Nucleic acid amplification method

Abstract

A reciprocating flow type nucleic acid amplification method is characterized in that a sample liquid reciprocates between two spatially independent temperature zones in a microchannel to perform thermal circulation, the microchannel is connected with the two temperature zones, and the two temperature zones are a denaturation temperature zone and an extension-annealing temperature zone; the micro-channel at least comprises a bent channel corresponding to the denaturation temperature zone, a bent channel corresponding to the extension-annealing temperature zone and a linear or bent intermediate channel, and the intermediate channel is connected with the bent channel corresponding to the denaturation temperature zone and the bent channel corresponding to the extension-annealing temperature zone; and a connector connected to a liquid delivery mechanism capable of moving the sample liquid; in one embodiment, a method includes moving a sample liquid within a microchannel by a liquid delivery mechanism, opening the liquid delivery mechanism to atmospheric pressure when liquid delivery is stopped, and measuring a fluorescence intensity for each thermal cycle at a predetermined point on the channel corresponding to a denaturation temperature zone and a predetermined point on the channel corresponding to an extension-annealing temperature zone, and carrying out real-time PCR.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number: 202080021631.5, application date: March 13, 2020, and invention name “Nucleic Acid Amplification Method”. Technical Field

[0002] Cross-references to related patent applications This application claims the benefit of priority to Japanese Patent Application No. 2019-049009 filed on March 15, 2019, the entire contents of which are incorporated herein by reference. The present invention relates to a method for amplifying a nucleic acid. Background Art

[0003] Nucleic acid detection is central to many fields including drug development, forensics, clinical testing, and identification of crop and pathogenic microbial species. The ability to detect a variety of diseases (such as cancer), microbial infections, and genetic markers through molecular phylogenetic analysis is a versatile technology for diagnosis of disease and developmental risks, biomarker searches, food and environmental safety assessments, criminal evidence, and many other technical aspects.

[0004] One of the most powerful basic technologies for detecting small amounts of nucleic acids (genes) with high sensitivity is the analysis of products obtained by exponential replication and amplification of a portion or the entire nucleic acid sequence.

[0005] Polymerase chain reaction (PCR) is a powerful technique for selectively amplifying specific regions of DNA. Using PCR, millions of copies of DNA fragments can be generated from a single template DNA for a target DNA sequence in the template DNA. PCR repeats the following reactions in sequence using two-phase or three-phase temperature conditions (called "thermal cycling"): DNA denaturation into single strands, primer annealing to form denatured DNA single strands, and extension of primers by thermostable DNA polymerase. The cycle is repeated until enough copies are obtained for analysis. In principle, the number of copies can be doubled in a single cycle of PCR. In fact, as the thermal cycle continues, the concentration of the required reaction reagents decreases, and the accumulation of the amplified DNA products will eventually stop. For general details of PCR, see Clinical Applications of PCR, Dennis Lo (ed.), Humana Press (Totowa, New Jersey) (1998); and PCR Protocols: A Guide to Methods and Applications, MA Innis et al. (ed.), Academic Press Inc. (San Diego, California) (1990).

[0006] Although PCR is a powerful technique that can selectively amplify target DNA, the amplified DNA needs to be confirmed by another operation performed separately, such as gel electrophoresis, after PCR is completed. In order to improve PCR, real-time PCR was developed to generate or quench fluorescence according to the amount of amplified target DNA. Real-time PCR simplifies the confirmation of whether the target DNA is present in the sample. In conventional PCR technology, template DNA exceeding the predetermined amount in the sample before PCR often causes the amplified amount of DNA after PCR to reach a plateau; this hinders the quantification of the amount of template DNA before PCR. However, real-time PCR is able to detect the amount of amplified DNA in real time during the PCR process before the amount of amplified DNA reaches a plateau; therefore, the amount of template DNA before PCR can be quantified from the progress of DNA amplification. This is why real-time PCR is also called "quantitative PCR."

[0007] Quantification of the amount of target DNA by real-time PCR is particularly useful in clinical practice, for example, in monitoring changes in virus levels to confirm the efficacy of treatment for viral infections such as AIDS virus (HIV), etc. DNA quantification by real-time PCR is also effective in the diagnosis of opportunistic infections such as herpes simplex virus (HHV) (many patients are often subclinically infected since infancy and develop infections due to the proliferation of the virus when the body is weak).

[0008] Although PCR and real-time PCR are powerful techniques for exponentially amplifying genes through thermal cycling, conventional thermal cyclers employed in PCR are slow in temperature control due to the large heat capacity of the aluminum block used as a heater; and typically require 1 to 2 hours, or even longer in some cases, to perform 30 to 40 cycles of PCR. Therefore, even with the most advanced genetic testing equipment, analysis typically still requires more than 1 hour in total. Speeding up PCR operations has been a huge challenge since the advent of the technology.

[0009] For the purpose of accelerating the PCR operation, the inventors of the present invention developed a reciprocating flow type nucleic acid amplification apparatus which uses a micro blower or the like as a liquid transport mechanism (PTL 1).

