JP2015186453A - nucleic acid amplification chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect fluorescence efficiently.SOLUTION: A nucleic acid amplification chip 1 includes: a substrate 2 in which a plurality of wells 3 are formed for amplification of a double stranded nucleic acid. The well makes the fluorescence progress above the well, in which the fluorescence is generated by irradiating the double stranded nucleic acid in the well with excitation light from above the well. The aspect ratio of a depth b of the well divided by a diameter a of the well is set to be larger than 0.5.

Description

  The present invention relates to a nucleic acid amplification chip that amplifies double-stranded nucleic acid.

  As a technique for selectively amplifying a specific gene sequence from a huge number of gene sequences, a technique called a PCR (Polymerase Chain Reaction) method is known. In the PCR method, double-stranded DNA is denatured into single-stranded DNA, and then only specific double-stranded DNA is amplified using a primer that binds only to a specific site of specific single-stranded DNA. is there. When a fluorescent molecule is bound to a specific double-stranded DNA that has been amplified, the amount of fluorescence increases as the number of double-stranded DNA amplified increases. By measuring this amount of fluorescence, the amplified double-stranded DNA is It is possible to identify pathogens and the like.

  In the PCR method, the time required for amplification is as short as about 2 hours, and the amplification process is simple. Therefore, amplification and identification of double-stranded DNA can be performed with a fully automatic desktop PCR measurement device. In a conventional PCR measuring apparatus, for example, a sample, a plurality of reagents, an enzyme, a primer, and the like are housed in a tube, and the amount of fluorescence is measured. In the PCR method, when denaturing double-stranded DNA into single-stranded DNA, it is necessary to set a high temperature, and after the primer is bound to the single-stranded DNA, it is necessary to cool, and the temperature is rapidly changed. This is important for shortening the measurement time.

  From such a background, a contrivance has been made to reduce the amount of reagent to be used by using a nucleic acid amplification chip having a microchannel and a microwell (see Patent Documents 1 to 6).

JP 2010-516281 A JP 2004-97200 A JP 2009-60859 A Japanese Unexamined Patent Publication No. 2007-189962 JP 2006-345798 A JP-A-5-317030

  However, in the micro flow path, the reagent passes through a predetermined flow path, so that the amplification state cannot be adjusted. In addition, since there is usually only one flow path, a separate chip must be provided for each primer, which is troublesome.

  Further, as a common problem between the microchannel and the microwell, since the amount of reagent used is very small, the amount of fluorescence emitted by the excitation light applied to the double-stranded DNA amplified with nucleic acid is not so strong. Therefore, it is desirable to be able to detect fluorescence as efficiently as possible. Patent Documents 1 to 6 described above do not disclose any device or technique for efficiently detecting fluorescence.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a nucleic acid amplification chip capable of efficiently detecting fluorescence emitted by excitation light irradiated to nucleic acid-amplified double-stranded DNA. It is to provide.

In order to solve the above-described problem, in one embodiment of the present invention, a substrate having a plurality of wells for amplification of double-stranded nucleic acid is provided.
The well advances fluorescence generated by irradiating the double-stranded nucleic acid in the well with excitation light from above the well.
A nucleic acid amplification chip is provided in which the aspect ratio obtained by dividing the well depth b by the well diameter a is set to be greater than 0.5.

  The surface roughness Ra of the inner wall surface of the well may be 5 μm or less.

  The reflectance for the fluorescence incident on the inner wall surface of the well may be 50% or more.

  The substrate may be made of polypropylene as a base material and containing an organic material having a higher reflectance than the polypropylene.

  The inner wall surface of the well may be a curved surface.

  The cross-sectional shape in the depth direction of the well may be a curve or a parabola that is less than half of the ellipse obtained by cutting the ellipse with a line parallel to the minor axis.

