JP7259279B2 - DEVICE, INSPECTION APPARATUS PERFORMANCE EVALUATION PROGRAM, INSPECTION APPARATUS PERFORMANCE EVALUATION METHOD, AND INSPECTION APPARATUS PERFORMANCE EVALUATION APPARATUS - Google Patents

Description

The present invention relates to a device, a performance evaluation program for an inspection apparatus, a performance evaluation method for an inspection apparatus, and a performance evaluation apparatus for an inspection apparatus.

In recent years, real-time polymerase chain reaction (real-time PCR) has become more and more important in the qualitative and quantitative analysis of genetic tests, and its performance evaluation, especially performance evaluation between devices and facilities, is important. At present, a calibration curve based on an external standard using absorbance is used, but it does not accurately define the absolute number of nucleic acids, and its influence is particularly pronounced at low copy numbers.

Nucleic acid sample series for such a calibration curve are prepared by serial dilution of nucleic acid samples of known concentrations. For example, a method has been proposed in which a DNA fragment having a specific base sequence is subjected to limiting dilution, and from the results of real-time PCR of the resulting diluted solution, a diluted solution containing the desired copy number is selected (see, for example, Patent Document 1).
Recently, a method for preparing a sample containing one copy of a DNA having a target base sequence has been proposed by introducing a DNA fragment of a specific copy number into cells by genetic recombination technology and isolating the cultured cells by manipulation. (See, for example, Patent Document 2).

SUMMARY OF THE INVENTION An object of the present invention is to provide a device capable of properly evaluating the in-plane uniformity of an inspection apparatus, the performance between inspection apparatuses, and the performance between inspection facilities.

A device of the present invention as a means for solving the above problems is a group of wells having a plurality of wells in which a specific copy number of an amplifiable reagent is arranged, wherein a specific copy of the amplifiable reagent is It has two or more groups of the wells whose numbers are different from each other.

ADVANTAGE OF THE INVENTION According to this invention, the device which can evaluate the in-plane uniformity of an inspection apparatus, the performance between inspection apparatuses, and the performance between inspection facilities appropriately can be provided.

FIG. 1 is a perspective view showing an 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 plan view showing an example of the device of the present invention. FIG. 4 is a plan view showing another example of the device of the present invention. FIG. 5 is a plan view showing another example of the device of the present invention. FIG. 6 is a plan view showing another example of the device of the present invention. FIG. 7 is a plan view showing another example of the device of the present invention. 8 is a diagram showing an example of a calibration curve created using the device of FIG. 7. FIG. FIG. 9 is a plan view showing another example of the device of the present invention. FIG. 10 is a plan view showing another example of the device of the present invention. FIG. 11 is a plan view showing another example of the device of the present invention. FIG. 12 is a plan view showing another example of the device of the present invention. FIG. 13 is a plan view showing another example of the device of the present invention. FIG. 14 is a graph showing an example of the relationship between the frequency of DNA-replicated cells and fluorescence intensity. FIG. 15A is a schematic diagram showing an example of an electromagnetic valve type ejection head. FIG. 15B is a schematic diagram showing an example of a piezo ejection head. FIG. 15C is a schematic diagram of a modification of the piezoelectric ejection head in FIG. 15B. FIG. 16A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. FIG. 16B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 17A is a schematic diagram showing an example of the state of droplets. FIG. 17B is a schematic diagram showing an example of the state of droplets. FIG. 17C is a schematic diagram showing an example of the state of droplets. FIG. 18 is a schematic diagram showing an example of a dispensing device for causing liquid droplets to land sequentially in wells. FIG. 19 is a schematic diagram showing an example of a droplet forming device. FIG. 20 is a diagram illustrating hardware blocks of control means of the droplet forming apparatus of FIG. 21 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. 19. FIG. FIG. 22 is a flow chart showing an example of the operation of the droplet forming device. FIG. 23 is a schematic diagram showing a modification of the droplet forming device. FIG. 24 is a schematic diagram showing another modification of the droplet forming device. FIG. 25A is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 25B is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 26 is a diagram illustrating the relationship between the luminance value Li when particles do not overlap and the luminance value Le that is actually measured. FIG. 27 is a schematic diagram showing another modification of the droplet forming device. FIG. 28 is a schematic diagram showing another example of the droplet forming device. FIG. 29 is a schematic diagram showing an example of a method of counting cells passing through a microchannel. FIG. 30 is a schematic diagram showing an example of a method of acquiring an image of the vicinity of the nozzle portion of the ejection head. FIG. 31 is a graph showing the relationship between the probability P (>2) and the average number of cells. FIG. 32 is a block diagram showing an example of a hardware configuration of a performance evaluation device for an inspection device; 33 is a diagram illustrating an example of a functional configuration of a performance evaluation device for an inspection device; FIG. FIG. 34 is a flow chart showing an example of performance evaluation program processing of the inspection apparatus. FIG. 35 is a graph showing the relationship between the copy number with variations based on the Poisson distribution and the coefficient of variation CV.

