Automated gene testing device

Abstract

This automated gene testing device enables accurate and rapid determination of gene amplification reactions within a reaction vessel through precise dispensing operations controlled by the rotation of a microfluidic chip and precise heating operations controlled by the temperature of the microfluidic chip. It allows for simple and rapid genetic testing of multiple viruses and other pathogens at the genetic level, even in on-site settings, without requiring special expertise or skills. [Solution] A gene amplification reaction is performed using a disc-shaped test chip equipped with a microfluidic centrifugal dispensing function, comprising a liquid delivery unit, a heating unit, an imaging unit, a display unit, and a control unit. The test chip comprises an injection reservoir set with a liquid sample and multiple reaction chambers set with primers. The liquid delivery unit comprises a drive source and a rotating shaft, and the heating unit comprises a heater and a lifting device. The imaging unit comprises a light source and a camera, and the display unit comprises a determination device and a display device.

Description

Technical Field

[0001] The present invention relates to a gene automatic inspection device, and more particularly to a gene automatic inspection device that automates gene inspection using a microfluidic chip that utilizes centrifugal force generated by the rotation of a substrate.

Background Art

[0002] In various fields including human infectious diseases (e.g., novel coronavirus, influenza virus), food allergies (e.g., wheat, buckwheat), food poisoning pathogens (e.g., Salmonella, norovirus), and viral diseases of agricultural crops (e.g., tomato pathogenic virus, cucurbit pathogenic virus), gene diagnosis is an important theme. In these gene diagnoses, it is required to perform multiple virus tests (gene amplification reactions) to accurately and quickly determine the target virus. In particular, in the agricultural field, as a support technology for efficiently and stably producing high-quality agricultural crops, it is desired to develop a diagnostic technology that allows general agricultural producers without specialized knowledge or skills to easily and quickly perform tests on multiple viruses and the like at the gene level on-site.

[0003] As one of the gene amplification reactions, there is the LAMP (Loop-Mediated Isothermal Amplification) method. Since the LAMP method can perform gene amplification at a constant temperature, it is a simple assay method that does not require an expensive precision temperature control device like the PCR test (the most popular gene diagnosis technology) that became famous during the COVID-19 pandemic and can be implemented on-site. However, in the conventional LAMP method, in order to perform virus diagnosis of multiple items, there was the complexity of adjusting specimens and reagents for each number of test targets and performing gene amplification reactions. Also, specialized knowledge and skills were required for the work, and it could not be easily performed.

[0004] Therefore, Non-Patent Document 1 below proposes a method that applies microfluidic chip technology as an industry-academia-government collaborative project. By introducing a mixture of an extremely small amount of sample (an extract containing target DNA or target RNA) extracted from the object to be tested and reagents (including DNA polymerase for the LAMP reaction and dyes for DNA amplification detection) into a microfluidic channel through the inlet of a diagnostic device in a single operation, the device autonomously dispenses the sample and reagents evenly and with high precision into multiple reaction vessels, and then heats them in hot water (60-65°C for about 30 minutes to 1 hour), thereby enabling simultaneous diagnosis of multiple types of viruses.

[0005] On the other hand, testing devices using microfluidic chips are configured to form microchannels and supply a predetermined test liquid to reaction vessels, allowing for reactions with small amounts of sample. Furthermore, in order to supply the same test liquid to multiple reaction vessels simultaneously, a main channel and branch channels are configured, and devices have been developed in which the test liquid is supplied to multiple reaction vessels via the main channel (see Patent Documents 1 and 2).

[0006] These dispensing devices require the injection of the test liquid into the supply channel at a predetermined pressure, which is typically done by forced injection using a syringe or the like. However, a device has been developed that pre-stores the test liquid in a test kit and uses centrifugal force generated by rotation to move the test liquid and supply it to the desired chamber, without the need for forced injection using a syringe or the like (a liquid delivery device utilizing a centrifugal supply mechanism) (see Patent Document 3). This device does not require forced injection at a constant pressure using a syringe or the like, and allows for virus testing with simpler operation. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2009-284769 [Patent Document 2] Japanese Patent Publication No. 2022-080026 [Patent Document 3] International Publication No. W02008-139697 [Non-patent literature]

[0008] [Non-Patent Document 1] Press Release from Toyohashi University of Technology (Attachment 2) (https: / / www.tut.ac.jp / docs / 200728kisyakaiken.pdf) [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] By the way, in order to obtain simple, rapid, and accurate test results using a liquid delivery device that utilizes a centrifugal supply mechanism such as the one described in Patent Document 3 above, it is necessary to automate the genetic testing. However, there was a problem in that rotational wobble occurred in the microfluidic tip when rotating it to dispense the test liquid, making stable dispensing impossible. In addition, there was a problem in that it was not possible to accurately heat the reaction vessel to the appropriate temperature when heating the microfluidic tip for gene amplification.

