JP2017163918A - Nucleic acid amplification reaction cartridge, nucleic acid amplification apparatus, and nucleic acid amplification method - Google Patents

Abstract

The present invention provides a cartridge for nucleic acid amplification reaction that can perform PCR efficiently even if the speed of PCR is increased. A reaction liquid and a liquid into which the reaction liquid is different in specific gravity and immiscible with the reaction liquid are introduced, and the container has a flow path through which the reaction liquid moves, The reaction solution includes a modified fluorescent probe, and the ratio (L / D) between the diameter L of the droplet of the reaction solution and the inner width D of the container that is the width of the flow path is 0.28 or more and 0. A cartridge for nucleic acid amplification reaction, which is 87 or less. [Selection] Figure 1

Description

  The present invention relates to a cartridge for nucleic acid amplification reaction, a nucleic acid amplification apparatus, and a nucleic acid amplification method.

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention due to the development of gene utilization technology, and many methods using genes for variety discrimination and variety improvement have been developed in the field of agriculture and livestock. . As a technique for utilizing a gene, a technique such as a PCR (Polymerase Chain Reaction) method is widely used. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a method of amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. The thermal cycle is a process in which two or more stages of temperature are periodically applied to the reaction solution. In the PCR method, a method of applying a two-stage or three-stage thermal cycle is common.

  For example, in Patent Document 1, a reaction vessel filled with reaction droplets and a liquid (oil or the like) that is immiscible with the reaction droplets and has a specific gravity smaller than that of the reaction solution is rotated around the rotation axis, and the specific gravity is increased. A nucleic acid amplification apparatus is described in which a reaction solution is moved by the difference to perform thermal cycling.

JP 2012-115208 A

  In the nucleic acid amplification apparatus as described above, there is a demand for shortening the product generation time by PCR. However, in the technique described in Patent Document 1, when the PCR reaction time (denaturation time or annealing / extension time) is shortened (when PCR is speeded up), the reaction solution can be brought to a desired temperature. In some cases, the nucleic acid amplification efficiency may be reduced.

  One of the objects according to some aspects of the present invention is to provide a cartridge for nucleic acid amplification reaction that can perform PCR efficiently even if the speed of PCR is increased. Another object of some aspects of the present invention is to provide a nucleic acid amplification apparatus that can perform PCR efficiently even if the PCR speed is increased. Another object of some aspects of the present invention is to provide a nucleic acid amplification method capable of performing PCR efficiently even if the PCR speed is increased.

The cartridge for nucleic acid amplification reaction according to the present invention comprises:
A reaction liquid and a liquid into which the reaction liquid is different in specific gravity and immiscible with the reaction liquid,
The container has a flow path through which the reaction solution moves,
The reaction solution includes a modified fluorescent probe,
The ratio (L / D) between the diameter L of the droplets of the reaction liquid and the inner width D of the container, which is the width of the flow path, is 0.28 or more and 0.87 or less.

  With such a cartridge for nucleic acid amplification reaction, it is possible to perform PCR efficiently even if the PCR speed is increased (for details, see “3. Experimental Example” described later)).

In the cartridge for nucleic acid amplification reaction according to the present invention,
The volume of the reaction solution may be 0.1 μL or more and 2.0 μL or less.

  Such a cartridge for nucleic acid amplification reaction can perform PCR efficiently even if the speed of PCR is increased.

In the cartridge for nucleic acid amplification reaction according to the present invention,
A reagent used for nucleic acid amplification may be lyophilized in the container.

  In such a cartridge for nucleic acid amplification reaction, a reagent used for nucleic acid amplification can be stored in a lyophilized state.

In the cartridge for nucleic acid amplification reaction according to the present invention,
The modified fluorescent probe may include an artificial nucleic acid.

  In such a cartridge for nucleic acid amplification reaction, the fluorescence intensity of the reaction solution can be increased.

In the cartridge for nucleic acid amplification reaction according to the present invention,
The artificial nucleic acid may be LNA, 3′-amino-2 ′, 4′-BNA, 2 ′, 4′-BNA ^COC , 2 ′, 4′-BNA ^NC , PNA, GNA, or TNA.

  In such a cartridge for nucleic acid amplification reaction, the fluorescence intensity of the reaction solution can be increased.

In the cartridge for nucleic acid amplification reaction according to the present invention,
The artificial nucleic acid may be LNA.

  In such a cartridge for nucleic acid amplification reaction, the fluorescence intensity of the reaction solution can be increased.

In the cartridge for nucleic acid amplification reaction according to the present invention,
The modified fluorescent probe may include a minor group binder molecule.

  In such a cartridge for nucleic acid amplification reaction, the fluorescence intensity of the reaction solution can be increased.

The nucleic acid amplification device according to the present invention comprises:
A mounting portion to which the cartridge for nucleic acid amplification reaction according to the present invention can be mounted;
A temperature gradient forming part for forming a temperature gradient in the flow path of the cartridge for nucleic acid amplification reaction;
including.

