JP7326778B2 - Nucleic Acid Sample Containing Container, Nucleic Acid Sample Containing Container Manufacturing Method, Nucleic Acid Sample Containing Container Manufacturing Device, Nucleic Acid Sample Containing Container Manufacturing Program, and Nucleic Acid Sample - Google Patents

Description

The present invention relates to a nucleic acid sample-containing container, a nucleic acid sample-containing container manufacturing method, a nucleic acid sample-containing container manufacturing apparatus, a nucleic acid sample-containing container manufacturing program, and a nucleic acid sample.

Nucleic acid analysis technology is widely used in various fields such as food testing, blood testing, and DNA (deoxyribonucleic acid) identification. Typical nucleic acid analysis techniques include, for example, real-time polymerase chain reaction (PCR) methods.

In the real-time PCR method, the fluorescence corresponding to nucleic acid amplification is timely detected in the process of PCR, and the standard sample series whose copy number is determined relative to the detected value of the nucleic acid in the target sample. A method for quantifying the amount of nucleic acids in a sample.
However, when quantifying nucleic acid in a sample, it is necessary to extract only the nucleic acid from PCR reaction inhibitors such as blood cells, sugar, and fat in the sample. , requires reverse transcription to cDNA. For this reason, in order to quantify nucleic acids in a sample, it is necessary to consider nucleic acid molecule extraction efficiency such as nucleic acid extraction efficiency and reverse transcription efficiency.
Therefore, conventionally, in order to consider the nucleic acid extraction efficiency, for example, after adding a standard nucleic acid sample with a defined concentration to the reaction system as an internal standard, extraction, reverse transcription, and real-time PCR are performed. It is common practice to quantify the efficiency of

Conventionally, in the nucleic acid standard sample used as an internal standard, in order to prepare a standard reagent for a region where the nucleic acid concentration is low, for example, a DNA fragment having a specific base sequence is subjected to limiting dilution, and the obtained diluted solution is quantitatively analyzed. A method has been proposed in which a diluent containing a desired copy number is selected from the results of PCR (see, for example, Patent Document 1).
There is also a method for producing a standard reagent containing a desired copy number of a DNA fragment by introducing a DNA fragment of a specific copy number into cells by gene recombination technology and manually isolating the cells into which the DNA fragment has been introduced. It has been proposed (see, for example, Patent Document 2).
Furthermore, a method of amplifying a region having a target nucleic acid base sequence in a droplet or gel droplet and sorting (selecting) the droplet or gel droplet in which the nucleic acid amplification reaction has occurred has been proposed (e.g. , see Patent Document 3).

An object of the present invention is to provide a nucleic acid sample-containing container containing a desired number of molecules (known number of copies) of a nucleic acid containing a predetermined number of target base sequences.

The nucleic acid sample-containing container of the present invention as a means for solving the above-mentioned problems comprises a predetermined number of first nucleic acid molecules having a target base sequence and a detection base sequence different from the target base sequence; and a second nucleic acid molecule that does not have the target nucleotide sequence and has the detection nucleotide sequence.

According to the present invention, it is possible to provide a nucleic acid sample-containing container containing a desired number of molecules (known number of copies) of a nucleic acid containing a predetermined number of target base sequences.

FIG. 1 is a diagram showing the relationship between copy number and coefficient of variation CV. FIG. 2 is a diagram showing an example of the nucleic acid sample-containing container of the present invention. FIG. 3 is a diagram showing an example of a dispersed phase forming means for dividing a solution into dispersed phases in a microchannel. FIG. 4 is a diagram showing another example of a dispersed phase forming means for dividing a solution into dispersed phases in a microchannel. FIG. 5 is a diagram showing an example of the amplifying means. FIG. 6 is a diagram showing an example of a sorting mechanism in the discriminating means and the sorting means. FIG. 7 is a diagram showing an example of a discharge mechanism in the dispensing means. FIG. 8 is an explanatory diagram showing an embodiment of a discharge mechanism and a counting mechanism in the dispensing means. FIG. 9 is a diagram illustrating hardware blocks of a control unit. FIG. 10 is a diagram illustrating functional blocks of a control unit; FIG. 11 is a flow chart showing an example of a processing procedure of a nucleic acid sample-containing container manufacturing program stored in a non-transitory recording medium. FIG. 12 is a diagram illustrating another example of functional blocks of the control unit; FIG. 13 is a flow chart showing an example of the operation of the ejection mechanism and counting mechanism in the dispensing means. FIG. 14 is an explanatory diagram showing an example of a state immediately after a dispersed phase containing a nucleic acid having a target base sequence is dispensed into a container. FIG. 15 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. 16 is a flow chart showing another example of the processing procedure of the nucleic acid sample-containing container manufacturing program stored in the non-transitory recording medium. FIG. 17 is a diagram showing an example of the results related to the production of the nucleic acid sample-containing container of the present invention. FIG. 18 is a diagram showing another example of the results related to the production of the nucleic acid sample-containing container of the present invention. 19A and 19B are diagrams showing an example of the operation of the sorting means in manufacturing the nucleic acid sample-containing container of the present invention. FIG. 20 is a diagram showing an example of the operation of the sorting means in manufacturing the nucleic acid sample-containing container of the present invention. FIG. 21 is a diagram showing an example of the results related to the production of the nucleic acid sample-containing container of the present invention. FIG. 22 is a diagram showing an example of the results of manufacturing the nucleic acid sample-containing container of the present invention.

(Container containing nucleic acid sample)
The nucleic acid sample-containing container of the present invention has a predetermined number of first nucleic acid molecules having a target base sequence and a base sequence for detection of a base sequence different from the target base sequence, and does not have the target base sequence. , a second nucleic acid molecule having the base sequence for detection, a solution, and optionally other materials.

The inventors of the present invention examined a nucleic acid sample-containing container containing a desired number of molecules (known number of copies) of a nucleic acid containing a predetermined number of target base sequences, and obtained the following findings.
As shown in the prior art, the standard nucleic acid sample used in the quantification method using real-time PCR contains a specific number of molecules (copy number) from the results of real-time PCR for a diluted solution of nucleic acid having the target base sequence. When sorting a liquid, when diluting nucleic acids with less than 100 molecules (copy numbers), the influence of Poisson distribution increases the uncertainty of the number of molecules (copy numbers) of the nucleic acids contained in the diluted solution. There is a problem that it is difficult to prepare a standard reagent containing an arbitrary number of molecules of nucleic acid.
In addition, conventional methods require complex operations such as genetic recombination and cell culture when using cells into which the desired nucleotide sequence has been introduced using genetic recombination technology, and preparation of standard nucleic acid samples in a short period of time. The problem is that it is difficult to In addition, since a nucleic acid having a target nucleotide sequence is introduced into cells and the isolated cells are treated to prepare a standard nucleic acid sample, the prepared standard nucleic acid sample must contain contaminants (proteins, lipids, etc.) derived from cells. is mixed in, there is a problem that the reaction system may be contaminated.
Further, the conventional method describes a method of individually amplifying multiple kinds of nucleic acids by microcompartmentalization and sorting (selecting) the microcompartments (droplets or gel droplets) in which the nucleic acid amplification reaction has occurred. Since microcompartments contain multiple types of nucleic acids, it is difficult to ensure the presence of only one type of nucleic acid molecule in a positively detected microcompartment, and the desired number of molecules (predetermined number of copies There is a problem that it is difficult to obtain a nucleic acid sample having a target base sequence of a certain number (also referred to as specific copy number).
Furthermore, standard nucleic acid samples produced by these conventional techniques exist in the form of solutions or suspensions, making it difficult for users to add the required amount of standard nucleic acid samples to any reaction system. There's a problem.

In the nucleic acid sample-containing container of the present invention, by containing a predetermined number of the first nucleic acid molecules in the container, the first nucleic acid molecules, which are nucleic acid samples, are excellent in storage stability and user operability, and further contain Since the number of nucleic acid molecules (the number of copies (units) of the target base sequence) is known, a minute amount of the nucleic acid sample can be added to any reaction system. Here, the term "trace amount" means the number of molecules of about 1 molecule or more and 1,000 molecules or less.

In the present invention, a predetermined base sequence may be used as a basic unit and may be referred to as a copy, and the number may be counted using a predetermined base sequence as a unit (for example, one target base sequence has one copy, etc.). ). Furthermore, one unit of a nucleic acid that includes this predetermined base sequence and other base sequences that are linked together is sometimes referred to as, for example, one molecule.

The copy number means the number of target or specific base sequences (the number of sense strands) in the nucleic acid contained in the well.
The target nucleotide sequence refers to at least the nucleotide sequences of the primer and probe regions that have been determined, and in particular, those that have determined the entire length of the nucleotide sequence are also referred to as specific nucleotide sequences.
A specific copy number means that the number of target base sequences among the copy numbers is specified with at least a certain degree of accuracy.
That is, it can be said to be known as the number of target base sequences actually contained in a container (eg, particles, wells, etc.). That is, the specific copy number in the present application has higher accuracy and reliability as a number than the copy number (calculated estimated value) obtained by conventional serial dilution, especially in a low copy number region of 1,000 or less. is also a controlled value independent of the Poisson distribution. For the controlled value, the coefficient of variation CV, which represents the uncertainty, is generally within the range of either CV < 1/√x or CV ≤ 20% with respect to the average copy number x. preferable. Therefore, by using a container (e.g., particles, wells, etc.) containing the specific copy number of the target nucleotide sequence, it is possible to perform qualitative and quantitative analysis of a sample having the target nucleotide sequence more accurately than before. Inspection can be performed.
In the case of a nucleic acid molecule such as a double-stranded DNA in which a target nucleotide sequence (sense strand) and a complementary strand (antisense strand) having a complementary nucleotide sequence are integrated, simply the sense strand The total number of copies of the "target nucleotide sequence in the sense strand" and the "complementary strand of the target nucleotide sequence in the antisense strand" is present in the reaction system, rather than the number of copies of the target nucleotide sequence alone. should be thought of as the "copy number" of the target sequence
In addition, in the present invention, the specific copy number of a nucleic acid is also referred to as the predetermined number or absolute number of the nucleic acid.
The specific copy number of the nucleic acid is preferably 1 copy or more and 1,000 copies or less, preferably 100 copies or less, more preferably 20 copies or less, and even more preferably 10 copies or less.

-nucleic acid-
Nucleic acid means a macromolecular organic compound in which nitrogen-containing bases derived from purines or pyrimidines, sugars, and phosphoric acids are regularly linked, and includes fragments of nucleic acids, analogs of these nucleic acids or fragments thereof, and the like.
Nucleic acids are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include DNA, RNA, cDNA and the like. Plasmids can also be used as nucleic acids. Nucleic acids may be modified or mutated.
Nucleic acid or nucleic acid fragment analogs include nucleic acids or nucleic acid fragments bound to non-nucleic acid components, and nucleic acids or nucleic acid fragments labeled with fluorescent dyes, isotopes, and other labeling agents (e.g., fluorescent labeling dyes, radioactive (labeled primers and probes), artificial nucleic acids (eg, PNA, BNA, LNA, etc.) in which the chemical structure of some of the nucleotides constituting the nucleic acid or nucleic acid fragment is changed.
As for the nucleic acid, when the same molecule has a plurality of target base sequences to be described later, at least one artificial nucleic acid that cannot be amplified by a natural nucleic acid synthetase (such as DNA polymerase) is placed between each target base sequence. It is preferred to have a base. In the case where one molecule has multiple target base sequences by having at least one base between each target base sequence and an artificial nucleic acid that cannot be amplified by natural nucleic acid synthetase (DNA polymerase, etc.) Also, since the target base sequence is amplified one copy at a time, multiple amplification products can be obtained at once even when the number of nucleic acid molecules having the target base sequence is small.
These may be natural products obtained from living organisms or processed products thereof, products produced using genetic recombination technology, artificial synthetic nucleic acid molecules chemically synthesized, and the like. These may be used individually by 1 type, and may use 2 or more types together. By using an artificially synthesized nucleic acid molecule, impurities can be reduced and the molecule can be made low-molecular-weight, thereby improving the initial reaction efficiency.
The term "artificially synthesized nucleic acid molecule" means a nucleic acid obtained by artificially synthesizing a nucleic acid composed of components (bases, deoxyribose, phosphoric acid) similar to those of naturally occurring DNA or RNA. Artificially synthesized nucleic acid molecules include, for example, not only nucleic acids having base sequences that encode proteins, but also nucleic acids having arbitrary base sequences.

