Nucleic acid amplification method

Abstract

To provide a real-time PCR method that is rapid and has reduced noise even when performing multiplex PCR.SOLUTION: In a reciprocal-flow-type nucleic acid amplification method in which two spatially separated temperature zones are connected by a microchannel and thermal cycling is performed by reciprocating a sample liquid between the two temperature zones in the microchannel, the two temperature zones are a denaturation temperature zone and an elongation-annealing temperature zone. The microchannel includes at least a curved channel corresponding to the denaturation temperature zone, a curved channel corresponding to the elongation-annealing temperature zone, a linear or curved intermediate channel that connects the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the elongation-annealing temperature zone, and a connector connectable to a liquid delivery mechanism for enabling movement of the sample liquid. Movement of the sample liquid in the microchannel is carried out by the liquid delivery mechanism, which is open to atmospheric pressure when liquid delivery is stopped, and real-time PCR is performed by measuring the fluorescence intensity for each thermal cycle at a predetermined position on the channel corresponding to the denaturation temperature zone and at a predetermined position on the channel corresponding to the elongation-annealing temperature zone.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority based on Japanese Patent Application No. 2019-049009, filed on March 15, 2019, the entire disclosure of which is incorporated herein by reference. The present invention relates to a method for amplifying nucleic acids. [Background technology]

[0002] Nucleic acid detection is central to various fields, such as pharmaceutical research and development, forensic medicine, clinical testing, and identification of agricultural crops and pathogenic microorganisms. 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 acids, such as genes, is a method of exponentially replicating and amplifying part or all of a nucleic acid sequence and analyzing the amplified product.

[0004] PCR is a powerful technique for selectively amplifying specific regions of DNA. Using PCR, millions of copies of a target DNA sequence can be generated from a single template DNA. PCR involves repeated thermal cycling, a three- or two-phase process: denaturation of DNA into single strands, annealing of the denatured DNA single strands with primers, and primer extension by a thermostable DNA polymerase enzyme. This cycle is repeated until a sufficient number of copies is obtained for analysis. In principle, a single PCR cycle can double the copy number. In practice, as thermal cycling continues, the concentration of the necessary reaction reagents decreases, eventually halting the accumulation of amplified DNA products. 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 for selectively amplifying target DNA. However, to confirm the amplified DNA, a separate procedure such as gel electrophoresis was required after PCR was completed. Therefore, as an improvement over PCR, real-time PCR, which generates or quenches fluorescence in response to the amount of target DNA amplified, has been developed, allowing for easy confirmation of the presence or absence of target DNA in a sample. In conventional PCR, once the amount of template DNA in a sample exceeds a certain amount before PCR, the amount of amplified DNA after PCR often reaches a plateau, making it impossible to quantify the amount of template DNA before PCR. However, real-time PCR allows for real-time detection of the amount of amplified DNA during PCR before the plateau is reached, making it possible to quantify the amount of template DNA before PCR based on the DNA amplification pattern. For this reason, real-time PCR is also known as quantitative PCR.

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

[0007] PCR and real-time PCR are powerful techniques that exponentially amplify genes through thermal cycling, 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 the heater, and traditionally, 30 to 40 PCR cycles take 1 to 2 hours, and in some cases even longer. As a result, even with the latest genetic testing equipment, analysis typically takes more than an hour in total, and speeding up PCR operations has been a major challenge since the technology was introduced.

[0008] The present inventors have developed a reciprocal flow type nucleic acid amplification device that uses a microblower or the like as a liquid delivery mechanism in order to speed up PCR operations (Patent Document 1).

[0009] Meanwhile, multiplex PCR, which uses multiple primer pairs in a single PCR reaction system to simultaneously amplify multiple gene regions, has been gaining attention. Real-time multiplex PCR, an advanced version of multiplex PCR, aims to distinguish and detect multiple different target genes without compromising sensitivity and to obtain quantitative results, while minimizing the influence of each target (crosstalk) on other targets. However, it has been reported that quantitative multiplex reactions involving two or more types are often difficult due to issues such as the types of fluorescent substances that can be labeled and overlapping fluorescence wavelengths.

