JP2021019581A - Template nucleic acid molecule for preparing standard nucleic acid sample - Google Patents

Abstract

To provide a method for producing a container comprising a standard nucleic acid sample, which dispenses a desired number of copies of a nucleic acid molecule comprising a predetermined number of target base sequence regions into a container for a standard nucleic acid sample.SOLUTION: Provided is a method for dispensing into a container. The method comprises: forming more than one dispersion phase comprising a template nucleic acid molecule for preparing a standard nucleic acid sample including a target base sequence region, a base sequence region for inhibition, and a base sequence region for detection in order from the 5' end side on the same molecule, a restriction enzyme and/or a blocking oligonucleotide, and a primer pair for amplifying a base sequence region for detection; amplifying only the base sequence for detection; and subsequently dispensing a positive dispersion phase containing an amplified product into a container for standard nucleic acid sample.SELECTED DRAWING: None

Description

The present invention relates to a template nucleic acid molecule for preparing a standard nucleic acid sample, a kit for preparing a standard nucleic acid sample, and a method for producing a container containing a standard nucleic acid sample.

Nucleic acid analysis technology for amplifying a trace amount of nucleic acid present in a sample and then analyzing the obtained amplification product in detail is widely used in various fields such as food testing, blood testing, and DNA testing. Typical nucleic acid analysis techniques include, for example, the real-time polymerase chain reaction (PCR) method. The "real-time PCR method" is a PCR process in which fluorescence corresponding to the amplified nucleic acid amount is detected in a timely manner, and the detection value of the nucleic acid in the target sample is relatively compared with a standard sample series in which the copy number is specified. This is a method for quantifying nucleic acid in the sample.

In general, in order to accurately quantify a target nucleic acid by a nucleic acid amplification method such as PCR, it is necessary to remove nucleic acid amplification reaction inhibitors such as blood cells, sugars, and fats in a sample and extract only the nucleic acid. is there. In addition, when the nucleic acid of interest is RNA, a reverse transcription reaction to cDNA is required.
Therefore, in quantification, nucleic acid acquisition efficiency such as nucleic acid extraction efficiency and reverse transcription efficiency must be taken into consideration.

Conventionally, the nucleic acid acquisition efficiency has been determined by, for example, adding a standard nucleic acid sample having a defined concentration to the reaction system as an internal standard, and then performing extraction, reverse transcription reaction, and real-time PCR to determine the initial amount of nucleic acid and the efficiency of each step. Was generally considered by the method of quantifying. In this case, a standard nucleic acid sample in a region where the nucleic acid concentration is extremely low may be required as the internal standard. However, such low copy number standard nucleic acid samples are prone to variability and it is very difficult to keep the copy number contained per container in production lots constant. Therefore, various techniques have been developed for making containers containing low copy standard nucleic acid samples.

For example, in Patent Document 1 (Japanese Unexamined Patent Publication No. 2014-33658), a template nucleic acid molecule containing a target base sequence region and a detection base sequence region in one nucleic acid molecule is subjected to limit dilution, and a diluted solution obtained is obtained. A method of selecting a diluent having only one copy from the result of quantitative PCR to obtain one copy of a standard nucleic acid sample is disclosed. However, this method has a problem that only one copy of a standard nucleic acid sample can be prepared, and it is difficult to prepare a standard sample containing two or more copies of nucleic acid. In addition, there is a possibility that even the target base sequence region may be amplified due to the overrun of DNA polymerase generated when the detection base sequence region is amplified. Therefore, in Patent Document 1, a sufficiently long spacer sequence is introduced between the target base sequence region and the detection base sequence region to prevent overrun. However, if the spacer sequence becomes long, the risk of cleavage increases, and even if the detection base sequence region is detected, a state may occur in which the target base sequence region is not included. Therefore, a new problem arises in which the uncertainty as a standard sample increases.

Further, in Patent Document 2 (Japanese Unexamined Patent Publication No. 2015-195735), a DNA fragment having a specific copy number is introduced into a cell by a gene recombination technique, and the cell is manually isolated to obtain the desired copy number. A method for producing a standard reagent containing a DNA fragment is disclosed. However, this method requires a complicated process until the production of the standard nucleic acid reagent, is difficult to produce in a short time, and has a problem that the production cost is high. Furthermore, since cell-derived impurities can be mixed in the prepared standard nucleic acid sample, contamination of the inhibitor in the nucleic acid amplification reaction cannot be excluded.

Further, in Patent Document 3 (Japanese Unexamined Patent Publication No. 2008-245612), a target region of a plurality of types of template nucleic acids is amplified in a micro-compartment such as a droplet or a gel droplet, and a nucleic acid amplification reaction occurs in the micro-compartment. Is disclosed as a method of sorting by sorting. However, in this method, since a plurality of types of nucleic acids are contained in the microcompartment, it is difficult to guarantee the presence of only one type of nucleic acid molecule in the microcompartment detected positive, and the target base sequence is used. There is a problem that it is difficult to obtain a standard nucleic acid sample having a region having a desired number of molecules.

In addition, the standard nucleic acid samples produced by the above techniques are all present in solution or suspension. Therefore, there is a problem that it is difficult for the user to add a required amount of a standard nucleic acid sample to an arbitrary reaction system.

An object of the present invention is to provide a method for producing a standard nucleic acid sample-containing container containing a predetermined number of nucleic acid molecules containing a desired base sequence region in a desired number of molecules (known number of copies).

As a result of diligent research, the present inventors have succeeded in developing a novel template nucleic acid molecule that can solve the above-mentioned problems. By performing a nucleic acid amplification reaction in a container containing the template nucleic acid molecule in a desired number of copies, amplification of the target base sequence region on the template nucleic acid molecule is suppressed, and only the detection base sequence region is amplified. Is possible. Thereby, a container containing a desired base sequence region as a standard nucleic acid sample in a desired number of molecules can be produced, for example, only one copy. The present invention has been completed based on the above development results, and provides the following.

That is, the present invention provides a template nucleic acid molecule for preparing a standard nucleic acid sample, a method for producing a standard nucleic acid sample-containing container using the template nucleic acid molecule, and the like. Specifically, the target base sequence region, the inhibitory base sequence region, and the detection base sequence region are included in this order from the 5'end side, and the target base sequence region is the base sequence of the target nucleic acid as a standard nucleic acid. The inhibitory nucleotide sequence region is a region containing a region to which the blocking oligonucleotide complementarily binds and / or a restriction enzyme target site that the restriction enzyme specifically recognizes and cleaves, and is a region containing the above-mentioned target. It consists of a base sequence and a base sequence that is different from the detection base sequence region and does not self-anneal, and the detection base sequence region is a target region for nucleic acid amplification and is the same as the base sequence of the target base sequence region. Provided are template nucleic acid molecules having a composition consisting of different base sequences.

According to the template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention and the method for producing a standard nucleic acid sample-containing container using the same, a standard nucleic acid sample-containing container containing a predetermined number of nucleic acid molecules containing a predetermined number of target base sequences in a desired number of copies. Can be manufactured and provided.

FIG. 1-1 shows the configuration of the template nucleic acid molecule (110) for preparing a standard nucleic acid sample of the present invention and the amplification mechanism of the base sequence region for detection when a blocking oligonucleotide (170) is used for the template nucleic acid molecule. It is a conceptual diagram which shows. FIG. 1-2 is a conceptual diagram showing the amplification mechanism of the base sequence region for detection when a restriction enzyme (190) is used for the template nucleic acid molecule (110) for preparing a single-stranded standard nucleic acid sample of the present invention. .. FIG. 1-3 shows the amplification of the base sequence region for detection when the restriction enzyme (190) is used for the template nucleic acid molecule of the present invention when the template nucleic acid molecule (110) for preparing the standard nucleic acid sample is double-stranded. It is a conceptual diagram which shows the mechanism. FIG. 2 is a diagram showing the relationship between the copy number and the coefficient of variation CV. FIG. 3 is a conceptual diagram showing an example of a container containing a standard nucleic acid sample of the present invention. FIG. 4 is a conceptual diagram showing an example of a dispersed phase forming means for dividing a solution into dispersed phases in a microchannel. FIG. 5 is a conceptual diagram showing another example of the dispersed phase forming means for dividing the solution into dispersed phases in the microchannel. FIG. 6 is a conceptual diagram showing an example of the amplification means. FIG. 7 is a conceptual diagram showing an example of the discriminating means and the sorting mechanism in the sorting means. FIG. 8 is a conceptual diagram showing an example of a discharge mechanism in the dispensing means. FIG. 9 is an explanatory diagram showing an embodiment of a discharge mechanism and a counting mechanism in the dispensing means. FIG. 10 is a diagram illustrating a hardware block of the control unit. FIG. 11 is a diagram illustrating a functional block of the control unit. FIG. 12 is a flowchart showing an example of a processing procedure of a production program of a standard nucleic acid sample-containing container stored in a non-transient recording medium. FIG. 13 is a diagram illustrating another example of the functional block of the control unit. FIG. 14 is a flowchart showing an example of the operation of the discharge mechanism and the counting mechanism in the dispensing means. FIG. 15 is an explanatory diagram showing an example of a state immediately after the dispersed phase containing the nucleic acid having the target base sequence is dispensed into the container. FIG. 16 is an explanatory diagram showing an example of a state after a dispersed phase containing a nucleic acid having a target base sequence is dispensed into a container and the dispersed phase is dissolved. FIG. 17 is a flowchart showing another example of the processing procedure of the manufacturing program of the standard nucleic acid sample-containing container stored in the non-transient recording medium. FIG. 18 is a diagram showing an example of a result relating to the production of the standard nucleic acid sample-containing container of the present invention. FIG. 19 is a diagram showing another example of the result relating to the production of the standard nucleic acid sample-containing container of the present invention. FIG. 20 is a diagram showing an example of the operation of the sorting means according to the production of the standard nucleic acid sample-containing container of the present invention. FIG. 21 is a diagram showing an example of the operation of the sorting means according to the production of the standard nucleic acid sample-containing container of the present invention. FIG. 22 is a diagram showing an example of a result relating to the production of the standard nucleic acid sample-containing container of the present invention. FIG. 23 is a diagram showing an example of a result relating to the production of the standard nucleic acid sample-containing container of the present invention.

1. 1. Template nucleic acid molecule for standard nucleic acid sample preparation 1-1. Overview A first aspect of the present invention is a template nucleic acid molecule for preparing a standard nucleic acid sample. This template nucleic acid molecule is characterized by having a configuration including an inhibitory base sequence region between a target base sequence region and a detection base sequence region.

As shown in FIGS. 1-1 to 1-3 (hereinafter, these are often collectively referred to as "FIG. 1"), the template nucleic acid molecule (110) for preparing a standard nucleic acid sample of the present invention (in the present specification). Often simply abbreviated as "template nucleic acid molecule"), along with blocking oligonucleotides (170) and / or restriction enzymes (190), as well as predetermined primer pairs (150, 160), in a vessel containing a dispersed phase (100). It is subjected to a nucleic acid amplification reaction. Thereby, the blocking oligonucleotide hybridizes to the inhibitory base sequence region (130) in FIG. 1-1, and the inhibitory base sequence region (130) is restricted in FIGS. 1-2 and 1-3. Overrun of the amplified fragment extended from the reverse primer (160) during amplification of the detection base sequence region (140) due to cleavage of the enzyme (190) (extension reaction to the downstream region beyond the detection base sequence region) Can be suppressed or prevented. As a result, only the detection base sequence region is amplified, and a standard nucleic acid sample-containing container having a desired base sequence region (120) in a desired copy number can be produced.

1-2. Definitions The following terms that are frequently used herein are defined. The definitions of the following terms apply to all applicable terms described herein, unless otherwise noted.

(Nucleic acid)
As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a biopolymer in which nucleotides are, in principle, constituent units, and they are linked by phosphodiester bonds. Usually, DNA (including cDNA), RNA, or a combination of natural nucleic acids in which only natural nucleotides are linked is applicable, but in the present specification, non-natural nucleotides and the like are included in whole or in part. It may also contain native nucleic acids. Therefore, in the present invention, a natural nucleic acid or a non-natural nucleic acid can be appropriately selected depending on the intended purpose.

As used herein, the term "natural nucleotide" refers to a nucleotide that exists in nature. Specifically, a deoxyribonucleotide that constitutes DNA and has any of the bases of adenine, guanine, cytosine and thymine, and a ribonucleotide that constitutes RNA and has any of the bases of adenine, guanine, cytosine and uracil Applicable.

As used herein, the term "non-natural nucleotide" refers to an artificially constructed or artificially chemically modified nucleotide that does not exist in nature. In general, in addition to nucleotide-like substances having properties and / or structures similar to the natural nucleotides, non-natural nucleosides or base analogs having properties and / or structures similar to the natural nucleosides or bases, or Nucleotides containing modified bases are applicable. "Non-natural nucleosides" include, for example, debased nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and other nucleosides with sugar modifications. The "base analog" includes, for example, 2-oxo (1H) -pyridin-3-yl group, 5-position substituted-2-oxo (1H) -pyridin-3-yl group, 2-amino-6-. Examples thereof include (2-thiazolyl) purine-9-yl group, 2-amino-6- (2-thiazolyl) purine-9-yl group, 2-amino-6- (2-oxazolyl) purine-9-yl group and the like. Be done. "Modified bases" include, for example, modified pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, and 4-thiouracil, modified purines such as 6-methyladenine, and 6-thioguanosine, and other heterocycles. Ring base and the like can be mentioned.

As used herein, the term "non-natural nucleic acid" refers to an artificially constructed nucleic acid analog having a structure and / or properties similar to those of a natural nucleic acid. Examples thereof include crosslinked nucleic acids (BNA / LNA: Bridged Nucleic Acid / Locked Nucleic Acid), peptide nucleic acids (PNA: Peptide Nucleic Acid), peptide nucleic acids having phosphate groups (PHONA), morpholino nucleic acids and the like. It may contain chemically modified nucleic acids such as methylphosphonate type DNA / RNA, phosphorothioate type DNA / RNA, phosphoramidate type DNA / RNA, 2'-O-methyl type DNA / RNA, and nucleic acid analogs.

As used herein, nucleic acids may be labeled with a phosphate group, sugar and / or base, if desired. The labeling position of the labeling substance may be appropriately determined according to the characteristics of the labeling substance and the purpose of use, and is not limited, but usually 5'end and / or 3'end is preferable. As the labeling substance, any substance known in the art can be used. For example, radioisotopes, fluorescent substances, quenchers, chemiluminescent substances, DIG, biotin, magnetic beads and the like can be mentioned. The "radioactive isotope" refers to an element that emits radiation among isotopes having different mass numbers. For example, ³² P, ³³ P, or ³⁵ S can be mentioned.

The "fluorescent substance" refers to a substance having the property of being excited by absorbing excitation light of a specific wavelength and emitting fluorescence when returning to the original ground state. For example, FITC, Texas, Texas Red (registered trademark), cy3, cy5, cy7, FAM, HEX, VIC (registered trademark), JOE, ROX, TET, Bodypy493, NBD, TAMRA, Quasar (registered trademark) 670, Quasar (registered trademark). 705, CAL Fluor® Red610, SYBR Green®, Eva Green®, SYSTEM Green®, fluorescein or derivatives thereof, fluorescein or derivatives thereof, azos, or rhodamine or Examples thereof include its derivatives, coumarin or its derivatives, pyrene or its derivatives, cyanine or its derivatives and the like. These may be used alone or in combination of two or more.

The "quencher" refers to a substance having a property of absorbing the excitation energy of the fluorescent substance and suppressing fluorescence. For example, AMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 and the like can be mentioned. The "chemiluminescent substance" refers to a substance having the property of emitting differential energy as light when returning to the ground state after being excited by a chemical reaction. For example, acridinium ester and the like can be mentioned.

As used herein, the shape of the nucleic acid is not limited. Appropriate shape can be taken as needed. For example, it may be a linear nucleic acid such as an oligonucleotide, or it may be a cyclic nucleic acid such as a plasmid.

In the present specification, the nucleic acid may be in a state of being encapsulated in a dispersed phase or a state of being supported in a dispersed phase.

As used herein, the term "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several tens of hydroxyl groups and phosphate groups of a sugar moiety between adjacent nucleotides.

As used herein, the term "standard nucleic acid sample" refers to a standard substance composed of nucleic acids. A "reference material" (RM) is a reference substance for determining a measured value in the measurement of a chemical substance.

(copy)
In the art, "copy" refers to a nucleic acid molecule that is a replica of a given base sequence as a basic unit.

In the present invention, the "copy number" means the number of basic units when the predetermined base sequence is used as the basic unit. Usually, the number of template-replicating nucleic acid molecules obtained by the nucleic acid amplification method using the basic unit as a template corresponds. However, in the present specification, not only the replicated nucleic acid molecule but also the template nucleic acid molecule is counted as one copy. As a general rule, the sense strand is counted, but before the nucleic acid amplification reaction, the double-stranded nucleic acid molecule is counted as two copies because both the sense strand and the antisense strand can serve as templates.

In addition, in this specification, a copy can be paraphrased as a "molecule". The term "molecule" in this case is used to mean the same basic unit as a copy. For example, having one copy of the target base sequence region can be paraphrased as having one molecule of the target base sequence region (dispersed phase).
As used herein, the term "dispersed phase" refers to compartments divided into minute sizes, in which individual compartments can be dispersed in space. For example, the section indicated by "100" in FIG. 1 corresponds to this.

