JP6641706B2 - Assurance method of gene analysis system - Google Patents

Description

  The present invention relates to a reaction container, a nucleic acid analysis device provided with the reaction container, and a nucleic acid analysis method.

  “Made-to-order medicine,” which provides effective treatment and medication according to the patient's genetic background based on epidemiological and scientific evidence, not only improves the quality of life (QOL) of each individual but also It is also expected that the increase in medical costs due to side effects of drugs will be suppressed. There are various methods for determining the effects, side effects, and the like of drugs, but genetic diagnosis, which analyzes differences in genes among individuals, is one of the effective methods. It is desirable that custom-made medical treatment can be performed easily and quickly in the field, and μ-TAS (Total Analysis System), which is a reaction device for processing a small amount of a sample solution containing a gene, as a means for realizing them. And a technology called Lab-on-Chip.

This is provided with a plurality of reaction chambers and flow paths in one chip or cartridge, and can analyze a plurality of samples or simultaneously process a plurality of types of items. These techniques have various merits such as minimizing the size of the chip and the cartridge so that the amount of chemicals handled can be reduced. The main line of gene analysis is to extract or purify nucleic acids for gene analysis from biological samples such as blood, oral mucosa, and sputum collected from patients, and to extract and purify the gene region or nucleic acid sequence to be examined by PCR, etc. It consists of a process of detecting an array specific to the object to be inspected, such as a type.
Patent Literature 1, Patent Literature 2, and Non-Patent Literature 1 disclose a gene diagnosis apparatus in which the steps of amplification and detection proceed automatically after nucleic acid removal from a biological sample.
Patent Documents 10 to 12 disclose a small and highly efficient gene diagnosis apparatus in which the position and shape of an incubator for performing a nucleic acid amplification step are devised.
Patent Literature 9 discloses a nucleic acid extraction / purification apparatus that is automatically advanced.
Patent Literatures 3 to 7 and Non-Patent Literature 2 disclose a staining method using an asymmetric cyanine dye as a rapid, high-sensitivity, and selective method capable of detecting nucleic acids.
Non-Patent Document 3 discloses a nucleic acid detection method utilizing an invader method.

JP 2004-221050 A JP-A-2004-093297 JP-A-7-196930 U.S. Pat. No. 5,436,134 U.S. Pat. No. 5,658,751 U.S. Pat. No. 5,321,130 U.S. Pat. No. 5,582,977 Japanese Patent No. 552154 U.S. Pat. No. 8,633,032 Japanese Patent No. 5343801 Japanese Patent No. 4996950 Japanese Patent No. 5555367

Clinical Chemistry 51: 882-890, 2005 Nucleic Acids Res. 31: 6227-6234, 2003. Molecular Diagnostics 4: 353-364, 1999

When the nucleic acid extraction step is independent of the subsequent amplification and detection steps, the amount of the nucleic acid can be confirmed by an operation using an external device. However, in the devices disclosed in Patent Literature 1, Patent Literature 2, and Non-Patent Literature 1, the steps of amplification and detection after nucleic acid extraction from a biological sample are fully automated, and the amount of the obtained nucleic acid cannot be confirmed. For this reason, the amount of the extracted nucleic acid is unclear, and is not sufficient when the test object is to detect a gene mutation or the like.
For example, in the case of a gene mutation typified by cancer or the like, 99.9% in total is a normal wild type, and when it is desired to detect the remaining 0.1% of the mutant type, the amount of nucleic acid required before amplification is 1,000 copies. Otherwise, it cannot be guaranteed that the mutation is 0.1%.
Also, in the analysis of the presence or absence of a specific nucleic acid sequence or gene polymorphism, when the results were output irregularly, the amount of nucleic acid was insufficient, or a reaction inhibitor etc. was mixed in the system It is also conceivable that the phenomenon is indistinguishable.
As described above, in the fully automatic gene analysis system, the amount of the nucleic acid is unclear, and the reliability of the analysis result may depend on the characteristics of the test item in some cases.

  The present invention has been made in view of the above circumstances, and is suitably used in a fully automatic gene analysis system, in which the quantification of a nucleic acid obtained by extraction from a biological sample and the analysis of the nucleic acid are performed in the same well. An object of the present invention is to provide a possible reaction vessel, a nucleic acid analysis device provided with the reaction vessel, and a nucleic acid analysis method.

