WO2019009297A1

WO2019009297A1 - Analysis system, analysis method, program, and storage medium

Info

Publication number: WO2019009297A1
Application number: PCT/JP2018/025232
Authority: WO
Inventors: 本間　務; 淳 ▼高▲橋; 南　昌人; 矢野　哲哉
Original assignee: キヤノン株式会社
Priority date: 2017-07-06
Filing date: 2018-07-03
Publication date: 2019-01-10

Abstract

The present invention concerns analysis of the concentration of a to-be-analysed object within a sample. A plurality of reaction sites is generated by dividing a fluid comprising the sample. A distribution of reaction site sizes is divided into a plurality of partitions. A partition used to derive the concentration is determined, per partition, on the basis of the number or the proportion of positive reaction sites, or the number or the proportion of negative reaction sites. The concentration of the to-be-analysed object is derived on the basis of data from the determined partition.

Description

Analysis system, analysis method, program, and storage medium

The present invention relates to an analysis system, an analysis method, a program, and a storage medium.

Digital PCR (dPCR; digital Polymerase Chain Reaction) has attracted attention as a method of quantitatively analyzing a nucleic acid (target nucleic acid) having a specific base sequence as an analyte.

In digital PCR, a sample containing a target nucleic acid is mixed and diluted with an amplification reagent for amplifying the target nucleic acid, a fluorescent reagent for detecting the target nucleic acid, etc., and divided into a large number of physically independent reaction fields. . At this time, the sample is diluted (hereinafter, such dilution is referred to as “limit dilution”) such that the number of target nucleic acids contained in each reaction field is either 1 or 0. Then, PCR is independently generated in each of the plurality of reaction fields to amplify the target nucleic acid so as to be detectable. Thereby, the concentration of the target nucleic acid in the sample is obtained from the number of reaction fields in which the signal is detected after amplification (the number of positive reaction fields) and / or the number of reaction fields in which the signal is not detected after amplification (the number of negative reaction fields) can do.

A method of forming droplets of a reaction solution in oil as a method of dividing a reaction solution containing a sample into a large number of physically independent reaction fields in digital PCR, that is, a water-in-oil emulsion (W / O emulsion) There is a way to form In this method, each single droplet in a water-in-oil emulsion is used as a reaction site (Patent Document 1).

In addition, even in the case of limiting dilution, one reaction site may contain a plurality of analytes. As a method of correcting an error resulting from this, there is known a method of stochastically calculating the number or concentration of an analyte based on a Poisson model.

Japanese Patent Application Publication No. 2012-503773

As a method of forming a water-in-oil emulsion, there is a method using a microchannel device, a method using mechanical agitation, etc. When the water-in-oil emulsion is formed at high speed, the size of the droplets, That is, the size of the reaction site tends to vary.

The probability that each reaction field contains an analyte depends on the size of the reaction field. Therefore, there is a problem that when there is variation in the size of the reaction field, the reliability of the analysis result may not be sufficiently high only by performing the calculation based on the conventional Poisson model.

Therefore, in the present invention, in view of the above-mentioned problems, it is an object of the present invention to increase the reliability of analysis results even when there is variation in the size of reaction sites.

An analysis system according to one aspect of the present invention is an analysis system that analyzes the concentration of an analyte in a sample, and the plurality of reactions for a plurality of reaction fields generated by dividing a liquid containing the sample. A size information acquisition unit for acquiring information on each size of the field, an information on acquisition of the presence of the analysis object in each of the plurality of reaction fields, an analysis object information acquisition unit, and a distribution of sizes of the reaction fields The information is divided into a plurality of sections, and information on the number of positive reaction fields which are reaction fields in which the analyte is detected, and negative reaction fields which are reaction fields in which the analyte is not detected. Distribution data including at least one information selected from the group consisting of information related to the number is acquired by the size information acquisition unit and the signal information acquisition unit Of the concentration based on at least one information selected from the group consisting of: a distribution data generation unit generated based on the information, information on the number of positive reaction fields, and information on the number of negative reaction fields And a concentration deriving unit for deriving the concentration of the analyte in the sample based on data of the section determined by the section determining unit among the distribution data. It is characterized by having.

According to the present invention, even in the case where there is variation in the size of the reaction site, the reliability of the analysis result can be enhanced.

It is a figure showing composition of an analysis system typically. It is a figure which shows the hardware constitutions of an information processing unit typically. It is a graph which shows the number of reaction fields containing an analyte and the relationship of "the uncertainty of measurement" in digital analysis. It is a flowchart which shows the procedure of the analysis process by an analysis system. 8 is a fluorescence microscope image of Emulsion 1 after thermal cycling. 8 is a fluorescence microscope image of Emulsion 2 after thermal cycling. 8 is a fluorescence microscope image of Emulsion 3 after thermal cycling. 7 is a fluorescence microscope image of Emulsion 4 after thermal cycling. 7 is a fluorescence microscope image of Emulsion 5 after thermal cycling. 7 is a fluorescence microscope image of Emulsion 6 after thermal cycling. 7 is a fluorescence microscope image of Emulsion 7 after thermal cycling. 8 is a fluorescence microscope image of Emulsion 8 after thermal cycling. It is a graph which shows the relationship between relative dilution magnification and the calculation result of the concentration of the analyte in the sample in Comparative Example 1. 5 is a graph showing the relationship between relative dilution factor and calculation result of concentration of an analyte in a sample in Example 1. FIG. FIG. 16 is a graph showing the relationship between relative dilution factor and calculation result of concentration of analyte in sample in Example 2. FIG. It is a graph which shows the relationship between relative dilution magnification and the calculation result of the concentration of the analyte in the sample in Comparative Example 2. FIG. 16 is a graph showing the relationship between relative dilution factor and calculation result of concentration of analyte in sample in each of Example 3. FIG. FIG. 16 is a graph showing the relationship between relative dilution factor and calculation result of concentration of analyte in sample in each of Example 4. FIG. 8 is a fluorescence microscope image of Emulsion 9 after thermal cycling. 8 is a fluorescence microscopy image of Emulsion 10 after thermal cycling. 7 is a fluorescence microscope image of emulsion 11 after thermal cycling. 8 is a fluorescence microscopy image of Emulsion 12 after thermal cycling. 7 is a fluorescence microscope image of emulsion 13 after thermal cycling. It is a graph showing the relationship between the preparation result in each of comparative example 3 and Example 5, and the calculation result of the DNA concentration in a sample. 15 is a graph showing the relationship between the calculation results of the concentrations of each of the emulsions 9 to 11 and the calculation results of the relative dilution ratio and the concentration of the analyte in the sample at the concentration of a commercially available device.

Hereinafter, an analysis system according to an embodiment of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments, and appropriate modifications may be made to the following embodiments based on the ordinary knowledge of those skilled in the art without departing from the spirit of the present invention. Those to which improvements and the like have been added are also included in the scope of the present invention.

[Analysis system configuration]
FIG. 1 is a view schematically showing a configuration of an analysis system according to the present embodiment. The analysis system 1 according to the present embodiment includes a reaction field generation unit U1, a reaction unit U2, a detection unit U3, and an information processing unit U4. The units may be partially or totally connected via a network such as a LAN or the Internet.

<Reaction field generation unit>
The reaction field generation unit U1 is a unit that divides a liquid such as a reaction liquid containing an analyte in a sample to generate a plurality of reaction fields physically independent of one another.

(Reaction site)
In the present specification, the reaction site refers to a space surrounded by at least one interface selected from the group consisting of a liquid-liquid interface, a gas-liquid interface, and a solid-liquid interface. The reaction in the reaction site takes place in this closed space and proceeds independently of the other reaction sites. In other words, the reaction in one reaction site involves only the substance confined in the space defined by the above-mentioned interface.

For example, when the reaction solution is respectively dispensed to a plurality of wells on a plate such as a microplate, each reaction solution dispensed to each well serves as a reaction site. In this case, the reaction site is surrounded by the solid-liquid interface between the wall of the well and the reaction liquid, and the gas-liquid interface between the atmosphere and the reaction liquid. Alternatively, when the reaction liquid forms droplets in an emulsion such as a water-in-oil emulsion (W / O emulsion), each droplet in the emulsion is a reaction site. In this case, the reaction site is surrounded by the liquid-liquid interface between the continuous phase and the dispersed phase.

(Analyte)
As used herein, an analyte refers to a compound or particle contained in a sample and subjected to quantitative analysis. The analyte according to the present embodiment is not particularly limited as long as it can be detected by a reaction in a reaction site described later, and examples thereof include nucleic acids, peptides, proteins, enzymes, etc. .

In the present specification, “to make detectable” means to make a signal derived from an analyte detectable by a reaction in a reaction unit U2 described later. For example, an originally undetectable weak signal can be detected by enhancing the signal by increasing the number or concentration of analytes by the amplification reaction in the reaction unit U2. The analyte can also be made detectable by the fact that a substance that emits a predetermined signal due to the reaction is generated from the analyte. Alternatively, the analyte can be made detectable even if it changes to emit a signal, such as a chemical change.

The nucleic acid, which will be described in detail later, is used as an amplification reagent for amplifying the nucleic acid, a fluorescent reagent which interacts with the nucleic acid to emit fluorescence, and an agent for making the nucleic acid detectable, and a nucleic acid amplification reaction such as PCR. Can be made detectable. In addition, peptides and proteins can be made detectable by ELISA (Enzyme-Linked Immunosorbent Assay) method or the like. The analyte may be a substance containing the above-described nucleic acid, peptide, protein or the like. For example, molecules, microparticles, nanoparticles, cells and the like in which at least any one of nucleic acids, peptides, and proteins are covalently bound or attached, etc. can be mentioned.

For example, if blood collected from human or nucleic acid extracted therefrom is used as a sample and nucleic acid containing genes involved in diseases such as cancer and infectious diseases that can be contained in the sample is the analysis target It can be expected that useful information can be obtained for the diagnosis of diseases. Moreover, if a food is used as a sample, food inspection such as evaluation of genetically modified crops (GMO) can be performed. Alternatively, environmental monitoring can be performed by using soil and water in the environment as samples.

In the present embodiment, when the nucleic acid is an analyte, the nucleic acid is not particularly limited as long as it is a template nucleic acid to be amplified, and may be DNA (DeoxyriboNucleic Acid) or RNA (RiboNucleic Acid). May be The form of the nucleic acid is also not particularly limited, and may be a linear nucleic acid or a circular nucleic acid. The nucleic acid may be a single type of nucleic acid having a single base sequence, or may be a plurality of types of nucleic acids (for example, complementary DNA library etc.) each having various base sequences.

(Sample)
As used herein, a sample is a source of a sample collected or extracted from an organism, food, environment or the like. In general, the concentration of the analyte in the quantitatively analyzed sample is converted to the concentration in the sample and then used for various purposes such as medical diagnosis and evaluation of food and environment.

