Nucleic acid amplification method and nucleic acid amplification reaction system

Abstract

To provide high sensitivity and reliable data when performing PCR at high speed.SOLUTION: While applying thermal cycles at high speed reduces amplification efficiency and detection sensitivity, performing multiple thermal cycles to maximize an amount of amplified products in a short time also increases an amount of non-specific amplified products, causing a decrease in S / N and false positives, reducing the reliability of analysis results. For this reason, by changing the thermal cycle midway, it is possible to increase an amount of target products amplified at high speed and suppress non-specific amplification. More specifically, this disclosure proposes a nucleic acid amplification technique that includes a first phase in which a denaturation reaction is performed on a sample solution for a seconds, followed by an annealing and extension reaction for b seconds, and a second phase (b≠d) in which a denaturation reaction is performed on the sample solution after a first phase process is performed, for c seconds, followed by an annealing and extension reaction for d seconds.SELECTED DRAWING: Figure 2

Description

[Technical field]

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

[0002] In recent years, the importance of infectious disease testing has increased due to the increase in deaths from sepsis caused by drug-resistant bacteria and the social damage caused by COVID-19. Infectious disease testing requires easy operation because the operators are laboratory technicians and nurses. In addition, shortening the testing time directly leads to improved treatment efficacy and reduced treatment costs. Furthermore, since it is often difficult to predict the causative respiratory pathogen from clinical findings in infectious diseases, there is a demand for multiplex detection that can simultaneously test for multiple types of pathogens.

[0003] Rapid genetic testing systems using disposable cartridges have been proposed to realize 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, and environmental measurement. For example, Patent Document 1 discloses a technology that enables multiplex nucleic acid measurement by providing a sample inlet, purification section, PCR section, and detection section in the primary flow path within the cartridge.

[0004] PCR (Polymerase Chain Reaction) is a method for amplifying nucleic acids. PCR amplifies the target DNA by repeating three steps at different temperatures: DNA denaturation (dissociation of double-stranded DNA into single-stranded DNA), annealing (binding of a complementary primer 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 performed at a constant temperature of about 35-65°C.

[0005] For PCR, a thermal cycler device is widely used, in which a reaction tube containing a reaction solution is inserted into a well placed in a metal block, and the reaction temperature and reaction time are controlled by controlling the temperature of the metal block. To reduce temperature variation, the metal block has a volume equal to or larger than that of the reaction tube or microplate placed in it. This increases the heat capacity of the metal block, limiting the rate at which the temperature can be increased or decreased, so performing 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 a thermal cycle that repeats two temperature zones, denaturation and annealing. In two-step PCR, the annealing step and the extension step are performed simultaneously at the same temperature (e.g., 60 to 68°C), which has the advantage of shortening the time and making it easier to control. In order to further speed up two-step PCR or to build a disposable cartridge-type genetic analysis system, a method has been proposed in which PCR is performed by applying a thermal cycle at high speed on a microfluidic device with wells or fine channels formed on a substrate.

[0007] For example, Patent Document 2 discloses a nucleic acid amplification device that can carry out a thermal cycle in about 10 minutes by contacting a microchannel chip having a meandering channel on a heat block set to two different temperature zones, and by simply maintaining the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction solution does not depend on the heating and cooling speed of the heat block, but is determined by the channel length and the flow rate of the reaction solution. For example, Patent Document 3 discloses a nucleic acid amplification device that can carry out a thermal cycle in about 6 to 8 minutes by contacting a microchannel chip having one channel on a heat block set to two different temperature zones, and by moving the PCR reaction solution in the channel back and forth between the two different temperature zones. Such a method is called a reciprocating channel PCR, and by simply maintaining the temperature of each heat block at a predetermined temperature. Therefore, the temperature transition of the reaction solution does not depend on the heating and cooling speed of the heat block, but is determined by the time the reaction solution is held in the temperature zone. Furthermore, for example, Non-Patent Document 1 refers to 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 detect nucleic acid amplification cycle by cycle, and end-point detection methods, which detect the amount of nucleic acid after the reaction is completed. The lateral flow assay method, which is one of the end-point detection methods, is characterized by its low cost compared to real-time detection methods and the ease of constructing a small, simultaneous, multi-item detection system. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2012 / 0178091 [Patent Document 2] US Patent Application Publication No. 2010 / 0167288 [Patent Document 3] JP 2018-139765 A [Patent Document 4] Special Publication No. 2008-516613 [Non-patent literature]

[0010] [Non-Patent Document 1] Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic, Trends Anal. Chem. 143, 116377(2021) [Non-Patent Document 2] Molecular Cloning: A laboratory manual, Volume 2,” Sambrool and Russell (2001). Summary of the Invention [Problem to be solved by the invention]

[0011] One of the challenges of testing using nucleic acids is to speed up the test and obtain reliable data. Generally, nucleic acid amplification techniques such as PCR are highly sensitive because they exponentially amplify and detect the nucleic acid to be detected, but slight changes in temperature, reagent concentration, etc. often cause a decrease in amplification efficiency and amplification accuracy (selectivity of the sequence to be amplified). When thermal cycling is performed at high speed, the reaction time of each step becomes insufficient, resulting in a decrease in amplification efficiency and a decrease in detection sensitivity. At the same time, the amplification accuracy also decreases because it becomes impossible to precisely control the temperature change given to the reaction solution, and non-specific amplification products other than the target product, such as primer dimers, are produced. This is an undesirable event that leads to failures in testing, such as false negatives due to insufficient sensitivity and false positives due to non-specific amplification products. Insufficient sensitivity can be relatively easily improved by improving amplification efficiency or increasing the number of thermal cycles. Non-Patent Document 1 lists several examples of high-speed PCR technology that achieve high sensitivity.

