JP2023110116A - Nucleic acid measuring apparatus and nucleic acid measuring method - Google Patents

Abstract

To provide a nucleic acid measuring apparatus capable of shortening a temperature cycle and downsizing the apparatus, and capable of performing high-accuracy detection.SOLUTION: A nucleic acid measuring apparatus 1 comprises: a temperature cycle imparting member 33 which is arranged on the side of a container 51 housing a nucleic acid sample and a reaction solution 52 for amplifying the nucleic acid sample, and gives a predetermined temperature cycle to the container 51 in order to amplify the nucleic acid sample to obtain an amplified product having fluorescent label; a light-emitting element 11 which is arranged above the container 51 and emits light including the wavelength of excitation light of the fluorescent label; and a light-receiving element 12 which is arranged below the container 51 and receives fluorescence from the side of the container 51, where the nucleic acid measuring apparatus further comprises a first condenser lens 13 which is arranged between the container 51 and the light-emitting element 11 and condenses emission light from the light-emitting element 11 to irradiate the inside of the container with the emission light.SELECTED DRAWING: Figure 1

Description

The present invention relates to a nucleic acid measuring device and a nucleic acid measuring method. In particular, nucleic acids such as DNA extracted from specimens and nucleic acid test solutions containing reagents for nucleic acid amplification are stored in containers and amplified (proliferated) by PCR (Polymerase Chain Reaction). The present invention relates to a nucleic acid measuring device and nucleic acid measuring method suitable for real-time PCR detection.

Conventionally, nucleic acid amplification of DNA (DeoxyriboNucleic Acid) by PCR amplification is known. As a method therefor, 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 specimen. Real-time PCR is excellent in that it can be quantified in real time. Various proposals have been made with the aim of further improving this real-time PCR.
As a document disclosing real-time PCR, there is Patent Document 1, for example. Patent Document 1 discloses a configuration in which a Peltier element (315) for applying a temperature cycle is arranged below a container (sample plate 305 in FIG. 3 of the same document) that stores a reaction solution (sample). Also, FIG. 5C discloses a configuration using a liquid metal, a Peltier element, or the like. Also disclosed in FIG. 6 is a configuration using a dichroic mirror (611). Thus, Patent Document 1 discloses various configurations for real-time PCR.

Japanese Patent Publication No. 2009-537152

If the temperature cycle can be shortened in real-time PCR, the nucleic acid measurement time, which is generally long, can be shortened. In addition, if the nucleic acid measuring device (real-time PCR device) can be downsized, the convenience will be improved.
Here, as in Patent Document 1, if a Peltier element (315) is arranged under the sample plate (Fig. 3, 305 of the same document), a large number of samples must be placed on the sample plate (305). While the temperature cycle can be applied to a large number of samples at once, it is difficult to heat and cool the sample plate and the samples thereon, making it difficult to shorten the temperature cycle.
In addition, if a liquid metal is used to apply a temperature cycle to a sample (see FIG. 5C, summary column, etc. in the above document), miniaturization of a nucleic acid measuring device is difficult.
In addition, if a dichroic mirror (Fig. 6, 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 exits (outgoing direction) can be reversed. In this case, the light-emitting part and the light-receiving part can be placed on the same side, but this complicates the optical system and makes it difficult to miniaturize the nucleic acid measuring apparatus.
Accordingly, it is an object of the present invention to provide a nucleic acid measurement apparatus and a nucleic acid measurement method that enable shortening of the temperature cycle and downsizing of the apparatus, and that have high detection accuracy.

[1] The nucleic acid measurement device of the present invention is arranged on the side of a container storing a nucleic acid and a nucleic acid test solution containing a nucleic acid amplification reagent. a temperature cycle imparting member that applies a predetermined temperature cycle to a container; a light emitting element that is arranged above the container and emits light including the wavelength of the excitation light of the fluorescent label; a light-receiving element for receiving fluorescence emitted from the container side, the nucleic acid measuring apparatus for measuring the amount of nucleic acid amplification based on the fluorescence received by the light-receiving element, wherein the nucleic acid measuring apparatus further includes the container and the luminescence. It is characterized by comprising a first condensing lens disposed between the elements for condensing light emitted from the light emitting element and irradiating the inside of the container.

Here, "nucleic acid" means, for example, DNA (target DNA, DNA template, detection target DNA, detection target DNA region) and RNA (RiboNucleic Acid). In the case of RNA, DNA converted from RNA by reverse transcription reaction is used.
The term "nucleic acid amplification reagent" refers to reagents used for nucleic acid amplification, including primers for DNA to be detected, fluorescent substances, enzymes (thermostable DNA polymerase), dNTPs, and the like.
The term “fluorescent label” refers to a label possessed by an amplified nucleic acid (target DNA amplicon) that emits fluorescence when excited by excitation light, ie, a so-called fluorescent substance.
As a fluorescent substance, for example, in the annealing stage (step), a DNA probe bound with a fluorescent dye and a quencher (quencher) is bound to the template DNA, and in the extension (reaction) stage, this DNA probe is cleaved, There is a fluorescent substance (fluorescent dye and quencher) used in a method (probe method) for detecting fluorescence from a fluorescent dye that has been suppressed by a quencher. In addition, there is a dye (fluorescent dye) used in a method using a dye that enters between strands of double-stranded DNA and emits fluorescence (intercalator method).
The nucleic acid test solution (or nucleic acid amplification reagent) contains a buffer, which is a solvent for stabilizing the reagent, Mg, etc., as necessary.
The term “nucleic acid amplification amount” refers to the amount of amplified nucleic acid (target DNA) (DNA amount).
"Nucleic acid measurement device" mainly means a real-time PCR device.

