Nucleic acid detection system and nucleic acid detection method

Abstract

To provide a nucleic acid detection system and a nucleic acid detection method that have simple configuration and enable liquid feeding to be more appropriately controlled.SOLUTION: A nucleic acid detection system comprises: flow path structure; a pair of pressure adjustment portions connected to both ends of the flow path structure; and a liquid feeding control mechanism connected to the pair of pressure adjustment portions. The flow path structure comprises: a first flow path; a second flow path connected to the first flow path; and a detection paper installed in the second flow path. The nucleic acid detection system performs: a step in which the liquid feeding control mechanism moves sample solution within the first flow path and nucleic acid amplification reaction is performed; a step in which after the nucleic acid amplification reaction, the liquid feeding control mechanism starts moving the sample solution to the second flow path; and a step in which after the sample solution reaches one end of the detection paper, the liquid feeding control mechanism stops the movement of the sample solution depending on a predetermined stopping condition being satisfied.SELECTED DRAWING: Figure 2

Description

[[Technical Field]]

[0001] The present invention relates to a nucleic acid detection system and a nucleic acid detection method. [[Background Art]]

[0002] In recent years, the importance of infectious disease testing has increased due to factors including an increase in the number of sepsis deaths caused by drug-resistant bacteria and social damage caused by COVID-19. In infectious disease testing, since tests are operated by clinical technologists or nurses, simple operability is required. In addition, shortening test time is directly linked to improving therapeutic effects and reducing treatment costs. Furthermore, since it is often difficult to predict causative pathogens from clinical findings for infectious diseases, multiplex detection that enables simultaneous testing for multiple types of pathogens is required.

[0003] For infectious disease diagnosis, rapid genetic testing systems using disposable cartridges have been proposed for performing assays including nucleic acid amplification and detection at clinical sites. Patent Document 1 proposes a technology that enables multiplex nucleic acid measurement, in which a sample inlet, a purification unit, a PCR unit and a detection unit are provided in a primary flow path within a cartridge.

[0004] As a method for amplifying nucleic acids, there is the PCR (Polymerase Chain Reaction) method. The PCR method amplifies a target DNA by repeating three steps—denaturation of DNA (separation of double-stranded DNA into single-stranded DNA), annealing (binding of complementary primers to single-stranded DNA), and extension (replication of double-stranded DNA via complementary strand synthesis)—each at different temperatures. Typically, thermal denaturation is performed at approximately 95°C, annealing at approximately 60 to 65°C, and extension at approximately 72°C. In the case of isothermal amplification methods, reactions corresponding to these cycles are performed at a constant temperature of approximately 35 to 65°C.

[0005] The PCR method widely employs a thermal cycling apparatus, in which reaction tubes containing the reaction solution are inserted into wells 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 variations, the metal block has a volume equal to or greater than that of the reaction tubes or microplates placed inside. This increases the heat capacity of the metal block, limiting the heating and cooling rate, so PCR requires at least 30 minutes, and typically 1 to 2 hours.

[0006] In recent years, methods have been proposed to accelerate PCR by using microfluidic devices (fine channels) formed on a substrate. Furthermore, a method called two-step PCR has been proposed, which performs PCR using a thermal cycle that repeats two temperature zones: denaturation and annealing. Since the annealing step and extension step are performed simultaneously, the time required can be reduced.

[0007] Several high-speed PCR methods have been proposed that combine the aforementioned microfluidic device with two-step PCR.

[0008] Patent Document 2 describes a nucleic acid amplification apparatus in which a microfluidic chip having a meandering channel is brought into contact with heat blocks set to two different temperature zones, and a thermal cycle is performed according to the number of meandering cycles of the channel. This method is called meandering channel PCR, and it only requires 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 rate of the heat blocks, but is determined by the channel length and the flow rate of the reaction solution.

[0009] Patent Document 3 describes a nucleic acid amplification apparatus that performs thermal cycling by bringing a microfluidic chip with a single channel into contact with heat blocks 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 only requires 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 rate of the heat blocks, but is determined by the time the reaction solution is held in the temperature zone.

[0010] Patent documents 4 and 5 propose a gene testing system using a disposable cartridge equipped with a reaction chamber for pre-treatment such as nucleic acid amplification and lateral flow assay paper. In Patent Document 4, a sample placed in the sample port of a vertically installed cartridge is sequentially delivered from the reaction chamber to the lateral flow assay paper by gravity and operation of an in-flow valve, and the assay is completed by the capillary force of the lateral flow assay paper drawing up the sample. In Patent Document 5, delivery to the reaction chamber or lateral flow assay paper is achieved by operation of an in-flow valve. Furthermore, Patent Document 6 proposes a deployment method in which a sample is guided to the lateral flow assay paper by perforating the sample chamber in the flow channel device with a lancet.