--First Nucleic Acid Molecule--
The first nucleic acid molecule has a target base sequence and a base sequence for detection of a base sequence different from the target base sequence.
Also, the nucleic acid sample-containing container of the present invention has a predetermined number of first nucleic acid molecules.
The predetermined number of the first nucleic acid molecules is not particularly limited, can be appropriately selected according to the purpose, and is preferably 100 molecules or less.
The form of the first nucleic acid molecule is not particularly limited and can be appropriately selected depending on the purpose. Examples include nucleic acid molecules in which double-stranded nucleic acid molecules and single-stranded nucleic acid molecules are mixed. A nucleic acid molecule in which a double-stranded nucleic acid molecule and a single-stranded nucleic acid molecule are mixed is a nucleic acid molecule in which a double-stranded portion and a single-stranded portion are mixed in the first nucleic acid molecule. means.

--- Target base sequence ---
The target base sequence is not particularly limited as long as it is different from the base sequence for detection described below, and can be selected as appropriate. Hereinafter, the target base sequence may be referred to as a base sequence different from the detection base sequence (different sequence). Examples of target nucleotide sequences include the following.
Base sequences used for infectious disease testing, non-natural base sequences that do not exist in nature, base sequences derived from animal cells, base sequences derived from plant cells, and the like can be mentioned. These may be used individually by 1 type, and may use 2 or more types together.

When using a non-natural base sequence, the GC content is preferably 30% or more and 70% or less of the target base sequence.

The base length of the target base sequence is not particularly limited and can be appropriately selected depending on the purpose. is mentioned.

The form of the target base sequence is not particularly limited and can be appropriately selected depending on the purpose. Examples include nucleic acids in which nucleic acid molecules and single-stranded nucleic acid molecules are mixed. Among these, the target base sequence is preferably a single-stranded nucleic acid. When the target base sequence is a single-stranded nucleic acid, only the sense strand of the target base sequence can be used, and compared to the case of using a double-stranded nucleic acid that also contains an antisense strand, the amount of the nucleic acid sample-containing container can be reduced. The copy number of the target base sequence can be controlled with higher precision.

When using a nucleotide sequence used for infectious disease testing, there is no particular restriction as long as it contains a nucleotide sequence specific to that infectious disease, and it can be selected as appropriate according to the purpose. It preferably contains a nucleotide sequence that is

The target base sequence to be contained in the nucleic acid sample-containing container of the present invention can be arbitrarily selected according to the application of the user. For example, base sequences used in known standard nucleic acid samples are preferable. By selecting a nucleotide sequence used in a known standard nucleic acid sample, it can be used, for example, as an internal standard for real-time PCR.

--- Nucleotide sequence for detection ---
The base sequence for detection is a base sequence different from the target base sequence described above, and is a base sequence used to confirm the presence or absence of a nucleic acid having the target base sequence. As the base sequence for detection, at least the primer region and the probe region are known base sequences when the present invention is carried out. It is a base sequence that does not amplify.
The base sequence for detection is not particularly limited as long as it is a base sequence different from the target base sequence, and can be appropriately selected according to the purpose.
Since the base sequence for detection is amplified by the amplification means described later, the GC content of the base sequence for detection is preferably 30% or more and 70% or less.
The base length of the base sequence for detection is not particularly limited and can be appropriately selected depending on the purpose.
The number of copies (units) of the base sequence for detection is not particularly limited, and can be appropriately selected depending on the purpose.
The form of the base sequence for detection is not particularly limited and can be appropriately selected depending on the purpose. Examples include nucleic acids in which nucleic acid molecules and single-stranded nucleic acid molecules are mixed. Among these, the base sequence for detection is preferably a single-stranded nucleic acid (ssDNA or ssRNA). When the base sequence for detection is a single-stranded nucleic acid, the first nucleic acid molecule can be detected without changing the number of copies of the target base sequence in the method for producing a nucleic acid sample-containing container of the present invention, which will be described later. Therefore, it is possible to improve the accuracy of the copy number of the target base sequence to be contained in the nucleic acid sample-containing container of the present invention. In addition, that the base sequence for detection is a single-stranded nucleic acid means that the base sequence for detection in the first nucleic acid molecule is at least a single-stranded nucleic acid.
The position of the base sequence for detection in the first nucleic acid molecule is not particularly limited and can be appropriately selected depending on the purpose.
In the method for producing a nucleic acid sample-containing container of the present invention, which will be described later, because the base sequence for detection is a single-stranded nucleic acid molecule and the base sequence for detection is present on the 5' end side of the target base sequence, Since the target base sequence can be detected without changing the copy number of the target base sequence in the first nucleic acid molecule, the accuracy of the target base sequence copy number to be contained in the nucleic acid sample-containing container of the present invention can be improved. In addition, due to the synergistic effect of the above configuration, it is possible to further improve the accuracy of the number of copies of the target base sequence to be contained in the nucleic acid sample-containing container of the present invention.
In addition, the distance between the target base sequence and the base sequence for detection is not particularly limited as long as it is sufficiently separated, and can be appropriately selected according to the purpose. preferably. However, this does not apply when the base sequence for detection is a single-stranded nucleic acid and exists on the 5' side of the target base sequence. Since the distance between the target base sequence and the detection base sequence is sufficiently far, the number of copies of the target base sequence in the first nucleic acid molecule can be increased in the method for producing a nucleic acid sample-containing container of the present invention, which will be described later. Since the target base sequence can be detected without being changed, the accuracy of the number of copies of the target base sequence to be contained in the container containing the nucleic acid sample of the present invention can be improved.

--Second Nucleic Acid Molecule--
The second nucleic acid molecule is a nucleic acid molecule that does not have the above-described target base sequence but has a detection base sequence.
The base sequence for detection in the second nucleic acid molecule is a base sequence amplified using the base sequence for detection in the first nucleic acid molecule as a template in the production of the nucleic acid sample-containing container of the present invention.

In the nucleic acid sample-containing container of the present invention, nucleic acids are preferably in a dry state or in a state dispersed in a solution. A dry state can reduce the possibility of the nucleic acid being decomposed by an enzyme, and can be stored at room temperature.

-solution-
The solution is not particularly limited as long as it can be dispersed without affecting the nucleic acid, and can be appropriately selected according to the purpose. Examples include buffer solutions, organic substance solutions, organic solvents, polymer gel solutions, colloidal dispersions, aqueous electrolyte solutions, aqueous inorganic salt solutions, aqueous metal solutions, and mixed liquids thereof. Among these, water and buffers are preferred, and water, nuclease-free water, phosphate buffered saline (PBS), Tris-EDTA buffer (TE) and the like are preferred. These may be used individually by 1 type, and may use 2 or more types together.

The solution may contain a nucleic acid amplification reagent used in the method for producing a nucleic acid sample-containing container of the present invention, which will be described later. Since the nucleic acid amplification reagent and the like will be described in detail in the manufacturing method of the nucleic acid sample-containing container of the present invention, which will be described later, the description thereof is omitted.

In addition, the first nucleic acid and the second nucleic acid of the nucleic acid sample-containing container of the present invention may be in a state of being included in the dispersed phase or in a state of being supported by the dispersed phase.

-dispersed phase-
By dispersed phase is meant micro-sized compartments.
The dispersed phase is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a liquid dispersed phase and a solid dispersed phase.

Dispersed phases in the liquid phase include, for example, water-in-oil droplets (W/O type) or oil-in-water droplets (O/W type) forming a water-oil emulsion, micelles, and the like.
The volume of the dispersed phase depends on the flow rate of the continuous phase, the flow rate of the dispersed phase, the width of the channel, the height of the channel, and the amount of surfactant in the preparation of the dispersed phase in the method for manufacturing a nucleic acid sample-containing container of the present invention, which will be described later. It can be controlled by the concentration, the viscosity of the solution, the pressure loss of the channel, and the like. The volume of the dispersed phase is preferably 1 fL or more and 1 μL or less.

Examples of the solid-phase dispersed phase include those in which the nucleic acid in the nucleic acid-containing liquid is captured on particles capable of capturing nucleic acid in units of one molecule.
The particles are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include silica particles that adsorb nucleic acids in a solid phase in the presence of such particles.

Particles whose surface is modified with a nucleic acid probe capable of specifically binding to a nucleic acid having a desired base sequence may be those particles having a surface modified with a nucleic acid probe capable of specifically binding to a nucleic acid having a desired base sequence. It is not particularly limited and can be appropriately selected depending on the purpose. For example, metals such as gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, nickel; alloys such as silicon; glass materials such as glass, quartz glass, fused quartz, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive glass; polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), plastics such as nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin; agarose, dextran, cellulose, polyvinyl alcohol, nitrocellulose, Examples include chitin and chitosan. These may be used individually by 1 type, and may use 2 or more types together. Furthermore, by using magnetic beads that are magnetized as particles, contain a magnetic material, or are magnetizable, separation processing and the like can be automated, efficient, or accelerated.

The number of particles having a surface modified with a nucleic acid probe capable of specifically binding to a nucleic acid having a target base sequence is such that one particle or less contains one molecule or less of a nucleic acid having a target base sequence. A ratio is preferable, and for example, it is preferable to adjust the number of particles so that the number of particles is about 1 to 10 times the number of nucleic acids having the target base sequence.

The method of modifying the particle surface with a nucleic acid probe capable of specifically binding to a nucleic acid having a desired base sequence is not particularly limited and can be appropriately selected depending on the purpose. , physical adsorption, and biological binding (for example, binding of biotin to avidin or streptavidin, binding of antigen to antibody, etc.).
A nucleic acid probe that can specifically bind to a nucleic acid having a target nucleotide sequence may be immobilized on particles via a spacer sequence, for example, a hydrocarbon group containing 1 to 10 carbon atoms.

For immobilization of a nucleic acid probe capable of specifically binding to a nucleic acid having a target base sequence to particles via a covalent bond, for example, a nucleic acid probe capable of specifically binding to a nucleic acid having a target base sequence may be functionalized. It can be carried out by introducing a group and a functional group reactive with the functional group to the particle surface and reacting them. For example, introducing an amino group into a nucleic acid probe capable of specifically binding to a nucleic acid having a target nucleotide sequence, and introducing an active ester group, epoxy group, aldehyde group, carbodiimide group, isothiocyanate group, or isocyanate group into particles. can form a covalent bond.
Alternatively, a mercapto group may be introduced into a nucleic acid probe capable of specifically binding to a nucleic acid having a target nucleotide sequence, and an active ester group, maleimide group or disulfide group may be introduced into the particles.
Active ester groups include, for example, p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group and the like.

Examples of methods for introducing functional groups to the surface of particles include a method of treating particles with a silane coupling agent having a predetermined functional group.
Silane coupling agents include, for example, γ-aminopropyltriethoxysilane, N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, N-β-(aminoethyl)-β-aminopropylmethyldimethoxysilane. , γ-glycidoxypropyltrimethoxysilane, and the like.

Plasma treatment is another method for introducing functional groups into the particles that serve as binding sites. Such plasma treatment can introduce functional groups such as hydroxyl groups and amino groups onto the surface of the solid phase. Plasma treatment can be performed using equipment known to those skilled in the art.

As a method for immobilizing a nucleic acid probe capable of specifically binding to a nucleic acid having a target base sequence by physical adsorption to particles, particles surface-treated with polycations (polylysine, polyallylamine, polyethyleneimine, etc.) are coated with oligo ( dT) can be used for electrostatic coupling.
Moreover, the shape of the dispersed phase is not particularly limited, and examples thereof include a spherical shape.

When the dispersed phase is a liquid phase, it is preferable that the dispersed phase is divided into microcompartments of the above solution by a continuous phase.
Continuous phase means a liquid in a dispersed system in which other liquids are dispersed. Here, it is synonymous with a dispersion medium, and means a liquid used for dividing a solution into dispersed phases and transporting the divided dispersed phases.

The continuous phase is not particularly limited as long as it can separate the solution, and can be appropriately selected according to the purpose. For example, when the dispersed phase is aqueous, fluorocarbon oil, mineral oil, etc. be done.

Further, when an aqueous solution is divided into dispersed phases using an oily continuous phase, it is preferable to add a surfactant to either the solution or the continuous phase. Addition of a surfactant can improve the stability of the separated dispersed phase.

The surfactant is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include ionic surfactants and nonionic surfactants. These may be used individually by 1 type, and may use 2 or more types together.

Examples of ionic surfactants include sodium fatty acid, potassium fatty acid, sodium alpha sulfo fatty acid ester, sodium linear alkylbenzene sulfonate, sodium alkyl sulfate, sodium alkyl ether sulfate, and sodium alpha olefin sulfonate. These may be used individually by 1 type, and may use 2 or more types together.

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 ( 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 individually by 1 type, and may use 2 or more types together.

-container-
The container is not particularly limited and can be appropriately selected according to the purpose. ) and the like. Among these, a particulate container is preferable. If the container is a particulate container, it can be a nucleic acid sample-containing container that is excellent in storability and operability and that contains a known number of copies (units) of the target base sequence.

