JP6004486B2 - Nucleic acid amplification method using microfluidic device - Google Patents

Description

  The present invention relates to a method for amplifying nucleic acid in a meandering flow path at an ultra-high speed. More specifically, the present invention provides a flow condition and a channel design for accurately controlling temperature and residual time using a continuous flow microfluidic system, and an ultrafast polymerase chain reaction (PCR). On how to perform.

  Polymer chain reaction (PCR), which uses a microchannel chip fabricated using semiconductor microfabrication technology, to amplify the target gene sequence region from a small amount of DNA sample at high speed and easily, has attracted attention. .

  As a conventional technique, for example, a nucleic acid amplification device having a meandering microchannel, which has a heat denaturation temperature zone (about 94 ° C.), an annealing temperature zone (about 60 ° C.), and an extension temperature zone (about 72 ° C.) In addition, a nucleic acid amplification device (Patent Documents 1 and 2) in which a PCR sample solution is fed into a meandering channel by a pump in the shape of a sample plug sandwiched between gases, and the reflux of the PCR sample solution is optimized. A method (Patent Document 3) that achieves high-speed gene amplification is disclosed.

In Patent Documents 1 and 2, high-speed temperature control is achieved by utilizing the vapor pressure difference generated at the interface with the gas before and after the PCR sample plug solution flowing in the flat microfluidic device.
(i) When the sample plug liquid is fed by gas discharge from the inlet, the continuous serpentine micro-channel causes a high internal pressure rise, so the flow velocity is near the inlet, near the center, and near the outlet of the serpentine channel. Remarkably different. Therefore, a complicated fluid control method is required to effectively perform fluid control based on the difference in vapor pressure.
(ii) When the sample plug liquid is sent by gas suction from the outlet, the high vapor pressure in the microchannel is released by pressure reduction due to suction from the outlet, so the vapor pressure difference in the microchannel is reduced. .
(iii) The effect of the vapor pressure difference is greatly affected by variations in PID control of the heater block in contact with the device and non-uniform heat conduction between the device and the heater contact surface.
(iv) When a plurality of sample plug liquids are sent by the segment flow method in a state separated by one cycle or more, each sample plug liquid is passed through the micro flow path where the vapor pressure difference is generated. However, it is technically difficult to continue liquid feeding while realizing the fluid control.

  In Patent Document 3, by utilizing the reflux of the sample solution itself, accurate temperature control and quick raising / lowering operation are possible. However, a complicated control system is necessary, and other biochemical reaction units are used. However, there is a problem that the degree of freedom is structurally difficult to handle.

  With the conventional technology, it is impossible to perform rapid and simple PCR that can be processed in the field, and a method capable of performing ultra-high-speed amplification has been desired.

JP2011-200193 JP 2013-055921 JP2011-250800

  An object of this invention is to provide the method for amplifying the nucleic acid in a flow path at ultra high speed.

  Therefore, in order to solve the above problems, the present inventor devised the structure of the flow path so that the viscosity of the fluid can be utilized, a sufficient reaction time is ensured in the PCR extension part, and the reaction liquid in the cooling part is Passing was performed in a short time so that a satisfactory nucleic acid elongation reaction could be performed with simple control.