The form of the dispersed phase is not particularly limited and can be appropriately selected depending on the intended purpose. For example, a liquid phase dispersed phase, a solid phase dispersed phase, and the like can be mentioned. In the present specification, the dispersed phase of the liquid phase is often referred to as "liquid phase dispersed phase". In addition, the dispersed phase of the solid phase is often referred to as "solid phase dispersed phase".

In the case of a liquid phase dispersed phase, it is preferable that the solution divided into minute sections such as droplets forms a dispersed phase by a continuous phase.

"Continuous phase" refers to a liquid present in a dispersion system in which other liquids are dispersed. In the present specification, it means a dispersion medium in which a liquid phase dispersed phase is dispersed. The continuous phase can be used to transport the liquid phase dispersed phase.

The continuous phase is not particularly limited as long as the solution can be divided, and can be appropriately selected depending on the intended purpose. For example, when the dispersed phase is aqueous, fluorocarbon-based oil, mineral oil and the like can be mentioned.

When an aqueous solution is classified into a dispersed phase using an oily continuous phase, it is preferable to add a surfactant to either the solution or the continuous phase. By adding a surfactant, the stability of the partitioned dispersed phase can be improved.

The surfactant is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include an ionic surfactant and a nonionic surfactant. These may be used alone or in combination of two or more.

Examples of the ionic surfactant include fatty acid sodium, fatty acid potassium, alpha sulfo fatty acid ester sodium, linear alkylbenzene sulfonate sodium, alkyl sulfate ester sodium, alkyl ether sulfate sodium, alpha olefin sulfonate sodium, Pico-Surf1 and the like. Can be mentioned. These may be used alone or in combination of two or more.

Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (Brij series, etc.), octylphenol ethoxylates (Triton X series, Igepal CA series, Nonidet P series, Nikkol OP series, etc.), polysorbates (, etc.) (Tween series such as Tween 20), sorbitan fatty acid ester, polyoxyethylene fatty acid ester, alkylmaltoside, sucrose fatty acid ester, glycoside fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, fatty acid monoglyceride and the like. These may be used alone or in combination of two or more.

Specific examples of the liquid phase dispersed phase formed by the continuous phase include water droplets in oil (W / O type) or oil droplets in water (O / W type) forming a water-oil emulsion, micelles and the like. In the case of the W / O type, the oily solvent corresponds to the continuous phase, and the water droplets of the aqueous solution correspond to the liquid phase dispersed phase. Further, the micelle corresponds to the liquid phase dispersed phase formed by the addition of the surfactant when the liquid phase dispersed phase is W / O type.

Examples of the solid phase dispersed phase include a substance that captures nucleic acid in a dispersed phase solution on a molecular basis on particles capable of capturing nucleic acid. The particles capable of capturing nucleic acid are not particularly limited and can be appropriately selected depending on the intended purpose. For example, particles having a surface modified with a capturing nucleic acid probe, silica particles that adsorb nucleic acid in a solid layer in the presence of a chaotropic substance, and the like can be mentioned.

The "capture nucleic acid probe" refers to a nucleic acid probe capable of capturing a nucleic acid molecule by specifically binding to a target base sequence region in the template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention.

The "particles having the capture nucleic acid probe modified on the surface" are not particularly limited as long as the capture nucleic acid probe is modified on the surface, and can be appropriately selected depending on the intended purpose. For example, metals, silicon, glass materials, synthetic resins, polysaccharides and the like can be mentioned. The metal includes pure metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium and nickel, and alloys such as stainless steel, Hastelloy, Inconel, Monel and duralumin. Glass materials include quartz glass, fused silica, synthetic quartz, alumina, sapphire glass, ceramics, forsterite, photosensitive glass and the like. Synthetic resins include polyester resin, polystyrene, polyethylene resin, polypropylene resin, polyvinyl alcohol, ABS resin (Acrylonirile Butadiene Style resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, and melamine resin. , Epoxy resin, vinyl chloride resin, etc. are included. Polysaccharides include agarose (agar), mannan, dextran, cellulose, chitin, chitosan and the like, as well as derivatives such as nitrocellulose. These may be used alone or in combination of two or more. Further, in order to automate, improve the efficiency, or speed up the separation process and the like, magnetized particles, particles containing a magnetic substance, or magnetizable magnetic beads can also be used.
The shape of the dispersed phase is not particularly limited, and examples thereof include a spherical shape such as a water droplet.

1-3. Structure The structure of the template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention is shown in “110” in FIG. As shown in this figure, the template nucleic acid molecule (110) for preparing a standard nucleic acid sample of the present invention has a target base sequence region (120) and an inhibitory base sequence region (120) in order from the 5'terminal side as essential components. 130) and the base sequence region for detection (140). Hereinafter, each component will be specifically described with reference to FIG.

1-3-1. Target Base Sequence Region The “target base sequence region” (often referred to as “target base sequence region” in the present specification) (120) is a region containing the base sequence of the target nucleic acid, and is shown in FIG. It corresponds to the region indicated by "A" in FIGS. 4 to 8 and 16.

The "target nucleic acid" is a nucleic acid that should be used as a standard nucleic acid sample in the present specification. The base sequence of the target nucleic acid can be any base sequence required as a standard nucleic acid sample. Therefore, there is no limitation, and a desired sequence can be appropriately selected according to the intended use. For example, it may be a base sequence used in a known standard nucleic acid sample, a whole base sequence or a part of a base sequence of a desired gene, a part of a base sequence on the genome, or an arbitrary base sequence that does not exist in nature. You may. More specifically, for example, a base sequence of a gene or the like used for an infectious disease test, or a base sequence of a nucleic acid extracted from an animal cell or a plant cell can be mentioned. When using a base sequence such as a gene used for an infectious disease test, there is no particular limitation as long as it contains a base sequence peculiar to the infectious disease causative gene, but the base sequence specified by the official method or the notification method can be used. It is more preferable if it is included.

The base length of the target base sequence is not particularly limited, and a desired length can be appropriately selected depending on the purpose. For example, if it is short, it may be several bases (for example, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases), and if it is long, it may be genomic DNA. It may range from 5 million bases to 3 billion bases. Usually, a range of 20 bases or more and 10000 bases or less, 100 bases or more and 8000 bases or less, or 500 bases or more and 5000 bases or less is appropriate.

The target nucleic acid contained in one target base sequence region may be one or two or more. When two or more target sequences are contained, they may be the same or different, and when three or more are contained, they may be a combination thereof. It is preferably one.

In the standard nucleic acid sample preparation template nucleic acid molecule, since the detection base sequence (140) described later is an amplification target, the number of copies increases before and after the nucleic acid amplification reaction, but the target base sequence region existing in the same molecule is the present invention. In principle, the number of copies does not change before and after the amplification reaction because it is not an amplification target.

The target base sequence region is arranged on the most 5'terminal side of the above three essential components in the template nucleic acid molecule for preparing a standard nucleic acid sample.

1-3-2. Nucleotide Sequence Region for Inhibition The "nucleotide sequence region for inhibition" (130) is a region in which a blocking oligonucleotide (170) binds complementarily and / or a region containing a restriction enzyme target site, and the nucleotide sequence thereof is It consists of a base sequence that is different from the target base sequence and the base sequence region for detection and is not self-annealed. In FIGS. 1, 4 to 8, and 16, it corresponds to a short black-painted small area C existing between AB.

The "restriction enzyme target site" is a target site for the restriction enzyme (190) to recognize and cleave, and refers to a so-called restriction site. The restriction enzyme target site is, in principle, strictly determined by the type of restriction enzyme that recognizes and cleaves it. Therefore, the base sequence and the number of bases of the restriction enzyme target site may be appropriately determined depending on the type of restriction enzyme used when preparing a standard nucleic acid sample using the template nucleic acid molecule of the present invention. Although not limited, the restriction sites of type II restriction enzymes frequently used in the field of molecular biology generally have a palindromic sequence and are 4 bases, 6 bases, 8 bases, 10 bases, or 12 bases. Often configured. Therefore, the restriction enzyme target site may have a similar configuration. The specific restriction sites of each restriction enzyme are known in the field of molecular biology, and the product catalogs of each life science manufacturer that handles restriction enzymes may be referred to.

The restriction enzyme target site may be composed of either single-strand or double-strand. The restriction site of the restriction enzyme to be used may be appropriately determined according to whether it is single-strand recognition or double-strand recognition. In many cases, restriction enzymes recognize double-stranded restriction sites. In that case, as shown in FIG. 1-3, at least the restriction enzyme target site of the inhibitory base sequence region in the template nucleic acid molecule of the present invention should be composed of double strands. However, this double-stranded region may be formed at the time of preparation of the standard nucleic acid sample. For example, when the template nucleic acid molecule is alone, it is composed of a single strand, and when preparing a standard nucleic acid sample, the mixed blocking oligonucleotides hybridize to form a double-stranded region containing a restriction enzyme target site. It may be configured in. When the restriction enzyme target site is composed of two strands, the cut end by the restriction enzyme is a cohesive / sticky end or a blunt end, but in the template nucleic acid molecule of the present invention, Restriction enzyme If the target site is cleaved by the restriction enzyme, it may be any end.

"Different from the target base sequence and the detection base sequence region" means that the base sequence of the inhibition base sequence region is 4 or more bases continuous with the base sequence of the target base sequence region and the detection base sequence region. It means that it does not contain the same base sequence of bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, or 10 bases or more. As a result, when the template nucleic acid molecule for preparing the standard nucleic acid sample of the present invention, the blocking oligonucleotide and / or the restriction enzyme, and the primer set (150, 160) are mixed, the target base sequence on the template nucleic acid molecule for preparing the standard nucleic acid sample is obtained. Blocking oriconucleotides cannot bind non-specifically to regions and detection base sequence regions, and restriction enzymes cannot cleave those regions. As a result, blocking oligonucleotides and restriction enzymes act only on the inhibitory base sequence region.

"Self-annealing" means base pairing of complementary base sequences within a single-stranded nucleic acid molecule. As a result, the single-stranded nucleic acid molecule self-folds to form a secondary structure. The inhibitory base sequence region is a region to which the blocking oligonucleotide complementarily binds and / or is a region containing a restriction enzyme target site that the restriction enzyme recognizes and cleaves. Therefore, the base sequence must not suppress or inhibit the binding of blocking oligonucleotides and / or the recognition by restriction enzymes by self-folding.

The base length of the inhibitory base sequence region is not particularly limited. However, when the number of bases is 100 or more, the risk of non-specific cleavage by endonuclease or the like increases. On the contrary, if it is 6 bases or less, the hybridization stability by the blocking oligonucleotide becomes low, and if it is 3 bases or less, specific recognition and cleavage by the restriction enzyme become difficult. Therefore, DNA polymerase is used when amplifying the detection base sequence region described later. There is a high risk that the overrun will not be prevented. Therefore, when it contains a restriction enzyme target site, it is 4 bases or more and less than 100 bases, or 6 bases or more and less than 100 bases, and when it contains a region to which a blocking oligonucleotide complementarily binds, it is 7 bases or more and less than 100 bases, and in any of the above cases, 8 Bases to 90 bases, 9 bases to 80 bases, 10 bases to 70 bases, 15 bases to 65 bases, 18 bases to 60 bases, 20 bases to 60 bases, 23 bases to 55 bases, or 25 It is preferably bases or more and 50 bases or less.

The base sequence of the inhibitory base sequence region is not particularly limited. However, if a blocking oligonucleotide is used when preparing a standard nucleic acid sample using the template nucleic acid molecule of the present invention, the blocking oligonucleotide can stably hybridize during the amplification reaction of the detection base sequence region. As described above, the GC content is preferably 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more. If a restriction enzyme is used, it may contain the base sequence of the restriction enzyme target site of the restriction enzyme used as described above.

The inhibitory base sequence region usually needs to be configured so that one blocking oligonucleotide can hybridize, but if necessary, two or more blocking oligonucleotides of the same and / or different types are used. It may be configured to be hybridizable.

In addition, the inhibitory base sequence region usually needs to contain one restriction enzyme target site, but may include a plurality of the same and / or different restriction enzyme target sites, if necessary. When a plurality of restriction enzyme target sites are contained, they may be present separately from each other on the inhibitory base sequence region, or may be present in whole or in part in duplicate.

The inhibitory base sequence region is arranged between the target base sequence region and the detection base sequence region in the standard nucleic acid sample preparation template nucleic acid molecule. However, when the plasmid nucleic acid molecule of the present invention exhibits a cyclic structure, for example, when the 5'end of the target base sequence region described above and the 3'end of the detection base sequence region described below are linked. Alternatively, when it is included in a cyclic nucleic acid, for example, when it is included in a vector such as a plasmid, a second is provided between the 3'end of the detection base sequence region and the 5'end of the target base sequence region. Can include a base sequence region for inhibition of. In this case, the basic configuration of the second inhibitory base sequence region conforms to the configuration of the inhibitory base sequence region. The specific configurations of the inhibitory base sequence region and the second inhibitory base sequence region may be the same or different.

The composition of the blocking oligonucleotide and the restriction enzyme will be described in detail in the second aspect.

1-3-3. Nucleotide sequence region for detection The "nucleotide sequence region for detection" (140) is a region targeted for nucleic acid amplification in a template nucleic acid molecule (110) for preparing a standard nucleic acid sample, and the base sequence thereof is a target base sequence region. It has a base sequence different from that of (120), and corresponds to the region indicated by "B" in FIGS. 1, 4 to 8, and 16.

"Different from the target base sequence region" means that the base sequence of the detection base sequence region is 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more continuous with the base sequence of the target base sequence region. , 9 bases or more, or 10 bases or more does not contain the same base sequence. As a result, the amplification primer pair (150, 160) of the detection base sequence region cannot hybridize to the target base sequence region, so that the target base sequence region is not amplified in the nucleic acid amplification reaction, and the detection base is not amplified. Only the sequence region is amplified (180).

The base length of the base sequence region for detection is not particularly limited, but if it is less than 20 bases, the primer pair cannot be stably hybridized, and there is a high possibility that the amount of nucleic acid sufficient for detection cannot be amplified in the nucleic acid amplification reaction. Considering that the amplification efficiency decreases when the number of bases exceeds, and that there is no need to amplify such a long base length in the first place, 20 bases or more and 1000 bases or less, 30 bases or more and 900 bases or less, 40 bases or more and 800 bases Hereinafter, 50 bases or more and 700 bases or less, 60 bases or more and 600 bases or less, or 70 bases or more and 500 bases or less are preferable.

The base sequence for detection is not particularly limited as long as a primer pair capable of amplifying the region can hybridize. It may be either the entire base sequence or a partial base sequence of the desired gene, or any base sequence that does not exist in nature. However, in the template nucleic acid molecule for preparing a standard nucleic acid sample, the base sequence region for detection is the base sequence region that is the target of the nucleic acid amplification reaction. Therefore, the GC content is 30% or more and 70% or less, 40% or more and 68% or less, and 45% so that primer hybridization (annealing) and denaturation of the double-stranded amplified fragment are smoothly performed during the acid amplification reaction. It is preferably 65% or more, or 50% or more and 60% or less.

The detection base sequence region may include a plurality of amplification target base sequences to be detected in the region. In that case, each base sequence for amplification target may contain the same and / or different base sequences. When the detection base sequence region contains two or more base sequences for amplification and their base sequences are different from each other, a plurality of sets of primer pairs that specifically recognize each base sequence are configured to be able to hybridize. .. Further, even when only one base sequence for amplification is present in the detection base sequence region, it may be configured so that it can be amplified by a plurality of sets of primer pairs such as a nested primer.

The base sequence region for detection is arranged on the most 3'terminal side of the three essential components in the template nucleic acid molecule for preparing a standard nucleic acid sample.

1-3-4. Other Components The template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention may contain other components as selective components in addition to the above three essential components. Specifically, for example, a spacer region and a connecting region can be mentioned. In addition, the template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention can contain a labeling substance. Hereinafter, each component will be described.

(1) Spacer region The “spacer region” is a region for preventing overrun of DNA polymerase during a nucleic acid amplification reaction of a detection base sequence region in a template nucleic acid molecule for preparing a standard nucleic acid sample. Therefore, in the standard nucleic acid sample preparation template nucleic acid molecule, the target base sequence region is placed at a position physically separated from the detection base sequence region, that is, between the target base sequence region and the detection base sequence region. Similarly, the position with the inhibitory base sequence region arranged between those regions does not matter. It may be arranged on the 5'end side of the inhibitory base sequence region, or may be arranged on the 3'end side. Preferably, it is on the 3'end side closer to the detection base sequence region.

The base length of the spacer region is not particularly limited, but as described above, when the spacer region is 500 bases or more, the risk of cleavage increases, and the template nucleic acid molecule for preparing a standard nucleic acid sample is the target base sequence region and the detection base. It is divided between the sequence regions. Therefore, if it is less than 500 bases, 400 bases or less, 300 bases or less, 200 bases or less, or 100 bases or less, this is preferable.

(2) Linkage region The “linkage region” refers to the connection of each of the regions (target base sequence region, inhibition base sequence region, detection base sequence region, and spacer region) in the template nucleic acid molecule for standard nucleic acid sample preparation. An intervening area. Normally, the regions are directly linked by a phosphodiester bond, but all or part of the linking portion thereof may be indirectly linked via this linking region.

