JP2011250714A - Amplification apparatus and detecting device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an amplification apparatus capable of accurately amplify a nucleic acid.SOLUTION: In the amplification apparatus, a reaction vessel wherein a plurality of recesses for storing a reaction sample including the nucleic acid are arranged is placed. The amplification apparatus includes a heat conductive member having higher heat conductivity than that of aluminum and a control device for heating and cooling the heat conductive member at a predetermined heat cycle so that the nucleic acid is amplified.

Description

  The present invention relates to an amplification device and a detection device.

  As a device for inspecting DNA by causing polymerase chain reaction (hereinafter, PCR: Polymerase Chain Reaction) on nucleic acid such as DNA (Deoxyribonucleic Acid), there is a real-time PCR device (see, for example, Patent Document 1). . A real-time PCR apparatus (hereinafter referred to as a PCR apparatus) amplifies DNA by controlling the temperature of a reaction sample containing DNA, a fluorescent substance, or the like. In general, a PCR apparatus irradiates excitation light that excites a reaction sample, and measures amplified DNA based on fluorescence generated from the reaction sample at that time.

JP 2010-81898 A

  By the way, in a general PCR apparatus, a resin reaction vessel provided with a plurality of depressions for accommodating reaction samples is used. The reaction vessel is generally placed on a base made of aluminum, and the temperature of the base is controlled. Although DNA is amplified by repeating a predetermined temperature cycle, it is desirable that the temperature is exactly the same at any position in order to amplify in the same manner in all the depressions of the reaction vessel. However, conventionally, there has been a temperature difference of about 0.5 ° C. depending on the position of the container, that is, the position on the substrate. For this reason, variation in the amplification factor occurs in each depression, which causes a decrease in efficiency.

  This invention is made | formed in view of the said subject, and it aims at providing the amplifier which can amplify a nucleic acid accurately.

  In order to achieve the above object, an amplification device according to one aspect of the present invention is equipped with a reaction vessel in which a plurality of pits containing reaction samples containing nucleic acids are arranged, and has a thermal conductivity higher than that of aluminum. And a control device that heats / cools the heat conducting member with a predetermined heat cycle so that the nucleic acid is amplified.

  An amplification device and a detection device that can amplify nucleic acid with high accuracy can be provided.

It is a side view of PCR apparatus 10 which is one embodiment of the present invention. 1 is a plan view of a PCR device 10. FIG. It is a perspective view of the rotation apparatus 31 with which the filter apparatus 32 was mounted | worn. 3 is a perspective view of a reaction unit 21. FIG. 2 is a cross-sectional view of a reaction unit 21. FIG. 3 is a plan view of a plate 101. FIG.

  At least the following matters will become apparent from the description of this specification and the accompanying drawings.

  FIG. 1 is a side view of a PCR device 10 according to an embodiment of the present invention. FIG. 2 is a plan view of the PCR apparatus 10. The PCR apparatus 10 is an apparatus that amplifies DNA by controlling the temperature of a liquid reaction sample containing DNA, a fluorescent substance, and the like, and detects the state of the amplified DNA by an optical measurement method. The PCR device 10 (detection device) includes a main body 15 and a cover 16 attached to the front of the main body 15. In FIGS. 1 and 2, some of the blocks are depicted in cross-sectional views.

  The main body 15 is provided with a reaction vessel 20, a reaction unit 21, a cover 22, a control device 24, a light source 30, a rotation device 31, filter devices 32 and 33, a motor 34, and a camera 35.

  The cover 16 is installed in the main body 15 so as to be movable back and forth so that the user can change the reaction container 20, and is stored inside the main body 15 when moving backward. The cover 16 in FIG. 1 is in a state of being moved forward. For example, by attaching a light shielding member (not shown) between the main body 15 and the cover 16, the internal space of the PCR device 10 is shielded from light. Further, a reflecting mirror 25 and a Fresnel lens 26 are installed inside the cover 16.

  The reaction container 20 is formed with a total of 60 depressions for accommodating liquid reaction samples containing DNA, fluorescent substances, etc., for example, six vertically and ten horizontally at equal intervals. The reaction vessel 20 is made of a thin resin plate in order to efficiently transmit the heat of the substrate to be described later to the reaction sample, and the opposite side of the recess has a convex shape corresponding to the recess. In this embodiment, when the fluorescent substance is excited, the DNA is fluorescently labeled so that, for example, two types of fluorescence with different wavelengths (fluorescence L1 and L2) are generated.

