JP2015050968A - Nucleic acid amplification system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel nucleic acid amplification system.SOLUTION: The nucleic acid amplification system includes: a nucleic acid detection control apparatus, the nucleic acid detection control apparatus comprising a rotating body having a mounting section mountable with a cartridge comprising a tube which has a plug containing a reaction liquid which elutes a nucleic acid from a nucleic acid binding solid-phase carrier bound with the nucleic acid, and a nucleic acid amplification reaction container which communicates with the tube and contains an oil phase-separated from the reaction liquid, heaters for forming temperature gradient within the nucleic acid amplification reaction container, and a fluorescence measuring device for measuring the fluorescences which are emitted from fluorescent substances contained in the reaction liquid within the nucleic acid amplification reaction container and serve as indices of the nucleic acid amplification reaction; and a nucleic acid amplification apparatus comprising a control section for controlling the fluorescence measuring device, wherein the control section, when having the fluorescence measuring device measure three fluorescences with different peak wavelengths, superposes at least part of the time for measuring a first fluorescence and the time for measuring a third fluorescence if the three fluorescences are called the first fluorescent, the second fluorescent, and the third fluorescent in the ascending order of the peak wavelengths.

Description

  The present invention relates to a nucleic acid amplification system.

In Patent Documents 1 and 2, the reaction liquid is moved inside the biochip by rotating the biochip filled with the reaction liquid and oil having a specific gravity smaller than that of the reaction liquid phase-separated from the reaction liquid. An apparatus for applying a thermal cycle is disclosed.

JP 2009-136250 A JP 2012-115208 A

  An object of the present invention is to provide a novel nucleic acid amplification system.

A nucleic acid amplification system according to an embodiment of the present invention is a nucleic acid amplification system including the following (1) nucleic acid amplification device and (2) nucleic acid detection control device.
(1) a tube having a plug containing an eluate from which the nucleic acid is eluted from a nucleic acid-binding solid phase carrier to which the nucleic acid is bound; a nucleic acid amplification reaction vessel containing oil that communicates with the tube and phase-separates from the eluate; , A rotating body having a mounting portion on which a cartridge can be mounted, a heater that forms a temperature gradient inside the nucleic acid amplification reaction vessel, and a fluorescent material contained in the reaction solution in the nucleic acid amplification reaction vessel, A nucleic acid amplification apparatus comprising: a fluorescence measuring device that detects fluorescence that serves as an index of a nucleic acid amplification reaction;
(2) A control unit that controls the fluorescence measuring device is provided, and the control unit first sets the three fluorescences from the shorter peak wavelength when the fluorescence measuring device measures three fluorescences having different peak wavelengths. When the fluorescent light, the second fluorescent light, and the third fluorescent light are used, at least a part of the time for measuring the first fluorescent light and the time for measuring the third fluorescent light is overlapped. Detection control device. Here, the control unit may cause the fluorescence measuring instrument to measure the first fluorescence and the third fluorescence, and then measure the second fluorescence.
The nucleic acid detection method according to another embodiment of the present invention includes a tube having a plug containing an eluate that elutes the nucleic acid from a nucleic acid-binding solid phase carrier to which a nucleic acid is bound, and a tube that communicates with the tube. A rotating body having a mounting portion to which a cartridge equipped with a nucleic acid amplification reaction vessel containing oil for phase separation, a heater for forming a temperature gradient inside the nucleic acid amplification reaction vessel, and a nucleic acid amplification reaction vessel In a nucleic acid amplification device comprising a fluorescence measuring device that detects fluorescence emitted from a fluorescent substance contained in a reaction solution and serves as an index of a nucleic acid amplification reaction, the peak wavelengths contained in the reaction solution in the nucleic acid amplification reaction vessel are different. A nucleic acid detection method comprising a step of detecting three fluorescences emitted from three fluorescent substances and serving as an index of a nucleic acid amplification reaction, wherein the three fluorescences have a shorter peak wavelength. A nucleic acid characterized by overlapping at least part of the time for measuring the first fluorescence and the time for measuring the third fluorescence when the first fluorescence, the second fluorescence, and the third fluorescence are used. It is a detection method. Here, the first fluorescence and the third fluorescence may be measured, and then the second fluorescence may be measured.
A nucleic acid amplification system according to a further embodiment of the present invention includes a tube having a plug containing an eluate that elutes the nucleic acid from a nucleic acid-binding solid phase carrier to which the nucleic acid is bound, and a tube that communicates with the tube and is in phase with the eluate. A nucleic acid amplification reaction vessel containing oil to be separated, a mounting portion that can be equipped with a cartridge, and fluorescence emitted from a fluorescent substance contained in a reaction solution in the nucleic acid amplification reaction vessel and used as an indicator of a nucleic acid amplification reaction A fluorescence measuring instrument that controls the fluorescence measuring instrument, and when the controller causes the fluorescence measuring instrument to measure three fluorescences having different peak wavelengths, the three fluorescences have a short peak wavelength. When the first fluorescence, the second fluorescence, and the third fluorescence are used from the side, at least a part of the time for measuring the first fluorescence and the time for measuring the third fluorescence are overlapped. The symptom is a nucleic acid amplification system. Here, the control unit may cause the fluorescence measuring instrument to measure the first fluorescence and the third fluorescence, and then measure the second fluorescence.

1A and 1B are explanatory views of the cartridge 1. 2A to 2C are operation explanatory views of the cartridge 1. 3A to 3D are explanatory views of the tank 3. FIG. 4 is an explanatory diagram of the fixing claw 25, the guide plate 26 and the mounting portion 62. 5A and 5B are explanatory diagrams of the periphery of the PCR container 30. FIG. FIG. 6A is a perspective view of the internal configuration of the PCR device 50. FIG. 6B is a side view of the main configuration of the PCR device 50. FIG. 7 is a block diagram of the PCR device 50. FIG. 8A is an explanatory diagram of the rotating body 61. FIG. 8B is an explanatory diagram of a state where the cartridge 1 is mounted on the mounting portion 62 of the rotating body 61. 9A to 9D are explanatory diagrams of the state of the PCR device 50 when the cartridge 1 is mounted. FIG. 10 is a conceptual diagram of the behavior of the magnetic beads 7 when the magnet 71 is moved downward. 11A to 11C are explanatory diagrams of the nucleic acid elution process. FIG. 12 is a conceptual diagram of the behavior of the magnetic beads 7 when the magnet 71 is swung. FIG. 13 is a table showing whether or not the magnet 71 swings. 14A to 14C are explanatory diagrams of the droplet forming process. 15A and 15B are explanatory diagrams of the thermal cycle process. 16A and 16B are explanatory diagrams of the cartridge 1 according to the second embodiment. FIG. 17A is an explanatory diagram of an initial state of the cartridge 1 according to the second embodiment. FIG. 17B is a side view when the plunger 10 is pushed from the state of FIG. 17A and the seal 12A comes into contact with the lower syringe 22. FIG. 17C is an explanatory diagram of the cartridge 1 of the second embodiment after the plunger 10 is pushed. 18A and 18B are explanatory diagrams of the periphery of the PCR container 30 of the second embodiment. It is a figure which shows the kit for nucleic acid extraction used in one Example, and the apparatus after the assembly. It is a graph which shows the result of real-time PCR in one Example.

Hereinafter, embodiments of the present invention will be described in detail. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments of the present invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation, and the present invention is not limited thereto. Based on the description of the present specification, various changes and modifications can be made within the spirit and scope of the present invention disclosed in the present specification.
It will be apparent to those skilled in the art.
=== First Embodiment ===
First, after describing the cartridge attached to the PCR device 50 (nucleic acid amplification reaction device), the configuration and operation of the PCR device 50 of the present embodiment will be described.

<Cartridge 1>
1A and 1B are explanatory views of the cartridge 1. 2A to 2C are operation explanatory views of the cartridge 1. FIG. 2A is an explanatory diagram of the initial state of the cartridge 1. 2B.
FIG. 2B is a side view when the plunger 10 is pushed from the state of FIG. 2A and the seal 12A comes into contact with the lower syringe 22. FIG. 2C is an explanatory diagram of the cartridge 1 after the plunger 10 is pushed.

The cartridge 1 is a container that performs a nucleic acid elution process for eluting nucleic acid from the magnetic beads 7 to which the nucleic acid is bound, and a container that performs a thermal cycle process for a polymerase reaction on the reaction solution 47 that becomes a PCR solution.

The nucleic acid extraction process is performed in the tank 3 and purified while passing through the tube 20. Tube 2
The material of 0 is not particularly limited, but may be, for example, a resin such as glass or plastic, or a metal. In particular, when transparent glass or resin is selected as the material of the tube 20, the inside can be observed from the outside of the tube 20, which is more preferable. In addition, when a material that transmits magnetic force or a non-magnetic material is selected as the material of the tube 20, the magnetic particles are moved by applying a magnetic force from the outside of the tube 20 when passing the magnetic particles through the tube 20. Is preferable because it is facilitated. Moreover, since the heater (displacement heater 65A and high temperature side heater 65B described later) is disposed in the vicinity of the material of the tube, it is preferable that the tube has heat resistance of at least 100 ° C. or more. The material of the tube 20 is
It may be the same as the material of the tank.

The tube 20 includes a cleaning liquid plug 45, a reaction liquid plug 47, and an oil plug. Since the magnetic beads 7 to which the nucleic acid is bound are attracted to an external magnet, the magnetic beads 7 move in the tube 20 by moving the magnet outside along the tube 20, pass through the washing liquid plug 45, and then the reaction solution. The plug 47 is reached. The nucleic acid bound to the magnetic beads 7 is washed with the washing liquid plug 45 and eluted with the reaction liquid plug 47. Here, the “plug” means a liquid when a specific liquid occupies one section in the tube 20. For example, in FIG. 2A to FIG.
It is called a “plug”. Oil is phase-separated from other solutions (not miscible with other solutions). Therefore, the plug made of oil has a function of preventing the water-soluble plugs on both sides from mixing with each other. . It is preferable that there are no bubbles or other liquids in or between the plugs, but bubbles or other liquids may exist as long as the magnetic beads 7 can pass through the plug.

The type of oil is not particularly limited, and mineral oil, silicone oil (2CS silicone oil, etc.), vegetable oil, etc. can be used. However, by making the oil more viscous, the nucleic acid at the interface with the upper plug is used. When the binding solid phase carrier is moved, the “wiping effect” by the oil can be enhanced. As a result, when the nucleic acid-binding solid phase carrier is moved from the upper plug to the plug made of oil, water-soluble components attached to the nucleic acid-binding solid phase carrier are made less likely to be brought into the oil. Can do.

The thermal cycle process is performed in the PCR container 30 of the cartridge 1. The PCR container 30
Since the reaction liquid 47 is filled with oil and phase-separates with the oil, when the reaction liquid plug 47 is pushed out from the tube 20 into the PCR container 30, it becomes droplets and has a higher specific gravity than the oil. Therefore, the reaction liquid 47 in the form of droplets settles. When the high temperature region 36A and the low temperature region 36B are formed in the PCR container 30 by the external heater and the entire cartridge 1 is turned upside down together with the heater, the reaction liquid in the form of droplets is formed between the high temperature region 36A and the low temperature region 36B. 47 alternately move, and the reaction solution 47, which is a PCR solution, is subjected to a two-step temperature treatment.

Although the material of the PCR container 30 is not specifically limited, For example, resin, metals, such as glass and a plastic, can be used. Moreover, since the material of the PCR container 30 has the high temperature side heater 65B in the vicinity, it is preferable to have heat resistance of at least 100 ° C. or more. PC
It is preferable to select a transparent or translucent material for the R container 30 because fluorescence measurement (fluorescence intensity measurement) becomes easy. However, the entire region of the PCR container 30 does not need to be transparent or translucent, and at least a part facing the fluorescence measuring instrument 55 (for example, the bottom 35A of the PCR container 30).
May be transparent or translucent. The material of the PCR container 30 may be the same as that of the tank 3 and the plunger 10.

The cartridge 1 includes a tank 3 and a cartridge main body 9. In the kit constituting the cartridge 1, the adapter 5 is prepared in advance together with the tank 3 and the cartridge main body 9. The cartridge 1 is assembled by connecting the tank 3 and the cartridge main body 9 via the adapter 5. However, the tank 3 can be configured to be directly attached to the cartridge body 9.

In the following description of the components of the cartridge 1, as shown in FIG. 2A, the direction along the long cartridge 1 is “longitudinal direction”, the tank 3 side is “upstream side”, and the PCR container 30 side is “ “Downstream”. The upstream side may be simply expressed as “upper” and the downstream side may be expressed as “lower”.

(1) Tank FIGS. 3A to 3D are explanatory views of the tank 3.

A tank 3 prepared in advance in the kit contains a solution 41 and magnetic beads 7. A removable lid 3A is attached to the opening of the tank 3 (see FIG. 3A).
As the dissolving solution 41, 5M guanidine thiocyanate, 2% Triton X-100,
50 mM Tris-HCl (pH 7.2) is used. The operator removes the lid 3A, opens the opening of the tank 3 (see FIG. 3B), immerses the swab with the virus attached thereto in the lysis solution 41 in the tank 3, and collects the virus in the lysis solution 41 (see FIG. 3C). ). When the liquid in the tank 3 is agitated, the tank 3 may be shaken in the state of FIG. 3C. However, since the solution 41 easily overflows, the adapter 5 with a lid 5A as shown in FIG. 3D. It is preferable that the tank 3 is shaken after being attached to the opening of the tank 3. As a result, the substance in the tank 3 is agitated, the virus particles are dissolved by the solution 41, the nucleic acid is released, and the silica coated on the magnetic beads 7 adsorbs the nucleic acid. The magnetic beads 7 correspond to a nucleic acid binding solid phase carrier. Thereafter, the operator removes the cover 5A of the adapter 5 attached to the opening of the tank 3, and attaches the tank 3 to the cartridge body 9 via the adapter 5 (see FIG. 2A).

The tank 3 is made of a flexible resin, and the tank 3 can expand. When the plunger 10 slides and changes from the state shown in FIG. 2A to the state shown in FIG. 2B, the tank 3 expands, so that the pressure of the liquid in the tube 20 is suppressed from rising too much. Extrusion to the downstream side is suppressed. It is desirable to form the deformed portion 3B in the tank 3 so that the tank 3 is easily expanded.

The sample from which the nucleic acid is extracted / amplified is not limited to a virus but may be a cell. The origin of the cell is not particularly limited, and it may be a microorganism or a tissue piece or blood of a higher organism.