(sample)
In the present specification, the sample means one to be subjected to analysis according to the present embodiment, and in the present embodiment, the concentration of the analyte in the sample is measured. The sample may be the sample itself, or may be subjected to pretreatment or adjustment for analysis such as purification or concentration of the sample, chemical modification or fragmentation of the analyte, or the like. The concentration (number per unit volume) of analyte in the sample is not particularly limited, but when multiple reaction sites are generated, the number of analytes contained in each of the multiple reaction sites is one or zero. Preferably, the amount is By doing this, the reliability of the analysis result can be improved.

The reaction field generation unit U1 includes a sample injection unit 101, a reaction field generation unit 102, and a container 103.

(Sample injection unit)
The sample injection unit 101 is a unit for injecting a reaction liquid, which is a liquid containing a sample, into the reaction field generation unit 102.

The reaction solution injected from the sample injection unit 101 is sent to the reaction field generation unit 102. At this time, the reaction liquid may be fed by a liquid feeding means (not shown) such as a pump. In addition, the reaction liquid injected from the sample injection unit 101 may be mixed with oil as a continuous phase for forming an emulsion while being sent to the reaction site generation unit 102. Alternatively, while only the sample is injected from the sample injection unit 101 and sent to the reaction field generation unit 102, the reaction solution may be generated by mixing with a drug or the like for detecting an analyte.

(Reaction liquid)
In the present specification, the reaction liquid refers to a liquid containing at least a sample containing an analyte and an agent for making the analyte detectable. The reaction solution is preferably an aqueous liquid containing water.

(A drug to make an analyte detectable)
When the analyte is a nucleic acid, the analyte can be made detectable by amplifying the nucleic acid using a nucleic acid amplification reaction using an enzyme, as typified by the PCR method. Here, as a nucleic acid amplification reaction, a reaction method is carried out by subjecting the reaction site to a thermal cycle, a PCR method for advancing the reaction, a LCR (Ligase Chain Reaction) method, or a reaction field by temperature control without subjecting it to a thermal cycle. Preferably, an SDA (Strand Displacement Amplification) method, an ICAN (Isothermal and Chimeric primer-Initiated Amplification of Nucleic acids) method, a LAMP (Loop-Mediated Isothermal Amplification) method, etc. can be used.

When a nucleic acid amplification reaction is used, it is used as an amplification reagent for amplifying a nucleic acid, a fluorescence reagent that interacts with the nucleic acid to emit fluorescence, and an agent for making the nucleic acid detectable.

The amplification reagent is one or a pair of primers (forward primer and reverse primer) having a base sequence complementary to a predetermined base sequence of the target nucleic acid to be analyzed, and a biocatalyst promoting a nucleic acid synthesis reaction And a polymerase. The polymerase is preferably a heat-resistant polymerase, and more preferably a heat-resistant DNA polymerase. In addition, the amplification reagent contains ribonucleic acid such as dNTP (DeoxyriboNucleotide-5'-TriPhosphate) as a raw material of nucleic acid. Furthermore, the amplification reagent preferably contains a buffer or buffer for controlling the hydrogen ion concentration (pH) in the reaction solution, or a salt. In addition, you may use the commercially available kit which contains said each component as an amplification reagent.

The primer is not particularly limited as long as it is an oligonucleotide that hybridizes with the base sequence of a partial region of the target nucleic acid under stringent conditions and can be used for a nucleic acid amplification reaction. Here, stringent conditions mean that the primer can specifically hybridize to the template nucleic acid when there is at least 90% or more, preferably 95% or more, of sequence identity between the primer and the template nucleic acid. It is. The primers can be appropriately designed based on the base sequence of the target nucleic acid. Also, it is desirable that the primers be designed according to the type of nucleic acid amplification method. The length of the primer is usually 5 to 50 nucleotides, preferably 10 to 40 nucleotides. In addition, a primer can be produced | generated by the nucleic acid synthesis method generally used in the molecular biology area | region.

As the buffer or buffer, any suitable buffer or buffer can be used. The buffer or buffer is preferably configured to maintain the hydrogen ion concentration (pH) of the reaction solution at or near the pH at which the desired reaction can efficiently occur. When PCR is carried out, the pH of the reaction solution can be arbitrarily selected according to each component of the amplification reagent to be used, for example, between 6.5 and 9.0. As the buffer or the type of buffer, those commonly used in the molecular biology area can be used, for example, Tris (Tris (hydroxymethyl) aminomethane) buffer, HEPES (4- (2-hydroxyethyl) -1-Piperazineethanesulfonic acid) buffer, MES (2-morpholinoethanesulfonic acid) buffer, etc. can be used.

As the salt, for example, a salt appropriately selected from CaCl ₂ , KCl, MgCl ₂ , MgSO ₄ , NaCl, and a combination thereof can be used.

A fluorescent reagent is an agent that fluoresces by interacting with a nucleic acid, and a fluorescent intercalator (fluorescent dye) or a probe for a probe assay (fluorescently labeled probe) generally used in a PCR method can be used. As a fluorescent intercalator, ethidium bromide, SYBR Green I ("SYBR" is a registered trademark of Molecular Probes), LC Green, etc. can be used suitably. The fluorescently labeled probe is an oligonucleotide (probe) that specifically hybridizes to a target nucleic acid, and one end (5 'end) is modified with a reporter, and the other end (3' end) is a quencher. Those modified with can be used. As a reporter, a fluorescent substance such as FITC (Fluorescein-5-IsoThioCyanate) or VIC can be used, and as a quencher, a fluorescent substance such as TAMARA, Eclipse, DABCYL, MGB or the like can be used. As a fluorescently labeled probe, TaqMan ("TaqMan" is a registered trademark of Roche Diagnostics) probe or the like can be used. In addition, although the case where a fluorescence reagent was used was demonstrated here, you may use the luminescence reagent which utilizes light emission other than fluorescence.

On the other hand, when the analyte is a peptide or a protein, the analyte is obtained by an antigen-antibody reaction and an enzyme reaction using an antibody (or an antigen) that specifically reacts with the analyte, such as ELISA. Can be made detectable. More specifically, for example, an antibody (or an antigen) labeled with an enzyme is conjugated to an analyte by an antigen-antibody reaction, and a colored or luminescent substance generated by the enzyme reaction of this enzyme is detected. The analyte and the antibody (or antigen) that causes an antigen-antibody reaction may not be previously labeled with an enzyme, and may be labeled with an enzyme after the antigen-antibody reaction.

When the ELISA method is used, a reagent containing an antibody (or an antigen) and an enzyme is used as an agent for making an analyte detectable. A commercially available kit may be used as a reagent for use in the ELISA method.

In addition, if a plurality of types of drugs that can be detected so that a plurality of analytes can be distinguished, for example, different wavelengths of fluorescence to be generated, a plurality of types of analytes are collectively detected in one analysis You can also.

(Reaction site generator)
The reaction field generation unit 102 divides the reaction solution injected from the sample injection unit 101 to generate a plurality of reaction fields physically independent of each other. As a method of dividing | segmenting a reaction liquid and producing | generating several reaction fields, the following method is mentioned, for example.

As a first method, there is a method of dispensing a reaction solution to each well using a glass or resin substrate having a plurality of minute wells formed on a substrate such as a microwell plate. Thereby, the inside of each of the minute wells becomes a reaction site.

As a second method, there is a method of applying a reaction liquid on a substrate using a glass or resin substrate which has been subjected to a water repellent treatment or an oil repellent treatment in a predetermined pattern shape on the surface. For example, when an aqueous reaction solution is applied on a glass substrate water-repellently treated in the form of a grid, droplets are formed inside each grid, and each of the plurality of droplets serves as a reaction site.

As a third method, an emulsion in which a reaction liquid is dispersed in the form of droplets from a reaction liquid and a liquid incompatible with the reaction liquid (hereinafter referred to as "non-phase solution") in the non-phase solution. Methods to form This method is, in other words, a method of forming an emulsion in which the non-phase solution is a continuous phase and the reaction liquid is a dispersed phase. For example, if a reaction liquid which is an aqueous liquid containing water and an oily liquid (oil) are mixed to form a water-in-oil emulsion (W / O emulsion), the reaction liquid dispersed in the oil Each of the liquid droplets is a reaction site.

Among these, the reaction field generation unit 102 forms a reaction field by the third method, that is, a method of forming an emulsion in which the reaction liquid is dispersed in the form of droplets from the reaction liquid and the nonphase solution in the nonphase solution. It is preferred to produce That is, the reaction field generation unit 102 is preferably an emulsion generation unit that generates an emulsion from the reaction liquid and the non-phase solution body incompatible with the reaction liquid.

(Emulsion part)
There is no particular limitation on the method of producing the emulsion, and conventionally known emulsification methods can be used. For example, there is a mechanical emulsification method in which an emulsion is formed by applying mechanical energy with a stirring device or an ultrasonic crushing device. In addition, a method using a microchannel device such as a microchannel emulsification method or a microchannel bifurcation emulsification method, a membrane emulsification method using an emulsification film, and the like can be mentioned. These methods may be used alone or in combination of two or more. Among these, mechanical emulsifying method and membrane emulsifying method are preferable because they can form an emulsion with good throughput, although dispersion (dispersion) of droplet size tends to be larger as compared with the method using a microchannel device. In addition, the membrane emulsification method is particularly preferable because the apparatus configuration of the apparatus for forming the emulsion can be simplified and the emulsion having relatively small variation in droplet size can be formed. That is, the reaction site generation unit 102 is more preferably a membrane emulsification means or a mechanical emulsification means, and particularly preferably a membrane emulsification means.

The membrane emulsification method is a method of forming an emulsion by permeating a dispersed phase or a continuous phase, or a mixture of a dispersed phase and a continuous phase to an emulsion membrane having a plurality of pores and slits. The number of times the dispersed phase or the continuous phase, or the mixture of the dispersed phase and the continuous phase is allowed to permeate through the emulsion membrane in the membrane emulsification method is not particularly limited, and may be once or plural times.

As the membrane emulsification method, a direct membrane emulsification method, a pumping emulsification method or the like can be used. The direct membrane emulsification method is a method of forming an emulsion in a continuous phase flowing slowly on the extruded side by extruding the dispersed phase at a constant pressure through the emulsification membrane. The pumping emulsification method is a method of preparing an emulsion by sandwiching the emulsification membrane with a syringe from which the continuous phase is collected and a syringe from which the dispersion phase is collected, and alternately pushing out the liquid from the two syringes to pass through the emulsification membrane. is there. In the pumping emulsification method, a mixture of the continuous phase and the dispersed phase may be collected in one of two syringes, and the other syringe may be emptied. In the pumping emulsification method, it is possible to use a pumping emulsification device in which an emulsification film is sandwiched between a pair of connectors that can be connected to a syringe.