The base length of the linking region is not particularly limited, but 1 base or more and 15 bases or less, 2 bases or more and 12 bases or less, or 3 bases or more and 10 bases or less is sufficient.

The base sequence of the linking region is not particularly limited, but when it is composed of 4 or more bases, the base sequence is not included in each of the regions.

(3) Labeling substance Each region of the template nucleic acid molecule for preparing a standard nucleic acid sample may be labeled with the above-mentioned labeling substance, if necessary. The labeling position and type of the labeling substance may be appropriately determined according to the characteristics of the labeling substance and the purpose of use, and is not limited.

1-3-5. Structure of the entire template nucleic acid molecule for preparing a standard nucleic acid sample The form of the template nucleic acid molecule (110) for preparing a standard nucleic acid sample is not particularly limited, and various forms can be appropriately selected depending on the intended purpose. For example, all of the template nucleic acid molecules for preparing a standard nucleic acid sample may be composed of a single strand as shown in FIGS. 1-1 and 1-2, or may be composed of a double strand as shown in FIG. 1-3. It may be composed of. It may be configured in a form in which single strand and double strand are mixed. When composed of a single strand, it may be either ssDNA, ssRNA, or a hybrid of ssDNA and ssRNA. Similarly, when it is composed of double strands, it may be any of dsDNA, dsRNA, double strands in which DNA and RNA are mixed, double strands consisting of ssDNA strands and ssRNA strands, and the like.

As a form in which single-stranded and double-stranded are mixed, for example, when the inhibitory base sequence region contains a restriction enzyme target site of a restriction enzyme for double-strand recognition as described above, only the inhibitory base sequence region is present. In some cases, it is composed of double strands and the other regions are composed of single strands. Alternatively, there is a case where the target base sequence region and the inhibitory base sequence region are composed of a single strand, and the detection base sequence region is composed of a double strand. Although not limited, the preferred form of the template nucleic acid molecule for preparing a standard nucleic acid sample is at least when the target base sequence region is single-strand. This is because a single strand having one copy of the target base sequence region can provide a standard nucleic acid sample with higher accuracy.

The base length of the standard nucleic acid sample preparation template nucleic acid molecule is the sum of the above regions. Although not limited, usually 45 bases or more and 3000 bases or less, 50 bases or more and 2500 bases or less, 80 bases or more and 2300 bases or less, 100 bases or more and 2000 bases or less, 200 bases or more and 1800 bases or less, 400 bases or more and 1500 bases or less, or It is 500 bases or more and 1200 bases or less.

1-4. Effect According to the template nucleic acid molecule for preparing a standard nucleic acid sample of the present invention, in the method for producing a standard nucleic acid sample-containing container according to the fifth aspect described later, only the base sequence region for detection is used without changing the number of copies of the target base sequence region. Can be amplified and detected. Thereby, the copy number of the target base sequence to be included in the standard nucleic acid sample-containing container of the present invention can be controlled with high accuracy.

2. 2. Kit for preparing standard nucleic acid samples 2-1. Overview A second aspect of the present invention is a standard nucleic acid sample preparation kit. For this kit, the template nucleic acid molecule for preparing the standard nucleic acid sample described in the first aspect, the blocking oligonucleotide, and the primer pair capable of amplifying the base sequence region for detection are essential components, and the container for the standard nucleic acid sample is selected. Included as a component.
According to the standard nucleic acid sample preparation kit of the present invention, a desired standard nucleic acid sample can be easily prepared.

2-2. Structure The standard nucleic acid sample preparation kit of the present invention includes a template nucleic acid molecule for standard nucleic acid sample preparation, a blocking oligonucleotide, and a primer pair as essential components, and a container for standard nucleic acid samples and the like as selective components. Hereinafter, each component will be specifically described.

2-2-1. The template nucleic acid molecule for preparing a standard nucleic acid sample The “template nucleic acid molecule for preparing a standard nucleic acid sample” has the constitution of the template nucleic acid molecule for preparing a standard nucleic acid sample according to the first aspect. Therefore, a specific description here will be omitted.

2-2-2. Blocking oligonucleotide The "blocking oligonucleotide" in this embodiment is an oligonucleotide that can complementarily bind to the inhibitory base sequence region of the template nucleic acid molecule for preparing a standard nucleic acid sample.

In principle, the base length and base sequence of the blocking oligonucleotide are determined by the base length and base sequence of the inhibitory base sequence region. However, when the blocking oligonucleotide undergoes an extension reaction in the nucleic acid amplification reaction of the detection nucleic acid sequence region, its base length and base sequence can be varied as long as it can continue to bind to the inhibitory base sequence region. For example, the base length of the blocking oligonucleotide may be shorter or longer than the base length of the inhibitory base sequence region. As an example of a long case, there is a case where the nucleotide sequence has such that a loop is formed in the blocking oligonucleotide when it is complementarily bound to the inhibitory nucleotide sequence region. In addition, the complement ratio of the inhibitory base sequence region to the base sequence is such that the blocking oligonucleotide can continue to bind to the inhibitory base sequence region via base pairing during the extension reaction in the nucleic acid amplification reaction of the detection nucleic acid sequence region. It is sufficient as long as it can provide the nucleotide. For example, complementarity is 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95. It may be% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The preferred complement ratio is 100%.

The blocking oligonucleotide consists of native nucleotides and / or non-natural nucleotides. In order to avoid the blocking oligonucleotide being used as a primer, it is desirable that the blocking oligonucleotide is composed of a non-natural nucleic acid having a full length or a part of the non-natural nucleotide. When partly composed of non-natural nucleotides, the non-natural nucleotides in the blocking oligonucleotide are preferably located at the 3'end and / or in the vicinity and have 3'→ 5'exonuclease resistance. When the blocking oligonucleotide contains two or more non-natural nucleotides, they can be of the same or different types. Although not particularly limited, it is usually preferably any one or more of BNA, LNA, and PNA. A particularly preferred form of blocking oligonucleotide is an oligonucleotide whose entire sequence is composed of PNA.

The blocking oligonucleotide increases the Tm value by complementarily binding to the inhibitory base sequence region, and functions as a stopper for preventing overrun of DNA polymerase during the nucleic acid amplification reaction of the detection base sequence region. Thereby, in the method for producing a standard nucleic acid sample-containing container according to the fifth aspect, the accuracy of the copy number of the target base sequence region contained in one container can be improved.

2-2-3. Restriction enzyme The "restriction enzyme" in this embodiment refers to an endonuclease capable of specifically recognizing and cleaving a restriction enzyme target site in the inhibitory base sequence region of the template nucleic acid molecule for preparing a standard nucleic acid sample. It may be either a DNA endonuclease or an RNA endonuclease. It is preferably a DNA endonuclease. Various types of restriction enzymes are known, but any restriction enzyme can be used by incorporating the base sequence of the restriction site (restriction enzyme target site) recognized by the restriction enzyme used into the inhibitory base sequence region. Can be done. Although not limited, various types of type II restriction enzymes, which are often used in the field of molecular biology, are commercially available, and the most appropriate conditions for determining the conditions have been clarified, which is convenient. In the template nucleic acid molecule of the present invention, as a general rule, only the restriction enzyme target site is cleaved by the restriction enzyme of the present embodiment. Therefore, a restriction enzyme with high specificity is preferable to a restriction enzyme with low specificity for the restriction enzyme target site.

When a restriction enzyme is used in the standard nucleic acid sample preparation kit of this embodiment, the restriction enzyme reagent required for the restriction enzyme to cleave the restriction enzyme target site of the template nucleic acid molecule is used as a selective component. Can be included. For example, when a type II restriction enzyme is used as a restriction enzyme, Mg ²⁺ or a Basal Buffer with an optimum salt concentration may be contained as the restriction enzyme reagent.

2-2-4. Primer pair The "primer pair" in this embodiment means a primer pair capable of amplifying the detection base sequence region of a template nucleic acid molecule for preparing a standard nucleic acid sample.

The primer pair is composed of a forward primer (referred to as "Fw primer" in the present specification) and a reverse primer (referred to as "Rv primer" in the present specification). Each primer is composed of native and / or non-natural nucleotides. It is usually composed of DNA or natural nucleotides of RNA. Among them, DNA having high stability, easy synthesis and low cost is particularly preferable. In addition, natural nucleotides of DNA and RNA can be combined, and non-natural nucleotides such as LNA can be partially combined, if necessary.

The base sequence and base length of the Fw primer and Rv primer are not particularly limited as long as they are configured to be capable of amplifying the detection base sequence region. Normally, each base sequence and base length are designed so that the region to be amplified in the detection base sequence region is sandwiched between both primers. Although not limited, the Tm value should be in the range of 55 ° C. or higher and 80 ° C. or lower, preferably 60 ° C. or higher and 75 ° C. or lower, and continuous 18 to 35 bases or 19 bases in the detection base sequence region. A base length and base sequence that can be hybridized to a base sequence of 34 bases or less, 20 bases or more and 33 bases or less, 21 bases or more and 32 bases or less, 22 bases or more and 31 bases or less, or 23 bases or more and 30 bases or less. , Preferably designed.

In the standard nucleic acid sample preparation kit, two or more sets of primer pairs may be included. The case where a plurality of primer pairs are required is not particularly limited, but includes a case where the detection base sequence region is amplified by a nested primer or a case where the detection base sequence region includes a plurality of detection target base sequences. There are cases.

The standard nucleic acid sample preparation kit of this embodiment can include, together with the primer pair, an amplification reagent necessary for amplifying the detection base sequence region of the standard nucleic acid sample preparation template nucleic acid molecule as a selective component. .. For example, DNA polymerase (thermostable DNA polymerase), dNTPs, PCR Buffer, Mg ^{2+ and the} like can be mentioned. In addition, when an RT reaction is required, such as when the detection base sequence region is composed of RNA, reverse transcriptase can also be included.

2-2-5. Container for standard nucleic acid sample A "container for standard nucleic acid sample" is a container that constitutes a container containing a standard nucleic acid sample and can contain a standard nucleic acid sample, and is a selective component in the kit for preparing a standard nucleic acid sample of the present invention. is there. The shape, material, color, size / volume of the standard nucleic acid sample container are not particularly limited and can be appropriately selected depending on the intended purpose.

(1) Shape The shape of the standard nucleic acid sample container is not particularly limited as long as an amplifyable reagent can be arranged, and can be appropriately selected according to the purpose. For example, flat bottom, round bottom, U bottom, V. Examples thereof include a concave container having a bottom and the like, a flat plate container having a section, a spherical container and the like. Further, the container may be either an open container having an opening / closing portion or a closed container.

(Concave container)
The concave container is not particularly limited, and examples thereof include a tube, a well, and a dish. The number of recesses in the concave container may be plural. The number of recesses in the standard nucleic acid sample container is preferably a plurality. 5 or more is more preferable, and 25 or more, or 48 or more is further preferable. When there are a plurality of recesses, each recess can be connected by a base material. As a specific example of a concave container having a plurality of recesses, a microtube, a multi-well plate, or the like can be preferably used. The microtube may be, for example, any of 2 stations, 3 stations, 4 stations, 6 stations, 8 stations, 12 stations, 16 stations, 24 stations, or 48 stations. The multi-well plate may be, for example, 24 holes, 48 holes, 96 holes, 384 holes, or 1536 holes.

(Flat container)
The flat plate container is not particularly limited, and examples thereof include chips and plates.
The number of compartments in the flat plate container may be plural. The number of compartments for the standard nucleic acid sample container is preferably a plurality. 5 or more is more preferable, and 25 or more, or 48 or more is further preferable. The flat plate container may have a structure in which a section is formed on a flat base material.

Examples of the spherical container include a particulate container and the like. The particulate container is particularly preferable as a container shape because it is excellent in storage stability and operability, and by including the copy number of a predetermined base sequence region per particle, the copy number can be easily adjusted by the number of particles. ..

(Particulate container)
As the particulate container, an outer shell container, an inclusion container, or the like can be used.
An "outer shell container" is composed of particles having a space inside which can contain the contents. For example, capsule-shaped particles can be mentioned.

The "encapsulation container" consists of solid or gel particles that can take up the contents. Examples of the solid particles include tablet-shaped particles containing dispersed phases containing the above-mentioned essential components. Specifically, for example, pharmaceutical capsules, liposomes, microcapsules and the like are applicable. Examples of the gel-like particles include particles containing sodium alginate, agarose gel and the like. Specifically, for example, artificial salmon roe and the like can be mentioned.

The particulate container may have a dispersed phase including the above-mentioned essential components, a continuous phase, and a protective member containing the dispersed phase and the continuous phase.

(2) Material The material of the standard nucleic acid sample container is not particularly limited and can be appropriately selected depending on the intended purpose. For example, water-soluble materials, water-insoluble materials and the like can be mentioned.

As used herein, the term "water-soluble material" refers to a material that is composed of a substance that is soluble in water and is solid in a dry environment. "Dry environment" means an environment under standard conditions (atmospheric pressure conditions of 15 ° C. or higher and 25 ° C. or lower) and humidity of 50% or lower, preferably 40% or lower, 30% or lower, 20% or lower, or 10% or lower. To say. Specific examples of the water-soluble material include sodium alginate, polysaccharides (including agar, starch, cellulose, mannan and the like), gelatin, starch, pullulan and the like. The water-soluble material is not limited, but may be a combination of two or more kinds of water-soluble materials. Further, the water-soluble material may be not only soluble in water (pure water) but also soluble in an aqueous solution containing one or more solutes.

As used herein, the term "water-insoluble material" refers to a material that is composed of a substance that is insoluble in water and is a solid material under normal temperature and pressure. Examples of the water-insoluble material include natural resin, synthetic resin, synthetic rubber and the like, and any one of them or a combination thereof may be used. Examples of the natural resin include lacquer, rosin, latex (natural rubber), shellac and the like. Synthetic resins are also called plastics, and include thermosetting resins and thermoplastic resins. Examples of the thermosetting resin include epoxy resin, unsaturated polyester resin, vinyl ester resin, phenol resin and the like. Examples of the thermoplastic resin include polyethylene, polypropylene, polyester, polystyrene, polyvinyl chloride, methacrylic resin, fluororesin, polycarbonate, polyurethane, polyethylene terephthalate, aromatic polyetherketone resin, and polyphenylene sulfide resin. Examples of synthetic rubber include butadiene rubber, chloroprene rubber, styrene butadiene rubber, isoprene rubber, ethylene propylene rubber, nitrile rubber, silicone rubber, acrylic rubber, fluororubber, and urethane rubber.

(3) Color The color of the standard nucleic acid sample container is not particularly limited. For example, transparent, translucent, colored, completely shaded and the like. When the container for the standard nucleic acid sample is colored, the user can visually recognize the information of the container for the standard nucleic acid sample by coloring, and the operability can be improved.

(4) Size / Volume The size and volume of the standard nucleic acid sample container are not particularly limited and can be appropriately selected according to the purpose.

For example, the size is not limited when the container for the standard nucleic acid sample is a particulate container, but for example, in the case of a particulate container, the minor axis length is larger than 0.0 mm and 5.0 mm or less, 0. It is preferably greater than 0 mm and 3.0 mm or less, and preferably 0.1 mm or more and 1.0 mm or less. When the minor axis length of the particulate container is 0.01 mm or more, the container can be made into a container having excellent operability such as picking by the user.

The volume is not particularly limited and can be appropriately selected depending on the intended purpose. For example, when a container for a standard nucleic acid sample is used as a reaction container for a nucleic acid test device, it is preferably 10 μL or more and 1000 μL or less in consideration of the amount of the sample. However, when a particulate container is added to the reaction system as a container for a standard nucleic acid sample, 200 μL or less is preferable, 20 μL or less is more preferable, and 2 μL or less is further preferable. This is because the volume change of the reaction system in the container can be reduced by reducing the volume of the standard nucleic acid sample container to 200 μL or less.

In the standard nucleic acid sample container of the present invention, the nucleic acid is preferably in a dry state or in a state of being dispersed in a solution. In the dry state, the possibility that nucleic acid is decomposed by an enzyme can be reduced, and the nucleic acid can be stored at room temperature.

(5) Base material A container for a standard nucleic acid sample can be constructed on a base material.
The "base material" refers to a substance that has rigidity in itself and functions as a support capable of imparting a certain shape to a container for a standard nucleic acid sample. For example, when the container for a standard nucleic acid sample is in a thin layer state, the container itself does not have rigidity. Further, when the concave container described above has a plurality of recesses, the recesses are not integrated. In such a case, by arranging the container for the standard nucleic acid sample on the surface of the base material or connecting the recesses via the base material, a certain shape including rigidity and integration is imparted.

The material of the base material is not particularly limited as long as it has a rigidity sufficient to maintain a certain shape, and can be appropriately selected depending on the intended purpose. For example, semiconductors, glass, quartz glass, plastics, synthetic rubber, ceramics, metals, stones, plant pieces (including paper and cellulose), animal pieces (including bone pieces, shells and sponges) or carbon-based materials (carbon fiber, (Including carbon rods). Plastics are preferred because they are easy to process into the desired shape, do not rot, are durable, and are light. Specific examples of the plastic follow the description of the material of the standard nucleic acid sample container.

