JP2011239742A - Microfluidic chip, microfluidic chip set and nucleic acid analysis kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microfluidic chip, a microfluidic chip set and a nucleic acid analysis kit that can easily determine with high degree of sensitivity and high degree of accuracy whether a plurality kinds of genes are expressed at a predetermined dosage.SOLUTION: The microfluidic chip includes: reagents 30, 31, and 32 which are nucleic acids amplifiable by a common primer with a target nucleic acid, and include a known amount of an internal standard nucleic acid having a different sequence from that of the target nucleic acid and the primer common to the target nucleic acid and the internal standard nucleic acid; and a fluorescence probe exhibiting different fluorescence changes from when binding to a part of an amplified product of the target nucleic acid and the internal standard nucleic acid and binding to the amplified product of the target nucleic acid and from when binding to the amplified product of the internal standard nucleic acid. The amount of primers and fluorescence probes included in wells 16 are different from each wells 16. The amount of the internal standard nucleic acid is the same as the amount of the target nucleic acid included in a reaction solution introduced in the wells 16 when the reaction solution of a reference specimen as a criterion to determine whether the target nucleic acid are expressed at the predetermined dosage is introduced in the wells 16.

Description

  The present invention relates to a microfluidic chip, a microfluidic chip set, and a nucleic acid analysis kit.

  In recent years, DNA chips (DNA microarrays) have attracted attention in order to analyze the expression of a large number of genes at once. The nucleic acid detection / quantification method using a DNA chip is a nucleic acid detection / quantification method using a hybridization reaction based on DNA complementarity. A DNA chip is characterized in that many types of DNA fragments (probe DNA) are immobilized on a flat substrate. As a specific method of use, an expressed gene contained in a cell to be studied or examined can be expressed with a fluorescent dye. There is a method in which the amount of gene expression in a sample is quantified by labeling, binding to a DNA fragment on a flat substrate, and measuring the fluorescence intensity with a dedicated device.

  However, as described in Non-Patent Document 1, the DNA chip has several problems.

  First, there is a problem regarding sensitivity. The detection capability (sensitivity) of the DNA chip is not sufficient for low-expressing genes.

  Secondly, there is a problem regarding accuracy. In order to detect a low-expressed gene with a DNA chip, it is necessary to ensure a sufficient amount of specimen. If the amount of the sample is small and not sufficient for the reaction, the gene is subjected to an amplification reaction such as PCR (Polymerase Chain Reaction) or IVT (in-vitro transcription) to increase the amount of nucleic acid in the sample. There is a need. In PCR, a mixture of target nucleic acid and reagent is put in a PCR tube (reaction vessel), and a temperature control device called a thermal cycler is used, for example, to change temperature in three steps of 55 ° C, 72 ° C, and 94 ° C for several minutes. The reaction is repeated in a cycle, and only the target nucleic acid can be amplified by about 2 times per temperature cycle by the action of the enzyme (DNA polymerase). This amplification process has a problem that the quantitative accuracy is lowered because the ratio of the amount of genes contained in the sample changes.

  In addition, the accuracy is also lowered by nucleic acid having a sequence similar to the target nucleic acid erroneously bound to the probe (cross hybridization). Furthermore, it has been pointed out that the binding of a specific gene may be biased by the sequence of the probe. Similarly, fluorescent labeling of the target nucleic acid may affect the accuracy.

  Thirdly, there is a problem regarding reproducibility. The results differ depending on the DNA chip platform (DNA chip product). There are various factors, but the biggest cause is considered to be the difference in the length and arrangement of the probes.

  As methods for analyzing gene expression with high accuracy and high sensitivity used in clinical examinations, there are an absolute quantification method of real-time PCR and a competitive PCR method. If quantification is performed by these techniques and careful calibration is performed based on the results, some of the problems of sensitivity and accuracy of the DNA chip can be improved. However, the absolute quantification method of real-time PCR needs to create a calibration curve from data of at least 5 points, and the competitive PCR method also needs to create a plurality of calibration curves. This work is complicated and takes a lot of time and effort. Furthermore, the calibration itself is a complicated operation. Therefore, it has been difficult to apply these techniques to many kinds of genes.

Sorin Draghici et.al., "Reliability and reproducibility issues in DNA microarray measurement", TRENDS in Genetics Vol.22 No.2 February 2006, P101-109

  The present invention has been made in view of the above problems, and according to some aspects of the present invention, whether or not a plurality of types of genes are expressed in a predetermined amount is determined with high sensitivity and high sensitivity. It is possible to provide a microfluidic chip, a microfluidic chip set, and a nucleic acid analysis kit that can be easily performed with accuracy.

(1) A microfluidic chip according to the present invention comprises:
A microfluidic chip used for amplification of a target nucleic acid contained in a specimen and determination of whether or not the target nucleic acid is expressed in a predetermined amount,
Including a plurality of reaction vessels,
The reaction vessel is
A known amount of an internal standard nucleic acid that is a nucleic acid that can be amplified with a primer common to the target nucleic acid, and that has a different sequence from the target nucleic acid;
A primer common to the target nucleic acid and the internal standard nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the internal standard nucleic acid, and exhibits a different fluorescence change when bound to the amplification product of the target nucleic acid and the amplification product of the internal standard nucleic acid; ,
Including
Among the reaction vessels, the types of the primer and the fluorescent probe contained in the first reaction vessel and the second reaction vessel are different from each other,
The amount of the internal standard nucleic acid is introduced into the reaction vessel when a reaction solution of a reference sample, which is a sample serving as a reference for determining whether or not a predetermined amount of the target nucleic acid is expressed, is introduced into the reaction vessel. It is the same as the reference amount that is the amount of the target nucleic acid contained in the reaction solution.

  According to the present invention, among the reaction containers, the types of primers and fluorescent probes contained in the first reaction container and the second reaction container are different from each other, so that the expression of a plurality of types of genes can be easily and accurately determined. A microfluidic chip can be realized.

  Further, according to the present invention, the amount of the internal standard nucleic acid is determined when the reaction liquid of the reference sample, which is a sample serving as a reference for determining whether or not the target nucleic acid is expressed in a predetermined amount, is introduced into the reaction vessel. The amount of target nucleic acid expressed in the sample to be measured is about the same as the amount of target nucleic acid expressed in the reference reference sample It is possible to realize a microfluidic chip capable of determining whether or not with high sensitivity and high accuracy.

(2) This microfluidic chip is
The reference amount may be at least one of an amount representative when the reference sample is a sample collected from a healthy individual and an amount representative when the reference sample is a sample collected from a sick individual.

  This makes it highly sensitive whether the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in at least one of a sample collected from a healthy individual and a sample collected from a diseased individual, Can be determined with high accuracy.

(3) This microfluidic chip is
A reaction container under a first condition, wherein the reference amount is a representative amount when the reference sample is a sample collected from the healthy individual;
A reaction container under a second condition in which the reference amount is a representative amount when the reference sample is a sample collected from the diseased individual; and
May be included.

  Thus, with one microfluidic chip, whether the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in a sample collected from a healthy individual and a sample collected from a sick individual Can be determined with high sensitivity and high accuracy.

(4) This microfluidic chip is
A reaction container having a first condition in which the reference amount is an amount representative of a case where the reference sample is a sample collected from an individual in a first progressing state of the disease;
A reaction container under a second condition in which the reference amount is an amount representative of a case where the reference sample is a sample collected from an individual in a second advanced state of the disease;
May be included.

  Thus, with one microfluidic chip, the target nucleic acid expression level in the sample to be measured is the target in the sample collected from the individual in the first advanced state of disease and the sample collected from the individual in the second advanced state of disease. It can be determined with high sensitivity and high accuracy whether the expression level is the same as the nucleic acid expression level.

(5) A microfluidic chip set according to the present invention includes:
A reaction container under a first condition, wherein at least part of the plurality of reaction containers, the reference amount is a quantity representative of a case where the reference specimen is a specimen collected from the diseased first progressing individual. Including the above-described microfluidic chip;
A reaction container having a second condition, wherein at least a part of the plurality of reaction containers, the reference amount is an amount representative of a case where the reference sample is a sample collected from the diseased second progressive individual. Including the above-described microfluidic chip;
including.

  ADVANTAGE OF THE INVENTION According to this invention, the microfluidic chip set which can select easily the reference amount used as the criterion of determination according to a use is realizable.

(6) The nucleic acid analysis kit according to the present invention comprises:
A nucleic acid analysis kit used for amplification of a target nucleic acid contained in a specimen and determination of whether or not the target nucleic acid is expressed in a predetermined amount,
A microfluidic chip;
An internal standard reagent that is a liquid containing a known amount of an internal standard nucleic acid, which is a nucleic acid that can be amplified with a primer common to the target nucleic acid and has a sequence different from the target nucleic acid;
Including
The microfluidic chip includes a plurality of reaction vessels,
The reaction vessel is
A primer common to the target nucleic acid and the internal standard nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the internal standard nucleic acid, and exhibits a different fluorescence change when bound to the amplification product of the target nucleic acid and the amplification product of the internal standard nucleic acid; ,
Including
Among the reaction vessels, the types of the primer and the fluorescent probe contained in the first reaction vessel and the second reaction vessel are different from each other,
The molar concentration of the internal standard nucleic acid is the same as the reference molar concentration, which is the molar concentration of the target nucleic acid contained in a reference sample that is a reference for determining whether or not a predetermined amount of the target nucleic acid has been expressed.

  According to the present invention, among the reaction containers, the types of primers and fluorescent probes contained in the first reaction container and the second reaction container are different from each other, so that the expression of a plurality of types of genes can be easily and accurately determined. A nucleic acid analysis kit can be realized.

  Further, according to the present invention, the molar concentration of the internal standard nucleic acid is the same as the molar concentration of the target nucleic acid contained in the reference sample, which is a sample used as a criterion for determining whether or not the target nucleic acid is expressed in a predetermined amount. Thus, it is possible to realize a nucleic acid analysis kit that can determine with high sensitivity and high accuracy whether or not the expression level of the target nucleic acid in the sample to be measured is comparable to the expression level of the target nucleic acid in the reference reference sample.

(7) This nucleic acid analysis kit is
The reference molar concentration may be at least one of a molar concentration representative when the reference specimen is a specimen collected from a healthy individual and a molar concentration representative when the specimen is a specimen collected from a sick individual. .

  This makes it highly sensitive whether the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in at least one of a sample collected from a healthy individual and a sample collected from a diseased individual, Can be determined with high accuracy.

(8) This nucleic acid analysis kit is
As the internal standard reagent,
A first internal standard reagent containing the internal standard nucleic acid, wherein the reference molar concentration is a molar concentration representative of a case where the reference sample is a sample collected from an individual in a first advanced state of the disease;
A second internal standard reagent containing the internal standard nucleic acid, wherein the reference molar concentration is a molar concentration representative of a case where the reference sample is a sample collected from an individual in the first advanced state of the disease;
May be included.

  Thereby, the expression level of the target nucleic acid in the sample to be measured is comparable to the expression level of the target nucleic acid in the sample collected from the individual in the first advanced state of disease and the sample collected from the individual in the second advanced state of disease. It can be determined with high sensitivity and high accuracy.