[2] In the nucleic acid measurement apparatus of the present invention, the first condenser lens is configured to have a focal point positioned above the standard liquid level when the standard amount of the nucleic acid test solution is stored in the container. preferably.

[3] In the nucleic acid measurement apparatus of the present invention, the area of the standard liquid surface irradiated by the light beam condensed at the focal point of the first condenser lens spreads and becomes substantially equal to the area of the standard liquid surface. It is preferably configured as follows.

[4] In the nucleic acid measurement apparatus of the present invention, the container has a vertically long tube shape with a gradually tapering lower portion, and has a container opening at the upper portion. The cap has a substantially flat upper surface and an annular convex portion on the lower surface side. The nucleic acid measurement device further comprises a heater, which is laminated on the cap so as to have a heater opening in at least a part of the container opening. preferably.

[5] In the nucleic acid measurement apparatus of the present invention, the nucleic acid measurement apparatus further includes a first parallel light lens that converts the light emitted from the light emitting element into parallel light and emits the light toward the first light collecting lens; a first filter disposed between the light-emitting element and the container for passing excitation light having a wavelength that excites the fluorescent label; and a second parallel light lens for converting fluorescence emitted from the container side into parallel light. , a second condenser lens for condensing parallel light emitted from (to be emitted) from the second parallel light lens and emitted to the side of the light receiving element; 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 condenser lens, and the first filter in a state in which they are arranged in the vertical direction; It is preferable to include a second optical cylinder that holds the second parallel light lens, the second condenser lens, the second filter, and the light receiving element in a state in which they are vertically arranged.
In this specification, "between A and B" may be described as "between A and B".

[6] In the nucleic acid measurement device of the present invention, 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 the focus of the plano-convex lens is is positioned above the tip of the convex portion of the light emitting element.

[7] In the nucleic acid measurement apparatus of the present invention, it is preferable that the second condenser lens is configured such that its focal point is located above the light receiving element.

[8] The method for measuring nucleic acid of the present invention comprises the steps of: preparing a container for storing a nucleic acid and a nucleic acid test solution containing a reagent for nucleic acid amplification; 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 a container, a step of subjecting the container to a temperature cycle from the side of the container, and a step of irradiating the fluorescent label with excitation light from above the container; A nucleic acid measurement method comprising a step of receiving fluorescence from the side of the container below the container, and a step of measuring a nucleic acid amplification amount from the received fluorescence, wherein the step of irradiating the excitation light comprises: It is characterized in that the excitation light is condensed and irradiated to the inside of the container from above the container.

According to the present invention, since the temperature cycle imparting member is arranged on the side of the container storing the nucleic acid test solution, heating and cooling can be easily performed, and the temperature cycle can be shortened. In addition, since the light-emitting element is arranged above the container and the light-receiving element is arranged below, the optical system does not become complicated, and the size of the nucleic acid measurement apparatus can be reduced. In addition, since the first condenser lens is arranged between the container and the light-emitting element, and the emitted light from the light-emitting element is collected and irradiated to the inside of the container, the fluorescence label is easily excited, and the nucleic acid has high detection accuracy. It becomes possible to provide a measurement device (or nucleic acid measurement method).

1 is a schematic explanatory diagram of a nucleic acid measurement device 1 according to Embodiment 1. FIG. 1 is an exploded explanatory view of the nucleic acid measuring device 1 according to Embodiment 1. FIG. FIG. 2 is an explanatory diagram of a temperature cycle in the nucleic acid measuring device 1 according to Embodiment 1; FIG. 2 is an explanatory diagram of fluorescence amount detection in the nucleic acid measurement device 1 according to Embodiment 1; FIG. 4 is an explanatory diagram of irradiation of excitation light to a nucleic acid test solution 52 in the nucleic acid measurement device 1 according to Embodiment 1; 2 is an explanatory diagram of the vicinity of a light-emitting element 11 in the nucleic acid measurement device 1 according to Embodiment 1. FIG. 2 is an explanatory diagram of the vicinity of a light receiving element 12 in the nucleic acid measurement device 1 according to Embodiment 1. FIG. FIG. 10 is an explanatory diagram of a temperature cycle in the nucleic acid measuring device according to Embodiment 2;

The nucleic acid measuring apparatus and nucleic acid measuring method of the present invention will be described below with reference to FIGS. 1 to 8. FIG. Each figure described below is a schematic diagram showing the actual shape, configuration, characteristics, etc. in a simplified manner.

