Sample pretreatment system and sample pretreatment method

Abstract

To enable a sample pretreatment to be efficiently performed at low cost.SOLUTION: This disclosure provides a sample pretreatment system comprising: a test chip having a charging port for charging a sample solution, a first flow path, and a second flow path, and in which the charging port, the first flow path, and the second flow path are arranged in this order; a first temperature controller that adjusts the temperature of the first flow path; and a liquid feeding unit that moves the sample solution between the first flow path and the second flow path, the first temperature controller adjusting the temperature of the first flow path to a first temperature higher than that of the second flow path, in the first channel, nucleic acids in the sample solution being extracted, and in the second channel, the nucleic acids in the sample solution after the nucleic acids are extracted being dissociated.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a sample pretreatment system and a sample pretreatment method.

Background Art

[0002] As methods for amplifying nucleic acids, there are isothermal amplification methods such as the PCR (Polymerase Chain Reaction) method and the LAMP (Loop-Mediated Isothermal Amplification) method. The PCR method is a method for amplifying a target DNA by repeating three steps, namely denaturation of DNA (dissociation of double-stranded DNA into single-stranded DNA), annealing (binding of a complementary primer to single-stranded DNA), and extension (double-stranded DNA replication by complementary strand synthesis), 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, the reactions corresponding to these cycles are performed at a constant temperature of approximately 35 to 65°C.

[0003] In recent years, rapid genetic testing systems using a nucleic acid amplification and detection device and a disposable dedicated device have been proposed. For example, as a dedicated device for nucleic acid amplification reactions, a microchannel formed on a substrate (microchannel chip) has been proposed. By moving a sample in a microchannel chip placed on heat blocks set to different temperature zones, the temperature changes required for PCR can be achieved simply by maintaining each heat block at a predetermined preset temperature.

[0004] Infectious diseases caused by viruses such as influenza virus and novel coronavirus require rapid testing. Therefore, there is high demand for performing high-speed nucleic acid amplification by methods such as PCR and LAMP and conducting testing at the site where the sample is collected, such as in clinics and pharmacies.

[0005] Regarding rapid testing, for example, Patent Document 1 discloses a microfluidic device in which a reaction reagent is supported on the inner wall of a channel, allowing the reaction reagent to be supported at a specific location without introducing impurities. Patent Document 2 discloses a nucleic acid extraction device that has a structure to prevent the nucleic acid sample from leaking out and can contain various reagents for long-term storage. Patent Document 3 discloses a configuration in which reagents and sample solutions are introduced and mixed from each port, and then quantitatively dispensed. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-141685 [Patent Document 2] Patent No. 4489088 [Patent Document 3] Japanese Patent Publication No. 2008-151771 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] One of the challenges in nucleic acid-based testing is the need for full automation and cost reduction. While nucleic acid amplification and detection mechanisms and dedicated devices come in various forms, a typical testing flow consists of the steps of collecting a sample, preparing a solution (sample) from which the nucleic acid of the pathogen has been extracted, introducing that solution into a dedicated device, amplifying the target gene in a nucleic acid amplification reactor, and detecting the amplified product. The conventional testing process had challenges such as being time-consuming and labor-intensive because it involved a manual step of preparing the sample, which depended on the skill of the person performing the test.

[0008] On the other hand, devices that automate sample preparation often contain numerous components in the cartridge, such as valves for moving the sample solution, a chamber and a liquid delivery mechanism with complex pathways, and a mechanism for mixing with reagents. This complexity leads to high costs and large cartridges and devices. In light of these circumstances, this disclosure proposes a technology that enables efficient and low-cost sample pretreatment. [Means for solving the problem]

[0009] To solve the above problems, this disclosure proposes a sample pretreatment system, as an example, comprising a test chip having an inlet for introducing a sample solution, a first channel, and a second channel, with the inlet, the first channel, and the second channel arranged in this order; a first temperature controller for controlling the temperature of the first channel; and a liquid transfer unit for moving the sample solution between the first channel and the second channel, wherein the first temperature controller controls the temperature of the first channel to a first temperature higher than that of the second channel, nucleic acids in the sample solution are extracted in the first channel, and nucleic acids in the sample solution after extraction are dissociated in the second channel.

[0010] Further features relating to this disclosure will become apparent from the description herein and the accompanying drawings. Furthermore, aspects of this disclosure are achieved and realized by elements and various combinations of elements and the modes of the claims described herein and the accompanying claims. The descriptions herein are typical examples only and do not limit in any way the claims or applications of this disclosure. [Effects of the Invention]

[0011] The technology described herein enables efficient and low-cost sample preparation. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows a schematic configuration example of the sample measurement system 100 according to the first embodiment. [Figure 2] This is a flowchart illustrating the sample measurement process performed by the sample measurement system 100 according to the first embodiment. [Figure 3] This figure shows a schematic configuration example of the sample measurement system 300 according to the second embodiment. [Figure 4] This is a flowchart illustrating the sample measurement process performed by the sample measurement system 300 according to the second embodiment. [Figure 5] This figure shows an example configuration of the sample measurement system 500 according to the third embodiment. [Figure 6] This figure shows how a sample solution containing air bubbles is moved within the test chip 501 according to the third embodiment. [Figure 7] This figure shows a schematic configuration example of a sample measurement system 500 having a different low-temperature flow path configuration in the third embodiment. [Figure 8] This figure shows an example configuration of the sample measurement system 800 according to the fourth embodiment. [Figure 9] This is a flowchart illustrating the sample measurement process performed by the sample measurement system 800 according to the fourth embodiment. [Modes for carrying out the invention]