[0012] On the other hand, no solution has been proposed in the prior art for the occurrence of false negatives 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 make multiplex analysis, one of the applications in which non-specific amplification products are particularly likely to be produced and often pose a serious problem, feasible at a practical level.

[0013] In general, the touchdown PCR method as exemplified in Non-Patent Document 2 is the standard method for suppressing the production of non-specific amplification products. Touchdown PCR is a method in which the annealing and extension temperatures are gradually lowered while performing thermal cycling, taking advantage of the fact that the binding specificity of primers is usually increased when the annealing and extension temperatures are set high. However, when applying touchdown PCR to high-speed PCR technology, the heater temperature must be lowered rapidly in accordance with the thermal cycle. In order to achieve temperature control that can lower the temperature by 1 to 3 degrees Celsius within a few seconds and stabilize it at a constant temperature, it is necessary to reduce the amount of reaction solution or the chip device to reduce the heat capacity sufficiently, and then perform precise temperature control using a large Peltier element and a heat sink. This limits the use of analysis, and the device becomes large and expensive. In addition, the touchdown PCR method is premised on operation in a PCR system with a sufficiently high amplification efficiency (approximately 85% or more). For this reason, in a high-speed PCR system with low amplification efficiency, the target product is more likely to dissociate, which may reduce sensitivity.

[0014] Furthermore, other methods proposed to suppress the production of non-specific amplification products include 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).

[0015] However, these methods rely on the modification of reagents including primers, and therefore have a large limitation in the scope of application, and depending on the sample to be amplified and the extension speed of DNA polymerase, they may not be suitable for high-speed thermal cycling. In view of these circumstances, the present disclosure proposes a PCR technique that is fast and highly reliable (does not produce false negatives or false positives). [Means for solving the problem]

[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 be apparent from the description of the present specification and the accompanying drawings. Also, the aspects of the present disclosure are achieved and realized by the elements and combinations of various elements and the following detailed description and the appended claims. It should be understood that the description of the present specification is merely a typical example and does not limit the scope or application of the present disclosure in any way. Effect of the Invention

[0018] According to the technology of the present disclosure, PCR can be performed quickly and reliably (without producing false negatives or false positives). [Brief description of the drawings]

[0019] [Figure 1A] FIG. 1 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. [Figure 1B] FIG. 2 is a diagram showing a cross-sectional configuration of the section AA in FIG. 1A. [Diagram 2]4 is a flowchart for explaining details of a nucleic acid amplification reaction process according to the first embodiment. [Diagram 3] 10 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 3-step PCR and the second phase (S208 to S211 in FIG. 3) is a 2-step PCR. [Figure 4A] 13 is a diagram showing an example of the upper surface configuration of a flow channel device 101 used in a second embodiment. FIG. [Figure 4B] 4B is a diagram showing a configuration example taken along the cross section AA of FIG. 4A. FIG. [Figure 5A] FIG. 1 is a diagram showing an example of the configuration of a nucleic acid amplification reaction system 201 according to a second embodiment. [Figure 5B] FIG. 5B is a diagram showing an example of the cross-sectional structure AA in FIG. 5A. [Figure 6A] FIG. 2 is a diagram showing an example of the composition of a nucleic acid amplification reaction reagent. [Figure 6B] 1A and 1B are diagrams showing an example of the upper surface configuration of a nucleic acid chromatograph 302 and an example of detection of a target nucleic acid. [Figure 7] 10 is a flowchart illustrating a nucleic acid amplification reaction process according to a second embodiment. [Figure 8A] FIG. 1 shows a bright-field image of nucleic acid chromatography. [Figure 8B] FIG. 1 shows the target nucleic acid concentration semi-quantified by electrophoresis. [Figure 9] 13 is a flowchart for explaining a nucleic acid amplification reaction process for cycle switching PCR in which thermal cycle conditions are changed in three stages according to a third embodiment of the present disclosure. [Figure 10] FIG. 13 is a diagram showing an example of a cross-sectional configuration of a nucleic acid amplification reaction system 201 and a flow channel device 101 capable of performing real-time PCR according to a fourth embodiment. [Figure 11A] 1 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]1 is a graph showing the dependence of amplification efficiency and amplification rate on annealing and extension time. [Figure 12A] This figure shows the relationship between the total reaction time (target reaction time) and the amount of target nucleic acid under various conditions in which the annealing and extension times were changed. [Figure 12B] FIG. 1 is a diagram showing a schematic representation of the relationship between the total reaction time and the amount of target nucleic acid in cycle switching PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] An embodiment of the present disclosure relates to a highly reliable and rapid PCR processing technique (cycle switching PCR) that performs multiple phase 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 indicated by the same numbers. 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 by no means used to interpret the present disclosure in a limiting manner.