[0010] Patent Document 1 reports, as an example of multiplex PCR, the results of measurement using a multicolor fluorescence detector capable of simultaneously measuring three types of fluorescence, with one point on a linear microchannel as the detection point. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2016 / 006612 Summary of the Invention [Problem to be solved by the invention]

[0012] However, when multiplex PCR is performed using a multicolor fluorescence detector as in Patent Document 1, and the fluorescence intensities of multiple probes are measured simultaneously using a single point on a linear flow path as the detection point, the spectrum of the fluorescence wavelength emitted from one of the probes partially overlaps with the spectrum of the fluorescence wavelength from another probe that is measured simultaneously, or with the spectrum of the wavelength of the light source that excites the fluorescent dye labeled on another probe, making it impossible to distinguish between them. Therefore, in order to accurately measure the fluorescence intensity, it is desirable to avoid such interference between fluorescence light and excitation light.

[0013] To prevent this interference, it is also possible to arrange three measurement points on a linear flow path and measure the fluorescence intensity by turning on the excitation light source at different times. However, this method has the disadvantage of requiring a slower flow rate to avoid interference.

[0014] An object of the present invention is to provide a real-time PCR method that is rapid and has reduced noise even when multiplex PCR is performed. [Means for solving the problem]

[0015] As a result of extensive research to solve the above problems, the present inventors have found that the above problems can be solved by measuring the fluorescence intensity for each thermal cycle at predetermined positions in a microchannel with two spatially separated temperature ranges when performing real-time PCR. The present invention was completed as a result of further research based on this discovery.

[0016] The present invention includes the following aspects.

[0017] Item 1: A reciprocal flow 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 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 connecting part connectable to a liquid transfer mechanism for realizing the movement of the 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. A method for performing real-time PCR by measuring fluorescence intensity for each thermal cycle at predetermined positions in the flow channel corresponding to the denaturation temperature range and the flow channel corresponding to the extension / annealing temperature range.

[0018] Item 2. A nucleic acid amplification method comprising the following steps: Step 1: A heater capable of forming a denaturation temperature zone and an extension / annealing temperature zone. a fluorescence detector capable of measuring the fluorescence intensity of the sample solution 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 solution delivery mechanism that enables the transfer of the sample solution between the denaturation temperature range and the extension / annealing temperature range and that is released to atmospheric pressure when the solution delivery is stopped; a substrate on which a nucleic acid amplification chip can be mounted; a control mechanism that controls 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; On the substrate of a reciprocal flow type nucleic acid amplification device characterized by performing real-time PCR by measuring the fluorescence intensity for each thermal cycle, a step of placing a nucleic acid amplification chip having at least one curved flow channel 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 micro-flow channel provided with a connector at one or both ends of the flow channel that can be connected to a liquid supply mechanism in the nucleic acid amplification device; Step 2: connecting the liquid delivery mechanism connecting portion of the microchannel to the liquid delivery mechanism; Step 3: performing thermal cycling by moving the sample liquid back and forth between the two curved flow paths of the microchannel using the liquid delivery mechanism; and Step 4: A step of measuring the fluorescence intensity of the sample solution for each thermal cycle by the fluorescence detector at predetermined positions in the flow channel corresponding to the denaturation temperature range and the flow channel corresponding to the extension / annealing temperature range.

[0019] Item 3. The nucleic acid amplification method according to Item 1 or 2, wherein the liquid-transporting mechanism is a microblower or a blower.

[0020] Item 4. The nucleic acid amplification method according to any one of Items 1 to 3, wherein the intermediate channel connecting the curved channels is linear.

[0021] Item 5. The nucleic acid amplification method according to Item 4, further comprising a fluorescence detector capable of measuring the fluorescence intensity of the sample solution passing through the intermediate flow path connecting the curved flow path of the denaturation temperature zone and the extension / annealing temperature zone, and measuring the fluorescence intensity of the sample solution for each thermal cycle using the fluorescence detector at a predetermined position in the intermediate flow path.

