JP7317311B2 - Library preparation device - Google Patents

Description

The present invention relates to a device for library preparation.

2. Description of the Related Art In the technology of decoding DNA base sequences, next-generation sequencers (NGS) are widely used for genetic testing and the like because they can acquire a large amount of base sequence data from DNA extracted from specimen samples. In particular, in recent years, there has been a flourishing of research to detect extremely small amounts of samples with high accuracy using next-generation sequencers.

For example, in order to accurately quantify the 16S rRNA gene of microorganisms, an internal standard nucleic acid sample has been proposed that can be amplified with primers that amplify the 16S rRNA gene, but has a base sequence that is clearly distinguishable from the 16S rRNA gene ( For example, see Patent Document 1).

An object of the present invention is to provide a nucleic acid analysis method that enables easy and highly accurate analysis even if the number of nucleic acids to be analyzed is extremely small.

The nucleic acid analysis method of the present invention as a means for solving the above problems comprises a library preparation step of preparing a library containing a standard nucleic acid of a specific copy number and a nucleic acid to be analyzed in the same system; a calibration curve data creating step of creating calibration curve data based on the number of copies of the standard nucleic acid; and specifying the base sequence of the nucleic acid to be analyzed and the number of base sequences of the nucleic acid to be analyzed using the calibration curve data. and an analysis target nucleic acid analysis step for specifying.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a nucleic acid analysis method that enables easy and highly accurate analysis even if the number of nucleic acids to be analyzed is extremely small.

FIG. 1A is a perspective view showing an example of the device of the present invention. FIG. 1B is a perspective view showing another example of the device of the present invention. FIG. 2 is a side view showing an example of the device of the present invention. FIG. 3 is a diagram showing an example of positions of wells filled with nucleic acids in the device of the present invention. FIG. 4 is a diagram showing another example of positions of wells filled with nucleic acids in the device of the present invention. FIG. 5 is a graph showing an example of the relationship between the frequency of DNA-replicated cells and fluorescence intensity. FIG. 6A is a schematic diagram showing an example of an electromagnetic valve type ejection head. FIG. 6B is a schematic diagram showing an example of a piezo ejection head. FIG. 6C is a schematic diagram of a modification of the piezoelectric ejection head in FIG. 6B. FIG. 7A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. FIG. 7B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 8A is a schematic diagram showing an example of the state of droplets. FIG. 8B is a schematic diagram showing an example of the state of droplets. FIG. 8C is a schematic diagram showing an example of the state of droplets. FIG. 9 is a schematic diagram showing an example of a dispensing device for causing droplets to land sequentially in wells. FIG. 10 is a schematic diagram showing an example of a droplet forming device. FIG. 11 is a diagram illustrating hardware blocks of control means of the droplet forming apparatus of FIG. 12 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. 10. FIG. FIG. 13 is a flow chart showing an example of the operation of the droplet forming device. FIG. 14 is a schematic diagram showing a modification of the droplet forming device. FIG. 15 is a schematic diagram showing another modification of the droplet forming device. FIG. 16A is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 16B is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 17 is a diagram illustrating the relationship between the luminance value Li when particles do not overlap each other and the luminance value Le that is actually measured. FIG. 18 is a schematic diagram showing another modification of the droplet forming device. FIG. 19 is a schematic diagram showing another example of the droplet forming device. FIG. 20 is a schematic diagram showing an example of a method of counting cells that have passed through the microchannel. FIG. 21 is a schematic diagram showing an example of a method of acquiring an image near the nozzles of the ejection head. FIG. 22 is a graph showing the relationship between the probability P (>2) and the average number of cells. FIG. 23 is a graph showing the relationship between the number of copies with variations based on the Poisson distribution and the coefficient of variation CV. FIG. 24 is a block diagram showing an example of the hardware configuration of a nucleic acid analyzer. FIG. 25 is a diagram showing an example of the functional configuration of a nucleic acid analyzer. FIG. 26 is a flowchart showing an example of nucleic acid analysis program processing. FIG. 27 is a graph showing an example of the results of the example. FIG. 28 is a graph showing another example of the results of the example. FIG. 29 is a graph showing another example of the results of the example.

(Nucleic acid analysis method and nucleic acid analysis program)
The nucleic acid analysis method of the present invention includes a library preparation step of preparing a library containing a standard nucleic acid with a specific copy number and a nucleic acid to be analyzed in the same system, and calibration based on the copy number of the standard nucleic acid with a specific copy number. a calibration curve data creation step of creating line data; an analysis target nucleic acid analysis step of specifying the base sequence of the analysis target nucleic acid and specifying the number of base sequences of the analysis target nucleic acid using the calibration curve data; and optionally other steps.
The nucleic acid analysis program of the present invention generates a specific number of copies of a standard nucleic acid and a nucleic acid to be analyzed in a library prepared in the same system by means of a calibration curve data creation means. Based on the number, the standard curve data of the standard nucleic acid is created, the base sequence of the analysis target nucleic acid is specified by the analysis target nucleic acid analysis means, and the number of base sequences of the analysis target nucleic acid using the calibration curve data is specified, the computer is caused to execute processing, and other processing is executed as necessary.
The nucleic acid analysis method can be suitably performed by a nucleic acid analysis apparatus according to the nucleic acid analysis method, the library preparation step can be suitably performed by the library preparation means, and the calibration curve data acquisition step can be suitably performed by the calibration curve data acquisition means. The analysis target nucleic acid analysis step can be preferably carried out by the analysis target nucleic acid analysis means, and the other steps can be carried out by other means.

The inventors of the present invention have studied a nucleic acid analysis method that enables easy and highly accurate analysis even if the number of nucleic acids to be analyzed is very small and of a plurality of types, and obtained the following findings.
In the prior art, a sample with a known concentration is a value obtained by measuring the concentration of the nucleic acid itself, and when used, a standard sample for quantitative analysis was prepared by serially diluting it. In the case of an extremely small amount of standard sample, there is no guarantee that a diluted solution with the desired copy number is accurately prepared, and it has been difficult to perform accurate quantification when the amount of nucleic acid to be analyzed is extremely small. The present invention is based on the above findings.
Furthermore, with respect to the internal standard gene for quantifying the 16S rRNA gene of microorganisms, it is based on the finding that it is unclear whether or not accurate quantification can be performed when the amount of nucleic acid to be analyzed is very small.

The nucleic acid analysis apparatus according to the nucleic acid analysis method of the present invention operates as an apparatus for executing the nucleic acid analysis method of the present invention by reading and executing the nucleic acid analysis program of the present invention. That is, the nucleic acid analysis apparatus according to the nucleic acid analysis method of the present invention has the nucleic acid analysis program of the present invention that causes a computer to perform the same functions as the nucleic acid analysis method of the present invention. The nucleic acid analysis program of the present invention is not limited to being executed by the nucleic acid analysis apparatus according to the nucleic acid analysis method of the present invention. For example, the nucleic acid analysis program of the present invention may be executed by another computer or server, and any of the nucleic acid analysis apparatus, other computer, and server according to the nucleic acid analysis method of the present invention may be executed in cooperation. may
In other words, the nucleic acid analysis apparatus according to the nucleic acid analysis method of the present invention is synonymous with carrying out the nucleic acid analysis method of the present invention. Details of the nucleic acid analyzer will also be clarified. In addition, since the nucleic acid analysis program of the present invention is realized as a nucleic acid analysis method according to the nucleic acid analysis method of the present invention by using a computer or the like as hardware resources, the present invention can be understood through the explanation of the nucleic acid analysis method of the present invention. The details of the nucleic acid analysis program will also be clarified.

<Library Preparation Step and Library Preparation Means>
The library preparation step is a step of preparing a library by arranging a standard nucleic acid with a specific number of copies and a nucleic acid to be analyzed in the same system. The library preparation step is preferably performed by library preparation means.

A library means a library containing nucleic acids obtained by treating nucleic acids to be analyzed so that nucleic acid analysis is possible. The number of nucleic acids to be analyzed contained in the library is preferably 1 or more, more preferably 2 or more. When the number of nucleic acids to be analyzed contained in the library is two or more, it can be suitably used, for example, in environmental surveys for identifying species.

A standard nucleic acid means a nucleic acid of a specific copy number used for obtaining calibration curve data, which will be described later, in nucleic acid analysis. Analysis means identifying the base sequence and identifying the number of copies for each base sequence. Note that the specific copy number will be described in detail later in the description of the device used in the nucleic acid analysis method of the present invention, so the description is omitted.
The nucleic acid to be analyzed means a nucleic acid (nucleotide sequence) to be analyzed, which is a sample. may be used, or two or more may be used in combination. Moreover, the number thereof is not particularly limited, and can be appropriately selected according to the purpose. One type may be used alone, or two or more types may be used in combination.
The nucleic acid to be analyzed is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include DNA, RNA and cDNA. The nucleic acid to be analyzed may contain two or more nucleic acids (fragments) having different base sequences.

There is no particular limitation on treating the nucleic acid to be analyzed so that it is ready for nucleic acid analysis, and it can be appropriately selected depending on the purpose. .

The treatment for binding the adapter sequence is not particularly limited and can be appropriately selected depending on the purpose. A treatment for binding an oligonucleotide to at least one of the 5′ end and the 3′ end of the nucleic acid to be analyzed, a treatment for binding a peptide or protein, and the like are included.
The treatment for binding oligonucleotides is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include a method of preparing a library using different primers for the nucleic acid and the nucleic acid to be analyzed. By using the method of preparing the library using the same primers for the standard nucleic acid and the nucleic acid to be analyzed, the difference in amplification efficiency can be almost ignored. Furthermore, by using a method of preparing a library using different primers for the standard nucleic acid and the nucleic acid to be analyzed, primers can be selected without depending on the base sequence of the nucleic acid to be analyzed, so versatility is improved. can be improved.
Treatments for binding other oligonucleotides include, for example, a method using a transposon, a method using a ligase, a method using homologous recombination, and the like. As a treatment for binding other oligonucleotides, for example, http://www. epibio. com/docs/default-source/forum-archive/forum-16-3---nextera-technology-for-ngs-dna-library-preparation---simultaneous-fragmentation-and-tagging-by-in- in vitro transportation. pdf? The method described in sfvrsn=4 and the like can be preferably used.

The treatment for binding peptides or proteins is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include Rapid Sequencing Kit of MinION (Oxford Nanopore Technologies).
As processing for binding peptides or proteins, for example, https://store. nanopore tech. com/catalog/product/view/id/219/s/rapid-sequencing-kit/category/28/ and the like can be preferably used.
The adapter sequence is not particularly limited and can be appropriately selected depending on the purpose.

The treatment for nucleic acid amplification is not particularly limited as long as it can amplify a specific base sequence of interest (for example, a gene) among the nucleic acids to be analyzed contained in the sample, and can be appropriately selected according to the purpose. can do.

In the nucleic acid analysis method of the present invention, since the number of standard nucleic acids is specified by amplifying the standard nucleic acid and the nucleic acid to be analyzed in the same system, the reliability of the results of the nucleic acid to be analyzed can be improved. .
Here, when the standard nucleic acid and the nucleic acid to be analyzed are included in the same system, a mode in which standard nucleic acids having different base sequences are included in the same system and a mode in which standard nucleic acids having the same base sequence are included in different systems. are mentioned.
In the embodiment in which standard nucleic acids having different base sequences are included in the same system, the standard nucleic acids having different base sequences are included in the same system in specific copy numbers different from each other, that is, two or more types of standard nucleic acids are included in the same system. It means that the nucleic acids are contained in specific copy numbers different from each other, and the reliability of the analysis result of the nucleic acid to be analyzed can be improved. In the same system, including a specific copy number different from each other means, for example, in A, B, and C, which are base sequences different from each other, the base sequence A is 1 (copy) and the base sequence B is 10 (copies). , 3 levels of 50 (copies) of base sequence C are amplified in the same system. "Level" means that when a certain specific copy number is "1", another specific copy number is "10", and another specific copy number is "50", it is expressed as "3 levels". means.
In an aspect in which standard nucleic acids having the same base sequence are included in different systems, standard nucleic acids having the same base sequence are used, that is, a system exists for each level of the standard nucleic acid (for each specific number of copies), and each It means that the same nucleic acid to be analyzed is contained within the system. By using standard nucleic acids having the same base sequence, the number of types of standard nucleic acids to be used can be reduced.

The library preparation process is not particularly limited and can be appropriately selected according to the purpose. -u.ac.jp/news/NGS1.pdf), Non-Patent Document 1, Analysis Method in Sequence Using Nanopore Device (Oxford Nanopore Technologies.), Analysis Method in Sequence Using PacBio RS II/Sequel System (Pacific BioSciences, Inc.), analysis methods in the Ion Torrent ^™ semiconductor sequence system series (ThermoFisher SCIENTIFIC, Inc.), and the like can be referred to.

Here, the fact that the specific copy number of the standard nucleic acid is specified in the nucleic acid analysis method of the present invention will be described in detail. The nucleic acid analysis method of the present invention is premised on using a device in which a specific copy number of a standard nucleic acid is specified.

-device-
The device used in the nucleic acid analysis method of the present invention has at least one filling site, and contains a specific number of copies of the standard nucleic acid in at least one filling site.

By using the device used in the nucleic acid analysis method of the present invention, even if the number of nucleic acids to be analyzed is extremely small, it is possible to analyze (quantify) them with high accuracy. In the present invention, the term "extremely small amount" means "extremely small amount" of nucleic acids, for example, 1,000 or less.

A specific copy number means the number of target or specific nucleic acids (or base sequences) in the standard nucleic acid contained in the filling site.
The target base sequence refers to at least the base sequence of the primer region that has been determined, and in particular, the base sequence of which the full length has been determined is also referred to as a specific base sequence.
A specific number means that the number of target nucleic acids (base sequences) among the number of nucleic acids (base sequences) is specified with a certain or more accuracy.
That is, it can be said that it is known as the number of target nucleic acids (base sequences) actually contained in the filling site. That is, the specific copy number in the present application has higher accuracy and reliability as a number than the predetermined number (calculated estimated value) obtained by conventional serial dilution, is also a controlled value independent of the Poisson distribution. The controlled value is that the coefficient of variation CV, which represents the uncertainty, falls within the magnitude of either CV < 1/√x or CV ≤ 20% with respect to the average specific copy number x. is preferred. Therefore, by using a device having a filling site containing the specific copy number of the target nucleic acid (base sequence), the sample having the target nucleic acid (base sequence) can be tested more accurately than before qualitatively and quantitatively. It is possible to do
Here, when the number of copies for each base sequence of the target and the number of molecules of the nucleic acid having that sequence match, the "specific number of copies" may be associated with the "number of molecules".
Specifically, for example, in the case of norovirus, if the number of viruses = 1, the number of nucleic acid molecules = 1 and the number of copies = 1. In the case of yeast in the GI stage, if the number of yeast = 1, the number of nucleic acid molecules (same chromosome number) = 1, copy number = 1, and in the case of G0/GI phase human cells, if the human cell number = 1, then the nucleic acid molecule number (identical chromosome number) = 2, copy number = 2.
Furthermore, in the case of yeast in the GI stage into which the target base sequence has been introduced at two sites, if the number of yeast = 1, the number of nucleic acid molecules (the number of identical chromosomes) = 1 and the number of copies = 2.
In addition, in the present invention, the specific copy number of a nucleic acid is also referred to as the predetermined number or absolute number of the nucleic acid.
The specific copy number of the nucleic acid is preferably 1 (copy) or more and 1,000 (copy) or less, preferably 100 (copy) or less, more preferably 20 (copy) or less, and even more preferably 10 (copy) or less.
The specific copy number of a nucleic acid is preferably two or more different integers.
Combinations of specific copy numbers of nucleic acids include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 3, 5, 7, 9, 2, 4, 6, 8, 10 and the like.
Further, the combination of specific copy numbers of nucleic acids may be, for example, 1, 10, 50, 100, and 500 in the case of four levels of 1, 10, 100, and 1,000. A calibration curve can be prepared using the device in the nucleic acid analysis method of the present invention that combines a plurality of different specific copy numbers.
Filling sites containing multiple different specific copy numbers of nucleic acid may be the same or different. However, when there are a plurality of filling sites containing nucleic acids, it is necessary to add the same nucleic acid to be analyzed to each filling site.

The method of disposing the library prepared in the library preparation step in a device having a specific number of copies of the standard nucleic acid is not particularly limited, and can be appropriately selected according to the purpose. It is preferable to fill a specified amount of multiple levels of solutions/dispersions prepared by serial dilution of the above, or to fill based on counting the number of microregions and carriers in which the number of nucleic acid molecules in the microregions is known. . For these selections, it is preferable to select the best method according to the filling accuracy and filling time required for each level. The uncertainty assigned to each filling site is preferably calculated appropriately by the above-described filling method or serial dilution preparation method.

