Nucleic acid amplification method and nucleic acid amplification reaction system

Abstract

In the present disclosure, during performance of PCR at a high speed, in order to provide data with high sensitivity and high reliability, application of a thermal cycle at a high speed reduces the amplification efficiency and reduces detection sensitivity, whereas performing multiple thermal cycles in order to maximize the amplification product amount in a short time also increases the non-specific amplification product amount, causes an S / N reduction and / or pseudo-positive determination, and reduces reliability of an analysis result. Thus, an increase in amount of amplification of the target product amount and suppression of non-specific amplification are achieved at a high speed by changing the thermal cycle midway. More specifically, the present disclosure proposes a nucleic acid amplification technology comprising: a first phase in which a sample solution is subjected to a denaturation reaction for a second(s) and then subjected to an annealing and extension reaction for b second(s); and a second phase in which the sample solution that has been subjected to the first phase step is subjected to the denaturation reaction for c second(s) and then subjected to the annealing and extension reaction for d second(s) (b≠d) (see fig. 2).

Description

Nucleic acid amplification method and nucleic acid amplification reaction system

[0001] The present disclosure relates to a nucleic acid amplification method and a nucleic acid amplification reaction system.

[0002] In recent years, the importance of infectious disease testing has increased due to factors such as the increase in deaths from sepsis caused by drug-resistant bacteria and the social damage caused by COVID-19. Since infectious disease testing is performed by laboratory technicians and nurses, ease of operation is required. Furthermore, shortening testing time directly leads to improved treatment efficacy and reduced treatment costs. Furthermore, because it is often difficult to predict the respiratory pathogen responsible for infectious diseases from clinical findings, there is a demand for multiplex detection, which allows for simultaneous testing of multiple pathogens.

[0003] Rapid genetic testing systems using disposable cartridges have been proposed to achieve rapid and reliable infectious disease diagnosis, or to enable anyone to easily perform assays including nucleic acid amplification and detection in various fields such as forensic medicine, livestock farming, environmental measurement, etc. For example, Patent Document 1 discloses a technology that enables multiplex nucleic acid measurement by providing a sample inlet, a purification section, a PCR section, and a detection section in the primary flow path within the cartridge.

[0004] One method for amplifying nucleic acids is the polymerase chain reaction (PCR). PCR amplifies target DNA by repeating three steps at different temperatures: DNA denaturation (dissociation of double-stranded DNA into single-stranded DNA), annealing (binding of complementary primers to single-stranded DNA), and extension (replication of double-stranded DNA by complementary strand synthesis). Typically, thermal denaturation is performed at about 95°C, annealing at about 55-65°C, and extension at about 72°C. In the case of isothermal amplification, the reactions corresponding to these cycles are carried out at a constant temperature of about 35-65°C.

[0005] A thermal cycler is widely used for PCR, in which reaction tubes containing reaction solutions are inserted into wells in a metal block, and the reaction temperature and reaction time are controlled by controlling the temperature of the metal block. To reduce temperature variations, the metal block has a volume equal to or greater than that of the reaction tubes or microplates in which it is placed. This increases the heat capacity of the metal block, limiting the rate at which the temperature can be increased or decreased, so PCR requires at least 30 minutes, typically 1 to 2 hours.

[0006] In recent years, in order to speed up PCR, a method called two-step PCR has been proposed, in which PCR is performed using thermal cycles that alternate between two temperature ranges: denaturation and annealing. Two-step PCR has the advantages of shortening the time required and making it easier to control, since the annealing step and extension step are performed simultaneously at the same temperature (e.g., 60-68°C). To further speed up two-step PCR or to construct a disposable cartridge-type genetic analysis system, a method has also been proposed in which PCR is performed by applying a high-speed thermal cycle on a microfluidic device in which wells or fine channels are formed on a substrate.

[0007] For example, Patent Document 2 discloses a nucleic acid amplification device that can perform a thermal cycle in approximately 10 minutes by contacting a microchannel chip having a serpentine channel with a heat block set to two different temperature zones and performing a thermal cycle corresponding to the number of meanders of the channel. This method is called serpentine channel PCR, and it is only necessary to maintain the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction solution is not dependent on the heating and cooling rate of the heat block, but is determined by the channel length and the flow rate of the reaction solution. Furthermore, Patent Document 3 discloses a nucleic acid amplification device that can perform a thermal cycle in approximately 6 to 8 minutes by contacting a microchannel chip having a single channel with a heat block set to two different temperature zones and moving the PCR reaction solution in the channel back and forth between the two different temperature zones. This method is called reciprocating channel PCR, and it is only necessary to maintain the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction solution is not dependent on the heating and cooling rate of the heat block, but is determined by the time the reaction solution is maintained in the temperature zone. Furthermore, for example, Non-Patent Document 1 mentions various high-speed PCR techniques and discloses the reaction time, reaction solution volume, and detection sensitivity of each method.

[0008] Technologies for detecting nucleic acids amplified by PCR can be broadly divided into real-time detection methods, which use reagents containing fluorescent dyes to sequentially detect nucleic acid amplification for each cycle, and endpoint detection methods, which detect the amount of nucleic acid after the reaction has finished. Lateral flow assays, one of the endpoint detection methods, are characterized by their lower cost compared to real-time detection methods and the ease with which a small, simultaneous multi-item detection system can be constructed.

[0009] US Patent Application Publication No. 2012 / 0178091 US Patent Application Publication No. 2010 / 0167288 JP 2018-139765 A JP 2008-516613 A

[0010] Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic, Trends Anal. Chem. 143, 116377(2021)Molecular Cloning:A laboratory manual, Volume 2,”Sambrool and Russell(2001).

[0011] One of the challenges in nucleic acid-based testing is to speed up the testing while obtaining reliable data. Generally, nucleic acid amplification technologies, such as PCR, are highly sensitive because they exponentially amplify and detect target nucleic acids. However, even slight changes in temperature, reagent concentration, etc. often result in a decrease in amplification efficiency and amplification accuracy (selectivity of the amplified sequence). When thermal cycling is performed at high speed, the reaction time for each step is insufficient, resulting in a decrease in amplification efficiency and a decrease in detection sensitivity. At the same time, the inability to precisely control the temperature changes applied to the reaction solution also reduces amplification accuracy, resulting in the production of non-specific amplification products other than the target product, such as primer dimers. This is undesirable in testing, as it can lead to false negatives due to insufficient sensitivity and false positives due to non-specific amplification products. Insufficient sensitivity can be relatively easily remedied by improving amplification efficiency or increasing the number of thermal cycles. Non-Patent Document 1 provides several examples of high-speed PCR technologies that achieve high sensitivity.

[0012] On the other hand, no solution has been proposed in the prior art for the occurrence of false negative results due to non-specific amplification products in high-speed PCR technology. This is also supported by the fact that there are very few examples of high-speed PCR technology that aim to enable practical implementation of multiplex analysis, which is one application in which non-specific amplification products are particularly likely to be produced and often pose a serious problem.

[0013] Generally, touchdown PCR, as exemplified in Non-Patent Document 2, is a standard method for suppressing the production of nonspecific amplification products. Touchdown PCR utilizes the fact that primer binding specificity is enhanced when the annealing and extension temperatures are set high, and involves performing thermal cycling while gradually lowering the annealing and extension temperatures. However, when applying touchdown PCR to high-speed PCR technology, the heater temperature must be rapidly lowered in tandem with the thermal cycle. To achieve temperature control that can lower the temperature by 1-3°C within a few seconds and stabilize it at a constant temperature, precise temperature control using a large Peltier element and heat sink is required, including reducing the reaction solution volume or downsizing the chip device. This limits analytical applications and results in large, expensive equipment. Furthermore, touchdown PCR is intended for use in PCR systems with sufficiently high amplification efficiency (generally 85% or higher). Therefore, in high-speed PCR systems with low amplification efficiency, target product dissociation is more likely to occur, potentially reducing sensitivity.

[0014] Furthermore, as other methods for suppressing the production of non-specific amplification products, methods such as halting the activity of DNA polymerase until just before the start of thermal cycling (hot start PCR) and selecting a sequence that is less prone to mispriming (Patent Document 4) have been proposed.

[0015] However, these methods rely on modifying reagents, including primers. Therefore, their applicability is significantly limited, and they may not be suitable for faster thermal cycling depending on the sample to be amplified and the extension rate of DNA polymerase. In light of this situation, the present disclosure proposes a high-speed, highly reliable PCR technology (without false negatives or false positives).

