JP6452965B2 - Nucleic acid amplification equipment - Google Patents

Description

  The present invention relates to a nucleic acid amplification apparatus for controlling and amplifying the temperature of a sample containing nucleic acid to determine the presence or absence of a target nucleic acid and to perform quantification.

  When a nucleic acid contained in a sample derived from a living body is examined, a nucleic acid amplification method that amplifies and detects the amount of the nucleic acid to a detectable level is used. As this nucleic acid amplification technique, for example, there is a technique using a Polymerase Chain Reaction (hereinafter referred to as PCR) method. In the PCR method, a desired base sequence can be selectively amplified by periodically controlling the temperature of a reaction solution in which a specimen and a reagent are mixed according to predetermined conditions. Other examples include those using a Transcription Mediated Amplification (hereinafter referred to as TMA) method or a Nucleic Acid Sequence Based Amplification (hereinafter referred to as NASBA) method. The TMA method and the NASBA method are classified as a constant temperature amplification method, and a base sequence can be amplified by controlling the temperature of a reaction solution in which a sample and a reagent are mixed to a predetermined constant temperature.

  In these nucleic acid amplification techniques, a change in fluorescence intensity emitted from a fluorescent dye labeled on the nucleic acid is analyzed in time series. Nucleic acid analyzers have been developed that control the temperature of a reaction solution composed of a specimen and a reagent used for nucleic acid amplification, measure changes in fluorescence intensity, and perform analysis.

  Japanese Patent Laid-Open No. 2002-318192 (Patent Document 1) discloses a fluorescence detection apparatus equipped with a sample holder provided with an insertion port for a sample container and a temperature adjusting means for controlling the sample to a predetermined temperature. . In this apparatus, the sample container containing a liquid sample is placed in the sample holder, and temperature control using a heater or the like is performed as a temperature control means, so that incubation at a predetermined temperature such as an enzyme reaction in the sample can be performed with high accuracy. It is described that it can be carried out and that the time-dependent change of the fluorescence signal accompanying the enzyme reaction can be monitored in real time.

  Japanese Unexamined Patent Application Publication No. 2009-139138 (Patent Document 2) discloses a reaction vessel including a plate-like body, a film member, and an upper lid member. The film member has a flat part placed on the surface of the plate state, and a bag body connected to the flat part and containing a reaction sample, and the upper lid member is placed in the shape of the flat part. The insertion opening of the film member is sealed. The bag body is made of a flexible material that deforms along the shape of the temperature adjustment member when it comes into contact with the temperature adjustment member, thereby improving the close contact between the temperature adjustment member and the film member. It is described that the conduction efficiency can be improved.

JP 2002-318192 A JP 2009-139138 A

  As a measure to solve the problem of improving the accuracy and rapidity of nucleic acid amplification methods such as PCR, which is a kind of enzymatic reaction, it is considered to improve the accuracy and rapidity of temperature control of specimen samples containing nucleic acids. It is done. According to Patent Document 1, a state in which a sample container that is a non-heating member is installed in a sample holder that is a heating member during analysis is disclosed. However, in general, both members are hard solids, and in order to improve heat conduction efficiency, a measure for increasing the degree of adhesion is required. When this adhesion is insufficient, there are problems such as insufficient heating and a long time for heating.

  The reaction container disclosed in Patent Document 2 improves the heat conduction efficiency by improving the close contact between the bag housing the sample and the temperature control member. On the other hand, there is no room for further improvement since a technique and structure for analyzing a nucleic acid amplification method such as a PCR method over time have not been presented. Furthermore, since the bag for storing individual samples is not independent (see FIG. 1), a new reaction container is installed on the nucleic acid amplification device to start the nucleic acid amplification reaction. A sample cannot be added, and a waiting time is imposed on a user who processes requests for nucleic acid amplification reactions that occur one after another. If nucleic acid amplification is to be performed with high accuracy, it is not preferable to stop the temperature control of the sample halfway. In addition, various nucleic acid amplification methods and conditions (protocols) such as reagents, temperatures, and times that differ for each base sequence to be amplified cannot be processed in parallel.

The present invention has been made in view of the above, and improves the close contact between the sample container and the temperature control member, enables the nucleic acid amplification method to be performed accurately and quickly, and has a plurality of different protocols. It is an object of the present invention to provide a reaction container and a nucleic acid amplification apparatus that can parallelize the specimens and can start the processing of another sample even if there is a process being executed.
In order to achieve the above object, the present invention provides a nucleic acid amplifying apparatus for amplifying nucleic acid in a reaction mixture in which a sample and a reagent are mixed.
A temperature control block for holding an air layer and a reaction solution and holding a sealed reaction vessel;
A temperature adjustment mechanism that is provided in a temperature control block and adjusts the temperature of the reaction solution,
The temperature control block includes a portion having the same inner diameter, a first taper portion having an inner diameter that is increased downward, a portion having the same inner diameter as the lower end of the first taper, and an inner diameter. It has a 2nd taper part which becomes small gradually toward the lower part in this order.

  According to the present invention, the temperature of the reaction solution stored in the reaction vessel can be adjusted accurately and quickly, and a plurality of types of specimens having different protocols can be processed in parallel and executed. The processing of another sample can be started even if there is processing in the middle.

It is a partial section perspective view showing a schematic structure of a nucleic acid amplification device of the present invention. It is a top view which shows schematic structure of the nucleic acid amplifier of this invention. It is a side view which shows schematic structure of the nucleic acid amplifier of this invention. It is a perspective view which extracts and shows the temperature control block of this invention. It is a figure which shows the temperature control block and reaction container of this invention. It is a figure which shows the temperature control block and reaction container of this invention. It is a figure which shows the temperature control block and reaction container of this invention. It is a top view which shows the nucleic acid amplifier of this invention. It is a perspective view which shows the nucleic acid amplifier of this invention. It is a figure which shows an example of the temperature control of PCR method. It is a figure which shows the temperature control block and reaction container of this invention. It is a figure which shows the temperature control block and reaction container of this invention. It is a figure which shows the temperature control block and reaction container of this invention. It is the schematic which shows the whole structure of the nucleic acid test | inspection apparatus provided with the nucleic acid amplification apparatus which concerns on embodiment of this invention. It is a figure which shows the temperature control block, reaction container, and clamp mechanism of this invention. It is a figure which shows the temperature control block, reaction container, and clamp mechanism of this invention. It is a figure which shows the temperature control block, reaction container, and clamp mechanism of this invention.

