JP2009002913A - Nucleic acid detection cassette - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid detection cassette constituted so as to realize the miniaturization of an apparatus, made detachable with respect to an apparatus main body and suitable for disposable use. <P>SOLUTION: The nucleic acid detection cassette 12 is equipped with: a PCR tank 51 wherein PCR is performed; a nucleic acid detection tank 52 provided with a nucleic acid microarray 75; a first reagent tank 54, a second reagent tank 55 and a washing liquid tank 56 which respectively store a first reagent, a second reagent and a washing liquid; a waste liquid tank 53 to which a waste liquid is discharged; the first and second flow channels 59 and 60 connected to the PCR tank 51; a third flow channel 61 for connecting the nucleic acid detection tank 52 and the waste liquid tank 53; fourth flow channels 65A, 65B, 69 and 70 connected to the first reagent tank 54, the second reagent tank 55 and the washing liquid tank 56; fifth flow channels 66-68 respectively connecting the first reagent tank 54, the second reagent tank 55, the washing liquid tank 56 and the nucleic acid detection tank 52; and a plurality of cam followers 85-87 for opening and closing the fifth flow channels 66-68. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a nucleic acid detection cassette suitably used for nucleic acid detection and detachable from a predetermined apparatus main body.

  In recent years, with the progress of genome sequencing, the entire base sequences of genomes possessed by various organisms are being clarified. And the technique which utilizes the elucidated base sequence usefully is expected. For example, nucleic acid detection using nucleic acid microarrays is a technology that can simultaneously analyze multiple genes, and has been developed not only as a base sequencing method but also as a method for efficiently examining gene expression levels and polymorphisms, The development of technologies for the identification of biological classifications such as fungi and the diagnosis of diseases is being made.

  In general, nucleic acid microarrays used for identification of tailor-made medicine and biological classification need only be capable of detecting a specific base sequence qualitatively. An inexpensive one suitable for use is expected.

  Prior to a nucleic acid detection reaction using a nucleic acid microarray, PCR is often performed to amplify a nucleic acid to be detected contained in a sample such as blood. In general, in order to detect a nucleic acid using PCR, a pretreatment for extracting a nucleic acid from a sample is performed in order to remove a PCR inhibitory substance contained in blood or the like. In recent years, a pretreatment reagent capable of performing this pretreatment (extraction) simultaneously with PCR has been developed and is commercially available (for example, trade name: Ampdirect Plus, manufactured by Shimadzu Corporation).

  If the three steps of extraction / amplification / detection in nucleic acid detection can be easily carried out in one cassette in which required reagents and nucleic acid microarrays are made into a kit, it is considered that the utility value is high in medical facilities and research facilities. . Further, if the cassette is downsized, the desired nucleic acid can be detected from a small amount of sample, thereby reducing the burden on the subject and reducing the size of the automatic analyzer loaded with the cassette. Space can also be realized.

  Further, if the pretreatment reagent described above is used, extraction and amplification in nucleic acid detection can be performed simultaneously, but detection using a nucleic acid microarray needs to be performed separately. In addition, it is necessary to supply a plurality of reagents, cleaning solutions, and the like used for hybridization to the reaction tank in a predetermined order and timing. Therefore, in the cassettes and automatic analyzers provided as kits, a reaction vessel for performing PCR and a reaction vessel for performing nucleic acid detection (hybridization, labeling) are provided independently, and a liquid containing a sample and a reagent is supplied to each reaction vessel. Must be moved sequentially.

  For example, if each reaction tank is connected by a flow path, the flow path must be openable and closable by a valve or the like in accordance with PCR or liquid movement. However, when a general valve is used, there is a problem that a mechanism for driving the valve becomes large, and as a result, the automatic analyzer becomes large. In particular, if there are a plurality of reagent tanks, there are a plurality of flow paths and valves, and this problem is remarkable. On the other hand, for example, the following has been proposed as a nucleic acid detection cassette suitable for disposable use.

  In the nucleic acid detection cassette disclosed in Patent Document 1, a flow path is configured by a combination of a fixing member and a flexible member. This flow path is opened and closed when the flexible member is deformed by the pressing mechanism (FIGS. 3 and 5 of Patent Document 1).

  In the disposable dual-chamber reaction container for amplification reaction disclosed in Patent Document 2, a connection conduit that connects the first chamber and the second chamber so that a fluid sample can flow is opened and closed by valve means (see Patent Document 2). 44, 45 etc.).

JP 2005-176836 A Japanese Patent Laid-Open No. 11-341975

  In the nucleic acid detection cassette disclosed in Patent Document 1, since the cassette is blocked according to each function, there is a problem that the size of the cassette is increased in a detection reaction using a plurality of reagents. Further, since a pressing mechanism is required for each cassette, there is a concern that the entire apparatus will be enlarged.

  In the disposable dual chamber reaction vessel for amplification reaction disclosed in Patent Document 2, as disclosed in paragraphs “0064” to “0084”, a control system such as a valve is required to open and close the conduit. The specific mechanism of this control system is not disclosed, and it is unclear how to realize the control system with a small and simple configuration.

  In addition, when using a pretreatment reagent capable of performing pretreatment and PCR at the same time as described above, it is not preferable that a sample such as blood and a reagent are mixed and stirred particularly in the initial reaction. In the initial stage of this reaction, it is considered that the nucleic acid amplification (PCR) inhibitory action derived from a sample such as blood is suppressed. If the sample and the reagent are mixed and stirred at the initial stage of the reaction, the efficiency of subsequent PCR is lowered, and there is a possibility that amplification of the nucleic acid necessary for detection by the nucleic acid microarray becomes incomplete.

  The nucleic acid detection cassette disclosed in Patent Document 1 and the disposable dual chamber reaction vessel for amplification reaction disclosed in Patent Document 2 are considered to perform PCR after the step of extracting nucleic acid from a sample. The configuration suitable for the pretreatment described above is not adopted.

  The present invention has been made in view of these circumstances, and an object of the present invention is to realize a nucleic acid detection cassette that can be miniaturized and can be attached to and detached from the apparatus body and is suitable for disposable use. Is to provide. Another object of the present invention is to provide a cassette-type kit suitable for sample pretreatment, PCR, and nucleic acid detection.

  (1) The nucleic acid detection cassette according to the present invention is attached to a device main body having a pump for sending gas, and a first actuator, a second actuator, and a third actuator, each of which can change its posture into at least two different postures. The

This nucleic acid detection cassette is
(a) a PCR tank having an opening through which a sample and a PCR reagent can be injected, and capable of storing a liquid containing the sample and the PCR reagent;
(b) a nucleic acid detection tank provided with a nucleic acid microarray on which nucleic acid probes are fixed;
(c) a first reagent tank in which a first reagent used in the first step of performing hybridization between the nucleic acid probe and a nucleic acid to be detected in a sample is stored;
(d) a second reagent tank storing a second reagent used in the second step of reacting the nucleic acid probe hybridized with the nucleic acid to be detected and an optically detectable substance;
(e) a cleaning liquid tank storing a cleaning liquid used in a cleaning step for removing the unreacted liquid after each of the above steps;
(f) a waste liquid tank in which the waste liquid in each of the above steps is discharged;
(g) The PCR tank and the pump are connected to each other so that the gas sent from the pump can flow therethrough, and at least partly has a first deformable portion that can be elastically deformed, and based on the posture change of the first actuator A first flow path opened and closed by
(h) The PCR tank and the nucleic acid detection tank are connected so that the liquid stored in the PCR tank can be circulated, and at least part of the second tank is elastically deformable, and the posture of the second actuator A second flow path opened and closed based on the change;
(i) The nucleic acid detection tank and the waste liquid tank are connected so that the liquid stored in the nucleic acid detection tank can be circulated, and at least a part of the third deformation part is elastically deformable. A third flow path that is opened and closed based on a change in posture;
(j) a plurality of fourth flow paths respectively connecting the first reagent tank, the second reagent tank, the cleaning liquid tank, and the pump so that the gas sent from the pump can flow.
(k) The first reagent tank, the second reagent tank, the washing liquid tank, and the nucleic acid detection tank are connected to each other so that the first reagent, the second reagent, or the washing liquid can be circulated, and elastically at least partially. A plurality of fifth flow paths each having a deformable fourth deformable portion;
(l) A first posture in which each fourth deforming portion of each of the plurality of fifth flow paths is elastically deformed to close the fifth flow path, and each fourth deforming portion is elastically returned to each of the fifth flow paths. A plurality of first pressing members that change posture to a second posture that opens the road;
(m) An elastic member that elastically biases each of the first pressing members to a first posture.

  The nucleic acid detection cassette is used by being mounted on the apparatus main body. The first flow path is closed by closing the first actuator. The second flow path is closed by closing the second actuator. Thereby, the PCR tank is brought into a state in which no gas or liquid flows in and out through the first channel and the second channel. A sample and a reagent are injected into the PCR tank in this state from the opening. Then, a predetermined PCR is performed. After the PCR, the first actuator is brought into an open posture, whereby the first flow path is opened. Thereby, the gas sent out from the pump can be given to the PCR tank as a pressure for causing the liquid stored in the PCR tank to flow out. Further, the second flow path is opened by opening the second actuator. Thereby, the liquid stored in the PCR tank flows out to the nucleic acid detection tank through the second flow path by the gas.

