WO2021261322A1 - Nucleic acid measurement device and nucleic acid measurement method - Google Patents

Abstract

A nucleic acid measurement device according to the present invention is a nucleic acid measurement device 1 comprising a temperature cycle application member 33 which is arranged at the side of a container 51 for storing both of a nucleic acid sample and a reaction solution 52 for amplifying the nucleic acid sample therein and applies a predetermined temperature cycle to the container 51 for the purpose of amplifying the nucleic acid sample to produce an amplification product having a fluorescent label, a light-emitting element 11 which is arranged above the container 51 and emits light containing a wavelength of excitation light for the fluorescent label, and a light-receiving element 12 which is arranged below the container 51 and receives fluorescent light coming from the side of the container 51, the nucleic acid measurement device further including a first light-condensing lens 13 which is arranged between the container 51 and the light-emitting element 11, condenses the light emitted from the light-emitting element 11 and emits the condensed light to the inside of the container.　According to the present invention, a nucleic acid measurement device can be provided, in which the temperature cycle can be shortened, the size of the device can be reduced, and high detection accuracy can be achieved.

Description

Nucleic acid measuring device and nucleic acid measuring method

The present invention relates to a nucleic acid measuring device and a nucleic acid measuring method. In particular, a nucleic acid test solution containing nucleic acid such as DNA extracted from a sample and a nucleic acid amplification reagent is stored in a container and PCR (Polymerase Chain Reaction) amplification (proliferation) is performed, and the amount is used as the amount of fluorescence in real time. The present invention relates to a nucleic acid measuring device and a nucleic acid measuring method suitable for real-time PCR for detection.

Conventionally, nucleic acid amplification of DNA (DeoxyriboNucleic Acid) by the PCR amplification method is known. As a method for this, there is real-time PCR in which the amount of nucleic acid amplified by PCR is measured in real time and analyzed to quantify the nucleic acid extracted from the sample. Real-time PCR is excellent in that it can be quantified in real time. Various proposals have been made for this real-time PCR with the aim of further improvement.
For example, Patent Document 1 is a document disclosed about real-time PCR. Patent Document 1 discloses a configuration in which a Perche element (315) for giving a temperature cycle is arranged below a container (FIG. 3, sample plate 305 of the same document) for storing a reaction solution (sample). Further, FIG. 5C discloses a configuration using a liquid metal, a Pelche element, or the like. Further, FIG. 6 discloses a configuration using a dichroic mirror (611). As described above, Patent Document 1 discloses various configurations of real-time PCR.

Special Table 2009-537152

If the temperature cycle can be shortened in real-time PCR, the time for nucleic acid measurement, which generally takes a long time, can be shortened. Further, if the nucleic acid measuring device (real-time PCR device) can be miniaturized, the convenience is improved.
Here, if the configuration is such that the perche element (315) is arranged under the sample plate (FIG. 3, 305 of the same document) as in Patent Document 1, a large number of samples are placed on the sample plate (305). Therefore, while it is possible to give a temperature cycle to a large number of samples at one time, it is difficult to heat and cool the sample plate and the sample on it, and it is difficult to shorten the temperature cycle.
Further, if a temperature cycle is given to the sample using a liquid metal (see FIG. 5C of the above document, a summary column, etc.), it is difficult to miniaturize the nucleic acid measuring device.
Further, when a dichroic mirror (FIGS. 6 and 611 of the above document) is used, the direction in which the excitation light enters the sample (incident direction) and the direction in which the excited fluorescence is emitted (exit direction) are reversed. Therefore, the light emitting part and the light receiving part can be on the same side, but the optical system becomes complicated and it is difficult to miniaturize the nucleic acid measuring device.
Therefore, an object of the present invention is to provide a nucleic acid measuring device and a nucleic acid measuring method which can shorten the temperature cycle and miniaturize the device and have high detection accuracy.

[1] The nucleic acid measuring apparatus of the present invention is arranged on the side of a container containing a nucleic acid test solution containing nucleic acid and a nucleic acid amplification reagent, and is described above in order to amplify the nucleic acid to obtain an amplified product having a fluorescent label. A temperature cycle imparting member that imparts a predetermined temperature cycle to the container, a light emitting element that is arranged above the container and emits light including the wavelength of the excitation light of the fluorescence label, and a light emitting element that is arranged below the container and described above. A nucleic acid measuring device comprising a light receiving element that receives fluorescence emitted from the side of the container and measuring the amount of nucleic acid amplification by the fluorescence received by the light receiving element. The nucleic acid measuring device further comprises the container and the light emission. It is characterized by comprising a first condensing lens which is arranged between the elements and condenses the emitted light from the light emitting element and irradiates the inside of the container.

Here, the "nucleic acid" refers to, for example, DNA (target DNA, DNA template, detection target DNA, detection target DNA region) or RNA (RiboNucleic Acid, ribonucleic acid). In the case of RNA, DNA converted from RNA by reverse transcription reaction is used.
The "nucleic acid amplification reagent" refers to a reagent used for nucleic acid amplification, which contains a primer and a fluorescent substance for the DNA to be detected, an enzyme (heat-resistant DNA polymerase), dNTP and the like.
The "fluorescent label" is a label possessed by the amplified product by binding to an amplified nucleic acid (amplified product of the target DNA), and refers to a label that emits fluorescence by excitation light, a so-called fluorescent substance.
As the fluorescent substance, for example, a DNA probe in which a fluorescent dye and a quencher (quenching substance) are bound is bound to the template DNA at the annealing stage (step), and this DNA probe is cleaved at the extension (reaction) stage. There are fluorescent substances (fluorescent dyes and quenching substances) used in a method (probe method) for detecting fluorescence from a fluorescent dye suppressed by a quencher. In addition, there is a dye (fluorescent dye) used in a method (intercalator method) or the like using a dye that enters between the strands of double-stranded DNA and emits fluorescence.
The nucleic acid test solution (or nucleic acid amplification reagent) appropriately contains a buffer, which is a solvent for stabilizing the reagent, Mg, or the like, if necessary.
The "nucleic acid amplification amount" means the amount (DNA amount) of the amplified nucleic acid (target DNA).
"Nucleic acid measuring device" mainly means a real-time PCR device.

[2] In the nucleic acid measuring apparatus of the present invention, the first condensing lens is configured so that the focal point is located above the standard liquid level when the standard amount of the nucleic acid test liquid is stored in the container. It is preferable to have.

[3] In the nucleic acid measuring apparatus of the present invention, the area where the light flux focused at the focal point of the first condensing lens spreads and irradiates the standard liquid surface becomes substantially equal to the area of the standard liquid surface. It is preferable that it is configured as such.