(device)
The device of the present invention is a group of wells having a plurality of wells, wherein a specific copy number of an amplifiable reagent is arranged, wherein the specific copy number of the amplifiable reagent is different from each other. has two or more.

In the conventional dilution method, the device of the present invention has a part that is not stochastically filled and the absolute number of copies to be arranged is only 1 copy, so it cannot be arranged according to the design value. It is based on the knowledge that it cannot be used for evaluation.
In addition, the device of the present invention is a technology related to a sample solution preparation method in the conventional method using a manipulator, and does not describe or suggest performance evaluation of an inspection device. It is based on the knowledge that there is

According to the device of the present invention, a group of wells in which a specific copy number of an amplifiable reagent is placed in a plurality of wells, and two groups of wells in which the specific copy number of the amplifiable reagent is different from each other are provided. By having the above, it is possible to properly evaluate the in-plane uniformity of the inspection device, the performance between inspection devices, and the performance between inspection facilities.

<Well>
The shape, number, volume, material, color, etc. of the wells are not particularly limited, and can be appropriately selected according to the purpose.
The shape of the well is not particularly limited as long as a reagent containing a reagent capable of amplifying a specific copy number can be placed therein, and can be appropriately selected according to the purpose. Recesses such as bottoms, compartments on a substrate, and the like.
The number of wells is preferably 2 or more, more preferably 5 or more, and even more preferably 50 or more.
A multiwell plate having two or more wells is preferably used.
Multiwell plates include, for example, 24, 48, 96, 384, or 1,536 well plates.
The volume of the well 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 10 μL or more and 1,000 μL or less.
The material of the well is not particularly limited and can be appropriately selected depending on the purpose. .
Well colors include, for example, transparent, translucent, colored, and completely shaded.
The wettability of the well is not particularly limited and can be appropriately selected according to the purpose. For example, water repellency is preferable. If the wettability of the well is water repellent, adsorption of the amplifiable reagent to the inner wall of the well can be reduced. In addition, when the wettability of the well is water repellent, the amplifiable reagent, primers, and amplification reagent within the well can move in a solution state.
The method for imparting water repellency to the inner wall of the well is not particularly limited and can be appropriately selected depending on the intended purpose. In particular, by applying a water-repellent treatment with a contact angle of 100° or more, it is possible to suppress the decrease in the amplifiable reagent and the increase in uncertainty (or coefficient of variation) due to spillage of the liquid.

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

<Identification Means>
The device preferably has identification means capable of identifying specific copy number information of the amplifiable reagent.
The identification means is not particularly limited and can be appropriately selected depending on 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 the amplifiable reagent, analysis results (e.g., activity value, luminescence intensity, etc.), the number of amplifiable reagents (e.g., the number of cells ), life and death of cells, number of copies of a specific nucleotide sequence, which well among multiple wells is filled with an amplifiable reagent, the type of amplifiable reagent, the date and time of measurement, the name of the person doing the measurement, etc. .
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 counting droplet forming 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 depending on the purpose. Examples thereof include a sealing member.

-sealing member-
The device preferably has a sealing member to prevent contamination of the wells, spillage of the filler, and the like.
It is preferable that the sealing member can seal at least one well and be separable by a perforation line so that each well can be individually sealed or unsealed.
The shape of the sealing member is preferably a cap-like shape that matches the inner wall diameter of the well, 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 wells at once. In addition, it is preferable that the wells requiring re-opening and the wells not requiring re-opening have different adhesive strengths so as to reduce misuse by the user.

A device of the present invention has a plurality of wells and reagents containing a specific copy number of an amplifiable reagent are placed in the plurality of wells.

