JP2002171997A - Method and apparatus for assaying nucleic acid amplification reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus for quantitatively assaying a target nucleic acid without using a labeled substance. SOLUTION: This apparatus 100 for assaying a nucleic acid amplification reaction is equipped with an installing part 142 of a reaction part for holding the reaction part 120 and a temperature controlling part 144 of the reaction part for controlling a temperature at the reaction part 120. The reaction part 120 is a closed type cell and has a space for storing a reaction solution containing an assayed substance, a reagent, etc. The nucleic acid amplification reaction by LAMP(lysosome-associated membrane protein) method is carried out in the reaction part 120. The apparatus 100 for assaying the nucleic acid amplification reaction is further equipped with a light source part 102 for emitting a light beam to the reaction part 120, a polarizer 104, an analyzer 106 and a photodetector 108 for detecting the light transmitted through the analyzer 106. The light source part 102 and the polarizer 104 are arranged so that the optical axis takes a prescribed angle to the surface of the reaction part 120. The analyzer 106 and the photodetector 108 are arranged so that the optical axis takes the same angle thereto to the surface of the reaction part 120 in the same way.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for measuring a target nucleic acid.

[0002]

BACKGROUND OF THE INVENTION DNA or RN for genetic testing
The amplification reaction of a nucleic acid such as A is important,
(Polymerase Chain Reaction) method is widely used. Conventional techniques for measuring nucleic acid amplification reactions involve using a labeling substance such as a fluorescent dye, combining with a separation technique such as electrophoresis, or adding a step of performing a binding reaction with a labeled binding reagent. This enables quantitative measurement. Some measurement techniques that do not use a labeling substance have also been proposed. For example, Japanese Patent Application Laid-Open No. 2000-508168 discloses that a nucleic acid that competes with a target nucleic acid is immobilized on a solid support, and the target nucleic acid and a nucleic acid that competes with the target nucleic acid are specified. The nucleic acid sequences that specifically bind are reacted. The reaction at this time is measured using the surface plasmon resonance method to determine the presence and amount of the target nucleic acid.

[0003]

The conventional techniques for measuring the nucleic acid amplification reaction are of a practical level when using a labeling substance.
It is widely used because it enables quantitative measurement. However, the labeling substance requires a step of removing excess labeling reagent to improve the S / N ratio. Further, the labeling substance may change over time or depending on the use environment so that the labeling performance is degraded. Also, labeling substances are generally expensive and can be harmful to the human body or the natural environment. Generally, a nucleic acid sequence that specifically binds to a target nucleic acid has a base sequence of 7 to
It has a size of 24, and the change in weight on the solid support due to the binding of a nucleic acid sequence that specifically binds is very small, which is problematic in terms of sensitivity.

It is an object of the present invention to provide a method and an apparatus for quantitatively measuring a target nucleic acid at a practical level without using a labeling substance.

Another object of the present invention is to provide a highly accurate measuring method and a measuring apparatus capable of measuring a sample with high sensitivity.

[0006]

Means for Solving the Problems The present inventors have proposed WO 0
0/28082 and EP-1020534A1 and Nucl
eic Acids Research Vol. 28, No. 12, e63 (2000) proposes a novel nucleic acid amplification reaction technique called the LAMP method. According to this nucleic acid amplification reaction technology, since the reaction has the effect of repeatedly amplifying and extending the nucleic acid base sequence to be amplified, individual amplification products are further polymerized as compared with the conventional PCR method and the like. The molecular weight is much higher than that of a product obtained by the PCR method. As described above, further polymerization and higher molecular weight change the optical properties (eg, refractive index) of the reaction solution. This change in optical properties can be detected by measuring the change in the polarization state of light passing through the reaction solution.

[0007] In one aspect, the present invention relates to a method for measuring a nucleic acid polymerized by the above-described novel nucleic acid amplification reaction technique without using a labeling substance. It is characterized by measuring polarized light.