[0013] This disclosure discloses sample pretreatment techniques, specifically techniques for extracting, amplifying, and detecting nucleic acids from samples such as cells and viruses. Each embodiment proposes performing nucleic acid dissociation, extension, and amplification by moving the sample solution (sample solution) after reagent mixing back and forth between a high-temperature controlled channel (high-temperature channel) and a medium-temperature controlled channel (medium-temperature channel).

[0014] Hereinafter, embodiments of the present disclosure and respective examples will be described with reference to the accompanying drawings. In the accompanying drawings, functionally identical elements may be denoted by the same reference numerals. The accompanying drawings show specific embodiments and implementation examples in accordance with the principles of the present disclosure, but these are provided for the understanding of the present disclosure and are by no means intended to be used for a restrictive interpretation of the present disclosure.

[0015] In the embodiments of the present disclosure, for convenience, the description is divided into a plurality of sections or embodiments when necessary, but unless otherwise explicitly stated, these are not independent of each other, and one is in a relationship of being a modification, detailed description or supplementary explanation of part or all of the other. In addition, in the following embodiments, when the number of elements (including the number, numerical value, amount, range, etc.) is mentioned, it is not limited to a specific number except where explicitly stated or where it is clearly limited to a specific number in principle, and may be more than or less than the specific number.

[0016] Furthermore, it goes without saying that in the following embodiments, the constituent elements (including element steps and the like) are not necessarily essential unless otherwise explicitly stated or where they are clearly considered essential in principle. Similarly, in the following embodiments, when reference is made to the shape, positional relationship and the like of constituent elements, it is intended to include those substantially approximated or similar to the shape and the like unless otherwise explicitly stated or where it is clearly considered otherwise in principle. This also applies to the above numerical values and ranges.

[0017] It should be noted that the drawings show specific examples in accordance with the principles of the present invention, but these are provided for the understanding of the present invention and are not intended to be used for a restrictive interpretation of the technology of the present disclosure. Ribonucleic acid (RNA) is exemplified as a biological substance to be analyzed, but it is not limited to RNA, and may be a nucleic acid such as deoxyribonucleic acid (DNA). In addition, the chip used for testing (testing chip) may also be referred to as a cartridge, a device, or the like in other cases, which are synonymous.

[0018] (1) First Embodiment The first embodiment discloses a basic sample measurement system 100. The sample measurement system 100 is a system that can handle both real-time measurement and endpoint measurement. The sample measurement system 100 according to the first embodiment will be described below with reference to Figures 1 and 2.

[0019] <Example configuration of sample measurement system 100> Figure 1 shows a schematic configuration example of a sample measurement system 100 according to the first embodiment. The sample measurement system 100 includes a test chip 101, temperature controllers 107 to 109, a temperature control driver 110, a tube 111, and a pump 112. The sample measurement system 100 is configured as a system that assumes an operator (user) manually operates the temperature control driver 110 and the pump 112. However, as in the sample measurement systems according to the second and fourth embodiments described later, a controller (computer) that controls the operation of the temperature control driver 110 and the pump 112 may be provided when automatic temperature control and pump control are performed. In addition, the sample solution may be delivered using a flux control device that delivers liquid by creating an airflow, rather than the pump 112 as a pressure regulating device.

[0020] The test tip 101 includes a sample inlet 102, a medium temperature channel 103, a high temperature channel 104, a low temperature channel 105, and a pressure adjustment port 106.

[0021] The medium-temperature channel 103, the high-temperature channel 104, and the low-temperature channel 105 are in contact with temperature controllers 107 to 109, respectively, and are temperature-controlled to medium, high, and low temperatures. The medium temperature range is suitable for DNA annealing and extension, for example, in the range of 55-65°C. The high temperature range is suitable for DNA dissociation, for example, in the range of 90-100°C. The low temperature range is suitable for RNA reverse transcription, for example, in the range of 40-50°C. Each of the temperature controllers 107 to 109 is controlled by a temperature control driver 110.

[0022] The sample solution containing the sample to be detected is introduced into the sample inlet 102. The sample solution is drawn into the test tip 101 using a pump 112 (suction) and moved to the high-temperature channel 104. The medium-temperature channel 103 simply passes through at this stage before reaching the high-temperature channel 104. The sample solution is heat-treated in the high-temperature channel 104. The purpose of this heat treatment is, for example, to extract nucleic acids from cells. By heating, the cell walls of the sample cells are destroyed, and the nucleic acids inside can be extracted from the cells. Alternatively, the process can be used to extract nucleic acids not only from cells but also from viruses.

