JP2012034617A - Nucleic acid amplification reaction apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reaction apparatus capable of detecting both a turbidity material and a fluorescent material.SOLUTION: The nucleic acid amplification reaction apparatus has: a first light source emitting light for exciting the fluorescent material; a second light source emitting light in a wavelength range which overlaps with the wavelength of the fluorescent generated from the fluorescent material; and a control part for switching to emit the first light source and the second light source. A reaction zone as a reaction field of the nucleic acid amplification reaction is irradiated with the light from the first light source and the second light source passed on the overlapped light path, and thus the quantity of light by the turbidity material generated according to the progression of the amplification reaction and the strength of the fluorescent generated from the fluorescent material excited by the light according to the progression of the amplification reaction can be detected.

Description

  The present invention relates to a nucleic acid amplification reaction apparatus. More specifically, the present invention relates to a nucleic acid amplification reaction apparatus used for gene analysis such as gene expression analysis, infectious disease inspection, and SNP analysis.

In recent years, verification by performing a gene amplification reaction by a PCR method (Polymerase Chain Reaction) or a LAMP method (Loop-Mediated Isothermal Amplification) has become a standard method for quantitative analysis of a small amount of nucleic acid. By such methods, gene analysis such as gene expression analysis, genetic disease, canceration, infectious diseases such as microorganisms and viruses, and SNP analysis has been promoted.
Here, in the method for detecting the amplification product of nucleic acid, a fluorescence detection method using a fluorescent substance such as an intercalator method or a fluorescent labeling probe method, or a metal ion such as magnesium ion is used as a byproduct pyrophosphate in the amplification process. A turbidity detection method using a turbidity substance in which a salt that is insoluble or hardly soluble in water is formed is mainly used (Patent Documents 1 to 3).
Fluorescence detection devices and turbidity detection devices for detecting fluorescent materials and turbidity materials as described above are widely available on the market.

JP 2008-237207 A International Publication No. 01/83817 Pamphlet Japanese Patent No. 413347

In a conventional nucleic acid amplification fluorescence detection apparatus, in order to detect a fluorescent component weaker than excitation light, an excitation light wavelength filter (hereinafter referred to as “excitation filter”) having a desired transmission region in consideration of the absorption spectrum of the fluorescent material in the excitation system. ”). However, in this method, turbidity detection that detects a change in the amount of light transmitted through the sample due to internal scattering by amplification by-products generated in the nucleic acid amplification reaction cannot be realized in the same system as fluorescence detection.
Further, even in a conventional nucleic acid amplification turbidity detection device, if an excitation filter for fluorescence detection is provided, turbidity cannot be detected, and thus fluorescence detection cannot be realized with the same optical system.
Accordingly, the main object of the present invention is to provide a nucleic acid amplification reaction apparatus capable of detecting fluorescence and detecting turbidity in the same system.

  In order to solve the above problems, the present invention provides a first light source that emits light that excites the fluorescent material, a second light source that emits light in a wavelength region that matches the wavelength region of fluorescence generated from the fluorescent material, An optical system having a control unit that switches between the first light source and the second light source to emit light, and the light from the first light source and the light from the second light source overlap at the light guide member The reaction area that is the reaction field of the amplification reaction of nucleic acid is irradiated through the road, and the amount of light caused by the turbidity substance that accompanies the amplification reaction and the fluorescent substance that is excited by light as the amplification reaction proceeds A nucleic acid amplification reaction apparatus capable of detecting the intensity of generated fluorescence is provided.

In addition, a light guide member that guides light from these light sources on the same optical system path is disposed on the optical system path between the first light source and the second light source and the reaction region, It is preferable that a fluorescent filter is disposed on the optical system path between the reaction region and the photodetector.
The first light source is preferably a laser light source.

A light guide for guiding light from the second light source onto the optical path of the first light source on the optical path between the first light source and the second light source and the reaction region. Preferably, a member is disposed, and a fluorescent filter is disposed on the optical system path between the reaction region and the photodetector.
An excitation filter that transmits light from the first light source and the light guide member that transmits light from the excitation filter are disposed on an optical system path between the first light source and the reaction region. Is preferred.

  The nucleic acid amplification detection apparatus according to the present invention can detect fluorescence and turbidity with the same optical system by switching light sources.