(1) A nucleic acid analysis method using a reaction container or a nucleic acid analysis device including the reaction container,
The reaction vessel,
A substrate, on which a nucleic acid analysis reagent used for nucleic acid analysis and a quantitative reagent capable of specifically detecting nucleic acid are arranged, and at least one or more control reaction wells,
And a plurality of other reaction wells in which the nucleic acid analysis reagent is arranged,
The nucleic acid-containing sample is configured to be distributed to the control reaction well and the other plurality of reaction wells,
b1) injecting a sample containing nucleic acids into the control reaction well and the other plurality of reaction wells;
b2) forming an association state in which the nucleic acid contained in the sample and the quantification reagent are associated in the control reaction well;
b3) a measuring step of measuring a signal emitted in the association state;
b4) calculating the amount of nucleic acid contained in the sample that can be analyzed in the control reaction well and the other plurality of reaction wells from the signal measured in the measurement step;
A nucleic acid quantification step B comprising:
After the step B, a nucleic acid analysis step of injecting a sample containing the nucleic acid into the control reaction well and the other plurality of reaction wells to identify the nucleic acid in the control reaction well and the other plurality of reaction wells C and
Only including,
A nucleic acid analysis method , wherein the concentration of the quantitative reagent is a concentration that does not inhibit the reaction performed in the nucleic acid analysis step C.
(2) The nucleic acid analysis method according to (1), wherein the nucleic acid analysis device further includes an extraction unit for extracting a nucleic acid.
(3) Prior to the nucleic acid quantification step B, the method further includes a nucleic acid extraction step A for extracting nucleic acids from a biological sample, wherein the nucleic acids to be quantified and analyzed in the nucleic acid quantification step B and the nucleic acid analysis step C are: The nucleic acid analysis method according to (1) or (2), which is the nucleic acid obtained in the nucleic acid extraction step A.
(4) The nucleic acid analysis method according to (3), further including a nucleic acid purification step P after the nucleic acid extraction step A and before the nucleic acid quantification step B.
(5) The nucleic acid analysis method according to any one of (1) to (4), wherein the nucleic acid analysis device further includes a purification unit that purifies the nucleic acid.
(6) After the nucleic acid quantification step B and before the nucleic acid analysis step C, the method further includes a nucleic acid purification step P, and the nucleic acid analysis apparatus further includes a purification unit for purifying the nucleic acid (1) to (1). The nucleic acid analysis method according to any one of 3).
(7) A step J ′ of judging whether or not to shift to the nucleic acid purification step P based on the amount of nucleic acid calculated in the nucleic acid determination step B is performed after the nucleic acid purification step B and before the nucleic acid purification step P. The nucleic acid analysis method according to (4) or (6), further comprising:
(8) After the nucleic acid quantification step B, a step D of judging the validity of the analysis result obtained in the nucleic acid analysis step C based on the amount of the nucleic acid calculated in the nucleic acid quantification step B is included (1) to ( The nucleic acid analysis method according to any one of 7).
(9) Based on the amount of nucleic acid calculated in the nucleic acid quantification step B and the analysis result obtained in the nucleic acid analysis step C, at the same time with the step D of judging the validity of the analysis result, a defect of the nucleic acid analyzer is detected. The nucleic acid analysis method according to any one of (1) to (8), further comprising a step E of:
(10) The step J of judging whether or not transfer to the nucleic acid analysis step C is possible based on the amount of nucleic acid calculated in the nucleic acid quantification step B is further performed after the nucleic acid quantification step B and before the nucleic acid analysis step C. The nucleic acid analysis method according to any one of (1) to (9).
( 11 ) The nucleic acid analysis method according to any one of (1) to ( 10 ), wherein the base material is made of a resin having optical transparency.
( 12 ) The base material further includes a flow path and an injection port through which a solution can be injected, and the flow path allows the nucleic acid contained in the sample to flow through the control reaction well and the other plurality of reaction wells. The nucleic acid analysis method according to any one of (1) to ( 11 ), which can be distributed into
( 13 ) The nucleic acid analysis method according to any one of (1) to ( 12 ), wherein the quantification reagent is a fluorescent substance.
( 14 ) The nucleic acid analysis method according to any one of (1) to ( 13 ), wherein the quantification reagent and / or the nucleic acid analysis reagent is arranged in a dry state.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to confirm the quantity of the nucleic acid obtained by nucleic acid extraction from a biological sample, and it can improve the reliability with respect to the analysis result of a nucleic acid. Further, it is possible to detect a defect of the nucleic acid analyzer, which can be used for maintenance of the apparatus.

It is a perspective view of a reaction container of a first embodiment of the present invention. (A) (b) It is the schematic diagram which shows one aspect of the flow path in the reaction container of this invention. (A) (b) (c) It is the schematic plan view and side view of the reaction container of 2nd embodiment of this invention. 1 is a flowchart illustrating a nucleic acid analysis method according to a first embodiment of the present invention. 5 is a flowchart illustrating a second embodiment of the nucleic acid analysis method of the present invention. 5 is a flowchart illustrating a third embodiment of the nucleic acid analysis method of the present invention. 9 is a flowchart illustrating a fourth embodiment of the nucleic acid analysis method according to the present invention. 9 is a flowchart illustrating a fifth embodiment of the nucleic acid analysis method of the present invention. 13 is a flowchart illustrating a nucleic acid analysis method according to a sixth embodiment of the present invention. 13 is a flowchart illustrating a nucleic acid analysis method according to a seventh embodiment of the present invention. It is a graph which shows the relationship between the density | concentration of the standard DNA amplified by PCR, and the fluorescence intensity by Redmond Red (fluorescence of 595 nm).

≪Reaction vessel≫
[First embodiment]
In the reaction vessel of the first embodiment of the present invention, a base material is provided with a quantification reagent capable of specifically detecting nucleic acid and a nucleic acid analysis reagent used for nucleic acid analysis, and the amount of nucleic acid in a sample containing nucleic acid is determined. At least one or more control reaction wells capable of quantifying and analyzing the nucleic acid contained in the sample and the nucleic acid analysis reagent are arranged, and the nucleic acid contained in the sample can be analyzed. A plurality of other reaction wells, and the nucleic acid contained in the sample can be distributed to the control reaction well and the other plurality of reaction wells.

  The reaction vessel according to the first embodiment of the present invention will be described in more detail with reference to FIG. As shown in FIG. 1, a reaction container 10 is provided with a nucleic acid analysis reagent 3 used for nucleic acid analysis, and is capable of analyzing a sample containing nucleic acid. Only one is described, but there may be more than one.), And the nucleic acid analysis reagent 3 and the quantitative reagent 4 capable of specifically detecting the nucleic acid are arranged, and the nucleic acid contained in the sample is A control reaction well 2 capable of quantifying an amount and analyzing a nucleic acid, a flow channel 5, and an injection port 6 capable of injecting a solution are provided in the base material 7. Can be distributed to the control reaction well 2 and the other plurality of reaction wells 1.

  In the present invention, “nucleic acid” refers to DNA, RNA or an analog thereof, and may be natural or synthetic. Examples of the analog include artificial nucleic acids such as PNA and LNA. Natural nucleic acids include, for example, genomic DNA, mRNA, tRNA, rRNA, hnRNA and the like recovered from living organisms. Examples of the synthesized nucleic acid include DNA synthesized by a known chemical synthesis method such as a β-cyanoethyl phosphoramidite DNA solid phase synthesis method, nucleic acid synthesized by a known nucleic acid amplification method such as PCR, and inversion. Examples include cDNA synthesized by a transcription reaction.

