JP5717235B2 - Nucleic acid amplification method - 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 relates to a method for performing polymerase chain reaction (PCR) using a continuous flow microfluidic system.

  Nucleic acid detection is central to various fields such as pharmaceutical research and development, forensic medicine, clinical testing, and identification of crops and pathogenic microorganisms. The ability to detect various diseases including cancer, microbial infections, genetic markers based on molecular phylogenetic analysis, disease and onset risk diagnosis, marker search, food and environmental safety assessment, crime verification, and It has become a universal technology for many other technologies.

  One of the most powerful basic techniques for detecting a small amount of nucleic acid with high sensitivity is a technique for analyzing a product obtained by exponentially replicating and amplifying a part or all of a nucleic acid sequence. PCR is probably the most widely used of the various amplification techniques.

PCR is a powerful technique for selectively amplifying a specific region of DNA. Using PCR, millions of copies of DNA fragments can be generated from a single template DNA for a target DNA sequence in the template DNA. PCR repeats a three-phase temperature condition called a thermal cycle to denature DNA into single strands, anneal denatured DNA single strands and primers, and extend primers with a thermostable DNA polymerase enzyme. Individual reactions are repeated sequentially. This cycle is repeated until a sufficient copy number is obtained for analysis. In principle, it is possible to double the number of copies in one cycle of PCR. In fact, as the thermal cycle continues, the concentration of the amplified DNA product eventually stops as the concentration of the required reaction reagent decreases. For general details of PCR, see "Clinical Applications of PCR", Dennis Lo (edited), Humana Press (Totowa, NJ) (1998), and "PCR Protocols A Guide."
to Methods and Applications ", M. et al. A. Innis et al. (Edit), Academic Press Inc. (San Diego, Calif.) (1990).

  In the last 10 years, for example, many high-productivity approaches for performing analysis and chemical reaction using a microfluidic device manufactured by applying semiconductor microfabrication technology and the like have been developed and reported. Under these trends, microfluidic devices for DNA amplification by PCR have also been developed, and thermal cycling of samples is usually done in one of three ways. In the first method, a sample solution is introduced into the device, and temperature cycling is performed over time while the solution is held in the same portion. This is very similar to a conventional PCR instrument. In the second method, a plurality of spatially separated temperature zones are connected by a micro flow channel, and the sample solution is alternately moved so as to stop on these temperature zones for a predetermined time. . Similarly, the third method is a method called continuous flow PCR in which the sample solution moves through a plurality of temperature zones that are spatially separated through a microchannel, but the sample solution is continuously fed without stopping. .

As an example of the first method, Lagally et al. (Anal Chem 73: 565-570 (2001)), Minutani and Nagai et al. (Patent No. 3041423 and Anal Chem 73: 1015-1019 (2001)) are: 280nl and 86p respectively
It is demonstrated that template DNA can be amplified and detected in a very small PCR chamber such as 1. In these references, the heat capacity is reduced by reducing the amount of sample and the thermal cycle is aimed at speeding up. However, since there is a limit to reducing the heat capacity of the chamber and the heater itself, in order to perform a sufficient amplification reaction, At least about 30 seconds were required per cycle.

  As a second method, for example, Enzelberger et al. (US Pat. No. 6,960,437) describes a microfluidic device comprising a circular rotating channel having three temperature zones. Excellent in that each temperature time can be set freely and can be thermally cycled, and a number of integrated valves and pumps are used to introduce samples and pump them into their temperature zones using a pump. Is used.

  Shenderov et al. (PCT Published Patent Application No. WO2006 / 124458) use electrostatic droplet driving to alternate sample droplets to two or more temperature zones provided in a microchannel. A PCR device based on the method of moving to is described. This technology uses droplet drive with a valve for injecting and stopping the sample solution and an electrode array with insulation coating instead of the valve. Both technologies are based on precise program control. .

  On the other hand, as a third method, Kopp et al. (Science 280: 1046-1048 (1998)) have three meandering channels with 95 ° C. (denaturation), 72 ° C. (extension), and 60 ° C. (annealing). Continuous flow PCR is demonstrated using a glass substrate that passes through a constant temperature zone. Since this PCR technique is not based on heating the entire surface of the reaction vessel, it is determined by the flow rate, not the heating / cooling rate that is affected by the heat capacity of the flow path or the heater itself. In PCR, the process of denaturation and annealing is known to be a very fast reaction that is completed within 1 second, so it is expected that high-speed PCR can be realized by increasing the flow rate. Since the amplification efficiency is remarkably lowered because the reaction time necessary for the extension reaction cannot be sufficiently taken, after all, one minute or more is required per cycle to perform sufficient amplification.