[0010] Meanwhile, multiplex PCR, which uses multiple primer pairs to simultaneously amplify multiple gene regions in a single PCR reaction system, has attracted attention. Real-time multiplex PCR, which evolved from multiplex PCR, aims to distinguish and detect multiple different target genes and obtain quantitative results while being less affected by other target genes (crosstalk) and without reducing sensitivity. However, it is reported that quantitative multiplex reactions for more than two targets are generally difficult due to problems such as the limited combination of fluorescent substances that can be used for labeling to avoid overlapping fluorescence wavelengths.

[0011] PTL 1 reports the results of measurement at one point (detection point) on a linear channel of a microchannel as an example of multiplex PCR by employing a multicolor fluorescence detector capable of simultaneously measuring three types of fluorescence.

[0012] Reference List Patent Literature PTL 1: WO2016 / 006612A Summary of the invention

[0013] Problems to be solved by the invention However, if a multicolor fluorescence detector is used in multiplex PCR to simultaneously measure the fluorescence intensity of a plurality of probes at one detection point on a linear channel as in PTL 1, the spectrum of the fluorescence wavelength emitted from one of the probes partially overlaps with the spectrum of the fluorescence wavelength from another probe measured simultaneously or the spectrum of the wavelength of the light source used to excite the fluorescent dye labeled with the other probes, making these spectra indistinguishable from each other. Therefore, in order to accurately measure the fluorescence intensity, it is desirable that there is no such interference between the fluorescence emissions or between the fluorescence emissions and the excitation light.

[0014] To prevent such interference, three measurement points can be arranged on the linear channel, and the excitation light source can be turned on at different time intervals to measure the fluorescence intensity. However, inconveniently, the liquid delivery speed must be slowed down to avoid interference.

[0015] An object of the present invention is to provide a real-time PCR method that is rapid and low-noise even when performing multiplex PCR.

[0016] Technical solutions to solve problems To achieve the above object, the inventors found after in-depth research that the object can be achieved by measuring the fluorescence intensity of each thermal cycle at predetermined points in two spatially independent temperature zones of a microchannel when performing real-time PCR. Based on this discovery, the inventors further conducted research and completed the present invention.

[0017] The present invention includes the following embodiments.

[0018] Item 1. A reciprocating flow type nucleic acid amplification method, wherein the method performs thermal cycling by reciprocating a sample liquid between two spatially independent temperature zones in a microchannel, wherein the microchannel connects the two temperature zones. The two temperature zones are a denaturation temperature zone and an extension-annealing temperature zone, The microchannel comprises at least: The tortuous channel corresponding to the denaturation temperature zone, The curved channel corresponding to the extension-annealing temperature zone, 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 connector that can be connected to a liquid delivery mechanism capable of moving the sample liquid; The method comprises The sample liquid in the microchannel is moved by the liquid delivery mechanism, wherein when the liquid delivery is stopped, the liquid delivery mechanism is opened to the atmospheric pressure, and The fluorescence intensity of each thermal cycle is measured at a predetermined point on the channel corresponding to the denaturation temperature zone and at a predetermined point on the channel corresponding to the extension-annealing temperature zone to perform real-time PCR.

[0019] Item 2: A nucleic acid amplification method comprising the following steps: Step 1: Mounting a nucleic acid amplification chip on a substrate of a reciprocating flow type nucleic acid amplification device capable of performing real-time PCR by measuring the fluorescence intensity of each thermal cycle, The reciprocating flow type nucleic acid amplification device comprises: a heater forming a denaturation temperature zone and an extension-annealing temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the denaturation temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the extension-annealing temperature zone; a liquid delivery mechanism that allows the sample liquid to move between the denaturation temperature zone and the extension-annealing temperature zone, and when the liquid delivery is stopped, the liquid delivery mechanism is open to the atmospheric pressure; a substrate on which a nucleic acid amplification chip is mounted; and A control mechanism, which receives an electrical signal related to the movement of the sample liquid from the fluorescence detector to control the driving of the liquid delivery mechanism; The nucleic acid amplification chip comprises at least one microchannel, and the at least one microchannel comprises: The tortuous channel corresponding to the denaturation temperature zone, The curved channel corresponding to the extension-annealing temperature zone, 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 connector that connects the liquid delivery mechanism of the nucleic acid amplification device to one or both ends of the microchannel; Step 2: Connecting a connector of a liquid delivery mechanism on a microchannel to the liquid delivery mechanism; Step 3: using the liquid transport mechanism to allow the sample liquid to reciprocate between the two curved channels of the microchannel to perform thermal cycling; and Step 4: Measure the fluorescence intensity of the sample liquid for each thermal cycle at a predetermined point on the curved channel corresponding to the denaturation temperature zone and at a predetermined point on the curved channel corresponding to the extension-annealing temperature zone by a fluorescence detector.

[0020] Item 3. The nucleic acid amplification method according to Item 1 or Item 2, wherein the liquid delivery mechanism is a micro blower or a fan.

[0021] 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 a straight channel.