  Such a nucleic acid amplification apparatus can perform PCR efficiently even if the PCR speed is increased.

The nucleic acid amplification method according to the present invention comprises:
Forming a temperature gradient in the flow path of the container;
A step of moving a reaction liquid containing nucleic acid in the flow path to give a temperature cycle to the reaction liquid;
Including
The reaction solution includes a modified fluorescent probe,
The ratio (L / D) between the diameter L of the droplets of the reaction solution and the inner width D of the container serving as the flow path is 0.28 or more and 0.87 or less.

  In such a nucleic acid amplification method, it is possible to perform PCR efficiently even if PCR is accelerated.

1 is a cross-sectional view schematically showing a cartridge for nucleic acid amplification reaction according to the present embodiment. 1 is a cross-sectional view schematically showing a cartridge for nucleic acid amplification reaction according to the present embodiment. 1 is a cross-sectional view schematically showing a cartridge for nucleic acid amplification reaction according to the present embodiment. The figure for demonstrating the modification fluorescence probe containing a minor group binder molecule | numerator. The perspective view which shows typically the nucleic acid amplifier which concerns on this embodiment. The perspective view which shows typically the nucleic acid amplifier which concerns on this embodiment. 1 is an exploded perspective view schematically showing a nucleic acid amplification device according to the present embodiment. 1 is a cross-sectional view schematically showing a nucleic acid amplification device according to an embodiment. 1 is a cross-sectional view schematically showing a nucleic acid amplification device according to an embodiment. The graph which shows the PCR result at the time of using the modified fluorescent probe containing artificial nucleic acid. The graph which shows the PCR result at the time of using the modified fluorescent probe containing a minor group binder molecule. The graph which shows the PCR result at the time of using a TaqMan (trademark) fluorescent probe.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Nucleic Acid Amplification Reaction Cartridge First, the nucleic acid amplification reaction cartridge according to this embodiment will be described with reference to the drawings. FIG. 1 is a cross-sectional view schematically showing a nucleic acid amplification reaction cartridge 10 according to this embodiment.

  The nucleic acid amplification reaction cartridge 10 includes a container 12 and a cap 14 as shown in FIG.

  As shown in FIG. 1, the container 12 has, for example, a cylindrical side wall portion 12a and a hemispherical bottom portion 12b. The cap 14 closes the opening at the end facing the bottom 12 b of the container 12. The cap 14 can be attached to and detached from the container 12. The material of the container 12 and the cap 14 is, for example, glass, polymer, metal or the like.

  In the cartridge 10 for nucleic acid amplification reaction, a reaction solution 20 and a liquid 30 immiscible with the reaction solution 20 are introduced. The nucleic acid amplification reaction cartridge 10 has a flow path 16 through which the reaction solution 20 moves. The flow path 16 is formed by the container 12. The channel 16 extends along the central axis (not shown) of the cylindrical side wall portion 12a. Hereinafter, the reaction solution 20 and the liquid 30 accommodated in the cartridge 10 for nucleic acid amplification reaction will be described.

1.1. Reaction solution The reaction solution 20 is disposed in the flow path 16. The reaction liquid 20 is held in the liquid 30 in the form of droplets. In the illustrated example, the shape of the reaction solution 20 is spherical. The reaction liquid 20 has a higher specific gravity than the liquid 30. The reaction solution 20 moves relative to the container 12 through the flow path 16 as the nucleic acid amplification reaction cartridge 10 moves.

  The ratio L (D / D) of the diameter L of the droplet of the reaction liquid 20 and the inner width D that is the width of the flow path 16 is 0.28 or more and 0.87 or less, preferably 0.49 or more and 0. .87 or less, more preferably 0.70 or more and 0.79 or less. By setting the ratio (L / D) within the above range, PCR can be performed efficiently (for details, see “3. Experimental Examples” described later). The inner wall of the side wall portion 12a preferably has water repellency to such an extent that the reaction solution 20 does not adhere.

  The inner width D is the diameter (inner diameter) of the cross section when the cross section of the container 12 in a direction orthogonal to the extending direction of the flow path 16 is circular, and the cross section is elliptic when the cross section is elliptical. It is the length of the short axis of the cross section, and when the cross section is a polygon, it is the minimum length of the diagonal lines of the cross section.

  The volume of the reaction solution 20 is, for example, 0.1 μL or more and 2.0 μL or less, preferably 0.5 μL or more and 2.0 μL or less, and more preferably 1.5 μL or more and 2.0 μL or less. By setting the volume of the reaction solution 20 within the above range, PCR can be performed efficiently (for details, refer to “3. Experimental Example” described later).

