JP7132158B2 - Temperature controller and nucleic acid amplifier - Google Patents

Description

The present disclosure relates to a temperature adjustment device and a nucleic acid amplification device.

The PCR (Polymerase Chain Reaction) method is widely used as a method for amplifying nucleic acids. The PCR method is a method of amplifying the DNA of interest by repeating a thermal cycle (temperature cycle), in which one cycle consists of the following three consecutive steps (A) to (C).
(A) A step of thermally denaturing a double-stranded DNA as a template into a single-stranded DNA (thermal denaturation).
(B) Binding of complementary primers to the single-stranded DNA (annealing).
(C) A step of replicating double-stranded DNA by allowing a thermostable DNA polymerase to synthesize a complementary strand from the primer (elongation).

FIG. 1(a) is a schematic diagram showing the thermal cycle of PCR. As shown in FIG. 1(a), the temperature and reaction time of the reaction solution (nucleic acid solution) are controlled in each step of thermal denaturation, annealing and extension. Typically, heat denaturation is performed at about 94°C, annealing at about 60-65°C, and extension at about 72°C.

Conventionally, a thermal cycler has been used as a PCR device that automatically controls the reaction temperature and reaction time of the thermal cycle. In the thermal cycler, a reaction tube containing a reaction solution is inserted into wells of a microplate placed on a metal block, and the temperature of the metal block is controlled so as to meet preset conditions. The metal block is designed to accommodate reaction tubes and microplates, and has a volume equal to or greater than that of the microplates to reduce temperature variations. As a result, the heat capacity of the metal block increases, so that it takes time to raise or lower the temperature, and it takes at least 30 minutes, typically 1 to 2 hours, to perform PCR.

In order to speed up PCR or to perform analysis directly after PCR, a method of performing PCR using a microfluidic device having a channel through which a reaction solution flows has been proposed. For example, in Non-Patent Document 1, a microfluidic device having a meandering channel is brought into contact with a heat block set to three temperature zones corresponding to "denaturation", "annealing" and "elongation", and the meandering of the channel A nucleic acid amplifier that performs thermal cycling according to the number of times is described. Such a method is called continuous flow PCR, and it is only necessary to maintain the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction liquid is determined by the channel length and the flow rate of the reaction liquid, without depending on the heating and cooling speeds of the heat block.

A method of performing PCR in a thermal cycle in which two temperature zones of denaturation and annealing are repeated has also been proposed. This method is called 2-step PCR (shuttle PCR), is useful when the Tm of the primer is high, and can shorten the time because the annealing step and extension step are performed simultaneously. In continuous flow PCR employing two-step PCR, complementary strand synthesis is performed to some extent by a thermostable polymerase in the annealing step. Alternatively, complementary strand synthesis is carried out when the temperature of the reaction solution reaches the elongation temperature zone while the temperature is rising from the annealing temperature zone to the denaturation temperature zone.

In Patent Document 1, in a nucleic acid amplification device that performs continuous flow PCR employing 2-step PCR, one end of a microfluidic device having a meandering flow path becomes a high temperature region (denaturation temperature zone), and the other end becomes It is described that a heater and a cooler are installed so as to provide a low temperature range (annealing temperature range) (see FIG. 13, etc. of the same document). Also in the method of Patent Document 1, since it is not necessary to change the temperature of each heat block, the temperature of the reaction liquid flowing through the flow channel can be changed at high speed without depending on the heating or cooling rate of the heat block.

U.S. Patent Application Publication No. 2010/0167288

Kopp et al., Science, vol.280, 1998, p.1046-1048

However, in the continuous flow PCR described in Non-Patent Document 1, etc., there is a problem that the same thermal cycle as in a conventional thermal cycler cannot be performed.

FIG. 1(b) is a schematic diagram showing a conventional nucleic acid amplifier and its thermal cycle. As shown in the upper diagram of FIG. 1(b), a denaturation temperature zone, an annealing temperature zone, and an elongation temperature zone were set for a microfluidic device 105 having a meandering channel 102 in the same manner as in Non-Patent Document 1. When the heat blocks 111 to 113 are brought into contact with each other, a thermal cycle of "denaturation, elongation, annealing, elongation, . The conventional cycle of "denaturation, annealing, elongation, ..." cannot be reproduced.

That is, when the heat block for the extension temperature zone is installed between the heat block for the denaturation temperature zone and the heat block for the annealing temperature zone, the reaction solution not only passes through the extension temperature zone during the temperature rise from annealing to denaturation, The reaction solution also passes through the elongation temperature zone when the temperature is lowered from denaturation to annealing. As a result, in the process of moving the reaction mixture from the denaturation temperature zone to the extension temperature zone, nonspecific amplification may occur due to the extension reaction due to nonspecific hybridization.

Further, in the continuous flow PCR employing the two-step PCR described in Patent Document 1, there is a problem that the elongation reaction is insufficient and the amplification efficiency is lowered.

FIG. 1(c) is a schematic diagram showing a conventional nucleic acid amplification device employing two-step PCR and its thermal cycle. As shown in the upper part of FIG. 1(c), heat blocks 111 and 112 set in the denaturation temperature zone and the annealing temperature zone, respectively, are applied to a microfluidic device 105 having a meandering channel 102 in the same manner as in Patent Document 1. , the continuous flow PCR is accelerated, and as shown in the lower diagram of FIG. As a result, complementary strand synthesis is not performed sufficiently, and the amplification efficiency of PCR is lowered. This is because complementary strand synthesis in the elongation step of PCR consists of binding of DNA polymerase and incorporation of substrate nucleotides, and elongation becomes insufficient if the reaction time is short. On the other hand, it is known that the dissociation of double-stranded DNA in the denaturation step of PCR and the hybridization of primers to single-stranded DNA in the annealing step are high-speed reactions that complete within one second.

In particular, speeding up is required when PCR is performed outside a large-scale laboratory such as an on-site inspection. However, as described above, the generation of non-specific amplification products and the decrease in PCR amplification efficiency have been problems as adverse effects of increasing the speed of continuous flow PCR.

Therefore, the present disclosure provides a technique for preventing deterioration of PCR amplification efficiency in continuous flow PCR.

A temperature adjustment device of the present disclosure has a first surface and is set to a denaturation temperature zone for nucleic acids, and has a second surface and is set to an annealing temperature zone for nucleic acids. 2 members, and a third member having a third surface and set to an elongation temperature zone of the nucleic acid, wherein the first surface, the second surface and the third surface There is a plane containing said first member, said third member and said second member are arranged in this order along a first direction parallel to said plane so as to be spaced apart from each other, said plane parallel to and orthogonal to the first direction is the length of the first surface and the length of the second surface in the second direction characterized by being shorter than the

Further features related to the present disclosure will become apparent from the description of the specification and the accompanying drawings. In addition, the aspects of the present disclosure will be achieved and attained by means of the elements and combinations of various elements and aspects of the detailed description that follows and the claims that follow.
It should be understood that the description herein is merely exemplary and is not intended in any way to limit the scope or application of this disclosure.

As described above, according to the present disclosure, it is possible to prevent a decrease in PCR amplification efficiency in continuous flow PCR.
Problems, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

FIG. 1(a) is a schematic diagram showing the thermal cycle of PCR, FIG. 1(b) is a schematic diagram showing a conventional nucleic acid amplification device and its thermal cycle, and FIG. 1(c) is a two-step PCR. It is a schematic diagram showing a conventional nucleic acid amplification device and its thermal cycle. FIG. 2(a) is a plan view showing the microfluidic device according to the first embodiment, and FIG. 2(b) is a plane showing a state where the heat block and the microfluidic device according to the first embodiment are in contact. It is a diagram. FIG. 3(a) is a perspective view showing the structure of the heat block according to the first embodiment, and FIG. 3(b) shows a state in which the heat block and the microfluidic device are in contact with each other according to the first embodiment. It is a perspective view. It is a perspective view which shows the structure of the temperature control apparatus which concerns on 2nd Embodiment. FIG. 11 is a plan view showing a state in which the heat block and the microfluidic device are in contact with each other according to the third embodiment; FIG. 6(a) is a perspective view showing the structure of the heat block according to the third embodiment, and FIG. 6(b) shows a state where the heat block and the microfluidic device according to the third embodiment are in contact with each other. It is a perspective view. FIG. 11 is a plan view showing a state in which a heat block and a microfluidic device are in contact with each other according to the fourth embodiment; FIG. 8(a) is a perspective view showing the structure of a heat block according to the fourth embodiment, and FIG. 8(b) shows a state in which the heat block and the microfluidic device are in contact with each other according to the fourth embodiment. It is a perspective view. FIG. 9(a) is a perspective view showing the structure of a heat block according to a fifth embodiment, and FIG. 9(b) is a perspective view showing a nucleic acid amplifier according to the fifth embodiment. FIG. 10(a) is a plan view showing the microfluidic device according to the sixth embodiment, and FIG. 10(b) is a plane showing a state where the heat block and the microfluidic device according to the sixth embodiment are in contact. It is a diagram. FIG. 11(a) is a perspective view showing the structure of the heat block according to the sixth embodiment, and FIG. 11(b) shows a state in which the heat block and the microfluidic device according to the sixth embodiment are in contact with each other. It is a perspective view. 4 is a graph showing temperatures of reaction liquids in Example 1 and Comparative Example 1. FIG. 1 is a diagram showing the results of electrophoresis of PCR amplification products in Example 1 and Comparative Example 1. FIG. 4 is a graph showing temperatures of reaction liquids in Example 2 and Comparative Example 2. FIG. 4 is a graph showing the temperature of the reaction solution in Example 3 and Comparative Example 3. FIG. 4 is a graph showing the temperature of the reaction solution in Example 3 and Comparative Example 3. FIG. FIG. 2 shows the results of electrophoresis of PCR amplification products in Example 3, Example 3-2 and Comparative Example 3. FIG. 4 is a graph plotting the fluorescence intensities of the reaction solutions in Example 4 and Comparative Example 4 for each cycle.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, these embodiments are merely examples for realizing the present disclosure, and do not limit the technical scope of the present disclosure. In addition, the same reference numerals are given to common configurations in each figure.