[0022] Item 6. The nucleic acid amplification method according to Item 5, wherein the distance between the fluorescence measurement point (P2) on the intermediate flow path and the fluorescence measurement point (P1) on the denaturation temperature zone is 8 mm or more. [Effects of the Invention]

[0023] According to the present invention, it is possible to provide a real-time PCR method that is rapid and has reduced noise, even when multiplex PCR is performed. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a diagram showing an example of the device configuration of a nucleic acid amplification device (two heaters). [Figure 2] FIG. 1 is a diagram showing an example of the device configuration of a nucleic acid amplification device (three heaters). [Figure 3] FIG. 1 shows an example of curved channels for the denaturation temperature zone and extension / annealing temperature zone of a PCR chip, and a linear intermediate channel connecting these channels. [Figure 4] This is a graph of multiplex qPCR (Cy5 detection). [Figure 5]Graph of multiplex qPCR (FAM detection). [Figure 6] Graph of multiplex qPCR (ABY detection). DETAILED DESCRIPTION OF THE INVENTION

[0025] The nucleic acid amplification method of the present invention will be described in detail below.

[0026] The nucleic acid amplification method of the present invention is a method of thermal cycling in which a sample solution is moved back and forth between two temperature zones in a microchannel. This nucleic acid amplification method is sometimes called a reciprocal flow type nucleic acid amplification method.

[0027] The nucleic acid amplification method of the present invention is a real-time PCR (polymerase chain reaction) that can monitor the status of gene amplification during the PCR reaction. PCR amplifies nucleic acids by utilizing multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension, which results in an exponential increase in the copy number of the target nucleic acid sequence.

[0028] To achieve real-time PCR, fluorescence intensity is measured at each thermal cycle. Specifically, the change in fluorescence intensity with each cycle, which increases as the target DNA is amplified by thermal cycling, is recorded over time. 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 manner in which gene amplification occurs—i.e., the number of cycles at which the gene product grows exponentially—depends on the amount of the template. Therefore, the amount of target gene present in a sample can be calculated by comparing the gene amplification status with that when an external standard DNA of known concentration is used as a template.

[0029] 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 performed 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 performed using a nucleic acid amplification device configured as shown in Figure 2, where complementary DNA (cDNA) is first generated (reverse transcribed) from mRNA using a reverse transcriptase. 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 allows reverse transcription and PCR cycling to be carried out quickly and easily in one step.

[0030] In one preferred embodiment, the nucleic acid amplification method of the present invention is a multiplex PCR in which multiple gene regions are simultaneously amplified by using multiple primer pairs in a single PCR reaction system. Multiplex PCR in which two or three gene regions are simultaneously amplified is particularly preferred.

[0031] The nucleic acid amplification method of the present invention is carried out by moving a sample liquid through a microchannel. The microchannel for carrying out the nucleic acid amplification method of the present invention includes at least curved channels corresponding to a denaturation temperature range and an extension / annealing temperature range, respectively, a straight or curved intermediate channel connecting the curved channel corresponding to the denaturation temperature range with the curved channel corresponding to the extension / annealing temperature range, and a connector that can be connected to a liquid delivery mechanism for realizing the movement of the sample liquid. The microchannel can also include an opening for introducing the sample liquid. The opening for introducing the sample liquid can optionally be made sealable with a seal, valve, etc.

[0032] 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 thermosetting and photosetting resins such as cycloolefin polymer (COP). Furthermore, from the perspective of performing fluorescence detection, a transparent material that is highly transmissive to light (especially excitation light and emitted light for fluorescence detection) (i.e., has little absorption, diffusion, reflection, etc.) is preferred.

[0033] The microchannel can have a structure in which grooves are formed in a material by, for example, machining such as NC cutting, injection molding, nanoimprinting, soft lithography, or the like, and then sealed with a seal (preferably a transparent seal made of, for example, polyolefin). Alternatively, the microchannel can be formed by three-dimensional printing. The cross-sectional shape of the microchannel is not particularly limited and can be semicircular, circular, rectangular, wedge-shaped, trapezoidal, polygonal, or the like. The cross-section of the microchannel can have a width of, for example, about 10 to 1000 μm and a depth of, for example, about 10 to 1000 μm. The width and depth of the microchannel can each be constant, or the width or depth can vary partially.

[0034] The shapes of the curved flow channel corresponding to the denaturation temperature range and the curved flow channel corresponding to the elongation / annealing temperature range provided in the microchannel can be a serpentine flow channel having a loop shape, a spiral flow channel, etc. The intermediate flow channel connecting the curved flow channel corresponding to the denaturation temperature range and the curved flow channel corresponding to the elongation / annealing temperature range can be either linear or curved.