The present invention provides the following nucleic acid amplification method and nucleic acid amplification apparatus.
Item 1. A meandering channel capable of performing a PCR reaction, an annealing temperature zone corresponding to a loop portion on the opposite side to a loop portion on one side of the meandering channel, an annealing temperature zone and a heat denaturing temperature zone A nucleic acid amplification apparatus comprising a heater capable of forming an extension temperature zone, and further a heat denaturation temperature zone, an extension temperature zone, and an annealing temperature zone, wherein the meandering channel has an extension portion, a heat denaturation portion, a cooling portion, and an annealing The cross-sectional area of the flow path is extended portion <cooling portion <heat-denatured portion ≦ annealing portion, heat-denatured portion is placed in the heat-denatured temperature zone, and stretched portion and cooled portion are placed in the stretched temperature zone. The nucleic acid amplification device in which the annealing part is disposed in the annealing temperature zone.
Item 2. Item 2. The nucleic acid amplification device according to Item 1, wherein an end of the heat denaturing part disposed in the heat denaturing temperature zone on the side of the extending part has the same flow path cross-sectional area as that of the extending part.
Item 3. The nucleic acid amplification device according to claim 1 or 2, wherein a PCR reaction solution discharge pump is connected to the meandering channel inlet, and a PCR reaction solution suction pump is connected to the meandering channel outlet.
Item 4. Items 1 to 3, wherein the heater is a flat plate heater made of stainless steel, and a flat plate microfluidic substrate having meandering channels is sandwiched between the flat plate heater and a magnet, and a stable contact between the heater and the substrate is ensured by magnetic force. The nucleic acid amplification device according to any one of the above.
Item 5. Item 5. The nucleic acid amplification method for performing PCR reaction by supplying a PCR sample solution to the meandering flow path of the nucleic acid amplification device according to any one of Items 1 to 4, wherein the PCR sample solution is in the form of a sample plug sandwiched between gases by a pump. A method for amplifying a nucleic acid, wherein the method is fed into a meandering channel and supplied into the meandering channel in a state separated by one cycle of PCR or more by a gas.
Item 6. Item 6. The nucleic acid amplification method according to Item 5, wherein the plurality of sample plugs are fed into the meandering channel so as to be located in the same channel selected from the group consisting of an extension unit, a heat denaturing unit, a cooling unit, and an annealing unit. .

  Realization of a system capable of testing the presence or absence of a gene with high sensitivity from a biological sample collected in a small amount at a medical site, such as genetic testing, is required. In the present invention, in PCR, which is a gene amplification technique, the form (shape) of a sample plug sandwiched between air is used, and the cross-sectional area of the meandering channel is defined as an extension portion <cooling portion <thermal denaturation portion ≦ annealing portion. Thus, the high-speed and high-efficiency compatibility that has been a problem in the continuous flow PCR using the flat plate-type microfluidic device up to now can be achieved with the vapor pressure of the sample liquid itself generated at the interface with the air before and after the sample plug. This can be realized by utilizing the viscosity of the fluid. As a result, there is a feature that contributes to the miniaturization and speeding up of the continuous flow PCR system that makes the best use of the advantages of the microfluidic device without requiring a special external device.