[0023] Subsequently, the sample solution is moved to the low-temperature channel 105. Mixing of the sample solution and reagents takes place at this location. Specifically, mixing can be performed by allowing the sample solution to remain in the low-temperature channel 105 for a predetermined time. Alternatively, a mechanism to vibrate the low-temperature channel 105 may be provided to promote mixing. The reagents may be placed in the low-temperature channel 105 in advance, or they may be introduced from another location. One method for placing the reagents in the channel in advance is to freeze-dry the reagents. Alternatively, a reagent holder connected to the channel may be provided, and the reagents may be pushed from the holder into the channel to mix the sample solution and reagents. Furthermore, if the target of measurement is RNA, reverse transcription to cDNA is performed. If reverse transcription is not performed, this low-temperature region may be at room temperature. In this case, the temperature controller 109 for temperature control of the low-temperature channel 105 is unnecessary. Alternatively, temperature control of the low-temperature channel 105 may not be performed at all.

[0024] The sample solution mixed with the reagent (hereinafter sometimes referred to as the reagent-mixed sample solution) is moved again to the high-temperature channel 104 using pump 112 (pressure extrusion). In the high-temperature channel 104, the reagent-mixed sample solution is heated to perform a hot start (enzyme activation) of the enzymes contained in the reagent. If the reagent contains an enzyme for reverse transcription, that enzyme is also deactivated. At this temperature, the dissociation reaction of double-stranded DNA takes place.

[0025] Subsequently, the reagent-mixed sample solution is moved to the medium-temperature channel 103 using pump 112 (pressure extrusion). Here, annealing and extension reactions of the dissociated single-stranded DNA take place. Specifically, the single-stranded DNA to be detected binds to the primer, is extended by the enzyme, and DNA with a double-stranded amplification region is generated. Furthermore, this solution moves back and forth between the high-temperature channel 104 and the medium-temperature channel 103, repeating the DNA dissociation, annealing, and extension reactions to carry out the PCR amplification reaction. The amplification reaction is repeated for approximately 30 to 50 cycles.

[0026] The PCR-amplified DNA is detected in this manner. For real-time detection (detection during PCR amplification), a method using a TaqMan probe to detect the fluorescent dye produced during the extension reaction is possible. Alternatively, the PCR amplification product may be detected at the endpoint (detection after PCR amplification is complete). Endpoint detection methods include fluorescence measurement, nucleic acid chromatography, and hybridization. Fluorescence measurement can be performed, for example, by a detector (not shown) located on the medium-temperature channel 103 or the high-temperature channel 104.

[0027] <Sample Measurement Processing> Figure 2 is a flowchart illustrating the sample measurement process performed by the sample measurement system 100 according to the first embodiment. Each step of the sample measurement process is described as being performed by an operator manually, but the basic operation is the same even when performed automatically by a controller (not shown), only the controller becomes the primary operator.

[0028] (i)S201 The operator introduces the pre-mixed reagent sample solution into the test tip 101 through the sample inlet 102. For example, the operator can introduce the pre-mixed reagent sample solution into the test tip 101 by placing the pre-mixed reagent sample solution in an input container, connecting the solution outlet of the container to the sample inlet 102, and performing a suction operation with the pump 112.

[0029] (ii) S202 After adding the sample solution before reagent mixing, the operator continues to operate the pump 112 to move the solution to the high-temperature channel 104 and heat it.

[0030] (iii) S203 After heating the sample solution before reagent mixing, the operator operates the pump 112 (suction) to move the solution to the low-temperature region 105 and mix it with the reagent. The mixing operation is as described above.

[0031] (iv) S204 The operator operates the pump 112 (pressure extrusion) to move the reagent-mixed sample solution to the high-temperature region (high-temperature channel 104). The operator then repeatedly uses the pump 112 to aspirate and pressurize the sample solution, moving it back and forth between the high-temperature channel 104 and the medium-temperature channel 103 to perform PCR amplification. The amplification product is detected by a detector (not shown) located near (above or below) the medium-temperature channel 103 or the high-temperature channel 104, as described above (supporting both real-time and endpoint measurement).

[0032] <Effects of the first embodiment, etc.> In this first embodiment, the high-temperature channel 104 and the temperature controller 108 are shared between the step of extracting nucleic acids from cells or viruses and the step of heating the solution by PCR amplification. This makes it possible to miniaturize and reduce the cost of the test chip 101 and the sample measurement system 100. Furthermore, according to the first embodiment, nucleic acid extraction and mixing of reagents and sample solutions can be performed within the test chip 101, thereby reducing manual labor.

[0033] The structure of the flow path is not limited to this. A flow path configuration that repeatedly moves between high-temperature and medium-temperature regions in a meandering flow path to perform PCR may be used (not a configuration that moves back and forth, but a test chip configured with multiple high-temperature and medium-temperature flow paths arranged in a line). Alternatively, a configuration in which the solution is placed in a single chamber and the temperature of the chamber is raised and lowered to perform PCR may be used. Furthermore, although the first embodiment shows an example in which the sample solution is drawn in by the pump 112, it is not limited to this. Other liquid delivery means may be used, such as pressurizing the solution with the pump 112 to push it in.

[0034] (2) Second embodiment The second embodiment discloses a sample measurement system 300 equipped with a detection chamber (detection chamber) for detecting PCR amplification products at an endpoint on a test chip. The sample measurement system 300 is an endpoint measurement system. The sample measurement system 300 according to the second embodiment will be described below with reference to Figures 3 and 4.