  In the nucleic acid detection tank, two steps of hybridization and optical detection are performed. The first reagent, the second reagent, and the cleaning liquid used at a predetermined timing in the hybridization and optical detection are stored in the first reagent tank, the second reagent tank, and the cleaning liquid tank, respectively. The first reagent tank, the second reagent tank, the cleaning liquid tank, and the nucleic acid detection tank are independently connected by a plurality of fifth flow paths. Through each of the fifth flow paths, the first reagent, the second reagent, and the cleaning liquid are discharged to the nucleic acid detection tank. The fifth flow path can be opened and closed by elastic deformation of the fourth deforming portion, and the fifth flow path is opened and closed based on the posture change of the first pressing member. Each first pressing member is elastically biased by the elastic member and is in the first posture. By changing the posture of each first pressing member at a predetermined timing, any one of the first reagent, the second reagent, and the cleaning liquid flows out to the nucleic acid detection tank at a predetermined timing.

  The third flow path is closed by setting the third actuator to the closed posture. Then, when the first pressing member is set to the first posture and the fifth flow path is closed, the nucleic acid detection tank is closed. Then, by supplying the first reagent, the second reagent, or the cleaning liquid at a predetermined timing, the hybridization as the first step and the optical detection or the cleaning step as the second step are performed. After each of these steps, the third flow path is opened by opening the third actuator. In addition, the first flow path and the second flow path are opened by opening the first actuator and the second actuator. Thereby, the gas sent out from the pump can be given to the nucleic acid detection tank as the pressure at which the liquid (waste liquid) stored in the nucleic acid detection tank flows out. By applying this gas, the waste liquid in the nucleic acid detection tank is discharged to the waste liquid tank through the third channel.

  (2) The first reagent tank, the second reagent tank, and the cleaning liquid tank are preferably sealed except for the fourth flow path and the fifth flow path. This prevents the first reagent, the second reagent, and the cleaning liquid from leaking out during storage or transportation of the nucleic acid detection cassette.

  (3) It is preferable that the fourth channel is connected to the vicinity of the upper end of the first reagent tank, the second reagent tank, or the cleaning liquid tank. Thereby, while being able to flow in gas from a 4th flow path to each tank, the leakage of a 1st reagent, a 2nd reagent, and a washing | cleaning liquid is prevented.

  (4) The apparatus main body further includes a drive source that generates a rotational drive force, and the nucleic acid detection cassette includes a rotation shaft that rotates based on drive transmission from the drive source, and the plurality of second pressing members. Corresponding to each of the rotating shafts, each of the pressing members corresponding to each of the rotating shafts is changed in posture as the rotating shaft rotates, and all the first pressing members at the predetermined rotating position of the rotating shaft are And a plurality of cams in one posture.

  For example, a cam follower is used as the first pressing member, and each cam corresponding thereto changes the posture of the first pressing member (cam follower) against the elastic biasing by the elastic member. Thereby, opening and closing of the plurality of fifth flow paths is controlled based on the rotational positions of the plurality of cams. Further, when the rotation axis is set to a predetermined rotation position, each cam has all the corresponding first pressing members (cam followers) in the first posture. Thereby, all the plurality of fifth flow paths are closed. Thereby, when the nucleic acid detection cassette is stored or transported, the first reagent, the second reagent, and the cleaning liquid are prevented from flowing out to the nucleic acid detection tank.

  (5) It is preferable that the plurality of cams change the posture of the first pressing member, which is driven by any one of the cams, to the second posture according to the rotational position of the rotation shaft. Thereby, a desired flow path can be opened according to the rotational position of the rotating shaft.

  (6) The PCR tank preferably has a lid that closes the opening. Thereby, it can prevent that a liquid evaporates from a PCR tank in PCR.

  (7) The lid may be held in a posture to close the opening by being pressed by the second pressing member. Thereby, even if a PCR tank is heated in PCR and the internal pressure increases, the opening can be reliably closed.

  (8) The second pressing member may be a heater provided in the apparatus main body for heating the PCR tank. Thereby, simplification and size reduction of an apparatus structure are implement | achieved.

  According to the present invention, the first flow path, the second flow path, and the third flow path for flowing the liquid out of the PCR tank can be opened and closed by the first actuator, the second actuator, and the third actuator. Downsizing is realized. In addition, since the opening for injecting the sample and the reagent is provided in the PCR tank, the injection of the sample and the reagent is easy. In particular, when sample pretreatment and PCR are simultaneously performed in a PCR tank, the sample and the reagent can be gently injected into the PCR tank so as not to be mixed and stirred. Therefore, a cassette type kit suitable for sample pretreatment and PCR can be realized.

  In addition, according to the present invention, since the opening and closing of the fifth flow path is controlled based on the change in the posture of the first pressing member, the opening and closing of a plurality of flow paths can be realized with a simple structure, and the device Miniaturized. Furthermore, since the first pressing member is biased to the first posture by the elastic member, the first reagent tank, the second reagent tank, and the cleaning liquid tank are filled with the first reagent, the second reagent, and the cleaning liquid in advance. Even if the nucleic acid detection cassette is stored or transported, the first reagent, the second reagent, and the cleaning liquid do not flow out to the nucleic acid detection tank.

  Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.

  FIG. 1 is a block diagram showing a schematic configuration of the apparatus main body 11 of the nucleic acid detection apparatus 10. FIG. 2 is a block diagram showing a schematic configuration of the nucleic acid detection cassette 12. FIG. 3 is a plan view showing an external configuration of the nucleic acid detection cassette 12. FIG. 4 is a partial cross-sectional view showing the IV-IV cross section in FIG. FIG. 5 is a partial cross-sectional view showing a VV cross section in FIG. 3. FIG. 6 is a partial cross-sectional view showing a VI-VI cross section in FIG. 3. FIG. 7 is a flowchart showing a detection method in the present embodiment.

[Entire Configuration of Nucleic Acid Detection Device 10]
The nucleic acid detection device 10 includes a device main body 11 and a nucleic acid detection cassette 12 that can be attached to the device main body 11. The apparatus main body 11 is a so-called analyzer, and is repeatedly used with the nucleic acid detection cassette 12 mounted every time it is used. The nucleic acid detection cassette 12 is a so-called disposable cassette that is discarded when used in one nucleic acid detection.

[Device Main Body 11]
The apparatus main body 11 mainly includes a heating unit 21, a drive unit 22, an air supply unit 23, an optical detection unit 24, and a control unit 25. Although not shown in FIG. 1, the apparatus main body 11 also has a known configuration such as a power supply unit, an operation unit, a liquid crystal display unit, and an interface for connecting to an external information device. The heating unit 21 heats the PCR tank 51 and the nucleic acid detection tank 52 of the nucleic acid detection cassette 12. The heating unit 21 includes a Peltier element 31, a counter heater 32, and a ceramic heater 33. The Peltier element 31 and the counter heater 32 are used for temperature adjustment of the PCR tank 51. The Peltier element 31 and the counter heater 32 are arranged so as to sandwich the PCR tank 51 of the nucleic acid detection cassette 12 mounted on the apparatus main body 11 from above and below (see FIG. 4). The Peltier element 31 is disposed below the PCR tank 51, and the counter heater 32 is disposed above the PCR tank 51. The counter heater 32 is movable in the vertical direction, and is separated from the PCR tank 51 when the nucleic acid detection cassette 12 is detached. A predetermined temperature cycle suitable for PCR is realized in the PCR tank 51 by the pair of Peltier elements 31 and the counter heater 32. The counter heater 32 corresponds to the second pressing member in the present invention. The ceramic heater 33 is used for heating the inside of the nucleic acid detection tank 52.

  The drive unit 22 includes a first microactuator 34, a second microactuator 35, a third microactuator 36, and a stepping motor 37. The first microactuator 34, the second microactuator 35, and the third microactuator 36 correspond to the first actuator, the second actuator, and the third actuator in the present invention. The stepping motor 37 corresponds to the drive source in the present invention. The first microactuator 34, the second microactuator 35, and the third microactuator 36 have the same configuration except for the arrangement in the apparatus main body 11 corresponding to each flow path. Is explained.

  As shown in FIG. 5, the first microactuator 34 is disposed on the lower side with respect to the nucleic acid detection cassette 12 mounted on the apparatus main body 11. The first microactuator 34 has a motor 38 and a piston 39. Although not shown in detail in FIG. 5, the piston 39 is connected to the output shaft of the motor 38 via a rotation-linear motion conversion mechanism. This rotation-linear motion conversion mechanism also functions as a so-called speed reducer. As an example of such a speed reducer, there is a small speed reducer disclosed in JP-A-2006-349013. When a predetermined drive current is applied to the first microactuator 34 and the motor 38 is driven, the piston 39 moves up and down based on the rotation direction of the motor 38. By this vertical movement, the first microactuator 34 changes its posture to an open posture that opens each channel or a closed posture that closes each channel. The output shaft of the stepping motor 37 is connected to a gear 99 (see FIG. 3) of a rotating shaft 88 described later.

  The air supply unit 23 includes an air pump 40 and a switching valve 41. The air pump 40 is a pump that sends out compressed air (hereinafter also simply referred to as “air”), and is connected to the switching valve 41 by a flow path through which air can flow. When the nucleic acid detection cassette 12 is attached to the apparatus main body 11, the switching valve 41 is connected to the PCR tank 51, the first reagent tank 54, the second reagent tank 55, and the cleaning liquid tanks 56 and 57 of the nucleic acid detection cassette 12, respectively. Are connected so that they can be sent out. By switching the flow path of the switching valve 41, the air sent from the air pump 40 is selectively sent to either the PCR tank 51, the first reagent tank 54, the second reagent tank 55, or the cleaning liquid tanks 56, 57. .