The conceptual diagram which looked at side view of 1st embodiment in the nucleic acid amplification reaction apparatus concerning this invention is shown. The conceptual diagram which looked at 2nd embodiment in the nucleic acid amplification reaction apparatus concerning this invention from the side is shown. An example of the specific excitation light for the fluorescence detection of 1st embodiment (a) and 2nd embodiment (b) is shown.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

1. Nucleic acid amplification reaction device (1) First light source (2) Second light source (3) Control unit (4) Light guide member (5) Fluorescence filter and photodetector (6) Reaction region (a) Nucleic acid amplification reaction ( b) Detection method of nucleic acid amplification First Embodiment of Nucleic Acid Amplification Reaction Apparatus According to the Present Invention (1) Configuration of First Embodiment (2) Operation of First Embodiment (a) Fluorescence Detection (a-1) Operation of RT-PCR Apparatus ( b) Turbidity detection (b-1) Operation of LAMP device Second embodiment of nucleic acid amplification reaction apparatus according to the present invention (1) Configuration of second embodiment (2) Operation of second embodiment (a) Fluorescence detection (b) Turbidity detection

<1. Nucleic acid amplification reaction apparatus>
1 and 2 are conceptual views of the first and second embodiments of the nucleic acid amplification reaction apparatus according to the present invention viewed from the side.
Note that, in the drawings described below, for the convenience of explanation, the configuration of the apparatus and the like are simplified.

The general configuration of the nucleic acid amplification reaction apparatus according to the present invention will be described below.
A nucleic acid amplification reaction apparatus according to the present invention includes a first light source and a second light source that emit light to irradiate a reaction region, a control unit that switches between these light sources, a light guide member, a fluorescent filter, and a photodetector. I have. The reaction region is detachable, and the reaction region becomes a reaction field for nucleic acid amplification reaction. The nucleic acid amplification reaction apparatus of the present invention can detect the amount of light caused by a turbid substance that accompanies the amplification reaction and the intensity of the fluorescence generated from the fluorescent substance excited by the progress of the amplification reaction.

(1) First light source The first light source emits light that excites a fluorescent substance.
The excitation light has a specific wavelength range corresponding to the absorption spectrum of a desired fluorescent material, and has a wavelength range that does not overlap with the wavelength range of fluorescence generated from the fluorescent material.
The excitation light reaches the reaction region, and excites the fluorescent material generated as the amplification reaction proceeds in the reaction region. Then, the intensity of the fluorescence generated from the fluorescent material is detected by a photodetector through a fluorescent filter.

(2) Second light source The second light source emits light in a wavelength region that coincides with light having a light emission wavelength in a light emission wavelength region of fluorescence generated from a fluorescent material and a transmission wavelength region of a fluorescent filter.
The light has light in a wavelength region that coincides with light having an emission wavelength in the emission wavelength region of fluorescence generated from a desired fluorescent material and the transmission wavelength region of the fluorescence filter. For example, the light is emitted from a normal light source. The light etc. with which such various wavelengths are mixed are mentioned. In addition, although the said wavelength range which corresponds is a long wavelength range, 450 nm or more, for example, 450-780 nm is mentioned especially, for example.
The light reaches the reaction region and is reflected or absorbed by the turbid substance generated as the amplification reaction proceeds in the reaction region. Then, the amount of light scattered or transmitted by the turbid substance is detected by a photodetector.

(3) Controller The switching between the first light source and the second light source is controlled by the controller. For example, in the nucleic acid amplification, the control unit considers a light source that emits a light component suitable for detection by fluorescence or turbidity, irradiates light from one light source, and emits light from the other light source. Control not to irradiate. Further, the control unit may control the amount of light emitted from the light source.
In addition to controlling the light source, the control unit may control various operations related to the apparatus of the present invention (for example, nucleic acid amplification reaction, detection light amount calculation, monitoring, etc.).

(4) Light guide member The light guide member is provided with a light incident end, and the light incident end is emitted from one or more of the first light source and / or the second light source. Light is incident. Inside the light guide member, there are provided members (for example, prisms, reflectors, projections and depressions, etc.) that direct the incident light in the direction of the reaction region. By using this light guide member, the number of light sources can be reduced, and uniform irradiation and low power consumption can be achieved. Furthermore, the entire apparatus can be reduced in size, particularly reduced in thickness.

(5) Fluorescent filter and light detector The fluorescent filter preferably transmits fluorescence, scattered light and transmitted light from the reaction region. Specifically, according to the fluorescent substance and turbidity substance used in the nucleic acid amplification reaction, a substance that efficiently transmits these wavelength bands and blocks other wavelength bands may be selected as appropriate. An example is a long pass filter (LRF). Further, the wavelength band is preferably a long wavelength region, and the specific wavelength band is 450 nm or more, more specifically 450 to 780 nm.
The photodetector is not particularly limited as long as at least fluorescence and turbidity can be detected. For example, an area imaging device such as a CCD or CMOS device, a PMT (photomultiplier tube), a photodiode, or the like. Is mentioned.
In addition, what is necessary is just to arrange | position a condensing lens suitably between each structure mentioned above.

(6) Reaction Area The reaction area is an area that serves as a reaction field for nucleic acid amplification reaction, and is formed in a reaction vessel such as a microchip for nucleic acid amplification reaction.
The reaction region is formed by one or a plurality of reaction substrates. The reaction substrate can be formed by wet etching or dry etching of a glass substrate layer, or by nanoimprinting, injection molding, or cutting of a plastic substrate layer. At this time, the shape of the reaction region can be set as appropriate, and may be, for example, well or tubular.
The material of the reaction substrate is not particularly limited, and is preferably selected as appropriate in consideration of the detection method, processability, durability, and the like. The material is a light-transmitting material and may be appropriately selected according to a desired detection method. Examples thereof include glass and various plastics (polypropylene, polycarbonate, cycloolefin polymer, polydimethylsiloxane, etc.).
Further, when forming the reaction vessel, the reaction region may be appropriately filled with reagents necessary for the nucleic acid amplification reaction.

(A) Nucleic Acid Amplification Reaction In the present invention, the “nucleic acid amplification reaction” includes a conventional PCR (polymerase chain reaction) method in which a temperature cycle is performed, and various isothermal amplification methods not involving a temperature cycle. As isothermal amplification methods, for example, LAMP (Loop-Mediated Isothermal Amplification) method, SMAP (SMartAmplification Process) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic acids) method (Registered trademark), TRC (transcription-reverse transcription concerted) method, SDA (strand displacement amplification) method, TMA (transcription-mediated amplification) method, RCA (rolling circle amplification) method and the like.
In addition, the “nucleic acid amplification reaction” broadly encompasses nucleic acid amplification reactions by temperature change or isothermal for the purpose of nucleic acid amplification. These nucleic acid amplification reactions also include reactions involving quantification of amplified nucleic acid chains such as real-time PCR (RT-PCR) method and RT-RAMP method.

The “reagent” is a reagent necessary for obtaining an amplified nucleic acid chain in the nucleic acid amplification reaction described above, specifically, an oligonucleotide primer having a base sequence complementary to the target nucleic acid chain, Nucleic acid monomers (dNTP), enzymes, reaction buffer (buffer) solutes and the like are included.

In the PCR method, amplification cycles of “thermal denaturation (about 95 ° C.) → primer annealing (about 55-60 ° C.) → extension reaction (about 72 ° C.)” are continuously performed.
The LAMP method is a method for obtaining dsDNA as an amplification product from DNA or RNA at a constant temperature by using DNA loop formation. As an example, components (i), (ii) and (iii) are added, the inner primer can form a stable base pair bond with a complementary sequence on the template nucleic acid, and the strand displacement polymerase is an enzyme. It proceeds by incubating at a temperature that can maintain activity. In this case, the incubation temperature is preferably 50 to 70 ° C. and the time is preferably about 1 minute to 10 hours.
Component (i) 2 types of inner plummer, or 2 types of outer primer, or 2 types of loop primer; Component (ii) strand displacement polymerase; Component (iii) substrate nucleotide.

(B) Method for detecting nucleic acid amplification (product) The method for detecting nucleic acid amplification is not particularly limited, and examples thereof include a method using a fluorescent substance, a turbid substance, a chemiluminescent substance, and the like.

Examples of the method using the fluorescent substance or the chemiluminescent substance include an intercalation method using a fluorescent dye (derivative) that specifically inserts into a double-stranded nucleic acid and emits fluorescence, an oligo specific to the nucleic acid sequence to be amplified. Examples thereof include a labeled probe method using a probe in which a fluorescent dye is bound to a nucleotide.
Examples of the labeled probe method include a hybridization (Hyb) probe method and a hydrolysis (TaqMan) probe method.
The Hyb probe method is a method using two types of probes, a probe labeled with a donor dye and a probe labeled with an acceptor dye, which are previously designed so that the two types of probes are close to each other. When the two types of probes hybridize to the target nucleic acid, the acceptor dye excited by the donor dye emits fluorescence.
The TaqMan probe method is a method using a probe labeled so that the reporter dye and the quencher dye are close to each other. Then, the probe is hydrolyzed during nucleic acid extension, and at this time, the quencher dye and the reporter dye are separated from each other, and emits fluorescence when the reporter dye is excited.

Examples of the fluorescent dye (derivative) used in the method using the fluorescent substance include SYBR (registered trademark) Green I, SYBR (registered trademark) Green II, SYBR (registered trademark) Gold, YO (Oxazole Yellow), and TO (Thiazole Orange). ), PG (Pico (registered trademark) Green), ethidium bromide and the like.
Examples of the organic compound used in the method using the chemiluminescent substance include luminol, lophine, lucigenin, and oxalate.

Moreover, as a method using the said turbidity substance, the method of using the turbidity substance produced | generated by the pyrophosphoric acid produced as a result of nucleic acid amplification reaction and the metal ion which can be couple | bonded with this, etc. are mentioned, for example. The metal ion is a monovalent or divalent metal ion, and forms a turbid substance by forming an insoluble or hardly soluble salt in water when combined with pyrophosphoric acid.
Specific examples of the metal ions include alkali metal ions, alkaline earth metal ions, and divalent transition metal ions. Among these, for example, alkaline earth metal ions such as magnesium (II), calcium (II) and barium (II); zinc (II), lead (II), manganese (II), nickel (II) and iron (II 1 type or 2 or more chosen from bivalent transition metal ions, such as). More preferred are magnesium (II), manganese (II), nickel (II) and iron (II).
The concentration when the metal ion is added is preferably in the range of 0.01 to 100 mM. The detection wavelength is preferably 300 to 800 nm.

  The nucleic acid amplification reaction apparatus according to the present invention is preferably provided with temperature control means (not shown) for heating the reaction region. The temperature control means is installed so that light to the reaction region and light from the reaction region pass through. The arrangement of the temperature control means is preferably the upper, lower and / or side portions of the reaction region. Examples of the temperature control means include a heater such as a Peltier or a light-transmitting ITO heater. Thereby, since the reaction temperature, reaction time, etc. of nucleic acid amplification can be controlled, nucleic acid amplification reactions, such as PCR method and LAMP method, can be performed satisfactorily.

  Hereinafter, the nucleic acid amplification reaction apparatus according to the present invention will be described more specifically. The configuration described above is omitted.

<2. First Embodiment of Nucleic Acid Amplification Reaction Apparatus According to the Present Invention>
(1) Configuration of First Embodiment A first embodiment will be described with reference to FIG. In addition, although one optical system path is demonstrated, it is not limited to this, A plurality may exist.
The first embodiment of the nucleic acid amplification detection apparatus 1 according to the present invention includes a first light source 2, a second light source 3, a light guide member 4, fluorescent filters 9 and 11, and a photodetector (hereinafter referred to as “light detector”) And a control unit (not shown) that switches between the first light source 2 and the second light source 3 to emit light.

The first light source 2 may be any light source that emits light that excites a fluorescent material. For example, a laser light source is preferable, and a laser light source that emits laser light having a narrow line width is more preferable. The laser light source is not particularly limited depending on the type of laser light, but a light source that emits an argon ion (Ar) laser, a helium-neon (He-Ne) laser, a die (dye) laser, a krypton (Cr) laser, or the like. If it is. The laser light sources can be used alone or in combination of two or more. The excitation light excites the fluorescent material generated as the amplification reaction proceeds, and the fluorescence intensity increases.
The first light source 2 may be either a single light source or a plurality of light sources.
This eliminates the need to use an excitation filter (see FIG. 3A), and turbidity can be detected by the second light source 3 described later. Further, the number of members related to the excitation filter can be reduced, and the entire apparatus can be reduced in size, particularly reduced in thickness.

The second light source 3 may be a light source that emits light in a wavelength region that overlaps the wavelength region of fluorescence generated from the fluorescent material. For example, a white or monochromatic light emitting diode (LED), a mercury lamp, a tungsten lamp, or the like may be used. Of these, LEDs are preferred. The light will be reflected and absorbed by the surface of the turbid material. Thereby, the amount of scattered light increases and the amount of transmitted light in the optical axis direction decreases.
The second light source 3 may be either a single light source or a plurality of light sources.

