JP2016192931A - Cartridge for nucleic acid amplification reaction, and nucleic acid amplification apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten creation time of a PCR product while suppressing reduction of amplification efficiency.SOLUTION: A cartridge 1 for nucleic acid amplification reaction of the present invention comprises: a container 10 into which solution containing template nucleic acid is introduced; and liquid 12 which is housed in the container 10, has different specific gravity from the solution, and causes phase separation. A ratio between a diameter D2 of droplets of the solution which is obtained by causing phase separation to the liquid 12 and an inner diameter D1 of a cylindrical part of the container 10 being a flow channel through which the droplets move is 0.36 or more and 0.89 or less.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a cartridge for nucleic acid amplification reaction and a nucleic acid amplification apparatus, and is suitable for shortening the production time of a PCR product by shortening the amplification reaction period.

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

  As a nucleic acid amplification apparatus using such a PCR method, a PCR apparatus of Patent Document 1 below has been proposed by the present applicant. In the biochip attached to the PCR device of Patent Document 1 below, a flow path is formed in which a droplet-like reaction liquid containing a target nucleic acid or the like moves, and the flow path contains the reaction liquid, A liquid having a specific gravity smaller than that of the reaction liquid and immiscible with the reaction liquid is filled.

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

JP 2012-115208 A

  By the way, there is a demand to further accelerate the PCR product generation time in the above-described PCR apparatus.

  Therefore, an object of the present invention is to shorten the generation time of PCR products through shortening of the amplification reaction period.

  In the amplification reaction period, the time during which the droplet moves between the first region and the second region, the time during which the temperature of the droplet moved to the region changes, and the reaction proceeds in the droplet. Broadly divided into time. The inventors of the present invention paid attention to the fact that the time during which the droplets move is effective in shortening the amplification reaction period. Therefore, in order to achieve the above object, the present inventors diligently studied the moving speed of the liquid droplets, and as a result, have reached the cartridge for nucleic acid amplification reaction and the nucleic acid amplification apparatus of the present invention.

  The cartridge for nucleic acid amplification reaction of the present invention comprises a container into which a solution containing a template nucleic acid is introduced, and a liquid that is contained in the container and has a specific gravity different from that of the solution and phase-separates. The ratio between the diameter of the droplet of the solution to be phase-separated and the inner diameter of the cylindrical portion of the container serving as a flow path through which the droplet moves is 0.36 or more and 0.89 or less. .

  The nucleic acid amplification apparatus of the present invention heats the first region of the container in the cartridge for nucleic acid amplification reaction to the denaturation temperature of the target nucleic acid, and the second region of the container independent of the first region is the target nucleic acid. A heater for heating to the synthesis temperature of the liquid, and a droplet that phase-separates with the liquid in the liquid contained in the container, from the first region to the second region, or from the second region to the first region. The moving mechanism is controlled so as to repeat a plurality of cycles including a moving mechanism that moves the liquid droplets to the first region, a modification step that retains the droplets in the first region, and a synthesis step that retains the droplets in the second region. A controller, and the droplet is added with a template nucleic acid and a reagent used for amplification of the target nucleic acid in the template nucleic acid, thereby forming a diameter of the droplet and a flow path through which the droplet moves. Circle of the container The ratio of the inner diameter of the part is 0.36 or more than 0.89, characterized in that.

It is a figure which shows the cross section of the cartridge for nucleic acid amplification reaction. It is a figure which shows a mode that the template nucleic acid solution is introduce | transduced in the container of the cartridge for nucleic acid amplification reaction. It is a figure which shows a mode when the reagent of a freeze-dried state returns to the original state with the template nucleic acid solution. It is a block diagram of a nucleic acid amplifier. It is a figure which shows the mode of a rotation mechanism. It is a figure which shows a mode that the cartridge for nucleic acid amplification reaction was mounted | worn with the mounting part. It is a figure which shows the mode of a heat cycle process. It is a graph which shows an amplification curve.

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

(1) Embodiment As an embodiment, after describing a cartridge for nucleic acid amplification reaction mounted on a nucleic acid amplification device for amplifying a nucleic acid, the nucleic acid amplification device will be described.

=== Cartridge for nucleic acid amplification reaction ===
FIG. 1 is a view showing a cross section of a cartridge 1 for nucleic acid amplification reaction. As shown in FIG. 1, the nucleic acid amplification reaction cartridge 1 includes a container 10 in which a reagent 11 and oil 12 are accommodated. In the present embodiment, the side wall 10A of the container 10 is cylindrical, and the bottom wall 10B of the container 10 is hollow hemispherical.