[0016] In order to solve the above problems, the present disclosure proposes a nucleic acid amplification method for amplifying nucleic acids contained in a sample solution contained in a flow path device, the method including: a first phase step in which the sample solution is subjected to a denaturation reaction for a seconds, followed by an annealing and extension reaction for b seconds; and a second phase step (b≠d) in which the sample solution after the first phase step is subjected to a denaturation reaction for c seconds, followed by an annealing and extension reaction for d seconds.

[0017] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Furthermore, aspects of the present disclosure are achieved and realized by the elements and combinations of various elements, as well as the aspects of the following detailed description and the appended claims. It should be understood that the description of this specification is merely exemplary and does not limit the scope or application of the claims of the present disclosure in any way.

[0018] According to the technology of the present disclosure, PCR can be performed quickly and with high reliability (without producing false negatives or false positives).

[0019] 5A is a diagram showing an example of a schematic configuration of a flow channel device 101 and a nucleic acid amplification reaction system 201 according to a first embodiment. FIG. 1A is a diagram showing a cross-sectional configuration taken along line A-A in FIG. 1A. FIG. 1B is a flowchart for explaining details of a nucleic acid amplification reaction process according to the first embodiment. FIG. 1C is a flowchart for explaining an example of a nucleic acid amplification process in which the first phase (S202 to S207 in FIG. 3) is a three-step PCR and the second phase (S208 to S211 in FIG. 3) is a two-step PCR. FIG. 5B is a diagram showing an example of a top view configuration of a flow channel device 101 used in a second embodiment. FIG. 4A is a diagram showing an example of a cross-sectional configuration taken along line A-A in FIG. 4A. FIG. 5C is a diagram showing an example of a cross-sectional configuration taken along line A-A in FIG. 5A. FIG. 5D is a diagram showing an example of a composition of a nucleic acid amplification reaction reagent. FIG. 5E is a diagram showing an example of a top view configuration of a nucleic acid chromatograph 302 and an example of detection of a target nucleic acid. FIG. 5F is a flowchart for explaining a nucleic acid amplification reaction process according to the second embodiment. FIG. 5G is a diagram showing a bright-field image of a nucleic acid chromatograph. FIG. 5G is a diagram showing the concentration of a target nucleic acid semi-quantified by electrophoresis. 1 is a flowchart for explaining a nucleic acid amplification reaction process related to a cycle switching PCR method in which thermal cycle conditions are changed in three stages according to a third embodiment of the present disclosure. It is a diagram showing an example of the cross-sectional configuration of a nucleic acid amplification reaction system 201 and a flow path device 101 capable of performing real-time PCR according to a fourth embodiment. It is a table showing the results of a simple evaluation of the effects of hot start time (Hot start) and DNA denaturation time (Denature) on amplification efficiency (PCR efficiency). It is a graph showing the dependency of amplification efficiency and amplification rate on annealing and extension time (Anneal & Extension). It is a diagram showing the relationship between total reaction time (target reaction time) and target nucleic acid amount under each condition in which the annealing and extension time is changed. It is a diagram schematically showing the relationship between total reaction time and target nucleic acid amount in cycle switching PCR.

[0020] An embodiment of the present disclosure relates to a highly reliable and fast PCR processing technique (cycle switching PCR) that performs multiple phases of steps (e.g., a first phase step and a second phase step) with different annealing and extension times.

[0021] Hereinafter, embodiments and specific examples of the present disclosure will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be designated by the same numerals. Note that the accompanying drawings show specific embodiments and implementation examples according to the principles of the present disclosure, but these are for understanding the present disclosure and are not to be used to interpret the present disclosure in any way as being limiting.

[0022] Although the present embodiment has been described in sufficient detail to enable those skilled in the art to practice the present disclosure, it should be understood that other implementations and forms are possible, and that changes in configuration and structure and substitutions of various elements are possible without departing from the scope and spirit of the technical ideas of the present disclosure. Therefore, the following description should not be interpreted as being limited thereto.

[0023] (1) First Embodiment <Configuration Example of Nucleic Acid Amplification Reaction System> Fig. 1A is a diagram showing a schematic configuration example of a flow channel device 101 and a nucleic acid amplification reaction system 201 according to a first embodiment. Fig. 1B is a diagram showing the AA cross-sectional configuration of Fig. 1A.

[0024] The flow channel device 101 includes a substrate 102 having a flow channel structure formed thereon, a flow channel lid material 103 attached to the surface of the substrate 102, and a nucleic acid amplification unit 107. The nucleic acid amplification unit 107 may be connected to a flow channel structure such as a sample introduction channel or a vent. The nucleic acid amplification reaction system 201 includes the flow channel device 101, a temperature adjustment mechanism 214 that can change the temperature of a sample solution 301 in the nucleic acid amplification unit 107 at any desired value, and a control device (computer) 300 that controls the temperature adjustment function of the temperature adjustment mechanism 214.

[0025] The temperature control mechanism 214 is required to be capable of rapidly heating and cooling (temperature control) at least the nucleic acid amplification unit 107. The temperature control mechanism 214 may control the temperature of the entire flow channel device 101 as shown in FIG. 1 , or may control the temperature of only the nucleic acid amplification unit 107, based on the control of the control device 300. The temperature control mechanisms 214 may also be arranged above and below the flow channel device 101 so as to sandwich it. The sample solution 301 may be an aqueous solution containing a mixture of an analyte, a PCR reagent capable of rapidly amplifying nucleic acids, and a primer having a sequence capable of identifying the target nucleic acid at the start of the nucleic acid amplification reaction, and is not particularly limited as long as it is used in the relevant field. For example, the PCR reagent and primer may be pre-immobilized in the nucleic acid amplification unit 107 and then dissolved in an aqueous solution containing the analyte to prepare the sample solution 301.

[0026] <Details of Nucleic Acid Amplification Reaction Process> Figure 2 is a flowchart for explaining the details of the nucleic acid amplification reaction process according to the first embodiment. The amplification reaction process includes a first phase in which the introduction of a sample solution (S101) and steps S102 to S105 are repeated m times, and a second phase in which steps S106 to S109 are repeated n times, where m≦n. The control device 300 also loads a program corresponding to the flowchart in Figure 2 from a memory (not shown) (a memory within the control device or an external storage device) and executes the program.

[0027] (i) S101: The sample solution 301 is introduced into the nucleic acid amplification unit 107. This operation may be performed in advance by an operator using a dedicated tool such as a micropipette before installing the flow path device 101 in the nucleic acid amplification reaction system 201, or a liquid delivery mechanism that enables the sample solution 301 to be supplied from outside to the nucleic acid amplification reaction system 201 and the flow path device 101 may be provided.

[0028] (ii) S102 The control device 300 controls the temperature adjustment mechanism 214 to set the temperature of the nucleic acid amplification unit 107 to a first temperature (for example, 98° C.) and raise the temperature of the sample solution 301.

[0029] (iii) S103 The control device 300 controls the temperature adjustment mechanism 214 so that the temperature of the sample solution 301 is maintained at the first temperature for a seconds (for example, 1 second; it may be less than 1 second), and performs a denaturation reaction (standby at the first temperature for a seconds).

[0030] (iv) S104 The control device 300 controls the temperature adjustment mechanism 214 to change the temperature of the nucleic acid amplification unit 107 to a second temperature (for example, 64° C.), and lowers (cools) the temperature of the sample solution 301.

[0031] (v) S105 The control device 300 controls the temperature regulation mechanism 214 so that the temperature of the sample solution 301 is maintained at the second temperature for b seconds (for example, 3 seconds), and performs an annealing / extension reaction (standby at the second temperature for b seconds).

[0032] (vi) Repeated Processing The control device 300 repeats steps S102 to S105 m times (for example, 20 times), thereby completing the first phase of the nucleic acid amplification reaction process, and then the nucleic acid amplification reaction process proceeds to the second phase.

[0033] (vii) S106 The control device 300 controls the temperature adjustment mechanism 214 to set the temperature of the nucleic acid amplification unit 107 to a first temperature (for example, 98° C.).

[0034] (viii) S107 The control device 300 controls the temperature adjustment mechanism 214 so that the temperature of the sample solution 301 is maintained at the first temperature for c seconds (for example, 1 second; it may be less than 1 second), and performs a denaturation reaction (standby at the first temperature for c seconds).

[0035] (ix) S108 The control device 300 controls the temperature adjustment mechanism 214 to change the temperature of the nucleic acid amplification unit 107 to a second temperature (for example, 64° C.), and lowers (cools) the temperature of the sample solution 301.