  The optical detection unit 24 includes a light source 42 and a detection camera 43. The light source 42 and the detection camera 43 are arranged corresponding to the nucleic acid detection tank 52. The type of the light source 42 is selected corresponding to a second reagent described later. In this embodiment, a light source capable of irradiating light with a wavelength of 200 to 400 nm, which is the excitation wavelength of the intercalator, is selected. The detection camera 43 is a CCD camera, and a camera having a predetermined number of pixels or more capable of analyzing a nucleic acid microarray image is employed.

  The control unit 25 is mainly configured as an arithmetic device such as a CPU, a ROM, or a RAM, and controls operations and image analysis of the heating unit 21, the drive unit 22, the air supply unit 23, and the optical detection unit 24. Although not shown in FIG. 1, the control unit 25 includes hardware such as a drive circuit as necessary. A part of the control unit 25 may be configured by an external information device such as a personal computer connected to the apparatus main body 11.

[Nucleic acid detection cassette 12]
As shown in FIGS. 2 and 3, the nucleic acid detection cassette 12 is configured in a cassette type that can be attached to and detached from the apparatus main body 11. This nucleic acid detection cassette 12 is provided with reagents and reaction vessels necessary for detecting a desired nucleic acid to be detected from a sample by PCR and hybridization. That is, by attaching the nucleic acid detection cassette 12 to the apparatus main body 11, one nucleic acid detection can be performed.

  The nucleic acid detection cassette 12 includes a PCR tank 51, a nucleic acid detection tank 52, a waste liquid tank 53, a first reagent tank 54, a second reagent tank 55, and two cleaning liquid tanks 55 and 56 formed in the cassette body 50, respectively. The cassette body 50 is generally tray-shaped, and is formed integrally with the above-described tanks recessed at predetermined positions. The shape and size of the cassette body 50 are not particularly limited, but a size and shape that are easy to handle when being attached to and detached from the apparatus body 11 are employed. Moreover, it is preferable that the cassette body 50 has a shape in which the attaching / detaching direction of the nucleic acid detection cassette 12 is easily recognized.

  Specifically, a PCR tank 51, a nucleic acid detection tank 52, and a waste liquid tank 53 are formed side by side from the right side to the left side on the front side (lower side in FIG. 3) of the cassette body 50. A first reagent tank 54, a second reagent tank 55, and two cleaning liquid tanks 55 and 56 are formed side by side from the left side to the right side on the back side of the cassette body 50 (upper side in FIG. 3). As shown in FIG. 3, a first joint portion 58 is formed on the right rear side of the cassette body 50. The first joint portion 58 is connected to the air pump 40 via the switching valve 41 when the nucleic acid detection cassette 12 is attached to the apparatus main body 11.

  As shown by a dotted line in FIG. 3, a first flow path 59 through which air sent from the air pump 40 can flow from the first joint portion 58 to the PCR tank 51 is formed. The first flow path 59 is configured by a tube that connects the first joint portion 58 and the opening of the PCR tank 51. The tube is exposed and arranged on the bottom side of the cassette body 50. The material of the tube is not particularly limited, but a tube that is elastically deformable by a predetermined pressure and can be crushed, has heat resistance with respect to the PCR temperature cycle, and is not reactive with blood, reagents, or the like is suitable. is there. By this tube, the 1st flow path and the 1st deformation part in the present invention are realized.

  As shown by a dotted line in FIG. 3, a second flow path 60 through which air and liquids such as blood and reagents can flow is formed from the PCR tank 51 to the nucleic acid detection tank 52. The second flow path 60 is configured by a tube that connects the opening of the PCR tank 51 and the opening of the nucleic acid detection tank 52. This tube is the same as the tube constituting the first flow path 59, and is exposed and arranged on the bottom side of the cassette body 50. With this tube, the second flow path and the second deformation portion in the present invention are realized.

  As shown by a dotted line in FIG. 3, a third flow path 61 is formed from the nucleic acid detection tank 52 to the waste liquid tank 53 through which air and liquids such as blood and reagents can flow. The third flow path 61 is configured by a tube that connects the opening of the nucleic acid detection tank 52 and the opening of the waste liquid tank 53. This tube is the same as the tube constituting the first flow path 59, and is exposed and arranged on the bottom side of the cassette body 50. With this tube, the third flow path and the third deformation portion in the present invention are realized.

  As shown in FIG. 3, a second joint portion 62 is formed on the left back side of the cassette body 50. The second joint portion 62 is connected to the air pump 40 via the switching valve 41 when the nucleic acid detection cassette 12 is attached to the apparatus main body 11. A fourth flow path 69 through which air can flow is formed from the second joint portion 62 to the first reagent tank 54. The fourth channel 69 is connected to the vicinity of the upper end of the first reagent tank 54. A filter is provided at the opening of the second joint portion 62 so that the first reagent does not flow out.

  As shown in FIG. 3, a third joint portion 63 is formed at the left center portion of the cassette body 50. The third joint portion 63 is connected to the air pump 40 via the switching valve 41 when the nucleic acid detection cassette 12 is attached to the apparatus main body 11. A fourth flow path 70 through which air can flow is formed from the third joint portion 63 to the second reagent tank 55. The fourth flow path 70 is connected to the vicinity of the upper end of the second reagent tank 55. A filter is provided at the opening of the third joint portion 63 so that the second reagent does not flow out.

  As shown in FIG. 3, a fourth joint portion 64 is formed on the right rear side of the cassette body 50. The fourth joint portion 64 is connected to the air pump 40 via the switching valve 41 when the nucleic acid detection cassette 12 is attached to the apparatus main body 11. A fourth flow path 65 </ b> A through which air can flow is formed from the fourth joint portion 64 to the cleaning liquid tank 57. The fourth flow path 65 </ b> A is connected to the vicinity of the upper end of the cleaning liquid tank 57. A filter is provided at the opening of the fourth joint portion 64 so that the cleaning liquid does not flow out. Between the cleaning liquid tank 56 and the cleaning liquid tank 57, a fourth flow path 65B through which air and the cleaning liquid from the air pump 40 can flow is formed. The plurality of fourth flow paths in the present invention are realized by the above-described fourth flow paths 65A, 69, and 70.

  As shown by a dotted line in FIG. 3, a fifth channel 66 through which air and the first reagent can flow is formed from the first reagent tank 54 to the nucleic acid detection tank 52. The fifth channel 66 is constituted by a tube that connects the opening of the first reagent tank 54 and the opening of the nucleic acid detection tank 52. This tube is the same as the tube constituting the first flow path 59. The tube constitutes the fifth flow path and the fourth deformation portion in the present invention.

  As shown by a dotted line in FIG. 3, a fifth flow path 67 through which air and the second reagent can flow is formed from the second reagent tank 55 to the nucleic acid detection tank 52. The fifth channel 67 is configured by a tube that connects the opening of the second reagent tank 55 and the opening of the nucleic acid detection tank 52. This tube is the same as the tube constituting the first flow path 59. The tube constitutes the fifth flow path and the fourth deformation portion in the present invention.

  As shown by a dotted line in FIG. 3, a fifth flow path 68 through which air and cleaning liquid can flow is formed from the cleaning liquid tank 56 to the nucleic acid detection tank 52. The fifth flow path 68 is configured by a tube connecting the opening of the cleaning liquid tank 56 and the opening of the nucleic acid detection tank 52. This tube is the same as the tube constituting the first flow path 59. The tube constitutes the fifth flow path and the fourth deformation portion in the present invention. The plurality of fifth flow paths in the present invention are realized by the fifth flow paths 66, 67, 68 described above.

  Below, the detailed structure of each part of the nucleic acid detection cassette 12 is demonstrated. As shown in FIG. 4, the PCR tank 51 is a triple tank-shaped tank having an open top, and can store a predetermined volume of liquid. The triple saddle shape is formed by forming a continuous space in which a circular hole 71, a large bowl 72 and a small bowl 73 are overlapped from the upper side to the lower side. The hole 71 is press-fitted with a lid 74 that closes the upper opening of the large collar 72. The lid 74 closes in close contact with the upper opening of the large bowl 72. The lid 74 has a shape in which a central portion thereof is recessed along the hole 71. When the nucleic acid detection cassette 12 is attached to the apparatus main body 11, the counter heater 32 is fitted into the recessed portion of the lid 74. Thereby, even if the vapor pressure inside the PCR tank 51 increases, the posture in which the lid 74 is press-fitted into the hole 71 is maintained. In FIG. 4, the Peltier element 31 and the counter heater 32 of the apparatus main body 11 are shown, but these do not constitute the nucleic acid detection cassette 12.

  The large ridge 72 and the small ridge 73 are spaces for storing liquid in a state where the lid 74 is press-fitted into the hole 71. A first flow path 59 and a second flow path 60 formed in the horizontal direction are connected to the boundary between the large bowl 72 and the small bowl 73, respectively. Although not shown in FIG. 3, the first flow path 59 and the second flow path 60 are closed by clips 76 and 77 described later. That is, in a state where the lid 74 is press-fitted into the hole 71, the space formed by the large ridge 72 and the small ridge 73 is a closed space. When the lid 74 is removed from the hole 71, the upper opening of the large bowl 72 is opened. From this upper opening, the sample and the sample pretreatment reagent can be injected into the large basket 72 and the small basket 73. Since the gavel 73 is located below the position (opening) where the first flow path 59 and the second flow path 60 are connected, the liquid stored in the gavel 73 is unlikely to flow into the second flow path 60. Therefore, solids such as blood coagulum generated in the PCR accumulate in the gavel 73. This prevents the second flow path 60 from being clogged with solids.