[0010] Therefore, the present invention aims to address the above-mentioned problems by providing an automated gene testing device that can accurately and quickly determine the gene amplification reaction in a reaction vessel through precise dispensing operations by controlling the rotation of a microfluidic chip and precise heating operations by controlling the temperature of a microfluidic chip, and that can perform simple and rapid genetic testing of multiple viruses, etc., even in on-site settings, without requiring special expertise or skills. [Means for solving the problem]

[0011] In order to solve the above problems, the inventors diligently conducted research and, as a result, improved the rotation mechanism of the microfluidic chip and the contact and temperature measurement / control method between the microfluidic chip and the heater, leading to the completion of the present invention.

[0012] That is, according to the description in claim 1, the automated gene testing device 100 according to the present invention is, An automated gene testing device having a liquid delivery unit 30, a heating unit 40, an imaging unit 50, a display unit 60, and a control unit 70, which performs gene amplification reactions using a disc-shaped test chip 10 equipped with a centrifugal dispensing function via a microfluidic channel, and automatically determines viruses, bacteria, and biological species in multiple samples. The inspection chip comprises an injection reservoir 11 and a plurality of reaction chambers 12. When a liquid sample is set in the injection reservoir, a primer is set in the reaction chamber, and the inspection chip is heated to a predetermined temperature, a light-transmitting first heat-insulating disk 21 and a first heat-transmitting disk 22 are fixed to the upper and lower surfaces of the inspection chip, respectively, to maintain a uniform temperature of the liquid sample in the reaction chambers, thereby forming a fixing unit 20. The liquid delivery unit comprises a drive source 32 and a rotating shaft 33 controlled by the control unit, and by fixing the center of the fixed unit with the rotating shaft and rotating the fixed unit, the liquid sample set in the injection reservoir of the inspection chip is filled into the interior of the multiple reaction chambers via the microchannel by the centrifugal force of the rotation. The heating unit comprises a heater 41 and a lifter 42 controlled by the control unit, the heater is configured with a second heat-transferring disc 44 fixed to the upper surface of a disc-shaped heater 43 and a third heat-transferring disc 45 fixed to the lower surface, and when heating the test chip, the heater is raised by the lifter to bring the second heat-transferring disc of the heater into contact with the first heat-transferring disc of the fixing unit, and the test chip is heated to a predetermined temperature by heating from the disc-shaped heater, thereby causing the liquid sample filled inside the reaction chamber of the test chip and the primer that was previously fixed inside the reaction chamber to react and the gene amplification reaction to proceed. The imaging unit comprises a light source 51 and a camera 52 controlled by the control unit, and during the operation of the heating unit, it images the state inside the reaction chamber of the test chip via the light-transmitting first heat-insulating disk to record the presence or absence of a gene amplification reaction and the progress of the reaction. The display unit comprises a determination device and a display device controlled by the control unit, wherein the determination device determines the state of the gene amplification reaction during heating of the test chip by the heating unit, and the display device displays the result.

[0013] Furthermore, according to claim 2, the present invention is an automated gene testing device as described in claim 1, The liquid transfer unit fixes the rotating shaft from both the upper and lower surfaces of the substrate that fixes the rotating shaft via bearings 36, In fixing the central part of the fixing unit with the rotating shaft, a polygonal spigot structure is adopted as a joining method that combines the concave joint portion 24 of the fixing unit with the convex projection 35 of the rotating shaft. The invention is characterized by eliminating the loss of rotational force transmitted from the drive source to the rotating shaft, reducing frictional resistance between the rotating shaft and the fixed unit, and ensuring stable rotation of the inspection chip.

[0014] Furthermore, according to claim 3, the present invention is an automated gene testing device according to claim 1 or 2, The heating unit has a temperature measuring instrument 48 for detecting the temperature of the test chip heated by the heater, and the temperature measuring junction 48b of the thermocouple 48a of the temperature measuring instrument is installed on the lower surface of the third heat-transferring disk which constitutes the lower surface of the heater. When heating the fixing unit, the first heat-transferring disc that constitutes the lower surface of the inspection chip of the fixing unit and the second heat-transferring disc that constitutes the upper surface of the heater are brought into contact and fixed together. The sum of the thickness of the first heat conductive disk that is abutted and fixed to the lower surface of the inspection chip and the thickness of the second heat conductive disk that is abutted and fixed thereto is the same as the thickness of the third heat conductive disk that is fixed to the lower surface portion of the disk-shaped heater that constitutes the warmer.

[0015] Also, according to the description of claim 4, the present invention is a gene automatic inspection apparatus according to claim 3, The warmer includes a plurality of cushion supports 46, and the cushion support fixes one end 47a to the second heat insulating disk and abuts the other end 47c against the lower surface portion of the first heat conductive disk, characterized in that the adhesion accuracy between the first heat conductive disk that constitutes the fixing unit and the second heat conductive disk that constitutes the warmer is improved.