[0036] (x) S109 The control device 300 controls the temperature regulation mechanism 214 so that the temperature of the sample solution 301 is maintained at the second temperature for d seconds (for example, 1 second), and performs an annealing / extension reaction (standby at the second temperature for d seconds).

[0037] (xi) Repeated Processing The control device 300 repeats the steps from S106 to S109 n times (for example, 20 times).

[0038] (xii) S110 All steps of the nucleic acid amplification reaction process are completed by the above. After the nucleic acid amplification reaction is completed, the sample solution 301 can be collected and examined for the presence or absence of amplification of the target nucleic acid by an endpoint detection method such as a lateral flow assay or electrophoresis. Alternatively, the amplification of the target nucleic acid can be confirmed by a real-time detection method during the nucleic acid amplification reaction.

[0039] <Relationship between First Phase and Second Phase> The greatest feature of this embodiment is that the time required for one cycle of the first phase (S102 to S105 in FIG. 2) is different from the time required for one cycle of the second phase (S106 to S109 in FIG. 2) (a+b≠c+d), and further, the latter is set to be shorter (a+b>c+d), and more preferably, the annealing / extension time (d) of the second phase is shorter than the annealing / extension time (b) of the first phase (b>d).

[0040] It has generally been reported that the amplification efficiency of PCR is highly dependent on the annealing / extension time. As a result of extensive research, the present inventors have found that the shorter the annealing / extension time, the less amplified the target nucleic acid is, and at the same time, the more likely it is that the amount of non-specific amplification products such as primer dimers is produced. This tendency is particularly pronounced when the initial nucleic acid concentration of the analyte is low (generally 1,000 copies of DNA / test or less), when the DNA strand length of the target nucleic acid to be amplified is long (generally 150 bp or more), or when the number of multiplexes is large (generally two or more types). The technology disclosed herein has been devised in consideration of these research results. By setting the annealing / extension time (b) longer in the early stages of the cycle when the target nucleic acid concentration is low, mispriming is suppressed, minimizing the production of non-specific amplification products and accurately amplifying the target nucleic acid. After the target nucleic acid has been amplified to a certain extent (controlled by time) (first phase), the cycle time (c+d) is shortened to perform multiple cycles at high speed, thereby increasing the amount of target nucleic acid in a short period of time (second phase). This makes it possible to realize high-speed PCR that achieves both high amplification accuracy and amplification efficiency.

[0041] <Example of Nucleic Acid Amplification Process Using Three-Step PCR> In the above example, a nucleic acid amplification process using two-step PCR to maximize speed has been described, but it can also be applied to three-step PCR. Figure 3 is a flowchart for explaining an example of a nucleic acid amplification process in which the first phase (S202 to S207 in Figure 3) is three-step PCR and the second phase (S208 to S211 in Figure 3) is two-step PCR. Note that the control device 300 loads a program corresponding to the flowchart in Figure 3 from a memory (not shown) (a memory within the control device or an external storage device) and executes the program.

[0042] In Figure 3, the first temperature corresponds to the DNA denaturation temperature (e.g., 98°C), the second temperature corresponds to the annealing / extension temperature of two-step PCR (e.g., 64°C), the third temperature corresponds to the annealing temperature of three-step PCR (e.g., 60°C), and the fourth temperature corresponds to the DNA extension temperature of three-step PCR (e.g., 72°C).

[0043] Even when applying three-step PCR, high-speed PCR that combines high amplification accuracy and amplification efficiency can be achieved by setting the reaction time so that: (i) the sum of the annealing time and DNA extension time is the annealing / extension time (b or d), and the time required for one cycle of the first phase is different from the time required for one cycle of the second phase (a + b ≠ c + d); (ii) further, the latter is shorter (a + b > c + d); and (iii) more preferably, the annealing / extension time of the second phase is shorter than that of the first phase (b > d).

[0044] Note that three-step PCR does not have to be applied only to the first phase; the second phase may also be three-step PCR, or only the second phase may be three-step PCR. Generally, three-step PCR has a longer reaction time than two-step PCR, and often achieves higher amplification accuracy and efficiency. Therefore, by using three-step PCR for the first cycle (first phase), where mispriming is likely to occur, and two-step PCR for the second cycle (second phase), once the reaction has progressed to a certain extent, reliable data can be obtained quickly even in difficult analyses. Furthermore, the method for determining the parameters a to d and m and n of the thermal cycle conditions will be described later (in the fourth embodiment).

[0045] (2) Second Embodiment A nucleic acid amplification reaction system 201 and a flow path device 101 according to a second embodiment will be described below. Here, the features of the second embodiment will be described using as an example a method for amplifying nucleic acids from a simulated influenza B virus specimen (equivalent to 100 copies of DNA per test) in about 5 minutes using a reagent set capable of identifying influenza A and B viruses, and detecting the nucleic acids by a lateral flow assay.

[0046] Fig. 4A is a diagram showing an example of the top configuration of a flow channel device 101 used in the second embodiment, and Fig. 4B is a diagram showing an example of the configuration in the AA cross section of Fig. 4A.

[0047] 4A , the flow channel device 101 includes a substrate 102 having a flow channel structure formed thereon, a flow channel lid material 103 attached to the surface of the substrate 102, a pair of connection ports 104 and 105, two connection flow channels 106 corresponding to the pair of connection ports 104 and 105, and a nucleic acid amplification unit 107. The first connection port 104 and the second connection port 105 are each located at the end points of the connection flow channels 106 and are processed as through-holes on the substrate 102, with the undersides closed by sealing with sealing tape (flow channel lid material 103). The two connection flow channels 106 are both formed on the underside of the substrate 102 and are connected to both ends of the nucleic acid amplification unit 107. The nucleic acid amplification unit 107 is a single-path flow channel formed on the substrate 102. When the flow path device 101 is installed in the nucleic acid amplification reaction system 201, the nucleic acid amplification section 107 is composed of a first temperature region 108 whose temperature is controlled by a first external heat source 206, a second temperature region 109 whose temperature is controlled by a second external heat source 207, and an intermediate flow path 110 connecting the first temperature region 108 and the second temperature region 109.

[0048] For example, the flow channel device 101 can be fabricated by forming a first connection port (vent) 104 with a diameter of 0.8 mm, a connecting flow channel 106 with a width of 0.8 mm and a depth of 0.6 mm, and a nucleic acid amplification section 107 on a polycarbonate (PC) substrate 102 measuring 80 mm wide, 30 mm long, and 3 mm thick. The flow channel of the nucleic acid amplification section 107 has a serpentine shape that is continuously connected to the connecting flow channel 106. This allows for compact placement of the external heat sources 206 and 207, while simultaneously agitating the solution in the serpentine section to homogenize the concentration of the sample solution 301 and stabilize the nucleic acid amplification reaction. Furthermore, a polyolefin (PO) sealing tape can be attached as a flow channel lid 103 to the surface on which the flow channel is formed, forming a tubular flow channel.

[0049] The material of the flow channel device 101 does not have to be PC; it can be any material that can withstand a heat temperature of approximately 100°C or higher, can be formed into a flow channel structure by machining or injection molding, and does not adsorb nucleic acids or inhibit nucleic acid amplification reactions. For example, resins such as cycloolefin polymer (COP), polydimethylsiloxane (PDMS), cyclic cycloolefin copolymer (COC), and acrylic (PMMA), or non-resin materials such as quartz glass, can be used. Similarly, the material of the flow channel lid material 103 does not have to be PO. It can be selected from resins that can withstand a heat temperature of approximately 100°C or higher and do not adsorb nucleic acids or inhibit nucleic acid amplification reactions, such as polycarbonate (PC), cycloolefin polymer (COP), cyclic cycloolefin copolymer (COC), polypropylene (PP), and polyethylene (PE). Furthermore, the assembly of the flow channel lid material 103 and the flow channel device 101 can be performed using adhesive bonding, thermal bonding, ultrasonic bonding, chemical bonding, or the like, with a strength of approximately 1 N / cm. ２ The above-mentioned method of maintaining the adhesive force stably can be used, thereby obtaining a pressure resistance sufficient for carrying out general liquid transfer operations.

[0050] <Configuration Example of Nucleic Acid Amplification Reaction System> Fig. 5A is a diagram showing a configuration example of a nucleic acid amplification reaction system 201 according to the second embodiment. Fig. 5B is a diagram showing a cross-sectional configuration example taken along line AA in Fig. 5A.

[0051] The nucleic acid amplification reaction system 201 includes a liquid delivery mechanism 202 , a device holder 203 , a cover 204 , a temperature control circuit 205 , external heat sources 206 and 207 , piping tubes 208 , and a control device (computer) 300 .