[0008] In another aspect, the present invention relates to a nucleic acid polymerized by the novel nucleic acid amplification reaction technique described above,
An apparatus for measuring without using a labeling substance, a reaction section installation section for holding the reaction section, a reaction section temperature control section for controlling the temperature of the reaction section, and a light beam directed toward the reaction section. A light source unit that emits light, a polarizer that causes only a specific polarization component to enter the reaction unit, an analyzer that extracts a specific polarization component from the light reflected by the reaction unit, and a light that passes through the analyzer The reaction section has a space for accommodating a reaction solution containing a test substance, a reagent, and the like, in which a nucleic acid amplification reaction by the LAMP method is performed. It is characterized by the following. In the nucleic acid amplification reaction by the LAMP method, the reaction for increasing the molecular weight proceeds while the sample solution is kept at an isothermal temperature, for example, at 65 ° C., and repeatedly extended. Here, since the temperature at which the reaction proceeds is lower than that of the conventional PCR method, there is no need to use a reagent having heat resistance, for example, a heat-resistant polymerase. Further, there is an advantage that the temperature control unit is simplified.

[0009]

Embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment] As shown in FIG. 1, a nucleic acid amplification reaction measuring apparatus 100 according to the present embodiment is shown.
Emits a light beam toward the reaction section 120, a reaction section installation section 142 for holding the reaction section 120 containing the reaction solution, a reaction section temperature control section 144 for controlling the temperature of the reaction section 120. A light source unit 102, a polarizer 104 for causing only a specific polarization component to enter the reaction unit 120,
An analyzer 106 for extracting a specific polarization component from the light reflected by the reaction unit 120 and a photodetector 108 for detecting light transmitted through the analyzer 106 are provided.

The light source unit 102 includes a light source, for example, a solid laser, a gas laser, a semiconductor laser, a xenon lamp, a halogen lamp, and the like. The light source unit 102
In the case where a light source of a semiconductor laser is provided, a collimator lens for collimating the emitted light is preferably further provided.

The photodetector 108 is connected via a cable 110 to a data processing unit 112 such as a computer.

The polarizer 104 includes a light source unit 102 and a reaction unit 1.
The analyzer 106 is disposed between the reaction unit 120 and the photodetector 108.

The polarizer 104 and the analyzer 106 are both optimally a Glan-Thompson prism in terms of the extinction ratio, but may be other optical elements such as a polarizing plate, a polarizing beam splitter, and a Gran laser prism. .

The polarizer 104 is fixed so as to transmit linearly polarized light having a polarization plane inclined at −45 ° with respect to the plane of incidence with respect to the reaction section 120. As used herein, the term “incident surface” is a plane determined by an incident ray and an incident normal, and more specifically, a wavefront normal of light incident on the reaction section and a normal set on the surface of the reaction section. Include plane. The polarizer 104 may be held rotatably around the optical axis so that the angle direction around the optical axis can be changed.

The analyzer 106 is fixed so as to transmit linearly polarized light having a polarization plane inclined at −45 ° with respect to the plane of incidence with respect to the reaction section 120. More preferably, the analyzer 106 is rotatably held around the optical axis so that the angle direction around the optical axis can be changed.

The photodetector 108 includes a photoelectric conversion element, for example, a photodiode, an avalanche photodiode, a photomultiplier, a CCD camera, a SIT camera, and the like.

The light source unit 102 and the polarizer 104 are arranged so that the optical axis forms a predetermined angle with respect to the surface of the reaction unit 120. Similarly, the analyzer 106 and the photodetector 108
Are arranged such that the optical axis forms the same angle with the surface of the reaction section 120.

The reaction section installation section 142 can securely fix the reaction section 120 by air suction or pressing.

The reaction section temperature control section 144 can heat and maintain the reaction section 120 at a desired constant temperature as needed. The reaction unit temperature control unit 144 performs heating by a Peltier element, a resistor, a lamp, or the like, and performs cooling by heat radiation, water cooling, or air cooling.