[0022] Item 5. The nucleic acid amplification method according to Item 4, wherein the reciprocating flow type nucleic acid amplification device further comprises a fluorescence detector, which measures the fluorescence intensity of the sample liquid flowing through the intermediate channel, wherein the intermediate channel connects the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension-annealing temperature zone, The method comprises the step of measuring the fluorescence intensity of the sample liquid for each thermal cycle at a predetermined point of the intermediate channel by the fluorescence detector.

[0023] Item 6. The nucleic acid amplification method according to Item 5, wherein the distance between the fluorescence measurement point (P2) on the intermediate channel and the fluorescence measurement point (P1) on the denaturation temperature zone is greater than 8 mm.

[0024] Beneficial Effects The present invention provides a real-time PCR method that is rapid and low-noise even when performing multiplex PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 An example of the structure of a nucleic acid amplification device (two heaters) is shown.

[0026] Figure 2 An example of the structure of a nucleic acid amplification device (three heaters) is shown.

[0027] Figure 3 An embodiment of the curved channels of the denaturation temperature zone and the extension-annealing temperature zone on the PCR chip and the straight intermediate channels connecting these curved channels is shown.

[0028] Figure 4 is a graph of multiplex qPCR (Cy5 detection).

[0029] Figure 5 is a graph of multiplex qPCR (FAM detection).

[0030] Figure 6 is a graph of multiplex qPCR (ABY assay). DETAILED DESCRIPTION

[0031] The nucleic acid amplification method according to the present invention is described in detail below.

[0032] The nucleic acid amplification method according to the present invention performs thermal cycling by reciprocating the sample liquid between two temperature zones in a microchannel. This type of nucleic acid amplification method is sometimes referred to as a "reciprocating flow type nucleic acid amplification method."

[0033] In the PCR (polymerase chain reaction) technology, the nucleic acid amplification method according to the present invention is a real-time PCR capable of monitoring the gene amplification status during the PCR reaction. PCR amplifies nucleic acids by using multiple cycles of denaturation, annealing of primer pairs to form opposite chains, and primer extension, resulting in an exponential increase in the number of copies of the target nucleic acid sequence.

[0034] In order to realize real-time PCR, the fluorescence intensity of each thermal cycle is measured. Specifically, the initial amount of the target DNA can be quantified by recording the change of the fluorescence intensity of each cycle over time, wherein the fluorescence intensity increases with the target DNA amplified by thermal cycling, and calculating the cycle number (Ct value) when the fluorescence intensity exceeds the threshold. In PCR, the way of gene amplification (i.e., the cycle number of the logarithmic increase of the gene product) depends on the amount of the basic template. Therefore, the amount of the target gene present in the sample can be calculated by comparing the amplification of the gene with the amplification of an external standard DNA with a known concentration.

[0035] The nucleic acid amplification method of the present invention can be carried out using DNA or RNA as a template. In the case of using DNA as a template, by using Figure 1 In the case of using RNA as a template (real-time RT-PCR), firstly, the nucleic acid amplification device is configured to perform PCR. Figure 2 The nucleic acid amplification device is configured to generate (reverse transcribe) complementary DNA (cDNA) from mRNA by reverse transcriptase, and then perform PCR. The kit and protocol used in PCR can be selected from a series of known kits and protocols. When the nucleic acid amplification method according to the present invention is real-time RT-PCR, a one-step RT-PCR can be used, which can achieve rapid and simple reverse transcription and cycling in one step in PCR.

[0036] In a preferred embodiment, the nucleic acid method according to the present invention is multiplex PCR, which simultaneously amplifies multiple gene regions by using multiple primer pairs in a single PCR reaction system. Particularly preferably, the nucleic acid amplification method according to the present invention is multiplex PCR for simultaneously amplifying two or three different gene regions.

[0037] The nucleic acid amplification method of the present invention is performed by allowing the sample liquid to move through a microchannel. The microchannel used to perform the nucleic acid amplification method according to the present invention includes at least a curved channel corresponding to a denaturation temperature zone, a curved channel corresponding to an extension-annealing temperature zone, 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 connector that can be connected to a liquid transport mechanism capable of moving the sample liquid. The microchannel may also include an opening for introducing the sample liquid. The opening for introducing the sample liquid can be sealed by an arbitrarily selected seal, valve or similar component.

[0038] Preferably, the microchannel is formed of a material that meets some or all of the following requirements: (i) relatively high thermal conductivity, (ii) stability within the temperature range required for PCR, (iii) resistance to corrosion caused by electrolyte solutions or organic solvents, and (iv) low adsorption of nucleic acids and proteins. Specific examples of materials include glass, quartz, silicon, and various thermosetting or photocurable resins, such as cycloolefin polymers (COPs). From the perspective of detecting fluorescence, the material preferably has high light transmittance (especially excitation light and emission light used for fluorescence detection) (i.e., the material does not absorb, diffuse or reflect a large amount of light) and is transparent.

[0039] The microchannel may have a structure in which a groove is formed in a material by, for example, mechanical processing such as CNC machining cutting, injection molding, nanoimprinting, or soft lithography; and in which the groove is sealed by a seal (preferably a transparent seal formed of, for example, polyolefin). Alternatively, the microchannel may be formed by three-dimensional printing. The cross-section of the microchannel may be any shape; the shape may be, for example, semicircular, circular, rectangular, wedge-shaped, trapezoidal, or polygonal. The cross-section of the microchannel may be, for example, about 10 to 1000 μm wide and about 10 to 1000 μm deep. The width and depth of the microchannel may be constant, or the width or depth may be partially changed.