[0022] In the present embodiment, the description is given in sufficient detail for a person skilled in the art to implement the present disclosure, but 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 <Example of nucleic acid amplification reaction system configuration> 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 a cross-sectional configuration taken along line AA in 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 flow channel for introducing a sample or an air vent. The nucleic acid amplification reaction system 201 includes the flow channel device 101, a temperature adjustment mechanism 214 that can give any temperature change to a sample solution 301 in the nucleic acid amplification unit 107, and a control device (computer) 300 that controls the temperature adjustment function of the temperature adjustment mechanism 214.

[0025] The temperature adjustment mechanism 214 may be capable of heating and cooling (temperature adjustment) at least the nucleic acid amplification section 107 at high speed. The temperature adjustment 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 section 107, based on the control of the control device 300. The temperature adjustment mechanisms 214 may be arranged above and below the flow channel device 101 so as to sandwich it. The sample solution 301 may be an aqueous solution in which an analyte, a PCR reagent capable of amplifying nucleic acid at high speed, and a primer having a sequence capable of identifying a target nucleic acid are mixed 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 the primer may be fixed in the nucleic acid amplification section 107 in advance, and may be dissolved in an aqueous solution containing the analyte to prepare the sample solution 301.

[0026] <Details of nucleic acid amplification reaction process> 2 is a flowchart for explaining details of the nucleic acid amplification reaction process according to the first embodiment. The amplification reaction process includes the introduction of a sample solution (S101), a first phase in which 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 reads a program corresponding to the flowchart in FIG. 2 from a memory (not shown in the figure, a memory in 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 can supply the sample solution 301 from the outside to the nucleic acid amplification reaction system 201 and the flow path device 101 may be provided.

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

[0029] (iii) S103 The controller 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: may be less than 1 second), and performs a denaturation reaction (standby at the first temperature for a seconds).

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

[0031] (v)S105 The controller 300 controls the temperature adjustment mechanism 214 so that the temperature of the sample solution 301 is maintained at the second temperature for b seconds (eg, 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 the steps from 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 section 107 to a first temperature (for example, 98° C.).

[0034] (viii) S107 The controller 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: may be less than 1 second), and performs a denaturation reaction (standby at the first temperature for c seconds).

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

[0036] (x)S109 The controller 300 controls the temperature adjustment 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) Iterative 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 the presence or absence of amplification of the target nucleic acid can be examined by an end-point 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 the first and second phases> 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 of the second phase (d) is shorter than the annealing / extension time of the first phase (b) (b>d).

[0040] It has been reported that the amplification efficiency of PCR is generally highly dependent on the annealing and extension time. As a result of intensive studies, the inventors of the present invention have clarified that the shorter the annealing and extension time, the less the amount of amplification of 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, and that this tendency is particularly pronounced when the initial nucleic acid concentration of the analyte is low (generally 1000 copies / test or less), when the DNA chain 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). The technology disclosed herein has been devised in consideration of these results of studies, and in the early stages of the cycle when the target nucleic acid concentration is low, the annealing and extension time (b) is set to be longer to suppress mispriming, and the target nucleic acid is amplified with high accuracy while minimizing the production of non-specific amplification products, and 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, and the amount of target nucleic acid is increased in a short period of time (second phase). This makes it possible to realize high-speed PCR that combines high amplification accuracy and efficiency.

[0041] <Example of nucleic acid amplification using 3-step PCR> In the above example, a nucleic acid amplification process using two-step PCR to maximize the speed has been described, but it can also be applied to three-step PCR. Fig. 3 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 three-step PCR and the second phase (S208 to S211 in Fig. 3) is two-step PCR. The control device 300 reads a program corresponding to the flowchart in Fig. 3 from a memory (memory in the control device or an external storage device) not shown, 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 3-step PCR, (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) Furthermore, so that the latter is shorter (a+b>c+d), (iii) More preferably, the annealing / extension time of the second phase is shorter than that of the first phase (b>d), By setting the reaction time, it is possible to achieve high-speed PCR that combines high amplification accuracy and efficiency.

[0044] It is not necessary to apply 3-step PCR only in the first phase, but the second phase may also be 3-step PCR, or only the second phase may be 3-step PCR. In general, 3-step PCR has a longer reaction time than 2-step PCR, and often achieves high amplification accuracy and efficiency. For this reason, if the first cycle (first phase), where mispriming is likely to occur, is 3-step PCR, and the second cycle (second phase), where the reaction has progressed to a certain extent, is 2-step PCR, reliable data can be obtained quickly even in difficult analyses. In addition, a 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 The following describes a nucleic acid amplification reaction system 201 and a flow path device 101 according to the second embodiment. Here, the characteristics of the method are described by taking as an example a method of amplifying nucleic acids of a simulated influenza B virus sample (equivalent to 100 DNA copies / test) in about 5 minutes using a reagent set capable of identifying influenza A and B viruses, and detecting the same by a lateral flow assay.

[0046] Fig. 4A is a diagram showing an example of the upper surface 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 a cross section taken along line AA in Fig. 4A.

[0047] 4A, the flow channel device 101 includes a substrate 102 having a flow channel structure formed thereon, a flow channel cover 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 located at the end points of the connection flow channels 106, and are processed as through holes on the substrate 102, and the lower surface is closed by sealing with a sealing tape (flow channel cover material 103). The two connection flow channels 106 are both formed on the lower surface 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 regulated by a first external heat source 206, a second temperature region 109 whose temperature is regulated 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 connection 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 with dimensions of 80 mm wide x 30 mm long x 3 mm thick. The flow channel of the nucleic acid amplification section 107 is formed in a meandering shape that is continuously connected to the connection flow channel 106. This allows the external heat sources 206 and 207 to be compactly arranged, and at the same time, the solution is stirred in the meandering section to make the concentration of the sample solution 301 uniform, resulting in a stable nucleic acid amplification reaction. Furthermore, a sealing tape made of polyolefin (PO) can be attached as a flow channel cover material 103 to the surface on which the flow channel is formed, to form a tubular flow channel.

