JP2011112636A - Detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection capable of uniformly irradiating each reaction sample, to a plurality of reaction vessels with an excitation light. <P>SOLUTION: The detection deice includes a plurality of reaction vessels arranged to house reaction samples containing fluorescent material; a light source generating light; a first optical filter selecting excitation light among lights from the light source to excite reaction samples; a reflective transmission plate transmitting fluorescence generated from the reaction samples excited, while reflecting excitation light from the first optical filter so as to irradiate the reaction samples with the excitation light; a second optical filter transmitting fluorescence among the lights entered so as to observe fluorescence transmitted through the reflective transmission plate with an observing instrument, and a diffusion plate prepared between the light source and the reflective transmission plates in the optical path, from the light source to the reaction samples to scatter light entered. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a detection device.

  As a device for inspecting DNA by causing polymerase chain reaction (hereinafter referred to as PCR) to 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 2007-46904 A

  For example, as disclosed in Patent Document 1, a PCR apparatus is often provided with a plurality of reaction containers for storing reaction samples. Even if the amount of DNA in all the reaction vessels is the same, if the intensity of the excitation light to each reaction vessel is different, a difference occurs in the intensity of fluorescence from the reaction sample in each reaction vessel. As a result, there arises a problem that DNA amplification cannot be detected with high accuracy.

  This invention is made | formed in view of the said subject, and it aims at providing the detection apparatus which can irradiate excitation light uniformly to each reaction sample of several reaction container.

  In order to achieve the above object, a detection apparatus according to one aspect of the present invention includes a plurality of reaction containers arranged to store a reaction sample containing a fluorescent substance, a light source that generates light, and light from the light source. A first optical filter that transmits excitation light for exciting the reaction sample, and the excitation light from the first optical filter is reflected and excited to irradiate the reaction sample with the excitation light. A reflection / transmission plate that transmits fluorescence generated from the reaction sample, a second optical filter that transmits the fluorescence of the input light so that the fluorescence transmitted through the reflection / transmission plate can be observed by an observation device, and the light source A diffusion plate that is provided between the light source and the reflection / transmission plate in the optical path from to the reaction sample and diffuses input light.

  It is possible to provide a detection device that can uniformly irradiate each reaction sample in a plurality of reaction vessels with excitation light.

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 rotating device 31 to which the filter device 32 is attached. FIG. 4 is a cross-sectional view of a main part of the filter device 32 showing details of the structure when the diffusion plate 71 and the optical filter 72 are fixed to the filter cube 70. It is an example of the observation result which observed fluorescence L1 using the PCR apparatus. It is the observation result which observed fluorescence L1 using the general PCR apparatus.

  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. A reflection plate 25 and a Fresnel lens 26 are installed inside the cover 16.

  The reaction container 20 stores a liquid reaction sample containing DNA, a fluorescent substance, and the like. The reaction vessel 20 is configured to include 96 reaction vessels arranged in a vertical manner and 12 in a horizontal manner. The reaction vessels are arranged at intervals of, for example, 9 mm both vertically and horizontally. In the present embodiment, when the fluorescent substance is excited, the DNA is fluorescently labeled so as to generate, for example, two types of different wavelengths of fluorescence (fluorescence L1, L2). Here, the intensity of the fluorescence L1 increases with an increase in the amount of DNA, for example, and the fluorescence L2 has a predetermined intensity regardless of the amount of DNA, for example.

  The reaction unit 21 accommodates the reaction container 20. The reaction unit 21 controls the temperature of the reaction vessel 20 to amplify DNA based on an instruction from the control device 24 described later.

  The cover 22 is provided to prevent the reaction sample from evaporating when the reaction vessel 20 is overheated, for example. 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. The control device 24 controls, for example, the temperature of the reaction unit 21, the turning on / off of the light source 30, and the rotation of the motor 34.

  The reflecting mirror 25 reflects, for example, excitation light output 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 filter device 32, for example.

