Nucleic Acid Amplification Method

Abstract

To provide a real-time PCR method using a new sample liquid-feeding control method using a microchip.SOLUTION: There is provided a reciprocal flow type nucleic acid amplification method in which two spatially separated temperature zones are connected by a microchannel to reciprocate sample liquid between the two temperature zones in the microchannel to perform thermal cycling. The two temperature zones are a denaturation temperature zone and an extension / annealing temperature zone, respectively. The microchannel comprises at least: a curved flow path corresponding to the denaturation temperature zone, a curved flow path corresponding to the extension / annealing temperature zone, a linear or curved intermediate flow path connecting the curved flow path corresponding to the denaturation temperature zone and the curved flow path corresponding to the extension / annealing temperature zone, and a connection part that can be connected to a liquid-feeding mechanism to realize the movement of the sample liquid. The movement of the sample liquid in the microchannel is performed by the liquid-feeding mechanism in which an atmospheric pressure is released when the liquid feeding is stopped.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for amplifying nucleic acid. [Background technology]

[0002] Nucleic acid detection is central to many fields, including pharmaceutical research and development, forensic medicine, clinical testing, identification of agricultural crops and pathogenic microbial species, etc. The ability to detect various diseases including cancer, microbial infections, and genetic markers based on molecular phylogenetic analysis has made it a universal technology for disease and risk diagnosis, marker discovery, food and environmental safety assessment, criminal proof, and many other technologies.

[0003] One of the most powerful basic technologies for highly sensitive detection of small amounts of nucleic acid, such as genes, is a method in which a part or all of a nucleic acid sequence is exponentially replicated and amplified, and then the amplified product is analyzed.

[0004] PCR is a powerful technique for selectively amplifying a specific region of DNA. Using PCR, millions of copies of a DNA fragment of a target DNA sequence can be generated from a single template DNA. PCR involves repeated three- or two-phase temperature conditions called thermal cycles, which are repeated in sequence to perform the individual reactions of denaturing DNA into a single strand, annealing the denatured DNA single strand with a primer, and elongating the primer with a thermostable DNA polymerase enzyme. This cycle is repeated until a sufficient number of copies are obtained for analysis. In principle, it is possible to double the number of copies in one cycle of PCR. In practice, as thermal cycles continue, the concentration of the necessary reaction reagents decreases, and the accumulation of amplified DNA products eventually stops. For general details of PCR, see Clinical Applications of PCR, Dennis Lo (ed.), Humana Press, Totowa, NJ (1998), and PCR Protocols A Guide to Methods and Applications, MA Innis et al. (eds.), Academic Press Inc., San Diego, CA (1990).

[0005] PCR is a powerful method that can selectively amplify the target DNA, but in order to confirm the amplified DNA, a separate confirmation step such as gel electrophoresis was required after the end of PCR. Therefore, as an improvement to PCR, real-time PCR, which generates or quenches fluorescence according to the amount of amplified target DNA, has been developed, making it easy to confirm the presence or absence of target DNA in a sample. In conventional PCR, when the amount of template DNA in a sample before PCR exceeds a certain amount, the amount of amplified DNA after PCR often reaches a plateau, making it impossible to quantify the amount of template DNA before PCR. However, in real-time PCR, the amount of amplified DNA during PCR can be detected in real time before the plateau is reached, making it possible to quantify the amount of template DNA before PCR from the state of DNA amplification. Therefore, real-time PCR is also called quantitative PCR.

[0006] The quantitative nature of target DNA by real-time PCR is particularly useful in clinical practice, and is used to monitor the progression of viral load when confirming the effectiveness of treatment for viral infections such as AIDS virus (HIV). DNA quantification by real-time PCR is also effective in diagnosing opportunistic infections such as herpes virus (HHV), many of which are asymptomatically infected from early childhood but which grow and develop as a result of physical decline.

[0007] PCR and real-time PCR are powerful methods that exponentially amplify genes through thermal cycles, but the general-purpose thermal cycler devices used in PCR have slow temperature control due to the huge heat capacity of the aluminum block that acts as a heater, and traditionally it takes 1 to 2 hours, and sometimes even longer, to perform 30 to 40 PCR cycles. Therefore, even with the latest genetic testing devices, a total analysis usually takes more than an hour, and speeding up PCR operations has been a major challenge since the technology was introduced.

[0008] As a method for solving the problem of increasing the speed of PCR operation, there has been a report of a reciprocal flow type nucleic acid amplifier that uses a microblower or the like as a liquid delivery mechanism (Patent Document 1).

[0009] Patent Document 1 discloses a method for controlling the stopping position of a sample liquid in a nucleic acid amplification device by controlling the liquid flow using as a clue the confirmation of the passage of the sample liquid at a point between the denaturation temperature zone and the extension / annealing temperature zone. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2016 / 006612 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a real-time PCR method using a microchip and utilizing a new method for controlling the delivery of sample liquid. [Means for solving the problem]

[0012] As a result of extensive research to solve the above problems, the inventors have found that when performing real-time PCR, the accuracy of liquid flow control can be improved by detecting fluorescence for each thermal cycle at predetermined positions in microchannels with two spatially separated temperature zones and in an intermediate channel connecting these temperature zones, and by taking into account the movement speed of the sample liquid as a control factor.

[0013] The present invention includes the following aspects.