[4] In the nucleic acid measuring apparatus of the present invention, the container is vertically long, has a tube shape with a gradually narrowing lower portion, and has a container opening at the upper portion, and the container further comprises the container opening. The cap has a substantially flat upper surface and an annular convex portion on the lower surface side, and when closing the container opening, the outer peripheral surface of the annular convex portion is inside the container opening. The nucleic acid measuring device is further configured to be in close contact with the peripheral surface, and the heater is laminated on the cap so as to have a heater opening in at least a part of the container opening. It is preferable to have.

[5] In the nucleic acid measuring apparatus of the present invention, the nucleic acid measuring apparatus further comprises a first parallel light lens that makes the emitted light of the light emitting element parallel and emits light to the side of the first condensing lens. A first filter arranged between the light emitting element and the container and passing the excitation light having a wavelength that excites the fluorescent label, and a second parallel light lens that converts the fluorescence emitted from the side of the container into parallel light. A second condensing lens that collects (emits) parallel light emitted from the second parallel light lens and emits it to the light receiving element side, and is arranged between the container and the light receiving element and has a predetermined wavelength. A second filter that allows fluorescence to pass through, a first optical cylinder that holds the light emitting element, the first parallel light lens, the first condensing lens, and the first filter in a vertically arranged state. It is preferable to include the second parallel light lens, the second condensing lens, the second filter, and the second optical cylinder that holds the light receiving element in a vertically arranged state.
In this specification, "between A and B" may be described as "between A and B".

[6] In the nucleic acid measuring device of the present invention, the light emitting element is a bell-shaped LED element having a convex portion on the lower side, and the first parallel light lens is a plano-convex lens, which is the focal point of the plano-convex lens. Is preferably configured to be located above the tip of the convex portion of the light emitting element.

[7] In the nucleic acid measuring apparatus of the present invention, it is preferable that the second condensing lens is configured so that the focal point is located above the light receiving element.

[8] The nucleic acid measuring method of the present invention comprises a step of storing a nucleic acid test solution containing nucleic acid and a nucleic acid amplification reagent, and preparing a container for amplifying the nucleic acid to obtain an amplified product having a fluorescent label. A step of storing the nucleic acid and the nucleic acid amplification reagent in the container, a step of giving a temperature cycle to the container from the side of the container, and a step of irradiating the excitation light of the fluorescent label from above the container. A nucleic acid measuring method comprising a step of receiving fluorescence from the side of the container below the container and a step of measuring the amount of nucleic acid amplification by the received fluorescence, in the step of irradiating the excitation light. It is characterized in that the excitation light is focused and irradiated from above the container to the inside of the container.

According to the present invention, since the temperature cycle imparting member is arranged on the side of the container for storing the nucleic acid test solution, it is easy to heat and cool, and the temperature cycle can be shortened. Further, since the light emitting element is arranged above the container and the light receiving element is arranged below the container, the optical system is not complicated and the nucleic acid measuring device can be miniaturized. Further, since the first condensing lens is arranged between the container and the light emitting element, the light emitted from the light emitting element is condensed and irradiated inside the container, the fluorescent label is easily excited and the nucleic acid has high detection accuracy. It becomes possible to provide a measuring device (or a nucleic acid measuring method).

It is a schematic explanatory drawing of the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is a disassembly explanatory view of the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing of the temperature cycle in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing of the fluorescence amount detection in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing about the irradiation of the excitation light to the nucleic acid test liquid 52 in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing of the vicinity of a light emitting element 11 in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing of the vicinity of the light receiving element 12 in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing for supplementary explanation about the fluorescence amount detection in the nucleic acid measuring apparatus 1 which concerns on Embodiment 1. FIG. It is explanatory drawing of the temperature cycle in the nucleic acid measuring apparatus which concerns on Embodiment 2. FIG.

Hereinafter, the nucleic acid measuring apparatus and the nucleic acid measuring method of the present invention will be described with reference to FIGS. 1 to 9. It should be noted that each figure described below is a schematic diagram showing the actual shape, configuration, characteristics and the like in a simplified manner.

[Embodiment 1]
The nucleic acid measuring apparatus 1 according to the first embodiment will be described with reference to FIGS. 1 to 8.
FIG. 1 is a schematic explanatory view of the nucleic acid measuring device 1 according to the first embodiment, and FIG. 2 is an exploded explanatory view of the nucleic acid measuring device 1. The nucleic acid measuring device 1 includes eight containers 51 (wells) (see FIG. 2). The eight containers 51 are connected in the vertical direction (left-right direction in FIG. 2) of the paper in FIG. FIG. 1 shows a cross section of one of the containers 51 of the containers 51 having eight consecutive cross sections of the nucleic acid measuring device 1 (the direction of the central axis AA'of the container 51 in FIG. 2 and the connection of the eight consecutive containers 51. It is shown along the BB'direction perpendicular to the direction in which the container 51 is used and the cross section determined by (however, FIG. 1 is a view showing a state in which the container 51 is sealed with a cap 54), whereas FIG. Is a diagram showing a state in which the cap 54 is opened in order to facilitate understanding of the outline of the nucleic acid measuring device 1).

The nucleic acid measuring device 1 has a hardware component 2 (hardware component) shown on the left side of FIG. 1 and a control unit 3 shown on the right side.
The hardware component 2 includes a temperature cycle imparting unit 33 including a temperature cycle imparting member 33 arranged on the side of the container 51, a light emitting side optical system including a light emitting element 11 arranged above the container 51, and a container 51. It includes a light receiving side optical system including a light receiving element 12 arranged below.
The control unit 3 includes a light receiving / emitting element controlling means 41 that controls the light receiving / emitting of the light emitting element 11 and the light receiving element 12, a temperature cycle controlling means 42 that controls the temperature of the temperature cycle imparting member 33, and a light receiving element 12 that receives fluorescence. A / D conversion means 45 (Analog-Digital conversion means) for converting the output of the device, a calculation means 44 for calculating the output, a storage means 43 for storing the calculation result, and an overall control for controlling the entire nucleic acid measuring device 1. Means 46 and.

Explaining the outline of the nucleic acid measurement, the container 51 (nucleic acid test solution 52 containing the target DNA) is heated and cooled by heating and cooling the temperature cycle imparting member 33 under the control of the control unit 3 (providing a temperature cycle). Will be). Then, the nucleic acid (target DNA) in the container 51 (nucleic acid test solution 52) is amplified. When the light emitting element 11 is driven to emit light under the control of the control unit 3 and the filtered light having a predetermined wavelength (fluorescence excitation light) is irradiated to the nucleic acid test solution 52, the amount of amplified target DNA (nucleic acid amplification) is applied. The fluorescence of the amount of light corresponding to the amount (amount, amount of DNA) is excited. The light receiving element 12 is driven by the control unit 3 to receive the excited fluorescence. If the target DNA is present in the nucleic acid test solution 52, it is amplified in a predetermined temperature cycle, so that the fluorescence of the light amount corresponding to the amplification amount (nucleic acid amplification amount, DNA amount) is received. In the absence of target DNA, no fluorescence is received. In this way, it becomes possible to amplify and measure (detect) a small amount of target DNA.