The reaction section installation section 142 is a reaction section temperature control section 1
44 functions may be included. As shown in the figure,
When the reaction section installation section 142 and the reaction section temperature control section 144 are separate bodies, the reaction section installation section 142 may be made of a material having high thermal conductivity, for example, a metal such as copper, gold, brass, or aluminum. .

As shown in FIGS. 2 and 3, the reaction section 120 is a closed cell having a reaction solution support 122, a side plate section 124, and an upper plate section 126. And a space 130 for accommodating a reaction solution containing a reagent and the like. Here, it is preferable that the space 130 has a volume capable of accommodating a sufficient amount of the reaction solution, and a capillary force is generated in a part or the whole of the space 130 when a small amount of the reaction solution is measured. The size may be set to a degree. The reaction section 120 includes a reaction solution support 122 and an upper plate section 126.
Are preferably arranged so as to be parallel to the horizontal plane, but they may be appropriately inclined or installed perpendicular to the horizontal plane.

A through hole 128 is formed in the upper plate portion 126 to allow the reaction solution to enter the space portion 130.
The through hole 128 may be formed anywhere as long as the position is not irradiated with the measurement light. The illustrated reaction unit 120 has one through hole 128, but may have a plurality of through holes to facilitate supply and discharge of the liquid to and from the space 130. Further, a liquid injection protrusion (not shown) may be connected to the through-hole 128 in order to easily put a reaction solution.

The reaction solution support 122, the side plate portion 124, and the upper plate portion 126 are made of alkali ion-containing glass such as borosilicate glass. For cost reduction, it may be made of a colorless and transparent plastic such as polystyrene, polypropylene, or polyethylene. In particular, it is desirable that the upper plate portion 126 has high transparency.

The reaction solution support 122 and the side plate 124 are joined by anodic bonding or an adhesive. Side plate 124
The upper plate part 126 is also joined by anodic bonding or an adhesive. The reaction liquid support 122 and the side plate portion 124 or the side plate portion 124 and the upper plate portion 126 may be formed integrally.

On the upper surface of the reaction solution support 122, a coating such as a silicon film (not shown) is applied by CVD or the like to totally reflect light.

Further, on the upper surface of the reaction solution support 122,
Specific affinity binding reagents (probes and primers) that specifically react with antibodies to the test substance, target DNA, etc.
Are physically or chemically bonded. That is,
Specific affinity binding reagent immobilized on top of reaction solution support 122
(Immobilized). The specific affinity binding reagent may be bound to the entire upper surface of the reaction solution support 122, or may be bound to only a part of the upper surface of the reaction solution support 122. In the latter case, light for measurement is applied to a part of the region.

The specific affinity binding reagent is used in the reaction solution support 12.
2 may be coupled to the lower surface of the upper plate portion 126 instead of to the upper surface, or may be coupled to both the upper surface of the reaction solution support 122 and the lower surface of the upper plate portion 126. Of course, the specific affinity binding reagent may also be bound to the inner surface of the side plate 124. Further, the binding of the specific affinity binding reagent may be reversibly bound, and after measurement, may be replaced with an unreacted specific affinity binding reagent for the same or a different measurement item.

Further, a sensor chip (support having a probe) on which a specific affinity binding reagent is immobilized is provided in the reaction section 120.
It may be embedded in. The type in which such a sensor chip is embedded has an advantage that a manufacturing space can be reduced and mass production is easy. Further, the type that does not use a sensor chip has an advantage that the cost can be reduced because the structure is simple.