[Embodiment 1]
A nucleic acid measurement device 1 according to Embodiment 1 will be described with reference to FIGS. 1 to 7. FIG.
FIG. 1 is a schematic explanatory view of a nucleic acid measuring device 1 according to Embodiment 1, and FIG. 2 is an exploded explanatory view of the nucleic acid measuring device 1. FIG. The nucleic acid measurement device 1 includes eight containers 51 (wells) (see FIG. 2). The eight containers 51 are arranged in a row in the vertical direction of the paper surface of FIG. 1 (horizontal direction in FIG. 2). FIG. 1 shows the cross section of one container 51 among the containers 51 with the cross section of the nucleic acid measurement device 1 having eight series (the central axis AA′ direction of the container 51 in FIG. 2 and the connection of the eight series of containers 51). BB' direction perpendicular to the direction in which the container 51 is placed and the cross section determined by the direction of is a diagram showing a state in which the cap 54 is opened for easy understanding of the outline of the nucleic acid measurement device 1).

The nucleic acid measurement device 1 has a hardware configuration section 2 (hardware configuration section) illustrated on the left side of FIG. 1 and a control section 3 illustrated on the right side.
The hardware configuration part 2 includes a temperature cycle imparting part including a temperature cycle imparting member 33 arranged on the side of the container 51 , a light emitting side optical system including the light emitting element 11 arranged above the container 51 , and the container 51 . and a light-receiving side optical system including a light-receiving element 12 disposed below.
The control unit 3 includes a light receiving/emitting element control means 41 for controlling light receiving/emitting of the light emitting element 11 and the light receiving element 12, a temperature cycle control means 42 for controlling the temperature of the temperature cycle applying member 33, and the light receiving element 12 which received fluorescence. A/D conversion means 45 (Analog-Digital conversion means) for converting the output of, calculation means 44 for calculating the output, storage means 43 for storing calculation results, and overall control for controlling the entire nucleic acid measurement apparatus 1 means 46;

To explain the outline of nucleic acid measurement, the temperature cycle applying member 33 is heated and cooled under the control of the control unit 3, thereby heating and cooling the container 51 (the nucleic acid test solution 52 containing the target DNA). be done). Then, the nucleic acid (target DNA) in the container 51 (nucleic acid test solution 52) is amplified. Under the control of the control unit 3, the light emitting element 11 is driven to emit light, and when the nucleic acid test solution 52 is irradiated with filtered light of a predetermined wavelength (fluorescence excitation light), the amount of amplified target DNA (nucleic acid amplification Fluorescence is excited with an amount of light corresponding to the amount of DNA). The light receiving element 12 is driven by the controller 3 and receives the excited fluorescence. If there is a target DNA in the nucleic acid test solution 52, it will be amplified in a predetermined temperature cycle, so fluorescence with an amount of light corresponding to the amplification amount (nucleic acid amplification amount, DNA amount) is received. No fluorescence is received in the absence of target DNA. In this way, it becomes possible to amplify and measure (detect) small amounts of target DNA.

[Temperature cycle imparting part]
First, the temperature cycle imparting section will be described.
On the side of the container 51 (place where it is placed) are a temperature cycle applying member 33 composed of a heat conducting member 31 and a Peltier element 32, heat radiating fins 34 outside thereof, and a cooling fan 35 (heat radiating fan) outside thereof. and are placed. The heat conducting member 31 and the heat radiating fins 34 are made of, for example, a metal having excellent heat conductivity such as aluminum or copper, or a material containing such metal as a main component. The radiation fins 34 have an uneven shape, thereby increasing the heat transfer area and increasing the heat exchange efficiency.

Regarding the temperature cycle imparting member 33, one surface of the Peltier element 32, for example, the heat dissipation surface (or cooling surface), is arranged in contact with the heat conducting member 31, and the other surface, for example, the cooling surface (or heat dissipation surface) is arranged to dissipate heat. It is placed facing the fins 34 . When the Peltier element 32 is driven by the temperature cycle control means 42 to heat (heat radiation) or cool, the adjacent heat conducting member 31 is heated (heat radiation) or cooled. Then, the container 51 (and the nucleic acid test solution 52 therein) is subjected to a temperature cycle (see FIG. 3), and the nucleic acid in the nucleic acid test solution 52 is amplified. Here, the Peltier element 32 can reverse the heat radiation surface and the cooling surface (heat absorption surface) by reversing the direction of the drive current.
The temperature cycle control means 42 installs a temperature sensor 57 on the heat conducting member 31 to detect the temperature of the heat conducting member 31. According to the detection result, the container 51 (and the nucleic acid test solution 52 therein) is heated to a predetermined temperature. The Peltier element 32 may be driven to provide cycles.

[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. By irradiating the inside of the container 51 (nucleic acid test solution 52) with excitation light (fluorescence excitation light), the nucleic acid amplified by the temperature cycle. Fluorescence corresponding to the amplified amount (nucleic acid amplified amount, DNA amount) of the amplified product (the nucleic acid amplified product has a fluorescent label) is excited.

1 and 2, above the container 51, a light-emitting element 11 (LED element) that emits light including the wavelength of the excitation light of the fluorescent label and the emitted light from the light-emitting element 11 side are arranged in parallel. The first parallel light lens 15 that converts the light into light and emits it to the first condenser lens 13 side, and the parallel light from the first parallel light lens 15 side is condensed and irradiated (emitted) toward the inside of the container 51 . A first condenser lens 13 and a first filter 16 that is arranged between the light emitting element 11 and the container 51 (nucleic acid test solution 52) and that allows passage of excitation light having a wavelength that excites the fluorescent label is arranged. The first optical tube 21 holds the light emitting element 11, the first collimating lens 15, the first condensing lens 13, and the first filter 16 in a state in which they are vertically arranged inside the tube.
In this embodiment, the inner peripheral surface of the first optical tube 21 is provided with a concave portion 21A formed along the circumferential direction centering on the optical tube axis. By fitting the outer peripheral portions of the parallel light lens 15, the first filter 16, and the first condenser lens 13, these (they have a circular shape when viewed from above) are vertically arranged inside the cylinder. They are kept in their original condition.