The information on the specific copy number of the nucleic acid is not particularly limited as long as it is information on the nucleic acid in the device, and can be appropriately selected according to the purpose. information, etc.

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

Furthermore, in the device of the present invention, the coefficient of variation of the nucleic acid loaded into the loading site may be used as uncertainty information.
The coefficient of variation means the relative value of the variation in the number of nucleic acids filled in each recess that occurs when nucleic acids are filled into the recesses. That is, the coefficient of variation means the filling accuracy of the number of nucleic acids filled in the recesses. The coefficient of variation is a value obtained by dividing the standard deviation σ by the average x of the number of nucleic acids. Here, if the coefficient of variation CV is the value obtained by dividing the standard deviation σ by the average number of copies of the number of nucleic acids (average number of filled copies) x, the relational expression of Equation 1 below is obtained.

In general, nucleic acids are randomly distributed in a Poisson distribution in a dispersion. Therefore, in the serial dilution method, that is, in the state of random distribution in the Poisson distribution, the standard deviation σ can be considered to satisfy the relational expression of the following formula 2 with the average copy number x of the number of nucleic acids. From this, when the nucleic acid dispersion is diluted by the serial dilution method, the coefficient of variation CV (CV value) of the average number of nucleic acid copies x is calculated from the standard deviation σ and the average copy number x of the number of nucleic acids by the above formula. 3 derived from 1 and 2, the results are shown in Table 1 and FIG. Note that the CV value of the copy number variation coefficient having variation based on the Poisson distribution can be obtained from FIG.

From the results of Table 1 and FIG. 23, for example, when 100 (copy number) nucleic acids are filled in the filling site by a serial dilution method, the average of the standard nucleic acid (base sequence) finally filled in the reaction solution It can be seen that the copy number has a coefficient of variation (CV value) of at least 10%, ignoring other accuracies.

As the specific copy number of the nucleic acid, the CV value of the coefficient of variation and the average specific copy number x of the nucleic acid preferably satisfy the following formula, CV < 1/√x, and CV < 1/2 √x. is more preferred.

As for the uncertainty information, it is preferable to have uncertainty information for the device as a whole, based on the specific number of copies of the nucleic acid contained in the filling sites, where there are multiple wells containing the nucleic acid.

There are several factors that cause uncertainty. (including the inkjet device, or the results of the operation of each part of the device such as the timing of the operation of the device), the frequency with which the cells are placed in the proper position of the device, the cell breakage in the cell suspension. Contamination (contamination of contaminants, hereinafter sometimes referred to as "contamination") due to contamination of the cell suspension with nucleic acids due to the addition of the cell suspension.

Information on nucleic acids includes, for example, information on the number of nucleic acids, including information on the uncertainty of the number of nucleic acids contained in the device.

<Filling part>
There are no particular restrictions on the shape, number, volume, material, color, etc. of the filling sites, and they can be appropriately selected according to the purpose. A fill site is sometimes treated synonymously with a well.
The shape of the filling site is not particularly limited as long as nucleic acid or the like can be placed thereon, and can be appropriately selected according to the purpose. divisions, etc. The shape of the filling site preferably matches the shape of a mold for a general thermal cycler.
The number of filling sites is at least one, preferably two or more, more preferably five or more, and still more preferably fifty or more.
For example, a PCR tube or the like can be given as an example of a tube having one filling site.
A multi-well plate, for example, is suitably used as one having two or more filling sites.
Multiwell plates include, for example, 24, 48, 96, 384, or 1,536 well plates.
The volume of the filling site is not particularly limited and can be appropriately selected according to the purpose. Considering the sample volume used in a general nucleic acid testing apparatus, for example, it is preferably 1 μL or more and 1,000 μL or less.
The material of the filling portion is not particularly limited and can be appropriately selected according to the purpose. mentioned.
Examples of the color of the filling site include transparent, translucent, colored, and completely shaded.
The wettability of the filled portion is not particularly limited and can be appropriately selected according to the purpose. For example, it is preferably water repellent. When the wettability of the filling site is water repellent, adsorption of nucleic acid to the inner wall of the filling site can be reduced. In addition, when the wettability of the filling site is water repellent, the nucleic acid, primer, and amplification reagent in the filling site can move in a solution state.
The method for imparting water repellency to the inner wall of the filling portion is not particularly limited and can be appropriately selected depending on the intended purpose. In particular, by applying a water-repellent treatment that provides a contact angle of 100° or more, it is possible to reduce the risk of a decrease in nucleic acids and an increase in uncertainty (or coefficient of variation) due to spillage of the liquid.

<Base material>
The device is preferably plate-shaped with filling sites provided on a substrate, but may be a combination of connected well tubes such as octet tubes and unconnected wells.
The material, shape, size, structure, etc. of the substrate are not particularly limited, and can be appropriately selected according to the purpose.
The material of the substrate is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include semiconductors, ceramics, metals, glass, quartz glass, and plastics. Among these, plastics are preferred.
Examples of plastics include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
The shape of the substrate is not particularly limited and can be appropriately selected depending on the intended purpose.
The structure of the base material is not particularly limited and can be appropriately selected depending on the intended purpose. For example, it may be a single layer structure or a multi-layer structure.

<Identification Means>
Preferably, the device has identification means capable of identifying the specific copy number of the nucleic acid and the uncertainty information thereof.
The identification means is not particularly limited and can be appropriately selected according to the purpose. ), color coding, printing, etc.
The positions at which the identification means are provided and the number of identification means are not particularly limited, and can be appropriately selected according to the purpose.
Information to be stored in the identification means includes, in addition to information on the specific copy number of nucleic acid and its uncertainty, for example, analysis results (activity value, luminescence intensity, etc.), number of nucleic acids (e.g., number of cells), number of cells Life and death, the number of copies of a specific nucleotide sequence, which filling site of a plurality of filling sites is filled with nucleic acid, the type of nucleic acid, the date and time of measurement, the name of the measurer, and the like.
Information stored in the identification means can be read using various reading means. For example, if the identification means is a barcode, a barcode reader is used as the reading means.

The method of writing information in the identification means is not particularly limited, and can be appropriately selected according to the purpose. Examples include a method of writing data directly from the device, transfer of data stored in a server, transfer of data stored in the cloud, and the like.

<Other parts>
The other member is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a sealing member.

-sealing member-
The device preferably has a sealing member to prevent contamination of the filling site, leakage of the filling material, and the like.
It is preferable that the sealing member is configured to be capable of sealing at least one filling site and to be separable by a perforation line so that each filling site can be individually sealed or unsealed.
As for the shape of the sealing member, it is preferable to have a cap-like shape that matches the inner wall diameter of the filling site, or a film-like shape that covers the well opening.
Examples of materials for the sealing member include polyolefin resin, polyester resin, polystyrene resin, and polyamide resin.
The sealing member is preferably in the form of a film that can seal all the filled portions at once. In addition, it is preferable that the adhesive strength is different between a filling portion that requires re-opening and an unnecessary filling portion so that misuse by the user can be reduced.

The filling site preferably contains at least one of primers and amplification reagents.
Primers are synthetic oligonucleotides having 18 to 30 base complementary base sequences specific to the template DNA in the polymerase chain reaction (PCR). One place (pair) is set.
Examples of amplification reagents in polymerase chain reaction (PCR) include DNA polymerase as an enzyme, four types of bases (dGTP, dCTP, dATP, dTTP) as substrates, Mg ²⁺ (2 mM magnesium chloride), and optimum pH (pH 7.0). 5 to 9.5), and the like.

Preferably, the device has a negative control loading site with 0 copies of the nucleic acid and a positive control loading site with 10 or more copies of the nucleic acid.
When detection is detected in the negative control and when non-detection is detected in the positive control, it is suggested that there is an abnormality in the detection system (reagent or device). By providing a negative control and a positive control, the user can immediately notice when a problem occurs, stop the measurement, and check where the problem lies.

The states of the nucleic acids, primers, and amplification reagents in the filling site are not particularly limited, and can be appropriately selected according to the purpose. For example, they may be in either a solution or solid state. From the viewpoint of usability, it is particularly preferable to be in a solution state. Being in solution, it is ready for testing by the user. From the viewpoint of transportation, the solid state is particularly preferable, and the dry state is more preferable. In the solid dry state, the reaction rate of decomposition of reagents that can be used as amplification reagents by degrading enzymes or the like can be reduced, and the storage stability of nucleic acids, primers, and amplification reagents can be improved.
In addition, it is desirable that the solid dry device is filled with appropriate amounts of nucleic acids, primers, and amplification reagents so that it can be used immediately as a reaction solution by dissolving it in a buffer or water immediately before use. .
The drying method is not particularly limited and can be appropriately selected depending on the intended purpose. is mentioned.

Here, FIG. 1A is a perspective view showing an example of a device (also referred to as a nucleic acid sample filling container) 1 according to the nucleic acid analysis method of the present invention. FIG. 1B is a perspective view showing another example of the device 1 according to the nucleic acid analysis method of the present invention. FIG. 2 is a side view of the device 1 of FIG. 1B. In the device 1, a plurality of filling sites (wells) 3 are provided on a base material 2, and the inside of the filling sites (well) 3 (the inner space surrounded by the wall surfaces of the filling sites (wells) constituting the filling sites (wells) region) is filled with a specific copy number of nucleic acid 4 as a nucleic acid (sometimes referred to as a nucleic acid sample filling region). This device 1 is associated with a specific copy number of a nucleic acid and information about the uncertainty of the specific copy number of said nucleic acid. 1B and 2 show an example in which the device 1 covers the opening of the filling site (well) 3 with the sealing member 5. FIG.
For example, as shown in FIGS. 1B and 2, information on the number of reagents to be filled in each filling site (well) 3 and the uncertainty (probability) of the number, or information associated with these information is stored. An IC chip or bar code (identification means 6) is arranged between the sealing member 5 and the substrate 2 and at a position other than the opening of the filling site (well). This is suitable for preventing unintended modification of the identification means.
In addition, the device having the identification means can be distinguished from a general filling site (well) plate having no identification means. Therefore, it is possible to prevent mix-ups.

FIG. 3 is a diagram showing an example of positions of filling sites (wells) for filling nucleic acids in a device according to the nucleic acid analysis method of the present invention. The numbers within the filling sites (wells) in FIG. 3 represent the specific copy number of the nucleic acid. Filling sites (wells) not numbered in FIG. 3 are filling sites (wells) for sample and control measurements.
FIG. 4 is a diagram showing another example of positions of filling sites (wells) for filling nucleic acids in the device according to the nucleic acid analysis method of the present invention. The numbers within the filling sites (wells) in FIG. 4 represent the specific copy number of the nucleic acid. Filling sites (wells) not numbered in FIG. 4 are filling sites (wells) for sample and control measurements.

-nucleic acid-
Nucleic acid means a macromolecular organic compound in which nitrogen-containing bases derived from purines or pyrimidines, sugars, and phosphoric acids are regularly bound, and also includes fragments of nucleic acids, analogs of these nucleic acids or fragments thereof, and the like. .
Nucleic acids are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include DNA, RNA, cDNA and the like.

The nucleic acid or nucleic acid fragment may be a natural product obtained from an organism or a processed product thereof, a product produced using genetic recombination technology, or an artificial synthetic nucleic acid chemically synthesized. good. These may be used individually by 1 type, and may use 2 or more types together. By using an artificially synthesized nucleic acid, impurities can be reduced and the molecular weight can be reduced, so that the initial reaction efficiency can be improved.
The term "artificially synthesized nucleic acid" refers to a nucleic acid obtained by artificially synthesizing a nucleic acid composed of components (bases, deoxyribose, phosphoric acid) similar to those of naturally occurring DNA or RNA. Artificially synthesized nucleic acids include, for example, not only nucleic acids having base sequences encoding proteins, but also nucleic acids having arbitrary base sequences.

Nucleic acid or nucleic acid fragment analogs include nucleic acids or nucleic acid fragments bound to non-nucleic acid components, and nucleic acids or nucleic acid fragments labeled with fluorescent dyes, isotopes, and other labeling agents (e.g., fluorescent dyes, radioactive isotopes, primers and probes labeled with ), and artificial nucleic acids (eg, PNA, BNA, LNA, etc.) in which the chemical structure of some of the nucleotides constituting the nucleic acid or nucleic acid fragment is changed.

The form of the nucleic acid is not particularly limited and can be appropriately selected according to the purpose. Examples include double-stranded nucleic acids, single-stranded nucleic acids, partially double-stranded or single-stranded nucleic acids, Circular or linear plasmids can also be used.
Nucleic acids may also be modified or mutated.

The nucleic acid preferably has a target base sequence.
The target nucleotide sequence is not particularly limited and can be appropriately selected according to the purpose. sequences, plant cell-derived base sequences, fungal cell-derived base sequences, bacterium-derived base sequences, virus-derived base sequences, and the like. These may be used individually by 1 type, and may use 2 or more types together.
When a non-natural base sequence is used, the GC content is preferably 30% or more and 70% or less of the target base sequence, and the GC content is preferably constant (see, for example, SEQ ID NO: 6).
The base length of the target base sequence is not particularly limited and can be appropriately selected depending on the purpose. is mentioned.
When using a nucleotide sequence used for infectious disease testing, there is no particular restriction as long as it contains a nucleotide sequence specific to that infectious disease, and it can be selected as appropriate according to the purpose. It preferably contains a nucleotide sequence that is

The nucleic acid may be a nucleic acid derived from the cells used, or a nucleic acid introduced by gene transfer. When a nucleic acid introduced by gene transfer or a plasmid is used as the nucleic acid, it is preferable to confirm that one copy of the nucleic acid has been introduced into one cell. The method for confirming that one copy of the nucleic acid has been introduced is not particularly limited and can be appropriately selected according to the purpose. can.

The type of nucleic acid having a target base sequence to be introduced by gene transfer may be one type or two or more types. Even when the number of nucleic acids to be introduced by gene introduction is one, similar base sequences may be introduced in tandem depending on the purpose.

The method of gene introduction is not particularly limited as long as the target copy number of a specific nucleic acid sequence can be introduced into the target location, and can be appropriately selected according to the purpose. Cpf1, TALEN, zinc finger nuclease, Flip-in, Jump-in and the like. Among these, in the case of yeast, homologous recombination is preferable from the viewpoint of high efficiency and ease of control.

-Carrier-
Nucleic acids are preferably handled while being carried on a carrier. When the nucleic acid is a nucleic acid, it is preferable that the nucleic acid is supported (more preferably included) in a particle-shaped carrier (carrier particles).
The carrier is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include cells, resins, liposomes, microcapsules, metal particles, magnetic particles, ceramic particles, polymer particles, protein particles and the like. .

--cell--
Cells refer to structural and functional units that contain nucleic acids (eg, nucleic acids) and form an organism.
Cells are not particularly limited and can be appropriately selected according to the purpose. For example, all cells can be used regardless of whether they are eukaryotic cells, prokaryotic cells, multicellular biological cells, or unicellular biological cells. . These may be used individually by 1 type, and may use 2 or more types together.

Eukaryotic cells are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, fungi, algae, and protozoa. These may be used individually by 1 type, and may use 2 or more types together. Among these, animal cells and fungi are preferred.

Adhesive cells may be primary cells directly collected from a tissue or organ, or primary cells directly collected from a tissue or organ that have been subcultured for several generations. Examples include differentiated cells and undifferentiated cells.

Differentiated cells are not particularly limited and can be appropriately selected according to the purpose. For example, hepatocytes, which are parenchymal cells of the liver; Endothelial cells such as cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; Epithelial cells such as epithelial cells; breast cells; pericytes; muscle cells such as smooth muscle cells and myocardial cells; renal cells; .

The undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. Unipotent stem cells such as vascular endothelial progenitor cells having unipotency; iPS cells and the like.

The fungus is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include molds, yeasts, and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, yeast is preferable because it can regulate the cell cycle and can use a haploid.
The cell cycle means a process in which cell division occurs when cells multiply, and cells (daughter cells) produced by cell division become cells (mother cells) that undergo cell division again to produce new daughter cells.

The yeast is not particularly limited and can be appropriately selected depending on the intended purpose. For example, yeast that is synchronously cultured in synchronization with the G0/G1 phase and fixed in the G1 phase is preferred.
As the yeast, Bar-1-deficient yeast, which has increased sensitivity to pheromones (sex hormones) that regulate the cell cycle in the G1 phase, is preferred. When the yeast is Bar-1-deficient yeast, the abundance of yeast whose cell cycle is not controlled can be reduced, so the number of specific nucleic acids of cells accommodated in the filling site (well) increase can be prevented.