The shape of the reaction section 120 and the space section 130 is not necessarily limited to a flat rectangular shape as shown in the figure, but a plane on which a measurement light beam can reflect under a specific polarization condition. It may have any shape, for example, a flat circular shape, a flat elliptical shape, or an elongated tube shape. Further, instead of the through hole, an opening may be provided at one or more positions of the side plate portion 124, and the supply and discharge of the liquid may be performed from the side through the opening. Since the side opening does not obstruct the optical path of the measurement light, such a reaction section 120 also has an advantage that it can be further miniaturized.

Next, the procedure for measuring the nucleic acid amplification reaction will be described.

First, a reaction solution is prepared by mixing a biological material (test substance) and a specific reagent. Next, after a sufficient amount of the reaction liquid is put into the space 130 of the reaction section 120 through the through hole 128, the reaction section 120 is installed in the reaction section installation section 142. After the reaction section 120 is installed in the reaction section installation section 142, a sufficient amount of the reaction solution may be put into the space 130 of the reaction section 120 through the through hole 128.

The reaction section 1 is controlled by the reaction section temperature control section 144.
20 is heated to a predetermined temperature to perform a nucleic acid amplification reaction by the LAMP method. The nucleic acid amplification reaction by the LAMP method is described in detail in JP-A-10-337186 and Nikkei Biotech 448, pages 11 to 13. These documents are incorporated herein by reference.

FIGS. 5 and 6 schematically show the state before and after the nucleic acid amplification reaction by the LAMP method in the reaction section 120, respectively.

As shown in FIG. 5, the reaction solution 150 contained in the reaction section 120 contains a target DNA 152, a non-target DNA 154, a primer (reagent for starting NDA synthesis) 156, and dNTP (reagent for DNA synthesis). 158, DN
A synthase 160 and the like. In addition, a reagent that specifically reacts with the amplification product is provided on the upper surface of the reaction solution support 122.
(Probes and primers) 134 are immobilized (immobilized). That is, the reaction section 120 has the reagent 134 immobilized on the solid phase.

As a result of the nucleic acid amplification reaction by the LAMP method, as shown in FIG. 6, a film 170 is formed as a region containing an amplification product composed of the target DNA 164 polymerized with the reagent 134. The polymerized target DNA 164 constituting the membrane 170 of the amplification product is obtained by binding the polymerized target DNA 164 to the reagent 134;
One bound to reagent 134 during polymerization, reagent 13
4 and polymerized. The optical properties (eg, refractive index) of the amplification product film 170 are measured by examining the polarization of light passing through the amplification product film 170.

In the nucleic acid amplification reaction by the LAMP method, the DNA 154 other than the target DNA is not polymerized and the target DNA 1
Only 52 has a high molecular weight. In addition, the products having a high molecular weight are condensed with each other in the reaction section 120, particularly in a two-dimensional region where the reagent 134 is disposed, thereby contributing to the increase in the density of the amplification product film 170. Such densification of the amplification product film 170 changes the polarization component of the light before and after passing through it, and the three-dimensional volume increase of each product due to the increase in molecular weight significantly changes the polarization component. To increase.

As described above, the reagent 134 may be bonded to the entire upper surface of the reaction solution support 122 or may be bonded to only a part of the upper surface. However, the latter is more preferable. Conceivable. This is because the area where the reagent 134 exists is reduced, and the reagent 134 located in the limited area is reduced.
This is because it is expected that the ratio of the target DNA bound before and after the polymerization is increased, and the efficiency of forming the amplification product film 170 is increased.

In FIG. 1, a light beam emitted from the light source unit 102 passes through a polarizer 104,
The beam is converted into a linearly polarized beam inclined at −45 ° with respect to the incident surface of the reaction unit 120, and reflected by the reaction unit 120 fixed to the reaction unit installation unit 142. The reflection converts the linearly polarized beam into an elliptically polarized beam according to the optical properties of the film of the amplification product.