  The amplification efficiency of DNA is also reduced by the formation of by-products other than the target DNA fragment, which wastes the necessary reaction reagent and inhibits the production of the DNA fragment to be originally amplified. Although the annealing temperature in PCR is determined based on the GC (guanine and cytosine) content of the primer DNA sequence, undesirable by-products in such PCR are from a denaturation temperature of 95 ° C. to an annealing temperature of about 60 ° C. In the middle of the process, the DNA sequence is generated as an unintended DNA sequence that binds stably even at a temperature higher than the annealing temperature, a primer dimer that is generated by annealing between primers and the extension reaction proceeds.

  In continuous flow PCR, since it is easy to form each temperature region uniformly on a glass substrate, the temperature zones of 95 ° C., 72 ° C., and 60 ° C. are arranged in order. The temperature region is arranged in the center. Therefore, if the temperature range of 72 ° C. is extended in order to perform a sufficient extension reaction, the process of lowering the temperature from 95 ° C. to about 60 ° C. is also prolonged, and by-products such as primer dimers are amplified. The amplification efficiency of the DNA fragment is significantly reduced. Therefore, in the high-speed and high-efficiency continuous flow PCR, among the three-phase temperature conditions in the thermal cycle, the extension reaction at 72 ° C. sandwiched between about 60 ° C. and 95 ° C. is made slow, while 95 on the meandering channel. It is necessary to feed the process at high speed until it returns in the order of ° C, 72 ° C, and about 60 ° C.

Park et al. (Anal Chem 75: 6029-6033 (2003)) is a continuous flow PCR using a polyimide coated fused silica capillary with an inner diameter of 100 μm spirally wound around three cylindrically formed temperature control blocks. Explains the device. Unlike a flat microfluidic device used in general continuous flow PCR, this shape is not suitable for disposable applications because the capillary and the heater are integrated, but it is between 95 ° C and about 60 ° C. It is worth noting that an ideal thermal cycle is possible because a temperature range of 72 ° C. does not enter the temperature range.

Patent No. 3041423 US Pat. No. 6,960,437 PCT Published Patent Application No. WO2006 / 124458

“Clinical Applications of PCR”, Dennis Lo (edited), Humana Press (Totowa, NJ) (1998) “PCR Protocols A Guide to Methods and Applications”, M.M. A. Innis et al. (Edit), Academic Press Inc. (San Diego, California) (1990) Lagally et al. (Anal Chem 73: 565-570 (2001)) Nagai et al. (Anal Chem 73: 1015-1019 (2001)) Kopp et al. (Science 280: 1046-1048 (1998)) Park et al. (Anal Chem 75: 6029-6033 (2003))

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

  As a result of repeated investigations in view of the above problems, the present inventor has made use of the form of a sample plug sandwiched between air, which was not found in continuous flow PCR, so that continuous flow PCR using a conventional flat plate type microfluidic device can be used. It was clarified that both high speed and high efficiency, which were problems in, can be realized by utilizing the vapor pressure of the sample liquid itself.

The present invention provides the following nucleic acid amplification method.
Item 1. In a nucleic acid amplification method for performing a PCR reaction by supplying a PCR sample solution to a nucleic acid amplification device having a meandering flow path capable of performing at least one PCR cycle, the nucleic acid amplification device corresponds to a loop portion on one side. The PCR sample solution has one denaturation temperature zone and one or two temperature zones for annealing and / or extension, and the PCR sample solution is separated by gas for one cycle or more in the meandering channel. A method for amplifying nucleic acid, comprising:
Item 2. The meandering flow path has a loop portion on both sides and a straight portion therebetween, and corresponds to one denaturing temperature zone corresponding to the loop portion on one side, an annealing temperature zone corresponding to the other loop portion, and a straight portion. Item 2. The method for amplifying a nucleic acid according to Item 1, comprising three temperature zones of an extension temperature zone.
Item 3. The meandering channel has a loop portion on both sides and a straight portion between the loop portions, one denaturing temperature zone corresponding to the loop portion on one side, and an annealing temperature corresponding to the other loop portion and the straight portion from the middle of the denaturing temperature. Item 2. The method for amplifying nucleic acid according to Item 1, wherein the nucleic acid amplification method is composed of two temperature zones of the temperature zone where the extension process is performed at the temperature of.
Item 4. Item 4. The nucleic acid amplification method according to any one of Items 1 to 3, wherein the PCR sample solution is fed into the meandering channel by a pump in a segment flow method.
Item 5. The movement rate of the PCR sample solution from the lower temperature annealing region to the higher temperature denaturation region is reduced by the vapor pressure of the denaturation region, and the movement rate of the PCR sample solution from the denaturation region to the annealing region is the vapor pressure of the high temperature denaturation region. Item 5. The nucleic acid amplification method according to any one of Items 1 to 4, which is increased by