The light guide member 4 guides light from these light sources on the same optical system path between the first light source 2 and the second light source 3 and the reaction region A1. It is. The light guide member 4 is provided with a light incident end 41, and the first light source 2 and the second light source 3 are arranged along the light incident end 41. A member such as a prism or a reflector that directs light incident from the light incident end 41 toward the reaction region A1 is provided. The member such as the prism is preferably arranged so that the light from these light sources irradiates the reaction region A1 (for example, the surface of the reaction substrate 7) substantially perpendicularly.
As a result, even if the number of light sources of the first light source and the second light source is reduced, uniform irradiation can be obtained and power consumption can be reduced. Moreover, since the said light guide member 4 can be utilized also as a support body for supporting a light source, the member for supporting the light source in an apparatus can be reduced. Therefore, it is possible to reduce the size of the entire apparatus, particularly to reduce the thickness.
A condensing lens 5 and a diaphragm 6 are preferably disposed between the light guide member 4 and the reaction area A1. The light from the introduction member 4 is irradiated to the reaction region A1 through these.

The reaction region A1 has a well shape and is formed by the reaction substrate 7.
The reaction substrate 7 allows light L1 and L2 from the light guide member 4 to pass therethrough and light L11 and L21 transmitted through the reaction region A1.
Furthermore, it is preferable that a measurement substrate 8 for setting the reaction region A1 is provided below the reaction substrate 7. The substrate in the measurement substrate 8 is not particularly limited as long as it transmits light from the reaction region A1. For example, a hole may be provided in the substrate or a transparent substrate may be used. Further, the measurement substrate 8 may be provided with the temperature control means as described above.

The fluorescent filters 9 and 11 are preferably disposed on an optical system path between the reaction region A1 and the photodetector 13. The fluorescent filter may be appropriately singular or may be further added.
Further, one or more condenser lenses may be disposed between the fluorescent filter 9 and the fluorescent filter 11 and / or between the fluorescent filter 11 and the optical detector 13. For example, you may arrange | position like the condensing lenses 10 and 12. FIG.
The optical detector 13 is preferably supported by a support 14 and capable of detecting at least desired fluorescence and turbidity.

The control unit switches the first light source 2 and the second light source 3 to emit light according to detection of desired fluorescence or turbidity. For example, when detecting the fluorescence, the control unit emits the excitation light from the first light source 2 while preventing the light from the second light source 3 from being emitted. On the other hand, when detecting the turbidity, the light from the second light source 3 is emitted while the excitation light from the first light source 2 is not emitted.
At this time, the control unit is generated from the amount of light scattered or transmitted by the turbid substance generated with the progress of the nucleic acid amplification reaction, and the fluorescent substance generated with the progress of the nucleic acid amplification reaction is excited by light. It is also possible to calculate the amount of nucleic acid from the fluorescence intensity.
The control unit can also control each nucleic acid amplification reaction (temperature and time) in order to detect nucleic acid amplification in real time.

(2) Operation of First Embodiment FIG. 1 shows an optical system path of the first embodiment of the present invention.

(A) Fluorescence detection The case where fluorescence detection is selected in the nucleic acid amplification reaction in the reaction region A1 will be described below. At this time, the control unit performs control so that light from the second light source 3 is not emitted.
The first light source 2 emits light L1 (specific excitation light) that excites the fluorescent substance to be detected. The light L <b> 1 enters the light guide member 4 from the optical incident end 41. The incident light L1 is irradiated by a member such as a prism inside the light guide member 4 so as to reach the reaction region A1. The irradiated light L1 passes through the condenser lens 5 and then the diaphragm 6, passes through the upper surface of the reaction substrate 7, and is irradiated to the fluorescent material generated as the nucleic acid amplification reaction proceeds in the reaction region A1. Light L11 (fluorescence) generated from the fluorescent material excited by the irradiated light L1 passes through the fluorescent filter 9 and becomes light L12. The light L12 passes through the condenser lens 10 and further passes through the fluorescent filter 11, and becomes light L13 (specific fluorescent component) of a desired fluorescent component. The light L13 passes through the condenser lens 12 and is detected by the optical detector 13. Thereby, the desired fluorescence amount from the reaction region A1 can be measured.