  The reaction solution 20 is formed, for example, when the template nucleic acid solution 22 comes into contact with the reagent 24. Specifically, as shown in FIG. 2, a freeze-dried reagent 24 is fixed to the bottom 12 b of the container 12. When the template nucleic acid solution 22 is introduced into the nucleic acid amplification reaction cartridge 10 using, for example, the pipette 2, the introduced template nucleic acid solution 22 settles down to the bottom 12b as shown in FIG. The reagent 24 is lyophilized by the moisture of the template nucleic acid solution 22 and taken into the template nucleic acid solution 22 to become a reaction solution 20 as shown in FIG. Therefore, the reaction solution 20 contains the components of the template nucleic acid and the reagent 24, and serves as a place for advancing the nucleic acid amplification reaction.

  The template nucleic acid solution 22 is a solution containing a template nucleic acid. When the template nucleic acid solution 22 is introduced into the container 12, the cap 14 is removed from the container 12 and attached to the container 12 again after the template nucleic acid solution 22 is introduced.

  The template nucleic acid solution 22 is obtained as follows, for example. That is, specimens such as cells or viruses derived from organisms such as humans and bacteria are collected using a collection tool such as a cotton swab, and a template nucleic acid is extracted from the specimen using a known extraction technique. Thereafter, the template nucleic acid solution is purified to a predetermined concentration using a known purification method. In addition, the solution in the template nucleic acid solution 22 is, for example, water (distilled water, sterilized water) or a Tris-EDTA (ethylenediamine acetic acid) solution (TE).

  The reagent 24 is lyophilized on the bottom 12 b of the container 12. The reagent 24 is a reagent used for nucleic acid amplification reaction. Reagent 24 includes a primer, a polymerase, dNTPs, and a modified fluorescent probe.

  The primer is designed to anneal to the template nucleic acid. The reagent 24 includes a forward primer (Forward Primer) that anneals to one template nucleic acid (single-stranded DNA) having a single-stranded structure after the template nucleic acid (double-stranded DNA) having a double-stranded structure is denatured, and the other. Reverse primer that anneals to single-stranded DNA. The concentration of the primer contained in the reaction solution 20 is, for example, 0.4 μM or more and 3.2 μM or less.

  Although it does not specifically limit as a polymerase, DNA (Deoxyribonucleic Acid) polymerase is mentioned. The DNA polymerase polymerizes a nucleotide complementary to the base of the template nucleic acid at the end of the primer annealed to the template nucleic acid having a single-stranded structure (single-stranded DNA). As the DNA polymerase, a heat-resistant enzyme or a PCR enzyme is preferable. For example, there are a large number of commercially available products such as Taq polymerase, Tfi polymerase, Tth polymerase, or improved versions thereof. Is preferred. The amount of DNA polymerase contained in the reaction solution 20 is, for example, 0.5 U or more and 4 U or less.

  dNTP represents a mixture of four types of deoxyribonucleotide triphosphates. That is, dNTP is dATP (Deoxyadenosine triphosphate), dCTP (Deoxycydine triphosphate), dGTP (Deoxyguanosine triphosphate), and dTTP (Thymidine triphosphate). The DNA polymerase joins dATP, dCTP, dGTP, and dTTP to the ends of the annealed primers to form new DNA. The concentration of dNTP contained in the reaction solution 20 is, for example, 0.125 mM or more and 1.0 mM or less.

  The modified fluorescent probe is a fluorescently labeled probe (fluorescent probe) used for quantifying the amount of nucleic acid amplified, and is a fluorescently labeled probe containing an artificial nucleic acid or a minor group binder molecule. The concentration of the modified fluorescent probe contained in the reaction solution 20 is, for example, 0.15 μM or more and 1.2 μM or less.

  The artificial nucleic acid contained in the modified fluorescent probe refers to a nucleic acid molecule other than a natural nucleic acid molecule that can bind to a base of DNA or RNA by hydrogen bonding. The type of artificial nucleic acid contained in the modified fluorescent probe is not particularly limited, but, for example, an artificial nucleic acid that increases the Tm value between the probe and the complementary strand and increases the binding force with the template nucleic acid as compared with a probe consisting only of a natural nucleic acid. Nucleic acids are preferred. Specifically, an artificial nucleic acid is 2 ′, 4′-BNA (2′-O, 4′-C) in which a 2′-position oxygen atom and a 4′-position carbon atom of a nucleic acid ribose ring are methylene-bridged. -Methano-bridged nucleic acid (alias LNA (Locked Nucleic Acid)) is preferable. The chemical formula of LNA is shown in the following formula (1).

  In Formula (1), Base (base) includes T (thymine), C (cytosine), G (guanine), A (adenine), and the like, but is not particularly limited. The base may be a base modified by methylation or acetylation.

  The Tm value is a temperature at which 50% of the double-stranded DNA is dissociated into single-stranded DNA, that is, a melting temperature. The method for calculating the Tm value is not particularly limited, and for example, the GC% method, the closest base pair method, the Wallace method, or the like is used.