  In continuous-flow PCR, characteristics suitable for miniaturization of the system are required as an advantage of microfluidic devices, so a relatively small amount of PCR reagent is used while aiming for high-speed and high-efficiency amplification as much as possible. It is desirable to use less energy for temperature cycling. In the present invention, a high-speed and high-efficiency problem, which has been a problem in continuous-flow PCR using a flat plate type microfluidic device, has been achieved by utilizing a form of a sample plug sandwiched in air that was not found in continuous-flow PCR. Coexistence can be realized by utilizing the vapor pressure of the sample liquid itself. As a result, there is a feature that contributes to the miniaturization of a continuous flow PCR system that does not require a new special external device and takes full advantage of the microfluidic device.

  The conventional continuous liquid feeding method requires a large amount of sample liquid, and it is necessary to prevent the generation of bubbles occurring in the 95 ° C. region, whereas the present invention uses a segment flow method for liquid feeding by an ordinary pump, and 95 ° C. Vapor pressure from the region can be additionally used as the liquid feeding force in the microchannel, and a liquid feeding system capable of high-speed PCR was obtained. In other words, unnecessary PCR time loss under flow-through can be minimized, and the extension reaction process required for a long time can be secured stably without any special operation.

It is a figure which shows the microfluidic device for continuous flow PCR. In the continuous flow PCR microfluidic device shown in FIG. 1, it is a figure which shows the method of controlling temperature for every area | region of a meandering flow path for a thermal cycle. It is a figure which shows the fluorescence intensity obtained by amplification of a DNA fragment as a result of performing continuous flow PCR, changing a reagent liquid quantity and a liquid feeding speed using the microfluidic device shown in FIG. It is a figure which shows the result of having measured each moving time in the reciprocation of a high temperature area | region and a low temperature area | region, changing the liquid quantity of a PCR reagent. It is a figure which shows the result of having performed continuous flow PCR, changing the liquid feeding speed about each of two types of toxin related PA genes and CAP genes used for anthrax detection. (A) The case where the interval between two samples in the segment flow is one cycle or more is shown. (B) When moving from the annealing region to the higher temperature denaturing region, the flow becomes slow due to gas expansion, and when moving from the denaturing region to the lower temperature annealing region, the gas expansion (expansion) ) Is a schematic diagram showing that the flow becomes faster due to

  The present invention relates to a method for amplifying a nucleic acid by polymerase chain reaction (PCR) very rapidly and efficiently in a microchannel. More specifically, in the present invention, in a flat plate microfluidic system for continuous flow PCR, a small amount of sample liquid is sent as a sample plug (droplet introduced in the form of gas-liquid-gas). As a result, a difference in vapor pressure occurs between both ends of the sample plug, and the sample is moved locally at a low speed in the flow path during the temperature rising process and at a high speed during the temperature lowering process, so that nucleic acid amplification can be performed at high speed and high efficiency. On how to perform.

  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.

  A schematic diagram of a microfluidic device 4 for continuous flow PCR for carrying out the present invention is shown in FIG. This device includes a PCR reagent inlet 1, a meandering channel 2, and a reservoir 3 for storing a solution after the PCR reaction. One cycle of PCR can be performed by one unit consisting of a pair of loop portions on both sides (corresponding to a high temperature denaturation region and a low temperature annealing region) and two linear portions connecting the loop portions. In the microfluidic device used in the present invention, this unit may be formed by one meandering channel 3 or may be formed by a meandering channel 3 in which a large number of the units are connected. When one PCR sample solution advances in the meandering channel, one cycle of PCR reaction or multiple cycles of PCR reaction is performed according to the number of units in the meandering channel, and the necessary number of PCR products are formed. Released into the reservoir.