The shape of the base material is not particularly limited and can be appropriately selected depending on the intended purpose. It is usually selected according to the shape of the standard nucleic acid sample container. For example, it can be flat plate-shaped (plate-shaped).

The structure of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may have a single-layer structure or a multi-layer structure.

2-3. Effect According to the standard nucleic acid sample preparation kit of the present invention, a desired standard nucleic acid sample can be easily prepared.

3. 3. Method for discriminating the dispersed phase containing a standard nucleic acid sample 3-1. Outline A third aspect of the present invention is a method for discriminating a standard nucleic acid sample-containing dispersed phase. According to the discrimination method of the present invention, after the dispersed phase solution is subjected to the nucleic acid amplification reaction, it can be determined whether the dispersed phase solution subjected to the reaction is positive or negative based on the presence or absence of the amplification product.

3-2. Method The method for discriminating a standard nucleic acid sample-containing dispersed phase of the present invention includes an amplification step and a discrimination step as essential steps, and also includes a dispersed phase forming step as a selection step. Hereinafter, each step will be specifically described.

3-2-1. Dispersed phase forming step The “dispersed phase forming step” is a step of forming a dispersed phase including a dispersed phase solution, and is a selection step in this method. The dispersed phase forming step is executed prior to the amplification step described below.

A "dispersed phase solution" is a solution that is included in or surrounds a dispersed phase. For example, if the dispersed phase is a liquid phase dispersed phase, it is included in the phase, and if it is a solid phase dispersed phase, it surrounds the phase. The dispersed phase solution of the method contains the template nucleic acid molecule for preparing a standard nucleic acid sample according to the first aspect, the blocking oligonucleotide and / or the restriction enzyme according to the second aspect, and a primer pair.

In this method, the dispersed phase solution is a nucleic acid amplification reagent for amplifying the detection base sequence region in the amplification step described later, and if necessary, the restriction necessary for cleaving the restriction enzyme target site of the inhibition base sequence region. Includes enzyme reagents and labeling reagents.

Nucleic acid amplification reagents include, for example, a buffer such as nucleic acid polymerase, dNTP (dGTP, dCTP, dATP, dTTP, or dUTP), Mg ²⁺ , Tris-HCl that holds the optimum pH (pH 7.5 or more and pH 9.5 or less). Examples include nuclease-free water.

The labeling reagent is not particularly limited as long as it can discriminate the amplification of the detection base sequence as a label, and may be appropriately selected depending on the intended purpose. Those capable of optically labeling the dispersed phase, such as a fluorescent label and a luminescent label, are preferable. The reagent for performing optical labeling is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a nucleic acid probe such as Taq Man® probe, Molecular Beacon, or a commercially available labeling reagent such as an intercalator such as SYBR Green® or Eva Green can also be used.

When an intercalator is used, a method of adding the intercalator to the dispersed phase solution after amplifying the detection base sequence is also available.

The method for dividing the dispersed phase solution to form the dispersed phase is not particularly limited as long as the dispersed phase solution can be micropartitioned, and can be appropriately selected. For example, a method of forming a dispersed phase using an automated device such as a droplet generator can be mentioned. As the droplet generator, a known one can also be used. For example, an Automation Doopert Generator manufactured by BioRad, an emulsion (dispersed phase) production apparatus described in Japanese Patent No. 3746766, and the like can be mentioned. In the latter manufacturing apparatus, the dispersed phase can be manufactured by the shearing force of the continuous phase by discharging the dispersed phase in a direction intersecting the flow of the continuous phase with respect to the continuous phase flowing through the microchannel (microchannel). it can. Further, as a means for dividing the dispersed phase solution into a plurality of dispersed phases at high speed, a method of generating droplets using a microchannel and a method of filling a micro-nanoscale well with the dispersed phase solution can be mentioned. Further, the solution can be separated at a higher speed by continuously passing a plurality of splitters through the dispersed phase solution.

The dispersed phase may be either single-formed or plural-formed.
When the dispersed phase solution is classified into the dispersed phase, the standard nucleic acid sample preparation template nucleic acid molecule is contained in one dispersed phase at most 1 copy (1 molecule), that is, 0 copy or 1 copy, without particular limitation. As described above, it is preferable to adjust the concentration of the prepared template nucleic acid molecule for the standard nucleic acid sample in the dispersed phase solution. The concentration of the template nucleic acid molecule for preparing a standard nucleic acid sample is, for example, such that the number of dispersed phases containing the template nucleic acid molecule for preparing a standard nucleic acid sample is one with respect to 10 to 100 dispersed phases. preferable. That is, at such concentrations, it is expected that 10 to 100 or 9 to 99 dispersed phases will not contain template nucleic acid molecules for standard nucleic acid sample preparation. When one dispersed phase contains one copy of a template nucleic acid molecule for preparing a standard nucleic acid sample, if the template nucleic acid molecule for preparing a standard nucleic acid sample contains one copy of the target base sequence region, the target base sequence region is also a dispersed phase. One copy will be included per copy. By discriminating it in the discriminating step described later, the number of copies of the standard nucleic acid sample preparation template nucleic acid molecule per dispersed phase and the target base sequence region contained therein can be controlled more accurately.

3-2-2. Amplification step The "amplification step" is a step of amplifying the detection base sequence region by a nucleic acid amplification method using a dispersed phase solution, and is an essential step in this method.

As described in the dispersed phase forming step, the dispersed phase solution of this method is, in principle, the template nucleic acid molecule for preparing a standard nucleic acid sample described in the first aspect, and the blocking oligonucleotide and / or the blocking oligonucleotide described in the second aspect. A restriction enzyme, a primer pair, a nucleic acid amplification reagent for amplifying a detection base sequence region contained in a standard nucleic acid sample preparation template nucleic acid molecule, and, if necessary, an inhibitory base sequence region contained in the template nucleic acid molecule. Restriction enzyme Contains restriction enzyme reagents required to cleave the target site, and labeling reagents. Therefore, by subjecting the dispersed phase to the nucleic acid amplification reaction in this step, the base sequence region for detection of the amplification target contained in the template nucleic acid molecule for preparing a standard nucleic acid sample in the dispersed phase solution can be amplified.

The nucleic acid amplification method can be any method as long as it is a method of repeatedly amplifying a specific region on a target nucleic acid (detection base sequence region) sandwiched between Fw / Rv primers by repeating a nucleic acid extension reaction with a nucleic acid polymerase. May be good. For example, any known nucleic acid amplification method can be used. A quantitative nucleic acid amplification method in which the amplification product can be measured over time using the quantitative nucleic acid amplification method is particularly preferable. Examples of the nucleic acid amplification method include a PCR method and an isothermal amplification method. Examples of the isothermal amplification method include the NASBA method, LAMP method, RCA method, SMAP method, RPA method, HCR method, and ICAN method. Specific procedures for each nucleic acid amplification method are known in the art and may be carried out accordingly. In this step, the preferred nucleic acid amplification method is the PCR method. This is because the PCR method is the most widely used method in the field, various applied techniques have been developed, and optimized reagents, kits, reaction instruments, and the like are available. Examples of the quantitative nucleic acid amplification method include a real-time PCR method (real-time RT-PCR method).

As the reaction conditions of the nucleic acid amplification reaction, for example, in the case of amplification by the PCR method, the base length of the base sequence region for detection, the amount of the template nucleic acid molecule for preparing a standard nucleic acid sample, and the amount of the template nucleic acid molecule for preparing a standard nucleic acid sample are used based on the known PCR method. It may be appropriately determined in consideration of conditions such as the base length and Tm value of the primer, the optimum reaction temperature and the optimum pH of the nucleic acid polymerase to be used. As an example, in the case of the 3-step PCR method, the enzyme activity reaction is usually carried out at 94 to 95 ° C. for 1 minute to 10 minutes, the denaturation reaction is carried out at 94 to 95 ° C. for 5 seconds to 1 minute, and the annealing reaction is carried out for 50 minutes. The extension reaction is carried out at ~ 70 ° C. for 10 seconds to 1 minute and the extension reaction is carried out at 68 to 72 ° C. for 30 seconds to 3 minutes, and the modification reaction to the extension reaction are repeated for about 15 to 40 cycles as one cycle. Finally, the enzyme deactivation reaction is carried out at about 98 ° C. for 30 seconds to 10 minutes. When a commercially available PCR kit or the like is used, the reaction conditions may be, in principle, according to the protocol attached to the kit. The nucleic acid amplification reaction can be achieved by using a thermal cycler capable of precise temperature control, a real-time PCR device, a hot plate, or the like.

In the real-time PCR method, a reaction system in which the amplification product produced by the nucleic acid amplification reaction emits an optical label such as fluorescence is usually used. Therefore, it is preferable to carry out the nucleic acid amplification reaction using a thermal cycler equipped with a fluorescence detection device such as a real-time PCR device. The real-time PCR method is excellent in that the amount of product in the reaction can be monitored in real time without sampling, and the result can be regression-analyzed by a computer. Examples of the method for labeling the amplification product include a method using a fluorescently labeled probe such as the TaqMan (registered trademark) probe method, or an intercalation method. The TaqMan probe method uses a probe modified with a quencher at one end and a fluorescent substance at the other end. Normally, the quencher suppresses the fluorescence of the fluorescent substance on the probe, but in the nucleic acid extension reaction in PCR, the probe is degraded by the 5'→ 3'exonuclease activity of Taq polymerase, which causes the quencher. Since the suppression of the substance is released, it becomes fluorescent. The intercalation method is a method of fluorescently labeling an amplification product using an intercalator that specifically binds to double-stranded DNA. The intercalator is the above-mentioned SYBR Green (registered trademark), Eva Green, or the like. Available. In the TaqMan probe method and the intercalation method, the amount of fluorescence generated reflects the amount of amplification product.

In the amplification step, by amplifying only the detection base sequence region on the standard nucleic acid sample preparation template nucleic acid molecule in the dispersed phase, the number of copies of the standard nucleic acid sample preparation template nucleic acid molecule including the target base sequence region can be accurately copied. Can be controlled. For example, when the dispersed phase before this step contains one template nucleic acid molecule for preparing a standard nucleic acid sample having one copy of the target base sequence region, it is after the detection base sequence region is amplified in this step. However, the target base sequence region in the dispersed phase remains one copy. Therefore, in the dispensing step according to the fifth aspect, the dispersed phase containing the template nucleic acid molecule for preparing the standard nucleic acid sample in which the number of copies of the target base sequence region is specified is dispensed into the container for the standard nucleic acid sample or the like. It becomes possible to control so that the target base sequence region is contained in the container for a standard nucleic acid sample in a desired number of copies.

Further, in the amplification step, the detection base sequence contained in the dispersed phase may be amplified on the microchannel, or the detection base sequence contained in the dispersed phase separated into the tube may be amplified.

3-2-3. Discrimination step The "discrimination step" is a step of detecting an amplification product based on a detection base sequence region after the amplification step, and when the amplification product is detected, discriminating the dispersed phase solution as a positive dispersed phase. This is an essential process in this method.

In this step, the method for detecting the amplified product is not particularly limited and can be appropriately selected depending on the intended purpose. For example, the signal emitted by the amplification product may be detected. Specifically, when the dispersed phase contains a labeling reagent for optical labeling, the amplification product can be detected by an optical detection method. For optical detection, for example, the dispersed phase containing the amplification product may be determined by detecting the optical signal emitted from the dispersed phase. As a specific optical discrimination method, for example, a threshold value is set for the intensity of the optical signal emitted from the dispersed phase, and when the intensity of the detected optical signal exceeds the threshold value, the dispersed phase contains an amplification product. There is a method of determining that the signal is generated. The amplification product is a nucleic acid fragment in which the detection base sequence region on the template nucleic acid molecule for preparing a standard nucleic acid sample is amplified.

The detection of the optical signal is not particularly limited and can be appropriately selected according to the purpose. For example, a method of irradiating the dispersed phase with light (excitation light) from a light source and detecting an optical signal (fluorescence) emitted from the dispersed phase according to the light with a light receiving element can be mentioned.

The light source is not particularly limited as long as it can excite the optical signal of the dispersed phase, and can be appropriately selected depending on the intended purpose. Use a general light source such as a mercury lamp, HID, or halogen lamp with a filter that allows only specific wavelength light to pass through, or a light source that emits specific wavelength light such as an LED (Light Emitting Diet) or laser. Is possible. However, when the dispersed phase is formed of minute droplets of 1 nL or less, it is necessary to irradiate a narrow region with light of high light intensity, so it is preferable to use a laser. As the laser light source, various generally known and known lasers such as a solid-state laser, a gas laser, and a semiconductor laser can be used.

The light receiving element is not particularly limited as long as it is an element capable of receiving an optical signal emitted from the dispersed phase, and can be appropriately selected depending on the intended purpose. However, the droplet is irradiated with light having a specific wavelength and the droplet is dropleted. An optical sensor that receives an optical signal from the dispersed phase inside is preferable. Examples of the light receiving element include a one-dimensional element such as a photodiode and a photosensor, but when highly sensitive measurement is required, it is preferable to use a photomultiplier tube or an avalanche photodiode. As the light receiving element, for example, a two-dimensional element such as a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), or a gate CCD may be used.

Further, the discriminating means not only discriminates the dispersed phase in which the detection base sequence region as an amplification product is detected as the positive dispersed phase, but also discriminates the dispersed phase in which the detection base sequence is not amplified as the negative dispersed phase. It can also be determined as.

The dispersed phase determined to be a positive dispersed phase in this step contains only one template nucleic acid molecule for preparing a standard nucleic acid sample with a probability of 90% or more per dispersed phase.

3-3. Effect In the preparation of a standard nucleic acid sample, it is possible to determine whether or not the dispersed phase is a positive dispersed phase containing a target base sequence region that can be used as a standard nucleic acid sample, based on the presence or absence of an amplification product in the dispersed phase solution. ..

4. Method for selecting a dispersed phase containing a standard nucleic acid sample 4-1. Outline A fourth aspect of the present invention is a method for selecting a standard nucleic acid sample-containing dispersed phase. This method is a method of selecting a positive dispersed phase and a negative dispersed phase based on the result of the method for discriminating a standard nucleic acid sample-containing dispersed phase according to the third aspect. According to the present invention, after the amplification step, a positive dispersed phase containing a template nucleic acid molecule for preparing a standard nucleic acid sample and a negative dispersed phase not containing the template nucleic acid molecule can be selected, and only the positive dispersed phase can be selected.

4-2. Method The method for selecting a standard nucleic acid sample-containing dispersed phase of the present invention includes an amplification step, a discrimination step, and a sorting step as essential steps, and also includes a dispersed phase forming step as a selection step. Hereinafter, each step will be specifically described.

4-2-1. Dispersed phase forming step Since the dispersed phase forming step of this embodiment basically conforms to the dispersed phase forming step described in the third aspect, redundant description will be omitted here. In the dispersed phase forming step of this embodiment, a plurality of dispersed phases are formed.

4-2-2. Amplification step Since the amplification step of this embodiment basically conforms to the amplification step described in the third aspect, redundant description will be omitted here. In the amplification step of this embodiment, a nucleic acid amplification reaction is carried out on a plurality of dispersed phases.

4-2-3. Discrimination Step Since the discrimination step of this embodiment basically conforms to the discrimination step described in the third aspect, duplicate description will be omitted here. In the discrimination step of this embodiment, the presence or absence of the amplification product in each dispersed phase is detected based on the detection base sequence region after the amplification step, and the dispersed phase in which the amplification product is detected and the dispersed phase in which the amplification product is not detected are detected, respectively. Discriminate as a positive dispersed phase and a negative dispersed phase.

4-2-4. Sorting step The "sorting step" is a step of sorting the positive dispersion phase and the negative dispersion phase discriminated in the discrimination step. In the selection, a positive dispersed phase may be selected and the other dispersed phase may be a negative dispersed phase, or conversely, a negative dispersed phase may be selected and the other dispersed phase may be a positive dispersed phase.

The sorting method is not particularly limited and can be appropriately selected according to the purpose. For example, a method of electrodynamically sorting, a method of deforming a flow path to control the flow of fluid for sorting, a method of manually sorting using a mechanical valve or the like, and the like can be mentioned. By synchronizing these methods with the optical signal detected in the discrimination step, the dispersion phase can be more preferably selected.

Examples of the electrodynamic selection method include a method of forming an unequal electric field and attracting only the target dispersed phase by dielectrophoresis. This method can select the positive dispersed phase at high speed.

4-3. Effect In the preparation of a standard nucleic acid sample, it is possible to select and select only the positive dispersed phase whose dispersed phase contains a target base sequence region that can be used as a standard nucleic acid sample based on the presence or absence of an amplification product in the dispersed phase solution. it can.

5. Method for manufacturing standard nucleic acid sample-containing container 5-1. Outline A fifth aspect of the present invention is a method for producing a standard nucleic acid sample-containing container. This method uses the method for discriminating the standard nucleic acid sample-containing dispersed phase according to the third aspect and the method for selecting the standard nucleic acid sample-containing dispersed phase according to the fourth aspect, and further includes a standard nucleic acid sample in a predetermined number of copies. By controlling and dispensing the phase into a container for a standard nucleic acid sample so as to have a desired number of copies per container, a container containing the standard nucleic acid sample containing the desired number of copies can be produced. it can.