  As shown in FIG. 3, the nucleic acid detection tank 52 is formed as a circular hole in the cassette body 50 and stores a predetermined volume of liquid. A nucleic acid microarray 75 is fixed to the bottom of the nucleic acid detection tank 52. The upper part of the nucleic acid detection tank 52 is closed by a lid. The lid of the nucleic acid detection tank 52 is composed of a translucent member through which light emitted from the light source 42 can be transmitted in the vertical direction. The second flow path 60 and the third flow path 61 are connected to the nucleic acid detection tank 52. The second flow path 60 and the third flow path 61 are closed by clips 77 and 78 described later. ing. Further, the fifth flow paths 66, 67, and 68 are connected to the nucleic acid detection tank 52. The fifth flow paths 66, 67, and 68 are closed by cam followers 85, 86, and 87 described later. ing.

  The nucleic acid microarray 75 is a substrate in which a single-stranded nucleic acid probe capable of hybridizing with a nucleic acid to be detected in a sample to be detected is immobilized on a substrate, and is also referred to as a DNA chip. As the substrate, plastic or glass is used, and the shape is not particularly limited, but a flat plate of several tens to several thousand square millimeters is common. For the fixation of the nucleic acid probe to the substrate, physical treatment or chemical treatment may be employed. Examples of the physical treatment include a spotting method in which a solution such as a nucleic acid probe is spotted on a substrate by an extrusion method using a dispenser or an ink jet method. Examples of the chemical treatment include a method of modifying a functional group capable of forming a covalent bond with a substrate at the end of the nucleic acid probe, and a method of modifying a functional group capable of covalent bonding to the end of the nucleic acid probe and the substrate. Moreover, the base material may be subjected to plasma treatment as necessary.

  As shown in FIG. 3, the waste liquid tank 53 is formed as a square hole in the cassette body 50 and stores a predetermined volume of liquid. The waste liquid tank 53 has a capacity sufficient to store the waste liquid from the nucleic acid detection tank 52. Although the waste liquid tank 53 is sealed, an opening is formed in the vicinity of the upper end of the waste liquid tank 53 so as to be open to the atmosphere, and a filter is provided in the opening so that the waste liquid does not flow out.

  As shown by dotted lines in FIG. 3, clips 76, 77, and 78 are provided in the first flow path 59, the second flow path 60, and the third flow path 61, respectively. Each of the clips 76 to 78 has the same configuration except that the flow path to be opened and closed is different, and thus the detailed configuration will be described by taking the clip 76 provided in the first flow path 59 as an example.

  As shown in FIG. 5, the clip 76 is provided on the bottom surface side of the cassette body 50 and opens and closes the first flow path 59. The clip 76 is a bar material in which a shaft 80 is supported by a bearing portion 79 suspended from the bottom surface side of the cassette body 50 and both ends thereof perform a seesaw motion. The first end 81 of the clip 76 is bent upward and can be brought into contact with and separated from the tube constituting the first flow path 59. The second end 82 that is opposite to the first end 81 is also bent upward. When the nucleic acid detection cassette 12 is disposed in the apparatus main body 11, the first microactuator 34 is positioned below the second end portion 82. In FIG. 5, the first microactuator 34 is shown, but the first microactuator 34 does not constitute the nucleic acid detection cassette 12.

  A support plate 83 is disposed below the first end portion 81 with a predetermined space therebetween. The support plate 83 is formed integrally with the cassette body 50. A coil spring 84 is interposed between the first end 81 and the support plate 83 with the upper surface of the support plate 83 as a spring seat. The first end 81 is urged upward by the coil spring 84. That is, in FIG. 5, the clip 76 is biased counterclockwise about the shaft 80 (hereinafter, when the clip 76 is simply referred to as clockwise or counterclockwise, it indicates the state in FIG. 5). . The urging force of the coil spring 84 is strong enough to crush the tube constituting the first flow path 59. Therefore, the clip 76 is urged counterclockwise, and the first end portion 81 crushes the tube (first flow path 59) so as to sandwich the cassette body 50. As a result, the first flow path 59 is closed.

  The first microactuator 34 changes its posture between a closed posture shown in FIG. 5 (A) and an open posture shown in FIG. 5 (B). The driving force of the first microactuator 34 is strong enough to rotate the clip 76 clockwise against the biasing force of the coil spring 84. Therefore, when the first microactuator 34 pushes up the piston 39 to the open position, the second end 82 is pushed upward by the piston 39 and the clip 76 rotates clockwise against the biasing force of the coil spring 84. . As a result, the first end portion 81 is lowered and separated from the tube (first flow path 59), the tube is elastically restored, and the first flow path 59 is opened.

  The clip 77 is not described in detail with reference to the drawings. However, as with the clip 76, the second flow path 60 is closed when the second actuator 35 is in the closed position, and the second actuator 35 is in the open position. Then, the second flow path 60 is opened. Similarly, the clip 77 closes the third flow path 61 when the third actuator 36 is in the closed posture, and opens the third flow path 61 when the third actuator 36 is in the open posture.

  As indicated by dotted lines in FIG. 3, cam followers 85, 86, and 87 are provided in the fifth flow paths 66, 67, and 68, respectively. The cam followers 85, 86, and 87 correspond to a plurality of first pressing members in the present invention. Three cams 89, 90, 91 are provided on the rotary shaft 88 corresponding to the cam followers 85, 86, 87. The cam followers 85, 86, 87 and the cams 89, 90, 91 have the same configuration except that the flow paths to be opened and closed are different. Therefore, the detailed configuration of the cam follower 86 and the cam 90 provided in the fifth flow path 67 is taken as an example. Is explained.

  As shown in FIG. 6, the cam follower 86 is provided on the bottom surface side of the cassette body 50 and opens and closes the fifth flow path 67. The cam follower 86 is rotatably supported by a shaft 92 provided on the bottom surface side of the cassette body 50. The cam follower 86 has a generally disc shape having two convex portions 93 and 94. The convex portion 93 projects upward and can be brought into contact with and separated from the tube constituting the fifth flow path 67. The convex portion 94 projects laterally and contacts the cam 90.

  A support plate 95 is disposed above the convex portion 94 with a predetermined space therebetween. The support plate 95 is formed integrally with the cassette body 50. A coil spring 96 is interposed between the convex portion 94 and the support plate 95 with the lower surface of the support plate 95 as a spring seat. The convex portion 94 is urged downward by the coil spring 96. The coil spring 96 corresponds to the elastic member in the present invention. That is, in FIG. 6, the cam follower 86 is urged clockwise about the shaft 92 (hereinafter, the cam follower 86 is simply referred to as clockwise or counterclockwise indicates the state in FIG. 6). The urging force of the coil spring 96 is strong enough to crush the tube constituting the fifth flow path 67. Therefore, the cam follower 86 is urged clockwise, and the convex portion 93 crushes the tube (fifth flow path 67) so as to sandwich the cassette body 50. Thereby, the fifth channel 67 is closed. The posture of the cam follower 86 that closes the fifth flow path 67 is referred to as a first posture in the present invention.

  The cam 90 has a shape in which the radius of the circumferential surface thereof is continuously changed by continuously forming a short small diameter portion 97 and a long large diameter portion 98 in the radial direction of the rotary shaft 88. The ratio of the large diameter portion 98 to the entire circumferential surface is ¼ or less. That is, the small-diameter portion 97 always exists at a position rotated 90 ° from the large-diameter portion 98 in any rotation direction of the rotary shaft 88. The ratio between the length of the large diameter portion 98 and the length of the small diameter portion 97 in the radial direction of the rotating shaft is set as a dimension necessary for changing the posture of the cam follower 86.

  As shown in FIG. 3, three cams 89 to 91 are fixed to the rotating shaft 88 while being separated in the axial direction. The cams 89 and 91 are not described in detail here with reference to the drawings, but each has the same small diameter portion and large diameter portion as the cam 90. The three cams 89 to 91 are coaxially fixed to the rotation shaft 88 and are rotated together with the rotation of the rotation shaft 88. The directions in which the large-diameter portions of the cams 89 to 91 protrude with respect to the rotary shaft 88 are different, but will be described later. The rotating shaft 88 is rotatably supported by a bearing (not shown) provided on the bottom surface side of the cassette body 50 with its axial direction intersecting with the fifth flow paths 66, 67, 68.

  A gear 99 is provided at one end of the rotating shaft 88. The gear 99 is connected to the output shaft of the stepping motor 37 when the nucleic acid detection cassette 12 is arranged in the apparatus main body 11. When the stepping motor 37 is rotated, the rotation is transmitted to the rotating shaft 88, and the cams 89 to 91 are rotated to change their posture.

  As shown in FIG. 6 (A), the convex portion 93 of the cam follower 86 crushes the tube (the fifth flow path 67), and the posture of the cam 90 that can close the fifth flow path 67 is the closed position. . As shown in FIG. 6B, when the rotating shaft 88 rotates, the cam 90 changes its posture to the open posture. At this time, the large diameter portion 98 of the cam 90 comes into contact with the convex portion 94 of the cam follower 86. As a result, the convex portion 94 is pushed upward against the biasing force of the coil spring 96, and the cam follower 86 rotates counterclockwise. Thereby, the convex part 93 falls and leaves | separates from a tube (5th flow path 67), a tube returns elastically and the 5th flow path 67 is open | released. The posture of the cam follower 86 that opens the fifth flow path 67 is referred to as a second posture in the present invention.

  Although the detailed description using drawings is abbreviate | omitted about the other cam followers 85 and 87 and the cams 89 and 91, when the cam 89 becomes a closed attitude | position like the cam follower 86 and the cam 90, the cam follower 85 will be 5th flow path. 66 is closed, and the cam follower 86 opens the fifth flow path 66 when the cam 89 is in the open posture. Similarly, the cam follower 87 closes the fifth channel 68 when the cam 91 is in the closed posture, and the cam follower 87 opens the fifth channel 68 when the cam 91 is in the open posture.