[First embodiment]
<Configuration of microfluidic device>
FIG. 2(a) is a plan view showing the microfluidic device 5 of the nucleic acid amplifier according to the first embodiment. The nucleic acid amplification apparatus according to this embodiment is an apparatus for amplifying nucleic acids by continuous flow PCR, and includes a microfluidic device 5 and a temperature control device having heat blocks 11 and 12, which will be described later.

A microfluidic device 5 comprises an inlet 1 , a tortuous channel 2 , an outlet 3 and an alignment guide 4 . The meandering flow path 2 includes a flow path (first flow path) in which the reaction liquid flows in the positive direction of the X axis, a flow path (second flow path) in which the reaction liquid flows in the negative direction of the X axis, and a bent portion connecting them. , meandering in the X-axis direction at a constant length and at constant intervals. The reaction solution contains, for example, template DNA, primers, deoxyribonucleotide triphosphates, thermostable polymerase and buffer.

An inlet 1 and an outlet 3 are provided at the beginning and end of the meandering flow path 2, respectively. A reaction liquid is introduced from an inlet 1 into a meandering flow path 2, discharged from an outlet 3, and recovered. A tube such as a silicon tube and a pump such as a syringe pump may be connected to the injection port 1 , and the reaction liquid can be introduced into the meandering flow path 2 by applying pressure with the pump. The reaction liquid may be introduced so as to flow continuously through the meandering flow path 2, or may be introduced in the form of droplets. A tube such as a silicon tube may be connected to the outlet 3 .

As the material of the microfluidic device 5, commonly used resin (for example, cycloolefin copolymer, etc.), glass, or the like can be used. The meandering flow path 2 can be created by processing a meandering groove in a plate-like substrate that will be the microfluidic device 5 and attaching a film-like material. For simplification of the drawing, illustration of the film is omitted. The material of the film may be the same as or different from that of the substrate. The film is attached to the substrate by heat sealing or the like so as to cover the entire surface on which the grooves are formed, for example. Alternatively, the meandering flow path 2 can also be created by bonding substrates on which meandering grooves are formed. After the meandering flow path 2 is formed as described above, holes are formed on the upper surface of the substrate at positions corresponding to both ends of the meandering flow path 2 to form an inlet 1 and an outlet 3, respectively.

<Configuration of temperature control device>
FIG. 2(b) is a plan view showing a state in which the heat blocks 11 and 12 and the microfluidic device 5 are in contact with each other. As indicated by the dotted line in FIG. 2B, the heat blocks 11 and 12 are arranged along the X-axis positive direction (first direction) so as to be separated from each other. The upper surface (first surface) of the heat block 11 and the upper surface (second surface) of the heat block 12 are in the XY plane (the plane including the first surface and the second surface), and the lower surface of the microfluidic device 5 come into contact with

The heat block 11 (first member) is set to a nucleic acid denaturation temperature range (eg, 94° C. to 98° C.), and the heat block 12 (second member) is set to a nucleic acid annealing temperature range (eg, 55° C. to 65° C.). °C). Thus, in the thermal cycle of PCR in the nucleic acid amplification apparatus of this embodiment, a temperature field is formed in the reaction liquid flowing through the meandering channel 2 by bringing the microfluidic device 5 and the heat blocks 11 and 12 into contact with each other. carried out in The details of the positional relationship between the microfluidic device 5 and the heat blocks 11 and 12 will be described later.

Although not shown, the temperature control device of the present embodiment includes, in addition to the heat blocks 11 and 12, a heater such as a Peltier element, wiring, a temperature sensor, and other mechanisms necessary for forming a temperature field. As a material for the heat blocks 11 and 12, a metal having a high thermal conductivity can be used, such as aluminum, copper, silver, or the like.

In the case where the meandering flow path 2 is formed by grooves and films as described above, heat from the heat blocks 11 and 12 can be effectively absorbed by bringing the surfaces to which the films are attached into contact with the heat blocks 11 and 12. can be directly transferred to the reaction solution.

The alignment guide 4 is a mark for accurately positioning the microfluidic device 5 and the heat blocks 11 and 12 when they are brought into contact with each other.

FIG. 3(a) is a perspective view showing the structure of the heat blocks 11 and 12. FIG. As shown in FIG. 3A, the heat block 11 is formed in a comb shape having a body portion 11a and a plurality of protrusions 11b, and the heat block 12 has a body portion 12a and a plurality of protrusions 12b. It is formed in a comb shape with The main bodies 11a and 12a are substantially rectangular parallelepipeds extending in the Y-axis direction. Each convex portion 11b projects from the main body portion 11a in the positive direction of the X axis, and each convex portion 12b projects from the main body portion 12a in the negative direction of the X axis, facing each convex portion 11b via an air layer serving as a heat insulating layer. . The plurality of protrusions 11b and the plurality of protrusions 12b are arranged in the Y-axis direction at predetermined intervals. The arrangement interval of the plurality of convex portions 11b and the plurality of convex portions 12b in the Y-axis direction can be twice the interval between two adjacent channels of the meandering channel 2 . Thereby, each convex part 11b and 12b can contact every other flow path of the meandering flow path 2. As shown in FIG. The number of protrusions 11b and protrusions 12b is not limited to the illustrated number, and can be appropriately set according to the number of meandering of the meandering flow path 2 .

<Configuration of nucleic acid amplification device>
FIG. 3(b) is a perspective view showing a state in which the heat blocks 11 and 12 and the microfluidic device 5 are brought into contact with each other. As shown in FIG. 3(b), the microfluidic device 5 is arranged on the heat blocks 11 and 12 such that the straight portion of the meandering flow path 2 is along the arrangement direction (X-axis direction) of the heat blocks 11 and 12. be done. In the microfluidic device 5, the reaction solution introduced from the injection port 1 first passes over the heat block 11 in the denaturation temperature zone, and then travels straight in the positive direction of the X-axis (first direction) to reach the annealing temperature zone. It passes over the heat block 12, meanders, and is arranged so as to face in the negative direction of the X-axis (opposite direction of the first direction).

Thus, in the present embodiment, when the reaction solution flows through the meandering flow path 2 in the X-axis positive direction (first direction), the temperature is lowered from the denaturation temperature zone to the annealing temperature zone, and the meandering flow path 2 The temperature rises from the annealing temperature zone to the denaturation temperature zone when flowing in the negative axial direction (opposite direction to the first direction).

The body portion 11a contacts the end portion of the meandering flow path 2 on the negative side of the X-axis, and the body portion 12a contacts the end portion of the meandering flow path 2 on the positive side of the X-axis. The protrusions 11b and 12b contact straight portions in the meandering channel 2 where the reaction liquid flows in the positive direction of the X-axis, but do not contact straight portions in which the reaction liquid flows in the negative direction of the X-axis. That is, the projections 11b and 12b only contact the channel (first channel) through which the reaction solution flows when it moves from the denaturation temperature zone to the annealing temperature zone.

In other words, the contact length of the flow path (first flow path) through which the reaction solution flows in the positive direction of the X-axis to the heat blocks 11 and 12 is is longer than the contact length of the heat blocks 11 and 12. As a result, when the temperature of the reaction solution is lowered from the denaturation temperature zone to the annealing temperature zone, the temperature of the reaction solution is rapidly lowered, and when the temperature of the reaction solution is raised from the annealing temperature zone to the denaturation temperature zone, the reaction solution is gradually raised. Warm up.

The length of each projection 11b and 12b in the Y-axis direction is equal to or greater than the width of the meandering flow path 2 in the Y-axis direction, and the projections 11b and 12b do not contact the flow path adjacent to the flow path. Any length is acceptable.

Further, as shown in FIG. 3B, the main body portion 12a of the heat block 12 is provided with alignment pins 14 that can penetrate the microfluidic device 5. As shown in FIG. When the microfluidic device 5 and the heat blocks 11 and 12 are brought into contact with each other, they are positioned by the alignment pin 14 and the alignment guide 4 , and the alignment pin 14 is passed through the microfluidic device 5 . As a result, the heat blocks 11 and 12 and the microfluidic device 5 are brought into contact with each other while maintaining a correct positional relationship, and the temperature field formed by the heat blocks 11 and 12 can be correctly transmitted to the meandering channel 2 . Note that the alignment pin 14 may be provided on the heat block 11 as well.