[0052] The liquid delivery mechanism 202 includes a syringe pump 210, a first liquid delivery connection port 211 connected to the first connection port 104 of the flow path device 101 via a piping tube 208, and a second liquid delivery connection port 212 connected to the second connection port 105. The first liquid delivery connection port 211 is piped inside the liquid delivery mechanism 202, connected to an air vent 213, and opened to the atmosphere. The second liquid delivery connection port 212 is piped inside the liquid delivery mechanism 202 and connected to the syringe pump 210. In response to the pressurizing and suction operations of the syringe pump 210, the sample solution 301 can be transported within the flow path of the flow path device 101 via the second connection port 105.

[0053] The first external heat source 206 and the second external heat source 207 are maintained at temperatures suitable for PCR by the control device 300 and the temperature control circuit 205, respectively. The flow channel device is held so that the first external heat source 206 contacts the first temperature region 108 of the flow channel device 101, and the first external heat source 206 contacts the second temperature region 109.

[0054] The first external heat source 206 and the second external heat source 207 can be fabricated, for example, by closely attaching copper blocks measuring 15 mm wide, 20 mm long, and 2 mm thick to both sides of a small ceramic heater measuring 10 mm wide, 20 mm long, and 1 mm thick, and attaching a thermocouple to the side that does not contact the flow channel device 101. The temperature control circuit 205 performs feedback control by referencing the temperatures of the first external heat source 206 and the second external heat source 207 acquired by the thermocouples, and sets the first external heat source 206 to a temperature suitable for DNA denaturation (e.g., in the range of 90 to 100°C, for example, 98°C), and the second external heat source 207 to a temperature suitable for DNA annealing and elongation (e.g., in the range of 55 to 65°C, for example, 64°C). A slit 215 may be provided in the device holder between the first external heat source 206 and the second external heat source 207 to thermally isolate them. The insulating effect of the air in the slits 215 makes it difficult for thermal equilibrium to occur between the heat sources, allowing for more efficient temperature control of the first temperature zone 108 and the second temperature zone 109 .

[0055] The power source of the liquid delivery mechanism 202 does not have to be a syringe pump, but may be any power source and pressure control mechanism capable of applying any solution delivery pressure at any timing. For example, a diaphragm pump, a microblower, a compressor, or the like may also be used.

[0056] Furthermore, at least one of the first liquid supply connection port 211 and the second liquid supply connection port 212 may be connected to a liquid supply power source. For example, the second liquid supply connection port 212 may be connected to the vent port 213, or both may be connected to a liquid supply power source to adjust pressure. At least one of the first connection port 104 and the second connection port 105 may be connected to either the first liquid supply connection port 211 or the second liquid supply connection port 212, whichever is connected to the liquid supply power source, and does not necessarily have to be connected to the vent port 213.

[0057] The first external heat source 206 and the second external heat source 207 may be any heat source capable of maintaining the first temperature region 108 and the second temperature region 109 of the flow path device at constant temperatures, and there are no particular limitations on the material or characteristics as long as they are those used in the relevant technical field. The set temperatures of the first external heat source 206 and the second external heat source 207 may be reversed. In this case, the operational steps in Figure 6 may be reversed, i.e., all of the suction and pressurization operations of the liquid delivery mechanism 202 may be reversed, or the first connection port 104 may be connected to the second liquid delivery connection port 212, or the flow path device 101 itself may be rotated 180°.

[0058] <Example of Nucleic Acid Amplification Reaction Reagent> Figure 6A is a diagram showing an example of the composition of a nucleic acid amplification reaction reagent. In the second embodiment, the sample to be detected (Sample) is a simulated specimen of influenza B virus (equivalent to 100 copies of DNA per test), and the nucleic acid amplification reaction reagent is a KAPA 3G Plant PCR kit (KAPA BIOSYSTEMS). In this embodiment, in order to detect the product of the nucleic acid amplification reaction using nucleic acid chromatography 302, primers can be used that are modified at the 5' end of sequences published by the National Institute of Infectious Diseases as primers for identifying influenza A and B viruses, with the forward primer modified with biotin for binding to latex particles for color development on nucleic acid chromatography 302, and the reverse primer modified with a tag sequence for binding to the influenza B virus detection line on nucleic acid chromatography.

[0059] FIG. 6B shows an example of the top surface configuration of the nucleic acid chromatograph 302 and an example of target nucleic acid detection. The nucleic acid chromatograph 302 is made of paper and is composed of a sample pad 3001, a conjugate pad 3002, a detection paper 3003, and an absorbent pad 3004. These components are overlapped by approximately 1 mm so that the sample solution 301 flowing into the sample pad 3001 is transported sequentially by capillary force. The conjugate pad 3002 is composed of blue latex particles modified with streptavidin, which bind to biotin in the target amplification product. The detection paper 3003 includes a first detection line 3005 and a second detection line 3006, to which oligo sequences capable of binding to the tag sequence of the target amplification product by hybridization are immobilized. In the nucleic acid chromatograph 302, for example, an oligo sequence capable of detecting an amplification product derived from influenza A virus is fixed to the first detection line 3005, and an oligo sequence capable of detecting an amplification product derived from influenza B virus is fixed to the second detection line 3006.

[0060] The sample may contain nucleic acid derived from the target of detection, such as DNA, cDNA, RNA, cells, organelles, bacteria, or viruses. The reagent may be selected from pretreatments to enable nucleic acid amplification reactions, and enzymes and reaction systems with an extension rate that enables rapid nucleic acid amplification. The primer may have a sequence that can identify the target of detection, and is preferably one that is resistant to nonspecific binding. The tag sequence and biotin of the target amplification product may be modified to either the forward primer or the reverse primer. However, it is desirable to avoid modifying both the forward primer and the reverse primer (tag sequence and biotin). This allows only amplification products containing both primers to be detected, thereby reducing the probability of nonspecific amplification products binding to the detection line and improving detection accuracy. The nucleic acid chromatograph 302 may be any suitable one used in the relevant field, and the combination, number, and arrangement of the tag sequence modified to the primer and the oligo sequences of the detection lines 3005 and 3006 may be freely selected.

[0061] <Nucleic Acid Amplification Reaction Processing> Fig. 7 is a flowchart for explaining the nucleic acid amplification reaction processing according to the second embodiment. The control device 300 reads a program corresponding to the flowchart in Fig. 7 from a memory (not shown) (a memory within the control device or an external storage device) and executes the program.

[0062] (i) S301 The sample solution 301 is introduced into the flow channel device 101 through the vent 213.

[0063] (ii) S302 The control device 300 controls the suction operation of the syringe pump 210 to draw the sample solution 301 into the connection channel 106.

[0064] (iii) S303 The control device 300 continues to control the syringe pump 210 to move the sample solution 301 to the first temperature region 108. Note that the injection of the sample solution 301 (S301) may be performed directly from the first connection port 104 detached from the piping tube 208. In this case, the sample solution 301 does not pass through the inside of the liquid delivery mechanism 202, thereby minimizing carryover between tests. Alternatively, the operations of S301 to S303 in FIG. 7 may be performed in advance using a dedicated tool such as a micropipette before installing the flow path device 101 in the nucleic acid amplification reaction system 201.

[0065] (iv) S304: The control device 300 controls the first external heat source 206 via the temperature control circuit 205 to adjust (temperature control) the temperature of the first temperature region 108 to a predetermined temperature (e.g., 98°C). The control device 300 then controls the operation of the syringe pump 210 to keep the sample solution 301 in the first temperature region 108 (e.g., 98°C) for a predetermined time (e.g., 15 seconds) (temperature control), thereby raising the sample temperature. Adding this operation not only suppresses mispriming when using a DNA polymerase compatible with the hot start method, but also effectively carries out the first cycle of denaturation reaction by sufficiently raising the initial temperature of the sample solution 301, thereby achieving stable nucleic acid amplification even in a high-speed reaction system. When using a DNA polymerase that is not compatible with the hot start method, S304 is not an essential processing step, and omitting S304 allows the series of reactions to be completed more quickly.

[0066] (v) S305 The control device 300 then controls the operation of the syringe pump 210 to make the sample solution 301 wait (stay) (adjust the temperature) in the first temperature region 108 for a seconds (e.g., 1 second; it may be shorter than 1 second) to carry out a denaturation reaction.

[0067] (vi) S306 The control device 300 controls the operation of the syringe pump 210 to move the sample solution 301 to the second temperature region (for example, 64° C.) 109.