5-2. Method The method for producing a standard nucleic acid sample-containing container of the present invention includes an amplification step, a discrimination step, a sorting step, and a dispensing step as essential steps, and also includes a dispersed phase forming step and a counting step as selection steps.
Hereinafter, each step will be specifically described.

5-2-1. Dispersed phase forming step Since the dispersed phase forming step of this embodiment basically conforms to the dispersed phase forming step described in the third aspect, the description thereof is omitted here.

5-2-2. Amplification step Since the amplification step of this embodiment basically conforms to the amplification step described in the third aspect, the description thereof is omitted here.

5-2-3. Discrimination step Since the discrimination step of this embodiment basically conforms to the discrimination step described in the fourth aspect, the description thereof is omitted here.

5-2-4. Sorting step Since the sorting step of this embodiment basically conforms to the sorting step described in the fourth aspect, the description thereof is omitted here.

5-2-5. Counting step The "counting step" is discriminated by the discriminating step in the method for discriminating the standard nucleic acid sample-containing dispersed phase of the third aspect, or is sorted by the sorting step in the sorting method of the standard nucleic acid sample-containing dispersed phase of the fourth aspect. In addition, it is a step of counting the positive dispersed phases, which is a selection step in the production method of this embodiment. The counting step is executed at the same time as or after the discriminating step or the sorting step.

The method for counting the positive dispersed phase is not particularly limited and can be appropriately selected depending on the intended purpose. For example, when the positive dispersed phase is dispensed from the sorting mechanism via the discharging mechanism, the number of positive dispersed phases may be counted in the flow path to the discharging mechanism after sorting by the sorting mechanism, or in the discharging mechanism. The number of dispersed phases may be counted, or the number of positive dispersed phases discharged by the discharge mechanism may be counted. When counting the number of positive dispersed phases discharged by the discharge mechanism, the continuous phase discharged by the discharge mechanism may be irradiated with light to count the positive dispersed phases contained in the droplets in the continuous phase. At this time, the light may continuously irradiate the region through which the droplets pass, or may be pulsed at a timing with a predetermined time delay with respect to the droplet ejection operation in synchronization with the droplet ejection. You may.

By counting the number of discharged positive dispersed phases, the number of positive dispersed phases actually contained in the ejected droplets can be specified, so that the number of positive dispersed phases can be determined when dispensing into a container. It can be controlled accurately.

5-2-6. Dispensing step The "dispensing step" is the selection of the positive dispersed phase obtained after the discrimination step in the method for discriminating the standard nucleic acid sample-containing dispersed phase of the third aspect, or the sorting of the standard nucleic acid sample-containing dispersed phase of the fourth aspect. This is a step of dispensing the positive dispersed phase obtained after the step into a container for a standard nucleic acid sample.

The volume of the positive dispersed phase dispensed into the standard nucleic acid sample container is the flow rate of the continuous phase, the flow rate of the positive dispersed phase, the width of the flow path, the height of the flow path, the concentration of the surfactant, the viscosity of the solution, and the flow path. It can be controlled in consideration of the pressure loss of. The volume of the specific positive dispersion phase is not limited, but is preferably 1 fL or more and 1 μL or less, 100 fL or more and 0.5 μL or less, or 500 fL or more and 1 nL or less.

The method for dispensing the positive dispersed phase into the standard nucleic acid sample container is not particularly limited and can be appropriately selected depending on the intended purpose. A general dispensing method is a method using a discharge mechanism. The discharge mechanism can dispense the positive dispersed phase into a predetermined standard nucleic acid sample container so that the number of copies of the standard nucleic acid sample preparation template nucleic acid molecule having the target base sequence region is desired.

Specific examples of the discharge mechanism include, but are not limited to, a flow path capable of transporting a positive dispersed phase to a container for a standard nucleic acid sample (referred to as “positive dispersed phase transport flow path” in the present specification), on-demand. Examples include a method and a continuous method. Of these, the positive dispersed phase transport channel is preferable.

The positive dispersed phase transport flow path is not particularly limited and can be appropriately selected depending on the intended purpose.
For example, a tube capable of carrying a liquid and the like can be mentioned.

The on-demand discharge mechanism is not particularly limited and can be appropriately selected according to the purpose. For example, a discharge head and the like can be mentioned. A typical example of the ejection head is an inkjet method. The ejection head of the inkjet method includes an electrostatic method, a thermal method, a pressure application method and the like.

In the electrostatic method, it is necessary to install the electrode facing the ejection mechanism that holds the positive dispersed phase and forms droplets. In the standard nucleic acid sample-containing container manufacturing apparatus described later, since the discharge mechanism can be arranged so as to face the standard nucleic acid sample container, it is preferable that the electrodes are not arranged in order to increase the degree of freedom in the arrangement of the positive dispersed phase. However, it is not necessary to exclude it from the discharge mechanism of this step.

In the thermal method, since local heating is generated, there is a concern about the influence on the nucleic acid, which is a biomaterial, and the scorching (cogation) on the heater part. However, it is not necessary to exclude the influence of heat from the discharge mechanism of this step because it depends on the use of the dispersion phase content and the dispensed material.

Examples of the pressure application method include a method of applying pressure to a liquid using a piezo element, a method of applying pressure by a valve such as an electromagnetic valve, and the like. The pressure application method is the most preferable among the above three inkjet methods in that it does not require the installation of electrodes and there is no concern about scorching on the heater portion as compared with the thermal method.

The container for a standard nucleic acid sample used at the time of dispensing in this method is not limited, but a plate such as a 1-hole microtube, an 8-hole tube, or a 96-hole or 384-hole well plate is preferable. According to this step, even if the plate has a plurality of holes or wells, the target nucleotide sequence region can be dispensed into each hole or well with the same copy number, or with different copy numbers. It is also possible to do. If necessary, the negative dispersed phase may be dispensed to prepare holes or wells that do not contain the target base sequence region.

When a standard nucleic acid sample-containing container obtained by the production method of the present invention is used in the evaluation of a real-time PCR device or a digital PCR device that quantitatively evaluates nucleic acids, the template nucleic acid molecule for preparing the standard nucleic acid sample (that is, the target base sequence region) is used. It is preferred to use standard nucleic acid sample-containing containers dispensed in multiple levels. For example, it is possible to prepare a plate in which nucleic acids are dispensed at 7 levels of approximately 1, 2, 4, 8, 16, 32, and 64. By using such a plate, the quantitativeness, linearity, lower limit of evaluation, etc. of the real-time PCR device and the digital PCR device can be investigated, and it can be used as a reference in the expression level analysis of the next-generation sequencer.

In this step, a container for a standard nucleic acid sample may be pre-filled with an aqueous liquid. Examples of the aqueous liquid to be filled include nuclease-free water, nucleic acid amplification reagent, nucleic acid storage reagent and the like. By filling the standard nucleic acid sample container with an aqueous liquid, the nucleic acid sample after this step can be immediately used for the next reaction.

In this step, the label contained in the dispersed phase can also be detected at the time of dispensing. Detection can be achieved, for example, by equipping the discharge mechanism with a detector. The detector is not particularly limited and can be appropriately selected according to the purpose. For example, the optical detection method used in the discrimination step can be used.

This step controls the number of positive dispersed phases dispensed per standard nucleic acid sample container. As described in the discrimination step, the number of copies of the target base sequence region in the positive dispersion phase is controlled to a predetermined number, and as a result, according to the production method of the present invention, the produced standard The number of copies of the target base sequence region contained in the nucleic acid sample-containing container can also be controlled to a predetermined number of copies.

The “predetermined number of copies” refers to a template nucleic acid molecule for preparing a standard nucleic acid sample, that is, a number of target base sequence regions contained therein having an accuracy of a certain level or more. It can be said that it is known as the number of target base sequence regions actually contained in the container. That is, the predetermined number of copies in the present application is more accurate and reliable as a number than the number of copies (calculated estimated value) obtained by conventional serial dilution, and is particularly in a low copy number region of less than 1000. However, it is a controlled value that does not depend on the Poisson distribution. It is preferable that the controlled values generally have a coefficient of variation CV representing uncertainty within the values of CV <1 / √x and CV <20% with respect to the average copy number x. For example, it is preferable that the target base sequence region included in the standard nucleic acid sample container after the dispensing step is less than 1000 copies, and the CV value of the copy number is less than 20%. The predetermined number of copies included in the standard nucleic acid sample-containing container produced by this production method is preferably 1 copy or more and 1000 copies or less, preferably 1 copy or more and 100 copies or less, and more preferably 1 copy or more and 20 copies or less. More preferably, 1 copy or more and 10 copies or less.

The average value and standard deviation of the number of copies of the nucleic acid dispensed in the container containing the standard nucleic acid sample is the average value and standard deviation of the number of copies of the target base sequence region contained in one positive dispersed phase. It can be obtained from the calculation based on the following equations 1 to 5. By using Equations 1 to 5, the average value and standard deviation of the copy number of the target base sequence region contained in one positive dispersion phase can be obtained in consideration of the Poisson distribution.

In formula 1, k is the number of copies contained in one positive dispersed phase, λ is the ratio of the positive dispersed phase (Posi) among the total dispersed phases measured in the sample, and P is the target base in the all positive dispersed phases. The probability of a positive dispersed phase in which the sequence region contains a predetermined number of copies, the left side in Equation 2 is the average value of the number of copies contained in one positive dispersed phase, and σ1 in Equation 3 is within one positive dispersed phase. The standard deviation of the number of copies included, N in Equations 4 and 5 is the specific number of compartments of the positive dispersion phase, m is the average value of the number of copies included in the positive dispersion phase of the specific number of compartments, and σm in the equation is It means the standard deviation of the number of copies contained in the positive dispersion phase of a specific number of compartments.

The positive dispersed phase means the dispersed phase in which the label in the dispersed phase is detected by the detector of the discharge mechanism described in the method for producing the standard nucleic acid sample-containing container of the present invention, and the negative dispersed phase (Nega) is defined as. It means the dispersed phase in which the label in the dispersed phase was not detected by the detector of the discharge mechanism. In addition, the fact that the label was not detected means that the threshold value for detecting the label was not exceeded.

In addition, the probability that the positive dispersed phase contains only one target base sequence region is preferably 90% or more. The probability that the positive dispersion phase contains only one target base sequence region is the probability that each positive dispersion phase includes only one target base sequence region among the probabilities P of the number of discrete copies included. Means the probability of.

The above-mentioned "uncertainty" is "a parameter that characterizes the variability of the value that can be reasonably linked to the measured quantity associated with the measurement result". ISO / IEC uide99: 2007 [International metric measurement term-basic and It is defined in General Concepts and Related Terms (VIM)].

Here, "a value that can be reasonably linked to a measured quantity" means a candidate for a true value of the measured quantity. That is, the uncertainty means information on the variation in the measurement result due to the operation, equipment, etc. related to the manufacture of the measurement target. The greater the uncertainty, the greater the expected variability in the measurement results.

The uncertainty may be, for example, a standard deviation obtained from the measurement result, or may be a value that is half the confidence level expressed as the range of values in which the true value is included with a predetermined probability or more.

As a method of calculating uncertainty, Guide to the Expression is used.
It can be calculated based on the guidelines for measurement uncertainty in the of Enterprise in Quality in Measurement (GUM: ISO / IEC Guide98-3) and the Japan Accreditation Board Note 10 test. As a method for calculating uncertainty, for example, a type A evaluation method using statistics such as measured values, and uncertainty information obtained from a calibration certificate, manufacturer's specifications, published information, etc. are used. Two methods of the type B evaluation method used can be applied.

Uncertainty can be expressed at the same confidence level by converting all the uncertainty obtained from factors such as operation and measurement into standard uncertainty. Standard uncertainty refers to the variability of the mean value obtained from the measured values.

As an example of the method of calculating the uncertainty, for example, the factors causing the uncertainty are extracted and the uncertainty (standard deviation) of each factor is calculated. Furthermore, the uncertainty of each calculated factor is combined by the sum of squares method to calculate the combined standard uncertainty. Since the sum of squares method is used in the calculation of the composite standard uncertainty, the factor that causes the uncertainty and whose uncertainty is sufficiently small can be ignored. For the uncertainty, the coefficient of variation (CV value) obtained by dividing the composite standard uncertainty by the expected value may be used.

The coefficient of variation is the number of nucleic acids (or the number of nucleic acids) having the target base sequence region to be filled in each container generated when the container (for example, particles, wells, etc.) is filled with the nucleic acid having the target base sequence region. ) Means the relative value of the variation. That is, the coefficient of variation means the filling accuracy of the number of nucleic acids having the target base sequence region packed in the container. The coefficient of variation is a value obtained by dividing the standard deviation σ by the mean value x. Here, assuming that the value obtained by dividing the standard deviation σ by the average copy number (average filled copy number) x is the coefficient of variation CV, the relational expression of the following equation 6 is obtained.

In general, nucleic acids have a randomly distributed Poisson distribution in the dispersion. Therefore, in the serial dilution method, that is, in the random distribution state in the Poisson distribution, the standard deviation σ can be regarded as satisfying the relational expression between the average number of copies x and the following equation 7. From this, when the dispersion liquid of nucleic acid is diluted by the serial dilution method, the coefficient of variation CV (CV value) of the average number of copies x is derived from the standard deviation σ and the average number of copies x from the above formula 6 and the following formula 7. The results are shown in Table 1 and FIG. 2 when calculated using the following equation 8. The CV value of the coefficient of variation of the copy number with variation based on the Poisson distribution can be obtained from FIG.

From the results of Table 1 and FIG. 2, for example, when the container is filled with 100 copies of nucleic acid by the serial dilution method, the predetermined number of copies of the nucleic acid finally filled in the reaction solution has other accuracy. It can be seen that even if is ignored, it has a coefficient of variation (CV value) of at least 10% (Fig. 2: indicated by a broken line arrow).

The filling accuracy (CV value) of the number of target base sequence regions contained in the standard nucleic acid sample-containing container is not particularly limited and can be appropriately selected depending on the purpose. For example, less than 20% (in FIG. 2 in FIG. 2). A gray area that does not include a curve) is preferable.

The standard nucleic acid sample container may be provided with information on the nucleic acid contained in the standard nucleic acid sample container, information on the dispersed phase, and the like. There is no particular limitation on the method of giving information, and it may be selected as appropriate according to the purpose. For example, a method of coloring a container containing a standard nucleic acid sample according to the content of information, a method of printing the content of information on particles, and the like can be mentioned. By using the method of coloring the standard nucleic acid sample-containing container according to the content of the information, the user can visually recognize the information of the standard nucleic acid sample-containing container, and the operability can be improved.

The information on the nucleic acid contained in the standard nucleic acid sample container is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the sequence in the target base sequence region, the number of molecules of the standard nucleic acid sample preparation template nucleic acid molecule having the target base sequence region, the uncertainty of the number of molecules of the included standard nucleic acid sample preparation template nucleic acid molecule, and the standard nucleic acid sample preparation. Examples include the type of template nucleic acid molecule. These may be given alone or in combination of two or more.

Information on the dispersed phase includes the number of copies of the dispensed nucleic acid, the type of solution in which the nucleic acid is dispersed, the uncertainty of the dispersed phase, the filling accuracy of the dispersed phase, and the like. These may be given alone or in combination of two or more.

5-3. Specific Example of Standard Nucleic Acid Sample-Containing Container FIG. 3 is a diagram showing an example of the standard nucleic acid sample-containing container of the present invention. In this figure, the standard nucleic acid sample-containing container is shown in a capsule shape, but the standard nucleic acid sample-containing container of the present invention is not limited to this shape.

As shown in FIG. 3, the standard nucleic acid sample-containing container (300) contains a plurality of dispersed phases (100) containing a template nucleic acid molecule (110) for preparing a standard nucleic acid sample. Further, as shown in FIG. 3, one dispersed phase (100) has one molecule (one copy) of a template nucleic acid molecule (110) for preparing a standard nucleic acid sample, and one template nucleic acid molecule for preparing a standard nucleic acid sample is Since one copy of the target nucleotide sequence region (A) is contained, one container of the standard nucleic acid sample-containing container (300) can be controlled by controlling the number of dispersed phases (100) to be dispensed into the standard nucleic acid sample-containing container (300). The number of copies of the target base sequence region (A) contained in the area can be arbitrarily controlled.

In the standard nucleic acid sample-containing container of the present invention, the operability can be improved by shaping the standard nucleic acid sample-containing container (300) into a particle shape. By improving the operability of the nucleic acid sample, a required amount of a template nucleic acid molecule for preparing a standard nucleic acid sample containing a target base sequence region can be added to any experimental system. This makes it possible to create a reaction system that meets the needs of the user. Here, the extremely small amount means the number of molecules of 1 or more and 1000 or less.

Further, the standard nucleic acid sample-containing container (300) shown in FIG. 3 has excellent nucleic acid storage stability because the nucleic acid sample is encapsulated in particles.

5-4. Effect According to the production method of the present invention, a standard nucleic acid sample-containing container containing a target base sequence region in a predetermined copy number can be produced. As a result, it becomes possible to perform a qualitative and quantitative test of a sample having a target base sequence more accurately than a conventional standard nucleic acid. As a result, it can be suitably used in various fields such as food tests, blood tests, and DNA tests.