[0035] The length of each of the curved channel corresponding to the denaturation temperature range and the curved channel corresponding to the extension / annealing temperature range is preferably 20 mm or more.

[0036] The curved flow path corresponding to the denaturation temperature range and the curved flow path corresponding to the extension / annealing temperature range provided in the microchannel are each maintained at the corresponding temperature, and the temperature of the sample liquid that has moved into that temperature range is changed to the temperature of that temperature range.

[0037] The denaturation temperature zone is maintained at a temperature required for the DNA denaturation reaction in PCR. The temperature in 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 required for the DNA annealing reaction and extension reaction in PCR. The temperature in the extension / annealing temperature zone is preferably about 40 to 75°C, and more preferably about 55 to 65°C.

[0038] The denaturation temperature zone and the extension / annealing temperature zone are 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 is in contact with the microchannel. Specific examples of the heat source include a cartridge heater, a film heater, a Peltier heater, etc.

[0039] In the nucleic acid amplification method of the present invention, the sample liquid moves in the form of a plug through the microchannel. The volume of the sample liquid moving through the microchannel is not particularly limited, but is preferably about 5 to 50 μL, more preferably about 15 to 20 μL.

[0040] The sample solution contains components necessary for the 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 the PCR reaction such as template nucleic acid (which may be either DNA or RNA), polymerase, enzymes such as reverse transcriptase, various deoxyribonucleotide triphosphates which may be labeled, and primer sets corresponding to target gene regions; components necessary for fluorescence detection such as fluorescent probes such as TaqMan probes, Cycleave probes, and E-probes (registered trademark), and dyes such as SYBR GREEN; and buffer components for adjusting pH and salt concentration.

[0041] When the PCR of the present invention is multiplex PCR, the sample solution contains two or more primer sets. Generally, a "primer set" refers to a combination of a forward primer and a reverse primer, and typically 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 if the reverse primer is combined with two or more forward primers (as a primer pair) to generate amplification products that each correspond to a different gene region.

[0042] Examples of dyes (fluorochromes) 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.

[0043] The dyes used in the multiplex PCR of the present invention 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.

[0044] In the nucleic acid amplification method of the present invention, the movement of sample liquid is achieved by a liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped. In other words, rather than using a mechanism that requires the inside of the flow channel to be a closed system to prevent pressure from escaping, such as a syringe pump, 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, immediately stopping the liquid delivery. Therefore, accurate position control is possible even without multiple pressure-relief valves for controlling the position of the sample liquid.

[0045] Examples of the liquid delivery mechanism that is released to atmospheric pressure when liquid delivery is stopped include a micro-blower and a fan.

[0046] A microblower (also called a piezoelectric microblower) is a well-known device that draws in and discharges air and is characterized by not having a sealed structure (no check valve). In a typical microblower, the diaphragm is bent and deformed by applying a voltage to a piezoelectric element, thereby achieving the draw and discharge of air. Examples of microblower that can be used include those manufactured by Murata Manufacturing Co., Ltd. (MZB1001T02, MZB3004T04).

[0047] 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.

[0048] In the nucleic acid amplification method of the present invention, reciprocal flow type nucleic acid amplification is carried out by performing the following steps [1] to [4] as one cycle. [1] A step of operating a liquid delivery mechanism to move the sample liquid from the elongation / annealing temperature range to the denaturation temperature range via an intermediate flow path; [2] A step of 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 range through the intermediate flow path to the extension / annealing temperature range; and [4] A process in which the liquid delivery mechanism is operated to maintain the sample liquid within the elongation / annealing temperature range for a certain period of time.

[0049] 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 target gene, etc.

[0050] 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 path, 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.

[0051] The time for which the sample solution is held in the denaturation temperature range and the time for which the sample solution is held in the extension / annealing temperature range can each be appropriately set depending on the target gene region (type of gene, length of region, etc.) For example, the time for which the sample solution is held in the denaturation temperature range can be about 2 to 10 seconds, and the time for which the sample solution is held in the extension / annealing temperature range can be about 2 to 60 seconds.

[0052] The liquid delivery mechanism is connected to the microchannel via, for example, a connection portion.

[0053] In one embodiment of the present invention, two liquid delivery mechanisms are connected to the two ends of the microchannel, one at each end. That is, a first liquid delivery mechanism connected to deliver liquid from the elongation / annealing temperature range to the denaturation temperature range is operated in the above step [1], and a second liquid delivery mechanism connected to deliver liquid from the denaturation temperature range to the elongation / annealing temperature range is operated in the above step [3].