  An embodiment of the present invention will be described with reference to the drawings.

  FIG. 14 is a diagram schematically showing the overall configuration of the nucleic acid test apparatus 100 according to the present embodiment. In FIG. 14, the nucleic acid test apparatus 100 adds a plurality of sample containers 101 containing specimens containing nucleic acids to be amplified, a sample container rack 102 containing a plurality of sample containers 101, and added to the specimens. A plurality of reagent containers 103 in which various reagents are stored, a reagent container rack 104 in which a plurality of reagent containers 103 are stored, a reaction container 105 for mixing a specimen and a reagent, and an unused reaction container 105 Reaction container rack 106 in which a plurality of reaction containers are accommodated and an unused reaction container 105 are mounted, and a reaction solution is adjusted to dispense a specimen and a reagent from the sample container 101 and the reagent container 103 to the reaction container 105, respectively. A capping unit that seals the position 107 and a reaction container 105 containing a reaction liquid, which is a mixed liquid of a specimen and a reagent, with a lid member (not shown). 08, a stirring unit 109 for stirring the accommodated in sealed reaction vessel 105 reaction solution are provided.

The nucleic acid test apparatus 100 includes a robot arm X axis 110 provided on the nucleic acid test apparatus 100 so as to extend in the X axis direction (left and right direction in FIG. 14), and the Y axis direction (up and down direction in FIG. 14). A robot arm device 112 having a robot arm Y-axis 111 provided on the robot arm X-axis 110 so as to be movable in the X-axis direction, and a robot arm Y-axis 111 movable in the Y-axis direction. A gripper unit 113 that holds the reaction container 105 and conveys it to each part in the nucleic acid test apparatus 100 and a robot arm Y-axis 111 are provided so as to be movable in the Y-axis direction. A dispensing unit 114 for aspirating and discharging (dispensing) the reagent to the reaction container 105 placed at the reaction liquid adjustment position 107; and a dispensing unit 114 Nucleic acid amplification for subjecting a reaction solution stored in a reaction vessel 105 to a nozzle chip 115 mounted in a portion that comes into contact with a specimen or a reagent, a nozzle chip rack 116 in which a plurality of unused nozzle chips 115 are stored, and nucleic acid amplification A nucleic acid amplification apparatus comprising the apparatus 1, a disposal box 117 for discarding a used nozzle chip 115 and a used (tested) reaction container 105, an input device 118 such as a keyboard and a mouse, and a display device 119 such as a liquid crystal monitor 1 and a control device 120 that controls the overall operation of the nucleic acid testing device 100 including the number 1.

  Each sample container 101 is managed by identification information such as a barcode for each contained specimen, and is managed by position information such as coordinates assigned to each position of the sample container rack 102. Similarly, each reagent container 103 is managed by identification information such as a barcode for each stored reagent, and managed by position information such as coordinates assigned to each position of the reagent container rack 104. These identification information and position information are registered and managed in the control device 120 in advance. Further, each reaction vessel 105 is similarly managed by identification information and position information.

  Next, details of the nucleic acid amplification device 1 will be described with reference to FIGS.

   1 to 3 are a partial cross-sectional perspective view, a plan view, and a side view, respectively, showing a schematic configuration of the nucleic acid amplification device 1 according to the first embodiment of the present invention. FIG. 4 is a perspective view showing the temperature control block 10 of the holder 3 extracted and enlarged. 2 and 3, the cover 7 is omitted for the sake of explanation.

  1 to 3, the nucleic acid amplification device 1 is accommodated in a base 2, a holder 3 provided with a plurality of temperature control blocks 10 having a configuration for holding a reaction vessel 105, and a reaction vessel 105. A fluorescence detector 6 that detects fluorescence of the reaction solution and a cover 7 that covers the holder 3 and the fluorescence detector 6 are roughly provided.

  The holder 3 is provided with a disc-shaped holder base 4 arranged with the central axis facing upward (upward in FIG. 3), and around the central axis of the holder base 4 along the inner periphery. And a plurality of temperature control blocks 10 provided. The holder base 4 is provided so as to be rotatable in a circumferential direction around a rotation shaft 5a provided at the center thereof, and is rotationally driven by a stepping motor 5 which is a rotation drive device.

  The holder base 4 is formed using, for example, a member having excellent heat insulation, such as plastic, and is configured so that the temperatures between the plurality of temperature control blocks 10 are unlikely to interfere with each other. In addition, it is good also as a structure which forms a heat insulation layer by heat insulating materials, such as a polyurethane foam, between the holder base 4 and the temperature control block 10, and further reduces temperature interference.

As shown in FIG. 4, the temperature control block 10 includes a base 11 serving as a base of the temperature control block 10, and a hole-like installation position 12 provided through the base 11 in the vertical direction (vertical direction in FIG. 3). The temperature of the reaction liquid in the reaction vessel 105 is detected by detecting the temperature in the vicinity of the installation position 12 provided in the base 11 and the Peltier element 14 and the radiation fin 13 provided as a temperature adjusting device provided below the base 11. The temperature sensor 15 is provided. As the temperature sensor 15, a thermistor, a thermocouple, a resistance temperature detector, or the like is used.

  The base 11 is made of a heat conductor such as copper, aluminum, or various alloys, for example. By heating or cooling the base portion 11 with the Peltier element 14, the temperature of the reaction vessel 105 held at the installation position 12 of the base portion 11 is adjusted. Further, the heat radiation fins 13 are provided on the surface of the Peltier element 14 opposite to the base 11 to enhance the heat dissipation efficiency of the Peltier element 14. By inserting the reaction vessel 105 into the installation position 12 of the base 11 from above, the bottom of the reaction vessel 105 is held in a state of being exposed from the temperature control block 10.

  Returning to FIGS.

   One or more fluorescence detectors 6 (for example, four in the present embodiment) are provided, and are arranged at equal intervals along the outer periphery of the holder 3. The fluorescence detector 6 is disposed below the reaction vessel 105 (below the flow line of the reaction vessel 105), and performs fluorescence detection when the reaction vessel 105 passes above due to the rotation of the holder 3. When there are a plurality of fluorescence detectors 6, the reaction liquid in the reaction vessel 105 is detected or measured independently of each other.