The lysing solution 41 is not particularly limited as long as it contains a chaotropic substance, but it may contain a surfactant for the purpose of disrupting the cell membrane or denaturing proteins contained in the cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells or the like. Specifically, a Triton surfactant such as Triton-X or Tw
Nonionic surfactants such as tween surfactants such as een20 and anionic surfactants such as sodium N-lauroyl sarcosine (SDS) can be mentioned. It is preferable to use it in the range of 1 to 2%. Furthermore, it is preferable that the solution contains a reducing agent such as 2-mercaptoethanol or dithiothreitol. The lysis solution may be a buffer solution, but is preferably neutral at pH 6-8. In consideration of these matters, specifically, it contains 3 to 7 M guanidine salt, 0 to 5% nonionic surfactant, 0 to 0.2 mM EDTA, 0 to 0.2 M reducing agent and the like. It is preferable to do.

A chaotropic substance generates chaotropic ions (monovalent anions having a large ionic radius) in an aqueous solution and has an action of increasing the water solubility of hydrophobic molecules, contributing to adsorption of nucleic acids to a solid phase carrier. If it is a thing, it will not specifically limit. Specific examples include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate and the like. preferable. The concentration of these chaotropic substances used varies depending on each substance. For example, when guanidine thiocyanate is used, it is in the range of 3 to 5.5M, and when guanidine hydrochloride is used, it is used at 5M or more. Is preferred.

Moreover, the instrument which collects a sample is not specifically limited, What is necessary is just to select according to a use, such as a spatula, a stick | rod, and a scraper, instead of a cotton swab.

Although the internal volume of the tank 3 is not specifically limited, For example, it can be 0.1 mL or more and 100 mL or less. Although the material of the tank 3 is not specifically limited, For example, resin, metals, such as glass and a plastic, can be used. In particular, when transparent glass or resin is selected as the material of the tank, the inside can be observed from the outside of the tank 3, which is more preferable. The tank 3 and each tube 20 may be integrally formed or detachable. If a material having flexibility such as rubber, elastomer, polymer, etc. is used as the material of the tank 3, the inside of the tank 3 can be pressurized by deforming the tank 3 with the lid attached to the tank 3. it can. Thereby, the contents of the tube 20 can be pushed out from the inside of the tube from the inside to the outside.

(2) Cartridge Main Body The cartridge main body 9 includes a plunger 10, a tube 20, and a PCR container 30.

(2-1) Plunger Hereinafter, the plunger 10 will be described with reference to FIGS. 2A to 2C.

The plunger 10 is a movable pusher that pushes out liquid from the downstream side of the tube 20 that functions as a syringe. The plunger 10 has a function of pushing a predetermined amount of liquid in the tube 20 from the end of the tube 20 to the PCR container 30. The plunger 10 also has a function of attaching the tank 3 via the adapter 5.

The plunger 10 has a cylindrical portion 11 and a rod-like portion 12. The cylindrical part 11 is on the tank 3 side (
The rod-shaped part 12 is provided on the tube 20 side (downstream side). The rod-like portion 12 is supported by two plate-like ribs 13 from the inner wall on the downstream side of the tubular portion 11.
The downstream side of the rod-shaped part 12 protrudes from the cylindrical part 11 to the downstream side.

The cylindrical part 11 is opened upstream and downstream, and the inner wall of the cylindrical part 11 serves as a liquid passage. An adapter 5 is fitted into an opening on the upstream side (tank 3 side) of the cylindrical portion 11. The plunger 10 of the cartridge main body 9 prepared in advance in the kit may have a removable lid attached to the opening on the upstream side of the cylindrical portion 11. The opening on the downstream side of the cylindrical portion 11 is located inside the upper syringe 21 of the tube 20. The magnetic beads 7 introduced from the opening on the upstream side of the tubular part 11 pass through the inside of the tubular part 11, pass through the front and back of the rib 13, exit from the opening on the downstream side of the tubular part 11, and It will be introduced into the upper syringe 21.

The downstream side of the cylindrical portion 11 is fitted to the inner wall of the upper syringe 21 of the tube 20. The cylindrical portion 11 can slide in the longitudinal direction with respect to the upper syringe 21 while inscribed in the upper syringe 21 of the tube 20.

A mounting base 11A to which the adapter 5 is attached is formed around the opening on the upstream side of the cylindrical portion 11. The mounting base 11 </ b> A is also a part that is pushed when the plunger 10 is pushed. When the mounting base 11A is pushed, the plunger 10 slides with respect to the tube 20 and changes from the state of FIG. 2A to the state of FIG. 2C. When the plunger 10 moves downstream, the mounting base 11A contacts the upper edge of the tube 20 (see FIG. 2C). That is, the distance between the mounting base 11 </ b> A of the plunger 10 and the upper edge of the tube 20 becomes the slide length of the plunger 10.

In the initial state, the rod-shaped portion 12 is located inside the upper syringe 21 of the tube 20 and is separated from the lower syringe 22 (see FIG. 2A). When the plunger 10 slides with respect to the tube 20, the rod-shaped portion 12 is inserted into the lower syringe 22 of the tube 20, and the rod-shaped portion 12 slides in the downstream direction with respect to the lower syringe 22 while being inscribed in the lower syringe 22 ( 2B and 2C).

The shape of the cross section orthogonal to the longitudinal direction of the rod-shaped part 12 is circular. However, the cross-sectional shape of the rod-shaped portion 12 can be a circle, an ellipse, or a polygon as long as it can be fitted to the inner wall of the lower syringe 22 of the tube 20, and is not particularly limited.

A seal 12 </ b> A is formed at the downstream end of the rod-shaped portion 12. When the seal 12 </ b> A is fitted to the lower syringe 22, the liquid in the downstream tube 20 is prevented from flowing back to the upper syringe 21. When the plunger 10 is pushed from the state shown in FIG. 2B to the state shown in FIG. 2C, the liquid in the tube 20 is pushed out from the downstream side by the amount corresponding to the volume of the seal 12A slid in the lower syringe 22 during that time. Become.

Note that the volume in which the seal 12A slides in the lower syringe 22 (the amount by which the liquid in the tube 20 is pushed from the downstream side) is larger than the total of the reaction liquid plug 47 and the third oil plug 48 in the tube 20. Thereby, the liquid in the tube 20 can be pushed out so that the reaction solution 47 does not remain in the tube 20.

The material of the plunger 10 is not particularly limited, and may be, for example, a resin such as glass or plastic, or a metal. Moreover, the cylindrical part 11 and the rod-shaped part 12 of the plunger 10 may be integrally formed with the same material, and may be formed with another material. Here, the plunger 10 is formed by separately molding the cylindrical portion 11 and the rod-shaped portion 12 with resin and joining the cylindrical portion 11 and the rod-shaped portion 12 via the ribs 13.

An oil 42 and a first cleaning liquid 43 are stored in advance in the plunger 10.
Since the oil 42 in the plunger 10 has a specific gravity smaller than that of the first cleaning liquid 43, when the tank 3 is attached to the cartridge body 9, when the cartridge body 9 is stood with the mounting base 11A of the plunger 10 up, FIG. As shown, the oil 42 is disposed between the liquid in the tank 3 and the first cleaning liquid 43 of the cartridge body 9. As the oil 42, 2CS silicone oil is used, and as the first cleaning liquid 43, 8M guanidine hydrochloride and 0.7% Triton X-100 are used.

The first cleaning liquid 43 may be any liquid that phase-separates when mixed with both the oil 42 and the oil 44. The first cleaning solution 43 is preferably water or a low salt concentration aqueous solution, and in the case of a low salt concentration aqueous solution, it is preferably a buffer solution. The salt concentration of the low salt concentration aqueous solution is
100 mM or less is preferable, 50 mM or less is more preferable, and 10 mM or less is most preferable. The lower limit of the low salt concentration aqueous solution is not particularly limited, but is preferably 0.1 mM or more.
. More preferably, it is 5 mM or more, and most preferably 1 mM or more. This solution may also contain a surfactant such as Triton, Tween, SDS,
H is not particularly limited. The salt for making the buffer solution is not particularly limited, but Tris, Hepes,
Salts such as pipes and phosphoric acid are preferably used. In addition, this cleaning solution contains alcohol,
It is preferable to include an amount that does not inhibit adsorption of a nucleic acid to a carrier, reverse transcription reaction, PCR reaction or the like. In this case, the alcohol concentration is not particularly limited, but may be 70% or less.
It may be 0% or less, 50% or less, 40% or less, 30
% Or less, 20% or less, or 10% or less, but 5%
Or less, preferably 2% or less, more preferably 1% or less or 0.5% or less, and most preferably 0.2% or less or 0.1% or less.

The first cleaning solution 43 may contain a chaotropic agent. For example, the first cleaning liquid 4
When guanidine hydrochloride is contained in 3, the particles and the like can be washed while maintaining or enhancing the adsorption of the nucleic acid adsorbed to the particles and the like. As the concentration when guanidine hydrochloride is contained,
For example, 3 mol / L to 10 mol / L, preferably 5 mol / L to 8 mol / L
L or less. If the concentration of guanidine hydrochloride is within this range, other impurities can be washed while more stably adsorbing the nucleic acid adsorbed on the particles and the like.

(2-2) Tube Hereinafter, the tube 20 will be described with reference to FIGS. 2A to 2C.

The tube 20 has a cylindrical shape that allows a liquid to flow in the longitudinal direction. The tube 20 has an upper syringe 21, a lower syringe 22, and a capillary 23, and the inner diameters of the respective parts differ stepwise.

The upper syringe 21 has a cylindrical shape that can circulate liquid in the longitudinal direction. The cylindrical portion 11 of the plunger 10 is slidably inscribed on the inner wall of the upper syringe 21,
The upper syringe 21 functions as a syringe for the cylindrical portion 11 of the plunger 10.

The lower syringe 22 has a cylindrical shape that allows the liquid to flow in the longitudinal direction. The inner wall of the lower syringe 22 can be slidably fitted with the seal 12A of the rod-shaped portion 12 of the plunger 10, and the lower syringe 22 functions as a syringe for the rod-shaped portion 12 of the plunger 10.

The capillary 23 has a thin tubular shape that allows a liquid to flow in the longitudinal direction.
The inner diameter of the capillary 23 is a size that allows the liquid to maintain the shape of the plug.
. 0 mm. At the end of the capillary 23 (the end on the downstream side of the tube 20), the inner diameter is smaller than this, and here it is 0.5 mm. The inner diameter of the end of the capillary 23 is set smaller than the diameter (1.5 to 2.0 mm) of a droplet-like reaction liquid described later.
As a result, when the reaction solution plug 47 is pushed out from the end of the capillary 23, it is possible to avoid the droplet-like reaction solution from adhering to the end of the capillary 23 or entering back into the capillary 23.

The capillary 23 only needs to have a cylindrical shape that has a hollow inside and can circulate liquid in the longitudinal direction, and may be bent in the longitudinal direction, but is linear. Is preferred. The cavity inside the tube is not particularly limited in size and shape as long as the liquid can maintain the shape of the plug in the tube. The size of the cavity in the tube and the shape of the cross section perpendicular to the longitudinal direction may vary along the longitudinal direction of the tube.
The shape of the cross section perpendicular to the longitudinal direction of the outer shape of the tube is not limited. Furthermore, the thickness of the tube (the dimension from the side surface of the internal cavity to the external surface) is not particularly limited. When the tube is cylindrical, the inner diameter (the diameter of the circle in a cross section perpendicular to the longitudinal direction of the internal cavity) is, for example,
It can be 0.5 mm or more and 2 mm or less. When the inner diameter of the tube is in this range, it is easy to form a liquid plug in a wide range of tube materials and liquid types. The tip is preferably tapered more and can be 0.2 mm or more and 1 mm or less. Further, by reducing the inner diameter of the end of the capillary 23 (opening diameter of the capillary 23), it is possible to prevent the reaction liquid 47 formed in droplets in the PCR container 30 from adsorbing to the opening of the capillary 23 and not being separated. However, if the inner diameter of the end of the capillary 23 is made too small, a large number of small droplets of the reaction solution 47 are formed. If the diameter of the capillary 23 is reduced to the portion other than the end, similarly to the end, the cartridge 1 becomes longer due to the necessity of securing the volume of each plug, which is not desirable.

The capillary 23 includes a first oil plug 44, a cleaning liquid plug 45, a second oil plug 46, a reaction liquid plug 47, and a third oil plug 48 in that order from the upstream side. That is, oil plugs are arranged on both sides of a water-soluble plug (cleaning liquid plug 45 or reaction liquid plug 47).

The upper syringe 21 and the lower syringe 22 upstream of the first oil plug 44 are
Oil 42 and cleaning liquid 43 are stored in advance (see FIG. 2A). The inner diameters of the upper syringe 21 and the lower syringe 22 are larger than the inner diameter of the capillary 23, and the upper syringe 21 and the lower syringe 22 cannot maintain the liquid (oil 42 and cleaning liquid 43) in a columnar shape like a plug, but the first oil plug 44 Is held in the shape of a plug by the capillary 23, the oil constituting the first oil plug 44 is restrained from moving upstream.

The cleaning liquid plug 45 may be composed of a 5 mM Tris-HCl buffer that is the second cleaning liquid.
The second cleaning liquid may basically have the same configuration as described in the first cleaning liquid, and may be the same as or different from the first cleaning liquid, but is preferably a solution that does not substantially contain a chaotropic substance. . This is because the chaotropic substance is not brought into the later solution. As described above, this washing solution also preferably contains alcohol in an amount that does not inhibit adsorption of nucleic acid to a carrier, reverse transcription reaction, PCR reaction, and the like. In this case, the alcohol concentration is not particularly limited, but may be 70% or less, 60% or less, 50% or less, 40% or less, 30% Or 20% or less, or 10% or less, but preferably 5% or less or 2% or less.
% Or less or 0.5% or less is more preferable, and 0.2% or less or 0.1% or less is most preferable.

The cleaning liquid plug 45 may be composed of a plurality of plugs separated by an oil plug. When the cleaning liquid plug 45 includes a plurality of plugs, the liquid in each plug may be the same or different. If there is at least one plug of cleaning liquid therein, the liquid of other plugs is not particularly limited, but it is preferable that all plugs are cleaning liquid. The number of the cleaning liquid plugs 45 divided can be appropriately set in consideration of, for example, the length of the tube 20 and the object to be cleaned.