[0054] In another aspect of the present invention, one liquid delivery mechanism is connected to two ends of a microchannel via branched connecting channels equipped with switching valves. That is, in step [1], the liquid delivery mechanism is operated with the channel configured via the switching valve to deliver liquid from the elongation / annealing temperature range to the denaturation temperature range, and in step [3], the liquid delivery mechanism is operated with the channel configured via the switching valve to deliver liquid from the denaturation temperature range to the elongation / annealing temperature range. In the case of a three-way switching valve, the switching valve is connected to a microblower or fan that is released to atmospheric pressure when liquid delivery is stopped. Two three-way valves are located at both ends of the air channel that branches into two directions at the branch point, and alternately blow air through two connecting channels leading to the two ends of the microchannel, thereby enabling the sample liquid to be delivered back and forth within the microchannel. In this case, the three-way valve allows the sample liquid to be delivered by blowing air with one valve closed and the other valve open. Although it is preferable to have one three-way valve, a combination of two-way valves or a multi-way valve such as a three-way, four-way or five-way valve may be used depending on the combination of connecting flow paths.

[0055] In another aspect 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 the switching valve to deliver liquid from the extension / annealing temperature zone toward the denaturation temperature zone, and in the above step [3], the liquid delivery mechanism (air suction means) is operated via the switching valve to deliver liquid from the denaturation temperature zone toward the extension / annealing temperature zone.

[0056] In a typical embodiment of the nucleic acid amplification method of the present invention, the fluorescence intensity of the sample solution is measured for each thermal cycle at predetermined positions in at least two of the three flow paths: the flow path corresponding to the denaturation temperature range; the flow path corresponding to the extension / annealing temperature range; and the linear or curved intermediate flow path. For example, in one embodiment of the present invention, the fluorescence intensity of the sample solution is measured for each thermal cycle at predetermined positions in the flow path corresponding to the denaturation temperature range and the flow path corresponding to the extension / annealing temperature range, and optionally, in the linear or curved intermediate flow path. The predetermined position in the flow path corresponding to the denaturation temperature range is not particularly limited, but is preferably a position that has turned back one to several times (two to four times) or passed through a curved section from the intermediate flow path, such as P1 in Figure 3. The predetermined position in the flow path corresponding to the extension / annealing temperature range is not particularly limited, but is preferably a position that has turned back one to several times (two to four times) or passed through a curved section from the intermediate flow path, such as P3 in Figure 3. The predetermined position in the linear intermediate flow path is not particularly limited, but is preferably P2 in Figure 3. As described above, real-time PCR is achieved by measuring the fluorescence intensity for each thermal cycle.

[0057] When measuring the fluorescence intensity at two positions, the positions at which the fluorescence intensity is measured can be the channel corresponding to the extension / annealing temperature range and the linear or curved intermediate channel.

[0058] Furthermore, when measuring fluorescence intensity at three positions, the positions at which fluorescence intensity is measured are 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.

[0059] The detection of the fluorescence intensity at one position preferably involves the detection of one type of fluorescent species (one type of wavelength).

[0060] At least one of the measurements of fluorescence intensity is preferably for detecting the movement of the sample solution in addition to monitoring the state of gene amplification during the PCR reaction. For example, the movement of the sample solution can be detected in the denaturation temperature range and the extension / annealing temperature range, and the drive of the liquid delivery mechanism can be controlled by the electrical signal from the fluorescence detector related to the movement of the sample solution.

[0061] An example of sample liquid delivery control is shown below. [1] A step of turning on a light source (LED) that illuminates the flow path in the denaturation temperature range, and moving the sample liquid from the extension / annealing temperature range through the intermediate flow path to the denaturation temperature range using a liquid transport mechanism; [2] a step in which the control mechanism receives an electrical signal from the fluorescence detector indicating that the sample liquid in the denaturation temperature range has been detected, and stops the liquid delivery mechanism; [3] A step of turning on a light source (LED) that illuminates the flow path in the extension / annealing temperature range, and moving the sample liquid from the denaturation temperature range through the intermediate flow path to the extension / annealing temperature range by a liquid transport mechanism; [4] A step in which the control mechanism receives an electrical signal from the fluorescence detector indicating that the sample liquid has been detected in the elongation / annealing temperature range, and stops the liquid delivery mechanism.