[0040] The curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension-annealing temperature zone in the microchannel can be a serpentine microchannel having an annular shape, or a spiral curved microchannel. The intermediate channel connecting the curved channel corresponding to the denaturation temperature zone to the curved channel corresponding to the extension-annealing temperature zone can be a straight line or a curved shape.

[0041] Preferably, the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension-annealing temperature zone each have a length of 20 mm or more.

[0042] Each of the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension-annealing temperature zone in the microchannel is maintained at a predetermined temperature. The temperature of the sample liquid transported to the temperature zone is changed to the temperature of the temperature zone.

[0043] The denaturation temperature zone is maintained at a temperature required for the DNA denaturation reaction in PCR. Preferably, the temperature of the denaturation temperature zone is about 90 to 100° C., and more preferably about 95° C. The extension-annealing temperature zone is maintained at a temperature required for the annealing and extension reactions of the DNA in PCR. Preferably, the temperature of the extension-annealing temperature zone is about 40 to 75° C., and more preferably about 55 to 65° C.

[0044] Preferably, the denaturation temperature zone and the extension-annealing temperature zone are each maintained at a constant temperature. The temperature maintenance can be achieved by a heat source. The heat source, for example, is placed in or in contact with the microchannel. Specific examples of the heat source include a cartridge heater, a film heater, and a Peltier heater.

[0045] In the nucleic acid amplification method according to the present invention, the sample liquid flows through the microchannel in a plug-like form. The sample liquid flowing through the microchannel can be of any volume, preferably about 5 to 50 μL, more preferably about 15 to 20 μL.

[0046] The sample liquid contains components required for PCR reaction and components required for fluorescence detection to realize real-time PCR. For example, the sample liquid contains an aqueous matrix mainly composed of: water, enzymes such as template nucleic acid (DNA or RNA) polymerase and reverse transcriptase, optionally labeled deoxynucleotide adenosine triphosphate, and a primer set of the corresponding target gene region as components required for PCR reaction; and fluorescent probes (such as TaqMan probes, Cycleave probes, E-probes (registered trademark)) and dyes (such as SYBR GREEN) as components required for fluorescence detection. The sample liquid may contain a buffer component to adjust pH and salt concentration.

[0047] When the PCR of the present invention is a multiplex PCR, the sample liquid contains two or more types of primer sets. Generally speaking, a "primer set" refers to a combination of a forward primer and a reverse primer; typically, one type of forward primer and one type of reverse primer are used for one target gene region. Even if the primer set of the present invention contains only one reverse primer and is combined with two or more forward primers (as a primer pair) to produce an amplification product corresponding to the corresponding gene region, the primer set can be used as a primer set for multiplex PCR.

[0048] Examples of dyes (fluorescent dyes) used in 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 (such as 5-FAM, 6-FAM), FITC, GPF, 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.

[0049] Preferably, in the present invention, the dye used in the multiplex PCR comprises at least one member selected from ABY and HEX, such as a combination of ABY, Cy5 and FAM; and a combination of HEX, Cy5 and FAM.

[0050] The movement of the sample liquid in the nucleic acid amplification method according to the present invention is realized by a liquid delivery mechanism, wherein when the liquid delivery stops, the liquid delivery mechanism is open to atmospheric pressure. In particular, instead of using a mechanism (e.g., a syringe pump) that needs to form the interior of the channel as a closed system to prevent pressure from escaping, the present invention uses a liquid delivery mechanism that is configured to form an open system even during liquid delivery. This liquid delivery mechanism, when the airflow stops, makes the pressure in the channel equal to the pressure outside the channel in an instant, and eliminates the pressure acting on the plug-shaped sample liquid, thereby stopping liquid delivery at once. This can achieve accurate position control without the need for multiple pressure relief valves for controlling the position of the sample liquid.

[0051] Examples of liquid delivery mechanisms that are open to atmospheric pressure when liquid delivery is stopped include micro-blowers and fans.

[0052] A micro blower, also called a "piezoelectric micro blower," is a known device for sucking and ejecting air, characterized by its non-sealed structure (no check valve). A typical micro blower causes a piezoelectric element to bend and deform its diaphragm by applying a voltage to the element to suck and eject air. For example, micro blowers (MZB1001T02, MZB3004T04) manufactured by Murata Manufacturing Co., Ltd. can be used.

[0053] A fan is a device that blows out air through the rotation of an impeller. Due to the structural characteristics of the impeller, the channel is not a closed system.

[0054] In the nucleic acid amplification method according to the present invention, reciprocating flow type nucleic acid amplification is performed by the following [1] to [4] as one cycle: [1] a step of operating a liquid transport mechanism to move the sample liquid from the extension-annealing temperature zone to the denaturation temperature zone through the intermediate channel, [2] a step of stopping the liquid delivery mechanism to keep the sample liquid in the denaturation temperature zone for a predetermined period of time, [3] a step of operating the liquid transport mechanism to move the sample liquid from the denaturation temperature zone through the intermediate channel to the extension-annealing temperature zone, and [4] A step of operating the liquid delivery mechanism to maintain the sample liquid in the extension-annealing temperature zone for a predetermined period of time.