Copy number refers to the number of targets or specific base sequences in the amplifiable reagents contained in the well.
The target nucleotide sequence refers to at least the nucleotide sequences of the primer and probe regions that have been determined, and in particular, those that have determined the entire length of the nucleotide sequence are also referred to as specific nucleotide sequences.
A specific copy number means that the number of target base sequences among the copy numbers is specified with at least a certain degree of accuracy.
That is, it can be said that it is known as the number of target base sequences actually contained in the well. That is, the specific copy number in the present application has higher accuracy and reliability as a number than the predetermined copy number (calculated estimated value) obtained by conventional serial dilution, especially in a low copy number region of 1,000 or less. Even if there is, it will be a controlled value that does not depend on the Poisson distribution. As for the controlled value, it is preferable that the coefficient of variation CV, which represents the uncertainty, falls within either CV < 1/√x or CV ≤ 20% with respect to the average copy number x. . Therefore, by using a device having wells containing the specific copy number of the target nucleotide sequence, it becomes possible to perform qualitative and quantitative testing of a sample having the target nucleotide sequence more accurately than before. .
Here, when the number of target base sequences and the number of molecules of the nucleic acid having that sequence match, the "number of copies" and the "number of molecules" may be associated.
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 the present invention, the specific copy number of the amplifiable reagent is also referred to as the absolute number of the amplifiable reagent.
However, if there are multiple wells containing amplifiable reagents, the specific copy number of amplifiable reagents contained within each well may be the same.
Identical specific copy numbers of the amplifiable reagents means that variations in the number of amplifiable reagents that occur when the device is filled with the amplifiable reagents are within an acceptable range. Whether the variation in the number of amplifiable reagents is within the allowable range can be determined based on the uncertainty information shown below.

The information on the specific copy number of the amplifiable reagent is not particularly limited as long as it is information on the amplifiable reagent in the device, and can be appropriately selected according to the purpose. Examples include carrier information, amplifiable reagent information, and the like.

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 Measurement Uncertainty in Tests. . Methods for calculating uncertainty include, for example, Type A evaluation methods using statistics such as measured values, and uncertainty information obtained from calibration certificates, manufacturer's specifications, published information, etc. 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 average value obtained from the measurements.
As an example of a method of calculating uncertainty, for example, factors that cause uncertainty are extracted and the uncertainty (standard deviation) of each factor is calculated. Furthermore, the calculated uncertainty of each factor is combined by the sum of squares method to calculate the combined standard uncertainty. Since the sum of squares method is used in the calculation of the combined standard uncertainty, it is possible to ignore factors with sufficiently small uncertainty among the factors that cause uncertainty.

Furthermore, in the device of the present invention, the coefficient of variation of the amplifiable reagents filled in the wells may be used as uncertainty information.
The coefficient of variation means the relative value of variation in the number of cells (or the number of reagents that can be amplified) filled in each recess when cells are filled in the recess. That is, the coefficient of variation means the filling accuracy of the number of cells (or amplifiable reagents) filled in the recess. A coefficient of variation is a value obtained by dividing the standard deviation σ by the average value x. Here, if the value obtained by dividing the standard deviation σ by the average number of copies (average number of filled copies) x is the coefficient of variation CV, the relational expression of Equation 1 below is obtained.

Generally, the cells (or amplifiable reagents) are randomly distributed in a Poisson distribution in the dispersion. Therefore, in the stepwise dilution method, that is, in a random distribution state in the Poisson distribution, the standard deviation σ can be considered to satisfy the relational expression of the average copy number x and Equation 2 below. From this, when the dispersion of cells (or an amplifiable reagent) is diluted by a serial dilution method, the coefficient of variation CV (CV value) of the average copy number x is calculated from the standard deviation σ and the average copy number x by the above formula 3 derived from Equations 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 in Table 1 and FIG. 35, for example, when 100 copies of an amplifiable reagent are filled in a well by a serial dilution method, the copy number of the amplifiable reagent finally filled in the reaction solution is Even ignoring other accuracies, it can be seen that the coefficient of variation (CV value) is at least 10%.

As the copy number of the amplifiable reagent, the CV value of the coefficient of variation and the average specific copy number x of the amplifiable reagent preferably satisfy the following formula, CV < 1/√x, CV < 1/2 It is more preferable to satisfy √x.