The artificial nucleic acid contained in the modified fluorescent probe may be an LNA analog modified with LNA, specifically, 3′amino-2 ′, 4′-BNA, 2 ′, 4′-BNA ^COC , or 2 It may be ', 4'-BNA ^NC . The artificial nucleic acid contained in the modified fluorescent probe may be PNA (Peptide Nucleic Acid), GNA (Glycol Nucleic Acid), TNA (Threose Nucleic Acid), or an analog obtained by modifying these.

  The number of artificial nucleic acids contained in the modified fluorescent probe is not particularly limited, and one modified fluorescent probe may contain a plurality of artificial nucleic acids.

  FIG. 4 is a diagram for explaining a modified fluorescent probe containing a minor group binder (MGB) molecule. As shown in FIG. 4, the modified fluorescent probe containing the MGB molecule includes a probe P1, a reporter P2 added to the 5 ′ end side of the probe P1, and a quencher P3 added to the 3 ′ end side of the probe P1. , And an MGB molecule P4 added to the quencher P3.

  By using a modified fluorescent probe containing an artificial nucleic acid or MGB molecule as described above, the rise of nucleic acid amplification can be detected with higher sensitivity and nucleic acid amplification for the same number of cycles compared to the case of using a general hydrolyzed probe. The reaction product can be detected with high sensitivity.

1.2. Liquid The liquid 30 is arranged in the flow path 16 as shown in FIG. In the illustrated example, the flow path 16 is filled (filled) with the reaction liquid 20 and the liquid 30. The liquid 30 is a liquid that is not miscible with the reaction liquid 20, that is, does not mix. The liquid 30 has a specific gravity different from that of the reaction liquid 20. Specifically, the liquid 30 has a specific gravity smaller than that of the reaction liquid 20. Therefore, the reaction liquid 20 moves in the direction in which gravity acts by the action of gravity. The liquid 30 is, for example, dimethyl silicone oil or paraffin oil.

  In the above, the freeze-dried reagent 24 is fixed to the bottom 12 b of the container 12, the template nucleic acid solution 22 is introduced into the container 12, and the template nucleic acid solution 22 comes into contact with the reagent 24 to thereby react the reaction solution 20. An example in which is formed has been described. However, as the reaction solution 20, a solution containing the template nucleic acid and the reagent 24 used for amplification of the target nucleic acid in the template nucleic acid is prepared outside the container 12, and the solution is filled in the container 12 filled with the liquid 30. The reaction solution 20 may be disposed in the container 12 by being introduced into the container 12.

2. Nucleic acid amplification apparatus 2.1. Configuration Next, the nucleic acid amplification apparatus according to the present embodiment will be described with reference to the drawings. 5 and 6 are perspective views schematically showing the nucleic acid amplification device 100 according to this embodiment. FIG. 5 shows a state in which the lid 70 is opened, and FIG. 6 shows that the lid 70 is closed. Indicates the state. FIG. 7 is an exploded perspective view schematically showing the main body 50 of the nucleic acid amplification device 100 according to the present embodiment. 8 and 9 are cross-sectional views of the AA ship of FIG. 6 schematically showing the nucleic acid amplification device 100 according to the present embodiment. For convenience, FIG. 5 shows a simplified nucleic acid amplification reaction cartridge 10.

  As shown in FIGS. 5 to 9, the nucleic acid amplification device 100 includes a main body 50, a moving mechanism 60, a lid 70, and a fluorescence measuring device 80. For convenience, the fluorescence measuring device 80 is not shown in FIGS. 5 and 6. The nucleic acid amplification device 100 is, for example, a lift PCR device.

  As shown in FIG. 7, the main body 50 includes a mounting part 51, a first heating part 52, a second heating part 53, a spacer 54, a bottom plate 55, a flange 56, and a fixing plate 57. doing.

  The mounting portion 51 has a structure in which the nucleic acid amplification reaction cartridge 10 can be mounted. As shown in FIG. 5, the mounting portion 51 has a slot structure in which the nucleic acid amplification reaction cartridge 10 is inserted (inserted). In the example shown in FIG. 7, the mounting portion 51 is a through hole that penetrates the first heat block 52 b of the first heating portion 52, the spacer 54, and the second heat block 53 b of the second heating portion 53. The number of mounting parts 51 may be plural, and is 20 in the example of the illustrated example.

  The first heating unit 52 heats the first region 16 a of the flow channel 16 to the first temperature when the nucleic acid amplification reaction cartridge 10 is mounted on the mounting unit 51. In the example illustrated in FIG. 7, the first heating unit 52 is located closer to the lid body 70 than the second heating unit 53.