[0014] Item 1. A reciprocal flow type nucleic acid amplification method in which two spatially separated temperature zones are connected by a microchannel, and a sample liquid is moved back and forth between the two temperature zones in the microchannel to perform thermal cycling, The two temperature zones are a denaturation temperature zone and an extension / annealing temperature zone, the microchannel comprises at least a curved channel corresponding to a denaturation temperature range, a curved channel corresponding to an elongation / annealing temperature range, a straight or curved intermediate channel connecting the curved channel corresponding to the denaturation temperature range and the curved channel corresponding to the elongation / annealing temperature range, and a connection part connectable to a liquid delivery mechanism for realizing the movement of a sample liquid; The movement of the sample liquid in the microchannel is carried out by a liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped. The detection of fluorescence for each thermal cycle is performed by a fluorescence detector installed so as to be capable of measuring at a predetermined position in the flow path corresponding to the denaturation temperature zone, the flow path corresponding to the extension / annealing temperature zone, and the intermediate flow path; A method for amplifying nucleic acid, comprising the following step of transferring the sample liquid: (A) when the sample liquid is moved from the denaturation temperature zone to the extension / annealing temperature zone by the liquid delivery mechanism, measuring a time t1 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone; The distance (length of the microchannel) from the predetermined position (P2) where the fluorescence detection in the intermediate channel is performed to the predetermined position (P3) where the fluorescence detection in the elongation / annealing temperature range is performed is defined as L1 [mm], The length of the sample liquid in the microchannel is Ls1 [mm], Calculate the moving speed V1 [mm / s] of the sample liquid based on t1 [s] and L1 [mm], calculating a waiting time t3 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone until when the liquid delivery mechanism is stopped based on Ls1 [mm] and V1 [mm / s]; stopping the liquid delivery mechanism t3 [s] after the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone; (B) when the sample liquid is moved from the extension / annealing temperature zone to the denaturation temperature zone by the liquid delivery mechanism, measuring a time t2 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the sample liquid at a predetermined position (P1) in the denaturation temperature zone; The distance (length of the microchannel) from the predetermined position (P2) where the fluorescence detection in the intermediate channel is performed to the predetermined position (P1) where the fluorescence detection in the denaturation temperature range is performed is defined as L2 [mm]; The length of the sample liquid in the microchannel is Ls2 [mm], Calculate the moving speed V2 [mm / s] of the sample liquid based on t2 [s] and L2 [mm], Calculate a waiting time t4 [s] from when the fluorescence detector detects the sample liquid at the predetermined position (P1) in the denaturation temperature zone until when the liquid delivery mechanism is stopped based on Ls2 [mm] and V2 [mm / s]; a step of stopping the liquid delivery mechanism t4 [s] after the fluorescence detector detects the sample liquid at a predetermined position (P1) in the denaturation temperature zone. V1=L1 / t1 (1) V2=L2 / t2 (2) t3=Ls1 / V1-tc1 (3) t4=Ls2 / V2-tc2 (4) (tc1 and tc2 are device-specific fixed times [s]) In the nucleic acid amplification method according to Item 2 or Item 1, A nucleic acid amplification method comprising the step of transferring the sample liquid, wherein waiting time t3 of formula (3) is replaced by waiting time t3 of formula (3'), and waiting time t4 of formula (4') is replaced by waiting time t4 of formula (4). t3=(Ls1-Lp1) / V1 (3') t4=(Ls2-Lp2) / V2 (4') (Lp1 and Lp2 are the device-specific lengths [mm]) In the nucleic acid amplification method according to Item 3, Item 1 or 2, a step (A) in which time t1 is a time from when the fluorescence detector detects the passage of the front part of the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the passage of the front part of the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone, and time t3 is a waiting time from when the fluorescence detector detects that the front part of the sample liquid has passed through the predetermined position (P3) in the extension / annealing temperature zone until when the liquid sending mechanism is stopped; Step (B), in which time t2 is a time from when the fluorescence detector detects the passage of the front part of the sample liquid at the predetermined position (P2) of the intermediate flow path to when the fluorescence detector detects the passage of the front part of the sample liquid at the predetermined position (P1) in the denaturation temperature zone, and time t4 is a waiting time from when the fluorescence detector detects that the front part of the sample liquid has passed through the predetermined position (P1) in the denaturation temperature zone until when the liquid sending mechanism is stopped. A nucleic acid amplification method comprising the step of transferring the sample liquid. Item 4: A nucleic acid amplification chip having a microchannel through which a sample liquid moves; a liquid delivery mechanism for moving and stopping the movement of the sample liquid in the microchannel; A temperature control means for providing a denaturation temperature zone and an extension / annealing temperature zone to the microchannel; At least three fluorescence detectors; A nucleic acid amplification device comprising: the microchannel has a curved channel corresponding to the denaturation temperature range, a curved channel corresponding to the elongation / annealing temperature range, and a straight or curved intermediate channel connecting these curved channels; The liquid delivery mechanism is a mechanism that is released to atmospheric pressure when liquid delivery is stopped, the fluorescence detector detects the passage of the sample liquid at each detection position of the curved flow path corresponding to the denaturation temperature zone, the curved flow path corresponding to the extension / annealing temperature zone, and a straight or curved intermediate flow path connecting these curved flow paths; A nucleic acid amplifier, characterized in that a liquid delivery mechanism is controlled by receiving an electrical signal from each of the fluorescence detectors. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram showing an example of a device configuration of a nucleic acid amplification device (two heaters). [Diagram 2] FIG. 1 is a diagram showing an example of a device configuration of a nucleic acid amplification device (three heaters). [Diagram 3] FIG. 1 shows an example of curved flow channels for the denaturation temperature zone (High) and extension / annealing temperature zone (Low) of a PCR chip, and a straight intermediate flow channel connecting these. [Figure 4] FIG. 1 shows the length L1 of a microchannel from a predetermined position P2 in the intermediate channel of a PCR chip to a predetermined position P3 in the extension / annealing temperature zone. [Diagram 5] FIG. 13 is a diagram showing the length L2 of a microchannel from a predetermined position P2 in the intermediate channel of a PCR chip to a predetermined position P1 in the denaturation temperature zone. [Figure 6] FIG. 13 is a diagram showing the length Ls1 of the sample liquid in the microchannel in the extension / annealing temperature range of the PCR chip. [Figure 7] FIG. 13 is a diagram showing the length Ls2 of the sample liquid in the microchannel in the denaturation temperature range of the PCR chip. [Figure 8] FIG. 1 shows one embodiment of the stopping position of a sample liquid in the extension / annealing temperature range of a PCR chip. [Figure 9] FIG. 13 is a diagram showing another embodiment of the stopping position of the sample liquid in the extension / annealing temperature zone of the PCR chip. [Figure 10] FIG. 1 shows an embodiment of the stopping position of a sample liquid in the denaturation temperature range of a PCR chip. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The nucleic acid amplification method of the present invention will now be described in detail.

[0017] The nucleic acid amplification method of the present invention is a method of performing thermal cycling by moving a sample liquid back and forth between two temperature zones in a microchannel. This type of nucleic acid amplification method is sometimes called a reciprocal flow type nucleic acid amplification method.