The light-emitting element 11 is an LED element that emits light including the wavelength of the excitation light of the fluorescent label (the wavelength that excites fluorescence, the wavelength of 490 nm or less). The light emitting element 11 is driven by the light emitting/receiving element control means 41 every time each temperature cycle ends (at the end of the extension step in FIG. 3 to be described later) or all the time to emit light.
Each of the first parallel light lens 15 and the first condenser lens 13 is a plano-convex lens in which one surface is a flat surface (including a substantially flat surface) and the other surface is a convex surface. The plane side of the first parallel light lens 15 faces the direction of the light emitting element 11 , and the plane side of the first condenser lens 13 faces the direction of the container 51 .
The first filter 16 is a first filter 16 (a low-pass filter that allows passage of light with a wavelength of 490 nm or less) that allows passage of light with the wavelength of the excitation light. 1 and 2, the first filter 16 is arranged between the first parallel light lens 15 and the first condenser lens 13. , for example, between the light emitting element 11 and the first parallel light lens 15, between the first condenser lens 13 and the container 51 (nucleic acid test solution 52), and so on.

[Light receiving side optical system]
Next, the light receiving side optical system will be described.
A light-receiving optical system including the 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 acids are detected (identified) by detecting the amount of fluorescence.

1 and 2, below the container 51, fluorescent light scattered (emitted) from the container 51 (nucleic acid test solution 52) side (fluorescence emission port 31B) is converted into parallel light. A second parallel light lens 17 that emits light to the side of the second condenser lens 14 and a second parallel light lens 17 that converges (emitted) parallel light emitted from the second parallel light lens 17 and emits the light to the side of the light receiving element 12 . Condensing lens 14, light-receiving element 12 for receiving light condensed by second condensing lens 14, and arranged between container 51 and light-receiving element 12, downward from container 51 (nucleic acid test solution 52) side and a second filter 18 that passes light of a specific wavelength out of emitted (emitted) light. The second optical tube 22 holds the second collimating lens 17, the second condensing lens 14, the second filter 18, and the light receiving element 12 in a state in which they are vertically arranged inside the tube.
In this embodiment, the inner peripheral surface of the second optical tube 22 is provided with a concave portion 22A formed along the circumferential direction centering on the optical tube axis. 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 are circular when viewed from above) are arranged vertically inside the cylinder. Orientation holds them in place.

The second filter 18 is a band-pass filter that allows passage of light with a wavelength of fluorescence, allows passage of light with a wavelength of 515 nm to 535 nm, and blocks passage of light with 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. As long as it is between, for example, between the container 51 (nucleic acid test solution 52) and the second parallel light lens 17, or between the second condenser lens 14 and the light receiving element 12, it may be arranged anywhere.

Each of the second parallel light lens 17 and the second condenser lens 14 is a plano-convex lens in which one surface is a flat surface (including a substantially flat surface) and the other surface is a convex surface. The planar side of the second collimating lens 17 faces the container 51 , and the planar side of the second condenser lens 14 faces the light receiving element 12 .

The light receiving element 12 is a photodiode element that receives (detects) excited fluorescence (center wavelength of about 525 nm). The light-receiving element 12 is driven by the light-receiving/light-receiving element control means 41 every time each temperature cycle is completed or constantly to receive fluorescence and output an analog electric signal corresponding to the amount of received light. This analog electrical signal is input to the A/D conversion means 45 and converted into a digital electrical 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 apparatus 1 including the respective means (41, 42, . . . ) in FIG.

The inner surfaces of the first optical tube 21 and the second optical tube 22 are mirror-finished so as to reflect light. The mirror-finished first optical tube 21 and the second optical tube 22 are made of, for example, a metal plate of aluminum, silver, stainless steel, or the like, whose surfaces corresponding to the inner surfaces are mirror-finished, and whose surfaces corresponding to the inner surfaces are made of aluminum, silver, or the like. It can be composed of a plastic molding or the like which is mirror-finished by forming a metal film by plating, sputtering, or the like.

[Container 51]
A container 51 for storing a nucleic acid test solution 52 and its surroundings will be described (see FIGS. 1, 2, and 5).
The container 51 (well) and its cap 54 are made of polypropylene and substantially transparent. As used herein, the term "transparent" means to allow light to pass through. The term "substantially transparent" is intended to include cases in which light is completely transmitted, as well as cases in which most of the light is transmitted, though not completely. In addition to being colorless, it also includes cases with colors (including white) that do not block the passage of light. In actual industrial products, it is difficult to allow light to pass completely even if they are transparent, and most of the light is often allowed to pass through, but substantially transparent includes such products.
The container 51 is vertically long and has a tubular shape with a gradually tapering lower portion. 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 top through which the nucleic acid test solution 52 is taken in and out. During measurement (including during DNA amplification by temperature cycling), the container opening is closed with a cap 54 .