[0035] <Sample Measurement System> Figure 3 shows a schematic configuration example of a sample measurement system 300 according to the second embodiment. The sample measurement system 300 comprises an inspection chip 301, temperature controllers 107 to 109, a temperature control driver 110, a tube 111, a pump 112, a detector 305, and a controller 306.

[0036] The test chip 301 includes a sample inlet 102, a medium temperature channel 103, a high temperature channel 104, a low temperature channel 105, a pressure adjustment port 106, a branch section 302, a detection chamber 303, and a pressure adjustment section 304.

[0037] Similar to the first embodiment, in the second embodiment, the sample solution (before reagent mixing) is introduced into the sample inlet 102 and processed while moving through high-temperature, low-temperature, and medium-temperature channels. In the PCR amplification reaction, the nucleic acid is dissociated in the high-temperature channel (high-temperature channel 104), and then annealing and extension reactions are performed in the medium-temperature channel (medium-temperature channel 103). Therefore, when detecting the amplification product at the endpoint, the solution is located in the medium-temperature channel 103. In the sample measurement system 300 according to the second embodiment, the solution after the PCR amplification reaction is moved to the detection chamber 303. This movement is carried out by pushing out air with the pump 112.

[0038] A branching section 302 is provided in the flow path from the sample inlet to the medium-temperature flow path 103, and the branched flow path is connected to the detection chamber 303. The PCR-amplified sample solution is moved to the detection chamber 303 through the branching section 302. The branching section 302 includes a valve mechanism and is configured so that the movement of the sample solution can be controlled by opening and closing the valve mechanism. Alternatively, the direction of movement may be controlled by the width of the flow path, or by adjusting the pressure of the pressure adjustment section 304. The pressure adjustment section 304 may be a simple opening for releasing air, or it may be a pump that applies a constant pressure.

[0039] The PCR-amplified sample solution is moved to the detection chamber 303, where the presence and concentration of the amplification product are measured by the detector 305. Specifically, for example, the amount of amplification product is measured (detected) by measuring the fluorescence intensity using the detector 305. Alternatively, nucleic acid chromatography may be used, and the amplification product may be measured using an image sensor (CCD or CMOS sensor). By measuring the sample solution after the amplification reaction in this way, it is possible to determine the presence and concentration of the amplification product, that is, whether the sample contained the nucleic acid to be measured.

[0040] The controller 306 controls temperature, sample solution movement, and detection of amplification product quantity. Specifically, the controller 306 controls the temperature control driver 110 to adjust the temperature controllers 107 to 109 to a predetermined temperature. The controller 306 also controls the pump 112 to control the solution movement, its speed, and timing. Furthermore, the controller 306 controls the detector 305 to detect amplification products and perform the final determination.

[0041] <Sample Measurement Processing> Figure 4 is a flowchart illustrating the sample measurement process performed by the sample measurement system 300 according to the second embodiment. In the second embodiment, the controller 306 is basically the main operator of the sample measurement process, but as in the first embodiment, an operator may perform the sample measurement manually.

[0042] (i)S401 When the operator places the sample solution (before reagent mixing) in a container into the sample inlet 102 and inputs a measurement start command (for example, by pressing the measurement start button), the controller 306 controls the pump 112 to introduce the sample solution into the test chip 101.

[0043] (ii) S402 The controller 306 continues to control the pump 112, moving the introduced sample solution to the high-temperature region (high-temperature channel 104), where the sample solution is heated. During this process, nucleic acids are extracted from cells and viruses in the sample solution. The heating time varies depending on the sample type, but is typically between 30 seconds and 5 minutes.

[0044] (iii) S403 The controller 306 controls the pump 112 to move the sample solution after nucleic acid extraction to a low-temperature region (low-temperature channel 105), where it performs processing such as mixing the sample solution with reagents. The mixing operation is as described in the first embodiment.

[0045] (iv) S404 The controller 306 controls the pump 112 to move the reagent-mixed sample solution back into the high-temperature region (high-temperature channel 104) and perform a hot start (activate) of the enzymes contained in the reagents.

[0046] (v)S405 After activating the enzymes contained in the reagent, the controller 306 controls the pump 112 again, causing the reagent-mixed sample solution to move back and forth between the high-temperature region (high-temperature channel 104) and the medium-temperature region (medium-temperature channel 103). This back-and-forth movement repeats the DNA dissociation, annealing, and extension reactions, thereby carrying out the PCR amplification reaction.

[0047] (vi)S406 The controller 306 controls the pump 112 to move the PCR-amplified solution to the detection area (detection chamber 303). The controller 306 then controls the detector 305 to detect the amplified product.

[0048] <Effects of the second embodiment, etc.> In this way, by performing the nucleic acid extraction process and the nucleic acid dissociation process in PCR amplification in the same flow path, it is possible to simplify and miniaturize the test chip. Furthermore, since high-temperature processing is performed in the same location within the chip, the temperature controllers 107 to 109 and the necessary heat blocks and control drivers can be shared, contributing to the miniaturization and cost reduction of the device. Moreover, as shown in this embodiment, since the entire process from sample solution pretreatment to detection can be performed automatically on a single chip, a system that allows for measurement with simple operation can be provided.