[0011] In infectious disease diagnosis using genetic testing, reducing testing time and achieving multiplex detection are crucial challenges. The step of detecting amplification products can be broadly classified into two types: real-time detection, which sequentially detects nucleic acid amplification cycle by cycle using reagents containing fluorescent dyes, and endpoint detection, which detects the amount of nucleic acid after the reaction is complete. Lateral flow assay, one of the endpoint detection methods, has the advantage of being less expensive and easier to construct as a compact, simultaneous multi-parameter detection system compared to real-time detection. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent Application Publication No. 2012 / 0178091 [Patent Document 2] U.S. Patent Application Publication No. 2010 / 0167288 [Patent Document 3] International Publication No. 16 / 006612 brochure [Patent Document 4] International Publication No. 18 / 172724 brochure [Patent Document 5] Special Publication No. 2016-533185 [Patent Document 6] Japanese Patent Publication No. 2021-167822 [Overview of the project] [Problems that the invention aims to solve]

[0013] The systems described in Patent Documents 4-6 are characterized in that the only force acting on the sample during lateral flow assay paper is capillary force. On the other hand, high-speed PCR systems such as those shown in Patent Documents 1-3 do not use lateral flow assay paper, and high-speed liquid delivery with precise positional control is essential, requiring liquid delivery control by pressurization and suction using a pump or the like. In this case, the sample solution may leak out or the sample may remain in the flow path due to insufficient liquid delivery, which may lead to detection failure or other problems.

[0014] Furthermore, in the method of installing a valve in the flow channel between the pre-processing unit and the lateral flow assay paper, the flow channel structure and the configuration of the flow channel device related to valve installation become complicated, increasing the cost of the flow channel device and apparatus.

[0015] Therefore, the present invention aims to provide a system and method for nucleic acid detection using detection paper that simplifies the configuration while providing more appropriate liquid delivery control. [Means for solving the problem]

[0016] An example of a nucleic acid detection system according to the present invention is: Flow channel structure, a pair of pressure adjusting units connected to both ends of the flow channel structure, a liquid feeding control mechanism connected to the pair of pressure adjusting units, A nucleic acid detection system comprising: the flow channel structure comprises: a first flow channel, a second flow channel connected to the first flow channel, a detection paper disposed in the second flow channel, comprising, the nucleic acid detection system: a step in which the liquid feeding control mechanism moves a sample solution within the first flow channel to perform a nucleic acid amplification reaction; after the nucleic acid amplification reaction, a step in which the liquid feeding control mechanism starts moving the sample solution to the second flow channel; after the sample solution reaches one end of the detection paper, a step of stopping the movement of the sample solution by the liquid feeding control mechanism in response to a predetermined stop condition being satisfied; is executed.

[0017] An example of the nucleic acid detection method according to the present invention is: A nucleic acid detection method using a nucleic acid detection system, wherein the nucleic acid detection system comprises: a flow channel structure, a pair of pressure adjusting units connected to both ends of the flow channel structure, a liquid feeding control mechanism connected to the pair of pressure adjusting units, comprising, the flow channel structure comprises: a first flow channel, a second flow channel connected to the first flow channel, a detection paper disposed in the second flow channel, comprising, the nucleic acid detection method comprises: a step of moving a sample solution within the first flow channel to perform a nucleic acid amplification reaction; after the nucleic acid amplification reaction, a step of starting moving the sample solution to the second flow channel; After the sample solution reaches one end of the detection paper, the movement of the sample solution is stopped when a predetermined stopping condition is met. Includes. [Effects of the Invention]

[0018] According to the nucleic acid detection system and nucleic acid detection method of the present invention, it is possible to simplify the configuration while performing more appropriate liquid delivery control.

[0019] For example, it becomes possible to provide a high-speed nucleic acid amplification and detection device that is inexpensive, simple, and capable of achieving high-speed reactions through high-speed and precise liquid delivery control, while appropriately delivering the sample solution to lateral flow assay paper, thereby enabling stable operation.

[0020] Other issues, configurations, and effects will be clarified by the following description of the embodiments. [Brief explanation of the drawing]

[0021] [Figure 1] Conceptual diagram of a nucleic acid detection system according to Example 1 of the first embodiment. [Figure 2] Flowchart of the nucleic acid detection method according to Example 1 of the first embodiment. [Figure 3] A plan view of a flow channel device according to Example 2 of the first embodiment. [Figure 4] Configuration of the detection paper according to Example 2 of the first embodiment. [Figure 5] This figure shows the flow channel device according to Example 2 of the first embodiment installed in the device holding section of a nucleic acid amplification reaction apparatus. [Figure 6] A cross-sectional view drawn from a plane along line AA in Figure 5. [Figure 7] An example of the shape of the second channel according to Embodiment 4 of the first embodiment. [Figure 8] A schematic diagram of the portion including the first channel when the channel device according to the second embodiment is installed in the device holding section of a nucleic acid amplification reaction apparatus. [Figure 9] The PCR portion of the operation flow in the second embodiment. [Figure 10] A schematic diagram of the portion including the first channel when the channel device according to the third embodiment is installed in the device holding section of a nucleic acid amplification reaction apparatus. [Modes for carrying out the invention]

[0022] Embodiments of the present invention will be described below with reference to the drawings.

