JP5593205B2 - Nucleic acid analyzer - Google Patents

Description

  The present invention relates to a nucleic acid analyzer for analyzing a biological sample by amplifying a nucleic acid contained in the biological sample.

  Analysis of nucleic acids contained in biological samples such as blood, plasma, and tissue fragments not only in academic research such as biology, biochemistry, and medicine, but also in various fields including industries such as diagnosis, crop varieties improvement, and food inspection It is done in The most widely used method for analyzing nucleic acids is a technique called PCR (Polymerase Chain Reaction), which specifically amplifies the nucleic acid in the region to be analyzed. As an application of PCR, it is possible to detect a very small amount of nucleic acid with high sensitivity by attaching a fluorescent label to the nucleic acid to be analyzed, irradiating excitation light and measuring the fluorescence intensity over time.

  In PCR, a solution containing a nucleic acid and a reagent for amplifying it is heated to about 95 ° C to thermally denature the nucleic acid, and then cooled to about 60 ° C to proceed with nucleic acid annealing and extension reaction. Repeated ~ 40 times. In the current mainstream PCR apparatus, a reaction plate called a microtiter plate having 96 to 386 reaction wells is arranged on a Peltier element, and a temperature cycle is given by raising and lowering the temperature of the Peltier element. In this method, since it takes time to change the temperature of the Peltier element itself, it has been a big problem for shortening the analysis time.

  In addition, in the above method, it is necessary to perform batch processing in which a plurality of samples set in 96 to 386 reaction wells are processed at once. Once processing is started, the next processing is completed until the batch is completed. There was also a problem that could not start.

  Non-Patent Document 1 discloses a structure in which a disk-type reaction plate having reaction wells contacts and rotates on a heater set in advance to a plurality of temperatures in order to solve the problem of speeding up the temperature cycle. . In this example, it is not necessary to change the temperature of the heater, and the temperature of the reaction plate can be changed quickly. Moreover, in order to make it easy to receive the heat from the temperature control apparatus arrange | positioned at the upper surface and the lower surface side, the device which spreads a sample solution to a plane direction is also carried out.

JP 2008-185389 A US Patent Publication 2009/0068064 A1

Tsuguto Fujimoto, et al., Jpn. J. Ingect. Dis., 63, 31-35 (2010)

  Patent Document 1 describes an example of a temperature control device having a mechanism for rotating a disk-shaped microchip having a plurality of wells. U.S. Patent No. 6,057,049 describes an apparatus configured to load a fan-shaped plate insert into a disc assay plate.

  In the conventional nucleic acid analyzer, the next reaction plate cannot be loaded until the analysis of the loaded reaction plate is completed. In addition, the reaction plate was loaded at any time, and the reaction plate could not be unloaded at any time. Conventional nucleic acid analyzers cannot perform nucleic acid analysis efficiently because the degree of freedom of sample loading and unloading is not high.

  An object of the present invention is to provide a technique capable of efficiently analyzing a sample with a high degree of freedom in loading and unloading of a reaction plate in nucleic acid analysis.

  According to the present invention, the nucleic acid analyzer includes a plurality of temperature control devices arranged along the circumferential direction, and a rotation mechanism that rotates the reaction plate assembly arranged on the temperature control device along the circumferential direction. A carry-in base and a discharge base provided on the outer peripheral side of the temperature control device, a carry-in drive mechanism for carrying the reaction plate assembly from the carry-in base onto the temperature control device, and a discharge base from the top of the temperature control device. A discharge drive mechanism that discharges the sample to the reaction plate, and a detection device that optically detects the sample loaded in the reaction plate assembly.

  According to the present invention, the rotating mechanism includes a rotating shaft and a plurality of pressing portions that rotate around the rotating shaft. The reaction plate assembly is configured to move along the circumferential direction on the temperature control device while being pressed onto the temperature control device by the pressing portion.

  The carry-in drive mechanism moves the reaction plate assembly disposed on the carry-in base inward in the radial direction, and carries it in between the pressing unit and the temperature control device.

  The discharge drive mechanism moves the reaction plate assembly disposed on the temperature control device radially outward and discharges the reaction plate assembly on the discharge base from between the pressing portion and the temperature control device.

  According to the present invention, it is possible to provide a nucleic acid analyzer capable of efficiently analyzing a sample with a high degree of freedom in loading and unloading reaction plates.

It is a figure which shows the plane structure of the principal part of the nucleic acid analyzer of this invention. It is a figure which shows the example of the temperature cycle set to the temperature control apparatus of the nucleic acid analyzer of this invention. It is a figure which shows the example of the temperature cycle set to the temperature control apparatus of the nucleic acid analyzer of this invention. It is a figure which shows the example of the reaction plate of the nucleic acid analyzer of this invention. It is a figure which shows the example of the reaction plate of the nucleic acid analyzer of this invention. It is a figure which shows the example of the reaction plate of the nucleic acid analyzer of this invention. It is a figure which shows the example of the reaction plate of the nucleic acid analyzer of this invention. It is a figure which shows the cross-sectional structure of the reaction plate of FIG. 3D. It is a figure which shows the cross-sectional structure of the temperature control apparatus of the nucleic acid analyzer of this invention. It is a figure explaining the holding mechanism of the reaction plate of the temperature control apparatus of the nucleic acid analyzer of this invention. It is a figure explaining the wiring for temperature control of the holding mechanism of the reaction plate assembly of the temperature control apparatus of the nucleic acid analyzer of this invention. It is a figure explaining the wiring for temperature control of the holding mechanism of the reaction plate assembly of the temperature control apparatus of the nucleic acid analyzer of this invention. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is explanatory drawing explaining operation | movement of the holding | suppressing part of the rotation mechanism of the nucleic acid analyzer of this invention, and a carrying-in drive mechanism. It is a schematic diagram which shows the structure of the detection optical system of the nucleic acid analyzer of this invention. It is a figure which shows the structure of the whole nucleic acid analyzer of this invention.

  A nucleic acid analyzer according to the present invention will be described with reference to FIG. FIG. 1 shows a planar configuration of the main part of the nucleic acid analyzer. The nucleic acid analyzer of this example includes first to eighth temperature control devices 20A to 20H arranged along the circumferential direction, and detection devices 23A to 23C arranged above the eighth temperature control device 20H. , A carry-in base 25 disposed on the outer peripheral side of the first temperature control device 20A, and a discharge base 27 disposed on the outer peripheral side of the fifth temperature control device 20E.