  In the present invention, the “sample containing a nucleic acid” may be a sample extracted or purified from an organism such as an animal, a plant, a microorganism, or a cultured cell, or a sample containing a nucleic acid amplified by a nucleic acid amplification reaction. Known methods such as the phenol / chloroform method can be used to extract nucleic acids from animals. Examples of the nucleic acid amplification reaction include PCR, LAMP, SMAP, NASBA, and RCA. When the amplified nucleic acid is a double-stranded nucleic acid, it can be used after being made single-stranded by heat denaturation treatment or chemical denaturation treatment.

The `` quantitative reagent capable of specifically detecting a nucleic acid '' is a substance that emits a signal indicating an association state with the nucleic acid, and the amount of the nucleic acid is changed by a change in the signal according to the amount of the nucleic acid in the sample solution. The reagent is not particularly limited as long as it is a quantifiable reagent, and is preferably a substance which specifically associates with nucleic acid and emits fluorescence, and more preferably a substance which associates specifically with double-stranded DNA and then emits fluorescence. . As the substance that emits fluorescence, a compound that emits fluorescence in the range of 300 to 800 nm is preferable. The temperature for specifically associating double-stranded DNA is preferably from 0 to 80 ° C.
Examples of the above substances include SYBR Green, Pico Green, Eva Green, ethidium bromide, Hoechst 33258 (CAS No. 23491-45-4; phenol (4- [5- (4-methyl-1-piperazinyl)] [2,5′-bi -1H-benzimidazol] -2'-yl])-trihydrochloride), TOTO-1, YOYO-1, YO-PRO-1, BEBO, BETO, BOXTO and the like.

  "Analysis of nucleic acid" refers to the identification of nucleic acid information such as the nucleotide sequence of nucleic acids and nucleic acid modifications contained in the sample. Identification of information to be shown is also included. The information indirectly indicating the information includes, for example, detection of the presence or absence of double-stranded nucleic acid formation resulting from a difference in base sequence, and the like. The information on the nucleotide sequence of the nucleic acid to be identified is preferably a gene sequence composed of a sequence containing the nucleotide sequence of the nucleic acid to be identified, and more preferably a gene mutation of the gene is identified. The gene mutation is preferably a difference in the nucleotide sequence of a gene existing between individuals of the same species. Specifically, one or more bases in the base sequence are substituted / deleted / inserted, resulting in a difference in the base sequence. Such gene mutations include, for example, SNP and CNV (Copy Number Variation) polymorphisms, base deletion mutations, base insertion mutations, translocation mutations, and the like. In addition, in the present invention, a gene mutation refers to a somatic mutation such as a somatic mutation which is a difference in the base sequence of a gene existing between cells in the same individual, in addition to an innate mutation such as a genetic polymorphism such as SNP. Also includes acquired mutations. In the analysis, it is preferable that the nucleic acid to be analyzed is amplified.

The “nucleic acid analysis reagent” is not particularly limited as long as it is a reagent used for identifying the above-described nucleic acid information, and is preferably a reagent used for nucleic acid amplification for identification. The reagent includes an oligo primer set, a nucleic acid synthase, a nucleoside and the like, and various combinations necessary for general nucleic acid amplification may be used.
In addition, the nucleic acid analysis reagent may contain a substance capable of detecting the presence or absence of double-stranded nucleic acid formation, in addition to the molecule used for nucleic acid amplification. The substance includes a substance capable of specifically detecting the nucleic acid described above.

“Reagent is disposed” means that the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells before the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells. A reagent may be arranged in a well, and after the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells, the reagent is placed in the control reaction well or the reaction well. It means that the nucleic acid contained in the sample may be arranged beforehand, and is preferably arranged before the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells.
As a method in which the reagent is arranged after the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells, for example, each reagent well containing a reagent may be placed in the control reaction well. Wells or the other plurality of reaction wells are provided separately, and after the nucleic acid is distributed to the wells, each reagent well is communicated with the control reaction well or the other plurality of reaction wells, and reagents are arranged. Method.
As a method in which the reagent is arranged before the nucleic acid contained in the sample is distributed to the control reaction well or the other plurality of reaction wells, the reagent is placed in the control reaction well or the other plurality of reaction wells. A method of drying and fixing to the inner wall of the well may be used.
Therefore, in the present embodiment, it is preferable that the quantitative reagent and / or the nucleic acid analysis reagent are arranged in a dry state. Since the reagent is arranged in a dry state, deterioration of the reagent can be reduced.
In addition, all the substances contained in the reagent may not be provided with the reagent before the nucleic acid is distributed to the control reaction well or the other plurality of reaction wells, and the nucleic acid is used in the control reaction. It may be arranged separately before and after distribution into wells or the other plurality of reaction wells. Examples of such a method include, for example, after injecting a sample into the control reaction well and the other plurality of reaction wells in which a substance used for nucleic acid amplification among nucleic acid analysis reagents is preliminarily fixed, and then amplifying the nucleic acid. And injecting a substance capable of detecting the amount of the amplified nucleic acid into the control reaction well and the other plurality of reaction wells.