  The well includes a specimen, a reagent for extracting a double-stranded nucleic acid contained in the specimen, a primer that binds to a specific site of a specific single-stranded nucleic acid, and the number of double-stranded nucleic acids when irradiated with excitation light. And a reagent that generates fluorescence according to the above and an enzyme that promotes transcription of the double-stranded nucleic acid may be housed.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescence light-emitted by the excitation light irradiated to the double stranded DNA which carried out nucleic acid amplification can be detected efficiently.

1 is a top view of a nucleic acid amplification chip 1 according to an embodiment of the present invention. AA sectional view taken on the line AA of FIG. (A) The example whose aspect ratio of the well 3 is small, (b) is a figure which shows the example whose aspect ratio of the well 3 is large. (A)-(c) is a figure explaining surface roughness Ra of the well 3. FIG. (A) is a graph which shows the reflectance about three types of materials 1-3, (b) is a graph which shows the transmittance | permeability about three types of materials 1-3. The figure which put together the characteristic of the graph of Fig.5 (a) and FIG.5 (b). The figure which shows the cross-sectional shape of the well 3 of the example of a parabola or an ellipse. (A) shows the optical path trajectory of fluorescence traveling diagonally upward from the fluorescent substance bound to the double-stranded nucleic acid, and (b) shows the optical path trajectory of fluorescence traveling diagonally downward from the fluorescent substance bound to the double-stranded nucleic acid. FIG. (A) is an example in which the opening side of the well 3 is a flat surface, and (b) is a diagram showing an example in which the bottom surface of the well 3 is a flat surface. The flowchart which shows an example of the process sequence of PCR method using the nucleic acid amplification chip | tip 1 by this embodiment. The figure which shows the double stranded DNA couple | bonded complementarily. The figure which shows that double stranded DNA couple | bonded complementarily is isolate | separated into single stranded DNA. The figure which shows a mode that the primer couple | bonded with single strand DNA. The figure which shows a mode that two double stranded DNA was produced | generated.

  Hereinafter, embodiments of the present invention will be described in detail. In the drawings attached to the present specification, for convenience of illustration and understanding, the scale, the vertical / horizontal dimensional ratio, and the like are appropriately changed or exaggerated from those of the actual ones.

  FIG. 1 is a top view of a nucleic acid amplification chip 1 according to an embodiment of the present invention, and FIG. 2 is a cross-sectional view taken along line AA of FIG. The nucleic acid amplification chip 1 according to the present embodiment performs nucleic acid amplification of double-stranded DNA by, for example, a PCR method, and is arranged separately along one main surface of the substrate 2 as shown in FIG. A plurality of wells 3 are provided. Although FIG. 1 shows an example in which four wells 3 are provided on one main surface, the number of wells 3 is not particularly limited.

  The nucleic acid amplification chip 1 is configured using, for example, a rectangular substrate 2, and one side of the substrate 2 is about 10 to 15 mm and the thickness is about 0.5 to 3.0 mm. Moreover, the opening diameter of the well 3 is about 1-2 mm, and the depth of the well 3 is about 1.0-2.0 mm. The vertical and horizontal sizes and thickness of the nucleic acid amplification chip 1 and the opening diameter and depth of the well 3 are not particularly limited. However, in this embodiment, the amount of reagents that can be stored in one well 3 is kept in mind as much as possible. For example, the total amount of reagents stored in the well 3 is 0.5-3. It is assumed to be suppressed to about 0 μl.

  As will be described later, since the nucleic acid amplification chip 1 needs to raise or lower the temperature in the well 3 by 50 ° C. or more in a short time, it is excellent in thermal conductivity as a material of the substrate 2 and, for example, 0-100 Heat resistance in the temperature range of about ℃ is required.

  The nucleic acid to be amplified by the nucleic acid amplification chip 1 according to the present embodiment includes DNA (deoxyribonucleic acid), RNA (ribonucleic acid), other oligonucleotides, polynucleotides, and the like. Below, the example which amplifies DNA is demonstrated.