[Temperature cycle imparting unit]
First, the temperature cycle imparting unit will be described.
On the side of the container 51 (where it is placed), a temperature cycle applying member 33 composed of a heat guiding member 31 and a Pelche element 32, a heat radiation fin 34 on the outside thereof, and a cooling fan 35 (heat radiation fan) on the outside thereof. And are arranged. The heat conductive member 31 and the heat radiating fin 34 are made of, for example, aluminum, copper, or other metal having excellent heat transfer properties, or a material containing the metal as a main component. The heat radiating fin 34 has an uneven shape, whereby the heat transfer area is widened and the heat exchange efficiency is improved.

Regarding the temperature cycle imparting member 33, one surface of the Pelche element 32, for example, a heat radiation surface (or a cooling surface) is arranged in contact with the heat guiding member 31, and the other surface, for example, a cooling surface (or a heat radiation surface) is dissipated. Arrange toward the fin 34. When the Pelche element 32 is driven by the temperature cycle control means 42 to be heated (heat radiated) or cooled, the adjacent heat guiding member 31 is heated (heat radiated) or cooled. Then, a temperature cycle (see FIG. 3) is given to the container 51 (and the nucleic acid test solution 52 in the container 51), and the nucleic acid in the nucleic acid test solution 52 is amplified. Here, the Pelche element 32 can reverse the heat dissipation surface and the cooling surface (endothermic surface) by reversing the direction in which the drive current flows.
The temperature cycle control means 42 installs a temperature sensor 57 on the heat guide member 31 to detect the temperature of the heat guide member 31, and according to the detection result, a predetermined temperature is placed in the container 51 (and the nucleic acid test liquid 52 in the container 51). The Pelche element 32 may be driven so as to give a cycle.

[Light emitting side optical system]
Next, the light emitting side optical system will be described.
A light emitting side optical system including a light emitting element 11 is formed above the container 51, and a nucleic acid amplified by a temperature cycle is formed by irradiating the inside of the container 51 (nucleic acid test solution 52) with excitation light (fluorescence excitation light). The fluorescence corresponding to the amplification amount (nucleic acid amplification amount, DNA amount) of the amplification product (the nucleic acid amplification product has a fluorescence label) is excited.

Explaining with reference to FIGS. 1 and 2, above the container 51, a light emitting element 11 (LED element) that emits light including the excitation light of the fluorescent label and the light emitted from the light emitting element 11 side are parallel to each other. The first parallel light lens 15 that is converted into light and emitted to the first condensing lens 13 side and the parallel light from the first parallel light lens 15 side are condensed and irradiated (emitted) toward the inside of the container 51. A first condensing lens 13 and a first filter 16 arranged between the light emitting element 11 and the container 51 (nucleic acid test liquid 52) and passing the excitation light having a wavelength for exciting the fluorescent label are arranged. The first optical cylinder 21 holds the light emitting element 11, the first parallel light lens 15, the first condensing lens 13, and the first filter 16 in a state of being vertically arranged inside the cylinder.
In this embodiment, a recess 21A formed along the circumferential direction around the optical cylinder axis is provided on the inner peripheral surface of the first optical cylinder 21, and the light emitting element 11 (the circuit board 61 thereof), the first By fitting the outer peripheral portions of the parallel light lens 15, the first filter 16, and the first condenser lens 13, these (these have a circular shape when viewed from above) are arranged vertically inside the cylinder. It keeps these in the state that it was done.

The light emitting element 11 is an LED element that emits light including the wavelength of the excitation light of the fluorescence label (wavelength that excites fluorescence, wavelength of 490 nm or less). The light emitting element 11 is driven by the light receiving / receiving element control means 41 at the end of each temperature cycle (at the end of the extension step of FIG. 3 to be described later) or at all times to emit light.
Both the first parallel light lens 15 and the first condenser lens 13 are plano-convex lenses in which one surface is a flat surface (including a substantially flat surface) and the other surface is a convex surface. In the first parallel light lens 15, the plane side faces the light emitting element 11 direction, and in the first condenser lens 13, the plane side faces the container 51 direction.
The first filter 16 is a first filter 16 (a low-pass filter that allows light having a wavelength of 490 nm or less) to pass through light having a wavelength of excitation light. In FIGS. 1 and 2, the first filter 16 is arranged between the first parallel light lens 15 and the first condenser lens 13, but if it is between the light emitting element 11 and the container 51 (nucleic acid test liquid 52). For example, it may be arranged anywhere, such as between the light emitting element 11 and the first parallel light lens 15, and between the first condensing lens 13 and the container 51 (nucleic acid test liquid 52).

[Light receiving side optical system]
Next, the light receiving side optical system will be described.
A light receiving side optical system including a light receiving element 12 and the like is formed below the container 51, and receives fluorescence excited inside the container 51 (nucleic acid test solution 52). Nucleic acid is detected (specified) by detecting the amount of fluorescence.

Explaining with reference to FIGS. 1 and 2, below the container 51, the fluorescence scattered (emitted) from the side (fluorescence emission port 31B) of the container 51 (nucleic acid test liquid 52) is converted into parallel light. Then, the second parallel light lens 17 emitted to the second condensing lens 14 side and the second parallel light emitted (emitted) from the second parallel light lens 17 are collected and emitted to the light receiving element 12 side. The condensing lens 14, the light receiving element 12 that receives the light collected by the second condensing lens 14, and the light receiving element 12 are arranged between the container 51 and the light receiving element 12, and are arranged downward from the side of the container 51 (nucleic acid test liquid 52). A second filter 18 that allows light of a specific wavelength to pass through the emitted (emitted) light is arranged. The second optical cylinder 22 holds the second parallel light lens 17, the second condenser lens 14, the second filter 18, and the light receiving element 12 in a state of being vertically arranged inside the cylinder.
In this embodiment, a recess 22A formed along the circumferential direction around the optical cylinder axis is provided on the inner peripheral surface of the second optical cylinder 22, and the second parallel optical lens 17, the second filter 18, and the like are provided therein. By fitting the outer peripheral portions of the second condenser lens 14 and the light receiving element 12 (the circuit board 62 of the light receiving element 12), these (these have a circular shape when viewed from above) move up and down inside the cylinder. They are held in a state of being arranged in the direction.