  The fluorescence detector 6 includes an excitation light source for irradiating excitation light to the bottom (exposed portion) of the reaction vessel 105 held at the installation position 12 of the temperature control block 10, and a detection element for detecting fluorescence from the reaction solution. (Not shown). The reaction liquid stored in the reaction container 105 is fluorescently labeled with the base sequence to be amplified by the reagent, and the fluorescence from the reaction liquid generated by the excitation light irradiated to the reaction container 105 from the excitation light source is detected by the fluorescence detector 6. By detecting in step 3, the base sequence to be amplified in the reaction solution is quantified over time. The detection result is sent to the control device 120. As the excitation light source, for example, a light emitting diode (LED), a semiconductor laser, a xenon lamp, or a halogen lamp is used. As the detection element, a photodiode, a photomultiplier, a CCD, or the like is used.

  The cover 7 covers the holder 3 and the fluorescence detector 6 together with the base 2, and aims at a light shielding effect for suppressing the incidence of external light to the fluorescence detector 6 of the nucleic acid amplification device 1. The cover 7 is provided with a gate 7a that can be opened and closed (see FIG. 14), and the reaction vessel 105 is exchanged between the inside and outside of the cover 7 (that is, inside and outside of the nucleic acid amplification device 1) via the gate 7a. Is called. In FIG. 1, the gate 7a of the cover 7 is omitted.

  The control device 120 controls the entire operation of the nucleic acid test device 100, and uses various software stored in advance in a storage unit (not shown) based on the protocol set by the input device 118. The nucleic acid amplification process is performed, and the analysis result such as the fluorescence detection result and the movement status of the nucleic acid test apparatus 1 are stored in the storage unit or displayed on the display device 119.

  The operation in the present embodiment configured as described above will be described.

  First, as preparation for performing a nucleic acid amplification process, a sample container 101 containing a sample containing a nucleic acid to be amplified is stored in a sample container rack 102 of a nucleic acid test apparatus 100, and predetermined in a reagent container rack 103 by a protocol. The reagent container 103 containing various reagents to be added to each specimen is stored. Further, an unused reaction container 105 is stored in the reaction container rack 106, and an unused nozzle chip 115 is stored in the nozzle chip rack 116. In this state, the nucleic acid amplification process is started by operating the control device 120.

  When the start of the nucleic acid amplification process is instructed, the necessary number of unused reaction vessels 105 are first transported to the reaction solution adjustment position 107 by the gripper unit 113. Subsequently, an unused nozzle tip 115 is attached to the dispensing unit 114, and the specimen is dispensed from the predetermined sample container 101 to the reaction container 105. Thereafter, the used nozzle tip 115 is discarded in the disposal box 117 to prevent contamination. Subsequently, the reagent is also dispensed into a predetermined reaction vessel 105 in the same procedure, and mixed with the specimen to generate a reaction solution.

  When the required number of dispensings are completed, the reaction vessel 105 containing the reaction liquid is conveyed to the closing unit 108 by the gripper unit 113 and sealed by the lid member, and further conveyed to the agitation unit 109 for agitation processing. The The stirred reaction vessel 105 is transported by the gripper unit 113 and is inserted and held at a predetermined installation position 12 of the holder 3 through the gate 7a of the cover 7 in the stirring amplification device 1. At this time, the holder 3 is driven to rotate, and is controlled such that a predetermined installation position 12 is positioned at the position of the gate 7a. When there are a plurality of reaction vessels 105 to be processed, each of them is subjected to sealing and agitation processing by a lid member, and sequentially conveyed to a predetermined installation position 12.

  Here, the Peltier element 114 of the temperature adjusting device is controlled based on the protocol corresponding to the specimen accommodated in the reaction container 105 held in the holder 3, and the temperature of the reaction container 105 is controlled periodically and stepwise. And nucleic acid amplification treatment is performed. As described above, in the PCR method, which is a kind of nucleic acid amplification method, the temperature of a reaction solution in which a sample and a reagent are mixed is periodically changed stepwise based on a protocol corresponding to each sample, thereby obtaining a desired base. The sequence is selectively amplified. Even when a plurality of reaction vessels 105 are processed in parallel, when each reaction vessel 105 is held at the installation position 12, the nucleic acid amplification process is sequentially started and periodically stepwise based on the protocol corresponding to each sample. Change the temperature. During the nucleic acid amplification process, the holder 3 is driven to rotate, the fluorescence detector 6 detects fluorescence, and the fluorescence from the reaction solution is detected by the fluorescence detector 6, whereby the base sequence to be amplified in the reaction solution is detected. Quantification is performed over time. The detection results are sequentially sent to the control device 120.

  When the predetermined nucleic acid amplification process is completed, the reaction vessel 105 is transported to the disposal box 117 by the gripper unit 113 via the gate 7a and discarded.

  The effect in this Embodiment comprised as mentioned above is demonstrated.

  In the nucleic acid amplification technique using the PCR method, conditions (protocols) such as reagents, temperature, and time used differ depending on the base sequence to be amplified. Therefore, when a plurality of types of samples having different base sequences to be amplified are processed in parallel, it is necessary to individually set the temperature and the time defined in the protocol for each sample. However, in the prior art, there is only one type of protocol that can be handled at a time, and parallel processing for processing a plurality of types of samples with different protocols in parallel is not possible. In addition, since the samples having the same protocol cannot be processed with different start times, it is not possible to newly start another sample processing until the processing being executed is completed.

  On the other hand, in the present embodiment, the temperature adjusting device provided with each of the temperature control blocks 10 includes the holder 3 provided with a plurality of temperature control blocks 10 for holding the reaction vessel 105 containing the reaction liquid. Because the temperature of the reaction solution is adjusted by the method, multiple types of samples with different protocols can be processed in parallel, and processing of another sample can be started even if there is a process in progress. Can be greatly improved.

  Each temperature control block 10 is detachable from the holder base 4, and when a certain temperature control block 10 breaks down, the temperature control block 10 can be easily inspected and replaced. Further, by changing the shape of the erection position 12 provided at the base of the temperature control block 10, reaction vessels having different shapes can be erected on the holder base 4 at the same time. In addition, an arbitrary temperature control block 10 can be mounted on the holder base 4 by optimizing the base 11, the temperature adjustment device 14, and the temperature sensor 15 in order to correspond to a specific analysis item. Accordingly, various analysis items can be performed with the same holder 4 in a state in which the apparatus state is optimized with respect to the specified temperature. Note that a fan may be installed to promote heat exchange at the radiating fins 13 and forced air cooling may be performed. Further, air from the fan may be guided to a desired position by a duct so as to increase heat radiation efficiency. You may comprise.