As a method for detecting an amplification product obtained by PCR using the nucleic acid amplification apparatus of the present embodiment, there is a method in which a probe labeled with a fluorescent substance or the like is included in the reaction liquid and the flow path is observed by fluorescence, A method of collecting from the fluidic device and performing electrophoresis can be adopted.

<Technical effect>
As described above, in the temperature adjustment device of the present embodiment, the heat block 11 set to the denaturation temperature zone has the convex portion 11b, the heat block 12 set to the annealing temperature zone has the convex portion 12b, and the convex portion 11b and 12b contact only the channel through which the reaction solution flows when the reaction solution is cooled from the denaturation temperature zone to the annealing temperature zone. By adopting such a configuration, when the temperature of the reaction solution is lowered from the denaturation temperature zone to the annealing temperature zone, the temperature of the reaction solution is rapidly lowered, and when the temperature is raised from the annealing temperature zone to the denaturation temperature zone, the reaction solution is gradually cooled. The temperature rises to This makes it possible to suppress the production of non-specific amplification products in continuous flow PCR.

[Second embodiment]
<Configuration of temperature control device>
FIG. 4 is a perspective view showing the configuration of the temperature control device according to the second embodiment. As shown in FIG. 4, the temperature control device of this embodiment includes a printed circuit board 200, heat blocks 211 and 212, comb electrodes 221 and 222 (first and second members), and metal wirings 231 and 232. is provided, which is different from the first embodiment.

The heat blocks 211 and 212 are formed in a substantially rectangular parallelepiped shape extending in the Y-axis direction. The heat block 211 is set in the denaturation temperature range of nucleic acids, and the heat block 212 is set in the annealing temperature range of nucleic acids. Although not shown, the heat blocks 211 and 212 are in contact with heaters such as Peltier elements, and heat from the heaters is transferred to the heat blocks 211 and 212 .

The printed board 200 is formed of, for example, a PCB board or the like, and comb-shaped electrodes 221 and 222 and metal wirings 231 and 232 are provided on the printed board 200 . A metal wiring 231 connects the heat block 211 and the comb-shaped electrode 221 , and a metal wiring 232 connects the heat block 212 and the comb-shaped electrode 222 . The comb-shaped electrodes 221 and 222 and the metal wirings 231 and 232 can be made of a metal with a small heat capacity, such as copper. The heat capacities of the comb electrodes 221 and 222 and the metal wirings 231 and 232 are sufficiently smaller than that of the printed circuit board 200, and the temperature of the printed circuit board 200 is approximately room temperature. Thereby, the heat of the heat blocks 211 and 212 is supplied to the comb-shaped electrodes 221 and 222, and a temperature field corresponding to the shape of the comb-shaped electrodes 221 and 222 can be formed.

The upper surface shape of the comb-shaped electrode 221 is the same as the upper surface shape of the heat block 11 of the first embodiment, and the upper surface shape of the comb-shaped electrode 222 is the same as the upper surface shape of the heat block 12 of the first embodiment. . Although not shown, the same microfluidic device 5 as in the first embodiment can be used as the microfluidic device. As in the first embodiment, the temperature of the reaction solution flowing through the meandering flow path 2 can be adjusted to carry out thermal cycling of PCR.

<Technical effect>
As described above, the temperature control device of this embodiment is configured to control the temperature of the reaction solution by the comb-shaped electrodes 221 and 222 having the same shape as the upper surfaces of the heat blocks 11 and 12 of the first embodiment. is employed. Thereby, there can exist an effect similar to 1st Embodiment. Further, the shape of the comb-shaped electrodes 221 and 222 can be finely designed, and the heat blocks 211 and 212 do not need to be formed into complicated shapes, so the temperature control device of this embodiment is easy to manufacture.

[Third embodiment]
FIG. 5 is a plan view showing the nucleic acid amplification device according to the third embodiment. As shown in FIG. 5, the nucleic acid amplification apparatus of this embodiment includes a microfluidic device 5 and a temperature control device having heat blocks 311 to 313, which will be described later. FIG. 5 shows a state in which the heat blocks 311 to 313 and the microfluidic device 5 are in contact with each other. Since the microfluidic device 5 is the same as in the first embodiment, the description is omitted.

<Configuration of temperature control device>
As shown in FIG. 5, the temperature control device of this embodiment includes heat blocks 311 to 313 (first to third members). As indicated by dotted lines in FIG. 5, the upper surface (first surface) of the heat block 311, the upper surface (second surface) of the heat block 312, and the upper surface (third surface) of the heat block 313 are in the XY plane (the third surface). 1 plane, the second plane, and the third plane) and contacts the bottom surface of the microfluidic device 5 . The heat blocks 311, 313 and 312 are arranged in this order along the positive direction of the X-axis so as to be spaced apart from each other. The microfluidic device 5 is in contact with the heat blocks 311 to 313 so that the straight portion of the meandering flow path 2 is aligned with the arrangement direction of the heat blocks 311, 313 and 312 (X-axis direction).

The heat block 311 (first member) is set to a nucleic acid denaturation temperature range (eg, 94° C. to 98° C.), and the heat block 312 (second member) is set to a nucleic acid annealing temperature range (eg, 55° C. to 65° C.). ° C.), and the heat block 313 (third member) is set to a nucleic acid elongation temperature range (eg, 70° C. to 90° C.).

FIG. 6(a) is a perspective view showing the structure of the heat blocks 311-313. As shown in FIG. 6A, the heat blocks 311 and 312 are formed in a substantially rectangular parallelepiped shape extending in the Y-axis direction.

The heat block 313 includes a substantially rectangular parallelepiped body portion 313a extending in the Y-axis direction, and a plurality of convex portions 313b (third members). The convex portions 313b protrude from the body portion 313a in the Z-axis positive direction and are arranged in the Y-axis direction at predetermined intervals. The arrangement interval of the protrusions 313b in the Y-axis direction can be twice the interval between two adjacent channels of the meandering channel 2 . As a result, the convex portion 313 b can come into contact with every other flow path of the meandering flow path 2 .

The length of the upper surface of each projection 313b in the Y-axis direction is shorter than the length of the heat blocks 311 and 312 in the Y-axis direction. In addition, the length of the upper surface of each projection 313b in the Y-axis direction can be equal to or greater than the width of the meandering flow path 2 in the Y-axis direction, and the flow path adjacent to the flow path with which each projection 313b is in contact is in contact with the flow path. The length should be such that it does not occur. The number of protrusions 313b is not limited to the illustrated number, and can be appropriately set according to the number of meandering of the meandering flow path 2. FIG.

<Configuration of nucleic acid amplification device>
FIG. 6(b) is a perspective view showing a state in which the heat blocks 311 to 313 and the microfluidic device 5 are brought into contact with each other. As shown in FIG. 6B, the heat block 311 contacts the end of the meandering flow path 2 on the negative side of the X axis, and the heat block 312 contacts the end of the meandering flow path 2 on the positive side of the X axis. come into contact with Each convex portion 313b contacts only the intermediate portion of the channel (second channel) through which the reaction liquid flows in the negative direction of the X-axis. In the microfluidic device 5, the reaction solution introduced from the injection port 1 first passes over the heat block 311 in the denaturation temperature zone, and then travels straight in the positive direction of the X axis (first direction) to reach the annealing temperature zone. It is arranged to pass over the heat block 312, meander, go straight in the X-axis negative direction (the opposite direction to the first direction), and pass over the heat block 313 in the elongation temperature zone.

After passing over the heat block 311 in the denaturation temperature zone, the reaction solution does not contact any of the heat blocks 311 to 313 until it reaches the heat block 312 in the annealing temperature zone, and does not contact air acting as a heat insulating layer. in contact with Therefore, the temperature of the reaction solution drops as in the conventional continuous flow PCR using two heat blocks shown in FIG. 1(c). On the other hand, when the reaction solution passes over the heat block 312 in the annealing temperature zone and moves toward the heat block 311 in the denaturation temperature zone, it passes over the heat block 313 set in the elongation temperature zone on the way. Therefore, as in the conventional continuous flow PCR using three heat blocks shown in FIG. Rise.

Further, when the microfluidic device 5 and the heat blocks 311 to 313 are brought into contact with each other, they can be positioned by the alignment pins 14 and the alignment guides 4 as in the first embodiment.

In this embodiment, it is sufficient that the temperature of each convex portion 313b of the heat block 313 can be set in the extension temperature range, and the body portion 313a is not necessarily required.

<Technical effect>
As described above, the temperature control device of the present embodiment is set to the elongation temperature zone and includes the heat block 313 having the projections 313b. contact the middle part of the channel where the By adopting such a configuration, it is possible to secure a sufficient time for reaching the elongation temperature zone while the temperature of the reaction solution is rising from the annealing temperature zone to the denaturation temperature zone. This makes it possible to prevent a decrease in PCR amplification efficiency in continuous flow PCR.

[Fourth embodiment]
FIG. 7 is a plan view showing the nucleic acid amplification device according to the fourth embodiment. As shown in FIG. 7, the nucleic acid amplification device of this embodiment includes a microfluidic device 5, heat blocks 11 and 12 of the first embodiment, and a heat block 313 of the third embodiment. Since the microfluidic device 5 is the same as in the first embodiment, the description is omitted.