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

  A template nucleic acid solution is introduced into such a container 10. The template nucleic acid solution is a solution containing a template nucleic acid. When the template nucleic acid solution is introduced into the container 10, the cap 10C is removed from the container 10, and the cap 10C is attached to the container 10 again after the introduction.

  The template nucleic acid solution introduced into the container 10 is obtained, for example, as follows. That is, a sample such as cells or virus particles derived from organisms such as humans and bacteria is collected by a collection tool such as a cotton swab, and a template nucleic acid is extracted from the sample using a known extraction technique. Thereafter, the template nucleic acid solution is purified to a predetermined concentration using a known purification method. The composition of the template nucleic acid solution is, for example, water (distilled water, sterilized water) or a Tris-EDTA (ethylenediaminetetraacetic acid) solution (TE).

  The reagent 11 is a reagent used for the amplification reaction of the target nucleic acid, and is freeze-dried and fixed to the bottom wall portion 10B of the container 10. As this reagent 11, DNA (deoxyribonucleic acid) polymerase, a primer, dNTP (deoxyribonucleotide triphosphate), and a buffer solution are contained. When the reagent 11 is freeze-dried, the moisture in the reagent 11 disappears, so ions such as magnesium ions and potassium ions contained in the buffer solution are fixed to the bottom wall portion 10B of the container 10.

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

  When the template nucleic acid is RNA (ribonucleic acid), reverse transcriptase, a reverse transcriptase primer, and the like are also included as the reagent 11 used for the amplification reaction of the target nucleic acid in order to obtain cDNA of the RNA. When quantifying target nucleic acid amplification using the real-time PCR method, probes for quantification such as TaqMan probes, probes that bind to fluorescent dyes such as Molecular Beacon and cycling probes, and intercalator fluorescent dyes such as SYBR Green are also available. , Contained as a reagent 11 used in the amplification reaction of the target nucleic acid.

  The oil 12 has a specific gravity smaller than that of the template nucleic acid solution introduced into the container 10 and is a liquid that is phase-separated from the template nucleic acid solution, such as 2CS silicone oil or mineral oil.

  FIG. 2 is a view showing a state where a template nucleic acid solution is introduced into a container of a cartridge for nucleic acid amplification reaction, and FIG. 3 is a view showing a case where a freeze-dried reagent is returned to its original state by the template nucleic acid solution. FIG.

  As shown in FIG. 2, when a template nucleic acid solution is added to the oil 12 contained in the container 10 of the cartridge 1 for nucleic acid amplification reaction 1, the template nucleic acid solution acts to reduce the surface area of the interface, The template nucleic acid solution is phase-separated from the oil 12 in the container 10 to form droplets 20. Since the specific gravity of the droplet 20 is larger than the specific gravity of the oil 12, the droplet 20 settles along the side wall portion 10 </ b> A in the container 10. That is, the cylindrical side wall portion 10 </ b> A in the container 10 becomes a flow path for the droplet 20.

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

  When the reagent 11 changed from the freeze-dried state to the original state is taken into the droplet 20, the droplet 20 contains the template nucleic acid and the reagent 11 used for amplification of the target nucleic acid in the template nucleic acid. The droplet 20 becomes a place for advancing the nucleic acid amplification reaction.

  By the way, in the case of the present embodiment, the ratio of the inner diameter D1 of the side wall portion 10A that becomes a cylindrical flow path in which the droplet 20 moves in the container 10 to the diameter D2 of the droplet 20 is 0.36 or more and 0.00. 89 or less. The liquid volume of the droplet 20 is preferably 0.2 μL or more and 2 μL or less. Moreover, it is preferable that the inner wall of 10 A of side wall parts has the water repellency of the grade which the droplet 20 does not adhere.

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

<Rotation mechanism>
FIG. 5 is a diagram illustrating a state of the rotation mechanism. FIG. 5 is a side view of the rotation mechanism 60. In the following description of the nucleic acid amplification device 50, as shown in the figure, the top, bottom, front, back, left and right are defined. That is, the vertical direction when the base 51 of the nucleic acid amplification device 50 is installed horizontally is defined as “vertical direction”, and “upper” and “lower” are defined according to the direction of gravity. Further, the axial direction of the rotation axis of the cartridge 1 for nucleic acid amplification reaction 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”.

  As shown in FIG. 5, the rotating mechanism 60 includes a rotating body 61 and a rotating motor 66 (FIG. 4) that rotates the rotating body 61. The rotator 61 is provided with a heater portion 65 having an insertion hole 64 into which the cartridge 1 for nucleic acid amplification reaction can be attached and detached. The rotating body 61 is supported by a support base 52 fixed to the base 51 without changing the relative position between the heater section 65 and the nucleic acid amplification reaction cartridge 1 mounted in the insertion hole 64 of the heater section 65. Rotate around an axis.