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

[0050] <Example of nucleic acid amplification reaction system configuration> 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 configuration example of a cross section 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, a piping tube 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 suctioning operations of the syringe pump 210, the sample solution 301 can be transported in 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 path device is held such that the first external heat source 206 contacts the first temperature region 108 of the flow path 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 bonding copper blocks of 15 mm wide x 20 mm long x 2 mm thick to both sides of a small ceramic heater of 10 mm wide x 20 mm long x 1 mm thick without any gaps, and attaching a thermocouple to the surface that does not contact the flow channel device 101. The temperature control circuit 205 performs feedback control by referring to the temperatures of the first external heat source 206 and the second external heat source 207 acquired by the thermocouple, and sets the first external heat source 206 to a temperature suitable for DNA denaturation (for example, 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 (for example, in the range of 55 to 65°C, for example, 64°C). In order to thermally isolate the first external heat source 206 and the second external heat source 207, a slit 215 may be provided in the device holder between the first external heat source 206 and the second external heat source 207. 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 liquid delivery 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, etc. 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 the 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 is not necessarily required 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 are not particularly limited in terms of their materials and characteristics as long as they are 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 which case the operation steps of Fig. 6 may be reversed, i.e., all of the suction and pressurizing 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] <Examples of nucleic acid amplification reaction reagents> 6A is a diagram showing an example of the composition of a nucleic acid amplification reaction reagent. In the second embodiment, a sample to be detected is a simulated specimen of influenza B virus (equivalent to DNA 100 copies / test), and a KAPA 3G Plant PCR kit (KAPA BIOSYSTEMS) is used as a nucleic acid amplification reaction reagent. In this embodiment, in order to detect the product after the nucleic acid amplification reaction by nucleic acid chromatography 302, the primers can be modified at the 5' end of the sequence published by the National Institute of Infectious Diseases as a primer for identifying influenza A virus and B virus, with biotin modified as the forward primer for binding to latex particles for coloring on nucleic acid chromatography 302, and a tag sequence modified as the reverse primer for binding to the influenza B virus detection line on nucleic acid chromatography.

[0059] FIG. 6B is a diagram showing an example of the upper surface configuration of the nucleic acid chromatograph 302 and an example of detection of the target nucleic acid. 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 pasted together with an overlap of about 1 mm so that the sample solution 301 flowing into the sample pad 3001 is transported in sequence by capillary force. The conjugate pad 3002 is composed of blue latex particles modified with streptavidin, which are immobilized and 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 an oligo sequence capable of binding to the tag sequence of the target amplification product by hybridization is fixed. 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 detection target, and may be, for example, DNA, cDNA, RNA, cells, cell organelles, bacteria, or viruses. The reagent may be selected from pretreatment for enabling nucleic acid amplification reaction, and an enzyme and reaction system having an extension rate capable of amplifying nucleic acid at high speed. The primer may be a sequence capable of identifying the detection target, and is preferably one that is less prone to non-specific 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 preferable to avoid modifying both (tag sequence and biotin) to either the forward primer or the reverse primer. This allows only amplification products containing both primers to be detected, thereby reducing the probability of non-specific amplification products binding to the detection line and improving detection accuracy. The nucleic acid chromatograph 302 may be one used in the 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 treatment> Fig. 7 is a flowchart for explaining the nucleic acid amplification reaction process 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 in the control device or an external storage device) and executes the program.

[0062] (i) S301 A sample solution 301 is introduced into the flow path 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 flow 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 removed from the piping tube 208. In this case, the sample solution 301 does not pass through the inside of the liquid delivery mechanism 202, so that carryover between tests can be minimized. Alternatively, before the flow path device 101 is installed in the nucleic acid amplification reaction system 201, the operations from S301 to S303 in FIG. 7 may be performed in advance using a dedicated tool such as a micropipette.

[0065] (iv) S304 The control device 300 controls the first external heat source 206 via the temperature control circuit 205 to adjust (regulate) the temperature of the first temperature region 108 to a predetermined temperature (for example, 98°C). The control device 300 then controls the operation of the syringe pump 210 to make the sample solution 301 stay (regulate) in the first temperature region 108 (for example, 98°C) for a predetermined time (for example, 15 seconds) to raise the temperature of the sample. By adding this operation, when a DNA polymerase compatible with the hot start method is used, the effect of suppressing mispriming can be obtained, and by sufficiently raising the initial temperature of the sample solution 301, the denaturation reaction of the first cycle can be effectively performed, and stable nucleic acid amplification can be obtained even in a high-speed reaction system. When a DNA polymerase not compatible with the hot start method is used, S304 is not an essential processing step, and a series of reactions can be completed more quickly by omitting S304.

[0066] (v) S305 The control device 300 then 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 a seconds (for example, 1 second: it may be less 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 (eg, 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 (eg, 3 seconds), thereby carrying out an annealing and 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) Loop processing The control device 300 repeats the processes from S305 to S308 m times (for example, 20 times), thereby completing the first phase of processing.