  The first heating unit 52 includes, for example, a mechanism that generates heat, and a member that transmits the generated heat to the cartridge 10 for nucleic acid amplification reaction. In the illustrated example, the first heating unit 52 includes a first heater 52a and a first heat block 52b. The first heater 52 a is, for example, a cartridge heater, and is connected to an external power source (not shown) by a conducting wire 58. The 1st heater 52a is inserted in the hole provided in the 1st heat block 52b, and the 1st heat block 52b is heated when the 1st heater 52a generates heat. The first heat block 52b is a member that transfers heat generated from the first heater 52a to the cartridge 10 for nucleic acid amplification reaction. The first heat block 52b is, for example, an aluminum block. The first region 16a of the flow path 16 is a region surrounded by the first heat block 52b.

  The second heating unit 53 heats the second region 16b of the flow channel 16 to a second temperature different from the first temperature when the nucleic acid amplification reaction cartridge 10 is mounted to the mounting unit 51. The second heating unit 53 includes a second heater 53a and a second heat block 53b. The second heating unit 53 has the same structure and function as the first heating unit 52 except that the region of the nucleic acid amplification reaction cartridge 10 to be heated and the heating temperature are different from those of the first heating unit 52. The second region 16b of the flow path 16 is a region surrounded by the second heat block 53b.

  The temperature of the 1st heating part 52 and the 2nd heating part 53 is controlled by the temperature sensor which is not shown in figure and the control part mentioned later. For example, by controlling the first heating unit 52 to the first temperature and the second heating unit 53 to the second temperature, the first region 16a of the nucleic acid amplification reaction cartridge 10 is set to the first temperature, and the second region 16b is set to the second temperature. It can be heated to a second temperature. The temperature sensor in this embodiment is, for example, a thermocouple.

  The spacer 54 is provided between the first heating unit 52 and the second heating unit 53. The spacer 54 has a function of insulating between the first heating unit 52 and the second heating unit 53.

  The bottom plate 55 is a member that holds the nucleic acid amplification reaction cartridge 10. The bottom plate 55 determines the position of the cartridge 10 for nucleic acid amplification reaction in the height direction. That is, the nucleic acid amplification reaction cartridge 10 can be held at a predetermined position with respect to the heating units 52 and 53 by inserting the nucleic acid amplification reaction cartridge 10 to a position where it contacts the bottom plate 55. In the example shown in FIG. 7, the bottom plate 55 is provided with a through hole 55 a through which excitation light from the fluorescence measuring device 80 and fluorescence of the reaction liquid 20 pass.

  The flange 56 and the fixing plate 57 are members for fixing the heating units 52 and 53 and the spacer 54. In the illustrated example, two fixing plates 57 are fitted to the flange 56, and the heating units 52 and 53, the spacer 54, and the bottom plate 55 are fixed to the fixing plate 57.

  As shown in FIGS. 5 and 6, the moving mechanism 60 is a mechanism that rotates the main body 50. The moving mechanism 60 has, for example, a motor and a drive shaft (not shown). The drive shaft is connected to the flange 56 of the main body 50. The drive shaft is provided perpendicular to the longitudinal direction of the mounting portion 51, and when the motor is operated, the main body portion 50 rotates with the drive shaft as a rotation axis.

  The lid 70 covers the mounting portion 51. In the example shown in FIG. 5, the fixing plate 57 is provided with a magnet portion 72, and the lid body 70 can be fixed to the main body portion 50 by the magnet portion 72. In addition, the material of the spacer 54, the bottom plate 55, the flange 56, the fixing plate 57, and the lid 70 is, for example, a heat insulating material.

  The fluorescence measuring instrument 80 is a measuring instrument that measures the fluorescence intensity (fluorescence luminance) of the reaction solution 20 accommodated in the nucleic acid amplification reaction cartridge 10. As shown in FIGS. 8 and 9, the fluorescence measuring device 80 is disposed to face the bottom 12 b of the nucleic acid amplification reaction cartridge 10 with a predetermined distance therebetween. The fluorescence measuring instrument 80 irradiates the reaction liquid 20 with excitation light corresponding to the fluorescent dye of the modified fluorescent probe contained in the reaction liquid 20 based on an input signal from a control unit (not shown). The fluorescence intensity emitted is measured. The fluorescence measuring instrument 80 may measure the fluorescence intensity corresponding to one fluorescent dye, or may measure the fluorescence intensity corresponding to a plurality of fluorescent dyes.

  The nucleic acid amplification device 100 includes a control unit (not shown). The control unit controls the moving mechanism 60 and the fluorescence measuring device 80. The control unit includes a processor such as a CPU (Central Processing Unit) and a storage device such as a ROM (Read Only Memory) and a RAM (Random Access Memory). The storage device stores various programs, data, and the like for the above control. The storage device further has a work area for temporarily storing data being processed and results of various processes.

2.2. Thermal Cycle Processing Next, thermal cycle processing using the nucleic acid amplification device 100 will be described with reference to FIGS. 8 and 9. 8 and 9, the direction of arrow g is the direction in which gravity acts (the direction of gravity action). 8 shows a state in which the arrangement of the heating units 52 and 53 is the first arrangement, and FIG. 9 shows a state in which the arrangement of the heating units 52 and 53 is the second arrangement.