<Configuration of temperature control device>
FIG. 8(a) is a perspective view showing the heat blocks 11, 12 and 313. FIG. The structures of the heat blocks 11 and 12 are the same as in the first embodiment, and the structure of the heat block 313 is the same as in the third embodiment. As shown in FIG. 8A, the convex portion 313b (third member) of the heat block 313 is arranged outside the area between the facing surfaces of the convex portions 11b and 12b. That is, the convex portions 11b and 12b and the convex portion 313b are alternately arranged in the Y-axis direction. The length of the projection 313b in the X-axis direction may be longer or shorter than the interval between the projections 11b and 12b.

<Configuration of nucleic acid amplification device>
FIG. 8(b) is a perspective view showing a state in which the heat blocks 11, 12 and 313 and the microfluidic device 5 are in contact with each other. As shown in FIG. 8(b), the convex portions 11b and 12b are in contact with the linear portions in which the reaction liquid flows in the positive direction of the X-axis in the meandering channel 2, and are in contact with the linear portions in which the reaction liquid flows in the negative direction of the X-axis. , no contact. Therefore, the projections 11b and 12b contact only the channel (first channel) in which the temperature of the reaction solution is lowered from the denaturation temperature range to the annealing temperature range.

In addition, the convex portion 313b of the heat block 313 contacts the intermediate portion of the channel (second channel) through which the reaction liquid flows in the negative direction of the X-axis in the meandering channel 2 . That is, the convex portion 313b contacts only the intermediate portion of the channel where the temperature of the reaction solution is raised from the annealing temperature range to the denaturation temperature range. As a result, when the temperature of the reaction solution falls from the denaturation temperature zone to the annealing temperature zone, the temperature of the reaction solution drops sharply, and when the reaction solution rises from the annealing temperature zone to the denaturation temperature zone, the elongation temperature zone The temperature rises over time.

<Technical effect>
As described above, in the temperature adjustment device of the present embodiment, the heat block 11 for the denaturing temperature zone has the convex portion 11b, the heat block 12 for the annealing temperature zone has the convex portion 12b, and the convex portions 11b and 12b are , contact only the channel through which the reaction solution flows when the temperature is lowered from the denaturation temperature zone to the annealing temperature zone. Further, the temperature control device of the present embodiment is set to the elongation temperature zone and includes a heat block 313 having a convex portion 313b. Touch the middle part of the road. By adopting such a configuration, it is possible to suppress the production of non-specific amplification products and prevent a decrease in PCR amplification efficiency.

[Fifth embodiment]
<Configuration of temperature control device>
FIG. 9(a) is a perspective view showing a temperature control device according to a fifth embodiment. As shown in FIG. 9( a ), the temperature adjustment device of this embodiment includes heat blocks 511 and 512 . The heat block 511 is set in the denaturation temperature range of nucleic acids, and the heat block 512 is set in the annealing temperature range of nucleic acids.

The heat block 511 has a body portion 511a and a plurality of protrusions 511b, and the heat block 512 has a body portion 512a and a plurality of protrusions 512b. The convex portion 511b and the convex portion 512b are formed in a tapered shape that tapers away from the main body portion 511a and the main body portion 512a, respectively. Since other points are the same as those of the first embodiment, description thereof is omitted.

Note that the heat blocks 511 and 512 of the present embodiment and the heat block 313 of the third embodiment can be used in combination.

<Configuration of nucleic acid amplification device>
FIG. 9(b) is a perspective view showing the nucleic acid amplification device according to the fifth embodiment. The nucleic acid amplification apparatus of this embodiment includes heat blocks 511 and 512, a microfluidic device 5, and an imaging section 50 (detection section). Since the microfluidic device 5 is the same as in the first embodiment, the description is omitted. Note that the meandering flow path 2 and the like of the microfluidic device 5 are omitted from the drawing.

The imaging unit 50 is arranged above the microfluidic device 5 and includes a light source 31 , a lens 32 , an optical filter 33 , a CCD 34 , a dichroic mirror 35 , an optical filter 36 and a lens 37 . Light emitted from the light source 31 passes through the lens 32 and the optical filter 33 , is reflected by the dichroic mirror 35 , and illuminates the microfluidic device 5 . When a fluorescence-labeled probe is added to the reaction solution and introduced into the meandering channel 2, the fluorescence from the reaction solution passes through the dichroic mirror 35, the optical filter 36 and the lens 37, and is detected by the CCD 34. In this way, by adding the fluorescently labeled probe to the reaction solution and photographing the fluorescence from the reaction solution with the CCD 34, the amplification of the object to be amplified can be confirmed, and real-time PCR can be performed.

Note that the position of the imaging unit 50 is not limited to above the microfluidic device 5, and may be any position where the meandering flow path 2 can be photographed. Similarly, the nucleic acid amplification apparatuses according to the first to fourth embodiments can also be provided with an imaging unit 50 to perform real-time PCR.

<Technical effect>
As described above, in the temperature adjustment device of this embodiment, the heat block 511 set in the denaturation temperature zone has a tapered convex portion 511b, and the heat block 512 set in the annealing temperature zone has a tapered convex portion. It has a portion 512b, and the convex portions 511b and 512b are in contact only with the channel through which the reaction solution flows when the temperature is lowered from the denaturation temperature zone to the annealing temperature zone. By adopting such a configuration, the same effect as in the first embodiment can be obtained. Further, even if the convex portions 511b and 512b are tapered, a temperature field can be formed in the meandering flow path 2 as in the first embodiment. It can be manufactured inexpensively and easily as compared with the case of forming. Furthermore, since the nucleic acid amplification apparatus of this embodiment can arrange the imaging part 50 in the position which can image the meandering flow path 2, real-time PCR can be implemented.

[Sixth embodiment]
<Configuration of microfluidic device>
FIG. 10(a) is a plan view showing the microfluidic device 65 of the nucleic acid amplification apparatus according to the sixth embodiment. The nucleic acid amplification apparatus of this embodiment includes a microfluidic device 65 and a temperature control device having heat blocks 611 to 613, which will be described later.

As shown in FIG. 10( a ), the microfluidic device 65 includes an inlet 1 , an outlet 3 , an alignment guide 4 , a reciprocating channel 6 , and valves 7 and 8 .

An inlet 1 and an outlet 3 are provided at the beginning and end of the reciprocating flow path 6, respectively. A reaction liquid is introduced from the inlet 1 into the reciprocating flow path 6, discharged from the outlet 3, and recovered.

The reciprocating flow path 6 meanders in the Y-axis direction between the inlet 1 and the valve 7 and between the valve 8 and the outlet 3 . Further, the reciprocating flow path 6 branches into two flow paths 61 and 62 between the valves 7 and 8 . By switching the valves 7 and 8 , the reaction liquid can be controlled to flow through either the flow path 61 or 62 .

The inlet 1 and the outlet 3 of the reciprocating flow path 6 are connected to pumps such as syringe pumps (not shown). The drive of the valves 7 and 8 and the drive of the pump are controlled by a drive mechanism (not shown) so that the reaction solution reciprocates in the reciprocating flow path 6 when thermal cycling of PCR is performed.

In this embodiment, the reaction liquid passes through the flow path 61 (first flow path) in the positive direction of the X axis when heading from the inlet 1 to the outlet 3, and the reaction liquid flows from the outlet 3 to the inlet 1. The driving of the valves 7 and 8 and the pump is controlled so that the flow path 62 (second flow path) is passed in the negative direction of the X-axis. More specifically, the reaction liquid introduced from the injection port 1 into the reciprocating channel 6 passes through the valve 7, the channel 61 and the valve 8, reaches the outlet 3, and is connected to the outlet 3. Reverse flow due to pump pressure. After that, the valves 7 and 8 are switched, and the reaction liquid passes through the valve 8, the flow path 62 and the valve 7 and reaches the injection port 1 again. After that, the valves 7 and 8 are switched again, and the pressure of the pump connected to the injection port 1 causes the reaction liquid to flow back, pass through the channel 61 and flow to the discharge port 3 . By repeating the above steps, the reaction liquid reciprocates in the reciprocating flow path 6 .

The alignment guide 4 is a mark for accurately positioning the microfluidic device 65 and the heat blocks 611 to 613 when they are brought into contact with each other.

<Configuration of temperature control device>
FIG. 10(b) is a plan view showing a state in which the heat blocks 611 to 613 and the microfluidic device 65 are in contact with each other. As indicated by dotted lines in FIG. 10B, the upper surface of the heat block 611 (first surface), the upper surface of the heat block 612 (second surface), and the upper surface of the heat block 613 (third surface) are XY planes. It lies within a plane (the plane including the first, second and third surfaces) and contacts the bottom surface of the microfluidic device 65 . The heat blocks 611, 613 and 612 are arranged in this order along the positive direction of the X-axis so as to be spaced apart from each other.

The microfluidic device 65 contacts the heat blocks 611 to 613 so that the straight portions of the channels 61 and 62 are aligned along the direction of arrangement of the heat blocks 611, 613 and 612 (X-axis direction). Heat block 611 (first member) is set to a nucleic acid denaturation temperature range, heat block 612 (second member) is set to a nucleic acid annealing temperature range, and heat block 613 (third member) is set to , is set to the elongation temperature range of the nucleic acid.

