 31 is provided with a preferably cylindrical opening 31a provided on the upper surface of the container for taking in and out the solution, and a V-shaped bottom surface 31b protruding downward in a V-shape. When the solution in the emulsion container 31 is sucked with the tip of the dispenser 21, the last remaining solution collects in the V-shaped bottom surface 31b. In this state, by sucking the tip of the tip of the dispenser 21 in accordance with the lower end of the V-shaped bottom surface 31b, most of the solution can be sucked, and loss during solution movement can be reduced. .

  An elastic material layer 33a such as rubber is formed on the surface of the vibrating body 33 facing the emulsion container 31. When the vibrating body 33 vibrates, the vibration is transmitted to the emulsion container 31 and the emulsion container 31 is not damaged by vibration. 31 is protected. A vibrating body drive unit 35 is connected to the vibrating body 33 via an arm. The vibrator driving unit 35 generates an emulsion by generating vibration at a predetermined frequency, amplitude, and time by a machine control unit 1100 described later. The vibration body driving unit 35 can use any vibration generating device such as an eccentric cam type, an unbalanced mass type, a hydraulic type, or an electrodynamic type. The eccentric cam type is to vibrate the vibrator driving unit by driving the eccentric cam with a motor such as a stepping motor. The unbalanced mass type vibration generating device is a device that attaches a mass (weight) returned to the motor and generates vibration by centrifugal force generated by the rotation of the motor, and is used for vibrators of mobile phones and smartphones. The hydraulic vibration generating device generates vibration by driving a piston with hydraulic pressure. The electrodynamic type uses the same principle as vibration of a speaker to generate vibration using a force generated by passing a current through a coil in a magnetic field.

  In the present embodiment, the emulsion means that a hydrophilic micro water droplet containing a single bead is enclosed in a micelle surrounded by a hydrophobic liquid. The beads encapsulated in the emulsion can be formed from a polymer, such as polystyrene. Furthermore, it can also be set as the fluorescent bead which made the bead contain the fluorescent pigment | dye. As such fluorescent beads, fluorescent nanosilica can also be used (International Publication No. 2007/074722 is incorporated by reference). The particle size of the beads can be arbitrarily selected, but the particle size of the beads is preferably 1 to 20 μm, more preferably 1 to 3 μm, and still more preferably about 1.5 μm. By using the fluorescent beads, the fluorescent beads to which the amplified nucleic acid is attached can be used by using a fluorescence measuring apparatus of a system such as “tube and quantitative system for measuring biological substances” described in International Publication No. 2010/122990 by the present applicant. Quantification and / or detection can be easily performed by measuring fluorescence intensity. WO 2010/122990 is incorporated by reference. Nucleic acids attached to the fluorescent beads can be quantified and / or detected by capturing them with the target substance capturing beads contained in the biological substance measuring tube. However, if the fluorescent bead has a large particle size, it becomes difficult to capture with the target substance-capturing bead. Therefore, the bead particle size can be preferably in the range of 1 to 20 μm. Furthermore, when the fluorescent beads to which the amplified nucleic acid is attached are analyzed with a DNA chip, the amount of the nucleic acid can be quantified by measuring the fluorescence intensity of the fluorescent beads.

(PCR unit)
FIG. 4 shows a PCR unit (nucleic acid amplification unit) 40 that amplifies nucleic acid by emulsion PCR. The PCR unit 40 includes a flat PCR container 41 that has a wide surface and a narrow surface to form a thin reaction space, and a pair of Peltier element accommodating portions 42 that are in contact with a pair of wide surfaces of the PCR container 41 so as to be capable of transferring heat, respectively. Each of the pair of Peltier elements 42 a included in the pair of Peltier element accommodating portions 42 includes a pair of heat sinks 43 that come into contact with each other so that heat can be transferred, and a pair of fans 45 that blow air to each of the pair of heat sinks 43. The Peltier element accommodating part 42 is a Peltier element 42a covered with a heat insulating material, and the Peltier element 42a inside the Peltier element accommodating part 42a is a PCR container 41 according to a temperature and time predetermined by a machine control part 1100 described later. A thermal cycle is performed by heating or cooling the solution contained in the container.