[0062] The fluorescence intensity can be measured by detecting, with a fluorescence detector, the emitted light (fluorescence) generated by excitation light irradiated from a light source onto the sample solution in the microchannel. When measuring fluorescence intensity in the intermediate flow path, it is preferable to measure it from the time the sample solution starts passing through the fluorescence intensity detection position (P2) in the intermediate flow path until it passes through the fluorescence intensity detection position (P1 or P3) in the denaturation or extension / annealing temperature range. By specifying the fluorescence intensity measurement time (period) as described above, stable measurement results with less noise can be obtained compared to when fluorescence intensity is obtained until after the sample solution has passed P1 or P3.

[0063] The nucleic acid amplification method of the present invention can be carried out, for example, using the following combination of a nucleic acid amplification device and a nucleic acid amplification chip: [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 solution 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 liquid transfer mechanism that enables the transfer of the sample liquid between the two temperature zones and is released to atmospheric pressure when the liquid transfer is stopped; a substrate on which a nucleic acid amplification chip can be mounted; a control mechanism that controls 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] A nucleic acid amplification chip having at least one curved flow channel corresponding to the denaturation temperature range and the extension / annealing temperature range, respectively, a straight or curved intermediate flow channel connecting the curved flow channels, and a micro-flow channel provided with a connection part at one or both ends of the flow channel that can be connected to a liquid delivery mechanism in the nucleic acid amplification device.

[0064] Specifically, this can be carried out by the following steps 1 to 4: Step 1: placing the nucleic acid amplification chip on the 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 microchannel using the liquid transfer mechanism. Step 4: A step of measuring the fluorescence intensity of the sample solution for each thermal cycle by the fluorescence detector at predetermined positions in the flow channel corresponding to the denaturation temperature range and the flow channel corresponding to the extension / annealing temperature range.

[0065] Examples of a nucleic acid amplification device and a nucleic acid amplification chip will be described below with reference to the drawings.

[0066] As shown in Figure 1, the nucleic acid amplification device can include a substrate (not shown) for placing the 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.

[0067] In Figure 1, the temperature control unit for the nucleic acid amplification chip is configured by arranging two cartridge heaters in parallel with a gap of 10 mm between them so that they are in contact with the sealing surface sides 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 heater to control the temperature of the two heaters.

[0068] Cartridge heater 1 is controlled by a control computer to maintain the temperature required for the DNA denaturation reaction. Cartridge heater 2 is controlled by a control computer to maintain the temperatures required for the DNA annealing reaction and extension reaction. The temperature ranges for the DNA denaturation reaction and the annealing reaction and extension reaction can be maintained at constant temperatures using, for example, PID (proportional-integral-derivative) control.

[0069] The fluorescence detector is positioned to measure fluorescence intensity at detection points on each of the straight lines of the microchannels in the denaturation temperature range and the extension / annealing temperature range (P1, P3 in Figure 3) and one point on the intermediate channel (P2 in Figure 3). When the sample liquid sent from one curved channel section by pressure reaches detection point P1 or P3 or immediately thereafter, the liquid sending mechanism is stopped, and the sample liquid can be retained in the other curved channel section for a certain period of time.

[0070] The control computer is capable of programmably controlling the liquid delivery mechanism, and while continuously monitoring the fluorescence intensity at the detection point in the center of each microchannel, it alternately switches the liquid delivery mechanism to perform thermal cycling so that the sample liquid moves alternately to the curved channel section on each heater for a set time.The control computer also simultaneously records the change in fluorescence intensity for each cycle that increases as the target DNA is amplified by thermal cycling in real-time PCR, 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.

[0071] A nucleic acid amplification chip equipped with microchannels 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. [Example]

[0072] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0073] [Example] Quantitation of pneumonia-causing bacteria Using a PCR chip for high-speed real-time PCR and the device of the present invention, target genes of pneumonia pathogenic bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae) were quantified. High-speed real-time PCR was performed using commercially available control DNAs, AMPLIRUN® STREPTOCOCCUS PNEUMONIAE DNA CONTROL, AMPLIRUN® HAEMOPHILUS DNA CONTROL, and AMPLIRUN® MYCOPLASMA PNEUMONIAE DNA CONTROL, as template DNAs for Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae, with sterilized water mixed in instead as a negative control (NTC).