As an emulsification membrane used in the membrane emulsification method, a membrane of a porous body having a plurality of pores or a membrane having a slit can be used. Specifically, a porous glass membrane such as SPG (Shirasu porous glass), a polycarbonate membrane filter, a polytetrafluoroethylene (PTFE) membrane filter, or the like can be used. Moreover, it is more preferable that the surface of the emulsion film is subjected to a hydrophobization treatment. The pore diameter of the emulsion film can be selected according to the size of droplets in the water-in-oil emulsion to be formed, and is preferably 0.2 μm to 100 μm, and is 5 μm to 50 μm. More preferable.

(oil)
When the reaction liquid is an aqueous liquid containing water, an oily liquid (oil) can be used as a non-phase solution incompatible with the reaction liquid. In this case, the W / O emulsion is generated by the reaction field generation means 102.

As the oil, hydrocarbon oil, silicone oil, fluorine oil and the like can be used. Examples of hydrocarbon oils include mineral oils; oils derived from animals and plants such as squalane oil and olive oil; paraffin hydrocarbons having 10 to 20 carbon atoms such as n-hexadecane; olefin hydrocarbons having 10 to 20 carbon atoms Etc. can be used. As a commercial item of hydrocarbon-based oil, for example, TEGOSOFT DEC (diethylhexyl carbonate) (manufactured by Evonik, "TEGOSOFT" is a registered trademark of Evonik) can be used. As the fluorine-based oil, HFE-7500 (2- (trifluoromethyl) -3-ethoxydodecafluorohexane) can be used. As commercially available products of fluorinated oils, for example, FLUORINERT FC-40, FLUORINERT FC-40, FLUORINERT FC-3283 (manufactured by 3M, “FLUORINERT” is a registered trademark of 3M) can be used. Moreover, you may use combining hydrocarbon oil, silicone oil, and fluorine oil suitably.

(Other additives)
A surfactant may be further added in forming the emulsion. By adding a surfactant, effects such as control of the size of droplets in the emulsion and keeping the emulsion stable can be expected. As the surfactant, conventionally known surfactants generally used in emulsification treatment can be used. For example, it is preferable to use nonionic surfactant, fluorocarbon resin, phosphocholine containing resin, etc. . As a nonionic surfactant, a hydrocarbon surfactant, a silicone surfactant, or a fluorine surfactant can be used.

Commercially available hydrocarbon non-ionic surfactants include, for example, Pluronic F-68 (polyoxyethylene-polyoxypropylene block copolymer) (manufactured by Sigma-Aldrich, “Pluronic” is a registered trademark of BASF), Span 60 (Sorbitan Monostearate) (manufactured by Tokyo Chemical Industry Co., Ltd., "Span" is a registered trademark of Croda International), Span 80 (Sorbitan monooleate) (manufactured by Sigma-Aldrich, "Span" is a registered trademark of Croda International), Triton- X100 (polyoxyethylene (10) octyl phenyl ether) (manufactured by Sigma-Aldrich, “Triton” is a registered trademark of Union Carbide), Tween 20 (polyoxyethylene sorbitan monolaur) ), Tween 80 (polyoxyethylene sorbitan monooleate) (or, Sigma - Aldrich, "Tween" can be used registered trademark), and the like of Croda International. As silicone type nonionic surfactant, ABIL EM90 (Cetyl dimethicone copolyol (Cetyl PEG / PPG10-1 Dimethicone)), ABIL EM 120 (Bis- (glyceryl / lauryl) glyceryl lauryl dimethicone), ABIL EM180 (Cetyl PEG / PPG10-1 dimethicone), ABIL WE09 (polyglyceryl isostearate-4, cetyl dimethicone copolyol, hexyl laurate) (manufactured by Evonik, “ABIL” is a registered trademark of Evonik), and the like can be used. As the fluorine-based resin, Krytox-AS ("Krytox" is a registered trademark of Chemers) can be used. As the phosphocholine-containing resin, Lipidure-S (manufactured by NOF Corporation, “Lipidure” is a registered trademark of NOF, etc.) can be used.

The concentration of the surfactant in the emulsion is not particularly limited, but is preferably 0.01% by mass to 10% by mass and more preferably 0.1% by mass to 8% by mass, and more preferably 1% by mass. It is more preferable to set it as% or more and 4 mass% or less.

The volume ratio of the non-phase solution (continuous phase) to the reaction liquid (dispersed phase) in the emulsion is not particularly limited, but is preferably 1 or more and 300 or less, and more preferably 1 or more and 150 or less.

The size of the droplets in the emulsion is not particularly limited, but the diameter is preferably 1 μm or more and 300 μm or less, more preferably 1 μm or more and 200 μm or less, and still more preferably 20 μm or more and 150 μm or less. By setting the droplet diameter to 300 μm or less, the number of droplets (the number of reaction sites) even when the sample amount or sample amount is as small as several tens to several hundreds of μL, as in clinical examinations and the like The accuracy of the analysis can be increased. Moreover, the stability of an emulsion can be improved by the diameter of a droplet being 300 micrometers or less.

The distribution of droplet sizes in the emulsion is preferably polydispersed. In the present specification, polydispersion means not being monodisperse, that is, the size of droplets is not uniform but is dispersed. The distribution of the sizes of droplets in the emulsion may be a distribution close to monodispersion (for example, a coefficient of variation (CV) of droplet diameter is several% or less), and droplets of a plurality of sizes are mixed. It is also good. Generally, the distribution of droplet sizes in emulsions produced by mechanical emulsification or membrane emulsification has a distribution in which the variation coefficient (CV) of droplet diameter is about 10 to 20% or more in many cases. . In particular, when the emulsion is formed at high speed, the size of droplets in the emulsion tends to be polydispersed. According to the present embodiment, even if there is variation in droplet size, highly reliable quantitative analysis can be performed.

The number of droplets in the emulsion is preferably 100 or more and 1,000,000,000 or less, more preferably 100 or more and 20,000,000 or less, and 2,000 or more and 20 or more. It is more preferable that the number is, 000,000 or less. As described later, in the estimation by the present inventors, in order to ensure the reliability of the analysis result in the digital analysis, it is preferable that it is determined that at least 100 droplets or more contain the analyte. The number of droplets is preferably 100 or more. For example, in the case of clinical examination, the volume of the reaction solution is generally set to about 0.01 mL to 0.5 mL in many cases, and when the droplet size is about 10 μm to 200 μm, the number of droplets is approximately It will be set between 2,000 and 1,000,000,000.

(container)
The container 103 is a container for holding a plurality of reaction fields generated by the reaction field generation unit 102. When the reaction site generation unit 102 is an emulsion generation unit, the container 103 is a container for containing the emulsion generated by the emulsion generation unit. Also, in the case where the reaction field generation unit 102 generates a plurality of reaction fields by dispensing reaction liquid in each well using a glass or resin substrate in which a plurality of minute wells are formed on the substrate. The substrate on which the well is formed is the container 103.

The container 103 is transported by the transport unit (not shown) from the reaction field generation unit U1 to the reaction unit U2 and to the detection unit U3 while holding a plurality of reaction sites. In addition, although the case where the container 103 is conveyed between units by a conveyance unit (not shown) is demonstrated, it is not limited to this, The operator of the analysis system 1 may convey the container 103. FIG.

The container 103 is preferably configured to be removable from the reaction field generation unit U1 together with part or all of the sample injection unit 101 and the reaction field generation unit 102. That is, it is preferable that the reaction field production | generation unit U1 is a structure in which the cartridge provided with each function of the sample injection part 101, reaction field production | generation part 102, and the container 103 is detachable from the main body of reaction field production | generation unit U1. Such a configuration can prevent contamination between samples. The cartridge may include a reaction controller 201 described later.

<Reaction unit>
The reaction unit U2 has a reaction controller 201, and is a unit that causes a reaction to proceed in each of the plurality of reaction fields generated by the reaction field generation unit U1. This reaction can make the analyte contained in each of the plurality of reaction sites detectable.

(reaction)
When the analyte is a nucleic acid, as described above, it is possible to detect the analyte by amplifying the nucleic acid using a nucleic acid amplification reaction using an enzyme, as typified by the PCR method. Out. As the nucleic acid amplification reaction, as described above, PCR method, LCR method, SDA method, ICAN method, LAMP method and the like can be preferably used. When performing these nucleic acid amplification reactions, the reaction field is subjected to a thermal cycle, maintained at a constant temperature, or given a temperature with a predetermined profile, etc. to control the reaction field temperature. It is preferable to control. Alternatively, there is also known a method of controlling a reaction by flowing a reaction field in a microchannel of a microchannel device having a microchannel of a predetermined shape (Science, 280, 1046 (1998)).

When the analyte is a peptide or a protein, as described above, the analyte can be made detectable by a molecular biological technique combining an enzyme reaction and an antigen-antibody reaction such as ELISA. . In this case, it is preferable to adjust the temperature of the reaction site so as to maintain the temperature of the reaction site at a predetermined temperature.

(Reaction controller)
The reaction controller 201 controls the reaction in each of the plurality of reaction sites in the vessel 103. The reaction controller 201 can also be referred to as a reaction unit that causes a reaction to proceed in each of a plurality of reaction sites in the vessel 103. There is no particular limitation on the method for controlling the reaction in each of the plurality of reaction fields by the reaction controller 201. For example, the reaction may be controlled by controlling the temperature of the reaction field, or the reaction in the microchannel The reaction site may be controlled by controlling the position and velocity of the field. That is, the reaction controller 201 may have a temperature controller such as a heater or a cooler, or may have a pump connected to a microchannel.

In addition, the reaction controller 201 applies at least one selected from the group consisting of heat, magnetic field, electric field, current, light, and radiation to each of the plurality of reaction fields according to the type of reaction in the reaction field. It may be one. A commercially available thermal cycler can also be used as the reaction unit U2.

<Detection unit>
The detection unit U3 is a unit that performs detection of the size of each of the plurality of reaction fields and detection of an analyte for each of the plurality of reaction fields. The detection unit U3 acquires an analysis target object information acquisition unit 301 that acquires information on the presence of the analysis target in each of the plurality of reaction fields, and a size information acquisition unit 302 that acquires information on the respective sizes of the plurality of reaction sites. And.

Note that although the detection by the detection unit U3 may be performed by taking out some of the reaction fields among the plurality of reaction fields held in the container 103, it is preferable to carry out the detection for all the reaction fields that can be measured. . Thereby, the number of reaction fields to be detected can be increased, and the reliability of the analysis result can be improved.