The light beam reflected by the reaction unit 120 is
Directed to analyzer 106. Since the analyzer 106 transmits only the specific axis component of the elliptically polarized light, only the beam of the specific axis component of the elliptically polarized light passes through the analyzer 106 and reaches the photodetector 108. The analyzer 106 may have its transmission axis fixed in a specific direction during the measurement, or may be rotated around the optical axis. Further, a λ / 4 plate set at 45 ° may be added between the polarizer 104 and the reaction unit 120. In this case, during the DNA amplification reaction, polarization analysis may be performed from the angle of the polarizer or analyzer. It is also possible to extinguish only the initial time of the sample and analyze the change over time of the output. However, when rotating the polarizer, a light source that emits randomly polarized light, non-polarized light, or circularly polarized light, or a light source that directly outputs polarized light is used to keep the output of light passing through the polarizer constant. If so, it must be rotated in phase with the polarizer.

The light entering the photodetector 108 is photoelectrically converted. The electric signal output from the photodetector 108 is transmitted via a cable 110 to a data processing unit 11 such as a computer.
2 and subjected to a predetermined arithmetic processing. As a result, for example, measurement data reflecting changes in the optical characteristics (for example, refractive index) of the film of the amplification product is obtained.

As can be seen from the above description, the reaction section 12
0 is always arranged on the reaction section installation section 142 even during the nucleic acid amplification reaction. In other words, the nucleic acid amplification reaction is performed while being placed on the nucleic acid amplification reaction measurement device 100. In other words, the reaction unit 120 is not removed from the nucleic acid amplification reaction measurement device 100, and remains at a predetermined position. Therefore, the nucleic acid amplification reaction can be continuously measured. Further, the angle adjustment of the reaction unit 120 is required only once.

According to the present embodiment, it is possible to measure even a reaction in which an amplification product, which cannot be measured by electrophoresis, becomes a polymer. In addition, a nucleic acid amplification product can be measured without adding a detection compound such as a fluorescent dye. Furthermore, by continuously measuring after starting the nucleic acid amplification reaction, detection can be performed at the initial stage of amplification, and the final change amount can be calculated from the change amount per unit time of the change. Accordingly, there is provided an unprecedentedly high-speed, high-sensitivity, low-cost method and apparatus for measuring a nucleic acid amplification reaction.

By filling the reaction solution between the reaction solution support 122 and the upper plate 126 which are parallel to each other,
Not only can the reaction volume be measured accurately, the reaction results can be measured accurately, but also the optical conditions for the incidence and reflection of measurement light, which are important for polarization measurement, can be maintained with high accuracy, and the reaction due to evaporation or spilling of the reaction solution Variations in conditions and contamination to the outside can be effectively avoided.

[Second Embodiment] As shown in FIG. 4, a measuring apparatus 200 according to this embodiment includes a reaction unit 2
A reaction section installation section 242 for holding the reaction section 20;
Reaction unit temperature control unit 244 for controlling the temperature of unit 20
A target reaction section installation section 262 for holding the target reaction section 280; and a target reaction section temperature control section 264 for controlling the temperature of the target reaction section 280. The target reaction unit 280 is used as a comparison target with respect to the reaction unit 220 during the polarization analysis processing.

Both the reaction section 220 and the target reaction section 280
This is the same structure as the reaction unit 120 described with reference to FIGS. 2 and 3 in relation to the first embodiment. The reaction section 220 contains a reaction solution containing a test substance, a reagent, and the like. The target reaction section 280 contains a solution in which only the test substance is removed from the solution contained in the reaction section 220.

The reaction section setting section 242 and the target reaction section setting section 2
Reference numeral 62 denotes the same structure, and the reaction unit 220 and the target reaction unit 280 can be reliably fixed to each other by air suction or pressing.

The reaction section temperature control section 244 and the target reaction section temperature control section 264 are the same functional body, and maintain the reaction section 220 and the target reaction section 280 at desired constant temperatures, respectively. A desired temperature cycle can be applied to the reaction section 280. Reaction unit temperature control unit 24
4 and the target reaction unit temperature control unit 264 perform heating by a Peltier element, a resistor, a lamp, or the like, and perform cooling by heat radiation, water cooling, or air cooling.