[0074] The following primer and probe sequences were used for the three targets: the forward primer sequence for Streptococcus pneumoniae was 5'-AACTCTTACGCAATCTAGCAGATGAA-3' (SEQ ID NO: 1), the reverse primer sequence was 5'-CGTGCAATACTCGTGCGTTTTA-3' (SEQ ID NO: 2), and the TaqMan® probe sequence was 5'-CCGAAAACGCTTGATACA-3' (SEQ ID NO: 3). The forward primer sequence for Haemophilus influenzae was 5'-GGAATCCCAATGCACAAGAAC A-3' (SEQ ID NO: 11), the reverse primer sequence was 5'-GCTTTG GTCAACACATCAACCTT-3' (SEQ ID NO: 12), and the TaqMan® probe sequence was 5'-CATTATTAGTTGCAGGTTCT-3' (SEQ ID NO: 13). The forward primer sequence for Mycoplasma pneumoniae was 5'-CTTGGTCTC CATACTTAACTAAATAAAAAACTC-3' (SEQ ID NO: 21), the reverse primer sequence was 5'-GAACTACAAGCCGCTAATGCAG-3' (SEQ ID NO: 22), and the TaqMan (registered trademark) probe sequence was 5'-GCCTTGAAGGCTGGGTTTGCGCTA-3' (SEQ ID NO: 23).

[0075] Cy5-, FAM-, and ABY-labeled TaqMan® probes were used as fluorescent probes for Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae, respectively, and the final concentrations in the PCR solution were 200 nM each.

[0076] The final concentrations of the two forward primers and reverse primer for Streptococcus pneumoniae and Haemophilus influenzae in the PCR solution were each 1.5 μM, and the final concentrations of the forward primer and reverse primer for Mycoplasma pneumoniae in the PCR solution were each 2.0 μM. Other reagents included Takara Bio's SpeedSTAR® HS DNA polymerase at a final concentration of 0.15 U / μL, and the included FAST Buffer I and dNTP Mixture were mixed at the concentrations specified in the manual to prepare a PCR premix.

[0077] 5.0x10 to 11μl of PCR premixture 3 Three types of control DNA were prepared at copies / μL, and 2 μL of each was used for real-time multiplex PCR. Alternatively, 6 μL of sterile water was added as NTC, and a total volume of 20 μL was used.

[0078] The thermal cycle conditions were a hot start of 10 seconds at 98°C, followed by 50 cycles of 2 seconds at 98°C and 6 seconds at 62°C. The thermal cycle time for 50 cycles under these conditions was 8 minutes and 54 seconds.

[0079] The light source (LED) used to illuminate the denaturation temperature range flow path (P1 in Figure 3) had an excitation wavelength of 525 nm, the light source (LED) used to illuminate the intermediate flow path (P2 in Figure 3) had an excitation wavelength of 470 nm, and the light source (LED) used to illuminate the extension / annealing temperature range flow path (P3 in Figure 3) had an excitation wavelength of 630 nm.

[0080] The results of multiplex PCR using high-speed real-time PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae are shown in Figures 4 to 6. The amplification curve for Streptococcus pneumoniae shows the change in fluorescence intensity of a Cy5-labeled TaqMan® probe, while the amplification curve for Haemophilus influenzae shows the change in fluorescence intensity of a FAM-labeled TaqMan® probe, and the amplification curve for Mycoplasma pneumoniae shows the change in fluorescence intensity of an ABY-labeled TaqMan® probe, as shown by the solid lines in Figure 6, with the NTC results superimposed by the dashed lines. As shown by the solid lines, when any of the three types of control DNA was included, a clear amplification was obtained compared to the fluorescent signal of NTC, shown by the dashed line, making it possible to simultaneously measure multiple items from the same sample.

Claims

[Claim 1] A reciprocal flow 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 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 connecting part connectable to a liquid transfer mechanism for realizing the movement of the 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. A method for performing real-time PCR by measuring the fluorescence intensity for each thermal cycle at predetermined positions in the flow channel corresponding to the denaturation temperature range and the flow channel corresponding to the extension / annealing temperature range.

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