FIG. 3 is a plan view schematically showing the substrate 10 of the microfluidic chip 100 according to the first embodiment. The figure which represented typically the cross section of the AA line of FIG. FIG. 2 is a plan view schematically showing the microfluidic chip 100 according to the first embodiment. The figure which represented typically the cross section of the BB line of FIG. FIG. 3 is a schematic diagram showing an enlarged cross section near the well 16 of the microfluidic chip 100. FIG. 6A is a plan view schematically showing the microfluidic chip 100a according to the second embodiment, and FIG. 6B is a plan view schematically showing the microfluidic chip 100b according to the third embodiment. FIGS. 7A to 7C are plan views schematically showing microfluidic chips 101, 101a, 101b according to modifications. The figure which shows typically a mode that the inertial force was applied in the state which introduce | transduced the test liquid 80 into the reservoir | reserver 15 of the microfluidic chip | tip 100. FIG. The figure which showed typically a mode that the well 16 of the microfluidic chip | tip 100 was sealed with the roller 90. FIG. The top view which shows typically the microfluidic chip set 200 which concerns on 4th Embodiment. The figure which shows typically the nucleic acid analysis kit 300 which concerns on 5th Embodiment. The figure which shows typically the nucleic acid analysis kit 300a which concerns on 6th Embodiment. 13A and 13B schematically show nucleic acid analysis kits 301 and 301a according to modifications. Sequence examples of target nucleic acid and internal standard nucleic acid. The sequence example of the primer corresponding to the target nucleic acid shown in FIG. 14, and an internal standard nucleic acid. The example of a sequence of the fluorescent probe corresponding to the target nucleic acid and internal standard nucleic acid which are shown by FIG. Other sequence examples of target nucleic acid and internal standard nucleic acid. The sequence example of the primer corresponding to the target nucleic acid and internal standard nucleic acid which are shown by FIG. The example of a sequence of the fluorescent probe corresponding to the target nucleic acid and internal standard nucleic acid which are shown by FIG.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. Also, not all of the configurations described below are essential constituent requirements of the present invention.

1. Microfluidic chip 1-1. Configuration of Microfluidic Chip According to First Embodiment The microfluidic chip 100 according to the first embodiment amplifies a target nucleic acid contained in a sample to be measured and determines whether or not a predetermined amount of the target nucleic acid is expressed. And a plurality of reaction vessels. In the example described below, the microfluidic chip 100 includes a substrate 10 and a cover 20, and a plurality of wells 16 serving as reaction containers are provided on the substrate 10.

  In the following description, “when viewed in plan”, “when viewed in plan”, and “when viewed in plan” are when viewed from a direction orthogonal to the first surface 11 of the substrate 10. Shall point to.

1-1-1. Substrate FIG. 1 is a plan view schematically showing the substrate 10 of the microfluidic chip 100 according to the first embodiment. FIG. 2 is a diagram schematically showing a cross section taken along line AA of FIG.

  The substrate 10 is a plate-like member that becomes the base of the microfluidic chip 100. As shown in FIG. 2, the substrate 10 has a first surface 11 and a second surface 12 that have a front-back relationship. The shape of the substrate 10 is a plate shape, and the height of the well region 14 on the first surface 11 of the substrate 10 and the height of the surface of the substrate surrounding the well region 14 coincide with the height of the opening surface of the well opening 16. If it does not specifically limit. The thickness of the substrate 10 (distance between the first surface 11 and the second surface 12) is not particularly limited, but is preferably 0.5 mm or more and 5 mm or less in terms of ease of handling and resistance to breakage. The outer shape of the substrate 10 viewed in plan is not particularly limited, and may be a rectangle, a circle, or the like. In the present embodiment, an example in which the shape of the substrate 10 viewed in plan is a rectangle.

  The material of the substrate 10 is not particularly limited, and examples thereof include inorganic materials (for example, single crystal silicon, Pyrex (registered trademark) glass) and organic materials (for example, resins such as polycarbonate and polypropylene), and composite materials thereof. It may be. When the microfluidic chip 100 is used for an application involving fluorescence measurement, the substrate 10 is preferably formed of a material having a small spontaneous fluorescence. Examples of such a material having a small spontaneous fluorescence include polycarbonate and polypropylene. The substrate 10 is preferably made of a material that can withstand heating in PCR.

  Further, the material of the substrate 10 includes carbon black, graphite, titanium black, aniline black, Ru, Mn, Ni, Cr, Fe, Co or Cu oxide, Si, Ti, Ta, Zr or Cr carbide. A black substance such as can be blended. By blending such a black material into the material of the substrate 10, the spontaneous fluorescence of the resin or the like can be further suppressed. In addition, when the microfluidic chip 100 is used for an application (for example, real-time PCR or the like) in which a well 16 or the like described later is observed from the outside of the microfluidic chip 100, the material of the substrate 10 is made transparent as necessary. Can be. The material of the substrate 10 is preferably a material that hardly adsorbs nucleic acids and proteins and does not inhibit an enzyme reaction such as polymerase.

  In the case where the substrate 10 is formed of an inorganic material, it can be formed and processed by performing dry etching using a photolithography method. In addition, when the substrate 10 is formed using a resin as a main component, it can be molded and processed by a method such as mold molding, injection molding, or hot embossing.

1-1-2. Reservoir Region and Well Region As shown in FIG. 1, the substrate 10 is provided with a reservoir region 13 and a well region 14 in plan view. The reservoir region 13 and the well region 14 are provided adjacent to each other. The reservoir region 13 and the well region 14 may be provided adjacent to each other. A plurality of reservoir regions 13 and well regions 14 may be provided. The reservoir region 13 and the well region 14 are provided inside a fixing region 21 of the cover 20 described later in plan view. The positions where the reservoir region 13 and the well region 14 are provided on the substrate 10 are such that when an inertial force such as a centrifugal force is applied to the substrate 10, the inertial force acts in a direction from the reservoir region 13 toward the well region 14. However, there is no particular limitation as far as possible. The shape of the reservoir region 13 and the well region 14 viewed in plan is not particularly limited, and may be a rectangle, a circle, or the like.

1-1-3. Cover FIG. 3 is a plan view schematically showing the microfluidic chip 100 according to the first embodiment. FIG. 4 is a diagram schematically showing a cross section taken along line BB in FIG.

  In the following description, “laying” refers to a state where the cover 20 is laid on the first surface 11 of the substrate 10, and “adhesion” refers to laying on the first surface 11 of the substrate 10. Refers to the fixed state. That is, the term “laying” is used in the meaning including the case where the cover 20 and the first surface 11 of the substrate 10 are fixed (fixed) and the case where they are not fixed. Therefore, the state in which the cover 20 is laid and has the fixing region 21 includes a portion where it is easy to form a gap between the substrate 10 and the cover 20 and a portion where the substrate 10 and the cover 20 are difficult to separate. In this state, the portion where the latter substrate 10 and the cover 20 are difficult to separate is expressed as a fixed region 21 that is fixed.

  The cover 20 is laid on the first surface 11 side of the substrate 10. The cover 20 has a film or sheet shape. Although the thickness of the cover 20 is not specifically limited, For example, it can be 0.01 mm or more and 5 mm or less. The outer shape of the cover 20 viewed in a plane may or may not coincide with the outer shape of the substrate 10 viewed in a plane. In the example of FIG. 3, the outer shape of the cover 20 viewed in plan is the same as the outer shape of the substrate 10 viewed in plan. The cover 20 is in contact with the substrate 10 in a static state, but when an inertial force such as a centrifugal force is applied to the microfluidic chip 100, a gap is formed between the substrate 10 and the cover 20. It has a degree of flexibility or elasticity. In addition, the cover 20 closes an opening 16a of a well 16 (described later) provided in the substrate 10 in contact with the substrate 10, so that the well 16 can be an independent space (reaction vessel). When the cover 20 is pressed against the substrate 10, it is preferable that the cover 20 is not easily bent in the vicinity of the opening 16 a of the well 16 and has elasticity that does not cause a large change in the volume of the well 16. The degree of elasticity of the cover 20 is appropriately designed by selecting a material in consideration of the thickness of the cover 20, the size of the opening 16 a of the well 16, and the pressure when contacting the substrate 10. Can.

  Examples of the material of the cover 20 include those having elasticity that can be bent by an inertial force such as centrifugal force. For example, organic materials (resins such as polycarbonate and polypropylene and various rubbers), organic materials, A composite material of an inorganic material can be given. When the microfluidic chip 100 is used for an application involving fluorescence measurement, the cover 20 is desirably formed of a material having a small spontaneous fluorescence. Examples of such a material having a small spontaneous fluorescence include polycarbonate and polypropylene. The material of the cover 20 is preferably a material that hardly adsorbs nucleic acids and proteins and does not inhibit an enzyme reaction such as polymerase. In addition, the cover 20 is preferably made of a material that can withstand heating in PCR.

  The material of the cover 20 is carbon black, graphite, titanium black, aniline black, Ru, Mn, Ni, Cr, Fe, Co or Cu oxide, Si, Ti, Ta, Zr or Cr carbide. A black substance such as can be blended. By blending such a black substance into the material of the cover 20, the spontaneous fluorescence of the resin or the like can be further suppressed. Further, when the microfluidic chip 100 is used for the purpose of observing the well 16 described later from the outside of the microfluidic chip 100, the material of the cover 20 can be made transparent as necessary. The cover 20 can be molded and processed by a method such as film molding, sheet molding, injection molding, or press molding.

  The cover 20 is laid so that one surface 20 a faces the substrate 10. That is, the surface 20 a is provided so as to face the first surface 11 of the substrate 10. The surface 20a of the cover 20 may have adhesiveness. When the surface 20a has adhesiveness, it is preferable that the surface 20a and the first surface 11 have such an adhesive force that can be peeled off when an inertial force such as centrifugal force is applied to the microfluidic chip 100. However, this is not limited to this after the application of an inertial force such as centrifugal force to the microfluidic chip 100, and the adhesive force is sufficient to adhere (fix) the surface 20a and the first surface 11 so that they are not easily separated. May be.

  The surface 20a of the cover 20 may not have an adhesive force. In such a case, in order to fix the surface 20a to the first surface 11, an adhesive can be used or heat fusion can be performed. Further, the surface 20a may have a property of exhibiting an adhesive force by pressurization without exhibiting an adhesive force in a state where the cover 20 is not pressed against the substrate 10. An example of such a surface 20a is a porous surface. In such a method, since only pressurization is performed, no heat is generated when fixing, the temperature increase of the microfluidic chip 100 can be suppressed, and the influence of heat on the liquid or the like can be suppressed. can do. As a specific example of the cover 20 having such a surface 20a, trade name: LightCycler 480 Sealing Foil, model name: 04 729 757 001, manufactured by Roche Diagnostics, trade name: polyolefin microplate sealing tape, model name : 9793-3M, trade name: amplification tape 96, model name: 232702, manufactured by Nunc, etc.

  Furthermore, the surface 20a may have a potential adhesion. That is, the surface 20a does not have an adhesive force before an inertial force such as a centrifugal force is applied to the microfluidic chip 100, and an energy ray (for example, an ultraviolet ray) at a desired time after the inertial force is applied. , An electron beam, etc.) may be used to exert adhesive force.