Prokaryotic cells are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include eubacteria and archaea. These may be used individually by 1 type, and may use 2 or more types together.

Dead cells are preferred as the cells. Dead cells can prevent cell division after collection.
Cells are preferably cells that can emit light when receiving light. If the cells are capable of emitting light when receiving light, it is possible to control the number of cells with high precision and land them in the filling site (well).
Receiving means receiving light.
An optical sensor uses a lens to collect visible light that can be seen by the human eye, near-infrared rays with longer wavelengths, short-wave infrared rays, and light up to the thermal infrared region, and then picks up the target cells. It means a passive sensor that acquires the shape of the object as image data.

--Cells that can emit light upon receiving light--
Cells that can emit light when receiving light are not particularly limited as long as they can emit light when receiving light, and can be appropriately selected depending on the purpose. Protein-expressing cells, cells labeled with fluorescent-labeled antibodies, and the like are included.
A site stained with a fluorescent dye, a site expressing a fluorescent protein, or a site labeled with a fluorescent-labeled antibody in a cell is not particularly limited, and includes whole cells, cell nuclei, cell membranes, and the like.

--Fluorescent dye--
Examples of fluorescent dyes include fluoresceins, azos, rhodamines, coumarins, pyrenes, cyanines and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, fluoresceins, azos, and rhodamines are preferred, and eosin, Evans blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferred.

Commercially available products can be used as fluorescent dyes, and commercial products include, for example, trade name: EosinY (manufactured by Wako Pure Chemical Industries, Ltd.), trade name: Evans Blue (manufactured by Wako Pure Chemical Industries, Ltd.), commercial products Name: Trypan Blue (manufactured by Wako Pure Chemical Industries, Ltd.), Trade Name: Rhodamine 6G (manufactured by Wako Pure Chemical Industries, Ltd.), Trade Name: Rhodamine B (manufactured by Wako Pure Chemical Industries, Ltd.), Trade Name: Rhodamine 123 (manufactured by Wako Pure Chemical Industries, Ltd.) manufactured by Wako Pure Chemical Industries, Ltd.).

--fluorescent protein--
Examples of fluorescent proteins include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP , Venus , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Turb oFP602, mRFP1, JRed, KillerRed, mCherry, mPlum, PS - CFP, Dendra2, Kaede, EosFP, KikumeGR, etc. These may be used individually by 1 type, and may use 2 or more types together.

--Fluorescent-labeled antibody--
The fluorescently labeled antibody is not particularly limited as long as it is fluorescently labeled, and can be appropriately selected depending on the purpose. Examples include CD4-FITC and CD8-PE. These may be used individually by 1 type, and may use 2 or more types together.

The volume-average particle size of cells in a free state is preferably 30 μm or less, more preferably 10 μm or less, and particularly preferably 7 μm or less. If the volume average particle diameter is 30 μm or less, it can be suitably used for droplet ejection means such as an inkjet method and a cell sorter.

The volume average particle size of cells can be measured, for example, by the following measuring method.
The volume average particle size can be measured by removing 10 μL from the prepared dyed yeast dispersion, placing it on a PMMA plastic slide, and using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen). The number of cells can also be obtained by a similar measurement method.

The concentration of cells in the cell suspension is not particularly limited and can ^be appropriately selected depending on the ^purpose . ⁴ or more/mL and 5×10 ⁷ or less/mL are more preferable. When the cell count is 5×10 ⁴ cells/mL or more and 5×10 ⁸ cells/mL or less, cells can be reliably included in the ejected droplet. The number of cells can be measured using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen) in the same manner as the method for measuring the volume average particle diameter.

The number of nucleic acid-bearing cells is not particularly limited as long as it is plural, and can be appropriately selected according to the purpose.

-resin-
The material, shape, size, and structure of the resin are not particularly limited as long as they can support nucleic acid (for example, nucleic acid), and can be appropriately selected according to the purpose.

-Liposomes-
Liposomes are lipid vesicles formed from lipid bilayers containing lipid molecules. means an enclosed lipid-containing endoplasmic reticulum with an open space.
A liposome is a closed endoplasmic reticulum formed of a lipid bilayer membrane using lipids, and has an aqueous phase (inner aqueous phase) within the space of the closed vesicle. The inner water phase includes water and the like. Liposomes can be single lamellar (unilamellar, unilamellar, single bilayer membrane) or multilamellar (multilamellar, many bilayer membranes with an onion-like structure, each of which is a water-like layer). partitioned).
As the liposome, a liposome capable of encapsulating nucleic acid (for example, nucleic acid) is preferable, and the form thereof is not particularly limited. “Encapsulation” means that the liposome takes a form in which the nucleic acid is contained in the internal aqueous phase and the membrane itself. For example, there are a form in which the nucleic acid is enclosed in a closed space formed by a membrane, a form in which the nucleic acid is enclosed in the membrane itself, and the like, and a combination thereof is also possible.
The size (average particle size) of the liposome is not particularly limited as long as it can encapsulate a nucleic acid (for example, nucleic acid), but it is preferably spherical or nearly spherical.
Components (membrane components) that constitute the lipid bilayer of liposomes are selected from lipids. Any lipid can be used as long as it dissolves in a mixed solvent of a water-soluble organic solvent and an ester organic solvent. Specific examples of lipids include phospholipids, lipids other than phospholipids, cholesterols, derivatives thereof, and the like. These components may consist of a single type or multiple types of components.

－Micro Capsules－
A microcapsule means a minute particle having a wall material and a hollow structure, and can enclose a nucleic acid (for example, a nucleic acid) in the hollow structure.
The microcapsules are not particularly limited, and the wall material, size, etc. can be appropriately selected depending on the purpose.
Examples of wall materials for microcapsules include polyurethane resins, polyurea, polyurea-polyurethane resins, urea-formaldehyde resins, melamine-formaldehyde resins, polyamides, polyesters, polysulfonamides, polycarbonates, polysulfinates, epoxies, acrylic acid esters. , methacrylate, vinyl acetate, and gelatin. These may be used individually by 1 type, and may use 2 or more types together.
The size of the microcapsules is not particularly limited as long as the nucleic acid (for example, nucleic acid) can be encapsulated, and can be appropriately selected according to the purpose.
The method for producing the microcapsules is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include in-situ method, interfacial polymerization method and coacervation method.

Other forms of nucleic acid may be a solution of the aforementioned nucleic acid molecules or a dispersion micro-compartmentalized by micro-domains. The medium for the solution or dispersion is preferably water or a water-soluble solvent such as ethanol, DMSO, acetone, or DMF. Examples of minute domains include droplets and emulsions.

<Device manufacturing method>
A method for manufacturing a device using cells having a specific nucleic acid as the nucleic acid will be described below.
A method for manufacturing a device according to the nucleic acid analysis method of the present invention includes a cell suspension generation step of generating a cell suspension containing a plurality of cells having a specific nucleic acid and a solvent, and a cell suspension as droplets. A droplet landing step in which droplets are sequentially landed in filling sites (wells) of a plate by discharging; including a cell counting step of counting the number of cells contained by a sensor, and a nucleic acid extraction step of extracting nucleic acid from the cells in the filling site (well), a step of calculating the uncertainty of each step, an output step, and a recording It is preferable to include steps, and further include other steps as necessary.

<<Cell suspension generation process>>
A cell suspension production step is a step of producing a cell suspension containing a plurality of cells having a specific nucleic acid and a solvent.
Solvent means the liquid used to disperse the cells.
Suspension in a cell suspension means a state in which cells are dispersed in a solvent.
Generate means to produce.

-cell suspension-
A cell suspension contains a plurality of cells having a specific nucleic acid, a solvent, preferably additives, and optionally other components.
Multiple cells with specific nucleic acids are described above.

--solvent--
The solvent is not particularly limited and can be appropriately selected depending on the intended purpose. Liquids, aqueous electrolyte solutions, aqueous inorganic salt solutions, aqueous metal solutions, mixed liquids thereof, and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, water and buffer solutions are preferable, and water, phosphate-buffered saline (PBS) and Tris-EDTA buffer (TE) are more preferable.

--Additive--
Additives are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include surfactants, nucleic acids, resins and the like. These may be used individually by 1 type, and may use 2 or more types together.

Surfactants can prevent aggregation of cells and improve continuous ejection stability.

The surfactant is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include ionic surfactants and nonionic surfactants. These may be used individually by 1 type, and may use 2 or more types together. Among these surfactants, nonionic surfactants are preferred because they do not denature or inactivate proteins, depending on the amount added.

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

Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (Brij series, etc.), octylphenol ethoxylates (Triton X series, Igepal CA series, Nonidet P series, Nikkol OP series, etc.), polysorbates ( Tween series such as Tween 20), sorbitan fatty acid ester, polyoxyethylene fatty acid ester, alkylmaltoside, sucrose fatty acid ester, glycoside fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, fatty acid monoglyceride, and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, polysorbates are preferred.

The content of the surfactant is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 0.001% by mass or more and 30% by mass or less with respect to the total amount of the cell suspension. When the content is 0.001% by mass or more, the effect of addition of the surfactant can be obtained, and when it is 30% by mass or less, cell aggregation can be suppressed, so the cell suspension The copy number of nucleic acids in a can be tightly controlled.

The nucleic acid is not particularly limited as long as it does not affect the detection of the nucleic acid to be detected, and can be appropriately selected according to the purpose. Examples thereof include ColE1 DNA. If it is a nucleic acid, the nucleic acid having the target base sequence can be prevented from adhering to the wall surface of the filling site (well).

The resin is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include polyethyleneimide.

--Other Materials--
Other materials are not particularly limited and can be appropriately selected depending on the intended purpose. mentioned.

[Method for dispersing cells]
The method for dispersing cells is not particularly limited and can be appropriately selected according to the purpose. method, etc. These may be used individually by 1 type, and may use 2 or more types together. Among these methods, the ultrasonic method is more preferable because it causes little damage to cells. In the media method, the disintegration ability is strong, and the cell membrane and cell wall may be destroyed, and the media may be mixed as contamination.

[Cell screening method]
The cell screening method is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include wet classification, cell sorter, and screening using a filter. These may be used individually by 1 type, and may use 2 or more types together. Among these, screening using a cell sorter or a filter is preferred because it causes little damage to cells.

It is preferable to estimate the number of nucleic acids having the target base sequence from the number of cells contained in the cell suspension by measuring the cell cycle of the cells.
Measuring the cell cycle means quantifying the number of cells due to cell division.
Estimating the number of nucleic acids means determining the number of nucleic acid copies from the number of cells.

The object to be counted may be the number of target base sequences, not the number of cells. Usually, the target nucleotide sequence is selected to contain one region per cell or is introduced by genetic recombination, so the number of target nucleotide sequences can be considered to be equal to the number of cells. However, in order for cells to undergo cell division in a specific cycle, nucleic acid replication takes place within the cells. The cell cycle varies depending on the type of cell, but by extracting a predetermined amount of solution from the cell suspension and measuring the cycle of multiple cells, the expected value and uncertainty for the number of target base sequences contained in one cell can be calculated. This is possible, for example, by observing nuclear-stained cells with a flow cytometer.
Uncertainty means information about variations in measurement results due to operations, equipment, and the like involved in the manufacture of the object to be measured.
Calculation means to calculate and obtain a numerical value.

FIG. 5 is a graph showing an example of the relationship between the frequency of DNA-replicated cells and fluorescence intensity. As shown in FIG. 5, two peaks appear on the histogram depending on whether or not the target base sequence is replicated, so it is possible to calculate the percentage of cells that have undergone DNA replication. From this calculation result, it is possible to calculate the average number of target base sequences contained in one cell, and by multiplying the above-mentioned cell count result, it is possible to calculate the estimated number of target base sequences. It is possible.
In addition, it is preferable to perform a treatment to control the cell cycle before preparing the cell suspension. can be calculated more accurately from

It is preferable to calculate the uncertainty of the estimated specific copy number. By calculating the uncertainty, it is possible to express the uncertainty as a variance or standard deviation based on these numerical values and output it. When summing the effects of multiple factors, it is possible to use the commonly used square root sum of squares of standard deviations. For example, it is possible to use the correct answer rate of the number of discharged cells, the number of DNA in the cells, and the landing rate of the discharged cells in the filling site (well) as factors. It is also possible to select and calculate an item having a large influence from among these.

<<Droplet landing process>>
The droplet landing step is a step of ejecting the cell suspension as droplets and causing the droplets to land sequentially in filling sites (wells) of the device.
A droplet means a mass of liquid held together by surface tension.
Ejection means ejecting the cell suspension as droplets.
Sequential means in order one after another.
Landing means making the droplet reach the filling site (well).

As the ejecting means, means for ejecting the cell suspension as droplets (hereinafter sometimes referred to as "ejection head") can be preferably used.

Examples of methods for ejecting the cell suspension as droplets include an on-demand method and a continuous method in the inkjet method. Among these, in the case of the continuous method, idle ejection until reaching a stable ejection state, adjustment of the droplet amount, continuous droplet formation even when moving between filling sites (wells), etc. For this reason, the cell suspension used tends to have a large dead volume. In the present invention, it is preferable to reduce the influence of dead volume from the viewpoint of adjusting the number of cells. Therefore, the on-demand method is more suitable among the above two methods.

The on-demand method includes, for example, a pressure application method in which liquid is ejected by applying pressure to the liquid, a thermal method in which liquid is ejected by film boiling due to heating, and a droplet is formed by pulling the droplet by electrostatic attraction. A plurality of known methods such as an electrostatic method can be used. Among these, the pressure application method is preferable for the following reasons.

In the electrostatic method, it is necessary to set an electrode so as to face the discharge section that holds the cell suspension and forms droplets. In the plate generation method of the present invention, the plates for receiving droplets are arranged facing each other, and it is preferable that no electrodes be arranged in order to increase the degree of freedom in plate configuration.
Since the thermal method generates localized heating, there are concerns about the effect on the cells, which are biomaterials, and the burning (kogation) of the heater. Since the influence of heat depends on the contents and the application of the plate, it is not necessary to exclude it unconditionally.

Examples of the pressure applying method include a method of applying pressure to the liquid using a piezo element, a method of applying pressure using a valve such as an electromagnetic valve, and the like. 6A to 6C show configuration examples of droplet generation devices that can be used to eject droplets of a cell suspension.
FIG. 6A is a schematic diagram showing an example of an electromagnetic valve type ejection head. The electromagnetic valve type ejection head has an electric motor 13a, an electromagnetic valve 112, a liquid chamber 11a, a cell suspension 300a, and a nozzle 111a.
As the electromagnetic valve type ejection head, for example, a dispenser manufactured by TechElan can be preferably used.
FIG. 6B is a schematic diagram showing an example of a piezo ejection head. The piezoelectric ejection head has a piezoelectric element 13b, a liquid chamber 11b, a cell suspension 300b, and a nozzle 111b.
Cytena's single-cell printer or the like can be suitably used as the piezo-type ejection head.
Any of these ejection heads can be used, but since droplets cannot be formed repeatedly at high speed with the pressure application method using an electromagnetic valve, the piezo method should be used in order to increase the throughput of plate generation. is preferred. In addition, in a piezo-type ejection head using a general piezoelectric element 13b, sedimentation may cause unevenness in cell concentration and nozzle clogging.
Therefore, the configuration shown in FIG. 6C and the like can be cited as a more preferable configuration. FIG. 6C is a schematic diagram of a modification of the piezoelectric ejection head using the piezoelectric element in FIG. 6B. The ejection head of FIG. 6C has a piezoelectric element 13c, a liquid chamber 11c, a cell suspension 300c, and a nozzle 111c.
In the ejection head of FIG. 6C, by applying a voltage to the piezoelectric element 13c from a control device (not shown), compressive stress is applied in the horizontal direction of the paper, and the membrane can be deformed in the vertical direction of the paper.

Methods other than the on-demand method include, for example, a continuous method in which droplets are continuously formed. In the continuous method, when droplets are pressurized and pushed out from a nozzle, periodic fluctuations are given by a piezoelectric element or a heater, thereby making it possible to continuously produce minute droplets. Furthermore, it is also possible to select whether the droplets are landed in the filled portion (well) or collected in the collection portion by controlling the application of a voltage in the discharge direction of the flying droplets. Such a method is used in a cell sorter or a flow cytometer, and for example, a device name: Cell Sorter SH800 manufactured by Sony Corporation can be used.

FIG. 7A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. Moreover, FIG. 7B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 7A shows drive voltages for droplet formation. Droplets can be formed by varying the strength of the voltages (V _A , V _B , V _C ). FIG. 7B shows voltages for agitating the cell suspension without droplet ejection.

By inputting multiple pulses that are not strong enough to eject droplets during the period in which droplets are not ejected, it is possible to stir the cell suspension in the liquid, and the concentration distribution due to cell sedimentation can be changed. can be suppressed.