The reaction section setting section 242 and the target reaction section setting section 2
62 may include the functions of the reaction section temperature control section 244 and the target reaction section temperature control section 264, respectively. As shown in the figure, the reaction section installation section 242 and the reaction section temperature control section 24
4. Target reaction unit installation unit 262 and target reaction unit temperature control unit 2
In the case where 64 is a separate body, the reaction section installation section 242 and the target reaction section installation section 262 may be made of a material having high thermal conductivity, for example, a metal such as copper, gold, brass, or aluminum.

The positions of the reaction section 220 and the target reaction section 280 may be interchanged. That is, the reaction section installation section 242
The positions of the target reaction unit installation unit 262 and the target reaction unit installation unit 262 may be exchanged with each other. Such exchange has no effect on the optical properties or the measurement results.

The measuring apparatus 200 includes a light source section 202 for emitting a light beam toward the target reaction section 280 and a target reaction section 2
Polarizer 2 for allowing only a specific polarization component to enter 80
04, a folding prism 270 that folds the light beam reflected by the target reaction unit 280 in parallel at intervals and directs the light beam to the reaction unit 220, and a detection unit for extracting a specific polarization component from the light reflected by the reaction unit 220. Photon 206 and photodetector 20 for detecting light transmitted through analyzer 206
8 is provided.

The light source unit 202 includes a light source, for example, a solid laser, a gas laser, a semiconductor laser, a xenon lamp, a halogen lamp, and the like. The light source unit 202
In the case where a light source of a semiconductor laser is provided, a collimator lens for collimating the emitted light is preferably further provided.

The photodetector 208 is connected to a data processing unit 212 such as a computer via a cable 210.

From another point of view, the measuring apparatus 200 includes a first optical path or an outgoing optical path extending from the light source section 202 to the return prism 270 via the target reaction section 280 and the reaction section 220 from the return prism 270. A second optical path or return optical path extending to the photodetector 208 via the first and second optical paths, both extending parallel and spaced apart;
It is communicated via a folding prism 270.

The polarizer 204 is disposed between the light source unit 202 and the target reaction unit 280 on the outgoing optical path.
6 is the reaction unit 220 and the photodetector 208 on the return optical path.
It is located between.

The polarizer 204 and the analyzer 206 are both optimally a Glan-Thompson prism in terms of the extinction ratio, but may be other optical elements such as a polarizing plate, a polarizing beam splitter, and a Glan laser prism. . Both are
The polarizer 204 and the analyzer 206 are arranged so that the planes of polarization of linearly polarized light passing therethrough are orthogonal to each other. That is, the polarizer 204 and the analyzer 206 are arranged so as to satisfy the crossed Nicols with each other.

The polarizer 204 is fixed so as to transmit linearly polarized light having a polarization plane inclined at −45 ° with respect to the plane of incidence with respect to the target reaction section 280. The polarizer 204 may be held rotatably around the optical axis so that the angle direction around the optical axis can be changed.

The analyzer 206 is fixed so as to transmit linearly polarized light having a polarization plane inclined at + 45 ° with respect to the plane of incidence with respect to the reaction section 220. The analyzer 206 may be held rotatably around the optical axis so that the angular direction around the optical axis can be changed.

The photodetector 208 includes a photoelectric conversion element, for example, a photodiode, an avalanche photodiode, a photomultiplier, a CCD camera, a SIT camera, and the like.

The folding prism 270 internally reflects the incident light beam four times, and emits the emitted light beam in parallel with the incident light beam at a predetermined interval. Such a folded prism 270 is made, for example, by combining two right-angle prisms.