  In order to suppress an increase in the ambient temperature inside the nucleic acid amplification device 1 covered with the cover 7, an intake fan that sends outside air into the cover 7 and an exhaust fan that discharges the outside air may be installed. Thereby, the atmospheric temperature inside the nucleic acid amplification device 1 can be kept constant, and the temperature change of the holder base 4 and the temperature control block 10 can be continuously performed.

  Furthermore, in order to promote heat dissipation such as Joule heat generated by energization of Peltier elements and sensors, the holder base 4 and the rotating shaft 5a are made of a material having excellent heat conductivity such as aluminum, and the surface area is increased. It may be widely used, heat conductive grease may be used for the joint surfaces of the members, or the surface roughness of the joint surfaces of the members may be reduced to improve the adhesion between the members.

  Further, a heat pipe may be incorporated in the holder base 4 or the rotary shaft 5a to positively move the heat from the holder base 4 or the rotary shaft 5a to other members, in addition to fins or fans. The heat dissipation efficiency can be further improved by appropriately installing a duct and a water cooling mechanism. Further, by controlling the rotation speed (relative rotation speed) of the holder base 4 with respect to the fluorescence detector 6, the relative speed between the reaction vessel 105 and the fluorescence detector 6 at the time of fluorescence measurement can be controlled. The relative speed may be a constant speed, or fluorescence detection may be performed by temporarily stopping at a position where the reaction vessel 105 and the fluorescence detector 6 face each other.

  FIG. 8 is a plan view showing the nucleic acid amplification device 1 according to the present embodiment, and FIG. 9 is a perspective view. In the figure, the same members as those described in the first embodiment are denoted by the same reference numerals and description thereof is omitted. In this embodiment, in the temperature control block 10 of the holder 3 in the first embodiment, the temperature control block 10 is disposed on the outer periphery of the holder base 4, and heat insulation is provided between the temperature control blocks 10. This is an embodiment in which a notch 16 is provided as a space.

  8 and 9, the holder 3B according to the present embodiment is arranged in the circumferential direction on the outer side of the outer periphery of the holder base 4B and the disc-shaped holder base 4B arranged with the plane portion facing upward. And a plurality of temperature control blocks 10B provided. The holder base 4B and the temperature control block 10B are formed of a heat conductor such as aluminum, copper, or various alloys, for example. The temperature control block 10B is formed integrally with the holder 3B, and a cut extending from the outer periphery of the holder base 4B toward the center is provided between the temperature control blocks 10B in the circumferential direction of the holder base 4B. A notch portion 16 is provided. As described above, the space is provided in the temperature control blocks 10B arranged side by side in the circumferential direction of the holder base 4B, so that the heat insulation ability between the temperature control blocks 10 is increased. A temperature sensor 15 that detects the temperature of the reaction liquid in the reaction vessel 105 by detecting the temperature in the vicinity of the installation position 12 and the Peltier element 17 as a temperature adjusting device is provided for each temperature control block 10B. Yes. The Peltier element 17 is attached with one surface in close contact with the temperature control block 10 and the other surface in close contact with the holder base 4B. Here, since the temperature for nucleic acid amplification is generally higher than room temperature, the holder base 4B is cooler than the temperature control block 10 for adjusting the temperature of the reaction vessel. Thereby, when lowering the temperature of the temperature control block 10, the movement of heat from the temperature control block 10 to the holder base 4B is promoted, so that the temperature can be lowered more quickly. In this embodiment, the volume of the holder base 4B can be easily increased as compared with the temperature control block 10. If the material of the holder base 4B and the temperature control block 10 is made of, for example, the same aluminum, the heat capacity of the holder base 4B can be sufficiently increased, so that the heat dissipation efficiency in each temperature control block 10 is improved. be able to. Furthermore, when performing heat exchange with a plurality of temperature control blocks 10 at the same time, heat exchange between the holder base 4B and a certain temperature control block 10 is performed by heat exchange between the holder base 4B and another temperature control block 10. Can be minimized.

  Further, in the central portion of the holder base 4B, a Peltier element 18 as a temperature adjusting device, a temperature sensor 15a for detecting the temperature in the vicinity thereof, a radiating fin 41 connected to the Peltier element 18, and an air flow to the radiating fin 41 A fan 40 is provided. For this reason, by maintaining the temperature of the holder base 4B constant (for example, 40 ° C.) by the temperature adjusting device 18, the efficiency of heat dissipation and heat absorption of the Peltier element 17 of each temperature control block 10B can be further improved.

  When the PCR method, which is one of the nucleic acid amplification methods, is performed, the temperature control block 10 repeats and loads the prescribed temperature cycle consisting of the rise and fall of the temperature on the reaction vessel. By appropriately setting the temperature, it is possible to improve the temperature changing speed and control the balance between the rising speed and the falling speed. For example, if the holder base 4B is controlled to a temperature lower than the temperature range implemented by the temperature control block 10, the speed at which the temperature is lowered can be increased, and the temperature can be controlled inside the temperature range (between the upper limit and the lower limit). For example, the maximum temperature and the rate of increase can be increased. In the NASBA method, which is one of the nucleic acid amplification techniques, the temperature control block 10 keeps the reaction vessel at a constant temperature (41 ° C.), but the temperature of the holder base 4 can be set appropriately to achieve precise temperature control. can do.

  Furthermore, by providing a fan in the base 2 and the cover 7, an air flow is forcibly generated inside the nucleic acid amplification device 1, and this air flow passes through the cut portion 16, thereby improving the heat insulation effect. it can.

  Other configurations are the same as those of the first embodiment.

  Also in the present embodiment configured as described above, the same effects as those of the first embodiment can be obtained.

  The reaction vessel and the temperature control block will be described with reference to FIG.

  FIG. 5 is a diagram showing the configuration of the reaction vessel and the temperature control block of this example. As shown in FIG. 5, this reaction container contains a reaction liquid consisting of a specimen and a reagent, and is then closed with a lid 62 by a closing unit 108, stirred by a stirring unit 109, and installed on a temperature control block 10B.