[0055] This cycle is performed at least once, preferably about 30 to 50 times, and more preferably about 35 to 50 times to perform thermal cycling. The number of cycles can be appropriately determined according to the concentration of the template nucleic acid, the type of the target gene, and the like.

[0056] In the nucleic acid amplification method of the present invention, the speed of movement of the sample liquid, especially the speed of the sample liquid flowing through the intermediate 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.

[0057] The time period during which the sample liquid is kept in the denaturation temperature zone and the time period during which the sample liquid is kept in the extension-annealing temperature zone can be appropriately determined according to the target gene region (such as the type of gene and the length of the gene region). For example, the time period during which the sample liquid is kept in the denaturation temperature zone can be about 2 to 10 seconds, and the time period during which the sample liquid is kept in the extension-annealing temperature zone can be about 2 to 60 seconds.

[0058] The liquid delivery mechanism is connected to the microchannel via, for example, a connector.

[0059] In one embodiment of the present invention, two liquid delivery mechanisms are connected to the microchannel, one mechanism connected to one end of the microchannel and the other mechanism connected to the other end. Specifically, the first liquid delivery mechanism connected to deliver liquid from the extension-annealing temperature zone to the denaturation temperature zone is operated in the above step [1], and the second liquid delivery mechanism connected to deliver liquid from the denaturation temperature zone to the extension-annealing temperature zone is operated in the above step [3].

[0060] In another embodiment of the present invention, a single liquid delivery mechanism is connected to both ends of the microchannel through a branch connection channel having a switching valve. Specifically, in step [1], the liquid delivery mechanism is operated through a passage formed by the switching valve, so that the liquid is delivered from the extension-annealing temperature zone to the denaturation temperature zone. In step [3], the liquid delivery mechanism is operated through the passage structure, so that the liquid is delivered from the denaturation temperature zone to the extension-annealing temperature zone via the switching valve. If the switching valve is a 3-way valve, the 3-way valve is connected to a micro-blower or fan that is open to atmospheric pressure when the liquid delivery stops. By alternately supplying air to the two connecting passages leading to the two ends of the microchannel through two 3-way valves located at both ends of the air flow path that is divided into two directions at the branch point, the sample liquid can be reciprocated in the microchannel. In this case, the 3-way valve supplies air to allow the sample liquid to flow when one valve mouth is closed and the remaining valve mouth is open. Although it is preferred to use a single 3-way valve, a combination of 2-way valves may be used, or multi-way valves such as 3-way, 4-way or 5-way valves, depending on the combination of connecting channels.

[0061] In another embodiment of the present invention, two liquid delivery mechanisms (air injection device and air suction device) are connected to one end (on the extension-annealing temperature zone side) of the microchannel via a branch connection channel having a switching valve. Specifically, in step [1], the liquid delivery mechanism (air injection device) is operated to deliver the liquid from the extension-annealing temperature zone to the denaturation temperature zone through the switching valve. In step [3], the liquid delivery mechanism (air suction device) is operated to deliver the liquid from the denaturation temperature zone to the extension-annealing temperature zone through the switching valve.

[0062] In a typical embodiment, in the nucleic acid amplification method according to the present invention, the fluorescence intensity of the sample liquid of each thermal cycle is measured at predetermined points on at least two of the three channels, the three channels being: a channel corresponding to the denaturation temperature zone, a channel corresponding to the extension-annealing temperature zone, and a straight or curved intermediate channel. For example, in an embodiment of the present invention, the fluorescence intensity of the sample liquid of each thermal cycle is measured at a predetermined point on the channel corresponding to the denaturation temperature zone and at a predetermined point on the channel corresponding to the extension-annealing temperature zone, and optionally at a predetermined point on the straight or curved intermediate channel. The predetermined point on the channel corresponding to the denaturation temperature zone is not particularly limited. However, preferably, the predetermined point is a position 1 to several (2 to 4) turns or curved portions away from the intermediate channel, for example Figure 3 The predetermined point on the channel corresponding to the extension-annealing temperature zone is not particularly limited. However, preferably, the predetermined point is a position after 1 to several (2 to 4) turns or bends of the intermediate channel, for example Figure 3 The predetermined point on the linear intermediate channel is not particularly limited and can be Figure 3 As described above, the measurement of fluorescence intensity during each thermal cycle enables real-time PCR.

[0063] If the fluorescence intensity is measured at two points, the points at which the fluorescence intensity is measured may also be a point on the channel corresponding to the extension-annealing temperature zone and a point on the straight or curved intermediate channel.

[0064] If the fluorescence intensity is measured at three points, the points at which the fluorescence intensity is measured are a point on the channel corresponding to the denaturation temperature zone, a point on the channel corresponding to the extension-annealing temperature zone, and a point on the straight or curved intermediate channel.