  Each of the cams 89 to 91 has a timing at which the corresponding cam followers 85 to 87 are opened to be different from the rotation timing of the rotary shaft 88. In other words, at different rotational positions on the rotating shaft 88, each cam 89 changes the posture of the corresponding cam follower 85 to 87 to the open posture. Therefore, according to the rotational position of the rotating shaft 88, one of the three fifth flow paths 66, 67, 68 is opened, and the others are closed. Further, when the rotary shaft 88 reaches a predetermined rotational position, all of the cams 89 to 91 maintain the corresponding cam followers 85 to 87 in a closed posture. In other words, at the rotational position of the rotation shaft 88, all the three fifth flow paths 66, 67, 68 are closed. This rotational position is set as the initial position of the rotary shaft 88.

  The rotation positions of the rotary shaft 88 are such that the cam followers 85 to 87 driven by the cams 89 to 91 are changed to the open postures with respect to a certain rotational direction, and the fifth flow paths 66, 67 and 68 are opened. The order is arranged in consideration of the order of each step included in the nucleic acid detection reaction described later.

  When the rotation shaft 88 rotates in a certain rotation direction, that is, in either the clockwise direction or the counterclockwise direction, the corresponding cams 89 to 91 correspond to any one at each rotation position (No. 1 to 4) that sequentially arrives. The cam followers 85 to 87 are changed in posture to the open posture, and any of the three fifth flow paths 66, 67, and 68 are opened. That is, the direction from which each large diameter part protrudes in each cam 89-91 is either of each rotation position (No. 1-4). In the present embodiment, the rotation direction of the rotation shaft 88 in which each rotation position arrives in ascending order (from 1 to 4) is referred to as normal rotation, and the rotation direction of the rotation shaft 88 that arrives in descending order (from 4 to 1). This is called reversal.

  As shown in Table 1, no. The rotational position indicated by 1 is the initial position of the rotary shaft 88 described above, and none of the cams 89 to 91 opens the corresponding flow path at this rotational position. No. At the rotational position indicated by 2, the cam 89 changes the posture of the cam follower 85 and opens the fifth flow path 66. That is, the large diameter portion of the cam 89 is at a position rotated by 90 ° in the forward rotation direction from the initial position. No. 3, the cam 90 changes the posture of the cam follower 86 and opens the fifth flow path 67. That is, the large-diameter portion 98 of the cam 90 is at a position rotated 180 ° from the initial position in the forward rotation direction. No. At the rotational position indicated by 4, the cam 91 changes the posture of the cam follower 87 and opens the fifth flow path 68. That is, the large diameter portion of the cam 97 is at a position rotated by 270 ° in the forward rotation direction from the initial position.

  When any of the fifth flow paths 66, 67, 68 is opened, as described later, the first reagent tank 54, the second reagent tank 55, or the cleaning liquid tanks 56, 57 corresponding to the flow path are respectively stored. The first reagent, the second reagent, or the washing solution thus made flows out to the nucleic acid detection tank 52. As shown in Table 1, the rotational position of the rotary shaft 88 is No. When forward rotation is performed so as to sequentially change from 1 to 4, the flow paths are opened in the order of the fifth flow path 66, the fifth flow path 67, and the fifth flow path 68, and the first reagent, the second reagent, Each liquid flows out to the nucleic acid detection tank 52 in the order of the cleaning liquid. The order of the first reagent, the second reagent, and the washing solution that flow out to the nucleic acid detection tank 52 is set based on the order of each step included in the nucleic acid detection reaction described later.

  That is, the rotational position No. which is the initial position. Rotation position No. 1 adjacent to 2 and 4, the fifth channel 66 or the fifth channel 68 is set to be opened. Therefore, when the rotary shaft 88 is rotated from the initial position in either the forward rotation direction or the reverse rotation direction, the fifth flow path 66 or the fifth flow path 68 is opened without opening the other flow paths. As a result, the first reagent or the cleaning liquid flows out to the nucleic acid detection tank 52. The first reagent is a reagent used in the first (first step) in the nucleic acid detection reaction among the reagents filled in the nucleic acid detection cassette 12, and the cleaning liquid is a reagent used in the second. . And the 2nd reagent used for the 3rd (2nd step) is rotation position No. which is not adjacent to an initial position. 3, the fifth flow path 67 is set to be opened.

  The first reagent, the second reagent, and the cleaning solution are prefilled in the nucleic acid detection cassette 12. The first reagent tank 54 is filled with the first reagent. The first reagent is a liquid serving as a reaction field for forming a double strand between the nucleic acid to be detected in the sample and the nucleic acid probe fixed to the nucleic acid microarray 75, and is also referred to as a hybridization solution. A 1st reagent is a solution mainly containing surfactant, The kind is not specifically limited, A well-known thing can be used. The first reagent tank 54 is filled with the first reagent in a state where all but the fourth channel 69 and the fifth channel 66 are sealed.

  The second reagent tank 55 is filled with the second reagent. The second reagent is a liquid that can optically detect the nucleic acid to be detected that is hybridized and fixed to the nucleic acid microarray 75. The second reagent mainly includes mono-modified cyclodextrin. A mono-modified cyclodextrin is a compound in which an intercalator is modified to a cyclodextrin via a linker. The second reagent tank 55 is filled with the second reagent in a state where all but the fourth channel 70 and the fifth channel 67 are sealed. In this embodiment, a nucleic acid hybridized with a mono-modified cyclodextrin is detected. This nucleic acid detection is performed by a known method such as an enzyme coloring method, a chemiluminescence method, a fluorescent labeling method, a radioactive substance labeling method, or the like. A known reagent according to these methods can be used as the second reagent.

  Each of the cleaning liquid tanks 56 and 57 is filled with a cleaning liquid. The washing liquid is a liquid for washing the nucleic acid microarray 75 to remove unreacted reagents during the respective reactions of hybridization and adsorption of mono-modified cyclodextrin. The cleaning solution is not particularly limited, and a known one can be used. For example, water or a buffer solution can be used as the cleaning solution. Each of the cleaning liquid tanks 56 and 57 is filled with the cleaning liquid in a state where the parts other than the fourth flow paths 65A and 65B and the fifth flow path 68 are sealed.

[Nucleic acid detection method]
Hereinafter, a nucleic acid detection method using the nucleic acid detection apparatus 10 will be described in detail.
As shown in FIG. 7, the nucleic acid detection cassette 12 is first attached to the apparatus main body 11. In the nucleic acid detection cassette 12, the rotation shaft 88 is positioned at the initial position (rotation position No. 1). Therefore, each cam 89-91 has each corresponding cam follower 85-87 closed. As a result, all of the fifth flow paths 66, 67, 68 are closed, so that the reagent or the cleaning liquid flows out from the first reagent tank 54, the second reagent tank 55, and the cleaning liquid tanks 56, 57 to the nucleic acid detection tank 52. There is no. In the apparatus main body 11, the first microactuator 34, the second microactuator 35, and the third microactuator 36 are all closed. Therefore, even if the nucleic acid detection cassette 12 is attached to the apparatus main body 11, the clips 76 to 78 are in the closed posture, and the first flow path 59, the second flow path 60, and the third flow path 61 are closed.

  Subsequently, the lid 74 of the PCR tank 51 is removed, the large basket 72 and the small basket 73 (see FIG. 4) are opened, and the sample, the sample pretreatment reagent, and the PCR reagent are sequentially injected (S2). The sample is, for example, human blood, but is not limited to blood as long as it contains a nucleic acid to be detected, and may be a pretreated nucleic acid or cells, a liquid containing cells, and the like. Mouthwash, urine, saliva, and other secretions. Further, the sample is dissolved in a pH adjusting solution or the like as necessary.

  The sample pretreatment reagent is a reagent that inactivates a PCR inhibitor contained in blood or the like, and a commercially available product (for example, trade name: Ampdirect Plus, manufactured by Shimadzu Corporation) may be used. As the PCR reagent, a known reagent can be used as long as it includes an amplification primer that forms a double strand with the nucleic acid to be detected and a nucleic acid polymerase. In addition, the sample pretreatment reagent and the PCR reagent may be prepared as one solution.

  It is not preferable that the sample pretreatment reagent is bubbled or mixed and stirred with the sample. Therefore, the sample or the pretreatment reagent is gently injected so as to overlap the solution already injected into the PCR tank 51. For example, after injecting the sample into the small basket 73, the pretreatment reagent is gently injected into the large basket 74 so as to overlay the sample. After sequentially injecting a sample, a sample pretreatment reagent, and a PCR reagent into the PCR tank 51, the PCR tank 51 is sealed with a lid 74.

  The order of mounting the nucleic acid detection cassette 12 to the apparatus main body 11 (S1) and the injection of the sample, the sample pretreatment reagent and the PCR reagent into the PCR tank 51 (S2) may be reversed. When the nucleic acid detection cassette 12 is moved, there is a risk that the liquid level may swell or the sample may be mixed in the PCR tank 51. Therefore, it is preferable to attach the nucleic acid detection cassette 12 to the apparatus body 11 first.

  Subsequently, the nucleic acid detection apparatus 10 is operated to perform preprocessing and PCR (S3). Processing steps for preprocessing and PCR are input to the control unit 25 of the apparatus main body 11 in advance. Based on this processing step, the control unit 25 operates the Peltier element 31 and the counter heater 32 to perform preprocessing and PCR in the PCR tank 51. The counter heater 32 is moved so as to press the lid 74 of the PCR tank 51 in this processing step. This processing step is controlled mainly by the temperature cycle and the holding time. First, as a pretreatment, the PCR tank 51 is heated to about 80 ° C. and held for 15 minutes. Thereby, the PCR inhibitory substance in a sample is deactivated.