The present nucleic acid measuring apparatus 1 has a configuration in which a heater 56 for heating the cap 54 is stacked on the cap 54 when the container 51 is stored in a container storing hole 31A (described later) of the heat conducting member 31 . The heater 56 is configured by, for example, attaching an insulating film on which a resistance is printed to a copper plate, a stainless steel plate, or the like. Although the heater 56 itself blocks the passage of light, a heater opening is provided at a location corresponding to the opening of the container to allow the passage of light. The heater 56 is controlled by the controller 3 so as to always heat the cap 54 at, for example, 100 to 105.degree. 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 "substantially flat" is intended to include not only perfect planes but also planes with some irregularities, curves, and the like. An annular projection 54A is provided on the lower surface side, and when closing the container opening, the outer peripheral surface of the annular projection 54A is brought into close contact with the inner peripheral surface of the container opening. In addition, in order to increase the adhesion between the cap 54 and the inner surface of the container 51, the thickness of the annular convex portion 54A in the radial direction (horizontal direction) from the center of the annular circle is less than the thickness of the upper surface of the cap 54 (vertical direction). , is thickly formed. All or most of the light incident on the container 51 preferably passes through the upper surface of the cap 54 . Also, it is preferable that all or most of it does not come into contact with the annular projection 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 capacity, but the standard storage amount (standard amount, recommended storage amount) is 20 μl (10% of the maximum capacity). This is for speeding up the heating and cooling of the nucleic acid test solution 52, and the like.

A container storage hole 31A having a shape (concave shape) corresponding to the shape of the container (conical shape) is formed in the heat conducting member 31 described above. 51 stands on its own. 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 conforming to the standard container) is naturally used.
The upper portion of the container storage hole 31A has a shape that substantially conforms to the shape of the container 51, but the hole diameter is larger than the outer diameter of the container 51, and a gap 36 ( space) is formed (herein, "diameter" means diameter). Although the gap 36 may be a space, a heat insulating member such as expanded polystyrene may be disposed there.
As described above, the lower portion of the container housing hole 31A has a mortar shape, and the bottom is provided with a fluorescence emission port 31B for emitting fluorescence downward. The diameter 73 (inner diameter) of the fluorescence emission port 31B is, for example, approximately 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 portion (mortar-shaped portion) of the container storage hole 31A excluding the fluorescence emission port 31B (the portion may include a part of the cylindrical portion that is continuous with the mortar-shaped portion). The surface of the container storage hole 31A on the container 51 side is mirror-finished so that the excitation light incident on the container 51 and the fluorescent light of the nucleic acid test solution 52 are reflected.

The inner diameter 72 of the first optical tube 21 is substantially the same as the inner diameter 71 of the top of the container (see FIG. 1). Also, the inner diameter 74 of the second optical tube 22 is substantially the same as the inner diameter 71 of the inner diameter 71 of the upper portion of the container, but may be slightly smaller (see FIG. 1).
With this configuration, when the containers 51 are arranged at a predetermined pitch such as eight, even if the first optical cylinder 21 and the second optical cylinder 22 are arranged according to the pitch of the containers 51, the distance between the first optical cylinders 21 is small. Alternatively, the diameter of the first optical tube 21 (outer diameter and inner diameter) and the diameter of the second optical tube 22 (outer diameter and inner diameter) can be increased. Further, if the inner diameter 72 of the first optical tube 21 is large, the light emitting element 11 with a large outer diameter and a large amount of emitted light can be inserted into the first optical tube 21 and used. Further, when the inner diameter 74 of the second optical tube 22 is large, it is easy to widely collect fluorescence from the container 51 side (fluorescence emission port 31B side).

[Temperature cycle]
FIG. 3 is an explanatory diagram of the temperature cycle of the nucleic acid measuring device 1 according to Embodiment 1. FIG. In the nucleic acid measurement device 1, the control unit 3 controls the temperature of the temperature cycle imparting member 33 to apply temperature cycles to the container 51 (nucleic acid test solution 52), thereby amplifying a large amount of DNA. One cycle T in the temperature cycle has each step (process, stage) of thermal denaturation, annealing and elongation, as shown in FIG.

In the thermal 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. to bind the primers to the adjacent regions of the target DNA, and the DNA probe bound with the fluorescent dye and quencher (quenching substance) is attached to the template DNA. Bind (probe method).
In the elongation step, the container 51 (nucleic acid test solution 52) is reheated to 72° C., and DNA polymerase (an enzyme that synthesizes a DNA strand complementary to the template nucleic acid) extends the DNA starting from the primer. to synthesize a complementary strand of DNA (complementary sequence). The extension step cleaves the DNA probe and emits fluorescence from the fluorochrome that was suppressed by the quencher. The light-receiving element 12 receives this fluorescence, and the A/D conversion means 45 A/D-converts the output of the light-receiving element 12 .
After the elongation step, the heat denaturation step is performed again.
Target DNA is amplified exponentially by repeating such temperature cycles 25 to 40 times.

Here, transition time TA from heat denaturation (step) to annealing (step), transition time TB from annealing (step) to extension (step), and transition time TC from extension (step) to heat denaturation (step) may be shortened, and if these transition times are shortened, it becomes possible to shorten the total time of temperature cycles that are repeated 25 to 40 times until the amount of fluorescence is saturated.
In the nucleic acid measurement device 1 of Embodiment 1, since the heat conducting member 31 and the Peltier 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 times TA, TB, and TC for heating and cooling the container 51 can be shortened. Therefore, it is possible to shorten the total time of the temperature cycle.