The reaction liquid plug 47 is made of, for example, the following reaction liquid.
0.2u / μL AMV reverse transcriptase (Nippon Gene)
0.125u / μL Gene Taq NT PCR enzyme (Nippon Gene)
0.5 mM dNTP
1.0μM primer (forward)
1.0μM primer (reverse)
0.5μM probe (Taq man)
4.0 mg / mL BSA
x1 buffer (MgCl ₂ 7 mM; Tris pH 9.0 25 mM; KCl 50 mM)

The reaction solution 47 refers to a liquid in which the nucleic acid adsorbed on the nucleic acid-binding solid phase carrier is eluted from the carrier into the solution and subjected to reverse transcription reaction and polymerase reaction. Therefore, the reaction solution 47 after elution of the nucleic acid is prepared in advance so that it becomes a buffer solution used for the reverse transcription reaction and the polymerase reaction as it is. The reaction solution 47 contains an eluate for eluting nucleic acids.

The reaction solution 47 contains a reverse transcriptase, dNTP, and three or more kinds of primers (oligonucleotide) for reverse transcriptase for the reverse transcription reaction, and for the polymerase reaction,
Real-time P such as TaqMan probe, Molecular Beacon, Cycling probe, including DNA polymerase and DNA polymerase primer (oligonucleotide), and 3 or more kinds of fluorescent substances for multiplex PCR
Fluorescent materials for intercalators such as CR probes and SYBR green may be included. Furthermore, it is preferable to contain BSA (bovine serum albumin) or gelatin as a reaction inhibition inhibitor. The solvent is preferably water, and more preferably an organic solvent such as ethanol or isopropyl alcohol and a substance that does not substantially contain a chaotropic substance. Moreover, it is preferable to contain a salt so that it may become a buffer for reverse transcriptase and / or a buffer for DNA polymerase. The salt for making the buffer solution is not particularly limited as long as it does not inhibit the enzyme reaction, but salts such as Tris, Hepes, Pipes, and phosphoric acid are preferably used. The reverse transcriptase is not particularly limited. For example, Avian Myeloblast virus (Avian Mye)
lobous virus), ras associated virus type 2 (Ras Assoc)
iated Virus type 2), mouse Moloney Muleen Leukemia virus (Mo
Use Molecule Murine Leukemia Virus), reverse transcriptase derived from human immunodeficiency virus type 1 (Human Immunodefective Virus type 1), and the like can be used, but a thermostable enzyme is preferred. The DNA polymerase is not particularly limited, but is preferably a heat-resistant enzyme or a PCR enzyme, such as Taq polymerase, T
There are a large number of commercially available products such as fi polymerase, Tth polymerase, or improved versions thereof, but a DNA polymerase capable of hot start is preferred.

The primer for DNA polymerase for multiplex PCR can easily determine an appropriate sequence for each of three or more types of DNA to be detected. Usually, in order to amplify one kind of DNA, a primer pair of a 5′-side primer and a 3′-side primer may be included.

The concentration of dNTP or salt contained in the reaction solution may be a concentration suitable for the enzyme to be used. Usually, dNTP is 10 to 1000 μM, preferably 100 to 500 μM, Mg2 + is 1 to 100 mM, preferably 5 to 10 mM, Cl − is 1 to 2000 mM, preferably 20
The total ion concentration is not particularly limited, but may be a concentration higher than 50 mM, preferably a concentration higher than 100 mM, more preferably a concentration higher than 120 mM, and a concentration higher than 150 mM. Preferably, a concentration higher than 200 mM is more preferable. The upper limit is preferably 500 mM or less, more preferably 300 mM or less, and 2
More preferred is 00 mM or less. Each of the primer oligonucleotides is 0.1 to
10 μM, preferably 0.1 to 1 μM is used. The concentration of BSA or gelatin is 1
If it is less than mg / mL, the effect of preventing reaction inhibition is small, and if it is more than 10 mg / mL, reverse transcription reaction and subsequent enzyme reaction may be inhibited, so 1-10 mg / mL is preferable. When gelatin is used, its origin can be exemplified by cow skin, pig skin, and cow bone, but is not particularly limited.
If gelatin is difficult to dissolve, it may be dissolved by heating.
The volume of the reaction solution plug 47 is not particularly limited, and can be set as appropriate using the amount of particles or the like adsorbed with nucleic acid as an index. For example, when the volume of the particles or the like is 0.5 μL, it is sufficient that the volume of the reaction solution plug 47 is 0.5 μL or more, and 0.8 μL or more and 5 μL.
L or less is preferable, and 1 μL or more and 3 μL or less is more preferable. If the volume of the reaction solution plug 47 is within these ranges, for example, the volume of the nucleic acid-binding solid phase carrier is set to 0.00.
Even with 5 μL, the nucleic acid can be sufficiently eluted from the carrier.

A downstream portion of the capillary 23 is inserted into the PCR container 30. As a result, the reaction liquid 47 in the tube 20 is pushed out of the tube 20, thereby allowing the reaction liquid 47 to flow into P.
It can be introduced into the CR container 30.

When the annular convex portion on the outer wall of the capillary 23 comes into contact with the inner wall of the PCR container 30, an upper seal portion is formed. Further, when the outer wall of the capillary 23 on the downstream side of the upper seal part comes into contact with the inner wall of the PCR container 30, a lower seal part is formed. The upper seal part and the lower seal part will be described later.

The tube 20 further includes a fixed claw 25 and a guide plate 26. FIG. 4 is an explanatory diagram of the fixing claw 25, the guide plate 26 and the mounting portion 62.

The fixing claw 25 is a member that fixes the cartridge 1 to the mounting portion 62. When the cartridge 1 is inserted into the mounting portion 62 until the fixing claw 25 is caught, the cartridge 1 is fixed at a normal position with respect to the mounting portion 62. In other words, the cartridge 1 is attached to the mounting portion 62.
When the fixing claw 25 is in an abnormal position, the fixing claw 25 is not caught by the mounting portion 62.

The guide plate 26 is a member that guides the cartridge 1 when the cartridge 1 is mounted on the mounting portion 62 of the PCR device 50. A guide rail 63A is formed in the mounting portion 62 of the PCR device 50, and the cartridge 1 is inserted into the mounting portion 62 and fixed while the guide plate 26 of the tube 20 is guided along the guide rail 63A. Although the cartridge 1 has a long shape, the cartridge 1 is inserted into the mounting portion 62 while being guided by the guide plate 26.
It becomes easy to fix the cartridge 1 at a normal position with respect to the mounting portion 62.

The fixing claw 25 and the guide plate 26 are plate-like members protruding from the left and right sides of the capillary 23. When the magnetic beads 7 in the tube 20 are moved by a magnet, the magnet is brought close to the plate-shaped fixed claw 25 and the guide plate 26 from the perpendicular direction. Thereby, the distance of the magnet and the magnetic bead 7 in the tube 20 can be made close. However, the magnet and the magnetic beads 7 in the tube 20
The fixing claw 25 and the guide plate 26 may have other shapes as long as the distance between them is close.

(2-3) PCR Container FIGS. 5A and 5B are explanatory diagrams of the periphery of the PCR container 30. FIG. 5A is an explanatory diagram of an initial state. FIG. 5B is an explanatory diagram of a state after the plunger 10 is pushed. Hereinafter, the PCR container 30 will be described with reference to FIGS. 2A to 2C.

The PCR container 30 is a container that receives the liquid pushed out from the tube 20 and that contains the reaction liquid 47 during the thermal cycle process. The PCR container corresponds to a nucleic acid amplification reaction container.

The PCR container 30 has a seal formation part 31 and a flow path formation part 35. Seal forming part 31
Is a part into which the tube 20 is inserted, and is a part that suppresses the oil overflowing from the flow path forming part 35 from leaking to the outside. The flow path forming part 35 is a part on the downstream side of the seal forming part 31 and is a part that forms a flow path in which the liquid reaction liquid 47 moves. The PCR container 30 has a tube 2 at two locations, an upper seal portion 34A and a lower seal portion 34B.
Fixed to zero.

  The seal forming part 31 has an oil receiving part 32 and a step part 33.

The oil receiving portion 32 is a cylindrical portion and functions as a reservoir that receives oil overflowing from the flow path forming portion 35. There is a gap between the inner wall of the oil receiving portion 32 and the outer wall of the capillary 23 of the tube 20, and this gap becomes an oil receiving space 32 </ b> A that receives oil overflowing from the flow path forming portion 35. The volume of the oil receiving space 32 </ b> A is larger than the volume in which the seal 12 </ b> A of the plunger 10 slides with the lower syringe 22 of the tube 20.

When the inner wall on the upstream side of the oil receiving portion 32 comes into contact with the annular convex portion of the tube 20, the upper seal portion 34A is formed. The upper seal portion 34A is a seal that suppresses leakage of oil in the oil receiving space 32A to the outside while allowing passage of air. Upper seal part 3
4A has a vent hole to the extent that oil does not leak due to the surface tension of the oil. The vent of the upper seal portion 34A may be a gap between the convex portion of the tube 20 and the inner wall of the oil receiving portion 32, or may be a hole, a groove, or a notch formed in the convex portion of the tube 20. Further, the upper seal portion 34A may be formed of an oil absorbing material that absorbs oil.

The step portion 33 is a stepped portion provided on the downstream side of the oil receiving portion 32. The inner diameter of the downstream portion of the step portion 33 is smaller than the inner diameter of the oil receiving portion 32. The inner wall of the stepped portion 33 is in contact with the outer wall on the downstream side of the capillary 23 of the tube 20. When the inner wall of the stepped portion 33 and the outer wall of the tube 20 are in contact, the lower seal portion 34B is formed. Lower seal part 3
4B is a seal that resists the flow while allowing the oil in the flow path forming portion 35 to flow into the oil receiving space 32A. Due to the pressure loss at the lower seal part 34B, the flow path forming part 3
5 becomes higher than the external pressure, so that even if the liquid in the flow path forming unit 35 is heated during the thermal cycle process, bubbles are hardly generated in the liquid in the flow path forming unit 35.

The flow path forming unit 35 is a tubular part and serves as a container for forming a flow path through which the droplet-like reaction liquid 47 moves. The flow path forming portion 35 is filled with oil. The upstream side of the flow path forming part 35 is closed by the end of the tube 20, and the end of the tube 20 opens toward the flow path forming part 35. The inner diameter of the flow path forming portion 35 is larger than the inner diameter of the capillary 23 of the tube 20 and larger than the outer diameter when the liquid having the capacity of the reaction solution plug 47 becomes spherical. It is desirable that the inner wall of the flow path forming part 35 has water repellency to such an extent that the water-soluble reaction liquid 47 does not adhere.

The upstream side of the flow path forming unit 35 is heated to a relatively high temperature (for example, about 95 ° C.) by the external high temperature side heater 65B to form a high temperature region 36A. Flow path forming part 35
The downstream side is heated to a relatively low temperature (for example, about 60 ° C.) by an external low-temperature heater 65C to form a low-temperature region 36B. The bottom 35A (downstream end) of the PCR container 30 is included in the low temperature region 36B. Thereby, a temperature gradient is formed in the liquid in the flow path forming unit 35.

As shown in FIG. 5A, in the initial state, the flow path forming part 35 of the PCR container 30 is filled with oil. The oil interface is located relatively downstream of the oil receiving space 32A. The volume upstream of the oil interface in the oil receiving space 32 </ b> A is larger than the volume in which the seal 12 </ b> A of the plunger 10 slides with the lower syringe 22 of the tube 20.

As shown in FIG. 5B, when the plunger 10 is pushed, the liquid in the tube 20 is pushed out to the flow path forming part 35. The flow path forming portion 35 is filled with oil in advance,
Since the liquid in the tube 20 is pushed into the gas, no gas flows into the flow path forming portion 35.

When the plunger 10 is pushed, first, the third oil plug 48 of the tube 20 flows into the flow path forming portion 35, and the inflowed oil flows into the oil receiving space 32A from the flow path forming portion 35, and the oil receiving space 32A. The oil interface rises. At this time, the pressure of the liquid in the flow path forming portion 35 increases due to the pressure loss of the lower seal portion 34B. After the third oil plug 48 is pushed out from the tube 20, the reaction solution plug 47 flows from the tube 20 into the flow path forming part 35. Since the inner diameter of the flow path forming portion 35 is larger than the inner diameter of the capillary 23, the tube 20
The reaction solution 47 in the form of a plug (columnar) in the form of droplets in the oil of the flow path forming unit 35.
Note that the volume upstream of the oil interface in the initial state in the oil receiving space 32A is
Since the volume of the seal 12A of the plunger 10 that is slid by the lower syringe 22 of the tube 20 is larger, the oil does not overflow from the oil receiving space 32A.

<PCR device 50>
FIG. 6A is a perspective view of the internal configuration of the PCR device 50. FIG. 6B is a side view of the main configuration of the PCR device 50. FIG. 7 is a block diagram of the PCR device 50. The PCR device 50 performs nucleic acid elution processing and thermal cycle processing using the cartridge 1.

In the following description of the PCR apparatus 50, as shown in the figure, vertical, front / rear, and left / right are defined. That is, the vertical direction when the base 51 of the PCR device 50 is installed horizontally is defined as “up and down direction”, and “up” and “down” are defined according to the direction of gravity. Further, the axial direction of the rotation axis of the cartridge 1 is referred to as “left-right direction”, and the vertical direction and the direction perpendicular to the left-right direction are referred to as “front-rear direction”. When viewed from the rotation axis of the cartridge 1, the side of the cartridge insertion port 53 </ b> A is “rear”, and the opposite side is “front”. The right side in the left-right direction when viewed from the front side is “right”, and the left side is “left”.

The PCR device 50 includes a rotation mechanism 60, a magnet moving mechanism 70, a pressing mechanism 80, a fluorescence measuring instrument 55, and a controller 90.

(1) Rotating mechanism 60
The rotation mechanism 60 is a mechanism that rotates the cartridge 1 and the heater. Rotating mechanism 60
However, when the cartridge 1 and the heater are turned upside down, the reaction liquid 47 in the form of droplets moves in the flow path forming part 35 of the PCR container 30 and a thermal cycle process is performed.

The rotating mechanism 60 includes a rotating body 61 and a rotating motor 66. FIG. 8A shows the rotating body 6
FIG. FIG. 8B is an explanatory diagram of a state where the cartridge 1 is mounted on the mounting portion 62 of the rotating body 61.