[0016] Also, according to the description of claim 5, the present invention is a gene automatic inspection apparatus according to claim 3, The thermometer includes a plate spring-like pressing tool 49a that facilitates the detachment of the temperature measurement contact of the thermocouple from the third heat conductive disk and presses against the surface of the third heat conductive disk during attachment.

Effect of the Invention

[0017] According to the above configuration, the gene automatic inspection apparatus according to the present invention has a liquid feeding unit, a warming unit, an imaging unit, a display unit, and a control unit, and performs a gene amplification reaction using a disk-shaped inspection chip having a centrifugal dispensing function by a microchannel, and automatically determines the virus, bacteria, and species of a plurality of specimens.

[0018] The inspection chip includes an injection reservoir and a plurality of reaction chambers. A liquid sample is set in the injection reservoir, and a primer is set in the reaction chamber. When the inspection chip is heated to a predetermined temperature, in order to keep the temperature of the liquid sample in the reaction chamber uniform, a light-transmissive first heat insulating disk and a first heat conductive disk are abutted and fixed to the upper and lower surfaces of the inspection chip, respectively, to form a fixing unit.

[0019] The liquid delivery unit includes a drive source and a rotating shaft controlled by a control unit. The central part of the fixing unit is fixed by the rotating shaft to rotate the fixing unit. Thereby, the liquid sample set in the injection reservoir of the test chip is filled into the interiors of a plurality of reaction chambers through a microchannel by the centrifugal force of rotation.

[0020] The heating unit includes a heater and a lifter controlled by a control unit. The heater is configured by fixing a second heat conductive disk to the upper surface of a disk-shaped heater and fixing a third heat conductive disk to the lower surface thereof. When heating the test chip, the lifter raises the heater to abut and fix the second heat conductive disk of the heater and the first heat conductive disk of the fixing unit, and the test chip is heated to a predetermined temperature by the heating from the disk-shaped heater. Thereby, the liquid sample filled into the interior of the reaction chamber of the test chip reacts with the primer previously fixed in the interior of the reaction chamber, and the gene amplification reaction proceeds.

[0021] The imaging unit includes a light source and a camera controlled by a control unit. During the operation of the heating unit, the state inside the reaction chamber of the test chip is imaged through a light-transmissive first heat insulating disk to record the presence or absence of the gene amplification reaction and the progress state of the reaction. Further, the display unit includes a determination device and a display device controlled by a control unit. The state of the gene amplification reaction during the heating of the test chip by the heating unit is determined by the determination device, and the result is displayed by the display device.

[0022] Thereby, by the accurate dispensing operation by the rotation control of the microfluidic chip and the accurate heating operation by the temperature control of the microfluidic chip, the gene amplification reaction in the reaction vessel can be accurately and quickly judged, and without requiring special expertise or skills, it is possible to provide a gene automatic inspection device capable of simply and quickly performing inspections of a plurality of viruses and the like at the gene level even at the site.

[0023] Furthermore, according to the above configuration, the fluid transfer unit fixes the rotating shaft via bearings from both the upper and lower surfaces of the substrate that fixes the rotating shaft, and when fixing the center of the fixing unit with the rotating shaft, a polygonal spigot structure is used as the joining method, which combines the concave joint of the fixing unit with the convex projection of the rotating shaft. This eliminates the loss of rotational force transmitted from the drive source to the rotating shaft, reduces frictional resistance between the rotating shaft and the fixing unit, and ensures stable rotation of the inspection chip. This makes the above effects more concrete and effective.

[0024] Furthermore, according to the above configuration, the heating unit has a temperature measuring instrument that detects the temperature of the inspection chip heated by the heater, and the temperature sensing junction of the thermocouple equipped with the temperature measuring instrument is inserted into the lower surface of the third heat-transferring disc that constitutes the lower surface of the heater. When heating the fixed unit, the first heat-transferring disc that constitutes the lower surface of the inspection chip of the fixed unit and the second heat-transferring disc that constitutes the upper surface of the heater are brought into contact and fixed together so that the sum of the thickness of the first heat-transferring disc that is in contact and fixed to the lower surface of the inspection chip and the thickness of the second heat-transferring disc that is in contact and fixed together is the same as the thickness of the third heat-transferring disc that is fixed to the lower surface of the disc-shaped heater that constitutes the heater. This makes it possible to exert the above effects more concretely and effectively.

[0025] Furthermore, according to the above configuration, the heater is equipped with multiple cushion supports, each cushion support having one end fixed to the second heat-insulating disc and the other end in contact with the lower surface of the first heat-transferring disc. This improves the degree of contact between the first heat-transferring disc constituting the fixing unit and the second heat-transferring disc constituting the heater. As a result, the above effects can be exerted more concretely and effectively.