[Fluorescence measurement]
FIG. 4 is an explanatory diagram of fluorescence amount detection (fluorescence amount detection curve) in the nucleic acid measurement 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 this example, the amount of fluorescence begins to increase corresponding to the nucleic acid amplification amount (DNA amount) at around 20 temperature cycles (number), and saturates at 25 to 40 times.
The measurement accuracy of the nucleic acid measurement device 1 will be described.
Curve A in FIG. 4 is a fluorescence amount detection curve (standard curve) previously measured using a nucleic acid (DNA) and a nucleic acid test solution 52 (standard amount) containing a nucleic acid amplification reagent.
Curves A1 and A2, curves B1 and B2, and curves C1 and C2 on the left and right of curve A are obtained by storing standard amounts of the same nucleic acid test solution 52 as in the case of curve A in eight containers 51, and performing the same temperature cycle at the same time. It is a fluorescence amount detection curve showing the variation with respect to the A curve (standard curve) when measured by giving . The A1 and A2 curves are close to the A curve, but the B1 and B2 curves are further away from the A curve, and the C1 and C2 curves are further away.
The measurement accuracy of the nucleic acid measurement 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 curve A (standard curve).
The detection accuracy is "excellent" when the above eight curves fall within the A1 curve/A2 curve range near the A curve (standard curve). On the other hand, if the eight curves do not fall within the range of curves A1 and A2 and extend to the range of curves B1 and B2 that are far from curve A, the detection accuracy is "lower" than in the above case. If the above eight curves do not fall within the A1 curve/A2 curve range and the B1 curve/B2 curve range, but extend to the C1 curve/C2 curve range, it is "very inferior" compared to 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 diagram for explaining irradiation of excitation light to the nucleic acid test solution 52 with and without the first condenser 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 solution 52 is stored.

FIG. 5( a ) is a diagram showing a case where the nucleic acid measurement device 1 is provided with the first condenser lens 13 and its focal point F1 is located above the standard liquid surface 53 . The luminous flux condensed at the focal point F1 of the first condenser lens 13 spreads again and irradiates the standard liquid surface 53, and the irradiation area is approximately equal to the area of the standard liquid surface 53. FIG.

FIG. 5(b) is a diagram showing a case (comparative example 1) in which the nucleic acid measurement apparatus is provided with the first condenser lens 13 and the focal point F1 is located at the standard liquid surface 53. FIG. In this case, only one point on the standard liquid surface 53 is irradiated with the concentrated intense light.

FIG. 5(c) shows a case where the nucleic acid measurement apparatus does not include the first parallel light lens 15 and the first condenser lens 13 (see FIG. 1), and scattered light irradiates the standard liquid surface 53 (comparison FIG. 10 is a diagram showing Example 2). Since the first parallel light lens 15 and the first condenser lens 13 are not provided, the standard liquid surface 53 is irradiated with the scattered light (not condensed light) that has passed through the cap 54 among the light in the first optical cylinder 21 .

FIG. 5(d) is a diagram showing a case (comparative example 3) in which the nucleic acid measurement apparatus does not include the first condenser lens 13 (see FIG. 1) and the standard liquid surface 53 is irradiated with parallel light. Since the first parallel light lens 15 is provided but the first condenser lens 13 is not provided, 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 and Comparative Examples 1 to 3) in which standard amounts of nucleic acid test solutions 52 containing the same nucleic acids and nucleic acid amplification reagents are stored in eight containers 51 and the detection accuracy is evaluated.
Note that the conditions (temperature cycle, etc.) other than the conditions described in Table 1 (see "Focus F1" column) are the same in Example 1 and Comparative Examples 1-3.