[0023] <First Embodiment>

[0024] [Example 1] The following describes a nucleic acid amplification reactor and a flow channel device related to Example 1 of the first embodiment of the present invention. The nucleic acid amplification reactor and flow channel device constitute a nucleic acid detection system and perform the nucleic acid detection method described herein.

[0025] Figure 1 shows a conceptual diagram of the nucleic acid detection system related to Example 1, and Figure 2 shows a flow chart of the nucleic acid detection method. The flow channel device 101 has a flow channel structure. The flow channel structure has the following components. - Sample inlet 110 (sample solution inlet) - Sample introduction channel 109 - First channel 103 (nucleic acid amplification reaction section) for carrying out the nucleic acid amplification reaction - The second channel 104 is connected to the first channel 103 and stores the detection paper 201. - Intermediate channel 108 connecting the first channel 103 and the second channel

[0026] A first pressure adjustment unit 105 and a second pressure adjustment unit 106 are connected to both ends of the flow path structure (i.e., one side of the first flow path 103 and one side of the second flow path 104) via connecting flow paths 107, respectively, to form a pair of pressure adjustment units.

[0027] The material of the flow channel device 101 is preferably a resin that does not cause nucleic acid adsorption or inhibition of nucleic acid amplification reactions at a heat resistance temperature of about 100°C or higher, such as polycarbonate (PC), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), or cyclic cycloolefin copolymer (COC).

[0028] The sample inlet 110 and the sample introduction channel 109 may be installed at any location between the first pressure adjustment unit 105 and the intermediate channel 108. In particular, the sample introduction channel 109 may be provided within the first channel 103, or it may be provided on the opposite side of the second channel from the first channel 103 (the position shown in Figure 1). The sample inlet 110 and the first pressure adjustment unit 105 may have the same or partially overlapping structure. Also, the sample introduction channel 109 and the connecting channel 107 may have the same or partially overlapping structure.

[0029] The shape of the sample inlet 110 can be cylindrical, conical, countersunk, etc. Preferably, the sample inlet 110 has no corners when viewed from above (such as circular or elliptical), and it is also preferable that the shape allows the sample solution in the sample inlet 110 to be drawn into the sample introduction channel 109. By making the sample inlet 110 have no corners when viewed from above, unexpected sample leakage due to capillary action at corners can be prevented.

[0030] The nucleic acid amplification reactor 301 is equipped with a liquid delivery control mechanism 302. The liquid delivery control mechanism 302 is connected to a first pressure adjustment unit 105 and a second pressure adjustment unit 106. The liquid delivery control mechanism 302 contains at least one liquid delivery power source and is capable of applying pressure to either or both of the first pressure adjustment unit 105 or the second pressure adjustment unit 106. This enables the sample solution introduced into the flow path device 101 to be moved quickly and precisely.

[0031] The first channel 103 is, for example, a tubular channel without branches (the specific shape is omitted in Figure 1). Multiple temperature regions are provided in the first channel 103. By moving the sample solution through each temperature region and allowing it to remain there, a predetermined temperature change can be applied to the sample solution.

[0032] The second channel 104 is, for example, a tubular channel without branches. The channel structure of the channel device 101 includes a detection paper 201, which is placed in the second channel 104. The detection paper 201 is provided with a detection line 207.

[0033] The following describes each operation step with reference to Figure 2. First, the sample solution is introduced into the sample inlet 110 (Figure 2(a)). The flow channel device 101 then contains the sample solution within the sample inlet 110. The sample inlet 110 may then be closed. Next, the liquid delivery control mechanism 302 draws the sample solution into the sample introduction channel 109 (Figure 2(b)). Next, the liquid delivery control mechanism 302 moves the sample solution to the first channel 103 (Figure 2(c)). The steps in Figures 2(a) to (c) allow the sample solution to be smoothly moved to the first channel 103. Note that the operations in Figures 2(a) to (c) may also be performed using a dedicated instrument such as a micropipette.

[0034] Next, the liquid delivery control mechanism 302 moves the sample solution in the first channel 103 to a first temperature range (e.g., 98°C) (Figure 2(d)). Then, the sample solution is allowed to remain for a predetermined time (e.g., 15 seconds) to raise its temperature (Figure 2(e)).

[0035] Next, the PCR cycle is initiated by repeating the process. The PCR cycle is described below. During the PCR cycle, the sample solution is moved to the first temperature region in the first channel 103 (Figure 2(f)). Note that during the first PCR cycle, the sample solution has already moved to the first temperature region, so the step in Figure 2(f) is omitted.

[0036] Next, wait for a certain period of time (e.g., 1 second) in the first temperature range to allow the denaturation reaction to occur (Figure 2(g)). The step in Figure 2(g) may be omitted when performing the first PCR cycle.

[0037] Next, the sample solution is moved to a second temperature range (e.g., 60°C) (Figure 2(h)). Then, the sample solution is allowed to remain for a predetermined time (e.g., 6 seconds) (Figure 2(i)). This allows the annealing and extension reaction to occur.