[0049] (3) Third Embodiment The third embodiment discloses a sample measurement system 500 in which a test chip is equipped with a mechanism to remove bubbles generated when the sample solution is heated. Similar to the sample measurement system 100 according to the first embodiment, the sample measurement system 500 is a system that can handle both real-time measurement and endpoint measurement. The sample measurement system 500 according to the third embodiment will be described below with reference to Figures 5 to 7.

[0050] <Example configuration of sample measurement system 500> Figure 5 shows an example configuration of a sample measurement system 500 according to the third embodiment. The sample measurement system 500 includes a test chip 501, temperature controllers 107 to 109, a temperature control driver 110, a tube 111, and a pump 112. Similar to the first embodiment, the sample measurement system 500 is configured as a system that assumes an operator (user) manually operates the temperature control driver 110 and the pump 112. However, as in the sample measurement systems of the second and fourth embodiments, if automatic temperature control and pump control are to be performed, a controller (computer) that controls the operation of the temperature control driver 110 and the pump 112 may be provided. The test tip 501 includes a sample inlet 102, a medium temperature channel 502, a high temperature channel 503, a low temperature channel 504, and a pressure adjustment port 106. The transfer of the sample solution, temperature control, and detection of the amplified product are the same as in the first embodiment.

[0051] <Bubble formation and countermeasures> One of the challenges in measurement systems using microfluidic channels is the generation of bubbles. In the first and second embodiments described above, a solution is placed in a high-temperature channel to extract nucleic acids from cells, viruses, etc. In this process, since the sample solution is placed in the microfluidic channel (high-temperature channel 104) for a relatively long time, bubbles are generated. If bubbles are generated and expand, causing the sample solution to move out of the temperature-controlled channel range, it becomes impossible to properly heat the sample solution, and the generation of many bubbles leads to unstable liquid delivery.

[0052] Therefore, in the third embodiment, the effect of these bubbles is suppressed by devising the shape of the microchannel in the inspection chip 501. Specifically, a section (channel 505) where the cross-sectional area of ​​the channel changes is provided between the high-temperature channel 503 and the low-temperature channel 504. As shown in Figure 5, the cross-sectional area of ​​the low-temperature channel 504 is configured to be larger than the cross-sectional area of ​​the high-temperature channel 503. The cross-sectional area of ​​channel 505 may be the same as the cross-sectional area of ​​the high-temperature channel 503, and the cross-sectional area may be configured to increase from the low-temperature channel 504, or the cross-sectional area of ​​channel 505 may be the same as the cross-sectional area of ​​the high-temperature channel 503 at the connection point with the high-temperature channel 503, and the cross-sectional area may be the same as the cross-sectional area of ​​the low-temperature channel 504 at the connection point with the low-temperature channel 504, so that the cross-sectional area gradually changes (increases).

[0053] Figure 6 shows how a sample solution 601 with bubbles formed in it is moved within the test chip 501 according to the third embodiment. Since bubbles are mainly formed when heated, when the sample solution 601 is heated in the high-temperature channel 503, bubbles 602a, 602b, 602c, ... are formed (Figure 6A). This sample solution 601 is moved toward the low-temperature channel 504. Specifically, the sample solution 601 is moved by suction using the pump 112, and the liquid surface 603 moves toward the low-temperature channel 504 side (Figure 6B). As the sample solution moves, the bubbles 602 also move. As the movement continues, the bubbles 602 remain near the part 505 where the channel cross-sectional area increases (near the entrance to the low-temperature channel 504) (Figure 6C). This is because the bubbles do not cover the entire channel (they only cover the inside of the channel), and the sample solution moves between the bubbles that adhere to the inside and the side wall of the channel. Furthermore, because the flow velocity is slower on the inside and faster on the outside of the curved channel, bubbles accumulate on the inside while the sample solution flows on the outside. As movement continues, the bubbles 602 generated during heating move behind the liquid surface 604 of the sample solution 601. In other words, the bubbles 602 can be removed from the sample solution 601 (Figure 6D).

[0054] In the example configuration of the inspection chip 501 shown in Figures 5 and 6, the angle θ between the central axis 5051 of the flow path 505 and the central axis 5041 of the low-temperature flow path 504 is approximately 90 degrees. However, in Figure 7, which shows a sample measurement system 500 with a different low-temperature flow path configuration, the angle θ between the central axis 5051 and the central axis 5041 is less than 90 degrees. Furthermore, the inventors' verification revealed that this angle θ can be, for example, 30 degrees or more.