  As shown in the schematic diagram of FIG. 5, the PCR container 41 is provided on the top surface of the container to take in and out the solution, and preferably has a cylindrical opening 41a, and preferably has a V-shaped bottom surface protruding downward in a V-shape. 41b. The capacity of the PCR container 41 is preferably 5 to 10 ml, more preferably about 7 ml, and the width of the narrow surface of the PCR container 41 sandwiched between the pair of Peltier elements 42a is preferably 2 to 5 mm, more preferably about 3 mm.

  In such a preferable volume and shape of the PCR container 41, it is difficult for the dispenser chip normally used to suck the solution from the PCR container. Therefore, as shown in FIG. 5, when the solution in the PCR container 41 is sucked with the elongated capillary chip 21a attached to the dispenser 21, the last remaining solution is collected in the V-shaped bottom surface 41b. In this state, by sucking the tip of the capillary tip 21a of the dispenser 21 in accordance with the lower end of the V-shaped bottom surface 41b, most of the solution can be sucked, and the loss during solution movement can be reduced. Can do.

(Purification and concentration unit)
As shown in FIG. 2, the purification and concentration unit 50 includes a filter unit 52 including first and second filter tubes 53 and 54, and an enrichment tube 57. As shown in the conceptual diagram of FIG. 6, the filter unit 52 surrounds the first filter tube 53, the second filter tube 54, and the lower portions of the first filter tube 53 and the second filter tube 54 and heats them. A heat block (heating member) 56, a first filter tube 53, a second filter tube 54, a vibration body (stirring body) 58 that applies vibration V to the heat block 56, and a vibration body drive unit ( (Stirring body driving unit) 59. One or a plurality of the first filter tubes 53 and the second filter tubes 54 can be provided. The capacity of the first filter tube 53 is preferably larger than the capacity of the second filter tube 54. An elastic material layer 58a such as rubber is preferably formed on the surface of the vibrating body 58 facing the heat block 56, and the vibration is transmitted to the first and second filter tubes 53 and 54 when the vibrating body 58 vibrates. However, the first and second filter tubes 53 and 54 are protected from damage by vibration. Furthermore, the vibrating body 58 and the vibrating body driving unit 59 of the purification concentration unit 50 can be the same as the vibrating body 33 and the vibrating body driving unit 35 of the emulsion generation unit 30.

  Further, a filtering structure by the filter unit 52 will be described with reference to FIG. A first filter 53a and a second filter 54a each having a hole diameter smaller than the diameter of the nucleic acid binding beads are provided at the bottoms of the first filter tube 53 and the second filter tube 54, respectively. The first filter 53a and the second filter 54a can preferably be membrane filters. A pipe 55 is connected to the lower side of the first filter 53a and the second filter 54a, and the pipe 55 is connected to a vacuum pump via a vacuum trap (waste liquid bottle 11). Preferably, at least a part of the pipe 55 is formed of a flexible member, so that damage and liquid leakage due to vibration of the vibrating body 58 can be prevented.

  As shown in FIG. 7, a magnet 57 a is disposed outside the side surface of the enrichment tube (concentration container) 57. The magnet 57a can move between an approach position A and a position B away from the side surface of the enrichment tube 57 by a magnet moving mechanism 57b. Impurities can be separated by adsorbing magnetic particles contained in the solution using the magnet 57a. The magnet moving mechanism 57b is not limited, but preferably a pneumatic actuator, a cam mechanism, or the like can be used.

(Block Diagram)
Control blocks of the nucleic acid amplification automation apparatus 1 will be described with reference to FIG. The nucleic acid amplification automation system 1 preferably includes a machine control unit 1100 and a PC (personal computer) 1200 connected to the machine control unit 1100 via a communication line. The machine control unit 1100 preferably has an X-axis motor 1101 that drives the X-axis direction moving unit 23 of the dispenser unit 20, and a Y-axis motor 1102 that drives the Y-axis direction moving unit 24 of the dispenser unit 20. The Z-axis motor 1103 for driving the Z-axis direction moving unit 25 of the dispenser unit 20, the dispenser motor 1104 for pumping the dispenser 21, and the first or the second (vortex) for controlling the vibration or agitation (vortex) of the emulsion unit 40. 1 vortex motor 1105, a second vortex motor 1106 for controlling vibration or stirring (vortex) of the refining and concentrating unit 50, and a magnet moving motor 1107 for driving the magnet moving mechanism 57b of the refining and concentrating unit 50. Furthermore, the machine control unit 1100 includes a storage unit including a RAM and a ROM for storing control information and programs. Further, an input display 1221 having a display unit and an input device may be connected to the PC 1200. Note that the display unit and the input device may be connected to the PC 1200 as independent devices.