[0038] This completes one PCR cycle. From Figure 2(f) to Figure 2(i), the liquid delivery control mechanism 302 constitutes the step of moving the sample solution in the first channel 103 and carrying out the nucleic acid amplification reaction.

[0039] To start the next PCR cycle, return the sample solution to the first temperature range (Figure 2(f)) and repeat the PCR cycle.

[0040] During the step of keeping the sample solution 401 within a predetermined temperature range (Figure 2(e), (g), (i)), the liquid delivery operation of the liquid delivery control mechanism 302 may be stopped, or the liquid delivery control may be continued to prevent the sample solution from spilling out of each temperature range.

[0041] By repeating the operations shown in Figures 2(f) through (i) a predetermined number of times (for example, 40 times), a specific region of the DNA to be detected is amplified. In this way, the nucleic acid amplification reactor 301 achieves the nucleic acid amplification reaction by moving the sample solution through multiple temperature ranges. This allows PCR to be performed at high speed (for example, in about 8 minutes).

[0042] After the PCR cycle is complete, the liquid delivery control mechanism 302 initiates the transfer of the sample solution, which has been processed in the first channel 103, to the second channel 104 via the intermediate channel 108 (Figure 2(j)). This causes the sample solution to move to the second channel 104 and reach the detection paper 201 placed in the second channel 104.

[0043] The nucleic acid amplification reactor 301 may be equipped with a sensor that detects when the sample solution has reached one end (for example, the tip) of the detection paper 201. The specific type and installation location of the sensor can be appropriately designed by those skilled in the art.

[0044] The liquid delivery control mechanism 302 stops the movement of the sample solution after the sample solution reaches one end of the detection paper 201, depending on whether predetermined stopping conditions are met (Figure 2(k)). For example, the liquid delivery control mechanism 302 stops applying pressure to the first pressure adjustment unit 105 and the second pressure adjustment unit 106. Even after the movement of the sample solution by the liquid delivery control mechanism 302 is stopped, the movement of the sample solution by means other than the liquid delivery control mechanism 302 (for example, movement by capillary force of the detection paper 201) continues.

[0045] The above stopping condition can be, for example, when the trailing end of the sample solution reaches the leading end of the detection paper 201. That is, when the entire volume of the sample solution reaches the detection paper 201, the movement by the liquid delivery control mechanism 302 is stopped. In this way, a sufficient amount of sample solution can be more reliably delivered to the detection paper 201.

[0046] In this specification, "leading edge of detection paper" refers to, for example, the part or end of the detection paper that first comes into contact with the sample solution. In the example shown in Figure 4 below, the end on the side of the sample pad 204 is the leading edge. Also, "leading edge of sample solution" refers to, for example, the part of the sample solution that first comes into contact with the detection paper. Furthermore, "rear end of sample solution" refers to, for example, the part of the sample solution that last comes into contact with the detection paper. That is, "the rear end of the sample solution reaching the leading edge of the detection paper" means that the entire volume of the sample solution reaches the detection paper.

[0047] Furthermore, the stopping condition may be defined as a predetermined time elapsed after the sample solution reaches one end of the detection paper 201. This makes it easier to determine the conditions.

[0048] Furthermore, if the detection paper 201 is equipped with a specific detection unit (for example, a detection unit 203 described in relation to Figure 4), the stopping condition may be that the tip of the sample solution reaches the detection unit. In this case, the spread of the sample solution in the detection unit is carried out solely by the capillary force of the detection unit, thus suppressing overflow of the sample solution in the detection unit.

[0049] After Figure 2(k), a predetermined time (e.g., 2 minutes) is waited, and then the detection line 207 on the detection paper is judged (Figure 2(l)). Before judgment, it may be confirmed that the sample solution has spread sufficiently within the detection paper 201 by capillary force. This judgment makes it possible to rapidly amplify and detect the target DNA in the sample solution (e.g., in about 10 minutes), and to determine the presence or absence of the target DNA.

[0050] Thus, according to the nucleic acid detection system and nucleic acid detection method of Example 1 of the first embodiment, it is possible to simplify the configuration while performing more appropriate liquid delivery control.

[0051] [Example 2] The following describes Example 2 of the first embodiment. In Example 2, a more detailed explanation is given for a modified nucleic acid detection system. The following explanation may omit details common to both Example 1 and Example 1.

[0052] Figure 3 shows a plan view of the flow channel device 101. First, the following structure was fabricated by cutting a PC plate measuring 80 x 30 mm with a thickness of 3 mm. - Sample introduction channel 109 with a width of 0.3 mm and a depth of 0.3 mm - First channel 103 with a width of 0.8 mm and a depth of 0.6 mm, connecting channel 107 and intermediate channel 108 - Second channel 104 with a width of 2.1 mm, a depth of 2 mm, and a length of 50 mm. - Sample inlet 110 with a diameter of 3 mm - First pressure adjustment section 105 and second pressure adjustment section 106 with a diameter of 1 mm - Filter housing section 112 with a diameter of 0.5 mm and a depth of 0.6 mm

[0053] In this embodiment, the first channel 103 and the second channel 104 are tubular. This allows for more efficient movement of the sample solution.

[0054] The second channel 104 is 2 mm deep and has a bottom support projection 113 (described later in relation to Figure 6) with a height of 1 mm to hold the detection paper. The bottom support projection 113 makes it easy to align the detection paper 201 and, at the same time, creates space within the second channel 104, which improves the stability of liquid delivery and airflow.

[0055] A detection paper 201 measuring 2 mm in width and 40 mm in length was placed in the second channel 104, and a polyethylene (PE) filter 111 measuring 0.5 mm in diameter and 0.6 mm in thickness was placed in the filter storage section 112.

[0056] Subsequently, a 0.1 mm thick polyolefin (PO) sealing tape (not shown) was pressed onto the entire surface of the channel cutting surface to form the channel. The sample inlet 110, the first pressure adjustment section 105, and the second pressure adjustment section 106 were processed as through holes in the sealing tape and opened on the opposite surface of the channel cutting surface.

[0057] The sealing tape does not have to be made of PO; any resin (such as PP) that can withstand temperatures of around 100°C or higher and does not cause nucleic acid adsorption or inhibition of nucleic acid amplification reactions is acceptable.

[0058] The sample inlet 110 and the sample introduction channel 109 may be located between the filter storage section 112 and the second channel 104. Furthermore, they may be placed at any position within the sequence of operations where the sample solution does not come into contact with the filter 111.

[0059] The filter 111 is preferably made of a porous material with a pore size of about 10 to 100 μm, which allows air to pass through but does not allow nucleic acids to pass through. For example, it may be made of resin such as PP, glass fiber material, or a material containing a mixture. By placing the filter 111 between the first channel 103 and the first pressure adjustment unit 105, it is possible to prevent the nucleic acids amplified in the first channel 103 from leaking from the channel device 101 into the nucleic acid amplification reactor 301 and the environment, thereby preventing contamination.

[0060] Figure 4 shows the configuration of the detection paper 201. Figure 4(a) shows a top view, and Figure 4(b) shows a cross-sectional view along line AA in Figure 4(a). Note that when the detection paper 201 is used, it is positioned upside down from the orientation shown in Figure 4(a) (see Figure 6).

[0061] The detection paper 201 can be made entirely of paper. The detection unit 203 is attached with adhesive to the center of the backing sheet 202. The detection unit 203 is the part that reacts with the target to be detected (for example, DNA). A detection line 207 is provided on the detection unit 203.

[0062] As shown in Figure 2(b), the sample pad 204 and the conjugate pad 205 are attached to one end in the longitudinal direction (the side into which the sample flows, i.e., the upstream side). Furthermore, the absorption pad 206 is attached to the other end in the longitudinal direction (i.e., the downstream side). The sample pad 204 and the conjugate pad 205 are attached so that they overlap by about 1 mm, and similarly, the detection unit 203 and the conjugate pad 205 are attached so that they overlap by about 1 mm. Furthermore, the detection unit 203 and the absorption pad 206 are also attached so that they partially overlap.

[0063] The conjugate pad 205 was immobilized with blue latex particles modified with streptavidin. The detection unit 203 had an oligo sequence, which can bind to a tag attached to the target DNA by hybridization, coated and immobilized at the capture site of the target DNA.

[0064] The sample pad 204, conjugate pad 205, and absorbent pad 206 do not have to be made of paper; any porous, highly absorbent material (such as glass fiber) is acceptable. The sample pad 204 and absorbent pad 206 control the flow rate of the sample solution flowing into the detection unit 203, contributing to stable line detection.

[0065] The backing sheet 202 maintains the relative positions of the detection unit 203, sample pad 204, conjugate pad 205, and absorption pad 206, contributing to ease of manufacturing and ease of handling when stored in the flow channel device 101. The backing sheet 202 is preferably made of a material with a certain degree of rigidity, and may be made of a material other than paper, such as polyethylene terephthalate (PET).

[0066] Figure 5 shows the flow channel device 101 installed in the device holder 303 of the nucleic acid amplification reactor 301. Figure 6 shows a cross-sectional view in plan along line AA in Figure 5. The nucleic acid amplification reactor 301 comprises a device holder 303 made of PEEK, a cover 304 made of PC, a liquid delivery control mechanism 302, and a line detection mechanism 315.

[0067] The device holder 303 includes a connection part 305, a ventilation hole 306, a first external heat source 310, a second external heat source 311, and an observation window 314. While materials other than PEEK can be used for the device holder 303, a material with a minimum heat resistance of 100°C and low thermal conductivity is preferred. The cover 304 prevents the flow channel device 101 installed in the device holder 303 from floating up and fixes it in place. The material of the cover 304 is preferably one with a minimum heat resistance of around 70°C and low thermal conductivity.

[0068] The first external heat source 310 and the second external heat source 311 each have a thermocouple and a heater built in, and are controlled by the temperature controller 309 to maintain the surface at a predetermined temperature. In the flow channel device 101, the area in contact with the first external heat source 310 becomes the first temperature region, and the area in contact with the second external heat source 311 becomes the second temperature region.

[0069] In relation to the second channel 104, a bottom support projection 113 is provided. This facilitates the alignment of the detection paper 201 and, at the same time, creates space within the second channel 104, thereby improving the stability of liquid delivery and airflow.

[0070] In this embodiment, a two-step PCR method was adopted to accelerate PCR by repeating denaturation and annealing / extension in two temperature ranges. A first external heat source 310 and a second external heat source 311 made of nickel-plated copper were used, with the target temperature of the first temperature range being the denaturation temperature range (e.g., 98°C) and the target temperature of the second temperature range being the annealing / extension temperature range (e.g., 64°C).

[0071] In this embodiment, a specific example of the liquid delivery control mechanism 302 is a syringe pump 308 and a vent hole 306. The first pressure adjustment unit is connected to the syringe pump 308 via a piping tube 307 and a connection part 305. The second pressure adjustment unit 106 is open to the atmosphere via the vent hole 306. This allows the sample solution in the flow path device 101 to be moved by the pressurizing or suctioning operation of the syringe pump 308. Instead of the syringe pump 308, a diaphragm pump or a blower may be used.

[0072] The line detection mechanism 315 comprises an objective lens 318, a half mirror 321, and a camera 316, and is connected to a light source 319 and a signal processing unit 317. The light source 319 is controlled by a light source control unit 320. A high-brightness red LED is used as an example of the light source 319.

[0073] The sample solution in this example was a PCR reaction mixture containing one or more types of DNA to be detected, a primer that specifically reacts with the target DNA, a thermostable enzyme, and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP). The 5' end of the forward primer was modified with biotin. The 3' end of the reverse primer was modified via a linker with a tag having a sequence complementary to the oligo sequence coated on the detection unit 203.

[0074] In this example, 30 μL of sample solution was introduced into the flow channel device 101, and detection was performed using the entire volume of the sample solution after PCR. The operation flow from sample introduction to target DNA detection can be modified from the operation flow of Example 1 shown in Figure 2 to match the flow channel structure in Figure 3.

[0075] The step in Figure 2(b) was achieved by drawing 30 μL of sample solution from the first channel 103 towards the connecting channel 107 using the suction action of the syringe pump 308. The step in Figure 2(c) was achieved by adjusting the pressure action of the syringe pump 308 so that the entire volume of sample solution is present in the first temperature region of the first channel 103.

[0076] The steps in Figures 2(f) and 2(g) were achieved by aspirating a predetermined amount using the syringe pump 308, stopping the aspiration operation when the entire sample solution was contained within the first temperature range, and waiting for a predetermined time (e.g., 2 seconds).

[0077] Similarly, the steps in Figure 2(h)(i) were achieved by pressurizing the syringe pump 308 by a predetermined amount, stopping the pressurization operation when the entire sample solution was contained in the second temperature region, and waiting for a predetermined time (e.g., 4 seconds). In other words, PCR was achieved by moving the sample solution back and forth between the first and second temperature regions 40 times.

[0078] The sample solution in the first channel 103 was initiated to move to the second channel 104 via the intermediate channel 108 by the dispensing operation of the syringe pump 308 (Figure 2(j)). Just before the sample solution reached the tip of the detection paper, the dispensing amount of the syringe pump 308 was adjusted so that the inflow rate of the sample solution into the detection paper 201 (i.e., the infiltration rate into the sample pad 204) did not exceed the inflow rate of the sample solution into the detection unit 203 (i.e., the infiltration rate from the conjugate pad 205 into the detection unit 203).

[0079] Immediately after the trailing end of the sample solution reached the leading end of the detection paper, the pressure application operation by the liquid delivery control mechanism 302 was stopped (Figure 2(k)). After that, a predetermined time (e.g., 2 minutes) was waited to confirm that the sample solution had sufficiently spread to the detection unit 203, and the detection line 207 of the detection paper was determined (Figure 2(l)). As a result, it became possible to amplify and detect the target DNA in the sample solution in about 10 minutes, and to determine the presence or absence of the target DNA.

[0080] The fluid delivery control mechanism 302 may be configured such that the syringe pump 308 is connected to the second pressure adjustment unit 106 and the vent hole 306 is connected to the first pressure adjustment unit 105. In that case, the discharge operation and the suction operation should be swapped in the operation flow described in this embodiment. Alternatively, the first temperature range may be the annealing / extension temperature range and the second temperature range may be the denaturation temperature range. In that case, the operation flow in Figure 2 should be configured by swapping the first and second temperature ranges.

[0081] Thus, according to the nucleic acid detection system and nucleic acid detection method of Example 2 of the first embodiment, it is possible to simplify the configuration while performing more appropriate liquid delivery control.

[0082] [Example 3] Example 3 modifies the stopping conditions shown in Figure 2(k) in Example 1 or 2. The following explanation may omit details common to both Example 1 and Example 2.