The uncertainty information preferably has uncertainty information for the device as a whole, based on the specific number of copies of the amplifiable reagents contained in the wells, where there are multiple wells containing the amplifiable reagents.

There are several factors that cause uncertainty. (e.g., the cell cycle of a cell, etc.), the means for placing cells in a device (inkjet device, or the result of the operation of each part in the device, such as the timing of the operation of the device. For example, the cell suspension is a droplet. (e.g., number of cells contained in droplets when lysed), frequency with which cells were placed in appropriate locations in the device (e.g., number of cells placed in wells), and number of cells disrupted in the cell suspension. Contamination (contamination of contaminants, hereinafter sometimes referred to as "contamination") due to contamination of the cell suspension with a reagent capable of proliferating by contamination.

Information on the amplifiable reagents includes, for example, information on the number of amplifiable reagents. Information on the number of amplifiable reagents includes, for example, information on the uncertainty of the number of amplifiable reagents contained in the device.

The wells contain primers, amplification reagents, etc., as well as specific copies of amplifiable reagents.
A primer is a synthetic oligonucleotide having a complementary nucleotide sequence of 18 to 30 bases 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.

The states of the amplifiable reagents, primers, and amplification reagents in the wells 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, it is particularly preferably in a solid state, and more preferably in a dry state. In the solid dry state, the reaction rate of decomposition of the amplification reagent by a degrading enzyme or the like can be reduced, and the storage stability of the amplification reagent, the primer, and the amplification reagent can be improved.
In addition, the solid dry state device is filled with appropriate amounts of amplifiable reagents, primers, and amplification reagents so that it can be used as a reaction solution immediately by dissolving in buffer or water immediately before use. is desirable.
The drying method is not particularly limited and can be appropriately selected depending on the intended purpose. is mentioned.

In the device of the present invention, the specific copy number of the amplifiable reagent in one well and the specific copy number of the amplifiable reagent in the other well are preferably two or more different from each other, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; 1, 3, 5, 7, 9; 2, 4, 6, 8, 10;
Also, the device of the present invention has a specific copy number of amplifiable reagent in one well of 10 ^N1 and a specific copy number of amplifiable reagent in the other well of 10 ^N2 (with the proviso that N1 and N2 are consecutive integers). be done. As a result, the device of the present invention can easily prepare a calibration curve over a wide range from low copy numbers to high copy numbers.

Devices of the invention have groups of two or more wells that differ in specific copy numbers of amplifiable reagents. For example, if the substrate of the device is a plate with a plurality of wells, each group will form a group "area" on the plate. In addition, the “regions” formed by two or more groups having different specific copy numbers of amplifiable reagents may be adjacent to each other or may be separated from each other.
As a result, for example, in the results obtained by performing real-time PCR, which is one of the performance evaluations of inspection equipment, using the device of the present invention, wells with the same specific copy number at different positions are compared, and wells that are not suitable for use If there are (nonconforming wells), it is possible to determine whether to recalibrate the real-time PCR or exclude the samples in the nonconforming wells from the actual samples. Further, by periodically measuring the inspection apparatus using the device of the present invention, it is possible to obtain information on the change in the Ct value over time at the in-plane position of the inspection apparatus. properties can be evaluated. Furthermore, by using devices with the same specific number of copies placed, performance can be compared between inspection systems.

In addition, in the device of the present invention, at least one of the groups of wells in which a specific copy number of the amplifiable reagent is arranged is a group in which the specific copy number of the amplifiable reagent is near the detection limit. preferable.

Limit of Detection (LOD) refers to the minimum number of copies of an amplifiable reagent that can be detected in a method that can detect an amplifiable reagent (e.g., nucleic acid), and is not particularly limited, depending on the assay method. can be selected as appropriate, for example, average +3σ.
In the present specification, when there is 1 undetected sample in 21 samples with a specific copy number (equivalent to 2σ determined with a probability of 95%), the number corresponding to the minimum copy number is detected. Can be used as a limit.