The reaction container of the present embodiment includes a flow path that can distribute the nucleic acid contained in the sample to the control reaction well or the other plurality of reaction wells. Although the position, direction, and the like of the branch of the flow path are not particularly limited, for example, embodiments shown in FIGS. 2A to 2C can be exemplified. From the viewpoint that the amount of nucleic acid obtained by nucleic acid extraction from a biological sample can be quantified before analysis of the sample, the control reaction well 2 is closer to the injection port than the other reaction wells 1. Preferably, they are arranged. Thereby, the sample containing the nucleic acid can be more easily distributed to the control reaction wells than to the other reaction wells.
For example, in the case of FIG. 2 (b) or (c), a flow path on a side of a plurality of other reaction wells with respect to a branch point between a flow path connected to a control reaction well and a flow path connected to another plurality of reaction wells. In addition, by providing a blocking means for the sample solution that can open and close the flow path, it is determined whether the liquid can be sent to other reaction wells based on the amount of nucleic acid calculated in the control reaction well, and the liquid sending is performed. Can be controlled.

  The base material 7 of the reaction container of the present embodiment is preferably a resin having optical transparency. As a material of the base material 7, a resin having light transmittance can be suitably used. In addition, when optical analysis (fluorescence measurement, colorimetric measurement, or the like) is performed on the solution in the other plurality of reaction wells 1 or the control reaction well 2, the transparency of the substrate 7 may be high. preferable. For example, the material of the base material 7 is not particularly limited as long as it does not affect the sample. In particular, when a resin material containing any of polypropylene, polycarbonate, and acryl is used, good visible light transmittance is secured. can do. As the polypropylene, a homopolypropylene or a random copolymer of polypropylene and polyethylene can be used. As the acryl, polymethyl methacrylate or a copolymer of methyl methacrylate with other monomers such as methacrylate, acrylate, and styrene can be used. When these resin materials are used, the heat resistance and strength of the reaction vessel can be ensured. Other than the resin material, metal materials such as aluminum, copper, silver, nickel, brass, and gold can be used. When a metal material is used, it is additionally excellent in thermal conductivity and sealing performance.

  By making the bottom of the other plurality of reaction wells 1 or the control reaction wells 2 of the base material 7 transparent, detection and analysis of fluorescence and the like can be performed from the outside. In the present embodiment, “transparent” and “light-transmitting” preferably mean that the total average transmittance in the light range (wavelength 230 to 780 nm) when formed is 70% or more.

  As a processing method of the base material 7, in the case of a resin material, various resin molding methods such as injection molding and vacuum molding, mechanical cutting, and the like can be used. In the case of a metal material, it can be formed by grinding or etching using a thick base material, or pressing or drawing a thin metal sheet.

  Further, when the thickness of the base material 7 is in the range of 50 μm to 3 mm, favorable light transmittance, heat resistance, and strength can be ensured, and processing of the concave portion can be reliably performed, which is preferable.

[Second embodiment]
In the reaction container of the present embodiment, a quantification reagent capable of specifically detecting a nucleic acid and a nucleic acid analysis reagent used for analysis of the nucleic acid are arranged, and the amount of the nucleic acid in the sample containing the nucleic acid is quantified. At least one or more control reaction wells capable of analyzing the contained nucleic acid, and the nucleic acid analysis reagent is arranged, and a plurality of other reaction wells capable of analyzing the nucleic acid contained in the sample. Wherein the nucleic acid contained in the sample can be distributed to the control reaction well and the other plurality of reaction wells. Although details are omitted because they have been described in the first embodiment, a reaction vessel having a plurality of wells in a reaction vessel as shown in FIGS. 3A to 3C and having a circular outer shape may be used.

The reaction vessel main body 21 includes a plurality of other reaction wells 1 and control reaction wells 2 arranged side by side in the circumferential direction of the reaction vessel main body 21 so as to be equidistant from the center of the reaction vessel main body 21. And a flow path 23 provided in communication with each of the other plurality of reaction wells 1 and the control reaction well 2. In the present embodiment, a total of 23 wells, that is, 20 other plural reaction wells 1 and 3 control reaction wells 2, are provided in the reaction container main body 21.
The number of the control reaction wells 2 and the well to be used as the control reaction well 2 in the reaction vessel main body 21 can be appropriately determined in accordance with the method of confirming the reaction in the reaction vessel 10 and analyzing. .

  In addition, an injection port 26 for sending a nucleic acid solution containing purified nucleic acid to the other plurality of reaction wells 1 and control reaction wells 2 is provided at a central portion of the reaction container body 21. And an outlet 27 formed in the vicinity. The inlet 26 and the outlet 27 communicate with the channel 23. Further, the reaction container main body 21 is provided with a protruding wall portion 28 formed so as to protrude from the outer surface of the reaction container main body 21 so as to surround the inlet 26 and the outlet 27.

  The material of the reaction vessel body 21 may be the same as the material of the base material 7 of the above-described first embodiment, and at least other parts of the reaction well 1 and the control reaction well 2 are the same as those of the above-described first embodiment. It is preferable to have light transmittance.

  The flow path 23 is provided by forming a concave portion on the reaction container body 21 radially inside the other reaction wells 1 and the control reaction wells 2. The flow path 23 has a main flow path 24 and a branch flow path 25. The main flow path 24 extends in the circumferential direction of the reaction vessel main body 21 while meandering between the center of the reaction vessel main body 21 and the radial outside of the reaction vessel main body 21. The branch channel 25 is formed by branching from the main channel 24 so as to connect the main channel 24 with each of the reaction well 1 and the control reaction well 2.

  One end 24A of the main flow path 24 communicates with the inlet 26, and the other end 24B of the main flow path 24 communicates with the outlet 27. Further, the main flow path 24 is formed so as to have a chevron 24C protruding along the central axis O between the other plurality of reaction wells 1 and the control reaction wells 2 which are adjacent to each other in the circumferential direction of the reaction vessel main body 21. I have. The reaction vessel 10 was rotated around the central axis O by having a mountain shape 24C in the direction of the central axis O between the plurality of other reaction wells 1 and the control reaction wells 2 adjacent in the circumferential direction of the reaction vessel main body 21. At this time, the liquid stored in the main flow path 24 is interrupted at the apex 24D of the mountain shape 24C, and the liquid is sent to each of the reaction well 1 and the control reaction well 2.