The standard nucleic acid sample-containing container obtained by the production method of the present invention can strictly control the number of copies of the template nucleic acid molecule for preparing a standard nucleic acid sample having a target base sequence region in the production process. It is possible to accurately grasp the number of copies of the target base sequence region contained in.

6. Manufacturing equipment for standard nucleic acid sample-containing containers 6-1. Outline A sixth aspect of the present invention is an apparatus for producing a standard nucleic acid sample-containing container. The manufacturing apparatus of the present invention is an apparatus that embodies the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect, and includes means capable of executing each step of the standard nucleic acid sample-containing container manufacturing method. According to the production apparatus of the present invention, a standard nucleic acid sample-containing container containing a standard nucleic acid sample in a desired number of copies can be easily produced with high accuracy.

6-2. Configuration The apparatus for producing a standard nucleic acid sample-containing container of the present invention may include amplification means, discrimination means, and dispensing means as essential components, and dispersed phase forming means and counting means as selective components. Hereinafter, each means will be described.

6-2-1. Dispersed phase forming means The “dispersed phase forming means” is a means configured to realize the dispersed phase forming step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the dispersed phase forming means is described in the dispersed phase forming step described in the third aspect, the description thereof is omitted here.

6-2-2. Amplification means The "amplification means" is a means configured to realize the amplification step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the amplification means is described in the amplification step described in the third aspect, the description thereof is omitted here.

6-2-3. Discrimination means The "discrimination means" is a means configured to realize the discrimination step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the discriminating means is described in the amplification step described in the third aspect, the description here will be omitted. The manufacturing apparatus of the present invention also includes a configuration for discriminating the positive dispersed phase and selecting the positive dispersed phase. That is, this means is also configured to realize the sorting step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the sorting means is described in the sorting step described in the fourth aspect, the description here will be omitted.

6-2-4. Counting Means The "counting means" is a means configured to realize the counting step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the counting means is described in the counting step described in the fifth aspect, the description here will be omitted.

6-2-5. Dispensing Means The "dispensing means" is a means configured to realize the dispensing step in the standard nucleic acid sample-containing container manufacturing method according to the fifth aspect. Since the specific configuration of the dispensing means is described in the dispensing step described in the fifth aspect, the description here will be omitted.

6-3. Specific Examples of Manufacturing Equipment and Manufacturing Method of Standard Nucleic Acid Sample-Containing Container Hereinafter, a manufacturing method of the standard nucleic acid sample-containing container using the standard nucleic acid sample-containing container manufacturing equipment of the present invention will be specifically described with reference to the drawings. .. Here, an example is used in which the dispersed phase solution is divided using a microchannel to form a dispersed phase, but the apparatus for producing the standard nucleic acid sample-containing container of the present invention and the method for producing the same are limited to the following methods. It is not something that is done.

FIG. 4 is a diagram showing an example of a dispersed phase forming means (401) that divides the solution X, which is a dispersed phase solution, into a dispersed phase in the microchannel (402). As shown in FIG. 4, the solution X containing the template nucleic acid molecule (110) for preparing a standard nucleic acid sample having the target base sequence region (A) and the detection base sequence region (B) is the dispersed phase forming means (401). It is flowing through the upper left channel. Further, the solution Y containing the nucleic acid amplification reagent (including the primer set and the blocking oligonucleotide) and the fluorescent labeling reagent flows through the lower left flow path of the dispersed phase forming means (401). Solution X and solution Y merge and are mixed in one flow path.

Further, when the solution X and the solution Y are mixed on the micro flow path, a flow path capable of stirring the solution X and the solution Y after the two flow paths are united with the one flow path is provided. You may. Examples of the flow path capable of stirring the solution X and the solution Y include a meandering flow path, a pillar array flow path, a spreading flow path, and a narrowing flow path.

Next, the mixed solution Z obtained by mixing the solution X and the solution Y is divided into dispersed phases (100, 101) as droplets by the shearing force from the oil O, which is a continuous phase fluid flowing from the upper and lower centers of FIG. It is transported to the downstream side (right side) of the flow path in FIG.

In FIG. 4, a solution having a nucleic acid concentration such that at most one template nucleic acid molecule (110) for preparing a standard nucleic acid sample is contained in one dispersed phase is used. Therefore, depending on the dispersed phase, a dispersed phase (100) containing one template nucleic acid molecule (110) for preparing a standard nucleic acid sample and a dispersed phase (101) containing no template nucleic acid molecule (110) for preparing a standard nucleic acid sample are formed. Will be done.

By using the microchannel (402) as the dispersed phase forming means (401), the mixed solution Z can be divided into dispersed phases at high speed. Specifically, the dispersed phase can be formed at a rate of about 1000 per second.

FIG. 5 is a diagram showing another example of the dispersed phase forming means (401) that divides the solution X into dispersed phases in the microchannel (402).

The solution X containing the template nucleic acid molecule (110) for preparing a standard nucleic acid sample having the target base sequence region (A) and the detection base sequence region (B) flows through the upper right flow path of the dispersed phase forming means (401). There is. In addition, the solution Y containing the nucleic acid amplification reagent (including the primer set and the blocking oligonucleotide) and the fluorescent labeling reagent flows through the upper left flow path of the dispersed phase forming means (401). The solution X and the solution Y merge in one flow path and are mixed to form a mixed solution Z.

Next, the mixed solution Z is divided into dispersed phases (100, 101) as droplets by the shearing force of the oil O, which is a continuous phase fluid flowing from the lower left of the center of the dispersed phase forming means (401), and the right side of FIG. It is transported downstream of the flow path.

FIG. 6 is a diagram showing an example of amplification means. The amplification means (601) carries out a nucleic acid amplification reaction using a PCR method on a dispersed phase (100) containing a template nucleic acid molecule (110) for standard nucleic acid sample preparation, thereby carrying out a nucleic acid amplification reaction using a standard nucleic acid sample preparation template nucleic acid molecule. Only the detection base sequence region (B) contained in (110) is amplified, and the dispersed phase (100) is optically labeled.

Specifically, the amplification means (601) contains a template nucleic acid molecule (110) for preparing a standard nucleic acid sample, a nucleic acid amplification reagent (including a primer set and a blocking oligonucleotide), and a dispersed phase (100) containing a fluorescent labeling reagent. , The temperature of the dispersed phase (100) is raised or lowered by circulating it in a meandering flow path having a plurality of temperature zones (not shown). When the dispersed phase (100) moves back and forth between a plurality of temperature zones, only the detection base sequence region (B) of the standard nucleic acid sample preparation template nucleic acid molecule (110) contained in the dispersed phase (100) is amplified. As a result, the dispersed phase (100) includes a template nucleic acid molecule (110) for preparing a standard nucleic acid sample and an amplified dispersed phase (102) containing an amplified fragment of the detection base sequence region (B). .. The amplified dispersion phase (102) is in a state of being optically labeled by reacting the fluorescent labeling reagent with the amplified detection base sequence region (B). The optically labeled amplified dispersed phase (102) is an empty dispersed phase (104) (“negative dispersed phase”) that does not contain a template nucleic acid molecule (110) for preparing a standard nucleic acid sample when irradiated with excitation light. It emits stronger fluorescence than (also called). Therefore, it is also referred to as a "positive dispersed phase". As described above, the positive dispersion phase (103), which is an optically labeled amplified dispersion phase, and the empty dispersion phase (negative dispersion phase) (104) can be easily distinguished by the fluorescence intensity. ..

FIG. 7 shows a discriminating means (701) for discriminating the amplified dispersed phase (102) including a plurality of amplified detection base sequence regions (B) as a positive dispersed phase (103), and sorting for the dispersed phase by dielectrophoresis. It is a figure which shows an example of the discrimination mechanism and the sorting mechanism in means (702).

The discriminating means (701) detects the fluorescence from the dispersed phase, and the positive dispersed phase (103) containing the template nucleic acid molecule (110) for preparing a standard nucleic acid sample in which the detection base sequence region (B) is amplified and the standard nucleic acid. The negative dispersed phase (104) containing no template nucleic acid molecule (110) for sample preparation is discriminated.

When the optical detection mechanism of the discriminating means (701) performs fluorescence detection, it has a light source and a light receiving element inside, and the positive dispersed phase (103) of the light receiving element absorbs the light from the light source as excitation light. Receives the emitted fluorescence. Since the fluorescence is emitted from the positive dispersed phase (103) in all directions, the light receiving element can be arranged at an arbitrary position where the fluorescence can be received. At this time, in order to improve the contrast, it is preferable to arrange the light receiving element at a position where the light emitted from the light source is not directly incident.

The discriminating means (701) discriminates between the positive dispersion phase (103) and the negative dispersion phase (104) based on the intensity of fluorescence received by the light receiving element.

The sorting mechanism (702) comprises a cathode (702a) and an anode (702b), and a positive dispersion phase (103) and a negative dispersion phase (104) are sorted by dielectrophoresis.

The sorting mechanism (702) applies an appropriate voltage to the cathode (702a) and the anode (702b) shown in the upper part of FIG. 7 as the positive dispersion phase (103) passes around the sorting mechanism (702). Therefore, an unequal electric field is formed around the sorting mechanism (702). The positive dispersed phase (103) polarized by the unequal electric field is dielectrophoresed by the unequal electric field, attracted to the sorting mechanism (702) side, flows through the upper right flow path of FIG. 7, and is transported to the discharge mechanism. ..

The sorting mechanism (702) does not apply a voltage to the cathode (702a) and the anode (702b) when the negative dispersion phase (104) passes around the sorting mechanism (702). Therefore, the negative dispersed phase (104) flows to the lower right flow path of FIG. 7 and is discarded without dielectrophoresis. At this time, the flow path (703) through which the positive dispersion phase (103) selected by the sorting mechanism (702) flows is 1.2 times larger than the disposal flow path (704) through which the negative dispersion phase (104) flows. It is preferable to have 1.5 times the fluid resistance. By using the above-mentioned fluid resistance, all the dispersed phases flow into the waste flow path (704) when no electric field is applied, so that the positive dispersed phase (103) can be selected more reliably.

FIG. 8 is a diagram showing an example of the discharge mechanism. As shown in FIG. 8, the discharge mechanism (801) has a detection mechanism (802) having the same function as the discriminating means (701) of FIG. 7, and a flow path (803) capable of transporting the dispersed phase. There is. As shown in FIG. 8, the discharge mechanism (801) dispenses the positive dispersion phase (103) into a capsule (300) which is a standard nucleic acid sample-containing container. At this time, the number of positive dispersed phases (103) dispensed into the capsule (300) is controlled based on the label detected by the detection mechanism (802).

Next, the dispensing means of the method for producing the standard nucleic acid sample-containing container of the present invention will be described more specifically with reference to FIG. Here, when the ejection mechanism of the inkjet method is used as the dispensing means, the dispersed phase is dispensed into the container. Since the method up to the selection of the dispersed phase is the same as the method shown in FIGS. 4 to 7, the description here will be omitted.

(Discharge mechanism)
FIG. 9 is an explanatory diagram showing an embodiment of a discharge mechanism and a counting mechanism.
The discharge mechanism (910) includes a liquid chamber (911), a membrane (912), and a driving element (913). The liquid chamber (911) has an atmospheric opening portion (915) that opens the inside of the liquid chamber (911) to the atmosphere at the upper part, and discharges air bubbles mixed in the oil (970) from the atmospheric opening portion (915). It is configured to be possible. The membrane (912) is a film-like member fixed to the lower end of the liquid chamber (911). A nozzle (940), which is a through hole, is formed in the substantially center of the membrane (912), and the oil (970) held in the liquid chamber (911) is liquid from the nozzle (940) due to the vibration of the membrane (912). It is discharged as drops (971). Since droplets (971) are formed by the inertia of the vibration of the membrane (912), even oil (970) having a high surface tension (high viscosity) can be discharged. The planar shape of the membrane (912) can be, for example, circular, but may be elliptical, quadrangular, or the like.

The material of the membrane (912) is not particularly limited, but if it is too soft, the membrane (912) easily vibrates. In that case, it is difficult to immediately suppress the vibration when not discharging, so it is preferable to use a material having a certain degree of hardness. As the material of the membrane (912), for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.

The nozzle (940) is preferably formed as a substantially perfect circular through hole in the substantially center of the membrane (912). In this case, the diameter of the nozzle (940) is not particularly limited, but it should be at least twice the size of the positive dispersion phase (103) in order to prevent the positive dispersion phase (103) from clogging the nozzle (940). Is preferable.

On the other hand, if the droplets become too large, it becomes difficult to achieve the purpose of forming fine droplets, so the diameter of the nozzle (940) is preferably 200 μm or less. That is, in the discharge mechanism (910), the diameter of the nozzle (940) is typically in the range of 10 μm or more and 200 μm or less.

The driving element (913) is formed on the lower surface side of the membrane (912). The shape of the driving element (913) can be designed according to the shape of the membrane (912). For example, when the planar shape of the membrane (912) is circular, it is preferable to form a driving element (913) having an annular (ring-shaped) planar shape around the nozzle (940).

The driving means (920) drives a discharge waveform that vibrates the membrane (912) to form a droplet (971) and a stirring waveform that vibrates the membrane (912) within a range that does not form a droplet (971). (913) can be selectively (for example, alternately) imparted.

For example, the ejection waveform and the stirring waveform are both rectangular waves, and the driving voltage of the stirring waveform is made lower than the driving voltage of the ejection waveform so that the droplets (971) are not formed by the application of the stirring waveform. it can. That is, the vibration state (degree of vibration) of the membrane (912) can be controlled by the level of the drive voltage.

In the discharge mechanism (910), since the drive element (913) is formed on the lower surface side of the membrane (912), when the membrane (912) is vibrated by the drive element (913), the drive element (912) is vibrated from the lower direction of the liquid chamber (911). It is possible to generate a flow in the upward direction.

That is, the driving means (920) adds the discharge waveform to the driving element (913) and controls the vibration state of the membrane (912) to eject the oil (970) held in the liquid chamber (911) to the nozzle (940). ) Can be ejected as droplets (971).

Further, in the discharge mechanism (910), air bubbles may be mixed in the oil (970) in the liquid chamber (911). Even in this case, since the discharge mechanism (910) is provided with the air opening portion (915) above the liquid chamber (911), the air bubbles mixed in the oil (970) are passed through the air opening portion (915). Can be discharged to the outside air. This makes it possible to continuously and stably form droplets (971) without discarding a large amount of liquid for discharging bubbles.
The membrane (912) may be vibrated at a timing when the droplets are not formed within a range where the droplets are not formed, and the bubbles may be positively moved above the liquid chamber 11.

(Counting mechanism)
The counting mechanism is composed of a light source (930), a light receiving element (950), and a control unit (960). The light source (930) can irradiate the flying droplets (971) with light L. The term “in flight” means a state in which the droplet (971) is ejected from the ejection mechanism (910) until it is deposited on the container. The flying droplet (971) is substantially spherical at the position where the light L is irradiated. Further, the beam shape of the light L is substantially circular.

Here, it is preferable that the beam diameter of the light L is about 10 times or more and 100 times or less with respect to the diameter of the droplet (971). This is because the light L from the light source (930) is surely irradiated to the droplet (971) even when the position variation of the droplet (971) is present. However, it is not preferable that the beam diameter of the light L greatly exceeds 100 times the diameter of the droplet (971). This is because the energy density of the light applied to the droplet (971) decreases, so that the amount of fluorescent Lf emitted using the light L as excitation light decreases, and it becomes difficult for the light receiving element (950) to detect it.

The light L emitted from the light source (930) is preferably pulsed light, and for example, a solid-state laser, a semiconductor laser, a dye laser, or the like is preferably used. When the light L is pulsed light, the pulse width is preferably 10 μS or less, more preferably 1 μS or less. The energy per unit pulse largely depends on the optical system such as the presence or absence of light collection, but is generally preferably 0.1 μJ or more, and more preferably 1 μJ or more.

Since the fluorescence Lf emitted by the positive dispersion phase (103) is weaker than the light L emitted by the light source (930), the wavelength range of the light L is attenuated in the front stage (light receiving surface side) of the light receiving element (950). A filter may be installed. As a result, it is possible to obtain an image of the positive dispersion phase (103) having a very high contrast in the light receiving element (950). As the filter, for example, a notch filter that attenuates a specific wavelength region including the wavelength of light L can be used.

Further, as described above, the light L is preferably pulsed light, but may be continuously oscillated light. In this case, the light receiving element (950) is controlled so that the light receiving element (950) can take in the light at the timing when the continuously oscillating light is irradiated on the flying droplet (971), and the fluorescent Lf is applied to the light receiving element (950). It is preferable to receive light.

(Control unit)
The control unit (960) has a function of controlling the driving means (920) and the light source (930). Further, the control unit (960) obtains information based on the amount of light received by the light receiving element (950), and the number of positive dispersed phases (103) contained in the droplet (971) (including the case where it is zero). Has a function of counting.

From the above, by using the method for producing a standard nucleic acid sample-containing container of the present invention, a desired number of dispersed phases (103) can be dispensed into the capsule (300), and thus the capsule (300) is sealed. The number of copies of the dispersed phase (103), that is, the target base sequence region, contained in the container 1 produced in this manner can be controlled to a known number (predetermined number). FIG. 10 is a diagram illustrating a hardware block of the control unit (960) shown in FIG. FIG. 11 is a diagram illustrating a functional block of the control unit. FIG. 12 is a flowchart showing an example of the operation of the discharge mechanism and the counting mechanism.