Near the detection limit means a copy number in the range of ±1 of the copy number of the detection limit.
When the device of the present invention has at least a group in which the specific copy number of the amplifiable reagent is near the detection limit, for example, the specific copy number of the amplifiable reagent is "1", "2", In the case of having groups of "3", "4", and "5", performance evaluation is performed on a certain testing device, and the test device is capable of amplifying the amplifiable reagent in groups of "3" or more. However, in the group of "2" or less, the amplifiable reagent cannot be amplified, and the lower limit of the detection limit for the copy number of the amplifiable reagent in the inspection device is "3". can be Further, when performance evaluation is performed on another similar testing device, the test device can amplify the amplifiable reagent in the group of "4" or more, but the reagent that can be amplified in the group of "3" or less can be clarified that the lower limit of detection for the number of copies of the amplifiable reagent in the test device is "4", and the minimum detectable amplification of the test device Specific copy numbers of possible reagents can be determined.

In addition, at least one of the groups of wells in which the amplifiable reagent is arranged at a specific copy number is preferably a group in which the specific copy number of the amplifiable reagent exceeds the lower limit of quantification.

The term "Limit of qualification (LOQ)" refers to the minimum quantifiable amplifiable reagent copy number in a method capable of quantifying an amplifiable reagent (e.g., nucleic acid), and is not particularly limited. It can be appropriately selected according to the measurement method.

In the present specification, a standard curve is prepared from a sample consisting of a plurality of molecular species (for example, a plurality of nucleic acid samples with different specific copy numbers), and the value of the copy number that deviates from the linearity of the standard curve is used as the lower limit of quantification. good too. Alternatively, the uncertainty of the calibration curve is represented by the CV value, the horizontal axis is the number of copies, and the vertical axis is the Ct value. copy number) can be used as the lower limit of quantitation.
In the case of quantitative evaluation, the copy number (copy number or concentration) corresponding to the Ct value can be obtained from the calibration curve and PCR efficiency instead of the Ct value itself. ) may be used to set the lower limit of quantitative determination.

When the device of the present invention has at least a group in which a specific copy number of an amplifiable reagent (e.g., nucleic acid) exceeds the lower limit of quantitation, for example, a specific copy number of an amplifiable reagent in a test device is 10 or more, quantitative detection can be guaranteed. A specific copy number of the smallest amplifiable reagent can be determined that can guarantee accurate detection.

More preferably, in the device of the present invention, at least one of the groups of wells is a negative control group in which the specific copy number of the amplifiable reagent is zero.
In the device of the present invention, at least one of the groups of wells is a negative control group in which the specific copy number of the amplifiable reagent is 0, so that the amplifiable reagent can be detected in the negative control group. If it does, it suggests that there is something wrong with the detection system (reagents or equipment). By providing a negative control group within the device, when a problem occurs, the user can immediately notice the problem and stop the measurement to check where the problem is.

Moreover, in the device of the present invention, at least one of the groups of wells is preferably a positive control group in which the amplifiable reagent has a specific copy number of 100 or more.
In the device of the present invention, at least one of the groups of wells is a positive control group in which the specific copy number of the amplifiable reagent is 100 or more, so that the amplifiable reagent is not present in the positive control group. When detected, it suggests that there is an abnormality in the detection system (reagent or device). By having a positive control group in the device, the user can immediately notice when a problem occurs and stop the measurement to check where the problem is.

In the device of the present invention, at least one of the groups of wells is the group with the lowest copy number (lowest copy number group) excluding the negative control group, and the lowest copy number group is at least the device It is more preferable that the wells are arranged in substantially the outer circumference of the well.
Here, the substantially outer circumference means an inner number row including at least the outermost circumference of the wells arranged two-dimensionally on the device. Examples of the inner number row including the outermost circumference include 1 row or more and 47 rows or less.
Unlike the wells located near the center of the device, the wells located at the outermost periphery of the device do not have wells on the outside and serve as a boundary between the device and the outside of the device. (2) being arranged around the outer periphery of the temperature control member, which is a component of the PCR device, is susceptible to temperature fluctuation factors of the device; Therefore, in the device of the present invention, at least one of the groups of wells is the group with the lowest copy number (the lowest copy number group) excluding the negative control group, and the lowest copy number group is , at least in the wells substantially on the periphery of the device, it is possible to detect malfunctions of the device with higher sensitivity.

Preferably, the amplifiable reagent is a nucleic acid. Preferably, the nucleic acid is integrated into the nucleus of the cell.

-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 includes fragments of nucleic acids, analogs of these nucleic acids or fragments thereof, and the like. .
Nucleic acids are not particularly limited and can be appropriately selected depending on the intended purpose. Examples thereof include DNA, RNA, cDNA and the like.