Thus, in the present embodiment, when the temperature of the reaction solution is lowered from the denaturation temperature zone to the annealing temperature zone, the reaction solution as a whole flows in the positive direction of the X axis (first direction), and rises from the annealing temperature zone to the denaturation temperature zone. The microfluidic device 65 and the heat blocks 611 to 613 are brought into contact with each other so that the heat flows in the X-axis negative direction (opposite direction to the first direction) as a whole when heated.

FIG. 11(a) is a perspective view showing the structure of heat blocks 611 to 613 according to the sixth embodiment. As shown in FIG. 11A, the heat block 611 has a main body portion 611a and convex portions 611b, and the heat block 612 has a main body portion 612a and convex portions 612b. The body portions 611a and 612a are substantially rectangular parallelepipeds. The convex portion 611b extends in the positive direction of the X-axis from the main body portion 611a. The convex portion 612b extends in the negative direction of the X-axis from the main body portion 612a and faces the convex portion 611b via an air layer (heat insulating layer).

The heat block 613 has a body portion 613a and a convex portion 613b. The convex portion 613b extends in the Z-axis positive direction from the main body portion 613a. The convex portion 613b (third member) is arranged outside the region between the facing surfaces of the convex portions 611b and 612b. The length of the projection 613b in the X-axis direction may be longer or shorter than the interval between the projections 611b and 612b. The length of the convex portion 613b in the Y-axis direction is equal to or greater than the width of the flow path 62 in the Y-axis direction, and the length of the body portion 611a of the heat block 611 in the Y-axis direction and the length of the body portion 612a of the heat block 612 in the Y-axis direction less than the direction length.

FIG. 11(b) is a perspective view showing a state in which the heat blocks 611 to 613 and the microfluidic device 65 according to the sixth embodiment are in contact with each other. As shown in FIG. 11(b), on the body portion 611a of the heat block 611, the channel between the injection port 1 and the valve 7 and the ends of the channels 61 and 62 on the negative direction side of the X axis are formed. On the body portion 612a of the heat block 612, the channel between the valve 8 and the discharge port 3 and the ends of the channels 61 and 62 on the positive side of the X axis are arranged. Further, the flow path 61 is in contact with the projections 611b and 612b, and the intermediate portion of the flow path 62 is in contact with the projection 613b. Therefore, when the temperature of the reaction solution is lowered from the denaturation temperature zone to the annealing temperature zone, the temperature is rapidly lowered, and when the temperature is raised from the annealing temperature zone to the denaturation temperature zone, the temperature is raised through the elongation temperature zone.

Further, as shown in FIG. 11B, the body portion 612a of the heat block 612 is provided with alignment pins 14 that can pass through the microfluidic device 65. As shown in FIG. When the microfluidic device 65 and the heat blocks 611 to 613 are brought into contact with each other, they can be positioned by the alignment pins 14 and the alignment guides 4 .

<Technical effect>
As described above, in the present embodiment, the heat block 611 for the denaturation temperature zone and the heat block 612 for the annealing temperature zone have convex portions 611b in contact with the flow path 61 in which the temperature of the reaction solution is lowered from the denaturation temperature zone to the annealing temperature zone. and 612b. The extension temperature zone heat block 613 has a protrusion 613b in contact with the intermediate portion of the channel 62 where the temperature of the reaction solution is raised from the annealing temperature zone to the denaturation temperature zone. With such a configuration, when the temperature of the reaction solution drops from the denaturation temperature zone to the annealing temperature zone, the temperature of the reaction solution drops sharply, and when the temperature rises from the annealing temperature zone to the denaturation temperature zone, the reaction solution The temperature is raised while ensuring a sufficient time to reach the elongation temperature zone. As a result, it is possible to suppress the production of non-specific amplification products and prevent the decrease in PCR amplification efficiency.

In this embodiment, as in the third embodiment, it is also possible to adopt a configuration in which the convex portion 611b of the heat block 611 and the convex portion 612b of the heat block 612 are not provided. In this case, the same effects as those of the third embodiment can be obtained. Also in this embodiment, as in the fifth embodiment, the imaging unit 50 can be provided above the microfluidic device 65, and a fluorescent labeled probe can be added to the reaction solution to perform real-time PCR.

[Modification]
The present disclosure is not limited to the embodiments described above, and includes various modifications. For example, the above-described embodiments have been described in detail in order to explain the present disclosure in an easy-to-understand manner, and do not necessarily include all the configurations described. Also, part of an embodiment can be replaced with the configuration of another embodiment. Moreover, the configuration of another embodiment can be added to the configuration of one embodiment. Moreover, a part of the configuration of each embodiment can be added, deleted or replaced with a part of the configuration of another embodiment.

As a modification, for example, the second embodiment can be combined with the third to sixth embodiments. In this case, the shape of the comb-shaped electrode 23 of the second embodiment is formed to be the same as the top surface shape of the heat block of each embodiment.

In addition, in the first to sixth embodiments, PCR for amplifying DNA has been described as an example, but each embodiment can also be applied to RT-PCR for detecting RNA such as viruses. Furthermore, instead of continuously flowing the reaction solution through the flow channel, droplets of the reaction solution are suspended in oil, and the oil is introduced into the flow channel to perform PCR (droplet PCR). can also be applied.

In the nucleic acid amplifiers of the first to sixth embodiments, the upper surface of the heat block is located on the XY plane and contacts the lower surface of the microfluidic device, but the orientation of the nucleic acid amplifier is not limited to this. The orientation of the nucleic acid amplification device may be, for example, upside down from the illustrated state, or may be rotated 90° from the illustrated state.

EXAMPLES Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are examples and the technical scope of the present disclosure is not limited to these examples.

[Example 1 and Comparative Example 1]
Using the nucleic acid amplification device of the first embodiment (Example 1) shown in FIG. 3B and the nucleic acid amplification device according to the conventional example (Comparative Example 1) shown in FIG. An experiment in which asflow PCR was performed will be described.

<Creation of microfluidic device>
A flat plate material made of cycloolefin copolymer was cut to obtain a substrate of 25 mm in width (X-axis direction)×92 mm in length (Y-axis direction)×2 mm in thickness (Z-axis direction). The length in the X-axis direction was 21 mm, the width (in the Y-axis direction) was 600 µm, the height (in the Z-axis direction) was 300 µm, and the grooves were meandered 40 times so that the interval between adjacent channels in the Y-axis direction was 1400 µm. formed on the substrate. A cycloolefin copolymer film having a thickness of 100 μm was adhered to the grooved surface by heat-sealing to form a meandering flow path. Thus, microfluidic devices according to Example 1 and Comparative Example 1 were obtained.

<Dimensions and layout of heat block>
In Example 1, a temperature control device having comb-shaped heat blocks 11 (denaturing temperature zone) and heat blocks 12 (annealing temperature zone) according to the first embodiment was prepared. The film of the microfluidic device was formed so that the body portions 11a and 12a were in contact with portions (bending portions) of 4 mm from both ends of the meandering channel, and the convex portions 11b and 12b were in contact only with the channel flowing in the positive direction of the X-axis. The heat blocks 11 and 12 were brought into contact with the surface to which the was attached.

The protrusions 11b and 12b were designed to protrude 6 mm from the body portions 11a and 12a, respectively. Therefore, in the channel in the positive direction of the X-axis, the length of contact between the heat block 11 in the denaturing temperature zone and the heat block 12 in the annealing temperature zone is 10 mm from both ends of the meandering channel, and the heat blocks 11 and 12 are in contact with each other. The length of the flow path (interval between the projections 11b and 12b) is 1 mm. On the other hand, in the channel in the negative direction of the X-axis, the length of contact between the heat block 11 in the denaturing temperature zone and the heat block 12 in the annealing temperature zone is 4 mm, respectively, and the length of the channel not in contact with the heat blocks 11 and 12 is 4 mm. The height is 13 mm.

In Comparative Example 1, a temperature control device having a substantially rectangular parallelepiped heat block 111 (denaturation temperature zone) and heat block 112 (annealing temperature zone) shown in FIG. 1(c) was used. The heat blocks 111 and 112 were brought into contact with the microfluidic device so that they were in contact with 4 mm portions (bending portions) from both ends of the meandering channel. That is, in both the channels flowing in the positive direction and the negative direction of the X axis, the length of the channel adjusted to the denaturation temperature zone and the annealing temperature zone is set to 4 mm, respectively, and the channel is not in contact with these temperature zones. was 13 mm.

<Preparation of reaction solution>
The human EGFR gene was extracted as template DNA for PCR amplification. Using TOYOBO's KOD DNA polymerase as a PCR enzyme, the PCR buffer attached to the kit was prepared so as to contain 3 mM Mg ²⁺ , and the human EGFR gene and 300 nM primer (forward side: 5'-cttggaggaccgtcgcttggt -3' (SEQ ID NO: 1), reverse side: 5'-tgcctccttctgcatggtattctttct-3' (SEQ ID NO: 2)) were added to prepare reaction solutions A according to Example 1 and Comparative Example 1.

<Implementation of PCR>
In Example 1, the heat block 11 was set at 95°C and the heat block 12 was set at 60°C. A silicon tube and a syringe pump were connected to the injection port of the microfluidic device, and continuous flow PCR was performed while flowing the reaction solution A at a flow rate of 1 μL/s. A silicon tube was connected to the outlet, and the reaction solution A was recovered.