The light source unit 202, the polarizer 204 and the analyzer 20
6 and the photodetector 208 are both arranged so that the optical axis is inclined at a predetermined angle with respect to the surfaces of the reaction section 220 and the target reaction section 280. Similarly, folded prism 270
Are arranged so that the optical axis forms the same angle with respect to the surfaces of the reaction section 220 and the target reaction section 280.

The measurement is performed in the same procedure as in the first embodiment. That is, first, a test solution and a specific reagent are mixed to prepare a reaction solution, a sufficient amount of the reaction solution is charged into the reaction unit 220, and then the reaction unit 220 is installed in the reaction unit installation unit 242. Also, a solution exactly the same as the above-mentioned reaction solution was prepared except that it did not contain a test substance,
After putting a sufficient amount into 0, the target reaction unit 280 is set in the target reaction unit setting unit 262.

The reaction section 2 is controlled by the reaction section temperature control section 244.
20 is heated to a predetermined temperature to perform a nucleic acid amplification reaction by the LAMP method. As a result of the nucleic acid amplification reaction by the LAMP method, as described in the first embodiment, the amplification product consisting of the polymerized target DNA bound to the reagent (probe or primer) fixed on the upper surface of the reaction solution support is obtained. A film is formed. The optical properties of the amplification product film are measured by examining the polarization of light passing through the amplification product film. At this time, by controlling the temperatures of the target reaction section 280 and the reaction section 220 to be the same, the target reaction section 2
It is possible to exclude the influence of a change in the optical characteristics (for example, the refractive index) depending on the temperature of the solution in the reaction unit 220 and the solution 80.

In FIG. 4, the light beam emitted from the light source unit 202 passes through the polarizer 204,
The beam is converted into a linearly polarized beam inclined at −45 ° with respect to the incident surface of the target reaction unit 230, and is reflected by the target reaction unit 280 fixed to the target reaction unit installation unit 262. By reflection
The linearly polarized beam is changed to an elliptically polarized beam according to the optical characteristics of the film of the amplification product.

The elliptically polarized beam is reflected by the folding prism 2
By 70, it is folded back in parallel at intervals. The beam of light turned back from the turning prism 270 is
As a result of being reflected four times inside the folding prism 270,
With respect to the elliptically polarized light before being folded, the elliptically polarized light has the same magnitude and the same direction (for example, the major axis) of the major axis component and the minor axis component but has the opposite rotation direction.

The light beam returned from the return prism 270 is applied to the reaction section 2 fixed to the reaction section installation section 242.
The light is reflected at 20 and directed to the analyzer 206. Analyzer 2
Since 06 transmits only the short-axis component of elliptically polarized light, only the beam of the short-axis component of elliptically polarized light passes through the analyzer 206 and reaches the photodetector 208.

The light entering the photodetector 208 is photoelectrically converted. The electric signal output from the photodetector 208 is transmitted via a cable 210 to a data processing unit 21 such as a computer.
2 and is subjected to predetermined arithmetic processing. As a result,
For example, measurement data such as optical characteristics of a film of an amplification product can be obtained.

If the target reaction section 280 and the reaction section 220 are exactly the same, the effects of the two on the light beam cancel each other out, so that the light detector 208 is in the extinction state in which no light is detected at all.

As can be seen from the above description, in this embodiment, as in the first embodiment, the reaction section 220
Remains at a predetermined position without being detached from the measurement device 200 even during the nucleic acid amplification reaction. Therefore, the nucleic acid amplification reaction can be continuously measured. Further, the angle adjustment of the reaction section 220 is required only once.

According to the present embodiment, it is possible to measure even a reaction in which an amplification product, which cannot be measured by electrophoresis, becomes a polymer. In addition, a nucleic acid amplification product can be measured without adding a detection compound such as a fluorescent dye. Furthermore, by continuously measuring after starting the nucleic acid amplification reaction, detection can be performed at the initial stage of amplification, and the final change amount can be calculated from the change amount per unit time of the change. Accordingly, there is provided an unprecedentedly high-speed, high-sensitivity, low-cost method and apparatus for measuring a nucleic acid amplification reaction.