[0071] (x)S309 After the first phase process is completed, the control device 300 continuously shifts the process 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 less 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 (eg, 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) Iterative processing The control device 300 repeats the process from S309 (keeping the sample solution 301 in the first temperature region 108 for c seconds (adjusting the temperature) to perform a denaturation reaction) to S312 (moving the sample solution 301 back to the first temperature region 108) n times (e.g., 20 times), thereby completing the second phase process and the nucleic acid amplification reaction process.

[0076] When performing the processes (S304, S305, S307, S309, and S311) of making the sample solution 301 stay (adjust the temperature) in the first temperature region 108 or the second temperature region 109, the operation of the liquid delivery mechanism 202 may be stopped, or delivery may be continued so that the sample solution 301 does not protrude from each temperature region. This can prevent the sample solution 301 from moving from a predetermined position due to the pressure difference in the flow channel at both ends, and also has the effect of stirring and homogenizing the 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 path device 101 into a microtube.

[0078] (xvi)S314 The operator inserts the nucleic acid chromatograph 302 into the microtube in which the sample solution 301 has been collected 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 the target DNA at high speed (for example, in about 8 minutes), and enables the presence or absence of the target DNA to be determined with high accuracy.

[0080] <Demonstration experiment results> FIG. 8 is a diagram showing the results of a demonstration experiment of the nucleic acid amplification reaction process according to the second embodiment. In the demonstration experiment, in addition to the nucleic acid chromatography, 2 μL of the sample solution 301 after the nucleic acid amplification reaction was taken and analyzed using an automatic electrophoresis system (for example, Agilient's TapeStation). FIG. 8A is a diagram showing a bright-field image of the nucleic acid chromatography. FIG. 8B is a diagram showing the target nucleic acid concentration semi-quantified by electrophoresis. Note that FIG. 8A and FIG. 8B show the results of a conventional high-speed PCR under slightly slow conditions, in which 40 cycles of PCR are completed in about 6.5 minutes. Also, FIG. 8A(i) shows the results (negative control) of the sample solution 301 to which the sample to be detected (simulated specimen of influenza type B virus) was not added. As shown in FIG. 8A(i), in the case where the sample to be detected was not included, neither the first detection line 3005 nor the second detection line 3006 was colored in the sample solution 301 after the nucleic acid amplification reaction. On the other hand, in FIG. 8A(ii), only the second detection line 3006 indicating the presence of influenza B virus was vividly colored, and the target substance contained in the sample solution 301 could be correctly detected.

[0081] 8A(iii) to (v) show the results of ultrafast PCR, which is completed in about 5 minutes. FIG. 8A(iii) shows the results of conventional fast PCR, in which the number of cycles is maximized, and FIG. 8A(iv) shows the results of conventional fast PCR, in which the annealing and extension times are maximized. FIG. 8A(v) shows the results of cycle-switching fast PCR, which is performed using the nucleic acid reaction processing method of this embodiment. In FIG. 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, is colored (false positive). This is thought to be because the amplification accuracy is reduced by increasing the annealing and extension time, which makes it easier to produce nonspecific amplification products, and nonspecific amplification products involving the primers for identifying influenza A virus are produced and are exponentially amplified together with the target nucleic acid during the cycles.

[0082] On the other hand, in FIG. 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 was colored (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 copies / μL, and the detection limit of nucleic acid chromatography 302 under the conditions of the demonstration experiment is 10 9 This is supported by the fact that the target nucleic acid concentration of the sample solution 301 in FIG. 8A(iv) is below the lower detection limit of the nucleic acid chromatography in this demonstration experiment, as shown in FIG. 8B.

[0083] In the nucleic acid chromatograph 302 in which the sample solution 301 in FIG. 8A(v) was analyzed, only the second detection line 3006 was colored, and the target substance could be correctly detected without any false positive or false negative.

[0084] As described above, this embodiment can effectively speed 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 and multiplex analysis.

[0085] In this embodiment, the amplified target nucleic acid is detected by a lateral flow assay (nucleic acid chromatography), but the nucleic acid detection method is not particularly limited as long as it is a method used in the relevant field. The lateral flow assay is a rapid and simple method suitable for application to a multiplex analysis system, but other methods such as end-point detection methods such as electrophoresis, absorbance measurement, sequencing, and microarray, or real-time detection methods that detect target nucleic acid during nucleic acid amplification reaction using a fluorescent probe, can also be used. In addition, this embodiment can be applied to a sample-to-answer type fully automated genetic testing system in which nucleic acid amplification and nucleic acid detection, or amplification pretreatment and nucleic acid amplification, or both are performed in the same flow path device 101 and the same device. According to this embodiment, there is no need to devise complicated reagent compositions or system configurations, and high effectiveness can be obtained simply by changing the thermal cycle conditions midway through the process.

[0086] (3) Third embodiment FIG. 9 is a flowchart for explaining a nucleic acid amplification reaction process according 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. 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 a similar one, can be used. The nucleic acid amplification reaction process according to the third embodiment differs from the nucleic acid amplification reaction process according to the first and second embodiments in that the third phase is performed after the second phase is completed. The control device 300 reads a program corresponding to the flowchart of FIG. 9 from a memory (not shown in the figure, a memory in 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-adjusted) to a first temperature (e.g., 98°C) (S410) and waits for a predetermined time (e.g., 1 second) (S411). Thereafter, the sample solution 301 is cooled to a second temperature (e.g., 64°C) (S412) and waits for a predetermined time (e.g., 1 second) (S413). The process from S410 to S413 is repeated o times (e.g., 20 times: o≧n≧m), thereby completing the third phase. At this time, the reaction time per cycle is set so that the later phase is shorter (a+b>c+d>e+f; in short, the time condition is b>d>f). In this way, by adding intermediate conditions (relatively fast conditions that provide a relatively high degree of amplification accuracy and efficiency) before transitioning from reliable target nucleic acid amplification using long annealing and extension times immediately after the start of the reaction to high-speed nucleic acid amplification using ultra-fast cycles in the latter half of the reaction, it is possible to achieve a balanced improvement in both the speed and accuracy of amplification.

[0088] In the present disclosure, the number of phases may be two or more, and it is also possible to change the thermal cycle conditions to four or more stages by adding a subsequent phase. 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 have 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 capable of performing real-time PCR and a flow path device 101. In addition to the nucleic acid amplification reaction system 201 and the flow path device 101 capable of performing real-time PCR, a method for determining the parameters a to d and m, n of the thermal cycle conditions in the above-mentioned cycle switching PCR method will be described.

[0090] <Examples of nucleic acid amplification reaction systems and flow path devices> 10 is a diagram showing an example of a cross-sectional configuration 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 FIG. 4). The nucleic acid amplification reaction system 201 has a configuration that is generally the same as that of the nucleic acid amplification reaction system 201 according to the second embodiment (FIG. 5), but is different from the nucleic acid amplification reaction system 201 of FIG. 5 in that an observation window 216 that allows observation of the intermediate flow channel 110 from below is provided in the device holder 203 and an optical detection mechanism 2001 that can detect fluorescence of a fluorescent probe is provided below the observation window 216. The optical detection mechanism 2001 includes a lens 2004, a camera 2002 that detects fluorescence contained in the sample solution 301, a signal processor 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 every 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] <Considerations on the results of amplification efficiency measurements> 11A and 11B, the results of measuring the amplification efficiency in the nucleic acid amplification reaction system 201 and the flow path device 101 will be discussed. Fig. 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). Fig. 11B is a graph showing the dependence of amplification efficiency and amplification rate on annealing and extension time (Anneal & Extension).

[0092] As shown in FIG. 11A, the amplification efficiency was almost 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 the amplification efficiency is not affected by the hot start time and the DNA denaturation time in this range, and can be shortened to at least 0 seconds and 1 second, respectively. Therefore, in the technology disclosed herein, both DNA denaturation times (states A and C) can be minimized, specifically to about 1 second or less. This allows PCR to be performed at high speed without reducing the amplification efficiency.

[0093] Figure 11B shows the results of investigating 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 30-40% with shortening of the annealing and extension time. On the other hand, referring to this amplification efficiency and cycle time, the DNA derived from the analyte (100 copies / test) was 10 9 When calculating the time required to amplify to copies / test, it can be seen that, despite the low amplification efficiency, the shorter the annealing and extension time, the faster the amplification can be completed (the longer the annealing and extension time, the higher the efficiency but the longer it takes to amplify, and the shorter the annealing and extension time, the lower the efficiency but the faster the amplification). This means that, 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 affected by the number of cycles rather than the amplification efficiency. In other words, in order to obtain a certain amount of target nucleic acid at high speed, it is effective to apply multiple cycles to the sample solution 301 at high speed even if it sacrifices some amplification efficiency. For this reason, in the technology disclosed herein, the annealing and extension time of the first phase is set to a range in which the amplification accuracy does not decrease (for example, b=3 seconds), while the second phase is specifically set to about 1 second or less. This makes it possible to speed up PCR without decreasing the amplification efficiency. From another perspective, as described above, when the annealing and extension time is shortened, the amplification efficiency decreases, but the amplification can be performed at a high speed. However, it is known that when the amplification efficiency decreases, the target nucleic acid decreases and non-specific amplification products are likely to 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 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 FIG. 12. FIG. 12A is a diagram showing the relationship between the total reaction time and the amount of target nucleic acid under each condition in which the annealing and extension times are changed. As described in FIG. 11B, the target nucleic acid increases exponentially with each cycle, and it can be seen that the higher the amplification efficiency (the longer the annealing and extension times), the smaller 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 are switched) taking into consideration the slope of this graph and the ease of producing non-specific amplification products in the PCR reaction system used.