[0026] Furthermore, according to the above configuration, the temperature measuring instrument is equipped with a leaf spring-shaped pressing device that facilitates the attachment and detachment of the thermocouple's temperature sensing junction to the third heat-transferring disk, and presses against the surface of the third heat-transferring disk when attached. This allows the above effects to be exerted more concretely and effectively. [Brief explanation of the drawing]

[0027] [Figure 1] (A) A plan view and (B) a front view of the microfluidic chip used in this embodiment. [Figure 2] This is a schematic diagram showing the internal structure of the automated gene testing device according to this embodiment. [Figure 3] This is a side cross-sectional view showing the configuration of the fixed unit. [Figure 4] Figure 3 is a bottom perspective view showing the fixed unit. [Figure 5] This is a schematic side view showing the drive source and rotating shaft that make up the liquid transfer unit. [Figure 6] Figure 5 is an enlarged view of the side showing the axis of rotation. [Figure 7] Figure 6 is a perspective view showing a convex projection located at the tip of the rotating shaft. [Figure 8] This is a perspective view showing the heater and elevator that make up the heating unit. [Figure 9] Figure 8 is a perspective view of the heating unit from the back. [Figure 10] This is a schematic diagram showing the side view of the configuration of the heating unit and the stationary unit, which are separated before heating. [Figure 11] This is a schematic side view showing the configuration of the heating unit and the fixing unit in contact during heating. [Figure 12] Figure 8 shows (A) a schematic cross-sectional view of the side of the heater that makes up the heating unit, showing the part with the cushion support attached, and (B) a perspective view of the cushion support used therefor. [Figure 13] This is a side view showing the relationship between the thermocouple and the thermocouple mounting base of the heater, and is a perspective view showing (A) the configuration of the thermocouple mounting base, (B) the thermocouple mounting base attached to the lower surface of the third heat transfer disk, and (C) the state in which the thermocouple's temperature sensing junction is attached to the thermocouple mounting base. [Modes for carrying out the invention]

[0028] The automated gene testing apparatus according to the present invention will be described below with reference to embodiments. However, the present invention is not limited to the embodiments described below.

[0029] First, the microfluidic chip used in the automated gene testing apparatus according to the present invention will be described. The microfluidic chip used in the present invention injects the test liquid into the reaction vessel via a supply channel using a centrifugal supply mechanism. Centrifugal supply mechanisms include those that supply the test liquid from the rotation center side toward the outer circumference side (such as in the above-mentioned Patent Document 3), and those that supply the test liquid from the outer circumference side toward the rotation center, depending on the direction of centrifugal force. Either type may be used in the present invention. In this embodiment, a microfluidic chip that supplies the test liquid from the outer circumference side toward the rotation center, which is considered to provide stable injection of the test liquid into the reaction vessel, is used.

[0030] Figure 1 shows (A) a plan view and (B) a front view of the microfluidic chip used in this embodiment. The microfluidic chip (hereinafter also referred to as the "inspection chip") is a disk in which injection reservoirs, reaction chambers, and microchannels are formed from a transparent heat-insulating resin (e.g., silicone resin, acrylic resin) by a transfer process from a mold (template) fabricated using microfabrication technology (e.g., lithography technology, 3D printing technology).

[0031] In Figure 1(A), the circular surface of the test tip 10 is provided with multiple injection reservoirs 11 (four in Figure 1), and each injection reservoir 11 is provided with multiple reaction chambers 12 (five for each injection reservoir in Figure 1). In addition, multiple microchannels are formed on the surface of the test tip 10. The number of injection reservoirs 11, the number of reaction chambers 12, and the connection of these to the multiple microchannels are not particularly limited as long as they enable accurate centrifugal dispensing functionality.

[0032] In Figure 1(A), the microchannels first flow from the injection reservoir 11 towards the outer circumference of the inspection tip 10, and then a main channel 13 is formed along the outer circumference of the inspection tip 10. From the main channel 13 along the outer circumference of the inspection tip 10, a first branch channel 14 is formed toward each reaction chamber 12 on the rotational center side of the inspection tip 10. Subsequently, a second branch channel 14 formed toward the rotational center side of the inspection tip 10 from each reaction chamber 12 connects to a discharge channel 15 formed on the rotational center side of the inspection tip 10, and the discharge channel 15 is formed toward the injection reservoir 11.

[0033] In the gene amplification reaction, the injection reservoir 11 is filled with a liquid sample containing a gene sample obtained by extracting and purifying the genes of the virus or other organism to be tested, mixed with a gene amplification reagent. The reaction chamber 12 is pre-filled with primers (short DNA strands that serve as the starting point for amplification) for amplifying the viral gene, and these primers are dried beforehand. Since the test chip 10 in this embodiment has four injection reservoirs 11, four different gene samples can be tested simultaneously. Furthermore, since five reaction chambers 12 are formed for each injection reservoir 11, five different viral primers can be set for each gene sample.

[0034] Next, the automated gene testing apparatus according to the present invention will be described in detail. Figure 2 is a schematic diagram showing the interior of the automated gene testing apparatus according to this embodiment. In Figure 2, the automated gene testing apparatus 100 is housed in a housing 101 (walls are not shown in Figure 2), and has a fixing unit 20, a liquid delivery unit 30, a heating unit 40, an imaging unit 50, a display unit 60, and a control unit 70 inside.