  The reaction vessel has a convex shape 61 on the outer peripheral surface. The temperature control block 10B has a concave shape 64 at the erection position 12 of the reaction vessel. When the reaction vessel is installed at the temperature control block installation position from above, the convex shape of the reaction vessel made of a soft plastic material suitable for the nucleic acid amplification method such as polypropylene elastically deforms the insertion port 66 at the installation position. Then, it is pushed down further and is securely fixed to the temperature control block in a state where it is in contact with the concave shape of the installation position and the upper contact surface 65. The distance between the upper contact surface and the lower contact surface in the reaction vessel is made to be longer than the distance between the upper contact surface and the lower contact surface of the installation position. Furthermore, the taper angle of the reaction vessel and the taper angle of the installation position are made the same at the lower contact surface. For this reason, the force that pushes the reaction vessel downward acts on the upper contact surface 65, and the force that pushes the reaction vessel upward acts on the lower contact surface 67 that is a taper structure, so that the reaction vessel is against the temperature control block. Securely fixed. The reaction solution accommodated in the reaction vessel is located near the lower contact surface that is the bottom of the reaction vessel. For this reason, the heat conduction efficiency in the vicinity of the reaction solution is increased, and the temperature control accuracy of the reaction solution can be improved.

  When the reaction vessel is installed on the temperature control block with the gripper unit, the pressing force between the reaction vessel and the lower contact surface of the installation position is not stable due to mechanical factors such as vibration of the device and manufacturing errors. It is not always easy to stably bring the container and the temperature control block into close contact with each other. If the contact between the reaction vessel and the temperature control block is inadequate, the air layer between the reaction vessel and the temperature control block intervenes, reducing the heat transfer efficiency and reducing the temperature control accuracy of the reaction solution. End up. In this method, the temperature control block is made of a metal material that has sufficient hardness and durability that does not deform even when a reaction vessel is installed, and that has excellent heat conduction efficiency. use. Compared to this temperature control block, the reaction vessel material is sufficiently soft, and the reaction vessel is deformed to match the shape of the installation position near the lower contact surface due to the force pushing the reaction vessel downward. The air layer between the reaction vessel and the temperature control block is effectively removed.

  As another embodiment, a buffer material 68 such as acrylic rubber or silicon rubber may be additionally installed on the temperature control block so as to form a lower contact surface of the temperature control block. The buffer material softer than the metal material of the temperature control block is deformed in accordance with the outer surface shape of the reaction vessel, and the air layer between the temperature control block and the reaction vessel can be more effectively removed. It is desirable that the buffer material is excellent in heat conduction efficiency.

The temperature control block is provided with an excitation light irradiation window 69 and an optical detection window 70 for measuring a change with time of nucleic acid amplification. A method of effectively removing the air layer using this excitation light irradiation window will be described. The reaction vessel is set so that the taper angle of the lower contact surface of the reaction vessel is θ _c °, the taper angle of the buffer material is θ _b °, and the magnitude relationship between θ _c and θ _b is θ _c > θ _b And cushioning material. When the reaction vessel is installed in the temperature control block, the space formed by the shape of the taper of the reaction vessel and the shape of the taper of the buffer material is gradually reduced, and air is discharged through the excitation light irradiation window. The air layer between the reaction vessel and the temperature control block can be effectively removed.

  When removing the reaction vessel that has been subjected to the nucleic acid amplification process from the nucleic acid amplification device, remove the reaction vessel that uses a soft material to such an extent that the catch is released at the upper contact surface between the reaction vessel and the temperature control block. It can be pulled out upwards with a convex shape. Due to the deformation at this time, the position and height of the convex shape can be optimized so that the sealing plug of the reaction vessel and the lid is not broken and the reaction liquid accommodated in the reaction vessel does not leak from the reaction vessel. Specifically, by providing the convex shape of the reaction container at a position lower than the height fitting with the upper lid of the reaction container, a sufficiently large bend can be ensured. In addition, the height is formed so that the catch height at the upper contact surface is about 1/100 of the outer dimensions of the reaction vessel, and the reaction vessel is sufficiently pressed against the temperature control block, and the reaction vessel Both easiness of erection and extraction can be achieved.

  FIG. 6 is a view showing a first modification of the structure in which the temperature control block is in close contact with the reaction container of the present embodiment.

  In the first modification, certain conditions are given to the outer diameter of the convex portion of the reaction vessel, the outer diameter of the insertion port at the installation position, and the outer diameter of the concave portion.

The outer diameter of the convex portion of the reaction vessel is φL _c, and the inner diameter of the insertion port at the installation position is φL _b . Although the convex outer diameter φL _c varies depending on the temperature, the general environmental temperature in which the nucleic acid amplification apparatus is installed is kept at about 30 ° C. or less, and the convex outer diameter of the reaction vessel at this time is φL _c30 If the outer diameter of the convex portion at 50 ° C., which is a general lower temperature when processing the PCR method, is _{φL c50} , the magnitude relationship with the insertion port inner diameter φL _b of the installation position is
φL _c30 <φL _b <φL _c50
It shape | molds so that it may become. Since the reaction vessel is a soft plastic, the expansion rate with respect to temperature change is large. In addition, not only the reaction liquid but also an air layer is accommodated inside the sealed reaction vessel, and when this air layer is sealed, the volume and pressure rise by heating the temperature control block, Generates a strong positive pressure. These work, outer径凸outer diameter is enlarged from .phi.L _c30 to .phi.L _c50. Temperature control block is a metal material such as aluminum material, the insertion opening inner diameter .phi.L _b fits relatively small changes.