It is a figure which shows two types of microfluidic devices for PCR which utilized the shape of the microchannel for the fluid control of the sample plug liquid. The heater is brought into contact with the surface on which the microchannel is designed. FIG. 2 is a diagram illustrating a method of controlling the temperature for each region of a meandering channel for thermal cycling in the two types of PCR microfluidic devices shown in FIG. 1. In FIG. 2, temperature control 1 (about 95 ° C.) is a DNA heat denaturing heater block, temperature control 2 (about 72 ° C.) is an extension reaction heater block, and temperature control 3 (about 30-60 ° C.) is Heating block for annealing. It is a figure which shows the uniformity of heat conduction when using the stainless steel heater for a heater block, and making the flat-plate PCR microfluidic device shown in FIG. 1 contact by pinching with a magnet. It is a figure explaining the process which PCR sample plug liquid flows through the inside of the microfluidic device for PCR which is contacting the heater block, and performs a thermal cycle. It is a figure explaining the flow principle of the sample plug liquid which flows the inside of the microfluidic device for PCR under room temperature. Residual time distribution of the flowing sample plug liquid in a PCR microfluidic device that uses the shape of the flow path for fluid control and a PCR microfluidic device that does not use it for fluid control at room temperature (about 20 ° C) and under discharge FIG. Residual time distribution of the flowing sample plug liquid in a PCR microfluidic device that uses the shape of the flow path for fluid control and a PCR microfluidic device that does not use it for fluid control at room temperature (about 20 ° C) and under discharge It is the table | surface and figure which compared these. It is a figure explaining the flow principle of the PCR sample plug liquid which flows in the microfluidic device for PCR under a thermal cycle. We compared the residual time distribution of the flowing PCR sample plug liquid between the PCR microfluidic device that uses the shape of the flow path for fluid control and the PCR microfluidic device that does not use it for fluid control under thermal cycling and discharge. FIG. We compared the residual time distribution of the flowing PCR sample plug liquid between the PCR microfluidic device that uses the shape of the flow path for fluid control and the PCR microfluidic device that does not use it for fluid control under thermal cycling and discharge. It is a table | surface and a figure. It is the figure which compared the residual time distribution of the PCR sample plug liquid when changing the cross-sectional area of the cooling part of a microchannel, and the cross-sectional area of an expansion | extension part under thermal cycle and discharge. Residual time distribution of the flowing sample plug fluid in a PCR microfluidic device that uses the shape of the flow path for fluid control and a PCR microfluidic device that does not use fluid control at room temperature (approximately 20 ° C) and under suction FIG. Residual time distribution of the flowing sample plug fluid in a PCR microfluidic device that uses the shape of the flow path for fluid control and a PCR microfluidic device that does not use fluid control at room temperature (approximately 20 ° C) and under suction It is the table | surface and figure which compared these. Comparison of the residual time distribution of the flowing PCR sample plug liquid between the PCR microfluidic device that uses the shape of the flow path for fluid control and the PCR microfluidic device that does not use the fluid control under thermal cycling and suction FIG. Comparison of the residual time distribution of the flowing PCR sample plug liquid between the PCR microfluidic device that uses the shape of the flow path for fluid control and the PCR microfluidic device that does not use the fluid control under thermal cycling and suction It is a table | surface and a figure. It is the figure which compared the residual time distribution of the PCR sample plug liquid when changing the cross-sectional area of the cooling part of a microchannel, and the cross-sectional area of an expansion | extension part under thermal cycle and attraction | suction. Using a microfluidic device for PCR that uses the shape of the flow channel for fluid control and a microfluidic device for PCR that does not use the shape of the flow channel for fluid control, change the volume of the PCR sample plug solution and discharge from the inlet It is a figure which shows the comparison of the fluorescence intensity obtained with amplification of the DNA fragment of a real-time PCR kit as a result of performing PCR. Using a microfluidic device for PCR that utilizes the shape of the flow channel for fluid control and a microfluidic device for PCR that does not utilize the shape of the flow channel for fluid control, the amount of the PCR sample plug solution is changed and suctioned from the outlet. It is a figure which shows the comparison of the fluorescence intensity obtained with amplification of the DNA fragment of a real-time PCR kit as a result of performing PCR. Using a PCR microfluidic device that utilizes the shape of the flow path for fluid control and a PCR microfluidic device that does not utilize the shape of the flow path for fluid control, the residual time distribution of the sample plug fluid flowed by the segment flow is calculated. It is the figure compared. Results of PCR using the segment flow shown in Fig. 19 using a PCR microfluidic device that utilizes the shape of the flow channel for fluid control and a PCR microfluidic device that does not utilize the shape of the flow channel for fluid control It is a figure which shows the comparison of the fluorescence intensity obtained with amplification of the DNA fragment of a real-time PCR kit, and the result by electrophoresis.

  The present invention relates to a method and apparatus for amplifying a nucleic acid by polymerase chain reaction (PCR) at a very high speed in a microchannel that repeatedly passes through a plurality of temperature zones while meandering. More specifically, the present invention relates to a plate-type microfluidic system for continuous flow PCR, using a meandering flow path, a heat denaturing portion and an extension for a PCR sample solution sandwiched between gases and flowing as a sample plug. The flow path design for accurately controlling the temperature and residence time in each reaction section of the cooling section, cooling section, and annealing section, and the liquid feed conditions are set appropriately to amplify the nucleic acid in the flow path at ultra-high speed The present invention relates to an apparatus and a method.

  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 to which this invention is related. Next, the present invention will be specifically described with reference to embodiments, but the present invention is not limited to these embodiments, and is similar or equivalent to that described in this specification. Any of a number of methods and materials can be used in practicing the present invention. Preferred materials and methods are described below.

  In the present specification, examples of the plurality of temperature zones include three temperature zones: a heat denaturing temperature zone, an annealing temperature zone, and an extension temperature zone. These three temperature zones may be clearly distinguished by a heating device (heater) or a cooling device, etc., and adjacent temperature zones (thermal denaturation temperature zone and extension temperature zone, or extension temperature zone and annealing temperature zone). The boundary may not be clear. In the specific embodiment shown in the drawings, the heat denaturation temperature zone is set to 95 ° C., the extension temperature zone is set to 72 ° C., and the annealing temperature zone is set to 30 ° C. However, as long as the nucleic acid amplification reaction (PCR) proceeds, The temperature can be raised or lowered. Although the extension temperature zone and the annealing temperature zone may seem to be one temperature zone that is apparently integrated, in this specification, even in such a case, the higher temperature part is referred to as the extension temperature zone. The lower temperature part is expressed as an annealing temperature zone.

  The meandering channel of the present invention is formed in a PCR microfluidic substrate. This substrate is preferably flat. The nucleic acid amplification substrate of the present invention comprises a PCR microfluidic substrate and a heater (preferably a flat plate heater). In a preferred embodiment, the PCR microfluidic substrate is sandwiched between a heater and a magnet and is opposite to the heat denaturation temperature zone. The temperature in the annealing temperature zone and the heat denaturing temperature zone extension temperature zone corresponding to the loop portion of the above is stably held at a predetermined temperature.

  In the embodiment shown in FIG. 3a, a magnet is provided on a planar PCR microfluidic substrate on a planar stainless steel heater. With this magnet, the substrate and the heater are in close contact, and a uniform temperature can be realized.

  A schematic view of a nucleic acid amplification apparatus for carrying out the present invention is shown in FIG. This apparatus includes an inlet for a PCR reagent, a meandering channel, and an outlet for a solution (sample plug solution) after the PCR reaction. One cycle of PCR can be performed by one unit consisting of a pair of loop parts (high temperature heat denaturing part and low temperature annealing part) on both sides and two linear parts (extension part and cooling part) connecting the loop parts. it can.

  As shown in FIG. 4, the heat denaturing part corresponds to “thermal denaturing temperature zone (95 ° C. heater block)”, and the annealing part corresponds to “annealing temperature zone (30 ° C. heater block)”. The two straight line sections (extension part and cooling part) connecting the two lines correspond to the “extension temperature zone (72 ° C. heater block)”. The nucleic acid amplification apparatus used in the present invention performs a nucleic acid amplification reaction in a meandering channel in which a large number of these units are connected. When one PCR sample solution advances through the meandering channel, a number of cycles of PCR reaction are performed according to the number of units in the meandering channel, and a necessary number of PCR products are formed from the outlet of the meandering channel. Released.

  Since the elongated portion of the meandering channel of the present invention has the smallest channel cross-sectional area and has enough space, the volume of the elongated portion can be increased by meandering the elongated portion design as shown in FIG. 2b, for example. Can do. Since the channel cross-sectional area of the extension portion is small, the sample plug liquid slowly flows through the extension portion due to the viscous force. If a large amount of sample plug solution is flowed to the heat-denaturing part (95 ° C), it may remain in the heat-denaturing part (95 ° C) for a long time (for example, 1 minute or more) and bubbles may be generated. If the flow time is such that no bubbles are generated (usually 30 seconds or less, preferably 20 seconds or less, more preferably 10 seconds or less), the nucleic acid amplification reaction may be a problem even if the amount of the sample plug solution increases. Can be done without.

  If the volume of the sample plug solution increases, nucleic acid amplification takes longer and the remaining time in the annealing section also becomes longer. Therefore, the temperature of the annealing section is increased from 30 ° C (Fig. 2) to about 55 ° C. Just do it. The temperature of the sample plug solution supplied from the annealing part to the extension part is preferably about 55 to 60 ° C., and the temperature of the annealing part can be appropriately set so as to be such a temperature. For example, the temperature of the annealing part may be set in a temperature range of about 30 ° C. to 60 ° C. according to the amount of the sample plug solution.