  Further, as a method for fixing the surface 20a and the first surface 11 of the substrate 10, an ultrasonic welding method may be used. For example, a jig having a shape corresponding to the shape of the region to be fixed may be pressed against the portion of the cover 20 to be fixed to the substrate 10 and ultrasonically vibrated to weld (fix) the cover 20 and the substrate 10. Good. In this way, since the substrate 10 and the cover 20 are welded (fixed) by ultrasonic irradiation, heating of the sample can be suppressed, and damage to the sample can be reduced. For example, a decrease in the activity of the reagent contained in the well 16 can be suppressed. Further, when the fixing region 21 is formed by the ultrasonic welding method, the bonding force between the substrate 10 and the cover 20 by the welding is relatively strong as compared with, for example, a method by pressurization. Can be reliably prevented.

  The cover 20 has a fixing region 21 fixed to the substrate 10. The fixing region 21 is disposed so as to surround the reservoir region 13 and the well region 14 of the substrate 10 in plan view. That is, the reservoir region 13 and the well region 14 are disposed inside the fixing region 21 when viewed in plan. The fixing region 21 is a region in which the laid cover 20 and the substrate 10 are not easily separated even when an inertial force such as a centrifugal force is applied to the microfluidic chip 100. When an inertial force such as centrifugal force is applied to the microfluidic chip 100, the surface 20a and the first surface 11 can be separated from the region other than the fixing region 21 of the laid cover 20. In the fixing region 21, for example, the cover 20 and the substrate 10 may be welded or bonded with an adhesive, an adhesive, or the like in accordance with the property of the surface 20 a of the cover 20.

  As one of the functions of the fixing region 21, when an inertial force such as a centrifugal force is applied to the microfluidic chip 100 to form a gap between the cover 20 and the substrate 10, the gap is formed inside. For example, a bag-like structure may be formed. This allows the reservoir region 13 and the well region 14 to communicate with each other on the first surface 11 side of the substrate 10 between the substrate 10 and the cover 20, and allows the liquid to flow between both regions.

  As already described, the inertial force is applied to the microfluidic chip 100 from the reservoir region 13 side toward the well region 14 side. Therefore, the fixing region 21 is continuously provided at least at a position surrounding the well region 14. Therefore, when the liquid is introduced into the gap between the cover 20 and the substrate 10, the liquid is prevented from leaking from the well region 14 side to the outside of the fixing region 21 due to inertial force. Further, the fixing region 21 can be continuous in an annular shape so as to surround both the reservoir region 13 and the well region 14 of the substrate 10 in a plan view. In this way, it is possible to prevent the liquid from leaking outside the fixing region 21 when the liquid is introduced into the gap between the cover 20 and the substrate 10. Note that the fixing region 21 can have a non-continuous portion on the reservoir region 13 side. When such a portion is provided, the discontinuous portion can function as a vent hole when an internal pressure is generated in the gap between the cover 20 and the substrate 10. Further, when the fixing region 21 is continuous in an annular shape so as to surround the reservoir region 13 and the well region 14 of the substrate 10 in plan view, a hole (not shown) is formed in the cover 20 at an appropriate position on the reservoir region 13 side. By providing this, the hole can function as a vent hole when an internal pressure is generated in the gap between the cover 20 and the substrate 10. The vent hole is preferably provided at a position and a size at which leakage of the liquid introduced into the reservoir 15 and the well 16 does not occur.

  In order to form the fixing region 21, for example, the fixing region 21 can be formed by applying an adhesive in the shape of the fixing region 21 on the first surface 11 of the substrate 10 and laying the cover 20. . In addition, for example, when the surface 20a of the cover 20 does not exert an adhesive force in a state where the cover 20 is not pressed against the substrate 10, but has a property of exerting an adhesive force by applying pressure, The fixing region 21 can be formed by preparing a jig or the like corresponding to the shape and applying pressure to the cover 20 against the substrate 10 with the jig. Further, for example, the fixing region 21 can be formed by ultrasonic welding using a jig having a shape corresponding to the shape of the fixing region 21.

1-1-4. Reservoir A reservoir 15 having an opening in the first surface 11 is formed in the reservoir region 13 of the substrate 10. A plurality of reservoirs 15 may be formed in the reservoir region 13. The shape of the reservoir 15 as viewed in plan can be a circle, a rectangle, or the like. The reservoir 15 may be formed as a through-hole through which a test liquid (liquid containing a sample to be measured) can be circulated in both directions on the first surface 11 side and the second surface 12 side. In this case, the test liquid can be introduced into the reservoir 15 from the second surface 12 side.

1-1-5. In the well region 14 of the well substrate 10, a well 16 having an opening 16a on the first surface 11 side of the substrate 10 is formed (see FIGS. 1 to 4). FIG. 5 is a schematic diagram showing an enlarged cross section near the well 16 of the microfluidic chip 100.

  A plurality of wells 16 are formed in the well region 14. The well 16 has a container shape having an opening 16 a on the first surface 11 side of the substrate 10. The well 16 can hold a test solution therein. In addition, the test solution can be reacted in the well 16. That is, the well 16 functions as a reaction container.

  The shape of the well 16 is not particularly limited as long as it is a container shape, and can take various forms. For example, the shape of the well 16 may be any of a cylinder, a prism, a truncated cone and a truncated pyramid, a shape in which they are inclined, and a combination of these. In the present embodiment, a case will be described in which the well 16 has a cylindrical shape (a circular shape in a plan view and a rectangular shape in a sectional view) as shown in FIGS.

  The opening 16 a of the well 16 can be blocked by the cover 20. Thereby, the well 16 can be an independent sealed container. By thus sealing the wells 16 independently, mixing with the contents of other wells 16 and contamination can be suppressed. The well 16 can be an independent sealed container when the cover 20 is laid and fixed around the opening 16 a of the well 16.

  In the well 16, reagents for use in amplification of the target nucleic acid contained in the specimen and determination of whether or not a predetermined amount of the target nucleic acid is expressed are arranged in advance. The state of the arranged reagent is preferably solid or liquid. In the example illustrated in FIG. 5, among the plurality of wells 16 provided in the microfluidic chip 100, the reagent 30 is disposed in the well 160, the reagent 31 is disposed in the well 161, and the reagent 32 is disposed in the well 162.

  In the case where a reagent 30, 31, 32 having a high stability with respect to drying or the like is used, the reagent 30, 31, 32 may be dried in the well 16, and for example, applied to the inner wall surface of the well 16 as shown in FIG. And may be placed in a dry state. As a method of applying the reagents 30, 31, and 32 to the inner wall surface of the well 16 as described above, for example, a method of applying the reagents 30, 31, and 32 by a liquid ejecting head or the like used for ink jet printing is exemplified. .

  The position where the reagent 30, 31, 32 is arranged in the well 16 is not particularly limited, but the reagent 30, 31, 32 flows out of the well 16 together with the introduced liquid as the position is farther from the opening 16a. It is more preferable in that the possibility of being reduced.

  Reagents 30, 31, and 32 contained in the well 16 (reaction vessel) are nucleic acids that can be amplified with a primer common to the target nucleic acid and have a different sequence from the target nucleic acid. And a primer common to the target nucleic acid and the internal standard nucleic acid, and binding to a part of the amplification product of the target nucleic acid and the internal standard nucleic acid, binding to the amplification product of the target nucleic acid and binding to the amplification product of the internal standard nucleic acid. And a fluorescent probe that exhibits different fluorescence changes.

  In addition, in the well 16 (reaction vessel), the types of primers and fluorescent probes contained in the first reaction vessel and the second reaction vessel are different from each other. For example, when the reagent 30 and the reagent 31 shown in FIG. 5 are different from each other, the well 160 and the well 161 containing these reagents correspond to the first reaction container and the second reaction container, respectively. This explanation also applies to any other two wells (reaction containers). Thereby, the expression of a plurality of types of genes can be determined with one microfluidic chip 100.

  In addition, when the types of primers and fluorescent probes are different for each well 16 (reaction vessel), different genes are amplified for each well 16 (reaction vessel). For example, other genes such as cross-contamination The influence can be suppressed.

  Further, when the microfluidic chip 100 includes a plurality of wells 16 (reaction containers) containing the same type of primer and fluorescent probe, the amounts of internal standard nucleic acids contained in the plurality of wells 16 (reaction containers) are different from each other. Also good. Thereby, a single microfluidic chip 100 can easily compare a single type of gene with a multi-condition gene expression state.

  Further, the amount of the internal standard nucleic acid contained in the reagents 30, 31, and 32 is the case where a reaction solution of a reference sample, which is a sample serving as a reference for determining whether or not a predetermined amount of the target nucleic acid is expressed, is introduced into the reaction vessel 16. In addition, the reference amount is the same as the amount of the target nucleic acid contained in the reaction solution introduced into the reaction vessel 16. For example, the copy number of the internal standard nucleic acid may be the same as the copy number of the target nucleic acid introduced into the reaction container 16 when the reaction solution of the reference specimen is introduced into the reaction container 16. For example, the molar concentration of the internal standard nucleic acid per volume of the reaction vessel 16 may be the same as the molar concentration of the target nucleic acid contained in the reference specimen. The reaction liquid of the reference specimen may be a liquid containing cDNA obtained by reverse transcription of mRNA contained in the reference specimen, for example.

  Here, the “same” range is not limited to being completely identical, but may be within a range including an error that can satisfy the required determination accuracy. For example, it may be within a range that can be quantified by one calibration curve determined by the amount of the internal standard nucleic acid in the reaction container 16.

  The reference sample is a sample that is selected according to the purpose of determining whether or not a predetermined amount of gene has been expressed. For example, the reference sample is a sample that serves as a reference for determining whether or not a predetermined amount of gene has been expressed. Since the measured value of the target nucleic acid based on the reference sample directly expresses the difference from the state to be referred to, it has a feature that the difference from the reference amount can be known as an absolute amount. For this reason, it is easy to compare with the result using a DNA chip (DNA microarray).

  Such a reference sample may be, for example, at least one of a sample representative of a sample collected from a healthy individual and a sample representative of a sample collected from a sick individual. A sample representative of a sample collected from a healthy individual may be a sample collected from one healthy individual, or a virtual sample derived from a sample collected from a plurality of healthy individuals by a statistical method It may be. Similarly, a sample representative of a sample collected from a diseased individual may be a sample collected from one diseased individual, or a virtual derived from a sample collected from a plurality of diseased individuals by a statistical method. May be a typical specimen. The “individual” described above may be a human or an animal or plant other than a human.

  By selecting such a reference sample and setting the amount of the internal standard nucleic acid contained in the reagents 30, 31, and 32, that is, when the reference amount is a sample collected from a healthy individual as a reference sample. By using the same amount as the representative amount and at least one of the representative amounts collected from a diseased individual, the expression level of the target nucleic acid in the sample to be measured is the same as the sample and disease collected from a healthy individual. It is possible to determine with high sensitivity and high accuracy whether or not the expression level of the target nucleic acid in at least one of the samples collected from the individual is the same.

  As described above, according to the microfluidic chip 100 according to the first embodiment, among the reaction containers, the types of the primers and the fluorescent probes included in the first reaction container and the second reaction container are different from each other. It is possible to realize a microfluidic chip that can easily determine whether or not a predetermined amount of types of genes are expressed.