This effect will be described. In a nucleic acid analyzer, the temperature of a reaction solution is generally adjusted in a temperature range higher than the environment in which the nucleic acid amplifier is installed in order to perform a nucleic acid amplification process. For example, in the PCR method, a temperature range of about 45 to 100 ° C. is set, and in the NASBA method, a temperature of about 41 ° C. is set. Before the reaction vessel is installed in the temperature control block of the nucleic acid amplification device, the reaction solution should be adjusted at a temperature lower than the temperature range used for nucleic acid amplification treatment so that the nucleic acid amplification reaction does not proceed unintentionally. There is a need to do. It is during this process that the cap is closed by the closing unit, and the inside of the sealed reaction vessel is maintained at about atmospheric pressure. In the process of installing the reaction vessel on the temperature control block, the insertion port inner diameter φL _b is larger than the reaction vessel outer diameter φL _c30 , and the insertion port can be passed through without load and placed against the lower contact surface. it can. After that, when the temperature control block is heated to adjust the temperature of the reaction vessel and the reaction solution, the reaction vessel outer diameter _{φL c50} becomes larger than the insertion port inner diameter _{φL b.} It is fixed by force, and the close contact with the lower contact surface is maintained. Similarly, _{φL c95} is larger than _{φL b} . As a method for further enhancing the effect of the adhesion, a concave shape can be formed at the installation position. As shown in FIG. 7, the inside of the heated reaction vessel becomes positive pressure, so that the side wall of the reaction vessel swells into a concave space, and the effect of fixing the reaction vessel to the temperature control block at the point of action is generated. To do. At this time, since the reaction solution stays in the vicinity of the taper shape of the reaction vessel, the air layer in the region surrounded by the reaction vessel and the concave shape of the installation position does not affect the heat treatment efficiency of the reaction solution. In addition, the temperature control block that holds the reaction vessel that has finished the nucleic acid amplification process quickly reduces the temperature to the standby state, and the reaction vessel contracts. For this reason, catching and friction in the concave shape of the reaction container and the installation position are eliminated, and the reaction container can be easily pulled out by the gripper unit. Preferably, a communication port that communicates the void with the outside is provided so that the void formed by the concave shape and the reaction vessel becomes a sealed space, and the closed air layer does not generate a positive pressure by heating. The communication port may have a groove shape provided upward toward the opening of the reaction vessel, a groove shape provided at the insertion port of the temperature control block, or a hole. Preferably, when the lid of the reaction vessel is inserted into the reaction vessel and used as a hermetic stopper by compression, at a position different from the position where the reaction container and the lid are fitted, so that the hermetic stopper does not loosen, It is better to provide a concave shape.

  According to this method, it is easy to place the reaction vessel on the temperature control block before starting the heat treatment for nucleic acid amplification treatment and to remove the reaction vessel from the temperature control block after the treatment is completed. In the heat treatment process, the reaction vessel can be stably adhered to the temperature control block, and the temperature can be adjusted accurately and quickly.

As a modification of this method, the concave shape of the temperature control block can be provided in a taper shape. Since the temperature control block and the reaction vessel are placed in contact with each other on the taper-shaped lower contact surface, excessive or insufficient catching due to the swelling of the reaction vessel to the concave shape caused by dimensional error during the production of the reaction vessel The influence can be reduced. Reaction vessel Te - and path angle theta _c °, Te of the temperature control block - by adjusting the path angle theta _b °, it is possible to improve the accuracy of the caught. For example, by setting θ _c ° <θ _b °, the contact between the reaction vessel and the temperature control block can be ensured by the concave shape provided in the upper portion of the taper shape.

  Another advantage of providing the concave shape of the temperature control block described in the present embodiment is that the volume of the temperature control block is reduced. When the volume of the temperature control block decreases, the heat capacity also decreases, so that the speed of temperature change when the temperature of the temperature control block is raised or lowered can be increased. In addition, as a heat transfer path at a site close to the reaction liquid, a path that penetrates the region of the reaction container sandwiched between the temperature control block and the reaction liquid in the normal direction of the surface of the reaction container is mainly, Since the influence of the path from the vicinity of the concave shape to the reaction solution via the reaction vessel can be almost ignored, it is not necessary to consider the deterioration of the heat conduction efficiency due to the provision of the concave shape. In nucleic acid amplification methods typified by the PCR method, speeding up the treatment is an issue, and this improvement can be achieved by the above-described effects.

  FIG. 11 is a diagram showing a second modification of the structure in which the reaction container and the temperature control block of the present embodiment are brought into close contact with each other.

  In Modification 2, a member having a high coefficient of expansion due to heat is added to the insertion port of the temperature control block.

A fixing member 72 made of a material having a high expansion coefficient such as silicon rubber is fixed to the insertion opening of the temperature control block by fitting. An adhesive can be applied to make the fixation stronger. The convex outer diameter of the reaction vessel is φL _c, and the inner diameter of the fixing member is φL _f . Although the fixed part inner diameter φL _f varies depending on the temperature, the general environmental temperature where the nucleic acid amplification apparatus is installed is kept at about 30 ° C. or less. The outer diameter of the fixed member at this time is _{φL f30} , and the PCR method _Assuming that the outer diameter of the fixing member at 50 ° C., which is a general lower limit temperature when treating _Φ , is φL _f50 , the magnitude relationship with the outer diameter φL _c30 of the reaction vessel at 30 ° C. is
φL _b50 <φL _c30 <φL _b30
The insertion port for the installation position and the fixing member are formed so that

  According to this method, it is easy to place the reaction vessel on the temperature control block before starting the heat treatment for nucleic acid amplification treatment and to remove the reaction vessel from the temperature control block after the treatment is completed. In addition, the reaction vessel can be brought into close contact with the temperature control block, and the temperature can be adjusted accurately and quickly.

  In this embodiment, in order to increase friction on the surface of the insertion port of the temperature control block and the outer surface of the reaction vessel, it is possible to roughen the surface accuracy and to provide uneven shapes such as knurled eyes.

  The reaction container and temperature control block which are the 2nd Example are demonstrated using FIG.

  FIG. 12 is a diagram showing the configuration and operation of the gripper unit, reaction vessel, and temperature control block. As shown in FIG. 12, a gripper unit holds a reaction vessel that contains a prepared reaction solution, is tightly stoppered, and is stirred. The reaction vessel, the temperature control block, the fixing member, and the like are the same as those in each of the embodiments and modifications described in the first embodiment.

  Initially, the temperature control block is installed in an empty position, the temperature control block is not heated, and is stable at a temperature equivalent to the installation environment temperature of the nucleic acid analyzer, or by nucleic acid amplification such as 35 ° C. The standby temperature is adjusted to be lower than the temperature range to be used.

  When an instruction to start the nucleic acid amplification process of the reaction vessel is issued by the nucleic acid amplification device, the gripper unit starts moving while holding the reaction vessel, and the reaction vessel is placed in the temperature control block. At this time, the gripper unit continues to be pressed against the temperature control block with a constant force while holding the reaction vessel. Next, heating for nucleic acid amplification processing in the temperature control block is started, and the temperature of the temperature control block converted from the output value from the temperature sensor 15 is, for example, a general lower limit for processing the PCR method. After exceeding a certain temperature such as 50 ° C., the gripper unit releases the reaction vessel, releases the pressing force, and then moves to a predetermined position.