[0065] The detection of fluorescence intensity at one point is preferably the detection of one type of fluorescence (one wavelength).

[0066] In addition to monitoring the state of gene amplification during the PCR reaction, it is preferred that at least one measurement of fluorescence intensity is used for the detection of sample liquid movement. For example, the movement of the sample liquid can be detected in the denaturation temperature zone and in the extension-annealing temperature zone, and the driving of the liquid sample transport mechanism can be controlled by the electrical signal related to the movement of the sample liquid emitted by the fluorescence detector.

[0067] An example of control of sample liquid delivery is shown below.

[0068] [1] turning on a light source (LED) for illuminating the denaturation temperature zone channel, and moving the sample from the extension-annealing temperature zone to the denaturation temperature zone through the intermediate channel by a liquid transport mechanism; [2] a step of receiving, in the control mechanism, an electrical signal from the fluorescence detector indicating detection of the sample liquid within the denaturation temperature zone, and stopping the liquid delivery mechanism; [3] turning on a light source (LED) that illuminates the extension-annealing temperature zone channel, and moving the sample from the denaturation temperature zone through the intermediate channel to the extension-annealing temperature zone by a liquid transport mechanism; and [4] A step of receiving, in the control mechanism, an electrical signal from the fluorescence detector indicating detection of the sample liquid within the extension-annealing temperature zone, and stopping the liquid delivery mechanism.

[0069] The fluorescence intensity can be measured by detecting the emission light (fluorescence) generated by the excitation light irradiated by the light source to the sample liquid in the microchannel using a fluorescence detector. When measuring the fluorescence intensity in the intermediate channel, it is preferred to measure the fluorescence intensity from the time point when the sample liquid starts to pass through the fluorescence intensity detection point (P2) on the intermediate channel to the time point when the sample liquid passes through the fluorescence intensity detection point (P1 or P3) on the denaturation or extension-annealing temperature zone. The measurement result obtained by specifying the measurement time (period) of the fluorescence intensity as described above can be stable and less noisy than the result obtained by measuring the fluorescence intensity even after the sample liquid has passed through P1 or P3.

[0070] The nucleic acid amplification method of the present invention can be performed, for example, by using the nucleic acid amplification device and the nucleic acid amplification chip described below.

[0071] Nucleic acid amplification device.

[0072] A reciprocating flow type nucleic acid amplification device, comprising: a heater capable of forming a denaturation temperature zone and an extension-annealing temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the denaturation temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the extension-annealing temperature zone; a liquid delivery mechanism that allows the sample liquid to move between two temperature zones and is open to atmospheric pressure when liquid delivery is stopped; a substrate on which a nucleic acid amplification chip is mounted; and a control mechanism that receives an electrical signal indicating movement of the sample liquid from the fluorescence detector and controls the driving of the liquid delivery mechanism; The reciprocating flow type nucleic acid amplification device performs real-time PCR by measuring the fluorescence intensity of each thermal cycle.

[0073] Chips for nucleic acid amplification.

[0074] Chips for nucleic acid amplification, including at least one microchannel, the at least one microchannel comprising: The tortuous channel corresponding to the denaturation temperature zone, The curved channel corresponding to the extension-annealing temperature zone, Straight or curved intermediate channels connecting the curved channels; and A connector connects the liquid delivery mechanism of the nucleic acid amplification device to one end or both ends of the microchannel.

[0075] Specifically, the nucleic acid amplification method of the present invention can be performed by following steps 1 to 4: Step 1: Mounting a nucleic acid amplification chip on a substrate of a nucleic acid amplification device; Step 2: Connecting a connector of a liquid delivery mechanism on one or both ends of the microchannel to the liquid delivery mechanism; Step 3: Using a liquid transport mechanism to reciprocate the sample liquid between two curved channels of the microchannel to perform thermal cycling; and Step 4: The fluorescence intensity of the sample liquid for each thermal cycle is measured by a fluorescence detector at a predetermined point on the channel corresponding to the denaturation temperature zone and at a predetermined point on the channel corresponding to the extension-annealing temperature zone.

[0076] Embodiments of a nucleic acid amplification device and a nucleic acid amplification chip are described below with reference to the drawings.

[0077] like Figure 1 As shown, the nucleic acid amplification device may include a substrate (not shown) on which a nucleic acid amplification chip is mounted, a temperature control unit of the nucleic acid amplification chip, a liquid delivery mechanism (taking a micro-blower as an example), a fluorescence detector, and a small battery for providing power to a control computer serving as a control mechanism.

[0078] exist Figure 1 In the embodiment, the temperature control unit of the nucleic acid amplification chip includes a cartridge heater, and the two cartridge heaters are arranged in parallel at an interval of 10 mm so as to contact the sealing surface side of the two curved channels of the nucleic acid amplification chip. In order to control the temperature of the two heaters, each heater is connected to a K-type thermocouple.

[0079] The cartridge heater 1 is controlled by a control computer to a temperature required for DNA denaturation reaction. The cartridge heater 2 is controlled by a control computer to a temperature required for DNA annealing reaction and extension reaction. The temperature zone for DNA denaturation reaction and the temperature zone for annealing reaction and extension reaction can be controlled by PID (proportional-integral-differential) and maintained at a constant temperature, for example.