  The meandering flow path of the nucleic acid amplification device of the present invention is composed of an extending part, a heat denaturing part, a cooling part, and an annealing part, and the cross-sectional area of the flow path is extending part <cooling part <thermal denaturing part ≦ annealing part. In particular, it is important that the cross-sectional area of the flow path is the extension portion <the cooling portion. By adopting such a configuration, the time for moving the extension part is lengthened, the time for moving the cooling part is shortened, a sufficient time is secured for the nucleic acid extension reaction in the extension part, and the liquid feeding in the cooling part is performed. Time is reduced. When the amount of the plug-like sample liquid is small, a linear flow path is sufficient as shown in FIG. 2a. However, when the amount of the plug-like sample liquid is large, the flow path of the elongated part is It may be meandered as shown in FIG. 2b. In the present invention, it is important that the plug-like sample solution can exist independently in each part (thermal denaturation part, extension part, cooling part, annealing part), and the volume in the extension part of the extension part having the smallest channel cross-sectional area. Needs to be larger than the volume of each plug-like sample solution. Since the elongated portion has the smallest channel cross-sectional area and the volume in the channel, the volume in the channel can be increased by meandering the channel as shown in FIG. 2b.

  As shown in FIG. 4a, the end of the heat denaturing part in the heater block at 95 ° C. has a channel cross-sectional area that is narrower than that of the extending part. This is because the sample solution slowly passes through the thin channel portion at 95 ° C. to ensure a sufficient time for heat denaturation. It is also shown in the cross-sectional views of FIGS. 1A and 1B that the cross-sectional areas of the extension part and the cooling part are smaller than the cross-sectional areas of the heat-denaturing part and the annealing part.

  FIG. 5 shows an outline of the liquid feeding principle in the apparatus of the present invention. In the annealing section, the PCR sample plug solution is moved and moved by the positive pressure of the pump (i), and the liquid feeding from the annealing section having a large channel cross-sectional area to the extension section having a small channel cross-sectional area moves quickly by capillary force. (ii). Since the cross-sectional area of the flow path is small in the extension part, the plug liquid moves slowly due to the viscosity, and a sufficient extension time is secured (iii). When the boundary between the extension portion and the heat denaturation portion is set in the heat denaturation temperature region, the liquid feeding in this region is performed very slowly, so that the time necessary for the heat denaturation is sufficiently secured in this portion (iv). When the plug liquid is sent to the heat-denaturing part having a large flow path cross-sectional area, the plug liquid quickly moves due to the positive pressure of the pump that is raised during the slow movement before the plug liquid (v). The movement from the heat denaturing part to the cooling part and the movement in the cooling part are quickly performed by a high positive pressure and capillary force (vi), (vii). Since the annealing section has a larger channel cross-sectional area, the sample plug liquid moves more slowly than the cooling section, and the time required for annealing is secured. As shown in FIGS. 6a and 9a, the meandering channel of the nucleic acid amplification device of the present invention has a cross-sectional area of the extension part <cooling part <thermal denaturing part ≦ annealing part so that the sample plug liquid moves. The time is extended part> annealing part> thermal denaturing part> cooling part, and the reaction time distribution is ideal for performing nucleic acid amplification reaction, so that nucleic acid amplification reaction can be performed reliably while realizing high speed. .

  The pump used in the present invention can be a normal pump, and is not particularly limited. For example, a small diaphragm pump (mp6 piezoelectric diaphragm micropump) can be preferably used. This diaphragm pump is a pump capable of both discharging and sucking gas and liquid, and can be used for both discharging at the inlet of the meandering channel and suction at the outlet.