(Analysis subject information acquisition unit)
The analyte information acquisition unit 301 is a part that detects an analyte for each of a plurality of reaction fields. The analyte information acquiring unit 301 detects a signal derived from the analyte for each of a plurality of reaction fields in which the reaction has progressed in the reaction unit U2. The analyte information acquisition unit 301 detects a signal derived from the analyte, and determines whether or not the analyte is contained for each of the plurality of reaction sites. Thereby, the analyte information acquisition unit 301 acquires information (analyte information) on the presence of the analyte in each of the plurality of reaction sites. Light is preferably used as the signal.

In the present specification, a reaction field in which a signal is detected by the analyte information acquiring unit 301, that is, a reaction field in which an analyte is contained is referred to as a “positive reaction field”. In addition, reaction fields in which no signal is detected by the analyte information acquiring unit 301, that is, reaction fields in which no analyte is contained are referred to as "negative reaction fields". In the present specification, when the intensity of the signal detected by the analyte information acquisition unit 301 is weaker than a preset threshold value, it is considered that the signal is not detected. That is, whether or not the reaction site contains the analyte is determined by comparing the intensity of the signal from the reaction site with a predetermined threshold.

For example, when the analyte is a nucleic acid, and an amplification reagent and a fluorescent reagent are used as agents for making the analyte detectable, the analyte information acquiring unit 301 uses the signal derived from the analyte as a signal. Preferably, fluorescence of a predetermined wavelength is detected.

When the analysis subject information acquisition unit 301 detects light such as fluorescence as a signal, the analysis subject information acquisition unit 301 may be configured of a light source 303a, a detector 304a, and a control unit (not shown). it can. The light source 303 a emits light of a wavelength according to the signal to be detected to each of the plurality of reaction fields held in the container 103. The detector 304a detects a signal emitted from each of the plurality of reaction fields irradiated with light. That is, the light source 303a functions as an excitation unit, and the detector 304a functions as a light detection unit.

As the detector 304a, a photodiode, a line sensor, an image sensor (image sensor) or the like can be used, and among them, it is preferable to use an image sensor in that signals can be detected collectively for a large number of reaction fields. . As the image sensor, a CCD (charge coupled device) or a CMOS (complementary metal oxide semiconductor image sensor) can be used. Alternatively, as the detector 304a, a digital camera provided with an image sensor may be used. When light is detected by the detector 304a, an optical filter may be used to adjust the wavelength of light from the reaction field.

The analyte information acquisition unit 301 may be a flow cytometer that sequentially detects a plurality of reaction fields flowing in the flow path. Alternatively, the analyte information acquisition unit 301 may two-dimensionally excite and detect a plurality of reaction fields. In other words, even if a plurality of reaction fields arranged in a plane are two-dimensionally irradiated with light to be excited, signals generated from the reaction fields are detected two-dimensionally using an image sensor. Good. This configuration is preferable because signal detection can be performed with high throughput for a large number of reaction sites.

Note that the light source 303a of the analysis target information acquisition unit 301 may be a light source that emits light of a plurality of different wavelengths to the reaction field. For example, the light source 303a may be a wavelength variable light source, or may have a plurality of light sources that respectively emit light having different wavelengths. As a result, by using a plurality of types of agents that allow detection so that a plurality of analytes can be distinguished, such as different wavelengths of generated fluorescence, a plurality of types of analytes are collectively detected in one analysis can do.

(Size information acquisition unit)
The size information acquisition unit 302 is a part that detects the size of each of a plurality of reaction fields. The size information acquisition unit 302 detects the size of each of the plurality of reaction fields for each of the plurality of reaction fields where the reaction has progressed in the reaction unit U2.

The size information acquisition unit 302 can be configured by a detector 304 b and a control unit (not shown). The detector 304 b detects light from the reaction field. A detector similar to the detector 304 a can be used as the detector 304 b. Also, the detector 304a may have the function of the detector 304b. The size detection unit 302 may further include a light source 303 b. The light source 303 b is preferably a light source that emits light having a wavelength different from that of the light source 303 a. When the light source 303 b is a wavelength variable light source, the light source 303 b may have the function of the light source 303 a.

Preferably, the size information acquisition unit 302 detects scattered light from the reaction field to detect the size of the reaction field. Further, as the detector 304 b of the size information acquisition unit 302, it is preferable to use an image sensor in that a large number of reaction fields can be detected at one time.

It is particularly preferable that the size information acquisition unit 302 acquires an image of a reaction field in which a large number of reaction fields are included in the field of view, and the controller (not shown) acquires the size of the reaction field from the image data. In this case, for example, generally used image analysis software can be used to analyze acquired image data to acquire information on the size of each reaction field. For example, in the case where the reaction field is a droplet, if the shape of the droplet can be regarded as a true sphere, acquiring at least one information consisting of the droplet radius, diameter, cross-sectional area, and volume from the image it can.

<Information processing unit>
The information processing unit U4 derives the concentration of the analyte in the sample based on the detection results of the detection unit U3 (information on the respective sizes of the reaction fields and the information on the presence of the analytes in each of the reaction fields). It is a unit.

FIG. 2 is a hardware block diagram of the information processing unit U4. The information processing unit U4 has the CPU 451, the ROM 452, the RAM 453, the storage 454, the input / output I / F 455, the communication I / F 456, and the image output I / F 457 in terms of hardware.

The CPU 451 executes a program stored in the ROM 452 or a program loaded to the RAM 453 and controls each part of the information processing unit U4. The ROM 452 is a non-volatile memory, and stores programs and the like necessary for the initial operation of the information processing unit U4. The RAM 453 is a volatile memory, and is used to read a program stored in the ROM 452 or storage 454 or an external storage device (not shown). The RAM 453 is also used as a work area of the CPU 451 when executing these programs.

The storage 454 has installed therein various programs to be executed by the CPU 451 such as an operating system and application programs, and data used to execute the programs. In the storage 454 is installed a program for analyzing measurement data supplied from the detection unit U3 and outputting an analysis result.

By loading a program stored in a storage medium such as the storage 454 into the RAM 453 and operating the CPU 451 according to the program loaded into the RAM 453, the functions of the units shown in FIG. 1 and the processes shown in FIG. To be executed.

The input / output interface (I / F) 455 is connected to an input unit 407 including a mouse, a keyboard, a touch panel, etc., and the user uses the input unit 407 to input data to the information processing unit U4. Be done. An image output interface (I / F) 457 is connected to the display unit 408 configured of a liquid crystal panel or the like, and outputs a video signal corresponding to the image data to the display unit 408. The display unit 408 displays an image based on the input video signal. The information processing unit U4 is connected to each of the reaction field generation unit U1, the reaction unit U2, the detection unit, and the transport unit (not shown) via a communication interface (I / F) 456. The information processing unit U4 can transmit and receive data to and from each unit described above through the communication interface 456.

The information processing unit U4 has, as its functions, a storage unit 401, a control unit 402, a distribution data generation unit 403, a section determination unit 404, and a concentration derivation unit 405, as shown in FIG.

The storage unit 401 is a portion that stores data received from the detection unit U3 or the input unit 407 or data generated by processing by the information processing unit U4. The control unit 402 is a part that controls the operation of each of the reaction field generation unit U1, the reaction unit U2, the detection unit U3, and the transport unit (not shown).

(Distribution data generation unit)
The distribution data generation unit 403 acquires, from the detection unit U3, information on the size of each reaction field and information on the presence of the analyte in each of the reaction fields, integrates these pieces of information, and obtains distribution data. Generate In the following description, data including information on the size of each of the reaction fields and information on the presence of the analyte in each of the reaction fields obtained from the detection unit U3 may also be referred to as detection data. is there. More specifically, the distribution data generation unit 403 divides the distribution of the size of the reaction field into a plurality of sections (classes), and for each section, information on the number of positive reaction sites and information on the number of negative reaction sites Generating distribution data including at least one information selected from the group consisting of In addition, with "the information regarding a number" here, a number itself, a ratio, etc. are mentioned, for example.

For example, it is assumed that the size of the reaction field is a minimum of 10 μm and a maximum of 210 μm as a sphere equivalent diameter in the case where the respective shapes of the plurality of reaction fields are substantially spherical. In this case, the distribution data generation unit 403 divides, for example, the distribution of the size of the reaction field into 10 sections in 20 μm steps. Then, the number of positive reaction fields and the number of negative reaction fields are totaled for a plurality of reaction fields having a size included in each section. At this time, the sum of the number of positive reaction fields and the number of negative reaction fields in each section, ie, the total number of reaction fields included in that section, or the total number of positive reaction fields for all reaction fields included in that section The ratio or the ratio of negative reaction sites may be counted. The number of sections and the width of the sections when the distribution data generation section 403 generates distribution data is not particularly limited, and may be determined according to the resolution of the size information acquisition section 302, or in statistical processing. It may be determined by a general method.

Note that the information on the size of the reaction field obtained from the detection unit U3 is linked to the information on whether the reaction field contained an analyte (whether it is a positive reaction field or a negative reaction field). Be acquired. In the case where the analysis object information acquisition unit 301 and the size information acquisition unit 302 both include means for acquiring image data using an image sensor, the image data acquired from the respective units are superimposed to obtain the above information. Can be tied.

(Section determination unit)
The section determination unit 404 determines, of the distribution data generated by the distribution data generation unit 403, data to be used for deriving the concentration in the concentration derivation unit 405 described later. More specifically, the section determining unit 404 configures distribution data based on at least one piece of information selected from the group consisting of information on the number of positive reaction sites and information on the number of negative reaction sites. From among the sections of, the section used to derive the concentration is determined.

In the present embodiment, the section determination unit 404 determines that the number or ratio of positive reaction fields or the number or ratio of negative reaction fields is included in a preset numerical range for each of a plurality of sections constituting the distribution data. It is determined whether or not Then, the section determining unit 404 determines a section determined to be included in a preset numerical range as a section to be used for deriving the concentration.

In digital analysis represented by digital PCR, the liquid containing an analyte is divided into a large number of reaction fields so that the number of analytes contained in each reaction field is either 1 or 0. Do. Thus, by counting the number of reaction fields in which the analyte is detected after the reaction in each reaction field, it is possible to measure the number of analytes contained in the liquid before division. However, even when limiting dilution is performed and then divided into a plurality of reaction sites, in fact, a case where two or more analytes are contained in one reaction site will also occur stochastically. Therefore, in such a case, the calculation result can be brought close to the true value and the reliability of the analysis result can be improved by calculating probabilistically based on the Poisson model. Details of such a conventional calculation method will be described later.

However, if there is a large variation in reaction field size, for example, each reaction field will have two or more of each reaction field in the large reaction field size even if the reaction area is close to the limiting dilution state in the small area. In some cases, it may contain an analyte of As a result of studies by the present inventors, in such a case, in the region where the size of the reaction field is small, the calculation based on the Poisson model has the effect of bringing the calculation result closer to the true value. Then, it turned out that the effect may not be obtained enough.