  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 installed on a side surface (side surface on the −Y side) of the main body 15 and includes a halogen lamp 50, a reflecting mirror 51, and an infrared cut filter 52. The halogen lamp 50 is a lamp that emits light including excitation light. The reflecting mirror 51 reflects the light from the halogen lamp 50 in the Y side direction of the main body 15. The infrared blocking filter 52 (blocking filter) absorbs and reflects infrared rays from the halogen lamp 50. The infrared cut filter 52 transmits light other than infrared light.

  The rotating device 31 (moving device) 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 (the rear side of the PCR device 10), 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 plate 25 side (the front side of the PCR device 10), 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 coincides with optical axes of a diffusion plate 71 and an optical filter 72 described later. FIG. 2 shows a state in which the filter device 32 faces the light source 30, for example.

  The filter device 32 (first device) is used when observing the fluorescence L1, and a diffuser plate 71, optical filters 72 and 74, and a dichroic mirror 73 are attached to a box-shaped filter cube 70.

  The diffusion plate 71 diffuses the light from the light source 30 and outputs it to the optical filter 72. For example, the diffusion plate 71 diffuses the input light by approximately 10 degrees and outputs it. For the diffusing plate 71, for example, frosted glass can be used, and the surface on the incident side of the frosted glass is provided with irregularities so that incident light can be diffused by approximately 10 degrees.

  The optical filter 72 (first optical filter) is a band-pass filter that transmits excitation light that excites the reaction sample among the light output from the diffusion plate 71.

  The dichroic mirror 73 (reflection / transmission plate) reflects the excitation light output from the optical filter 72 and transmits the fluorescence generated from the excited reaction sample so as to irradiate the reaction sample with excitation light. The excitation light reflected by the dichroic mirror 73 is further reflected by the reflecting plate 25, passes through the Fresnel lens 26, and is irradiated on the reaction vessel 20.

  The optical filter 74 (second optical filter) is a bandpass filter that transmits the fluorescence L <b> 1 in the light from the dichroic mirror 73.

  Here, the detailed structure when the diffusion plate 71 and the optical filter 72 are fixed to the filter cube 70 in the filter device 32 will be described with reference to the sectional view of FIG. Although not shown in FIGS. 1 and 2, the optical filter 72 has a holding frame 100. Further, the filter device 32 is provided with a fixing member 110 and four bolts (only two bolts 120a and 120b are shown in FIG. 4) for fixing the fixing member 110.

  In FIG. 4, for example, the filter cube 70 and the holding frame 100 of the optical filter 72 are drawn separately for convenience so that each structure can be easily understood. At 32, they are in contact. Further, the filter cube 70 and the fixing member 110 and the diffusion plate 71 and the fixing member 110 are in contact in the same manner.

  The optical filter 72 is provided with a holding frame 100 that surrounds the optical filter 72 and holds the optical filter 72. Further, the thickness of the holding frame 100 is larger than the thickness of the optical filter 72 in the optical path direction. In this embodiment, the optical filter 72 is provided on the upper side of the filter cube 70 (on the light source 30 side in FIG. 2) with the holding frame 100 in contact therewith. On the upper side of the holding frame 100, a diffusion plate 71 is installed in contact with the holding frame 100. In addition, a fixing member 110 for fixing the diffusion plate 71 and the holding frame 100 while being in contact with the diffusion plate 71 is provided on the upper side of the diffusion plate 71. The fixing member 110 is fixed to the filter cube 70 with bolts 120a and 120b. The other two bolts (not shown) are the same as the bolts 120a and 120b.

  The thickness of the diffusion plate 71 is about 0.2 mm, for example, and is thinner than the thickness of the optical filter 72 (for example, several mm). For this reason, even when the fixing member 110 fixes both the diffusion plate 71 and the optical filter 72, an increase in the size of the filter device 32 can be suppressed.

  The filter device 33 (second 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 72, the optical filter 81 (third optical filter) is a band-pass filter that transmits excitation light that excites the reaction sample in the input light. The dichroic mirror 82 (second reflection / transmission plate) is the same as the dichroic mirror 73 (first reflection / transmission plate). The optical filter 83 (fourth optical filter) is a bandpass filter that transmits the fluorescence L2 from the input light. For this reason, the filter device 33 has the same configuration as the filter device 32 excluding the diffusion plate 71.