[0018] The nucleic acid amplification method of the present invention is a real-time PCR (polymerase chain reaction) that can monitor the state of gene amplification during a PCR reaction. In PCR, nucleic acids are amplified by utilizing multiple cycles of denaturation, annealing of a primer pair to the opposite strand, and extension of the primers that results in an exponential increase in the copy number of the target nucleic acid sequence.

[0019] To realize real-time PCR, the fluorescence intensity is measured for each thermal cycle. That is, the change in fluorescence intensity for each cycle, which increases as the target DNA is amplified by thermal cycling, is recorded over time, and the initial amount of target DNA can be quantified by calculating the cycle number (Ct value) at which the fluorescence intensity exceeds a certain threshold. In PCR, the way in which genes are amplified, i.e., the number of cycles at which the gene product increases exponentially, depends on the amount of the original template, so the amount of target gene present in a sample can be calculated by comparing the state of gene amplification when an external standard DNA with a known concentration is used as a template.

[0020] The nucleic acid amplification method of the present invention may use either DNA or RNA as a template. When DNA is used as a template, PCR is carried out using a nucleic acid amplification device configured as shown in Figure 1. When RNA is used as a template (real-time RT-PCR), PCR is carried out after first generating (reverse transcribing) complementary DNA (cDNA) from mRNA using reverse transcriptase using a nucleic acid amplification device configured as shown in Figure 2.

[0021] Various known PCR kits and protocols can be used. When the nucleic acid amplification method of the present invention is real-time RT-PCR, one-step RT-PCR can be used, which can rapidly and easily perform reverse transcription and PCR cycling in one step.

[0022] In one preferred embodiment, the nucleic acid amplification method of the present invention is a multiplex PCR in which multiple gene regions are amplified simultaneously by using multiple primer pairs in one PCR reaction system.

[0023] The nucleic acid amplification method of the present invention is carried out by moving a sample liquid in a microchannel. The microchannel for carrying out the nucleic acid amplification method of the present invention at least comprises curved channels corresponding to a denaturation temperature zone and an extension / annealing temperature zone, respectively, a straight or curved intermediate channel connecting the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension / annealing temperature zone, and a connecting part connectable to a liquid delivery mechanism for realizing the movement of the sample liquid. The microchannel may also comprise an opening for introducing the sample liquid. The opening for introducing the sample liquid may be provided at the top of the microchannel, or may be provided on a branch channel extending from a branch point. In addition, the opening may be optionally made sealable by a seal, a valve, or the like.

[0024] The microchannel is preferably made of a material that satisfies some or all of the following requirements: (i) relatively high thermal conductivity, (ii) stability in the temperature range required for PCR, (iii) resistance to corrosion by electrolyte solutions and organic solvents, and (iv) low adsorption of nucleic acids and proteins. Specific examples include glass, quartz, silicon, and various resins such as cycloolefin polymer (COP). From the viewpoint of performing fluorescence detection, a transparent material that is highly permeable to light (especially excitation light and emitted light for performing fluorescence detection) (i.e., has little absorption, diffusion, reflection, etc.) is preferable.

[0025] The microchannel may have a structure in which a groove is formed in a material by a method such as machining such as cutting by NC machining, injection molding, nanoimprinting, soft lithography, or the like, and sealed with a seal (preferably a transparent seal made of polyolefin, for example). Alternatively, the microchannel may be formed by three-dimensional printing. The cross-sectional shape of the microchannel is not particularly limited, and may be a semicircular shape, a circular shape, a rectangular shape, a wedge shape, a trapezoid shape, a polygonal shape, or the like. The cross-section of the microchannel may have a width of about 10 to 1000 μm and a depth of about 10 to 1000 μm, for example. The width and depth of the microchannel may each be constant, or the width or depth may vary partially.

[0026] The shape of the curved flow channel corresponding to the denaturation temperature zone and the curved flow channel corresponding to the elongation / annealing temperature zone of the microchannel can be a meandering flow channel having a loop shape, a spiral flow channel shape, etc. The intermediate flow channel connecting the curved flow channel corresponding to the denaturation temperature zone and the curved flow channel corresponding to the elongation / annealing temperature zone can be a straight line or any of the above-mentioned curved flow channel shapes.

[0027] The length of each of the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension / annealing temperature zone may be 20 mm or more.

[0028] The curved flow path corresponding to the denaturation temperature zone and the curved flow path corresponding to the extension / annealing temperature zone of the microchannel are each maintained at a corresponding temperature, and the temperature of the sample liquid that has moved into that temperature zone is changed to the temperature of that temperature zone.

[0029] The denaturation temperature zone is maintained at a temperature necessary for the DNA denaturation reaction in PCR. The temperature of the denaturation temperature zone is preferably about 90 to 100°C, and more preferably about 95°C. The extension / annealing temperature zone is maintained at a temperature necessary for the DNA annealing reaction and extension reaction in PCR. The temperature of the extension / annealing temperature zone is preferably about 40 to 75°C, and more preferably about 55 to 65°C.

[0030] Each of the denaturation temperature zone and the extension / annealing temperature zone is preferably maintained at a constant temperature. The temperature can be maintained by a heat source. The heat source is, for example, built into the microchannel or in contact with the microchannel. Specific examples of the heat source include a cartridge heater, a film heater, a Peltier heater, and the like.

[0031] In the nucleic acid amplification method of the present invention, the sample liquid moves in the microchannel in the form of a plug. The volume of the sample liquid moving in the microchannel is not particularly limited, and may be preferably about 5 to 50 μL, more preferably about 15 to 20 μL.

[0032] The sample solution contains components necessary for PCR reaction, components necessary for fluorescence detection to realize real-time PCR, etc. For example, the sample solution contains, in an aqueous medium mainly composed of water, components necessary for PCR reaction such as template nucleic acid (which may be either DNA or RNA), enzymes such as reverse transcriptase, various deoxyribonucleotide triphosphates which may be labeled, primer sets corresponding to target gene regions, etc.; components necessary for fluorescence detection such as fluorescent probes such as TaqMan probe, Cycleave probe, E probe (registered trademark), and dyes such as SYBR GREEN, etc. In addition, the sample solution may contain buffer components for adjusting pH and salt concentration.