Therefore, in the present embodiment, the section determining unit 404 selects a section in which the number or ratio of positive reaction sites or the number or ratio of negative reaction sites is included in a preset numerical range, and the concentration is derived therefrom. Determined as the interval used for Then, the concentration deriving unit 405 selects data of at least a part of all the sections selected by the section determining unit 404 (information on the size of each reaction field and information on the presence of the analyte in each reaction field) Used to derive the concentration of analyte in the sample.

The section determining unit 404 preferably selects a section that can ensure the reliability of the calculation result based on the Poisson model. It is preferable that the interval determination unit 404 select an interval other than an interval in which the proportion of positive reaction fields is too high or an interval in which the proportion of negative reaction fields is too low. In a section where the proportion of positive reaction fields is too high or in a section where the proportion of negative reaction fields is too low, the probability that two or more analytes are included in one reaction field is too high, and calculation based on Poisson model There is a possibility that even if you do not get close to the true value.

In this case, the section determining unit 404 preferably selects a section where the percentage of positive reaction fields is less than 100%, more preferably selecting a section less than 90%, and selecting a section less than 80% More preferable. In addition, when the interval determination unit 404 selects an interval in which the proportion of positive reaction fields is less than 10%, the concentration derivation unit 405 can perform calculation without being based on the Poisson model. Thus, the reliability of the calculation result based on the Poisson model is improved by decreasing the upper limit of the proportion of positive reaction fields in the selected section or increasing the lower limit of the proportion of negative reaction fields. be able to. However, if the interval is narrowed too much, the number of reaction fields included in the interval decreases, and if the number of reaction fields used to derive the concentration decreases too much, the reliability of the calculation result is affected by the decrease. is necessary. The lower limit of the numerical range of the ratio of positive reaction fields in the section selected by the section determining unit 404 is not particularly limited, and may be 0% or more, or 5% or more. Alternatively, the interval determination unit 404 preferably selects an interval in which the proportion of negative reaction fields is greater than 0%, more preferably selects an interval larger than 10%, and further selects an interval larger than 20%. preferable. The upper limit of the numerical range of the ratio of negative reaction sites in the section selected by the section determining unit 404 is not particularly limited, and may be 100% or less or 95% or less. These numerical ranges regarding the proportion of positive reaction fields and the proportion of negative reaction fields are appropriately determined according to the accuracy required for analysis.

Alternatively, it is preferable that the interval determination unit 404 select an interval other than an interval in which the number of positive reaction fields is too small. If the number of positive reaction sites is too small, the reliability of the analysis results tends to decrease. Therefore, the concentration of the analysis results can be derived by using the data of the sections other than the section where the number of positive reaction sites is too small. Confidence can be increased. In order to know the relationship between the number of positive reaction fields and the dispersion of the analysis results, the present inventors have described “relative uncertainty” when the number of positive reaction fields is changed, as described in the technical literature Lab Chip , 14, 1176 (2014). The calculated results are shown in FIG. According to FIG. 3, in order to make the “measurement uncertainty” 10% or less, about 100 or more of the positive reaction fields in which the analyte is detected are required regardless of the size of the reaction fields. I understand that. Therefore, the interval determination unit 404 can increase the reliability of the analysis result to such an extent that “the uncertainty of measurement” becomes 10% or less by selecting the interval in which the number of positive reaction sites is 100 or more. In addition, the tolerance | permissible_range of the required "measurement uncertainty" may change with the objective of analysis, and is not limited to said numerical value. For example, if the required “measurement uncertainty” is 20% or less, the section determination unit 404 may select a section having 30 or more positive reaction fields.

As described above, the section determining unit 404 according to the present embodiment selects at least one section in which the number or ratio of positive reaction fields or the number or ratio of negative reaction fields is included in a predetermined range, and Determined as the interval to be used for the derivation of However, the section determination unit 404 according to the present invention is not limited to this, and the section not used for deriving the concentration may be discarded and determined as a section for use for deriving the concentration. That is, the section determination unit 404 may be a data selection unit that selects a part of the distribution data generated by the distribution data generation unit 403, or a data rejection unit that discards a part of the distribution data. It is also good.

When the section determining unit 404 discards a section not used for concentration derivation and determines it as a section used for concentration derivation, the section to be rejected may be determined by the same method as the method for selecting the above section. it can. More specifically, the section determining unit 404 does not include the number or proportion of positive reaction fields or the number or proportion of negative reaction fields in a predetermined range for each of a plurality of sections constituting distribution data. You may reject the data of the section.

For example, in this case, the section determining unit 404 preferably discards sections with a percentage of positive reaction fields of 100%, more preferably discards sections with 90% or more, and discards sections with 80% or more. Is more preferred. In addition, the section determining unit 404 may discard a section in which the proportion of positive reaction sites is 10% or more.

The section determination unit 404 may determine a section to be used for deriving the concentration, and extract data of the section from the distribution data generated by the distribution data generation unit 403. In this case, the section determined object 404 can also be referred to as a data processing unit that processes the distribution data generated by the distribution data generation unit 403. That is, the data processing unit generates the distribution data generated by the distribution data generation unit 403 based on at least one piece of information selected from the group consisting of information on the number of positive reaction sites and information on the number of negative reaction sites. Process

Note that the data processing unit selects at least one selected from the group consisting of information on the number of positive reaction fields and information on the number of negative reaction fields instead of determining the section used for deriving the concentration by the section determining unit 404. The weighting of each section may be changed based on one piece of information. For example, the data processing unit is a data correction factor based on at least one piece of information selected from the group consisting of information on the number of positive reaction fields and information on the number of negative reaction fields in the data included in each of the plurality of sections. Processing may be performed.

(Concentration deriving unit)
The concentration deriving unit 405 derives the concentration of the analyte in the sample based on the data of the section determined by the section determining unit 404 among the distribution data generated by the distribution data generating unit 403. That is, the concentration deriving unit 405 derives the concentration of the analyte in the sample using at least a part of the distribution data generated by the distribution data generating unit 403.

The concentration deriving unit 405 may perform part or all of the following calculation processing for each section of the distribution data. The concentration deriving unit 405 preferably derives, for each of the sections of the distribution data, the analyte included in the section based on the Poisson model. Then, it is preferable that the concentration deriving unit 405 derives the concentration by totaling the number of analytes in each of the derived sections and dividing it by the total volume of the reaction field used for the calculation. Alternatively, it is preferable that the concentration deriving unit 405 derives the concentration of the analyte for each section, weighted averages it, and derives the concentration of the analyte in the reaction liquid or the sample. Thus, the reliability of the analysis result can be enhanced by deriving the number or concentration of the analyte based on the Poisson model for each section.

Hereinafter, calculation processing when the concentration deriving unit 405 derives the concentration of the analyte in the sample will be described.

(Calculating the concentration of the analyte)
The calculation of the concentration of the analyte can be carried out using a conventional method of calculating the concentration in digital analysis. Before the reaction in the reaction unit U2, the case where each reaction field can be regarded as either one or zero analytes will be described. In this case, the number x of reaction fields (positive reaction fields) in which the analyte is detected is the number of analytes contained in the reaction solution of the volume Vs in which the detection unit U3 is the target of detection of the analyte. It can be regarded as Therefore, the concentration λ _r of the analyte in the reaction liquid can be calculated by the following formula (1). In addition, the volume Vs which the detection unit U3 made the detection object of the analysis object can be calculated based on the information regarding the size of the reaction field acquired from the detection unit U3.
λ _r = x / Vs (1)

Also, for example, if it can be considered that a plurality of analytes can enter one reaction site before the reaction in the reaction unit U2, calculate the concentration of the analyte by performing correction using a Poisson model. Can. In this case, the concentration of the analyte is calculated by estimating the average number C of the analytes contained in each reaction site before the reaction in the reaction unit U2. Specifically, assuming that the average number of analytes contained in one reaction field is C for the reaction fields for which the detection unit U3 is the target of detection of the analytes, n analysis targets in one reaction field The probability that an object is included is expressed as the following equation (2) from the Poisson model equation.

Here, the probability that one reaction field does not contain any one analyte is represented by the following formula (3) with n = 0 in formula (2).
P (0, C) = e- ^C equation (3)

If at least one analyte is contained in one reaction field before the reaction in reaction unit U2, a signal can be detected from the reaction field, but it is included in the reaction field before the reaction. Information on the number of analytes that have been stored is unknown. Therefore, equation (3) is used based on the ratio of reaction fields in which no analyte is detected (the ratio of reaction fields in which a signal is not detected) to the total number of reaction fields targeted by detection unit U3. The number of analytes contained in the reaction solution to be detected is estimated.

Specifically, the ratio F _{0 of} reaction fields in which no signal was detected from the number of reaction fields in which a signal was detected or the number of reaction fields in which a signal was not detected, and the total number of reaction fields to be detected. Calculate Then, from the following formula (4), the average number C of the analytes contained in one reaction field before the reaction in the reaction unit U2 is estimated as the reaction field to be detected.
C = −ln (F ₀ ) formula (4)

Here, assuming that the average volume of the reaction field to be detected by the detection unit U3 is v, the concentration λ _r of the analyte in the reaction liquid can be calculated by the following equation (5). In addition, the average volume v which the detection unit U3 made the detection object of the analysis object can be calculated based on the information regarding the size of the reaction field acquired from the detection unit U3.
λ _r = C / v formula (5)

The concentration λ _r of the analyte in the reaction solution is the total number of analytes obtained by multiplying the average number C of analytes by the number of reaction sites, the average volume v of reaction sites and the number of reaction sites It may be calculated based on the total volume of the reaction site obtained by multiplying

The concentration of the analyte in the reaction solution thus obtained is converted to the concentration of the analyte in the specimen or sample by using the dilution factor when adjusting the reaction solution from the specimen or sample. be able to.

[Analytical method]
Next, an analysis method using the analysis system 1 according to the present embodiment will be described with reference to FIG. FIG. 4 is a flow chart showing the procedure of analysis processing by the analysis system.

At S401, a sample for performing quantitative analysis of an analyte is prepared. Here, the sample is prepared by diluting and pretreating the sample. The preparation of the sample may be performed in the analysis system 1, or may be performed using an apparatus outside the analysis system 1, for example, a commercially available sample pretreatment apparatus.

At S402, the reaction field generation unit U1 divides the reaction solution containing the sample to generate a plurality of reaction fields independent of each other.

At S403, the reaction unit U2 causes the reaction to proceed in each of the plurality of reaction sites, making the analyte detectable.

At S404, the detection unit U3 performs detection of the analyte and detection of the size of each of the plurality of reaction fields for each of the plurality of reaction fields. Thereby, information on the size and information on the presence of the analyte are obtained for each of the plurality of reaction sites.