[0083] The rate at which the sample solution flows into the detection paper 201 is preferably always less than or equal to the rate at which the sample solution unfolds within the detection paper 201. The rate at which the sample solution unfolds within the detection paper 201 can be freely designed by the composition and material of the detection paper 201. For example, increasing the width of the detection paper 201 (i.e., the shorter dimension in Figure 4(a)) will increase the rate at which the sample solution unfolds. Alternatively, the rate at which the sample solution unfolds can be changed by using a material with a fast water absorption rate (such as a glass fiber absorbent pad) or by increasing the volume of the sample pad 204 or absorbent pad 206.

[0084] As described above, the deployment speed to the detection unit 203 is determined by the physical properties of the sample solution and the detection paper 201. Therefore, the inflow rate to the detection paper 201 should be set by referring to the deployment speed of the detection paper that has been designed in advance.

[0085] The timing for stopping the operation of the liquid delivery control mechanism 302 can be immediately after the trailing end of the sample solution reaches the leading end of the detection paper, as in Example 2. However, the liquid delivery operation may be continued after the trailing end of the sample solution has passed the leading end of the detection paper, as long as the amount of sample solution flowing into the detection paper 201 per unit time is always less than or equal to the amount of sample solution flowing out of the conjugate pad 205 to the detection unit 203 by capillary force.

[0086] Alternatively, even if the leading edge of the sample solution has reached the leading edge of the detection paper but the trailing edge of the sample solution has not yet reached the leading edge of the detection paper, some of the unreached portion may remain in the intermediate channel 108 if it is an amount that can be absorbed by the capillary force of the detection paper. In other words, the stopping condition in Figure 2(k) may be such that the portion of the sample solution that has not reached the detection paper 201 is an amount that can be absorbed by the detection paper 201 by the capillary force of the detection paper 201.

[0087] In this case, the amount of sample solution that may remain in the intermediate channel 108 is approximately 10% of the sample solution and depends on the water absorption rate of the detection paper 201, so it can be adjusted by the design of the detection paper 201. In this way, a sufficient amount of sample solution can reach the detection paper 201 while suppressing leakage of the sample solution due to unnecessary pressurization.

[0088] The amount that can be absorbed by the detection paper 201 can be determined by prior experiments. The nucleic acid amplification reactor 301 may also be equipped with a sensor to measure the amount of the sample solution that has not reached the detection paper 201. The specific type and placement of the sensor can be appropriately designed by those skilled in the art.

[0089] Thus, according to the nucleic acid detection system and nucleic acid detection method of Example 3 of the first embodiment, it is possible to simplify the configuration while performing more appropriate liquid delivery control.

[0090] [Example 4] Example 4 modifies the flow path structure in any of Examples 1 to 3. The following explanation may omit details common to any of Examples 1 to 3.

[0091] Figure 7 shows an example of the shape of the second channel 104 according to Embodiment 4. Figure 7 is a schematic diagram of the second channel 104 provided with side support protrusions 114 and an irradiation window 115. In Figure 7, multiple bottom support protrusions 113 are arranged, but only one is given a reference numeral as a representative. The side support protrusions 114, like the bottom support protrusions 113, contribute to the alignment of the detection paper 201 and to preventing misalignment. Furthermore, by providing the side support protrusions 114, the width of the second channel 104 can be designed to be wider than the width of the detection paper 201, thereby improving the operability when storing the detection paper 201.

[0092] Thus, the nucleic acid detection system and nucleic acid detection method according to Example 4 of the first embodiment can further improve operability.

[0093] <Second Embodiment>

[0094] The second embodiment of the present invention will be described below. The second embodiment realizes a three-step high-speed PCR that performs DNA denaturation, annealing, and extension in three temperature ranges. Parts common to the first embodiment may be omitted from the description.

[0095] Figure 8 is a schematic diagram of the portion including the first channel 103 when the channel device is installed in the device holding section of the nucleic acid amplification reaction apparatus. In this embodiment, the configuration of the channel device 101 and the detection paper 201 near the first channel 103 can be the same as those in Example 2 of the first embodiment.

[0096] The nucleic acid amplification reactor is a modified version of the nucleic acid amplification reactor 301 of Example 2 of the first embodiment, in which a third external heat source 312 is positioned in an intermediate region between the first external heat source 310 and the second external heat source 311.

[0097] The material and configuration of the third external heat source 312 can be the same as those of the first external heat source 310 and the second external heat source 311. The third external heat source 312 is also connected to the temperature controller 309. The surface temperature of each external heat source can be controlled so that the target temperature of the first temperature region is the denaturation temperature range (e.g., 92°C), the target temperature of the second temperature region is the annealing temperature range (e.g., 60°C), and the target temperature of the third temperature region related to the third external heat source 312 is the extension temperature range (e.g., 72°C). Note that the order of the temperature ranges does not have to be as shown in Figure 8.

[0098] Figure 9 shows the PCR portion of the operation flow in this embodiment. The operation flow other than the portion shown in Figure 9 can be the same as the flow shown in Figure 2. The operations from Figure 2(f) to (i) correspond to the operations from Figure 9(a) to (f).

[0099] Thus, according to the nucleic acid detection system and nucleic acid detection method of the second embodiment, a three-step high-speed PCR can be realized.