[0033] When the PCR of the present invention is a multiplex PCR, the sample solution contains a primer set containing two or more types of primers. In general, a "primer set" refers to a combination of a forward primer and a reverse primer, and usually one forward primer and one reverse primer are used corresponding to one target gene region. Even if the primer set of the present invention contains only one reverse primer, it can be used as a primer set for multiplex PCR when the reverse primer generates amplification products corresponding to separate gene regions in combination with two or more forward primers (as a primer pair).

[0034] Examples of dyes (fluorescent dyes) used for fluorescence detection include ABY, acridine, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, ATTO (ATTO-TEC fluorescent dye), BioSearch Blue, Cy3, Cy3.5, Cy5, Cy5.5, coumarin, DANSYL, FAM (e.g., 5-FAM, 6-FAM), FITC, GPF, and 5-HEX. , 6-HEX, JOE, JUN, Marina Blue, NED, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PET, Pulsar, Quasar 570, Quasar 670, Quasar 705, Rhodamine Green, Rhodamine Red, 5-ROX, 6-ROX, 5-TAMRA, 6-TAMRA, 5-TET, 6-TET, Texas Red, TRITC, and VIC.

[0035] The dyes used in multiplex PCR preferably include at least one selected from the group consisting of ABY and HEX, and examples thereof include a combination of ABY, Cy5, and FAM, and a combination of HEX, Cy5, and FAM.

[0036] In the nucleic acid amplification method of the present invention, the movement of the sample liquid is achieved by a liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped. That is, instead of using a mechanism that requires the inside of the flow channel to be a closed system so that the pressure of a syringe pump or the like does not escape, a liquid delivery mechanism that is configured to form an open system even during liquid delivery is used. By employing such a liquid delivery mechanism, when the air delivery is stopped, the pressure inside the flow channel instantly becomes equal to the pressure outside the flow channel, and the pressure acting on the plug-shaped sample liquid is lost, so that the liquid delivery immediately stops.

[0037] Examples of the liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped include a microblower and a fan.

[0038] A microblower (also called a piezoelectric microblower) is a known device that draws in and discharges air, and is characterized by not having a sealed structure (having no check valve). In a typical microblower, the diaphragm is bent and deformed by applying a voltage to a piezoelectric element, thereby achieving the drawing in and discharge of air. As a microblower, for example, those manufactured by Murata Manufacturing Co., Ltd. can be used (MZB1001T02, MZB3004T04).

[0039] A fan is a device that blows air by the rotational motion of an impeller. Due to the structural characteristics of the impeller, the flow path is not a closed system.

[0040] In the nucleic acid amplification method of the present invention, reciprocal flow type nucleic acid amplification is carried out by configuring the following [1] to [4] as one cycle. [1] A step of operating a liquid delivery mechanism to move the sample liquid from an elongation / annealing temperature range to a denaturation temperature range through an intermediate flow path; [2] stopping the liquid delivery mechanism and maintaining the sample liquid within the denaturation temperature range for a certain period of time; [3] Operate the liquid delivery mechanism to move the sample liquid from the denaturation temperature zone through the intermediate flow path to the extension / annealing temperature zone; and [4] A process in which the liquid delivery mechanism is stopped and the sample liquid is held within the extension / annealing temperature range for a certain period of time.

[0041] Thermal cycling is performed by repeating the above cycle at least once, preferably about 30 to 50 times, and more preferably about 35 to 50 times. The number of cycles can be appropriately set depending on the concentration of the template nucleic acid, the type of the target gene, etc.

[0042] In the nucleic acid amplification method of the present invention, the speed of movement of the sample liquid, particularly the speed at which the sample liquid moves through the intermediate flow channel, can be, for example, about 25 mm / sec to 2.2 m / sec, more preferably about 40 mm / sec to 1 m / sec, or about 60 mm / sec to 300 mm / sec.

[0043] The time for which the sample liquid is held in the denaturation temperature zone and the time for which the sample liquid is held in the extension / annealing temperature zone can each be appropriately set according to the target gene region (type of gene, length of region, etc.) For example, the time for which the sample liquid is held in the denaturation temperature zone can be about 2 to 10 seconds, and the time for which the sample liquid is held in the extension / annealing temperature zone can be about 2 to 60 seconds.

[0044] The liquid delivery mechanism is connected to the microchannel via, for example, a connection portion.

[0045] In one embodiment of the present invention, two liquid delivery mechanisms are connected to each of the two ends of the microchannel, i.e., a first liquid delivery mechanism connected to deliver liquid from within the elongation / annealing temperature zone to within the denaturation temperature zone is operated in the above step [1], and a second liquid delivery mechanism connected to deliver liquid from within the denaturation temperature zone to within the elongation / annealing temperature zone is operated in the above step [3].

[0046] In another aspect of the present invention, one liquid delivery mechanism is connected to two ends of a microchannel via a branched connecting flow path equipped with a switching valve. That is, in the above step [1], the liquid delivery mechanism is operated in a state in which the flow path is configured via a switching valve so that liquid is delivered from within the elongation / annealing temperature zone to within the denaturation temperature zone, and in the above step [3], the liquid delivery mechanism is operated in a state in which the flow path is configured via a switching valve so that liquid is delivered from within the denaturation temperature zone to within the elongation / annealing temperature zone. Note that, in the case of a three-way valve, the switching valve is connected to a microblower or fan that is released to atmospheric pressure when liquid delivery is stopped, and two three-way valves are arranged at both ends of the air flow path that branches into two directions at the branch point, and air is alternately delivered to two connecting flow paths that branch to the two ends of the microchannel, thereby enabling the sample liquid to be delivered back and forth in the microchannel. In this case, the three-way valve can deliver the sample liquid by delivering air with one valve closed and the other valve open. In addition, it is preferable to have one three-way valve, but depending on the combination of connecting flow paths, a combination of two-way valves or a multi-way valve such as three-way, four-way, five-way, etc. may also be used.

[0047] In another embodiment of the present invention, two liquid delivery mechanisms (air discharge means and air suction means) are connected to one end (the extension / annealing temperature zone side) of the microchannel via a branched connecting channel equipped with a switching valve. That is, in the above step [1], the liquid delivery mechanism (air discharge means) is operated via a switching valve so as to deliver liquid from the extension / annealing temperature zone to the denaturation temperature zone, and in the above step [3], the liquid delivery mechanism (air suction means) is operated via a switching valve so as to deliver liquid from the denaturation temperature zone to the extension / annealing temperature zone.