[0035] Here, we will explain, unit by unit, the process of performing a gene amplification reaction using the automated gene testing device 100 and determining the gene sample contained in the liquid sample.

[0036] ≪Fixed Unit 20≫ The fixing unit 20 is constructed by fixing two discs to the upper and lower surfaces of the inspection chip 10 in order to maintain a uniform temperature of the liquid sample in the reaction chamber 12 when the inspection chip 10 is heated to a predetermined temperature. Figure 3 is a side cross-sectional view showing the configuration of the fixing unit. In Figure 3, the fixing unit 20 has the inspection chip 10, a light-transmitting first heat-insulating disc 21 on the upper surface, and a first heat-transferring disc 22 on the lower surface in contact with the inspection chip 10, sandwiching it. The first heat-insulating disc 21, the inspection chip 10, and the first heat-transferring disc 22 are firmly fixed in contact with each other by ring-shaped cover screws 23 that cover the first heat-insulating disc 21 and the inspection chip 10 from above and are screwed to the outer circumference of the first heat-transferring disc 22. This makes it possible to maintain a uniform temperature of the liquid sample in the reaction chamber 12 when the inspection chip 10 is heated to a predetermined temperature. Figure 4 is a bottom perspective view showing the fixing unit of Figure 3. In Figure 4, a concave joint 24 is provided at the center of the bottom surface of the fixed unit 20 to receive the rotation axis of the liquid delivery unit 30, which will be described later.

[0037] In this embodiment, the first heat-insulating disk 21 is made of light-transmitting acrylic resin, and the presence or absence of gene amplification reactions and the progress of the reactions in each reaction chamber 12 can be photographed by the imaging unit 50, which will be described later. In this embodiment, the first heat-transferring disk 22 is made of aluminum and contacts the heating surface of the heating unit 40, which will be described later, to transfer heat from the heater to the test chip 10. As described above, the test chip 10 has a liquid sample injected into the injection reservoir 11 and a primer dropped and dried into the reaction chamber 12.

[0038] ≪Control Unit 70≫ The control unit 70 is an automatic control device such as a microcomputer, and has a control board 70a that controls the operation of all units other than the fixed unit 20 individually or in conjunction with each other. It also stores the judgment criteria for the display unit 60, which determines the state of the gene amplification reaction recorded by the imaging unit 50, as a database.

[0039] ≪Liquid delivery unit 30≫ The liquid delivery unit 30 rotates the fixed unit 20 by fixing its center on a rotating shaft, thereby filling the liquid sample set in each injection reservoir 11 of the inspection tip 10 into the interior of the multiple reaction chambers 12 via microchannels 13 and 14 using the centrifugal force of rotation. Here, the inspection tip 10 has a very precise fluid circuit, as it is called a microfluidic tip, and it is required to uniformly dispense the liquid sample from the injection reservoir 11 into the multiple reaction chambers 12. Therefore, if rotational wobble occurs in the inspection tip 10 when it is rotated by the liquid delivery unit 30, stable dispensing cannot be achieved. Thus, the present invention employs a method to control the rotational wobble of the inspection tip 10.

[0040] Figure 5 is a schematic side view showing the drive source and rotating shaft that constitute the liquid delivery unit 30. In Figure 5, the motor 32, which is the drive source, and the rotating shaft 33 are connected to the substrate 31 by a drive belt 34. The motor 32 is connected to the liquid delivery power supply 32a (see Figure 2) and is driven at a predetermined output by the control unit 70. The rotating shaft 33 fixes the rotation center of the stationary unit 20 and rotates the stationary unit 20 at a predetermined rotational speed by the control unit 70.

[0041] Figure 6 is an enlarged side view showing the rotating shaft of Figure 5. The rotating shaft 33 penetrates the fixed substrate 31 and is rotatably fixed from both its upper and lower surfaces via bearings 36. The type of bearing 36 that fixes the rotating shaft 33 is not particularly limited, but in this embodiment, a ball bearing 36 is used. Figure 7 is a perspective view showing a convex projection provided at the tip of the rotating shaft of Figure 6. In Figure 7, the convex projection 35 is fixed to the upper tip of the rotating shaft 33 and rotates together with the rotating shaft 33. This convex projection 35 fits into a concave joint 24 provided at the center of the bottom surface of the fixed unit 20 shown in Figure 4, employing a polygonal spigot structure that connects the liquid supply unit 30 and the rotating shaft 33. This eliminates the loss of rotational force from the motor 32 to the rotating shaft 33 via the drive belt 34, and reduces frictional resistance between the rotating shaft 33 and the fixed unit 20, enabling stable rotation of the fixed unit 20. The rotational speed of the liquid delivery unit 30 is adjusted as appropriate depending on the structure of the inspection tip 10, but is generally within the range of 1000 to 3000 rpm.