  First, the nucleic acid amplification reaction cartridge 10 is attached to the attachment portion 51 of the nucleic acid amplification apparatus 100. Specifically, after introducing the reaction solution 20 into the flow path 16 filled with the liquid 30, the nucleic acid amplification reaction cartridge 10 is attached to the attachment portion 51. For example, when the nucleic acid amplification reaction cartridge 10 is attached to the attachment part 51, the attachment part 51 is covered with the lid 70.

  Here, the arrangement of the heating units 52 and 53 is the first arrangement. As shown in FIG. 8, in the first arrangement, the first region 16a is located at the lowest part of the flow path 16 in the direction of gravity action. Therefore, the reaction liquid 20 having a higher specific gravity than the liquid 30 is located in the first region 16a.

  Next, when the control unit receives a signal to start the heat cycle process from the operation unit (not shown), the control unit controls the heating units 52 and 53, and the first region 16a of the cartridge 10 for nucleic acid amplification reaction and The second region 16b is heated. Specifically, the first heating unit 52 heats the first region 16a to the first temperature, and the second heating unit 53 heats the second region 16b to a second temperature lower than the first temperature. Thereby, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 16 a and the second region 16 b of the flow path 16. Here, the heating units 52 and 53 are temperature gradient forming units that form a temperature gradient in which the temperature decreases from the first region 16a toward the second region 16b. The first temperature is a temperature suitable for dissociation (denaturation reaction) of double-stranded DNA, and is, for example, 95 ° C. or higher and 100 ° C. or lower. The second temperature is a temperature suitable for the annealing reaction and the extension reaction, and is, for example, 50 ° C. or higher and 75 ° C. or lower.

  Since the arrangement of the heating units 52 and 53 is the first arrangement, when the cartridge 10 for nucleic acid amplification reaction is heated, the reaction solution 20 is heated to the first temperature. When the first heating unit 52 reaches the first temperature and the first time (first period) elapses, the control unit controls the moving mechanism 60 so that the arrangement of the heating units 52 and 53 is changed to the first. Switch from placement to second placement. The first time is a time for the denaturation reaction, and is, for example, 1 second or more and 10 seconds or less.

  As shown in FIG. 9, the second arrangement is an arrangement in which the second region 16b is positioned at the lowest part of the flow path 16 in the direction of gravity action. Specifically, the control unit controls the moving mechanism 60 to rotate the main body 50 by 180 degrees. Thereby, arrangement | positioning of the heating parts 52 and 53 is switched from 1st arrangement | positioning to 2nd arrangement | positioning.

  In the second arrangement, the positional relationship between the first area 16a and the second area 16b in the direction in which gravity acts is opposite to that in the first arrangement. Therefore, the reaction solution 20 moves from the first region 16a to the second region 16b by the action of gravity. The control unit stops the operation of the moving mechanism 60 after the heating units 52 and 53 are arranged in the second arrangement. Thereby, arrangement | positioning of the heating parts 52 and 53 is hold | maintained at 2nd arrangement | positioning. When the second time (second period) elapses in the second arrangement, the controller controls the moving mechanism 60 again to rotate the main body 50 by 180 degrees. The second time is a time for the annealing reaction and the extension reaction, and is, for example, 1 second or more and 10 seconds or less.

  Then, the control unit moves the reaction solution 20 forward through the flow path 16 by rotating the heating units 52 and 53 while switching the positions of the first arrangement and the second arrangement until the number of cycles reaches a predetermined number. (Reciprocating motion) to give the reaction solution 20 a temperature cycle. Thereby, a nucleic acid amplification reaction is performed. When the number of cycles reaches a predetermined number, the control unit ends the thermal cycle process.

  The control unit performs amplification analysis processing at the same time as the thermal cycle processing. Thereby, real-time PCR can be performed. Specifically, the control unit inputs a signal for giving a measurement instruction every second time (time for performing the annealing reaction and the extension reaction) to the fluorescence measuring device 80, and the measurement result of the fluorescence measuring device 80. Is stored in a storage unit (not shown). Further, the control unit reads data indicating the fluorescence intensity for the number of cycles set as the number of cycles to be repeated based on a signal input from the operation unit (not shown) from the storage unit, and cycles based on the data An amplification curve showing the transition of the fluorescence intensity with respect to the number may be generated. Then, when the amplification curve is generated, the control unit determines whether or not the reference amplification efficiency is acceptable based on the amplification curve, and displays either or both of the determination result and the amplification curve as appropriate (not shown). ) May be displayed.

3. Experimental Example An experimental example is shown below to describe the present invention more specifically. The present invention is not limited by the following experimental examples.