[0068] (vii) S307 The control device 300 then controls the operation of the syringe pump 210 to cause the sample solution 301 to stay (control the temperature) in the second temperature region 109 for b seconds (e.g., 3 seconds), thereby carrying out an annealing / extension reaction.

[0069] (viii) S308 The control device 300 controls the operation of the syringe pump 210 to move the sample solution 301 back to the first temperature region 108.

[0070] (ix) Repeated Processing The control device 300 repeats the processing from S305 to S308 m times (for example, 20 times), thereby completing the processing of the first phase.

[0071] (x) S309 After the first phase of processing is completed, the control device 300 continuously transitions the processing to the second phase (S309 to S312). First, the control device 300 controls the operation of the syringe pump 210 to make the sample solution 301 wait (reside) (adjust the temperature) in the first temperature region 108 for c seconds (for example, 1 second; it may be shorter than 1 second) to perform a denaturation reaction.

[0072] (xi) S310 The control device 300 controls the operation of the syringe pump 210 to move the sample solution 301 to the second temperature region (for example, 64° C.) 109.

[0073] (xii) S311 The control device 300 controls the operation of the syringe pump 210 to cause the sample solution 301 to stay (control the temperature) in the second temperature region 109 for d seconds (for example, 1 second), thereby performing an annealing / extension reaction. Note that the time condition is b>d, and the number of times condition is m≦n.

[0074] (xiii) S312 The control device 300 controls the operation of the syringe pump 210 to move the sample solution 301 back to the first temperature region 108.

[0075] (xiv) Repeated Processing The control device 300 repeats the processes from S309 (keeping the sample solution 301 in the first temperature region 108 for c seconds (by adjusting the temperature) to perform a denaturation reaction) to S312 (moving the sample solution 301 back to the first temperature region 108) n times (for example, 20 times). This completes the second phase processing and the nucleic acid amplification reaction processing.

[0076] When performing the processes (S304, S305, S307, S309, and S311) of causing sample solution 301 to stay (adjust the temperature) in first temperature region 108 or second temperature region 109, operation of solution delivery mechanism 202 may be stopped, or delivery may be continued so that sample solution 301 does not overflow from each temperature region. This prevents sample solution 301 from moving from a predetermined position due to a pressure difference within the flow channel at both ends, and also has the effect of stirring and homogenizing sample solution 301.

[0077] (xv) S313 After the nucleic acid amplification reaction is completed, for example, the operator discharges the sample solution 301 from the first connection port 104 and recovers it from the flow channel device 101 into a microtube.

[0078] (xvi) S314 The operator inserts the nucleic acid chromatograph 302 into the microtube containing the collected sample solution 301 so that the sample pad 3001 comes into contact with the sample solution 301, confirms that the sample solution 301 has been sufficiently spread on the detection paper 3003 by capillary force, and checks the colored detection lines 3005 and 3006 on the detection paper 3003.

[0079] The above process enables the amplification and detection of target DNA at high speed (for example, in about 8 minutes), and enables the presence or absence of target DNA to be determined with high accuracy.

[0080] <Demonstration Experiment Results> FIG. 8 shows the results of a demonstration experiment of the nucleic acid amplification reaction process according to the second embodiment. In this demonstration experiment, in addition to nucleic acid chromatography, 2 μL of sample solution 301 after the nucleic acid amplification reaction was taken and analyzed using an automated electrophoresis system (e.g., Agilent's TapeStation). FIG. 8A shows a bright-field image of nucleic acid chromatography. FIG. 8B shows the target nucleic acid concentration semi-quantified by electrophoresis. Note that FIGS. 8A and 8B show the results of conventional high-speed PCR under relatively slow conditions, completing 40 PCR cycles in approximately 6.5 minutes. FIG. 8A(i) shows the results (negative control) for sample solution 301 to which no target sample (simulated influenza B virus specimen) was added. As shown in FIG. 8A(i), in the sample solution 301 after the nucleic acid amplification reaction without the target sample, neither the first detection line 3005 nor the second detection line 3006 developed color. On the other hand, in FIG. 8A(ii), only the second detection line 3006, which indicates the presence of influenza B virus, was brightly colored, and the target substance contained in the sample solution 301 was correctly detected.

[0081] Figures 8A(iii) to 8A(v) show the results of ultrafast PCR, which was completed in approximately 5 minutes. Figure 8A(iii) shows the results of conventional fast PCR, where the number of cycles was maximized, and Figure 8A(iv) shows the results of conventional fast PCR, where the annealing and extension times were maximized. Figure 8A(v) shows the results of cycle-switching fast PCR using the nucleic acid reaction processing method of this embodiment. In Figure 8A(iii), not only the second detection line 3006 but also the first detection line 3005, which indicates the presence of influenza A virus not contained in the sample solution 301, was colored (false positive). This is thought to be because the increased annealing and extension times reduced the amplification accuracy, making it easier for nonspecific amplification products to be produced. These nonspecific amplification products, which involve the primers for identifying influenza A virus, were produced and exponentially amplified together with the target nucleic acid during the cycles.

[0082] On the other hand, in Figure 8A (iv), even though the sample solution 301 contained a simulated specimen of influenza B virus, neither the first detection line 3005 nor the second detection line 3006 developed a color (false negative). This is thought to be because the amount of target nucleic acid did not reach the lower limit of detection of nucleic acid chromatography 302 due to an insufficient number of cycles under conditions of insufficient amplification efficiency. In fact, the lower limit of detection of general nucleic acid chromatography is approximately 10 ７ ~10 １１ The lower limit of detection for nucleic acid chromatography 302 under the conditions for carrying out the demonstration experiment is about 10 ９ This is supported by the fact that, as shown in Figure 8B, the target nucleic acid concentration of the sample solution 301 in Figure 8A (iv) is below the lower limit of detection of nucleic acid chromatography in this demonstration experiment.

[0083] In the nucleic acid chromatograph 302 analyzing the sample solution 301 in FIG. 8A(v), only the second detection line 3006 developed color, and the target substance could be correctly detected without any false positives or false negatives.

[0084] As described above, this embodiment effectively speeds up PCR while suppressing both false negatives due to insufficient sensitivity and false positives due to non-specific amplification. This makes it possible to provide a high-speed nucleic acid amplification and detection device that can be stably operated even in cases where high amplification accuracy and amplification efficiency are required, such as analysis of low-concentration samples or multiplex analysis.

[0085] Although this embodiment illustrates an example in which the amplified target nucleic acid is detected using a lateral flow assay (nucleic acid chromatography), the nucleic acid detection method is not particularly limited as long as it is a method commonly used in the relevant field. Lateral flow assays are rapid, simple, and suitable for use in multiplex analysis systems. Other methods, such as endpoint detection methods such as electrophoresis, absorbance measurement, sequencing, and microarray analysis, or real-time detection methods that use fluorescent probes to detect target nucleic acids during nucleic acid amplification reactions, can also be used. This embodiment can also be applied to a fully automated sample-to-answer genetic testing system in which nucleic acid amplification and nucleic acid detection, or pre-amplification and nucleic acid amplification, or both, are performed within the same flow path device 101 and the same equipment. This embodiment achieves high effectiveness by simply changing the thermal cycle conditions during the process, without requiring complex reagent composition or system configuration.

[0086] (3) Third Embodiment FIG. 9 is a flowchart illustrating a nucleic acid amplification reaction process according to a third embodiment of the present disclosure, involving a cycle-switching PCR method in which thermal cycle conditions are changed in three stages. In the third embodiment, the nucleic acid amplification reaction system 201 and the flow path device 101 according to the first or second embodiment, or similar devices, can be used. The nucleic acid amplification reaction process according to the third embodiment differs from the nucleic acid amplification reaction processes according to the first and second embodiments in that a third phase is performed after completion of the second phase. The control device 300 loads a program corresponding to the flowchart of FIG. 9 from a memory (not shown) (a memory within the control device or an external storage device) and executes the program.

[0087] Specifically, after the first phase (S402 to S405) (e.g., a = 1, b = 5, m = 5) and the second phase (S406 to S409) (e.g., c = 1, d = 3, n = 10) are performed, the sample solution 301 is again heated (temperature-controlled) to a first temperature (e.g., 98°C) (S410) and waits for a predetermined time (e.g., 1 second) (S411). The sample solution 301 is then cooled to a second temperature (e.g., 64°C) (S412) and waits for a predetermined time (e.g., 1 second) (S413). The third phase is completed by repeating the steps S410 to S413 o times (e.g., 20 times: o ≥ n ≥ m). The reaction time per cycle is set so that the later the phase, the shorter the reaction time (a + b > c + d > e + f; in short, the time condition is b > d > f). In this way, by adding intermediate conditions (conditions that are relatively fast and achieve a certain degree of high amplification accuracy and efficiency) before transitioning from reliable target nucleic acid amplification using a long annealing and extension period immediately after the start of the reaction to high-speed nucleic acid amplification using ultra-high-speed cycles in the latter half of the reaction, it is possible to improve the speed and accuracy of amplification in a balanced manner.