The second filter 18 is a bandpass filter that allows light having a wavelength of fluorescence to pass through, and passes light having a wavelength of 515 nm to 535 nm and blocks light having other wavelengths. In FIGS. 1 and 2, the second filter 18 is arranged between the second parallel light lens 17 and the second condenser lens 14, but the light emitting element 11, the container 51 (nucleic acid test liquid 52), and the light receiving element 12 As long as it is between, for example, it may be arranged anywhere, such as between the container 51 (nucleic acid test liquid 52) and the second parallel light lens 17, and between the second condensing lens 14 and the light receiving element 12.

Both the second parallel light lens 17 and the second condenser lens 14 are plano-convex lenses in which one surface is a flat surface (including a substantially flat surface) and the other surface is a convex surface. In the second parallel light lens 17, the plane side faces the container 51 direction, and in the second condenser lens 14, the plane side faces the light receiving element 12 direction.

The light receiving element 12 is a photodiode element that receives (detects) the excited fluorescence (center wavelength of about 525 nm). The light receiving element 12 is driven by the light receiving / receiving element control means 41 at the end of each temperature cycle or at all times to receive fluorescence, and outputs an analog electric signal corresponding to the amount of light received. This analog electric signal is input to the A / D conversion means 45 and converted into a digital electric signal (see FIG. 1). This digital signal is calculated by a calculation means 44 such as a CPU (Central Processing Unit) and converted into a fluorescence amount. The converted fluorescence amount data is stored in a storage means 43 such as a semiconductor memory or a hard disk.
The overall control means 46 controls the entire nucleic acid measuring device 1 including each means (41, 42, ...) In FIG.

The inner surfaces of the first optical cylinder 21 and the second optical cylinder 22 are mirror-finished so as to reflect light. The mirror-finished first optical cylinder 21 and the second optical cylinder 22 are, for example, a metal plate having a mirror-finished surface corresponding to the inner surface of aluminum, silver, stainless steel or the like, and aluminum, silver or the like having a surface corresponding to the inner surface. It can be made of a plastic molded product or the like in which a metal film is formed by plating, sputtering or the like and mirror-finished.

[Container 51]
The container 51 for storing the nucleic acid test solution 52 and its peripheral portion will be described (see FIGS. 1, 2, and 5).
The container 51 (well) and its cap 54 are made of polypropylene and are substantially transparent. As used herein, "transparent" means that light passes through (passes through) well. The term "substantially transparent" is intended to include not only the case where light is completely passed through but also the case where most of the light is passed through although it is not completely passed through. In addition to being colorless, it also includes cases where it has a color (including white) that does not block the passage of light. In actual industrial products, it is difficult to completely pass light even if it is transparent, and in many cases, only most of the light is passed through, but substantially transparent includes such things.
The container 51 is vertically long and has a tube shape in which the lower portion gradually becomes thinner. More specifically, the upper side of the container 51 has a cylindrical shape, and the lower side has a mortar shape. The container 51 has a container opening at the upper part, from which the nucleic acid test solution 52 is taken in and out. At the time of measurement (including the time of DNA amplification by the temperature cycle), the container opening is closed with the cap 54.

The nucleic acid measuring device 1 has a configuration in which a heater 56 for heating the cap 54 is laminated on the cap 54 when the container 51 is stored in the container storage hole 31A (described later) of the heat conducting member 31. The heater 56 is configured by, for example, attaching an insulating film on which resistance is printed to a copper plate, a stainless plate, or the like. The heater 56 itself blocks the passage of light, but a heater opening is provided at a position corresponding to the container opening so that light can pass through. The heater 56 is controlled by the control unit 3, for example, so as to constantly heat the cap 54 at 100 to 105 ° C. This is to prevent the nucleic acid test solution 52 evaporated in the container 51 from condensing on the cap 54 or the like. The upper surface of the cap 54 is substantially flat. In the present specification, the term "substantial plane" is intended to include a plane having some irregularities, curves, etc., in addition to a perfect plane. An annular convex portion 54A is provided on the lower surface side, and when closing the container opening, the outer peripheral surface of the annular convex portion 54A is brought into close contact with the inner peripheral surface of the container opening. In order to improve the adhesion between the cap 54 and the inner surface of the container 51, the thickness in the radial direction from the center of the annular circle of the annular convex portion 54A (horizontal direction) is larger than the thickness of the upper surface of the cap 54 (vertical direction). , Is thickly formed. It is preferable that all or most of the incident light to the container 51 passes through the upper surface of the cap 54. Further, it is preferable that all or most of them do not hit the annular convex portion 54A.

The maximum capacity of each container 51 is 200 μl (microliter). However, the nucleic acid test solution 52 is not stored up to the maximum volume, and the standard storage amount (standard amount, recommended storage amount) is 20 μl (10% of the maximum volume). This is for speeding up heating and cooling of the nucleic acid test solution 52.

The heat-conducting member 31 described above is formed with a container storage hole 31A having a shape (concave shape) corresponding to the container shape (mortar shape), and when the container 51 enters the container storage hole 31A, it fits into the mortar-shaped portion of the container. 51 is independent. Due to the size and shape of the container storage hole 31A, a container 51 having a certain shape (a standard container or a container having a shape similar to the standard container) is naturally used.
The upper part of the container storage hole 31A has a shape substantially following the shape of the container 51, but the hole diameter is larger than the outer diameter of the container 51, and the gap 36 (the inner surface of the container storage hole 31A and the container 51) ( A space) is formed (in this specification, "diameter" means diameter). The gap 36 may be a space, but a heat insulating member such as styrofoam may be arranged therein.
As described above, the lower portion of the container storage hole 31A has a mortar shape, but the bottom is provided with a fluorescence emission port 31B that emits fluorescence downward. The diameter 73 (inner diameter) of the fluorescence emission port 31B is, for example, about 0.5 times the inner diameter 71 of the upper cylindrical portion of the container 51. It may be about 0.4 to 0.6 times. The container 51 is in contact with the lower part (mortar-shaped portion) of the container storage hole 31A excluding the fluorescence emission port 31B (may include a part of a cylindrical portion continuous with the mortar-shaped portion). The surface of the container storage hole 31A on the container 51 side is mirror-finished so that it is reflected when the excitation light incident on the container 51 or the fluorescence of the nucleic acid test solution 52 hits.

The inner diameter 72 of the first optical cylinder 21 is substantially the same as the inner diameter 71 of the upper part of the container (see FIG. 1). Further, the inner diameter 74 of the second optical cylinder 22 is substantially the same as the inner diameter 71 of the inner diameter 71 of the upper part of the container, but may be slightly smaller (see FIG. 1).
In this way, when the containers 51 are connected at a predetermined pitch such as 8 stations, even if the first optical cylinder 21 and the second optical cylinder 22 are arranged according to the pitch of the container 51, the first optical cylinders 21 are connected to each other. Or, the diameter (outer diameter and inner diameter) of the first optical cylinder 21 and the diameter (outer diameter and inner diameter) of the second optical cylinder 22 in a state where they do not interfere with each other (overlap) between the second optical cylinders 22. Inner diameter) can be increased. Further, when the inner diameter 72 of the first optical cylinder 21 is large, the light emitting element 11 having a large amount of emitted light and a large outer diameter can be used by being inserted into the first optical cylinder 21. Further, when the inner diameter 74 of the second optical cylinder 22 is large, it is easy to collect fluorescence from the container 51 side (fluorescence emission port 31B side) widely.

[Temperature cycle]
FIG. 3 is an explanatory diagram of the temperature cycle of the nucleic acid measuring device 1 according to the first embodiment. In the nucleic acid measuring device 1, the control unit 3 controls the temperature of the temperature cycle imparting member 33 and imparts a temperature cycle to the container 51 (nucleic acid test solution 52) to amplify a large amount of DNA. One cycle T during the temperature cycle has heat denaturation, annealing and elongation steps (steps, stages), as shown in FIG.

In the heat denaturation step, the container 51 (nucleic acid test solution 52) is heated to 95 ° C. to separate the double-stranded DNA into two single-stranded DNAs.
In the annealing step, the container 51 (nucleic acid test solution 52) is cooled to 55 ° C., the primer is bound to the adjacent region of the target DNA, and the DNA probe to which the fluorescent dye and the quencher (quenching substance) are bound is used as the template DNA. Combine (probe method).
In the extension step, the container 51 (nucleic acid test solution 52) is reheated to 72 ° C., and DNA is extended from the primer by a DNA polymerase (an enzyme that synthesizes a DNA strand complementary to the nucleic acid as a template). And synthesize the complementary strand (complementary sequence) of DNA. The elongation step cleaves the DNA probe and emits fluorescence from the fluorochrome that was suppressed by the quencher. The light receiving element 12 receives the fluorescence, and the A / D conversion means 45 A / D converts the output of the light receiving element 12.
After the extension step, the process proceeds to the heat denaturation step again.
When such a temperature cycle is repeated 25 to 40 times, the target DNA is amplified exponentially.

Here, the transition time TA from heat denaturation (step) to annealing (step), the transition time TB from annealing (step) to elongation (step), and the transition time TC from elongation (step) to heat denaturation (step). May be shortened, and if these transition times are shortened, the total time of the temperature cycle repeated 25 to 40 times until the amount of fluorescence is saturated can be shortened.
In the nucleic acid measuring apparatus 1 of the first embodiment, since the heat guiding member 31 and the Pelche element 32 (temperature cycle imparting member 33) are arranged on the side of the container 51, when these are arranged only in the lower part of the container 51. In comparison, the transition time TA, TB, and TC of heating and cooling of the container 51 can be shortened. Therefore, the total time of the temperature cycle can be shortened.

[Fluorescence measurement]
FIG. 4 is an explanatory diagram of fluorescence amount detection (fluorescence amount detection curve) in the nucleic acid measuring device 1 according to the first embodiment. The horizontal axis is the number of temperature cycles, and the vertical axis is the detected fluorescence amount (fluorescence light amount). The fluorescence amount detection curve is a curve connecting the fluorescence amounts detected for each temperature cycle. In the example here, when the temperature cycle (number) is about 20 times, the fluorescence amount starts to increase corresponding to the nucleic acid amplification amount (DNA amount), and is saturated at 25 to 40 times.
The measurement accuracy of the nucleic acid measuring device 1 will be described.
The A curve in FIG. 4 is a fluorescence amount detection curve (standard amount) measured in advance using a nucleic acid test solution 52 (standard amount) containing a certain nucleic acid (DNA) and a nucleic acid amplification reagent.
For the A1 and A2 curves, the B1 and B2 curves, and the C1 and C2 curves on the left and right of the A curve, the same nucleic acid test solution 52 as in the case of the A curve is stored in a standard amount in eight containers 51, respectively, and the same temperature cycle is performed at the same time. It is a fluorescence amount detection curve which shows the variation with respect to the A curve (standard curve) when it is measured by giving. The A1 and A2 curves are in the vicinity of the A curve, but the B1 and B2 curves are separated from the A curve, and the C1 and C2 curves are further separated.
The measurement accuracy of the nucleic acid measuring device 1 is evaluated as follows from the degree of variation of the eight fluorescence amount detection curves corresponding to the eight containers 51 with respect to the A curve (standard curve).
When the above eight curves are within the range of the A1 curve and the A2 curve near the A curve (standard curve), the detection accuracy is "excellent". On the other hand, when the eight curves do not fit in the A1 curve / A2 curve range and extend to the B1 curve / B2 curve range away from the A curve, the detection accuracy is “inferior” to the above case. When the above eight curves do not fit in the A1 curve / A2 curve range and the B1 curve / B2 curve range and extend to the C1 curve / C2 curve range, they are "very inferior" as compared with these cases.

[Focus F1]
FIG. 5 is an explanatory diagram of irradiation of the nucleic acid test solution 52 with excitation light (fluorescence excitation light). Specifically, it is a figure for demonstrating the irradiation of the nucleic acid test liquid 52 with the excitation light with and without the first condensing lens 13 (focus F1). The liquid level irradiated with the excitation light is the liquid level (standard liquid level 53) when the standard amount of the nucleic acid test liquid 52 is stored.

FIG. 5A is a diagram showing a case where the nucleic acid measuring device 1 is provided with a first condensing lens 13 and its focal point F1 is located above the standard liquid level 53. The luminous flux collected by the focal point F1 of the first condenser lens 13 spreads again to irradiate the standard liquid surface 53, and the irradiation area thereof is substantially equal to the area of the standard liquid surface 53.

FIG. 5B is a diagram showing a case where the nucleic acid measuring device is provided with the first condensing lens 13 but its focal point F1 is located at the standard liquid level 53 (Comparative Example 1). In this case, only one point on the standard liquid level 53 is irradiated with the focused and strong light.

FIG. 5 (c) shows a case where the nucleic acid measuring device does not have the first parallel light lens 15 and the first condensing lens 13 (see FIG. 1), and the scattered light illuminates the standard liquid surface 53 (comparison). It is a figure which shows the example 2). Since there is no first parallel light lens 15 and first condensing lens 13, scattered light (light that does not condense) that has passed through the cap 54 among the light in the first optical cylinder 21 irradiates the standard liquid surface 53.