  The branch flow path 25 is formed to be smaller than the flow path area of the main flow path 24 at a connection portion with the main flow path 24. The portion of the branch flow channel 25 connected to the main flow channel 24 is a flow restricting portion 25A that restricts the flow of the liquid from the main flow channel 24 to the branch flow channel 25.

  The lid 29 is provided on the side of the reaction vessel body 21 where the other plurality of reaction wells 1 and control reaction wells 2 and the flow path 23 are formed. By attaching the lid body 29 to the reaction vessel main body 21, the other plurality of reaction wells 1 and control reaction wells 2 and the concave portions serving as the flow paths 23 are covered, thereby forming independent reaction spaces and flow paths. ing.

  The lid 29 is preferably made of a material having a high thermal conductivity, for example, a metal material such as aluminum, copper, silver, nickel, brass, and gold. The portion having thermal conductivity may be at least a portion where the other plurality of reaction wells 1 and control reaction wells 2 are located. Further, heat is applied to the liquid inside the other plurality of reaction wells 1 and control reaction well 2 at a speed sufficient for a biochemical reaction (eg, PCR reaction) in the other plurality of reaction wells 1 and control reaction well 2. As long as transmission is possible, the lid 29 may be formed of a thin plate made of the same resin material as the above-described reaction vessel body 21.

  The reaction vessel 10 of the present embodiment sends the liquid stored in the main flow path 24 to each of the branch flow paths 25 by centrifugal force generated by the reaction vessel 10 rotating around the central axis O, and the branch flow path 25 The liquid is sent to each of the other plurality of reaction wells 1 and control reaction wells 2 through the control unit.

≪Nucleic acid analyzer≫
[First embodiment]
The nucleic acid analyzer of the present embodiment includes the above-described reaction container, an extraction unit for extracting nucleic acids, and a purification unit for purifying nucleic acids obtained in the extraction units. In addition, the nucleic acid analyzer of the present embodiment preferably includes a measurement unit that optically analyzes a solution in a control reaction well or a plurality of other reaction wells. The extraction unit and the purification unit may be provided on the substrate as a part of the reaction vessel of the present invention, but may have a different configuration from the reaction vessel. As the nucleic acid analyzer of the present embodiment, for example, an automatically processable nucleic acid analyzer described in Japanese Patent No. 50038445 is preferably used.
The nucleic acid analyzer equipped with the reaction container described above can easily, with high reliability, measure the amount of nucleic acid contained in the sample obtained in the extraction unit or the purification unit without removing the sample from the nucleic acid analyzer. Can be analyzed.
In addition to the automatic analysis performed by the above-described analyzer, the reaction is performed by manual dispensing using, for example, the reaction container of Embodiment 1 illustrated in FIG. 1 and the analysis is performed using a known fluorescence measurement device / software. It is also possible.

≪Nucleic acid analysis method≫
[First embodiment]
The nucleic acid analysis method of the present embodiment is a nucleic acid analysis method using the reaction container described above or the nucleic acid analysis device described above, and b1) the same as the nucleic acid that can be analyzed in the other plurality of reaction wells Injecting a sample containing the nucleic acid into the control reaction well, and b2) forming an association state in which the nucleic acid contained in the sample and the quantitative reagent are associated in the control reaction well, b3) a measuring step of measuring a signal emitted in the associated state; and b4) calculating an amount of a nucleic acid contained in the sample that can be analyzed in the other plurality of reaction wells, from the signal measured in the measuring step. A nucleic acid extraction step A for performing nucleic acid extraction from a biological sample prior to the nucleic acid determination step B; A nucleic acid analysis step C of injecting a sample containing a nucleic acid into the other plurality of reaction wells to identify the nucleic acid in the other plurality of reaction wells after the step A, wherein the nucleic acid is This is a nucleic acid analysis method which is a nucleic acid obtained in the nucleic acid extraction step A. "The same nucleic acid as the nucleic acid that can be analyzed in the other plurality of reaction wells" refers to a nucleic acid having the same composition to the extent that the amount of the nucleic acid that can be analyzed in the other plurality of reaction wells can be calculated. Point.

  In the present invention, the “nucleic acid analysis step” generally refers to a nucleic acid analysis method such as a real-time PCR method, a Taqman (registered trademark) method, a Cycleave (registered trademark) method, a Scopion method, an iFRET method, and an Invader (registered trademark) method. This is the method used for analysis.

  Hereinafter, the nucleic acid analysis method of the present embodiment will be described with reference to FIG. First, a nucleic acid is extracted from a biological sample to obtain a nucleic acid (Step A). The nucleic acid-containing sample obtained in step A is injected into another plurality of reaction wells, and the nucleic acid is identified using a nucleic acid analysis reagent (step C). Similarly, a sample containing the nucleic acid obtained in step A is injected into a control reaction well (step b1). The nucleic acid contained in the sample injected into the control reaction well and the quantification reagent associate to form an associated state (step b2). Next, a signal emitted from the quantification reagent that has become associated with the nucleic acid is measured (step b3). Based on the measured signal intensity, the amount of the nucleic acid contained in the sample before the amplification of the nucleic acid in step C is performed (step b4).

Since the nucleic acid analysis method includes the step B, the amount of the nucleic acid before amplification used for the nucleic acid analysis performed in the other plurality of reaction wells can be calculated, and the nucleic acid analysis method is performed in the other plurality of reaction wells. The reliability and accuracy of the nucleic acid analysis result can be improved.
In particular, since the amount of nucleic acid is important in the analysis of gene mutation, it is very significant to be able to confirm that the amount of nucleic acid is sufficient before step C.