  In this embodiment, excitation light having a predetermined wavelength is irradiated from above the nucleic acid amplification chip 1 toward each well 3 and the excitation light is bound to the double-stranded nucleic acid amplified in each well 3. The fluorescence emitted when hitting the substance is detected by a detector placed above the nucleic acid amplification chip 1. The detector can grasp the amplification degree of the double-stranded nucleic acid by detecting the amount of fluorescent light. One of the features of this embodiment is that a well 3 is provided that can cause the fluorescence generated by the nucleic acid amplification chip 1 to travel upward so that as much fluorescence as possible can be detected by the detector.

(Aspect ratio of well 3)
In the well 3 according to the present embodiment, as shown in FIG. 2, the aspect ratio b / a obtained by dividing the depth b by the diameter a of the well 3 is set to be larger than 0.5. The reason for setting such an aspect ratio b / a is that the emission direction of fluorescence generated in the well 3 is more easily dispersed as the depth of the well 3 is shallower. The excitation light is incident from the normal direction of the bottom surface of the well 3, that is, from above the well 3, but the fluorescence emitted when the excitation light strikes the fluorescent substance bound to the double-stranded nucleic acid is not necessarily in the well 3. It does not always go upward. When the aspect ratio b / a of the well 3 is small, as shown in FIG. 3A, the proportion of double-stranded nucleic acids distributed and arranged in the bottom direction rather than the depth direction of the well 3 is relatively increased. As a result, the ratio of the fluorescence traveling obliquely with respect to the normal direction of the bottom surface of the well 3 increases. That is, as the aspect ratio b / a of the well 3 is smaller, the proportion of fluorescence dispersed in various directions increases, and the fluorescence detected by the detector disposed above the nucleic acid amplification chip 1 also decreases.

  On the other hand, as shown in FIG. 3 (b), when the aspect ratio b / a of the well 3 is increased, the proportion of double-stranded nucleic acids dispersed and arranged in the depth direction rather than the bottom surface direction of the well 3 is relatively Therefore, the proportion of fluorescence that proceeds in the normal direction of the bottom surface of the well 3 also increases. Therefore, the higher the aspect ratio of the well 3, the easier it is for the fluorescence to travel upward.

  According to the examination result of the present inventor, when the total amount of reagents stored in the well 3 is 0.5 to 3.0 μL, the aspect ratio b / a with which the amount of fluorescence can be correctly detected by the detector. Was found to be greater than 0.5, more desirably greater than 1.0.

  Thus, the detection accuracy of the fluorescence light quantity can be improved by making the aspect ratio b / a of the well 3 larger than 0.5, more desirably larger than 1.0.

  The diameter a of the well 3 is the diameter when the opening of the well 3 is circular, the length of the long axis when it is elliptical, and the length of the diagonal line when it is square, for example. The depth b of the well 3 is the length from the opening surface forming the opening of the well 3 to the deepest portion in the vertical direction.

(Surface roughness Ra of well 3)
Since some of the fluorescence emitted when the excitation light strikes the fluorescent substance bound to the double-stranded nucleic acid, the fluorescence progresses obliquely from the normal direction of the bottom surface of the well 3. When the ratio b / a is larger than 0.5, there is a possibility that the fluorescence hits the side surface of the well 3. In this case, as shown in FIG. 4A, it is desirable that reflected fluorescent light be reflected above the well 3. A part of the fluorescence travels toward the bottom surface of the well 3 and hits the bottom surface, but it is desirable that the fluorescence hitting the bottom surface is also reflected upward. If the side surface and the bottom surface of the well 3 are flat as shown in FIG. 4B, the fluorescence hitting the side surface and the bottom surface is regularly reflected in the direction according to the incident direction, but the side surface and the bottom surface are as shown in FIG. If the surface is roughened in this way, the fluorescence hitting the side and bottom surfaces will be scattered in various directions. For this reason, when the side surface and bottom surface of the well 3 are roughened, the intensity of the fluorescence traveling above the well 3 is weakened.

  Therefore, it is desirable to improve the smoothness of the inner wall surface and to keep the surface roughness Ra as low as possible in order to easily reflect the fluorescence hitting the inner wall surface composed of the side surface and the bottom surface of the well 3 upward.