FIG. 5D is a diagram showing a case where the nucleic acid measuring device does not have the first condenser lens 13 (see FIG. 1) and the parallel light irradiates the standard liquid surface 53 (Comparative Example 3). Since there is a first parallel light lens 15 but no first condensing lens 13, only the parallel light that has passed through the cap 54 among the parallel light emitted from the first parallel light lens 15 irradiates the standard liquid surface 53.

　表１の「参照図」は、核酸測定装置が図５のいずれに対応するかを示す。「焦点Ｆ１」は、核酸測定装置が第１集光レンズ１３を備えている場合にはその焦点Ｆ１がどこに位置するかを示し、第１集光レンズ１３を備えていない場合には（焦点Ｆ１）「なし」としている。
　「８個の容器５１の８つの曲線」は、８個の容器５１に対する８つの蛍光量検出曲線の、Ａ曲線（標準曲線、図４参照）に対するばらつきの度合いを示す。例えば、「Ａ１曲線・Ａ２曲線範囲」は、８つの曲線が「Ａ１曲線・Ａ２曲線範囲」（内）にあることを示す。
　「検出精度」は、「８個の容器５１の８つの曲線」に基づく検出精度を示す。「○：優れる」、「△：劣る」、「Ｘ：大変劣る」で示している（［蛍光測定］参照）。「検出精度」については、実施例１の核酸測定装置１は「○：優れる」。これに対し、比較例１の核酸測定装置は「△：劣る」、比較例２及び３の核酸測定装置は「Ｘ：大変劣る」。 [Examples, comparative examples]
Table 1 is a comparison table (Example 1, Comparative Examples 1 to 3) in which a standard amount of a nucleic acid test solution 52 containing the same nucleic acid and a nucleic acid amplification reagent is stored in eight containers 51 and the detection accuracy is evaluated.
The conditions (temperature cycle, etc.) other than the conditions shown in Table 1 (see the “Focus F1” column) are the same in Example 1 and Comparative Examples 1 to 3.

The “reference diagram” in Table 1 shows which of FIG. 5 the nucleic acid measuring device corresponds to. "Focus F1" indicates where the focal point F1 is located when the nucleic acid measuring device is provided with the first focusing lens 13, and when the nucleic acid measuring device is not provided with the first focusing lens 13 (focus F1). ) "None".
"8 curves of 8 containers 51" indicates the degree of variation of the 8 fluorescence amount detection curves for 8 containers 51 with respect to the A curve (standard curve, see FIG. 4). For example, "A1 curve / A2 curve range" indicates that eight curves are in the "A1 curve / A2 curve range" (inside).
"Detection accuracy" indicates the detection accuracy based on "eight curves of eight containers 51". It is indicated by "○: excellent", "△: inferior", and "X: very inferior" (see [Fluorescence measurement]). Regarding "detection accuracy", the nucleic acid measuring device 1 of Example 1 is "◯: excellent". On the other hand, the nucleic acid measuring device of Comparative Example 1 is "Δ: inferior", and the nucleic acid measuring devices of Comparative Examples 2 and 3 are "X: very inferior".

[Focus F3]
FIG. 6 is an explanatory diagram of the vicinity of the light emitting device 11 in the nucleic acid measuring device 1 according to the first embodiment. The light emitting element 11 is an LED element, and includes an LED chip 11A (semiconductor), a substrate 11B on which the LED chip 11A is mounted, a substantially transparent mold resin 11D for encapsulating the LED chip 11A, and a lead terminal 11C. Have. The electrodes of the LED chip 11A are connected to the wiring on the substrate 11B by wire bonding or the like, and the wiring is connected to the lead terminal 11C by soldering or the like. The circuit board 61 is electrically connected to the light emitting element 11 (LED element) via the lead terminal 11C. The light emitting element 11 (LED element) has a bell shape or a cannonball shape having a convex portion on the lower side. The outer diameter of the light emitting element 11 (LED element) is substantially the same as the inner diameter 72 of the first optical cylinder 21.
The mold resin 11D is a resin such as epoxy and is substantially transparent. Most of the light emitted from the LED chip 11A is radiated to the outside through the mold resin 11D, but some of the light is reflected on the surface of the mold resin 11D.
The first parallel light lens 15 (plano-convex lens) incidents (radiates) the light emitted (radiated) from the light emitting element 11 (LED element), converts it into parallel light, and emits it.
Here, when the focal point F3 of the first parallel light lens 15 (plano-convex lens) is located above the tip portion 11E of the light emitting element 11 (the tip portion of the convex portion of the light emitting element 11), the detection accuracy can be further improved. It will be possible. This is probably because more light emitted from the light emitting element 11 can be captured. It is more preferable that the focal point F3 is located above the LED chip 11A.

[Focus F2]
FIG. 7 is an explanatory diagram of the vicinity of the light receiving element 12 in the nucleic acid measuring device 1 according to the first embodiment.
FIG. 7A is an enlarged view of the vicinity of the light receiving element 12 of FIG. The light receiving element 12 is a photodiode element, and is a photodiode chip 12A (semiconductor) having a surface having a photovoltaic power generation region 12G (see FIG. 7 (b) described later) having a square shape when viewed from above, and the chip. It has a shielding metal plate 12C surrounding the 12A, a substantially transparent glass plate 12D placed above the chip 12A and sealing the chip 12A, a substrate 12B on which the chip 12A is mounted, and a lead terminal 12E. The electrodes of the photodiode chip 12A are connected to the wiring on the substrate 12B by wire bonding or the like, and the wiring is connected to the lead terminal 12E by soldering or the like. The shield metal plate 12C surrounds the chip 12A so as to be higher than the upper surface of the chip 12A (the surface where the photovoltaic power generation region 12G is located).
As shown in FIG. 7A, it is preferable that the focal point F2 of the second condenser lens 14 is located above the light receiving element 12 (photodiode chip 12A). In this way, the luminous flux collected by the focal point F2 of the second condenser lens 14 spreads to irradiate the light receiving element 12, so that the light flux can be easily collected by the light receiving element 12 (photodiode chip 12A).
When the metal plate 12C for shielding surrounds the chip 12A so as to be higher than the upper surface of the chip 12A (the surface where the photovoltaic power generation region 12G is located), the light from the second condenser lens 14 passes through the focal point F2. If the chip 12A is to be directly irradiated before, the irradiation light to the edge of the chip 12A is easily blocked by the shielding metal plate 12C. On the other hand, if the light from the second condenser lens 14 is once focused by the focal point F2 and irradiated with the light passing through the focal point F2, even the edge of the chip 12A is blocked by the shielding metal plate 12C. It becomes easy to irradiate without being damaged.