[0042] Here, we will explain how the rotation of the fixed unit 20 uniformly fills the liquid sample set in each injection reservoir 11 of the inspection tip 10 into the interior of the multiple reaction chambers 12. In Figure 1, the liquid sample injected into the injection reservoir 11 flows along the outer circumference of the inspection tip 10 via the main channel 13 due to the centrifugal force of rotation. Next, the liquid sample flows uniformly into each reaction chamber 12 via the branch channels 14 from the main channel 13, which is provided along the outer circumference of the inspection tip 10, due to the pressure from the injection reservoir 11. Furthermore, once the interior of each reaction chamber 12 is filled with liquid sample, any excess liquid sample is returned to the injection reservoir 11 via the discharge channel 15. As a result, the liquid sample set in each injection reservoir 11 of the inspection tip 10 can be uniformly filled into the interior of the multiple reaction chambers 12 via the main channel 13 and branch channels 14 due to the centrifugal force of rotation.

[0043] ≪Heating Unit 40≫ The heating unit 40 uses a heater power supply to raise the temperature of a disc-shaped heater, heating the test chip 10 to a predetermined temperature, allowing the gene amplification reaction to proceed. Here, when heating the test chip 10, it is necessary to accurately heat the inside of the reaction vessel. Gene amplification reactions using the LAMP method are generally controlled to a predetermined temperature of ±1°C within the range of 60-65°C. Figure 8 is a perspective view showing the heater and elevator that make up the heating unit. Figure 9 is a perspective view of the heating unit of Figure 8 viewed from the back. In Figures 8 and 9, the heating unit 40 comprises a heater 41 and an elevator 42 that raises and lowers it. The bottom surface of the heater 41 is fixed to the arm 42a of the elevator 42. Furthermore, the control unit 70 controls the temperature of the disc-shaped heater 43 equipped in the heater 41 and the vertical movement of the elevator 42.

[0044] Figure 10 is a schematic side view showing the configuration of the heating unit and the fixed unit in a separate state before heating. In Figure 10, the fixed unit 20 is set on top of the heater 41. Although not shown in Figure 10, the rotating shaft 33 of the liquid supply unit 30 is joined to the fixed unit 20 from below. Therefore, although the heater 41 is disc-shaped, including the heater, it has a ring-like shape with an open center through which the rotating shaft 33 passes. Also, because the fixed unit 20 is joined to the rotating shaft 33 of the liquid supply unit 30, it cannot be raised or lowered. Therefore, the heater 41 is raised to the position of the fixed unit 20 using the elevator 42. This operation brings the lower surface of the fixed unit 20 into contact with the upper surface of the heater 41.

[0045] Figure 11 is a schematic side view showing the configuration of the heating unit and the fixing unit in contact during heating. Although not shown in Figure 11, the rotating shaft 33 of the liquid delivery unit 30 is joined to the fixing unit 20 from below. If the contact accuracy between the lower surface of the fixing unit 20 and the upper surface of the heater 41 is insufficient, the heating of the test chip 10 inside the fixing unit 20 will be uneven, making it impossible to perform a uniform and accurate gene amplification reaction. Therefore, the present invention employs a method for uniformly contacting the lower surface of the fixing unit 20 and the upper surface of the heater 41.

[0046] Figure 12 shows (A) a schematic cross-sectional view of the side of the heater that constitutes the heating unit in Figure 8, showing the portion with the cushion support attached, and (B) a perspective view of the cushion support used therefor. In Figure 12, in order to ensure uniform contact between the lower surface of the fixed unit 20 and the upper surface of the heater 41, the heater 41 is equipped with a plurality of cushion supports 46 made of cylindrical vibration-damping rubber on the arm 42a of the lifter 42. The cushion supports 46 are fixed between the third heat-transferring disc 45 and the arm 42a of the lifter 42 by support fixing screws 47. This improves the contact accuracy between the first heat-transferring disc 22 of the fixed unit 20 and the second heat-transferring disc 44 of the heater 41, resulting in uniform heating of the test chip 10 and enabling accurate gene amplification reactions.

[0047] Next, the heating control of the inspection chip 10 by the heater 41 will be described. In the present invention, in order to improve the accuracy of temperature measurement, a configuration is adopted that facilitates the attachment of the temperature sensing junction 48b of the thermocouple 48a provided by the temperature measuring instrument 48, and allows for accurate temperature measurement. Figure 13 is a side view showing the relationship between the thermocouple of the heater and the thermocouple mounting base, and is a perspective view showing (A) the configuration of the thermocouple mounting base, (B) the thermocouple mounting base attached to the lower surface of the third heat-transferring disk, and (C) the state in which the temperature sensing junction of the thermocouple is attached to the thermocouple mounting base. In Figure 13(A), a leaf spring-shaped pressing holder 49a is fixed to the thermocouple mounting base 49 for contacting and fixing the temperature sensing junction 48b of the thermocouple 48a to the measurement position. In Figure 13(B), the thermocouple mounting base 49 is fixed to the lower surface of the third heat-transferring disk 45, and the pressing holder 49a is in contact with the third heat-transferring disk 45. In Figure 13(C), the temperature sensing junction 48b of the thermocouple 48a is inserted between the third heat-transferring disk 45 and the thermocouple mounting base 49, and is pressed and fixed to the lower surface of the third heat-transferring disk 45 by the pressing holder 49a.