The rotating body 61 is a member that can rotate around a rotation axis. The rotating shaft of the rotating body 61 is supported by a support base 52 fixed to the base 51. The rotating body 61 is provided with a mounting portion 62 for mounting the cartridge 1 and heaters (elution heater 65A, high temperature side heater 65B and low temperature side heater 65C). When the rotating body 61 rotates, the cartridge 1 can be turned upside down while maintaining the positional relationship between the cartridge 1 and the heater. The rotation motor 66 is a power source that rotates the rotating body 61. The rotation motor 66 rotates the rotating body 61 to a predetermined position in accordance with an instruction from the controller 90. A transmission mechanism such as a gear may be interposed between the rotating motor 66 and the rotating body 61.

Note that the rotation axis of the rotating body 61 is located closer to the tube 20 than the PCR container 30 of the cartridge 1. In other words, the height of the rotating shaft of the rotating body 61 is positioned at the height of the tube 20 of the cartridge 1 mounted on the mounting portion 62. Tube 20 is PCR container 3
Since it is longer than 0, if the center of the PCR container 30 is set as the rotation axis (if the rotation axis of the rotation body 61 is positioned at the height of the PCR container), the rotation body 61 becomes large. Because.

The mounting portion 62 is a portion where the cartridge 1 is mounted. The mounting part 62 has a fixing part 63 in which a notch is formed. Also, heaters (elution heater 65A, high temperature side heater 65A
B and the insertion hole 64A formed in the low temperature side heater 65C) also function as the mounting portion 62.
The cartridge 1 is inserted into the notch of the fixing portion 63 with the PCR container 30 inserted into the insertion hole 64A.
When the fixed claw 25 is caught, the cartridge 1 is mounted on the rotating body 61 (see FIG. 4). Here, a part of the heater also serves as the mounting portion 62, but the mounting portion 6
2 and the heater may be separate. The mounting portion 62 is indirectly fixed to the rotating body 61 via the elution heater 65 </ b> A, but may be directly provided on the rotating body 61. Also,
The number of cartridges 1 that can be mounted on the mounting unit 62 is not limited to one, and may be plural.

The fixing portion 63 of the mounting portion 62 functions as a tube fixing portion that fixes the tube 20 of the cartridge 1, and the insertion hole 64 </ b> A functions as a PCR container fixing portion that fixes the PCR container 30. Thereby, the long cartridge 1 composed of the tube 20 and the PCR container 30 is stably fixed to the mounting portion 62.

A guide rail 63A is formed in the fixed portion 63 along the vertical direction (see FIG. 4).
. The guide rail 63A guides the guide plate 26 of the cartridge 1 in the insertion direction while restraining the guide plate 26 in the front-rear direction. Since the cartridge 1 is inserted into the mounting portion 62 while the guide plate 26 is guided by the guide rail 63A, the PCR container 30 of the cartridge 1 is guided to the insertion hole 64A, and the cartridge 1 is in a normal position with respect to the mounting portion 62. Fixed.

The PCR device 50 includes an elution heater 65A, and a high temperature side heater 65B and a low temperature side heater 65C as PCR heaters. Each heater includes a heat source (not shown) and a heat block. The heat source is, for example, a cartridge heater, and is inserted into the heat block. The heat block is a metal such as aluminum having high thermal conductivity, for example, and heats the liquid in the cartridge 1 with heat from a heat source while suppressing heat unevenness. In addition, the heat block does not attract the magnet 71 that moves the magnetic beads 7.
A non-magnetic material is desirable.

The elution heater 65 </ b> A is a heater that heats the reaction solution plug 47 of the cartridge 1. When the cartridge 1 is fixed at a normal position, the elution heater 65A
Opposite the zero reaction solution plug 47. For example, the elution heater 65A heats the reaction solution plug 47 to about 50 ° C., thereby promoting the release of nucleic acids from the magnetic beads.

The high temperature side heater 65 </ b> B is a heater that heats the upstream side of the flow path forming unit 35 of the PCR container 30. When the cartridge 1 is fixed at a normal position, the high temperature side heater 65B
It faces the upstream side (high temperature region 36A) of the flow path forming part 35 of the R container 30. For example, the high temperature side heater 65 </ b> B heats the liquid on the upstream side of the flow path forming unit 35 of the PCR container 30 to about 90 to 100 ° C.

The low temperature side heater 65 </ b> C is a heater that heats the bottom 35 </ b> A of the flow path forming unit 35 of the PCR container 30. When the cartridge 1 is fixed at a normal position, the low temperature side heater 65C
It faces the downstream side (low temperature region 36B) of the flow path forming part 35 of the CR container 30. For example, the low temperature side heater 65 </ b> C heats the liquid in the low temperature region 36 </ b> B of the PCR container 30 to about 50 to 75 ° C.

A spacer 65D is disposed between the high temperature side heater 65B and the low temperature side heater 65C. The spacer 65D suppresses heat conduction between the high temperature side heater 65B and the low temperature side heater 65C. The spacer 65D is composed of a high temperature side heater 65B and a low temperature side heater 6B.
It is also used to accurately determine the distance to 5C. Thereby, the high temperature side heater 6
A temperature gradient is formed in the liquid in the flow path forming part 35 of the PCR container 30 by 5B and the low temperature side heater 65C.

The heat blocks constituting the elution heater 65A, the high temperature side heater 65B, and the low temperature side heater 65C are respectively formed with through holes constituting the insertion holes 64A.
The outer wall of the bottom 35A of the PCR container 30 is exposed from the lower opening of the insertion hole 64A of the low temperature side heater 65C. The fluorescence measuring instrument 55 measures the luminance of the reaction solution 47 from the lower opening of the insertion hole 64A.

Each of the high temperature side heater 65B and the low temperature side heater 65C is provided with a temperature control device and can be set to a temperature suitable for each polymerase reaction.

(2) Magnet moving mechanism 70
The magnet moving mechanism 70 is a mechanism that moves the magnet 71. The magnet moving mechanism 70 draws the magnetic beads 7 in the cartridge 1 to the magnet 71 and moves the magnetic beads 7 in the cartridge 1 by moving the magnet 71. The magnet moving mechanism 70 includes a pair of magnets 71, an elevating mechanism 73, and a swing mechanism 75.

The magnet 71 is a member that attracts the magnetic beads 7. A permanent magnet, an electromagnet, or the like can be used as the magnet 71, but here a permanent magnet that does not generate heat is used. The pair of magnets 71 are held by the arms 72 so that the positions in the vertical direction are substantially the same so as to face each other in the front-rear direction. Each magnet 71 can be opposed from the front side or the rear side of the cartridge 1 mounted on the mounting portion 62. The pair of magnets 71 can sandwich the cartridge 1 mounted on the mounting portion 62 from the front-rear direction. Fixed claw 25 of cartridge 1 or guide plate 2
The distance between the magnetic beads 7 in the cartridge 1 and the magnets 71 can be made closer by making the magnets 71 face each other from the direction (here, the front-rear direction) perpendicular to the direction in which the 6 is provided (here, the left-right direction). .

The lifting mechanism 73 is a mechanism that moves the magnet 71 in the vertical direction. Since the magnet 71 attracts the magnetic beads 7, the magnetic beads 7 in the cartridge 1 can be attracted in the vertical direction by moving the magnet 71 in the vertical direction in accordance with the movement of the magnetic beads 7.

The elevating mechanism 73 includes a carriage 73A that moves in the vertical direction, and an elevating motor 73B. The carriage 73 </ b> A is a member that can move in the vertical direction.
It is guided by a carriage guide 73C provided on the side wall 53 with 3A so as to be movable in the vertical direction. Since the arm 73 that holds the pair of magnets 71 is attached to the carriage 73A, when the carriage 73A moves in the vertical direction, the magnet 71 moves in the vertical direction. The lifting motor 73B is a power source that moves the carriage 73A in the vertical direction. The lifting motor 73B moves the carriage 73A to a predetermined position in the vertical direction in accordance with an instruction from the controller 90. Lifting motor 73B is belt 7
Although the carriage 73A is moved in the vertical direction using the 3D and the pulley 73E, the carriage 73A may be moved in the vertical direction by another transmission mechanism.

When the carriage 73A is at the uppermost position (retracted position), the magnet 71 moves to the cartridge 1
It is located on the upper side. When the carriage 73A is in the retracted position, the lifting mechanism 73 does not contact the cartridge 1 even if the cartridge 1 rotates. Further, the elevating mechanism 73 can lower the position of the carriage 73A to a position where the magnet 71 faces the reaction plug. Thereby, the elevating mechanism 73 can move the magnet 71 so as to move the magnetic beads 7 in the tank 3 to the position of the reaction plug.

The swing mechanism 75 is a mechanism that swings the pair of magnets 71 in the front-rear direction. A pair of magnets 71
Is pivoted back and forth, the intervals between the magnets 71 and the cartridge 1 change alternately. Since the magnetic beads 7 are attracted toward the magnet 71 having a shorter distance, the magnetic beads 7 in the cartridge 1 are moved in the front-rear direction by swinging the pair of magnets 71 in the front-rear direction.

The swing mechanism 75 includes a swing motor 75A and a gear. The swing motor 75A and the gear are provided on the carriage 73A and can move in the vertical direction together with the carriage 73A. The power of the swing motor 75A is transmitted to the arm 72 through the gear, so that the arm 72 holding the magnet 71 is swung with respect to the carriage 73A.
Rotate around B. The swing mechanism 75 swings the magnet 71 in a range where the magnet 71 and the cartridge 1 do not contact with each other in order to prevent the magnet 71 from contacting the cartridge 1 and damaging the cartridge 1.

The swing rotation shaft 75B is a rotation shaft of the arm 72. The swing rotation shaft 75B is parallel to the left-right direction so that the magnet 71 can swing back and forth. When the swinging rotation shaft 75B is viewed from the right or left, the swinging rotation shaft 75B is disposed so as to be shifted to the front side or the rear side from the cartridge 1. Thereby, when the carriage 73A moves downward, the cartridge 1 and the arm 72 are moved.
Can be avoided. As long as the magnet 71 can be swung in the front-rear direction, the rocking rotation shaft 75B may be an axis parallel to the up-down direction.

(3) Press mechanism 80
The pressing mechanism 80 is a mechanism that presses the plunger 10 of the cartridge 1. When the plunger 10 is pressed by the pressing mechanism 80, the reaction liquid plug 47 of the cartridge 1 is pressed.
And the oil plug is pushed out into the PCR container 30, and a droplet-like reaction solution 47 is formed in the oil in the PCR container 30.

The pressing mechanism 80 includes a plunger motor 81 and a rod 82. The plunger motor 81 is a power source that moves the rod 82. The rod 82 is a member that pushes the mounting base 11 </ b> A of the plunger 10 of the cartridge 1. The reason for pressing the mounting base 11A instead of the tank 3 of the cartridge 1 is that the tank 3 is made of a flexible resin so as to be inflatable. When the tank 3 is not deformed, the pressing mechanism 80 may push the plunger 10 by pushing the tank 3.

The direction in which the rod 82 pushes the plunger 10 is 4 in the vertical direction, not in the vertical direction.
It is tilted 5 degrees. Therefore, when the plunger 10 is pressed by the pressing mechanism 80, the PCR
The apparatus 50 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 and then moves the rod 82. Since the direction in which the rod 82 pushes the plunger 10 is inclined 45 degrees with respect to the vertical direction, it is easy to arrange the pressing mechanism 80 so as not to interfere with the lifting mechanism 73. The rod 82 is connected to the plunger 10.
Since the direction of pushing is inclined 45 degrees with respect to the vertical direction, the vertical dimension of the PCR device 50 can be reduced.

(4) Fluorescence measuring instrument 55
The fluorescence measuring device 55 includes an excitation light source that irradiates the reaction solution 47 of the PCR container 30 with excitation light, and a fluorometer that measures the intensity of fluorescence emitted from the reaction solution 47. The fluorescence measuring device 55 is disposed below the rotating body 61 so as to face the bottom 35 </ b> A of the PCR container 30 of the cartridge 1. The fluorescence measuring instrument 55 measures the intensity of the fluorescence emitted from the reaction solution 47 in the bottom 35A of the PCR container 30 from the lower opening of the insertion hole 64A of the low temperature side heater 65C.

The fluorescence measuring device 55 is configured so that three or more types of nucleic acid amplification reactions can be detected independently. For example, an excitation light source for exciting fluorescence may be prepared for each excitation wavelength of each fluorescent substance contained in the reaction solution 47, but transmits only light of a specific wavelength and does not transmit other light. Such a bandpass filter may be prepared for each necessary wavelength region, and light having a necessary wavelength may be extracted from an excitation light source such as a mercury lamp.

Also in the fluorescence measurement, in order to detect the fluorescence of a specific wavelength emitted from the reaction solution 47 using a spectrofluorometer or the like, a band pass filter suitable for each fluorescence wavelength emitted by each fluorescent substance. It is preferable to measure fluorescence using

A person skilled in the art can easily select the type of these bandpass filters depending on the fluorescent material used.

(5) Controller 90
The controller 90 is a control device that controls the PCR device 50. The controller 90 includes a processor such as a CPU and a storage device such as a ROM and a RAM. Various programs and data are stored in the storage device. Further, the storage device provides an area for developing a program. Various processes are realized by the processor executing the program stored in the storage device.

For example, the controller 90 controls the rotation motor 66 to rotate the rotating body 61 to a predetermined rotation position. The rotation mechanism 60 is provided with a rotation position sensor (not shown), and the controller 90 drives and stops the rotation motor 66 according to the detection result of the rotation position sensor.

The controller 90 includes heaters (elution heater 65A, high temperature side heater 65
B and the low temperature side heater 65C) are controlled to cause each heater to generate heat. The heat block that constitutes the heater is provided with a temperature sensor (not shown).
The cartridge heater is turned on / off according to the detection result of the temperature sensor.

The controller 90 also controls the lifting motor 73B to move the magnet 71 in the vertical direction. The PCR device 50 is provided with a position sensor (not shown) that detects the position of the carriage 73A, and the controller 90 drives and stops the lifting motor 73B according to the detection result of the position sensor.

The controller 90 controls the swing motor 75A to swing the magnet 71 in the front-rear direction. The PCR device 50 is provided with a position sensor that detects the position of the arm 72 that holds the magnet 71, and the controller 90 drives and stops the swing motor 75A according to the detection result of the position sensor.

In addition, the controller 90 controls the fluorescence measuring device 55 to react with the reaction solution 4 in the PCR container 30.
The fluorescence intensity of 7 is measured. The controller 90 is configured such that the fluorescence measuring device 55 is connected to the cartridge 1
When facing the bottom 35 </ b> A of the CR container 30, the fluorescence measuring device 55 is made to perform measurement. The measurement result is stored in a storage device.