The “reference diagram” in Table 1 indicates which of FIG. 5 the nucleic acid measurement device corresponds to. "Focus F1" indicates where the focal point F1 is located when the nucleic acid measurement apparatus is equipped with the first condenser lens 13, and when the first condenser lens 13 is not provided (focus F1 ) is “none”.
"8 curves for 8 containers 51" indicates the degree of variation of the 8 fluorescence amount detection curves for 8 containers 51 with respect to curve A (standard curve, see FIG. 4). For example, "A1 curve/A2 curve range" indicates that 8 curves are in (within) "A1 curve/A2 curve range".
"Detection accuracy" indicates the detection accuracy based on "eight curves of eight containers 51". It is indicated by "○: excellent", "Δ: poor", and "X: very poor" (see [Fluorescence measurement]). As for the "detection accuracy", the nucleic acid measurement apparatus 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 element 11 in the nucleic acid measurement device 1 according to Embodiment 1. FIG. 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 that seals the LED chip 11A, and lead terminals 11C. have. The electrode of the LED chip 11A is 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 solder 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 cannonball shape with 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 tube 21 .
The mold resin 11D is resin such as epoxy and is substantially transparent. Most of the light emitted from the LED chip 11A is radiated outside through the mold resin 11D, but part of the light is reflected on the surface of the mold resin 11D.
The first parallel light lens 15 (plano-convex lens) receives light emitted (radiated) from the light emitting element 11 (LED element), converts the light into parallel light, and emits the parallel light.
Here, when the focal point F3 of the first parallel light lens 15 (plano-convex lens) is positioned 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 becomes possible. This is probably because more light emitted from the light emitting element 11 can be captured. In addition, 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 measurement device 1 according to Embodiment 1. FIG.
FIG. 7(a) is an enlarged view of the vicinity of the light receiving element 12 in FIG. The light-receiving element 12 is a photodiode element, and includes a photodiode chip 12A (semiconductor) having a square photodiode chip 12A (semiconductor) on which a photovoltaic generation region 12G (see FIG. 7B, which will be described later) is located on the upper side. It has a shielding metal plate 12C surrounding the chip 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 lead terminals 12E. The electrodes of the photodiode chip 12A are connected to wiring on the substrate 12B by wire bonding or the like, and the wiring is connected to lead terminals 12E by solder or the like. The shielding metal plate 12C surrounds the chip 12A so as to be higher than the upper surface of the chip 12A (the surface having the photovoltaic generating region 12G).
As shown in FIG. 7A, the second condenser lens 14 preferably has a focal point F2 above the light receiving element 12 (photodiode chip 12A). In this way, the light beam condensed at the focal point F2 of the second condenser lens 14 spreads and irradiates the light receiving element 12, so that the light beam can be easily collected on the light receiving element 12 (photodiode chip 12A).
When the shielding metal plate 12C surrounds the chip 12A so as to be higher than the upper surface of the chip 12A (the surface on which the photovoltaic generation region 12G is present), the light from the second condenser lens 14 passes through the focal point F2. If the chip 12A is directly irradiated with the light before, the irradiation light to the edge of the chip 12A is likely to be blocked by the shielding metal plate 12C. On the other hand, if the light from the second condenser lens 14 is condensed once at the focal point F2 and is irradiated with the light that has passed through the focal point F2, even the edge of the chip 12A is shielded by the shielding metal plate 12C. It becomes easy to irradiate without being exposed.

FIG. 7B shows a photovoltaic generation region 12G of a photodiode chip 12A (a large number of photodiodes are formed, and when irradiated with light, free electrons and free holes are generated to generate current and voltage). A region where an electromotive force effect occurs (the output of the photodiode chip 12A is a collection of outputs from a large number of these photodiodes) is shown illuminated with fluorescent light. While the photovoltaic force generation region 12G is almost the entire area (square region) of the upper surface of the chip 12A, the actual irradiation region 12H is a circular shape within this square light-receivable region (photovoltaic force generation region 12G). area.
Note that the regions other than the irradiation region 12H irradiated with the fluorescent light in the photovoltaic generation region 12G are regions that are not related to the fluorescent light. Then, since the photovoltaic generation region 12G and the irradiation region 12H are almost the same region, the SN ratio (signal noise ratio) can be further increased compared to the case without masking. Become.

According to the nucleic acid measurement device 1 and the nucleic acid measurement method according to the first embodiment, the temperature cycle imparting member 33 (31, 32) is arranged on the side of the container 51 that stores the nucleic acid test solution 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 container 51, the optical system is not complicated and the device can be made compact. In addition, since the first condenser lens 13 is arranged between the container 51 and the light emitting element 11, and the light emitted from the light emitting element 11 is collected and irradiated inside the container 51, the fluorescence label is easily excited and the cost is high. It is possible to provide the nucleic acid measurement device 1 (or nucleic acid measurement method) having detection accuracy.

[Embodiment 2]
The nucleic acid measuring apparatus (and nucleic acid measuring method) according to Embodiment 2 is basically the same as that of Embodiment 1, but differs in that the temperature cycle is changed to that shown in FIG. 8 instead of the temperature cycle shown in FIG.
In Embodiment 2, as shown in FIG. 8, annealing and elongation during one cycle T are performed at the same temperature of around 60.degree. By changing the enzyme or the like put into the nucleic acid test solution 52, these can be made to have the same temperature. The temperature transition periods in one cycle T are a period TA1 during which thermal denaturation transitions to annealing and a period TC1 during which elongation transitions to thermal denaturation.
Since the nucleic acid measurement apparatus (and nucleic acid measurement method) according to Embodiment 2 is the same as Embodiment 1 except that the temperature cycle is changed to that shown in FIG. 8 instead of the temperature cycle shown in FIG. Among the effects of the nucleic acid measurement device 1 (and the nucleic acid measurement method) of No. 1, it has the corresponding effect.

In this specification, "upper" and "lower" may be replaced with "light-emitting device direction" and "light-receiving device direction", respectively.

[Modification]
Although the present invention has been described based on the above embodiments, the present invention is not limited to the above embodiments. It is possible to implement it without departing from the gist thereof, and for example, the following modifications are also possible.

(1) The materials, shapes, positions, sizes, wavelengths, temperatures, etc. described in the above embodiments are examples, and can be changed within a range that does not impair the effects of the present invention.

(2) In the above embodiment, plano-convex lenses are used as the first condenser lens 13 and the second condenser lens 14, but this is an example. A lens is also possible.

(3) In the above embodiment, a fixed focus lens (plano-convex lens) was used as the first condenser lens 13 and the second condenser lens 14. A varifocal lens, a zoom lens, etc.) can also be used.

(4) In the above embodiment, the PCR amplified product was detected by fluorescence using the probe method. PCR amplification products may be detected by fluorescence using the intercalator method of inclusion or other methods.