FIG. 7B shows the photovoltaic power generation region 12G of the photodiode chip 12A (when a large number of photodiodes are formed and irradiated with light, free electrons and holes are generated to generate current and voltage. The region where the photovoltaic effect is generated. The output of the photodiode chip 12A is a collection of the outputs from many of these photodiodes), and the state of fluorescence irradiation is shown. While the photovoltaic power generation region 12G is an almost entire surface region (square region) on the upper surface of the chip 12A, the actual irradiation region 12H is a circular shape in this square light-receiving region (photovoltaic power generation region 12G). It is an area.
In the photovoltaic power generation region 12G, the region other than the irradiation region 12H where the fluorescent light is irradiated is a region unrelated to the fluorescent light, but the region other than the irradiation region 12H should be masked to block the received light. Then, since the photovoltaic power generation region 12G and the irradiation region 12H are almost the same region, it is possible to further increase the SN ratio (Signal Noise Ratio) as compared with the case without masking. Become.

According to the nucleic acid measuring apparatus 1 and the nucleic acid measuring method according to the first embodiment, the temperature cycle imparting member 33 (31, 32) is arranged on the side of the container 51 for storing the nucleic acid test liquid 52 containing the nucleic acid sample. Therefore, it is easy to heat and cool, and the temperature cycle can be shortened. Further, since the light emitting element 11 is arranged above the container 51 and the light receiving element 12 is arranged below, the optical system is not complicated and the device can be miniaturized. Further, since the first condensing lens 13 is arranged between the container 51 and the light emitting element 11, the light emitted from the light emitting element 11 is condensed and irradiated inside the container 51, so that the fluorescent label is easily excited and is high. It is possible to provide a nucleic acid measuring device 1 (or a nucleic acid measuring method) having detection accuracy.

　なお、図５（ｂ）の核酸測定装置１で発光素子１１（図１参照）の発光量（発光強度）を図５（ａ）より大きく（強く）し、図５（ｃ）や図５（ｄ）では図５（ｂ）より更に大きく（強く）すると、Ｂ’曲線やＣ’曲線の飽和値はＡ曲線の飽和値近傍の値をとり、図４に近いグラフとなる（測定精度は、表１や表２で説明したのと同様）。 [Supplementary explanation of fluorescence amount detection]
FIG. 8 is an explanatory diagram for supplementarily explaining the fluorescence amount detection in the nucleic acid measuring device 1 according to the first embodiment. In the explanation so far, FIG. 4 is used as an explanatory diagram of the fluorescence amount detection, but FIG. 4 shows the fluorescence amount detection in a schematic graph for the sake of clarity. Actually, as shown in FIG. 8, when the emission amount (emission intensity) of the light emitting element 11 (see FIG. 1) is the same in the nucleic acid measuring apparatus 1 of FIGS. 5 (a) to 5 (d), B' The curve tends to be on the right side of the A curve (the side with a larger number of temperature cycles), and the C'curve tends to be on the right side of the B'curve. (The B'curve is the B curve shifted to the right. The C'curve is the C curve shifted to the right. The same applies to other curves). Further, the B1'curve tends to be located on the right side of the A1'curve, the B2'curve tends to be located on the right side of the A2'curve, the C1'curve tends to be located on the right side of the B1'curve, and the C2'curve tends to be located on the right side of the B2'curve.
Further, the saturation value of the fluorescence amount of the B'curve tends to be smaller than the saturation value of the A curve, and the saturation value of the C'curve tends to be smaller than the saturation value of the B'curve.
However, regarding the measurement accuracy, the measurement accuracy (the range of the B1'curve and the B2'curve (the size in the horizontal axis direction of the range sandwiched between the two curves) is the same as the range of the B1 curve and the B2 curve, and is C1. The range of the'curve / C2'curve is the same as the range of the C1 curve / C2 curve, and the measurement accuracy is the same as in the case of FIG.
If a comparison table is created according to Table 1, it will be as shown in Table 2 (measurement accuracy is the same as the accuracy explained in Table 1).

In the nucleic acid measuring device 1 of FIG. 5 (b), the light emission amount (light emission intensity) of the light emitting element 11 (see FIG. 1) is made larger (stronger) than that of FIG. 5 (a), and FIGS. 5 (c) and 5 (see). In d), when it is made larger (stronger) than in FIG. 5 (b), the saturation value of the B'curve and the C'curve takes a value near the saturation value of the A curve, and the graph becomes close to FIG. 4 (measurement accuracy is: Same as described in Table 1 and Table 2).

[Embodiment 2]
The nucleic acid measuring apparatus (and nucleic acid measuring method) according to the second embodiment is basically the same as that of the first embodiment, except that the temperature cycle is as shown in FIG. 9 instead of the temperature cycle of FIG.
In the second embodiment, as shown in FIG. 9, annealing and elongation during one cycle T are performed at the same temperature of about 60 ° C. By changing the enzyme or the like to be put in the nucleic acid test solution 52, these can be set to the same temperature. The temperature transition period in one cycle T is the period TA1 for transitioning from thermal denaturation to annealing and the period TC1 for transitioning from elongation to thermal denaturation.
The nucleic acid measuring apparatus (and the nucleic acid measuring method) according to the second embodiment is the same as the first embodiment except that the temperature cycle is as shown in FIG. 9 instead of the temperature cycle of FIG. Among the effects of the nucleic acid measuring device 1 (and the nucleic acid measuring method) of No. 1, the corresponding effect is obtained.

In this specification, "upper" and "lower" may be paraphrased as "light emitting element direction" and "light receiving element direction", respectively.

[Modification example]
Although the present invention has been described above based on the above embodiment, the present invention is not limited to the above embodiment. It can be carried out within a range that does not deviate from the purpose, and for example, the following modifications are also possible.

(1) The materials, shapes, positions, sizes, wavelengths, temperatures, and the like described in the above embodiments are examples, and can be changed as long as the effects of the present invention are not impaired.

(2) In the above embodiment, a plano-convex lens is used as the first condensing lens 13 and the second condensing lens 14, but this is an example. For example, at least one of them is condensing with a convex lens, a Fresnel lens, or the like. It can also be a lens.

(3) In the above embodiment, a fixed focus lens (plano-convex lens) is used as the first condenser lens 13 and the second condenser lens 14, but at least one of them is a varifocal lens whose focal distance can be changed (3). It is also possible to use a varifocal lens, a zoom lens, etc.).