3.1. Sample preparation 3.1.1. Example 1
As a template nucleic acid, 25 copies of DNA extracted from a culture of Mycoplasma pneumonia (template DNA, manufactured by Vircell) were used. The template nucleic acid was added to a nucleic acid amplification reaction reagent to prepare the following mixed reagent solution. In addition, the liquid volume (volume) of each reagent in the following is a value with respect to 10 μL of the mixed reagent. That is, for example, polymerase is 0.4 μL with respect to 10 μL of the mixed reagent picture container.

<Composition of mixed reagent solution>
PlatinumTaq (polymerase) 0.4 μL
dNTP (10 mM) 0.5 μL
Buffer 2.0 μL
Forward primer for mycoplasma detection (20μM) 0.8μL
Reverse primer for mycoplasma detection (20μM) 0.8μL
Fluorescent probe for mycoplasma detection (10 μM) 0.6 μL
Template DNA 1.0 μL
Water up to 10μL

  The composition of the buffer is as follows.

MgCl ₂ : 25 mM
Tris-HCl (PH9.0): 250 mM
KCl: 125 mM

  Table 1 shows the sequences of the forward primer and the reverse primer.

  In Example 1, a modified fluorescent probe in which a part of the base of the TaqMan fluorescent probe was modified with LNA was used as the fluorescent probe. The sequence of the modified fluorescent probe is shown in Table 2. In Table 2, the base surrounded by a broken line is a base modified with LNA. That is, if the base (“C”) connected to “FAM” is the first base from the left, the eighth base from the left (“G”), the ninth base from the left (“A”), The tenth base (“A”) from the left was an artificial nucleic acid modified with LNA. In Table 2, “FAM” is a reporter, and “BHQ1” and “NFQ” are quenchers.

  A reaction solution was collected from the mixed reagent solution having the above composition. The amount of the reaction solution collected was changed to 0.1 μL, 0.2 μL, 0.5 μL, 1.0 μL, 1.5 μL, 2.0 μL, 2.8 μL, 3.0 μL, and 3.5 μL.

3.1.2. Example 2
The same as Example 1 except that a modified fluorescent probe containing an MGB molecule was used as the fluorescent probe (see Table 2).

3.1.3. Comparative Example 1
The same as Example 1 except that a TaqMan fluorescent probe was used as the fluorescent probe (see Table 2).

3.2. PCR results PCR was performed on the reaction solution as described above. Silicone oil was used as the liquid 30. The inner diameter of the container of the nucleic acid amplification reaction cartridge was 2 mm. The first temperature (temperature for the denaturation reaction) is 98 ° C., the first time (period for the denaturation reaction) is 4 seconds, the second temperature (temperature for the annealing reaction and extension reaction) is 54 ° C., The time of 2 (period for annealing reaction and extension reaction) was 6 seconds. Moreover, it heated at 98 degreeC for 10 second, and was hot-started.

  FIG. 10 is a graph showing the PCR results of Example 1. FIG. 11 is a graph showing the PCR results of Example 2. FIG. 12 is a graph showing the PCR results of Comparative Example 1. 10 to 12, the horizontal axis indicates the number of thermal cycles, and the vertical axis indicates the fluorescence intensity measured by the fluorescence measuring apparatus. In addition, the case where 1st temperature and 2nd temperature were provided once with respect to the reaction liquid was counted as 1 cycle (1 time).

  As shown in FIGS. 10 to 12, Experimental Example 1 using the modified fluorescent probe containing LNA and Experimental Example 2 using the modified fluorescent probe containing the MGB molecule are more than Comparative Example 1 using the TaqMan fluorescent probe. The fluorescence intensity was high. Thereby, it turned out that the fluorescence intensity in PCR can be made high by the modified fluorescent probe containing an artificial nucleic acid or an MGB molecule. Furthermore, Experimental Example 2 using the modified fluorescent probe containing MGB molecules had higher fluorescence intensity than Experimental Example 1 using the modified fluorescent probe containing LNA.

  Furthermore, as shown in FIGS. 10 and 11, the volume of the reaction solution is 0.1 μL or more and 2.0 μL or less, preferably 0.5 μL or more and 2.0 μL or less, more preferably 1.5 μL or more and 2.0 μL or less. As a result, it was found that the fluorescence intensity in PCR can be further increased and PCR can be performed efficiently.

  In Examples 1 and 2, no fluorescence was observed in the reaction solutions of 2.8 μL, 3.0 μL, and 3.5 μL. In Comparative Example 1, no fluorescence was confirmed in the reaction solutions of 0.1 μL, 2.8 μL, 3.0 μL, and 3.5 μL.

3.3. Measurement of reaction liquid drop time Based on the apparatus used in Example 1, a part of the heat block was provided with a slit to prepare a device capable of observing the movement of the reaction liquid. The reaction liquid dropped due to the difference in specific gravity with silicone oil. The movie was shot and converted to a still image. Then, from the still image, the timing at which the reaction solution reaches the end of the cartridge for nucleic acid amplification reaction is determined, and the number of frames from the start of rotation of the heating unit of the elevating PCR device to the end of the cartridge for nucleic acid amplification reaction From the time per frame, the drop (movement) time of the reaction solution was calculated. Furthermore, the ratio (L / D) between the diameter L of the droplet of the reaction solution and the inner diameter D of the container of the nucleic acid amplification reaction cartridge was calculated. Table 3 shows the calculation results.