  In addition, according to the microfluidic chip 100 according to the first embodiment, the amount of the internal standard nucleic acid is determined by using a reaction solution of a reference sample that is a sample serving as a reference for determining whether or not a predetermined amount of the target nucleic acid has been expressed. Is the same as the amount of target nucleic acid introduced into the reaction container, the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in the reference reference sample. It is possible to realize a microfluidic chip capable of determining whether or not with high sensitivity and high accuracy.

  In the principle of PCR, a target nucleic acid is amplified using two primer DNAs (forward primer and reverse primer). Therefore, by applying the principle of PCR to the microfluidic chip 100 according to the first embodiment, the specificity is higher than that of a DNA chip (DNA microarray) using one kind of probe DNA, and the measurement reliability is high. It becomes. In addition, since nucleic acid amplification is performed by PCR, it is possible to quantify even low expression genes that were impossible with a DNA chip (DNA microarray).

  As a method for quantifying the expression level of a gene, there is a competitive PCR method in which a known amount of an internal standard nucleic acid that can be amplified with a common primer is added and co-amplified. The competitive PCR method is a method for quantifying the target nucleic acid by comparing the amount of the amplification product derived from the target nucleic acid with the amount of the amplification product derived from the internal standard nucleic acid.

  If the principle of the competitive PCR method is used, the target nucleic acid and the internal standard nucleic acid can be regarded as having the same property except for the fluorescent reaction by the fluorescent probe, and therefore the amplification rate of both nucleic acids can be regarded as equal. Therefore, the ratio of the amplification product when the target nucleic acid and the internal standard nucleic acid are co-amplified in one reaction vessel reflects the ratio of both before amplification. Therefore, when the principle of the competitive PCR method is used using the microfluidic chip 100 according to the first embodiment, a quantifiable range (the amount of the target nucleic acid and the amount of the internal standard nucleic acid can be obtained by using a known amount of the internal standard nucleic acid. Is close to the range; the range that can be quantified with a single calibration curve), and the quantification accuracy is high, and absolute quantification is possible without requiring complicated work such as drawing a plurality of calibration curves. Thereby, the expression level of a gene can be absolutely quantified at low cost.

  Further, the reaction using the microfluidic chip 100 according to the first embodiment is not a reaction on a solid surface like a DNA chip (DNA microarray) but a reaction in a homogeneous liquid in the well 16. The reaction efficiency and accuracy will be high. Furthermore, since one type of amplification reaction is performed in one well 16, the accuracy of quantification due to cross-hybridization does not occur.

1-2. Configuration of Microfluidic Chip According to Second Embodiment FIG. 6A is a plan view schematically showing a microfluidic chip 100a according to the second embodiment. Further, the state in which the cross section near the well 16 of the microfluidic chip 100a is enlarged is the same as FIG. Compared to the microfluidic chip 100 according to the first embodiment, the microfluidic chip 100a according to the second embodiment includes an internal standard nucleic acid contained in the reagents 30, 31, and 32 disposed in the well 16 (reaction vessel). The reference sample that is the basis for the amount of the sample is different. Accordingly, the same components as those of the microfluidic chip 100 according to the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  In the microfluidic chip 100a according to the second embodiment, as at least a part of the plurality of reaction vessels, the reference amount is a well 16 (first number) representing a case where the reference sample is a sample collected from a healthy individual. A one-condition reaction container) and a well 16 (second-condition reaction container) whose reference amount is a representative amount when the reference sample is a sample collected from a diseased individual.

  In the example shown in FIG. 6A, the substrate 10 of the microfluidic chip 100a is provided with a health region 41 and a disease region 42 in plan view. The well 16 arranged in the health region 41 is a well 16 (reaction container of the first condition) whose reference amount is a representative amount when the reference sample is a sample collected from a healthy individual, and is sick. The well 16 arranged in the region 42 is the well 16 (reaction container of the second condition) whose reference amount is a representative amount when the reference sample is a sample collected from a sick individual.

  When the disease progresses and the gene expression level changes from the expression level when the gene is healthy, the expression level is significantly different from the normal level, so whether the level of expression is close to a healthy or diseased state. There is a possibility that the determination accuracy in the case of determining using the principle of the competitive PCR method is lowered. Therefore, the determination accuracy can be maintained by newly setting the amount of the internal standard nucleic acid to a value representative of the disease state based on the database in which the expression state of the gene appearing in the disease state is examined.

  In addition, from the viewpoint of improving the accuracy of determination as to whether the expression level is close to a healthy state or a diseased state, a sample representative of each state sample serving as a reference sample is a sample collected from one individual It is preferable to use a virtual specimen derived from an individual collected from a plurality of specimens by a statistical method.

  As described above, according to the microfluidic chip 100a according to the second embodiment, the well 16 (reaction under the first condition) in which the reference amount is a representative amount when the reference sample is a sample collected from a healthy individual. Container) and a well 16 (second reaction container) which is a quantity representative of the case where the reference sample is a sample collected from a sick individual. Highly sensitive and accurate whether the target nucleic acid expression level in the sample to be measured is the same as the target nucleic acid expression level in the sample collected from a healthy individual or a sample collected from a sick individual. Can be determined.

1-3. Configuration of Microfluidic Chip According to Third Embodiment FIG. 6B is a plan view schematically showing a microfluidic chip 100b according to the third embodiment. Further, the state in which the cross section near the well 16 of the microfluidic chip 100a is enlarged is the same as FIG. Compared to the microfluidic chip 100 according to the first embodiment, the microfluidic chip 100b according to the third embodiment includes an internal standard nucleic acid contained in the reagents 30, 31, and 32 disposed in the well 16 (reaction vessel). The reference sample that is the basis for the amount of the sample is different. Accordingly, the same components as those of the microfluidic chip 100 according to the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  In the microfluidic chip 100b according to the third embodiment, as at least a part of the plurality of reaction containers, the reference amount is an amount representative of a case where the reference sample is a sample collected from an individual in the first advanced state of disease. A well 16 (reaction container of the first condition) and a well 16 (reaction of the second condition) whose reference amount is a representative amount when the reference sample is a sample collected from an individual in the second advanced state of disease. Container).

  In the example shown in FIG. 6B, the substrate 10 of the microfluidic chip 100a is provided with a first progress state region 51 and a second progress state region 52 in plan view. The well 16 arranged in the first progress state region 51 is a well 16 (first condition) in which the reference amount is a representative amount when the reference sample is a sample collected from a diseased individual in the first progress state. The well 16 disposed in the second progress state region 52 has a reference amount that represents a case where the reference sample is a sample collected from an individual in the second progress state of the disease. A certain well 16 (second reaction container).

  When various expression states are predicted for a gene depending on the progress and type of disease, the quantitative accuracy of the target nucleic acid can be maintained by setting a plurality of amounts of the internal standard nucleic acid.

  Note that two arbitrarily selected progress states among the progress states of a plurality of diseases correspond to the first progress state of the disease and the second progress state of the disease, respectively. The specimen representing the specimen in the first advanced state of the disease may be a specimen collected from one individual in the first advanced stage of the disease, or collected from a plurality of individuals in the first advanced state of the disease It may be a virtual sample derived from a sample by a statistical method. Similarly, the specimen representing the specimen in the second advanced state of disease may be a specimen collected from one individual in the second advanced state of disease, or collected from a plurality of individuals in the second advanced state of disease. It is also possible to use a virtual specimen derived from the specimen by a statistical method. In addition, from the viewpoint of improving the determination accuracy as to which progression state of the disease is close to the expression level, the specimen representing the specimen in each state was collected from a plurality of specimens rather than the specimen collected from one individual. It is preferable to use a virtual specimen derived from an individual by a statistical method.

  As described above, according to the microfluidic chip 100b according to the third embodiment, the well 16 (the reference amount) is a representative amount when the reference sample is a sample collected from a diseased first individual. A first reaction container) and a well 16 (second reaction container) in which the reference amount is a representative amount when the reference sample is a sample collected from an individual in the second advanced state of illness. Therefore, in one microfluidic chip, the expression level of the target nucleic acid in the sample to be measured is a sample collected from an individual in a first diseased state and a sample collected from an individual in a second diseased state. It is possible to determine with high sensitivity and high accuracy whether the expression level is the same as the target nucleic acid expression level.

1-4. Modified Example In the microfluidic chip 100 according to the first embodiment, the microfluidic chip 100a according to the second embodiment, and the microfluidic chip 100b according to the third embodiment, the well 16 is provided for each well 16 (reaction vessel). The example in which at least one of the types of primers and fluorescent probes contained in the (reaction vessel) and the amount of the internal standard nucleic acid is different has been described, but the well 16 (reaction vessel) has a plurality of wells 16 (reaction vessel). And a primer contained in the well 16 (reaction vessel) belonging to the first reaction vessel group and the well 16 (reaction vessel) belonging to the second reaction vessel group, among the reaction vessel groups, At least one of the type of fluorescent probe and the amount of the internal standard nucleic acid may be different from each other. In other words, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group, and the amount of the internal standard nucleic acid may be the same.

  FIGS. 7A to 7C are plan views schematically showing microfluidic chips 101, 101a, 101b according to modifications. A microfluidic chip 101 shown in FIG. 7A is a modification of the microfluidic chip 100 according to the first embodiment, and a microfluidic chip 101b shown in FIG. 7B is a microfluidic chip 100a according to the second embodiment. A microfluidic chip 101b shown in FIG. 7C is a modified example of the microfluidic chip 100b according to the third embodiment.

  In the example shown in FIG. 7A, the well 16 (reaction container) constitutes reaction container groups 61, 62, 63, 64 and 65 each including ten wells 16 (reaction containers). For example, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group 61 and the well 16 (reaction vessel) belonging to the reaction vessel group 62 shown in FIG. When at least one of the amounts of the internal standard nucleic acid is different from each other, the reaction container group 61 and the reaction container group 62 correspond to the first reaction container group and the second reaction container group, respectively. This explanation also applies to any two other reaction container groups.

  In the example shown in FIG. 7A, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group are different for each of the reaction vessel groups 61, 62, 63, 64, and 65. In other words, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group, and the amount of the internal standard nucleic acid are the same.

  In the example shown in FIG. 7B, the well 16 (reaction vessel) includes reaction vessel groups 611, 612, 613, 614, 615, 621, 622, 623, 624 each including five wells 16 (reaction vessels). And 625. Among these, the reaction container groups 611, 612, 613, 614 and 615 are arranged in the health area 41, and the reaction container groups 621, 622, 623, 624 and 625 are arranged in the disease area 42. That is, the internal standard nucleic acid contained in the well 16 (reaction vessel) belonging to the reaction vessel groups 611, 612, 613, 614 and 615 and the well 16 (reaction vessel) belonging to the reaction vessel groups 621, 622, 623, 624 and 625 The amounts are different from each other. In the example shown in FIG. 7B, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group are different for each of the reaction vessel groups 611, 612, 613, 614 and 615. Furthermore, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group are different for each of the reaction vessel groups 621, 622, 623, 624 and 625. Also in the example shown in FIG. 7B, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group, and the amount of the internal standard nucleic acid are the same.