  The machine control unit 1100 preferably includes a PCR temperature control unit 1120 that controls the temperature of the PCR unit 40 and a heat block heating unit 1122 provided in the heat block 56. A temperature sensor 1120 a that measures the temperature of the PCR container 41 and a Peltier element 42 a of the Peltier element accommodating part 42 are connected to the PCR temperature control unit 1120. The heat block heating unit 1122 is provided with a temperature sensor 1122a, and the temperature of the heat block 56 is maintained in an appropriate range. Further, the machine control unit 1100 includes any one or more of a pressure sensor 1112 that measures the pressure downstream of the first and second filters 53a and 54a, a vacuum pump 1114, and a water level sensor 1116 of the waste liquid bottle. Can be provided with selection.

(Operation of nucleic acid amplification automation device)
A specific operation when emPCR is executed using the nucleic acid amplification automation apparatus 1 according to the present embodiment will be described with reference to FIG. In step S901, the dispenser 20 adjusts various reagents and solutions on the stage 10 and dispenses them to the emulsion unit 40. In step S902, the container 41 of the emulsion unit 40 is vibrated or stirred by the vibrating body 33, and an emPCR emulsion generation process is executed.

  In step S903, the dispenser 21 sucks the generated emulsion from the emulsion unit 40 and dispenses it into the PCR container 41 of the PCR unit 40. The dispenser 21 dispenses various reagents and solutions necessary for PCR into the PCR container 41. After the dispensing of the emulsion, reagent, and solution is completed, the pair of Peltier elements 42a heat or cool the PCR container 41 to execute the PCR process.

  Specifically, an example of a thermal cycle of PCR executed by the PCR unit 40 will be described. The emulsion solution transferred into the PCR container 41 using the dispenser 21 is a water-in-oil emulsion containing beads to which nucleic acids are bound, and further nucleic acid amplification such as primers, DNA polymerase, deoxynucleotide triphosphate, etc. Contains reagents. The reaction conditions for amplification are, for example, first reaction at 94 ° C. for 1 to 3 minutes (however, this reaction is optional), denaturation at 94 ° C. for 30 seconds to 1 minute, and 40 to 60 ° C. for 30 seconds to 1 Amplification of the nucleic acid is carried out for a predetermined number of cycles of annealing for 1 minute to 3 minutes within a temperature range (for example, about 72 ° C.) in which the polymerase reaction can be performed without separating the primer from the nucleic acid. . If necessary, the solution may be heated again at 72 ° C. for about 5 to 10 minutes after nucleic acid amplification.

  After completion of the PCR, the dispenser 21 moves the emulsion containing the amplified nucleic acid from the PCR unit 40 to the emulsion unit 30 and performs the breaking (braking) step of step S904. In step S <b> 904, the dispenser 21 breaks the emulsion by adding an emulsion breaking solution such as isopropanol to the emulsion containing the amplified nucleic acid and vibrating or stirring the emulsion container 31 using the vibrating body 33. After the destruction of the emulsion is completed, the dispenser 21 moves the solution in which the emulsion is broken from the emulsion container 31 to the first filter tube 53 of the filter unit 51, and executes the purification and concentration process in step S905.

  In step S905, the first filter tube 53 filters, heats, and cools the beads to which the amplified nucleic acid in the solution is attached. Further, the enriched tube 57 concentrates the amplified nucleic acid by binding the magnetic beads to the beads to which the amplified nucleic acid is attached. Hereinafter, the sub-steps included in step S905 will be described in detail.

  In a state where the solution containing the beads to which the amplified nucleic acid is attached is accommodated in the first filter tube 53, the beads downstream of the first filter 53a are decompressed to suck the unnecessary isopropanol and oil, and the beads to which the amplified nucleic acid is attached. Wash. Further, the mixed liquid in the first filter tube 53 is heated by the heat block 56 and cooled to room temperature, and the beads are washed again by reducing the pressure downstream of the first filter 53a, and the solution is added to the beads.