  Thereafter, as PCR, (1) a denaturation step of about 92 to 95 ° C. for about 0.1 second to 1 minute, (2) an annealing step of about 50 to 65 ° C. for about 0.1 second to 1 minute, (3) Three steps of a chain extension step of about 70 to 75 ° C. and about 0.1 second to 5 minutes are repeated about 1 to 40 times. In the denaturation step, the nucleic acid in the sample is denatured into single strands. In the annealing step, the amplification primer is bound to the single-stranded nucleic acid, and a double-stranded portion serving as a PCR reaction start point is created. In the strand extension step, the nucleic acid polymerase reacts to extend the double-stranded portion to create a double-stranded nucleic acid. Note that the pretreatment and PCR do not necessarily have to be clearly distinguished.

  When the preprocessing and the PCR are finished, the control unit 25 operates the first microactuator 34 and the second microactuator 35 and operates the air pump 40 and the switching valve 41 (S4). The first microactuator 34 and the second actuator 35 are each changed in posture from the closed posture to the open posture. In conjunction with this, the postures of the clips 76 and 77 are changed, and the first flow path 59 and the second flow path 60 are opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the first flow path 59. This air flows from the first flow path 59 into the PCR tank 51 while increasing the internal pressure. That is, the air in the first channel 59 and the PCR tank 51 is compressed by the air. Then, by being pressed by the air that has flowed in, the sample in the PCR tank 51, the sample pretreatment reagent, and the PCR reagent (hereinafter collectively referred to as “PCR solution”) flow out to the second flow path 60. Then, the PCR solution flows into the nucleic acid detection tank 52 through the second channel 60. The control unit 25 operates the first microactuator 34 and the second microactuator 35 after a predetermined time required for the PCR solution to flow out of the PCR tank 51 and closes the air pump 40. Stop. Thereby, the first flow path 59 and the second flow path 60 are closed again.

  Preferably, the PCR solution is heated in the nucleic acid detection tank 52, and the amplified nucleic acid is denatured into a single strand (S5). Processing steps for this nucleic acid denaturation are input to the control unit 25 of the apparatus body 11. Based on this processing step, the control unit 25 operates the ceramic heater 33. This processing step is mainly controlled by heating and holding time, and in this embodiment, the nucleic acid detection tank 52 is heated to about 90 ° C. and held for 5 minutes. Thereby, the nucleic acid in the PCR solution is denatured into a single strand.

  Subsequently, the control unit 25 operates the stepping motor 37 and operates the air pump 40 and the switching valve 41 (S6). When the stepping motor 37 is operated, the rotary shaft 88 is rotated. The rotation shaft 88 has a rotational position No. at which the fifth flow path 66 is opened. 90 ° forward rotation from the initial position to 2. As the rotation shaft 88 rotates, the cam 89 changes the posture of the cam follower 85 from the closed posture to the open posture, and as a result, the fifth flow path 66 is opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the second joint portion 62. The air flows from the second joint portion 62 into the first reagent tank 54 while increasing the internal pressure. That is, the air in the first reagent tank 54 is compressed by the air. Pressed by the inflowing air, the first reagent in the first reagent tank 54 flows out to the fifth channel 66. Then, the first reagent flows into the nucleic acid detection tank 52 through the fifth channel 66. After a predetermined time necessary for the first reagent to flow out of the first reagent tank 54 has elapsed, the control unit 25 operates the stepping motor 37 in the reverse direction to move the rotation shaft 88 to the initial position (rotation position No. 1). ) And the air pump 40 is stopped. As a result, the fifth channel 66 is closed again. During the forward and reverse rotation of the rotary shaft 88 accompanying the opening and closing of the fifth flow path 66, the fifth flow path 67 and the fifth flow path 68 are not opened.

  Subsequently, hybridization is performed in the nucleic acid detection tank 52 (S7). This step corresponds to the first step in the present invention. Processing steps for hybridization are input to the control unit 25 of the apparatus main body 11 in advance. Based on this processing step, the controller 25 operates the ceramic heater 33 to perform hybridization. The temperature condition for hybridization is set according to the heat denaturation temperature of the nucleic acid probe fixed to the nucleic acid microarray 75. For example, if the heat denaturation temperature of the nucleic acid probe is about 55 to 75 ° C., the ceramic heater 33 is controlled so that the inside of the nucleic acid detection tank 52 is heated to about 62 ° C. The hybridization time is set to about 1 to 30 minutes. By hybridization, the nucleic acid to be detected in the PCR solution and the nucleic acid probe fixed to the nucleic acid microarray 75 are hybridized. In order to perform hybridization efficiently, the PCR solution in the nucleic acid detection tank 52 may be stirred.

  When the hybridization is completed, the control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36, and operates the air pump 40 and the switching valve 41 (S8). The postures of the first microactuator 34, the second actuator 35, and the third microactuator 36 are changed from the closed posture to the open posture. In conjunction with this, the postures of the clips 76 to 78 are changed, and the first flow path 59, the second flow path 60, and the third flow path 61 are opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the first flow path 59. This air flows into the PCR tank 51 from the first flow path 59 and then flows into the nucleic acid detection tank 52 from the second flow path 60. Pressed by the inflowing air, the PCR solution and the first reagent in the nucleic acid detection tank 52 flow out to the third flow path 61 and are discharged to the waste liquid tank 53. The nucleic acid hybridized with the nucleic acid probe of the nucleic acid microarray 75 remains on the nucleic acid microarray 75. In the present invention, the PCR solution and the first reagent after hybridization are referred to as a waste solution. The control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36 after a predetermined time required for the PCR solution and the first reagent to be discharged to the waste liquid tank 53 has elapsed. The closed position and the air pump 40 are stopped. As a result, the first flow path 59, the second flow path 60, and the third flow path 61 are closed again.

  Subsequently, the control unit 25 operates the stepping motor 37 and operates the air pump 40 and the switching valve 41 (S9). When the stepping motor 37 is operated, the rotary shaft 88 is rotated. The rotation shaft 88 has a rotation position No. at which the fifth flow path 68 is opened. Reversed by 90 ° from the initial position to 4. As the rotation shaft 88 rotates, the cam 91 changes the posture of the cam follower 87 from the closed posture to the open posture, and as a result, the fifth flow path 68 is opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the fourth joint part 64. The air flows into the cleaning liquid tank 57 from the fourth joint portion 64 while increasing the internal pressure. That is, the air in the cleaning liquid tank 57 is compressed by the air. Pressed by the inflowing air, the cleaning liquid in the cleaning liquid tank 57 flows into the cleaning liquid tank 56 through the fourth flow path 64. Accordingly, the cleaning liquid in the cleaning liquid tank 56 flows into the nucleic acid detection tank 52 through the fifth flow path 68. After a predetermined time required for the cleaning liquid to flow out of the cleaning liquid tank 56 has elapsed, the control unit 25 operates the stepping motor 37 in the reverse direction to rotate the rotating shaft 88 forward to the initial position (rotating position No. 1). At the same time, the air pump 40 is stopped. Thereby, the fifth flow path 68 is closed again. Here, out of the cleaning liquid filled in the cleaning liquid tanks 56 and 57, the cleaning liquid corresponding to the capacity filled in the cleaning liquid tank 56 flows out to the nucleic acid detection tank 52. During the forward and reverse rotation of the rotary shaft 88 accompanying the opening and closing of the fifth flow path 68, the fifth flow path 66 and the fifth flow path 67 are not opened.

  When the nucleic acid detection tank 52 is filled with the cleaning liquid, the control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36, and operates the air pump 40 and the switching valve 41 (S10). ). In addition, in order to perform washing | cleaning efficiently, stirring may be performed after the nucleic acid detection tank 52 is filled with the washing | cleaning liquid. The postures of the first microactuator 34, the second actuator 35, and the third microactuator 36 are changed from the closed posture to the open posture. In conjunction with this, the postures of the clips 76 to 78 are changed, and the first flow path 59, the second flow path 60, and the third flow path 61 are opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the first flow path 59. This air flows into the PCR tank 51 from the first flow path 59 and then flows into the nucleic acid detection tank 52 from the second flow path 60. The cleaning liquid in the nucleic acid detection tank 52 flows out into the third flow path 61 and is discharged into the waste liquid tank 53 by being pressed by the inflowing air. Along with the cleaning liquid, the unreacted PCR liquid remaining in the nucleic acid detection tank 52 is also discharged to the waste liquid tank 53. The filling and discharging of the cleaning liquid into the nucleic acid detection tank 52 is referred to as a cleaning step in the present invention. In addition, a cleaning liquid containing an unreacted liquid is referred to as a waste liquid in the present invention. The control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36 to a closed posture after a predetermined time necessary for the cleaning liquid to be discharged to the waste liquid tank 53 has elapsed. At the same time, the air pump 40 is stopped. As a result, the first flow path 59, the second flow path 60, and the third flow path 61 are closed again.

  Subsequently, the control unit 25 operates the stepping motor 37 and operates the air pump 40 and the switching valve 41 (S11). When the stepping motor 37 is operated, the rotary shaft 88 is rotated. The rotation shaft 88 has a rotational position No. at which the fifth flow path 67 is opened. It is rotated forward 180 ° from the initial position up to 3. As the rotary shaft 88 rotates, the cam 90 changes the posture of the cam follower 86 from the closed posture to the open posture, and as a result, the fifth flow path 67 is opened. The air pump 40 sends air, and the switching valve 41 sends the air to the third joint part 63. This air flows into the second reagent tank 55 from the third joint portion 63 while increasing the internal pressure. That is, the air in the second reagent tank 55 is compressed by the air. Pressed by the inflowing air, the second reagent in the second reagent tank 55 flows out to the fifth channel 67. Then, the second reagent flows into the nucleic acid detection tank 52 through the fifth channel 67. After a predetermined time required for the second reagent to flow out of the second reagent tank 55 has elapsed, the control unit 25 operates the stepping motor 37 in the reverse direction to reverse the rotation shaft 88 to the initial position, and The pump 40 is stopped. Thereby, the fifth channel 67 is closed again.

  When the rotating shaft 88 is rotated forward and backward, the rotating shaft 88 is rotated at the rotational position No. 5, the fifth flow channel 66 is once opened, but since the first reagent has already flowed out from the first reagent tank 54, and no air is supplied to the first reagent tank 54, the nucleic acid detection tank No first reagent flows out to 52.

  Subsequently, the mono-modified cyclodextrin is adsorbed in the nucleic acid detection tank 52 (S12). The adsorption of the mono-modified cyclodextrin is referred to as the second step in the present invention. Processing steps for this adsorption are input in advance to the control unit 25 of the apparatus main body 11. Based on this processing step, the control unit 25 operates the ceramic heater 33. The temperature condition for adsorption of the mono-modified cyclodextrin is controlled by the ceramic heater 33 so that the inside of the nucleic acid detection tank 52 is heated to about 45 ° C., for example. Moreover, about 1 to 30 minutes are set as adsorption time. The mono-modified cyclodextrin intercalator is stably disposed at or near the π stacking structure in the double-stranded nucleobase bond on the nucleic acid microarray 75 and exhibits predetermined fluorescence characteristics. In addition, in a state where the mono-modified cyclodextrin is dissolved in the second reagent, the intercalator is disposed in or near the hydrophobic cavity of the cyclodextrin and becomes water-soluble. Of the two arrangement states of the intercalator, the arrangement state at or near the π stacking structure in the double-stranded nucleobase bond is more stable. In order to efficiently adsorb the mono-modified cyclodextrin, the second reagent in the nucleic acid detection tank 52 may be stirred.

  When the adsorption of the mono-modified cyclodextrin is completed, the control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36, and operates the air pump 40 and the switching valve 41 (S13). . The postures of the first microactuator 34, the second actuator 35, and the third microactuator 36 are changed from the closed posture to the open posture. In conjunction with this, the postures of the clips 76 to 78 are changed, and the first flow path 59, the second flow path 60, and the third flow path 61 are opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the first flow path 59. This air flows into the PCR tank 51 from the first flow path 59 and further flows into the nucleic acid detection tank 52 from the second flow path 60. Pressed by the inflowing air, the second reagent in the nucleic acid detection tank 52 flows out to the third flow path 61 and is discharged to the waste liquid tank 53. The second reagent after adsorption is referred to as waste liquid in the present invention. In addition, the mono-modified cyclodextrin arranged at or near the π stacking structure in the double-stranded nucleobase bond on the nucleic acid microarray 75 remains on the nucleic acid microarray 75. The control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36 after the predetermined time necessary for the second reagent to be discharged to the waste liquid tank 53 has elapsed to close the position. And the air pump 40 is stopped. As a result, the first flow path 59, the second flow path 60, and the third flow path 61 are closed again.

  Subsequently, the control unit 25 operates the stepping motor 37 and operates the air pump 40 and the switching valve 41 (S14). When the stepping motor 37 is operated, the rotary shaft 88 is rotated. The rotation shaft 88 has a rotation position No. at which the fifth flow path 68 is opened. Reversed by 90 ° from the initial position to 4. As the rotation shaft 88 rotates, the cam 91 changes the posture of the cam follower 87 from the closed posture to the open posture, and as a result, the fifth flow path 68 is opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the fourth joint part 64. This air flows from the fourth joint portion 64 into the cleaning liquid tank 56 through the cleaning liquid tank 57 while increasing the internal pressure. That is, the air in the cleaning liquid tanks 56 and 57 is compressed by the air. Accordingly, the cleaning liquid in the cleaning liquid tank 56 flows into the nucleic acid detection tank 52 through the fifth flow path 68. After a predetermined time necessary for the cleaning liquid to flow out of the cleaning liquid tank 56 has elapsed, the control unit 25 operates the stepping motor 37 to rotate the rotating shaft 88 to the initial position and stops the air pump 40. Thereby, the fifth flow path 68 is closed again. Here, of the cleaning liquid filled in the cleaning liquid tanks 56 and 57, the remaining cleaning liquid (corresponding to the capacity filled in the cleaning liquid tank 57) flows out to the nucleic acid detection tank 52. During the forward and reverse rotation of the rotary shaft 88 accompanying the opening and closing of the fifth flow path 68, the fifth flow path 66 and the fifth flow path 67 are not opened.

  When the nucleic acid detection tank 52 is filled with the cleaning liquid, the control unit 25 operates the first microactuator 34, the second microactuator 35, and the third microactuator 36, and operates the air pump 40 and the switching valve 41 (S15). ). In addition, in order to perform washing | cleaning efficiently, stirring may be performed after the nucleic acid detection tank 52 is filled with the washing | cleaning liquid. The postures of the first microactuator 34, the second actuator 35, and the third microactuator 36 are changed from the closed posture to the open posture. In conjunction with this, the postures of the clips 76 to 78 are changed, and the first flow path 59, the second flow path 60, and the third flow path 61 are opened. The air pump 40 sends out air, and the switching valve 41 sends out the air to the first flow path 59. This air flows into the PCR tank 51 from the first flow path 59 and then flows into the nucleic acid detection tank 52 from the second flow path 60. The cleaning liquid in the nucleic acid detection tank 52 flows out into the third flow path 61 and is discharged into the waste liquid tank 53 by being pressed by the inflowing air. Along with the cleaning liquid, the unreacted second reagent remaining in the nucleic acid detection tank 52 is also discharged to the waste liquid tank 53. Note that after the cleaning liquid is discharged to the waste liquid tank 53, the first flow path 59, the second flow path 60, and the third flow path 61 may be left open or closed. Either is acceptable.

  Subsequently, the control unit 25 irradiates the nucleic acid detection tank 52 with light from the light source 42 and images the nucleic acid microarray 75 with the detection camera 43 (S16). The light emitted from the light source 42 is the excitation wavelength of the mono-modified cyclodextrin intercalator. When the intercalator is pyrene or fluorescein, light of about 200 to 400 nm is irradiated. The intercalator arranged at or near the π stacking structure in the double-stranded nucleobase bond on the nucleic acid microarray 75 emits light of a specific color such as yellowish green or light blue upon receiving this light. The controller 25 performs image processing on the image of the nucleic acid microarray 75 photographed by the detection camera 43 and detects the light emission of the intercalator in the image, thereby optically detecting the mono-modified cyclodextrin adsorbed on the nucleic acid microarray 75. be able to.

[Modification]
In the above embodiment, the directions in which the large diameter portions of the three cams 89 to 91 fixed to the rotating shaft 88 protrude with respect to the rotating shaft 88 are different every 90 °. It is good also as arrangement | positioning which the direction which each large diameter part protrudes with respect to the rotating shaft 88 differs every 120 degrees. Table 2 shows the relationship between the rotational position of the rotary shaft 88 and the open / closed state of each flow path due to the arrangement of the cams 89 to 91.

  In this modified example, when the rotation shaft 88 rotates in a certain rotation direction, that is, in either the clockwise direction or the counterclockwise direction, the corresponding cams 89 to 91 are respectively corresponding to the rotation positions (No. I to VI) that sequentially arrive. Change any one of the cam followers 85 to 87 to the open posture to open any one of the fifth flow paths 66 to 68, but the rotational position while each flow path is opened, that is, the rotational position No. In I, III, and V, all of the fifth flow paths 66 to 68 are closed. In other words, the direction in which each large-diameter portion of each of the cams 89 to 91 protrudes is the rotational position No. Either II, IV, or VI. In this modification, the rotation direction of the rotation shaft 88 in which each rotation position arrives in ascending order (from I to VI) is referred to as normal rotation, and the rotation direction of the rotation shaft 88 that arrives in descending order (from VI to I). This is called reversal.

  According to this modification, the above-described opening and closing of the fifth channel 66 (S6 in FIG. 7) is performed as follows. The control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotation position No. From I (initial position) to rotational position No. Rotate 60 ° forward to II. As a result, the fifth channel 66 is opened. Then, as described above, after the first reagent in the first reagent tank 54 flows out to the fifth flow channel 66, the control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotational position No. Reverse 60 ° to I. As a result, the fifth channel 66 is closed again.

  When the hybridization (S7 in FIG. 7) and the discharge of the first reagent (S8 in FIG. 7) are finished, the above-described opening and closing of the fifth flow path 68 (S9 in FIG. 7) is performed as follows. The control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotation position No. From I, rotational position No. Reverse 60 ° to VI. As a result, the fifth flow path 68 is opened. Then, after the cleaning liquid in the cleaning liquid tanks 56 and 57 flows out to the HD tank 52 as described above, the control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotational position No. From VI, the rotation position No. Reverse 60 degrees to V. Thereby, the fifth flow path 68 is closed again.