(A-1) Operation of RT-PCR Device Here, the case where the first embodiment of the present invention is used as an RT-PCR device and a nucleic acid (for example, DNA) is quantified with a fluorescent substance is described below in Step Sp1. (Thermal denaturation), step Sp2 (primer annealing), step Sp3 (DNA extension) will be described.
In the heat denaturation step (step Sp1), the temperature control means controls the temperature in the well A1 to be 95 ° C. to denature the double-stranded DNA into a single-stranded DNA.
In the subsequent annealing step (Step Sp2), the primer is set to 55 ° C. so that the primer binds to the base sequence complementary to the single-stranded DNA.
In the next DNA extension step (Step Sp3), the inside of the well A1 is controlled to be 72 ° C., so that the primer is used as a starting point for DNA synthesis to advance the polymerase reaction to extend the cDNA.
By repeating such a temperature cycle of steps Sp1 to Sp3, DNA in each well is amplified.
For example, a fluorescent dye such as SYBR (registered trademark) Green I intercalates into ds (double-standed) -DNA generated during the DNA replication reaction. When the intercalated ds-DNA is irradiated with excitation light (light L1), it is excited to emit fluorescence (light L11) (excitation light 497 nm: emission wavelength 520 nm). The desired fluorescence component is obtained by the fluorescence filters 9 and 11, and the light emission amount of the fluorescence (light L13) is measured and quantified by the fluorescence detection unit 13 for each temperature cycle. The initial cDNA amount can also be obtained as the gene expression level based on the correlation between the number of temperature cycles and the corresponding fluorescence amount.
In the PCR method, nucleic acid can be quantified even if a turbidity substance is used. In this case, the turbidity may be detected according to (b) turbidity detection described later.

(B) Turbidity detection The case where turbidity detection is selected in the nucleic acid amplification reaction in the reaction region A1 will be described below. At this time, the control unit controls so as not to emit light from the first light source 2.
From the second light source 3, light L2 irradiated to the turbidity substance to be detected is emitted. The light L <b> 2 enters the light guide member 4 from the optical incident end 41. The incident light L2 is irradiated by a member such as a prism inside the light guide member 4 so as to reach the reaction region A1. The irradiated light L2 passes through the condenser lens 5 and then the diaphragm 6, passes through the upper surface of the reaction substrate 7, and is applied to the turbidity substance generated as the nucleic acid amplification reaction proceeds in the reaction region A1. The irradiated light L2 is reflected or absorbed by the surface of the turbid substance, and becomes light L21 (scattered light and transmitted light). The light L21 passes through the fluorescent filter 9 and becomes light L22. The light L22 passes through the condenser lens 10 and further passes through the fluorescent filter 11, and becomes the desired light L23 (scattered light component or transmitted light component). The light L23 that has become a desired light component passes through the condenser lens 12 and is detected by the optical detector 13. Thereby, the amount of scattered light and the amount of transmitted light in the reaction region A1 can be measured.

(B-1) Operation of LAMP Apparatus Here, the case where the first embodiment of the present invention is used as a LAMP apparatus and nucleic acid is quantified with a turbid substance will be described below in the procedure of Step S11.
In the temperature control step (step S11), the nucleic acid in each well is amplified by setting the well A1 to have a constant temperature (60 to 65 ° C.). In this LAMP method, heat denaturation from a single strand to a double strand is not necessary, and primer annealing and nucleic acid extension are repeated under this isothermal condition.
As a result of this nucleic acid amplification reaction, pyrophosphate is generated, and metal ions bind to this pyrophosphate to form an insoluble or hardly soluble salt, which becomes a turbid substance (measurement wavelength: 300 to 800 nm). When this turbidity substance is irradiated with incident light (light L2), it becomes scattered light (light L21). The amount of scattered light transmitted through the fluorescent filters 9 and 11 is measured by the fluorescence detector 13 in real time and quantified. It is also possible to quantify the amount of transmitted light.
As for the LAMP method, nucleic acid can be quantified using a fluorescent substance. In this case, this may be detected according to the above-described (a) fluorescence detection.

  In addition, 1st embodiment can be used as various nucleic acid amplification apparatuses other than the above-mentioned RT-PCR apparatus, a LAMP apparatus, etc.

  As described above, each light from the first light source 2 and the second light source 3 is placed on the same optical system path by the light guide member 4. More specifically, when the light from the second light source 3 enters the light guide member 4, the light from the second light source 3 is transmitted by the light guide member 4 to the first light source. The light is guided so as to overlap the optical system path of the light source 2.

  According to the first embodiment, each measurement of fluorescence detection and turbidity detection can be performed by switching the light source. In addition, since an optimal detection method can be selected, more accurate quantitative analysis and real-time detection are possible. In addition, since no excitation filter is required, it is easier to reduce the size of the device.

<3. Second Embodiment of Nucleic Acid Amplification Reaction Apparatus According to the Present Invention>
(1) Configuration of Second Embodiment A second embodiment will be described with reference to FIG. The configuration described above is omitted. In addition, although one optical system path is demonstrated, it is not limited to this, A plurality may exist.

  The second embodiment of the nucleic acid amplification reaction apparatus 1 according to the present invention includes a first light source 21, an excitation filter 24, a second light source 22, a light guide member 25, a first light source 21, and a second light source 21. And a controller that switches light source 22 to emit light. Although the second embodiment includes both the fluorescent filters 9 and 11 and the optical detector 13, it is omitted because it is the same as that of the first embodiment described above.

The first light source 21 and the second light source 22 may be the same as the second light source 3 described above. For example, LEDs are suitable.
The first light source 21 may be supported by a support 23, and may be arranged so that light enters the light guide member 4 as in the first embodiment. . It is preferable to adopt a configuration like the light guide member 4.

The excitation filter 24 is preferably disposed between the first light source 21 and the reaction region A1. Further, the excitation filter 24 is preferably one that converts the light output from the first light source 21 into excitation light having a specific wavelength that enables desired fluorescence detection.
Thereby, even if a normal light source (for example, LED) in which various wavelengths are mixed is used, the adverse effect on the fluorescence detection can be reduced (see FIG. 3B). When the first light source 2 described above is a laser light source, a laser with an effective oscillation wavelength may not be available depending on the fluorescent material. However, if the excitation filter system is used, excitation light having a desired specific wavelength can be appropriately obtained.

The light guide member 25 transmits the light from the second light source 22 to the first light source on the optical system path between the first light source 21 and the second light source 22 and the reaction region A1. The light is guided onto the optical system path 21. Furthermore, the light guide member 25 is preferably disposed between the excitation filter 24 and the reaction region A1.
The light guide member 25 is provided with a light incident end 251, and the second light source 22 is disposed along the incident light end 251. A member such as a prism or a reflector is provided to direct the light from the second light source 22 and the light from the first light source 21 incident from the light incident end 251 toward the reaction region A1. .
This member such as a prism irradiates light from the first light source 21 so as to irradiate light from the second light source 22 substantially perpendicularly to the reaction area A1 (for example, the surface of the reaction substrate 7). It is preferable that the reaction region A1 is disposed so as to pass through. The reaction region A1 may be irradiated with light from the introduction member 4 through a condenser lens (not shown) or a diaphragm 6.

  The control unit switches the first light source 21 and the second light source 22 to emit light according to detection of desired fluorescence and turbidity. For example, when detecting the fluorescence, the control unit emits light from the first light source 21 while preventing light from the second light source 22 from being emitted. On the other hand, when detecting the turbidity, the light from the second light source 22 is emitted while the excitation light from the first light source 21 is not emitted.

(2) Operation of Second Embodiment FIG. 2 shows an optical system path of the second embodiment of the present invention. In addition, about the part which overlaps with 1st embodiment mentioned above, it abbreviate | omits.

(A) Fluorescence detection The case where fluorescence detection is selected in the nucleic acid amplification reaction in the reaction region A1 will be described below. At this time, the control unit controls so as not to emit light from the second light source 22.
Light L3 for exciting the fluorescent material is emitted from the first light source 21. The light L3 passes through the condenser lens 5 and further passes through the excitation filter 24 to become light L31 (specific excitation light). The light L31 passes through the light guide member 25, the diaphragm 6, and then the upper surface of the reaction substrate 7, and is irradiated to the fluorescent material generated as the nucleic acid amplification reaction proceeds in the reaction region A1. Light L32 (fluorescence) generated from the fluorescent material excited by the irradiated light L31 passes through the fluorescent filter 9 and becomes light L33. The light L33 passes through the condenser lens 10 and further passes through the fluorescent filter 11, and becomes light L34 (specific fluorescent component) of a desired fluorescent component. The light L34 (fluorescence) passes through the condenser lens 12 and is detected by the optical detector 13. Thereby, the desired fluorescence amount from the reaction region A1 can be measured.