  The insertion hole 64 of the heater unit 65 also serves as a holding unit for detachably holding the nucleic acid amplification reaction cartridge 1. However, the nucleic acid amplification reaction cartridge 1 inserted into the insertion hole 64 is detachably held. A holding unit may be provided in the nucleic acid amplification device 50. Further, the number of nucleic acid amplification reaction cartridges 1 to which the holding unit can be attached is not limited to one and may be plural.

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

  FIG. 6 is a diagram showing a state in which the cartridge for nucleic acid amplification reaction is mounted. As shown in FIG. 6, the heater unit 65 has a high temperature side heater unit 65B for heating to a temperature at which the denaturation reaction of the target nucleic acid proceeds, and a temperature at which the target nucleic acid synthesis reaction (annealing reaction and extension reaction) proceeds. A low-temperature side heater unit 65C for heating.

  When the nucleic acid amplification reaction cartridge 1 is held in the insertion hole 64 of the heater section 65, the first region 36A corresponding to one end side of the side wall section 10A serving as the flow path of the droplet 20 in the container 10 of the nucleic acid amplification reaction cartridge 1 is used. Is surrounded by the high temperature side heater section 65B. The high temperature side heater unit 65B heats the first region 36A to, for example, 95 to 100 ° C.

  Further, when the nucleic acid amplification reaction cartridge 1 is held in the insertion hole 64 of the heater section 65, the second end corresponding to the other end side of the side wall section 10 </ b> A serving as the flow path of the droplet 20 in the container 10 of the nucleic acid amplification reaction cartridge 1. The two regions 36B are surrounded by the low temperature side heater portion 65C. The low temperature side heater unit 65C heats the second region 36B to, for example, 50 to 75 ° C.

  Thus, the first region 36A of the container 10 in the cartridge 1 for nucleic acid amplification reaction is heated to a temperature at which the target nucleic acid denaturation reaction proceeds, and the second region 36B of the container 10 at a temperature at which the target nucleic acid synthesis reaction proceeds. Heated.

  Note that a spacer 65D that suppresses heat conduction between the high temperature side heater unit 65B and the low temperature side heater unit 65C is disposed between the high temperature side heater unit 65B and the low temperature side heater unit 65C. Even in this spacer, a through hole is formed in the 65D at a position along the longitudinal direction of the insertion hole 64 of the high temperature side heater unit 65B and the low temperature side heater unit 65C, and the container of the nucleic acid amplification reaction cartridge 1 is inserted into the insertion hole 64. Preventing the insertion of 10 is prevented.

<Fluorescence measuring instrument>
The fluorescence measuring device 70 is a measuring device for measuring the fluorescence intensity of the droplet 20 accommodated in the container 10 in the cartridge 1 for nucleic acid amplification reaction, and is attached to the insertion hole 64 of the heater section 65 as shown in FIG. The nucleic acid amplification reaction cartridge 1 is arranged to face the end of the cartridge 1 with a predetermined distance.

  The fluorescence measuring instrument 70 irradiates excitation light corresponding to the fluorescent dye contained in the droplet 20 in accordance with a measurement instruction from the control unit 80 (FIG. 4), and measures the fluorescence intensity emitted from the droplet 20. . Further, the fluorescence measuring instrument 70 gives data indicating the fluorescence intensity obtained as a measurement result to the control unit 80. The fluorescence measuring instrument 70 may measure the fluorescence intensity corresponding to one fluorescent dye, or may measure the fluorescence intensity corresponding to a plurality of fluorescent dyes.

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

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

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

  That is, when the control unit 80 receives, for example, a command to be subjected to heat cycle processing from the input unit 92, the control unit 80 drives the high temperature side heater unit 65 </ b> B provided in the rotating body 61, and the first container 10 in the nucleic acid amplification reaction cartridge 1. The region 36A is heated to a temperature at which the target nucleic acid denaturation reaction proceeds. Further, the control unit 80 drives the low-temperature side heater unit 65C provided in the rotator 61 to heat the second region 36B of the container 10 in the cartridge 1 for nucleic acid amplification reaction to a temperature at which the target nucleic acid synthesis reaction proceeds. To do. Thereby, a temperature gradient is formed in the oil 12 filled in the container 10 of the cartridge 1 for nucleic acid amplification reaction.