Furthermore, the controller 90 is a nucleic acid detection control device, and includes a nucleic acid detection control unit.
The nucleic acid detection controller is configured to measure at least the time for measuring fluorescence with a small peak wavelength and the time for measuring fluorescence with a large peak wavelength when the fluorescence measuring instrument 55 measures three or more types of fluorescence with different wavelengths in multiplex PCR. The fluorescence measuring device 55 is controlled so that a part is overlapped, and then the fluorescence having an intermediate peak wavelength is measured.

The nucleic acid detection control device may be systematized as a nucleic acid amplification system together with any of the nucleic acid amplification devices described above.

<Description of operation>
(1) Mounting Operation of Cartridge 1 FIGS. 9A to 9D are explanatory diagrams of the state of the PCR device 50 when the cartridge 1 is mounted.
FIG. 9A is an explanatory diagram of an initial state before the cartridge 1 is mounted. FIG. 9B is an explanatory diagram of a standby state. FIG. 9C is an explanatory diagram immediately after the cartridge 1 is mounted. FIG. 9D is an explanatory diagram of an initial state when the cartridge 1 is mounted.

As shown in FIG. 9A, in the initial state before the cartridge 1 is mounted, the mounting direction of the mounting portion 62 is the vertical direction. In the following description, the rotational position of the rotating body 61 in this state is the reference (0
The rotation position of the rotating body 61 is indicated with a counterclockwise direction as viewed from the right as a positive direction.

As shown in FIG. 9B, the controller 90 drives the rotation motor 66 to rotate the rotating body 61 to −30 degrees. In this state, the operator inserts the cartridge 1 into the mounting portion 62 from the cartridge insertion port 53A. At this time, since the guide plate 26 is guided by the guide rail 63A and the cartridge 1 is inserted into the mounting portion 62, the PCR container 30 of the cartridge 1 is guided to the insertion hole 64A of the mounting portion 62. The operator inserts the cartridge 1 until the fixing claw 25 of the cartridge 1 is caught by the notch of the fixing portion 63. Thereby, the cartridge 1 is fixed at a normal position with respect to the mounting portion 62. Temporarily PCR container 3
When 0 is not inserted into the insertion hole 64A and the cartridge 1 is in an abnormal position with respect to the mounting portion 62, the fixing claw 25 of the cartridge 1 is not caught by the notch of the fixing portion 63, so that the cartridge 1 is abnormal. The operator can recognize that it is in the position.

As shown in FIG. 9C, when the cartridge 1 is fixed at a normal position with respect to the mounting portion 62, the reaction solution plug 47 of the tube 20 faces the elution heater 65 </ b> A and the flow path forming portion 35 of the PCR container 30. The upstream side (high temperature region 36A) of the PCR container 30 faces the high temperature side heater 65B, and the downstream side (low temperature region 36B) of the flow path forming part 35 of the PCR container 30 faces the low temperature side heater 65C. Since the rotating body 61 is provided with the mounting portion 62 and the heater, even if the rotating body 61 rotates, the positional relationship between the cartridge 1 and the heater is maintained as it is.

After the cartridge 1 is mounted on the mounting portion 62, as shown in FIG.
0 rotates the rotator 61 by 30 degrees and returns the position of the rotator 61 to the reference. The controller 90 may detect that the cartridge 1 is mounted on the mounting unit 62 by a sensor (not shown) or may be detected by an input operation from an operator.

(2) Nucleic Acid Elution Processing / Vertical Movement of Magnet 71 FIG. 10 is a conceptual diagram of the behavior of the magnetic beads 7 when the magnet 71 is moved downward.
The magnetic beads 7 in the cartridge 1 are attracted to the magnet 71. For this reason, when the magnet 71 moves outside the cartridge 1, the magnetic beads 7 in the cartridge 1 move together with the magnet 71.

11A to 11C are explanatory diagrams of the nucleic acid elution process. FIG. 11A is an explanatory diagram of the state of the PCR device 50 before the nucleic acid elution process. FIG. 11B is an explanatory diagram of the state of the PCR device 50 when the magnet 71 is moved to the reaction solution plug 47. FIG. 11C is an explanatory diagram of the state of the PCR device 50 when the magnet 71 is pulled up.

As shown in FIG. 11A, the cartridge 1 in the initial state has the tank 3 on the upper side and the longitudinal direction is parallel to the vertical direction. In this state, as shown in FIG. 2A, the cartridge 1 includes, in order from the top, a solution 41 (tank 3) containing the magnetic beads 7 and an oil 42 (plunger 10).
), Cleaning liquid 43 (upstream of tube 20), first oil plug 44 (capillary 23)
, Cleaning liquid plug 45 (capillary 23), second oil plug 46 (capillary 23)
, Reaction solution plug 47 (capillary 23), third oil plug 48 (capillary 23)
Oil (PCR container 30).

As shown in FIG. 11A, in the initial state, the carriage 73A is at the uppermost position (retracted position).
The magnet 71 is located above the cartridge 1. From this state, the controller 90 drives the elevating motor 73B to move the carriage 73A gradually downward and move the magnet 71 gradually downward. Since the longitudinal direction of the cartridge 1 is parallel to the vertical direction, the magnet 71 moves along the cartridge 1.

When the magnet 71 moves downward, the magnet 71 comes to face the tank 3, and the tank 3
The inner magnetic beads 7 are attracted to the magnet 71. The controller 90 moves the carriage 73 </ b> A downward at such a speed that the magnetic beads 7 can move together with the magnet 71.

When the magnet 71 moves from a position facing the tank 3 (height of the tank 3) to a position facing the plunger 10 (height of the plunger 10), the magnetic beads 7 are moved to the plunger 10
Passes through the opening on the upstream side of the cylindrical portion 11 and passes through the interface between the solution 41 in the tank 3 and the oil 42 on the upstream side of the cartridge body 9. Thereby, the magnetic beads 7 to which the nucleic acid is bound are introduced into the cartridge body 9. When the magnetic beads 7 pass through the interface with the oil 42, the solution 41 is wiped by the oil 42, so that the components of the solution 41 are not easily brought into the oil 42. Thereby, it can suppress that the component of the solution 41 is mixed in the cleaning solution or the reaction solution 47.

When the magnet 71 moves downward while facing the plunger 10, the magnetic beads 7 are
Passing through the inside of the cylindrical part 11, passing through the front and back of the rib 13, exiting from the opening on the downstream side of the cylindrical part 11
It is introduced into the upper syringe 21 of the tube 20. During this time, the magnetic beads 7 pass through the interface between the oil 42 and the cleaning liquid 43 in the plunger 10. When the magnetic beads 7 are introduced into the cleaning liquid 43, the nucleic acid bound to the magnetic beads 7 is cleaned by the cleaning liquid 43.

At this stage, since the rod-shaped portion 12 of the plunger 10 is not inserted into the lower syringe 22 of the tube 20, the position where the magnet 71 faces the capillary 23 from the position facing the upper syringe 21 (the height of the upper syringe 21). When moving to the height of the capillary 23), the magnetic beads 7 move from the upper syringe 21 to the lower syringe 22, and move from the lower syringe 22 to the capillary 23. A first oil plug 44 is provided on the upstream side of the capillary 23, and when the magnetic bead 7 moves from the lower syringe 22 to the capillary 23, the magnetic bead 7 is washed with the cleaning liquid 4.
3 passes through the oil interface. At this time, since the cleaning liquid 43 is wiped by the oil, the components of the cleaning liquid 43 are not easily brought into the oil. Thereby, it is possible to suppress the components of the cleaning liquid 43 from being mixed into the cleaning liquid plug 45 and the reaction liquid plug 47.

When the magnet 71 moves from the position facing the first oil plug 44 (the height of the first oil plug 44) to the position facing the cleaning liquid plug 45 (the height of the cleaning liquid plug 45), the magnetic beads 7 move between the oil and the cleaning liquid. And pass through the interface. When the magnetic beads 7 are introduced into the cleaning liquid plug 45, the nucleic acids bound to the magnetic beads 7 are cleaned with the cleaning liquid.

When the magnet 71 moves from the position facing the cleaning liquid plug 45 (the height of the cleaning liquid plug 45) to the position facing the second oil plug 46 (the height of the second oil plug 46), the magnetic beads 7 are moved into the magnetic field. The beads 7 pass through the interface between the cleaning liquid and the oil. At this time, since the cleaning liquid is wiped off by the oil, the components of the cleaning liquid are not easily brought into the oil. Thereby, it can suppress that the component of a washing | cleaning liquid mixes in the reaction liquid plug 47. FIG.

When the magnet 71 moves from the position facing the second oil plug 46 (the height of the second oil plug 46) to the position facing the reaction liquid plug 47 (the height of the reaction liquid plug 47), the magnetic beads 7 And the reaction liquid 47.

The controller 90 controls the elution heater 65 </ b> A to heat the reaction solution plug 47 to about 50 ° C. before the magnetic beads 7 are introduced into the reaction solution plug 47. In addition, by heating the reaction solution 47 before the magnetic beads 7 are introduced, the time from the introduction of the magnetic beads 7 to the reaction solution 47 until the elution of the nucleic acid is completed can be shortened.

As shown in FIG. 11B, the position where the magnet 71 faces the reaction liquid plug 47 (reaction liquid plug 4
7), the controller 90 stops the elevating motor 73B to stop the movement of the magnet 71 in the vertical direction, and when processed at 50 ° C. for 30 seconds, the magnetic beads 7
The nucleic acid bound to is released into the solution of the reaction solution plug 47, and the reverse transcription reaction proceeds. Reaction solution 47
Is heated to promote elution of nucleic acid from the magnetic beads 7 and reverse transcription reaction.

After the nucleic acid is eluted with the reaction solution plug 47, the controller 90 moves the elevating motor 73.
B is driven in the opposite direction, and the carriage 73A is gradually moved upward, and the magnet 71 is gradually moved upward. The controller 90 moves the carriage 73 </ b> A upward at a speed at which the magnetic beads 7 can move together with the magnet 71.

When the magnet 71 moves upward from the state shown in FIG. 11B, the magnetic beads 7 move from the reaction solution plug 47 to the second oil plug 46, and the magnetic beads 7 are removed from the reaction solution plug 47.

When the magnet 71 gradually moves to a position facing the upper syringe 21, the magnetic beads 7 also move to the upper syringe 21, and the magnetic beads 7 are on the upper side of the lower syringe 22. If the magnetic beads 7 are moved to this position, the magnetic beads 7 are not introduced into the PCR container 30 when the plunger 10 is pushed. For this reason, the controller 90 from FIG.
Until the state shown in C, the carriage 73A may be moved upward at such a speed that the magnetic beads 7 cannot follow the movement of the magnet 71. If the magnetic beads 7 are not introduced into the PCR container 30 when the plunger 10 is pushed, the carriage 73 is introduced at an earlier stage.
The moving speed of A may be increased.

Information related to the moving speed of the magnet 71 is stored in the storage device of the controller 90, and the controller 90 executes the above-described operation (operation for moving the magnet 71 up and down) according to this information.

-Swinging of the magnet 71 While moving the magnet 71 in the vertical direction, the controller 90 moves the swinging motor 75.
A may be driven to swing the pair of magnets 71 sandwiching the cartridge 1 in the front-rear direction.

  FIG. 12 is a conceptual diagram of the behavior of the magnetic beads 7 when the magnet 71 is swung.

While the magnet 71 moves in the vertical direction, the tube 20 is sandwiched between the pair of magnets 71 from the front-rear direction. Since the pair of magnets 71 are held by the arm 72, the distance in the front-rear direction of the pair of magnets 71 is substantially constant. For this reason, when one of the pair of magnets 71 approaches the tube 20, the other is separated from the tube 20.

Since the magnetic beads 7 are attracted toward the magnet 71 that is closer to the distance, when one magnet 71 is close to the tube 20, the magnetic beads 7 are attracted toward the magnet 71. Thereafter, when the magnet 71 moves away from the tube 20 and the opposite magnet 71 approaches the tube 20, the magnetic beads 7 are attracted to the opposite magnet 71 this time. Thereby, the magnetic beads 7 move in the front-rear direction. When the pair of magnets 71 are swung in the front-rear direction, the magnetic beads 7 reciprocate in the front-rear direction.

When the magnetic beads 7 reciprocate in the front-rear direction, the liquid easily comes into contact with the magnetic beads 7. In particular, since the liquid in the capillary 23 has almost no fluidity, the capillary 23
When it is desired to bring the liquid inside into contact with the magnetic beads 7 as much as possible, it is effective that the magnetic beads 7 reciprocate in the front-rear direction.

  FIG. 13 is a table showing whether or not the magnet 71 swings.

When the magnetic beads 7 move down the oil plug (the first oil plug 44 or the second oil plug 46), the controller 90 stops the swing motor and does not swing the magnet 71. At this time, the controller 90 moves the magnet 71 downward while keeping one of the pair of magnets 71 close to the tube 20. This is because the magnetic beads 7 can easily follow the movement of the magnet 71 as compared with the case where the distance between each magnet 71 and the tube 20 is made equal.

When the magnetic beads 7 move down the cleaning liquid plug 45, the controller 90 drives the swing motor to swing the magnet 71 in the front-rear direction. Thereby, magnetic beads 7
Moves downward while swinging in the cleaning liquid plug 45 in the front-rear direction, so that the cleaning efficiency of the magnetic beads 7 can be increased. Further, since the cleaning efficiency is increased, the amount of the cleaning liquid plug 45 can be suppressed, and the cartridge 1 can be downsized.

When the magnetic beads 7 pass through the interface between the cleaning liquid and the oil (second oil plug 46), the controller 90 stops the swing motor and does not swing the magnet 71. This
Since the magnetic beads 7 pass through the interface without swinging, it becomes difficult for the components of the cleaning liquid to be brought into the oil. The controller 90 moves the magnet 71 downward in a state where one of the pair of magnets 71 is brought close to the tube 20. As a result, the magnetic beads 7 are attracted and agglomerated by the magnets 71 that are close to each other, and the cleaning liquid adhering to the magnetic beads 7 is squeezed.

When the magnetic beads 7 are in the reaction solution plug 47, the controller 90 drives the swing motor to swing the magnet 71 in the front-rear direction. Accordingly, since the magnetic beads 7 swing in the reaction solution plug 47 in the front-rear direction, the elution efficiency of the nucleic acid bound to the magnetic beads 7 can be increased. In addition, since elution efficiency is increased, the time from the introduction of the magnetic beads 7 into the reaction solution 47 to the end of elution of nucleic acids can be shortened.