  After cooling, the dispenser 21 moves the solution containing the beads from the first filter tube 53 to the enrichment tube 57. And the dispenser 21 adds the solution containing a magnetic bead to the enrichment tube 57, and mixes with the solution containing the bead which the nucleic acid adhered. Since the magnetic beads have a high affinity with the beads to which the nucleic acids are attached, the magnetic beads and the beads to which the amplified nucleic acids are attached are aggregated in the enrichment tube 57. After the aggregate is formed, when the magnet 57a is moved to the approach position A in FIG. 7, the aggregate is attracted to the magnet 57a. In a state where the aggregate is adsorbed by the magnet 57 a, the dispenser 21 sucks a solution containing no nucleic acid from the bottom of the enrichment tube 57, dispenses it into the first filter tube 53, and discards it via the pipe 55. .

  Further, a plurality of bead washing operations described below are executed. First, the dispenser 21 adds the cleaning solution to the enrichment tube 57. Then, using the magnet moving part 57b, the magnet 57a is moved with respect to the enrichment tube 57 from the approach position A in FIG. When the magnetic force of the magnet 57 a does not reach the enrichment tube 57 due to the movement of the magnet 57 a, the aggregate is dispersed in the enrichment tube 57. The dispenser 21 repeats the suction and discharge of the dispersed solution a plurality of times to wash the beads to which the amplified nucleic acid is attached. Next, the position moves from the position B away from the magnet 57 a to the approach position A again, and the aggregate is adsorbed to the inner surface of the enrichment tube 57. In a state where the aggregate is adsorbed by the magnet 57 a, the dispenser 21 sucks a solution containing no nucleic acid from the bottom of the enrichment tube 57, dispenses it into the first filter tube 53, and discards it via the pipe 55. .

  In a state where the aggregate is adsorbed by the magnet 57a and the unnecessary solution is discarded, the dispenser 21 adds the solution to the enrichment tube 57, moves to the position B away from the magnet 57a, and disperses the aggregate. In a state where the separation liquid is added to the aggregate and the nucleic acid-adhered beads and the magnetic beads are separated, the magnet 57a is moved to the approach position A to adsorb only the magnetic beads in the solution to the inner surface of the enrichment tube 57. With the magnetic beads adsorbed, the dispenser 21 moves the solution containing the nucleic acid-attached beads from the enrichment tube 57 to the second filter tube 54. The second filter tube 54 sucks from the downstream side of the first filter 54 a to remove the solution, and the dispenser 21 adds the solution and sequencing primer to the remaining nucleic acid-adhered beads, and the solution is heated and cooled by the heat block 56. To do. Finally, the solution is removed from the downstream side of the filter 54 a by the second filter tube 54, the solution is removed, and the solution is added to the amplified nucleic acid-attached beads remaining in the dispenser 21, and this solution is dispensed into the output tube 18. And exit. The details of step S905 have been described above.

  The nucleic acid amplification automation apparatus 1 of the present embodiment automates steps S901 to S905 in FIG. 9, but when a nucleic acid amplification analyzer described later is used, step S906 is executed. In step S906, the amplified nucleic acid obtained in step S905 is detected and / or quantified using a nucleic acid analyzer (sequencer or DNA chip) described below.

[Second Embodiment]
The nucleic acid amplification analyzer of the second embodiment of the present invention is obtained by connecting a nucleic acid analyzer to the nucleic acid amplification automation apparatus 1 of the first embodiment. Specifically, the nucleic acid purified and concentrated by the nucleic acid amplification automation apparatus 1 is automatically moved to a sequencer (pyro sequencer) or a DNA chip (DNA microarray) by the dispenser 21 to automatically analyze the nucleic acid. Is what you do. This makes it possible to automatically execute a series of steps from nucleic acid amplification to analysis.

[Third Embodiment]
An automated apparatus and method for amplification and purification using emPCR and for analysis using a DNA chip according to the third embodiment of the present invention will be described with reference to FIG. Note that the beads a to c used in FIG. 10 are preferably fluorescent beads in which a phosphor is immobilized on the beads themselves.