  In the reaction unit 21, the reaction vessel 20 is placed, and the temperature of the reaction vessel 20 is controlled based on an instruction from a control device 24 described later. Details of the reaction unit 21 will be described later.

  The cover 22 is provided to prevent evaporation of the reaction sample when the reaction vessel 20 is heated. The cover 22 is made of a light transmissive film or the like so that excitation light for exciting the reaction sample and fluorescence from the reaction sample are not affected.

  For example, the control device 24 receives an instruction from a computer (not shown) provided outside the PCR device 10 and controls each block of the PCR device 10. Specifically, the control device 24 controls the temperature of the reaction unit 21 so that, for example, DNA is amplified. Further, the control device 24 controls turning on / off of the light source 30 and rotation of the motor 34.

The reflecting mirror 25 reflects, for example, excitation light from a filter device 32 described later toward the Fresnel lens 26. The reflecting mirror 25 reflects the fluorescence from the reaction sample to the camera 35.
The Fresnel lens 26 converges and transmits the excitation light reflected by the reflecting mirror 25 in a state parallel to the optical axis of the Fresnel lens.

  The light source 30 is, for example, a halogen lamp installed on the side surface (the side surface on the −Y side) of the main body 15 and irradiates light including excitation light.

  The rotating device 31 is a so-called turret type rotating device, and is equipped with filter devices 32 and 33 used when observing fluorescence from a reaction sample. FIG. 3 is a perspective view of the rotating device 31 to which the filter device 32 is attached. The rotating device 31 includes rotating plates 60 and 61 and a rotating shaft 62. For example, a maximum of six filter devices can be mounted between the rotating plate 60 and the rotating plate 61 at intervals of 60 degrees.

The rotating plate 60 is a plate on the camera 35 side, and is provided with six round windows through which fluorescence can pass at a position where the filter device is mounted.
The rotating plate 61 is a plate on the reflecting mirror 25 side, and is provided with six angular windows through which excitation light and fluorescence can pass at a position where the filter device is mounted.
The rotating shaft 62 is connected to the rotating plates 60 and 61 so as to rotate the rotating plates 60 and 61 and the attached filter device.

  In the present embodiment, the filter device 33 is mounted at a position 180 degrees away from the position where the filter device 32 is installed. Although details will be described later, the rotating device 31 causes either of the filter devices 32 and 33 to face the light source 30 and inputs light from the light source 30. Note that the filter device 32 faces the light source 30 means a state in which an optical axis of the light source 30 and an optical axis of an optical filter 72 of the filter device 32 described later coincide. FIG. 2 shows a state in which the filter device 32 is opposed to the light source 30, for example.

  The filter device 32 (optical device) is used when observing the fluorescence L1, and optical filters 71 and 73 and a dichroic mirror 72 are attached to a box-like filter cube 70.

The optical filter 71 is a band-pass filter that transmits excitation light that excites the reaction sample in the light from the light source 30.
The dichroic mirror 72 reflects the excitation light transmitted through the optical filter 71 and transmits the fluorescence L1 generated from the excited reaction sample so as to irradiate the reaction sample with excitation light. The excitation light reflected by the dichroic mirror 72 is further reflected by the reflecting mirror 25, passes through the Fresnel lens 26, and is irradiated onto the reaction vessel 20.
The optical filter 73 is a band-pass filter that selectively transmits the fluorescence L <b> 1 in the light from the dichroic mirror 72.

  The filter device 33 (optical device) is used when observing the fluorescence L2, and optical filters 81 and 83 and a dichroic mirror 82 are attached to a box-like filter cube 80.

  Similar to the optical filter 71, the optical filter 81 is a bandpass filter that transmits excitation light that excites the reaction sample among input light. On the other hand, the dichroic mirror 82 reflects the excitation light and transmits the fluorescence L2 from the reaction sample. The optical filter 83 is a bandpass filter that selectively transmits the fluorescence L2 from the input light.