  Since the well 3 has a small size, it is not easy to perform a processing for reducing the surface roughness Ra of the inner wall surface of each well 3 after the nucleic acid amplification chip 1 is manufactured. Therefore, it is desirable to form the well 3 by reducing the surface roughness Ra when the nucleic acid amplification chip 1 is manufactured. For example, when the nucleic acid amplification chip 1 having the well 3 is integrally formed by injection molding, the surface of the well 3 portion of the injection mold is made as flat as possible, and nucleic acid amplification produced using this mold It is desirable that the surface roughness Ra of the inner wall surface of each well 3 in the chip 1 be a predetermined value or less. The predetermined value is, for example, 5 μm. As a result of studies by the present inventor, if the surface roughness Ra of the side surface and the bottom surface of the well 3 is 5 μm or less, it is possible to suppress scattering when the fluorescent light hits and to increase the ratio of the fluorescent light reflected upward Thus, it was found that the amount of fluorescent light can be detected correctly by the detector.

  For example, a white interferometer (manufactured by Zygo) can be used to measure the surface roughness Ra.

(Surface reflectance of well 3)
Depending on the material of the nucleic acid amplification chip 1, the reflectance and transmittance for incident fluorescence are different, so that it may not always be possible to efficiently reflect the fluorescence upward simply by increasing the smoothness of the inner wall surface of the well 3. sell. Therefore, in order to further improve the reflection performance of the inner wall surface of the well 3, it is desirable to make the material of the nucleic acid amplification chip 1 excellent in the reflection performance.

  Fig.5 (a) is a graph which shows the reflectance about three types of materials 1-3. The horizontal axis of this graph is the wavelength of light, and the vertical axis is the reflectance. Since the fluorescence wavelength has a central wavelength of about 500 nm, the wavelength range on the horizontal axis is set to 400 to 600 nm.

  Material 1 is PP (polypropylene). Material 2 is obtained by adding wax to PP. Material 3 is made by adding wax and magnesium oxide (MgO) to PP. Wax is an organic substance having a higher reflectance than PP, and a specific material is, for example, a resin-modified silicone Dow Corning Toray BY27 series (manufactured by Toray Industries, Inc.). Magnesium oxide is a white solid, and by adding magnesium oxide, the whiteness of the nucleic acid amplification chip 1 is increased and the reflection performance is improved. The addition ratio of the wax in the material 2 is, for example, about 3%, and the addition ratio of magnesium oxide in the material 3 is, for example, about 50%.

  As can be seen from FIG. 5A, for light of 400 to 600 nm including the fluorescence wavelength range, the material 2 has a higher reflectance than the material 1, and the material 3 is more reflective than the material 2. The rate is high.

  FIG.5 (b) is a graph which shows the transmittance | permeability about the materials 1-3 mentioned above. The horizontal axis of this graph is the wavelength of light, and the vertical axis is the transmittance. As can be seen from FIG. 5 (b), the transmittance of the material 2 is lower than that of the material 1 and the material 3 is more transparent than the material 2 for light of 400 to 600 nm including the wavelength range of fluorescence. The rate is lowered.

  Note that the reflectance in FIG. 5A and the transmittance in FIG. 5B were measured using an ultraviolet-visible spectrophotometer UV2550 / 2450 (manufactured by Shimadzu Corporation).

  FIG. 6 is a graph summarizing the characteristics of the graphs of FIGS. 5 (a) and 5 (b). As shown in the figure, for light of 400 to 600 nm including the fluorescence wavelength range, the reflectance of the material 1 is 10 to 15%, the transmittance of the material 1 is 80 to 85%, and the reflectance of the material 2 is 50. The transmittance of the material 2 is 47 to 50%, the reflectance of the material 3 is 90 to 97%, and the transmittance of the material 3 is 7 to 12%.

  If the reflectance is less than 50%, it may depend on the number of double-stranded nucleic acids amplified in the well 3, but the detector may not be able to detect the amount of fluorescent light correctly. Therefore, judging from the results shown in FIG. 6, when wax or MgO is added to PP, the reflectance may increase. Therefore, as a material for the nucleic acid amplification chip 1, wax is added to PP or MgO is further added. May be.