The shape, size, volume, material, color, and number of nucleic acid sample-containing containers are not particularly limited, and can be appropriately selected according to the purpose.

The size of the nucleic acid sample-containing container is not particularly limited and can be appropriately selected according to the purpose. 3 mm is preferred, and 0.5 mm is more preferred. When the minor axis length of the particulate container is 0.1 mm or more, the particulate container can be made to be excellent in operability such as picking up by the user.

The shape of the nucleic acid sample-containing container is not particularly limited as long as it can contain an amplifiable reagent, and can be appropriately selected according to the purpose. , compartments on a substrate, spheres, and the like.

The number of recesses or compartments in the nucleic acid sample-containing container is preferably 2 or more, more preferably 5 or more, and even more preferably 50 or more.
As a nucleic acid sample-containing container, a microtube, a multiwell plate, or the like, which is connected by a base material having two or more recesses or compartments, can be preferably used.
Examples of microtubes connected by a substrate include 2, 3, 4, 6, 8, 12, 16, 24, and 48 microtubes.
Multi-well plates include, for example, 24-well, 48-well, 96-well, 384-well, or 1,536-well plates.

The volume of the nucleic acid sample-containing container is not particularly limited, and can be appropriately selected according to the purpose. More than 1,000 μL or less is preferable. However, when a particulate container is added to the reaction system as the nucleic acid sample-containing container, the volume is 200 μL or less, preferably 20 μL or less, and more preferably 2 μL or less. If the volume of the nucleic acid sample-containing container is 200 μL or less, the change in volume of the reaction system to which the nucleic acid sample-containing container is added can be reduced.

The material of the container containing the nucleic acid sample is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include insoluble and soluble materials.
Examples of insoluble materials include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
Soluble materials include, for example, sodium alginate, agarose gel, sugar and the like.

Examples of the color of the nucleic acid sample-containing container include transparent, translucent, colored, and completely light-shielding. When the nucleic acid sample-containing container is colored, the user can visually recognize the information of the nucleic acid sample-containing container by coloring, and the operability can be improved.

--Base material--
The substrate is preferably a plate-shaped substrate having a nucleic acid sample-containing container provided thereon, but may be a well tube of a connected type such as an octet tube.
The material, shape, size, structure, etc. of the base material are not particularly limited, and can be appropriately selected according to the purpose.
The material of the substrate is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include semiconductors, ceramics, metals, glass, quartz glass, and plastics. Among these, plastics are preferred.
Examples of plastics include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
The shape of the substrate is not particularly limited and can be appropriately selected depending on the intended purpose.
The structure of the base material is not particularly limited and can be appropriately selected depending on the intended purpose. For example, it may be a single layer structure or a multi-layer structure.

--particulate container--
As the particulate container, solid or gel particles having an outer shell containing the first nucleic acid molecule and the second nucleic acid molecule, or containing the first nucleic acid molecule and the second nucleic acid molecule are used. and so on.
Examples of the outer shell encapsulating the first nucleic acid molecule and the second nucleic acid molecule include capsule-like particles.

Further, the nucleic acid sample-containing container of the present invention has a dispersed phase containing nucleic acids including a first nucleic acid molecule and a second nucleic acid molecule, a continuous phase, and a protective member enclosing the dispersed phase and the continuous phase. You may have

Solid particles include, for example, tablet-like particles containing dispersed phases containing a first nucleic acid molecule and a second nucleic acid molecule. Specific examples include pharmaceutical capsules, liposomes, microcapsules and the like.
Gel particles include, for example, particles containing sodium alginate, agarose gel, and the like. Specifically, artificial salmon roe etc. are mentioned, for example.

The volume of the particulate container is not particularly limited and can be appropriately selected according to the purpose. If the volume is 200 μL or less, the change in volume of the reaction system to which the particulate container is added can be reduced.

The nucleic acid sample-containing container may be provided with information on the nucleic acid contained in the nucleic acid sample-containing container, information on the dispersed phase, and the like.

The information on the nucleic acid contained in the nucleic acid sample-containing container is not particularly limited and can be appropriately selected according to the purpose. Uncertainty in the number of nucleic acid molecules contained, the type of nucleic acid, and the like. These may be used singly 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 used singly or in combination of two or more.

The average value and standard deviation of the copy number of the nucleic acid dispensed in the container is the average value and standard deviation of the copy number of the target base sequence contained in one positive dispersed phase, from the following formula 1 to the formula 5 can be obtained. By using Formulas 1 to 5, the average value and standard deviation of the number of copies of the target nucleotide sequence contained in one positive variance can be obtained in consideration of the Poisson distribution.

In Equation 1, k is the number of copies contained in one positive dispersed phase, λ is the proportion of the positive dispersed phase (Posi) among the total dispersed phases in which the sample was measured, and P is the target copy number in the total positive dispersed phase. The probability of a positive dispersed phase containing a given number of copies of the base sequence, the left side in Equation 2 is the average number of copies contained in one positive dispersed phase, and σ ₁ in Equation 3 is in one positive dispersed phase. standard deviation of the number of copies contained in, N in Equations 4 and 5 is the specific number of compartments of the positive dispersed phase, m is the average number of copies contained in the positive dispersed phase of the specified number of compartments, σ _m denotes the standard deviation of the number of copies contained within the positive dispersed phase for the specified number of compartments.
The positive dispersed phase means a dispersed phase in which a label in the dispersed phase is detected by the detector of the ejection mechanism described in the method for producing a nucleic acid sample-containing container of the present invention, and the negative dispersed phase (Nega) means an ejected It means the dispersed phase in which no label was detected in the dispersed phase by the detector of the mechanism. Note that "no label was detected" means that the threshold for detecting the label was not exceeded.

Moreover, the probability that the positive dispersed phase contains only one target base sequence is preferably 90% or more. The probability that the positive dispersed phase contains only one target base sequence is the probability P of the number of discrete copies included in each positive dispersed phase, which contains only one target base sequence. means the probability of

ISO/IEC Guide 99: 2007 [International metrological terminology-basic and General Concepts and Related Terms (VIM)].
Here, "a value that can reasonably be associated with a measurand" means a candidate for the true value of the measurand. In other words, uncertainty means information about variations in measurement results due to operations, equipment, and the like involved in manufacturing the object to be measured. The greater the uncertainty, the greater the expected variability of the measurement results.
The uncertainty may be, for example, the standard deviation obtained from the measurement results, or may be half the confidence level expressed as the range of values in which the true value is included with a predetermined probability or more.
Uncertainty can be calculated based on the Guide to the Expression of Uncertainty in Measurement (GUM: ISO/IEC Guide 98-3) and the Japan Accreditation Board Note 10 Guideline on Measurement Uncertainty in Tests. . Methods for calculating uncertainty include, for example, Type A evaluation methods using statistics such as measured values, and uncertainty information obtained from calibration certificates, manufacturer's specifications, published information, etc. Two methods of Type B assessment can be applied.
Uncertainties can be expressed with the same level of confidence by converting all uncertainties from factors such as manipulation and measurement into standard uncertainties. Standard uncertainty refers to the scatter of the average value obtained from the measurements.
As an example of a method of calculating uncertainty, for example, factors that cause uncertainty are extracted and the uncertainty (standard deviation) of each factor is calculated. Furthermore, the calculated uncertainty of each 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 combined standard uncertainty, it is possible to ignore factors with sufficiently small uncertainty among the factors that cause uncertainty. Uncertainty may be the coefficient of variation (CV value) obtained by dividing the combined standard uncertainty by the expected value.

The coefficient of variation is the number of nucleic acids having the desired base sequence filled in each container (or the number of nucleic acids number) 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 filled in the container. A coefficient of variation is a value obtained by dividing the standard deviation σ by the average value x. Here, if the value obtained by dividing the standard deviation σ by the average number of copies (average number of filled copies) x is the coefficient of variation CV, the relational expression of Equation 6 below is obtained.

In general, nucleic acids are randomly distributed in a Poisson distribution in a dispersion. Therefore, in the stepwise dilution method, that is, in a random distribution state in the Poisson distribution, the standard deviation σ can be considered to satisfy the relational expression of the average copy number x and Expression 7 below. From this, when the nucleic acid dispersion is diluted by the serial dilution method, the coefficient of variation CV (CV value) of the average copy number x from the standard deviation σ and the average copy number x is derived from the above formulas 6 and 7. When calculated using the following formula 8, the results are shown in Table 1 and FIG. Note that the CV value of the coefficient of variation of the copy number with variations based on the Poisson distribution can be obtained from FIG.

From the results of Table 1 and FIG. 1, for example, when a container is filled with 100 copies of nucleic acid by the serial dilution method, the specific copy number of the nucleic acid finally filled in the reaction solution ignores other accuracies. However, it can be seen that the coefficient of variation (CV value) is at least 10%.

The filling accuracy (CV value) of the number of target base sequences contained in the nucleic acid sample-containing container is not particularly limited and can be appropriately selected according to the purpose, and is preferably less than 20%, for example.

The method for providing information on the nucleic acid sample-containing container is not particularly limited and can be appropriately selected according to the purpose. Examples include a method of printing on particles.
By using the method of coloring the nucleic acid sample-containing container according to the content of the information, the user can visually recognize the information of the nucleic acid sample-containing container, and the operability can be improved.

－Other materials－
Other materials are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include solutions other than the dispersed phase solution, fluorescent substances (fluorescent labeling reagents), and quenching substances.

Solutions other than the dispersed phase solution are not particularly limited and can be selected according to the purpose. Examples thereof include oil-based solvents that do not dissolve nucleic acids.
Examples of solutions other than the dispersed phase solution include protic polar solvents, aprotic polar solvents, and nonpolar solvents.

The fluorescent substance (fluorescent labeling reagent) is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include fluorescent dyes.

Examples of fluorescent dyes include fluoresceins, azos, rhodamines, coumarins, pyrenes, cyanines and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, fluoresceins, azos, and rhodamines are preferred, and SYBR Green, Eva Green, SYTOX Green, FAM, HEX, VIC (registered trademark), Texas Red (registered trademark), ROX, and FITC are more preferred.

The quenching substance is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include BHQ1, BHQ2, BHQ3, JOE and TAMRA.

FIG. 2 is a diagram showing an example of the nucleic acid sample-containing container of the present invention. In FIG. 2, the case where the nucleic acid sample-containing container is in the form of a capsule will be described, but the present invention is not limited to this form.
As shown in FIG. 2, the nucleic acid sample-containing container 1 contains a dispersed phase 110 having a nucleic acid 120 having a target base sequence. In addition, as shown in FIG. 2, one dispersed phase 110 contains one molecule of nucleic acid. It is possible to control the number of nucleic acids 120 having the desired base sequence to an arbitrary number.

The nucleic acid sample-containing container of the present invention can improve the operability by forming the nucleic acid sample into particles. Due to the improved operability of nucleic acid samples, it is possible to add a necessary amount of a very small amount of nucleic acid having the target base sequence to any experimental system, so that it is possible to create a reaction system that meets the needs of the user. can. Here, the extremely small amount means the number of molecules of about 1 molecule or more and 1,000 molecules or less.
In addition, the nucleic acid sample-containing container of the present invention is excellent in preservability of the nucleic acid because the nucleic acid sample is encapsulated in the particles.
Furthermore, the nucleic acid sample-containing container of the present invention can strictly control the number of copies (units) of the nucleic acid having the target base sequence contained in the nucleic acid sample-containing container during production. The number of copies (units) can be grasped.

(Method for Manufacturing Container Containing Nucleic Acid Sample, Apparatus for Manufacturing Container Containing Nucleic Acid Sample, and Program for Manufacturing Container Containing Nucleic Acid Sample)
The nucleic acid sample-containing container manufacturing apparatus of the present invention operates as a device for executing the nucleic acid sample-containing container manufacturing method of the present invention by reading and executing the nucleic acid sample-containing container manufacturing program of the present invention. That is, the apparatus for manufacturing nucleic acid sample-containing containers of the present invention has a program for manufacturing nucleic acid sample-containing containers of the present invention that causes a computer to perform the same functions as the method of manufacturing nucleic acid sample-containing containers of the present invention. The program for manufacturing nucleic acid sample-containing containers of the present invention is not limited to being executed by the apparatus for manufacturing nucleic acid sample-containing containers of the present invention. For example, the nucleic acid sample-containing container manufacturing program of the present invention may be executed by another computer or server, and any of the nucleic acid sample-containing container manufacturing apparatus of the present invention, the other computer, and the server may cooperate. may be executed
In other words, the apparatus for manufacturing a container containing a nucleic acid sample of the present invention is synonymous with carrying out the method for manufacturing a container containing a nucleic acid sample of the present invention. The details of the manufacturing method of the invention are also clarified. In addition, since the program for manufacturing a container containing a nucleic acid sample of the present invention can be realized as an apparatus for manufacturing a container containing a nucleic acid sample of the present invention by using a computer or the like as a hardware resource, the description of the manufacturing apparatus of the present invention will be used. The details of the production program for the nucleic acid sample-containing container of the invention will also be clarified.