At S405, the information processing unit U4 acquires information on the size of each of the reaction fields and information on the presence of the analyte in each of the reaction fields from the detection unit U3 and integrates these pieces of information, Generate distribution data.

At S406, the information processing unit U4 determines a section to be used for deriving the concentration of the analyte based on the number or proportion of positive reaction fields, or the number or proportion of negative reaction fields. At this time, the information processing unit U4 compares the number or ratio of positive reaction fields or the number or ratio of negative reaction fields with a preset numerical range and uses it for deriving the concentration of the analyte Determine the interval.

At S407, the information processing unit U4 derives the concentration of the analyte using the data of the section determined at S406.

Other Embodiments
Although analysis system 1 was explained as an embodiment of the present invention, the present invention is not limited to this, and the present invention can also be realized by an analysis system which consists of a part of each part which constitutes analysis system 1.

Furthermore, the present invention provides a program that implements one or more functions of the above-described embodiments to a system or apparatus via a network or computer readable storage medium, and one or more of the computer of the system or apparatus are provided. It can also be realized by a process in which a processor reads and executes a program. It can also be implemented by a circuit (eg, an ASIC) that implements one or more functions.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following.

(Preparation example 1)
<Formation of Emulsion 1>
2 μL of a 10-fold diluted solution of QuickPrimer Control DNA 5 (5 ng / μL, model number MR405, manufactured by Takara Bio Inc.), QuickPrimer Escherichia / Shigella group (16S rDNA) (forward primer, reverse primer 2.0 μM, model number MR201, Takara Bio ) And 10 μL of SYBR Premix Ex Taq (Tli RNase H Plus) (Model No. RR420, manufactured by Takara Bio Inc.) and 4 μL of sterile distilled water were prepared and mixed.

The mixture was subjected to thermal cycling under the following thermal cycling conditions to perform PCR to obtain an amplicon of E. coli 16S rDNA. Agarose gel electrophoresis confirmed that a 413 bp amplification product was obtained. The solution was diluted so that the concentration of amplicon was approximately 5 × 10 ⁴ copies / μL, and this was used as template 1.

[Thermal cycle conditions]
1) Initial denaturation (95 ° C for 2 minutes) 1 cycle 2) PCR (95 ° C for 20 seconds, 55 ° C for 20 seconds, 74 ° C for 20 seconds) 35 cycles 3) Holding (4 ° C) for 1 cycle

2 μL of the template 1 described above in 10 μL of a commercially available intercalator PCR reagent (ddPCR EvaGreen Supermix, manufactured by Bio-Rad Laboratories) 2 μL of QuickPrimer Escherichia / Shigella group (16S rDNA) (forward primer, reverse primer 2.0 μM each) Model No. MR201, manufactured by Takara Bio Inc.) and 1 μl of sterile distilled water were respectively added and mixed to obtain a dispersed phase.

50 μL of a commercially available oil for digital PCR (Droplet Generator Oil for Evagreen, manufactured by Bio-Rad Laboratories) was added as a continuous phase to the dispersed phase described above. The mixture was vortexed to produce Emulsion 1, a water-in-oil emulsion.

<PCR using emulsion>
The resulting emulsion 1 was subjected to thermal cycling under the following thermal cycling conditions to perform PCR.

[Thermal cycle conditions]
1) Enzyme activation (95 ° C. for 5 minutes) 1 cycle 2) PCR (95 ° C. for 30 seconds, 55 ° C. for 1 minute) 50 cycles 3) Signal stabilization (4 ° C. for 5 minutes, 90 ° C. for 5 minutes) 1 cycle holding (4 ° C) 1 cycle

<Measurement of droplet after thermal cycle>
20 μL of the emulsion 1 after thermal cycling was collected on a glass settling plate (MUR-300, Matsunami Glass Industrial Co., Ltd.) and observed using a fluorescence microscope (BZ-8000, Keyence Corporation). For observation, a visible light image and a fluorescence image (excitation wavelength 480/30 nm, absorption wavelength 510 nm) are taken with a built-in camera (imaging element: 1.5 million pixel CCD image sensor) in one field of view in the same field of view. I did in one of the visions. An example of the image | photographed image is shown to FIG. 5A.

The diameter of each droplet was measured from the obtained visible light image using image processing software. At this time, the resolution of measurement was 10 μm, and droplets having a diameter of 10 μm or less were excluded from measurement. Moreover, the presence or absence of fluorescence enhancement due to gene proliferation was determined for each droplet by visual determination based on the obtained fluorescence image, and it was determined whether or not an analyte was detected. By superimposing the visible light image and the fluorescence image, data in which the information on the size of each droplet was associated with the information on whether or not the analyte was detected was created.

For the obtained data, the droplet size was divided into a plurality of sections to create frequency distribution data. Specifically, the droplet diameter was divided into 18 segments, with the droplet diameter of 20 μm or more and less than 30 μm as one segment, and the measurement resolution of 10 μm as the segment width. Then, the number of droplets, the number of positive droplets, and the number of negative droplets were counted for each section. The results are summarized in Table 1.

In the table, the droplet diameter is the average diameter of droplets, Total is the total number of droplets, Positive is the number of droplets with fluorescence enhancement (positive droplets), Negative is the droplet without fluorescence enhancement (negative droplets The number of) is shown, respectively (following, it is the same).

(Preparation example 2)
In Preparation Example 1, template 1 was diluted 10 times to form template 2. That is, in the template 2, the concentration of the amplicon is approximately 5 × 10 ³ copies / μL. Emulsion 2 was produced in the same manner as in Preparation Example 1 except that template 2 was used instead of template 1 to form a dispersed phase.

The emulsion 2 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 1, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 1. An example of the image | photographed image is shown to FIG. 5B. The measurement results are summarized in Table 1 below.

(Preparation example 3)
In Preparation Example 1, template 1 was diluted 100 times to form template 3. That is, in the template 3, the concentration of the amplicon is approximately 5 × 10 ² copies / μL. Emulsion 3 was produced in the same manner as in Preparation Example 1 except that template 3 was used instead of template 1 to form a dispersed phase.

The emulsion 3 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 1, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 1. An example of the image | photographed image is shown to FIG. 5C. The measurement results are summarized in Table 1 below.

(Preparation example 4)
In Preparation Example 1, template 1 was diluted 1000 times to form template 4. That is, in the template 4, the concentration of the amplicon is approximately 5 × 10 copies / μL. Emulsion 4 was produced in the same manner as in Preparation Example 1 except that template 4 was used instead of template 1 to form a dispersed phase.

The emulsion 4 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 1, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 1. An example of the image | photographed image is shown to FIG. 5D. The measurement results are summarized in Table 1 below.

(Preparation example 5)
In Preparation Example 1, PCR was carried out in the same manner as in Preparation Example 1 except that Human β-Actin (Human ACTB Endogenous Control, Thermo Fisher Scientific Co., Ltd.) was used instead of 16S rDNA (QuickPrimer Escherichia / Shigella group). Did. The solution was diluted so that the concentration of human β-Actin amplicon was approximately 5 × 10 ⁴ copies / μL, and used as template 5. An emulsion 5 was produced in the same manner as in Preparation Example 1 except that template 5 was used instead of template 1 to form a dispersed phase.

The emulsion 5 was subjected to a thermal cycle in the same manner as in Preparation Example 1 to perform PCR, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 1. An example of the image | photographed image is shown to FIG. 6A. The measurement results are summarized in Table 2.

(Preparation example 6)
In Preparation Example 5, template 5 was diluted 10 times to form template 6. That is, in the template 6, the concentration of the amplicon is approximately 5 × 10 ³ copies / μL. Emulsion 6 was produced in the same manner as in Preparation Example 5, except that template 6 was used instead of template 5 to form a dispersed phase.

The emulsion 6 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 5, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 5. An example of the image | photographed image is shown to FIG. 6B. The measurement results are summarized in Table 2.

(Preparation example 7)
In Preparation Example 5, the template 5 was diluted 100 times to be a template 7. That is, in the template 7, the concentration of the amplicon is approximately 5 × 10 ² copies / μL. Emulsion 7 was produced in the same manner as in Preparation Example 5 except that template 7 was used instead of template 5 to form a dispersed phase.

The emulsion 7 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 5, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 5. An example of the image | photographed image is shown to FIG. 6C. The measurement results are summarized in Table 2.

(Preparation example 8)
In Preparation Example 5, template 5 was diluted 1000 times to form template 8. That is, in the template 8, the concentration of the amplicon is approximately 5 × 10 copies / μL. An emulsion 8 was produced in the same manner as in Preparation Example 5 except that the dispersed phase was formed using template 8 instead of template 5.

The emulsion 8 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 5, and the droplets after the thermal cycle were measured in the same manner as in Preparation Example 5. An example of the image | photographed image is shown to FIG. 6D. The measurement results are summarized in Table 2.

(Comparative example 1)
With respect to emulsions 1 to 4 of Preparation Examples 1 to 4, the concentration of the analyte (target nucleic acid) in the sample was calculated by a method commonly performed in digital PCR using droplets. Specifically, the percentage of negative droplets is calculated using the sum of the total number of droplets in all sections and the total number of negative droplets in all sections, and is included in one reaction field using equation (4) The average number C of analyzed analytes was calculated. Then, the total number of analytes contained in all droplets to be detected was calculated by multiplying the average number C and the sum of the total number of droplets in all sections. Thereafter, the volume of droplets contained in each section was calculated from the total number of droplets in each section and the droplet diameter of the droplet in that section, and the total volume of droplets to be detected was calculated. Then, the concentration in the reaction solution is calculated by dividing the total number of analytes contained in all droplets to be detected by the total volume of all droplets to be detected, and multiplying this by 10 times the dilution ratio Were converted to the concentration of the analyte in the sample. The results are shown in Table 3.

Example 1
For the emulsion 1, the concentration of the analyte in the sample was calculated using only the data in the interval of 0% or more and less than 100% of the distribution data among the distribution data. Specifically, as shown in Table 4, the data is rejected for the

sections

10, 14, 15 and 17 in which the percentage of positive droplets is 100%, and the calculation is performed using data of other sections. The For each of the sections that were not rejected, the number of analytes was calculated for each section by the same calculation as in Comparative Examples 1 to 4. Also, for each of the sections that were not rejected, the total volume of droplets in the section was calculated for each section. Then, the concentration in the reaction solution is calculated by dividing the sum of the number of analytes in each section obtained by the total volume of droplets in each section, and multiplying this by 10 times the dilution rate of the sample Thus, it was converted to the concentration of the analyte in the sample.

Calculations were performed on emulsions 2 to 3 in the same manner as in emulsion 1 to calculate the concentration of the analyte in the sample. The calculation results are summarized in Table 5.