The droplet formation operation of the ejection head that can be used in the present invention will be described below.
The ejection head can eject droplets by applying a pulsed voltage to upper and lower electrodes formed on the piezoelectric element. 8A to 8C are schematic diagrams showing states of droplets at respective timings.
In FIG. 8A, first, by applying a voltage to the piezoelectric element 13c, the membrane 12c is rapidly deformed, thereby generating a high pressure between the cell suspension held in the liquid chamber 11c and the membrane 12c. Then, the droplet is pushed out from the nozzle part by this pressure.
Next, as shown in FIG. 8B, the droplet continues to be extruded from the nozzle until the pressure relaxes upward, and the droplet grows.
Finally, as shown in FIG. 8C, when the membrane 12c returns to its original state, the liquid pressure near the interface between the cell suspension and the membrane 12c decreases, forming a droplet 310'.

In the device manufacturing method, a plate in which filling sites (wells) are formed is fixed on a movable stage, and droplets are sequentially landed in concave portions by combining stage driving and droplet formation from an ejection head. Let Here, the method of moving the plate as the movement of the stage has been shown, but it is a matter of course that the ejection head may be moved.

The plate is not particularly limited, and one having filling sites (wells) generally used in the biotechnology field can be used.
The number of filling sites (wells) in the plate is not particularly limited and can be appropriately selected depending on the purpose, and may be singular or plural.

FIG. 9 is a schematic diagram showing an example of a dispensing device 400 for sequentially landing droplets in filling sites (wells) of a plate.
As shown in FIG. 9 , a dispensing device 400 for landing droplets has a droplet forming device 401 , a plate 700 , a stage 800 and a control device 900 .

In the pipetting apparatus 400, the plate 700 is placed on a stage 800 that is movable. The plate 700 is formed with a plurality of filling portions (wells) 710 (concave portions) onto which droplets 310 ejected from the ejection head of the droplet forming device 401 land. The control device 900 moves the stage 800 and controls the relative positional relationship between the ejection head of the droplet forming device 401 and each filling site (well) 710 . As a result, the droplets 310 containing the fluorescently stained cells 350 can be sequentially discharged from the discharge head of the droplet forming device 401 into the filling sites (wells) 710 .

The control device 900 can be configured to include, for example, a CPU, a ROM, a RAM, a main memory, and the like. In this case, various functions of the control device 900 can be realized by reading a program recorded in a ROM or the like into a main memory and executing the program by the CPU. However, part or all of the control device 900 may be realized only by hardware. Also, the control device 900 may be physically composed of a plurality of devices or the like.

As for the droplets to be ejected, it is preferable to land the droplets in the filling sites (wells) so as to obtain a plurality of levels when the cell suspension is landed in the filling sites (wells).
By multiple levels is meant multiple criteria that serve as standards.
As the plurality of levels, it is preferable that a plurality of cells having a specific nucleic acid in the filling site (well) have a predetermined concentration gradient. By having a concentration gradient, it can be suitably used as a calibration curve reagent. Multiple levels can be controlled using values counted by the sensor.

As the plate, it is preferable to use a 1-hole microtube, 8-tube, 96-well, or 384-well filling site (well) plate. The filling sites (wells) can contain the same number of cells or different levels of numbers. There may also be filling sites (wells) that do not contain cells. For example, it is conceivable to prepare plates in which cells (or nucleic acids) are dispensed at 7 levels of approximately 1, 2, 4, 8, 16, 32, and 64 cells.

<<Cell count step>>
The cell counting step is a step of counting the number of cells contained in the droplet by a sensor after the droplet is ejected and before the droplet lands on the filling site (well).
Sensors are used to capture the mechanical, electromagnetic, thermal, acoustic, or chemical properties of natural phenomena and artifacts, or the spatial and temporal information indicated by them, by applying some scientific principles to human beings and human beings. It means a device that replaces a signal of another medium that is easy for a machine to handle.
Counting means counting numbers.

The cell counting step is not particularly limited as long as the number of cells contained in the droplet is counted by a sensor after the droplet is ejected and before the droplet lands on the filling site (well). It can be selected as appropriate, and may include a process of observing cells before discharge and a process of counting cells after landing.

Counting the number of cells contained in the droplet after the droplet is ejected and before the droplet lands on the filling site (well) predicts that the droplet will surely enter the filling site (well) of the plate. It is preferable to observe the cells in the droplet at a timing that is directly above the fill site (well) opening.

Methods for observing cells in droplets include, for example, an optical detection method, an electric/magnetic detection method, and the like.

-Method of optical detection-
The optical detection method will be described below with reference to FIGS. 10, 14 and 15. FIG.
FIG. 10 is a schematic diagram showing an example of the droplet forming device 401. As shown in FIG. 14 and 15 are schematic diagrams showing other examples of droplet forming devices 401A and 401B. As shown in FIG. 10 , the droplet forming device 401 has an ejection head (droplet ejection means) 10 , driving means 20 , light source 30 , light receiving element 60 and control means 70 .

In FIG. 10, a liquid obtained by fluorescently staining cells with a specific dye and then dispersing them in a predetermined solution is used as the cell suspension, and light having a specific wavelength emitted from a light source is applied to droplets formed from the ejection head. Counting is performed by detecting the fluorescence emitted from the irradiated cells with a light-receiving element. At this time, in addition to the method of staining the cells with a fluorescent dye, the autofluorescence emitted by the molecules originally contained in the cells may be used, or the cells may produce a fluorescent protein (e.g., GFP (Green Fluorescent Protein)). A gene for the purpose may be introduced in advance so that the cells emit fluorescence.
Irradiating with light means applying light.

The ejection head 10 has a liquid chamber 11, a membrane 12, and a driving element 13, and can eject a cell suspension 300 in which fluorescently stained cells 350 are suspended as droplets.

The liquid chamber 11 is a liquid holding portion that holds a cell suspension 300 in which fluorescence-stained cells 350 are suspended, and a nozzle 111 that is a through hole is formed on the lower surface side. The liquid chamber 11 can be made of metal, silicon, ceramic, or the like, for example. Examples of the fluorescently-stained cells 350 include inorganic fine particles and organic polymer particles dyed with a fluorescent dye.

The membrane 12 is a film-like member fixed to the upper end of the liquid chamber 11 . The planar shape of the membrane 12 can be, for example, circular, but may be elliptical, square, or the like.

The driving element 13 is provided on the upper surface side of the membrane 12 . The shape of the driving element 13 can be designed according to the shape of the membrane 12 . For example, when the planar shape of the membrane 12 is circular, it is preferable to provide circular drive elements 13 .

By supplying a driving signal from the driving means 20 to the driving element 13, the membrane 12 can be vibrated. By vibrating the membrane 12 , a droplet 310 containing fluorescently stained cells 350 can be ejected from the nozzle 111 .

When a piezoelectric element is used as the driving element 13, for example, a structure in which electrodes for applying a voltage are provided on the upper and lower surfaces of the piezoelectric material can be employed. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means 20, a compressive stress is applied in the horizontal direction of the paper, and the membrane 12 can be vibrated in the vertical direction of the paper. For example, lead zirconate titanate (PZT) can be used as the piezoelectric material. Various other piezoelectric materials can be used, such as bismuth iron oxide, metal niobate, barium titanate, or any of these materials plus metals or different oxides.

The light source 30 irradiates the light L onto the droplet 310 in flight. Note that "during flight" means a state from when the droplet 310 is ejected from the droplet ejection unit 10 to when the droplet 310 lands on the droplet-receiving object. The flying droplet 310 has a substantially spherical shape at the position where the light L is irradiated. Also, the beam shape of the light L is substantially circular.

Here, it is preferable that the beam diameter of the light L is about 10 to 100 times the diameter of the droplet 310 . This is to ensure that the droplets 310 are irradiated with the light L from the light source 30 even when the droplets 310 have positional variations.

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

The light L emitted from the light source 30 is preferably pulsed light, and for example, a solid-state laser, a semiconductor laser, a dye laser, or the like is preferably used. When the light L is pulsed light, the pulse width is preferably 10 μs or less, more preferably 1 μs or less. Although the energy per unit pulse largely depends on the optical system such as the presence or absence of light condensing, it is preferably about 0.1 μJ or more, more preferably 1 μJ or more.

The light-receiving element 60 receives fluorescence Lf emitted by the fluorescently-stained cells 350 absorbing the light L as excitation light when the droplet 310 in flight contains the fluorescently-stained cells 350 . Since the fluorescence Lf is emitted in all directions from the fluorescence-stained cells 350, the light-receiving element 60 can be placed at any position where the fluorescence Lf can be received. At this time, in order to improve the contrast, it is preferable to arrange the light receiving element 60 at a position where the light L emitted from the light source 30 does not directly enter.

The light receiving element 60 is not particularly limited as long as it can receive the fluorescence Lf emitted from the fluorescently stained cells 350, and can be appropriately selected according to the purpose. An optical sensor that receives fluorescence from cells within the droplet is preferred. Examples of the light receiving element 60 include one-dimensional elements such as photodiodes and photosensors, but if highly sensitive measurement is required, it is preferable to use a photomultiplier tube or an avalanche photodiode. As the light receiving element 60, for example, a two-dimensional element such as a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), or a gate CCD may be used.

Since the fluorescence Lf emitted by the fluorescently stained cells 350 is weaker than the light L emitted by the light source 30, a filter that attenuates the wavelength range of the light L may be provided in front of the light receiving element 60 (on the side of the light receiving surface). . As a result, an image of the fluorescently stained cells 350 with a very high contrast can be obtained in the light receiving element 60 . As the filter, for example, a notch filter or the like that attenuates a specific wavelength range including the wavelength of the light L can be used.

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

The control means 70 has a function of controlling the drive means 20 and the light source 30 . The control means 70 also has a function of obtaining information based on the amount of light received by the light receiving element 60 and counting the number of fluorescently stained cells 350 contained in the droplet 310 (including the case where the number is zero). there is The operation of the droplet forming apparatus 401 including the operation of the control means 70 will be described below with reference to FIGS. 11 to 16. FIG.

FIG. 11 is a diagram illustrating hardware blocks of control means of the droplet forming apparatus of FIG. 12 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. 10. FIG. FIG. 11 is a flow chart showing an example of the operation of the droplet forming device.

As shown in FIG. 11 , the control means 70 has a CPU 71 , a ROM 72 , a RAM 73 , an I/F 74 and a bus line 75 . The CPU 71 , ROM 72 , RAM 73 and I/F 74 are interconnected via a bus line 75 .

The CPU 71 controls each function of the control means 70 . The ROM 72, which is storage means, stores programs executed by the CPU 71 to control each function of the control means 70 and various information. A RAM 73, which is a storage means, is used as a work area for the CPU 71 and the like. Also, the RAM 73 can temporarily store predetermined information. The I/F 74 is an interface for connecting the droplet forming device 401 with other devices. The droplet forming device 401 may be connected to an external network or the like via the I/F 74 .

As shown in FIG. 12, the control means 70 has ejection control means 701, light source control means 702, and cell number counting means (cell number detection means) 703 as functional blocks.

Cell number (particle number) counting of the droplet forming device 401 will be described with reference to FIGS. 12 and 13 . First, in step S<b>11 , the ejection control means 701 of the control means 70 issues an ejection command to the drive means 20 . The driving means 20 that has received the ejection command from the ejection control means 701 supplies a driving signal to the driving element 13 to vibrate the membrane 12 . Vibration of the membrane 12 causes droplets 310 containing fluorescently stained cells 350 to be ejected from the nozzle 111 .

Next, in step S12, the light source control means 702 of the control means 70 controls the light source 30 in synchronization with the ejection of the droplets 310 (synchronization with the driving signal supplied from the driving means 20 to the droplet ejection means 10). Issue a turn-on command. As a result, the light source 30 is turned on to irradiate the light L onto the droplet 310 in flight.

Here, synchronizing does not mean emitting light at the same time as the droplet 310 is ejected by the droplet ejecting means 10 (at the same time as the driving means 20 supplies the driving signal to the droplet ejecting means 10), but droplets. It means that the light source 30 emits light at the timing when the droplet 310 is irradiated with the light L when the droplet 310 flies and reaches a predetermined position. That is, the light source control unit 702 emits light with a delay of a predetermined time with respect to ejection of the droplets 310 by the droplet ejection unit 10 (driving signal supplied from the driving unit 20 to the droplet ejection unit 10). Control the light source 30 .

For example, the velocity v of the droplets 310 to be ejected when the drive signal is supplied to the droplet ejection means 10 is measured in advance. Then, based on the measured speed v, the time t required for the droplet 310 to reach a predetermined position after being ejected is calculated, and the light source 30 emits light at the timing of supplying the driving signal to the droplet ejecting means 10. delay the timing by t. As a result, good light emission control becomes possible, and the droplet 310 can be reliably irradiated with the light from the light source 30 .

Next, in step S13, the cell number counting means 703 of the control means 70 counts the number of fluorescently stained cells 350 contained in the droplet 310 based on the information from the light receiving element 60 (including the case where it is zero). Count. Here, the information from the light receiving element 60 is the brightness value (light amount) and area value of the fluorescently stained cells 350 .

The cell number counting means 703 can count the number of fluorescently stained cells 350 by, for example, comparing the amount of light received by the light receiving element 60 with a preset threshold value. In this case, the light receiving element 60 may be a one-dimensional element or a two-dimensional element.

When a two-dimensional element is used as the light receiving element 60, the cell number counting means 703 performs image processing for calculating the luminance value or area of the fluorescently stained cells 350 based on the two-dimensional image obtained from the light receiving element 60. You may use the method to carry out. In this case, the cell number counting means 703 calculates the brightness value or area value of the fluorescently stained cells 350 by image processing, and compares the calculated brightness value or area value with a preset threshold to determine the fluorescence The number of stained cells 350 can be counted.

Note that the fluorescently stained cells 350 may be cells or stained cells. A stained cell means a cell stained with a fluorescent dye or a cell capable of expressing a fluorescent protein.

In the stained cells, the fluorescent dye is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include fluoresceins, rhodamines, coumarins, pyrenes, cyanines, azos and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, eosin, Evans blue, trypan blue, rhodamine 6G, rhodamine B, and rhodamine 123 are more preferred.
Examples of fluorescent proteins include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP , Venus , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, KusabiraOrange, mOrange, TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Turb oFP602, mRFP1, JRed, KillerRed, mCherry, mPlum, PS - CFP, Dendra2, Kaede, EosFP, KikumeGR, etc. These may be used individually by 1 type, and may use 2 or more types together.

As described above, in the droplet forming apparatus 401 , a driving signal is supplied from the driving means 20 to the droplet discharging means 10 holding the cell suspension 300 in which the fluorescently stained cells 350 are suspended, so that the fluorescently stained cells 350 are produced. The liquid droplets 310 contained therein are ejected, and the light L from the light source 30 is applied to the flying liquid droplets 310 . Then, the fluorescence-stained cells 350 contained in the flying droplet 310 emit fluorescence Lf using the light L as excitation light, and the light receiving element 60 receives the fluorescence Lf. Furthermore, based on the information from the light receiving element 60, the cell number counting means 703 counts the number of fluorescently stained cells 350 contained in the flying droplet 310. FIG.

In other words, in the droplet forming apparatus 401, the number of fluorescently stained cells 350 contained in the flying droplet 310 is actually observed on the spot, so that the counting accuracy of the number of fluorescently stained cells 350 can be improved more than conventionally. becomes possible. In addition, since the fluorescence-stained cells 350 contained in the flying droplet 310 are irradiated with the light L to emit the fluorescence Lf and the fluorescence Lf is received by the light-receiving element 60, an image of the fluorescence-stained cells 350 can be obtained with high contrast. , and the frequency of erroneous counting of the number of fluorescently stained cells 350 can be reduced.

FIG. 14 is a schematic diagram showing a modification of the droplet forming device 401 of FIG. As shown in FIG. 14, the droplet forming device 401A is different from the droplet forming device 401 (see FIG. 10) in that a mirror 40 is arranged in front of the light receiving element 60. As shown in FIG. Note that the description of the same components as those of the already described embodiments may be omitted.

As described above, in the droplet forming device 401A, by arranging the mirror 40 in front of the light receiving element 60, the degree of freedom of the layout of the light receiving element 60 can be improved.

For example, when the nozzle 111 and the object to deposit droplets are brought close to each other, interference may occur between the object to deposit droplets and the optical system (especially the light receiving element 60) of the droplet forming device 401 in the layout of FIG. By using the layout of FIG. 14, it is possible to avoid the occurrence of interference.