The method for producing a nucleic acid sample-containing container of the present invention comprises:
a dispersed phase forming step of forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence;
an amplification step of amplifying the base sequence for detection in the dispersed phase;
a discrimination step of discriminating between a positive dispersed phase in which the base sequence for detection has been amplified and a negative dispersed phase in which the base sequence for detection has not been amplified in the dispersed phase;
A sorting step of sorting the positive dispersed phase and the negative dispersed phase discriminated in the discriminating step;
A dispensing step of dispensing the positive dispersed phase selected in the screening step into a container;
and, if necessary, other steps.

The apparatus for manufacturing a nucleic acid sample-containing container of the present invention comprises:
Dispersed phase forming means for forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence;
an amplification means for amplifying the base sequence for detection in the dispersed phase;
a discrimination means for discriminating between a positive dispersed phase in which the base sequence for detection is amplified in the dispersed phase and a negative dispersed phase in which the base sequence for detection is not amplified;
a sorting means for sorting the positive dispersed phase and the negative dispersed phase discriminated by the discriminating means;
a dispensing means for dispensing the positive dispersed phase selected by the screening means into a container;
and, if necessary, other means.

The method for producing a nucleic acid sample-containing container of the present invention can be suitably carried out using an apparatus for producing a nucleic acid sample-containing container. That is, the dispersed phase forming step can be preferably performed by the dispersed phase forming means, the amplifying step can be preferably performed by the amplifying means, the discrimination step can be preferably performed by the discriminating means, and the sorting step can be preferably carried out by the sorting means. The dispensing step can be preferably performed by a dispensing means, and the other steps can be performed by other means.

The present invention is based on the finding that it is difficult to dispense a nucleic acid containing a predetermined number of target base sequences into a container in a short period of time with almost no contaminants in the conventional method for producing a container containing a nucleic acid sample. It is based on
Specifically, for example, in the prior art, when selecting a diluent containing a specific number of molecules from the results of real-time PCR for a diluent of a nucleic acid having a target base sequence, any number of molecules of nucleic acid is selected. There was a problem that it was difficult to prepare and dispense a standard reagent containing In this case, the time required to prepare the standard reagent and the amount of the standard reagent that can be prepared depend on the performance of the real-time PCR device, so the standard reagent cannot be prepared and dispensed in a short time.
In addition, for example, in the prior art, when using cells into which a desired nucleotide sequence has been introduced using gene recombination technology, complicated operations such as gene recombination and cell culture are required, and nucleic acids are dispensed in a short time. The problem is that it is difficult to In this case, cell-derived contaminants (proteins, lipids, etc.) may contaminate the dispensed container when isolating the cells into which the desired base sequence has been introduced.
Furthermore, in the prior art, in the method of amplifying a portion of a nucleic acid having a desired nucleotide sequence and sorting (selecting) the droplets or gel droplets in which the nucleic acid amplification reaction has occurred, the desired number of copies of the desired nucleotide sequence ( specific copy number) can be difficult to dispense.

The method for producing a nucleic acid sample-containing container of the present invention can dispense a nucleic acid containing a predetermined number of target base sequences into a container with little contaminants in a short period of time.

<Dispersed Phase Forming Means and Dispersed Phase Forming Step>
The dispersed phase forming means forms a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence.
In the dispersed phase forming step, the dispersed phase forming means forms a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence.

The means for forming the dispersed phase is not particularly limited as long as it can divide the nucleic acid-containing liquid described later to form a plurality of dispersed phases (micro-compartmentalization), and can be appropriately selected according to the purpose. etc. Examples of means for dividing a nucleic acid-containing liquid into a plurality of dispersed phases at high speed include droplet generation using a microchannel and a method of filling a nucleic acid-containing liquid into a micro/nanoscale well.
A droplet generator, for example, can also be used as a device that automates the formation of the dispersed phase of the nucleic acid-containing liquid.
As the droplet generator, a known one can be used, and examples thereof include an Automated Droplet Generator manufactured by Bio-Rad, an emulsion (dispersed phase) production apparatus described in Japanese Patent No. 3746766, and the like. The emulsion production apparatus described in Japanese Patent No. 3746766 discharges a dispersed phase into a continuous phase flowing through a microchannel (microchannel) in a direction that intersects the flow of the continuous phase, and the shear force of the continuous phase disperses the dispersed phase. to manufacture.
It is also preferred to partition the solution faster by passing the dispersed phase through a series of splitters.

<<Nucleic acid-containing solution>>
The nucleic acid-containing solution is not particularly limited, can be appropriately selected according to the purpose, and can be the same as the solution used for the nucleic acid sample-containing container of the present invention, so the explanation is omitted.

In addition, the nucleic acid-containing solution preferably contains primers, a nucleic acid amplification reagent, and a labeling reagent in order to amplify the base sequence for detection in the amplification step described later.

When the base sequence for detection is amplified using the PCR method, the primers are 18-mer to 30-mer synthetic oligonucleotides having a base sequence complementary to the base sequence for detection, and a forward primer and a reverse primer are arranged so as to sandwich the region to be amplified. is set in two places.
Nucleic acid amplification reagents include, for example, DNA polymerase, four types of bases (dGTP, dCTP, dATP, dTTP), Mg ²⁺ (2 mM magnesium chloride), a buffer that maintains the optimum pH (pH 7.5 to 9.5), dNTP, dUTP, Tris-HCl, nuclease-free water, and the like.
By including primers and nucleic acid amplification reagents for amplifying the base sequence for detection in the nucleic acid-containing solution, in the discrimination step described later, the number of copies of the target base sequence contained in the dispersed phase is not changed. contains the target base sequence.

The labeling reagent is not particularly limited as long as it becomes identifiable as a label by amplification of the base sequence for detection, and can be appropriately selected according to the purpose, and the dispersed phase is optically labeled (fluorescent labeling). It is preferable to be able to Reagents for optical labeling are not particularly limited and can be appropriately selected depending on the purpose. For example, nucleic acid probes such as Taq Man probe and Molecular Beacon, and intercalators such as SYBR Green and Eva Green are used. can.
When using an intercalator, a method of adding the intercalator to the nucleic acid-containing solution after amplifying the base sequence for detection can also be used.

<<dispersed phase>>
The dispersed phase means that the nucleic acid-containing liquid described above is divided into micro-sized units. Hereinafter, the dispersed phase may be referred to as "microcompartments".

The dispersed phase is a region containing a nucleic acid having a target base sequence, and means a micro-sized compartment in which the nucleic acid-containing solution in which the nucleic acid having the target base sequence is dispersed is divided. Detailed description of the dispersed phase is omitted because it is the same as the dispersed phase in the nucleic acid sample-containing container of the present invention.

When dividing the solution into dispersed phases by the dispersed phase forming means, at most one nucleic acid having the target base sequence and the detection base sequence before amplification is in one dispersed phase (one molecule). It is preferable to use a nucleic acid-containing solution having a concentration of the nucleic acid such that the nucleic acid is contained.
The concentration of the nucleic acid such that one dispersed phase contains at most one molecule of the nucleic acid having the desired nucleotide sequence is, for example, 10 to 100 dispersed phases containing the nucleic acid having the desired nucleotide sequence. A concentration of nucleic acid such that only one dispersed phase is included is preferred.
By adjusting the solution to a concentration of nucleic acid such that there is one dispersed phase containing a nucleic acid having a target base sequence, the number of nucleic acids contained in the dispersed phase is 0 (no nucleic acid is contained) or 1. Therefore, the number of nucleic acid molecules to be dispensed can be controlled more accurately when the dispersed phase is dispensed by the dispensing means described later.

--nucleic acid--
Since the same nucleic acids as those used in the nucleic acid sample-containing container of the present invention can be used, the description thereof is omitted.

--- Target base sequence ---
The target base sequence is not particularly limited, can be appropriately selected according to the purpose, and can be the same as those that can be used in the nucleic acid sample-containing container of the present invention, so that the description is omitted.

In the present invention, the number of the target base sequence itself is also referred to as copy number. Furthermore, one unit of a nucleic acid that has this target base sequence and also includes other base sequences (for example, the base sequence for detection described below) and is linked together is also referred to as, for example, one molecule. In the present invention, one copy of the target base sequence contained in one molecule of nucleic acid is preferred. In addition, in the present invention, since the detection base sequence is amplified for a nucleic acid having a target base sequence and a detection base sequence described later, the number of copies of the target base sequence before and after amplification is It does not change.

--- Nucleotide sequence for detection ---
The base sequence for detection is not particularly limited, can be appropriately selected according to the purpose, and can be the same as the one that can be used in the nucleic acid sample-containing container of the present invention, so the description is omitted.

<Amplification Means and Amplification Process>
The amplification means amplifies the base sequence for detection in the dispersed phase.
In the amplification step, the detection base sequence is amplified in the dispersed phase by an amplification means.
The amplification means is not particularly limited as long as the temperature can be precisely controlled, and can be appropriately selected according to the purpose. Examples thereof include thermal cyclers, real-time PCR devices, and hot plates.

The method for amplifying the base sequence for detection is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include PCR method and isothermal amplification method.
Isothermal amplification methods include, for example, the NASBA method, the LAMP method, the RCA method, the SMAP method, the RPA method, and the HCR method.
When using the RCA method, for example, first, in a nucleic acid having a base sequence for detection arranged at the 3' end, a probe having a base sequence complementary to the base sequences at the 3' and 5' ends is used to synthesize nucleic acids. is circularized and a ligation reaction is performed to obtain a circularized nucleic acid. Next, it is possible to amplify a nucleic acid fragment having a base sequence for detection by using the obtained extension reaction primer for the circle.
Also, a nucleic acid amplification reaction can be performed using a pre-circularized circular probe containing a base sequence complementary to the base sequence for detection placed at the 3′ end. In this case, it is preferable to use a label such as a probe that specifically binds to the base sequence for detection as an optical labeling reagent, which will be described later.
In addition, the amplification means may be in the form of amplifying the base sequence for detection contained in the dispersed phase on the microchannel or in the form of amplifying the base sequence for detection contained in the dispersed phase separated into tubes.

The amplification means can accurately control the number of nucleic acid molecules having the target base sequence contained in the dispersed phase by amplifying the base sequence for detection in the dispersed phase. For example, it is assumed that one molecule of nucleic acid having one copy of the target base sequence is contained in the dispersed phase before the amplifying means amplifies the base sequence for detection. In this case, the target nucleotide sequence contained in the dispersed phase after the amplification means has amplified the detection nucleotide sequence remains one copy, the same as before amplification. The dispensing means described later dispenses the dispersed phase containing the nucleic acid whose copy number of the target base sequence is specified to a container or the like described later, thereby obtaining the desired number of copies (predetermined number of copies) of the target base sequence. number) can be dispensed.

<Determination means and determination process>
The discrimination means discriminates a dispersed phase (positive dispersed phase) in which the base sequence for detection is amplified in the dispersed phase and a dispersed phase (negative dispersed phase) in which the base sequence for detection is not amplified.
In the discriminating step, the discriminating means in the dispersed phase discriminates between the dispersed phase in which the base sequence for detection is amplified (positive dispersed phase) and the dispersed phase in which the base sequence for detection is not amplified (negative dispersed phase).

The discriminating means is not particularly limited and can be appropriately selected according to the purpose. For example, when the dispersed phase contains a labeling reagent for optical labeling, an optical discriminating means having a light source and a light receiving element, etc. is mentioned.
By using the optical discrimination means as the discriminating means, the discriminating means can detect the optical signal generated by the dispersed phase and discriminate the dispersed phase containing the nucleic acid whose base sequence for detection is amplified. As a specific method of discriminating by the optical discriminating means, for example, a threshold is set for the intensity of the optical signal that causes the dispersed phase, and when the intensity of the detected optical signal exceeds the threshold, the target base is detected in the dispersed phase. A method for determining that a nucleic acid having a sequence is included is included.

The optical discriminating means is not particularly limited as long as it has a light receiving element capable of irradiating the dispersed phase with light from the light source and detecting the optical signal of the dispersed phase corresponding to the irradiated light, and is appropriately selected according to the purpose. You can choose.
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 according to the purpose. It is possible to use an LED (Light Emitting Diode), a laser, or the like. However, especially when forming minute droplets of 1 nL or less, it is necessary to irradiate a narrow region with high light intensity, so it is preferable to use a laser. As a laser light source, various commonly known lasers such as solid lasers, gas lasers, and semiconductor lasers 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 according to the purpose. Optical sensors that receive optical signals from the dispersed phase within are preferred. Examples of light-receiving elements include one-dimensional elements such as photodiodes and photosensors. When highly sensitive measurements are required, photomultiplier tubes and avalanche photodiodes are preferably used. 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.