In step S101 in FIG. 10, a sample solution containing a DNA fragment such as sample DNA, beads bound with primers (fluorescent beads such as plastic), PCR reagents and the like is prepared. Preferably, an adapter is linked to the sample DNA. Step S102 in FIG. 10 mixes oil for producing an emulsion with the sample solution. The sample solution includes first beads a, second beads b, and third beads c. A primer is fixed on each bead. Preferably, as the primer, a gene specific primer or a universal primer for an adapter can be used. In step S103 of FIG. 10, the sample solution is stirred to be emulsified. The emulsion includes a first emulsion a _em including the first beads a, a second emulsion b _em including the second beads b, and a third emulsion c _em including the third beads c. These emulsions contain substances necessary for PCR, such as primers, enzymes, and four types of deoxyribonucleotide triphosphates (dNTPs) for reaction. The first DNA fragment is hybridized (bound) to the primer of the first bead a. The DNA fragment is not hybridized with the primer of the second bead b. The third DNA fragment is hybridized to the primer of the third bead c.

In step S104 of FIG. 10, PCR is performed to amplify DNA within each emulsion. The first DNA fragment is amplified in the first emulsion a _em , the DNA fragment is not amplified in the second emulsion b _em , and the third DNA fragment is amplified in the third emulsion c _em . In step S105 of FIG. 11, the emulsion is braked in step S104 to extract beads with hybridized DNA fragments. In step S106, a solution containing beads hybridized with DNA fragments is supplied to the DNA chip. The DNA chip binds to the first DNA fragment hybridized to the first bead a at the first spot a ′ where the first gene-specific primer is immobilized, and the third gene-specific primer is immobilized on the first spot a ′. It binds to the third DNA fragment hybridized to the third bead c at 3 spots c ′. However, the second spot b ′ where the second gene-specific primer is immobilized does not bind to the second bead b. This is because the second bead b cannot bind to the second spot b ′ because the second DNA fragment is not bound to the second bead b.

  Finally, in a state where fluorescent beads are bound to each region, excitation light is preferably irradiated from a light source, and the fluorescence intensity emitted from the beads is measured. This enables detection and / or quantification of DNA fragments bound to each bead. Note that steps S101 and S102 in FIG. 10 can be executed in the same manner by the apparatus used in step S901 in FIG. Steps S103, S104, and S105 in FIG. 10 can be executed by the same devices as those used in steps S902, S903, and S904 in FIG.

[Fourth Embodiment]
A nucleic acid amplification analysis automation apparatus and method using digital PCR according to a fourth embodiment of the present invention will be described with reference to FIG. Digital PCR is the creation of minute (picoliter level) droplets (oil droplets) containing a single-molecule DNA fragment in a solution and performing PCR in the droplets. The fluorescence intensity is measured and target DNA is detected and / or quantified.

  As shown in FIG. 11, the droplet forming device 60 includes a sample solution supply unit 61 that supplies a sample solution containing a DNA fragment, a sample channel pipe 63 connected to the sample solution supply unit 61, mineral oil, and the like. A pair of oil supply pipes 65 that supply oil to the flow path 63, a connection part 67 that connects the oil supply pipe 65 to the flow path pipe 63, and a liquid droplet outflow part 69 that flows out the liquid droplets flowing through the flow path pipe 63 Is provided. The sample solution contains PCR reagents, primers, phosphors, and the like.

  In the droplet forming device 60, the DNA sample solution and the oil are mixed at the connection portion 67, and the mixed solution is emulsified (generates droplets). Since the DNA fragment has a Poisson distribution when the droplet is generated, the droplet D1 with the target DNA (first DNA fragment), the droplet D2 without the DNA fragment, and the DNA fragment other than the target DNA (second DNA fragment) And a droplet D3 containing. The solution containing these droplets is moved to the PCR container 71. The movement of the droplet solution from the droplet forming device 60 to the PCR container is performed automatically by gravity dropping with the PCR container 71 provided below the droplet outflow portion 69 or automatically by the dispenser 21. Also good. The PCR container 71 can also have the same structure as the PCR container 41 of the PCR unit 40 as shown in FIG. In this case, the DNA fragment in each droplet is amplified by the PCR unit 40 for each droplet. Droplets after PCR include target DNA, a primer D1 containing a primer that binds to the target DNA, and a phosphor, a simple droplet D2 that does not contain a DNA fragment, and a liquid that contains non-fluorescent or DNA fragments other than the target DNA. Drop D3.