  In the example shown in FIG. 7C, the well 16 (reaction vessel) includes reaction vessel groups 631, 632, 633, 634, 635, 641, 642, 643, 644 each including five wells 16 (reaction vessels). And 645. Among these, the reaction container groups 631, 632, 633, 634 and 635 are arranged in the first progress state area 51, and the reaction container groups 641, 642, 643, 644 and 645 are arranged in the second progress state area 52. ing. That is, the internal standard nucleic acids contained in the well 16 (reaction vessel) belonging to the reaction vessel groups 631, 632, 633, 634 and 635 and the well 16 (reaction vessel) belonging to the reaction vessel groups 641, 642, 643, 644 and 645 The amounts are different from each other. In the example shown in FIG. 7C, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group are different for each of the reaction vessel groups 631, 632, 633, 634 and 635. Furthermore, the types of primers and fluorescent probes contained in the well 16 (reaction vessel) belonging to the reaction vessel group are different for each of the reaction vessel groups 641, 642, 643, 644 and 645. Also in the example shown in FIG. 7C, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group, and the amount of the internal standard nucleic acid are the same.

  Thus, by including a plurality of wells 16 (reaction containers) of the same condition for each reaction container group, errors for each well 16 (reaction container) are not easily reflected in the determination result, and the reliability of the determination is improved. . Further, even when bubbles or foreign substances are mixed into one well 16 (reaction container) by mistake, the determination can be made using the results of other wells 16 (reaction containers) belonging to the same reaction container group.

  Moreover, since at least one of the types of primers and fluorescent probes contained in the well 16 and the amount of the internal standard nucleic acid is different for each reaction container group, a plurality of types of genes or A microfluidic chip that can determine with high sensitivity and high accuracy whether the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in the reference reference sample for multiple conditions realizable.

1-5. Next, a method of filling the test solution into the well 16 (reaction vessel) will be described using the microfluidic chip 100 according to the first embodiment as an example. Note that the microfluidic chip 100a according to the second embodiment, the microfluidic chip 100b according to the third embodiment, and the microfluidic chips 101, 101a, and 101b according to the modified examples are also supplied to the well 16 by the same method. (Reaction vessel) can be filled.

  In the following example, it is assumed that the surface 20a of the cover 20 has a property of exerting an adhesive force by pressurization without exhibiting an adhesive force in a state where the cover 20 is not pressed against the substrate 10. . In the following example, the fixing region 21 of the cover 20 is assumed to be continuous in an annular shape so as to surround both the reservoir region 13 and the well region 14 of the substrate 10 in plan view. In the following example, the cover 20 is formed of a transparent material. In this example, the well 16 has a cylindrical shape, and the bottom surface has a diameter of about 1 mm and a depth of about 0.5 mm. Further, in this example, the external shape of the substrate 10 and the cover 20 in a plan view is a rectangle having a long side of about 7.5 cm and a short side of about 2.5 cm.

  First, a test solution 80 containing a target nucleic acid is prepared. Examples of the target nucleic acid to be measured in the test solution 80 include cDNA reverse-transcribed from RNA extracted from a biological sample such as blood, urine, saliva, and cerebrospinal fluid. More specifically, for example, cDNA reversely transcribed from mRNA extracted from the cells contained in the biological sample described above can be mentioned. The amount of the test solution 80 is appropriately determined according to the entire volume of the well 16, and is preferably equal to or larger than the total volume of the plurality of wells 16, for example. More preferably, the sample volume 80 is larger than the total volume of the plurality of wells 16 in that the test solution 80 can be filled.

  Next, the test solution 80 is introduced into the reservoir 15 of the microfluidic chip 100. For example, the test solution 80 may be introduced before the cover 20 is attached. In the microfluidic chip 100 in which holes are formed in the region corresponding to the reservoir 15 of the cover 20, the cover 20 is attached. Later, the test solution 80 may be introduced from the hole. Furthermore, the test solution 80 may be introduced by removing the cover 20 from at least a part of the region corresponding to the reservoir 15. At this time, the test solution 80 is not introduced into the well 16 of the microfluidic chip 100.

  Next, an inertial force is applied to the microfluidic chip 100 from the reservoir region 13 side toward the well region 14 side. FIG. 8 is a diagram schematically illustrating a state in which an inertial force is applied in a state where the test solution 80 is introduced into the reservoir 15 of the microfluidic chip 100.

  In this embodiment, the inertia force is generated by a centrifuge. The rotation axis R of the centrifuge is disposed on the side opposite to the well region 14 when viewed from the reservoir region 13 of the microfluidic chip 100. When an inertial force (centrifugal force) is generated in the microfluidic chip 100 by operating the centrifuge, the test solution 80 in the reservoir 15 moves away from the rotation axis R as shown in FIG. Acceleration G (arrow in the figure) is received in the direction toward 14. In a state where acceleration is applied, a gap is generated between the substrate 10 and the cover 20, and the test solution 80 is transferred in the gap toward the well region 14. The size (thickness) of the gap at this time is not particularly limited, but FIG. 8 shows an example in which the size of the gap is very small. Then, the test solution 80 is introduced into the well 16 from the opening 16a of the well 16 formed in the well region 14 of the substrate 10 by being replaced with air in the well 16 by inertia. The test solution 80 introduced into the well 16 is mixed with the reagent 30 or the like previously arranged in the well 16.

  In the example shown in FIG. 8, the plane of the microfluidic chip 100 (for example, the first surface 11 of the substrate 10) is arranged and rotated along the direction perpendicular to the rotation axis R of the centrifuge. However, the arrangement of the microfluidic chip 100 with respect to the rotation axis R of the centrifuge is arbitrary as long as the test solution 80 inside the container 200 can move from the reservoir 15 to the well 16 by centrifugal force. is there. Such an arrangement can be appropriately set depending on the shape of the reservoir 15 and the arrangement of the wells 16 of the substrate 10.

  Next, the centrifuge is stopped and the application of inertia force is stopped. In this state, the test solution 80 is filled in the well 16. Further, for example, when the volume of the test solution 80 is larger than the total volume of the well 16, the test solution 80 exists in the well 16 and in the gap between the substrate 10 and the cover 20. ing. In any case, by pressing the cover 20 against the substrate 10 from the outside of the cover 20 with a jig such as a roller, a squeegee or a blade, the cover 20 is brought into close contact with the microfluidic chip 100 and the well 16 is sealed. The test solution 80 can be sealed in the well 16. That is, in a state where the test solution 80 is not present in the gap between the substrate 10 and the cover 20, the well 16 is sealed by pressing the cover 20 against the substrate 10, and the test solution 80 is sealed with the substrate 10 and the cover 20. In the state in which the well 16 exists, the well 16 can be sealed while pushing the excess test solution 80 back to the reservoir region 13 side. Thereby, all the surfaces which the board | substrate 10 and the cover 20 contact can be adhere | attached. In addition, the test solution 80 can be sealed in the well 16 with a volumetric accuracy that does not affect the determination result of whether or not the target nucleic acid is expressed in a predetermined amount.

  FIG. 9 is a diagram schematically showing how the well 16 of the microfluidic chip 100 is sealed by the roller 90. In the example shown in FIG. 9, the roller 90 moves the surface of the cover 20 from the far side to the near side (in the direction of the white arrow in FIG. 9) at a relative position with respect to the microfluidic chip 100. Thus, the state in which the well 16 is sealed while returning the excessive test solution 80 existing in the gap between the substrate 10 and the cover 20 from the reservoir 15 to the container 200 is shown.

  In this way, an accurate volume of the test solution 80 can be introduced into the well 16 of the microfluidic chip 100. The same applies to the case where a plurality of wells 16 are formed in the microfluidic chip 100, and the plurality of wells 16 become independent sealed spaces as a result of the above operation, and a desired amount of the test solution 80 is precisely measured. Can be dispensed. As a result, even a minute amount of the test solution 80, which is difficult with a pipette, can be accurately and easily dispensed into the well 16. Thereby, the time and cost spent for dispensing work can be suppressed.

  The method for analyzing the target nucleic acid after introducing the test solution 80 into the well 16 will be described in the section “4. Nucleic acid analysis method”.

2. Microfluidic Chip Set According to Fourth Embodiment FIG. 10 is a plan view schematically showing a microfluidic chip set 200 according to the fourth embodiment. A microfluidic chip set 200 according to the fourth embodiment includes a microfluidic chip 100c and a microfluidic chip 100d. A state in which the cross section of the microfluidic chip 100c and the microfluidic chip 100d in the vicinity of the well 16 is enlarged is the same as FIG. Compared to the microfluidic chip 100 according to the first embodiment, the microfluidic chip 100c and the microfluidic chip 100d are internal standard nucleic acids contained in the reagents 30, 31, and 32 disposed in the well 16 (reaction vessel). The reference sample that is the basis for the quantity is different. Accordingly, the same components as those of the microfluidic chip 100 according to the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  In the microfluidic chip 100c, as at least a part of the plurality of reaction containers, the reference amount is a well 16 (first amount) that represents a case where the reference sample is a sample collected from a diseased first progressing individual. It is a microfluidic chip including a reaction container under conditions. As shown in FIG. 10, the substrate 10 of the microfluidic chip 100c is provided with a first progress state region 51 in plan view. The well 16 arranged in the first progress state region 51 is a well 16 (first condition) in which the reference amount is a representative amount when the reference sample is a sample collected from a diseased individual in the first progress state. Reaction vessel).

  In the microfluidic chip 100d, as at least a part of the plurality of reaction vessels, the reference amount is a well 16 (second well) representing a case where the reference sample is a sample collected from a diseased second progressive individual. It is a microfluidic chip including a reaction container under conditions. As shown in FIG. 10, the substrate 10 of the microfluidic chip 100d is provided with a second progress state region 52 in plan view. The well 16 arranged in the second progress state region 52 represents a case where the reference amount is a sample collected from an individual in the second progress state of the disease, as at least a part of the plurality of reaction containers. It is the well 16 (the reaction container of the second condition) that is the amount to be.

  As described above, in the microfluidic chip set 200 according to the fourth embodiment, when at least a part of the plurality of reaction containers, the reference amount is the sample collected from the individual in the first advanced state of the disease as the reference sample. And a microfluidic chip 100c including a well 16 (reaction vessel under the first condition), which is a representative amount, and at least part of the plurality of reaction vessels, the reference amount is the reference specimen, the individual in the second advanced state of the disease A microfluidic chip 100d including a well 16 (second reaction container) that is representative of a sample collected from a microfluidic sample, thereby making it possible to easily select a reference amount as a criterion for determination. A chipset can be realized.

  In the microfluidic chip 100c and the microfluidic chip 100d, the test liquid can be filled by the method described in the section “1-4. Therefore, even a minute amount of test solution that is difficult with a pipette can be accurately and easily dispensed into the well 16.

  A method for analyzing the target nucleic acid after introducing the test solution into the well 16 will be described in “4. Nucleic Acid Analysis Method”.

3. Nucleic acid analysis kit 3-1. Nucleic Acid Analysis Kit According to Fifth Embodiment FIG. 11 is a diagram schematically showing a nucleic acid analysis kit 300 according to the fifth embodiment. A nucleic acid analysis kit 300 according to the fifth embodiment is a nucleic acid analysis kit used for amplification of a target nucleic acid contained in a specimen and determination of whether or not a predetermined amount of the target nucleic acid has been expressed. An internal standard reagent 110 that is a liquid containing a known amount of an internal standard nucleic acid, which is a nucleic acid that can be amplified with a primer common to the target nucleic acid and has a different sequence from the target nucleic acid. In the example shown in FIG. 11, the internal standard reagent 110 is a liquid accommodated in a lidded container 150. The microfluidic chip 100e includes a plurality of wells 16 (reaction containers).

  FIG. 11 is a plan view schematically showing the microfluidic chip 100e. Further, an enlarged view of the cross section in the vicinity of the well 16 of the microfluidic chip 100e is the same as in FIG. 5, and will be described with reference again. The microfluidic chip 100e is different from the microfluidic chip 100 according to the first embodiment in the reagents 30, 31, and 32 disposed in the well 16 (reaction vessel). Accordingly, the same components as those of the microfluidic chip 100 according to the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted.

  Reagents 30, 31, and 32 arranged in the well 16 of the microfluidic chip 100e bind to a primer common to the target nucleic acid and the internal standard nucleic acid, and a part of the amplification product of the target nucleic acid and the internal standard nucleic acid. And a fluorescent probe exhibiting a different fluorescence change when bound to the amplified product of the internal standard nucleic acid.

  In addition, in the well 16 (reaction vessel), the types of primers and fluorescent probes contained in the first reaction vessel and the second reaction vessel are different from each other. For example, when the reagent 30 and the reagent 31 shown in FIG. 5 are different from each other, the well 160 and the well 161 containing these reagents correspond to the first reaction container and the second reaction container, respectively. This explanation also applies to any other two wells. Thereby, it is possible to determine the expression of a plurality of types of genes with one microfluidic chip 100e.

  In addition, when the types of primers and fluorescent probes are different for each well 16 (reaction vessel), different genes are amplified for each well 16 (reaction vessel). For example, other genes such as cross-contamination The influence can be suppressed.

  In addition, the molar concentration of the internal standard nucleic acid contained in the internal standard reagent 110 is the reference molar that is the molar concentration of the target nucleic acid contained in the reference sample that is a reference for determining whether or not the target nucleic acid is expressed in a predetermined amount. Same as concentration.

  Here, the “same” range is not limited to being completely identical, but may be within a range including an error that can satisfy the required determination accuracy. Further, the user himself / herself can dilute the internal standard reagent 110 at a predetermined ratio or mix the internal standard reagent 110 and the test solution at a predetermined ratio in accordance with instructions in the manual or the like. When the standard reagent 110 is introduced into the well 16 (reaction vessel), the molar concentration of the internal standard nucleic acid may be adjusted to be the same as the reference molar concentration. Thereby, the reference molar concentration can be easily selected. In addition, the degree of freedom in selecting the reference molar concentration is increased.

  The reference sample is a sample that is selected according to the purpose of determining whether or not a predetermined amount of gene has been expressed. For example, the reference sample is a sample that serves as a reference for determining whether or not a predetermined amount of gene has been expressed. The measured value of the target nucleic acid based on the reference sample directly expresses the difference from the state to be referred to, so that the difference from the reference molar concentration can be known as an absolute amount. . For this reason, it is easy to compare with the result using a DNA chip (DNA microarray).

  Such a reference sample may be, for example, at least one of a sample representative of a sample collected from a healthy individual and a sample representative of a sample collected from a sick individual. A sample representative of a sample collected from a healthy individual may be a sample collected from one healthy individual, or a virtual sample derived from a sample collected from a plurality of healthy individuals by a statistical method It may be. Similarly, a sample representative of a sample collected from a diseased individual may be a sample collected from one diseased individual, or a virtual derived from a sample collected from a plurality of diseased individuals by a statistical method. May be a typical specimen. The “individual” described above may be a human or an animal or plant other than a human.

  Selecting such a reference sample and setting the molar concentration of the internal standard nucleic acid, i.e., the reference molar concentration is the molar concentration and the disease representative when the reference sample is a sample collected from a healthy individual. The target nucleic acid expression level in the sample to be measured is collected from a healthy sample and a diseased individual by making it the same as at least one of the representative molar concentrations of the sample collected from the individual. It can be determined with high sensitivity and high accuracy whether or not the expression level of the target nucleic acid in at least one of the specimens is comparable.

  The nucleic acid analysis kit 300 according to the fifth embodiment is included in the sample by adjusting a mixed solution obtained by mixing the test solution and the internal standard reagent 110 and then introducing the mixed solution into the well 16 (reaction vessel). Can be used to amplify the target nucleic acid and to determine whether the target nucleic acid is expressed in a predetermined amount.

  Thus, according to the nucleic acid analysis kit 300 according to the fifth embodiment, among the reaction containers, the types of primers and fluorescent probes included in the first reaction container and the second reaction container are different from each other. A nucleic acid analysis kit that can easily and accurately determine the expression of various genes can be realized.

  Further, according to the nucleic acid analysis kit 300 according to the fifth embodiment, the molar concentration of the internal standard nucleic acid is the target nucleic acid contained in the reference sample which is a sample used as a criterion for determining whether or not the target nucleic acid is expressed in a predetermined amount. Nucleic acid analysis that can determine with high sensitivity and high accuracy whether the target nucleic acid expression level in the sample to be measured is comparable to the target nucleic acid expression level in the reference reference sample A kit can be realized.

  Also in the microfluidic chip 100e, after preparing a mixed solution obtained by mixing the test solution and the internal standard reagent 110, the mixed solution is introduced into the reservoir 15, and "1-4. Method of filling the test solution" The mixed solution can be filled into the well 16 (reaction vessel) by the method described in the above section. Therefore, even a minute amount of the liquid mixture, which is difficult with a pipette, can be accurately and easily dispensed into the well 16.

  A method for analyzing the target nucleic acid after introducing a mixed solution obtained by mixing the test solution and the internal standard reagent 110 into the well 16 will be described in the section “4. Nucleic acid analysis method”.

3-2. Nucleic Acid Analysis Kit According to Sixth Embodiment FIG. 12 is a diagram schematically showing a nucleic acid analysis kit 300a according to the sixth embodiment. A nucleic acid analysis kit 300a according to the sixth embodiment is a nucleic acid analysis kit used for amplification of a target nucleic acid contained in a specimen and determination of whether or not a predetermined amount of the target nucleic acid has been expressed. The first internal standard reagent as an internal standard reagent that is a liquid containing a known amount of the internal standard nucleic acid, which is a nucleic acid that can be amplified with a primer common to the target nucleic acid and has a different sequence from the target nucleic acid 111 and a second internal standard reagent 112. In the example shown in FIG. 12, the first internal standard reagent 111 and the second internal standard reagent 112 are liquids contained in the lidded container 151 and the lidded container 152, respectively.

  The first internal standard reagent 111 includes an internal standard nucleic acid whose reference molar concentration is a molar concentration that represents a case where the reference sample is a sample collected from a diseased individual in an advanced state. That is, the molar concentration of the internal standard nucleic acid contained in the first internal standard reagent 111 is the same as the molar concentration of the target nucleic acid contained in the specimen representing the individual in the second diseased state.

  The second internal standard reagent 112 includes an internal standard nucleic acid whose reference molar concentration is a molar concentration representative of the case where the reference sample is a sample collected from an individual in the second advanced state of illness. That is, the molar concentration of the internal standard nucleic acid contained in the second internal standard reagent 112 is the same as the molar concentration of the target nucleic acid contained in the specimen representing the individual in the second progressive state of the disease.

  Here, the “same” range is not limited to being completely identical, but may be within a range including an error that can satisfy the required determination accuracy. In addition, the user himself / herself dilutes the first internal standard reagent 111 or the second internal standard reagent 112 at a predetermined rate according to an instruction in the manual or the like, or the test solution and the first internal standard reagent 112 at a predetermined rate. When the first internal standard reagent 111 or the second internal standard reagent 112 is introduced into the well 16 (reaction vessel) by mixing the internal standard reagent 111 or the second internal standard reagent 112, The molar concentration of the internal standard nucleic acid may be adjusted to be the same as the reference molar concentration. Thereby, the reference molar concentration can be easily selected. In addition, the degree of freedom in selecting the reference molar concentration is increased.

  The nucleic acid analysis kit 300a according to the sixth embodiment adjusts a mixed solution obtained by mixing the first internal standard reagent 111 or the second internal standard reagent 112 and the test solution, and then adds the mixed solution to the well 16 (reaction By introducing it into a container, it can be used for amplification of the target nucleic acid contained in the specimen and determination of whether or not a predetermined amount of the target nucleic acid has been expressed.

  As described above, in the nucleic acid analysis kit 300a according to the sixth embodiment, the internal standard nucleic acid whose reference molar concentration is a molar concentration representative of the case where the reference sample is a sample collected from an individual in the first advanced state of disease. A second internal standard nucleic acid containing a first internal standard nucleic acid having a molar concentration representative of the case where the reference sample is a sample collected from an individual in a second diseased state. The target nucleic acid expression level in the sample to be measured in the sample collected from the individual in the first diseased state and the sample collected from the individual in the second diseased state. It is possible to realize a nucleic acid analysis kit that can determine with high sensitivity and high accuracy whether or not the expression level is the same as the target nucleic acid expression level.

  In one microfluidic chip 100e, the well 16 (reaction vessel) using the first internal standard reagent 111 and the well 16 (reaction vessel) using the second internal standard reagent 112 may be used separately. Thus, using one microfluidic chip 100e, the expression level of the target nucleic acid in the sample to be measured was collected from the sample collected from the diseased first progressing individual and the diseased second progressing individual. It is possible to realize a nucleic acid analysis kit capable of determining with high sensitivity and high accuracy whether or not the expression level of the target nucleic acid in the sample is comparable.

3-3. Nucleic acid analysis kit according to modification In the nucleic acid analysis kit 300 according to the fifth embodiment and the nucleic acid analysis kit 300a according to the sixth embodiment, each well 16 (reaction vessel) is included in each well 16 (reaction vessel). Although the example in which the types of primers and fluorescent probes used are different has been described, the well 16 (reaction container) constitutes a reaction container group including a plurality of wells 16 (reaction containers), and the first of the reaction container groups is the first. The types of primers and fluorescent probes contained in the well 16 (reaction container) belonging to the reaction container group and the well 16 (reaction container) belonging to the second reaction container group may be different from each other. In other words, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group, and the amount of the internal standard nucleic acid may be the same.

  FIGS. 13A and 13B are diagrams schematically showing nucleic acid analysis kits 301 and 301a according to modifications. A nucleic acid analysis kit 301 shown in FIG. 13A is a modification of the nucleic acid analysis kit 300 according to the fifth embodiment, and a nucleic acid analysis kit 301a shown in FIG. 13B is a nucleic acid analysis kit 300a according to the sixth embodiment. It is a modified example of.