[0095] FIG. 12B is a diagram showing a schematic diagram of the relationship between the total reaction time and the amount of target nucleic acid in the cycle switching PCR method. As shown in FIG. 12B, the slope of the amplification line changes at least once during the reaction, and further, the slope of the first phase is smaller, which is a feature of the technology disclosed herein. To determine the parameters a to d and m and n of the appropriate thermal cycle conditions, first, the amount of DNA contained in the analyte (initial amount), the target amount of DNA to be finally obtained (target amount), and the target reaction time (total reaction time) are set. At this time, the target amount of DNA may be set taking into consideration the lower detection limit of the subsequent nucleic acid analysis method. Next, the annealing and extension times of the first and second phases are selected. At this time, the annealing and extension times of the second phase are set to about 1 to 2 seconds regardless of the reaction conditions, and the annealing and extension times of the first phase are set taking into consideration the ease of production of non-specific amplification products. This is because the initial reaction (first phase) is generally more susceptible to the influence of reaction conditions such as the analyte and the reagent composition. After the thermal cycle conditions for each phase are determined, 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 cycle condition. The technical significance of the second phase in the present disclosure is to significantly increase the amount of nucleic acid amplified by applying multiple cycles in a short period of time, that is, to obtain the effect of shortening the reaction time, so that the second phase is preferably set to a condition for at least about 10 cycles, that is, to obtain 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 time of approximately 15 minutes or more). In that case, however, the same effect may be obtained by extending the annealing and extension time. On the other hand, in high-speed PCR (reaction time 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 According to the technology disclosed herein, it is possible to provide a high-speed nucleic acid amplification and detection apparatus that can be stably operated by suppressing both false negatives due to insufficient sensitivity and false positives due to non-specific amplification using inexpensive and simple flow path devices and systems (apparatus).

[0098] (i) The nucleic acid amplification technique according to this embodiment includes at least a first phase in which a denaturation reaction is performed on a sample solution for a seconds, followed by an annealing and extension reaction for b seconds, and a second phase in which a denaturation reaction is performed on the sample solution after the first phase 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 process and the second phase process, which have different annealing and extension times, are performed, so that PCR processing can 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 seconds (S103), adjusting the temperature of the sample solution after the temperature adjustment for a seconds at a second temperature (e.g., 64° C.) lower than the first temperature for b seconds (lowering the temperature from the first temperature to the second temperature and then heating the sample solution) (S105), and repeating the steps of S103 and S105 m times. The second phase includes adjusting the temperature of 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 the temperature of the sample solution after the temperature adjustment (heating) for c seconds at the second temperature for d seconds (lowering the first temperature to the second temperature and heating the sample solution) (S109), and repeating the steps of S107 and S109 n times. Here, a to 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 many times, allowing PCR to be performed at high speed. In addition, since the annealing and extension time in the first phase is longer than that in the second phase, nucleic acids can be amplified with high efficiency, and the generation of non-specific amplification products can be suppressed, resulting in highly reliable PCR results (suppressing 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. Also, m and n may be natural numbers and n≧10, or m and n may be natural numbers and n≧m. Furthermore, even if the total time required for the first and second phases is set to less than 15 minutes (high speed), it is possible to amplify nucleic acids while suppressing the generation of non-specific amplification products.

[0101] (iii) The 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), 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 after the temperature adjustment (heating) for a seconds by a conveying operation of the syringe pump (S306), adjusting (heating) the sample solution at the second temperature region 109 for b seconds (heating at a second temperature lower than the first temperature) (S307), moving the sample solution after the temperature adjustment for b seconds to the first temperature region 108 by a conveying operation of the syringe pump (S308), and repeating the steps from S305 to S308 m times. The second phase includes adjusting (heating) the temperature of the sample solution after the completion of 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 conveying operation of the syringe pump (S310), adjusting the temperature of 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 conveying operation of the syringe pump (S312), and repeating the steps from S309 to S312 n times. Here, a to d are positive numbers, and b>d. Since the temperature region in the flow channel is divided to adjust (heat) the temperature of the sample solution, the temperature can be controlled accurately and easily, and further, the annealing and extension process can be performed accurately. Furthermore, similarly to the first embodiment, it is possible to increase the speed of 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, a third phase is carried out in which the sample solution after the second phase is subjected to a denaturation reaction for e seconds, followed by an 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 seconds (S402), adjusting the temperature of the sample solution after the temperature adjustment (heating) for a seconds at a second temperature (e.g., 64°C) lower than the first temperature for b seconds (reducing the first temperature to the second temperature and then heating the sample solution for b seconds) (S405), and repeating the steps of S402 and S405 m times. 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 the temperature adjustment (heating) for c seconds at the second temperature for d seconds (reducing the first temperature to the second temperature and then heating the sample solution) (S409), and repeating 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 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 the steps of 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 and performing annealing and extension processing under intermediate conditions (between an upper phase (e.g., the first phase) and a lower phase (e.g., the third phase)), it becomes possible to balance the 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 itself read from the storage medium realizes the functions of the above-mentioned embodiment, and the program code itself and the storage medium on which it is stored constitute the present disclosure. Examples of storage media for supplying such program code include flexible disks, CD-ROMs, DVD-ROMs, hard disks, optical disks, magneto-optical disks, CD-Rs, magnetic tapes, non-volatile memory cards, and ROMs.

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

[0106] Furthermore, the program code of the software that realizes the functions of the embodiments may be distributed over 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 out 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 may be implemented by a combination of components. Various types of general-purpose devices may also be added. A dedicated device may be constructed to execute the functions of the present embodiment. Various functions may also be formed by appropriately combining multiple components disclosed in the present embodiment. For example, some components may be deleted from all the components shown in the embodiment, or components across different embodiments may be appropriately combined.

[0108] In this disclosure, specific embodiments are described, but they are in all respects for the purpose of explanation (understanding the technology of the present disclosure) and not for the purpose of limitation. It is understood by those skilled in the art that there are many combinations of hardware, software, and firmware suitable for implementing the technology of the present disclosure. For example, the described software can be implemented in a wide range of program or script 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 of the control lines and information lines in the product are necessarily shown. All of the components may be connected to each other.