As shown in FIG. 10, the control unit (960) has a CPU (961), a ROM (962), a RAM (963), an I / F (964), and a bus line (965). There is. The CPU (961), ROM (962), RAM (963), and I / F (964) are connected to each other via a bus line (965).

The CPU (961) controls each function of the control unit (960). The ROM (962), which is a storage means, stores a program executed by the CPU (961) to control each function of the control unit (960) and various information. The RAM (963), which is a storage means, is used as a work area or the like of the CPU (961). Further, the RAM (963) can temporarily store predetermined information. The I / F (964) is an interface for connecting the discharge mechanism (910) shown in FIG. 9 to another device or the like. The discharge mechanism (910) may be connected to an external network or the like via the I / F (964).

As shown in FIG. 11, the control unit (960) includes a division control unit (970), a discrimination control unit (980), a sorting control unit (990), and a dispensing control unit (1000). There is.

The control unit (960) controls the entire manufacturing apparatus of the standard nucleic acid sample-containing container of the present invention.

Next, the control of the control unit (960) when the inkjet method is used as the dispensing means will be described with reference to FIGS. 13 to 17.

As shown in FIG. 13, the control unit (960) includes a division control unit (970), a discrimination control unit (980), a sorting control unit (990), and a dispensing control unit (1000). There is. The discrimination control unit (980) has a light source control unit (981) and a detection control unit (982). The sorting control unit (990) has an electric field control unit (991). The dispensing control unit (1000) includes a discharge control unit (1001), a dispersed phase counting unit (1002), and a light source control unit (1003).

The number of dispersed phases (number of particles) of the discharge mechanism (910) will be described with reference to FIGS. 13 and 14. First, in step S11, the discharge control unit (1001) of the control unit (960) issues a discharge command to the drive means (920). The drive means (920) that receives the discharge command from the discharge control unit (1001) supplies a drive signal to the drive element (913) to vibrate the membrane (912). Due to the vibration of the membrane (912), the droplet (971) containing the positive dispersed phase (103) is ejected from the nozzle (940).

Next, in step S12, the light source control unit (981) of the control unit (960) sends a drive signal supplied from the drive means (920) to the discharge mechanism (910) in synchronization with the discharge of the droplet (971). (Synchronized with) issues a lighting command to the light source (930). As a result, the light source (930) is turned on, and the flying droplet (971) is irradiated with the light L.

Here, "synchronizing" means emitting light at the same time as the droplet (971) is ejected by the ejection mechanism (910) (at the same time as the driving means (920) supplies the driving signal to the ejection mechanism (910)). This does not mean that the light source (930) emits light at the timing when the droplet (971) is irradiated with the light L when the droplet (971) flies and reaches a predetermined position. That is, the light source control unit (981) delays the ejection of the droplet (971) by the ejection mechanism (910) (the drive signal supplied from the driving means (920) to the ejection mechanism (910) by a predetermined time). The light source (930) is controlled so as to emit light.

For example, the velocity v of the droplet (971) ejected when the drive signal is supplied to the ejection mechanism (910) is measured in advance. Then, based on the measured velocity v, the time t for the droplet (971) to reach a predetermined position after being ejected is calculated, and the light source (930) emits light with respect to the timing of supplying the drive signal to the ejection mechanism 10. The timing of irradiation is delayed by t. As a result, good light emission control becomes possible, and the light from the light source (930) can be reliably irradiated to the droplet (971).

The dispersion phase counting unit (1002) of the control unit (960) is based on the information from the light receiving element (950), and the number of positive dispersion phases (103) contained in the droplet (971) (may be zero). Includes). Here, the "information from the light receiving element (950)" is a brightness value (light amount) or an area value of the positive dispersed phase (103).

The dispersed phase counting unit (1002) can count the number of positive dispersed phases (103) by comparing, for example, the amount of light received by the light receiving element (950) with a preset threshold value. In this case, a one-dimensional element or a two-dimensional element may be used as the light receiving element (950).

When a two-dimensional element is used as the light receiving element (950), the dispersed phase counting unit (1002) determines the brightness value or area of the positive dispersed phase (103) based on the two-dimensional image obtained from the light receiving element (950). A method of performing image processing for calculating the above may be used. In this case, the dispersion phase counting unit (1002) calculates the brightness value or area value of the positive dispersion phase (103) by image processing, and compares the calculated brightness value or area value with a preset threshold value. This makes it possible to count the number of positive dispersed phases (103).

In this way, a drive signal is supplied from the drive means (920) to the discharge mechanism (910) that holds the oil (970) in which the positive dispersion phase (103) is turbid, and the positive dispersion phase (103) is contained. The droplet (971) is discharged, and the flying droplet (971) is irradiated with light L from the light source (930). Then, the positive dispersion phase (103) contained in the flying droplet (971) emits fluorescence Lf using light L as excitation light, and the light receiving element (950) receives the fluorescence Lf. Further, based on the information from the light receiving element (950), the dispersed phase counting unit 742 counts the number of positive dispersed phases (103) contained in the flying droplets (971).

That is, since the number of positive dispersed phases (103) contained in the flying droplet (971) is actually observed on the spot, the counting accuracy of the number of positive dispersed phases (103) can be improved as compared with the conventional case. It will be possible. Further, since the positive dispersion phase (103) contained in the flying droplets (971) is irradiated with light L to emit fluorescent Lf and the fluorescent Lf is received by the light receiving element (950), the positive dispersion is performed with high contrast. An image of the phase (103) can be obtained, and the frequency of erroneous counting of the number of positive dispersed phases (103) can be reduced.

In this embodiment, the counting mechanism has a form of counting the number of dispersed phases contained in the droplets discharged by the discharging mechanism, but the form of the counting mechanism is not limited to this. The counting mechanism may be, for example, a form of counting in the flow path through which the positive dispersion phase (103) sorted by the sorting mechanism (702) of FIG. 7 flows, or a liquid chamber (911) of the discharge mechanism (910). ) May be used for counting.

FIG. 15 is an explanatory diagram showing an example of the state immediately after the positive dispersed phase (103) is dispensed into the standard nucleic acid sample-containing container (300).

The well (1500) corresponding to the standard nucleic acid sample-containing container is pre-filled with an aqueous solution (1501). When the specific gravity of the oil (970) is smaller than that of the aqueous solution (1501), immediately after the droplet (971) lands on the well (1500), the oil layer (1502) containing the positive dispersed phase (103) is shown as shown in FIG. And oil (970) are located above the aqueous solution (1501).

FIG. 16 shows an example of a state after a positive dispersed phase containing a template nucleic acid molecule (110) for preparing a standard nucleic acid sample having a target base sequence region is dispensed into a container containing a standard nucleic acid sample and the dispersed phase collapses. It is a figure.

The water-soluble component in the positive dispersed phase (103) contained in the droplet (971) landed on the well (1500) dissolves in the aqueous solution (1501). Therefore, the template nucleic acid molecule (110) for preparing a standard nucleic acid sample having the target base sequence region (A) contained in the positive dispersed phase (103) and the amplified base sequence region for detection (B) are added to the aqueous solution (1501). Dissolve.

When the specific gravity of the oil is larger than that of the aqueous solution (1501), the positive dispersed phase (103) sinks into the bottom of the aqueous solution (1501) together with the droplet (971), and the droplet (971) comes into contact with the interface of the oil. The positive dispersed phase (103) dissolves in the aqueous solution (1501).

When the positive dispersed phase (103) cannot come into contact with the aqueous solution (1501), the positive dispersed phase (103) is dissolved in the aqueous solution (1501) by performing centrifugation to form a layer separation based on the specific gravity. be able to.

Next, a program for producing a standard nucleic acid sample-containing container of the present invention, which causes a computer to perform the same functions as the method for producing a standard nucleic acid sample-containing container of the present invention, will be described. The processing according to the production program of the standard nucleic acid sample-containing container of the present invention can be carried out by using a computer having a control unit (960) for constructing the production apparatus of the standard nucleic acid sample-containing container of the present invention.

FIG. 12 is a flowchart showing an example of a processing procedure of a manufacturing program for a container containing a standard nucleic acid sample.

In S101, the control unit (960) includes a standard nucleic acid sample preparation template nucleic acid molecule (110) having a target base sequence region (A) and a detection base sequence region (B) by the dispersed phase forming means (401). Solution X is mixed with a nucleic acid amplification reagent (including a primer pair and a blocking oligonucleotide) and a solution Y containing a fluorescent labeling reagent, and the mixed solution Z is micropartitioned to form a dispersed phase, and then the treatment is carried out in S102. Transition.

In step S102, the control unit (960) uses the amplification means (601) to meander flow with respect to the dispersed phase (100) containing the template nucleic acid molecule (110) for preparing a standard nucleic acid sample, the nucleic acid amplification reagent, and the fluorescent labeling reagent. After raising and lowering the temperature in a plurality of temperature zones while flowing through the path, the process shifts to S103. By raising or lowering the temperature in a plurality of temperature zones, only the detection base sequence region (B) is amplified, and the state of the optically labeled amplified dispersion phase (102) is obtained.

In step S103, when the control unit (960) detects the fluorescence generated from the positive dispersion phase (103) in which the detection base sequence region (B) is amplified by the discrimination means (701), the process shifts to S104.

In step S104, the control unit (960) uses the sorting mechanism (702) to amplify the detection base sequence region (B) by dielectrophoresis based on the detection result of the discrimination means (701). Is selected, the process shifts to S105. Here, to select the positive dispersed phase (102) means that only the positive dispersed phase (102) is transported to the discharge mechanism (910) by the sorting mechanism (702) based on the intensity of the optical signal of the dispersed phase detected in S103. Means to do.

In step S105, the control unit (960) dispenses the positive dispersion phase (102) into the capsule (300) by the discharge mechanism (910), and then shifts the process to S106.

In step S106, the control unit (960) detects the positive dispersed phase (103) dispensed by the light source (930) and the light receiving element (950), and the number thereof is a desired number set by the user (for example, a capsule). If the number of dispersed phases to be dispensed in (300) has not been reached, the process is returned to S105. On the other hand, the control unit (960) ends this process when the counted number reaches a desired number set by the user.

Further, the nucleic acid may be dispensed into a plurality of capsules (300) by repeating the treatment procedure shown in FIG. 12 a plurality of times.

FIG. 17 is a flowchart showing another example of the processing procedure of the manufacturing program of the standard nucleic acid sample-containing container.

Further, since step S201 in FIG. 17 is the same process as S101, step S202 is S102, step S203 is S103, step S204 is S104, and step S206 is S105, the description thereof will be omitted.

In step S205, when the control unit (960) counts the number of positive dispersed phases (102) in the flow path in S204 by the light source (930) and the light receiving element (950), the process shifts to S206.

In step S207, the number of positive dispersion phases (102) dispensed in S206 is a desired number set by the user based on the number of positive dispersion phases (102) counted in S205 by the control unit (960). It is determined whether or not (for example, the number of dispersed phases to be dispensed into the well (1500)) has been reached. When the control unit (960) determines that the number of the dispensed positive dispersion phases (102) has not reached the desired number set by the user, the process returns to S206. On the other hand, when the control unit (960) determines that the number of the dispensed positive dispersion phases (102) has reached the desired number set by the user, the control unit (960) ends this process.

Further, the template nucleic acid molecule for preparing a standard nucleic acid sample may be dispensed into a plurality of wells (1500) by repeating the treatment procedure shown in FIG. 17 a plurality of times.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

<< Example 1: Preparation of a container containing a double-stranded DNA (dsDNA) sample >>
Based on the method for producing a standard nucleic acid sample-containing container of the present invention, a particulate container containing double-stranded DNA as a standard nucleic acid sample is prepared.
(1) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescent labeling reagent DNA600-G (manufactured by National Research and Development Corporation, Industrial Technology Research Institute, NMIJ CRM 6205-a, SEQ ID NO: 1) and nucleic acid as template nucleic acids Fw primer (SEQ ID NO: 2) and Rv primer (SEQ ID NO: 3) complementary to nucleic acid as amplification reagent, base sequence complementary to a part between Fw primer and Rv primer of nucleic acid as fluorescence labeling reagent, and fluorescent dye TaqMan probe (SEQ ID NO: 4) having TAMRA as a nucleic acid, Supermix (manufactured by Bio-Rad, ddPCR Supermix for Probes (no dUTP), 186-3025), NFW (Thermo Fisher).
Using UltraPure DNase / RNase-Free-Distilled Water, 10977-015) manufactured by Scientific, reagents were prepared at the mixing ratio shown in Table 2, and finally a plate for digital PCR (Bio-Rad, ddPCR96-Well). Plates, 1200125) were filled with 22 μL each. Each reagent was diluted with NFW to the concentration shown in Table 2 and used.

(2) Dispersion phase forming step The mixed solution prepared by using a droplet generator (manufactured by BioRad, whose device name is Automated Droplet Generator) was classified into dispersed phases. As the solvent for the mobile phase, Automated Droplet Generator Oil for Probes manufactured by BioRad was used. The prepared droplet suspension was subjected to the next amplification step immediately after aluminum sealing using a plate sealer (manufactured by BioRad, trade name: PX1 Plate Sealer).

(3) Amplification step The amplification step was carried out by a thermal cycler (manufactured by BioRad, device name: t100 thermal cycler) by the thermal cycle process shown in Table 3.

(4) Confirmation of Nucleic Acid Amplification and Calculation of Uncertainty The dispersed phase (also referred to as a positive dispersed phase) in which nucleic acid amplification was performed is fluorescently subjected to a droplet reader (manufactured by BioRad, device name: QX200 Droplet Reader). While confirming, based on the detection results (FIG. 18 and Table 4), the probability of each number of copies contained in the dispersed phase was calculated by a calculation process (the above equation 1) in consideration of the Poisson distribution. The calculated results (in the case of D02 in Table 4) are shown in Table 5.

(5) Discrimination and Sorting Step of Dispersed Phase The sorting of the nucleic acid-amplified dispersed phase (positive dispersed phase) and the dispensing into the particulate container are discriminated using, for example, the sorting mechanism (702) shown in FIG. Sorted. The detection result of the positive dispersed phase is shown in FIG. In addition, an applied waveform (dotted line) was applied to generate an unequal electric field with respect to the positive dispersed phase (solid line) (FIG. 20). FIG. 21 shows how the positive dispersed phase (102) is selected. The selected positive dispersed phase was introduced into the discharge mechanism in the dispensing step of the next step.

(6) Dispersed phase dispensing step A flow path for dispensing was provided inside the particulate container as shown in FIG. 8, and the dispersed phase was dispensed while detecting the number of selected dispersed phases. MP Capsule No. 5 (manufactured by AS ONE Corporation) was used as the capsule used as the particulate container. Information on the total number of copies of the target nucleotide sequence region in the particulate container and the uncertainty of the DNA copy number obtained from the number of copies of the target nucleotide sequence region contained in one drop in the calculated dispersed phase is provided in the dispensed capsule. It was displayed by coloring and made visible.

<< Example 2: Preparation of a double-stranded nucleic acid (dsDNA) sample-containing container >>
The dispersed phase was dispensed into the container in the same manner as in Example 1. As a container, MicroAmp ^TM Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used.

After dispensing the positive dispersed phase into the container, the container was vacuum dried. A barcode was attached to the side of the dried container (plate). The barcode includes the calculation result of the total number of copies of the target nucleotide sequence region dispensed into the container (calculated from the number of copies of the target nucleotide sequence region contained in one drop of the dispersed phase) and the dsDNA dispensed into the container. Information on the uncertainty of the number of copies that counted both the sense strand and the antisense strand of the above was made available by barcode.

<< Example 3: Preparation of a container containing a single-strand RNA (ssRNA) sample >>
(1) Preparation of RNA-DNA hybridizing nucleic acid solution-
The composition of RNA500-A (manufactured by National Institute of Advanced Industrial Science and Technology, NMIJ CRM 6204-a, see SEQ ID NO: 5) and ssDNA (single-stranded DNA) (SEQ ID NO: 6) as a template nucleic acid are shown in Table 6. After mixing with, the mixture was heat-denatured at 95 ° C. for 2 minutes, cooled to room temperature for 1 hour, and sufficiently hybridized to synthesize RNA-DNA double strands. Then, 5 μL of Exonucleoase I (manufactured by Takara Bio Inc.) was added, and the mixture was allowed to stand at room temperature for 30 minutes to remove single-stranded DNA. Then, it was inactivated at 80 ° C. for 15 minutes to inactivate Exonuclease I to obtain an RNA-DNA hybridized nucleic acid solution.

RNA-DNA hybrid solution as a template nucleic acid, Fw primer (SEQ ID NO: 7) and Rv primer (SEQ ID NO: 8) complementary to nucleic acid as nucleic acid amplification reagent, and Fw primer and Rv primer of nucleic acid as fluorescent labeling reagent. Nucleic acid / nucleic acid amplification in the same manner as in Example 1 except that a base sequence complementary to a part of the space between them, FAM as a fluorescent dye, and a TaqMan probe (SEQ ID NO: 9) having TAMRA as a photochromic substance were used. Preparation of a mixed solution of reagents and fluorescent labeling reagents, formation of dispersed phases, amplification steps, confirmation of nucleic acid amplification, and calculation of uncertainty were performed. The composition is shown in Table 7 below. The results are shown in FIGS. 22, 8 and 9 (for F05 in Table 8).