As the reaction liquid A progresses through the meandering flow path, it undergoes a thermal cycle by receiving heat from the denaturation temperature zone and the annealing temperature zone. Since the meandering flow path meanders 40 times, the thermal cycle is repeated 40 times. Therefore, the temperatures of the heat blocks 11 and 12 do not have to be changed during PCR.

In Comparative Example 1, PCR was performed in the same manner as in Example 1, with the heat block 111 set at 95° C. and the heat block 112 set at 60° C.

<Temperature measurement of reaction solution>
In each of Example 1 and Comparative Example 1, the temperature of reaction solution A is lowered from the denaturation temperature zone to the annealing temperature zone in one of the channels through which reaction solution A flows in the positive direction of the X-axis during PCR. The temperature of the reaction solution A was measured. Temperature measurements were taken at intervals of 0.5 mm.

FIG. 12 is a graph comparing the temperature of the reaction liquid A during cooling in Example 1 and Comparative Example 1. FIG. The horizontal axis in the graph of FIG. 12 indicates the distance from the end of the meandering channel on the annealing temperature zone side (positive direction of the X axis), and the vertical axis indicates the temperature of the reaction solution A. In FIG.

As shown in FIG. 12, in the channel of Comparative Example 1, the temperature reached 90° C. at a position 13 mm from the end on the annealing temperature zone side, and 70° C. at a position 2 mm from the end on the annealing temperature zone side. Thus, the temperature range was from 70° C. to 90° C. over the length of 11 mm of the channel located between them. When the reaction liquid A is moved at a flow rate of 1 μL/s, the reaction liquid A advances through the channel by approximately 5 mm per second. That is, the temperature of the reaction liquid A reached 70° C. to 90° C. for 2 seconds or longer.

In the flow channel of Example 1, the temperature was 90° C. at a position 10.5 mm from the end on the annealing temperature zone side, and 70° C. at a position 6.5 mm from the end on the annealing temperature zone side. Therefore, the length of the flow path to reach the temperature range of 70° C. to 90° C. was 4 mm, and the time was less than 1 second. From the above, it was found that in Example 1, compared with Comparative Example 1, the temperature range of 70° C. to 90° C. was less than half, and the time was also less than half.

<Electrophoresis of amplified products>
The PCR amplification products of the human EGFR gene in Example 1 and Comparative Example 1 were each subjected to electrophoresis.

13 shows the results of electrophoresis of the amplification products in Example 1 and Comparative Example 1. FIG. Lane M shown on the left end of FIG. 13 is a size marker, which indicates the migration length of fragments with a base length of 25 to 1500 nucleotides.

As shown in FIG. 13, in the lane of Example 1 and the lane of Comparative Example 1, the target product, the amplified product of the human EGFR gene, is observed around 150 bases. However, in the lane of Comparative Example 1, amplification of a non-specific amplification product of 100 bases or less was observed in addition to the target product. On the other hand, no such non-specific amplification products were observed in the lane of Example 1, indicating that the generation of non-specific amplification products could be suppressed.

[Example 2 and Comparative Example 2]
PCR was performed using the nucleic acid amplification device of the third embodiment (Example 2) shown in FIG. 6B and the nucleic acid amplification device (Comparative Example 2) according to the conventional example shown in FIG. I will describe the experiments that I conducted.

<Creation of microfluidic device>
In the same manner as in Example 1 and Comparative Example 1, microfluidic devices according to Example 2 and Comparative Example 2 were produced.

<Dimensions and layout of heat block>
In Example 2, a temperature control device having heat block 311 (denaturation temperature zone), heat block 312 (annealing temperature zone) and heat block 313 (extension temperature zone) according to the third embodiment was prepared. The microfluidic device was brought into contact with the heat blocks 311 and 312 in such a manner that the heat blocks 311 and 312 were in contact with 4 mm from both ends of the meandering channel. Further, the length of the projection 313b of the heat block 313 in the X-axis direction is set to 7 mm, and the projection 313b is formed in the center of the flow path in the negative direction of the X-axis so that the heat blocks 311 and 312 are separated from each other by 3 mm. made contact.

Therefore, in the process in which the reaction solution flows in the positive direction of the X axis and the temperature is lowered from the denaturation temperature zone to the annealing temperature zone, 4 mm from the end of the meandering flow path is in contact with the heat block 311 in the denaturation temperature zone, and 13 mm from the end of the meandering flow path is in contact with the heat block. No contact, 4 mm contact the heat block 312 in the annealing temperature zone. On the other hand, in the process in which the reaction solution flows in the negative direction of the X-axis and the temperature rises from the annealing temperature zone to the denaturing temperature zone, 4 mm from the end of the meandering flow path contacts the heat block 312 in the annealing temperature zone, and 3 mm from the end of the heat block. , 7 mm is in contact with the heat block 313 in the extension temperature zone, 3 mm is not in contact with the heat block, and 4 mm is in contact with the heat block 311 in the denaturation temperature zone.

In Comparative Example 2, the same heat blocks 111 and 112 as in Comparative Example 1 were used.

<Implementation of PCR>
In Example 2, the heat block 311 was set at 95° C., the heat block 312 was set at 60° C., and the heat block 313 was set at 72° C., and the above reaction solution A was used to perform PCR.

In Comparative Example 2, PCR was performed in the same manner as in Example 2, with the heat block 111 set at 95° C. and the heat block 112 set at 60° C.

<Measurement of temperature of reaction solution>
For each of Example 2 and Comparative Example 2, the temperature of reaction solution A was raised from the annealing temperature range to the denaturation temperature range in one of the channels through which reaction solution A flows in the negative direction of the X-axis during PCR. The temperature of the reaction solution A was measured in the case of Temperature measurements were taken at intervals of 0.5 mm.

FIG. 14 is a graph comparing the temperatures of reaction liquid A during heating in Example 2 and Comparative Example 2. FIG. The horizontal axis in the graph of FIG. 14 indicates the distance from the end of the meandering channel on the annealing temperature zone side, and the vertical axis indicates the temperature of the reaction liquid A. In FIG.

As shown in FIG. 14, in the channel of Comparative Example 2, the temperature reached 70° C. at a position 13 mm from the end of the annealing temperature zone, and 75° C. at a position 16.5 mm from the end of the annealing temperature zone. Over the channel length of 3.5 mm, the temperature range was 70° C. to 75° C. where the thermostable DNA polymerase has sufficient activity. When the reaction liquid A is moved at a flow rate of 1 μL/s, the reaction liquid A advances through the channel by approximately 5 mm per second. Therefore, it took less than 1 second for the reaction solution A to reach the temperature of 70 to 75°C.

In the flow channel of Example 2, the temperature reached 70° C. at a position 9.5 mm from the end on the annealing temperature zone side, and reached 75° C. at a position 15.5 mm from the end on the annealing temperature zone side. That is, the length of the flow path for the temperature range of 70.degree. C. to 75.degree. From the above, in Example 2, compared to Comparative Example 2, the temperature range of 70 ° C. to 75 ° C., which is the elongation temperature zone, is reached in the process of moving the reaction solution A from the annealing temperature zone to the denaturation temperature zone. It was found that the area was approximately doubled and the time was also approximately doubled.

[Example 3 and Comparative Example 3]
Using the nucleic acid amplification device of the fourth embodiment (Example 3) shown in FIG. 8B and the nucleic acid amplification device according to the conventional example (Comparative Example 3) shown in FIG. An experiment in which asflow PCR was performed will be described.

<Creation of microfluidic device>
In the same manner as in Example 1 and Comparative Example 1, microfluidic devices of Example 3 and Comparative Example 3 were produced.

<Dimensions and layout of heat block>
In Example 3, a temperature control device having heat block 11 (denaturation temperature zone), heat block 12 (annealing temperature zone) and heat block 313 (extension temperature zone) according to the fourth embodiment was prepared. The film of the microfluidic device was formed so that the body portions 11a and 12a were in contact with portions (bending portions) of 4 mm from both ends of the meandering channel, and the convex portions 11b and 12b were in contact only with the channel flowing in the positive direction of the X-axis. The heat blocks 11 and 12 were brought into contact with the surface to which the was attached.

The protrusions 11b and 12b were designed to protrude 6 mm from the body portions 11a and 12a, respectively. Therefore, in the channel in the positive direction of the X-axis, the length of contact between the heat block 11 in the denaturing temperature zone and the heat block 12 in the annealing temperature zone is 10 mm from both ends of the meandering channel, and the heat blocks 11 and 12 are in contact with each other. The length of the flow path (interval between the projections 11b and 12b) is 1 mm. On the other hand, in the channel in the negative direction of the X-axis, the length of contact between the heat block 11 in the denaturing temperature zone and the heat block 12 in the annealing temperature zone is 4 mm, respectively, and the length of the channel not in contact with the heat blocks 11 and 12 is 4 mm. The height is 13 mm.

In addition, the length of the protrusion 313b of the heat block 313 in the X-axis direction was set to 7 mm, and the heat blocks 11 and 12 were spaced 3 mm apart from each other by 3 mm at the center of the channel through which the reaction solution flows in the negative direction of the X-axis. In contrast, the convex portion 313b is brought into contact.