Nucleic acids or nucleic acid fragments may be natural products obtained from living organisms or processed products thereof, those produced using genetic recombination technology, chemically synthesized artificial synthetic nucleic acids, etc. 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 artificially synthesized nucleic acid means a nucleic acid obtained by artificially synthesizing a nucleic acid composed of components (bases, deoxyribose, phosphoric acid) similar to those of naturally occurring DNA or RNA. Artificially synthesized nucleic 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, labeled primers and probes), artificial nucleic acids (e.g., PNA, BNA, LNA, etc.) in which the chemical structure of some of the nucleotides that make up the nucleic acid or nucleic acid fragment is changed

The form of the nucleic acid is not particularly limited and can be appropriately selected depending on 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.

Nucleic acids preferably have a specific base sequence. Specific means specifically defined.
The specific 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 specific base sequence, and the GC content is preferably constant (see, for example, SEQ ID NO: 1).
The base length of the specific 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. (see, for example, SEQ ID NOs: 2 and 3).

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 specific 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-
The amplifiable reagent is preferably handled while being supported on a carrier. When the amplifiable reagent 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 purpose. Examples thereof include cells, resins, liposomes, microcapsules and the like.

--cell--
Cells refer to structural and functional units that contain amplifiable reagents (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 cannot be controlled can be reduced, preventing an increase in the number of specific nucleic acids in the cells accommodated in the well. be able to.

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, the number of cells can be controlled with high precision and landed in the 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 according to 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 the whole cell, cell nucleus, cell membrane, 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, rhodamines, and cyanines are preferred, and eosin, Evans blue, trypan blue, rhodamine 6G, rhodamine B, rhodamine 123, and Cy3 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、ＴｕｒｂｏＦＰ６０２、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 determined 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 volume average particle size measurement method.

The number of nucleic acid-containing 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 carry an amplifiable reagent (eg, 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 an amplifiable reagent (eg, nucleic acid) is preferred, and its form 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 combinations thereof are also possible.
The size (average particle size) of the liposome is not particularly limited as long as it can encapsulate an amplifiable reagent (eg, 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 an amplifiable reagent (eg, 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 microcapsule is not particularly limited as long as it can contain an amplifiable reagent (eg, nucleic acid), 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.

Here, FIG. 1 is a perspective view showing an example of the device of the present invention. 2 is a side view of the device of FIG. 1; FIG. The device 1 has a substrate 2 provided with a plurality of wells 3, and the wells 3 are filled with a specific copy number of nucleic acid 4 as an amplifiable reagent. In addition, 5 in FIG.1 and FIG.2 is a sealing member.
Also, for example, an IC chip or barcode (identification means 6) storing information on a specific number of copies of the nucleic acid 4 to be filled in each well 3 or information associated with such information is attached to the sealing member 5 and the base material 2. and at a position other than the opening of the well. This is suitable for preventing unintended modification of the identification means.
Moreover, since the device has identification means, it can be distinguished from a general well plate that does not have identification means. Therefore, it is possible to prevent mix-ups.

FIG. 3 is a plan view showing an example of the device of the present invention. In the device of FIG. 3, nucleic acids with a specific copy number of 10 copies and primers are arranged almost over the entire surface of a 96-well plate. In addition, 0 copies of 0 copies are placed at two positions as negative controls (NTC), and 1,000 copies of nucleic acids, primers and amplification reagents are placed at two positions as positive controls (PC) in the center.
Real-time PCR was performed using the device shown in FIG. 3, and the measured Ct (Threshold Cycle) values are shown in FIG. The average Ct value is 37.1, the standard deviation of the Ct value is 1.00, and the CV value [(standard deviation/average Ct value)×100] is 2.70. In addition, "UD" in FIG. 3 means that the Ct value is not detected.
For real-time PCR, for example, first, for each sample in the device, 1 μL of a master mix (manufactured by Thermo Fisher, TaqMan Universal PCR Master Mix), a forward primer and a reverse primer for amplifying a specific base sequence are added to 0 .5 nmol, add 0.4 nmol of probe. Amplification and detection can then be performed using a real-time PCR device (QuantStudio 7 Flex, manufactured by Thermo Fisher).