  FIG. 6 shows the residual time distribution of the sample plug liquid at room temperature, and FIG. 9A shows the residual time distribution during the thermal cycle. When the shape of the channel is not utilized, that is, when the channel cross-sectional area is the extension portion = cooling portion = thermal denaturing portion = annealing portion, the remaining time is all the same at room temperature (FIG. 6a). The remaining time varies greatly (FIG. 9a). When the flow path shape is utilized, the remaining time of the extension part and the heat-denaturing part becomes longer and the remaining time of the cooling part and the annealing part becomes shorter under room temperature conditions where the vapor pressure of the plug liquid does not contribute. This change in the residual time pattern is ideal for achieving high speed while reliably performing the nucleic acid amplification reaction. As shown in FIG. 9a, the remaining time of the extension part and the annealing part is greatly extended compared to room temperature (FIG. 6a) without using the flow path shape of the present invention during the thermal cycle, and the residual heat denaturation part. The time is slightly reduced and the remaining time of the cooling part is greatly reduced. This is an effect due to the vapor pressure in the meandering flow path. By superimposing the effect of the channel shape on this, the remaining time of the sample plug liquid in the extension part, the heat denaturing part, the cooling part, and the annealing part can be made more ideal.

As shown in FIG. 20, in the PCR using the nucleic acid amplification device of the present invention, an amplified nucleic acid is obtained, but the amplified nucleic acid is obtained when the device of FIG. Was not obtained. This is an effect of the present invention in which the remaining time in each cycle is constant.
As a PCR sample solution, a standard PCR kit can be used. For the amplification reaction (PCR) of nucleic acid, a real-time PCR method, an RT-PCR method or the like can be used.

  Next, using the nucleic acid amplification device of the present invention having a 40-cycle meandering channel and the comparison target nucleic acid amplification device having a constant channel cross-sectional area, the remaining in each cycle when the sample plug solution is fed at room temperature The time distribution is shown in FIG. As shown in FIGS. 7a and 7b, the nucleic acid amplification apparatus of the present invention has almost one cycle of remaining time from the inlet to the outlet and the remaining time of each of the cooling part, annealing part, extension part and heat denaturing part. Although it does not change, in a device with a constant channel cross-sectional area (FIG. 7b), the remaining time becomes the longest when the number of cycles is around 20, and the remaining time rapidly decreases when approaching the outlet (40 cycles). . This change in the remaining time similarly occurs in the case of liquid feeding under the thermal cycle (FIG. 10), and in the apparatus of the present invention, the variation in the remaining time becomes smaller under the thermal cycle (FIG. 7a, FIG. 10a comparison). In the device of the comparative example in which the flow path cross-sectional area is constant, the fluctuation of the remaining time is further larger under the thermal cycle (comparison of FIGS. 7b and 10b). As described above, the apparatus and method of the present invention can reliably amplify a nucleic acid when a large number of PCR cycles are performed. However, if the flow path cross-sectional area is constant, the required remaining time particularly near the outlet is obtained. Not only is it not ensured, but the amplification becomes poor, and the remaining time is longer than necessary near the center of the meandering flow path, which hinders speeding up.

The influence of the channel cross-sectional area of the extension part and the cooling part on the remaining time is shown in FIGS. Even if the flow path cross-sectional area of the extension part and the cooling part is the same (0.25 mm ² ), the remaining time of the cooling part is sufficiently short under the thermal cycle, but the extension part and the cooling part are from the viewpoint of speeding up the reaction. It is not sufficient if the flow path cross-sectional areas are the same, and it is important for speeding up the reaction that the cross-sectional area of the cooling part is made larger than that of the extension part and the remaining time of the cooling part is sufficiently shorter than that of the extension part.

  FIG. 12 shows the remaining time at room temperature and under suction. Compared with FIG. 6 (under room temperature discharge), the remaining time changes in the same manner due to suction and discharge, and the degree of change in the remaining time is slightly larger under discharge in FIG.

  Further, FIGS. 14 and 15 show changes in the remaining time under thermal cycling and suction. From FIG. 14, it can be seen that the remaining time of the cooling section is greatly shortened when the flow channel shape of the present invention is utilized. Furthermore, comparing FIG. 15 and FIG. 10, it can be seen that the rate of change in the remaining time depending on the number of cycles is smaller when the thermal cycle is sucked (FIG. 15) than when the thermal cycle is discharged (FIG. 10).