  Although FIG. 5 illustrates an example in which PP is used as the material of the substrate 2, PC (polycarbonate), PE (polyethylene), polyester, or the like may be used in addition to PP. Moreover, if the magnesium oxide mentioned above is used as an additive, not only a reflectance but thermal conductivity can also be improved. Moreover, materials other than magnesium oxide may be added for the purpose of improving thermal conductivity.

(Shape of well 3)
FIG. 2 shows an example in which the side surface of the well 3 extends in a substantially vertical direction with respect to the bottom surface. However, in order to reflect the fluorescence hitting the side surface upward, the side surface and the bottom surface of the well 3 are curved. desirable. Here, the curved surface means that the cross-sectional shape in all directions passing through the central portion of the bottom surface of the well 3 is a curve, and the specific cross-sectional shape of the curved surface is a partial curve of a parabola or an ellipse. Etc. The partial curve of an ellipse is a curve of half or less of the ellipse obtained by cutting the ellipse with a line parallel to the minor axis.

  When the cross-sectional shape of the well 3 is a parabolic or elliptical partial curve, as shown in FIG. 7, the closer to the opening of the well 3, the smaller the inclination angle of the tangent of the cross-section. That is, the closer to the opening of the well 3, the smaller the angle formed between the normal direction and the tangent of the bottom surface of the well 3. In FIG. 7, the angle formed on the opening side of the well 3 is θ1, and the angle formed on the bottom side of the well 3 is θ2, where θ2> θ1. Therefore, the reflection angle of the light hitting the side surface close to the opening is also reduced, and the fluorescence proceeds in an angle direction close to the normal direction, that is, upward.

  FIG. 8 is a cross-sectional view when the cross-sectional shape of the well 3 is an elliptical partial curve, and FIG. 8 (a) shows an optical path locus of fluorescence traveling obliquely upward from a fluorescent substance bound to a double-stranded nucleic acid. FIG. 8 (b) is a diagram showing an optical path locus of fluorescence traveling obliquely downward from a fluorescent substance bound to a double-stranded nucleic acid.

  In the case of FIG. 8A, the fluorescence traveling obliquely upward hits the left curved surface of the well 3, for example, and is reflected upward. On the other hand, in the case of FIG. 8B, the fluorescence traveling diagonally downward is reflected, for example, on the left curved surface of the well 3, and then further reflected on the right curved surface and travels upward. The optical path of the fluorescence is not limited to FIG. 8A or FIG. 8B, but the excitation light is applied to the fluorescent substance bound to the double-stranded nucleic acid in the well 3 by making the cross-sectional shape of the well 3 a curve. Most of the fluorescence generated by hitting the light proceeds above the well 3. Therefore, by making the cross-sectional shape of the well 3 a curve, the proportion of fluorescence detected by the detector can be increased.

  The entire side surface and bottom surface of the well 3 need not be curved. For example, as shown in FIG. 9A, the opening side of the well 3 may be a flat surface. Alternatively, as shown in FIG. 9B, at least a part of the bottom surface of the well 3 may be a flat surface. In other words, it is desirable that at least a part of the side surface portion that is highly likely to be irradiated with a fluorescent light be curved. According to the study of the present inventor, if about 67% or more of the well 3 is curved with respect to the total area of the side surface and the bottom surface, the fluorescence can be efficiently advanced upward. The reason for setting it to about 67% or more is as follows. If the side surface of the well 3 is a curved surface, the fluorescence easily proceeds above the well 3 as shown in FIG. If the aspect ratio b / a ≧ 0.5 and the bottom surface of the well 3 is a flat surface, the ratio of the curved surface to the total area of the side surface and the bottom surface of the well 3 is 67% or more.