(2) Dispersion Phase Discrimination and Sorting Step The obtained dispersed phase was discriminated and sorted in the same manner as in Example 1.

(3) Dispersed phase dispensing step The positive dispersed phase was dispensed into the container in the same manner as in Example 2. As a container, MicroAmp ^TM Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used. The positive dispersed phase was dispensed into a container (plate) and then dried by vacuum drying. A barcode was attached to the side of the dried container (plate). The barcode shows the calculation result of the total number of copies of the target nucleotide sequence region dispensed into the container (plate) (calculated from the number of copies of the target nucleotide sequence region contained in one drop of the dispersed phase) and the number of copies in the container. Information on the uncertainty of the number of RNA copies injected was made available by barcode.

<< Example 4: Preparation of a container containing a single-strand DNA (ssDNA) sample >>
(1) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescence labeling reagent k03_ssDNA (manufactured by Integrated DNA Technologies, SEQ ID NO: 10) as a template nucleic acid and a forward primer complementary to the nucleic acid (SEQ ID NO: 11) as a nucleic acid amplification reagent. ) And reverse primer (SEQ ID NO: 12), a base sequence complementary to a part between the forward primer and the reverse primer of nucleic acid as a fluorescent labeling reagent, FAM as a fluorescent dye, and ZEN and IowaBlack (registered trademark) as a photochromic substance. Preparation of a mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescence labeling reagent, formation of dispersed phase, amplification step, and nucleic acid amplification in the same manner as in Example 1 except that the TaqMan probe having the above (SEQ ID NO: 13) was used. Was confirmed and the uncertainty was calculated. The composition is shown in Table 10 below, and the thermal cycle process in the amplification step is shown in Table 11. The results are shown in FIGS. 23, 12 and 13 (for E11 in Table 12).

(2) Dispersion Phase Discrimination and Sorting Step The obtained dispersed phase was discriminated and sorted in the same manner as in Example 1.

(3) Dispersed phase dispensing step The positive dispersed phase was dispensed into the container in the same manner as in Example 2. As a container, MicroAmp ^TM Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used. The positive dispersed phase was dispensed into a container (plate) and then dried by vacuum drying. A barcode was attached to the side of the dried container (plate). The barcode shows the calculation result of the total number of copies of the target nucleotide sequence region dispensed into the container (plate) (calculated from the number of copies of the target nucleotide sequence region contained in one drop of the dispersed phase) and the number of copies in the container. Information on the uncertainty of the number of ssDNA copies injected was made available by barcode.

<< Example 5: Evaluation of effectiveness of blocking oligo >>
(1) Amplification of the base sequence region for detection (first-stage PCR reaction)
(A) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescence labeling reagent / blocking oligo It is present in the sequence of k03_ssDNA (manufactured by Integrated DNA Technologies, see SEQ ID NO: 10) as a template nucleic acid and k03_ssDNA as a nucleic acid amplification reagent. Fw primer (see SEQ ID NO: 14) and Rv primer (see SEQ ID NO: 15) complementary to the base sequence region for detection, Fw primer of nucleic acid as a fluorescent labeling reagent, and a base sequence complementary to a part between Rv primers. Complementary to the inhibitory nucleotide sequence (SEQ ID NO: 17) present in the sequences of TaqMan® probe (SEQ ID NO: 16), k03_ssDNA, which has HEX as a fluorescent dye, ZEN and IowaBlack® as a light-dissipating substance. PrimeTime Master Mix (Integrated), a blocking oligo (SEQ ID NO: 18) consisting of a peptide nucleic acid (PNA) having a unique base sequence.
Prime Time (registered trademark) Gene, manufactured by DNA Technologies, Inc.
Reagents were prepared using Expression Master Mix) and NFW (UltraPure DNase / RNase-Free-Distilled Water: 10977-015) manufactured by Thermo Fisher Scientific, in the mixing ratio shown in Table 14.

(B) First-stage PCR reaction The prepared reaction solution (30 μL) was placed in a thermal cycler (BioRad: T100), and the first-stage PCR reaction was performed according to the thermal cycle process shown in Table 15. I got 1. At the same time, the negative control to which no blocking oligo was added was also subjected to the first-stage PCR to obtain Reaction Solution 2.

(2) Amplification of target base sequence region (second stage PCR reaction)
(A) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescent labeling reagent Fw primer (SEQ ID NO: 11) and Rv primer (sequence number 11) complementary to the target base sequence region existing in the sequence of k03-ssDNA as a nucleic acid amplification reagent. No. 12), TaqMan® having a base sequence complementary to a part between the Nucleic acid Fw primer and Rv primer as a fluorescent labeling reagent, FAM as a fluorescent dye, and ZEN and IowaBlack® as a fluorescent dye. Probe (SEQ ID NO: 13), PrimeTime (registered trademark) Master Mix (manufactured by Integrated DNA Technologies), PrimeTime (registered trademark) Gene Expression Master Mix) and NFW (ThermoUniselect) and NFW (ThermoFirseSystem) Using 10977-015), a reagent was prepared at the mixing ratio shown in Table 16 (reaction solution 3). Then, 88 μL of the reaction solution 3 was filled in 8 wells. To wells 1 and 2, 30 μL of reaction solution 1 (k03_ssDNA = 1320 copies) and reaction solution 2 (k03_ssDNA = 1320 copies) were added, respectively. After adding 15 μL of Prime Time Master Mix and 7.5 μL of NFW to wells 3 to 8, k03_ssDNA dilution series (well 3: 132000 copies, well 4: 13200 copies, wells 5: 1320 copies, wells 3 to 8). Well 132 copies, well 7: 13.2 copies) or NFW (well 8) as NTC was added in 7.5 μL each. Wells 1 to 8 were well stirred with a vortex mixer and then spun down with a small centrifuge. Finally, MicroAmp ^TM Fast Optical 96-Well Reaction Plate, 0.1 mL (manufactured by Thermo Fisher Scientific) was filled with 20 μL of each of the solutions of wells 1 to 8 into 5 wells, spun down by a centrifuge, and the reaction plate. Was produced.

(B) Second-stage PCR reaction The prepared reaction plate was placed in real-time PCR (Quant Studio 7Flex manufactured by Thermo Fisher Scientific), and the second-stage PCR reaction was performed according to the thermal cycle process shown in Table 17, and each well was subjected to a second-stage PCR reaction. Ct value was obtained. Table 18 shows the copy number in the well of each sample, the average Ct value, and the copy number estimated from the k03_ssDNA dilution series calibration curve. According to this, it can be seen that the well 2 to which the blocking oligo is not added has a larger copy number than the original addition amount (1320 copies). That is, in the first-stage PCR reaction, an overrun of the PCR reaction occurs when the detection base sequence region is amplified, and the target base sequence region existing on the 5'end side of the detection base sequence region is amplified. It is shown that. On the other hand, in the well 1 to which the blocking oligo was added, the amount was almost the same as the original addition amount (1320 copies). Therefore, it was shown that the addition of blocking oligos can inhibit the overrun of the PCR reaction.

<< Example 6: Evaluation of Efficacy of Restriction Enzyme >>
(1) Amplification of detection base sequence region (first-stage PCR reaction)
(A) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescent labeling reagent / blocking oligo k03_dsDNA (manufactured by Integrated DNA Technologies, SEQ ID NO: 19 [only sense strand]) as a template nucleic acid and k03_dsDNA as a nucleic acid amplification reagent Fw primer (SEQ ID NO: 14) and Rv primer (SEQ ID NO: 15) complementary to the detection base sequence region present in the sequence, and a part of the nucleic acid Fw primer and Rv primer as fluorescent labeling reagents. Specific to the nucleotide sequence, HEX as the fluorescent dye, TaqMan® probe (SEQ ID NO: 16) having ZEN and IowaBlack® as the extinguishing substance, and the inhibitory nucleotide sequence (GGCC) present in the sequence of k03_dsDNA. Restrictions that act in a positive manner: HaeIII (New England Biolabs, HaeIII), ddPCR Supermix (Bio-Rad, ddPCR Supermix for Probes (no dUTP), NFW (Ther Primer)) and NFW (Therse Primer) -Distilled Water: 10977-015) was used to prepare reagents at the mixing ratios shown in Table 19.

(B) First-stage PCR reaction The prepared reaction solution (30 μL) was placed in a thermal cycler (BioRad: T100), and the first-stage PCR reaction was performed according to the thermal cycle process shown in Table 20, and the reaction solution was used. I got 1. At the same time, a negative control to which the restriction enzyme HaeIII was not added was also subjected to the first-stage PCR to obtain a reaction solution 2.

(2) Amplification of target base sequence region (second stage PCR reaction)
(A) Preparation of mixed solution of nucleic acid / nucleic acid amplification reagent / fluorescent labeling reagent Fw primer (SEQ ID NO: 11) and Rv primer (SEQ ID NO: 12) complementary to the target nucleotide sequence region existing in the sequence of k03_dsDNA as a nucleic acid amplification reagent. ), TaqMan® probe having a base sequence complementary to a part between the Fw primer and Rv primer of nucleic acid as a fluorescent labeling reagent, FAM as a fluorescent dye, and ZEN and IowaBlack® as a light-dissipating substance (registered trademark). SEQ ID NO: 13), ddPCR Supermix (Bio-Rad, ddPCR Supermix for Probes (no dUTP)) and NFW (Thermo Fisher Scientific, UltraPure DNase / RNase / RNase / RNase / RNase / RNase / RNase / RNase- Nucleic acids were prepared at the mixing ratios shown in Table 21 (reaction solution 3). Then, 88 μL of the reaction solution 3 was filled in 8 wells. To wells 1 and 2, 30 μL of reaction solution 1 (k03_dsDNA = 1480 copies) and reaction solution 2 (k03_dsDNA = 1480 copies) were added, respectively. To wells 3 to 8, 15 μL of ddPCR Supermix and 7.5 μL of NFW were added, and then k03_dsDNA dilution series (well 3:148,000 copies, wells 4: 14800 copies, wells 5: 1480 copies, wells) were added to each well. 6: 148 copies, well 7: 14.8 copies) or 7.5 μL of NFW (well 8) as NTC was added. Wells 1 to 8 were well stirred with a vortex mixer and then spun down with a small centrifuge. Finally, MicroAmp ^TM Fast Optical 96-Well Reaction Plate, 0.1 mL (manufactured by Thermo Fisher Scientific) was filled with 20 μL of each of the solutions of wells 1 to 8 into 5 wells, spun down by a centrifuge, and the reaction plate. Was produced.

(B) Second-stage PCR reaction The prepared reaction plate was placed in real-time PCR (Quant Studio 7Flex manufactured by Thermo Fisher Scientific), and the second-stage PCR reaction was performed according to the thermal cycle process shown in Table 22, and each well was subjected to a second-stage PCR reaction. Ct value was obtained. Table 23 shows the copy number in the well of each sample, the average Ct value, and the copy number estimated from the k03_dsDNA dilution series calibration curve. According to this, it can be seen that in the well 2 not subjected to the restriction enzyme treatment, a larger copy number than the original addition amount (1480 copies) is present. That is, in the first-stage PCR reaction, an overrun of the PCR reaction occurs when the detection base sequence region is amplified, and the target base sequence region existing on the 5'end side of the detection base sequence region is amplified. It is shown that. On the other hand, in the well 1 treated with the restriction enzyme, the amount was almost the same as the original addition amount (1480 copies). Therefore, it was shown that restriction enzyme treatment can inhibit the overrun of the PCR reaction.

Japanese Unexamined Patent Publication No. 2014-33658 JP-A-2015-195735 Japanese Unexamined Patent Publication No. 2008-245612

100 Distributed phase 110 Template nucleic acid molecule for standard nucleic acid sample preparation 300 Standard nucleic acid sample-containing container (particulate)
401 Dispersed phase forming means 601 Amplifying means 701 Discriminating means 702 Sorting mechanism 801 Discharge mechanism 960 Control unit

Claims (16)

A template nucleic acid molecule for preparing standard nucleic acid samples.
The template nucleic acid molecule contains a target base sequence region, an inhibitory base sequence region, and a detection base sequence region in this order from the 5'end side.
The base sequence region of interest contains the base sequence of the target nucleic acid as a standard nucleic acid sample.
The inhibitory base sequence region contains a region to which a blocking oligonucleotide complementarily binds and / or a restriction enzyme target site, and is different from the base sequence of the target base sequence region and the detection base sequence region. Consists of a base sequence that is not annealed
The detection base sequence region is a target region for nucleic acid amplification, and is a template nucleic acid molecule having a base sequence different from the base sequence of the target base sequence region. The template nucleic acid molecule according to claim 1, wherein each base sequence region does not contain the same base sequence of 4 or more consecutive bases. The template nucleic acid molecule according to claim 1 or 2, wherein the inhibitory base sequence region comprises 7 or more bases and less than 100 bases when the blocking oligonucleotide contains a region to which the blocking oligonucleotide is complementary. The template nucleic acid molecule according to claim 1 or 2, wherein the inhibitory base sequence region comprises 4 or more bases and less than 100 bases when containing the restriction enzyme target site. The template nucleic acid molecule according to any one of claims 1 to 4, wherein the detection base sequence region comprises 20 bases or more and 1000 bases or less. The template nucleic acid molecule according to any one of claims 1 to 5, wherein the target base sequence region contains two or more target sequences that are the same and / or different. A kit for preparing standard nucleic acid samples
The template nucleic acid molecule according to any one of claims 1 to 6.
A restriction enzyme that recognizes and cleaves a blocking oligonucleotide and / or a restriction enzyme target site that can complementarily bind to the inhibitory base sequence region of the template nucleic acid molecule, and can amplify the detection base sequence region of the template nucleic acid molecule. The kit, which comprises a suitable primer pair. The kit according to claim 7, wherein the blocking oligonucleotide comprises a natural nucleotide and / or a non-natural nucleotide. The kit according to claim 8, wherein the non-natural nucleotide is any one or more of BNA, LNA, and PNA. A method for discriminating a dispersed phase containing a standard nucleic acid sample.
Recognize and cleave the template nucleic acid molecule according to any one of claims 1 to 6, the blocking oligonucleotide and / or the restriction enzyme target site that can complementarily bind to the inhibitory base sequence region of the template nucleic acid molecule. An amplification step of amplifying the detection base sequence region using a dispersion phase solution containing a restriction enzyme and a primer pair capable of amplifying the detection base sequence region of the template nucleic acid molecule, and for detecting an amplification product after the amplification step. The method including a determination step of detecting based on a base sequence region and determining that the dispersed phase solution is a positive dispersed phase containing a standard nucleic acid sample when the amplified product is detected. A method for selecting a dispersed phase containing a standard nucleic acid sample.
The template nucleic acid molecule according to any one of claims 1 to 6, a blocking oligonucleotide capable of complementarily binding to the inhibitory base sequence region of the template nucleic acid molecule, and / or a restriction enzyme, a restriction enzyme capable of cleaving a target site. , And an amplification step of amplifying the detection base sequence region using a plurality of dispersed phase solutions containing a primer pair capable of amplifying the detection base sequence region of the template nucleic acid molecule.
Discrimination step in which the amplification product is detected based on the detection base sequence region after the amplification step, and the detected dispersed phase solution and the undetected dispersed phase solution of the amplification product are discriminated as a positive dispersed phase and a negative dispersed phase, respectively. The method including a sorting step of selecting a dispersed phase solution determined to be a positive dispersed phase in the discrimination step as a dispersed phase containing a standard nucleic acid sample. The method according to claim 10 or 11, wherein the detection in the discrimination step is to detect a signal emitted by the amplification product. The method according to any one of claims 10 to 12, wherein the positive dispersed phase is a dispersed phase containing only one template nucleic acid molecule per solution with a probability of 90% or more. A method for manufacturing a container containing a standard nucleic acid sample.
A copy of the target base sequence region contained in the template nucleic acid molecule with the positive dispersed phase obtained after the discrimination step in the discrimination method according to claim 10 or the positive dispersed phase obtained after the sorting step in the sorting method according to claim 11. The production method comprising a dispensing step of dispensing into a container so that the number is a predetermined number per container. The production method according to claim 14, wherein the target base sequence region included in each container is less than 1000 copies, and the coefficient of variation (CV value) of the copy number is less than 20%. A device for manufacturing standard nucleic acid sample-containing containers.
The template nucleic acid molecule according to any one of claims 1 to 6, a blocking oligonucleotide capable of complementarily binding to the inhibitory base sequence region of the template nucleic acid molecule, and / or a restriction enzyme, a restriction enzyme capable of cleaving a target site. , And an amplification means for amplifying the detection base sequence region using a dispersed phase solution containing a primer pair capable of amplifying the detection base sequence region of the template nucleic acid molecule.
After the amplification, the amplification product is detected based on the detection base sequence region, and when the amplification product is detected, the dispersion phase solution is determined to be a positive dispersion phase containing a standard nucleic acid sample, and a determination means. A dispensing means is provided for dispensing the dispersed phase solution determined to be the positive dispersed phase by the discrimination means into the container so that the number of copies of the target base sequence region contained in the template nucleic acid molecule is a predetermined number per container. The manufacturing apparatus.