[0048] In this state, the heater 41 rises due to the operation of the elevator 42, and the first heat-transferring disc 22 of the fixed unit 20 and the second heat-transferring disc 44 of the heater 41 come into contact. As a result, the inspection chip 10 of the fixed unit 20 is heated by the heat from the disc-shaped heater 43 of the heater 41 via the second heat-transferring disc 44 and the first heat-transferring disc 22. It is preferable to directly measure the temperature of the heated inspection chip 10. However, the fixed unit 20 is joined to the rotating shaft 33 of the liquid supply unit 30 and is in a state of rotation in the previous process. Therefore, it is not possible to attach the temperature-measuring junction 48b of the thermocouple 48a to the inspection chip 10 of the fixed unit 20.

[0049] Therefore, in this invention, a special laminated structure of the fixed unit 20 and the heater 41 is adopted to indirectly measure the temperature of the inspection chip 10. In other words, the total thickness of the first heat-conducting disc 22 and the second heat-conducting disc 44 located between the disc-shaped heater 43 and the inspection chip 10 is configured to be the same as the thickness of the third heat-conducting disc 45 located on the lower surface of the disc-shaped heater 43. Specifically, in this embodiment, the disc-shaped heater 43 consists of a nichrome wire and a stainless steel casing. The first heat-conducting disc 22 and the second heat-conducting disc are both 2 mm thick aluminum plates, with a total thickness of 4 mm. On the other hand, the third heat-conducting disc 45 is a 4 mm thick aluminum plate. In this invention, the structure of the disc-shaped heater 43 and the material and thickness of each heat-conducting disc are not particularly limited as long as they have a predetermined relationship.

[0050] As a result, the temperature of the first heat-transferring disk 22 on the side facing the inspection chip 10, which contacts the inspection chip 10 via the first heat-transferring disk 22 and the second heat-transferring disk from the disk-shaped heater 43, and the temperature of the third heat-transferring disk 45 on the side facing the second insulating disk 46, which contacts the second insulating disk 46 via the third heat-transferring disk 45 from the disk-shaped heater 43, are the same. Therefore, the temperature measured by the thermocouple 48a of the temperature measuring instrument 48, in which a temperature sensing junction 48b is inserted into the lower surface of the third heat-transferring disk 45, can be considered as the temperature of the inspection chip 10.

[0051] ≪Imaging Unit 50≫ The imaging unit 50 images the internal state of the reaction chamber 12 of the test chip 10 while the heating unit 40 is operating, and records the presence or absence of a gene amplification reaction and the progress of the reaction. In the LAMP reaction, the gene amplification reaction proceeds inside the reaction chamber 12 by the gene sample, primers, and DNA polymerase. In conjunction with this, the presence or absence of a gene amplification reaction and the progress of the reaction can be visually confirmed using a colorimetric reagent. The colorimetric reagent is not particularly limited, but in this embodiment, hydroxynaphthol blue (HNB) was used.

[0052] The imaging unit 50 includes a light source 51 (preferably an LED light source) and a camera 52 (see Figure 2). Under the control of the control unit 70, it captures and records images of the internal state of the reaction chamber 12 of the inspection chip 10 over time via a light-transmitting first heat-insulating disk 21 while the heating unit 40 is in operation. The camera 52 may be a CCD image sensor or a CMOS image sensor. The captured images are transmitted to the sequential display unit 60.

[0053] ≪Display Unit 60≫ The display unit 60 determines the state of the gene amplification reaction during heating of the test chip 10, which has been imaged by the imaging unit 50, and displays the result. The display unit 60 includes a determination device and a display device (neither of which are shown). The determination device stores determination criteria for determining the state of the gene amplification reaction as a database and determines the image sent from the imaging unit 50 under the control of the control unit 70. The display device also displays the state of the gene amplification reaction and the determination result on the monitor 61 (see Figure 2) under the control of the control unit 70.

[0054] Confirmation tests using the automated gene testing device configured as described above yielded very favorable results. Therefore, by precisely controlling the rotation of the microfluidic chip for accurate dispensing and precisely controlling the temperature of the microfluidic chip for accurate heating, the gene amplification reaction in the reaction vessel can be accurately and rapidly determined. This allows for the provision of an automated gene testing device that can easily and quickly perform genetic testing for multiple viruses and other pathogens, even in on-site settings, without requiring special expertise or skills. [Explanation of Symbols]