  The motor 34 rotates the rotating shaft 62 of the rotating device 31 according to an instruction from the control device 24. The motor 34 is, for example, a stepping motor.

  The camera 35 (observation apparatus) receives and captures the fluorescence L1 and L2. As a result, the user can detect the amount of amplified DNA and the like.

== Details of Reaction Unit 21 ==
Here, the detailed structure of the reaction unit 21 will be described with reference to the perspective view of FIG. 4 and the cross-sectional view of FIG.
The reaction unit 21 includes a base 100, a plate 101, a heat insulating member 102, a heat sink 103, and fixing members 104a and 104b.

  In FIG. 5, each member is drawn separately for convenience so that the structure of the reaction unit 21 can be easily understood. However, in the actually completed reaction unit 21, each member is in contact. .

  The base 100 (heat conducting member) is a member on which the reaction vessel 20 is placed, and a total of 60 holes of 6 × 10 are formed in the vertical and horizontal directions to accommodate the convex shape corresponding to the depression of the reaction vessel 20. Yes. In addition, copper having a higher thermal conductivity than aluminum, which is a general base material, is used as the base 100 material. Furthermore, the surface of the substrate 100 is plated with nickel after being blasted for matting to give fine irregularities.

  The plate 101 heats or cools the substrate 100 based on an instruction from the control device 24. For example, as shown in FIG. 6 which is a plan view, the plate 101 includes Peltier elements 120 to 125 for heating / cooling each of the regions obtained by dividing the base 100 into six equal parts, and temperature sensors 130 to 132. Is done.

  The temperature sensor 130 is, for example, a sensor that is provided between the Peltier elements 120 and 121 and controls the temperature of the Peltier elements 120 and 121. In the present embodiment, the control device 24 controls the temperatures of the Peltier elements 120 and 121 based on the output from the temperature sensor 130.

  The temperature sensors 131 and 132 are the same as the temperature sensor 130. That is, the temperature sensor 131 is a sensor for controlling the temperature of the Peltier elements 122 and 123, and the temperature sensor 132 is a sensor for controlling the temperature of the Peltier elements 124 and 125. Note that the temperature of the Peltier elements 122 to 125 is also controlled by the control device 24.

  The heat insulating member 102 is provided so as to surround the periphery of the substrate 100 and the plate 101 in order to suppress heat dissipation of the substrate 100.

  The heat sink 103 radiates heat generated by the Peltier elements 120 to 125. The fixing members 104a and 104b fix the base 100, the plate 101, the heat insulating member 102, and the heat sink 103.

  The control device 24 and the plate 101 correspond to a control device for heating or cooling the substrate 100, and the control device 24, the substrate 100, and the plate 101 correspond to an amplification device that amplifies DNA.

== Observation of fluorescence L1 using PCR device 10 ==
Details when the PCR apparatus 10 amplifies DNA and observes the fluorescence L1 will be described. As described above, when observing the fluorescence L1, the filter device 32 is used. For this reason, the control device 24 first rotates the rotation device 31 so that the light from the light source 30 is input to the filter device 32.

  Next, the control device 24 starts controlling the temperatures of the Peltier elements 120 to 125 so that the DNA is amplified. As a result, since the Peltier elements 120 to 125 repeat heating and cooling of the substrate 100 in a predetermined cycle, for example, DNA is amplified.

  Of the light from the light source 30, excitation light passes through the optical filter 71 and is reflected by the dichroic mirror 72. The reflected excitation light is further reflected by the reflecting mirror 25, passes through the Fresnel lens 26 and the cover 22, and is irradiated on the reaction sample in the reaction vessel 20. As a result, the reaction sample is excited and fluorescence L1 and L2 are generated. The fluorescence is reflected by the reflecting mirror 25 and then partially passes through the dichroic mirror 72. Since the fluorescence transmitted through the dichroic mirror 72 is input to the optical filter 73, only the fluorescence L 1 is selectively transmitted and output to the camera 35. As a result, the camera 35 can observe the fluorescence L1 in real time.

  In addition, although the observation result at the time of observing the fluorescence L1 using the PCR apparatus 10 was demonstrated here, it is the same also when observing the fluorescence L2. However, in that case, the filter device 33 is used instead of the filter device 32.