Note that after the nucleic acid is eluted with the reaction solution plug 47, when the magnet 71 is moved upward to pull up the magnetic beads 7, the controller 90 stops the swing motor to stop the magnet 71.
Do not rock. At this time, the controller 90 moves the magnet 71 downward while keeping one of the pair of magnets 71 close to the tube 20. Thereby, the magnetic beads 7 can easily follow the movement of the magnet 71, and the moving speed of the magnet 71 can be increased.

The storage device of the controller 90 stores information on the position of each plug of the capillary 23 and swing information as shown in FIG. 13, and the controller 90 performs the above-described operation (the magnet 71) according to this information. Oscillate).

(3) Droplet Formation Processing FIGS. 14A to 14C are explanatory diagrams of the droplet formation processing. FIG. 14A is an explanatory diagram of the state of the PCR device 50 when the magnet 71 is pulled up. FIG. 14B is an explanatory diagram of a state in which the rotating body 61 is rotated 45 degrees. FIG. 14C shows that the rod 82 of the pressing mechanism 80 is the plunger 10.
It is explanatory drawing of the state which pressed.

As shown in FIG. 14A, when the carriage 73A is in the retracted position, the cartridge 1
Does not come into contact with the cartridge 1 even if the rotation of. After this state, the controller 90 rotates the rotating body 61 by 45 degrees.

As shown in FIG. 14B, when the rotating body 61 rotates 45 degrees, the longitudinal direction of the cartridge 1 becomes parallel to the moving direction of the rod 82 of the pressing mechanism 80. The controller 90 drives the plunger motor 81 to move the rod 82. When the rod 82 further moves after the rod 82 contacts the mounting base 11A of the plunger 10 of the cartridge 1, the plunger 10 is pushed into the tube 20 side. The controller 90 moves the rod 82 to the state shown in FIG. 14C and pushes the plunger 10 until the mounting base 11A of the plunger 10 contacts the upper edge of the tube 20.

When the plunger 10 is pushed into the tube 20 side, the rod-shaped portion 12 of the plunger 10
The seal 12A is fitted into the lower syringe 22 of the tube 20 (see FIG. 2B). When the plunger 10 is further pushed in, the seal 12A slides in the lower syringe 22. Thereby, the liquid (the third oil plug 48, the reaction solution plug 47, etc.) on the downstream side of the tube 20 is equivalent to the volume corresponding to the volume of the seal 12A slid in the lower syringe 22.
It is pushed out to the zero flow path forming part 35.

First, the third oil plug 48 of the tube 20 flows into the flow path forming portion 35. Since the flow path forming portion 35 is filled with oil, the inflowed oil flows from the flow path forming portion 35 into the oil receiving space 32A, and the oil interface of the oil receiving space 32A rises. At this time, the pressure of the liquid in the flow path forming unit 35 becomes higher than the external atmospheric pressure (pressure in the oil receiving space 32A) due to the pressure loss in the lower seal portion 34B. After the third oil plug 48 is pushed out from the tube 20, the reaction solution plug 47 flows from the tube 20 into the flow path forming part 35. Since the inner diameter of the flow path forming portion 35 is larger than the inner diameter of the capillary 23, the reaction liquid 47 that is plug-shaped in the tube 20 becomes a droplet in the oil of the flow path forming portion 35.

The volume in which the seal 12A slides in the lower syringe 22 (the amount by which the liquid in the tube 20 is pushed out from the downstream side) is the reaction liquid plug 47 and the third oil plug 48 in the tube 20.
Therefore, after the reaction solution plug 47 is pushed out of the tube 20, a part of the second oil plug 46 is also pushed out to the flow path forming portion 35. As a result, the reaction solution 47 does not remain in the tube 20 and the entire amount of the reaction solution plug 47 becomes droplets. Further, when a part of the second oil plug 46 is pushed out from the downstream side of the tube 20, the droplet-like reaction solution 47 is easily separated from the tube 20 (the droplet-like reaction solution 47 enters the opening of the capillary 23. It becomes difficult to adsorb).

Since the inner diameter of the end of the capillary 23 (opening diameter of the capillary 23) is designed to be relatively small, the reaction liquid 47 that has become droplets in the PCR container 30 is difficult to adsorb to the opening of the capillary 23. The reaction solution 47 has a specific gravity greater than that of the oil in the PCR container 30. For this reason, the droplet-like reaction liquid 47 moves away from the end of the capillary 23 and flows into the flow path forming unit 3.
It sinks toward the bottom 35A using 5 as a flow path. However, at this stage, since the flow path of the flow path forming unit 35 is inclined at 45 degrees, the droplet-like reaction liquid 47 is likely to adhere to the inner wall of the flow path forming part 35. For this reason, it is necessary to return the flow path of the flow path forming unit 35 to the vertical direction.

After the droplet-like reaction liquid 47 is formed (after the plunger 10 is pushed), the controller 90 drives the plunger motor 81 in the opposite direction to move the rod 82 to the original position. return. In this state, even if the cartridge 1 rotates, the rod 82 of the pressing mechanism 80.
Does not come into contact with the cartridge 1. After making such a state, the controller 90 returns the rotating body 61 to the reference position. When the rotator 61 is at the reference position, the flow path of the flow path forming unit 35 is in the vertical direction, so that the liquid reaction liquid 47 is less likely to adhere to the inner wall of the flow path forming unit 35.

(4) Thermal Cycle Process FIGS. 15A and 15B are explanatory diagrams of the thermal cycle process. FIG. 15A is an explanatory diagram of a state in which the low temperature side temperature treatment is performed on the reaction solution 47. FIG. 15B is an explanatory diagram showing a state in which the temperature treatment on the high temperature side is performed on the reaction solution 47. The left side of each figure shows the state of the PCR device 50, and the right side of each figure shows the state inside the flow path forming part 35 of the PCR container 30.

When the cartridge 1 is fixed at a normal position with respect to the mounting part 62, the upstream side (high temperature region 36A) of the flow path forming part 35 of the PCR container 30 faces the high temperature side heater 65B, and the flow path of the PCR container 30 The downstream side (low temperature region 36B) of the formation unit 35 faces the low temperature side heater 65C.
During the heat cycle process, the controller 90 is connected to the high temperature side heater 6 provided on the rotating body 61.
The liquid in the high temperature region 36A on the upstream side of the flow path forming part 35 of the PCR container 30 is about 90% by 5B.
Heat to ~ 100 ° C. Further, the controller 90 causes the liquid in the low temperature region 36B on the downstream side of the flow path forming portion 35 to be about 50 to 75 ° C. by the low temperature side heater 65C provided on the rotating body 61.
Heat to. Thereby, a temperature gradient is formed in the liquid in the flow path forming part 35 of the PCR container 30 during the thermal cycle process. Since the mounting portion 62 and the heater are provided on the rotating body 61, even if the rotating body 61 rotates, the positional relationship between the cartridge 1 and the heater is maintained as it is.

During the thermal cycling process, the liquid in the PCR container 30 is heated. If bubbles are generated by heating the liquid in the PCR container 30, the temperature of the liquid in the flow path forming unit 35 varies, or the droplet-like reaction liquid 47 moves (sets down) in the flow path forming unit 35. ) May be hindered. However, in this embodiment, since the pressure of the liquid in the flow path forming unit 35 is higher than the external pressure due to the pressure loss in the lower seal portion 34B, bubbles are less likely to be generated in the liquid in the PCR container 30.

As shown in FIG. 15A, when the rotator 61 is at the reference position, the low temperature side heater 65C is positioned below the high temperature side heater 65B, and the bottom 35A of the PCR container 30 of the cartridge 1 is positioned downward. Since the droplet-like reaction liquid 47 has a specific gravity greater than that of oil, the droplet-like reaction liquid 47 settles in the flow path forming unit 35. When the droplet-like reaction liquid 47 settles in the flow path forming part 35, the PC
It reaches the bottom 35A of the R container 30, where it settles and stays in the low temperature region 36B. Thereby, the droplet-like reaction liquid 47 moves to the low temperature region 36B. The controller 90 is shown in FIG.
This state is maintained for a predetermined time, and the droplet-like reaction solution 47 is heated to about 50 to 75 ° C. in the low temperature region 36B (temperature treatment on the low temperature side is performed). During this time, the extension reaction of the polymerase reaction occurs.

When the controller 90 drives the rotation motor 66 from the state of FIG. 15A to rotate the rotating body 61 by 180 degrees, the state shown in FIG. 15B is obtained. When the rotating body 61 rotates 180 degrees from the reference position, the cartridge 1 is turned upside down and the high temperature side heater 65B and the low temperature side heater 65C are also turned upside down. That is, the high temperature side heater 65B is replaced with the low temperature side heater 65C.
The bottom 35A of the PCR container 30 of the cartridge 1 is on the upper side. When the droplet-like reaction liquid 47 settles in the flow path forming part 35, it reaches the end of the tube 20 (the end of the capillary 23), where it settles and remains in the high temperature region 36A. Thereby, the droplet-like reaction liquid 47 moves to the high temperature region 36A. The controller 90 maintains the state of FIG. 15B for a predetermined time, and heats the droplet-like reaction liquid 47 to about 90 to 100 ° C. in the high temperature region 36A (temperature processing on the high temperature side is performed). During this time, a denaturation reaction of the polymerase reaction occurs.

When the controller 90 drives the rotation motor 66 from the state of FIG. 15B to rotate the rotating body 61 by −180 degrees, the state returns to the state of FIG. 15A. In this state, when the droplet-like reaction liquid 47 settles in the flow path forming part 35, the droplet-like reaction liquid 47 moves to the low temperature region 36B and is heated again to about 50 to 75 ° C. in the low temperature region 36B. (Temperature treatment on the low temperature side is performed). In addition,
Since the inner diameter of the end of the capillary 23 (opening diameter of the capillary 23) is designed to be relatively small, the reaction solution 47 is less likely to be adsorbed to the opening of the capillary 23. FIG.
When the rotating body 61 rotates -180 degrees from the state B, the droplet-like reaction solution 47 leaves the tube 20 and settles toward the bottom 35A of the PCR container 30 without being adsorbed to the opening of the capillary 23.

The controller 90 drives the rotation motor 66 to set the rotation position of the rotating body 61 in FIG.
The state of 5A and the state of FIG. 15B are repeated for a predetermined number of cycles. As a result, the PCR device 50 can perform PCR thermal cycle processing on the reaction solution 47.

The storage device of the controller 90 includes the temperature of the high temperature side heater 65B and the low temperature side heater 6B.
The temperature of 5C, the time of holding in the state of FIG. 15A, the time of holding in the state of FIG. 15B, and the number of cycles (the number of repetitions of the state of FIG. 15A and the state of FIG. 15B) are stored. Performs the above processing according to the thermal cycle information.

(5) Fluorescence measurement in real-time PCR As shown in FIG. 15A, when the rotating body 61 is at the reference position, the fluorescence measuring device 55 faces the bottom 35A of the PCR container 30 of the cartridge 1. Therefore, at the time of measuring the fluorescence of the reaction solution 47, the controller 90 keeps the rotating body 61 at the reference position and the reaction solution 47 in the bottom 35A of the PCR container 30 from the lower opening of the insertion hole 64A of the low temperature heater 65C. The fluorescence measuring instrument 55 is made to measure the fluorescence intensity.

Immediately after the rotating body 61 is rotated 180 degrees to the reference position, the droplet-like reaction liquid 47 is transferred to the PC.
The flow path forming part 35 of the R container 30 is settled, and the droplet-like reaction liquid 47 may not reach the bottom 35A of the PCR container 30 in some cases. For this reason, the controller 90 waits for a predetermined time after the rotational position of the rotating body 61 reaches the state of FIG. 15A (from the state of FIG. 15A to the rotating body 6).
It is desirable to measure the fluorescence intensity just before rotating 1). Alternatively, the controller 90 may store the fluorescence intensity time history by causing the fluorescence measuring instrument 55 to measure the fluorescence intensity for a predetermined time from when the rotating body 61 is set to the reference position.

In the case of multiplex PCR, a plurality of fluorescent substances are used, but interference from other fluorescent substances is observed depending on characteristics such as an absorption spectrum to be absorbed and a fluorescence spectrum to be emitted. In particular, when there is an overlap between the excitation spectrum and the fluorescence spectrum for two fluorescent substances, interference occurs with each other, and accurate fluorescence intensity cannot be measured. Therefore, irradiation with excitation light of a specific wavelength and measurement of fluorescence of a specific wavelength are repeated for each fluorescent substance. However, when three or more fluorescent substances are detected one by one, it takes time, and it may be difficult to realize high-speed PCR, which is one of the advantages of this nucleic acid amplification apparatus. Patent Document 1,
In Section 2, sufficient consideration has been given to the problem of accelerating the entire process of enclosing (dispensing) the reaction solution inside the biochip and detecting the fluorescence in the monitoring of the nucleic acid amplification reaction. Absent.

Therefore, when detecting emission fluorescence from three or more fluorescent substances, for any three fluorescent substances, at least a part of the time for measuring fluorescence with a small peak wavelength and the time for measuring fluorescence with a large peak wavelength are overlapped. Thereafter, it is preferable to measure fluorescence having an intermediate peak wavelength.
For example, first, a fluorescent substance with a small peak wavelength and a fluorescent substance with a large peak wavelength are excited simultaneously to measure the emitted fluorescence, and then the fluorescent substance with an intermediate peak wavelength is excited to measure the emitted fluorescence. To do. Thereby, interference can be minimized and detection time can be shortened.

As real-time PCR for multiplex PCR, any method can be used as long as it uses fluorescence as an indicator of the amount of amplified DNA. Well, for example, intercalator method, TaqMan method, Molecular Beac
on method, cycling probe method and the like. The fluorescent material used is not particularly limited,
Alexa (registered trademark), FAM (registered trademark), VIC (registered trademark), SYBR gre
en (registered trademark), EvaGreen (registered trademark), TexRed (registered trademark), LCR
Examples thereof include ed (registered trademark), Cy3 (registered trademark), and Cy5 (registered trademark).

For example, as fluorescent substances, ATTO 655 (ATTO-TEC), bodipy FL (Invitrogen), Pci
When fic blue (Invitrogen) is used, excitation light is irradiated using a red filter (transmission wavelength 600-650 nm) and a blue filter (transmission wavelength 380-420 nm), respectively, and ATTO 655 and P
Cific blue is excited and fluorescence is measured using a red filter (transmission wavelength 655-730 nm) and a blue filter (transmission wavelength 440-530 nm). After that, the green filter (transmission wavelength 450-4
90nm) is used to excite bodipy FL by irradiating with excitation light, and a green filter (transmission wavelength 440)
-530) to measure fluorescence.