As shown in FIG. 14, by changing the layout of the light-receiving element 60, it becomes possible to reduce the distance (gap) between the nozzle 111 and the object on which the droplet 310 is to be deposited. can be suppressed. As a result, it is possible to improve the accuracy of dispensing.

FIG. 22 is a schematic diagram showing another modification of the droplet forming device 401 of FIG. As shown in FIG. 22, the droplet forming device 401B includes a light receiving element 60 that receives fluorescence _Lf1 emitted from the fluorescently stained cells 350, and a light receiving element 61 that receives fluorescence _Lf2 emitted from the fluorescently stained cells 350. It is different from the droplet forming device 401 (see FIG. 10) in that it is provided. Note that the description of the same components as those of the already described embodiments may be omitted.

Here, fluorescence Lf ₁ and Lf ₂ indicate part of the fluorescence emitted in all directions from the fluorescently stained cell 350 . The light-receiving elements 60 and 61 can be arranged at arbitrary positions where they can receive fluorescence emitted in different directions from the fluorescently-stained cells 350 . It should be noted that three or more light receiving elements may be arranged at positions capable of receiving fluorescence emitted from the fluorescently stained cells 350 in different directions. Further, each light receiving element may have the same specifications or may have different specifications.

When the number of light receiving elements is one, when a plurality of fluorescently stained cells 350 are included in the flying droplet 310, the fluorescently stained cells 350 overlap each other. There is a risk of erroneously counting the number of fluorescently stained cells 350 contained in (counting error occurs).

16A and 16B are diagrams illustrating a case where a flying droplet contains two fluorescently stained cells. For example, as shown in FIG. 16A, fluorescently stained cells 350 ₁ and 350 ₂ overlap, and as shown in FIG. 16B, fluorescently stained cells 350 ₁ and 350 ₂ do not overlap. obtain. By providing two or more light-receiving elements, it is possible to reduce the influence of overlapping fluorescently-stained cells.

As described above, the cell number counting means 703 calculates the brightness value or area value of the fluorescent particles by image processing, and compares the calculated brightness value or area value with a preset threshold to determine the fluorescence. The number of particles can be counted.

When two or more light-receiving elements are installed, it is possible to suppress the occurrence of count errors by adopting data indicating the maximum value among the luminance values or area values obtained from the respective light-receiving elements. This will be explained in more detail with reference to FIG.

FIG. 17 is a diagram illustrating the relationship between the luminance value Li when particles do not overlap each other and the luminance value Le that is actually measured. As shown in FIG. 17, Le=Li when there is no overlap between particles in the droplet. For example, if Lu is the luminance value of one cell, Le=Lu when the number of cells/droplet=1, and Le=nLu when the number of particles/droplet=n (n: natural number).

However, in reality, when n is 2 or more, particles may overlap each other, so the actually measured luminance values are Lu≦Le≦nLu (shaded area in FIG. 16A). Therefore, when the number of cells/droplet=n, for example, the threshold can be set as (nLu−Lu/2)≦threshold<(nLu+Lu/2). When a plurality of light-receiving elements are installed, it is possible to suppress the occurrence of count errors by adopting the data indicating the maximum value among the data obtained from the respective light-receiving elements. Note that an area value may be used instead of the luminance value.

Moreover, when installing a plurality of light receiving elements, the number of particles may be determined by an algorithm for estimating the number of cells based on a plurality of obtained shape data.
As described above, since the droplet forming device 401B has a plurality of light receiving elements that receive fluorescence emitted in different directions by the fluorescently stained cells 350, the frequency of erroneous counting of the number of the fluorescently stained cells 350 can be further reduced. can be reduced.

FIG. 18 is a schematic diagram showing another modification of the droplet forming device 401 of FIG. As shown in FIG. 18, a droplet forming device 401C differs from the droplet forming device 401 (see FIG. 10) in that the droplet discharging means 10 is replaced with a droplet discharging means 10C. Note that the description of the same components as those of the already described embodiments may be omitted.

The droplet ejection means 10C has a liquid chamber 11C, a membrane 12C, and a drive element 13C. The liquid chamber 11</b>C has an air opening portion 115 that opens the interior of the liquid chamber 11</b>C to the atmosphere, and air bubbles mixed in the cell suspension 300 can be discharged from the air opening portion 115 .

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

The material of the membrane 12C is not particularly limited, but if it is too soft, the membrane 12C easily vibrates, and it is difficult to immediately suppress the vibration when not ejecting, so it is preferable to use a material with a certain degree of hardness. . As the material of the membrane 12C, for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.

In particular, when cells are used as the fluorescently stained cells 350, it is preferable that the material has low adhesion to cells and proteins. Adhesion of cells is generally said to depend on the contact angle of the material with water, and the adhesion of cells is low when the material is highly hydrophilic or highly hydrophobic. Various metal materials and ceramics (metal oxides) can be used as highly hydrophilic materials, and fluorine resins and the like can be used as highly hydrophobic materials.

Other examples of such materials include stainless steel, nickel, aluminum, silicon dioxide, alumina, zirconia, and the like. In addition to this, it is also conceivable to reduce the cell adhesiveness by coating the material surface. For example, the surface of the material can be coated with the aforementioned metal or metal oxide material, or with a synthetic phospholipid polymer that mimics cell membranes (for example, Lipidure manufactured by NOF Corporation).

It is preferable that the nozzle 121 is formed as a substantially perfect circular through-hole substantially in the center of the membrane 12C. In this case, the diameter of the nozzle 121 is not particularly limited, but in order to avoid clogging the nozzle 121 with the fluorescently-stained cells 350, it is preferably at least twice the size of the fluorescently-stained cells 350. FIG. When the fluorescently stained cells 350 are, for example, animal cells, particularly human cells, the size of human cells is generally about 5 μm to 50 μm, so the nozzle 121 should have a diameter of 10 μm or more according to the cells to be used. It is preferably 100 μm or more, and more preferably 100 μm or more.

On the other hand, if the droplets are too large, it will be difficult to achieve the purpose of forming minute droplets, so the diameter of the nozzle 121 is preferably 200 μm or less. In other words, the diameter of the nozzle 121 is typically in the range of 10 μm to 200 μm in the droplet discharge means 10C.

13 C of drive elements are formed in the lower surface side of the membrane 12C. The shape of the drive element 13C can be designed according to the shape of the membrane 12C. For example, when the planar shape of the membrane 12C is circular, it is preferable to form the driving element 13C having an annular planar shape (ring shape) around the nozzle 121 . The driving method of the driving element 13C can be the same as that of the driving element 13. FIG.

The driving means 20 selectively (for example, alternately) applies a discharge waveform for vibrating the membrane 12C to form the droplets 310 and a stirring waveform for vibrating the membrane 12C within a range not forming the droplets 310 to the driving element 13C. ) can be granted.

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

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

At this time, the movement of the fluorescently-stained cells 350 is from bottom to top, and convection occurs in the liquid chamber 11C, causing agitation of the cell suspension 300 containing the fluorescently-stained cells 350 . Due to the flow from the bottom to the top of the liquid chamber 11C, the precipitated and aggregated fluorescently stained cells 350 are uniformly dispersed inside the liquid chamber 11C.

In other words, the driving means 20 applies the ejection waveform to the driving element 13C and controls the vibration state of the membrane 12C to eject the cell suspension 300 held in the liquid chamber 11C from the nozzle 121 as droplets 310. can be done. Further, the driving means 20 can stir the cell suspension 300 held in the liquid chamber 11C by applying a stirring waveform to the driving element 13C and controlling the vibration state of the membrane 12C. Note that the droplets 310 are not ejected from the nozzle 121 during stirring.

Thus, by stirring the cell suspension 300 while the droplets 310 are not formed, the fluorescently stained cells 350 are prevented from settling and aggregating on the membrane 12C, and the fluorescently stained cells 350 are prevented from being suspended in the cell suspension. It can be evenly dispersed in the turbid liquid 300 . This makes it possible to suppress clogging of the nozzle 121 and variations in the number of fluorescently stained cells 350 in the ejected droplet 310 . As a result, the cell suspension 300 containing the fluorescently-stained cells 350 can be continuously and stably ejected as droplets 310 for a long period of time.

Also, in the droplet forming device 401C, air bubbles may be mixed in the cell suspension 300 in the liquid chamber 11C. Even in this case, since the droplet formation device 401C is provided with the atmosphere opening portion 115 above the liquid chamber 11C, air bubbles mixed in the cell suspension 300 can be discharged to the outside air through the atmosphere opening portion 115. This makes it possible to continuously and stably form droplets 310 without discarding a large amount of liquid for discharging bubbles.

That is, when air bubbles are mixed in the vicinity of the nozzle 121 or when a large number of air bubbles are mixed on the membrane 12C, the ejection state is affected. Entrained air bubbles must be discharged. Normally, air bubbles entrapped on the membrane 12C move upward naturally or due to vibration of the membrane 12C. Ejection becomes possible. Therefore, even if air bubbles enter the liquid chamber 11C, it is possible to prevent non-ejection from occurring, and the droplets 310 can be continuously and stably formed.

It should be noted that the membrane 12C may be vibrated within a range in which droplets are not formed at the timing when the droplets are not formed to positively move the bubbles upward in the liquid chamber 11C.

-Electrical or magnetic detection method-
As a method of electrical or magnetic detection, as shown in FIG. A coil 200 for is installed as a sensor. Cells are covered with magnetic beads that are modified with specific proteins and can adhere to the cells. When the cells with the magnetic beads attached pass through the coil, the induced current generated by the cells in the flying droplets causes the flow of the cells. It is possible to detect the presence or absence. In general, cells have a cell-specific protein on their surface, and by modifying the magnetic beads with an antibody capable of adhering to this protein, it is possible to attach the magnetic beads to the cells. . As such magnetic beads, off-the-shelf products can be used. For example, Dynabeads (registered trademark) manufactured by Veritas Corporation can be used.

[Processing for Observing Cells Before Ejection]
The processing for observing cells before ejection includes a method of counting cells 350′ passing through a microchannel 250 shown in FIG. 20, a method of acquiring an image near the nozzle portion of the ejection head shown in FIG. 21, and the like. are mentioned. FIG. 20 shows a method used in a cell sorter apparatus. For example, a cell sorter SH800 manufactured by Sony Corporation can be used. In FIG. 20, laser light is irradiated from a light source 260 into a microchannel 250, and scattered light and fluorescence are detected by a detector 255 using a condenser lens 265 to identify the presence or absence of cells and the types of cells. It is possible to form droplets while By using this method, it is possible to estimate the number of cells that have landed in a predetermined filling site (well) from the number of cells that have passed through the microchannel 250 .
A single-cell printer manufactured by Cytena can be used as the ejection head 10' shown in FIG. In FIG. 21, it is estimated that the cells 350″ in the vicinity of the nozzle portion have been ejected from the result of image acquisition by the image acquisition portion 255′ through the lens 265′ in the vicinity of the nozzle portion before ejection, and images before and after ejection are shown. By estimating the number of cells that are considered to have been ejected from the difference from , it is possible to estimate the number of cells that have landed in a predetermined filling site (well). In the method of counting cells, droplets are generated continuously, whereas FIG. 21 is more preferable because droplet formation is possible on demand.

[Processing for counting cells after landing]
As processing for counting cells after landing, it is possible to adopt a method of detecting fluorescently-stained cells by observing filling sites (wells) in a plate with a fluorescence microscope or the like. This method is described, for example, in Sangjun et al. , PLoS One, Volume 6(3), e17455.

Although the method of observing cells before and after the droplets are ejected has the following problems, depending on the type of plate to be produced, it is most preferable to observe the cells in the droplets being ejected. In the method of observing cells before ejection, the number of cells that are thought to have landed is counted based on the number of cells that have passed through the channel and the image observation before (and after) ejection, so the cells are actually ejected. There is no confirmation of whether or not it has been done, and unexpected errors may occur. For example, when the nozzle section is dirty, droplets may not be ejected correctly and may adhere to the nozzle plate, which may cause the cells in the droplets not to land. In addition, problems may occur such as cells remaining in a narrow area of the nozzle portion, and cells moving more than expected due to the discharge operation and leaving the observation range.
There is also a problem with the method of detecting cells on the plate after impact. First, it is necessary to prepare a plate that allows microscopic observation. Plates with transparent and flat bottoms, especially plates with glass bottoms, are generally used as observable plates. I have a problem that makes it unusable. In addition, when the number of cells is large, such as several tens, there is also the problem that the cells overlap, making it impossible to perform accurate counting. Therefore, after the droplet is ejected and before the droplet lands on the filling site (well), the number of cells contained in the droplet is counted by a sensor and particle number (cell number) counting means. It is preferable to perform a process of observing cells before and a process of counting cells after landing.

As the light receiving element, a light receiving element having one or a small number of light receiving portions, such as a photodiode, an avalanche photodiode, or a photomultiplier tube, can be used. It is also possible to use a two-dimensional sensor such as a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), or a gate CCD.
When using a light-receiving element having one or a small number of light-receiving portions, it is conceivable to use a calibration curve prepared in advance to determine how many cells are contained from the fluorescence intensity. Binary detection of the presence or absence of cells is performed. When the cell concentration of the cell suspension is sufficiently low and the liquid droplet contains only 1 or 0 cells, it is possible to perform sufficiently accurate counting by binary detection. It is possible. Assuming that the cells are randomly arranged in the cell suspension, the number of cells in the flying droplet is considered to follow the Poisson distribution, and the probability that two or more cells are included in the droplet is P ( >2) is represented by the following formula (1). FIG. 22 is a graph showing the relationship between the probability P (>2) and the average number of cells. Here, λ is the average number of cells in the droplet, which is the cell concentration in the cell suspension multiplied by the volume of the ejected droplet.
P (>2) = 1 - (1 + λ) x e ^{- λ} Expression (1)

When counting the number of cells by binary detection, it is preferable that the probability P (> 2) is a sufficiently small value in order to ensure accuracy, and the probability P (> 2) is 1% or less λ <0.15 is preferred. The light source is not particularly limited as long as it can excite the fluorescence of cells, and can be appropriately selected according to the purpose. A filtered one, an LED (Light Emitting Diode), a laser, or the like can be used. However, especially when forming minute droplets of 1 nL or less, it is necessary to irradiate a narrow region with high light intensity, so it is preferable to use a laser. As a laser light source, various commonly known lasers such as solid lasers, gas lasers, and semiconductor lasers can be used. Further, the excitation light source may continuously irradiate the area through which the droplets pass, or may be synchronized with the ejection of the droplets and at a timing delayed by a predetermined time with respect to the droplet ejection operation. Pulse irradiation may be used.

<<Uncertainty calculation process>>
The uncertainty calculation step is a step of calculating the uncertainty in each step such as the cell suspension generation step, the droplet landing step, and the cell number counting step.
The uncertainty can be calculated in the same manner as the uncertainty in the cell suspension generation process.
In addition, the calculation timing of the uncertainty may be calculated collectively in the next step of the cell counting step, or at the end of each step such as the cell suspension generation step, the droplet landing step, and the cell counting step. , and each uncertainty may be combined and calculated in the next step of the cell counting step. In other words, the uncertainty in each of the above steps may be appropriately calculated before the composite calculation.

<<Output process>>
The output step is a step of outputting the number of cells contained in the cell suspension that landed in the filling site (well) by the particle number counting means based on the detection result measured by the sensor. .
The counted value means the number of cells contained in the filling site (well) by the particle counting means based on the detection result measured by the sensor.
Output means transmission of counted values to a server as external counting result storage means as electronic information, or printing of counted values as printed matter. means that

The output step observes or estimates the number of cells or the number of nucleic acids in each filling site (well) in the plate when the plate is generated, and outputs the observed value or estimated value to an external storage unit.
The output may be performed at the same time as the cell number counting process, or may be performed after the cell number counting process.

<<Recording process>>
The recording step is a step of recording the output observed value or estimated value in the output step.
The recording process can be preferably carried out in the recording section.
Recording may be performed simultaneously with the output process, or may be performed after the output process.
Recording means not only giving information to a recording medium, but also storing information in a recording unit.

<<Nucleic acid extraction step>>
The nucleic acid extraction step is a step of extracting nucleic acids from the cells in the filling site (well).
Extraction means destroying cell membranes, cell walls, etc., and drawing out nucleic acids.

As a method for extracting nucleic acids from cells, a method of heat treatment at 90° C. to 100° C. is known. Heat treatment at 90° C. or lower may not extract DNA, and heat treatment at 100° C. or higher may degrade DNA. At this time, it is preferable to add a surfactant and heat-treat.

The surfactant is not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include ionic surfactants and nonionic surfactants. These may be used individually by 1 type, and may use 2 or more types together. Among these surfactants, nonionic surfactants are preferred because they do not denature or inactivate proteins, depending on the amount added.