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

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

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

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

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

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

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

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

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

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

<Summary>
As described above, in the present embodiment, the diameter D2 of the template nucleic acid solution droplet 20 phase-separated from the oil 12 contained in the container 10 of the nucleic acid amplification reaction cartridge 1 and the flow path through which the droplet 20 moves. The ratio of the cylindrical side wall portion 10A to the inner diameter D1 is 0.36 or more and 0.89 or less. In this case, it has been found that the moving speed of the droplet 20 moving on the cylindrical side wall portion 10A in the container 10 increases without reducing the amplification efficiency. This is described by way of an example below.

  Therefore, according to the cartridge 1 for nucleic acid amplification reaction and the nucleic acid amplification apparatus 50 in this embodiment, the generation time of the PCR product can be shortened through shortening of the amplification reaction period.

  Further, when the ratio of the inner diameter D1 of the side wall portion 10A to the diameter D2 of the droplet 20 is not less than 0.89 and the liquid amount of the droplet 20 is not less than 0.2 μL and not more than 2 μL, the side wall portion 10A. It has been found that the amplification efficiency is further improved while increasing the moving speed of the droplet 20 moving on the substrate. This is also described by way of an example below.

(2) Modification In the above embodiment, the side wall 10A of the container 10 is cylindrical, and the bottom wall 10B of the container 10 is hollow hemispherical. However, if the portion that becomes the flow path through which the droplet 20 moves is a cylinder, there are various shapes such as the shape of the bottom wall portion 10B of the container 10 and the outer shape corresponding to the outer peripheral side of the side wall portion 10A of the container 10. can do.

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

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

  In the above embodiment, the lyophilized reagent 11 is fixed in the container 10, and the template nucleic acid solution is introduced into the oil 12 contained in the container 10 to obtain the template nucleic acid solution droplet 20. The reagent 11 was returned to the original state by the droplet 20 and taken into the droplet 20. That is, the droplet 20 containing the template nucleic acid and the reagent 11 used for amplification of the target nucleic acid in the template nucleic acid was formed in the container 10 and accommodated in the container 10. However, a solution containing the template nucleic acid and the reagent 11 used for amplification of the target nucleic acid in the template nucleic acid is prepared outside the container 10, and the solution is introduced into the oil 12 so that the liquid droplets are introduced into the container 10. 20 may be accommodated. In this case, placing the freeze-dried reagent 11 in the container 10 in advance is omitted. In short, a solution containing the template nucleic acid may be introduced into the container 10, and any method may be used for adding the reagent 11 used for amplification of the template nucleic acid and the target nucleic acid in the template nucleic acid to the droplet 20.

  In the above embodiment, the specific gravity of the droplets 20 is made larger than the specific gravity of the oil 12. However, the specific gravity of the droplet 20 may be smaller than the specific gravity of the oil 12. Even if it does in this way, there exists an effect similar to the said embodiment. In short, a liquid having a specific gravity different from the specific gravity of the solution introduced into the container 10 and phase-separating from the solution can be applied. When a solution is introduced into such a liquid, a solution droplet 20 is formed in the liquid as in the case of the above embodiment.

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

  In the above embodiment, the rotation mechanism 60 is employed as a mechanism for alternately moving the droplets 20 in the container 10 in the cartridge 1 for nucleic acid amplification reaction to the first region 36A and the second region 36B. However, the droplets 20 are alternately moved to the first region in the container 10 that is set to the denaturation temperature of the target nucleic acid and the second region that is independent from the first region and set to the synthesis temperature of the target nucleic acid. Various moving mechanisms other than the rotating mechanism 60 can be applied as long as the rotating mechanism 60 is used.

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

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

First, a template nucleic acid, a reagent 11 used for amplification of a target nucleic acid in the template nucleic acid, and a coloring ink were put into a test tube, and then distilled water was added to prepare a 10 μL solution.
The template nucleic acid was plasmid DNA (10 ⁵ copies / μL), which is a standard substance for pneumonia mycoplasma, and 1.0 μL was added to the test tube.

The DNA polymerase contained in the reagent 11 was PlatinumTaq manufactured by Life Technologies, and 0.4 μL was added to the test tube.
The dNTP contained in the reagent 11 was PCR Nucleotide Mix manufactured by Roche, and 0.5 μL was added to the test tube.
The primer contained in the reagent 11 was a mycoplasma detection forward primer and a mycoplasma detection reverse primer. 0.8 μL of the forward primer was added to the test tube, and 0.8 μL of the reverse primer was added to the test tube.
The buffer contained in Reagent 11 had a composition of MgCl ₂ of 25 mM, Tris-HCl (PH9.0) of 250 mM, and KCl of 125 mM, and 2.0 μL was charged into the test tube.
The probe contained in the reagent 11 was a TaqMan probe for mycoplasma detection, and 0.6 μL was charged into the test tube.