When radiated fluorescence from four or more fluorescent substances is detected by multiplex PCR, the time reduction effect can be obtained only by performing the method of the present invention for any three of them, but for other fluorescent substances, If there is little overlap between the excitation spectrum and the fluorescence spectrum, fluorescence measurement may be performed simultaneously.

=== Second Embodiment ===
In the second embodiment, a cartridge 1 having a configuration different from that of the first embodiment is attached to the PCR device 50.

<Cartridge 1>
16A and 16B are explanatory diagrams of the cartridge 1 according to the second embodiment. FIG. 17A is an explanatory diagram of an initial state of the cartridge 1 according to the second embodiment. FIG. 17B is a side view when the plunger 10 is pushed from the state of FIG. 17A and the seal 12A comes into contact with the lower syringe 22. FIG. 17C is an explanatory diagram of the cartridge 1 of the second embodiment after the plunger 10 is pushed. The tube 20 of the cartridge 1 of the second embodiment includes an eluate plug 47A, an oil plug 47B, and a nucleic acid amplification reaction solution plug 47C instead of the reaction solution plug 47 of the first embodiment. The oil plug 47B prevents the eluate plug 47A and the nucleic acid amplification reaction solution plug 47C, which are water-soluble plugs on both sides, from mixing with each other.

The cartridge 1 is a container that performs a nucleic acid elution process for eluting nucleic acid from the magnetic beads 7 to which the nucleic acid is bound, and a PCR that is a mixed liquid of the eluate 47A and the nucleic acid amplification reaction liquid 47C.
This is a container for performing a thermal cycle process for polymerase reaction on a solution.

The shapes of the tank 3 and the cartridge body 9 of the cartridge 1 are the same as those in the first embodiment. The solution 41 in the tank 3 of the cartridge 1 is the same as that in the first embodiment. The oil 42, the first cleaning liquid 43, the first oil plug 44, the cleaning liquid plug 45 (second cleaning liquid), and the second oil plug 46 of the cartridge body 9 are the same as in the first embodiment. Therefore, these descriptions are omitted.

The eluate plug 47A and the nucleic acid amplification reaction solution plug 47C are made of, for example, the following reaction solutions.

The eluate refers to a liquid in which a nucleic acid adsorbed on a nucleic acid-binding solid phase carrier is eluted from the carrier into the liquid and subjected to a reverse transcription reaction. For this reason, the eluate 47A after elution of nucleic acid is prepared in advance so as to be a buffer solution used for the reverse transcription reaction as it is. Further, the nucleic acid amplification reaction liquid refers to a liquid that performs a polymerase reaction.

A downstream portion of the capillary 23 is inserted into the PCR container 30. Accordingly, the nucleic acid amplification reaction solution 47C and the eluate 47A can be introduced into the PCR container 30 by pushing out the nucleic acid amplification reaction solution plug 47C and the eluate plug 47A in the tube 20 from the tube 20.

18A and 18B are explanatory diagrams of the periphery of the PCR container 30 of the second embodiment. FIG.
8A is an explanatory diagram of an initial state. FIG. 18B is an explanatory diagram of a state after the plunger 10 is pushed. Hereinafter, description will be made with reference to FIGS. 17A to 17C.

The PCR container 30 is a container that receives the liquid pushed out from the tube 20 and that stores a PCR solution that is a mixture of the nucleic acid amplification reaction solution 47C and the eluate 47A during the thermal cycle process.

When the plunger 10 is pushed, first, the third oil plug 48 of the tube 20 flows into the flow path forming portion 35, and the inflowed oil flows into the oil receiving space 32A from the flow path forming portion 35, and the oil receiving space 32A. The oil interface rises. At this time, the pressure of the liquid in the flow path forming portion 35 increases due to the pressure loss of the lower seal portion 34B.

After the third oil plug 48 is pushed out of the tube 20, the nucleic acid amplification reaction solution plug 47
C flows into the flow path forming part 35 from the tube. The inner diameter of the flow path forming portion 35 is the capillary 23
Since the inner diameter of the nucleic acid amplification reaction solution 47 is in the form of a plug (columnar) in the tube 20
C forms droplets in the flow path forming unit 35. Further, since the nucleic acid amplification reaction solution 47C has a specific gravity greater than that of oil, the droplet-like nucleic acid amplification reaction solution 47C settles.

After the nucleic acid amplification reaction solution plug 47C is pushed out of the tube 20, the oil plug 47B
Flows into the flow path forming portion 35. After the oil plug 47B is pushed out of the tube 20,
The eluate plug 47 </ b> A flows from the tube 20 into the flow path forming unit 35. Since the inner diameter of the flow path forming portion 35 is larger than the inner diameter of the capillary 23, the eluate 47 </ b> A that is plug-shaped (columnar) in the tube 20 becomes a droplet in the flow path forming portion 35. Further, since the eluate 47A has a specific gravity greater than that of the oil, the eluate 47A in the form of droplets settles. When the eluate 47A in the form of droplets settles, the droplet-shaped nucleic acid amplification reaction solution 4 that has already settled on the bottom of the flow path forming unit 35.
7C and oil are combined in oil to form a droplet-like PCR solution 47.

<Description of operation of PCR device 50>
The structure of the PCR device 50 of the second embodiment is the same as that of the first embodiment. PC
Structure of R device 50 (rotating mechanism 60, magnet moving mechanism 70, pressing mechanism 80, fluorescence measuring instrument 55,
The description of the controller 90 and the like is omitted, and here, the operation of the PCR device 50 when the cartridge 1 of the second embodiment is mounted will be described.

(1) Mounting Operation of Cartridge 1 The state when the cartridge 1 of the second embodiment is mounted is the same as in FIGS. 9A to 9D of the first embodiment. In the second embodiment, as shown in FIG. 9C, when the cartridge 1 is fixed at a normal position with respect to the mounting portion 62, the eluate plug 47A of the tube 20 is removed from the elution heater 65.
(In contrast, in the first embodiment, the reaction solution plug 47 of the tube 20 faces the elution heater 65A).

(2) Nucleic acid elution process The state at the time of the nucleic acid elution process of 2nd Embodiment is the same as that of FIG. 11A-FIG. 11C of 1st Embodiment.

In the second embodiment, as shown in FIG. 11B, after the magnet 71 has moved to a position facing the eluate plug 47A (the height of the eluate plug 47A), the controller 90 stops the elevating motor 73B. When the vertical movement of the magnet 71 is stopped and the treatment is performed at 50 ° C. for 30 seconds, the nucleic acid bound to the magnetic beads 7 is released into the solution of the eluate plug 47 and the reverse transcription reaction proceeds. By heating the eluate 47, elution and reverse transcription of nucleic acids from the magnetic beads 7 are promoted.

After the nucleic acid is eluted with the eluent plug 47A, the controller 90 moves the elevating motor 7
3B is driven in a direction opposite to the above, the carriage 73 is gradually moved upward, and the magnet 71 is gradually moved upward. When the magnet 71 moves upward from the state shown in FIG. 11B, the magnetic beads 7 move from the eluate plug 47A to the second oil plug 46, and the magnetic beads 7 are removed from the eluate plug 47A.

In the second embodiment, the magnetic beads 7 do not contact the nucleic acid amplification reaction solution 47C. For this reason, there is no possibility that the enzyme of the nucleic acid amplification reaction solution 47C will be adsorbed to the magnetic beads 7, and there is no possibility of reaction inhibition. Therefore, in the second embodiment, the nucleic acid elution process can be performed under higher efficiency conditions (higher temperature, higher frequency of oscillation, increased amount of magnetic beads, etc.) than in the first embodiment.

(3) Droplet formation process The state at the time of the droplet formation process of 2nd Embodiment is the same as that of FIG. 14A-FIG. 14C of 1st Embodiment.

When the plunger 10 is pushed, first, the third oil plug 48 of the tube 20 flows into the flow path forming portion 35, and the inflowed oil flows into the oil receiving space 32A from the flow path forming portion 35, and the oil receiving space 32A. The oil interface rises. At this time, the pressure of the liquid in the flow path forming portion 35 increases due to the pressure loss of the lower seal portion 34B.

After the third oil plug 48 is pushed out of the tube 20, the nucleic acid amplification reaction solution plug 47
C flows into the flow path forming part 35 from the tube. The inner diameter of the flow path forming portion 35 is the capillary 23
Since the inner diameter of the nucleic acid amplification reaction solution 47 is in the form of a plug (columnar) in the tube 20
C forms droplets in the flow path forming unit 35. Further, since the nucleic acid amplification reaction solution 47C has a specific gravity greater than that of oil, the droplet-like nucleic acid amplification reaction solution 47C settles.

After the nucleic acid amplification reaction solution plug 47C is pushed out of the tube 20, the oil plug 47B
Flows into the flow path forming portion 35. After the oil plug 47B is pushed out of the tube 20,
The eluate plug 47 </ b> A flows from the tube 20 into the flow path forming unit 35. Since the inner diameter of the flow path forming portion 35 is larger than the inner diameter of the capillary 23, the eluate 47 </ b> A that is plug-shaped (columnar) in the tube 20 becomes a droplet in the flow path forming portion 35. Further, since the eluate 47A has a specific gravity greater than that of the oil, the eluate 47A in the form of droplets settles. When the eluate 47A in the form of droplets settles, the droplet-shaped nucleic acid amplification reaction solution 4 that has already settled on the bottom of the flow path forming unit 35.
7C and oil are combined in oil to form a droplet-like PCR solution 47.

Since both the droplet-like eluent 47A and the nucleic acid amplification reaction solution 47C are small, the two droplets may not be united at the bottom of the flow path forming unit 35 and may remain separated. Therefore, after the droplet-like eluent 47A settles on the bottom of the flow path forming unit 35, the controller 90 drives the rotating mechanism 60 in small increments to vibrate the rotating body 61. As a result, two droplets are temporarily formed in the flow path forming unit 3.
Even in the state separated at the bottom of 5, the cartridge 1 vibrates so that the two droplets are combined into a droplet-like PCR solution 47. Alternatively, the droplets may be heated by the high temperature side heater 65B or the low temperature side heater 65C to reduce the viscosity of the droplets, thereby facilitating the combination of the two droplets.

After the droplet formation process, a thermal cycle process and a fluorescence measurement are performed. Since the thermal cycle process and fluorescence measurement of the second embodiment are the same as those of the first embodiment, description thereof is omitted here.

Also in the second embodiment, the PCR device 50 includes a rotating body 61 having a mounting part (fixing part 63 and insertion hole 64A) for mounting the cartridge 1, and a PCR container 3 which is a nucleic acid amplification reaction container.
PCR heaters (high temperature side heater 65B and low temperature side heater 65C) that form a temperature gradient inside 0, and the rotating body 61 rotates to change the posture of the cartridge 1, so that the inside of the PCR container 30 The reaction liquid 47 in the form of droplets introduced from the tube 20
Move. As a result, the PCR container 20 together with the tube 20 on which the nucleic acid elution process is performed.
Since the polymerase reaction can be performed by changing the posture of the substrate, the processing time can be shortened.

=== Others ===
The above-described embodiments 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 gist thereof, and it is needless to say that the present invention includes equivalents thereof.

<About PCR equipment>
The above-described PCR device 50 changes the posture of the cartridge 1 by a predetermined number of cycles as the thermal cycle process, and the PCR container 30 is turned upside down. However, the number of times of changing the posture of the cartridge 1 is not limited to a plurality of times, and may be one.

In the PCR apparatus described above, the rotating shaft of the rotating body is positioned closer to the tube than the PCR container. However, when the posture of the cartridge is changed by rotating the rotating body, the droplet can move inside the PCR container. In this case, the position of the rotating shaft of the rotating body is not limited to this.

In the above-described PCR apparatus, the fixing portion 63 having a notch and the insertion hole 64A are used as the cartridge mounting portion. However, the configuration is not limited as long as the cartridge can be mounted on the rotating body. Further, the mounting portion may have a structure in which the cartridge is fixed to the rotating body by fixing only the tube side, or a structure in which the cartridge is fixed to the rotating body by fixing only the PCR container side. good. However, the mounting portion needs to have a structure in which the cartridge is stably fixed to the rotating body even when the rotating body rotates and the posture of the cartridge changes.

The PCR device 50 includes a high temperature side heater 65B and a low temperature side heater 65C as PCR heaters. However, the PCR device 50 is not limited to this configuration as long as a temperature gradient can be formed inside the PCR container, which is a nucleic acid amplification reaction container. It is not something that can be done. For example, a heater may be provided only on the high temperature side. Alternatively, a heater may be provided on the high temperature side and a cooler may be provided on the low temperature side.

Further, the PCR device 50 described above is provided with a PCR heater (a high temperature side heater 65B and a low temperature side heater 65C) on the rotating body, but can form a temperature gradient inside the PCR container which is a nucleic acid amplification reaction container. If present, a PCR heater may be provided outside the rotating body. For example, when the PCR device is in a position where the rotating body 61 is at the reference position as shown in FIG.
A first PCR heater facing the R container and a second PCR heater facing the PCR container when the rotating body is rotated 180 degrees as shown in FIG. 15B are provided outside the rotating body. May be. Even in this case, a temperature gradient can be formed inside the PCR container which is a nucleic acid amplification reaction container. However, if the PCR heater is provided on the rotating body, the positional relationship between the PCR container of the cartridge and the heater can be maintained regardless of the rotational position of the rotating body.
desirable.

The PCR device 50 may include only the PCR heater (for example, the high temperature side heater 65B and the low temperature side heater 65C) without including the elution heater 65A. However, it is desirable that the PCR device 50 includes the elution heater 65A because release of nucleic acids from the magnetic beads is promoted.

The PCR device 50 may not include a magnet moving mechanism that moves the magnet along the tube. In this case, for example, the operator may have a magnet and move the magnet along the tube. However, it is desirable that the PCR device 50 includes a magnet moving mechanism because there is a possibility that the moving speed or the like of the magnetic beads, which are nucleic acid-binding solid phase carriers, varies depending on the operator.

Further, the magnet moving mechanism 70 of the PCR device 50 may not include the swing mechanism 75. In this case, although the magnet cannot be swung, the magnet can be moved along the tube, so that the magnetic beads to which the nucleic acid is bound can be moved to the plug containing the eluate.

The PCR device 50 may not include the pressing mechanism 80. In this case, for example, the operator may push the plunger of the cartridge by hand. When the plunger is not provided in the cartridge, the operator may pressurize the inside of the tank by deforming the tank and push the liquid from the tube to the PCR container.