The discriminating means preferably counts the positive dispersed phase at the same time as discriminating between the positive dispersed phase in which the base sequence for detection has been amplified and the negative dispersed phase in which the base sequence for detection has not been amplified in the dispersed phase. By counting the positive dispersed phases at the same time when the discriminating means discriminates between the positive dispersed phases and the negative dispersed phases, the number of positive dispersed phases to be dispensed in the later-described dispensing step can be adjusted.

<Sorting means and sorting process>
The sorting means sorts the positive dispersed phase and the negative dispersed phase discriminated by the discriminating means.
In the sorting step, sorting means sorts the positive dispersed phase and the negative dispersed phase discriminated in the discriminating step.

The sorting means is not particularly limited and can be appropriately selected according to the purpose. Examples include an electrodynamic mechanism, a mechanism for controlling the flow of fluid by deforming a flow path, a mechanism using a mechanical valve, and the like. is mentioned.
By using an electrodynamic mechanism as the sorting means, the sorting means can sort the positive dispersed phase at high speed. A specific electrodynamic mechanism is, for example, a method of forming a nonuniform electric field and attracting only the target dispersed phase by dielectrophoresis.
Further, by synchronizing with the optical signal detected by the discriminating means and the discriminating step, the dispersed phase can be appropriately selected.

The positive dispersed phase sorted by the sorting means is preferably transported to a later-described dispensing mechanism.

<Dispensing means and dispensing process>
The dispensing means dispenses the positive dispersed phase sorted by the sorting means into containers.
In the dispensing step, the dispensing means dispenses the positive dispersed phase selected in the screening step into the container.

The dispensing means has a discharge mechanism for dispensing into the container.
The ejection mechanism dispenses the dispersed phase containing the nucleic acid having the target base sequence selected by the selection mechanism into the container.
The discharge mechanism is not particularly limited and can be appropriately selected. Examples thereof include a channel capable of transporting the dispersed phase to a container, an on-demand system, a continuous system, and the like. Among these, the channel capable of transporting the dispersed phase to the container is preferred.
The channel capable of transporting the dispersed phase to the container is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include tubes capable of transporting the dispersed phase.
Examples of the on-demand ejection mechanism include an ejection head.

Examples of the ejection head system include an ink jet system.
Examples of ejection heads of the ink jet system include those of a pressure application system, a thermal system, an electrostatic system, and the like. Among these, the pressure application method is preferable for the following reasons.

In the electrostatic method, it is necessary to set an electrode facing a discharge mechanism that holds a dispersed phase and forms droplets. In the apparatus for manufacturing a nucleic acid sample-containing container of the present invention, the ejection mechanism may be arranged facing the container, and it is preferable that no electrode be arranged in order to increase the degree of freedom in arranging the dispersed phase.
Since the thermal method generates localized heating, there are concerns about the effect on the nucleic acid, which is a biomaterial, and the burning (kogation) of the heater. The influence of heat depends on the content of the dispersed phase and the application of the dispensed material, so it is not necessary to exclude it indiscriminately. preferable.

Examples of the pressure applying method include a method of applying pressure to the liquid using a piezo element, a method of applying pressure using a valve such as an electromagnetic valve, and the like.

The ejection mechanism may further include a detector capable of detecting the label contained in the dispersed phase.
The detector is not particularly limited and can be appropriately selected according to the purpose, and the same one as the optical amplification detection mechanism (discrimination means) can be used.

Various processes performed by the apparatus for manufacturing nucleic acid sample-containing containers described above are executed by a computer having a control unit that forms the apparatus for manufacturing nucleic acid sample-containing containers.
The computer is not particularly limited as long as it is equipped with devices such as storage, calculation, and control, and can be appropriately selected according to the purpose. Examples thereof include personal computers.

Since the number of copies (units) of the target base sequence dispensed into the container is controlled in the nucleic acid sample-containing container manufactured by the apparatus for manufacturing a nucleic acid sample-containing container of the present invention, it can be used for food inspection, blood test, etc. can be suitably used. Examples of the container include particles and a well-formed plate generally used in the biotechnology field. Also, the number of wells in the plate is not particularly limited and can be appropriately selected according to the purpose, and may be singular or plural.

The particles are not particularly limited and can be appropriately selected according to the purpose, and examples thereof include those described for the nucleic acid-containing container of the present invention.

As the plate, it is preferable to use a single-well microtube, an 8-well plate, a 96-well well plate, a 384-well well plate, or the like. It is also possible to dispense and to enter different levels of numbers. Also, there may be wells that do not contain nucleic acids. In particular, when preparing a plate used for evaluating a real-time PCR device or a digital PCR device that quantitatively evaluates the amount of nucleic acids, it is preferable to use a plate in which multiple levels of nucleic acids are dispensed. For example, it is conceivable to prepare a plate in which nucleic acids are dispensed at seven levels of approximately 1, 2, 4, 8, 16, 32, and 64. By using such a plate, it is possible to examine the quantification, linearity, evaluation lower limit value, etc. of a real-time PCR device and a digital PCR device, as well as use it as a reference for expression level analysis of next-generation sequencers.

The wells may be filled with an aqueous liquid in advance.
Examples of the aqueous liquid to fill the wells include nuclease-free water, nucleic acid amplification reagents, nucleic acid storage reagents, etc. By filling the wells with the aqueous liquid, the nucleic acid sample can be immediately prepared. It can be used for the following reactions.

<Other means and other steps>
Other steps are not particularly limited and can be appropriately selected according to the purpose, and examples thereof include a counting step.
The counting step can be suitably performed by counting means.

The counting means may, for example, count the number of dispersed phases in the channel between the sorting mechanism and the ejection mechanism described above, count the number of dispersed phases in the ejection mechanism, The number of dispersed phases ejected by the ejection mechanism may be counted. When counting the number of dispersed phases ejected by the ejection mechanism, the counting means counts the number of dispersed phases contained in droplets of the continuous phase ejected by the ejection mechanism.
The counting means is not particularly limited and can be appropriately selected according to the purpose. For example, when an optical label (fluorescent label) is used as the labeling reagent, an optical counting mechanism having a light source and a light receiving element can be mentioned. be done.

The optical counting mechanism is not particularly limited and can be appropriately selected according to the purpose, and the same mechanism as the optical amplifying detection mechanism (discrimination means) can be used.
When counting the number of dispersed phases contained in droplets of the continuous phase ejected by the ejection mechanism, the light emitted by the optical counting mechanism counts the number of dispersed phases contained in the droplets in flight. Therefore, it is preferable to continuously irradiate the area through which the droplets pass, or to irradiate in a pulsed manner in synchronization with the ejection of the droplets with a predetermined time delay with respect to the droplet ejection operation.

By counting the number of dispersed phases contained in the ejected droplets of the nucleic acid-containing liquid using the counting means, the number of dispersed phases contained in the actually ejected droplets can be specified. The number of dispersed phases dispensed can be precisely controlled.

In the method for producing a nucleic acid sample-containing container of the present invention, the nucleic acid is dispensed without the intervention of the cell, instead of the method of dispensing the nucleic acid by the method of isolating the cell after dispensing the cell containing the nucleic acid. , it is possible to prevent cell-derived contaminants (proteins, lipids, etc.) from entering the reaction system.

Hereinafter, the method for producing a nucleic acid sample-containing container of the present invention will be described in detail with reference to the drawings. In the following description, a case where a nucleic acid-containing liquid is divided using a microchannel to form a dispersed phase will be described as an example.

FIG. 3 is a diagram showing an example of a dispersed phase forming means for dividing a solution X, which is a nucleic acid-containing liquid, into dispersed phases in a microchannel.
As shown in FIG. 3, a solution X containing a nucleic acid 500 having a target base sequence A and a detection base sequence B for confirming the presence of the target base sequence A is distributed in the upper left channel of the dispersed phase forming means 601. is flowing. A solution Y containing a nucleic acid amplification reagent and a fluorescent labeling reagent flows through the lower left channel of the dispersed phase forming means 601 . Solution X and solution Y join and are mixed in one channel.

Further, when the solution X and the solution Y are mixed on the microchannel, after the two channels are merged into one channel, a channel is provided in which the solution X and the solution Y can be stirred. may Examples of the channel that can stir the solution X and the solution Y include a meandering channel, a pillar array channel, a widening channel, a narrowing channel, and the like.

Next, the mixed liquid obtained by mixing the solution X and the solution Y is divided into dispersed phases as droplets by the shearing force from oil, which is a continuous phase fluid flowing from the upper and lower sides of the center of FIG. Transported downstream (right).

In FIG. 3, a solution having a nucleic acid concentration such that at most one molecule of nucleic acid 500 is contained in one dispersed phase is used. Therefore, depending on the dispersed phase, a dispersed phase 510 containing one molecule of nucleic acid 500 and a dispersed phase 511 containing no nucleic acid 500 are formed.

By using a microchannel for the dispersed phase forming means 601, the mixture of solution X and solution Y can be separated into dispersed phases at a very high speed. Specifically, by dividing the nucleic acid-containing liquid into dispersed phases using microchannels, the dispersed phases can be formed at a high speed of about 1000 particles per second.

FIG. 4 is a diagram showing another example of the dispersed phase forming means for dividing the solution X into dispersed phases in the microchannel.
A solution X, which is a nucleic acid-containing liquid containing a nucleic acid 500 having a target base sequence A and a detection base sequence B for confirming the presence of the target base sequence A, flows through the upper right channel of the dispersed phase forming means 601. ing. A solution Y containing a nucleic acid amplification reagent and a fluorescent labeling reagent flows through the upper left channel of the dispersed phase forming means 601 . Liquid X and solution Y join and are mixed in one channel.

Next, the mixed liquid of solution X and solution Y is divided into dispersed phases as droplets by shearing force from oil, which is a continuous phase fluid, flowing from the lower left of the center of dispersed phase forming means 601, and the right side of FIG. is transported downstream of the flow path.

FIG. 5 is a diagram showing an example of the amplifying means.
The amplification means 602 amplifies the base sequence for detection B contained in the nucleic acid 500 by performing a nucleic acid amplification reaction using the PCR method on the dispersed phase 510 containing the nucleic acid 500, thereby optically amplifying the dispersed phase 510. to label.

Specifically, the amplification means 602 circulates the dispersed phase 510 containing the nucleic acid 500, the nucleic acid amplification reagent, and the fluorescent labeling reagent through a meandering flow path having a plurality of temperature zones to raise or lower the temperature of the dispersed phase 510. . As the dispersed phase 510 moves back and forth between a plurality of temperature zones, the base sequence of the detection base sequence B of the nucleic acid 500 contained in the dispersed phase 510 is amplified by the nucleic acid amplification reagent. As a result, dispersed phase 510 becomes dispersed phase 512 containing nucleic acid 500 in which base sequence B for detection is amplified. Due to the amplification of the base sequence for detection B, the dispersed phase 512 reacts with the fluorescent labeling reagent with the base sequence for detection B, and the dispersed phase 512 becomes optically labeled.
The optically labeled dispersed phase 512 emits stronger fluorescence than the dispersed phase 511 containing no nucleic acid 500 when irradiated with light from the light source of the discrimination means 603 shown in FIG. It becomes easy to distinguish between the dispersed phase 512 that contains the nucleic acid 500 and the dispersed phase 511 that does not contain the nucleic acid 500 .

FIG. 6 is a diagram showing an example of a discrimination mechanism and a selection mechanism in a discrimination means for discriminating a dispersed phase containing a nucleic acid having an amplified detection base sequence B and a selection means for sorting a dispersed phase by dielectrophoresis.
The discrimination means 603 detects fluorescence from the dispersed phase 512 in which the detection base sequence B is amplified, and the dispersed phase 512 containing the nucleic acid 500 in which the detection base sequence B is amplified and the dispersed phase 511 not containing the nucleic acid 500 determine.

When the optical detection mechanism of the discriminating means 603 performs fluorescence detection, it has a light source and a light receiving element. Fluorescence emitted by absorption of light from the light source as excitation light is received. Since fluorescence is emitted in all directions from the dispersed phase 512, the light receiving element can be arranged at any position where 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 does not directly enter.
The discriminating means 603 discriminates the dispersed phase 512 containing the nucleic acid in which the detection base sequence B is amplified and the dispersed phase 511 not containing the nucleic acid 500 based on the intensity of the fluorescence received by the light receiving element.