[Second embodiment]
The nucleic acid analysis method of the present embodiment, after the nucleic acid extraction step A and the nucleic acid quantification step B, determines the validity of the analysis result obtained in the nucleic acid analysis step C based on the amount of nucleic acid calculated in the nucleic acid quantification step B. Step D of determining is included.
Therefore, Step D is included after Step A, Step B, and Step C. FIG. 6 shows an example of the nucleic acid analysis method of the present embodiment including Step D. Since the nucleic acid analysis method includes the step D, the validity of the nucleic acid analysis result is determined, and it is possible to avoid adoption of an incorrect analysis result, promptly indicate the necessity of reanalysis, and the like.

[Third embodiment]
After the nucleic acid extraction step A and the nucleic acid quantification step B, the nucleic acid analysis method of the present embodiment performs the analysis based on the amount of nucleic acid calculated in the nucleic acid quantification step B and the analysis result obtained in the nucleic acid analysis step C. At the same time as the step D for judging the validity of the result, a step E for detecting a defect of the nucleic acid analyzer is included.
Therefore, the step E is included after the steps A, B and C, and is performed simultaneously with the step D for determining the validity of the analysis result. FIG. 7 shows an example of the nucleic acid analysis method of the present embodiment including the step E. Since the nucleic acid analysis method includes the step E, a defect of the nucleic acid analyzer is detected, and it is possible to determine a portion where the defect of the analyzer has occurred, to promptly deal with the defect, and the like.

[Fourth embodiment]
The nucleic acid analysis method of the present embodiment further includes a nucleic acid purification step P after the nucleic acid extraction step A and before the nucleic acid quantification step B. FIG. 8 shows an example of the nucleic acid analysis method of the present embodiment including the step P. Since the nucleic acid analysis method includes the step P, the purity of the nucleic acid in the sample is improved, and more accurate nucleic acid analysis can be performed.
After the fourth embodiment, whether to perform the analysis result determination step (D) and the analysis device failure detection step (E) may be arbitrary.

[Fifth embodiment]
In the nucleic acid analysis method of the present embodiment, the nucleic acid analysis step C is included after the nucleic acid quantification step B, and the nucleic acid purification step P is performed after the nucleic acid quantification step B and before the nucleic acid analysis step C. Including. FIG. 9 shows an example of the nucleic acid analysis method of the present embodiment including the step P. Since the step P is included after the nucleic acid quantification step B and before the nucleic acid analysis step C, nucleic acid purification is performed only on the sample analyzed in the step C. Can be reduced.

[Sixth embodiment]
In the nucleic acid analysis method of the present embodiment, the nucleic acid analysis step C is included after the nucleic acid quantification step B, and the transition to the nucleic acid analysis step C is performed based on the amount of the nucleic acid calculated in the nucleic acid quantification step B. A step J of judging whether or not the method is possible is further included after the nucleic acid quantification step B and before the nuclear analysis step C. FIG. 10 shows an example of the nucleic acid analysis method of the present embodiment including step J. Since the step J is included after the nucleic acid quantification step B and before the nuclear analysis step C, interruption of the nucleic acid analysis can be selected before performing the step C, and it is possible to avoid obtaining an inaccurate nucleic acid analysis result. . Further, reagents and the like required for the step C can be reduced.

[Seventh embodiment]
The nucleic acid analysis method according to the present embodiment includes a step J ′ of judging whether or not to shift to the nucleic acid purification step P based on the amount of the nucleic acid calculated in the nucleic acid quantification step B, after the nucleic acid quantification step B and the nucleic acid Further included before the purification step P. FIG. 11 shows an example of the nucleic acid analysis method of the present embodiment including the step J ′. Since the step J ′ is included after the nucleic acid quantification step B and before the nuclear analysis step P, interruption of the nucleic acid analysis can be selected before performing the step P, thereby avoiding obtaining an incorrect nucleic acid analysis result. it can. Further, reagents and the like necessary for the process P can be reduced.

The "concentration at which the quantification reagent does not inhibit the reaction performed in the nucleic acid analysis step C" is a concentration at which the nucleic acid quantification reagent can sufficiently quantify nucleic acid, and is carried out in the analysis step. The concentration does not inhibit the reaction in the nucleic acid amplification step and the nucleic acid detection step.
For example, with respect to SYBR Green I, it is preferable to dilute commercially available 10000 × SYBR Green I to 0.5 × SYBR Green I or more and less than 2 × SYBR Green I. More preferably, those diluted to 1 × SYBR Green I or more and less than 2 × SYBR Green I are preferable.

  Next, the present invention will be described in more detail with reference to examples. The following embodiments are specific examples to which the present invention is applied, and do not limit the present invention in any way.

≪Preparation for analysis≫
1) Selection of quantitative reagent As a quantitative reagent, SYBR Green I, which is a substance specifically associated with double-stranded DNA, was selected. SYBR Green I is a cyanine-based asymmetric fluorescent substance, which emits fluorescence having a maximum of 522 nm when irradiated with excitation light of 488 nm when entering between bases of double-stranded DNA (intercalation). are doing.

2) Selection of nucleic acid analysis reagent As the nucleic acid analysis step, an Invader Plus method combining an Invader (registered trademark) method and a PCR method was used. In the Invader Plus method, reagents for a PCR reaction and reagents for an Invader reaction are previously arranged in a reaction vessel. Redmond Red was selected as the luminescent dye. Redmond Red is 579 nm when the Cleavase (registered trademark) acts in the Invader reaction to separate the fluorescent dye and the fluorescent inhibitor of the FRET cassette which is a DNA substrate modified with a fluorescent dye and a fluorescent quencher. It is characterized in that it emits fluorescence having a maximum at 595 nm when irradiated with excitation light.

3) Selection of primer for PCR As a primer for PCR, a primer that amplifies a region that does not contain a polymorphism of TCF4, which is a housekeeping gene (a gene that is constantly expressed or functions), was selected.