  The same number of reaction plate assemblies 10A to 10H as the number of the temperature control devices 20A to 20H can be loaded on the temperature control devices 20A to 20H. The reaction plate assemblies 10A to 10H include a reaction plate and a transparent weight member disposed thereon. The reaction plate has a reaction well 102 and a hole 103. The weight member also has a hole at a position corresponding to the hole 103 of the reaction plate. The hole of the weight member and the hole 103 of the reaction plate are connected to form the guide pin hole of the reaction plate assembly. The structure of the reaction plate will be described later with reference to FIGS. 3A to 3E. The reaction plate and the weight member are formed of a material having high thermal conductivity.

  The reaction well 102 is injected with nucleic acid and necessary reagents. A thin transparent cover made of resin is mounted on the upper surface of the reaction plate so as to cover the reaction well. The reaction well is sealed with a transparent cover. The weight member is disposed on a transparent cover attached to the upper surface of the reaction plate. The weight member prevents the transparent cover from peeling off.

  The nucleic acid analyzer of this example further includes a rotation mechanism that rotates the reaction plate assemblies 10A to 10H on the temperature control devices 20A to 20H at a constant speed along the circumferential direction. The reaction plate assemblies 10A to 10H slide on the temperature control devices 20A to 20H in thermal contact with the temperature control devices 20A to 20H.

  The rotation mechanism includes a rotation shaft 33, a support member 32 connected to the rotation shaft, and pressing portions 31 </ b> A to 31 </ b> H supported by the support member 32. The pressing portions 31A to 31H are configured to press the reaction plate assemblies 10A to 10H onto the temperature control devices 20A to 20H. Windows 311 are formed in the holding portions 31A to 31H. From the window 311, the reaction well 102 of the reaction plate can be observed. An example of the structure of the pressing portions 31A to 31H will be described later with reference to FIG.

  The rotation mechanism of this example has eight pressing portions 31A to 31H. However, other structures may be used as long as the reaction plate assemblies 10A to 10H can be pressed onto the temperature control devices 20A to 20H. For example, the support member 32 is configured as a spoke-like member attached to the rotation shaft 33, but may have another structure.

  The rotating shaft 33 is rotated at a constant speed by a motor. When the rotating shaft 33 rotates, the reaction plate assemblies 10A to 10H pressed by the pressing portions 31A to 31H also rotate together with the pressing portions 31A to 31H. The reaction plate assemblies 10A to 10H move along the circumferential direction relative to the temperature control devices 20A to 20H. During the movement of the reaction plate assemblies 10A to 10H, the reaction plate is maintained at a predetermined temperature by the temperature control devices 20A to 20H.

  The nucleic acid analyzer of this example further includes a carry-in drive mechanism for carrying the reaction plate assembly from the carry-in base 25 onto the first temperature control device 20A.

  The carry-in base 25 includes a preheat heater 251 that is a heat source for preheating (enzyme activation), and a preheat cover 252. A reaction plate assembly can be disposed on the preheating heater 251. The preheat cover 252 is configured to cover the reaction plate assembly disposed on the preheat heater 251. The distance in the height direction between the preheat heater 251 and the preheat cover 252 is substantially equal to the thickness of the reaction plate assembly. Thereby, the preheat cover 252 adheres to the reaction plate assembly. Preferably, the temperature of the preheat cover 252 is kept the same as the temperature of the preheat heater 251.

  The carry-in drive mechanism includes a pair of introduction guide pin arms 255. The guide pin arm 255 for introduction is provided with a guide pin (see FIG. 7A). The guide pin extends below the introduction guide pin arm 255.

  In order to carry the reaction plate assembly into the temperature control device, a guide pin provided on the introduction guide pin arm 255 is inserted into a guide pin hole of the reaction plate assembly. Next, the introduction guide pin arm is moved radially inward (arrow 250). The distance between the two introduction guide pin arms 255 is slightly larger than the width of the pressing portions 31A to 31H. Accordingly, the distal ends of the two introduction guide pin arms 255 are movable inward in the radial direction so as to sandwich both sides of the pressing portions 31A to 31H. Along with the movement of the introduction guide pin arm 255, the reaction plate assembly moves radially inward and is disposed in the first temperature control device 20A.

  In the carry-in drive mechanism of the present example, the reaction plate assembly can be introduced into the temperature adjusting device without stopping the rotation of the rotating shaft 33. This will be described later with reference to FIGS. 7A to 7F. .

  The carry-in drive mechanism of the present example moves the reaction plate assembly on the preheating heater 251 and further carries the reaction plate assembly from the preheating heater 251 to the first temperature control device 20A. However, the carry-in drive mechanism performs only carry-in from the preheat heater 251 to the first temperature adjustment device 20A, and other drive mechanisms may be used to move the reaction plate assembly on the preheat heater 251.

  The nucleic acid analyzer of this example further includes a discharge drive mechanism that discharges the reaction plate assembly from the fifth temperature control device 20E onto the discharge base 27.

  The discharge base 27 includes a cover 272. The discharge drive mechanism includes a pair of discharge guide pin arms 275. The discharge guide pin arm 275 is provided with a guide pin (not shown). The guide pin extends below the discharge guide pin arm 275.

  In order to eject the reaction plate assembly from the temperature control device, the ejection guide pin arm 275 is moved radially inward. The distance between the two discharge guide pin arms 275 is slightly larger than the width of the pressing portions 31A to 31H. Accordingly, the distal ends of the two discharge guide pin arms 275 are movable inward in the radial direction so as to sandwich both sides of the pressing portions 31A to 31H. Next, the guide pin provided on the discharge guide pin arm 275 is inserted into the guide pin hole of the reaction plate assembly arranged in the fifth temperature adjusting device 20E. Next, the discharge guide pin arm 275 is moved radially outward (arrow 270). As the discharge guide pin arm 275 moves, the reaction plate assembly moves radially outward and is disposed on the discharge base 27.

  In the discharge drive mechanism of this example, the reaction plate assembly can be discharged from the temperature control device without stopping the rotation of the rotary shaft 33.