(5) In the above-described embodiment, the first filter 16 that passes the excitation light of the wavelength that excites the fluorescent label is used. 11 (LED element) may be used, or a cap 54 mixed with a substance that absorbs light of a predetermined wavelength may be used.
Further, instead of the second filter 18 for passing fluorescence of a predetermined wavelength, a substantially transparent glass plate 12D (for sealing the photodiode chip 12A) mixed with a substance that absorbs light of a predetermined wavelength (or the glass plate 12D is Alternatively, a substantially transparent encapsulating resin mixed with a substance that absorbs light of a predetermined wavelength may be used.

DESCRIPTION OF SYMBOLS 1... Nucleic acid measuring apparatus 2... Hardware structure part 3... Control part 11... Light emitting element 11A... LED chip 11B... Substrate 11C... Lead terminal 11D... Mold resin 11E... Tip part 12... Light receiving element , 12A... Photodiode chip, 12B... Substrate, 12C... Metal plate for shielding, 12D... Glass plate, 12E... Lead terminal, 12G... Photovoltaic generation area, 12H... Irradiation area, 13... First condenser lens, 14 2nd condenser lens 15 1st parallel light lens 16 1st filter 17 2nd parallel light lens 18 2nd filter 21 1st optical cylinder 21A recess 22 2nd Optical tube 22A Recessed portion 31 Heat conductive member 31A Container housing hole 31B Fluorescent emission port 32 Peltier element 33 Temperature cycle imparting member 34 Radiation fin 35 Cooling fan 36 Gap 41... Light emitting/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 projection 56 Heater 57 Temperature sensor 61, 62 Circuit board 71 Inside diameter of container top 72 Inside diameter of first optical cylinder 73 Fluorescence Diameter of emission port 74...Inner diameter of second optical cylinder 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 on the side of a container that stores a nucleic acid test solution containing a nucleic acid and a reagent for nucleic acid amplification, and applies a predetermined temperature cycle to the container in order to amplify the nucleic acid and obtain an amplified product having a fluorescent label. an imparting member;
a light-emitting element that is disposed above the container and emits light containing the wavelength of the excitation light of the fluorescent label;
a light-receiving element arranged below the container for receiving fluorescence emitted from the container;
A nucleic acid measurement device that measures the amount of nucleic acid amplification from the fluorescence received by the light receiving element,
The nucleic acid measurement device further comprises
A nucleic acid measurement apparatus, comprising: a first condensing lens disposed between the container and the light emitting element for condensing light emitted from the light emitting element and irradiating the inside of the container. In the nucleic acid measurement device according to claim 1,
The nucleic acid measurement apparatus, wherein the first condenser lens is configured to have a focal point above a standard liquid level when the standard amount of the nucleic acid test liquid is stored in the container. In the nucleic acid measurement device according to claim 2,
The nucleic acid, wherein the light beam condensed at the focal point of the first condensing lens spreads and irradiates the standard liquid surface so that the area of the standard liquid surface is substantially equal to the area of the standard liquid surface. measuring device. In the nucleic acid measurement device according to any one of claims 1 to 3,
The container has a vertically long tube shape with a gradually narrowing lower portion and a container opening at the upper portion,
The container further has a cap that closes the container opening,
The cap has a substantially flat upper surface and 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 closing the container opening. is composed of
The nucleic acid measurement device further comprises a heater,
A nucleic acid measuring apparatus, wherein the heater is stacked on the cap so as to have a heater opening in at least part of the opening of the container. In the nucleic acid measurement device according to any one of claims 1 to 4,
The nucleic acid measurement device further comprises
a first parallel light lens that converts light emitted from the light emitting element into parallel light and emits the light toward the first condenser lens;
a first filter disposed between the light-emitting element and the container, which passes excitation light having a wavelength that excites the fluorescent label;
a second parallel light lens for converting fluorescence emitted from the container side into parallel light;
a second condensing lens that collects the parallel light emitted from the second parallel light lens and emits the parallel light toward the light receiving element;
a second filter disposed between the container and the light-receiving element for passing fluorescence of a predetermined wavelength;
a first optical cylinder that holds the light emitting element, the first parallel light lens, the first condenser lens, and the first filter in a vertically arranged state;
a second optical cylinder that holds the second parallel light lens, the second condenser lens, the second filter, and the light receiving element in a state in which they are arranged in the vertical direction;
A nucleic acid measurement device comprising: In the nucleic acid measurement device according to claim 5,
The light emitting element is a bell-shaped LED element having a convex portion on the lower side,
The nucleic acid measurement device, wherein the first parallel light lens is a plano-convex lens, and 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 measurement device according to claim 5 or 6,
The nucleic acid measuring apparatus, wherein the second condenser lens is configured to have a focal point positioned above the light receiving element. preparing a container for storing a nucleic acid test solution containing a nucleic acid and a reagent for nucleic acid amplification, and 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;
applying a temperature cycle to the container from the side of the container;
a step of irradiating excitation light for the fluorescent label from above the container;
receiving fluorescence from the side of the container below the container;
a step of measuring the amount of nucleic acid amplification from the received fluorescence;
A nucleic acid measurement method comprising
In the step of irradiating the excitation light, the excitation light is condensed and irradiated into the interior of the container from above the container.

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