4) Preparation of mixed reagent i) Control reagent solution (0 × SYBR Green I)
20 mM MOPS buffer (pH 7.5)
0.4 mM dNTP
1 U / well polymerase 50 U / well Cleavase®
80 mM trehalose 0.8 μM allele probe oligo DNA
0.05μM Invader oligo DNA
0.7μM FRET cassette oligo DNA
0.9μM F primer oligo DNA
0.9 μM R primer oligo DNA
ii) Low concentration SYBR Green I-containing reagent solution 20 mM MOPS buffer (pH 7.5)
0.4 mM dNTP
1 U / well polymerase 50 U / well Cleavase®
80 mM trehalose 0.8 μM allele probe oligo DNA
0.05μM Invader oligo DNA
0.7μM FRET cassette oligo DNA
0.9μM F primer oligo DNA
0.9 μM R primer oligo DNA
Low concentration SYBR Green I
* "Low-concentration SYBER Green I" refers to the value of the fluorescence intensity obtained by measuring 0.1 ng / μL genomic DNA at an excitation wavelength of 488 nm and a fluorescence wavelength of 522 nm using the gene analyzer described in Patent Documents 9 to 12. SYBER Green I reagent prepared to be approximately 160,000.
iii) Highly concentrated SYBR Green I-containing reagent solution 20 mM MOPS buffer (pH 7.5)
0.4 mM dNTP
1 U / well polymerase 50 U / well Cleavase®
80 mM trehalose 0.8 μM allele probe oligo DNA
0.05μM Invader oligo DNA
0.7μM FRET cassette oligo DNA
0.9μM F primer oligo DNA
0.9 μM R primer oligo DNA
High concentration SYBR Green I
* "High-concentration SYBER Green I" refers to the value of the fluorescence intensity obtained by measuring 0.1 ng / μL genomic DNA at an excitation wavelength of 488 nm and a fluorescence wavelength of 522 nm using the gene analyzer described in Patent Documents 9 to 12. SYBER Green I reagent prepared to be approximately 280,000.
The oligo sequences used in this example are shown in Table 1.

5) Preparation of Reaction Container for Nucleic Acid Analysis (Analysis Chip) The reaction container having the flow channel shown in FIG. 3 was used. A total of 9 wells of the reagents having the compositions shown in the above i) to iii) were added to wells 1, 2, 3, 11, 12, 13, 20, 21, and 22 (numbered in order from the well closest to the inlet). Were prepared.

6) Preparation of sample genomic DNA solution When genomic DNA is obtained from a sample such as whole blood through the extraction step, the concentration of the genomic DNA differs each time the step is performed, even if the extract is from the same whole blood. . Therefore, a commercially available genomic DNA was prepared and measured as a standard so that the comparison could be established. The composition of the sample genomic DNA solution is as follows.
0.1 ng / μL genomic DNA
6.25 mM MgCl ₂
15 mM NaCl
The sample genomic DNA solution was fed from the inlet of the analysis chip by 270 μL per analysis chip to fill all the wells in which the reagents were arranged. 270 μL is the amount of solution that does not allow each well to connect through the solution.

≪Analysis process≫
7) Nucleic acid quantification step Each analysis chip was set on a nucleic acid analyzer (see Patent Documents 9 to 12), and analysis was started. First, the analysis chip was heated at 40 ° C. for 2 minutes. While maintaining the state at 40 ° C., the fluorescence of the wells of each analysis chip was measured at an excitation wavelength of 488 nm and a fluorescence wavelength of 522 nm for 5 cycles as one cycle of 30 seconds for a total of 2.5 minutes. Tables 2 to 4 show the values of the detected fluorescence intensities.

ｉ）の試薬を含む解析チップのＳＹＢＲ Ｇｒｅｅｎ Ｉによる蛍光強度

Fluorescence intensity of SYBR Green I of the analysis chip containing the reagent of i)

ｉｉ）の試薬を含む解析チップのＳＹＢＲ Ｇｒｅｅｎ Ｉによる蛍光強度

ii) Fluorescence intensity of SYBR Green I of the analysis chip containing the reagent of ii)

ｉｉｉ）の試薬を含む解析チップのＳＹＢＲ Ｇｒｅｅｎ Ｉによる蛍光強度

iii) Fluorescence intensity of SYBR Green I of the analysis chip containing the reagent of (iii)

8) Quantitative result Regarding i), since SYBER Green I, which is a fluorescent dye for quantifying nucleic acid, was not included, fluorescence at 522 nm was not detected.
Regarding ii) and iii), fluorescence at 522 nm was detected because it contains SYBER Green I, which is a fluorescent dye for quantifying nucleic acids. In particular, for iii), since high concentration of SYBER Green I was included, the fluorescence intensity was a high value of about 280,000.

9) Nucleic acid amplification step PCR was performed on the analysis chip containing the reagents of i) to iii) under the following conditions after the quantification step.
Initial denaturation 95 ° C: 2 minutes (denaturation 95 ° C: 30 seconds, extension 72 ° C: 16 seconds) x 35 cycles PCR stop 99 ° C: 2 minutes

10) Nucleic acid analysis step After passing through the amplification step, while maintaining the temperature at 63 ° C, the well part of each analysis chip was subjected to 20 cycles as one cycle of 30 seconds, a total of 10 minutes, at an excitation wavelength of 579 nm and a fluorescence wavelength of 595 nm. A fluorescence measurement was performed. FIG. 12 shows collectively plotted values of the detected fluorescence intensities of the analysis chips including the reagents of i) to iii).

11) Analysis results Regarding i) ii), 595 nm fluorescence of Redmond Red was detected, but for iii), fluorescence of 595 nm of Redmond Red was not detected (see FIG. 12, and in FIG. 12, i ) Control reagent solution is a curve plotted by a black circle, ii) Low concentration SYBER Green I-containing reagent solution is a white square, and iii) High concentration SYBER Green I-containing reagent solution is a curve plotted by a white triangle). This is probably because the concentration of SYBR Green I was high and the nucleic acid amplification step was inhibited.
In addition, by including at least one well containing the optimal concentration of the nucleic acid quantification reagent and the nucleic acid amplification reagent in the reaction vessel as in ii), it serves as a control of the nucleic acid analysis step, and the validity of the analysis results It is possible to detect the malfunction of the determination and analysis device.