[0055] <Effects of the third embodiment> As described above, in the third implementation system of the test chip 501, by changing the cross-sectional area of ​​the high-temperature channel 503 and the low-temperature channel 504, it is possible to retain bubbles near the point of cross-sectional area change, and bubbles generated during solution heating can be removed. Therefore, subsequent liquid delivery can be carried out stably. In particular, when heating a solution for a long time for purposes such as nucleic acid extraction, many bubbles are generated. When performing PCR amplification by moving the solution back and forth between channels, as in the technology of this disclosure, heat transfer from the channel of the chip to the solution is important. Increasing the surface area is effective in improving the efficiency of heat transfer, so it is good to narrow the channel and increase the contact area between the solution and the channel. In such a case, the sample solution can be heated efficiently, but bubbles are more likely to be generated. During PCR amplification, nucleic acid dissociation occurs at high speed, taking only a few seconds to more than ten seconds. On the other hand, nucleic acid extraction takes a longer time, so bubbles are particularly likely to be generated when performing nucleic acid extraction when sharing a channel. In such cases, by broadly changing and bending the cross-sectional area of ​​the channel as in this embodiment, bubbles can be removed, and stable liquid delivery becomes possible.

[0056] (4) Fourth Embodiment The fourth embodiment discloses a sample measurement system 800 equipped with a medium- and low-temperature channel 805 that combines a medium-temperature channel and a low-temperature channel. The sample measurement system 800 will be described below with reference to Figures 8 and 9.

[0057] <Example configuration of sample measurement system 800> Figure 8 shows an example configuration of a sample measurement system 800 according to the fourth embodiment. The sample measurement system 800 comprises an inspection chip 801, temperature controllers 808 and 809, a temperature control driver 812, a tube 810, a pump 811, a detector 813, and a controller 814.

[0058] The test chip 801 includes a sample inlet 802, a high-temperature channel 804, a medium- and low-temperature channel 805, pressure adjustment ports 803 and 807, a branching section 815, and a detection chamber 806.

[0059] Compared to the configurations of the test chips according to the first to third embodiments, the test chip 801 has a configuration in which the low-temperature and medium-temperature channels are shared, contributing to lower costs and miniaturization. The high-temperature channel 804 of the test chip 801 is in contact with the temperature controller 808, and its temperature is controlled by the controller 814 and temperature control driver 812. The medium- and low-temperature channel 805 is in contact with the temperature controller 809, and its temperature is controlled by the controller 814 and temperature control driver 812.

[0060] When a sample solution (after PCR amplification) is present in the detection chamber 806, the detector 813 detects the amplification product in the sample solution. The pump 811 is connected to the pressure adjustment port 807 via a tube 810. The pump 811 controls the pressure or flow rate, thereby controlling the movement of the sample solution within the test chip 801. The temperature control driver 812, detector 813, and pump 811 are controlled by the controller 814.

[0061] During PCR amplification, the sample solution is moved back and forth between the high-temperature channel 804 and the medium-to-low temperature channel 805, raising concerns that the amplification product may leak into the pump 811 or the air inside the device. Therefore, for example, by providing a pressure adjustment port 803 with a filter, and ensuring that the air movement after sample introduction passes through the filter, it is possible to prevent the sample solution from leaving the tip.

[0062] The reagents are kept, for example, in the medium-to-low temperature channel 805. The controller 814 controls the temperature control driver 812 to heat the sample solution introduced from the sample inlet 802 in the high-temperature channel 804. Next, the controller 814 controls the pump 811 to move the heated sample solution to the medium-to-low temperature channel 805 and mix it with the reagents. At this time, the medium-to-low temperature channel 805 is controlled by the controller 814 and the temperature control driver 812 to a temperature suitable for reagent mixing (low temperature).

[0063] <Sample Measurement Processing> Figure 9 is a flowchart illustrating the sample measurement process performed by the sample measurement system 800 according to the fourth embodiment. In the fourth embodiment, the controller 814 is basically the main operator of the sample measurement process, but as in the first embodiment, an operator may perform the sample measurement manually.

[0064] (i)S901 When the operator places the sample solution (before reagent mixing) in a container into the sample inlet 702 and inputs a measurement start command (for example, by pressing the measurement start button), the controller 714 controls the pump 811 to introduce the sample solution into the test chip 801.

[0065] (ii) S902 The controller 306 continues to control the pump 811, moving the introduced sample solution to the high-temperature region (high-temperature channel 804), where the sample solution is heated. During this process, nucleic acids are extracted from cells and viruses in the sample solution. The heating time varies depending on the sample type, but is typically between 30 seconds and 5 minutes.

[0066] (iii) S903 The controller 814 controls the pump 811 to move the sample solution after nucleic acid extraction to the medium-to-low temperature channel 805, where it performs processing such as mixing the sample solution with reagents. When mixing the sample solution with reagents, the controller 814 controls the medium-to-low temperature channel 805 to a low temperature suitable for mixing with reagents. The mixing operation is as described in the first embodiment.

[0067] (iv)S904 The controller 814 controls the pump 811 to move the sample solution back into the high-temperature channel 804 to perform nucleic acid dissociation.

[0068] (v)S905 While nucleic acid dissociation is occurring in the high-temperature channel 804, the controller 814 controls the temperature controller 809, which is in contact with the medium- and low-temperature channel 805, to adjust (heat) the temperature to a medium temperature (approximately 55-65°C) suitable for the nucleic acid extension reaction.

[0069] (vi)S906 The controller 814 controls the pump 811 to move the sample solution after nucleic acid dissociation to the medium-to-low temperature channel 805, where it performs annealing and extension reactions.