[0080] The fluorescence detector is used to measure a point on the linear channel of the microchannel in the denaturation temperature zone and a point on the linear channel of the microchannel in the extension-annealing temperature zone ( Figure 3 P1 and P3 in the middle), and a point on the intermediate channel ( Figure 3 At the time point when the sample liquid delivered reaches the detection point P1 or P3 from one curved channel due to the application of pressure, or immediately after this time point, the liquid delivery structure can be stopped to allow other curved channels to retain the sample liquid for a predetermined period of time.

[0081] The control computer can program the liquid delivery mechanism. While continuously monitoring the fluorescence intensity at the detection point at the center of each microchannel, the control computer alternately switches the liquid delivery mechanism to move the sample liquid alternately to each curved channel located on the heater for a set time, thereby performing thermal cycling. The control computer also simultaneously records the change in fluorescence intensity of each cycle, wherein the fluorescence intensity increases with the target DNA amplified by thermal cycling in real-time PCR, and calculates the cycle number (Ct value) when the fluorescence intensity exceeds a threshold value to quantify the initial amount of the target DNA.

[0082] Figure 3 FIG. 2 shows a chip for nucleic acid amplification equipped with a microchannel. Figure 3 In the nucleic acid amplification chip shown, two curved channels (meandering channels) are connected by a straight intermediate channel, one of which corresponds to the denaturation temperature zone and the other corresponds to the extension-annealing temperature zone; and the fluorescence of the sample liquid is detected at detection points P1, P2 and P3.

[0083] Example Hereinafter, the present invention is described in detail with reference to the embodiments, but the present invention is not limited to these embodiments.

[0084] Example: Quantification of pneumonia pathogenic bacteria Pneumonia pathogenic bacteria (Streptococcus pneumoniae ( Streptococcus pneumoniae )、Haemophilus influenzae( Haemophilus influenzae ), Mycoplasma pneumoniae) target genes were quantified. The template DNAs of Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae used were AmpliRun (registered trademark) Streptococcus pneumoniae DNA control, AmpliRun (registered trademark) Haemophilus influenzae DNA control, and AmpliRun (registered trademark) Mycoplasma pneumoniae DNA control, all of which are commercially available control DNAs. For the negative control (NTC), sterile water was used for mixing. High-speed real-time PCR was performed using them.

[0085] Primers and probes of the following sequences were used for three different targets. For S. pneumoniae, the forward primer sequence was 5'-AACTCTTACGCAATCTAGCAGATGAA-3' (SEQ ID NO: 1), the reverse primer sequence was 5'-CGTGCAATACTCGTGCGTTTTA-3' (SEQ ID NO: 2), and the TaqMan (registered trademark) probe sequence was 5'-CCGAAAACGCTTGATACA-3' (SEQ ID NO: 3). For Haemophilus influenzae, the forward primer sequence was 5'-GGAATCCCAATGCACAAGAAC A-3' (SEQ ID NO: 4), the reverse primer sequence was 5'-GCTTTGGTCAACACATCAACCTT-3' (SEQ ID NO: 5), and the TaqMan (registered trademark) probe sequence was 5'-CATTATTAGTTGCAGGTTCT-3' (SEQ ID NO: 6). For Mycoplasma pneumoniae, the forward primer sequence was 5'-CTTGGTCTC CATACTTAACTAAATAAAAAACTC-3' (SEQ ID NO: 7), the reverse primer sequence was '-GAACTACAAGCCGCTAATGCAG-3' (SEQ ID NO: 8) and the TaqMan (registered trademark) probe sequence was 5'-GCCTTGAAGGCTGGGTTTGCGCTA-3' (SEQ ID NO: 9).

[0086] The fluorescent probes for Streptococcus pneumoniae, Haemophilus influenzae and Mycoplasma pneumoniae were Cy5-labeled, FAM-labeled and ABY-labeled TaqMan (registered trademark) probes, respectively. The final concentration of each fluorescent probe in the PCR solution was 200 nM.

[0087] The final concentration of each forward primer for Streptococcus pneumoniae and Haemophilus influenzae and the final concentration of each reverse primer in the PCR solution were 1.5 μM. The final concentration of the forward primer for Mycoplasma pneumoniae and the final concentration of the reverse primer in the PCR solution were 2.0 μM. For other reagents, SpeedSTAR (registered trademark) HSDNA polymerase (Takara Bio Inc.) with a final concentration of 0.15 U / μL was used. The attached FAST buffer I and dNTP mixture were mixed at the concentrations indicated in the manual to prepare a PCR premix.

[0088] 5.0×10 3Three types of control DNA were prepared in the amount of copies / μL. 2 μl of each of the three control DNAs or 6 μl of sterile water as NTC was added to 11 μl of the premix for PCR, and a total of 20 μl was used for real-time multiplex PCR.

[0089] Thermocycling conditions were set to perform the following: hot start with heating at 98° C. for 10 seconds, followed by further heating at 98° C. for 2 seconds and 62° C. for 6 seconds for 50 cycles. Under these conditions, the time period for 50 cycles of the thermocycling was 8 minutes and 54 seconds.