  Here, from FIG. 10, fluorescence was confirmed in the reaction solution of 0.1 μL to 2.8 μL. Therefore, from Table 3, fluorescence was confirmed when the ratio (L / D) was 0.28 or more and 0.87 or less. That is, by setting the ratio (L / D) to 0.28 or more and 0.87 or less, PCR is efficiently performed even under a high-speed condition in which the second time is 10 seconds or less (even if the PCR is accelerated). I found out that I could do it. Furthermore, it has been found that PCR can be performed more efficiently even if the PCR speed is increased by preferably setting it to 0.49 or more and 0.87 or less, more preferably 0.70 or more and 0.79 or less.

  In the case of 0.1 μL of the reaction solution, the drop time took 17.5 seconds (see Table 3). Here, as shown in “3.2. PCR Results”, the first time in PCR is 4 seconds and the second time is 6 seconds. Therefore, in the case of a 0.1 μL reaction solution, the reaction solution is It has not moved to the end of the cartridge for nucleic acid amplification reaction. However, as shown in FIG. 10, fluorescence was confirmed in the 0.1 μL reaction solution. This is presumably because the sensitivity was increased to the extent that fluorescence could be detected without moving the reaction solution to the end of the cartridge for nucleic acid amplification reaction by using a modified fluorescent probe containing LNA.

  The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

DESCRIPTION OF SYMBOLS 2 ... Pipette, 10 ... Cartridge for nucleic acid amplification reaction, 12 ... Container, 12a ... Side wall part, 12b ... Bottom part, 14 ... Cap, 16 ... Channel, 16a ... 1st area | region, 16b ... 2nd area | region, 20 ... Reaction liquid , 22 ... template nucleic acid solution, 24 ... reagent, 30 ... liquid, 50 ... body part, 51 ... mounting part, 52 ... first heating part, 52a ... first heater, 52b ... first heat block, 53 ... second heating Part, 53a ... second heater, 53b ... second heat block, 54 ... spacer, 55 ... bottom plate, 55a ... through hole, 56 ... flange, 57 ... fixing plate, 58 ... conductor, 60 ... moving mechanism, 70 ... lid 72 ... Magnetic part, 80 ... Fluorescence measuring instrument, 100 ... Nucleic acid amplification device

SEQ ID NO: 1 is the sequence of Mycoplasma forward primer.
SEQ ID NO: 2 is the sequence of the reverse primer of Mycoplasma.
SEQ ID NO: 3 is the sequence of a Mycoplasma fluorescent probe.

Claims (9)

A reaction liquid and a liquid into which the reaction liquid is different in specific gravity and immiscible with the reaction liquid,
The container has a flow path through which the reaction solution moves,
The reaction solution includes a modified fluorescent probe,
The ratio (L / D) between the diameter L of the droplet of the reaction solution and the inner width D of the container, which is the width of the flow path, is 0.28 or more and 0.87 or less. cartridge. In claim 1,
The cartridge for nucleic acid amplification reaction, wherein the volume of the reaction solution is 0.1 μL or more and 2.0 μL or less. In claim 1 or 2,
A cartridge for nucleic acid amplification reaction, wherein a reagent used for nucleic acid amplification is lyophilized in the container. In any one of Claims 1 thru | or 3,
The modified fluorescent probe is a cartridge for nucleic acid amplification reaction containing an artificial nucleic acid. In claim 4,
The artificial nucleic acid is LNA, 3′-amino-2 ′, 4′-BNA, 2 ′, 4′-BNA ^COC , 2 ′, 4′-BNA ^NC , PNA, GNA, or TNA, nucleic acid amplification reaction Cartridge. In claim 4 or 5,
The cartridge for nucleic acid amplification reaction, wherein the artificial nucleic acid is LNA. In any one of Claims 1 thru | or 3,
The modified fluorescent probe is a cartridge for nucleic acid amplification reaction containing a minor group binder molecule. A mounting portion to which the cartridge for nucleic acid amplification reaction according to any one of claims 1 to 7 can be mounted;
A temperature gradient forming part for forming a temperature gradient in the flow path of the cartridge for nucleic acid amplification reaction;
A nucleic acid amplification apparatus comprising: Forming a temperature gradient in the flow path of the container;
A step of moving a reaction liquid containing nucleic acid in the flow path to give a temperature cycle to the reaction liquid;
Including
The reaction solution includes a modified fluorescent probe,
The nucleic acid amplification method, wherein the ratio (L / D) of the diameter L of the droplet of the reaction solution to the inner width D of the container serving as the flow path is 0.28 or more and 0.87 or less.

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