  The reaction vessel installed in the temperature control block receives the effect of heating the temperature control block described in Example 1, and is continuously given frictional force and pressing force acting between the reaction vessel and the temperature control block. . These forces initially continue to act to ensure that the gripper unit presses the reaction vessel against the temperature control block and keeps it in close contact, and always reacts while the temperature is adjusted for nucleic acid amplification. Adhere close contact between the container and the temperature control block.

  When the nucleic acid amplification process is completed, the temperature control block in which the reaction vessel is installed reduces the temperature of the temperature control block to a standby temperature such as 35 ° C., for example. The gripper unit moves, grasps the reaction vessel, pulls it out from the temperature control block, and removes it from the nucleic acid amplification device. FIG. 10 is a diagram showing a typical example of such a temperature control method.

  According to this method, it is easy to place the reaction vessel on the temperature control block before starting the heat treatment for nucleic acid amplification treatment and to remove the reaction vessel from the temperature control block after the treatment is completed. In addition, the reaction vessel can be brought into close contact with the temperature control block, and the temperature can be adjusted accurately and quickly.

  A reaction container and a temperature control block according to the third embodiment will be described with reference to FIG.

  FIG. 13 is a diagram showing the configuration and operation of the reaction vessel and the temperature control block. As shown in FIG. 13, the temperature control block has the shape of a recess 73, and the reaction vessel has the shape of a protrusion 74 and the shape of a knurl 75. The gripper unit has a grip that engages with the knurling of the reaction vessel, a drive source 7 for rotating the grasped reaction vessel around the central axis of the reaction vessel, a detection plate, and a photo interrupter. ing.

  The operation of this embodiment will be described. The gripper unit engages and grips the reaction container with the knurling and the gripper for preventing slipping. The gripper unit grips the reaction vessel from above the installation position so that the convexity of the reaction vessel and the notch of the recess of the temperature control block pass, and the lower contact surface of the reaction vessel and the temperature control block is in close contact Then, the reaction vessel is rotated by using a drive source so that the concave portion of the temperature control block and the convex portion of the reaction vessel are in contact with each other at the upper contact surface.

  The reaction vessel rack 106 is provided with a shape (not shown) that engages with the convexity so that all reaction vessels in which the direction of the convexity of the reaction vessel is accommodated are aligned. The convexity points in a certain direction regardless of the container. The detection plate and the photo interrupter of the gripper unit are used for control to pass through the convex portion of the reaction vessel and the slit of the concave portion of the temperature control block, and to engage with the concave portion.

  After performing the nucleic acid amplification process on the reaction vessel, the reaction vessel is removed from the temperature control block in the reverse order of the above operation and discarded.

  According to this method, the reaction vessel can be brought into close contact with the temperature control block, and the temperature can be adjusted accurately and quickly.

  As a modification of the present embodiment, a screw portion can be provided instead of the convex portion of the reaction vessel and the concave portion of the temperature control block. The reaction vessel gripped by the gripper unit is installed so that the reaction vessel and the screw portion of the temperature control block are engaged, and is installed in close contact with the temperature control block at the lower contact surface.

  As another modification, a luer lock can be used instead of the threaded portion.

  A reaction vessel and a temperature control block according to the fourth embodiment will be described with reference to FIG.

  FIG. 15 is a diagram showing the configuration and operation of the reaction vessel, the temperature control block, and the holder base. As shown in FIG. 15, the holder base includes a clamp mechanism 79 including a clamp 76, a fire spring 77, and a base 78. The gripper unit includes a release bar 80.

  The operation of this embodiment will be described. The gripper unit grips the reaction vessel and descends from above the installation position. When the gripper unit is stopped at a certain height and the release bar is lowered using the drive source 82 provided in the gripper unit and the reverse end of the clamp of the clamp mechanism is pushed in, the repelling force of the spring spring is received. However, the clamp is rotated around the hinge 81 to open the upper position of the installation position. The gripper unit is lowered again, the reaction vessel is placed on the temperature control block, and the reaction vessel is released. Then, the gripper unit is raised while the release bar keeps the clamp open. When the gripper unit is raised to a certain height, the release bar opens the reverse end of the clamp and the clamp holds the top of the reaction vessel. At this time, the clamp pushes the reaction container into the temperature control block.

  The drive source and the release bar move as a unit and are pressed by the spring 86 from the gripper unit housing toward the lower clamping mechanism. Further, it can move up and down along the guide 85. As a result, the release bar pushes the clamp roller 84, and the entire gripper unit moves downward to install the reaction vessel while keeping the installation position open. At this time, the release bar keeps pushing the roller by the action of the spring. For this reason, the reaction vessel and the gripper unit do not interfere with the clamp.

  In the process in which the gripper unit releases the reaction vessel and moves upward, the release bar keeps pushing the roller until the gripper unit moves to a certain height, so that the reaction vessel and the gripper unit do not interfere with the clamp.

  According to this method, the reaction vessel can be brought into close contact with the temperature control block, and the temperature can be adjusted accurately and quickly.

  As another modified example, the structure used as the clamp mechanism in the present modified example can be substituted with other generally known pressing structures such as a clamp, a clamp pin and a catcher, and a fastener.

  A reaction vessel and a temperature control block according to the fifth embodiment will be described with reference to FIGS. 16 and 17.

  FIG. 16 is a diagram showing the configuration and operation of the reaction vessel, temperature control block, holder base, cover 7 and gate 7a. FIG. 16 is a view showing a nucleic acid amplification device in a state where a gate is opened in order to install a reaction vessel on a temperature control block. As shown in FIG. 16, the cover includes a gate, a drive source 87, a guide 88, and a button 90, and the holder includes a clamp mechanism 79, a temperature control block, and a Peltier element.