  In the example shown in FIGS. 13A and 13B, the well 16 (reaction container) constitutes reaction container groups 61, 62, 63, 64 and 65 each including ten wells 16 (reaction containers). is doing. For example, primers and fluorescence contained in the well 16 (reaction container) belonging to the reaction container group 61 and the well 16 (reaction container) belonging to the reaction container group 62 shown in FIGS. 13 (A) and 13 (B). When the types of probes are different from each other, the reaction vessel group 61 and the reaction vessel group 62 correspond to the first reaction vessel group and the second reaction vessel group, respectively. This explanation also applies to any two other reaction container groups.

  In the example shown in FIGS. 13 (A) and 13 (B), for each reaction container group 61, 62, 63, 64 and 65, the primer and fluorescent probe contained in the well 16 (reaction container) belonging to the reaction container group. The types are different. In other words, the types of primers and fluorescent probes contained in the wells 16 (reaction containers) belonging to the same reaction container group are the same.

  Thus, by including a plurality of wells 16 (reaction containers) of the same condition for each reaction container group, errors for each well 16 (reaction container) are not easily reflected in the determination result, and the reliability of the determination is improved. . Further, even when bubbles or foreign substances are mixed into one well 16 (reaction container) by mistake, the determination can be made using the results of other wells 16 (reaction containers) belonging to the same reaction container group.

  In addition, since the types of primers and fluorescent probes contained in the wells 16 are different for each reaction container group, the target nucleic acid in the sample to be measured is targeted for a plurality of types of genes with one microfluidic chip. It is possible to realize a nucleic acid analysis kit that can determine with high sensitivity and high accuracy whether the expression level is comparable to the expression level of the target nucleic acid in the reference reference sample.

4). Next, an example of a nucleic acid analysis method using the microfluidic chip 100 according to the first embodiment will be described. Hereinafter, a process after the test solution is introduced into the well 16 (reaction vessel) will be described. In addition, also when the microfluidic chip | tip, microfluidic chip set, and nucleic acid analysis kit which concern on other embodiment mentioned above are used, a nucleic acid can be analyzed by the same method.

  In the nucleic acid analysis method described below, a step of preparing a test solution containing cDNA obtained by reverse transcription of mRNA contained in a specimen, and a nucleic acid amplification reaction (for example, PCR) in a plurality of wells 16 (reaction containers) are performed. The step of measuring, the step of measuring the fluorescence intensity emitted by the fluorescent probe bound to a part of the amplified nucleic acid in each well 16 (reaction vessel), and the measurement in each well 16 (reaction vessel) Based on the fluorescence intensity and the amount of the internal standard nucleic acid arranged (or introduced) in each well 16 (reaction vessel), the amount of the target nucleic acid contained in the test solution is estimated to be contained in the sample. estimating the amount of mRNA.

  In the well 16 (reaction vessel) of the microfluidic chip 100 according to the first embodiment, known amounts of internal standard nucleic acids, primers, and fluorescent probes are arranged as reagents 30, 31, and 32. Therefore, in the step of performing the nucleic acid amplification reaction in the well 16 (reaction container), the internal standard nucleic acid previously disposed in the well 16 (reaction container) and the test solution introduced into the well 16 (reaction container) are included. The target nucleic acid to be amplified is amplified in the well 16 (reaction vessel).

  In the nucleic acid amplification reaction, for example, the fluorescent probe can be bound to both the target nucleic acid and the internal standard nucleic acid, and further, quenching of the fluorescence can be generated when the fluorescent probe is bound to one of them. it can. In this state, the fluorescence intensity generated from each well 16 (reaction vessel) is measured. Next, based on the fluorescence intensity measured for each well 16 (reaction vessel) and the amount of the internal standard nucleic acid placed in each well 16 (reaction vessel), the amount of target nucleic acid contained in the test solution is determined. Can be estimated. Then, the amount of mRNA contained in the specimen can be estimated based on the estimated amount of target nucleic acid.

  In order to estimate the amount of the target nucleic acid using the fluorescence intensity measured for each well 16 (reaction vessel) and the formula (1) described later, data of positive control and negative control are prepared in advance.

  First, as a positive control, a well containing only the target nucleic acid TGT is prepared. In this well, all amplification products obtained in the nucleic acid amplification reaction are those obtained by amplifying the target nucleic acid TGT. In this case, the amount of change in fluorescence is Ft. This target nucleic acid TGT may be prepared in advance.

  When analyzing a plurality of types of nucleic acids with one microfluidic chip, a well for positive control is prepared for each type of target nucleic acid. It is not necessary to change the amount of the target nucleic acid depending on the amount of the internal standard nucleic acid.

  Next, as a negative control, a well containing only the internal standard nucleic acid CPT is prepared. In this well, all amplification products are those obtained by amplifying the internal standard nucleic acid CPT, and the amount of change in fluorescence in this case is Fc.

  The above-mentioned “well containing only the target nucleic acid TGT” and “well containing only the internal standard nucleic acid CPT” may be a part of the microfluidic chip actually used for nucleic acid analysis, or the same type. It may be the well of some other microfluidic chip.

  When both the target nucleic acid TGT and the internal standard nucleic acid CPT are present in the well, both amplification products are produced, and the fluorescence change amount F at that time can be expressed by the following formula (1).

F = Ft · X / (X + C) + Fc · C / (X + C)
= [C · (Fc−Ft) / (X + C)] + Ft (1)

  The fluorescence change amount F in the above formula (1) may be the fluorescence intensity itself emitted by the fluorescent probe, or (i) the fluorescence intensity measured before the nucleic acid amplification reaction and the fluorescence intensity measured after the nucleic acid amplification reaction. Ratio to intensity (amount of fluorescence change), or (ii) a state in which the amplified product and the fluorescent probe are heated to a dissociation temperature after nucleic acid amplification (the fluorescence probe is dissociated from the amplified nucleic acid after the nucleic acid amplification reaction) 1) and the temperature at which the fluorescent probe is bound (the second state in which the fluorescent probe is bound to a part of the amplified nucleic acid after the nucleic acid amplification reaction). It may be a ratio to the fluorescence intensity (fluorescence change amount).

  In the above formula (1), C is the amount of the internal standard nucleic acid CPT in the well 16 (reaction vessel) (copy number in the well 16 (reaction vessel)), and X is the amount of the target nucleic acid TGT in the well 16 (reaction vessel) ( The number of copies in the well 16 (reaction vessel)).

  Therefore, when Ft and Fc obtained from the fluorescence brightness of the positive control and the negative control and the amount C of the internal standard nucleic acid are substituted into the above formula (1), the amount X of the target nucleic acid can be obtained from the fluorescence change amount F. The steps from the measurement of the fluorescence intensity to the estimation of the amount of target nucleic acid (or the amount of mRNA contained in the sample) are the same as the amount of target nucleic acid (or sample) based on the fluorescence detection device and the fluorescence detection result thereby. It can also be performed in combination with a program that performs an operation for estimating the amount of mRNA contained.

  The fluorescent probe is not particularly limited as long as it binds to a part of the target nucleic acid amplified by PCR and distinguishes between the target nucleic acid and the internal standard nucleic acid and shows a fluorescence change. For example, Taqman probe (registered trademark), Hyb probe (Registered trademark), Molecular Beacon (registered trademark), Q-Probe (registered trademark), and the like can be used. Q-Probe is a probe that detects a target gene using a “fluorescence quenching phenomenon” in which fluorescence emitted decreases when a guanine base approaches a labeled fluorescent dye.

  Q-Probe has a fluorescently labeled cytosine at the end and is designed to specifically bind to the target gene. When Q-Probe binds to the target gene, the fluorescence is affected by guanine. Decrease. In order to distinguish the internal standard nucleic acid from the target nucleic acid using this phenomenon, when the internal standard nucleic acid has guanine as a base corresponding to the fluorescently labeled terminal base of Q-Probe, the target nucleic acid is Q -It is necessary that the base corresponding to the fluorescently labeled terminal base of Probe is a base other than guanine. As a result, when Q-Probe co-amplifies both the target nucleic acid and the internal standard nucleic acid, when the internal standard nucleic acid and Q-Probe hybridize, the fluorescence emission of the labeled fluorescent dye is reduced (quenched), while the target nucleic acid and When hybridized with Q-Probe, the fluorescence emission of the labeled fluorescent dye does not decrease. In addition, about said description, even if it replaces a target nucleic acid and an internal standard nucleic acid, it is materialized. Therefore, it is possible to select whether the quenching of fluorescence occurs when Q-Probe binds to the target nucleic acid or the internal standard nucleic acid.

  As described above, by using the above-described nucleic acid analysis method, a plurality of wells 16 (reaction containers) of the microfluidic chip 100 are used to compare gene expression levels for a plurality of types of genes from a single specimen and a plurality of expression states. The gene expression level can be analyzed.

  Moreover, high reproducibility is obtained compared with a DNA chip by performing the reaction in the well 16 (reaction vessel) in a liquid and analyzing the expression level after amplification by a nucleic acid amplification reaction.

  Furthermore, since the expression level of the gene can be analyzed by calculating the nucleic acid amplification reaction and the measurement result of the fluorescence intensity, the analysis can be realized with a simple device.

5). Examples Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the examples.

  In this example, the case where Q-Probe (Kurata et al., Nucleic acids Research, 2001, vol. 29, No. 6 e34) is used as a fluorescent probe is taken as an example, and a competitive PCR method using a microfluidic chip 100 is used. A method for quantifying cDNA obtained by reverse transcription of mRNA contained in a specimen as a target nucleic acid will be described.

  Q-Probe interacts with guanine contained in the bound nucleic acid, and its fluorescence is significantly quenched. Therefore, by allowing Q-Probe to bind to both the target nucleic acid and the internal standard nucleic acid, and further causing fluorescence quenching to occur when Q-Probe binds to either one, a known internal nucleic acid can be obtained. The relative amount of target nucleic acid relative to the amount of standard nucleic acid can be estimated.

  Target nucleic acid and internal standard nucleic acid were purchased from J-Bio21 (Co). In this example, human β-actin gene and human albumin gene were used as target nucleic acids. As a buffer, a mixed solution of 10 mM Tris-HCl buffer (pH: 8.3) and KCl: 50 mM, MgCl2: 1.5 mM was used.

  FIG. 14 is a sequence example of the target nucleic acid and the internal standard nucleic acid. In the example shown in FIG. 14, the target nucleic acid is a human β-actin gene. The sequence example shown in FIG. 14 represents the sense strands of the target nucleic acid and the internal standard nucleic acid. 15 is a sequence example of primers corresponding to the target nucleic acid and internal standard nucleic acid shown in FIG. 14, and FIG. 16 is a sequence example of fluorescent probes corresponding to the target nucleic acid and internal standard nucleic acid shown in FIG. .

  The fluorescent probe (Q-Probe) shown in FIG. 16 binds to the part of the antisense strand that is in a partially complementary relationship with the underlined sequences of the target nucleic acid and internal standard nucleic acid shown in FIG. Comparing the target nucleic acid and the internal standard nucleic acid, as shown in FIG. 14, immediately after the binding portion (underlined portion) of the fluorescent probe, the target nucleic acid is “cc” and the internal standard nucleic acid is “aa”. In other respects, the other sequences are identical. Therefore, when Q-Probe binds to the target nucleic acid, it reacts with guanine of the antisense strand that is complementary to cytosine (c) immediately after the binding portion (underlined portion) of the fluorescent probe, so that the fluorescence is quenched. To do.