  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 receives and captures the fluorescence L1 and L2. As a result, the user can detect the amount of amplified DNA and the like.

== Observation result of fluorescence L1 using PCR apparatus 10 ==
An observation result when the fluorescence L1 is observed using the PCR apparatus 10 will be described. Here, it is assumed that a reaction solution in which the brightness of the fluorescence L1 is constant when the intensity of the excitation light is constant is stored in the reaction vessel 20. Further, as described above, the filter device 32 is used when observing the fluorescence L1. For this reason, the rotating device 31 makes the filter device 32 face the light source 30 so that the light from the light source 30 is input to the filter device 32.

  First, light from the light source 30 is diffused when input to the diffusion plate 71. Of the diffused light, the excitation light passes through the optical filter 72 and is reflected by the dichroic mirror 73. The reflected excitation light is further reflected by the reflecting plate 25, passes through the Fresnel lens 26 and the cover 22, and is applied to the reaction solution in the reaction vessel 20. As a result, the reaction solution is excited and fluorescence L1 and L2 are generated. The fluorescent lights L1 and L2 are reflected by the reflecting plate 25 and then pass through the dichroic mirror 73. Since the fluorescence L1 and L2 from the dichroic mirror 73 are input to the optical filter 74, only the fluorescence L1 is transmitted and output to the camera 35. As a result, the camera 35 can observe the fluorescence L1.

  FIG. 5 is an example of observation results obtained by observing the fluorescence L1 using the PCR device 10. FIG. 6 shows observation results obtained by observing the fluorescence L1 under the same conditions by a general PCR apparatus, that is, a PCR apparatus that does not have the diffusion plate 71. In FIG. 6, for example, among the reaction vessels 20 in 12 rows, the brightness of the fluorescence L1 is significantly different from the other regions in the second, third, and tenth and eleventh rows from the left side of the page. However, when the PCR device 10 is used, the brightness of the fluorescence L1 from the reaction vessel 20 is uniform as shown in FIG. Thus, by using the PCR apparatus 10, the reaction vessel 20 can be irradiated with excitation light uniformly.

  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. In the PCR apparatus 10, a diffusion plate 71 is provided between the light source 30 and the dichroic mirror 73 in the optical path from the light source 30 to the reaction sample. For this reason, uniform and uniform excitation light that is diffused is incident on the dichroic mirror 73. Therefore, in this embodiment, as shown in FIG. 5, excitation light can be uniformly irradiated to each reaction sample of 96 reaction vessels 20.

  In the PCR apparatus 10, a diffusion plate 71 is disposed on the light source 30 side of the optical filter 72. For this reason, in this embodiment, the light diffused in advance can be incident on the optical filter 72.

  Further, the thickness of the holding frame 100 is thicker than the thickness of the optical filter 72, and the diffusion plate 71 is fixed by the fixing member 110 while being in contact with the light source 30 side of the holding frame 100. For example, the diffusing plate 71 can be fixed in direct contact with the optical filter 72. However, when the optical filter 72 and the diffusion plate 71 are fixed in direct contact with each other, a specific reaction vessel in the reaction vessel 20 may be irradiated with strong excitation light. In this embodiment, there is no such problem, and excitation light can be uniformly irradiated to each of the reaction vessels.

  The reaction vessel 20 includes 96 reaction vessels arranged vertically and 12 horizontally, and each reaction vessel is arranged, for example, every 9 mm. When irradiating such reaction vessel 20 with excitation light, a uniform excitation light can be irradiated as shown in FIG. 5 by using a diffusion plate 71 that diffuses light from the light source 30 by approximately 10 degrees.

  In general, the halogen lamp 50 or the like that generates light including excitation light generates infrared rays. When the diffusion plate 71 is provided on the light source 30 side as in the present embodiment, the temperature of the diffusion plate 71 rises. For this reason, depending on the material of the diffusing plate 71, the characteristics of the diffusing plate 71 may be deteriorated or deteriorated. In the present embodiment, since the infrared shielding filter 52 is provided between the halogen lamp 50 and the diffusion plate 71, the temperature rise of the diffusion plate 71 can be suppressed.