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

Examples of nonionic surfactants include alkyl glycosides, alkyl polyoxyethylene ethers (Brij series, etc.), octylphenol ethoxylates (Totiton X series, Igepal CA series, Nonidet P series, Nikkol OP series, etc.), polysorbates ( Tween series such as Tween 20), sorbitan fatty acid ester, polyoxyethylene fatty acid ester, alkylmaltoside, sucrose fatty acid ester, glycoside fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, fatty acid monoglyceride, and the like. These may be used individually by 1 type, and may use 2 or more types together. Among these, polysorbates are preferred.

The content of the surfactant is preferably 0.01% by mass or more and 5.00% by mass or less with respect to the total amount of the cell suspension in the filling site (well). When the content is 0.01% by mass or more, it can exert an effect on DNA extraction, and when it is 5.00% by mass or less, it is possible to prevent inhibition of amplification during PCR. As a numerical range in which the effect of (1) can be obtained, the above range of 0.01% by mass or more and 5.00% by mass or less is preferable.
With respect to cells that have cell walls, DNA may not be sufficiently extracted by the above method. In that case, for example, the osmotic shock method, the freeze-thaw method, the enzymatic digestion method, the use of a DNA extraction kit, the sonication method, the French press method, a method such as a homogenizer, and the like can be used. Among these methods, the enzymatic digestion method is preferable because loss of the extracted DNA is small.

<<Other processes>>
Other steps are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include an enzyme deactivation step.

-Enzyme deactivation step-
The enzyme deactivation step is a step of deactivating the enzyme.
Enzymes include, for example, DNase, RNase, enzymes used to extract nucleic acids in the nucleic acid extraction step, and the like.
The method for deactivating the enzyme is not particularly limited and can be appropriately selected depending on the purpose, and known methods can be suitably used.

A device for use in the nucleic acid analysis method of the present invention can be manufactured by the method described above.

<Calibration curve data creation process and calibration curve data creation unit>
The calibration curve data creation step is a step of creating calibration curve data of the standard nucleic acid based on the copy number of the standard nucleic acid having a specific copy number. The calibration curve data creation step is preferably performed by the calibration curve data creation unit and the calibration curve data creation means.
The calibration curve data means data relating to the number (copy number) of the base sequence of the standard nucleic acid, and may be the prepared data itself, and includes the calibration curve itself derived from the data.
A calibration curve means a relational expression between a parameter such as the amount and activity of a substance whose amount and activity to be used for analysis is known in advance and a parameter different from the parameter. The parameter is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include "copy number (copy)" of a specific base sequence, "read number" of a specific base sequence, and the like.
The calibration curve data can be prepared from the copy number data of the standard nucleic acids in the library obtained by the calibration curve data preparation means. The calibration curve data is not particularly limited as long as it is data relating to the copy number of the standard nucleic acid in the library obtained by the analysis equipment described later, and can be appropriately selected according to the purpose. The method for preparing calibration curve data from a standard nucleic acid with a specific copy number is not particularly limited and can be appropriately selected according to the purpose. Examples include a method of expressing the relationship between the amplification result and the original specific copy number as a relational expression.
The nucleic acid analysis method of the present invention uses a standard nucleic acid with a specific number of copies of the standard nucleic acid in the calibration curve data preparation step, so that even if the number of nucleic acids to be analyzed is extremely small, analysis (quantification) can be performed with high accuracy. be able to.

In the calibration curve data creation step, a standard nucleic acid with a specific copy number is included in two or more different systems with a specific copy number different from each other, and the calibration curve data of the obtained standard nucleic acid is normalized and combined for calibration. It is preferable to create lines. A standard nucleic acid with a specific copy number is included in two or more different systems with a specific copy number different from each other, and the standard curve data of the obtained standard nucleic acid is normalized and combined to create a standard curve. It is possible to reduce the number of types of standard nucleic acids to be used.

<Analysis target nucleic acid analysis step and analysis target nucleic acid analysis part>
The analysis target nucleic acid analysis step is a step of specifying the base sequence of the analysis target nucleic acid and specifying the number of base sequences of the analysis target nucleic acid. The analysis target nucleic acid analysis step is preferably performed by the analysis target nucleic acid analysis unit.
Identifying the base sequence of the nucleic acid to be analyzed means reading the base sequence of the nucleic acid to be analyzed.
Specifying the number of base sequences of the nucleic acid to be analyzed means that the number of copies of the base sequence contained in the nucleic acid to be analyzed is obtained by using the calibration curve prepared in the calibration curve data preparation step to obtain the measured value (number of reads, etc.) of the nucleic acid to be analyzed. ) means counting from When the nucleic acid to be analyzed contains two or more nucleic acids (fragments) having different base sequences, the base sequence and the number of base sequences are identified for each base sequence contained in the nucleic acid to be analyzed. . The read data of the nucleic acid to be analyzed may be managed in units of "read number" for each base sequence.
The number of nucleic acids to be analyzed is sometimes referred to as "copy number", "molecule number", and the like.
The analysis target acid analysis step below can also be performed (processed) in parallel with the calibration curve data creation step described above.

Regarding the analysis target nucleic acid analysis step, for example, the analysis method in the next-generation sequencer published by Illumina (https://www.address.ehime-u.ac.jp/news/NGS1.pdf), non- Patent Document 1, an analysis method in sequencing using a nanopore device (Oxford Nanopore Technologies.), an analysis method in sequencing using a PacBio RS II/Sequel system (Pacific BioSciences), an analysis method in the Ion Torrent ^TM semiconductor sequencing system series ( (ThermoFisher SCIENTIFIC)) can be referred to, and the analysis can be performed using the analysis equipment used for each analysis method.
The analysis equipment is not ^particularly limited and can be appropriately selected according to the purpose. etc. Commercially available products can be used as analytical instruments, and examples of commercially available products include Miseq (manufactured by Illumina); MiION, GridION, PromethION (manufactured by Oxford Nanopore Technologies); PacBio RS II (manufactured by Pacific BioSciences). , Ion Gene Studio S5 (ThermoFisher SCIENTIFIC), and the like.

The processing by the nucleic acid analysis program of the present invention can be executed using a computer having a controller that constitutes a nucleic acid analysis apparatus.
The hardware configuration and functional configuration of the nucleic acid analysis apparatus will be described below.

<Hardware Configuration of Nucleic Acid Analyzer>
FIG. 24 is a block diagram showing an example of the hardware configuration of the nucleic acid analysis device 100. As shown in FIG.
As shown in FIG. 24 , the nucleic acid analysis apparatus 100 has CPU (Central Processing Unit) 101 , main storage device 102 , auxiliary storage device 103 , output device 104 and input device 105 . Each of these units is connected via a bus 106 .
The CPU 101 is a processing device that performs various controls and calculations. The CPU 101 implements various functions by executing an OS (Operating System) and programs stored in the main storage device 102 or the like. That is, in this embodiment, the CPU 101 functions as the control section 130 of the nucleic acid analysis apparatus 100 by executing the nucleic acid analysis program.
Also, the CPU 101 controls the operation of the entire nucleic acid analysis apparatus 100 . In this embodiment, the CPU 101 is used as a device for controlling the operation of the entire nucleic acid analysis apparatus 100, but it is not limited to this, and may be, for example, an FPGA (Field Programmable Gate Array).

The nucleic acid analysis program and various databases do not necessarily have to be stored in the main storage device 102, the auxiliary storage device 103, or the like. The nucleic acid analysis program and various databases may be stored in another information processing apparatus connected to the nucleic acid analysis apparatus 100 via the Internet, LAN (Local Area Network), WAN (Wide Area Network), or the like. The nucleic acid analysis device 100 may acquire and execute a nucleic acid analysis program and various databases from these other information processing devices.
The main storage device 102 stores various programs, and stores data and the like necessary for executing the various programs.
The main memory device 102 has ROM (Read Only Memory) and RAM (Random Access Memory), which are not shown.
The ROM stores various programs such as BIOS (Basic Input/Output System).
The RAM functions as a working range expanded when various programs stored in the ROM are executed by the CPU 101 . The RAM is not particularly limited and can be appropriately selected according to the purpose. Examples of RAM include DRAM (Dynamic Random Access Memory) and SRAM (Static Random Access Memory).
The auxiliary storage device 103 is not particularly limited as long as it can store various types of information, and can be appropriately selected according to the purpose. Examples thereof include solid state drives and hard disk drives. Also, the auxiliary storage device 103 may be a portable storage device such as a CD (Compact Disc) drive, a DVD (Digital Versatile Disc) drive, or a BD (Blu-ray (registered trademark) Disc) drive.

A display, a speaker, or the like can be used as the output device 104 . The display is not particularly limited, and any known display can be used as appropriate. Examples thereof include liquid crystal displays and organic EL displays.
The input device 105 is not particularly limited as long as it can receive various requests to the nucleic acid analysis apparatus 100, and any known device can be used as appropriate. Examples thereof include a keyboard, a mouse, and a touch panel.
The processing functions of the nucleic acid analysis apparatus 100 can be realized by the hardware configuration as described above.

<Functional Configuration of Nucleic Acid Analyzer>
FIG. 25 is a diagram showing an example of the functional configuration of the nucleic acid analysis device 100. As shown in FIG.
As shown in FIG. 25, the nucleic acid analysis apparatus 100 has an input section 110, an output section 120, a control section 130, and a storage section 140.
The control unit 130 has a library preparation unit 131 , a calibration curve data creation unit 132 , and an analysis target nucleic acid analysis unit 133 . The control unit 130 controls the nucleic acid analysis apparatus 100 as a whole.
The storage unit 140 has a calibration curve database 141 and an analysis target nucleic acid analysis database 142 . Hereinafter, "database" may be referred to as "DB". Note that the data to be saved in the storage unit may be stored in either volatile or nonvolatile memory. Memory is sometimes referred to as "M" and is sometimes used interchangeably with "DB."

The library preparation unit 131 adjusts the reaction conditions for library preparation based on the information about the nucleic acid to be analyzed input from the input unit 110 .
The calibration curve data creation unit 132 creates calibration curve data of the standard nucleic acid based on the copy number data of the specific copy number of the standard nucleic acid. The control unit 130 stores the acquired calibration curve data in the calibration curve M141.
The analysis-target nucleic acid analysis unit 133 uses the analysis-target nucleic acid analysis data stored in the analysis-target nucleic acid analysis M142 of the storage unit 140 for the base sequence of the analysis-target nucleic acid, and identifies the base sequence of the analysis-target nucleic acid analysis. , by comparing the number of base sequences of the nucleic acid to be analyzed with the data of the calibration curve M141.

Next, the processing procedure of the nucleic acid analysis program of the present invention is shown. FIG. 26 is a flow chart showing an example of the processing procedure of the nucleic acid analysis program in the control section 130 of the nucleic acid analysis apparatus 100. As shown in FIG.

In step S101, the library preparation unit 131 of the control unit 130 of the nucleic acid analysis apparatus 100 outputs the reaction conditions for library preparation to the output unit 120 based on the information about the standard nucleic acid and the nucleic acid to be analyzed input from the input unit 110. , and the process proceeds to S102.
In step S102, the calibration curve data creation unit 132 of the control unit 130 of the nucleic acid analysis apparatus 100 creates calibration curve data of the standard nucleic acid based on the copy number of the standard nucleic acid of the specific copy number, and acquires the calibration curve M141. The result is recorded, and the process proceeds to S103. As the standard curve data, the "read number" of the standard nucleic acid in the system can be used.
In step S103, the analysis target nucleic acid analysis unit 133 of the control unit 130 of the nucleic acid analysis apparatus 100 identifies the base sequence of the analysis target nucleic acid, and uses the calibration curve data created from the calibration curve M141 to analyze the analysis target nucleic acid. is specified, the analyzed data is recorded in the analysis target nucleic acid M142, and this process is terminated.
Note that the processes of S102 and S103 may be performed in parallel.

Devices related to the nucleic acid analysis method of the present invention are widely used in bio-related industries, life science industries, medical industries, etc. It can be suitably used for accuracy control of sequence analysis equipment and the like.
As a device, the method stipulated in the official method, the notification method, etc. can be applied when it is carried out for infectious diseases.

(device for library preparation)
The library preparation device of the present invention is particularly suitable for library preparation used in the nucleic acid analysis method of the present invention, and has standard nucleic acids of a specific copy number.
Since the library preparation device of the present invention is the same as the device used in the nucleic acid analysis method of the present invention, description thereof will be omitted.
The library preparation device of the present invention can be suitably used in nucleic acid analysis involving next-generation sequencers.
In addition, the library preparation device of the present invention is represented by the coefficient of variation CV value obtained by dividing the uncertainty of the specific copy number of the standard nucleic acid by the average specific copy number, and the average specific copy number x of the standard nucleic acid. It is preferable to satisfy the following formula, CV<1/√x.

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

(Example 1)
<Fabrication of device for library preparation>
A device for library preparation was fabricated as follows.
-Preparation of standard nucleic acid-
--Design of artificial nucleotide sequences--
As an artificial base sequence, a concentrated nucleic acid sample DNA600-G (manufactured by National Institute of Advanced Industrial Science and Technology, NMIJ CRM 6205-a, see SEQ ID NO: 6) was prepared with a plasmid in which URA3 as a selection marker was arranged in tandem. made.
In addition, primers MiFish-U (see Non-Patent Document 1, manufacturer name: Fasmac Co., Ltd., see SEQ ID NOS: 7 and 8), a plasmid incorporating an artificial nucleotide sequence (see SEQ ID NOS: 1 to 5) synthesized to have a complementary nucleotide sequence was prepared. Thus, by having a base sequence complementary to the primer MiFish-U at both ends of the artificial base sequence, when analyzing the fish 12S rRNA contained in the nucleic acid to be analyzed, it has the same base sequence Standard nucleic acids and target nucleic acids can be analyzed using primers.

--Genetically modified yeast--
Saccharomyces cerevisiae YIL015W BY4741 (manufactured by ATCC, ATCC4001408) was used as a carrier cell for one copy of the specific nucleic acid sequence for preparation of the recombinant. By homologous recombination between the plasmid prepared above and the BAR1 region of the carrier cell, one copy of the specific artificial nucleic acid sequence was introduced into the yeast genomic DNA to prepare a genetically modified yeast. In addition, DNA600-G has uncertainty information regarding nucleic acid concentration as product information of DNA600-G.

--Culture and cell cycle control--
Dulbecco's phosphate-buffered saline (manufactured by Thermo Fisher Scientific Co., Ltd., 14190-144, hereinafter referred to as “DPBS”) was adjusted to 500 μg/mL α1-Mating Factor acetate salt (manufactured by Sigma-Aldrich, T6901-5MG, hereinafter referred to as “α factor”). 900 μL was added.
Next, using a bioshaker (BR-23FH, manufactured by Taitec Co., Ltd.), shaking speed: 250 rpm, temperature: 28 ° C. Incubate for 2 hours to synchronize the yeast to the G0 / G1 phase to obtain a yeast suspension. rice field.

-Immobilization-
Transfer 45 mL of the synchronized yeast suspension to a centrifuge tube (manufactured by AS ONE Corporation, VIO-50R) and centrifuge at a rotation speed of 3000 rpm for 5 minutes using a centrifuge (manufactured by Hitachi, Ltd., F16RN). , the supernatant was removed to obtain a yeast pellet.
4 mL of formalin (manufactured by Wako Pure Chemical Industries, Ltd., 062-01661) is added to the resulting yeast pellet, allowed to stand for 5 minutes, centrifuged to remove the supernatant, and 10 mL of ethanol is added to suspend. , to obtain an immobilized yeast suspension.

－Nuclear staining－
A 200 μL aliquot of the immobilized yeast suspension was taken, washed once with DPBS, and then resuspended in 480 μL of DPBS.
Next, 20 μL of 20 mg/mL RNase A (manufactured by Nippon Gene Co., Ltd., 318-06391) was added, followed by incubation at 37° C. for 2 hours using a bioshaker.
Next, 25 μL of 20 mg/mL proteinase K (Takara Bio Inc., TKR-9034) was added, and Petit Cool (Waken Bee Tech Co., Ltd., Petit Cool MiniT-C) was used to incubate at 50° C. for 2 hours. .
Finally, 6 μL of 5 mM SYTOX Green Nucleic Acid Stain (manufactured by Thermo Fisher Scientific, S7020) was added and stained for 30 minutes in the dark.

-dispersion-
The dyed yeast suspension was dispersed using an ultrasonic homogenizer (LUH150, manufactured by Yamato Scientific Co., Ltd.) at an output of 30% for 10 seconds to obtain a yeast suspension ink.