  The recommended ratios of preferred temperatures and times for annealing, elongation and denaturation are shown in Table 1 below.

The nucleic acid amplification device has three temperature zones, one denaturation temperature zone corresponding to the loop portion on one side, an annealing temperature zone corresponding to the other loop portion, and an extension temperature zone corresponding to the straight line portion connecting the loop portions on both sides. In this case, the recommended ratio in Table 1 above may be set, and one denaturation temperature zone corresponding to the loop portion on one side and a temperature zone where the extension process corresponding to the other loop portion / linear portion can be performed. When PCR is performed with two thermal temperature settings (the elongation process is performed at a temperature halfway from the annealing temperature to the denaturation temperature), it may be performed so as to correspond to the recommended ratios in Table 1.

  The microfluidic device for continuous flow PCR shown in FIG. 1 uses a NC processing machine to cut the flow channel shape designed on CAD on a flat plate of cycloolefin resin (COP) 80 mm long, 40 mm wide and 2 mm thick. Made by processing. The micro flow path was cut using a resin ball end mill having a diameter of 200 μm, and the cross-sectional shape of the flow path was a semicircle having a width of 400 μm and a depth of 500 μm. A straight groove having a width and depth of 0.65 mm is cut from the injection side end of the microchannel to the end of the COP plate, and a cylindrical reservoir having a diameter of 2 mm is connected to the end on the outlet side. Shaped. Insert a metal tube with an outer diameter of 0.65 mm into the groove on the injection side, drop the adhesive only on the outer periphery of the metal tube to prevent liquid leakage, and then apply a pressure sensitive adhesive coated microplate seal (3M A continuous-flow microfluidic device for PCR was produced by bonding 9795) to the entire surface. The seal at the portion covering the reservoir on the microchannel outlet side was cut.

Two aluminum blocks 5, 6 and 7 having a length of 150 mm, a width of 15 mm, and a height of 10 mm with a built-in heater are arranged in parallel as shown in FIG. 2 with an interval of about 0.5 mm to 1 mm.
The temperature was controlled individually and locally in the three contact areas by contacting the microfluidic device for continuous flow PCR produced thereon. Each heater has a performance capable of uniformly heating the aluminum block surface to 120 ° C. or higher, and has a function of cooling to 5 ° C. or lower by bonding a Peltier element to the bottom surface side.

As for the design of the micro flow path, the loop portion is a meandering flow path that folds continuously with an interval of 400 μm or more every 30 mm in length, and the three aluminum blocks and the meandering flow path are orthogonal to each other, and the loop portion of the meandering flow path is folded back. However, it designed so that it might contact 7 mm of heaters of both ends. Moreover, it was set as the design which can perform the thermal cycle required for PCR 40 times by making it the design which folds back 40 times each on the aluminum block of both ends among three aluminum blocks. 1 and 2 show an example using a meandering flow path capable of executing 40 thermal cycles and three temperature zones, but the meandering flow path may be designed so that a thermal cycle can be executed once. You may design so that there may be two temperature zones.

  The temperature of each aluminum block 5, 6 and 7 is kept uniform and constant by a heater or Peltier element by PID control based on a temperature sensor provided inside. The set temperatures of the individual aluminum blocks 5, 6, and 7 shown in FIG. 2 were set so that the temperature of the sample solution was 95 ° C., 72 ° C., and 55 ° C. necessary for PCR denaturation, extension, and annealing. It is not necessary to change the temperature of the aluminum block once set as long as continuous flow PCR is performed under the same conditions.

  As a PCR sample solution, a standard kit for real-time PCR (PCR Core Kit manufactured by Takara) was used. A 134 bp DNA sample (Positive Control) attached to the kit was used as an amplification target in PCR, and PCR reagents were prepared according to the kit. 1 μl to 30 μl of PCR reagent was injected from a metal tube of a microfluidic device for continuous flow PCR through a 0.5 mm ID silicon tube connected to a syringe pump (Harvard Model 11). At that time, the syringe pump is preliminarily filled with a sufficient amount of air so that the sample liquid can flow from the metal tube of the microfluidic device for continuous flow PCR to the reservoir at the outlet of the microchannel. The flow rate of the syringe pump was set to 65 to 225 μl / min, and the solution was continuously fed until the syringe pump was discharged to the reservoir at the outlet end of the microchannel. This corresponds to 1 sec / cycle to 10 sec / cycle as the time required to flow through the flow path length for one cycle in continuous flow PCR.