  A digital PCR analyzer is used to analyze the DNA in the droplet. The analysis apparatus for digital PCR includes a transparent capillary (elongated tube) having a diameter through which only one droplet can pass, a light source that is provided in the capillary and that irradiates the phosphor with excitation light, and a phosphor that is provided in the capillary. And a fluorescence measuring unit that measures the fluorescence intensity emitted by. The movement of the solution containing droplets from the PCR container 71 to the capillary of the PCR analyzer may be automatically executed by the dispenser 21.

  When the solution containing the droplets D1 to D3 flows through the capillary, the droplet D1 emits fluorescence by the excitation light irradiated with the excitation light from the light source, and the fluorescence intensity is measured by the fluorescence measurement unit. The droplet D2 does not emit light because it does not contain a phosphor. In order to detect and quantify the second DNA fragment contained in the droplet D3, a primer and a phosphor that bind to the second DNA fragment may be added in advance to the sample solution. Further, the respective phosphors can be selected so that the fluorescence spectrum of the phosphor for the first DNA fragment is different from the fluorescence spectrum of the phosphor for the second DNA fragment. As a result, the droplets can also be visually identified. Moreover, it is possible to calculate the abundance ratio of the first DNA fragment and the second DNA fragment by measuring the ratio of these fluorescence intensities. When measuring the fluorescence of the droplets D1 and / or D3, these droplets have the same function as the fluorescent beads described in the first embodiment, and thus the droplets can be regarded as fluorescent beads.

  In each embodiment of the present invention, the nucleic acid amplified by the nucleic acid amplification automation apparatus is not limited to DNA, but also includes RAN such as microRNA. In the case of amplifying RNA, a step of reverse transcription of RNA to DNA is added. In addition, multiplex detection can also be performed in the nucleic acid amplification analysis automation apparatus of each embodiment of the present invention. That is, the nucleic acid obtained from the nucleic acid amplification automation apparatus is analyzed in parallel by any of a plurality of analysis automation apparatuses (for example, a sequencer and a DNA chip). Such analysis enables multi-dimensional analysis of nucleic acid information.

  The sequencer used in the second embodiment is not limited, but a Roche 454 sequencing system or a GS junior system provided by Roche Applied Science can be used. As the DNA chip used in the second or third embodiment, LuBEA manufactured and sold by Precision System Science can be used. The technology relating to LuBEA has been filed by the present applicant as a “biologically related substance measuring tube and quantitative system” described in International Publication No. 2010/122990, the contents of which are incorporated herein by reference ( Incorporation by Reference). In the second or third embodiment, by using LuBEA as a DNA chip, DNA can be analyzed and / or quantified with a detection sensitivity equivalent to that of digital PCR.

  Furthermore, as the DNA chip (DNA microarray) used in the second or third embodiment, GeneChip and cDNA microarray (Stanford type) developed by Affymetrix can also be used. The analysis device (scanner) for the DNA chip preferably includes a light source that irradiates fluorescent beads with excitation light, and a fluorescence measurement device that measures fluorescence generated by the excitation light.

1 Nucleic acid amplification automation apparatus 10 Stage 20 Dispenser unit 21 Dispenser 30 Emulsion unit 40 PCR unit 50 Purification and concentration unit

Claims (19)