  Further, as described above, the intensity of the fluorescence L2 is constant regardless of the amount of DNA, for example. Even when such fluorescence L2 is observed, if the excitation light irradiated to the reaction sample is not uniform, a difference also occurs in the intensity of the fluorescence L2. However, generally, when observing the fluorescence L2, the difference in the intensity of the fluorescence L2 may be corrected using, for example, an offset. For this reason, for example, when observing the fluorescence L2, it is also possible to use the filter device 33 in which the diffusion plate 71 is not provided. Therefore, the PCR device 10 can reduce the cost as compared with the case where the diffusion plate is also provided in the filter device 33.

  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.

  For example, the diffusion plate 71 may be provided in contact with the dichroic mirror 73 side of the optical filter 72. Even in this case, the same effect as in the present embodiment can be obtained.

DESCRIPTION OF SYMBOLS 10 PCR apparatus 15 Main body 16,22 Cover 20 Reaction container 21 Reaction block 25,51 Reflector 26 Fresnel lens 30 Light source 31 Rotating device 32,33 Filter device 34 Motor 35 Camera 50 Halogen lamp 52 Infrared shielding filter 60,61 Rotating plate 62 Rotating shaft 70, 80 Filter cube 71 Diffusion plate 72, 74, 81, 83 Optical filter 73, 82 Dichroic mirror 100 Holding frame 110 Fixing member 120a, 120b Bolt

Claims (6)

A plurality of reaction vessels arranged to store reaction samples containing fluorescent materials;
A light source that generates light;
A first optical filter that transmits excitation light that excites the reaction sample out of light from the light source;
A reflection / transmission plate that reflects the excitation light from the first optical filter to irradiate the reaction sample with the excitation light and transmits fluorescence generated from the reaction sample to be excited;
A second optical filter that transmits the fluorescence of the input light so that the fluorescence transmitted through the reflection / transmission plate can be observed by an observation device;
A diffusion plate that is provided between the light source and the reflection / transmission plate in the optical path from the light source to the reaction sample, and diffuses input light;
A detection apparatus comprising: The detection device according to claim 1,
The diffusion plate is
Disposed on the light source side of the first optical filter, diffusing light from the light source and outputting the diffused light to the first optical filter;
A detection device characterized by. The detection device according to claim 2,
A thickness of the first optical filter in the optical path direction is larger than that of the first optical filter, and a holding frame that surrounds the first optical filter and holds the first optical filter;
A fixing member that fixes the holding frame and the diffusion plate in a state where the diffusion plate is in contact with the holding frame on the light source side of the holding frame;
Further comprising,
A detection device characterized by. A detection device according to claim 2 or claim 3, wherein
The diffusion plate is
Diffusing the light from the light source by approximately 10 degrees and outputting it to the first optical filter;
A detection device characterized by. The detection device according to any one of claims 2 to 4,
A cutoff filter that is provided between the light source and the diffusion plate in an optical path from the light source to the diffusion plate and blocks infrared rays from the light source;
A detection device characterized by. A plurality of reaction vessels arranged to store reaction samples containing fluorescent materials;
A light source that generates light;
A diffusion plate that diffuses and outputs input light; a first optical filter that transmits excitation light that excites the reaction sample out of light output from the diffusion plate; and the excitation light that is transmitted to the reaction sample. A first reflection / transmission plate that reflects the excitation light from the first optical filter to be irradiated and transmits the first fluorescence generated from the excited reaction sample, and the first fluorescence of the input light. A first optical device that transmits the second optical filter;
Of the input light, a third optical filter that transmits the excitation light and a second optical filter that reflects the excitation light from the third optical filter and transmits second fluorescence generated from the excited reaction sample. A second device including a second reflection / transmission plate and a fourth optical filter that transmits the second fluorescence of the input light;
When the first fluorescence is observed by an observation device that is equipped with the first and second devices and observes the input light, the first device is moved so that light from the light source is input to the diffuser plate. And a moving device that moves the second device so that light from the light source is input to the third optical filter when the observation device observes the second fluorescence;
A detection apparatus comprising: