JP2012060908A - Nucleic acid amplification reactor, substrate used for nucleic acid amplification reactor and reaction method for amplifying nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reactor having good detection sensitivity.SOLUTION: The nucleic acid amplification reactor includes a reaction region formed in a thin-tube shape as a nucleic acid amplification reaction site, a temperature controlling means for heating the reaction region, an irradiating means for irradiating light from one end of the reaction region into the lumen and a detecting means for detecting the quantity of the light emitted from the other end of the reaction region.

Description

  The present invention relates to a nucleic acid amplification reaction apparatus, a substrate used in the nucleic acid amplification reaction apparatus, and a nucleic acid amplification reaction method. More specifically, the present invention relates to a nucleic acid amplification reaction apparatus or the like provided with a reaction region that forms a reaction field for a nucleic acid amplification reaction in a thin tube shape.

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).
Nucleic acid amplification reaction apparatuses that can perform gene analysis such as gene expression analysis, infectious disease inspection, and SNP analysis using the method for detecting a nucleic acid amplification product as described above are widely commercially available.

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

  However, a conventional turbidity detection apparatus uses a well-like nucleic acid amplification reaction field, but further improvement in detection sensitivity is required.

  Therefore, the main object of the present invention is to provide a nucleic acid amplification reaction apparatus, a substrate used in the nucleic acid amplification reaction apparatus, and a nucleic acid amplification reaction method that can obtain higher detection sensitivity.

  In order to solve the above problems, the present invention provides a reaction region formed in a thin tube shape that serves as a reaction field for a nucleic acid amplification reaction, a temperature control means for heating the reaction region, and irradiates light into the lumen from one end of the reaction region. There is provided a nucleic acid amplification reaction apparatus comprising: irradiation means for performing detection; and detection means for detecting the amount of the light emitted from the other end of the reaction region.

In the nucleic acid amplification reaction apparatus, a plurality of the reaction regions are arranged, and the irradiation unit includes a light guide unit that guides the light emitted from one light source from one end of each reaction region into a lumen. Is preferred.
In the nucleic acid amplification reaction apparatus, it is preferable that the detection means includes a one-dimensional sensor that detects the amount of the light emitted from one end of each reaction region.
In the nucleic acid amplification reaction apparatus, it is preferable that the reaction regions are arranged in parallel to each other on the same plane, and the temperature control means is provided on a surface facing the plane.

The present invention also provides a substrate on which a reaction region formed in a thin tube shape serving as a reaction field for a nucleic acid amplification reaction is provided. Furthermore, it is preferable that a curved surface is set on the incident side and / or the emission side of the reaction region.
The present invention also detects the amount of light emitted from the other end of the reaction region by irradiating light into the lumen from one end of the reaction region formed in a narrow tube shape that serves as a reaction field for nucleic acid amplification reaction. Thus, there is provided a nucleic acid amplification reaction method for detecting the amount of light scattered by the turbidity substance generated as the amplification reaction proceeds.

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid amplification reaction apparatus, a board | substrate, and nucleic acid amplification reaction method which can obtain higher detection sensitivity are provided.

The conceptual diagram in the nucleic acid amplification reaction apparatus concerning this invention is shown. The conceptual diagram of the optical system path | route in the nucleic acid amplification reaction apparatus concerning this invention is shown. The example of the board | substrate concerning this invention 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 apparatus (1) Reaction region (a) Substrate (b) Nucleic acid amplification reaction (c) Nucleic acid amplification (product) detection method (2) Temperature control means (3) Irradiation means (4) Detection means Operation of nucleic acid amplification reaction apparatus (1) Modification (a) Operation of RT-LAMP apparatus (b) Operation of RT-PCR apparatus

<1. Nucleic acid amplification reaction apparatus>
FIG. 1 is a conceptual view of a nucleic acid amplification reaction apparatus according to the present invention viewed from the side. FIG. 2 is a conceptual diagram of an optical system path in the nucleic acid amplification reaction apparatus according to the present invention.
Note that, in the drawings described below, for the convenience of explanation, the configuration of the apparatus and the like are simplified.

A nucleic acid amplification reaction apparatus 1 according to the present invention comprises a reaction region 2, a temperature control means 3, an irradiation means 4, and a detection means 5 for amplifying and quantifying a nucleic acid by controlling a nucleic acid amplification reaction. .
In the nucleic acid amplification reaction apparatus 1 of the present invention, the temperature control means 3 and the detachable reaction region 2 (substrate 6) are arranged between the irradiation means 4 and the detection means 5.
Further, gaps 9 and 10 may be appropriately provided between the reaction region 2 and the irradiation unit 4 or between the reaction region 2 and the detection unit 5. A filter (not shown), a condensing lens (not shown), and a diaphragm 11 may be provided in the gaps 9 and 10 as appropriate in order to adjust the light amount, the light component, and the like.
In the nucleic acid amplification reaction apparatus 1 of the present invention, a control unit (for example, light control, temperature control, nucleic acid amplification reaction, detection control, detection light quantity calculation and monitoring, etc.) relating to the apparatus of the present invention is controlled. (Not shown).
Each configuration will be described in detail below.

(1) Reaction area | region The said reaction area | region 2 is an area used as the reaction field of the amplification reaction of a nucleic acid, and is formed in the shape of a thin tube. The reaction region 2 may be either a single region or a plurality of arrays, but it is preferable that a plurality of reaction regions 2 are arrayed. With a plurality of arrays, detection sensitivity and detection accuracy are good, and many samples can be measured efficiently in a compact manner.
In a conventional nucleic acid amplification reaction apparatus having a well-shaped reaction field for nucleic acid amplification reaction as in the prior art, the detection sensitivity is not very good because the light beam passing distance in the reaction field is short. In addition, since each configuration is two-dimensionally arranged, the number of parts such as the number of detectors such as a photodiode (PD) array and the number of light sources is likely to increase. For this reason, variations in individual characteristics are likely to occur, and the detection accuracy is likely to be reduced. Due to the two-dimensional arrangement of each component and the increase in the number of parts, the entire apparatus becomes larger and more expensive.
On the other hand, it is possible to detect even a small amount of nucleic acid in the reaction region by making the reaction field of the nucleic acid amplification reaction into a thin tube shape as described above. In addition, since it is possible to set the light beam passing distance in the reaction field to be longer, the detection sensitivity during the initial reaction (low scattering) is improved. This also improves the monitoring from the initial reaction.
Furthermore, since each component can be arranged one-dimensionally, the number of parts, such as the number of light sources and detectors, can be reduced, so that variations in individual characteristics can be reduced and detection accuracy is improved. Is also possible. This also makes it possible to reduce the size of the entire device, particularly to reduce the thickness and cost, while ensuring the performance and reliability of the device.

  Furthermore, when there are a plurality of the reaction regions 2, it is preferable that they are arranged in parallel to each other on the same plane. Thus, by making a plurality of reaction regions into a one-dimensional array, it is possible to provide a temperature control means as a member (surface) facing the plane (for example, the upper surface or the bottom surface) of the array. Thereby, many samples can be efficiently measured with a compact arrangement | positioning with favorable detection sensitivity and detection accuracy.

(A) Substrate The reaction region 2 is preferably formed in a reaction vessel (for example, a substrate) such as a nucleic acid amplification reaction microchip. With such a reaction vessel, it is easy to appropriately form an array such as a plurality of arrays in the width direction or a parallel array on the same plane as described above.
The reaction region 2 can be formed from a single or a plurality of substrates. In the formed substrate 6, it is preferable that the reaction region 2 serving as a reaction field of the nucleic acid amplification reaction is arranged in a single tube shape or a plurality of the reaction regions 2. The capillary shape at this time is not limited as long as the length in the longitudinal direction (optical axis direction) takes a three-dimensional shape longer than the height and width. Examples of the three-dimensional shape include a rectangular shape and a cylindrical shape.
In addition, it is preferable that the surfaces of both ends of the substrate 6 (the reaction region 2) on the optical axis are formed so that optical detection is possible. Specifically, one end of the substrate 6 (the reaction region 2) is a surface on which light is irradiated (incident), and the other end emits light that has passed through the reaction region (a light path in the reaction field). Surface. Regarding the configuration of the entrance surface and the exit surface, for example, the entrance surface and the exit surface should be opposed surfaces that are parallel to each other; the entrance surface and / or the exit surface should have an uneven curvature (see, for example, FIG. 3). Etc.

It is preferable to set a concave and convex curved surface on the incident side and / or the outgoing side of the substrate 6. The position for setting the curved surface is preferably set at the end of the substrate 6 (the reaction region 2) on the optical axis, specifically, at the entrance and / or exit.
More specifically, setting a curved surface on the incident side means that at least one of the incident surface 21 of the reaction region 2 and the incident surface 61 of the substrate 6 on the incident side is a concave curved surface or a convex curved surface. Is preferable. As a result, the amount of light guided through the lumen increases and the loss of light quantity at the incident end decreases, so that the light utilization efficiency is improved.
Also, setting a curved surface on the exit side preferably means that at least one of the exit surface 22 of the reaction region 2 on the exit side or the exit surface 62 of the substrate 6 is a concave curved surface or a convex curved surface. . This increases the light extraction efficiency and improves the sensitivity to the effects of internal scattering by optimizing the curvature for rays propagating at a certain angle in the lumen.
By setting curved surfaces on the incident side and / or the outgoing side in this way, the detection sensitivity, particularly the sensitivity of turbidity detection is improved.

Examples of the substrate 6 having such a curved surface include those shown in FIGS. 3A to 3D, but are not limited thereto.
More specifically, for example, a curved concave portion is a light-transmitting material, and the end portion is a flat surface (see, for example, FIG. 3D). For example, a curved concave portion is a space; a concave portion is a cavity, and a flat surface is formed of a light-transmitting material at the end (see, for example, FIG. 3B). Is done. In addition, for example, the convex portion of the curved surface is filled with a transparent material (for example, see FIG. 3A); the surface of the convex portion is a transparent material, and the inside is a cavity ( (For example, see FIG. 3C).

  In addition, when the emission side and the incident side of the substrate 6 (the reaction region 2) are flat, it is possible to increase the excitation length (excitation distance) in the case of fluorescence detection. This is preferable because the sensitivity to fluorescence is improved.

Examples of the three-dimensional shape of the substrate 6 include a rectangular shape and a cylindrical shape.
Of the three-dimensional shapes of the substrate 6, the cylindrical shape having a single reaction region 2 is preferable because it is easy to perform measurement at any time. At this time, since the LAMP method is an isothermal reaction, the measurement efficiency can be appropriately measured without unifying the measurement start time of the specimen, so that the working efficiency is further improved. Further, the cylindrical shape is preferable because it is easy to give the entrance surface and the exit surface curvature so as to improve detection sensitivity and detection accuracy.
In addition, a rectangular shape having one or a plurality of the reaction regions 2 is preferable because many samples can be measured at the same time, so that work efficiency is good. In the case of a rectangular shape having a plurality of the reaction regions 2, the exit surface and / or the entrance surface on each optical axis may have the above-described uneven curvature. By adopting the above-mentioned rectangular shape, the light passing distance in the reaction field can be increased, and the contact surface with the temperature control means 3 is wide, so that heating is easily transmitted. Therefore, since temperature control of the reaction region 2 is easy to perform, detection sensitivity and detection accuracy are improved.

The method of forming the reaction region 2 on the substrate 6 is not particularly limited, and is preferably performed by, for example, wet etching or dry etching of a glass substrate layer, or nanoimprinting, injection molding, or cutting of a plastic substrate layer. It is.
For example, as a method of forming the reaction region 2, a groove serving as a reaction region (light path) along the longitudinal direction is formed on one substrate by polishing cutting, molding, or the like. A thin tube-shaped lumen having both ends in the optical axis direction formed in one substrate, and sealing and polishing the both ends.
The material of the substrate 6 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.).
The reaction region 2 thus formed may be filled in advance with reagents necessary for the nucleic acid amplification reaction.
In addition, it is preferable that the formed reaction region 2 is in a reduced pressure state in which the reaction region 2 is negative with respect to atmospheric pressure. Thus, if the target nucleic acid or a reagent containing a reagent necessary for the nucleic acid amplification reaction is punctured and injected, the nucleic acid easily flows into the reaction region, so that the nucleic acid amplification reaction is easily performed. Furthermore, it is preferable that a puncture hole (not shown) for puncture injection is provided. It is preferable that the puncture hole is not disposed on the optical axis (preferably disposed on the side of the reaction region) and is connected to the reaction region 2 via a flow path.

As described above, when the substrate of the present invention is used, detection sensitivity and detection accuracy are improved. Further, since the plurality of reaction regions 2 are easily arranged one-dimensionally in the width direction, many samples can be efficiently measured with a compact arrangement.
Therefore, the detection sensitivity and detection accuracy of the nucleic acid amplification reaction apparatus are improved, and further, the entire apparatus can be reduced in size, particularly reduced in thickness and cost, while ensuring the performance and reliability of the apparatus.

(B) 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-LAMP 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.

(C) Nucleic Acid Amplification (Product) Detection Method Examples of the nucleic acid amplification detection method include a method using a turbidity substance, a fluorescent substance, a chemiluminescent substance, and the like.

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.

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, and is specific to a nucleic acid sequence to be amplified. And a labeled probe method using a probe in which a fluorescent dye is bound to a simple oligonucleotide.
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.

(2) Temperature control means The temperature control means 3 is for heating the reaction region 2. Compared with the case where the conventional reaction region is well-shaped, the reaction region 2 has a contact surface with the temperature control means 3 by making the reaction region 2 elongated in the longitudinal direction (on the optical axis) as in the present invention. It can be widened. As a result, since the heat application property is improved, it becomes easy to control the temperature of nucleic acid amplification, so that a nucleic acid amplification reaction such as a PCR method or a LAMP method can be favorably performed. Therefore, detection sensitivity and detection accuracy can be improved.
Although it does not specifically limit as said temperature control means 3, For example, heaters, such as Peltier, an ITO heater with a light transmittance, etc. are mentioned. Since the temperature control means 3 does not have to be disposed on the light path, the heater can be a low-cost impermeable one.
Examples of the shape of the temperature control means 3 include a thin film shape, a flat plate shape, a single or a plurality of cylinder shapes, a U shape, and the like.
The temperature control means 3 may be disposed so as to be in contact with the reaction region 2, for example, at any position such as an upper portion, a lower portion, a side portion, or an outer peripheral portion of the reaction region 2. Also good.
Among these, it is preferable that the temperature control means 3 is in the form of a thin film or a flat plate and is disposed at the upper part and / or the lower part of the reaction region 2. Thereby, while the installation of the reaction region 2 is simple, the contact surface with the reaction region 2 is increased, the heat application property is improved, and the detection sensitivity and accuracy are improved. Further, since the temperature control means 3 can be arranged one-dimensionally, the entire apparatus can be reduced in size, particularly reduced in thickness.
Further, the temperature control means 3 is preferably provided on a surface corresponding to the plane of the reaction region 2. The surface corresponding to the plane of the reaction region may be, for example, a support surface that supports the temperature control means 3 on the apparatus, or an inner surface of a casing of the apparatus.

(3) Irradiation means The irradiation means 4 includes one or more light sources 7 that irradiate light into the lumen from one end (specifically, the incident surface) of the reaction region 2.
Although the said light source 7 is not specifically limited, What emits the desired light which can detect the target nucleic acid amplification product favorably is suitable. Examples of the light source 7 include a laser light source, a white or monochromatic light emitting diode (LED), a mercury lamp, a tungsten lamp, and the like. Among these, the LED is preferable because it can reduce power consumption and cost. In addition, the LED is advantageous because a desired light component can be obtained by using various filters.
The laser light source is not particularly limited depending on the type of laser light, but emits argon ion (Ar) laser, helium-neon (He-Ne) laser, die laser, krypton (Cr) laser, etc. Any light source may be used. The laser light sources can be used alone or in combination of two or more.

Further, it is preferable that the irradiation means 4 is provided with a light guide means 8 for guiding light emitted from the light source 7 from one end of each reaction region 2 into the lumen. The light guiding means 8 is provided with a light incident end portion 81, and light emitted from one or more of the light sources 7 is incident on the light incident end portion 81. A member (for example, a prism, a reflecting plate, unevenness, or the like) is provided inside the light guide means 8 so as to guide the incident light from one end of each reaction region 2 into the lumen.
By disposing the light guide means 8, the number of light sources can be reduced. In particular, even with only one light source, each reaction region 2 can be irradiated with uniform light, and detection sensitivity and detection accuracy can be achieved. Is also good. In addition, by reducing the number of light sources, the entire apparatus can be reduced in size, particularly reduced in thickness, and power consumption can be reduced.

As shown in FIG. 2, the light L1 from the irradiation means 4 reaches the reaction region 2 and is reflected or absorbed by a turbid substance generated as the amplification reaction proceeds in the reaction region. The Then, the scattered light amount or transmitted light amount (light L21, 22) of the turbid substance passes through the diaphragm 11, the condensing lens, the fluorescent filter, and the like as appropriate, and is detected by the detection means 5 (optical detector). .
Examples of scattered light include forward scattered light, back scattered light, side scattered light, and the like, but it is advantageous because forward scattered light is easily detected and detection sensitivity is good in this apparatus. .

(4) Detection means The detection means 5 may be any mechanism that can detect the amount of the light L2 emitted from the other end (specifically, the emission surface) of the reaction region 2. The detection means 5 includes at least an optical detector.
The optical detector is not particularly limited, and examples thereof include a photodiode (PD) array, an area imaging device such as a CCD image sensor and a CMOS image sensor, a small optical sensor, a line sensor scan, and a PMT (photomultiplier tube). These may be combined as appropriate. It is possible to detect turbidity and the like with the optical detector.

The detection means 5 preferably includes a one-dimensional sensor that detects the amount of light emitted from one end of each reaction region 2.
Here, the one-dimensional sensor is a one-dimensional arrangement of the optical detectors. Examples of the one-dimensional center include one in which a plurality of the optical detectors are arranged so as to face the emission surfaces of the plurality of reaction regions 2 such as a line sensor. As an example, a CCD linear image sensor including a photodiode (light detection unit), a CCD (transfer unit), a signal detector (output unit), and the like can be given.
Here, since the arrangement of the optical detectors becomes a two-dimensional arrangement when the reaction region is formed in a well shape as in the prior art, the required area of the detection means increases even when an image sensor is adopted as the detection means. . On the other hand, since the reaction region 2 can be arranged one-dimensionally by making the reaction region 2 into a thin tube shape as in the present invention, the detection means 5 can also be a one-dimensional sensor system. By adopting this one-dimensional sensor method, many samples can be measured efficiently, and detection sensitivity and detection accuracy are also good. This also makes it possible to reduce the size of the entire apparatus, particularly to reduce the thickness, while ensuring low cost, performance, and reliability.

In addition, you may arrange | position an excitation filter and a fluorescence filter in the nucleic acid amplification reaction apparatus 1 of this invention. For example, an excitation filter may be provided between the light source 7 and the light guide unit 8 or in the gap 9, and a fluorescent filter may be provided in the gap 10.
With the excitation filter (not shown), a light component having a desired specific wavelength can be obtained or an unnecessary light component can be removed according to the detection method of the nucleic acid amplification reaction. The fluorescent filter (not shown) can be used to make light components (scattered light, transmitted light, and fluorescence) necessary for detection. Thereby, detection sensitivity and detection accuracy are improved.

<2. Operation of Nucleic Acid Amplification Reaction Apparatus 1>
Below, the operation of the nucleic acid amplification reaction apparatus 1 described above and the nucleic acid amplification reaction method for detecting the amount of light scattered by the turbid substance will be described.
Light L1 is emitted from the light source 7. The light L1 is incident on the light guide means 8 from the light incident end 81. The incident light L1 is irradiated by a member such as a prism inside the light guide member 8 so as to reach the entrance end surface of the reaction region 2.
The irradiated light L1 is irradiated to one end (incident surface) of the reaction region 2 formed in the shape of a thin tube that serves as a reaction field for nucleic acid amplification reaction, and enters the lumen (capillary shape). At this time, the light L1 is irradiated to a turbid substance that is generated in the reaction region 2 as the nucleic acid amplification reaction proceeds. The irradiated light L1 is reflected or absorbed by the surface of the turbid substance in the reaction region 2, and becomes light L2 (scattered light and transmitted light). This light L2 is emitted from the other end (exit surface) of the reaction region. At this time, the emitted light L2 may be appropriately converted into a desired light component (for example, a scattered light component or a transmitted light component) by a fluorescent filter. The emitted light L2 is detected by the detecting means 5 (optical detector) to detect the amount of the emitted light. That is, the amount of light scattered by the turbidity substance generated as the amplification reaction proceeds is detected.
As shown in FIG. 2, the amount of scattered light (signal amount A) of L21 at the beginning of the reaction decreases as the nucleic acid amplification reaction proceeds, and the amount of scattered light (signal amount) of L22 after the reaction ends. B).

Thus, in the nucleic acid amplification reaction by turbidity detection, by irradiating light into the lumen from one end of the reaction region formed in a thin tube shape, by detecting the amount of light emitted from the other end of the reaction region It is preferable to detect the amount of scattered light with a turbid substance generated as the amplification reaction proceeds.
By adopting a narrow tube shape, the light beam passage distance can be lengthened, and the waveguide condition (total reflection condition at the interface) is easily broken, so that the amount of scattered light (or the amount of transmitted light) reaching the exit surface (in the optical axis direction) ) Is likely to change (decrease), so that detection sensitivity and detection accuracy are improved.

(1) Modified Example The nucleic acid amplification reaction apparatus of the present invention can be used as a nucleic acid amplification detection apparatus by installing the reaction region 2 after completion of the reaction in the temperature control means 3 or the like.
In addition, it can be used as a LAMP device or a PCR device to quantify nucleic acids by turbidity substance detection.
(A) Operation of RT-LAMP Device Hereinafter, a method for detecting a nucleic acid in the procedure of Step S11 in the RT-LAMP device will be described.
In the temperature control step (step S11), the reaction region 2 is set to have a constant temperature (60 to 65 ° C.), whereby the nucleic acid in each reaction region 2 is amplified. 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 L1), it becomes scattered light (light L22). The amount of scattered light is measured by the detection means 5 in real time and quantified. It is also possible to quantify the amount of transmitted light.

(B) Operation of RT-PCR Device Here, a method for detecting a nucleic acid in the procedure of Step Sp1 (thermal denaturation), Step Sp2 (primer annealing), and Step Sp3 (DNA extension) in the RT-PCR device will be described. .
In the heat denaturation step (step Sp1), the temperature control means controls the temperature in the reaction region 2 to be 95 ° C. to denature the double-stranded DNA to form single-stranded DNA.
In the subsequent annealing step (step Sp2), the primer is bound to the single-stranded DNA and a complementary base sequence by setting the reaction region 2 to be 55 ° C.
In the next DNA extension step (Step Sp3), the reaction region 2 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, the DNA in each reaction region 2 is amplified. As a result of this nucleic acid amplification reaction, pyrophosphate is generated, and the turbid substance is detected as described above, and the amount of nucleic acid is quantified.

  A method of detecting by fluorescence detection is also possible. At this time, an excitation filter and a fluorescence filter corresponding to the fluorescent substance used in the nucleic acid amplification reaction may be provided, and fluorescence from the fluorescent substance may be detected according to the above-described operation to quantify the nucleic acid.

  Since the nucleic acid amplification reaction apparatus according to the present invention can set the light passing distance longer, it can measure with higher detection sensitivity than the conventional nucleic acid amplification reaction apparatus. In addition, since each component can be arranged one-dimensionally, the entire apparatus can be reduced in size, particularly a thin handy type.

  DESCRIPTION OF SYMBOLS 1 Nucleic acid amplification reaction apparatus; 2 Reaction area | region; 3 Temperature control means; 4 Irradiation means; 5 Detection means; 6 Substrate; 7 Light source;

Claims (9)

A reaction region formed in the shape of a thin tube that serves as a reaction field for a nucleic acid amplification reaction;
Temperature control means for heating the reaction zone;
Irradiation means for irradiating light into the lumen from one end of the reaction region;
Detection means for detecting the amount of light emitted from the other end of the reaction region;
A nucleic acid amplification reaction apparatus comprising: A plurality of the reaction regions are arranged;
2. The nucleic acid amplification reaction apparatus according to claim 1, wherein the irradiating means includes light guiding means for guiding the light emitted from one light source from one end of each reaction region into the lumen.   The nucleic acid amplification reaction apparatus according to claim 2, wherein the detection means includes a one-dimensional sensor that detects the amount of the light emitted from one end of each reaction region. The reaction regions are arranged parallel to each other on the same plane;
The nucleic acid amplification reaction apparatus according to claim 3, wherein the temperature control means is provided on a surface facing the flat surface.   The nucleic acid amplification reaction apparatus according to claim 4, wherein the reaction regions are arranged in a substrate.   6. The nucleic acid amplification reaction apparatus according to claim 5, wherein a curved surface is set on the incident side and / or the outgoing side of the reaction region.   A substrate on which a reaction region formed in a thin tube shape serving as a reaction field of a nucleic acid amplification reaction is disposed.   The substrate according to claim 7, wherein a curved surface is set on an incident side and / or an emission side of the reaction region.   Amplification reaction is performed by irradiating light into the lumen from one end of a reaction region formed in a capillary shape that serves as a reaction field for nucleic acid amplification reaction, and detecting the amount of light emitted from the other end of the reaction region. A nucleic acid amplification reaction method for detecting the amount of light scattered by a turbid substance generated as a result of the progress of.

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