That is, in the process in which the reaction solution flows in the negative direction of the X-axis and the temperature rises from the annealing temperature zone to the denaturing temperature zone, 4 mm from the end of the meandering flow path contacts the heat block 12 in the annealing temperature zone, and 3 mm from the heat block. , 7 mm is in contact with the heat block 313 in the extension temperature zone, 3 mm is not in contact with the heat block, and 4 mm is in contact with the heat block 11 in the denaturation temperature zone.

The heat blocks in the temperature control device of Comparative Example 3 are the same as the heat blocks 111 and 112 of Comparative Example 1, and thus the description thereof is omitted.

<Preparation of reaction solution>
The human KRAS gene was extracted as template DNA for PCR amplification. KAPA Biosystems KAPA Taq Extra DNA polymerase was used as a PCR enzyme, the PCR buffer attached to the kit was prepared to contain 2 mM Mg ²⁺ , and human KRAS gene and 500 nM primer (forward side: 5′- tcacattttcattatttttattataagg-3' (SEQ ID NO: 3), reverse side: 5'-gtatggtcaaggcactcttgcc-3' (SEQ ID NO: 4)) were added to prepare a reaction solution B.

<Implementation of PCR>
In Example 3, the heat block for the denaturation temperature zone was set at 95°C, the heat block for the annealing temperature zone was set at 60°C, and the heat block for the extension temperature zone was set at 72°C. Other conditions were the same as in Example 1, and PCR was performed while flowing the reaction solution B at a flow rate of 1 μL/s.

In Comparative Example 3, the heat block for the denaturation temperature zone was set at 95° C. and the heat block for the annealing temperature zone was set at 60° C., and PCR was performed on the reaction solution B in the same manner as above.

<Temperature measurement when the temperature of the reaction solution is lowered>
For each of Example 3 and Comparative Example 3, the temperature of reaction solution B is lowered from the denaturation temperature zone to the annealing temperature zone in one of the channels through which reaction solution B flows in the positive direction of the X-axis during PCR. The temperature of the reaction liquid B in the case was measured. Temperature measurements were taken at intervals of 0.5 mm.

FIG. 15 is a graph comparing the temperature of the reaction liquid B in Example 3 and Comparative Example 3 when the temperature was lowered. The horizontal axis in the graph of FIG. 15 indicates the distance from the end of the meandering channel on the annealing temperature zone side, and the vertical axis indicates the temperature of the reaction liquid B. In FIG.

As shown in FIG. 15, in the channel of Comparative Example 3, the temperature reaches 90° C. at a position 12.5 mm from the end of the annealing temperature zone, and 70° C. at a position of 1.5 mm from the end of the annealing temperature zone. Therefore, the temperature range is from 70° C. to 90° C. over the length of 11 mm of the channel located therebetween. When the reaction solution is moved at a flow rate of 1 μL/s, the reaction solution advances through the channel by approximately 5 mm per second. Therefore, the temperature of the reaction liquid reached 70° C. to 90° C. for about 2 seconds.

In the flow path of Example 3, the temperature is 90°C at a position 10.0 mm from the end of the annealing temperature zone, and 70°C at a position 6.0 mm from the end of the annealing temperature zone. is 4 mm, and the time is less than 1 second. From the above, it was found that in Example 3, the temperature range of 70° C. to 90° C. was less than half that of Comparative Example 3, and the time was also less than half.

<Temperature measurement during temperature rise of the reaction solution>
Furthermore, during PCR, in one of the channels in which the reaction solution B flows in the negative direction of the X axis, the temperature of the reaction solution B when the temperature of the reaction solution B is raised from the annealing temperature range to the denaturation temperature range is It was measured. Temperature measurements were taken at intervals of 0.5 mm.

FIG. 16 is a graph comparing the temperature of reaction liquid B during heating in Example 3 and Comparative Example 3. FIG. The horizontal axis in the graph of FIG. 16 indicates the distance from the end of the meandering channel on the annealing temperature zone side, and the vertical axis indicates the temperature of the reaction liquid B. In FIG.

As shown in FIG. 16, in the channel of Comparative Example 3, the temperature reached 70°C at a position 14.5 mm from the end of the annealing temperature zone, and 75°C at a position of 17.0 mm from the end of the annealing temperature zone. Over the length of 2.5 mm of the channel located between them, the temperature range was 70° C. to 75° C. where the thermostable DNA polymerase had sufficient activity. When the reaction solution B is moved at a flow rate of 1 μL/s, the reaction solution B advances through the channel by about 5 mm per second. Therefore, it took about 0.5 seconds for the temperature of the reaction liquid B to reach 70°C to 75°C.

In the channel of Example 3, the temperature is 70° C. at a position 11.0 mm from the end of the annealing temperature zone, and 75° C. at a position 15.5 mm from the end of the annealing temperature zone. was 4.5 mm, which was about twice that of Comparative Example 3. From the above, it was found that in Example 3, compared with Comparative Example 3, the temperature range of 70° C. to 75° C. was approximately doubled, and the time was also approximately doubled.

<Electrophoresis of amplified product>
Here, using the heat blocks 11, 12 and 313 according to the third embodiment, PCR was performed on the reaction solution B (human KRAS gene) under the same conditions as in Example 3, and did. Further, the PCR amplification products of the human KRAS gene in Example 3, Example 3-2 and Comparative Example 3 were each subjected to electrophoresis.

17 shows the results of electrophoresis of the amplification products of the human KRAS gene in Example 3, Example 3-2 and Comparative Example 3. FIG. Lane M is a size marker, showing the migration length of fragments with a base length of 25 to 1500 nucleotides.

As shown in FIG. 17, in Comparative Example 3, the target product, the amplified product of the human KRAS gene, is faintly observed around 100 bases. On the other hand, in Examples 3 and 3-2, the target product was clearly observed around 100 bases. From the above, in both the case of using the nucleic acid amplification device of the fourth embodiment (Example 3) and the case of using the nucleic acid amplification device of the third embodiment (Example 3-2), It was found that it is possible to improve the efficiency of PCR amplification.

[Example 4 and Comparative Example 4]
An experiment in which continuous flow PCR was performed using the nucleic acid amplification apparatus according to the sixth embodiment shown in FIG. 11(b) and amplification products were detected in real time will be described.

<Creation of microfluidic device>
A flat plate material made of cycloolefin copolymer was cut to obtain a substrate of 80 mm in width (X-axis direction)×30 mm in length (Y-axis direction)×2 mm in thickness (Z-axis direction). The length in the X-axis direction (the distance between the injection port 1 and the discharge port 3 in the X-axis direction) is 60 mm and the width (in the Y-axis direction) is 600 μm so that the reciprocating flow path 6 has the shape shown in FIG. 10(a). A groove with a height (in the Z-axis direction) of 300 μm was formed in the substrate. More specifically, between the inlet 1 and the valve 7 and between the valve 8 and the outlet 3, the grooves meander over 14 mm in the Y-axis direction. Between the valves 7 and 8, in order to form the flow paths 61 and 62, the grooves are divided into two so that the length of the straight portion in the X-axis direction is 20 mm and the interval between the adjacent flow paths is 1400 μm. branched. A cycloolefin copolymer film having a thickness of 100 μm was adhered to the substrate by heat sealing to the grooved surface. As a result, microfluidic devices according to Example 4 and Comparative Example 4 were obtained.

<Dimensions and layout of heat block>
In Example 4 and Comparative Example 4, a temperature control device having heat blocks 611 to 613 according to the sixth embodiment was prepared. The dimensions of the upper surfaces of the body portion 611a of the heat block 611 and the body portion 612a of the heat block 612 were 20 mm (in the X-axis direction)×20 mm (in the Y-axis direction). The convex portion 611b was designed to protrude from the body portion 611a in the positive direction of the X-axis by 8 mm, and the convex portion 612b was designed to protrude from the body portion 612a by 8 mm. The distance between the projections 611b and 612b was set to 4 mm. The length of the projection 613b of the heat block 613 in the X-axis direction is 12 mm, and the heat block 611 and the heat block 612 are arranged so as to be 4 mm apart from the main body 611a and the heat block 612, respectively. Therefore, the distance in the X-axis direction between the body portion 611a of the heat block 611 and the body portion 612a of the heat block 612 is 20 mm.

The microfluidic device and the heat blocks 611 to 613 were brought into contact with each other so that the straight portions of the flow paths 61 and 62 were arranged between the body portion 611a of the heat block 611 and the body portion 612a of the heat block 612. Therefore, the channel between the inlet 1 and the valve 7 contacts the body portion 611 a of the heat block 611 , and the channel between the valve 8 and the outlet 3 contacts the body portion 612 a of the heat block 612 . Further, the straight portion of the flow path 61 was brought into contact with the convex portions 611b and 612b, and the straight portion of the flow channel 62 was brought into contact with the convex portion 613b.