A sorting mechanism 604 consisting of a cathode 604a and an anode 604b sorts a dispersed phase 512 containing a nucleic acid in which the detection base sequence B is amplified by dielectrophoresis and a dispersed phase 511 not containing the nucleic acid 500. FIG.
The selection mechanism 604 applies an appropriate voltage to the cathode 604a and the anode 604b shown in the upper part of FIG. Thereby, a non-uniform electric field is formed around the sorting mechanism 604 . The dispersed phase 512 polarized by the nonuniform electric field is dielectrophoretically attracted to the sorting mechanism 604 by the nonuniform electric field, flows through the upper right channel in FIG. 6, and is transported to the ejection mechanism.
The sorting mechanism 604 applies no voltage to the cathode 604a and the anode 604b when the dispersed phase 511 that does not contain the nucleic acid 500 passes around the sorting mechanism 604 . Therefore, the dispersed phase 511 that does not contain the nucleic acid 500 flows into the lower right channel of FIG. 6 and is discarded without undergoing dielectrophoresis.
At this time, the channel through which the dispersed phase 512 in which the base sequence for detection B selected by the sorting mechanism 604 is amplified flows is 1.2 times larger than the channel for disposal through which the dispersed phase 511 containing no nucleic acid 500 flows. It is preferable to have a fluid resistance of 1.5 times from . By using the above-described fluid resistance, all the dispersed phases flow to the waste channel when no electric field is applied. can be selected.

FIG. 7 is a diagram showing an example of a discharge mechanism.
As shown in FIG. 7, the ejection mechanism 605 has a detection mechanism 606 similar to the determination means 603 and a channel 607 capable of transporting the dispersed phase. In the following description, the dispersed phase 512 containing the nucleic acid in which the base sequence for detection B is amplified may be referred to as the sorted dispersed phase 350.

As shown in FIG. 7, the ejection mechanism 605 dispenses the dispersed phase 350 sorted by the sorting mechanism 604 into the capsules 100 . At this time, the number of dispersed phases 350 to be dispensed into capsule 100 is controlled based on the label detected by detection mechanism 606 .

Next, as the dispensing means of the method for manufacturing a nucleic acid sample-containing container of the present invention, the case of dispensing a dispersed phase into a container using an ink jet ejection mechanism will be described with reference to the drawings. 3 to 6 are the same as the case of dividing the nucleic acid-containing liquid using a microchannel to form a dispersed phase, so the description is omitted.

FIG. 8 is an explanatory diagram showing an embodiment of a discharge mechanism and a counting mechanism.
The ejection mechanism 10 has a liquid chamber 11 , a membrane 12 and a drive element 13 . The liquid chamber 11 has an air opening portion 115 that opens the inside of the liquid chamber 11 to the atmosphere, and is configured to allow air bubbles mixed in the oil 300 to be discharged from the air opening portion 115 . In addition, hereinafter, the dispersed phase 512 containing the nucleic acid in which the detection base sequence B is amplified may be referred to as the sorted dispersed phase 350 .

The membrane 12 is a film-like member fixed to the lower end of the liquid chamber 11 . A nozzle 121 , which is a through hole, is formed substantially in the center of the membrane 12 , and the oil 300 held in the liquid chamber 11 is discharged as droplets 310 from the nozzle 121 by vibration of the membrane 12 . Since the droplets 310 are formed by the inertia of vibration of the membrane 12, even the oil 300 with high surface tension (high viscosity) can be discharged. The planar shape of the membrane 12 can be, for example, circular, but may be elliptical, square, or the like.

The material of the membrane 12 is not particularly limited, but if it is too soft, the membrane 12 easily vibrates, and it is difficult to immediately suppress the vibration when not ejecting, so it is preferable to use a material with a certain degree of hardness. . As the material of the membrane 12, 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 121 is preferably formed as a substantially perfect circular through-hole in the approximate center of the membrane 12 . In this case, the diameter of the nozzle 121 is not particularly limited, but in order to avoid clogging the nozzle 121 with the selected dispersed phase 350, it is preferably at least twice the size of the selected dispersed phase 350.

On the other hand, if the droplets are too large, it will be difficult to achieve the purpose of forming minute droplets, so the diameter of the nozzle 121 is preferably 200 μm or less. That is, in the ejection mechanism 10, the diameter of the nozzle 121 is typically in the range of 10 μm to 200 μm.

The driving element 13 is formed on the lower surface side of the membrane 12 . The shape of the driving element 13 can be designed according to the shape of the membrane 12 . For example, when the planar shape of the membrane 12 is circular, it is preferable to form the drive element 13 having an annular planar shape (ring shape) around the nozzle 121 .

The driving means 20 selectively (for example, alternately) causes the driving element 13 to apply a discharge waveform for vibrating the membrane 12 to form the droplets 310 and a stirring waveform for vibrating the membrane 12 within a range in which the droplets 310 are not formed. ) can be granted.

For example, both the ejection waveform and the agitation waveform are square waves, and the drive voltage for the agitation waveform is set lower than the drive voltage for the ejection waveform. That is, the vibration state (degree of vibration) of the membrane 12 can be controlled by changing the drive voltage.

In the ejection mechanism 10, the drive element 13 is formed on the lower surface side of the membrane 12. Therefore, when the membrane 12 is vibrated by the drive element 13, it is possible to generate a flow from the bottom direction to the top direction of the liquid chamber 11. be.

In other words, the driving means 20 applies the ejection waveform to the driving element 13 and controls the vibration state of the membrane 12 , thereby ejecting the oil 300 held in the liquid chamber 11 from the nozzle 121 as droplets 310 .

Also, in the ejection mechanism 10 , air bubbles may enter the oil 300 in the liquid chamber 11 . Even in this case, since the discharge mechanism 10 is provided with the atmosphere opening portion 115 above the liquid chamber 11 , bubbles mixed in the oil 300 can be discharged to the outside air through the atmosphere opening portion 115 . This makes it possible to continuously and stably form droplets 310 without discarding a large amount of liquid for discharging bubbles.

It should be noted that the membrane 12 may be vibrated within a range in which droplets are not formed at timings when droplets are not formed, and bubbles may be positively moved upward in the liquid chamber 11 .

The light source 30 of the counting mechanism irradiates the light L on the droplet 310 in flight. Note that "during flight" means a state from when the droplet 310 is ejected from the ejection mechanism 10 until it lands on the container. The flying droplet 310 has a substantially spherical shape at the position where the light L is irradiated. Also, 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 to 100 times the diameter of the droplet 310 . This is to ensure that the droplets 310 are irradiated with the light L from the light source 30 even when the droplets 310 have positional variations.

However, it is not preferable that the beam diameter of the light L greatly exceeds 100 times the diameter of the droplet 310 . This is because the energy density of the light with which the droplet 310 is irradiated decreases, so the amount of fluorescence Lf that emits the light L as excitation light decreases, and detection by the light receiving element 60 becomes difficult.

The light L emitted from the light source 30 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. Although the energy per unit pulse largely depends on the optical system such as the presence or absence of light condensing, it is preferably about 0.1 μJ or more, more preferably 1 μJ or more.

Since the fluorescence Lf emitted by the selected dispersed phase 350 is weaker than the light L emitted by the light source 30, a filter that attenuates the wavelength range of the light L is installed in front of the light receiving element 60 (on the side of the light receiving surface). good too. As a result, a very high-contrast image of the selected dispersed phase 350 can be obtained on the light receiving element 60 . As the filter, for example, a notch filter or the like that attenuates a specific wavelength range including the wavelength of the light L can be used.

Further, as described above, the light L emitted from the light source 30 is preferably pulsed light, but the light L emitted from the light source 30 may be continuous wave light. In this case, it is preferable that the light receiving element 60 is controlled so that it can receive light at the timing when the droplet 310 in flight is irradiated with the continuous wave light, and the light receiving element 60 receives the fluorescence Lf.

The control section 70 has a function of controlling the driving means 20 and the light source 30 . In addition, the control unit 70 obtains information based on the amount of light received by the light receiving element 60, and has a function of counting the number of selected dispersed phases 350 contained in the droplet 310 (including the case where it is zero). are doing.

As described above, by using the method for producing a nucleic acid sample-containing container of the present invention, it is possible to dispense a desired number of dispersed phases 350 into capsules 100. The contained dispersed phase 350, that is, the number of copies of the target base sequence can be controlled to a known number (predetermined number).

FIG. 9 is a diagram illustrating hardware blocks of a control unit. FIG. 10 is a diagram illustrating functional blocks of a control unit; FIG. 11 is a flow chart showing an example of the operation of the ejection mechanism and the counting mechanism.

As shown in FIG. 9 , the control section 70 has a CPU 71 , a ROM 72 , a RAM 73 , an I/F 74 and a bus line 75 . The CPU 71 , ROM 72 , RAM 73 and I/F 74 are interconnected via a bus line 75 .

The CPU 71 controls each function of the control section 70 . The ROM 72, which is storage means, stores programs executed by the CPU 71 to control each function of the control section 70 and various information. A RAM 73, which is a storage means, is used as a work area for the CPU 71 and the like. Also, the RAM 73 can temporarily store predetermined information. The I/F 74 is an interface for connecting the ejection mechanism 10 with other devices and the like. The ejection mechanism 10 may be connected to an external network or the like via the I/F 74 .

As shown in FIG. 10 , the control section 70 has a division control section 710 , a discrimination control section 720 , a selection control section 730 and a dispensing control section 740 .
The control unit 70 controls the entire nucleic acid sample-containing container manufacturing apparatus of the present invention.

Next, the control of the control unit 70 in the case of using the ink jet system for the dispensing means will be described with reference to FIGS. 12 to 16. FIG.

As shown in FIG. 12 , the control section 70 has a division control section 710 , a discrimination control section 720 , a selection control section 730 and a dispensing control section 740 . The sorting control section 730 has an electric field control section 731 . The dispensing control section 740 has a discharge control section 741 , a dispersed phase counting section 742 and a light source control section 743 .
The control unit 70 controls the entire nucleic acid sample-containing container manufacturing apparatus of the present invention.

The counting of the number of dispersed phases (the number of particles) of the ejection mechanism 10 will be described with reference to FIGS. 12 and 13. FIG. First, in step S<b>11 , the ejection control section 741 of the control section 70 issues an ejection command to the driving means 20 . Upon receiving the ejection command from the ejection control unit 741 , the drive unit 20 supplies a drive signal to the drive element 13 to vibrate the membrane 12 . Droplets 310 containing the selected dispersed phase 350 are ejected from the nozzle 111 by vibration of the membrane 12 .

Next, in step S12, the light source control section 743 of the control section 70 turns on the light source 30 in synchronization with the ejection of the droplet 310 (in synchronization with the driving signal supplied from the driving means 20 to the ejection mechanism 10). issue a command. As a result, the light source 30 is turned on to irradiate the light L onto the droplet 310 in flight.

Here, synchronizing does not mean that the droplets 310 are emitted at the same time as the ejection mechanism 10 ejects the droplets 310 (at the same time that the drive means 20 supplies the drive signal to the ejection mechanism 10), but that the droplets 310 fly. This means that the light source 30 emits light at the timing when the droplet 310 is irradiated with the light L when the droplet 310 reaches a predetermined position. That is, the light source control unit 743 controls the light source 30 to emit light with a predetermined time delay with respect to ejection of the droplets 310 by the ejection mechanism 10 (driving signal supplied from the driving means 20 to the ejection mechanism 10). do.

For example, the velocity v of the droplets 310 ejected when the drive signal is supplied to the ejecting mechanism 10 is measured in advance. Then, based on the measured velocity v, the time t required for the droplet 310 to reach a predetermined position after being ejected is calculated, and the timing at which the light source 30 irradiates the light relative to the timing at which the driving signal is supplied to the ejection mechanism 10 is calculated. is delayed by t. As a result, good light emission control becomes possible, and the droplet 310 can be reliably irradiated with the light from the light source 30 .
The dispersed phase counting unit 742 of the control unit 70 counts the number of selected dispersed phases 350 contained in the droplet 310 (including the case of zero) based on the information from the light receiving element 60 . Here, the information from the light receiving element 60 is the brightness value (light amount) and area value of the selected dispersed phase 350 .

The dispersed phase counting section 742 can count the number of selected dispersed phases 350 by comparing the amount of light received by the light receiving element 60 with a preset threshold, for example. In this case, the light receiving element 60 may be a one-dimensional element or a two-dimensional element.

When a two-dimensional element is used as the light receiving element 60, the dispersed phase counting unit 742 calculates the luminance value or area of the selected dispersed phase 350 based on the two-dimensional image obtained from the light receiving element 60. You may use the technique of processing. In this case, the dispersed phase counting unit 742 calculates the brightness value or area value of the dispersed phase 350 selected by image processing, and compares the calculated brightness value or area value with a preset threshold value. , the number of sorted dispersed phases 350 can be counted.