[0100] <Third Embodiment> A third embodiment of the present invention will be described below. The third embodiment utilizes a continuous transport type high-speed PCR. Parts common to the first or second embodiment may be omitted from the description.

[0101] Figure 10 is a schematic diagram of the portion including the first channel 103 when the channel device is installed in the device holding section of the nucleic acid amplification reactor. In this embodiment, the configuration of the channel device 101 and the detection paper 201 near the first channel 103 can be the same as those in Example 2 of the first embodiment.

[0102] In the nucleic acid amplification reactor, the arrangement of the first external heat source 310 and the second external heat source 311 is different compared to Example 2 of the first embodiment. As the sample solution moves through the first channel 103, it repeatedly passes alternately through the first temperature region and the second temperature region.

[0103] As shown in Figure 10, by positioning an external heat source, the sample solution can pass through different temperature ranges while the syringe pump continues to operate in one direction, enabling PCR to be achieved with simple fluid delivery control. [Explanation of Symbols]

[0104] 101... Fluid flow device 103...First channel 104...Second flow path 105...First pressure adjustment section 106...Second pressure adjustment section 107…Connection channel 108...Intermediate channel 109... Sample introduction channel 110... Sample inlet 111...filter 112...Filter storage section 113…Bottom support protrusion 114...Side support protrusion 115... Irradiation window 201...Detection paper 202... Backing sheet 203...Detection unit 204... Sample Pad 205...Conjugate pad 206… Absorbent pad 207...Detection line 301... Nucleic acid amplification reactor 302... Fluid delivery control mechanism 303...Device holding section 304...cover 305...Connection part 306...Ventilation holes 307... Piping tubes 308... Syringe pump 309... Temperature controller 310...First external heat source 311…Second external heat source 312…Third external heat source 314... Observation window 315... Line detection mechanism 316...Camera 317... Signal Processing Unit 318…Objective lens 319...Light source 320...Light source control unit 321... Half mirror

Claims

1. Flow channel structure, A pair of pressure adjustment units connected to both ends of the aforementioned flow path structure, A fluid supply control mechanism connected to the pair of pressure adjustment units, A nucleic acid detection system comprising, The aforementioned flow channel structure is The first channel and A second channel connected to the first channel, The detection paper placed in the second channel, Equipped with, The nucleic acid detection system is The liquid delivery control mechanism moves the sample solution within the first channel and carries out the nucleic acid amplification reaction, Following the nucleic acid amplification reaction, the liquid delivery control mechanism initiates the transfer of the sample solution to the second channel, After the sample solution reaches one end of the detection paper, the step of stopping the movement of the sample solution by the liquid delivery control mechanism when a predetermined stopping condition is met, Execute, The aforementioned termination conditions are: The portion of the sample solution that has not reached the detection paper is such that it can be absorbed by the detection paper due to its capillary action. or The detection paper is equipped with a detection unit that reacts with the object to be detected, and the tip of the sample solution reaches the detection unit so that the spread of the sample solution in the detection unit occurs solely by the capillary force of the detection unit. Nucleic acid detection system.

2. Multiple temperature regions are provided in the first channel. The nucleic acid detection system according to claim 1, wherein the nucleic acid amplification reaction is achieved by moving the sample solution to each of the plurality of temperature regions.

3. The nucleic acid detection system further includes a sample solution inlet, The sample solution inlet is provided within the first channel, or on the side opposite to the second channel relative to the first channel. The nucleic acid detection system, prior to the step of carrying out the nucleic acid amplification reaction, The steps include introducing the sample solution into the sample solution inlet, The liquid delivery control mechanism provides the step of moving the sample solution into the first channel, A nucleic acid detection system according to claim 1, which performs the following:

4. The nucleic acid detection system according to claim 1, wherein the stopping condition is that a predetermined time has elapsed after the sample solution has reached one end of the detection paper.

5. The nucleic acid detection system according to claim 1, wherein the first channel and the second channel are tubular.

6. A nucleic acid detection method using a nucleic acid detection system, The nucleic acid detection system is Flow channel structure, A pair of pressure adjustment units connected to both ends of the aforementioned flow path structure, A fluid supply control mechanism connected to the pair of pressure adjustment units, Equipped with, The aforementioned flow channel structure is The first channel and A second channel connected to the first channel, The detection paper placed in the second channel, Equipped with, The nucleic acid detection method described above is The steps include moving the sample solution within the first channel and carrying out a nucleic acid amplification reaction, The steps include: starting the transfer of the sample solution to the second channel after the nucleic acid amplification reaction; and stopping the transfer of the sample solution after it has reached one end of the detection paper, in accordance with the fulfillment of predetermined stopping conditions. Includes, The aforementioned termination conditions are: The portion of the sample solution that has not reached the detection paper is such that it can be absorbed by the detection paper due to its capillary action. or The detection paper is equipped with a detection unit that reacts with the object to be detected, and the tip of the sample solution reaches the detection unit so that the spread of the sample solution in the detection unit occurs solely by the capillary force of the detection unit. Nucleic acid detection methods.

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