  The detection devices 23A to 23C are arranged above the eighth temperature adjustment device 20H. The detection devices 23 </ b> A to 23 </ b> C may be optical detection devices that irradiate the sample loaded in the reaction well 102 with excitation light and measure the intensity of fluorescence from the sample loaded in the reaction well 102. Each of the detection devices 23A to 23C detects light emission of a different pigment. Here, since three detection devices are provided, it is possible to detect light emission of three kinds of dyes.

  When the reaction plate assembly slides over the eighth temperature control device 20H, the plurality of reaction wells of the reaction plate assembly pass directly under the detection devices 23A-23C. The plurality of reaction wells 102 are arranged in an arc shape on the reaction plate. The reaction plate assembly is disposed on the temperature control device such that the plurality of reaction wells 102 are disposed along the circumference of a circle centered on the rotation center of the rotation shaft 33. Thus, when the reaction plate assembly moves over the temperature control device along the circumferential direction, all reaction wells move along the same circumference.

  The operation of the nucleic acid analyzer of this example will be described. The nucleic acid analyzer can perform various types of analysis and analysis. Here, a case where PCR is performed will be described. A nucleic acid extracted from a sample comprising blood or tissue and a reagent (enzyme, primer, buffer, etc.) necessary for the PCR reaction are dispensed into the reaction well of the reaction plate. Reaction plate assemblies 10A to 10H are formed by sealing the reaction well with a transparent cover and placing a transparent weight member thereon.

  In the carry-in base 25, preheating (enzyme activation) is performed. The preheating heater 251 holds the reaction plate assembly at 95 ° C. Preferably, the temperature of the preheat cover 252 is also maintained at 95 ° C. The reaction plate assembly is held on the preheating heater 251 for about 10 minutes. This temperature and time are necessary to activate the enzyme added in the reaction well. This is the case when the extracted nucleic acid is DNA.

  When RNA is extracted, a reverse transcription step of holding at 40 ° C. for 5 minutes immediately before preheating (enzyme activation) is required. In this case, a 40 ° C. preheat heater (not shown), a preheat cover (not shown), and a guide pin (not shown) may be added in front of the illustrated carry-in base 25.

  When preheating (enzyme activation) is completed in the carry-in base 25, the reaction plate assembly is carried from the carry-in base 25 onto the first temperature control device 20A by the carry-in drive mechanism. The carry-in drive mechanism carries the reaction plate assembly into the first temperature adjustment device 20A without stopping the rotation of the rotary shaft 33. The reaction plate assembly slides sequentially on the temperature control devices 20A to 20H in thermal contact with the temperature control devices 20A to 20H. The temperatures of the first to eighth temperature control devices 20A to 20H are set according to a predetermined temperature cycle. That is, the nucleic acid held in the reaction well of the reaction plate is subjected to a temperature cycle constituted by the first to eighth temperature control devices 20A to 20H.

  In PCR, a temperature cycle including a step in which a solution containing a nucleic acid and an amplification reagent is heated to about 95 ° C. to thermally denature the nucleic acid, and then cooled to about 60 ° C. to perform nucleic acid annealing and extension reaction. Used. This temperature cycle is typically repeated 30-40 times.

Here, it is assumed that one temperature cycle is completed by one rotation of the reaction plate assembly. If the temperature cycle is repeated 30-40 times, the reaction plate assembly is rotated 30-40 times. The temperature of 1st-8th temperature control apparatus 20A-20H is set as follows, for example.
1st temperature control apparatus 20A ... 95 degreeC
2nd temperature control apparatus 20B ... 95 degreeC
3rd temperature control apparatus 20C ... 60 degreeC
4th temperature control apparatus 20D ... 60 degreeC
5th temperature control apparatus 20E ... 60 degreeC
6th temperature control apparatus 20F ... 60 degreeC
7th temperature control apparatus 20G ... 60 degreeC
Eighth temperature controller 20H ... 60 ° C

  The time (period) of one temperature cycle is 50 to 200 seconds, particularly 100 to 150 seconds. The time for one temperature cycle is determined by the PCR assay and reagents. When a reagent capable of high-speed reaction is used, the time for one temperature cycle can be shortened. Therefore, the nucleic acid analysis time can be shortened. Furthermore, the time for one temperature cycle will also vary depending on the material and structure of the reaction plate assembly. By using a material with high thermal conductivity as the material of the reaction plate assembly and increasing the heat transfer coefficient between the reaction plate assembly and the temperature control device, it becomes possible to improve the efficiency of nucleic acid analysis and shorten the nucleic acid analysis time. .

  The circumferential dimensions of the eight temperature control devices may be the same. In this example, the temperature control device at 95 ° C. is composed of two temperature control devices and has a size of 1/4 of the circumference. The temperature control device at 60 ° C. is composed of six temperature control devices and has a dimension of 3/4 of the circumference. When the rotation time of the rotary shaft 33 is t seconds, each reaction well stays in the temperature controller at 95 ° C. is (1/4) t seconds, and each reaction well has a temperature of 60 ° C. The time spent in the temperature control device is (3/4) t seconds. The time during which each reaction well stays in the temperature controller of 95 ° C. and the temperature controller of 60 ° C. is preset. By changing the rotation speed of the rotary shaft 33, the time during which each reaction well stays in the temperature controller at 95 ° C and the time during which the reaction well stays in the temperature controller at 60 ° C is set to a predetermined value. Can do.

  Each time one temperature cycle is completed, the sample loaded in the reaction well of the reaction plate is optically observed by the detection devices 23A to 23C arranged above the eighth temperature control device 20H. Here, since three detection devices are provided, it is possible to detect light emission of three kinds of dyes.

  When the temperature cycle is repeated a predetermined number of times, the reaction plate assembly is discharged from the fifth temperature controller 20E to the discharge base 27 by the discharge drive mechanism.

  In the nucleic acid analyzer of this example, it is not necessary to start the temperature cycle at the same time and end the temperature cycle at the same time for all the reaction plate assemblies arranged in the holding portions 31A to 31H. If any one of the holding parts 31A to 31H is empty, a new reaction plate assembly can be inserted there at any time. For example, when the reaction plate assembly arrives at the carry-in base 25 sequentially or at any time, it can be loaded into the empty holding part sequentially or at any time. After completion of the PCR reaction, the reaction plate assembly can be discharged sequentially or at any time. Thus, PCR can be efficiently performed by always inserting the reaction plate assembly into all the pressing portions 31A to 31H so that there is no space.