The Ct value is the cycle number at which the fluorescence signal of the reaction crosses the Threshold Line. Since the Ct value is proportional to the initial amount of target, the initial copy number of DNA can be calculated.
Threshold Line is the signal level at which a statistically significant increase is observed with respect to the calculated baseline signal, and means the threshold of the real-time PCR reaction.
In this way, the in-plane characteristics of the device are the average Ct value, the standard deviation of the Ct value, the CV value [(standard deviation / average Ct value) × 100], [(Ct value (Max) - Ct value (min)) / 2 average Ct value]×100.

In the device of FIG. 5, nucleic acids with a specific copy number of 3 copies, primers, and amplification reagents are placed almost over the entire surface of a 96-well plate, and negative control (NTC) 0 copies are placed at 4 locations in the center of the plate. .
Using the device of FIG. 5, real-time PCR was performed to measure the Ct value, and the average Ct value was calculated. If the Ct value of the specific copy number (3 copies) well is within 10% of the average Ct value, "○", and if the Ct value of the specific copy number (3 copies) well is greater than 10% of the average Ct value It is indicated by "x". The results are shown in FIG. In addition, "UD" in FIG. 6 indicates that the Ct value is not detected.
As a result, four wells on the periphery of the 96-well plate had values exceeding 10% of the average Ct value. These four peripheral locations were found to be unsuitable for use at low copy numbers. Therefore, it is possible to decide whether to calibrate the real-time PCR again or not to use these four outer wells in the actual sample.

For example, by using the devices described with reference to FIGS. 3 to 6 and performing measurements for a certain period of time, changes in the Ct value over time can be obtained. As a result, similar to the in-plane characteristics of the inspection equipment, if the Ct value of each well exceeds 10% of the average Ct value, the inspection equipment will be calibrated or the measurement location will not be used. can take action. In addition, since the specific number of copies arranged is an absolute value, it is possible to compare the performance between inspection apparatuses by using devices in which the same specific number of copies are arranged.

As shown in FIG. 7, a specific copy number of 2 copies, 3 copies, 5 copies, 10 copies, 20 copies, 50 copies, and 100 copies of nucleic acids, primers, and amplification reagents are placed in a 96-well plate, and a negative control is performed. 0 copy as (NTC) and 500 copies of nucleic acid and primer as positive control (PC) were placed in the center.
Real-time PCR was performed using the device of FIG. 7 to measure the Ct value. FIG. 8 shows a standard curve in which these Ct values are plotted on the vertical axis and the copy number on the horizontal axis. There is a correlation coefficient (R ² ), which is 0.99 in this case, as an index representing the performance of the inspection device. The correlation coefficient is a value that indicates how well the data match the standard curve, and reflects the linearity of the standard curve. In addition, since the minimum number of copies (in this case, two copies) is arranged on the outermost circumference, it can be used as an index of in-plane uniformity.

In the device of FIG. 9, nucleic acids with specific copy numbers of 2 copies, 10 copies, and 100 copies, primers, and amplification reagents are placed in the center of a 96-well plate, 0 copies as a negative control (NTC), and a positive control. As (PC), 500 copies of nucleic acid and primers are placed in the center. The minimum number of copies (in this case, 2 copies) can be used as an index of in-plane uniformity by arranging on the outermost periphery and on the innermost periphery, one horizontal row and two vertical rows.

In the device of FIG. 10, nucleic acids with specific copy numbers of 2 copies, 10 copies, and 100 copies, primers, and amplification reagents are placed in 20 wells, respectively, in a 96-well plate, and the minimum copy number (2 in this case) is placed on the outermost circumference. copies), and the well with a specific copy number of 100 copies also serves as a positive control (PC). The minimum number of copies (two copies in this case) is placed on the outermost periphery, which can be used as an index of in-plane uniformity.

The device in FIG. 11 arranges nucleic acids and primers with specific copy numbers of 3 copies, 5 copies, 10 copies, and 100 copies in a 96-well plate, and places the minimum copy number (2 copies in this case) on the outermost circumference and It is arranged in a cross. 0 copies of a negative control (NTC) and 500 copies of a nucleic acid, a primer, and an amplification reagent are placed as a positive control (PC).