  As described above, as a measure for causing the fluorescence generated in the well 3 to travel above the well 3, 1) aspect ratio b / a> 0.5, 2) surface roughness Ra <5 μm on the side surface and bottom surface of the well 3 3) The reflectance of the side surface and the bottom surface of the well 3 is 50%, and 4) the shape of the well 3 is a curved surface. Among these, 1) is an indispensable condition, and the remaining 2) to 4) are not necessarily indispensable conditions. If carried out in combination with the conditions of 1) as appropriate, the proportion of fluorescence that progresses further can be increased. it can.

  FIG. 10 is a flowchart showing an example of a gene analysis processing procedure by the PCR method using the nucleic acid amplification chip 1 according to the present embodiment. The flowchart of FIG. 10 is performed by a PCR measurement device (not shown).

  When the nucleic acid amplification chip 1 is set in the PCR measuring apparatus, a specimen to be subjected to gene analysis and a DNA extraction reagent are placed in a predetermined container and mixed, and a part of each sample is added to each well of the nucleic acid amplification chip 1. 3 is injected (step S1). It is assumed that a separate primer is stored in each well 3 in advance. The primer has a property of binding to a specific site of a specific single-stranded DNA. Thus, for example, if the nucleic acid amplification chip 1 has four wells 3 and each type of well 3 stores a different type of primer, any one of the single-stranded DNAs corresponding to the four types of primers is contained in the specimen. Whether it is included or not can be detected. In some cases, the same primer may be placed in two or more wells 3.

  In addition, in the well 3, a PCR reaction reagent containing a fluorescent material for generating a light amount of light according to the number of double-stranded DNA, or a polymerase that is an enzyme that promotes transcription of double-stranded DNA Is also stored. Thus, the well 3, at least the specimen, the DNA extraction reagent, the primer, the PCR reaction reagent and the enzyme are stored. The DNA extracted with the reagent for DNA extraction is a double-stranded DNA 11 that is complementarily bound as shown in FIG.

  Next, by raising the temperature in the well 3, as shown in FIG. 12, the double-stranded DNA in the specimen is separated into single-stranded DNA (step S2). The temperature at this time is set to 90 ° C. or higher. By separating the double-stranded DNA, two single-stranded DNAs 12 are generated.

  Next, the temperature is lowered to about 40 to 65 ° C., and the primer 13 is bound to a specific site of the separated single-stranded DNA as shown in FIG. 13 (step S3).

  Thereafter, the temperature is raised to about 70 ° C., and the double-stranded DNA in the primer 13 portion is extended as shown in FIG. 14 (step S4). Since each of these two single-stranded DNAs becomes a double-stranded DNA 11, the original double-stranded DNA 11 is doubled. When the process of step S4 ends, the process returns to step S2, and the processes of steps S2 to S4 are repeated until the predetermined number of times is reached.

  As described above, the double-stranded DNA can be doubled every time the processes of steps S1 to S4 in FIG. 10 are performed. In the PCR method, steps S1 to S4 are repeated about 30 times to amplify the number of double-stranded DNA. Since there is only one type of double-stranded DNA that amplifies in one well 3 depending on the primer accommodated therein, a plurality of wells 3 each containing a different primer have different double-stranded DNAs. Can be increased. Thus, for example, when examining whether or not a sample contains a specific pathogen, a primer that binds to a single-stranded DNA corresponding to this specific pathogen is placed in any well 3. In this case, whether or not a specific pathogen is contained in the specimen can be detected based on whether or not the number of double-stranded DNA in the well 3 increases.

  In the PCR measurement apparatus, each well 3 in the nucleic acid amplification chip 1 is irradiated with excitation light, and the amount of light emitted from the fluorescent substance bound to the double-stranded nucleic acid, that is, the intensity is measured. As the number of double-stranded DNAs increases, the amount of fluorescent light increases. Therefore, whether or not double-stranded DNA has been amplified in each well 3 can be determined based on the amount of fluorescent light. Since the PCR measurement apparatus knows the type of primer placed in each well 3, the gene sequence of a specific pathogen or the like contained in the specimen is specified by the type of primer in the well 3 where the amount of fluorescence is large. be able to.