In this way, a driving signal is supplied from the driving means 20 to the ejection mechanism 10 holding the oil 300 in which the selected dispersed phase 350 is suspended, and droplets 310 containing the selected dispersed phase 350 are ejected. , the droplet 310 in flight is irradiated with the light L from the light source 30 . Then, the selected dispersed phase 350 contained in the flying droplet 310 emits fluorescence Lf using the light L as excitation light, and the light receiving element 60 receives the fluorescence Lf. Furthermore, based on the information from the light receiving element 60, the dispersed phase counting unit 742 counts the number of the selected dispersed phases 350 contained in the flying droplets 310. FIG.

In other words, since the number of the selected dispersed phases 350 contained in the flying droplet 310 is actually observed on the spot, it is possible to improve the counting accuracy of the number of the selected dispersed phases 350 than before. Become. In addition, since the selected dispersed phase 350 contained in the flying droplet 310 is irradiated with the light L to emit the fluorescence Lf and the fluorescence Lf is received by the light receiving element 60, the dispersed phase 350 selected with high contrast is obtained. can be obtained, and the frequency of erroneous counting of the selected dispersed phases 350 can be reduced.

In this embodiment, the counting mechanism is configured to count the number of dispersed phases contained in droplets ejected by the ejection mechanism, but the configuration of the counting mechanism is not limited to this. As a form of the counting mechanism, for example, it may be a form in which counting is performed in a flow path through which the dispersed phase 512 in which the base sequence for detection B selected by the selection mechanism 604 is amplified flows, or in the liquid chamber 11 of the discharge mechanism 10. It may be in the form of counting.

FIG. 14 is an explanatory diagram showing an example of a state immediately after a dispersed phase containing a nucleic acid having a target base sequence is dispensed into a container.
The well 520 is pre-filled with an aqueous liquid 515 . When the specific gravity of the oil 300 is smaller than the aqueous liquid 515, immediately after the droplet 310 lands in the well 520, the oil layer 514 containing the selected dispersed phase 350 and the oil 513 form an aqueous liquid as shown in FIG. Located above the liquid 515 .

FIG. 15 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 destroyed.
The water-soluble component of the selected dispersed phase 350 contained in the droplet 310 that has landed on the well 520 dissolves in the aqueous nucleic acid-containing liquid. Therefore, the nucleic acid 500 having the target nucleotide sequence A and the amplified detection nucleotide sequence B contained in the sorted dispersed phase 350 are dissolved in the aqueous liquid 515 .

In addition, when the specific gravity of the oil 300 is greater than the aqueous liquid 515, the separated dispersed phase 350 sinks under the aqueous liquid 515 together with the oil 300, contacts and fuses with the interface of the oil 300, Dissolves in aqueous liquid 515 .
If the selected dispersed phase 350 cannot contact the aqueous liquid 515, centrifugation is performed to form layer separation based on specific gravity, thereby dissolving the selected dispersed phase 350 in the aqueous liquid 515. can be done.

Next, a program for manufacturing a nucleic acid sample-containing container of the present invention, which causes a computer to perform the same functions as the method for manufacturing a nucleic acid sample-containing container of the present invention, will be described. The processing by the program for manufacturing nucleic acid sample-containing containers of the present invention can be executed using a computer having a control unit 70 that constructs the apparatus for manufacturing nucleic acid sample-containing containers of the present invention.

FIG. 11 is a flow chart showing an example of a processing procedure of a manufacturing program for nucleic acid sample-containing containers.

In step S101, the control unit 70 causes the dispersed phase forming means 601 to generate a solution X containing a nucleic acid 500 having a target base sequence A and a detection base sequence B different from the target base sequence, a nucleic acid amplification reagent, and a fluorescent labeling reagent. is mixed with the solution Y containing the solution Y, and the mixture of the solution X and the solution Y is micro-compartmentized to form a dispersed phase, and then the process proceeds to S102.
In step S102, the control unit 70 raises or lowers the temperature of the dispersed phase 510 containing the nucleic acid 500, the nucleic acid amplification reagent, and the fluorescent labeling reagent to a plurality of temperature ranges by the amplification means 602 while flowing through the meandering channel. Then, the process proceeds to S103. By raising and lowering the temperature of the dispersed phase 510 in a plurality of temperature ranges, the detection base sequence B is amplified and the dispersed phase 512 is optically labeled.
In step S103, when the determining means 603 detects fluorescence generated from the positive dispersed phase 512 in which the detection base sequence B is amplified, the control unit 70 shifts the process to S104.
In step S104, the control unit 70 causes the selection mechanism 604 to select the positive dispersed phase 512 in which the base sequence for detection B is amplified by dielectrophoresis based on the detection result of the determination unit 603. Then, the process proceeds to S105. . Here, selecting the positive dispersed phase 512 in which the detection base sequence B is amplified means that the positive dispersed phase 512 in which the detection base sequence B is amplified by the selection mechanism 604 is based on the intensity of the optical signal of the dispersed phase detected in S103. It means that only the dispersed phase 512 is transported to the ejection mechanism 10 .
In step S105, the controller 70 causes the discharge mechanism 10 to dispense the positive dispersed phase 512 in which the detection base sequence B is amplified into the capsule 100, and then shifts the process to S106.
In step S106, the control unit 70 detects the dispensed positive dispersed phase 512 by the light source 30 and the light receiving element 60, and the number of detected positive dispersed phases 512 reaches the desired number set by the user (for example, capsules 520 number of disperse phases to be dispensed), the process returns to S105. On the other hand, when the counted number reaches the desired number set by the user, the control unit 70 terminates this process.

Alternatively, nucleic acids may be dispensed into a plurality of capsules 520 by repeating the procedure shown in FIG. 11 a plurality of times.

FIG. 16 is a flow chart showing another example of the processing procedure of the manufacturing program for the nucleic acid sample-containing container.

16, step S201 is the same as S101, step S202 is the same as S102, step S203 is the same as S103, step S204 is the same as S104, and step S206 is the same as S105, so description thereof will be omitted.

In step S205, the control unit 70 causes the light source 30 and the light receiving element 60 to count the number of the positive dispersed phases 512 selected in S204 in the channel, and then shifts the process to S206.

In step S207, based on the number of positive dispersed phases 512 counted in S205, the control unit 70 determines that the number of positive dispersed phases 512 dispensed in S206 is the desired number set by the user (for example, the number of wells 520 It is determined whether or not the number of dispersed phases to be dispensed) has been reached. When determining that the number of dispensed positive dispersed phases 512 has not reached the desired number set by the user, the control unit 70 returns the process to S206. On the other hand, when the controller 70 determines that the number of dispensed positive dispersed phases 512 has reached the desired number set by the user, the process ends.

Nucleic acids may be dispensed into a plurality of wells 520 by repeating the procedure shown in FIG. 16 a plurality of times.

(Nucleic acid sample)
The nucleic acid sample of the present invention comprises a predetermined number of first nucleic acid molecules having a base sequence for detection and a base sequence different from the base sequence for detection, and second nucleic acid molecules having a base sequence for detection, Furthermore, it has other members as needed.
The form of the nucleic acid sample of the present invention is not particularly limited, and examples thereof include a dispersed phase. Since the dispersed phase is the same as that described for the nucleic acid sample-containing container of the present invention, description thereof is omitted.

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

(Example 1)
<Preparation of Nucleic Acid (dsDNA) Sample-Containing Container (Particulate Container)>
-Preparation of mixed solution of nucleic acid, nucleic acid amplification reagent, and fluorescent labeling reagent-
DNA600-G (manufactured by National Institute of Advanced Industrial Science and Technology, NMIJ CRM 6205-a, see SEQ ID NO: 1) as a template nucleic acid, and a forward primer complementary to the nucleic acid as a nucleic acid amplification reagent (see SEQ ID NO: 2) and a reverse primer (see SEQ ID NO: 3), a TaqMan probe (sequence See No. 4), Supermix (manufactured by Bio-Rad, ddPCR Supermix for Probes (no dUTP), 186-3025), NFW ((manufactured by Thermo Fisher Scientific, UltraPure DNase/RNase-Free-Distilled Water, 10977 -015), reagents were prepared at the mixing ratios shown in Table 2, and finally filled into digital PCR plates (ddPCR96-Well Plates, 12001925, manufactured by Bio-Rad) at 22 µL each.Each reagent contained NFW. It was used after diluting to the concentration shown in Table 2.

- Dispersed phase formation step -
A mixed solution prepared using a droplet generator (manufactured by Bio-Rad, device name: Automated Droplet Generator) was divided into dispersed phases. As a solvent for the mobile phase, Automated Droplet Generator Oil for Probes manufactured by Bio-Rad was used. The prepared droplet suspension was sealed with aluminum using a plate sealer (manufactured by Bio-Rad, trade name: PX1 Plate Sealer), and immediately subjected to the next amplification step.

-Amplification process-
The amplification step was performed by the thermal cycle process shown in Table 3 using a thermal cycler (manufactured by Bio-Rad, device name: T100 thermal cycler).

－Confirmation of Nucleic Acid Amplification and Calculation of Uncertainty－
The dispersed phase (also referred to as positive dispersed phase) in which nucleic acid amplification was performed was confirmed for fluorescence by a droplet reader (manufactured by Bio-Rad, device name: QX200 Droplet Reader), and the detection results (FIG. 17, and Table 4), the probability of each copy number contained in the dispersed phase was calculated by calculation processing (Formula 1) considering the Poisson distribution. Table 5 shows the calculated results (for D02 in Table 4).

- Discrimination and selection process of dispersed phase -
The sorting of the nucleic acid-amplified dispersed phase (positive dispersed phase) and the dispensing into the particulate container were discriminated and sorted using, for example, the sorting mechanism 604 shown in FIG. FIG. 18 shows the detection results of the positive dispersed phase. Also, an applied waveform (dotted line) was applied to generate a nonuniform electric field for the positive dispersed phase (solid line) (FIG. 19). FIG. 20 shows how the positive dispersed phase 512 is screened. The selected positive dispersed phase was introduced into the ejection mechanism in the next dispensing step.

-dispensing process of dispersed phase-
A channel for dispensing was provided inside the particulate container as shown in FIG. 7, and dispensing was performed while detecting the number of selected dispersed phases. Capsules used as particulate containers were MP Capsules No. 5 (manufactured by AS ONE Corporation). The dispensed capsule contains information on the total number of copies of the target base sequence in the particulate container, which is calculated from the number of copies of the target base sequence contained in one drop in the dispersed phase, and the uncertainty of the DNA copy number. It was displayed by coloring and made visible.

(Example 2)
<Preparation of Nucleic Acid (dsDNA) Sample-Containing Container>
The dispersed phase was dispensed into containers in the same manner as in Example 1. MicroAmp ^™ Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used as the container.
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). In the barcode, the calculation result of the total number of copies of the target base sequence dispensed in the container (calculated from the number of copies of the target base sequence contained in one drop of the dispersed phase) and the dsDNA dispensed in the container Copy number uncertainty information was made available by barcode counting for both the sense and antisense strands.

(Example 3)
<Preparation of container containing RNA sample>
-Preparation of RNA-DNA Hybridizing Nucleic Acid Solution-
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) (see SEQ ID NO: 6) as template nucleic acids in Table 6 After mixing the components, the mixture was heat denatured at 95° C. for 2 minutes, cooled to room temperature over 1 hour, and allowed to sufficiently hybridize to synthesize RNA-DNA double strands. After that, 5 μL of Exonuclease I (manufactured by Takara Bio Inc.) was added and allowed to stand at room temperature for 30 minutes to remove single-stranded DNA. After that, inactivation treatment was performed at 80° C. for 15 minutes to inactivate Exonuclease I to obtain an RNA-DNA hybridized nucleic acid solution.

An RNA-DNA hybridizing solution as a template nucleic acid, a forward primer (see SEQ ID NO: 7) and a reverse primer (see SEQ ID NO: 8) complementary to the nucleic acid as nucleic acid amplification reagents, and a nucleic acid forward primer and reverse primer as fluorescent labeling reagents. A nucleic acid was prepared in the same manner as in Example 1 except that a base sequence complementary to a portion between the primers, FAM as a fluorescent dye, and a TaqMan probe (see SEQ ID NO: 9) having TAMRA as a quencher were used.・Preparation of a mixed solution of nucleic acid amplification reagent and fluorescent labeling reagent, formation of dispersed phase, amplification process, confirmation of nucleic acid amplification, and calculation of uncertainty were performed. The composition is shown in Table 7 below. The results are shown in Figures 18, 21, Table 8 and Table 9 (for F05 in Table 8).

- Discrimination and selection process of dispersed phase -
The obtained dispersed phase was discriminated and screened in the same manner as in Example 1.