[0070] (vii)S907 The controller 814 controls the pump 811 to move the sample solution back and forth between the high-temperature channel 804 and the medium-to-low temperature channel 805, thereby performing PCR amplification.

[0071] (viii)S908 Since the PCR cycle ends at a moderate temperature, the controller 814 then controls the pump 811 to move the PCR-amplified sample solution to the detection chamber 806. The controller 814 then controls the detector 813 to detect the presence and concentration of the amplification product.

[0072] <Effects of the fourth embodiment> As described above, according to the fourth embodiment, by sharing the high-temperature channel for nucleic acid extraction by heating and for the nucleic acid dissociation reaction, the chip size can be made smaller, simpler, and the cost reduced. Furthermore, by sharing the medium-temperature channel and the low-temperature channel, and switching between the medium and low temperatures on the shared channel for temperature control, the temperature controller can be shared. In addition, the channels and temperature controllers for the low-temperature zone where reagents are mixed and the medium-temperature zone where annealing and extension reactions are carried out are shared. This makes it possible to further miniaturize the channels and the device, and reduce costs.

[0073] (5) Summary (i) In the sample measurement system (sample pretreatment system) according to the first to fourth embodiments, the sample solution (sample solution) introduced from the input port (102, etc.) of the test chip (101, etc.) is moved to a high-temperature channel (104, etc.: first channel), where the sample solution is heated to a high temperature to extract nucleic acids. The sample solution from which nucleic acids have been extracted is then moved to a low-temperature channel (105, etc.: second channel), where the sample solution and reagents are mixed. After the reagents are mixed, the sample solution is moved again to the high-temperature channel, where the nucleic acids in the sample solution are dissociated. In this way, the sample solution is moved between the high-temperature channel and the low-temperature channel until nucleic acid dissociation is performed. This allows the channels to be shared in each process, making it possible to pretreatment the sample with a simple configuration (miniaturization) and to provide an inexpensive sample measurement system. The low-temperature channel does not need to be heated by a temperature controller and may be kept at room temperature, or a temperature controller that heats to a predetermined low temperature may be brought into contact with the low-temperature channel. Reverse transcription of nucleic acids can be performed in the low-temperature channel.

[0074] Here, the reagents are pre-held in the low-temperature channel / medium-low-temperature channel, and after the sample solution is heated to a high temperature in the high-temperature channel, mixing of the sample solution and reagents takes place in the low-temperature channel / medium-low-temperature channel (when the sample solution is first introduced into the low-temperature channel / medium-low-temperature channel). This allows for easy mixing of the reagents.

[0075] (ii) In the first and second embodiments, the test chip (101 or 301) comprises a medium-temperature channel 103 (third channel), a high-temperature channel 104 (first channel), and a low-temperature channel 105 (second channel). In the test chip, the input port, the third channel, the first channel, and the second channel are arranged in this order. A temperature controller 107 is provided to control the temperature of the medium-temperature channel 103 (in the range of 55 to 65°C), a temperature controller 108 is provided to control the temperature of the high-temperature channel 104 (in the range of 90 to 100°C), and a temperature controller 109 is provided to control the temperature of the low-temperature channel 105. In this configuration, the pump 112 (liquid delivery unit) moves the sample solution in which the nucleic acid has been dissociated from the high-temperature channel 104 to the medium-temperature channel 103, where the nucleic acid extension reaction takes place. Here, each temperature controller 107 to 109 adjusts the temperature of each channel so that the temperature of the high-temperature channel 104 > the temperature of the medium-temperature channel 103 > the temperature of the low-temperature channel 105. Then, the pump 112 (liquid delivery unit) moves the sample solution back and forth between the high-temperature channel and the medium-temperature channel, thereby amplifying the nucleic acids contained in the sample solution (amplification products are generated).

[0076] Temperature control by a temperature controller and liquid delivery by a pump can be controlled by a controller. This makes it possible to automate the reagent measurement process.

[0077] (iii) In the third embodiment, the cross-sectional area of ​​the low-temperature channel 504 is made larger than that of the channel 505 (connecting channel) that connects the high-temperature channel 503 and the low-temperature channel 504. The channels are configured such that the angle between the central axis of the channel 505 and the central axis of the low-temperature channel 504 is 30 degrees or more. By doing so, bubbles generated by heating the sample solution in the high-temperature channel 503 can be eliminated (removed) near the entrance of the low-temperature channel 504.

[0078] (iv) In the fourth embodiment, a medium- and low-temperature channel 805 is provided, and the medium-temperature channel and the low-temperature channel are shared in a single channel. For example, the temperature controller 809 that controls the temperature of the medium- and low-temperature channel 805 controls the medium- and low-temperature channel 805 to a temperature suitable for reagent mixing (low temperature) when mixing reagents and sample solutions, and controls the medium- and low-temperature channel 805 to a temperature suitable for nucleic acid extension (medium temperature) when performing nucleic acid extension reactions. By sharing the low-temperature channel and the medium-temperature channel in this way, the device configuration can be simplified (miniaturized), and an inexpensive sample measurement system can be provided. [Explanation of Symbols]