The primer and probe sequences are shown in Table 1 below.

  “FAM” in Table 1 is an abbreviation for fluorescein aminohexyl and is one of reporter fluorescent dyes. In addition, “BHQ” in Table 1 is an abbreviation for Black Hole Quencher and is one of quencher fluorescent dyes.

Next, a plurality of containers 10 having an inner diameter D1 of the side wall portion 10A of 2 mm were prepared, and the above solutions were introduced into the containers 10 in different amounts, and the droplets 20 were accommodated in the containers 10. Thereafter, the container 10 was mounted in the insertion hole 64 of the heater unit 65 in the nucleic acid amplification device 50, and the thermal cycle process and the amplification analysis process were performed for each container 10 under the same conditions.
The number of cycles in the thermal cycle process is 50, the denaturation reaction period is 4 seconds, the synthesis reaction period is 6 seconds, the temperature of the high temperature side heater section 65B is 98 ° C., and the temperature of the low temperature side heater section 65C is 54 ° C. did.

Further, when the thermal cycle process is executed, the container 10 is photographed with a Casio digital video camera having a frame rate of 30 fps, and the side wall portion 10A of the container 10 is used by using data obtained as a result of the photographing. The moving time of the moving droplet 20 was measured. The measurement results are shown in Table 2 below, and the processing results (amplification curve) of the amplification analysis process executed simultaneously with the thermal cycle process are shown in FIG.
The moving time is the number of frames from the time when the rotating body 61 starts to rotate until the time when the droplet 20 reaches the second area 36B (or the second area 36B to the first area 36A) from the first area 36A. An average was obtained and obtained from the average and the frame rate.
Further, after confirming that the inner diameter D1 of the side wall 10A of the container 10 reflected in the frame is 2 mm, the diameter D2 of the droplet 20 reflected in the frame is acquired, and the inner diameter D1 and the droplet 20 of the side wall 10A are acquired. The ratio with the diameter D2 was determined.

  As shown in Table 2 and FIG. 8 above, the ratio of the inner diameter D1 of the cylindrical side wall portion 10A that becomes the flow path in which the droplet 20 moves in the container 10 to the diameter D2 of the droplet 20 is 0.36. When the ratio is 0.89 or less, it is confirmed that the moving speed of the droplet 20 is increased without reducing the amplification efficiency.

  Further, when the ratio of the inner diameter D1 of the side wall 10A to the diameter D2 of the droplet 20 is 0.36 to 0.89 and the liquid amount of the droplet 20 is 0.2 μL or more and 2 μL or less, the liquid It was confirmed that the amplification efficiency was further improved while the moving speed of the droplet 20 was increased.

  DESCRIPTION OF SYMBOLS 1 ... Nucleic acid amplification reaction cartridge, 10 ... Container, 10A ... Side wall part, 10B ... Bottom wall part, 10C ... Cap, 11 ... Reagent, 12 ... Oil, 20 ... droplet, 50 ... nucleic acid amplification device.

Claims (4)

A container into which a solution containing the template nucleic acid is introduced;
A liquid contained in the container and having a specific gravity different from that of the solution;
The ratio of the diameter of the droplet of the solution phase-separated from the liquid in the liquid to the inner diameter of the cylindrical portion of the container serving as a flow path through which the droplet moves is 0.36 or more and 0.89 or less. is there,
The cartridge for nucleic acid amplification reaction characterized by the above-mentioned. The liquid volume of the droplet is 0.2 μL or more and 2 μL or less.
The cartridge for nucleic acid amplification reaction according to claim 1. In the container, a reagent used for amplification of the target nucleic acid in the template nucleic acid is lyophilized and arranged.
The cartridge for nucleic acid amplification reaction according to claim 1 or 2. A heater for heating the first region of the container in the cartridge for nucleic acid amplification reaction to the denaturation temperature of the target nucleic acid, and heating the second region of the container independent of the first region to the synthesis temperature of the target nucleic acid;
A moving mechanism for moving droplets phase-separated from the liquid in the liquid contained in the container from the first region to the second region or from the second region to the first region;
A control unit for controlling the moving mechanism so as to repeat a modification step of retaining the droplets in the first region and a cycle through a synthesis step of retaining the droplets in the second region a plurality of times;
With
To the droplet, a template nucleic acid and a reagent used for amplification of the target nucleic acid in the template nucleic acid are added,
The ratio of the diameter of the droplet to the inner diameter of the cylindrical portion of the container that becomes a flow path through which the droplet moves is 0.36 or more and 0.89 or less.
A nucleic acid amplification device.

Images