  In the nucleic acid analyzer of this example, the reaction plate assembly after the completion of the PCR reaction can be discharged at any time. Therefore, it is not necessary to wait for the completion of the PCR reaction for all the reaction plate assemblies. Therefore, it is possible to shorten the time until the analysis result is reported. Furthermore, since all the reaction plate assemblies move along the same circumferential orbit, the carry-in base 25, the discharge base 27, and the detection devices 23A to 23C may be provided at one place. Therefore, the throughput can be improved while suppressing an increase in the overall size of the nucleic acid analyzer.

  In the above example, one reaction cycle assembly is completed by one revolution of the reaction plate assembly. However, the reaction plate assembly may be configured to complete a plurality of temperature cycles by one rotation. For example, two temperature cycles may be completed during one rotation. In this case, the temperature of the eight temperature control devices is set to a predetermined temperature so that one temperature cycle is completed in a half cycle.

  When a plurality of temperature cycles are completed while the rotation shaft of the rotation mechanism makes one rotation, it is necessary to reduce the rotation speed of the rotation shaft 33. For example, when the same temperature cycle is completed twice while the rotation shaft of the rotation mechanism makes one rotation, the rotation speed is halved. When the rotation speed is low, there is an advantage that the detection accuracy of the light emission in the detection device is increased.

  Furthermore, in the above-described example, eight temperature control devices 20A to 20H are provided on the circumference, but the number of temperature control devices is not limited to eight. The number of temperature control devices may be 8 or less, or 8 or more. Furthermore, the dimensions in the circumferential direction of the temperature control device may all be the same, but need not be the same. That is, the temperature adjusting device may be provided by equally dividing the circumference, but the temperature adjusting device may be provided by equally dividing the circumference.

  In this example, the planar dimensions of the temperature control devices 20A to 20H are larger than the planar dimensions of the reaction plate assemblies 10A to 10H. Accordingly, reaction plate assemblies having various planar dimensions and shapes can be used as long as they can be accommodated in the temperature control device. However, when only a predetermined reaction plate assembly is used, the planar size and shape of the temperature control device may be the same as the planar size and shape of the reaction plate assembly.

  In this example, since it has several temperature control apparatus 20A-20H, the change of a temperature cycle is easy. However, when it is not necessary to change the temperature cycle, it is not necessary to provide the plurality of temperature control devices 20A to 20H. For example, when only a temperature cycle including two set temperatures of 95 ° C. and 60 ° C. is executed as in the above example, two temperature control devices of 95 ° C. and 60 ° C. May be provided. In this case, the circumferential dimension of each temperature control device may be set so as to correspond to the time during which the temperature is maintained.

  In the above example, the reaction plate assemblies loaded in the nucleic acid analyzer are all subjected to the same temperature cycle. However, by stopping the rotation mechanism, the reaction plate assembly loaded in the nucleic acid analyzer can be subjected to different temperature cycles. The rotation mechanism is stopped, and each of the temperature control devices 20A to 20H may be operated at different temperature cycles by an independent temperature program.

  It is necessary to move the reaction plate assembly to the position of the detection devices 23A to 23C every time the temperature cycle is completed. Moreover, in order to carry in and discharge the reaction plate assembly, it is necessary to move the reaction plate assembly to the carry-in base or the discharge base of the reaction plate assembly. Therefore, in this case, a mechanism for moving the detection devices 23A to 23C, the carry-in base, and the discharge base around the temperature control device may be provided.

  In the above-described example, the case where the reaction plate assembly is loaded into the temperature control devices 20A to 20H of the nucleic acid analyzer has been described, but the reaction plate may be loaded into the temperature control devices 20A to 20H.

  2A and 2B show examples of temperature cycles. The temperature cycle shown in FIG. 2A includes heating at 95 ° C. and holding at 60 ° C. as described above. The temperature cycle shown in FIG. 2B includes three steps: 95 ° C. heating, 60 ° C. incubation, and about 72 ° C. incubation. In the temperature control device of this example, a desired temperature cycle can be configured by setting the temperatures of the first to eighth temperature control devices 20A to 20H to a predetermined temperature. That is, in the temperature control device of this example, an arbitrary temperature cycle can be set by setting the temperature of each of the eight temperature control devices to an arbitrary temperature.

  An example of a reaction plate according to the present invention will be described with reference to FIGS. 3A to 3E. In the example shown in FIG. 3A, a plurality of reaction wells 102 are arranged along the circumference 100 on the upper surface of the reaction plate 101. The circumference 100 corresponds to the locus of the reaction well 102 that rotates about the rotation axis 33. Guide pin holes 103 are provided at both ends of the reaction plate 101.

  In the example shown in FIG. 3B, two rows of reaction wells 102A and 102B are formed along concentric circles. In this example, a two-dimensional CCD image sensor may be used for the detection devices 23A to 23C. In the example shown in FIG. 3C, one reaction well 102 is formed on the upper surface of the reaction plate 101.

  Another example of the reaction plate 101 is shown in FIGS. 3D and 3E. 3D shows a planar configuration of the reaction plate 101, and FIG. 3E shows a cross-sectional configuration of the reaction plate 101 taken along line AA in FIG. 3D. On the upper surface of the reaction plate 101, a reaction well 102 formed of a recess is formed. After dispensing the sample and the reagent into the reaction well 102, a thin transparent cover 105 made of resin is attached thereon.

  With reference to FIG. 4, the example of the structure of the temperature control apparatus provided in the nucleic acid analyzer of this invention is demonstrated. Here, the structure of the first temperature control device 20A will be described. The first temperature adjustment device 20A includes a heat source 202, a heat conduction plate 201 disposed thereon, and a temperature sensor 203 that detects the temperature of the heat source 202. The reaction plate assembly 10 </ b> A is configured to slide on the heat conduction plate 201. The reaction plate assembly 10A includes a reaction plate 101, a transparent cover 105 thereon, and a transparent weight member 107 thereon.