[0088] In the present disclosure, the number of phases may be two or more, and it is also possible to add a subsequent phase to change the thermal cycling conditions to four or more stages. However, considering that the technical effect of the present disclosure is enhanced by prioritizing amplification efficiency and amplification accuracy until the target nucleic acid reaches a certain concentration or higher, and then maximizing the number of cycles per unit time, it is practically appropriate to use two or three phases, or at most four phases.

[0089] (4) Fourth Embodiment The fourth embodiment relates to a nucleic acid amplification reaction system 201 and a flow channel device 101 capable of performing real-time PCR. In this embodiment, in addition to the nucleic acid amplification reaction system 201 and the flow channel device 101 capable of performing real-time PCR, a method for determining the parameters a to d and m and n of the thermal cycle conditions in the cycle switching PCR method described above will be described.

[0090] <Configuration Example of Nucleic Acid Amplification Reaction System and Flow Channel Device> Figure 10 is a diagram showing a cross-sectional configuration example of a nucleic acid amplification reaction system 201 and a flow channel device 101 capable of performing real-time PCR according to the fourth embodiment. The flow channel device 101 has the same configuration as the flow channel device 101 according to the second embodiment (see Figure 4). The nucleic acid amplification reaction system 201 differs from the nucleic acid amplification reaction system 201 of Figure 5 in that it has an observation window 216 in the device holder 203 that allows observation of the intermediate flow channel 110 from below, and in that it includes an optical detection mechanism 2001 below the observation window 216 that can detect fluorescence from a fluorescent probe. The optical detection mechanism 2001 includes a lens 2004, a camera 2002 that detects fluorescence contained in the sample solution 301, a signal processing unit 2003, a light source 2006 that excites the fluorescent probe, a light source control circuit 2007, and a half mirror 2005 that aligns the optical paths of these components. According to the real-time PCR of the fourth embodiment, a fluorescent signal is acquired for each PCR cycle when the solution reaches the intermediate flow path 110, and from the plotted fluorescent intensity, an amplification curve of the target nucleic acid can be drawn, and further, the amplification efficiency can be calculated.

[0091] <Discussion on Measurement Results of Amplification Efficiency> The results of measuring the amplification efficiency in the nucleic acid amplification reaction system 201 and the flow channel device 101 will be discussed with reference to Figures 11A and 11B. Figure 11A is a table showing the results of a simple evaluation of the effects of hot start time and DNA denaturation time on amplification efficiency (PCR efficiency). Figure 11B is a graph showing the dependence of amplification efficiency and amplification rate on annealing and extension time.

[0092] As shown in FIG. 11A , the amplification efficiency was approximately the same when the hot start time was 60 seconds and the DNA denaturation time was 3 seconds (state A in FIG. 11A ) and when the hot start time was 0 seconds and the DNA denaturation time was 1 second (state C in FIG. 11A ). This means that within this range, the amplification efficiency is not affected by the hot start time or the DNA denaturation time, and can be shortened to at least 0 seconds and 1 second, respectively. Therefore, with the technology disclosed herein, the DNA denaturation time (state A and state C) can both be minimized, specifically, to about 1 second or less. This allows for faster PCR without reducing amplification efficiency.

[0093] Figure 11B shows the results of examining the amplification efficiency by changing the annealing and extension time under the conditions shown in Figure 11A. Figure 11B shows that the amplification efficiency decreases by 30 to 40% as the annealing and extension time is shortened. On the other hand, referring to this amplification efficiency and cycle time, the DNA derived from the analyte (100 copies / test) was used for 10 ９ When the time required for amplification to reach copies / test is calculated, it can be seen that, despite the low amplification efficiency, the shorter the annealing / extension time, the faster the amplification can be completed (the longer the annealing / extension time, the higher the efficiency but the longer it takes to amplify, and the shorter the annealing / extension time, the lower the efficiency but the faster the amplification). This is because, when the amplification efficiency is ρ (0≦ρ≦1) and the number of cycles is γ, the amount of target nucleic acid after γ cycles is ideally (1+ρ) γAs can be seen from the above, the amount of target nucleic acid is more affected by the number of cycles than by the amplification efficiency. In other words, in order to obtain a certain amount of target nucleic acid at high speed, it is effective to subject the sample solution 301 to multiple cycles at high speed, even if it means sacrificing some amplification efficiency. For this reason, in the technology disclosed herein, the annealing and extension time of the first phase is set within a range that does not reduce amplification accuracy (e.g., b = 3 seconds), while the second phase is specifically set to approximately 1 second or less. This allows for faster PCR without reducing amplification efficiency. From another perspective, as described above, shortening the annealing and extension time reduces amplification efficiency but enables faster amplification. However, it has been found that a decrease in amplification efficiency reduces the amount of target nucleic acid and makes it more likely that non-specific amplification products will increase. Therefore, in the technology disclosed herein, the amplification efficiency is increased in the first phase to suppress the generation of non-specific amplification products and generate a predetermined amount of target nucleic acid, and PCR is then performed at high speed and with multiple cycles in the second phase.

[0094] <Procedure for Determining Parameters> The procedure for determining each parameter will be described in detail with reference to Figure 12. Figure 12A shows the relationship between the total reaction time and the amount of target nucleic acid under various conditions in which the annealing and extension times are changed. As noted in Figure 11B, the target nucleic acid increases exponentially with each cycle, and it can be seen that the higher the amplification efficiency (longer the annealing and extension times), the lower the amount of nucleic acid amplified per unit time. When implementing the technology of the present disclosure, it is advisable to determine an appropriate cycle change point (the point at which the first and second phases switch) taking into account the slope of this graph and the likelihood of non-specific amplification products being produced in the PCR reaction system being used.

[0095] FIG. 12B is a schematic diagram showing the relationship between the total reaction time and the amount of target nucleic acid in cycle-switching PCR. As shown in FIG. 12B, the technology of the present disclosure is characterized in that the slope of the amplification line changes at least once during the reaction, and furthermore, the slope of the first phase is smaller. To determine appropriate thermal cycle condition parameters a to d and m and n, first, the amount of DNA contained in the analyte (initial amount), the target amount of DNA to be ultimately obtained (target amount), and the target reaction time (total reaction time) are set. The target DNA amount should be set taking into account the lower detection limit of the subsequent nucleic acid analysis method. Next, the annealing and extension times for the first and second phases are selected. The annealing and extension time for the second phase should be set to approximately 1 to 2 seconds regardless of the reaction conditions, and the annealing and extension time for the first phase should be set taking into account the likelihood of producing non-specific amplification products. This is because the initial stage of the reaction (first phase) is generally more susceptible to the influence of reaction conditions such as the analyte and reagent composition. After determining the thermal cycling conditions for each phase, the cycle change point is determined taking into consideration the slope of the amplification line, the target reaction time, and the target DNA amount for each thermal cycling condition. The technical significance of the second phase in the present disclosure is to significantly increase the amount of nucleic acid amplification by applying multiple cycles in a short period of time, i.e., to achieve a reaction time reduction effect. Therefore, the second phase is preferably set to at least about 10 cycles, i.e., conditions that allow at least 50- to 100-fold nucleic acid amplification within 1 minute in the second phase.

[0096] The technology disclosed herein can also be applied to PCR that does not require high speed (reaction times of approximately 15 minutes or more). In such cases, however, the annealing and extension times may be extended to achieve the same effect. On the other hand, in high-speed PCR (reaction times of approximately 1 to 15 minutes: less than 15 minutes), as described above, the technology disclosed herein can be an extremely effective solution.

[0097] (5) Summary The technology disclosed herein can provide a high-speed nucleic acid amplification and detection device that can be stably operated by using an inexpensive and simple flow path device and system (apparatus) to suppress both false negatives due to insufficient sensitivity and false positives due to non-specific amplification.

[0098] (i) The nucleic acid amplification technique according to this embodiment includes at least a first phase in which a sample solution is denatured for a second, followed by an annealing and extension reaction for b seconds, and a second phase in which the sample solution after the first phase is denatured for c seconds, followed by an annealing and extension reaction for d seconds. Here, the annealing and extension reaction time of the first phase (b seconds) is different from the annealing and extension reaction time of the second phase (d seconds) (b ≠ d). In this way, the first phase and second phase have different annealing and extension times, enabling PCR processing to be performed quickly and reliably.