－Dispensing and cell counting－
The number of yeast cells in the droplet was counted in the following manner, and one cell was discharged into each well to prepare a plate with a known number of cells. Specifically, using the droplet forming apparatus shown in FIG. 15, a piezoelectric application type ejection head as a droplet ejection means is applied to each well of a 96 plate (trade name: MicroAmp 96-well Reaction plate, manufactured by Thermofisher). (manufactured in-house) was used to sequentially eject yeast suspension ink at 10 Hz.
A high-sensitivity camera (manufactured by Tokyo Instruments Co., Ltd., sCMOS pco.edge) was used as a light-receiving means for the yeast in the ejected droplets. YAG laser (manufactured by Spectra Physics, Explorer ONE-532-200-KE) is used as the light source, and image processing is performed using Image J, which is image processing software, as a means for counting the number of particles in the photographed image to determine the number of cells. Counted and made a known plate of 1 cell number.

－Nucleic acid extraction－
ColE1 DNA (manufactured by Wako Pure Chemical Industries, Ltd., 312-00434) was adjusted to 5 ng/μL using Tris-EDTA (TE) Buffer to prepare ColE1/TE, and Zymolyase ^(R) was added using ColE1/TE. A Zymolyase solution was prepared so that 100T (Nacalai Tesque, 07665-55) was 1 mg/mL.
Add 4 μL of the Zymolyase solution to each well of the prepared plate with a known number of cells and incubate at 37.2° C. for 30 minutes to lyse the cell wall (nucleic acid extraction), followed by heat treatment at 95° C. for 2 minutes to obtain a reference device ( A library preparation device) was prepared.

Next, in order to consider the reliability of the results obtained from the plate with a known number of cells, a plate with a known number of 1 cell is manufactured and the uncertainty in the number of 1 cell is calculated. By using the method described below for each specific copy number, the uncertainty at various copy numbers can be calculated.

－Calculation of uncertainty－
In this example, the number of cells in a droplet, the copy number of nucleic acids in cells, the number of cells in a well, and contamination were used as factors of uncertainty.
The number of cells in a droplet is obtained by analyzing the image of the droplet ejected by the ejection means and counting the number of cells in the droplet. The number of cells obtained by microscopic observation was used.
Nucleic acid copy number in cells (cell cycle) was calculated using the proportion of cells corresponding to the G1 phase of the cell cycle (99.5%) and the proportion of cells corresponding to the G2 phase (0.5%). .
The number of cells in the well was counted by the number of ejected droplets that landed in the well, but all the droplets landed in the well in the counting of 96 samples, so the factor of the number of cells in the well is uncertain. were excluded from the calculation of thickness.
For contamination, 4 μL of the filtrate of the ink was subjected to real-time PCR to confirm whether or not nucleic acid other than nucleic acid in the cells was mixed in the ink liquid by three trials. As a result, the lower limit of detection was obtained in all three tests, so the factor of contamination was also excluded from the uncertainty.
For uncertainty, the standard deviation is obtained from the measured value of each factor, multiplied by the sensitivity coefficient, and the combined standard uncertainty is obtained by the sum of squares method of the standard uncertainty standardized in the unit of the measured quantity. Since the combined standard uncertainty only includes values in the range of about 68% of the normal distribution, the expanded uncertainty is double the combined standard uncertainty to obtain the uncertainty that takes into account the range of about 95% of the normal distribution. Obtainable. Results are shown in the budget sheet in Table 2 below.

In Table 2, "symbol" means an arbitrary symbol associated with an uncertainty factor.
In Table 2, "value (±)" is the experimental standard deviation of the average value, which is obtained by dividing the calculated experimental standard deviation by the square root of the number of data.
In Table 2, "probability distribution" is the probability distribution of the factors of uncertainty. In the case of A-type uncertainty evaluation, it is blank, and in the B-type uncertainty evaluation, normal distribution or rectangular distribution Enter one. In this embodiment, only the A-type uncertainty evaluation is performed, so the probability distribution column is blank.
In Table 2, "divisor" means a number that normalizes the uncertainty obtained from each factor.
In Table 2, "standard uncertainty" is a value obtained by dividing "value (±)" by "divisor".
In Table 2, "sensitivity coefficient" means a value used for unifying the unit of measurement quantity.

Next, the average specific copy number and uncertainty of the standard nucleic acid (nucleotide sequence) filled in the wells were calculated. Table 3 shows the results. Coefficient of variation CV values were calculated by dividing the uncertainty value by the mean specific copy number.

In the inkjet method, the specific copy number of the standard nucleic acid was 1, that is, the accuracy of dispensing one copy of the standard nucleic acid (base sequence) (one yeast) into the well was ±0.1281 copies. . When filling wells with more than one copy, the precision with which a specific number of copies of a standard nucleic acid (base sequence) is filled is determined by this precision stack.

From the above results, by storing the expanded uncertainty obtained as an index of measurement variability as data in the device, the person using the experiment can use the uncertainty index as a criterion for the reliability of the measurement results for each well. can be used as By using the reliability criteria described above, it is possible to highly accurately evaluate the performance of the analytical test.

- Uncertainty pricing for each filling site -
The uncertainties (or coefficients of variation) calculated above were assigned to each well.
From the above, the average specific copy number of the series of low-concentration nucleic acid samples, its uncertainty, and the coefficient of variation were calculated, and values were assigned to each well.

(Example 2)
<Implementation of Nucleic Acid Analysis Method 1: Calculation of 12S rRNA Copy Number of Fish Muscle Tissue>
In Example 2, NGS analysis was performed using a DNA sample extracted from fish muscle tissue.
First, three types of fish muscle tissues, red sea bream (Pagrus major), salmon (Oncorhynchus mykiss), and sardine (Sardinops melanoticus) were prepared, and DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN). This was used as the nucleic acid to be analyzed.

^-1st PCR reaction-
Three types of fish 12S rRNA sequences (see SEQ ID NOs: 1 to 3) of artificial base sequences (standard nucleic acids) 1 to 3 designed in Example 1 were used in the wells, and wells with a nucleic acid copy number of 1 copy (1 well yeast cells), 5 copy wells (containing 5 yeast cells), 10 copy wells (containing 10 yeast cells), and 50 copy wells (containing 50 yeast cells), respectively. prepared. Each well was filled with three types of artificial base sequences (hereinafter sometimes referred to as artificial 12S sequences). That is, a well with a nucleic acid copy number of 1 copy contains 1 yeast cell containing SEQ ID NO: 1, 1 yeast cell containing SEQ ID NO: 2, and 1 yeast cell containing SEQ ID NO: 3, respectively. Other wells are similar. After that, 5.0 μL of the nucleic acid to be analyzed was filled into each of the sample filling wells. After that, the nucleic acid to be analyzed and the fish 12S rRNA sequence of the artificial base sequence were subjected to an amplification reaction by the PCR method in the same well. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 1.6 μL
・KAPA HiFi HotStart ReadyMix (2X) 12.0 μL
・1 ^st PCR primer F (10 μM) 0.7 μL
・1 ^st PCR primer R (10 μM) 0.7 μL
・Fish muscle tissue-derived extracted DNA (sample) 5.0 μL
・ Yeast DNA (including artificial base sequences 1 to 3 and Zymolyase 0.4 U) 4.0 μL
Total 24.0 μL

The 1 ^st PCR primer is MiFish-U (see Non-Patent Document 1) to which a sequence that allows the annealing reaction of the 2 ^nd PCR primer is added. Nucleic acid amplification was performed by PCR using T100 ^™ Thermal Cycler (Bio-rad). First, incubation was performed at 95°C for 3 minutes. After that, 35 temperature cycles consisting of 3 steps of 98° C. for 20 seconds, 65° C. for 15 seconds, and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-2 ^nd PCR reaction: Binding of adapter sequence- A PCR reaction is performed to add a tag for distinguishing samples for sequencing and an adapter sequence for subjecting to a sequencing reaction to both ends of the obtained ^1st PCR amplified product, A 2 ^nd PCR reaction product was obtained. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 6.0 μL
・KAPA HiFi HotStart ReadyMix (2X) 12.0 μL
・^2nd PCR primer F (10 μM) 1.0 μL
・^2nd PCR primer R (10 μM) 1.0 μL
・ 2.0 μL of 1 ^st PCR product
Total 20.0 μL

Amplification of nucleic acids was performed by PCR using T100 ^™ Thermal Cycler (Bio-rad). First, incubation was performed at 95°C for 3 minutes. After that, 12 temperature cycles consisting of two steps of 98° C. for 20 seconds and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

Purification of PCR products by agarose gel electrophoresis Electrophoresis was performed at 100 V for 20 minutes using a 2% agarose gel. A band observed from 330 to 400 bp was excised, and a PCR product was generated using FastGene Gel/PCR Extraction Kit (manufactured by Nippon Genetics Co., Ltd.).

-Measurement of Concentration of Nucleic Acid Sample- 2 ^nd PCR products were quantified using Bioanalyzer 2100 (manufactured by Agilent Technologies). The kit used was the Agilent DNA7500 kit. Based on the quantitative results, the 2 ^nd PCR product was diluted with TE to 10 ng/μL. The diluted 2nd PCR products obtained from 4 wells were mixed in the same reaction solution.

-Sequencing reaction by next-generation sequencer (NGS)- Using a next-generation sequencer (device name: Miseq, manufactured by Illumina), the obtained ^2nd PCR product was analyzed. The data obtained by the next-generation sequencer was analyzed by sequence processing to obtain information on the base sequence and the number of reads. The data obtained are shown in Table 4. Numerical values in the table indicate the number of reads.

--Normalization of Reads-- Table 5 shows normalized reads, with the sum of reads other than the artificial 12S sequence being 100,000 reads. Numerical values in the table indicate the number of reads.

Based on the read numbers of the artificial 12S sequences 1, 2, and 3 in Table 5, a relational expression between the copy number (x) and the output read number (y) was derived, and the formula y = 31.343x was obtained (determined count ^R2 = 0.9612). Based on this formula, the copy number of each fish species was estimated, and Table 6 was obtained. Numerical values in the table indicate copy numbers.

(Example 3)
<Implementation of nucleic acid analysis method 2: Measurement of fish fauna using environmental DNA>
In Example 3, fish fauna was measured using environmental DNA in the Sagami River.
First, a water sample was collected from the Sagami River and filtered using a filter. DNA was extracted from the filtered filter using a DNA extraction kit (trade name: DNeasy Blood & Tissue kit, manufactured by QIAGEN). The nucleic acid concentration of the extracted DNA sample (analyzed nucleic acid) was quantified using a Qubit 3 fluorometer (Invitrogen ^™ ).

^-1st PCR reaction-
Five fish 12S rRNA sequences (see SEQ ID NOs: 1 to 5) of the artificial base sequences (standard nucleic acids) 1 to 5 designed in Example 1 were used in the wells, and artificial base sequence 1, the fish 12S rRNA sequence ( 1 (copy) of yeast containing artificial base sequence 2 (see SEQ ID NO: 1), 10 yeast (copy) containing fish 12S rRNA sequence (see SEQ ID NO: 2) that is artificial base sequence 2, and fish 12S rRNA that is artificial base sequence 3 50 (copies) of yeast containing the sequence (see SEQ ID NO: 3), 100 (copies) of yeast containing the fish 12S rRNA sequence (see SEQ ID NO: 4) that is the artificial base sequence 4, and the fish that is the artificial base sequence 5 500 copies (copy) of yeast containing the 12S rRNA sequence (see SEQ ID NO: 5) were packed in the same manner as in Example 1 above. 2.0 μL (0.25 ng/μL) of an extracted DNA sample collected from a water sample from the Sagami River was filled in the wells to prepare a device in which 5 levels of standard nucleic acids were arranged. Thereafter, the extracted DNA sample and the artificial base sequences 1 to 5 were subjected to an amplification reaction by PCR in the same well. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 1.6 μL
・KAPA HiFi HotStart ReadyMix (2X) 12.0 μL
・1 ^st PCR primer F (10 μM) 0.7 μL
・1 ^st PCR primer R (10 μM) 0.7 μL
・ Sagami River-derived environmental DNA extract (sample) 5.0 μL
・ Yeast DNA (including artificial base sequences 1 to 5 and Zymolyase 0.4 U) 4.0 μL
Total 24.0 μL

The 1 ^st PCR primer is MiFish-U (see Non-Patent Document 1) to which a sequence that allows the annealing reaction of the 2 ^nd PCR primer is added.
Amplification of standard nucleic acids (artificial base sequences 1 to 5) was carried out by PCR using a Thermal Cycler (apparatus name: T100 ^™ , manufactured by Bio-rad). First, incubation was performed at 95°C for 3 minutes. After that, 35 temperature cycles consisting of 3 steps of 98° C. for 20 seconds, 65° C. for 15 seconds, and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-Generation of PCR products by beads-
PCR product generation was performed using AMPure XP reagent (Beckman Coulter). First, the AMPure XP reagent was allowed to stand at room temperature for 30 minutes or longer before use. After mixing the AMPure XP reagent by inversion for 1 minute or more, 20 μL of the AMPure XP reagent was added to each well in which the PCR reaction was performed. The pipetting was repeated 10 times to thoroughly mix the PCR reaction solution and the AMPure XP reagent. After that, it was allowed to stand at room temperature for 5 minutes. Each well in which the PCR reaction was performed was placed on a Magnet Plate and allowed to stand for 2 minutes. The PCR reaction solution was removed using a pipette so as not to touch the magnetic beads contained in the AM Pure XP reagent while each well in which the PCR reaction was performed was placed on the Magnet Plate. 200 μL of 70% ethanol was added to each well and allowed to stand for 30 seconds. Ethanol was removed to wash the magnetic beads. The washing step was repeated once more. The washing step was performed while each well was placed on the Magnet Plate. Remove each well from the Magnet Plate, add 20 μL of elution buffer (purified water, Tris acetate pH 8.0, or Tris/EDTA solution) to each well, repeat pipetting 10 times, and remove magnetic beads and elution buffer sufficiently. mixed in. Each well was placed on the Magnet Plate and allowed to stand for 1 minute. With each well still on the Magnet Plate, the elution buffer was collected and transferred to another container. At this time, it is preferable to transfer to a PCR reaction vessel for the next step.

-2 ^nd PCR reaction: Binding of adapter sequences-
A PCR reaction was performed in which a tag for distinguishing samples for sequencing and an adapter sequence for sequencing reaction were added to both ends of the obtained ^1st PCR amplification product to obtain a ^2nd PCR reaction product. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 6.0 μL
・KAPA HiFi HotStart ReadyMix (2X) 10.0 μL
・^2nd PCR primer F (10 μM) 1.0 μL
・^2nd PCR primer R (10 μM) 1.0 μL
・2.0 μL of ^1st PCR product
Total 20.0 μL

A Thermal Cycler (apparatus name: T100 ^™ , manufactured by Bio-rad) was used for amplification of the ^2nd PCR product. First, incubation was performed at 95°C for 3 minutes. After that, 12 temperature cycles consisting of two steps of 98° C. for 20 seconds and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-Purification of PCR products by beads-
Since it is the same as the step performed after ^1st PCR, it is omitted.

-Concentration measurement of nucleic acid sample-
The 2 ^nd PCR product was quantified using Bioanalyzer 2100 (manufactured by Agilent Technologies). The kit used was the Agilent DNA7500 kit. Based on the quantitative results, the 2 ^nd PCR product was diluted with TE to 10 ng/μL.

- Sequencing reaction by next-generation sequencer (NGS) -
The obtained 2 ^nd PCR product was analyzed using a next-generation sequencer (device name: Miseq, manufactured by Illumina). The data obtained by the next-generation sequencer were analyzed by sequence processing to obtain information on the base sequence and the number of reads. The data obtained are shown in Table 7.

-Preparation and quantification of calibration curve-
Based on the results of amplification of artificial sequences (artificial base sequences) 1 to 5, which are standard nucleic acids shown in Table 7, a calibration curve was prepared. The prepared calibration curve is shown in FIG. In FIG. 27, the vertical axis represents the number of Miseq reads, and the horizontal axis represents the number of nucleic acids to be analyzed and standard nucleic acids (DNA) (copy number/well). The points represented by squares are the added artificial sequences, which are 1 copy, 10 copies, 50 copies, 100 copies and 500 copies, respectively. Based on these 5 points (5 levels), a calibration curve was created. Circled points are the number of reads obtained from standard nucleic acids plotted on the standard curve. As shown in FIG. 27, Numatitibu was the dominant species in the Sagami River and accounted for 70% of all samples. For other fish species, the number of reads was less than 4778, which is the number of reads of 500 copies, and quantification by interpolation was possible (reliable quantification using the numerical range used in the calibration curve was possible). The copy number of each fish species estimated from the standard curve is shown in Table 7.