  In this example, the well 16 (reaction vessel) of the microfluidic chip 100 has the internal standard nucleic acid having the sequence shown in FIG. 14, the primer having the sequence shown in FIG. 15, and the sequence shown in FIG. A fluorescent probe (Q-Probe) was placed as a reagent in the well 16 (reaction vessel). Q-Probe (purchased from J-Bio21) was fluorescently labeled using BODIPY FL (Molecular probes).

  FIG. 17 is another sequence example of the target nucleic acid and the internal standard nucleic acid. In the example shown in FIG. 17, the target nucleic acid is a human albumin gene. The sequence example shown in FIG. 17 represents the sense strands of the target nucleic acid and the internal standard nucleic acid. 18 is a sequence example of primers corresponding to the target nucleic acid and internal standard nucleic acid shown in FIG. 17, and FIG. 19 is a sequence example of fluorescent probes corresponding to the target nucleic acid and internal standard nucleic acid shown in FIG. .

  The fluorescent probe (Q-Probe) shown in FIG. 19 binds to the underlined portion of the target nucleic acid and internal standard nucleic acid sequences shown in FIG. When comparing the target nucleic acid and the internal standard nucleic acid, as shown in FIG. 17, the target nucleic acid is “gg” and the internal standard nucleic acid is “aa” immediately before the binding portion (underlined portion) of the fluorescent probe. In other respects, the other sequences are identical. Therefore, when Q-Probe binds to the target nucleic acid, it reacts with guanine (g) immediately before the binding portion (underlined portion) of the fluorescent probe, so that the fluorescence is quenched.

  In this example, the well 16 (reaction vessel) of the microfluidic chip 100 has the internal standard nucleic acid having the sequence shown in FIG. 17, the primer having the sequence shown in FIG. 18, and the sequence shown in FIG. A fluorescent probe (Q-Probe) was placed as a reagent in the well 16 (reaction vessel). Q-Probe (purchased from J-Bio21) was fluorescently labeled using BODIPY FL (Molecular probes).

  By the same method as described above, the well 16 of the microfluidic chip 100 is divided into two reaction container groups, and a primer and a different primer for each reaction container group corresponding to the two genes human β-actin gene and human albumin gene, respectively. A fluorescent probe was placed. The amount of the internal standard nucleic acid is determined when a reaction solution of a reference sample (specimen 1), which is a reference for determining whether or not the target nucleic acid is expressed in a predetermined amount, is introduced into the well 16 (reaction container). The reference amount, which is the amount of the target nucleic acid introduced into the well 16 (reaction vessel), was adjusted and placed in each well 16. The expression level of each gene in the reference sample was determined in advance by a real-time PCR method and is shown in Table 1.

  Subsequently, a test solution containing the target nucleic acid extracted from the sample (specimen 2) is prepared so as to be suitable for the PCR reaction, filled into the well 16 (reaction vessel), and PCR is performed with a thermal cycler (Master Cycler (Eppendorf)). Went. The fluorescence was measured before and after the amplification reaction at room temperature, and the ratio was calculated as the amount of change in fluorescence.

  Table 1 shows the results of quantification of the human β-actin gene and the human albumin gene by the competitive PCR method under the conditions described above. Symbols are shown corresponding to the above-described equation (1). From the measurement results of quantitative RT-PCR of the reference sample (sample 1), the reference amount was set to 5000 copy / μl for the β actin gene and 100 copy / μl for the albumin gene.

  From the results in Table 1, it can be seen that the amount of the target nucleic acid in the sample (specimen 2) can be appropriately quantified.

  If the amount of the internal standard nucleic acid is constant regardless of the type of sample, if the amount of the target nucleic acid in the sample is significantly different from the amount of the internal standard nucleic acid (as a guide, 1/10 or less or 10 times or more) Sufficient quantitative accuracy cannot be secured. Usually, since the expression level varies greatly depending on the type of gene, it is difficult to quantify all genes by competitive PCR with a constant concentration of internal standard nucleic acid. From the results in Table 1, it can be seen that by setting the reference amount in advance, it was possible to accurately quantify both of two types of genes whose expression levels differ by 100 times or more using one microfluidic chip 100.

  Thus, according to this example, by setting the amount of the internal standard nucleic acid in anticipation of the expression level of the target nucleic acid in advance, the expression level of the target nucleic acid is within the expected range. Can be accurately quantified. In addition, a plurality of genes with greatly different expression levels can be quantified with one microfluidic chip.

6). Application Example of the Present Invention According to the present invention, for example, a specimen can be collected from a diagnosis subject (patient) in a hospital or the like, and the gene expression state can be analyzed. For example, if the amount of the internal standard nucleic acid is a representative amount obtained from a sample obtained from a healthy individual, how much the gene expression state of the diagnosis subject is different from the expression state in a healthy state. Can be easily determined. In addition, for example, if the amount of the internal standard nucleic acid is a representative amount collected from an individual with a specific disease, the expression state of the gene of the diagnosis subject is compared with the expression state of the specific disease state. It can be easily determined how much difference there is. This makes it possible to diagnose a disease based on the gene expression level. Note that the criterion for determining whether or not the patient is ill is determined medically based on statistical data or the like.

  Further, according to the above-described method, it is possible to quantify the gene expression level by preparing a positive control and a negative control in advance. When the gene expression level changes so that it cannot be quantified with a single reference amount depending on the degree of disease, a well (reaction vessel) with a different amount of internal standard nucleic acid may be provided in one microfluidic chip, or the internal standard nucleic acid By using a plurality of microfluidic chips having different amounts, gene expression levels can be quantified.

  Even when the gene expression level changes so as not to be quantified as compared with the expression state in a healthy state, it can be used as an initial screening for whether or not the disease is ill. In this case, a more accurate diagnosis can be performed by changing the amount of the internal standard nucleic acid and reexamining the overranged gene as necessary.

  In addition, embodiment mentioned above and a modification are examples, Comprising: It is not necessarily limited to these. For example, a plurality of embodiments and modifications can be combined as appropriate.

  The present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the present invention includes substantially the same configuration (for example, a configuration having the same function, method and result, or a configuration having the same purpose and effect) as the configuration described in the embodiment. In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

10 Substrate, 11 First surface, 12 Second surface, 13 Reservoir region, 14 well region, 15 Reservoir, 16 well, 16a Opening, 20 Cover, 20a Surface, 21 Adhering region, 30, 31, 32 Reagent, 41 Health region , 42 disease region, 51 first progress state region, 52 second progress state region, 61, 62, 63, 64, 65 reaction vessel group, 80 test solution, 90 roller, 100, 100a, 100b, 100c, 100d, 100e, 100f, 101, 101a, 101b Microfluidic chip, 110 internal standard reagent, 111 first internal standard reagent, 112 second internal standard reagent, 150, 151, 152 container with lid, 160, 161, 162 well, 200 Microfluidic chip set, 300, 300a, 301, 301a Nucleic acid content Kit, 611,612,613,614,615,621,622,623,624,625,631,632,633,634,635,641,642,643,644,645 reaction vessel group, R rotation axis

Claims (8)

A microfluidic chip used for amplification of a target nucleic acid contained in a specimen and determination of whether or not the target nucleic acid is expressed in a predetermined amount,
Including a plurality of reaction vessels,
The reaction vessel is
A known amount of an internal standard nucleic acid that is a nucleic acid that can be amplified with a primer common to the target nucleic acid, and that has a different sequence from the target nucleic acid;
A primer common to the target nucleic acid and the internal standard nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the internal standard nucleic acid, and exhibits a different fluorescence change when bound to the amplification product of the target nucleic acid and the amplification product of the internal standard nucleic acid; ,
Including
Among the reaction vessels, the types of the primer and the fluorescent probe contained in the first reaction vessel and the second reaction vessel are different from each other,
The amount of the internal standard nucleic acid is introduced into the reaction vessel when a reaction solution of a reference sample, which is a sample serving as a reference for determining whether or not a predetermined amount of the target nucleic acid is expressed, is introduced into the reaction vessel. A microfluidic chip that is the same as a reference amount that is the amount of the target nucleic acid contained in the reaction solution. The microfluidic chip according to claim 1, wherein
The microfluidic chip, wherein the reference amount is at least one of an amount representative when the reference sample is a sample collected from a healthy individual and an amount representative when the reference sample is a sample collected from a sick individual. The microfluidic chip according to claim 1, wherein
A reaction container under a first condition, wherein the reference amount is a representative amount when the reference sample is a sample collected from the healthy individual;
A reaction container under a second condition in which the reference amount is a representative amount when the reference sample is a sample collected from the diseased individual; and
Including a microfluidic chip. The microfluidic chip according to claim 1, wherein
A reaction container having a first condition in which the reference amount is an amount representative of a case where the reference sample is a sample collected from an individual in a first progressing state of the disease;
A reaction container under a second condition in which the reference amount is an amount representative of a case where the reference sample is a sample collected from an individual in a second advanced state of the disease;
Including a microfluidic chip. 2. The microfluidic chip according to claim 1, wherein the reference amount includes a reaction container having a first condition that is representative of a case where the reference sample is a sample collected from the diseased first individual. 3. When,
2. The microfluidic chip according to claim 1, wherein the reference amount includes a second-condition reaction container whose amount is representative of a case where the reference sample is a sample collected from an individual in the second progressive state of the disease. When,
Including a microfluidic chipset. A nucleic acid analysis kit used for amplification of a target nucleic acid contained in a specimen and determination of whether or not the target nucleic acid is expressed in a predetermined amount,
A microfluidic chip;
An internal standard reagent that is a liquid containing a known amount of an internal standard nucleic acid, which is a nucleic acid that can be amplified with a primer common to the target nucleic acid and has a sequence different from the target nucleic acid;
Including
The microfluidic chip includes a plurality of reaction vessels,
The reaction vessel is
A primer common to the target nucleic acid and the internal standard nucleic acid;
A fluorescent probe that binds to a part of the amplification product of the target nucleic acid and the internal standard nucleic acid, and exhibits a different fluorescence change when bound to the amplification product of the target nucleic acid and the amplification product of the internal standard nucleic acid; ,
Including
Among the reaction vessels, the types of the primer and the fluorescent probe contained in the first reaction vessel and the second reaction vessel are different from each other,
The molar concentration of the internal standard nucleic acid is the same as the reference molar concentration, which is the molar concentration of the target nucleic acid contained in a reference sample that is a reference for determining whether or not the target nucleic acid is expressed in a predetermined amount. Nucleic acid analysis kit. The nucleic acid analysis kit according to claim 6,
The reference molar concentration is at least one of a molar concentration representative when the reference specimen is a specimen collected from a healthy individual and a molar concentration representative when the specimen is a specimen collected from a diseased individual. kit. The nucleic acid analysis kit according to claim 7,
As the internal standard reagent,
A first internal standard reagent containing the internal standard nucleic acid, wherein the reference molar concentration is a molar concentration representative of a case where the reference sample is a sample collected from an individual in a first advanced state of the disease;
A second internal standard reagent containing the internal standard nucleic acid, wherein the reference molar concentration is a molar concentration representative of a case where the reference sample is a sample collected from an individual in the first advanced state of the disease;
A nucleic acid analysis kit comprising:

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