[0099] (ii) In the first embodiment (see FIG. 2 ), the first phase includes adjusting (heating) the sample solution at a first temperature (e.g., 98°C) for a second (S103), adjusting (heating) the sample solution after adjusting (heating) for a second (e.g., 64°C) (S105) for b seconds (lowering the sample solution from the first temperature to the second temperature and then heating the sample solution), and repeating steps S103 and S105 m times. The second phase includes adjusting (heating) the sample solution after S105 at the first temperature for c seconds (heating from the second temperature of the first phase to the first temperature) (S107), adjusting (heating) the sample solution after adjusting (heating) for c seconds at the second temperature for d seconds (lowering the first temperature to the second temperature and then heating the sample solution) (S109), and repeating steps S107 and S109 n times. Here, a through d are positive numbers, and b>d. The annealing and extension time in the second phase is set shorter than that in the first phase, and the steps included in the second phase are repeated multiple times, allowing PCR to be performed at high speed. Furthermore, the annealing and extension time in the first phase is longer than that in the second phase, allowing nucleic acids to be amplified with high efficiency, preventing the generation of non-specific amplification products, and providing highly reliable PCR results (reducing false negatives and false positives).

[0100] As in other embodiments, a = c may be used, or a and c may be 1 second or less. Furthermore, m and n may be natural numbers, and n may be set to be greater than or equal to 10, or alternatively, m and n may be natural numbers, and n may be set to be greater than or equal to m. Furthermore, even if the total time required for the first and second phases is set to less than 15 minutes (high speed), nucleic acids can be amplified while suppressing the generation of non-specific amplification products.

[0101] (iii) A second embodiment relates to amplifying nucleic acids contained in a sample solution accommodated in a flow path device having a serpentine flow path (see FIG. 7 ). In this case, the first phase includes: moving the sample solution to a first temperature region 108 of the serpentine flow path by a conveying operation of a syringe pump (S303); adjusting (heating) the sample solution at a first temperature in the first temperature region 108 for a seconds (S305); after adjusting (heating) the sample solution for a second, moving the sample solution to a second temperature region 109 of the serpentine flow path at a second temperature lower than the first temperature by a conveying operation of the syringe pump (S306); adjusting (heating) the sample solution at the second temperature region 109 for b seconds (S307); moving the sample solution after adjusting (heating) the sample solution to the first temperature region 108 for b seconds by a conveying operation of the syringe pump (S308); and repeating steps S305 to S308 m times. The second phase includes adjusting (heating) the temperature of the sample solution after the first phase in the first temperature region 108 for c seconds (S309), moving the sample solution after the temperature adjustment (heating) for c seconds to the second temperature region 109 by the transport operation of the syringe pump (S310), adjusting (heating) the sample solution in the second temperature region 109 for d seconds (heating at a second temperature lower than the first temperature) (S311), moving the sample solution after the temperature adjustment (heating) for d seconds to the first temperature region 108 by the transport operation of the syringe pump (S312), and repeating steps S309 to S312 n times. Here, a to d are positive numbers, and b > d. The temperature adjustment (heating) of the sample solution is performed in separate temperature regions in the flow path, allowing for accurate and easy temperature control, and further enabling accurate annealing and extension processes. Furthermore, similar to the first embodiment, it is possible to speed up PCR while suppressing both false positives due to insufficient sensitivity and false positives due to non-specific amplification products (realizing high reliability).

[0102] (iv) In the third embodiment, in addition to the first and second phases, the sample solution after the second phase is subjected to a denaturation reaction for e seconds, followed by a third phase of annealing and extension reaction for f seconds.

[0103] In this case, the first phase includes adjusting (heating) the sample solution at a first temperature (e.g., 98°C) for a second (S402), adjusting the temperature of the sample solution after adjusting (heating) for a second at a second temperature (e.g., 64°C) lower than the first temperature for b seconds (lowering the first temperature to the second temperature and then heating the sample solution for b seconds) (S405), and repeating steps S402 and S405 m times. Also, the second phase includes adjusting (heating) the sample solution after the first phase at the first temperature for c seconds (S407), adjusting the temperature of the sample solution after adjusting (heating) for c seconds at the second temperature for d seconds (lowering the first temperature to the second temperature and then heating the sample solution) (S409), and repeating steps S407 and S409 n times. Furthermore, the third phase includes adjusting (heating) the sample solution after the second phase at the first temperature for e seconds (S411), adjusting the temperature of the sample solution after heating for e seconds at the second temperature for f seconds (lowering the first temperature to the second temperature and heating the sample solution) (S413), and repeating steps S411 and S413 o times. Here, a to f are positive numbers, and b > d > f. In this way, by increasing the number of phases, annealing and extension processing can be performed under intermediate conditions (between an upper phase (e.g., the first phase) and a lower phase (e.g., the third phase)), thereby achieving a balance between high speed and accuracy of amplification.

[0104] (v) The functions of the present embodiment can also be realized by software program code. In this case, a storage medium on which the program code is recorded is provided to a system or device, and the computer (or CPU or MPU) of the system or device reads the program code stored in the storage medium. In this case, the program code read from the storage medium itself realizes the functions of the above-described embodiment, and the program code itself and the storage medium on which it is stored constitute the present disclosure. Examples of storage media for providing such program code include flexible disks, CD-ROMs, DVD-ROMs, hard disks, optical disks, magneto-optical disks, CD-Rs, magnetic tape, non-volatile memory cards, and ROMs.

[0105] Furthermore, an operating system (OS) running on a computer may perform some or all of the actual processing based on instructions in the program code, and the functions of the above-described embodiments may be realized by this processing.Furthermore, after the program code is read from a storage medium and written to memory on the computer, a CPU of the computer may perform some or all of the actual processing based on instructions in the program code, and the functions of the above-described embodiments may be realized by this processing.

[0106] Furthermore, the program code of the software that realizes the functions of the embodiment may be distributed via a network and stored in a storage means such as a hard disk or memory of the system or device, or in a storage medium such as a CD-RW or CD-R, so that when used, the computer (or CPU or MPU) of the system or device reads and executes the program code stored in the storage means or storage medium.

[0107] The processes and techniques described herein are not inherently related to any specific device and can be implemented by a combination of components. Various types of general-purpose devices can also be added. A dedicated device may be constructed to perform the functions of the present embodiment. Various functions can also be formed by appropriately combining multiple components disclosed in the present embodiment. For example, some components may be omitted from all the components shown in the embodiments, or components from different embodiments may be appropriately combined.

[0108] Although specific embodiments are described in this disclosure, they are in all respects for the purpose of explanation (understanding the technology of the present disclosure) and not for the purpose of limitation. Those skilled in the art will recognize that there are many combinations of hardware, software, and firmware suitable for implementing the technology of the present disclosure. For example, the software described can be implemented in a wide variety of programming or scripting languages, such as assembler, C / C++, Perl, Shell, PHP, Java (registered trademark), etc.

[0109] Furthermore, in the above-described embodiment, the control lines and information lines are those that are considered necessary for the explanation, and not all control lines and information lines in the product are necessarily shown. All components may be interconnected.

[0110] In addition, other implementations of the present disclosure will be apparent to those skilled in the art from consideration of the present embodiments. The specification and examples are exemplary only, with the scope and spirit of the technology of the present disclosure being indicated by the following claims.

[0111] REFERENCE SIGNS LIST 101 Flow path device 102 Substrate 103 Flow path cover material 104 First connection port 105 Second connection port 106 Connection flow path 107 Nucleic acid amplification unit 108 First temperature region 109 Second temperature region 110 Intermediate flow path 201 Nucleic acid amplification reaction system 202 Liquid delivery mechanism 203 Device holder 204 Cover 205 Temperature control circuit 206 First external heat source 207 Second external heat source 208 Piping tube 209 Opening 210 Syringe pump 211 First liquid delivery connection port 212 Second liquid delivery connection port 213 Vent 214 Temperature control mechanism 215 Slit 216 Observation window 301 Sample solution 302 Nucleic acid chromatograph 2001 Optical detection mechanism 2002 Camera 2003 Signal processing unit 2004 Lens 2005 Half mirror 2006 Light source 2007 Light source control circuit 3001 Sample pad 3002 Conjugate pad 3003 Detection paper 3004 Absorbent pad 3005 First detection line (Flu A) 3006 Second detection line (Flu B)

Claims

1. A nucleic acid amplification method for amplifying nucleic acid contained in a sample solution contained in a flow path device, comprising: a first phase step of performing a denaturation reaction on the sample solution for a seconds, followed by an annealing and extension reaction for b seconds; and a second phase step of performing a denaturation reaction on the sample solution after the first phase step has been performed for c seconds, followed by an annealing and extension reaction for d seconds, wherein b and d satisfy the condition b ≠ d.