  Amplification of DNA by PCR is confirmed by collecting the sample solution that has reached the reservoir and determining the amount of change in fluorescence intensity before and after continuous flow PCR with a fluorescence plate reader (Thermochemical Fluorscan Ascent) set for FAM dye. did. As a result of continuous flow PCR, the increase in fluorescence intensity indicating amplification of the target DNA fragment was dependent on the time required for each cycle, as shown in FIG. This is because the heat conduction and reaction time necessary for each reaction of denaturation, annealing, and extension of PCR are sufficiently secured when the flow rate is decreased and the required time for each cycle is lengthened.

On the other hand, when continuous flow PCR was performed as a sample plug, which is a feature of the present invention, it became clear that the efficiency of PCR improved as the volume of the sample solution decreased. As shown in FIG. 4, when the amount of the reagent solution is 10 μl or more, in order to flow at a constant speed, the region of 72 ° C. between 55 ° C. and 95 ° C. contributing to the extension reaction, The travel time of the 72 ° C. region sandwiched during cooling to 55 ° C. is equal. However, since the flow path length of the straight part of the meandering flow path used is 30 mm and the volume corresponds to about 5.5 μl, when the volume of the sample solution is smaller than 5.5 μl, the sample plug is placed in the straight line part. The size will fit. Therefore, in the heating or cooling for the thermal cycle, the vapor pressure changes individually at the gas-liquid interface upstream and downstream of the sample plug and changes every moment depending on the position of the meandering channel. Thereby, in the process of the extension reaction from 55 ° C. to 95 ° C., the vapor pressure at the upstream gas-liquid interface increases from the downstream side, so that a force against the flow direction is generated, as shown in FIG. The sample plug moves at a low speed (see FIG. 6B). On the other hand, in the micro flow path from 95 ° C. to 55 ° C., the vapor pressure on the downstream side of the gas-liquid interface increases and works to accelerate the flow, so the sample plug moves at a rate of 2 to 3 times. .

  As is clear from FIG. 3, comparing the amount of fluorescence amplification by PCR when the volume of the sample solution is 5 μl or less and the case of 10 μl or more, the efficiency is about 2 to 6 times higher even at the same average flow rate. Met. Thus, by using the difference in vapor pressure at both ends of the sample plug, the extension reaction time of 72 ° C. following 55 ° C. is long, which is advantageous for PCR reaction. By passing through the extension region (72 ° C) unnecessary for PCR while returning from the (95 ° C) to the annealing region (55 ° C), an ideal thermal cycle without any by-products can be realized, and continuous flow PCR For the first time, both high speed and high efficiency were realized.

  As an example of the detection of other genes, amplification of DNA fragments by continuous flow PCR was examined for the toxin (PA) gene and capsule (CA) gene of Bacillus anthracis. Cyclea PCR anthrax Detection Kit Ver. 3.0, and the PCR reagents for each of the PA gene and CA gene were prepared as per kit. Each PCR reagent was individually subjected to continuous flow PCR. The prepared PCR reagent was injected into the microfluidic device for continuous flow PCR as three sample plugs of 1 μl each and was sent at a flow rate of 100 to 680 μl / min with a syringe pump (Model 11 manufactured by Harvard).

  As for the design of the micro flow path, the loop portion is a meandering flow path that folds continuously with an interval of 400 μm or more every 30 mm in length, and the three aluminum blocks and the meandering flow path are orthogonal to each other, and the loop portion of the meandering flow path is folded back. However, it designed so that it might contact 7 mm of heaters of both ends. Moreover, it was set as the design which can perform the thermal cycle required for PCR 40 times by making it the design which folds back 40 times each on the aluminum block of both ends among three aluminum blocks.

  The temperature of each aluminum block was set so that the temperature of the sample solution was 95 ° C., 72 ° C., and 55 ° C. required for PCR denaturation, extension, and annealing.