  The operation of this embodiment will be described. For the holder, select a single temperature control block that installs and / or removes the reaction vessel from among the multiple temperature control blocks provided as specified by the nucleic acid amplification device. The rotating shaft 5a is rotated and arranged at a specific position. The gate moves in the direction along the guide by the power of the drive source, and the insertion port 91 is opened. At this time, when the button fixed to the gate pushes the clamp mechanism, the upper part of the temperature control block is opened, and the reaction container can be installed and / or taken out. Of these operations, FIG. 17 shows a series of operations for installing the reaction vessel. The gripper unit 113 holding the reaction vessel and temporarily suspended above the gate descends after the gate is opened, and installs the reaction vessel on the temperature control block. Thereafter, the gripper unit is raised and the gate is closed. The inside of the cover where the gate is closed is shielded from light and kept warm, and the optical solution (not shown) is used for the reaction liquid stored in the installed reaction vessel by the holder rotating periodically. An optical measurement is performed. In addition, nucleic acid amplification processing is performed by temperature control using a Peltier element.

  According to this method, in the nucleic acid amplification device using fluorescence detection, the gate opening / closing operation and the opening / closing operation of the clamp mechanism for closely contacting the reaction container and the temperature control block are integrated, and the operation efficiency is high, and Therefore, it is possible to construct a system capable of realizing accurate temperature control. Since a reaction vessel can be installed in and / or taken out from any temperature control block, nucleic acid amplification processes of different protocols can be performed in parallel for each reaction vessel. Since the completed reaction container can be taken out and a reaction container to be the next target of the nucleic acid amplification process can be installed, an efficient operation with less waiting time is possible.

  As a modification of the present embodiment, the clamp mechanism has a structure that can keep the open state and the closed state independently, and an open / close mechanism that can mechanically switch between the open state and the closed state of the clamp mechanism is provided. be able to. For example, this clamping mechanism can be realized by providing a latch for keeping each of the opened state and the closed state. The operation of this modification will be described. Keep the lid of the temperature control block in the standby state without holding the reaction container, and keep the temperature control block in line with the timing when the reaction container prepared with the reaction solution for nucleic acid amplification treatment is transported Rotate the holder and open the gate so that is at the installation and removal position of the reaction vessel below the gate. The gripper mechanism installs the reaction vessel on the temperature control block and closes the gate. The holder is rotated, the temperature control block is moved to the opening / closing mechanism, the clamp mechanism is closed, and the reaction vessel is brought into close contact with the temperature control block. Thereafter, the rotation operation for normal optical measurement is repeated, and the temperature is adjusted to perform the nucleic acid amplification process. Further, the position at which the opening / closing mechanism opens / closes the clamp mechanism can be the same as the position at which the reaction container is installed or removed.

  According to this modification, the reaction containers of a plurality of temperature control blocks can be continuously installed or removed during a single gate opening / closing operation, thereby enabling efficient apparatus operation.

  As a second modification of the present embodiment, the opening and closing of the clamp mechanism can be electrically controlled. Electric actuators such as DC stepping motors and solenoids are installed independently in each clamp mechanism, and the opening and closing of the clamp mechanism of the temperature control block that installs or removes the reaction vessel is controlled by commands from the control unit.

  According to this modification, it is possible to freely open and close the clamp mechanism, and the nucleic acid amplification process can be efficiently performed by opening the clamp mechanism. Specifically, the clamping mechanism can be opened before or during the movement of the temperature control block to the lower part of the gate, and the reaction vessel can be installed or taken out more quickly.

  As a second modification of the present embodiment, a sensor for detecting that a reaction vessel is installed in the temperature control block is installed. A beam sensor or a reflective sensor is preferable, and the direction of the optical path for detection may be the horizontal direction or the height direction. If the reaction vessel is not installed at a predetermined position, it can be detected by a sensor and installed again using a robot arm, or a description to that effect is written in the analysis result. To notify the user. In the case of performing the installation again by the robot arm, the time allocated for performing the installation or the removal of the reaction container can be used in the analysis operation schedule when the holder stops the operation. As a result, it is possible to eliminate an error that the reaction vessel cannot be placed at a predetermined position without disturbing the processing of another reaction vessel that is already held in the temperature control block and that has been subjected to the nucleic acid amplification process.

1 Nucleic acid amplification device 2 Base 3, 3A, 3B, 3C, 3D Holder 4, 4A, 4B, 4C, 4D Holder base 5 Stepping motor 5a Rotating shaft 6 Fluorescence detector 7 Cover 7a Gate 10, 10A, 10B, 10C , 10D Temperature control block 10a Detection window 10b Fixed holder 11 Base 12 Installation position 13 Radiation fins 14, 17, 18, 19 Peltier element 15, 15a Temperature sensor 16 Notch 40 Fan 41 Radiation fins 51, 51a Detector base 52 , 52a Motor 53 Belt 54, 54a Detector base rotating shaft 55 Support member 100 Nucleic acid test apparatus 101 Sample container 102 Sample container rack 103 Reagent container 104 Reagent container rack 105 Reaction container 106 Reaction container rack 107 Reaction liquid adjustment position 108 Closing unit 109 Stir Uni DOO 110 robotic arm X-axis 111 the robot arm Y-axis 112 robotic arm device 113 gripper unit 114 dispensing unit 115 nozzle tip 116 nozzle tip rack 117 waste box 118 input device 119 display device 120 control unit

Claims (6)

In a nucleic acid amplification apparatus for amplifying nucleic acid in a reaction mixture in which a sample and a reagent are mixed,
A reaction vessel having a convex shape on the outer peripheral surface and capable of elastic deformation;
A temperature control block for holding the sealed the reaction vessel containing the air layer reaction solution,
A temperature adjustment mechanism that is provided in a temperature control block and adjusts the temperature of the reaction solution,
The temperature control block includes a portion having the same inner diameter, a first taper portion having an inner diameter that is increased downward, a portion having the same inner diameter as the lower end of the first taper, and an inner diameter. A nucleic acid amplification device having a second tapered portion that gradually decreases downward in this order. In claim 1,
A nucleic acid amplification apparatus comprising a buffer material made of a softer material than the temperature control block in the second taper portion. In claim 1,
The nucleic acid amplification device, wherein the taper angle of the second taper portion is smaller than the taper angle of the reaction vessel. In claim 1,
A nucleic acid amplification apparatus, wherein a window portion connected to the outside is provided at a part of a portion where the second taper portion is provided.   In claim 1,
  A nucleic acid amplification apparatus, wherein a communication port connected to the outside is provided in a part of the portion where the first taper portion is provided. In claim 1,
  Furthermore, the nucleic acid amplification apparatus has a gripper for transporting the reaction container, and the gripper presses the reaction container during temperature adjustment.