  17 and 18 show changes in the amount of PCR sample plug solution using a PCR microfluidic device that utilizes the shape of the flow channel for fluid control and a PCR microfluidic device that does not utilize the shape of the flow channel for fluid control. As a result of performing PCR by discharging from the inlet, a comparison of the fluorescence intensities obtained with the amplification of the DNA fragment of the real-time PCR kit is shown. The channel volume of the extension is 2 to 3 μL. In the case of a 1 μL or 2 μL sample plug solution, a sufficient fluorescence intensity can be obtained. However, when the amount of the sample plug solution is 3 μL or more, the remaining time in the extension portion is shortened, so that the fluorescence intensity gradually decreases. Therefore, a device having a channel shape as shown in FIGS. 2a and 2b can be used depending on the amount of the sample plug solution.

  FIG. 17 shows the case where the PCR sample plug solution is put into the inlet with a syringe and discharged from the inlet with a pump. FIG. 18 shows the results when the sample plug solution for PCR is put into the inlet with a syringe and sucked with a pump from the outlet. It was confirmed that a nucleic acid amplification product can be obtained by either discharging from the pump from the inlet or suction from the pump from the outlet.

  FIG. 19 compares the residual time distribution of the sample plug fluid flowed by the segment flow. The flow path structure of the present invention (FIG. 19a) has a longer remaining time than the flow path structure of the comparison target (FIG. 19b). It shows that it is excellent in the small fluctuation, and that it is important to send a plurality of sample plug liquids by the segment flow at the same interval (phase). FIG. 20 shows that a sufficient amount of the fluorescent substance as a nucleic acid amplification product is obtained in the sample liquids 1 and 2 according to FIG. 19a, whereas a sufficient amount of the fluorescent substance as a nucleic acid amplification product is obtained in the sample liquids 3 and 4 according to FIG. Indicates that it cannot be obtained. This result shows that control of the structure of the meandering channel (particularly the channel cross-sectional area) and the distance between the sample plug liquids is very important.

Claims (6)

A meandering channel capable of performing a PCR reaction, an annealing temperature zone corresponding to a loop portion on the opposite side to a loop portion on one side of the meandering channel, an annealing temperature zone and a heat denaturing temperature zone A nucleic acid amplification apparatus comprising a heater capable of forming an extension temperature zone, and further a heat denaturation temperature zone, an extension temperature zone, and an annealing temperature zone, wherein the meandering channel has an extension portion, a heat denaturation portion, a cooling portion, and an annealing The cross-sectional area of the flow path is extended portion <cooling portion <heat-denatured portion ≦ annealing portion, heat-denatured portion is placed in the heat-denatured temperature zone, and stretched portion and cooled portion are placed in the stretched temperature zone. The nucleic acid amplification device in which the annealing part is disposed in the annealing temperature zone. The nucleic acid amplification device according to claim 1, wherein an end of the heat denaturing part disposed in the heat denaturing temperature zone on the side of the extending part has the same channel cross-sectional area as the extending part. The nucleic acid amplification device according to claim 1 or 2, wherein a PCR reaction solution discharge pump is connected to the meandering channel inlet, and a PCR reaction solution suction pump is connected to the meandering channel outlet. The heater is a flat plate heater made of stainless steel, and sandwiches a flat plate microfluidic substrate having meandering flow paths with a flat plate heater and a magnet, and ensures a stable contact between the heater and the substrate by magnetic force. 4. The nucleic acid amplification device according to any one of 3. 5. A nucleic acid amplification method for performing a PCR reaction by supplying a PCR sample solution to the meandering flow path of the nucleic acid amplification device according to claim 1, wherein the PCR sample solution is pumped in the form of a sample plug sandwiched between gases. The nucleic acid amplification method is characterized in that the solution is fed into the meandering channel by the gas and supplied into the meandering channel in a state separated by one cycle of PCR or more by the gas. The nucleic acid amplification according to claim 5, wherein the plurality of sample plugs are fed into the meandering flow path so as to be located in the same flow path selected from the group consisting of an extension part, a heat denaturing part, a cooling part, and an annealing part. Method.