-dispensing process of dispersed phase-
In the same manner as in Example 2, the selected dispersed phase was dispensed into containers. MicroAmp ^™ Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used as the container. After dispensing the positive dispersed phase into a container (plate), it was dried by vacuum drying. A barcode was attached to the side of the dried container (plate). The barcode contains the calculation result of the total number of copies of the target base sequence dispensed into the container (plate) (calculated from the number of copies of the target base sequence contained in one drop of the dispersed phase), and the Information on the uncertainties of the noted RNA copy numbers was made available by barcode.

(Example 4)
<Preparation of Nucleic Acid (ssDNA) Sample-Containing Container>
-Preparation of mixed solution of nucleic acid, nucleic acid amplification reagent, and fluorescent labeling reagent-
k03_ssDNA (manufactured by Integrated DNA Technologies, see SEQ ID NO: 10) as a template nucleic acid, a forward primer (see SEQ ID NO: 11) and a reverse primer (see SEQ ID NO: 12) complementary to nucleic acids as nucleic acid amplification reagents, and a fluorescent labeling reagent A TaqMan probe (see SEQ ID NO: 13) having a base sequence complementary to a portion of the nucleic acid between the forward and reverse primers, FAM as a fluorescent dye, and ZEN and TAMRA as quenchers was used. In the same manner as in Example 1, preparation of a mixed solution of nucleic acid/nucleic acid amplification reagent/fluorescent labeling reagent, formation of dispersed phase, amplification step, confirmation of nucleic acid amplification, and calculation of uncertainty were carried out. 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 Figure 22, Table 12, and Table 13 (for E11 in Table 12).

- Discrimination and selection process of dispersed phase -
The obtained dispersed phase was discriminated and screened in the same manner as in Example 1.

-dispensing process of dispersed phase-
In the same manner as in Example 2, the selected dispersed phase was dispensed into containers. MicroAmp ^™ Optical 96-Well Reaction Plate with Barcode (manufactured by Thermo Fisher Scientific) was used as the container. After dispensing the positive dispersed phase into a container (plate), it was dried by vacuum drying. A barcode was attached to the side of the dried container (plate). The barcode contains the calculation result of the total number of copies of the target base sequence dispensed into the container (plate) (calculated from the number of copies of the target base sequence contained in one drop of the dispersed phase), and the Uncertainty information on the noted ssDNA copy number was made available by barcode.

Aspects of the present invention are, for example, as follows.
<1> a predetermined number of first nucleic acid molecules having a target base sequence and a detection base sequence different from the target base sequence;
and a second nucleic acid molecule that does not have the target base sequence and has the detection base sequence.
<2> The nucleic acid sample-containing container according to <1>, wherein the first nucleic acid molecule has a plurality of the target base sequences in the same molecule.
<3> When the first nucleic acid molecule has a plurality of the target base sequences,
The nucleic acid sample-containing container according to <2> above, comprising at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthase between one target base sequence and another target base sequence.
<4> Any one of <1> to <3>, wherein the number of target base sequences is less than 1,000 and the coefficient of variation (CV value) of the number of target base sequences is less than 20%. is a container containing a nucleic acid sample.
<5> The nucleic acid sample-containing container according to any one of <1> to <4>, wherein the nucleic acid molecule is an artificially synthesized nucleic acid molecule.
<6> The nucleic acid sample-containing container according to any one of <1> to <5>, which has an outer shell that encloses the first nucleic acid molecule and the second nucleic acid molecule.
<7> The nucleic acid sample-containing container according to any one of <1> to <5>, which is either solid particles or gel particles containing the first nucleic acid molecule and the second nucleic acid molecule. be.
<8> The nucleic acid sample-containing container according to any one of <6> to <7>, which is soluble.
<9> The nucleic acid sample-containing sample according to any one of <1> to <8>, which is colored.
<10> A nucleic acid sample-containing container comprising a dispersed phase containing a nucleic acid, a continuous phase, and a protective member containing the dispersed phase and the continuous phase.
<11> The nucleic acid sample-containing container according to any one of <1> to <10>, wherein the first nucleic acid molecule is a single-stranded nucleic acid molecule.
<12> The nucleic acid sample-containing container according to any one of <1> to <11>, which has the detection sequence on the 5' side of the target base sequence.
<13> a dispersed phase forming step of forming a plurality of dispersed phases containing a nucleic acid having a target base sequence and a detection base sequence different from the target base sequence;
an amplification step of amplifying the base sequence for detection in the dispersed phase;
a discrimination step of discriminating the dispersed phase in which the base sequence for detection is amplified;
a dispensing step of dispensing the dispersed phase containing the nucleic acid with the amplified base sequence for detection determined in the determination step;
A method for producing a nucleic acid sample-containing container, comprising:
<14> The method for producing a nucleic acid sample-containing container according to <13>, wherein the dispersed phase has a probability of containing one target base sequence of 90% or more.
<15> Any one of <13> to <14>, wherein the discriminating step detects an optical signal generated by the dispersed phase to discriminate the dispersed phase containing the nucleic acid in which the base sequence for detection is amplified. 3. A method for producing a nucleic acid sample-containing container according to .
<16> Any one of <13> to <15>, wherein, in the determining step, the dispensing step includes dispensing the dispersed phase containing the nucleic acid for which the number of the target base sequences is specified. is a method for manufacturing a container containing a nucleic acid sample.
<17> Any one of <13> to <16>, wherein the dispersed phase forming step is performed in a nucleic acid-containing solution having a concentration of the nucleic acid such that at most one molecule of the nucleic acid is contained in one dispersed phase. is a method for manufacturing a container containing a nucleic acid sample.
<18> The method for producing a nucleic acid sample-containing container according to any one of <12> to <15>, wherein the dispensing step is performed by an inkjet method.
<19> The method for producing a nucleic acid sample-containing container according to any one of <13> to <18>, wherein the dispensing step is performed by counting the number of the dispersed phases contained in the ejected droplets. .
<20> The method for producing a nucleic acid sample-containing container according to any one of <13> to <19>, wherein the dispensing step is performed by counting the number of the determined dispersed phases before dispensing.
<21> Dispersed phase forming means for forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence;
an amplification means for amplifying the base sequence for detection in the dispersed phase;
a discrimination means for discriminating the dispersed phase in which the base sequence for detection is amplified;
a dispensing means for dispensing into a container the dispersed phase containing the nucleic acid in which the base sequence for detection has been amplified and which has been determined by the determination means;
An apparatus for manufacturing a nucleic acid sample-containing container, characterized by comprising:
<22> Forming a plurality of dispersed phases containing a nucleic acid having a target base sequence and a detection base sequence different from the target base sequence,
amplifying the base sequence for detection in the dispersed phase;
Discriminating the dispersed phase in which the base sequence for detection is amplified,
Dispensing the dispersed phase containing the nucleic acid with the amplified base sequence for detection discriminated by the discriminating means into a container;
A non-transitory recording medium storing a nucleic acid sample-containing container manufacturing program characterized by having a computer execute control processing.
<23> a predetermined number of first nucleic acid molecules having a base sequence for detection and a base sequence different from the base sequence for detection;
and a second nucleic acid molecule having a base sequence for detection.

The nucleic acid sample-containing container according to any one of <1> to <12>, the method for producing the nucleic acid sample-containing container according to any one of <13> to <20>, and the nucleic acid sample according to <21>. According to the container manufacturing apparatus, the nucleic acid sample-containing container manufacturing program described in <22>, and the nucleic acid sample described in <23>, the above problems in the conventional art are solved, and the object of the present invention is achieved. can be achieved.

JP 2014-33658 A JP 2015-195735 A JP 2008-245612 A

1 Container containing nucleic acid sample (particulate)
11 dispersed phase 12, 500 nucleic acid 70 controller 410 ejection mechanism 601 sorting means 602 nucleic acid amplification mechanism 603 amplification detection mechanism 604 selection mechanism 605 ejection mechanism

Claims (18)

a predetermined number of first nucleic acid molecules having a target base sequence and a detection base sequence different from the target base sequence;
a second nucleic acid molecule that does not have the target base sequence and has the detection base sequence;
Consists of a nucleic acid-containing liquid containing a plurality of dispersed phases formed by a droplet generator,
the first nucleic acid molecule has a plurality of the target base sequences in the same molecule,
A nucleic acid sample-containing container characterized by having at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthase between one target base sequence and another target base sequence. Having a plurality of wells in which the nucleic acid-containing solution is arranged,
2. The number of the target base sequences in the plurality of wells is less than 1,000, and the coefficient of variation (CV value) of the number of the target base sequences in the plurality of wells is less than 20%. nucleic acid sample containing container. 3. The nucleic acid sample-containing container according to claim 1, wherein said nucleic acid molecule is an artificially synthesized nucleic acid molecule. 4. The nucleic acid sample-containing container according to any one of claims 1 to 3, which is either solid particles or gel particles containing said first nucleic acid molecule and said second nucleic acid molecule. 5. The nucleic acid sample-containing container according to claim 4, which is soluble. 6. The nucleic acid sample-containing container according to any one of claims 1 to 5, which is colored. 7. The nucleic acid sample-containing container according to any one of claims 1 to 6, wherein at least the target base sequence in said first nucleic acid molecule is a single-stranded nucleic acid. 8. The nucleic acid sample-containing container according to claim 7, which has the detection base sequence on the 5' side of the target base sequence. a dispersed phase forming step of forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence by a droplet generator;
an amplification step of amplifying the base sequence for detection in the dispersed phase;
a discrimination step of discriminating between a positive dispersed phase in which the base sequence for detection has been amplified and a negative dispersed phase in which the base sequence for detection has not been amplified in the dispersed phase;
A sorting step of sorting the positive dispersed phase and the negative dispersed phase discriminated in the discriminating step;
A dispensing step of dispensing the positive dispersed phase selected in the screening step into a container;
including
the nucleic acid has a plurality of the target base sequences in the same molecule,
A method for producing a nucleic acid sample-containing container, comprising at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthase between one target base sequence and another target base sequence. 10. The method for producing a nucleic acid sample-containing container according to claim 9, wherein the probability that the dispersed phase contains one of the target base sequences is 90% or more. 11. The method for manufacturing a nucleic acid sample-containing container according to any one of claims 9 to 10, wherein said discriminating step detects an optical signal generated by said dispersed phase to discriminate between said positive dispersed phase and said negative dispersed phase. 12. The nucleic acid sample-containing container according to any one of claims 9 to 11, wherein the dispensing step dispenses the dispersed phase containing the nucleic acid for which the number of target base sequences is specified in the determining step. Production method. 13. The nucleic acid sample-containing container according to any one of claims 9 to 12, wherein the dispersed phase forming step is performed with a nucleic acid-containing solution having a concentration of the nucleic acid such that at most one molecule of the nucleic acid is contained in one dispersed phase. Production method. 14. The method for manufacturing a nucleic acid sample-containing container according to any one of claims 9 to 13, wherein the dispensing step is performed by an inkjet method. 15. The method for manufacturing a nucleic acid sample-containing container according to any one of claims 9 to 14, wherein the dispensing step is performed by counting the number of the discriminated dispersed phases before dispensing. Dispersed phase forming means for forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence by a droplet generator;
an amplification means for amplifying the base sequence for detection in the dispersed phase;
a discrimination means for discriminating between a positive dispersed phase in which the base sequence for detection is amplified in the dispersed phase and a negative dispersed phase in which the base sequence for detection is not amplified;
a sorting means for sorting the positive dispersed phase and the negative dispersed phase discriminated by the discriminating means;
a dispensing means for dispensing the positive dispersed phase selected by the screening means into a container;
has
the nucleic acid has a plurality of the target base sequences in the same molecule,
An apparatus for producing a nucleic acid sample-containing container, characterized by having at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthase between one target base sequence and another target base sequence. Forming a plurality of dispersed phases containing a target base sequence and a nucleic acid having a detection base sequence different from the target base sequence by a droplet generator,
amplifying the base sequence for detection in the dispersed phase;
Discriminating between a positive dispersed phase in which the detection base sequence is amplified and a negative dispersed phase in which the detection base sequence is not amplified,
Selecting the determined positive dispersed phase and the negative dispersed phase,
dispensing the selected positive dispersed phase into a container;
cause the computer to execute the control process,
the nucleic acid has a plurality of the target base sequences in the same molecule,
A program for producing a nucleic acid sample-containing container, characterized by having at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthetase between one target base sequence and another target base sequence. a predetermined number of first nucleic acid molecules having a target base sequence and a detection base sequence different from the target base sequence;
a second nucleic acid molecule that does not have the target base sequence and has the detection base sequence,
the first nucleic acid molecule has a plurality of the target base sequences in the same molecule,
Having at least one base of an artificial nucleic acid that cannot be amplified by a natural nucleic acid synthetase between one target base sequence and another target base sequence,
A nucleic acid sample, characterized in that it is formed by a droplet generator.

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