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

[0111] 101 Flow Channel Device 102 Substrate 103 Channel cover material 104 First Connection Port 105 Second connection port 106 Connection Channel 107 Nucleic Acid Amplification Unit 108 First temperature region 109 Second temperature region 110 Intermediate Channel 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 supply connection port 212 Second liquid supply connection port 213 Vent 214 Temperature control mechanism 215 Slit 216 Observation Window 301 Sample solution 302 Nucleic Acid Chromatography 2001 Optical detection mechanism 2002 Camera 2003 Signal Processing Section 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 (FluA) 3006 Second detection line (FluB)

Claims

1. A nucleic acid amplification method for amplifying a nucleic acid contained in a sample solution contained in a flow path device, comprising: A first phase step in which the sample solution is subjected to a denaturation reaction for a seconds, followed by an annealing / extension reaction for b seconds; A second phase step in which a denaturation reaction is carried out for c seconds on the sample solution after the first phase step is carried out, followed by an annealing / extension reaction for d seconds; The method for amplifying a nucleic acid, wherein b and d satisfy the condition b≠d.

2. In claim 1, The first phase process comprises: 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 controlling the temperature of the sample solution after the temperature control for the a seconds at a second temperature lower than the first temperature for b seconds using the heat source; A first repeating step of repeating the first step and the second step m times, The second phase process comprises: a third step of controlling the temperature of the sample solution after the first repeating 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 the c seconds at the second temperature for d seconds using the heat source; A second repeating step of repeating the third step and the fourth step n times, The method for amplifying a nucleic acid, wherein a to d are positive numbers and b>d.

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

4. In claim 1, A method for amplifying a nucleic acid, wherein a and c are 1 or less.

5. In claim 2, A method for amplifying a nucleic acid, wherein m and n are natural numbers and n≧10.

6. In claim 2, A method for amplifying a nucleic acid, wherein m and n are natural numbers and n≧m.

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

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

9. In claim 1, further comprising: A nucleic acid amplification method comprising a third phase step in which the sample solution after the second phase step is subjected to a denaturation reaction for e seconds, followed by an annealing / extension reaction for f seconds.

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

11. A nucleic acid amplification reaction system for amplifying a nucleic acid contained in a sample solution, comprising: a flow path device that contains the sample solution; A heat source for adjusting the temperature of the sample solution; a computer that amplifies the nucleic acid contained in the sample solution according to a program for amplifying the nucleic acid; The computer includes: a first phase treatment in which a denaturation reaction is carried out for a second, followed by an annealing / extension reaction for b seconds; A second phase treatment is carried out on the sample solution after the first phase treatment, in which a denaturation reaction is carried out for c seconds, followed by an annealing / extension reaction for d seconds; The nucleic acid amplification reaction system, wherein b and d satisfy the condition b≠d.

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

13. In claim 11, Further, a syringe pump is provided, the flow path device has a serpentine flow path that accommodates the sample solution, The serpentine flow path includes a first temperature region for controlling a temperature at a first temperature and a second temperature region for controlling a temperature at a second temperature lower than the first temperature, In the first phase processing, the computer a first process of controlling a conveying operation of the syringe pump to move the sample solution to a first temperature region of the serpentine flow path; a second process of controlling the heat source to adjust the temperature of the sample solution in the first temperature range for a seconds; a third process of controlling a conveying operation of the syringe pump after the temperature adjustment for the a seconds to move the sample solution to the second temperature region; a second process of controlling the heat source to adjust the temperature of the sample solution in the second temperature range for b seconds; a third process of controlling a conveying operation of the syringe pump to move the sample solution after the temperature adjustment for the b seconds to the first temperature region; A first repetitive process is executed to repeat the first process to the third process m times; In the second phase treatment, a fourth process of controlling the heat source to adjust the temperature of the sample solution after the first phase process is completed in the first temperature range for c seconds; a fifth process of controlling a conveying operation of the syringe pump to move the sample solution after the temperature adjustment for the c seconds to the second temperature range; a sixth process of controlling the heat source to adjust the temperature of the sample solution in the second temperature range for d seconds; a seventh process of controlling a transport operation of the syringe pump to move the sample solution after the temperature adjustment for the d seconds to the first temperature region; A nucleic acid amplification reaction system, wherein a to d are positive numbers and b>d.

14. In claim 11, The computer further comprises: A nucleic acid amplification reaction system, which performs a denaturation reaction on the sample solution after the second phase treatment for e seconds, and then performs a third phase treatment of performing an annealing / extension reaction for f seconds.

15. In claim 14, In the first phase processing, the computer a first process of controlling the heat source to adjust the temperature of the sample solution at a first temperature for a seconds; a second process of controlling the heat source to adjust the temperature of the sample solution for b seconds after the temperature adjustment for the a seconds at a second temperature lower than the first temperature; A first repetitive process is executed in which the first process and the second process are repeated m times; In the second phase treatment, a third process of controlling the heat source to adjust the temperature of the sample solution after the first repeated process at the first temperature for c seconds; a fourth process 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; A second repetitive process is executed in which the third process and the fourth process are repeated n times; In the third phase treatment, a fifth process of controlling the heat source to adjust the temperature of the sample solution after the second repeating step at the first temperature for e seconds; a sixth process of controlling the heat source to adjust 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 is executed in which the fifth process and the sixth process are repeated o times; A nucleic acid amplification reaction system, wherein a to f are positive numbers and b>d>f.

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