(B) Turbidity detection The case where turbidity detection is selected in the nucleic acid amplification reaction in the reaction region A1 will be described below. At this time, the control unit performs control so that light from the first light source 21 is not emitted.
From the second light source 22, light L4 irradiated to the turbidity substance to be detected is emitted. The light L4 enters the light guide member 25 from the optical incident end 251. The incident light L4 is irradiated by a member such as a prism inside the light guide member 25 so as to reach the reaction region A1. The irradiated light L4 passes through the condenser lens 5, the diaphragm 6, and then the upper surface of the reaction substrate 7, and is irradiated to the turbidity substance generated as the nucleic acid amplification reaction proceeds in the reaction region A1. The irradiated light L4 is reflected or absorbed by the surface of the turbid substance, and becomes light L41 (scattered light component and transmitted light component). The light component L41 passes through the fluorescent filter 9 and becomes light L42. The light L42 passes through the condenser lens 10 and further passes through the fluorescent filter 11, and becomes light L43 (scattered light component or transmitted light component). The light L43 that has become a desired light component passes through the condenser lens 12 and is detected by the optical detector 13. Thereby, the amount of scattered light and the amount of transmitted light in the reaction region A1 can be measured.

  The second embodiment can be used as various nucleic acid amplification apparatuses such as an RT-PCR apparatus and a LAMP apparatus, as in the first embodiment described above.

  As described above, the light from the second light source 21 overlaps the optical system path of the first light source 22 by the light guide member 25. More specifically, when the light from the second light source 22 enters the light guide member 25, the light from the second light source 22 passes through the light guide member 25. The light is guided so as to overlap on the optical system path of one light source 21.

  According to the second embodiment, each measurement of fluorescence detection and turbidity detection can be performed by switching the light source. In addition, since an optimal detection method can be selected, more accurate quantitative analysis and real-time detection are possible. In addition, a wide range of fluorescent materials can be used by employing the excitation filter.

  According to the nucleic acid amplification reaction apparatus according to the present invention, detection by fluorescence, turbidity, and coloration is possible. Therefore, it is possible to cope with a nucleic acid amplification reaction according to the purpose with one apparatus.

  DESCRIPTION OF SYMBOLS 1 Nucleic acid amplification reaction apparatus 2,21 1st light source 3,22 2nd light source 4,25 Light guide member A1 reaction area | region 9,11 Fluorescence filter 13 Optical detector 24 Excitation filter

Claims (7)

A first light source that emits light that excites the fluorescent material;
A second light source that emits light in a wavelength region that matches a wavelength region of fluorescence generated from the fluorescent material;
A controller that switches between the first light source and the second light source to emit light;
The light from the first light source and the light from the second light source are irradiated onto a reaction region that becomes a reaction field of a nucleic acid amplification reaction through an optical system path that overlaps with a light guide member,
A nucleic acid amplification reaction apparatus capable of detecting the amount of light caused by a turbidity substance generated along with the progress of an amplification reaction and the intensity of fluorescence generated from a fluorescent substance excited by light as the amplification reaction proceeds. On the optical system path between the first light source and the second light source and the reaction region, a light guide member that guides light from these light sources onto the same optical system path is disposed,
The nucleic acid amplification reaction apparatus according to claim 1, wherein a fluorescent filter is disposed on an optical system path between the reaction region and the photodetector. A light guide member for guiding light from the second light source onto the optical path of the first light source on the optical path between the first light source and the second light source and the reaction region. Arranged,
The nucleic acid amplification reaction apparatus according to claim 1, wherein a fluorescence filter is disposed on an optical system path between the reaction region and the photodetector.   The nucleic acid amplification reaction apparatus according to claim 2, wherein the first light source is a laser light source.   An excitation filter that transmits light from the first light source and the light guide member that transmits light from the excitation filter are disposed on an optical system path between the first light source and the reaction region. The nucleic acid amplification reaction apparatus according to claim 3. Exciting the phosphor with light from the first light source;
The nucleic acid amplification reaction apparatus according to claim 4 or 5, wherein turbidity is detected by light from the second light source.   The nucleic acid amplification reaction apparatus according to claim 6, wherein the nucleic acid amplification reaction is performed by a PCR method or a LAMP method.