<Preparation of reaction solution>
The human EGFR gene was extracted as template DNA for PCR amplification. KAPA Biosystems KAPA Taq Extra DNA polymerase was used as a PCR enzyme, the PCR buffer attached to the kit was prepared to contain 2 mM Mg ²⁺ , and human KRAS gene and 500 nM primer (forward side: 5′- cctcacctccaccgtgca-3' (SEQ ID NO: 5), reverse side: 5'-aggcagccgaagggca-3' (SEQ ID NO: 6)) and FAM (fluorochrome)-labeled TaqMan (registered trademark) probe (5'-tcatcatgcagctc-3' (SEQ ID NO: 7)) was added to prepare a reaction solution C according to Example 4 and Comparative Example 4. By performing real-time PCR on such a reaction solution C, the T790M mutation of the EGFR gene can be detected.

<Implementation of real-time PCR>
In Example 4, the heat block 611 was set at 95° C. (denaturation temperature range), the heat block 612 was set at 60° C. (annealing temperature range), and the heat block 613 was set at 72° C. (extension temperature range). Moreover, an imaging unit similar to the imaging unit 50 shown in FIG.

PCR was performed by introducing the reaction solution C from the injection port 1, connecting a syringe pump to the injection port 1 and the discharge port 3, and switching the valves 7 and 8 45 times to cause the reaction solution C to reciprocate. When the temperature is lowered from the denaturation temperature zone to the annealing temperature zone by switching the valves 7 and 8, the reaction solution C flows through the channel 61, and when the temperature is raised from the annealing temperature zone to the denaturation temperature zone via the elongation temperature zone. , the reaction solution C was allowed to flow through the channel 62 . After holding the reaction solution C in the denaturation temperature zone for 3 seconds, the syringe pump switches the valves 7 and 8 to send the reaction solution C to the annealing temperature zone, holds it in the annealing temperature zone for 5 seconds, and then switches the valves 7 and 8. 8 was switched to send the reaction liquid C to the denaturing temperature zone. Real-time PCR was performed by measuring the fluorescence intensity of the reaction liquid C from above when the reaction liquid C passed through the elongation temperature zone by the imaging unit.

In Comparative Example 4, the heat block 613 was switched off so as not to supply heat to the flow path 62 . Other conditions were the same as in Example 4, and real-time PCR was performed on the reaction solution C.

18 is a graph showing the fluorescence intensity of reaction liquid C in Example 4 and Comparative Example 4. FIG. As shown in FIG. 18, in Example 4, amplification of the EGFR gene T790M mutant was confirmed by the rapid increase in fluorescence intensity after 20 cycles.

In Comparative Example 4, although the fluorescence intensity increased after the 20th cycle, the fluorescence intensity was about half that in Example 4. From this, in Example 4, when the reaction solution C is heated from the annealing temperature zone to the extension temperature zone to the denaturation temperature zone, the time to reach the extension temperature zone is sufficiently ensured, so the PCR amplification efficiency It was found that it is possible to improve the

Reference Signs List 1... inlet, 2... meandering channel, 3... outlet, 4... alignment guide, 5, 65... microfluidic device, 6... reciprocating channel, 7, 8... valve, 11, 12, 111, 112, 211, 212, 311 to 313, 511, 512, 611 to 613... Heat block 14... Positioning pin 200... Printed board 221, 222... Comb electrode 231, 232... Metal wiring 31... Light source 32 , 37... Lens, 33, 36... Optical filter, 34... CCD, 35... Dichroic mirror, 50... Imaging unit

Claims (16)

A temperature adjustment device for adjusting the temperature of a meandering flow path through which a reaction solution flows,
a first member having a first surface and set to a denaturation temperature zone for nucleic acids;
a second member having a second surface and set in the annealing temperature range of the nucleic acid;
a third member having a third surface and set to an elongation temperature zone of the nucleic acid;
A plane exists that includes the first surface, the second surface and the third surface, and
the first member, the third member and the second member are arranged in this order along a first direction parallel to the plane so as to be spaced apart from each other;
The length of the third surface in a second direction parallel to the plane and orthogonal to the first direction is the length of the first surface in the second direction and the length of the second surface. shorter than the length of
The third member is arranged so as to adjust only a portion of the meandering flow path in which the reaction liquid flows in a direction opposite to the first direction to the elongation temperature zone. temperature control device. The first member has a first protrusion projecting in the first direction,
the second member has a second protrusion that protrudes only in a direction opposite to the first direction and faces the first protrusion;
the first convex portion adjusts only the channel portion in which the reaction solution flows in the first direction in the meandering channel to the denaturation temperature zone;
the second convex portion adjusts only the channel portion in which the reaction solution flows in the first direction in the meandering channel to the annealing temperature range;
2. The temperature control device according to claim 1, wherein the third member is arranged outside the area between the facing surfaces of the first protrusion and the second protrusion. further comprising a plurality of said third members;
The first member and the second member extend in the second direction,
2. The temperature control device according to claim 1, wherein said plurality of said third members are arranged at predetermined intervals along said second direction. The first member has a plurality of first protrusions arranged in the second direction at the predetermined intervals and protruding in the first direction,
The second member has a plurality of second protrusions arranged in the second direction at the predetermined intervals and protruding in a direction opposite to the first direction to face the plurality of first protrusions. has
the first convex portion adjusts only the channel portion in which the reaction solution flows in the first direction in the meandering channel to the denaturation temperature zone;
the second convex portion adjusts only the channel portion in which the reaction solution flows in the first direction in the meandering channel to the annealing temperature range;
3. The plurality of first protrusions, the plurality of second protrusions, and the plurality of third members are alternately arranged in the second direction. 4. The temperature control device according to 3. The first member is a first electrode connected to a heat block set to the denaturing temperature zone,
The second member is a second electrode connected to a heat block set in the annealing temperature zone,
2. The temperature control device according to claim 1, wherein said third member is a third electrode connected to a heat block set in said elongation temperature zone. A temperature adjustment device according to claim 1;
a microfluidic device having the meandering flow path for a nucleic acid solution and in contact with the temperature control device in the plane;
The meandering channel is connected to a first channel through which the nucleic acid solution flows in the first direction, and a second channel is connected to the first channel and through which the nucleic acid solution flows in a direction opposite to the first direction. and a flow path of
Both ends of the first flow path are in contact with the first member and the second member, respectively;
Both ends of the second flow path are in contact with the first member and the second member, respectively;
A nucleic acid amplification device, wherein an intermediate portion of the second channel is in contact with the third member. The meandering flow path has a bend connecting an end of the first flow path and an end of the second flow path,
7. The nucleic acid amplifier according to claim 6, wherein said first member and said second member are in contact with said bent portion. The microfluidic device includes a valve that controls switching between the first channel and the second channel, and pumps connected to both ends of the channel,
7. The nucleic acid amplification apparatus according to claim 6, wherein the nucleic acid solution reciprocates in the meandering flow path by driving the valve and the pump. the nucleic acid solution contains a fluorescently labeled probe capable of binding to the nucleic acid;
7. The nucleic acid amplifier according to claim 6, further comprising a detection section for detecting fluorescence from said fluorescence-labeled probe bound to said nucleic acid. A temperature adjustment device for adjusting the temperature of a meandering flow path through which a reaction solution flows,
a first member having a first surface and set to a denaturation temperature zone for nucleic acids;
a second member having a second surface and set to a temperature range for annealing the nucleic acid, wherein a plane including the first surface and the second surface exists;
the first member and the second member are arranged along a first direction parallel to the plane so as to be spaced apart from each other;
The first member has a first protrusion projecting in the first direction,
The second member has a second protrusion projecting in a direction opposite to the first direction and facing the first protrusion ,
the first convex portion adjusts only the channel portion in which the reaction solution flows in the first direction in the meandering channel to the denaturation temperature zone;
The temperature control device , wherein the second convex portion adjusts only a portion of the meandering channel through which the reaction liquid flows in the first direction to the annealing temperature range . The first member has a plurality of first protrusions arranged at predetermined intervals in a second direction that is parallel to the plane and orthogonal to the first direction,
11. The temperature control device according to claim 10, wherein said second member has a plurality of said second projections arranged at said predetermined intervals in said second direction. The first member is a first electrode connected to a heat block set to the denaturing temperature zone,
11. The temperature control device according to claim 10, wherein said second member is a second electrode connected to a heat block set to said annealing temperature range. A temperature adjustment device according to claim 10;
a microfluidic device having the meandering flow path for a nucleic acid solution and in contact with the temperature control device in the plane;
The meandering channel is connected to the first channel through which the nucleic acid solution flows in the first direction, and the second channel is connected to the first channel and through which the nucleic acid solution flows in the opposite direction to the first direction. and a flow path of
Both ends of the first flow path are in contact with the first member and the second member, respectively;
Both ends of the second flow path are in contact with the first member and the second member, respectively;
The nucleic acid amplifier, wherein the first protrusion and the second protrusion are in contact only with the first channel. The meandering flow path has a bend connecting an end of the first flow path and an end of the second flow path,
14. The nucleic acid amplifier according to claim 13, wherein said first member and said second member are in contact with said bent portion. The microfluidic device includes a valve that controls switching between the first channel and the second channel, and pumps connected to both ends of the channel,
14. The nucleic acid amplification apparatus according to claim 13, wherein the nucleic acid solution reciprocates in the channel by driving the valve and the pump. the nucleic acid solution contains a fluorescently labeled probe capable of binding to the nucleic acid;
14. The nucleic acid amplifier according to claim 13, further comprising a detection section for detecting fluorescence from said fluorescence-labeled probe bound to said nucleic acid.

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