(4) In the above embodiment, the PCR amplification product was detected by fluorescence using the probe method, but a reagent that emits fluorescence by binding to double-stranded DNA (intercalator: TB Green, etc.).
The PCR amplification product may be detected by fluorescence using an intercalator method in which the nucleic acid test solution 52 is contained, or another method.

(5) In the above embodiment, the first filter 16 that allows the excitation light of the wavelength that excites the fluorescent label to pass through is used, but instead, the light emitting element in which the mold resin 11D is mixed with a substance that absorbs the light of a predetermined wavelength. 11 (LED element) may be used, or a cap 54 mixed with a substance that absorbs light having a predetermined wavelength may be used.
Further, instead of the second filter 18 that allows fluorescence of a predetermined wavelength to pass through, a substantially transparent glass plate 12D (for sealing the photodiode chip 12A) (or a glass plate 12D) in which a substance that absorbs light of a predetermined wavelength is mixed. Alternatively, a substantially transparent encapsulating resin mixed with a substance that absorbs light having a predetermined wavelength may be used.

1 ... nucleic acid measuring device, 2 ... hardware component, 3 ... control unit, 11 ... light emitting element, 11A ... LED chip, 11B ... substrate, 11C ... lead terminal, 11D ... mold resin, 11E ... tip, 12 ... light receiving element , 12A ... Photodioden chip, 12B ... Substrate, 12C ... Shielding metal plate, 12D ... Glass plate, 12E ... Lead terminal, 12G ... Photovoltaic power generation region, 12H ... Irradiation region, 13 ... First condenser lens, 14 ... 2nd condensing lens, 15 ... 1st parallel light lens, 16 ... 1st filter, 17 ... 2nd parallel light lens, 18 ... 2nd filter, 21 ... 1st optical cylinder, 21A ... concave, 22 ... 2nd Optical cylinder, 22A ... recess, 31 ... heat guide member, 31A ... container storage hole, 31B ... fluorescence emission port, 32 ... Pelche element, 33 ... temperature cycle imparting member, 34 ... heat dissipation fin, 35 ... cooling fan, 36 ... gap, 41 ... Light receiving / receiving element control means, 42 ... Temperature cycle control means, 43 ... Storage means, 44 ... Calculation means, 45 ... A / D conversion means, 46 ... Overall control means, 51 ... Container, 52 ... Nucleic acid test solution, 53 ... standard liquid level, 54 ... cap, 54A ... annular protrusion, 56 ... heater, 57 ... temperature sensor, 61, 62 ... circuit board, 71 ... inner diameter of upper part of container, 72 ... inner diameter of first optical cylinder, 73 ... fluorescence Diameter of emission port, 74 ... Inner diameter of second optical tube, F1 ... Focus of first condenser lens, F2 ... Focus of second condenser lens, F3 ... Focus of first parallel light lens

Claims (8)

A temperature cycle that is placed beside a container that stores a nucleic acid test solution containing nucleic acid and a nucleic acid amplification reagent, and gives a predetermined temperature cycle to the container in order to amplify the nucleic acid to obtain an amplified product having a fluorescent label. With the granting member
A light emitting element arranged above the container and emitting light including the wavelength of the excitation light of the fluorescent label, and a light emitting element.
A light receiving element arranged below the container and receiving fluorescence emitted from the side of the container, and a light receiving element.
A nucleic acid measuring device that measures the amount of nucleic acid amplification by the fluorescence received by the light receiving element.
The nucleic acid measuring device further
A nucleic acid measuring apparatus, which is arranged between the container and the light emitting element and includes a first condensing lens that collects the light emitted from the light emitting element and irradiates the inside of the container. In the nucleic acid measuring apparatus according to claim 1,
The first condensing lens is a nucleic acid measuring device characterized in that the focal point is located above the standard liquid level when the standard amount of the nucleic acid test liquid is stored in the container. In the nucleic acid measuring apparatus according to claim 2,
A nucleic acid characterized in that the area of the first condensing lens in which the light flux focused at the focal point spreads and irradiates the standard liquid surface is substantially equal to the area of the standard liquid surface. measuring device. In the nucleic acid measuring apparatus according to any one of claims 1 to 3.
The container is vertically long, has a tube shape in which the lower part is gradually tapered, and has a container opening in the upper part.
The container further has a cap that closes the container opening.
The upper surface of the cap is substantially flat and has an annular convex portion on the lower surface side so that the outer peripheral surface of the annular convex portion is in close contact with the inner peripheral surface of the container opening when the container opening is closed. It is composed and
The nucleic acid measuring device is further equipped with a heater.
The nucleic acid measuring apparatus, wherein the heater is laminated on the cap so as to have a heater opening in at least a part of the container opening. In the nucleic acid measuring apparatus according to any one of claims 1 to 4.
The nucleic acid measuring device further
A first parallel light lens that makes the light emitted by the light emitting element parallel and emits light to the side of the first condenser lens.
A first filter arranged between the light emitting element and the container and passing excitation light having a wavelength that excites the fluorescent label,
A second parallel light lens that converts fluorescence emitted from the container side into parallel light, and
A second condenser lens that collects parallel light emitted from the second parallel light lens and emits it to the light receiving element side.
A second filter, which is arranged between the container and the light receiving element and allows fluorescence of a predetermined wavelength to pass through,
A first optical cylinder that holds the light emitting element, the first parallel light lens, the first condensing lens, and the first filter in a vertically arranged state.
A second optical cylinder that holds the second parallel light lens, the second condensing lens, the second filter, and the light receiving element in a vertically arranged state.
A nucleic acid measuring device comprising. In the nucleic acid measuring apparatus according to claim 5.
The light emitting element is a bell-shaped LED element having a convex portion on the lower side.
The first parallel light lens is a plano-convex lens, and is a nucleic acid measuring device characterized in that the focal point of the plano-convex lens is positioned above the tip of the convex portion of the light emitting element. In the nucleic acid measuring apparatus according to claim 5 or 6.
The second condensing lens is a nucleic acid measuring device characterized in that the focal point is located above the light receiving element. A step of storing a nucleic acid test solution containing a nucleic acid and a reagent for nucleic acid amplification, and preparing a container for amplifying the nucleic acid to obtain an amplified product having a fluorescent label.
A step of storing the nucleic acid and the nucleic acid amplification reagent in the container, and
The process of giving a temperature cycle to the container from the side of the container,
The step of irradiating the excitation light of the fluorescent label from above the container and
A step of receiving fluorescence from the side of the container below the container,
The step of measuring the amount of nucleic acid amplification by the received fluorescence and
It is a nucleic acid measurement method including
In the step of irradiating the excitation light, a nucleic acid measuring method comprising condensing the excitation light and irradiating the inside of the container from above the container.

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