  The heat source 202 is preferably an electrical device such as a silicon rubber heater or Peltier. The heat conduction plate 201 is a plate-like rigid body. The heat conduction plate 201 is made of a material having high heat conductivity, for example, a metal such as aluminum or a ceramic, in order to make the heat of the heat source 202 uniform and efficiently transfer the heat to the reaction plate 101.

  The heat conducting plate 201 may be worn by sliding of the reaction plate assembly 10A. Therefore, in order to prevent wear, the heat conducting plate 201 may be subjected to a surface treatment. In the case of an aluminum plate, an alumite treatment may be performed. Although heat conductivity is sacrificed, a resin cover may be attached to the heat conduction plate 201 or a thin resin plate such as POM (polyacetal, polyoxymethylene) having high slidability may be adhered.

  The temperature sensor 203 is mounted so as to contact the heat source 202 or so as to contact both the heat source 202 and the heat conduction plate 201. The temperature of the heat source 202 or the heat conducting plate 201 detected by the temperature sensor 203 is sent to a control unit (not shown) of the temperature adjusting device. The control unit adjusts the current value supplied to the heat source 202 so that the detected temperature matches the predetermined temperature.

  Between the first temperature control device 20A and the second temperature control device 20B adjacent thereto, a heat insulating material 21 is filled to prevent heat interference between the temperature control devices.

  An example of the structure and operation of the reaction plate assembly holding mechanism provided in the nucleic acid analyzer of the present invention will be described with reference to FIG. The illustration of the heat conduction plate 201 is omitted. A reaction plate assembly 10A is disposed on the first temperature control device 20A. The reaction plate assembly 10A includes a reaction plate 101, a transparent cover 105 mounted on the reaction plate 101, and a transparent weight member 107 disposed thereon.

  A heater 251 of the carry-in base 25 is disposed adjacent to the temperature control device 20A. Further, a heater cover 252 is disposed on the heater 251. The distance between the temperature adjusting device 20A and the heater 251 of the carry-in base 25 is small, and both are in contact with each other. The upper surface of the temperature control device 20A and the upper surface of the heater 251 form a coplanar surface. A carry-in port 35 for carrying in the reaction plate assembly 10A is formed between the temperature adjusting device 20A and the pressing portion 31A. The carry-in port 35 is opened in a circumferential shape along the outer peripheral surface between the temperature adjusting device 20A and the pressing portion 31A. The dimension H1 in the height direction of the carry-in entrance 35 is larger than the dimension H2 of the gap between the heater 251 and the heater cover 252.

  The pressing portion 31A includes a pressing member 300A, a fulcrum 400, and a hook 401 that can be pivoted around the fulcrum. A protrusion 405 is formed on the lower surface of the outer end of the hook 401, and tapers 406 and 407 are formed on both sides of the protrusion. A recess 408 for pressing the reaction plate assembly 10A is formed inside the protrusion 405. A protrusion 403 for controlling the stroke of the hook is provided on the upper surface of the inner end of the hook 401.

  A spring 410 is mounted between the pressing member 300 </ b> A and the hook 401. Due to the elastic force of the spring 410, the hook 401 is given a pivotal force in the clockwise direction in the figure. Accordingly, the hook 401 is pressed onto the reaction plate assembly 10 </ b> A by the elastic force of the spring 410. The reaction plate assembly 10 </ b> A is pressed onto the temperature control device 20 </ b> A by the elastic force of the spring 410. When the reaction plate 101 is brought into close contact with the temperature adjusting device 20A in this way, the temperature of the reaction plate 101 becomes exactly the same as the temperature of the temperature adjusting device 20A.

  The weight member 107 is used to prevent the cover 105 attached to the reaction plate from being peeled off during the temperature cycle. Therefore, it is desirable that the weight member 107 has a flat surface so as to be in close contact with the cover 105 in a planar manner. Furthermore, it is formed of a transparent material so that the light emission of the nucleic acid in the reaction well of the reaction plate can be optically observed. As a material of the weight member 107, for example, glass having a predetermined transmittance may be used.

  The weight member 107 has a predetermined heat capacity. Therefore, even if the weight member 107 and the reaction plate 101 are in contact with each other via the cover 105, they may not be in a thermally uniform state, that is, at the same temperature. For example, when the weight member 107 is in a room temperature atmosphere and the reaction plate 101 is at a temperature of 95 ° C., a temperature difference occurs between the two. When the sample or reagent vaporized inside the reaction well comes into contact with the cover 105, the sample may be cooled and condensed. In order to avoid this, a temperature adjustment mechanism is provided in the rotation mechanism. The temperature adjustment mechanism provided in the rotation mechanism includes a temperature sensor and a heater provided in the hook 401 and the pressing portions 31A to 31H. The temperature sensor may be a thermistor or a thermocouple. The heater may be an electric resistance heater to which a DC 24V power supply is supplied. The temperature adjustment mechanism provided in the rotation mechanism holds the hook 401 and the pressing portions 31A to 31H at a predetermined temperature. Although the temperature of the reaction plate 101 changes according to the temperature set in the temperature control device, the temperature of the hook 401 and the pressing portions 31A to 31H may be maintained at a constant temperature, for example, 100 ° C.

  The holding portions 31 </ b> A to 31 </ b> H rotate around the rotation shaft 33. Therefore, in order to control the temperature of the hook 401 and the pressing portions 31A to 31H, a structure is required in which the wiring from the temperature control device does not break or get tangled by rotation. Therefore, the structure around the rotation axis will be described.

  With reference to FIG. 6A and FIG. 6B, the wiring for temperature control of the holding mechanism of the reaction plate assembly will be described. FIG. 6A shows a planar configuration of the rotation axis. Although eight support members are mounted on the rotating shaft, only one support member 32 is shown here. Around the rotating shaft 33, the same number of four electrodes 501 to 504 as the wiring are arranged. The four electrodes 501 to 504 are formed in an arc shape and are arranged concentrically. The four electrodes 501 to 504 are divided into eight segment electrodes having the same number as the support member. The eight segment electrodes are arranged corresponding to the eight temperature control devices.

  6B shows a cross-sectional configuration along line AA in FIG. 6A. A wiring 505 is disposed inside the support member 32. Here, it is assumed that four wires are arranged. A sliding electrode 506 is connected to the inner end of the wiring 505. The sliding electrodes 506 of the four wirings 505 are electrically connected to the four fixed electrodes 501 to 504, respectively.

  For example, the two electrodes 501 and 502 on the outer peripheral side may be used for the output of the temperature sensor provided on the hook 401 or the pressing portions 31A to 31H. The two electrodes 503 and 504 on the inner peripheral side may be used for supplying power to a heater provided on the hook 401 or the pressing portions 31A to 31H, for example, DC 24V and grounding.

  When the rotating shaft 33 rotates, the support member 32 also rotates. During the rotation of the support member 32, the sliding electrode 506 slides on the four electrodes 501 to 504 while making contact with each of the four electrodes 501 to 504.

  In this example, the four fixed electrodes 501 to 504 are divided into eight segment electrodes along the circumferential direction. When the support member 32 moves from one segment electrode to the next segment electrode, the sliding electrode of the support member 32 is not in contact with the fixed electrode. Therefore, at this time, the operation of the temperature sensors and heaters provided in the pressing portions 31A to 31H stops. However, since the distance between two adjacent segment electrodes is sufficiently small, the time during which the operation of the temperature sensors and heaters provided in the pressing portions 31A to 31H is stopped is short.

  When the temperature adjustment mechanism provided in the rotation mechanism is constituted by a single temperature sensor and heater, it is not necessary to divide the four fixed electrodes into eight segment electrodes. The temperature is detected by one temperature sensor representing the temperature of the eight hooks 401 or the holding parts 31A to 31H, the temperature is compared with a predetermined temperature, and the difference between the two is detected by the heaters of the hooks 401 or the holding parts 31A to 31H. Please give feedback to.

  Instead of providing a temperature sensor in the hook 401 or the holding portions 31A to 31H, a switch element that switches on and off in response to the temperature of a thermostat or the like may be provided in a wiring that supplies power to the heater instead. In this case, it is not necessary to divide the four fixed electrodes into eight segment electrodes.

  Next, with reference to FIGS. 7A to 7F, the operation of the pressing portion 31 </ b> A of the rotation mechanism and the carry-in drive mechanism will be described.

  FIG. 7A shows a state in which the reaction plate assembly 10 </ b> A is disposed on the carry-in preheating heater 251. The upper surface of the preheating heater 251 and the upper surface of the first temperature control device 20A are coplanar. A preheat cover 252 is disposed on the reaction plate assembly 10A. The carry-in drive mechanism has a guide pin arm 255 provided with a guide pin 256.

  In order to perform preheating (enzyme activation), the reaction plate assembly 10A is held on a preheating heater 251 at 95 ° C. for about 10 minutes.

  The pressing portion 31A includes a pressing member 300A, a fulcrum 400, and a hook 401 that is pivotably mounted around the branch 400. A carry-in port 35 is formed between the outer end of the pressing member 300A and the first temperature adjusting device 20A.

  A spring 410 is mounted between the pressing member 300 </ b> A and the hook 401. Due to the elastic force of the spring 410, the hook 401 is given the pivotal force in the clockwise direction shown in the figure. The protrusion 403 provided on the upper side of the inner end of the hook 401 is in contact with the pressing member 300A, and the hook cannot pivot further in the clockwise direction. Tapers 406 and 407 are formed on both sides of the protrusion 405 provided on the lower side of the outer end of the hook 401.

  FIG. 7B shows a state in which the guide pin 256 is lowered via the guide pin arm 255 connected to the drive unit. The guide pin 256 is inserted into the guide pin hole 10a of the reaction plate assembly 10A. The guide pin hole 10a is formed by connecting the hole of the weight member and the hole of the reaction plate.

  FIG. 7C shows a state where the guide pin arm 255 has moved inward in the radial direction of the rotation mechanism. When the guide pin arm 255 moves radially inward, the reaction plate assembly 10A also moves radially inward. The reaction plate assembly 10 </ b> A moves inward via the inlet 35. Since the upper surface of the preheating heater 251 and the upper surface of the first temperature control device 20A are coplanar, the reaction plate assembly 10A moves horizontally inward in the radial direction, so that the first from the upper surface of the preheating heater 251 is the first. Easily move to the upper surface of the temperature control device 20A. Here, it is assumed that the moving speed of the guide pin arm 255 is sufficiently higher than the moving speed in the circumferential direction of the pressing portion of the rotating mechanism. Therefore, it is possible to carry in the reaction plate assembly 10A without stopping the rotation mechanism.

  When the reaction plate assembly 10 </ b> A moves radially inward, the inner edge of the reaction plate assembly 10 </ b> A comes into contact with the outer taper 407 of the outer end of the hook 401.

  FIG. 7D shows a state where the guide pin arm 255 is further moved inward in the radial direction of the rotation mechanism. When the reaction plate assembly 10A further moves inward in the radial direction, the outer end of the hook 401 is pushed up by the reaction plate assembly 10A. The hook 401 pivots around the fulcrum 400 counterclockwise in this view.

  FIG. 7E shows a state where the guide pin arm 255 is further moved inward in the radial direction of the rotation mechanism. As the reaction plate assembly 10A further moves radially inward, the reaction plate assembly 10A is pushed under the recess 408 of the hook 401. Here, the hook 401 presses the reaction plate assembly 10 </ b> A by the elastic force of the spring 410. Thereby, the reaction plate assembly 10A is fixed.

  FIG. 7F shows a state where the guide pin arm 255 is raised and moved outward in the radial direction of the rotation mechanism. When the guide pin arm 255 is raised, the guide pin 115 is pulled out from the guide pin hole 10a of the reaction plate assembly 10A. Further, the guide pin arm 255 is moved outward in the radial direction to return to the initial position. Guide pin arm 255 waits to insert the next reaction plate assembly into the next empty hold.

  Note that the loading drive mechanism may be operated only when the reaction plate assembly is arranged on the loading base. However, even when the reaction plate assembly is not disposed on the carry-in base, the carry-in drive mechanism may be operated. Even if the guide pin arm 255 moves idle, the guide pin arm 255 does not collide with the pressing portion 31A or the hook 401.

  The operation of the discharge drive mechanism is the reverse of the operation of the carry-in drive mechanism. When the reaction plate assembly is discharged from the temperature control device to the discharge base by the discharge drive mechanism, the reverse operation of the above operation may be performed.

  An example of the structure of the detection device according to the present invention will be described with reference to FIG. The detection device 23 of this example includes a light source 801, a first lens 802, an excitation filter 803, a dichroic mirror 804, a second lens 805, a fluorescence filter 807, a third lens 808, and a detector 809.

  Excitation light from the light source 801 is condensed by the first lens 802 and becomes parallel light. An optimum excitation wavelength is selected by the excitation filter 803 from the parallel excitation light. The excitation light having the wavelength selected has its path changed by the dichroic mirror 804, is converged by the second lens 805, and reaches the reaction well 806. The fluorescent dye in the reaction well is excited by the excitation light and emits light.

  The light emitted from the reaction well (fluorescence) is collected by the second lens 805, becomes parallel light, and passes through the dichroic mirror 804. Based on the transmitted light, the fluorescent filter 807 selects fluorescence having a predetermined wavelength. The wavelength-selected fluorescence is focused by the third lens 808 and reaches the detector 809. The detector 809 detects fluorescence.

  The light source 801 generates excitation light that excites the fluorescent dye in the reaction well, and may be an LED or a halogen lamp. As the detector 809, a photodiode, a photomultiplier tube, or the like is used. Although not shown, a part of the excitation light transmitted through the excitation filter 803 may be divided by a beam splitter so as to monitor the excitation light.

  The light source 801, the excitation filter 803, and the fluorescence filter 807 can be changed according to the type of fluorescent dye to be detected. The nucleic acid analyzer of the present invention has three detection devices 23A to 23C, which are assumed to use three types of fluorescent dyes. A CCD image sensor may be used for the detection devices 23A to 23C.

  An example of the nucleic acid analyzer of the present invention will be described with reference to FIG. The nucleic acid analyzer of this example includes a nucleic acid extraction unit 901, a dispensing unit 902, and an amplification detection unit 903. The amplification detection unit 903 incorporates the above-described temperature control device.

  The nucleic acid extraction unit 901 is a part that extracts nucleic acid from a sample such as whole blood or tissue. The nucleic acid extraction unit 901 realizes the following steps. (1) The cells are disrupted or agarose gel is dissolved with a solution containing a chaotropic agent, and the target nucleic acid is eluted in the buffer. (2) The target nucleic acid is adsorbed on the particle surface by adding the magnetic beads (magnetic silica particles) to the dissolved sample and mixing them. (3) Unnecessary impurities such as nucleic acids and proteins are removed by repeating B / F (solid-liquid) separation in the washing solution. (4) After washing, DNA is eluted from the surface of the beads by suspending the magnetic beads in sterilized water or a low salt buffer. (5) The magnetic beads are removed from the eluate containing the target nucleic acid.

  The dispensing unit 902 is responsible for the step of dispensing the nucleic acid solution extracted in the previous step to the reaction well of the reaction plate. In the case of analysis focusing only on a single gene, a nucleic acid solution from one sample is dispensed into a single reaction well on the reaction plate. In the case of analysis focusing on a plurality of genes, a nucleic acid solution from one sample is dispensed into a plurality of reaction wells on a reaction plate. The reagent may be dispensed into each reaction well by the dispensing unit, or the manufacturer that provides the reaction plate may provide a reaction plate that has been dispensed in advance.

  After dispensing, attach a transparent cover to the top of the reaction plate. The cover is brought into close contact with the reaction plate by heat or pressure. A weight member is assembled to the dispensed and sealed reaction plate. Thus, a reaction plate assembly is formed.

  The amplification detection unit 903 generates a PCR reaction by the temperature control device, and detects this reaction in real time.

  Each process from the nucleic acid extraction unit 901 to the amplification detection unit 903 may be divided into independent devices, or may be a device that combines an amplification detection unit and an upstream dispensing unit, or an extraction unit. An integrated device from 901 to the amplification detector 903 may be used.

  Although the examples of the present invention have been described above, the present invention is not limited to the above-described examples, and it is easy for those skilled in the art to make various modifications within the scope of the invention described in the claims. Will be understood.

10A to 10H ... reaction plate assembly, 20A to 20H ... temperature control device, 21 ... heat insulating material, 23A-23C ... detection device, 25 ... carry-in base, 27 ... discharge base, 31A-31H ... presser, 32 ... support member, 33 ... Rotating shaft, 35 ... Loading port, 100 ... Circumference, 101 ... Reaction plate, 102, 102A, 102B ... Reaction well, 103 ... Guide pin hole, 105 ... Cover, 107 ... Weight member, 201 ... Heat conduction plate , 202 ... heat source, 203 ... temperature sensor, 251 ... preheat heater, 252 ... preheat cover, 255 ... guide pin arm for introduction, 256 ... pin, 272 ... cover, 275 ... guide pin arm for discharge, 400 ... fulcrum, 401 ... Hook, 405 ... Projection, 406, 407 ... Taper, 410 ... Spring, 501 to 504 ... Electrode, 505 Wiring, 506 ... sliding electrode, 801 ... light source, 802 ... lens, 803 ... excitation filter, 804 ... dichroic mirror, 805 ... lens, 807 ... fluorescent filter, 808 ... lens, 809 ... detector, 901 ... nucleic acid extraction unit, 902 ... Dispensing part, 903 ... Amplification detection part

Claims (19)

A plurality of temperature control devices arranged along the circumferential direction, a rotation mechanism for rotating the reaction plate assembly arranged on the temperature control device along the circumferential direction, and an outer peripheral side of the temperature control device A loading base and a discharging base provided; a loading drive mechanism for loading the reaction plate assembly from the loading base onto the temperature control device; and a discharge for discharging the reaction plate assembly from the temperature control device to the discharge base. In a nucleic acid analyzer having a drive mechanism and a detection device for optically detecting a sample loaded in a reaction plate assembly,
The rotating mechanism includes a rotating shaft and a plurality of pressing portions that rotate around the rotating shaft, and the reaction plate assembly is pressed onto the temperature adjusting device by the pressing portion, and the temperature It is configured to move along the circumferential direction on the adjusting device,
The carry-in drive mechanism moves the reaction plate assembly arranged on the carry-in base radially inward, and carries it between the pressing unit and the temperature control device,
The discharge driving mechanism moves the reaction plate assembly disposed on the temperature control device radially outward so as to discharge the reaction plate assembly on the discharge base from between the pressing portion and the temperature control device. The nucleic acid analyzer characterized by being comprised in this. The nucleic acid analyzer according to claim 1, wherein
The upper surface of the carry-in base and the upper surface of the temperature control device are coplanar, and the carry-in drive mechanism moves the reaction plate assembly from the carry-in base to the horizontal direction by moving the reaction plate assembly inward in the radial direction. A nucleic acid analyzer configured to be carried on a temperature control device. The nucleic acid analyzer according to claim 1, wherein
The upper surface of the discharge base and the upper surface of the temperature control device are coplanar, and the discharge drive mechanism moves the reaction plate assembly radially outward in a horizontal direction to move the reaction plate assembly to the temperature control device. A nucleic acid analyzer configured to be discharged from above into the discharge base. The nucleic acid analyzer according to claim 1, wherein
The carry-in drive mechanism includes a pair of arms movable in the radial direction with respect to the rotation axis, and a pin attached to the arms, and a distance between the pair of arms is determined by the pressing portion. greater than the circumferential width, said pins, nucleic acid analysis apparatus characterized by being configured to be inserted into the guide pin holes formed in the reaction plate assembly. The nucleic acid analyzer according to claim 1, wherein
The discharge drive mechanism includes a pair of arms movable in a radial direction with respect to the rotation axis, and a pin attached to the arms, and a distance between the pair of arms is determined by the pressing portion. greater than the circumferential width, said pins, nucleic acid analysis apparatus characterized by being configured to be inserted into the guide pin holes formed in the reaction plate assembly. The nucleic acid analyzer according to claim 1, wherein
The pressing portion includes a hook, a fulcrum that pivotally supports the hook, and a spring that imparts a pivotal force to the hook. The hook includes a protrusion at a radially outer end. The nucleic acid analyzer is configured to hold the reaction plate assembly inside the protrusion. The nucleic acid analyzer according to claim 6, wherein
A taper is formed on both sides of the protrusion, and the hook is configured to pivot about the fulcrum when the reaction plate assembly abuts against the taper. apparatus. The nucleic acid analyzer according to claim 6, wherein
A nucleic acid analyzer, wherein an inner end of the hook is provided with a protrusion for controlling a pivot stroke of the hook. The nucleic acid analyzer according to claim 1, wherein
A nucleic acid analyzer, wherein a window is formed in the pressing portion, and a sample loaded in the reaction plate assembly through the window is optically detected by the detection device. The nucleic acid analyzer according to claim 1, wherein
The nucleic acid analyzer according to claim 1, wherein the number of the pressing portions is the same as that of the temperature control device. The nucleic acid analyzer according to claim 1, wherein
The nucleic acid analyzer, wherein the plurality of temperature control devices can be independently temperature controlled according to a predetermined temperature cycle. The nucleic acid analyzer according to claim 1, wherein
The nucleic acid analyzer according to claim 1, wherein the carry-in base includes a preheater for maintaining the temperature of the reaction plate assembly at a predetermined temperature. The nucleic acid analyzer according to claim 1, wherein
A plurality of arc-shaped electrodes are concentrically arranged around the rotation axis, and each electrode is divided into a plurality of segments along the circumferential direction,
The nucleic acid analyzer is characterized in that a temperature control element is provided in the holding portion, and a sliding electrode that contacts the arc-shaped electrode is connected to an inner end of a wiring connected to the temperature control element. . The nucleic acid analyzer according to claim 1, wherein
The reaction plate assembly includes a reaction plate having a reaction well, a transparent cover that covers the reaction plate, and a transparent weight member that is arranged to cover the cover. The nucleic acid analyzer according to claim 14, wherein
A reaction well provided in a reaction plate of the reaction plate assembly disposed on the temperature control device is disposed along a circumference centering on a rotation axis of the rotation mechanism. Analysis equipment. The nucleic acid analyzer according to claim 1, wherein
A nucleic acid analyzer comprising: an extraction mechanism for extracting nucleic acid from a sample; a dispensing mechanism for dispensing the extracted nucleic acid into a reaction plate; and a detection unit for detecting PCR (polymerase chain reaction) of the nucleic acid . A plurality of temperature control devices arranged along the circumferential direction, a rotation mechanism for rotating the reaction plate assembly arranged on the temperature control device along the circumferential direction, and an outer peripheral side of the temperature control device A loading base and a discharging base provided; a loading drive mechanism for loading the reaction plate assembly from the loading base onto the temperature control device; and a discharge for discharging the reaction plate assembly from the temperature control device to the discharge base. In a nucleic acid analyzer having a drive mechanism and a detection device for optically detecting a sample loaded in a reaction plate assembly,
The carry-in drive mechanism moves the reaction plate assembly disposed on the carry-in base radially inward, and is placed on the temperature adjustment device via a carry-in port formed on the outer peripheral side of the temperature adjustment device. Carry in,
The discharge drive mechanism moves a reaction plate assembly disposed on the temperature control device radially outward and through a discharge port formed on an outer peripheral side of the temperature control device, Configured to drain on top ,
The rotating mechanism includes a rotating shaft and a plurality of pressing portions that rotate around the rotating shaft, and the reaction plate assembly is pressed onto the temperature adjusting device by the pressing portion, and the temperature It is configured to move along the circumferential direction on the adjusting device,
The nucleic acid analyzer, wherein the carry-in port and the discharge port are formed between the pressing portion and the temperature control device. The nucleic acid analyzer according to claim 17,
The upper surface of the carry-in base and the upper surface of the temperature control device are coplanar, and the carry-in drive mechanism moves the reaction plate assembly from the carry-in base to the horizontal direction by moving the reaction plate assembly inward in the radial direction. A nucleic acid analyzer configured to be carried on a temperature control device. The nucleic acid analyzer according to claim 17,
The upper surface of the discharge base and the upper surface of the temperature control device are coplanar, and the discharge drive mechanism moves the reaction plate assembly radially outward in a horizontal direction to move the reaction plate assembly to the temperature control device. A nucleic acid analyzer configured to be discharged from above into the discharge base.

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