[0048] In the nucleic acid amplification method of the present invention, the fluorescence of the sample liquid is detected for each thermal cycle at three predetermined positions in the flow channel corresponding to the denaturation temperature range, the flow channel corresponding to the extension / annealing temperature range, and the linear or curved intermediate flow channel. The predetermined position in the flow channel corresponding to the denaturation temperature range is not particularly limited, but is preferably a position that has turned back one to several times (e.g., 2 to 4 times) from the intermediate flow channel or has passed through a curved section, and an example of such a position can be P1 in FIG. 3. The predetermined position in the flow channel corresponding to the extension / annealing temperature range is not particularly limited, but is preferably a position that has turned back one to several times (e.g., 2 to 4 times) from the intermediate flow channel or has passed through a curved section, and an example of such a position can be P3 in FIG. 3. The predetermined position in the linear intermediate flow channel is not particularly limited, and an example of such a position can be P2 in FIG. 3.

[0049] Detection of the fluorescence intensity not only detects the movement of the sample liquid, but also makes it possible to monitor the state of gene amplification during the PCR reaction.

[0050] As one embodiment of the present invention, an example of sample liquid delivery control is shown below (Example 1). [1] A step of turning on a light source (LED) that illuminates the flow path in the extension / annealing temperature range and a light source (LED) that illuminates the intermediate flow path, and moving the sample liquid from the denaturation temperature range to the extension / annealing temperature range through the intermediate flow path by a liquid delivery mechanism; [2] a step in which the control mechanism receives an electrical signal from the fluorescence detector indicating that the front end of the sample liquid has passed through a predetermined position in the intermediate flow path; [3] A step in which the liquid delivery control mechanism receives an electrical signal from the fluorescence detector indicating that the front end of the sample liquid has passed through a predetermined position in the extension / annealing temperature zone; [4] A process of calculating the time t1 [s] required for the sample liquid to travel the distance (length) L1 [mm] of the microchannel from a predetermined position in the intermediate channel to a predetermined position in the elongation / annealing temperature zone from the time when an electrical signal is received from the fluorescence detector in the intermediate channel [2] and the time when an electrical signal is received from the fluorescence detector in the elongation / annealing temperature zone [3], and further calculating the travel speed V1 [mm / s] of the sample liquid (V1=L1 / t1); [5] calculating the time t5 [s] required for the tail of the sample liquid to pass a predetermined position in the elongation / annealing temperature zone when the front of the sample liquid passes through a predetermined position in the elongation / annealing temperature zone and the sample liquid moves at the moving speed V1 [mm / s] calculated in [4] from the length Ls1 [mm] of the sample liquid (t5 = Ls1 / V1); [6] A step of calculating a waiting time t3 [s] (t3 = t5 - tc1) based on an apparatus-specific time tc1 [s] that can be determined from a predetermined position in the extension / annealing temperature zone, a sample liquid stopping position set by the operator or the apparatus designer, etc., and stopping the liquid delivery mechanism after the waiting time t3 [s] has elapsed since the front end of the sample liquid passed the predetermined position in the extension / annealing temperature zone; [7] turning on a light source (LED) for illuminating the flow path in the denaturation temperature zone and a light source (LED) for illuminating the intermediate flow path, and moving the sample liquid from the extension / annealing temperature zone to the denaturation temperature zone through the intermediate flow path by a liquid delivery mechanism; [8] a step in which the liquid delivery control mechanism receives an electrical signal from the fluorescence detector indicating that the front end of the sample liquid has passed through a predetermined position in the intermediate flow path; [9] a step in which the control mechanism receives an electrical signal from the fluorescence detector indicating that the front end of the sample liquid has passed through a predetermined position in the denaturation temperature zone;

[10] A step of calculating the time t2 [s] required for the sample liquid to travel the distance (length) L2 [mm] of the microchannel from a predetermined position in the intermediate channel to a predetermined position in the denaturation temperature zone from the time when an electrical signal is received from the fluorescence detector in the intermediate channel [8] and the time when an electrical signal is received from the fluorescence detector in the denaturation temperature zone [9], and further calculating the travel speed V2 [mm / s] of the sample liquid (V2 = L2 / t2);

[11] calculating the time t6 [s] required for the tail of the sample liquid to pass a predetermined position in the denaturation temperature zone when the front of the sample liquid passes a predetermined position in the denaturation temperature zone and the sample liquid moves at the moving speed V2 [mm / s] calculated in

[10] from the length Ls2 [mm] of the sample liquid (t6 = Ls2 / V2);

[12] A process of calculating a waiting time t4 [s] taking into consideration an apparatus-specific time tc2 [s], which is determined from a predetermined position in the denaturation temperature zone and the sample liquid stopping position set by the person measuring or the apparatus designer, and stopping the liquid delivery mechanism after the waiting time t4 [s] has elapsed since the front of the sample liquid passed the predetermined position in the denaturation temperature zone.

[0051] As an embodiment of the present invention, a modified example of sample liquid delivery control is shown below (Example 2). In the sample liquid delivery control method of Example 1, step [6] is replaced with step [6-2], Step [12-2] is carried out instead of step

[12] . [6-2] A step of calculating a waiting time t3 [s] (t3 = t5 - Lp1 / V1) based on a length Lp1 [mm] specific to the device, which can be determined from a predetermined position in the elongation / annealing temperature zone and a sample liquid stopping position set by the operator or the device designer, and stopping the liquid delivery mechanism after the waiting time t3 [s] has elapsed since the front end of the sample liquid passed the predetermined position in the elongation / annealing temperature zone; [12-2] A process of calculating a waiting time t4 [s] taking into consideration an apparatus-specific length Lp2 [mm], which is determined from a predetermined position in the denaturation temperature zone and the sample liquid stopping position set by the person measuring or the apparatus designer, and stopping the liquid delivery mechanism after the waiting time t4 [s] has elapsed since the front of the sample liquid passed the predetermined position in the denaturation temperature zone.