<About the eluent plug 47A>
The eluate plug 47A may be divided into a reverse transcription reaction solution plug and an eluate plug. in this case,
The nucleic acid-binding solid phase carrier washed with the washing solution is moved to the reverse transcription reaction solution plug, and the reverse transcription reaction is carried out in the reverse transcription reaction solution plug. This reaction can be performed under conditions suitable for the reverse transcriptase used. For example, the reverse transcription reaction solution is heated to 30 to 50 ° C., preferably 42 to 45 ° C., and the nucleic acid-binding solid phase carrier is held therein for a certain period of time, so that the RNA remains bound to the carrier and is reversed. It can cause a photoreaction. The heating method is not particularly limited. For example, a method of contacting a heat medium such as a heat block at a position corresponding to the reverse transcription reaction solution plug of the tube, a method using a heat source such as a heater, a method using electromagnetic heating, etc. Can be illustrated. Although the practitioner can select the holding time as appropriate, it may be held for 10 seconds to 5 minutes, preferably 30 seconds to 1 minute. The cDNA synthesized at this stage is bound to the solid support in a state of being bound to RNA.
Thereafter, the nucleic acid-binding solid phase carrier is moved to the eluent plug. Here, it is preferable to heat the eluate plug in order to efficiently release nucleic acid, particularly cDNA, from the nucleic acid-binding solid phase carrier. The heating method is not particularly limited, but the same method as that used for heating the reverse transcription reaction solution plug can be used. The heating temperature should just be higher than 40 degreeC, 50 degreeC or more is preferable and 60 degreeC or more is more preferable. Although the upper limit of heating temperature is not specifically limited, 70 degreeC
The temperature is preferably 65 ° C. or lower, more preferably 60 ° C. or lower, still more preferably 60 ° C. or lower, and most preferably 60 ° C. or lower.
After releasing the nucleic acid from the nucleic acid-binding solid phase carrier, the nucleic acid-binding solid phase carrier is moved upward from the eluate plug using a magnet. If the nucleic acid-binding solid phase carrier is not mixed in the eluate plug, it may be moved anywhere.
Thus, by separating the reverse transcription reaction solution plug and the eluate plug, it is possible to perform the reverse transcription reaction and the nucleic acid elution under highly efficient conditions.

[Experimental example]
In this experimental example, as shown in FIG.
The configuration having the first plug 210 to the seventh plug 270 is used.
First, 375 μL of the adsorbing liquid and 1 μL of the magnetic bead dispersion were stored in a polyethylene container 130 having a capacity of 3 mL. The adsorbed liquid is 76% by mass of guanidine hydrochloride, 1.7% by mass.
Of ethylenediaminetetraacetic acid disodium dihydrogen dihydrate and 10% by mass of polyoxyethylene sorbitan monolaurate (manufactured by Toyobo, MagExtractor-Ge
name-, NPK-1). As the magnetic bead stock solution, a solution containing 50% by volume of magnetic silica particles and 20% by mass of lithium chloride was used.
Put 50 μL of blood collected from a human through the mouth 121 of the container 130 using a pipette,
The container 130 was covered with a lid 122 and shaken by hand for 30 seconds to stir. Thereafter, the lid 12 of the container 130
2 was removed and connected to the tube 200. The tube 200 has plugs 110 at both ends. The plug 110 on the first plug 210 side was removed and the container 130 was connected to the tube 200.
Here, the first plug 210, the third plug 230, the seventh plug 270, and the fifth plug 250.
Was silicon oil. The 1st washing | cleaning liquid of the 2nd plug 220 was made into 76 mass% guanidine hydrochloride aqueous solution. The second cleaning solution for the fourth plug 240 was a Tris-HCl buffer solution (solute concentration 5 mM) having a pH of 8.0. The eluate of the sixth plug 260 was sterilized water.
Then, the permanent magnet 410 is moved to move the magnetic beads 125 in the container 130 to the tube 20.
Introduced within 0. Then, the magnetic beads 125 were moved to the sixth plug 260. The time that the magnetic beads 125 existed in each plug in the tube 200 was approximately as follows.
1st, 3rd and 7th plugs: 3 seconds each, 2nd plug: 20 seconds, 4th plug: 20 seconds, 6th plug:
30 seconds. In the second plug 220 and the fourth plug 240, an operation such as vibrating the magnetic beads was not performed. The volumes of the second plug 220, the fourth plug 240, and the sixth plug 260 were 25 μL, 25 μL, and 1 μL, respectively.
Next, the stopper 110 on the seventh plug side of the tube was removed, the container 120 was deformed by hand, and the seventh plug 270 and the sixth plug 260 were discharged into the PCR reaction container. This operation was performed after the magnetic beads were moved by a permanent magnet and retracted to the second plug 220.
Then, 19 μL of a PCR reaction reagent was added to the extract, and real-time PCR was performed according to a conventional method. The breakdown of PCR reaction reagents is SYBR Green I (Life Technologies, Inc., S7563), 4 μL of Light Cycler 480 Genotyping Master (Roche Diagnostics 4 707 524), diluted 1000-fold with sterile water.
0.4 μL, 100 μM β-actin detection primer (F / R) each 0.06 μL, and sterile water 14.48 μL. The PCR amplification curve of Experimental Example 1 is shown in FIG. In addition, the vertical axis | shaft of FIG. 20 is fluorescence intensity, and a horizontal axis is the cycle number of PCR.

[Experiment 2]
In Experimental Example 2, nucleic acids were extracted by a general nucleic acid extraction method.
First, in a polyethylene container (Eppendorf tube) with a capacity of 1.5 mL, 375 μm
L adsorbent and 20 μL of magnetic bead dispersion were housed. The compositions of the adsorbing liquid and the magnetic bead dispersion are the same as in the above experimental example.
Next, 50 μL of blood collected from a human being is introduced from the mouth of the container using a pipette, the container is covered, stirred with a vortex mixer for 10 minutes, and a magnetic stand and pipette are operated to perform a B / F separation operation. It was. In this state, magnetic beads and a small amount of adsorbed liquid remained in the container.
Next, 450 μL of the first cleaning solution having the same composition as in Experimental Example 1 was introduced into the container, the cap was put on, and the mixture was stirred for 5 seconds by a vortex mixer, and the first cleaning solution was removed by operating the magnetic stand and pipette. This operation was repeated twice. In this state, the magnetic beads and a small amount of the first cleaning liquid remained in the container.
Next, 450 μL of the second cleaning solution having the same composition as that of Experimental Example 1 was introduced into the container, covered, stirred for 5 seconds by a vortex mixer, and the magnetic cleaning stand and pipette were operated to remove the first cleaning solution. This operation was repeated twice. In this state, the magnetic beads and a small amount of the second cleaning liquid remained in the container.
Then, 50 μL of sterilized water (eluate) was added to the container, the container was covered, and the mixture was stirred with a vortex mixer for 10 minutes, and the supernatant was recovered by operating a magnetic stand and pipette. This supernatant contains the target nucleic acid.
Then, 1 μL was dispensed from the extract, 19 μL of a PCR reaction reagent was further added, and real-time PCR was performed according to a conventional method. The breakdown of PCR reaction reagents is Light Cycler 480 Genotyping Master (Roche Diagnostics 4 707 5
24) SYBR Green I (Life Technologies, Inc., S7563) diluted 1000-fold with 4 μL of sterilized water 0.4 μL, 100 μM β-actin detection primer (F / R)
) 0.06 μL each and 14.48 μL sterilized water. The amplification curve at this time is shown in FIG.

[Experimental result]
The following can be understood from the above experimental example.

(1) Comparing the time required for the nucleic acid extraction process, which is a pretreatment of PCR, the time from the insertion of the specimen into the container until the introduction of the target nucleic acid into the PCR reaction container is approximately 2 minutes. In Experimental Example 2, it was approximately 30 minutes. Thus, the nucleic acid extraction method of Experimental Example 1 requires significantly less time for nucleic acid extraction than the nucleic acid extraction method of Experimental Example 2.

(2) The amount of each cleaning liquid was about 18 times smaller than that of Experimental Example 2 in Experimental Example 1. Further, the amount of the eluate was about 1/50 of that of Experimental Example 1 in Experimental Example 1. Therefore, in Experimental Example 1, a very small amount of the cleaning solution and the eluent is sufficient as compared with Experimental Example 2.

(3) Comparing the concentration of the target nucleic acid in the eluate in terms of the amount of the adsorbed solution and the eluate, it is considered that Experimental Example 1 is ideally 50 times more concentrated than Experimental Example 2. However, in this experimental example, the amount of nucleic acid contained in the blood sample is large and exceeds the adsorbable amount of 1 μL of magnetic beads, and the whole amount of nucleic acid contained in the blood sample cannot be recovered. The concentration 50 times that of Experimental Example 2 was not obtained. In the case of a specimen having a low nucleic acid content and not exceeding the adsorbable amount of 1 μL of magnetic beads, Experimental Example 1 can obtain a concentration 50 times that of Experimental Example 2.

(4) Referring to the graph of FIG. 20, even in a whole blood sample having a high nucleic acid content, the rise in nucleic acid amplification rate is about 0.6 cycles earlier in Experimental Example 1 than in Experimental Example 2. That is, the concentration of the target nucleic acid in the PCR reaction solution used in Experimental Example 1 is higher than that in the PCR reaction solution used in Experimental Example 2. That is, the concentration of the target nucleic acid in the eluate is higher in Experimental Example 1 than in Experimental Example 2.

1 cartridge,
3 tank, 3A lid, 3B deformation part,
5 Adapter, 5A lid, 7 Magnetic beads,
9 Cartridge body
10 Plunger, 11 Cylindrical part, 11A Mounting base,
12 rod-shaped part, 12A seal, 13 rib,
20 tube, 21 upper syringe, 22 lower syringe,
23 capillary, 25 fixed claw, 26 guide plate,
30 PCR container, 31 seal forming part,
32 oil receiving part, 32A oil receiving space,
33 Stepped portion, 34A Upper seal portion, 34B Lower seal portion,
35 channel formation part, 35A bottom,
36A high temperature region, 36B low temperature region,
41 solution, 42 oil, 43 cleaning solution,
44 first oil plug, 45 cleaning liquid plug, 46 second oil plug,
47 reaction solution (PCR solution),
47A lysis solution, 47B oil plug, 47C nucleic acid amplification reaction solution,
48 3rd oil plug,
50 PCR device, 51 base, 52 support base, 53 side wall,
53A cartridge insertion port, 55 fluorescence measuring instrument,
60 rotating mechanism, 61 rotating body,
62 mounting part, 63 fixing part,
63A guide rail, 64A insertion hole,
65 heater, 65A elution heater,
65B high temperature side heater, 65C low temperature side heater,
65D spacer, 66 motor for rotation,
70 magnet moving mechanism, 71 magnet, 72 arm,
73 Lifting mechanism, 73A carriage,
73B lifting / lowering motor, 73C carriage guide,
73D belt, 73E pulley,
75 swing mechanism, 75A swing motor, 75B swing rotating shaft,
80 pressing mechanism, 81 motor for plunger, 82 rod,
90 controller

Claims (6)

A tube having a plug containing an eluate that elutes the nucleic acid from a nucleic acid-binding solid phase carrier to which the nucleic acid is bound; and a nucleic acid amplification reaction vessel containing oil that communicates with the tube and phase-separates from the eluate. A rotating body having a mounting portion to which a cartridge can be mounted;
A heater that forms a temperature gradient inside the nucleic acid amplification reaction vessel;
A nucleic acid amplification apparatus comprising: a fluorescence measuring device that detects fluorescence emitted from a fluorescent substance contained in a reaction solution in the nucleic acid amplification reaction vessel and serves as an index of a nucleic acid amplification reaction;
A control unit that controls the fluorescence measuring device, wherein the control unit causes the fluorescence measuring device to measure the three fluorescences having different peak wavelengths from the one having the shorter peak wavelength, the first fluorescence, When the second fluorescence and the third fluorescence are used, the time for measuring the first fluorescence and the third fluorescence
A nucleic acid detection control device, characterized in that at least part of the time for measuring the fluorescence of
A nucleic acid amplification system comprising: The nucleic acid amplification system according to claim 1,
The nucleic acid amplification system, wherein the controller causes the fluorescence measuring instrument to measure the first fluorescence and the third fluorescence, and then measures the second fluorescence. A tube having a plug containing an eluate that elutes the nucleic acid from a nucleic acid-binding solid phase carrier to which the nucleic acid is bound; and a nucleic acid amplification reaction vessel containing oil that communicates with the tube and phase-separates from the eluate. A rotating body having a mounting portion to which a cartridge can be mounted;
A heater that forms a temperature gradient inside the nucleic acid amplification reaction vessel;
In a nucleic acid amplification apparatus comprising: a fluorescence measuring device that detects fluorescence emitted from a fluorescent substance contained in a reaction solution in the nucleic acid amplification reaction vessel and serves as an index of a nucleic acid amplification reaction, the reaction solution in the nucleic acid amplification reaction vessel A nucleic acid detection method comprising a step of detecting three fluorescences emitted from three fluorescent substances having different peak wavelengths and serving as an index of a nucleic acid amplification reaction,
When the three fluorescences are the first fluorescence, the second fluorescence, and the third fluorescence from the shorter peak wavelength, the time for measuring the first fluorescence and the time for measuring the third fluorescence A method for detecting a nucleic acid, comprising overlapping at least a part. In the nucleic acid detection method according to claim 3,
A method for detecting a nucleic acid, comprising measuring the first fluorescence and the third fluorescence, and then measuring the second fluorescence. A tube having a plug containing an eluate that elutes the nucleic acid from a nucleic acid-binding solid phase carrier to which the nucleic acid is bound; and a nucleic acid amplification reaction vessel containing oil that communicates with the tube and phase-separates from the eluate. A mounting section on which the cartridge can be mounted;
A fluorescence measuring instrument that detects fluorescence emitted from a fluorescent substance contained in a reaction solution in the nucleic acid amplification reaction vessel and serves as an indicator of a nucleic acid amplification reaction;
A control unit for controlling the fluorescence measuring instrument,
When the control unit causes the fluorescence measuring instrument to measure three fluorescences having different peak wavelengths, the control unit
When the first fluorescence, the second fluorescence, and the third fluorescence from the shortest peak wavelength,
A nucleic acid amplification system, wherein at least a part of a time for measuring the first fluorescence and a time for measuring the third fluorescence are overlapped. The nucleic acid amplification system according to claim 5,
The nucleic acid amplification system, wherein the controller causes the fluorescence measuring instrument to measure the first fluorescence and the third fluorescence, and then measures the second fluorescence.