(Example 4)
<Implementation of nucleic acid analysis method 3: Measurement of fish fauna using environmental DNA>
In Example 4, fish fauna was measured using environmental DNA in the Sagami River.
First, a water sample was collected from the Sagami River and filtered using a filter. DNA was extracted from the filtered filter using a DNA extraction kit (trade name: DNeasy Blood & Tissue kit, manufactured by QIAGEN). The nucleic acid concentration of the extracted DNA sample (analyzed nucleic acid) was quantified using a Qubit 3 fluorometer (Invitrogen ^™ ).

^-1st PCR reaction-
Using three types of fish 12S rRNA sequences (see SEQ ID NOs: 1 to 3) of artificial base sequences (standard nucleic acids) 1 to 3 designed in Example 1 above, wells with a nucleic acid copy number of 1 copy (1 well yeast cells), 5 copy wells (containing 5 yeast cells), 10 copy wells (containing 10 yeast cells), and 50 copy wells (containing 50 yeast cells), respectively. prepared. Each well was filled with triplicate artificial 12S sequences. That is, a well with a nucleic acid copy number of 1 copy contains 1 yeast cell containing SEQ ID NO: 1, 1 yeast cell containing SEQ ID NO: 2, and 1 yeast cell containing SEQ ID NO: 3, respectively. Other wells are similar. After that, 5.0 μL of the nucleic acid to be analyzed was filled into each of the sample filling wells. After that, the nucleic acid to be analyzed and the artificial 12S were subjected to an amplification reaction by the PCR method in the same well. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 1.6 μL
・KAPA HiFi HotStart ReadyMix (2X) 12.0 μL
・1 ^st PCR primer F (10 μM) 0.7 μL
・1 ^st PCR primer R (10 μM) 0.7 μL
・ Sagami River-derived environmental DNA extract (sample) 5.0 μL
・ Yeast DNA (including artificial base sequences 1 to 3 and Zymolyase (registered trademark) 0.4 U) 4.0 μL
Total 24.0 μL

The 1 ^st PCR primer is MiFish-U (see Non-Patent Document 1) to which a sequence that allows the annealing reaction of the 2 ^nd PCR primer is added.
Nucleic acid amplification was performed by PCR using T100 ^™ Thermal Cycler (Bio-rad). First, incubation was performed at 95°C for 3 minutes. After that, 35 temperature cycles consisting of 3 steps of 98° C. for 20 seconds, 65° C. for 15 seconds, and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-2 ^nd PCR reaction: Binding of adapter sequence-
A PCR reaction was performed in which a tag for distinguishing samples for sequencing and an adapter sequence for sequencing reaction were added to both ends of the obtained ^1st PCR amplification product to obtain a ^2nd PCR reaction product. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 6.0 μL
・KAPA HiFi HotStart ReadyMix (2X) 10.0 μL
・^2nd PCR primer F (10 μM) 1.0 μL
・^2nd PCR primer R (10 μM) 1.0 μL
・2.0 μL of ^1st PCR product
Total 20.0 μL

Amplification of nucleic acids was performed by PCR using T100 ^™ Thermal Cycler (Bio-rad). First, incubation was performed at 95°C for 3 minutes. After that, 12 temperature cycles consisting of two steps of 98° C. for 20 seconds and 72° C. for 15 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-Purification of PCR products by agarose gel electrophoresis-
Electrophoresis was performed at 100 V for 20 minutes using a 2% agarose gel. A band observed from 330 to 400 bp was excised, and a PCR product was generated using FastGene Gel/PCR Extraction Kit (Nippon Genetics Co., Ltd.).

-Concentration measurement of nucleic acid sample-
The 2 ^nd PCR product was quantified using Bioanalyzer 2100 (manufactured by Agilent Technologies). The kit used was the Agilent DNA7500 kit. Based on the quantitative results, the 2 ^nd PCR product was diluted with TE to 10 ng/μL. The diluted 2nd PCR products obtained from 4 wells were mixed in the same reaction solution.

- Sequencing reaction by next-generation sequencer (NGS) -
The obtained 2 ^nd PCR product was analyzed using a next-generation sequencer (device name: Miseq, manufactured by Illumina). The data obtained by the next-generation sequencer were analyzed by sequence processing to obtain information on the base sequence and the number of reads. The data obtained are shown in Table 8. Numerical values in the table indicate the number of reads.

- Read normalization -
Table 9 shows the normalized sum total of reads other than artificial 12S as 100,000 reads. Numerical values in the table indicate the number of reads. In addition, Table 10 shows the calculated mean values and coefficient of variation CV for the artificial 12S sequences 1, 2, and 3. Using the average value and the coefficient of variation CV in Table 10, accuracy control of the analysis can be appropriately performed.

Based on the number of reads of the artificial 12S sequences 1, 2, and 3 in Table 9, a relational expression between the number of copies (x) and the number of output reads (y) was derived. ^R2 = 0.9884). Based on this formula, the copy number of each fish species was estimated, and Table 12 was obtained. Numerical values in the table indicate copy numbers. Using the coefficient of determination ^R2 of the obtained calibration curve, the accuracy of the analysis can be appropriately controlled. Moreover, the prepared calibration curve is shown in FIG. For the sake of simplification, the fish species shown in FIG. 29 are averaged from four wells, and fish species estimated to have 1 copy or less are excluded. For reference, Table 11 shows the number of reads and the number of copies of each fish species used when creating FIG.

(Example 5)
<Implementation of nucleic acid analysis method 4: Measurement of microbiota using a mixed DNA sample of microorganisms>
In Example 5, microbial flora was measured using a mixed microbial DNA sample (ZymoBIOMICS Microbial Community DNA Standard (manufactured by Zymo Research)).
In Example 3, artificial base sequences 1 to 5, which are standard nucleic acids, were placed in the same well as the well in which the nucleic acid sample, which is the nucleic acid to be analyzed, was placed. A nucleic acid having an artificial base sequence of -G was arranged, and the amplification results were integrated to prepare a calibration curve.

- Preparation of nucleic acid to be analyzed (nucleic acid sample) -
The wells were filled with yeast containing DNA600-G prepared in Example 1 in the same manner as in Example 1 above. The yeast was dispensed into 4 wells with 1 (copy), 5 (copies), 10 (copies) and 50 (copies), respectively, into 4 different wells. Next, 2.0 μL (0.5 pg/μL) of the mixed DNA sample of the microorganism was manually filled into the wells containing the nucleic acid having the artificial base sequence of DNA600-G. After that, in the well, the mixed DNA sample of the microorganism and the DNA600-G were subjected to an amplification reaction by the PCR method in the same well. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 6.3 μL
・10x Ex Taq buffer 2.0 μL
・dNTP (2.5 mM) 1.6 μL
・ 1 ^st PCR primer F (10 μM) for microbial 16S amplification 1.0 μL
・ 1 ^st PCR primer R (10 μM) for microbial 16S amplification 1.0 μL
・ 1 ^st PCR primer F (10 μM) for 600 G amplification 1.0 μL
・^1st PCR primer R for 600G amplification (10 μM) 1.0 μL
・ Nucleic acid to be analyzed (mixed DNA sample of microorganisms) 2.0 μL
・Ex Taq (5 units/μL) 0.1 μL
・Standard nucleic acid (including DNA600-G, Zymolyase 0.4U) 4.0μL
Total 20.0 μL

The nucleic acid to be analyzed (mixed DNA sample of microorganisms) and the standard nucleic acid (DNA600-G, artificial sequence 6) were amplified by PCR using a Thermal Cycler (device name: T100 ^™ , Bio-rad). First, incubation was performed at 94°C for 2 minutes. After that, 23 temperature cycles consisting of 3 steps of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-Generation of PCR products by beads-
PCR product generation was performed using AMPure XP reagent (Beckman Coulter). First, the AMPure XP reagent was allowed to stand at room temperature for 30 minutes or longer before use. After mixing the AMPure XP reagent by inversion for 1 minute or longer, 20 μL of the reagent was added to each well in which the PCR reaction was performed. The pipetting was repeated 10 times to thoroughly mix the PCR reaction solution and the AMPure XP reagent. After that, it was allowed to stand at room temperature for 5 minutes. Each well in which the PCR reaction was performed was placed on a Magnet Plate and allowed to stand for 2 minutes. The PCR reaction solution was removed using a pipette so as not to touch the magnetic beads contained in the AM Pure XP reagent while each well in which the PCR reaction was performed was placed on the Magnet Plate. 200 μL of 70% ethanol was added to each well and allowed to stand for 30 seconds. Ethanol was removed to wash the magnetic beads. The washing step was repeated once more. The washing step was performed while each well was placed on the Magnet Plate. Remove each well from the Magnet Plate, add 20 uL of elution buffer (purified water, Tris acetate pH 8.0, or Tris/EDTA solution) to each well, repeat pipetting 10 times, and remove the magnetic beads and elution buffer. well mixed. Each well was placed on the Magnet Plate and allowed to stand for 1 minute. With each well still on the Magnet Plate, the elution buffer was collected and transferred to another container. At this time, it is preferable to transfer to a PCR reaction vessel for the next step.

-2 ^nd PCR reaction: Binding of adapter sequence-
A PCR reaction was performed in which a tag for distinguishing samples for sequencing and an adapter sequence for sequencing reaction were added to both ends of the obtained ^1st PCR amplification product to obtain a ^2nd PCR reaction product. The composition of the reaction solution is as follows.

[Reaction liquid composition]
・Distilled water 10.3 μL
・10x Ex Taq buffer 2.0 μL
・dNTP (2.5 mM) 1.6 μL
・^2nd PCR primer F (10 μM) 1.0 μL
・^2nd PCR primer R (10 μM) 1.0 μL
・ 4.0 μL of 1 ^st PCR product
・Ex Taq (5 units/μL) 0.1 μL
Total 20.0 μL

A Thermal Cycler (apparatus name: T100 ^™ , manufactured by Bio-rad) was used for amplification of the ^2nd PCR product. First, incubation was performed at 94°C for 2 minutes. After that, 8 temperature cycles consisting of 3 steps of 94° C. for 30 seconds, 50° C. for 30 seconds and 72° C. for 30 seconds were performed. After a final incubation at 72°C for 5 minutes, the reaction was terminated by cooling to 4°C.

-Purification of PCR products by beads-
Since it is the same as the step performed after ^1st PCR, it is omitted.

-Concentration measurement of nucleic acid sample-
The 2 ^nd PCR product was quantified using Bioanalyzer 2100 (manufactured by Agilent Technologies). The kit used was the Agilent DNA7500 kit. Based on the quantitative results, the 2 ^nd PCR product was diluted with TE to 10 ng/μL. Dilutions of the 2 ^nd PCR products obtained from 4 wells were mixed in the same reaction solution.

- Sequencing reaction by next-generation sequencer (NGS) -
The 2 ^nd PCR product was analyzed using a next-generation sequencer (device name: Miseq, manufactured by Illumina). The data obtained by the next-generation sequencer were analyzed by sequence processing to obtain information on the base sequence and the number of reads. The number of reads was normalized as 1 million reads obtained. The sum of normalized reads for the 4 samples is shown in Table 13.

-Preparation and quantification of calibration curve-
A standard curve was prepared based on the results of amplification of the standard nucleic acid DNA600-G (artificial sequence 6) shown in Table 13. The prepared calibration curve is shown in FIG. In FIG. 28, the vertical axis is the number of reads obtained by adding the number of reads obtained by normalizing the number of reads of Miseq to 1 million reads per well for 4 wells, and the horizontal axis is the number of DNA (copy number/4 wells). ). Square dots represent artificial sequences added to each well, 1 copy, 5 copies, 10 copies, and 50 copies, respectively. Based on these 4 points (4 levels), a calibration curve was drawn. Circled points are plotted on the standard curve based on the number of normalized reads obtained from nucleic acid samples. Regarding the 8 types of microorganisms contained in the mixed DNA sample of microorganisms that are nucleic acids to be analyzed, the number of reads is less than 92919 reads of 50 copies, and quantification by interpolation is possible (using the numerical range used in the calibration curve Reliable quantification was possible). The copy number of each microorganism estimated from the standard curve is listed in Table 13.

Embodiments of the present invention are, for example, as follows.
<1> A library preparation step of preparing a library containing a standard nucleic acid with a specific number of copies and a nucleic acid to be analyzed in the same system;
a calibration curve data creation step of creating calibration curve data based on the number of copies of the standard nucleic acid with a specific number of copies;
a nucleic acid analysis step of specifying the base sequence of the analysis target nucleic acid and specifying the number of base sequences of the analysis target nucleic acid using the calibration curve data. .
<2> The nucleic acid analysis method according to <1> above, wherein the standard nucleic acids having different base sequences are contained in the same system at different specific copy numbers.
<3> comprising the standard nucleic acid with different specific copy numbers in two or more different systems,
The nucleic acid analysis method according to any one of <1> to <2>, wherein the calibration curve data of the standard nucleic acids to be obtained are normalized and combined.
<4> The nucleic acid analysis method according to any one of <1> to <3>, wherein the standard nucleic acid is DNA.
<5> The nucleic acid analysis method according to any one of <1> to <4>, wherein the nucleic acid to be analyzed is at least one of DNA and cDNA.
<6> The nucleic acid analysis method according to any one of <1> to <5>, wherein the library is prepared using the same primers for the standard nucleic acid and the nucleic acid to be analyzed.
<7> The nucleic acid analysis method according to any one of <1> to <5>, wherein the preparation of the library is performed using different primers for the standard nucleic acid and the nucleic acid to be analyzed.
<8> For a library in which a standard nucleic acid with a specific number of copies and a nucleic acid to be analyzed are prepared in the same system,
creating standard curve data for the standard nucleic acid based on the data for the standard nucleic acid of a specific number of copies by the standard curve data creating means;
A computer is caused to execute a process of specifying the base sequence of the nucleic acid to be analyzed by means for analyzing the nucleic acid to be analyzed and specifying the number of base sequences of the nucleic acid to be analyzed using the calibration curve data. It is a nucleic acid analysis program.
<9> A library preparation device for use in the nucleic acid analysis method according to any one of <1> to <7>,
A library preparation device characterized by having a specific copy number of a standard nucleic acid.
<10> The following formula, represented by the coefficient of variation CV value obtained by dividing the uncertainty that the standard nucleic acid is the specific copy number by the average specific copy number, and the average specific copy number x of the standard nucleic acid, CV < 1 The device for library preparation according to <9> above, which satisfies /√x.
<11> The device for library preparation according to any one of <9> to <10>, wherein the standard nucleic acid of the specific number of copies is arranged by an inkjet method.

The nucleic acid analysis method according to any one of <1> to <7>, the nucleic acid analysis program according to <8>, and the library preparation device according to any one of <9> to <11>, , the above-mentioned problems in the conventional art can be solved and the object of the present invention can be achieved.

1 device 2 substrate 3 well 4 nucleic acid 5 sealing member

JP 2015-204813 A

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. Miya, et. al. , 2015

Claims (7)

A library preparation step of preparing a library containing a standard nucleic acid with a specific copy number and a nucleic acid to be analyzed in the same system;
a calibration curve data creation step of creating calibration curve data based on the number of copies of the standard nucleic acid with a specific number of copies;
an analysis target nucleic acid analysis step of specifying the base sequence of the analysis target nucleic acid and specifying the number of base sequences of the analysis target nucleic acid using the calibration curve data,
A library preparation device used in a nucleic acid analysis method in which the specific copy number is a controlled value that does not depend on Poisson distribution even if it is 1,000 or less,
having a specific copy number of a standard nucleic acid,
The following formula, represented by the coefficient of variation CV value obtained by dividing the uncertainty that the standard nucleic acid is the specific copy number by the average specific copy number, and the average specific copy number x of the standard nucleic acid, CV < 1/√x The filling,
A device for library preparation, wherein the standard nucleic acid is integrated into the genome of a cell whose cell cycle has been arranged . 2. The device for library preparation according to claim 1, wherein standard nucleic acids having different base sequences are included in the same system in specific copy numbers different from each other. comprising the standard nucleic acid at specific copy numbers different from each other in two or more different systems;
2. The device for library preparation according to claim 1, wherein the calibration curve data of the standard nucleic acids to be obtained are normalized and combined. 4. The library preparation device according to any one of claims 1 to 3, wherein the standard nucleic acid is DNA. 5. The library preparation device according to any one of claims 1 to 4, wherein the nucleic acid to be analyzed is at least one of DNA and cDNA. 6. The library preparation device according to any one of claims 1 to 5, wherein the library is prepared using the same primers for the standard nucleic acid and the nucleic acid to be analyzed. 6. The library preparation device according to any one of claims 1 to 5, wherein the library is prepared using different primers for the standard nucleic acid and the nucleic acid to be analyzed.