[0052] As an embodiment of the present invention, a modified example of sample liquid delivery control is shown below (Example 3). In the sample liquid delivery control method of Example 1, step [5] is replaced with step [5-3], step [6] is replaced with step [6-3], step

[11] is replaced with step [11-3], and step

[12] is replaced with step [12-3]. [5-3] A step of calculating the time t7 [s] required for the tail of the sample liquid to pass a predetermined position in the elongation / annealing temperature zone when the front of the sample liquid passes a predetermined position in the intermediate flow channel and the sample liquid moves at the moving speed V1 [mm / s] calculated in [4] from the distance (length) L1 [mm] of the micro flow channel from the predetermined position in the intermediate flow channel to the predetermined position in the elongation / annealing temperature zone and the length Ls1 [mm] of the sample liquid (t7 = L1 / V1 + Ls1 / V1); [6-3] A step of calculating a waiting time t3 [s] (t3 = t7 - tc1) based on an apparatus-specific time tc1 [s] that can be determined from a predetermined position in the extension / annealing temperature zone, a sample liquid stopping position set by an operator or an apparatus designer, etc., and stopping the liquid delivery mechanism after the waiting time t3 [s] has elapsed since the front end of the sample liquid passed the predetermined position in the extension / annealing temperature zone; [11-3] A step of calculating the time t8 [s] required for the tail of the sample liquid to pass a predetermined position in the denaturation temperature zone when the front of the sample liquid passes a predetermined position in the intermediate flow channel and the sample liquid moves at the moving speed V2 [mm / s] calculated in

[10] from the distance (length) L2 [mm] of the micro flow channel from the predetermined position in the intermediate flow channel to the predetermined position in the denaturation temperature zone and the length Ls2 [mm] of the sample liquid (t8 = L2 / V2 + Ls2 / V2); [12-3] A process of calculating a waiting time t4 [s] (t4 = t8 - tc2) taking into consideration an apparatus-specific time tc2 [s], which is determined from a predetermined position in the denaturation temperature zone and the sample liquid stopping position set by the person measuring or the apparatus designer, and stopping the liquid delivery mechanism after the waiting time t4 [s] has elapsed since the front of the sample liquid passed the predetermined position in the denaturation temperature zone.

[0053] As an embodiment of the present invention, a modified example of sample liquid delivery control is shown below (Example 4). In the sample liquid delivery control method of Example 1, step [5] is replaced with step [5-4], step [6] is replaced with step [6-4], step

[11] is replaced with step [11-4], and step

[12] is replaced with step [12-4]. [5-4] A step of calculating the time t7 [s] required for the tail of the sample liquid to pass a predetermined position in the elongation / annealing temperature zone when the front of the sample liquid passes a predetermined position in the intermediate flow channel and the sample liquid moves at the moving speed V1 [mm / s] calculated in [4] from the distance (length) L1 [mm] of the micro flow channel from the predetermined position in the intermediate flow channel to the predetermined position in the elongation / annealing temperature zone and the length Ls1 [mm] of the sample liquid (t7 = L1 / V1 + Ls1 / V1); [6-4] A step of calculating a waiting time t3 [s] (t3 = t7 - Lp1 / V1) taking into consideration a length time Lp1 [mm] specific to the device, which can be determined from a predetermined position in the elongation / annealing temperature zone and a sample liquid stopping position set by a measurer or an instrument designer, and stopping the liquid delivery mechanism after the waiting time t3 [s] after the front end of the sample liquid passes the predetermined position in the elongation / annealing temperature zone; [11-4] A step of calculating the time t8 [s] required for the tail of the sample liquid to pass a predetermined position in the denaturation temperature zone when the front of the sample liquid passes a predetermined position in the intermediate flow channel and the sample liquid moves at the moving speed V2 [mm / s] calculated in

[10] from the distance (length) L2 [mm] of the micro flow channel from the predetermined position in the intermediate flow channel to the predetermined position in the denaturation temperature zone and the length Ls2 [mm] of the sample liquid (t8 = L2 / V2 + Ls2 / V2); [12-4] A process of calculating a waiting time t4 [s] taking into consideration an apparatus-specific length Lp2 [mm], which is determined from a predetermined position in the denaturation temperature zone and the sample liquid stopping position set by the person measuring or the apparatus designer, and stopping the liquid delivery mechanism after the waiting time t4 [s] has elapsed since the front of the sample liquid passed the predetermined position in the denaturation temperature zone.

[0054] Fluorescence detection can be performed by detecting, with a fluorescence detector, emitted light (fluorescence) generated by excitation light irradiated from a light source toward a sample liquid in a microchannel.

[0055] The nucleic acid amplification method of the present invention includes, for example, a combination of the following nucleic acid amplification device and a nucleic acid amplification chip: This can be done using: [Nucleic acid amplification device] A heater that can create a denaturation temperature zone and an extension / annealing temperature zone. a fluorescence detector capable of measuring the fluorescence intensity of the sample liquid present in the denaturation temperature range; a fluorescence detector capable of measuring the fluorescence intensity of the sample solution present in the extension / annealing temperature range; a fluorescence detector capable of measuring the fluorescence intensity of a sample solution present in an intermediate flow path connecting the curved flow paths corresponding to the denaturation temperature zone and the extension / annealing temperature zone; a liquid transfer mechanism that enables the movement of the sample liquid between the two temperature zones and is released to atmospheric pressure when liquid transfer is stopped; A substrate on which a nucleic acid amplification chip can be mounted; a control mechanism for controlling the driving of the liquid delivery mechanism in response to an electrical signal from the fluorescence detector relating to the movement of the sample liquid; A reciprocal flow type nucleic acid amplification device that performs real-time PCR by measuring the fluorescence intensity for each thermal cycle. [Nucleic acid amplification chip] The nucleic acid amplification chip has at least one microchannel having curved flow channels corresponding to the denaturation temperature zone and the extension / annealing temperature zone, respectively, a straight or curved intermediate flow channel connecting the curved flow channels, and a connection portion at one or both ends of the flow channel that can be connected to a liquid delivery mechanism in the nucleic acid amplification device.