2. A method for amplifying nucleic acid according to claim 1, wherein the first phase step includes: a first step of adjusting the temperature of the sample solution to a first temperature for a seconds using a heat source; a second step of adjusting the temperature of the sample solution after the temperature adjustment for a seconds to a second temperature lower than the first temperature for b seconds using the heat source; and a first repetition step of repeating the first and second steps m times; and the second phase step includes: a third step of adjusting the temperature of the sample solution after the first repetition step at the first temperature for c seconds using the heat source; a fourth step of adjusting the temperature of the sample solution after the temperature adjustment for c seconds at the second temperature for d seconds using the heat source; and a second repetition step of repeating the third and fourth steps n times, wherein a to d are positive numbers and b>d.

3. A method for amplifying nucleic acid according to claim 1, wherein a=c.

4. The method for amplifying nucleic acid according to claim 1, wherein a and c are 1 or less.

5. The method for amplifying nucleic acid according to claim 2, wherein m and n are natural numbers and n≧10.

6. The method for amplifying nucleic acid according to claim 2, wherein m and n are natural numbers and n ≥ m.

7. The method for amplifying nucleic acid according to claim 1, wherein the total time required for the first phase step and the second phase step is less than 15 minutes.

8. The nucleic acid amplification method according to claim 1, wherein the nucleic acid amplification method is a method for amplifying nucleic acid contained in the sample solution accommodated in the flow path device having a serpentine flow path, and the first phase process includes: a first process of moving the sample solution to a first temperature region of the serpentine flow path by a conveying operation of a syringe pump; a second process of controlling the temperature of the sample solution at a first temperature in the first temperature region for a seconds by using a heat source; a third process of moving the sample solution to a second temperature region of the serpentine flow path at a second temperature lower than the first temperature by a conveying operation of the syringe pump after heating for the a seconds; a second process of controlling the temperature of the sample solution at the second temperature region for b seconds by using the heat source; a third process of moving the sample solution after temperature control for the b seconds to the first temperature region by a conveying operation of the syringe pump; and a first repeat process of repeating the processes from the second process to the third process m times, and the second phase process includes: a fourth step of controlling the temperature of the sample solution after completion of the first phase step in the first temperature region for c seconds using the heat source; a fifth step of moving the sample solution after heating for the c seconds to the second temperature region by a transporting operation of the syringe pump; a sixth step of controlling the temperature of the sample solution in the second temperature region for d seconds using the heat source; a seventh step of moving the sample solution after temperature control for the d seconds to the first temperature region by a transporting operation of the syringe pump; and a second repeating step of repeating the steps from the fourth step to the seventh step n times, wherein a to d are positive numbers and b>d.

9. A nucleic acid amplification method according to claim 1, further comprising a third phase step of carrying out a denaturation reaction for e seconds on the sample solution after the second phase step has been carried out, followed by an annealing and extension reaction for f seconds.

10. In claim 9, the first phase step includes a first step of controlling the temperature of the sample solution at a first temperature for a seconds using a heat source; a second step of controlling the temperature of the sample solution after the temperature control for a seconds at a second temperature lower than the first temperature for b seconds using the heat source; and a first repetition step of repeating the first step and the second step m times; the second phase step includes a third step of controlling the temperature of the sample solution after the first repetition step at the first temperature for c seconds using the heat source; a fourth step of controlling the temperature of the sample solution after the temperature control for c seconds at the second temperature for d seconds using the heat source; and a second repetition step of repeating the third step and the fourth step n times; and the third phase step includes a fifth step of controlling the temperature of the sample solution after the second repetition step at the first temperature for e seconds using the heat source; and a sixth step of controlling the temperature of the sample solution after the temperature control for e seconds at the second temperature for f seconds using the heat source. a third repeating step of repeating the fifth step and the sixth step o times, wherein a to f are positive numbers, and b>d>f.

11. A nucleic acid amplification reaction system for amplifying nucleic acid contained in a sample solution, comprising: a flow path device for accommodating the sample solution; a heat source for controlling the temperature of the sample solution; and a computer for amplifying the nucleic acid contained in the sample solution in accordance with a program for nucleic acid amplification processing, wherein the computer executes a first phase process for performing a denaturation reaction on the sample solution for a seconds, followed by an annealing and extension reaction for b seconds, and a second phase process for performing a denaturation reaction on the sample solution after the first phase process has been performed for c seconds, followed by an annealing and extension reaction for d seconds, wherein b and d satisfy the condition b ≠ d.

12. A nucleic acid amplification reaction system according to claim 11, wherein the computer executes, in the first phase processing, a first processing step of controlling the heat source to adjust the temperature of the sample solution at a first temperature for a seconds, a second processing step of controlling the heat source to adjust the temperature of the sample solution after the temperature adjustment for the a seconds at a second temperature lower than the first temperature for b seconds, and a first repetitive processing step of repeating the first processing step and the second processing step m times, and in the second phase processing, a third processing step of controlling the heat source to adjust the temperature of the sample solution after the first repetitive processing step at the first temperature for c seconds, a fourth processing step of controlling the heat source to adjust the temperature of the sample solution after the temperature adjustment for the c seconds at the second temperature for d seconds, and a second repetitive processing step of repeating the third processing step and the fourth processing n times, wherein a to d are positive numbers and b>d.

13. The method according to claim 11, further comprising a syringe pump, wherein the flow path device has a serpentine flow path that accommodates the sample solution, the serpentine flow path including a first temperature region for adjusting the temperature at a first temperature and a second temperature region for adjusting the temperature at a second temperature lower than the first temperature, and wherein the computer executes, in the first phase processing, a first processing step of controlling a transport operation of the syringe pump to move the sample solution to the first temperature region of the serpentine flow path, a second processing step of controlling the heat source to adjust the temperature of the sample solution at the first temperature region for a seconds, a third processing step of controlling the transport operation of the syringe pump to move the sample solution to the second temperature region after the temperature adjustment for the a seconds, a second processing step of controlling the heat source to adjust the temperature of the sample solution at the second temperature region for b seconds, and a third processing step of controlling the transport operation of the syringe pump to move the sample solution after the temperature adjustment for the b seconds to the first temperature region, and a first repetitive processing step of repeating the processing from the first processing step to the third processing step m times, and wherein, in the second phase processing, a fourth process of controlling the heat source to adjust the temperature of the sample solution in the first temperature region for c seconds after completion of the first phase processing; a fifth process of controlling a transport operation of the syringe pump to move the sample solution after the temperature adjustment for the c seconds to the second temperature region; a sixth process of controlling the heat source to adjust the temperature of the sample solution in the second temperature region for d seconds; and a seventh process of controlling the transport operation of the syringe pump to move the sample solution after the temperature adjustment for the d seconds to the first temperature region, wherein a to d are positive numbers and b>d.

14. A nucleic acid amplification reaction system according to claim 11, wherein the computer further executes a third phase process in which a denaturation reaction is performed for e seconds on the sample solution after the second phase process, followed by an annealing / extension reaction for f seconds.

15. According to claim 14, the computer executes, in the first phase processing, a first processing step of controlling the heat source and adjusting the temperature of the sample solution at a first temperature for a seconds, a second processing step of controlling the heat source and adjusting the temperature of the sample solution after the temperature adjustment for the a seconds at a second temperature lower than the first temperature for b seconds, and a first repetitive processing step of repeating the first processing step and the second processing step m times; and, in the second phase processing, a third processing step of controlling the heat source and adjusting the temperature of the sample solution after the first repetitive processing step at the first temperature for c seconds, a fourth processing step of controlling the heat source and adjusting the temperature of the sample solution after the temperature adjustment for the c seconds at the second temperature for d seconds, and a second repetitive processing step of repeating the third processing step and the fourth processing n times; and, in the third phase processing, a fifth processing step of controlling the heat source and adjusting the temperature of the sample solution after the second repetitive step at the first temperature for e seconds, and a sixth processing step of controlling the heat source and adjusting the temperature of the sample solution after the temperature adjustment for the e seconds at the second temperature for f seconds. a third repetitive process in which the fifth process and the sixth process are repeated o times, and the a to f are positive numbers, and b>d>f.

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