[0079] 100, 300, 500, 800 Sample Measurement System 101, 301, 501, 801 Test chips 102, 802 Sample input ports 103, 502 Medium temperature flow path 104, 503, 804 High temperature flow path 105, 504 Low-temperature channel 106, 803, 807 Pressure regulating ports 107, 108, 109, 808, 809 Temperature controller 110, 812 Temperature control driver 111, 810 tube 112, 811 pumps 302, 815 Branch 303, 806 Detection Chamber 304 Pressure Regulating Section 305, 813 detectors 306, 814 controllers 601 Sample Solution 602a, 602b, 602c bubbles 603, 604 liquid level 805 Medium and low temperature flow path

Claims

1. A test chip having an inlet for introducing a sample solution, a first channel, and a second channel, wherein the inlet, the first channel, and the second channel are arranged in this order, A first temperature controller for controlling the temperature of the first flow path, The system comprises a liquid delivery unit that moves the sample solution between the first channel and the second channel, The first temperature controller adjusts the temperature of the first flow path to a first temperature which is higher than that of the second flow path. In the first channel, nucleic acids in the sample solution are extracted, and the nucleic acids in the sample solution after extraction dissociate. Furthermore, the system includes a second temperature controller for controlling the temperature of the second flow path. Furthermore, the test chip has a third channel connected to the first channel and a third temperature controller for controlling the temperature of the third channel. In the aforementioned test chip, the input port, the third channel, the first channel, and the second channel are arranged in this order. The first temperature controller sets the temperature of the first flow path to a higher temperature than the temperature at which the third temperature controller sets the temperature of the third flow path. The second temperature controller sets the temperature of the second channel to a lower temperature than the temperature at which the third temperature controller sets the temperature of the third channel. The cross-sectional area of ​​the second channel is larger than the cross-sectional area of ​​the connecting channel that connects the first channel and the second channel. The angle between the central axis of the connecting channel and the central axis of the second channel is 30 degrees or more. Sample pretreatment system.

2. In claim 1, The second channel holds the reagent, A sample pretreatment system in which, when the sample solution is first introduced into the second channel, mixing of the sample solution and the reagent takes place in the second channel.

3. In claim 2, The sample solution contains the nucleic acid, A sample pretreatment system in which the reverse transcription of the nucleic acid is performed in the second channel.

4. In claim 3, The liquid delivery unit moves the sample solution, in which the nucleic acid has been dissociated, from the first channel to the third channel. In the third channel, the nucleic acid extension reaction takes place. Sample pretreatment system.

5. In claim 4, The liquid delivery unit moves the sample solution back and forth between the first channel and the third channel, and the nucleic acids contained in the sample solution are amplified by the back and forth movement, in a sample pretreatment system.

6. In claim 1, The first temperature controller controls the temperature of the first flow path in the range of 90 to 100°C. The third temperature controller is a sample pretreatment system that controls the temperature of the third flow path within a range of 55 to 65°C.

7. In claim 1, The aforementioned liquid delivery unit is a sample pretreatment system, which is a pressure adjustment device.

8. In claim 2, The second temperature controller controls the second flow path to a second temperature lower than the first temperature, and a third temperature lower than the second temperature that is suitable for mixing the reagent and the sample solution. A sample pretreatment system comprising: after the reagent and the sample solution are mixed in the second channel at the third temperature, the second temperature controller raises the temperature of the second channel from the third temperature to the second temperature for the nucleic acid extension reaction.

9. In claim 1, Furthermore, the system includes a controller that controls the operation of the first temperature controller and the liquid supply unit. The controller is a sample pretreatment system that controls the temperature control operation of the first channel by the first temperature controller and the movement of the sample solution between the first channel and the second channel by the liquid delivery unit.

10. The sample solution is placed into the input port of the test chip, The sample solution is moved to the first channel connected to the inlet, By controlling the temperature of the first channel holding the sample solution using the first temperature controller, nucleic acids are extracted from the sample solution. The sample solution after the extraction of the nucleic acid is sent to a second channel connected to the first channel and mixed with the reagent. The sample solution mixed with the reagent is sent from the second channel to the first channel. The process includes using the first temperature controller to re-temperature the sample solution in which the reagents have been mixed, thereby causing the nucleic acids contained in the sample solution to dissociate. The first channel is temperature-controlled to be higher than the second channel. Furthermore, the second flow path is temperature-controlled, The third flow path connected to the first flow path is temperature-controlled, Includes, The inlet, the third channel, the first channel, and the second channel are arranged in this order. The first channel is heated to a temperature higher than the temperature at which the third channel is heated, The second channel is temperature-controlled to a temperature lower than the temperature at which the third channel is temperature-controlled. The cross-sectional area of ​​the second channel is larger than the cross-sectional area of ​​the connecting channel that connects the first channel and the second channel. The angle between the central axis of the connecting channel and the central axis of the second channel is 30 degrees or more. Sample pretreatment method.

11. In claim 10, Furthermore, the sample solution from which the nucleic acid has been dissociated is sent back into the second channel to extend the nucleic acid. The sample solution after nucleic acid extension is moved back and forth between the first channel and the second channel to generate an amplified product. A sample pretreatment method, including the following.

12. In claim 11, Furthermore, a sample pretreatment method comprising detecting the amplification product with a detector.

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