[0090] Illuminate the denaturation temperature zone channel ( Figure 3 The light source (LED) in P1 has a wavelength of 525 nm, illuminating the intermediate channel ( Figure 3 The light source (LED) in P2 has a wavelength of 470 nm and illuminates the extension-annealing temperature zone channel ( Figure 3 The light source (LED) in P3) has a wavelength of 630nm.

[0091] Figures 4 to 6 Shown are the results of multiplex PCR using high-speed real-time PCR for S. pneumoniae, H. influenzae, and M. pneumoniae. Figure 4 The graph shows the fluorescence intensity change of the Cy5-labeled TaqMan (registered trademark) probe, wherein the solid line is the amplification curve of Streptococcus pneumoniae and the dotted line is the result of NTC. Figure 5 The fluorescence intensity change of the FAM-labeled TaqMan (registered trademark) probe is shown, wherein the solid line is the amplification curve of Haemophilus influenzae and the dotted line is the result of NTC. Figure 6 The graph shows the change in fluorescence intensity of the ABY-labeled TaqMan (registered trademark) probe, wherein the solid line is the amplification curve of Mycoplasma pneumoniae and the dotted line is the result of NTC.

[0092] As shown by the solid line, when any of the 3 types of control DNA was included, clear amplification was achieved compared to the amplification shown by the fluorescent signal of NTC shown by the dotted line. This indicates that multiple items of the same sample were measured simultaneously.

Claims

1. A reciprocating flow type nucleic acid amplification method, the method is to perform thermal cycling by reciprocating a sample liquid between two spatially independent temperature zones in a microchannel, the microchannel connecting the two temperature zones, wherein: The two temperature zones are a denaturation temperature zone and an extension-annealing temperature zone, The microchannel comprises at least: The tortuous channel corresponding to the denaturation temperature zone, The curved channel corresponding to the extension-annealing temperature zone, 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 connector that can be connected to a liquid delivery mechanism capable of moving the sample liquid; The method comprises: The sample liquid in the microchannel is moved by the liquid delivery mechanism, wherein when the liquid delivery is stopped, the liquid delivery mechanism is opened to the atmospheric pressure, and The fluorescence intensity of each thermal cycle is measured at a predetermined point on the channel corresponding to the denaturation temperature zone and at a predetermined point on the channel corresponding to the extension-annealing temperature zone to perform real-time PCR.

2. A nucleic acid amplification method comprising the following steps: Step 1: Mounting a nucleic acid amplification chip on a substrate of a reciprocating flow type nucleic acid amplification device capable of performing real-time PCR by measuring the fluorescence intensity of each thermal cycle, The reciprocating flow type nucleic acid amplification device comprises: a heater capable of forming a denaturation temperature zone and an extension-annealing temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the denaturation temperature zone; a fluorescence detector that measures the fluorescence intensity of the sample liquid present in the extension-annealing temperature zone; a liquid delivery mechanism that allows the sample liquid to move between the denaturation temperature zone and the extension-annealing temperature zone, and when liquid delivery is stopped, the liquid delivery mechanism is open to atmospheric pressure; a substrate on which the nucleic acid amplification chip is mounted; as well as A control mechanism, which receives an electrical signal related to the movement of the sample liquid from the fluorescence detector to control the driving of the liquid delivery mechanism; The nucleic acid amplification chip comprises at least one microchannel, and the at least one microchannel comprises: The tortuous channel corresponding to the denaturation temperature zone, The curved channel corresponding to the extension-annealing temperature zone, 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 connector that connects the liquid delivery mechanism of the nucleic acid amplification device to one or both ends of the microchannel; Step 2: Connecting a connector of a liquid delivery mechanism on a microchannel to the liquid delivery mechanism; Step 3: The sample liquid is caused to reciprocate between the two curved channels of the microchannel by the liquid transport mechanism to perform thermal cycling; and Step 4: measuring the fluorescence intensity of the sample liquid of each thermal cycle at a predetermined point on the curved channel corresponding to the denaturation temperature zone and at a predetermined point on the curved channel corresponding to the extension-annealing temperature zone by a fluorescence detector.

3. The nucleic acid amplification method according to claim 1, wherein The liquid conveying mechanism is a micro blower or a fan.

4. The nucleic acid amplification method according to claim 1, wherein The intermediate channel connecting the curved channels is a straight channel.

5. The nucleic acid amplification method according to claim 4, wherein The reciprocating flow type nucleic acid amplification device further comprises a fluorescence detector, which measures the fluorescence intensity of the sample liquid flowing through the intermediate channel, wherein the intermediate channel connects the curved channel corresponding to the denaturation temperature zone and the curved channel corresponding to the extension-annealing temperature zone, The method comprises the step of measuring the fluorescence intensity of the sample liquid for each thermal cycle at a predetermined point of the intermediate channel by the fluorescence detector.

6. The nucleic acid amplification method according to claim 5, wherein The distance between the fluorescence measurement point (P2) on the intermediate channel and the fluorescence measurement point (P1) on the denaturation temperature zone is greater than 8 mm.

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