  In FIG. 10, the gene amplification processing procedure by the PCR method has been described, but this embodiment can also be applied to gene amplification methods other than the PCR method. More specifically, this embodiment includes a SMAP (SMart Amplification Process) method, a LAMP (Loop-Mediated Isothermal Amplification) method, an ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) method, a NASBA (Nucleic Acid Sequence- It can be widely applied to the Based Amplification (SDA) method, the SDA (Strand Displacement Amplification) method, the TMA (Transcription-Mediated Amplification) method, the TRC (Transcription-Reverse transcription Concerted) method, and the like.

  In any of these methods, as a result, in order to amplify the double-stranded DNA, a reagent containing a fluorescent substance that binds to the double-stranded DNA is placed in the well 3, so that the PCR method Whether or not a specific gene sequence has been amplified can be determined.

  In the above-described embodiment, an example in which double-stranded DNA is amplified has been described. However, the present embodiment can also be applied to the case of amplifying RNA instead of DNA. That is, the nucleic acid amplification chip 1 according to the present embodiment is applicable when a double-stranded nucleic acid composed of DNA or RNA is amplified.

  Thus, in this embodiment, in order to make the aspect ratio of the well 3 greater than 0.5, the fluorescence emitted by the excitation light hitting the fluorescent substance bound to the double-stranded nucleic acid amplified in the well 3 is efficiently converted. It can be made to proceed well above the well 3, and the proportion of fluorescence detected by the detector can be increased. Therefore, the detector can detect the amount of fluorescent light with high accuracy. Further, by improving the smoothness of the inner wall surface of the well 3, it is possible to easily reflect the fluorescence hitting the inner wall surface upward. Furthermore, by using a material excellent in reflection performance as the material of the nucleic acid amplification chip 1, the reflectance on the inner wall surface of the well 3 can be increased, and more fluorescence can be advanced upward. In addition, by making the inner wall surface of the well 3 a curved surface, more fluorescence hitting the inner wall surface can be advanced upward.

  The aspect of the present invention is not limited to the individual embodiments described above, and includes various modifications that can be conceived by those skilled in the art, and the effects of the present invention are not limited to the contents described above. That is, various additions, modifications, and partial deletions can be made without departing from the concept and spirit of the present invention derived from the contents defined in the claims and equivalents thereof.

    1 Nucleic acid amplification chip, 2 substrate, 3 well, 11 double-stranded DNA, 12 single-stranded DNA, 13 primer

Claims (7)

A substrate on which a plurality of wells for amplification of double-stranded nucleic acid is formed;
The well advances fluorescence generated by irradiating the double-stranded nucleic acid in the well with excitation light from above the well.
A nucleic acid amplification chip in which an aspect ratio obtained by dividing the depth b of the well by the diameter a of the well is set to be larger than 0.5.   The nucleic acid amplification chip according to claim 1, wherein the surface roughness Ra of the inner wall surface of the well is 5 μm or less.   The nucleic acid amplification chip according to claim 1 or 2, wherein the reflectance of the fluorescence incident on the inner wall surface of the well is 50% or more.   The nucleic acid amplification chip according to claim 3, wherein the substrate contains polypropylene as a base material and contains an organic material having a higher reflectance than the polypropylene.   The nucleic acid amplification chip according to any one of claims 1 to 4, wherein an inner wall surface of the well is a curved surface.   6. The nucleic acid amplification chip according to claim 5, wherein the cross-sectional shape in the depth direction of the well is a curve or a parabola that is less than half of the ellipse obtained by cutting the ellipse with a line parallel to the minor axis.   The well includes a specimen, a reagent for extracting a double-stranded nucleic acid contained in the specimen, a primer that binds to a specific site of a specific single-stranded nucleic acid, and the number of double-stranded nucleic acids when irradiated with excitation light. The nucleic acid amplification chip according to any one of claims 1 to 6, wherein a reagent that generates fluorescence in accordance with an enzyme and an enzyme that promotes transcription of the double-stranded nucleic acid are housed.

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