  In the above, the PCR apparatus 10 which is one embodiment of this invention was demonstrated. The thermal conductivity of the copper substrate 100 of this embodiment is higher than the thermal conductivity of aluminum, which is a general substrate material. For this reason, for example, the temperature of the base 100 becomes more uniform as compared with the case where aluminum is used as the base material. In the experiment, the temperature difference between the samples accommodated in the respective depressions of the reaction vessel could be suppressed to about 0.2 ° C. at the maximum. Therefore, by using the control device 24, the base 100, and the plate 101, it is possible to amplify DNA with higher accuracy than a general DNA amplification device (for example, a thermal cycler).

  As metals having higher thermal conductivity than aluminum, for example, gold, silver, and copper are known. For this reason, as in the present embodiment, gold or silver can be used as the material of the substrate 100 in order to amplify DNA with high accuracy. However, in this embodiment, since copper is used as the material of the base 100, for example, the base 100 can be created at a lower cost than when gold, silver, or the like is used.

  Further, by using the control device 24, the base 100, and the plate 101 in the PCR device 10, the DNA amplification accuracy in the PCR device 10 can be improved.

  Moreover, the PCR apparatus 10 can be manufactured at low cost by using copper as the material of the base 100 in the PCR apparatus 10.

  In general, in a PCR apparatus, light reflected by the surface of the substrate mixed with fluorescence emitted from a reaction sample also enters the camera 35. When such a phenomenon occurs, the fluorescence L1 and L2 may not be detected with high accuracy. In the present embodiment, the surface of the substrate 100 is blasted. For this reason, since the reflection of the light on the surface of the base | substrate 100 can be suppressed, the detection accuracy of fluorescence L1, L2 can be improved. The blasting process is a process of spraying fine alumina particles to roughen the surface of the base so that light is diffusely reflected on the surface of the base, and is a type of so-called matting or satin treatment. Chemical treatment such as soaking may be used, and other methods may be used as long as the same effect is obtained.

  In the embodiment, the surface of the substrate 100 is plated with nickel. Therefore, since wear resistance and corrosion resistance are increased, it is possible to withstand friction when attaching and detaching the reaction vessel, and to prevent oxidation and corrosion. Note that the plating is not limited to nickel plating. Any other plating may be used as long as it has a similar effect.

  In addition, the said Example is for making an understanding of this invention easy, and is not for limiting and interpreting this invention. The present invention can be changed and improved without departing from the gist thereof, and the present invention includes equivalents thereof.

DESCRIPTION OF SYMBOLS 10 PCR apparatus 15 Main body 16,22 Cover 20 Reaction container 21 Reaction unit 25 Reflector 26 Fresnel lens 30 Light source 31 Rotating device 32, 33 Filter device 34 Motor 35 Camera 60, 61 Rotating plate 62 Rotating shaft 70, 80 Filter cube 71, 73, 81, 83 Optical filter 72, 82 Dichroic mirror 100 Base 101 Plate 102 Thermal insulation member 103 Heat sink 104a, 104b Fixing member 120-125 Peltier element 130-131 Temperature sensor

Claims (6)

A reaction vessel in which a plurality of depressions containing reaction samples containing nucleic acids are arranged, and a heat conducting member having a thermal conductivity higher than that of aluminum; and
A controller for heating / cooling the heat conducting member at a predetermined thermal cycle so that the nucleic acid is amplified;
An amplifying device comprising: The amplification device according to claim 1,
An amplifying device characterized in that the material of the heat conducting member is copper. A reaction vessel in which a plurality of depressions containing reaction samples containing a nucleic acid and a fluorescent substance are arranged; a heat conducting member having a thermal conductivity higher than that of aluminum; and
A controller for heating / cooling the heat conducting member at a predetermined thermal cycle so that the nucleic acid is amplified;
An optical device that transmits fluorescence generated from the reaction sample to an observation device when the reaction sample is irradiated with excitation light that excites the reaction sample;
A detection apparatus comprising: The detection device according to claim 3,
The material for the heat conducting member is copper. The detection device according to claim 4,
A detecting device, wherein the surface of the heat conducting member is matted. The detection device according to claim 5,
The surface of the said heat conductive member is plated, The detection apparatus characterized by the above-mentioned.

Images