  When the cleaning step (S10 in FIG. 7) is completed, the above-described opening and closing of the fifth flow path 67 (S11 in FIG. 7) is performed as follows. The control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotation position No. Rotation position no. Reverse 60 ° to IV. Thereby, the fifth channel 67 is opened. Then, after the second reagent in the second reagent tank 55 has flowed into the HD tank 52 as described above, the control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotational position No. Rotation position no. Rotate forward 60 ° to V. Thereby, the fifth channel 67 is closed again.

  When the adsorption of the mono-modified dextrin (S12 in FIG. 7) and the discharge of the second reagent (S13 in FIG. 7) are completed, the opening / closing of the fifth flow path 68 (S14 in FIG. 7) is performed as follows. . The control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotation position No. Rotation position no. Rotate forward 60 ° from VI. As a result, the fifth flow path 68 is opened. Then, after the cleaning liquid in the cleaning liquid tanks 56 and 57 flows out to the HD tank 52 as described above, the control unit 25 operates the stepping motor 37 to set the rotation shaft 88 to the rotational position No. From VI, the rotation position No. I or rotational position No. 60 ° forward or reverse to either V. Thereby, the fifth flow path 68 is closed again.

[Operational effects of this embodiment]
As described above, according to the nucleic acid detection cassette 12, the first flow path 59, the second flow path 60, and the third flow path 61 for flowing out the PCR solution from the PCR tank 51 are replaced with the first microactuator of the apparatus main body 11. 34, since the second microactuator 35 and the third microactuator 36 can be opened and closed, the nucleic acid detection apparatus 10 can be downsized. Moreover, since the opening for injecting the sample and the pretreatment reagent is provided in the PCR tank 51, the injection of the sample and the pretreatment reagent is easy. In particular, when sample pretreatment and PCR are simultaneously performed in the PCR tank 51, the sample and the reagent can be gently injected into the PCR tank 51 to prevent mixing and stirring.

  In addition, since the opening and closing of the fifth flow paths 66, 67, and 68 are controlled based on the posture change of the cam followers 85 to 87, the opening and closing of the three fifth flow paths 66, 67, and 68 are realized with a simple structure. Thus, the nucleic acid detection device 10 can be downsized. Furthermore, since the cam followers 85 to 87 are biased to the first posture by the coil spring 96 or the like, the first reagent tank 54, the second reagent tank 55, and the cleaning liquid tanks 56 and 57 are previously provided with the first reagent, the second reagent, and the cleaning liquid. Even if the nucleic acid detection cassette 12 is stored or transported in a state in which is filled, the first reagent, the second reagent, and the cleaning liquid do not flow out to the nucleic acid detection tank 52.

  The first reagent tank 54, the second reagent tank 55, and the cleaning liquid tanks 56, 57 are sealed except for the fourth flow paths 65A, 65B, 69, 70 and the fifth flow paths 66-68. When the cassette 12 is stored or transported, the first reagent, the second reagent, and the cleaning liquid are prevented from leaking out. In particular, the fourth flow path 65A, 65B, 69, 70 is connected near the upper end of the first reagent tank 54, the second reagent tank 55, or the cleaning liquid tank 56, so that the first reagent, the second reagent, and the cleaning liquid. Leakage prevention is effectively realized.

  Further, cam followers 85 to 87 are used as the first pressing members according to the present invention, and their postures are changed based on the rotational positions of the cams 89 to 91. When the rotary shaft 88 is set to a predetermined rotational position, The cams 89 to 91 have all the corresponding cam followers 85 to 87 as the first posture. As a result, the three fifth flow paths 66, 67, and 68 are all closed, so that the first reagent, the second reagent, and the cleaning liquid flow out to the nucleic acid detection tank 52 when the nucleic acid detection cassette 12 is stored or transported. Is prevented.

  Further, each of the cams 89 to 91 changes the posture of any one of the cam followers 85 to 87 to the second posture in accordance with the rotation position of the rotation shaft 88. Therefore, a desired fifth flow path is determined depending on the rotation position of the rotation shaft 88. 66, 67, 68 can be opened.

  Moreover, since the PCR tank 51 has the lid 74 for closing the opening, the liquid is prevented from evaporating from the PCR tank 51 in the PCR.

  Further, since the lid 74 is held in a posture to close the opening of the PCR tank 51 by being pressed by the counter heater 32, even if the PCR tank 51 is heated and the internal pressure increases in PCR, the opening is not opened. It can be reliably closed. In addition, by using the counter heater 32, the configuration of the nucleic acid detection device 10 can be simplified and downsized.

FIG. 1 is a block diagram showing a schematic configuration of the apparatus main body 11 of the nucleic acid detection apparatus 10. FIG. 2 is a block diagram showing a schematic configuration of the nucleic acid detection cassette 12. FIG. 3 is a plan view showing an external configuration of the nucleic acid detection cassette 12. FIG. 4 is a partial cross-sectional view showing the IV-IV cross section in FIG. FIG. 5 is a partial cross-sectional view showing a VV cross section in FIG. 3. FIG. 6 is a partial cross-sectional view showing a VI-VI cross section in FIG. 3. FIG. 7 is a flowchart showing a detection method in the present embodiment.

Explanation of symbols

11 ... Device body 12 ... Nucleic acid detection cassette 32 ... Counter heater (second pressing member)
34... First microactuator (first actuator)
35 ... Second microactuator (second actuator)
36 ... Third microactuator (third actuator)
37 ... Stepping motor (drive source)
40 ... Air pump (pump)
51 ... PCR tank 52 ... Nucleic acid detection tank 53 ... Waste liquid tank 54 ... First reagent tank 55 ... Second reagent tank 56, 57 ... Washing liquid tank 59 ... First flow Road (first deformation part)
60 ... 2nd flow path (2nd deformation part)
63 ... 3rd flow path (3rd deformation part)
65A, 65B, 69, 70 ... 4th flow path 66, 67, 68 ... 5th flow path (4th deformation part)
74: Lid 75 ... Nucleic acid microarray 85, 86, 87 ... Cam follower (first pressing member)
88 ... Rotating shafts 89, 90, 91 ... Cam 96 ... Coil spring (elastic member)

Claims (8)

A nucleic acid detection cassette attached to an apparatus main body having a pump for delivering gas and a first actuator, a second actuator, and a third actuator, each of which can change its posture into at least two kinds of postures,
A PCR tank having an opening through which a sample and a PCR reagent can be injected, and capable of storing a liquid containing the sample and the PCR reagent;
A nucleic acid detection tank provided with a nucleic acid microarray on which nucleic acid probes are fixed;
A first reagent tank in which the first reagent used in the first step of hybridizing the nucleic acid probe and the nucleic acid to be detected in the sample is stored;
A second reagent tank storing a second reagent used in the second step of reacting the nucleic acid probe hybridized with the nucleic acid to be detected and an optically detectable substance;
A cleaning liquid tank storing a cleaning liquid used in the cleaning step of removing the unreacted liquid after the first step;
Waste liquid tank in which the waste liquid in each of the above steps is discharged;
The PCR tank and the pump are connected to each other so that the gas sent from the pump can flow therethrough, and at least a part thereof has a first deformable portion that can be elastically deformed, and is opened and closed based on a change in posture of the first actuator. A first flow path,
The PCR tank and the nucleic acid detection tank are connected so that the liquid stored in the PCR tank can be circulated, and at least a part thereof has a second deformable portion that can be elastically deformed, and is based on a change in posture of the second actuator. A second flow path opened and closed by
The nucleic acid detection tank and the waste liquid tank are connected so that the liquid stored in the nucleic acid detection tank can be circulated, and at least a part of the third deformation part can be elastically deformed. A third flow path that is opened and closed based on;
A plurality of fourth flow paths respectively connecting the first reagent tank, the second reagent tank, the cleaning liquid tank, and the pump so that the gas sent from the pump can flow.
The first reagent tank, the second reagent tank, the washing liquid tank, and the nucleic acid detection tank are connected to each other so that the first reagent, the second reagent, or the washing liquid can be circulated, and can be elastically deformed at least partially. A plurality of fifth flow paths each having a fourth deformation portion;
A first posture that elastically deforms each fourth deforming portion of the plurality of fifth flow paths to close the fifth flow path, and elastically returns each fourth deformable section to open the fifth flow path. A plurality of first pressing members that change postures to a second posture;
A nucleic acid detection cassette comprising: an elastic member that elastically biases each of the first pressing members to a first posture.   The nucleic acid detection cassette according to claim 1, wherein the first reagent tank, the second reagent tank, and the cleaning liquid tank are sealed except for the fourth channel and the fifth channel.   The nucleic acid detection cassette according to claim 2, wherein the fourth channel is connected to the vicinity of the upper end of the first reagent tank, the second reagent tank, or the washing liquid tank. The apparatus main body further includes a drive source for generating a rotational drive force,
A rotating shaft that rotates based on drive transmission from the drive source;
The rotary shafts are respectively provided corresponding to the plurality of first pressing members, and each of the corresponding first pressing members is changed in posture with rotation of the rotary shafts, and a predetermined rotational position of the rotary shafts The nucleic acid detection cassette according to any one of claims 1 to 3, further comprising a plurality of cams having all the first pressing members in a first posture.   5. The nucleic acid detection cassette according to claim 4, wherein the plurality of cams change the posture of the first pressing member driven by any one of the cams to the second posture according to the rotational position of the rotation shaft.   The nucleic acid detection cassette according to any one of claims 1 to 5, wherein the PCR tank has a lid for closing the opening.   The nucleic acid detection cassette according to claim 6, wherein the lid is held in a posture to close the opening by being pressed by a second pressing member.   The nucleic acid detection cassette according to claim 7, wherein the second pressing member is a heater provided in the apparatus main body to heat the PCR tank.

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