  When continuous flow PCR is performed with multiple sample plugs introduced at the same time, if two or more sample plugs enter the length of one cycle on the microchannel, the vapor pressure required for an ideal thermal cycle can be obtained. The rhythm of the used liquid delivery is disturbed. Therefore, each sample plug was injected with a sufficient interval of one cycle or more (FIG. 6A). Thereby, in each sample plug, the extension process is decelerated, and the rhythm of the liquid feeding that the cooling process is accelerated is confirmed. In one continuous flow PCR, the number of sample plugs is not limited to one, It can handle a large amount of sample liquid.

  For the PCR reagent collected in the reservoir of the microfluidic device for continuous flow PCR, the amount of change in fluorescence intensity before and after continuous flow PCR is determined using a fluorescence plate reader (Thermochemical Ascent manufactured by Thermoscience) set for FAM dye. As shown in FIG. 5, amplification of DNA fragments by similar continuous flow PCR was confirmed in both the PA gene and the CAP gene. Focusing on the amplification rate of the DNA fragment, both the PA gene and the CAP gene were confirmed to be sufficiently amplified because the fluorescence intensity change was saturated under the condition that the 40-cycle continuous flow PCR time was 240 sec or more.

In these studies, a real-time PCR method for confirming amplification of DNA fragments by fluorescence change is used. However, the technique used in this specification is not limited to real-time PCR, and RT-PCR. It can be used for a nucleic acid amplification technique using a PCR method including

  Table 2 below shows the flow time (seconds / cycle) and product size (bp) by high-speed and high-amplification PCR using the PCR chip described in the present invention (1) and the literature (2-5).

Reference (2): Sang-Hoon et al. Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB), Journal of Invertebrate Pathology 99 (2008) 8-13
Reference (3): Pavel Neuzil et al. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes, Nucleic Acids Res. 2006; 34 (11): page1-9 of 9
Reference (4): Jeong Ah Kim1, et al. Fabrication and characterization of a PDMS-glass hybrid continuous-flow PCR chip, Biochemical Engineering Journal 29 (2006) 91-97
Reference (5): Martin UK et al. Chemical amplification continuous-flow PCR on a chip. Science 1998; 280: 1046-1048.

  From the above results, it became clear that the present invention can perform a PCR reaction at a very high speed.

1 Metal tube for PCR reagent injection 2 Meandering channel 3 Reservoir 4 Microfluidic device for continuous flow PCR 5 Aluminum block for annealing 6 Aluminum block for extension reaction 7 Aluminum block for DNA denaturation 8 Sample plug (sample)

Claims (4)

In a nucleic acid amplification method for performing a PCR reaction by supplying a PCR sample solution to a nucleic acid amplification device having a meandering flow path capable of performing at least one PCR cycle, the nucleic acid amplification device corresponds to a loop portion on one side. The PCR sample solution has one denaturation temperature zone and one or two temperature zones for annealing and / or extension, and the PCR sample solution is separated by gas for one cycle or more in the meandering channel. When the PCR sample solution is fed to the sample plug as a sample plug, a difference in vapor pressure occurs at both ends of the PCR sample solution, and the PCR sample solution moves from the lower temperature annealing region to the higher temperature denaturation region. Is reduced by the vapor pressure in the denaturing zone, and the rate of movement of the PCR sample solution from the denaturing zone to the annealing zone is increased by the vapor pressure in the hot denaturing zone. A nucleic acid amplification method characterized by the above. The meandering flow path has a loop portion on both sides and a straight portion therebetween, and corresponds to one denaturing temperature zone corresponding to the loop portion on one side, an annealing temperature zone corresponding to the other loop portion, and a straight portion. The nucleic acid amplification method according to claim 1, wherein the nucleic acid amplification method is composed of three temperature zones of an extension temperature zone. The meandering channel has a loop portion on both sides and a straight portion between the loop portions, one denaturing temperature zone corresponding to the loop portion on one side, and an annealing temperature corresponding to the other loop portion and the straight portion from the middle of the denaturing temperature. The nucleic acid amplification method according to claim 1, comprising two temperature zones of the temperature zone in which the extension process is carried out at a temperature of 1. The nucleic acid amplification method according to any one of claims 1 to 3, wherein the PCR sample solution is fed into the meandering flow path by a pump in a segment flow system.

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