[0055] 10...Inspection tip, 11...Injection reservoir, 12...Reaction chamber, 13...Main channel, 14...Branching channel, 15...Discharge channel, 20...Fixed unit, 21...First heat-insulating disc, 22...First heat-transferring disc, 23...Cover screw, 24...Concave joint, 30...Liquid supply unit, 31...Circuit board, 32...Motor, 32a...Power supply for liquid supply, 33...rotating shaft, 34...drive belt, 35...convex projection, 36...bearing 40...Heating unit, 41...Heater, 42...Elevator, 42a...Arm 43...Disc-shaped heater, 44...Second heat-transferring disc, 45...Third heat transfer disc, 46...Cushion support, 47...Support fixing screw, 48...Temperature measuring instrument, 48a...Thermocouple, 48b...Temperature sensing junction, 49... Thermocouple mounting base, 49a... Pressing holder, 50...imaging unit, 51...light source, 52...camera, 60...Display unit (judgment device, display device), 61...Monitor, 70...Control unit, 70a...Control board, 100...Automated gene testing device, 101...Housing.

Claims

1. An automated gene testing device having a liquid delivery unit, a heating unit, an imaging unit, a display unit, and a control unit, which performs gene amplification reactions using a disc-shaped test chip equipped with a centrifugal dispensing function via a microfluidic channel, and automatically determines viruses, bacteria, and biological species in multiple samples, The inspection chip comprises an injection reservoir and a plurality of reaction chambers. When a liquid sample is set in the injection reservoir, a primer is set in the reaction chamber, and the inspection chip is heated to a predetermined temperature, a light-transmitting first heat-insulating disk and a first heat-transmitting disk are fixed to the upper and lower surfaces of the inspection chip, respectively, to maintain a uniform temperature of the liquid sample in the reaction chambers, thereby forming a fixing unit. The liquid delivery unit comprises a drive source and a rotating shaft controlled by the control unit, and by fixing the center of the fixed unit with the rotating shaft and rotating the fixed unit, the liquid sample set in the injection reservoir of the inspection chip is filled into the interior of the plurality of reaction chambers via the microchannel by the centrifugal force of the rotation. The heating unit comprises a heater and a lifter controlled by the control unit, the heater is configured with a second heat-transferring disc fixed in contact with the upper surface of a disc-shaped heater and a third heat-transferring disc fixed in contact with the lower surface, and when heating the test chip, the heater is raised by the lifter to bring the second heat-transferring disc of the heater into contact with the first heat-transferring disc of the fixing unit, and the test chip is heated to a predetermined temperature by heating from the disc-shaped heater, thereby causing the liquid sample filled inside the reaction chamber of the test chip and the primer that was previously fixed inside the reaction chamber to react and the gene amplification reaction to proceed. The imaging unit comprises a light source and a camera controlled by the control unit, and during the operation of the heating unit, it images the state inside the reaction chamber of the test chip via the light-transmitting first heat-insulating disk to record the presence or absence of a gene amplification reaction and the progress of the reaction. The display unit comprises a determination device and a display device controlled by the control unit, wherein the determination device determines the state of the gene amplification reaction during heating of the test chip by the heating unit, and the display device displays the result.

2. The liquid transfer unit fixes the rotating shaft via bearings from both the upper and lower surfaces of the substrate that fixes the rotating shaft, In fixing the central part of the fixing unit with the rotating shaft, a polygonal spigot structure is adopted as the joining method, which combines the concave joint of the fixing unit with the convex projection of the rotating shaft. The automated gene testing apparatus according to claim 1, characterized in that it eliminates the loss of rotational force transmitted from the drive source to the rotating shaft, reduces frictional resistance between the rotating shaft and the fixed unit, and ensures stable rotation of the test chip.

3. The heating unit has a temperature measuring instrument for detecting the temperature of the test chip heated by the heater, and the temperature measuring junction of the thermocouple equipped with the temperature measuring instrument is installed on the lower surface of the third heat-transferring disk which constitutes the lower surface of the heater. When heating the fixing unit, the first heat-transferring disc, which constitutes the lower surface of the inspection chip of the fixing unit, and the second heat-transferring disc, which constitutes the upper surface of the heater, are brought into contact and fixed together. The automated gene testing apparatus according to claim 1 or 2, characterized in that the sum of the thickness of the first heat-transferring disk fixed in contact with the lower surface of the test chip and the thickness of the second heat-transferring disk fixed in contact with the first heat-transferring disk is the same as the thickness of the third heat-transferring disk fixed to the lower surface of the disk-shaped heater constituting the heating element.

4. The heater comprises a plurality of cushion supports, each of which has one end fixed to the second heat-insulating disc and the other end in contact with the lower surface of the first heat-transferring disc. The automated gene testing apparatus according to claim 3, characterized in that the contact accuracy between the first heat-transferring disk constituting the fixed unit and the second heat-transferring disk constituting the heater is improved.

5. The gene automated testing apparatus according to claim 3, characterized in that the temperature measuring instrument is equipped with a leaf spring-shaped pressing and holding device that facilitates the attachment and detachment of the thermometer's temperature sensing junction to the third heat-transferring disk and presses against the surface of the third heat-transferring disk when attached.

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