  As described above, the embodiments of the present invention have been described in detail with reference to the drawings and examples, but the specific configuration is not limited to these embodiments, and a design within a range not departing from the gist of the present invention. Changes are also included.

  According to the reaction container, the nucleic acid analysis device, and the nucleic acid analysis method of the present invention, the analysis result is validated by processing the nucleic acid analysis step using the biological sample and the nucleic acid analysis step using the biological sample in the same well. It is possible to judge the gender and detect the malfunction of the analyzer. Therefore, it is suitably used in a fully automatic gene analysis system in which it is difficult to confirm the amount of nucleic acid obtained from a biological sample. In addition, there is a possibility that it will be widely used in the medical field, drug discovery field, and the like, and improvement in the quality of personalized medicine is expected.

REFERENCE SIGNS LIST 1 reaction well 2 control reaction well 3 nucleic acid analysis reagent 4 quantitative reagent 5 flow path 6 inlet 7 base material 10 reaction vessel 21 reaction vessel main body 23 flow path 24 main flow path 24A one end 24B other end 24C mountain shape 24D vertex 25 branch flow path 25A Flow restriction part 26 Inlet 27 Outlet 28 Protruding wall part 29 Lid O Center axis (rotation axis)

Claims (14)

A reaction container, or a nucleic acid analysis method using a nucleic acid analysis device including the reaction container,
The reaction vessel,
A substrate, on which a nucleic acid analysis reagent used for nucleic acid analysis and a quantitative reagent capable of specifically detecting nucleic acid are arranged, and at least one or more control reaction wells,
And a plurality of other reaction wells in which the nucleic acid analysis reagent is arranged,
The nucleic acid-containing sample is configured to be distributed to the control reaction well and the other plurality of reaction wells,
b1) injecting a sample containing nucleic acids into the control reaction well and the other plurality of reaction wells;
b2) forming an association state in which the nucleic acid contained in the sample and the quantification reagent are associated in the control reaction well;
b3) a measuring step of measuring a signal emitted in the association state;
b4) calculating the amount of nucleic acid contained in the sample that can be analyzed in the control reaction well and the other plurality of reaction wells from the signal measured in the measurement step;
A nucleic acid quantification step B comprising:
After the step B, a nucleic acid analysis step of injecting a sample containing the nucleic acid into the control reaction well and the other plurality of reaction wells to identify the nucleic acid in the control reaction well and the other plurality of reaction wells C and
Only including,
A nucleic acid analysis method , wherein the concentration of the quantitative reagent is a concentration that does not inhibit the reaction performed in the nucleic acid analysis step C.   The nucleic acid analysis method according to claim 1, wherein the nucleic acid analysis device further includes an extraction unit for extracting a nucleic acid. Prior to the nucleic acid quantification step B, the method further includes a nucleic acid extraction step A of extracting nucleic acid from a biological sample,
The nucleic acid analysis method according to claim 1 or 2, wherein the nucleic acids quantified and analyzed in the nucleic acid quantification step B and the nucleic acid analysis step C are the nucleic acids obtained in the nucleic acid extraction step A.   The nucleic acid analysis method according to claim 3, further comprising a nucleic acid purification step P after the nucleic acid extraction step A and before the nucleic acid quantification step B.   The nucleic acid analysis method according to any one of claims 1 to 4, wherein the nucleic acid analysis device further includes a purification unit that purifies the nucleic acid. After the nucleic acid quantification step B and before the nucleic acid analysis step C, the method further includes a nucleic acid purification step P,
The nucleic acid analysis method according to any one of claims 1 to 3, wherein the nucleic acid analysis device further includes a purification unit that purifies the nucleic acid.   The method further comprising, after the nucleic acid quantification step B and before the nucleic acid purification step P, a step J ′ of judging whether or not to shift to the nucleic acid purification step P based on the amount of the nucleic acid calculated in the nucleic acid quantification step B. Item 7. The nucleic acid analysis method according to item 4 or 6. After the nucleic acid quantification step B,
The nucleic acid analysis method according to any one of claims 1 to 7, comprising a step D of judging the validity of the analysis result obtained in the nucleic acid analysis step C based on the amount of the nucleic acid calculated in the nucleic acid quantification step B. .   From the amount of nucleic acid calculated in the nucleic acid quantification step B and the analysis result obtained in the nucleic acid analysis step C, simultaneously with the step D of judging the validity of the analysis result, a step E of detecting a defect of the nucleic acid analyzer. The nucleic acid analysis method according to any one of claims 1 to 8, further comprising:   The method according to claim 1, further comprising, after the nucleic acid quantification step B and before the nucleic acid analysis step C, a step J of determining whether to shift to the nucleic acid analysis step C based on the amount of the nucleic acid calculated in the nucleic acid quantification step B. 10. The nucleic acid analysis method according to any one of 1 to 9. The nucleic acid analysis method according to any one of claims 1 to 10 , wherein the substrate is made of a resin having light transmittance. The base material further includes a flow path and an injection port capable of injecting a solution, and the flow path distributes the nucleic acid contained in the sample to the control reaction well and the other plurality of reaction wells. The nucleic acid analysis method according to any one of claims 1 to 11 , which can be performed. The nucleic acid analysis method according to any one of claims 1 to 12 , wherein the quantitative reagent is a fluorescent substance. The nucleic acid analysis method according to any one of claims 1 to 13 , wherein the quantification reagent and / or the nucleic acid analysis reagent are arranged in a dry state.

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