Although some embodiments have been described in detail with reference to the drawings, the present invention is not limited to the above-described embodiments, but may be performed within the scope of the invention. Including all implementations. For example, the reaction unit 120, the reaction unit 220, and the target reaction unit 280
May be a plate or a module in which a plurality of them are integrated. Further, the reaction unit 120, the reaction unit 220, and the target reaction unit 280 may be designed in a card type.

[0072]

According to the present invention, it is possible to measure even a reaction in which an amplification product, which cannot be measured by electrophoresis, becomes a polymer. In addition, a nucleic acid amplification product can be measured without adding a detection compound such as a fluorescent dye. Furthermore, by continuously measuring after starting the nucleic acid amplification reaction, detection can be performed at the initial stage of amplification, and the final change amount can be calculated from the change amount per unit time of the change. That is, a measurement method called a rate assay can be performed with an unlabeled and non-probe. Accordingly, there is provided an unprecedented high-speed, high-sensitivity, low-cost method and apparatus for measuring a nucleic acid amplification reaction.

[Brief description of the drawings]

FIG. 1 is a perspective view showing a configuration of a measuring device according to a first embodiment of the present invention.

FIG. 2 is a plan view of a reaction section shown in FIG.

FIG. 3 is a sectional view of a reaction section taken along line III-III of FIG. 2;

FIG. 4 is a perspective view showing a configuration of a measuring device according to a second embodiment of the present invention.

FIG. 5 schematically shows a state before a nucleic acid amplification reaction by a LAMP method in a reaction section.

FIG. 6 schematically shows a state after a nucleic acid amplification reaction by a LAMP method in a reaction section.

[Explanation of symbols]

 REFERENCE SIGNS LIST 100 nucleic acid amplification reaction measuring device 102 light source unit 104 polarizer 106 analyzer 108 photodetector 120 reaction unit 142 reaction unit installation unit 144 reaction unit temperature control unit

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Iwao Mizoguchi 2-43-2 Hatagaya, Shibuya-ku, Tokyo Inside Olympus Optical Industrial Co., Ltd. (72) Inventor Yasuyoshi Mori 1381-3 Shimoishigami, Otawara-shi, Tochigi Eiken Chemical Nasu Works F-term (reference) 4B024 AA11 BA80 CA01 CA11 HA11 4B029 AA07 BB20 FA12 4B063 QA01 QQ42 QQ52 QR08 QR32 QR62 QS25 QS36 QX01

Claims (3)

[Claims] 1. A nucleic acid amplification reaction measuring method for measuring a nucleic acid amplification reaction comprising a step of increasing a molecular weight while repeatedly extending a nucleic acid sequence to be amplified, wherein the method specifically reacts with a product obtained by the nucleic acid amplification reaction. Immobilizing the reagent to be performed on the surface in contact with the reaction solution, and measuring based on the change in the polarization component of light before and after passing through the region containing the product of high molecular weight while repeating the nucleic acid sequence to be amplified. Nucleic acid amplification reaction measurement method. 2. A nucleic acid amplification reaction measuring device for measuring a nucleic acid amplification reaction including a step of increasing a nucleic acid sequence while repeatedly extending a nucleic acid sequence to be amplified, comprising: Reaction section installation section, reaction section temperature control section for controlling the temperature of the reaction section, light source section for emitting a light beam toward the reaction section, and polarization for allowing only a specific polarization component to enter the reaction section A nucleic acid amplification reaction measurement device, comprising: a detector; an analyzer for extracting a specific polarization component from light reflected by the reaction unit; and a photodetector for detecting light transmitted through the analyzer. 3. The nucleic acid amplification reaction measuring device according to claim 2, wherein a reagent specifically reacting with a product obtained by the nucleic acid amplification reaction is fixed on a surface of the reaction section which is in contact with the reaction solution.