[0056] Specifically, this can be carried out by the following steps 1 to 4: Step 1: placing the nucleic acid amplification chip on a substrate of the nucleic acid amplification device; Step 2: connecting the liquid delivery mechanism to a liquid delivery mechanism connection part at one or both ends of the microchannel; Step 3: A step of performing thermal cycling by moving the sample liquid back and forth between the two curved flow paths of the micro flow path using the liquid delivery mechanism. Step 4: A step of measuring the fluorescence intensity of the sample solution for each thermal cycle using the fluorescence detector at predetermined positions in the flow path corresponding to the denaturation temperature zone, the flow path corresponding to the extension / annealing temperature zone, and an intermediate flow path connecting the flow paths corresponding to the denaturation temperature zone and the extension / annealing temperature zone.

[0057] Hereinafter, examples of a nucleic acid amplification device and a nucleic acid amplification chip will be described with reference to the drawings.

[0058] As shown in FIG. 1, the nucleic acid amplification device can include a substrate (not shown) for mounting a nucleic acid amplification chip, a nucleic acid amplification chip temperature control unit, a liquid delivery mechanism (for example, a microblower is shown as an example), a fluorescence detector, and a small battery for powering a control computer as a control mechanism.

[0059] In Figure 1, the temperature control unit for the nucleic acid amplification chip is configured by arranging two cartridge heaters in parallel with an interval of 10 mm so that they are in contact with the respective sealing surfaces of the two curved flow path sections of the nucleic acid amplification chip without any gaps, and a K-type thermocouple is attached to each of the two heaters to control the temperature.

[0060] Cartridge heater 1 is controlled by a control computer to maintain a temperature required for the DNA denaturation reaction. Cartridge heater 2 is controlled by a control computer to maintain a temperature required for the DNA annealing reaction and extension reaction. The temperature zone for the DNA denaturation reaction and the temperature zone for the annealing reaction and extension reaction can be kept constant by, for example, PID (proportional-integral-derivative) control.

[0061] The fluorescence detector is arranged to measure the fluorescence intensity at detection points (P1, P3 in Fig. 3) on the straight line of the microchannel in the denaturation temperature zone and the extension / annealing temperature zone, and at one point on the intermediate channel (P2 in Fig. 3). After the leading edge of the sample liquid sent from one curved channel section by pressurization reaches detection point P1 or P3, and a specific waiting time has elapsed, the liquid sending mechanism is stopped and the sample liquid can be held for a certain period of time in the other curved channel section.

[0062] Alternatively, after the front of the sample liquid sent from one curved flow path section by pressurization reaches detection point P2 and a specific waiting time has elapsed, the liquid sending mechanism can be stopped and the sample liquid can be retained in the other curved flow path section for a fixed period of time.

[0063] The control computer is capable of program-controlling the liquid delivery mechanism, and performs thermal cycling by alternately switching the liquid delivery mechanism so that the sample liquid moves alternately to the curved flow channel section on each heater for a set time while continuously monitoring the fluorescence intensity at the detection point at the center of each microchannel. The control computer further simultaneously records the change in fluorescence intensity for each cycle that increases as the target DNA is amplified by thermal cycling in the real-time PCR method, and can quantify the initial amount of target DNA by calculating the cycle number (Ct value) at which the fluorescence intensity exceeds a certain threshold.

[0064] A nucleic acid amplification chip equipped with a microchannel is shown in Figure 3. In the nucleic acid amplification chip shown in Figure 3, two curved channels (serpentine channels) corresponding to the denaturation temperature range and the extension / annealing temperature range are connected by a straight intermediate channel, and sample liquid fluorescence is detected at detection points P1, P2, and P3.

[0065] Figure 4 shows the length L1 of the microchannel from a predetermined position P2 in the intermediate channel of the PCR chip to a predetermined position P3 in the extension / annealing temperature zone. L1 indicates the length of the line from P2 to P3 that connects the centers (midpoints) of the channel width of the channel created on the surface of the PCR chip. In Figure 4, the line from P2 to P3 represents a straight line-curve-straight line-curve-straight line shape.

[0066] Figure 5 shows the length L2 of the microchannel from a predetermined position P2 in the intermediate channel of the PCR chip to a predetermined position P1 in the denaturation temperature zone. L2 indicates the length of the line from P2 to P1 that connects the centers (midpoints) of the channel width of the channel created on the surface of the PCR chip, and in Figure 5 the line from P2 to P1 represents a straight line-curve-straight line-curve-straight line shape.

[0067] The length Ls1 of the sample liquid in the microchannel in the extension / annealing temperature range of the PCR chip is shown in Figure 6. Ls1 is the length of the line that extends from P3 in the extension / annealing temperature range along the flow channel toward the air vent (on the right side) when the sample liquid is introduced into the microchannel of the PCR chip, and connects the centers (midpoints) of the flow channel width of the flow channel created on the surface of the PCR chip.

[0068] The length Ls2 of the sample liquid in the microchannel in the denaturation temperature range of the PCR chip is shown in Figure 7. Ls is the length of the line that extends from P1 in the denaturation temperature range along the flow channel toward the air communication port (left side) when the sample liquid is introduced into the microchannel of the PCR chip, and connects the centers (midpoints) of the flow channel width of the flow channel created on the surface of the PCR chip. When the cross-sectional area of ​​the microchannel corresponding to the denaturation temperature zone and the cross-sectional area of ​​the microchannel corresponding to the extension / annealing temperature zone are constant and equal, Ls1 and Ls2 are equal.

[0069] Figure 8 shows one mode in which the sample liquid stops in the extension / annealing temperature range of the PCR chip. The sample liquid stops at a distance of about 1 / 10 of Ls1 from the position P3. For example, by setting a fixed time tc1 = Ls1 x 1 / 10 x 1 / V1 specific to the device, calculating the waiting time t3, and controlling the liquid delivery mechanism, it is possible to stop the sample liquid at the desired position in the extension / annealing temperature range. The fixed time tc1 specific to the device is determined by the stop position of the microchannel in the extension / annealing zone, the amount of air blown by the liquid delivery mechanism, and the like, which are set by the person performing the measurement or the device designer.