(Example 2)
The concentration of the analyte in the sample was calculated in the same manner as in Example 1 except that the width of the droplet diameter section was changed from 10 μm to 20 μm in Example 1. Table 6 shows frequency distribution data and calculation results in the case where the width of the droplet diameter section of the emulsion 1 is changed from 10 μm to 20 μm.

Calculations were performed on emulsions 2 to 4 in the same manner as in emulsion 1 to calculate the concentration of the analyte in the sample. The calculation results are summarized in Table 7.

Comparison of Comparative Example 1 and Examples 1 and 2
As shown in Tables 3, 5, and 7, in each of Comparative Example 1 and Examples 1 and 2, the emulsions 1, 2, 3 and 4 had a relative dilution ratio to the emulsion 1 of 1, 10, respectively. It corresponds to 100 times and 1000 times. Therefore, the concentrations of analyte in the sample in emulsions 1, 2, 3 and 4 should be 1 times, 0.1 times, 0.01 times and 0.001 times that of emulsion 1, respectively. .

FIG. 7 is a graph showing the relationship between the relative dilution ratio and the calculation result of the concentration of the analyte in the sample in each of Comparative Example 1 and Examples 1 and 2. FIG. 7A shows the result of Comparative Example 1, FIG. 7B shows the result of Example 1, FIG. 7C shows the result of Example 2 by a double logarithm graph in which the horizontal axis represents relative dilution ratio and the vertical axis is calculation result of concentration. ing.

As described above, relative dilution factor and concentration are y = ax ^−1, where y is a relative dilution factor and x is a concentration. Therefore, in the double logarithm graph, the relationship between the two should be represented by a straight line with a slope of -1. In FIGS. 7A to 7C, the relationship between the relative dilution ratio and the concentration is shown by a dotted line on the assumption that the concentration of the analyte in the sample in Emulsion 1 is 5 × 10 ⁴ copies / μL. Further, in FIGS. 7A to 7C, solid lines indicate approximate curves when the results of Comparative Example 1 and Examples 1 and 2 are power-approximated in a double logarithm graph. When FIGS. 7A to 7C are compared, it is found that in FIGS. 7B and 7C, the slope of the solid line is closer to the slope of the dotted line than in FIG. 7A. Specifically, the slope of the approximate curve was −0.78 in Comparative Example 1, −0.86 in Example 1, and −0.86 in Example 2. From this, it was found that in Examples 1 and 2, the result closer to the true value was obtained than Comparative Example 1, that is, the reliability of the quantitative analysis was high. In Examples 1 and 2, it was found that the result close to the true value was obtained particularly in the low dilution ratio part, that is, in the high concentration part of the analyte in the sample. From the above, it has been found that according to the present invention, highly reliable analysis results can be obtained even when the droplet size varies.

(Comparative example 2)
The concentrations of the analytes in the samples of the emulsions 5 to 8 of Preparation Examples 5 to 8 were calculated in the same manner as in Comparative Example 1. The results are shown in Table 8.

(Example 3)
The concentrations of the analytes in the samples were calculated in the same manner as in Example 1 with respect to emulsions 5 to 8 of Preparation Examples 5 to 8. The results are shown in Table 9.

(Example 4)
The concentrations of the analyte in the samples were calculated in the same manner as in Example 2 for emulsions 5 to 8 in Preparation Examples 5 to 8. The results are shown in Table 10.

Comparison of Comparative Example 2 and Examples 3 and 4
As shown in Tables 8, 9 and 10, in each of Comparative Example 2 and Examples 3 and 4, the emulsions 5, 6, 7 and 8 have a relative dilution ratio to the emulsion 5 of 1, 10, respectively. It corresponds to 100 times and 1000 times. Thus, the concentrations of analyte in the sample in emulsions 5, 6, 7 and 8 should be 1 times, 0.1 times, 0.01 times and 0.001 times that of emulsion 5, respectively. .

FIG. 8 is a graph showing the relationship between the relative dilution ratio and the calculation result of the concentration of the analyte in the sample in each of Comparative Example 2 and Examples 3 and 4. FIG. 8A shows the result of Comparative Example 2, FIG. 8B shows the result of Example 3, FIG. 8C shows the result of Example 4 by a double logarithm graph in which the horizontal axis represents relative dilution factor and the vertical axis is calculation result of concentration. ing.

As described above, relative dilution factor and concentration are y = ax ^−1, where y is a relative dilution factor and x is a concentration. Therefore, in the double logarithm graph, the relationship between the two should be represented by a straight line with a slope of -1. In FIG. 8A to FIG. 8C, the relationship between relative dilution ratio and concentration is shown by a dotted line assuming that the concentration of the analyte in the sample in emulsion 5 is 5 × 10 ⁴ copies / μL. Further, in FIGS. 8A to 8C, a solid line indicates an approximate curve when the results of Comparative Example 2 and Examples 3 and 4 are power-approximated in a double logarithm graph. Comparing FIGS. 8A to 8C, it was found that in FIGS. 8B and 8C, the slope of the solid line is closer to the slope of the dotted line than in FIG. 8A. Specifically, the slope of the approximate curve was −0.73 in Comparative Example 2, −0.88 in Example 3, and −0.89 in Example 4. From this, it was found that in Examples 3 and 4, the result closer to the true value was obtained than Comparative Example 2, that is, the reliability of the quantitative analysis was high. In Examples 3 and 4, it was found that results close to the true value were obtained, particularly in the low dilution ratio part, that is, in the part where the concentration of the analyte in the sample is high. From the above, it has been found that according to the present invention, highly reliable analysis results can be obtained even when the droplet size varies.

(Preparation example 9)
<Formation of Emulsion 9>
25 copies / μL of deoxyribonucleic acid (DNA) aqueous solution for quantification (Model No. 6205-a, manufactured by National Institute of Advanced Industrial Science and Technology, Metric Standard Center) as the dispersed phase final concentration, primers designed to correspond to the DNA (forward primer, reverse Add 0.5 μM of each primer as the final concentration of the dispersed phase, and a probe labeled with FAM as a fluorescent dye for detecting DNA amplification to a final concentration of the dispersed phase of 0.25 μM, and add Premix Ex Taq (Model No. RR390A, The dispersed phase of emulsion 9 was prepared by further mixing with Takara Bio Inc. and sterile distilled water.

A surfactant, KF-6038 (manufactured by Shin-Etsu Chemical Co., Ltd.), was dissolved in Isopar L (manufactured by Exxon Mobil), which is an isoparaffin aliphatic hydrocarbon, to prepare Emulsion 9. In this example, the oil-based composition was prepared such that the concentration of the surfactant was 4% by mass when the entire oil-based composition was 100% by mass.

A shirasu porous glass (SPG) film (DC 20 U, manufactured by SPG Techno), which is an emulsified film, was connected to the tip of a syringe (08040, manufactured by Nipro) from which the aqueous composition was collected. Set the syringe to a syringe pump (SPS-1, manufactured by As One), immerse the emulsified film at the tip of the syringe in 9 mL of the above-mentioned oily composition, and after sucking up a small amount of the oily composition, emulsifying flow rate of 5 mL / h (aqueous composition The dispersed phase was injected at a substance injection rate to produce Emulsion 9, which is a water-in-oil emulsion.

<PCR using emulsion>
The obtained emulsion 9 was subjected to thermal cycling under the following thermal cycling conditions to perform PCR.

[Thermal cycle conditions]
1) Enzyme activation (95 ° C for 30 seconds) 1 cycle 2) PCR (95 ° C for 5 seconds, 60 ° C for 30 seconds) 40 cycles 3) Holding (4 ° C) 1 cycle

<Measurement of droplet after thermal cycle>
After thermal cycling, 20 μL of the emulsion 9 was collected on a glass settling plate (MUR-300, Matsunami Glass Industrial Co., Ltd.) and observed using a fluorescence microscope (BZ-8000, Keyence Corporation). For observation, a visible image and a fluorescence image (excitation wavelength 480/30 nm, absorption wavelength 510 nm) are taken with a built-in camera (imaging element: 1.5 million pixel CCD image sensor) in one field of view in the same field of view. I went over the field of view. An example of the image | photographed image is shown to FIG. 9A.

From the microscopic image of the obtained emulsion, the diameter of each droplet was measured using image processing software (ImageJ). At this time, since a droplet with a diameter of 40 μm or less was difficult to separate from noise, it was excluded from measurement. Furthermore, using the above-mentioned image processing software, the presence or absence of fluorescence enhancement by gene proliferation is determined for each droplet, and information on the size of each droplet and information on whether or not an analyte is detected I created the data that I matched.

For the obtained data, the droplet size was divided into a plurality of sections to create frequency distribution data. Specifically, the droplet diameter was divided into 19 segments, with the droplet diameter being 40 μm or more and less than 50 μm as one segment, and the measurement resolution of 10 μm as the segment width. Then, the number of droplets, the number of positive droplets, and the number of negative droplets were counted for each section. The results are summarized in Table 11.

In Table 11, the droplet diameter is the average diameter of droplets, Total is the total number of droplets, Positive is the number of droplets with fluorescence enhancement (positive droplets), and Negative is the droplet without fluorescence enhancement (negative Each indicates the number of droplets) (the same applies hereinafter).

(Preparation example 10)
<Formation of Emulsion 10>
An emulsion 10 was produced in the same manner as in Preparation Example 9 except that the DNA concentration was changed to 250 copies / μL.

The emulsion 10 was subjected to a thermal cycle and subjected to PCR in the same manner as in Preparation Example 9, and in the same manner as in Preparation Example 9, the total number of droplets after thermal cycling (Total), the number of positive droplets (Positive), and the negative droplets. The number (Negative) was measured. An example of the image | photographed image is shown to FIG. 9B. The measurement results are summarized in Table 11.

(Preparation example 11)
<Formation of Emulsion 11>
Emulsion 11 was produced in the same manner as in Preparation Example 9 except that the DNA concentration was changed to 2500 copies / μL.

The emulsion 11 was subjected to thermal cycling in the same manner as in Preparation Example 9 to conduct PCR, and the total number of droplets after thermal cycling, the number of positive droplets, and the number of negative droplets were measured in the same manner as in Preparation Example 9. An example of the image | photographed image is shown to FIG. 9C. The measurement results are summarized in Table 11.

(Preparation example 12)
<Formation of Emulsion 12>
Emulsion 12 was produced in the same manner as in Preparation Example 9 except that the DNA concentration was changed to 6250 copies / μL.

The emulsion 12 was subjected to thermal cycling in the same manner as in Preparation Example 9 to perform PCR, and the total number of droplets after thermal cycling, the number of positive droplets, and the number of negative droplets were measured in the same manner as in Preparation Example 9. An example of the photographed image is shown in FIG. 9D. The measurement results are summarized in Table 11.