An emulsion generating step of encapsulating nucleic acid-bound beads in a water-in-oil emulsion, a nucleic acid amplification step of amplifying the nucleic acid in the emulsion, an emulsion breaking step of destroying the emulsion, and purification and concentration of the amplified nucleic acid A nucleic acid amplification automation apparatus for performing a purification and concentration step,
A dispenser unit comprising a dispenser for dispensing solutions and / or reagents used in each step;
An emulsion unit for performing the emulsion generation step and the emulsion breaking step;
A nucleic acid amplification unit for performing the nucleic acid amplification step using the emulsion produced by the emulsion production step in the emulsion unit;
With a solution containing an amplified nucleic acid obtained by the emulsion breaking step for emulsion unit, possess a purified concentration unit to perform purification concentration step,
The emulsion unit includes an emulsion container that contains the emulsion, and a vibrating body that vibrates the emulsion container in a lateral direction in order to generate and destroy the emulsion, and the emulsion container is downwardly formed in a V shape. Ru with a V-shaped bottom surface that protrudes, nucleic acid amplification automated device. In the nucleic acid amplification automation apparatus according to claim 1,
A nucleic acid amplification automation apparatus, wherein an elastic material layer is formed on a surface of the vibrator facing the emulsion container . In the nucleic acid amplification automation apparatus according to claim 2,
The nucleic acid amplification automation apparatus, wherein the emulsion container has a thin space for containing the emulsion. In the nucleic acid amplification automation device according to any one of claims 1 to 3 ,
The nucleic acid amplification unit includes a PCR container containing the emulsion and a heating / cooling element for heating and cooling the PCR container. 5. The nucleic acid amplification automation apparatus according to claim 4 , wherein the PCR container has a thin space for accommodating the emulsion. 6. The nucleic acid amplification automation apparatus according to claim 5 , wherein the PCR container has a wide surface and a narrow surface, and the heating and cooling element heats and cools the wide surface. The nucleic acid amplification automation apparatus according to claim 6 , wherein the wide surfaces are a pair of wide surfaces, and the heating / cooling element is a pair of heating / cooling elements in contact with each of the pair of wide surfaces. Automation device. The nucleic acid amplification automation device according to any one of claims 4 to 7 , wherein the PCR container includes a V-shaped bottom surface protruding downward in a V shape. In the nucleic acid amplification automation device according to any one of claims 1 to 8 ,
The said purification concentration unit is a nucleic acid amplification automation apparatus provided with the filter part provided with the 1 or several filter tube for refine | purifying amplification nucleic acid, and the purification concentration container which concentrates amplification nucleic acid. The nucleic acid amplification automation apparatus according to claim 9 ,
The nucleic acid amplification automation apparatus, wherein the filter unit includes a heating member that heats the one or more filter tubes. The nucleic acid amplification automation apparatus according to claim 9 or 10 ,
The nucleic acid amplification automation apparatus, wherein the one or more filter tubes include a filter of a size that does not allow the beads to pass through, and performs filtering by reducing pressure from below the filter. In the nucleic acid amplification automation apparatus according to any one of claims 9 to 11 ,
The refinement concentration container includes a magnet that can move outside the refinement concentration container, and the magnetic beads coupled to the beads are attracted to the inner surface of the refinement concentration container by bringing the magnet closer to the outer surface of the refinement concentration container. A nucleic acid amplification automation device. In the nucleic acid amplification automation device according to any one of claims 9 to 12 ,
A nucleic acid amplification automation apparatus comprising an agitator that agitates the one or more filter tubes. The nucleic acid amplification automation apparatus according to claim 13 ,
The nucleic acid amplification automation device, wherein the stirring body is a vibrating body that vibrates the filter unit. The nucleic acid amplification automation device according to any one of claims 1 to 14 , wherein the dispenser has an elongated capillary tube that is attached to suck a solution from the V-shaped bottom surface. In the nucleic acid amplification automation device according to any one of claims 1 to 15 ,
A stage equipped with the emulsion unit, the nucleic acid amplification unit, and the purification and concentration unit; and the dispenser unit comprises a moving mechanism that allows the dispenser to move three-dimensionally on the stage. Amplification automation equipment. In the nucleic acid amplification automation device according to any one of claims 1 to 16 ,
The dispenser moves the emulsion from the emulsion unit to the nucleic acid amplification unit for the nucleic acid amplification step, and an emulsion containing amplified nucleic acid from the nucleic acid amplification unit to the emulsion unit for the emulsion breaking step. A nucleic acid amplification automation apparatus that moves and moves the solution containing the amplified nucleic acid obtained in the emulsion breaking step from the emulsion unit to the purification concentration unit for the purification and concentration step. A nucleic acid analysis automation apparatus comprising the nucleic acid amplification automation apparatus according to any one of claims 1 to 17 , and analyzing a nucleic acid obtained from the nucleic acid amplification automation apparatus.
A nucleic acid analysis automation device comprising a DNA chip or a sequencer for analyzing the nucleic acid. A nucleic acid analysis automation apparatus comprising the nucleic acid amplification automation apparatus according to any one of claims 1 to 17 , and analyzing a nucleic acid obtained from the nucleic acid amplification automation apparatus.
The beads are fluorescent beads, a DNA chip that binds to the amplified nucleic acid attached to the fluorescent beads, a light source that irradiates the fluorescent beads with excitation light, and a fluorescence measurement device that measures the fluorescence generated by the excitation light. An apparatus for automating nucleic acid analysis.

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