[0070] Figure 9 shows one mode in which the sample liquid stops in the extension / annealing temperature range of the PCR chip. The sample liquid stops at about 1 / 3 of Ls1 from the position P3. For example, by setting a fixed time tc1 = Ls1 x 1 / 3 x 1 / V1 specific to the device, calculating the waiting time t3, and controlling the liquid delivery mechanism, it is possible to stop the sample liquid at the desired position in the extension / annealing temperature range. The fixed time tc1 specific to the device is determined by the stop position of the microchannel in the extension / annealing zone, the amount of air blown by the liquid delivery mechanism, and the like, which are set by the person performing the measurement or the device designer.

[0071] Figure 10 shows one mode in which the sample liquid stops in the denaturation temperature range of the PCR chip. The sample liquid stops at a distance of about 1 / 4 of Ls2 from the position P1. For example, by setting a fixed time tc2 = Ls2 x 1 / 4 x 1 / V2 specific to the device, calculating the waiting time t4, and controlling the liquid delivery mechanism, it is possible to stop the sample liquid at the desired position in the denaturation temperature range. The fixed time tc2 specific to the device is determined by the person who performs the measurement or the device designer based on the stopping position of the micro flow path in the denaturation temperature range, the amount of air blown by the liquid delivery mechanism, and the like.

[0072] Another embodiment of the present invention includes a method in which the amount of sample liquid used is limited to a specific amount (e.g., 20 μL). In order to stop the front of the sample liquid when it has passed a predetermined position (P3) in the extension / annealing temperature zone and moved Lc1 [mm], the waiting time until the liquid delivery mechanism is stopped can be set as t3=Lc1 / V1. Also, in order to stop the front of the sample liquid when it has passed a predetermined position (P1) in the denaturation temperature zone and moved Lc2 [mm], the waiting time until the liquid delivery mechanism is stopped can be set as t4=Lc2 / V2. Here, 0

Claims

1. A reciprocal flow type nucleic acid amplification method in which two spatially separated temperature zones are connected by a microchannel, and a sample liquid is moved back and forth between the two temperature zones in the microchannel to perform thermal cycling, comprising: The two temperature zones are a denaturation temperature zone and an extension / annealing temperature zone, the microchannel comprises at least a curved channel corresponding to a denaturation temperature range, a curved channel corresponding to an extension / annealing temperature range, a straight or curved intermediate channel connecting the curved channel corresponding to the denaturation temperature range and the curved channel corresponding to the extension / annealing temperature range, and a connection part connectable to a liquid delivery mechanism for realizing the movement of a sample liquid; The movement of the sample liquid in the microchannel is carried out by a liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped. The detection of fluorescence for each thermal cycle is carried out by a fluorescence detector installed so as to be capable of measuring at a predetermined position in the flow path corresponding to the denaturation temperature zone, the flow path corresponding to the extension / annealing temperature zone, and the intermediate flow path; A method for amplifying nucleic acid, comprising the following step of transferring the sample liquid: (A) when the sample liquid is moved from the denaturation temperature zone to the extension / annealing temperature zone by the liquid delivery mechanism, measuring a time t1 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone; The distance (length of the microchannel) from the predetermined position (P2) where the fluorescence detection in the intermediate channel is performed to the predetermined position (P3) where the fluorescence detection in the elongation / annealing temperature range is performed is L1 [mm], The length of the sample liquid in the microchannel is Ls1 [mm], The moving speed of the sample liquid V1 [mm / s] is calculated based on t1 [s] and L1 [mm] using formula (1), Calculate a waiting time t3 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone until when the liquid delivery mechanism is stopped, based on Ls1 [mm] and V1 [mm / s], using formula (3); stopping the liquid delivery mechanism t3 [s] after the fluorescence detector detects the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone; (B) when the sample liquid is moved from the extension / annealing temperature zone to the denaturation temperature zone by the liquid delivery mechanism, measuring a time t2 [s] from when the fluorescence detector detects the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the sample liquid at a predetermined position (P1) in the denaturation temperature zone; The distance (length of the microchannel) from the predetermined position (P2) where the fluorescence detection in the intermediate channel is performed to the predetermined position (P1) where the fluorescence detection in the denaturation temperature range is performed is defined as L2 [mm], By setting the length of the sample liquid in the microchannel to Ls2 [mm], The moving speed of the sample liquid V2 [mm / s] is calculated based on t2 [s] and L2 [mm] using formula (2), Calculate a waiting time t4 [s] from when the fluorescence detector detects the sample liquid at the predetermined position (P1) in the denaturation temperature zone until when the liquid delivery mechanism is stopped based on Ls2 [mm] and V2 [mm / s] using formula (4); A process of stopping the liquid delivery mechanism t4 [s] after the fluorescence detector detects the sample liquid at a predetermined position (P1) in the denaturation temperature zone. V1=L1 / t1 (1) V2=L2 / t2 (2) t3=Ls1 / V1-tc1 (3) t4=Ls2 / V2-tc2 (4) (tc1 and tc2 are device-specific fixed times [s])

2. The nucleic acid amplification method according to claim 1, A nucleic acid amplification method comprising the step of transferring the sample liquid, wherein waiting time t3 of formula (3) is replaced with waiting time t3 of formula (3'), and waiting time t4 of formula (4') is replaced with waiting time t4 of formula (4). t3=(Ls1-Lp1) / V1 (3') t4=(Ls2-Lp2) / V2 (4') (Lp1 and Lp2 are the device-specific lengths [mm])

3. The method for amplifying nucleic acid according to claim 1 or 2, a step (A) in which time t1 is a time from when the fluorescence detector detects the passage of the front part of the sample liquid at a predetermined position (P2) in the intermediate flow path to when the fluorescence detector detects the passage of the front part of the sample liquid at a predetermined position (P3) in the extension / annealing temperature zone, and time t3 is a waiting time from when the fluorescence detector detects that the front part of the sample liquid has passed through the predetermined position (P3) in the extension / annealing temperature zone to when the liquid sending mechanism is stopped; Step (B), in which time t2 is a time from when the fluorescence detector detects the passage of the front part of the sample liquid at the predetermined position (P2) of the intermediate flow path to when the fluorescence detector detects the passage of the front part of the sample liquid at the predetermined position (P1) in the denaturation temperature zone, and time t4 is a waiting time from when the fluorescence detector detects that the front part of the sample liquid has passed through the predetermined position (P1) in the denaturation temperature zone until when the liquid sending mechanism is stopped. A nucleic acid amplification method comprising the step of transferring the sample liquid.

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