(Preparation example 13)
<Formation of Emulsion 13>
An emulsion 13 was produced in the same manner as in Preparation Example 9 except that the DNA concentration was changed to 20000 copies / μL.

The emulsion 13 was subjected to thermal cycling in the same manner as in Preparation Example 9 to conduct PCR, and the total number of droplets after thermal cycling, the number of positive droplets, and the number of negative droplets were measured in the same manner as in Preparation Example 9. An example of the image | photographed image is shown to FIG. 9E. The measurement results are summarized in Table 11.

(Comparative example 3)
With respect to the emulsions 9 to 13 of Preparation Examples 9 to 13, the total number of analytes contained in all droplets to be detected as in Comparative Example 1 and the concentration of the analyte in each sample were calculated. Furthermore, the divergence rate between the analyte concentration calculated for each sample and the dispersed phase concentration for each sample was calculated. The results are shown in Table 12.

(Example 5)
The concentration of the analyte in the sample was calculated in the same manner as in Example 1 for emulsions 9-12. The results are shown in Table 13.

Comparison of Comparative Example 3 and Example 5
FIG. 10 is a graph showing the relationship between the prepared concentration for each of the emulsions 9 to 13 in Comparative Example 3 and Example 5 and the calculation result of the concentration of the analyte in the sample. From the results of FIG. 10 and Tables 12 and 13, it is found that the deviation ratio with the sample concentration is less than 9% in the embodiment in which the droplet size is considered, and the fluctuation of the value is smaller even in comparison with the comparative example. . Furthermore, the DNA sample used this time allows an error of 9.2% in the 95% confidence interval to the true value. From the above, it was found that according to the present invention, even when the droplet size varies, calculation results close to the true value can be obtained.

(Comparative example 4)
A comparison of the two was performed using a commercially available drop-type digital PCR device (model: QX200 Droplet Digital PCR System, Bio-Rad Laboratories) for a DNA concentration of 25, 250, 2500 copies / μL corresponding to emulsions 9-11. The

The dispersed phase of the droplet was prepared by adding the primers (forward primer and reverse primer respectively) as the dispersed phase final concentration to 0.5 μM and the FAM labeled probe as the dispersed phase final concentration to 0.25 μM in the same manner as in Example 5. The mix (Model No. 186-3023, manufactured by Bio-Rad Laboratory) and sterile distilled water were further mixed to prepare a dispersed phase corresponding to emulsions 9-11.

The prepared dispersed phase was used to generate droplets in a droplet generator (Automated Droplet Generator system, manufactured by Bio-Rad Laboratory), and subjected to thermal cycling under the following thermal cycle conditions to conduct PCR.

[Thermal cycle conditions]
1) Enzyme activation (95 ° C. for 10 minutes) 1 cycle 2) PCR (95 ° C. for 30 seconds, 60 ° C. for 1 minute) 50 cycles 3) Holding (4 ° C.) 1 cycle Procedures for concentration measurement other than the above are system protocols I obeyed.

Table 14 shows the analysis results of the commercially available device. The relative dilution ratio shown in the table is 1 where 2500 copies / μL, which is the DNA concentration corresponding to emulsion 11, is 1, 250 copies / μL corresponding to emulsion 10 is 10, and 25 copies corresponding to emulsion 9 / μL is 100. From Table 4, it was found that the value obtained in the commercial apparatus was close to the true value, with the rate of deviation from the sample concentration being less than 9.2%.

Comparison of Emulsions 9 to 11 of Comparative Example 4 and this Example
FIG. 11 shows the results of the examples of the emulsions 9 to 11 and the results carried out in the comparative example 4, with the relative dilution factor on the horizontal axis and the double logarithm graph on the vertical axis as the calculation result of the concentration.

In the figure, the black dotted line indicates the approximate curve when the result of the comparative example in the commercial apparatus is approximated by a power approximation. Comparing the two, it was found that the slopes of the approximate curves were -1.002 and -0.966, respectively, and values close to the ideal -1 were obtained. Furthermore, since both plots were within 9% of the true value in the concentration range studied, it was found that both results close to the true value and high quantitativity were obtained for both. . From the above, it was found that according to the embodiment of the present invention, highly reliable analysis results can be obtained even when the droplet size varies.

The present invention is not limited to the above embodiment, and various changes and modifications can be made without departing from the spirit and scope of the present invention. Accordingly, the following claims are attached to disclose the scope of the present invention.

The present application claims priority based on Japanese Patent Application No. 2017-132911 filed on July 6, 2017 and Japanese Patent Application No. 2018-125187 submitted on June 29, 2018, The entire contents of the description are incorporated herein.

Claims

An analysis system for analyzing the concentration of an analyte in a sample, comprising:
A size information acquisition unit for acquiring information on the size of each of the plurality of reaction fields, for a plurality of reaction fields generated by dividing the liquid containing the sample;
An analyte information acquisition unit that acquires information on the presence of the analyte in each of the plurality of reaction sites;
The distribution of the size of the reaction field was divided into a plurality of sections, and for each section, information on the number of positive reaction sites which were reaction sites where the analyte was detected, and the analyte were not detected Distribution data including at least one piece of information selected from the group consisting of information on the number of negative reaction sites that are reaction sites, based on the information acquired by the size information acquiring unit and the analyte information acquiring unit A distribution data generation unit to generate
A section determination unit that determines a section to be used for concentration derivation based on at least one piece of information selected from the group consisting of information on the number of positive reaction sites and information on the number of negative reaction sites;
An analysis system comprising: a concentration deriving unit that derives the concentration of the analyte in the sample based on the data of the section determined by the section determining unit among the distribution data.
The section determining unit determines that the number or ratio of the positive reaction fields or at least one section in which the number or ratio of the negative reaction fields is included in a predetermined range is a section used for deriving the concentration. The analysis system according to claim 1, characterized in that
The section determining unit discards at least one section where the number or proportion of the positive reaction sites or the number or proportion of the negative reaction sites is not included in the predetermined range, and the section not rejected is concentration The analysis system according to claim 1, wherein the interval is determined to be used for the derivation of.
The analysis according to any one of claims 1 to 3, wherein the concentration deriving unit derives, for each of the sections, the number of the analytes included in the section based on a Poisson model. system.
The said size information acquisition part and the said analysis object information acquisition part contain the imaging means which images at least one part of these reaction fields, The any one of Claim 1 to 4 characterized by the above-mentioned. Analysis system.
The analysis system according to any one of claims 1 to 5, further comprising a reaction field generation unit that divides the liquid to generate the plurality of reaction fields.
7. The analysis according to claim 6, wherein the reaction field generating unit is a means for generating an emulsion in which the liquid is dispersed in the form of droplets in a second liquid incompatible with the liquid. system.
The analysis system according to claim 7, wherein the reaction field generation unit is a means for generating the emulsion by a membrane emulsification method or a mechanical emulsification method.
The liquid contains an agent for making the analyte detectable.
The reaction according to any one of claims 1 to 8, further comprising a reaction part which causes the reaction by the drug to proceed in each of the plurality of reaction fields and makes the analyte detectable. Analysis system.
The analysis system according to any one of claims 1 to 9, wherein the analyte is a nucleic acid.
The analysis system according to claim 10, wherein the agent comprises an amplification reagent for amplifying the nucleic acid, and a fluorescent reagent that interacts with the nucleic acid to emit fluorescence.
The analysis system according to claim 10 or 11, wherein the reaction comprises PCR.
The analysis system according to any one of claims 9 to 12, wherein the reaction unit includes a temperature controller that adjusts the temperature of each of the plurality of reaction sites.
The analysis system according to any one of claims 1 to 13, wherein the distribution of sizes of the plurality of reaction fields is polydispersion.
The section determining unit determines at least one section having a ratio of the positive reaction field of 0% or more and less than 100% as a section used for deriving a concentration. The analysis system according to any one of the preceding claims.
The section determining unit determines at least one section having a ratio of the positive reaction field of 0% or more and less than 90% as a section to be used for deriving a concentration. The analysis system according to any one of the preceding claims.
An analytical method for analyzing the concentration of an analyte in a sample, comprising:
A size information acquisition step of acquiring information on the size of each of the plurality of reaction fields, for the plurality of reaction fields generated by dividing the liquid containing the sample;
An analyte information acquisition step of acquiring information on the presence of the analyte in each of the plurality of reaction sites;
The distribution of the size of the reaction field was divided into a plurality of sections, and for each section, information on the number of positive reaction fields which were reaction sites where the analyte was detected, and the analyte were not detected. Distribution data including at least one piece of information selected from the group consisting of information on the number of negative reaction sites that are reaction sites, based on the information acquired in the size information acquiring step and the analyte information acquiring step Distribution data generation process to be generated;
Of the distribution data, the number or proportion of the positive reaction field or the number or proportion of the negative reaction field is based on data of a part of the plurality of sections included in a predetermined range, And D. a concentration deriving step of deriving the concentration of the analyte in the sample.
Information on the size of each of the plurality of reaction fields related to the plurality of reaction fields generated by dividing the liquid containing the sample containing the analyte on a computer, and the analyte in each of the plurality of reaction fields A program for executing processing of detection data including information on the existence of
The process is
The distribution of the size of the reaction field was divided into a plurality of sections, and for each section, information on the number of positive reaction fields which were reaction sites where the analyte was detected, and the analyte were not detected. A distribution data generation step of generating from the detection data distribution data including at least one information selected from the group consisting of information on the number of negative reaction sites that are reaction sites;
Of the distribution data, the number or proportion of the positive reaction field or the number or proportion of the negative reaction field is based on data of a part of the plurality of sections included in a predetermined range, And D. a concentration deriving step of deriving the concentration of the analyte in the sample.
A computer readable storage medium storing the program according to claim 18.
An analysis system for analyzing the concentration of an analyte in a sample, comprising:
A size information acquisition unit for acquiring information on the size of each of the plurality of reaction fields, for a plurality of reaction fields generated by dividing the liquid containing the sample;
An analyte information acquisition unit that acquires information on the presence of the analyte in each of the plurality of reaction sites;
The distribution of the size of the reaction field was divided into a plurality of sections, and for each section, information on the number of positive reaction sites which were reaction sites where the analyte was detected, and the analyte were not detected Distribution data including at least one piece of information selected from the group consisting of information on the number of negative reaction sites that are reaction sites, based on the information acquired by the size information acquiring unit and the analyte information acquiring unit A distribution data generation unit to generate
A data processing unit for processing the distribution data based on at least one piece of information selected from the group consisting of information on the number of positive reaction sites and information on the number of negative reaction sites;
An analysis system, comprising: a concentration deriving unit that derives the concentration of the analyte in the sample based on the distribution data processed by the data processing unit.