In the device of FIG. 12, nucleic acids and primers with specific copy numbers of 2 copies, 5 copies, 10 copies, and 100 copies are placed in a 96-well plate, and the minimum copy number (2 copies in this case) is placed on the outermost circumference. It is arranged. 0 copies of a negative control (NTC) and 500 copies of a nucleic acid and a primer as a positive control (PC) are placed in the center.

In the device of FIG. 13, nucleic acids with specific copy numbers of 2 copies, 10 copies, and 100 copies, primers, and amplification reagents are arranged in the center in a 384-well plate. copy). The minimum number of copies (in this case, 2 copies) can be used as an index of in-plane uniformity by arranging the number of copies on the outermost periphery and on the inner periphery of the outermost periphery in five horizontal rows and eight vertical rows.

By performing measurements for a certain period of time using the devices shown in FIGS. 7 and 9 to 13, changes in the Ct value over time can be obtained. As a result, in the same way as the in-plane characteristics of the inspection equipment, if a numerical value deviating from the quality control value is obtained, it is possible to take measures such as calibrating the inspection equipment or not using the measurement location. . In addition, since the number of arranged copies is an absolute value, performance can be compared between inspection apparatuses by using devices in which the same number of copies are arranged.

<Device manufacturing method>
A method for manufacturing a device using cells having a specific nucleic acid will be described below.
The device manufacturing method of the present invention includes a cell suspension generating step of generating a cell suspension containing a plurality of cells having a specific nucleic acid and a solvent, and a plate by discharging the cell suspension as droplets. and a cell count step of counting the number of cells contained in the droplets with a sensor after the droplets are ejected and before the droplets reach the wells. , a nucleic acid extraction step of extracting nucleic acids from the cells in the well, a step of calculating the likelihood of the number of nucleic acids to be estimated in the cell suspension generation step, the droplet landing step, and the cell number counting step, and an output step , preferably includes a recording step, and further includes 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 create.

-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, or mixed liquids thereof. 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 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 according to 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 tested, and can be appropriately selected according to the purpose. Examples thereof include ColE1 DNA. A nucleic acid can prevent a nucleic acid having a specific base sequence from adhering to the walls of a well or the like.

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 disaggregation ability is strong, and there is a possibility of destroying cell membranes and cell walls, and media may be mixed as contamination.

[Cell screening method]
The method of screening cells is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include wet classification, cell sorter, screening using a filter, and the like. 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 nucleotide 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 copy number of the nucleic acid 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 base sequence is selected to contain one region per cell or is introduced by genetic recombination, so the number of target base 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 likelihood of 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.
Probability means the probability of occurrence of a specific event when there is a possibility of occurrence of several events, and the degree of possibility of occurrence of a specific event is predicted in advance.
Calculation means to calculate and obtain a numerical value.

FIG. 14 is a graph showing an example of the relationship between the frequency of DNA-replicated cells and fluorescence intensity. As shown in FIG. 14, two peaks appear on the histogram depending on the presence or absence of DNA replication, 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 nucleotide sequences contained in one cell. 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 certainty (probability) of the estimated specific copy number. By calculating the likelihood (probability), it is possible to express the likelihood as 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 rate of correct answers for the number of ejected cells, the number of DNAs in cells, and the rate of impact of ejected cells in a well. 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 to sequentially land the droplets in the wells of the plate.
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 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, the following reasons are required, such as idle ejection until a stable ejection state is reached, adjustment of the amount of droplets, and continuous formation of droplets even when moving between wells. The cell suspension used tends to have a large dead volume. In the present invention, it is preferable to reduce the influence of the 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 device manufacturing method, the plates for receiving the droplets are arranged facing each other, and it is preferable that the electrodes are not arranged in order to increase the degree of freedom in the plate configuration.
Since the thermal method generates localized heating, there are concerns about the effect on cells, which are biomaterials, and kogation of the heater. Since the influence of heat depends on the contents and the application of the plate, it is not necessary to generally exclude it.

Examples of the pressure application method include a method of applying pressure to the liquid using a piezo element, a method of applying pressure by a valve such as an electromagnetic valve, and the like. 15A to 15C show configuration examples of droplet generation devices that can be used to eject droplets of cell suspension.
FIG. 15A 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. 15B 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. 16C and the like can be cited as a more preferable configuration. FIG. 15C is a schematic diagram of a modification of the piezoelectric ejection head using the piezoelectric element in FIG. 15B. The ejection head of FIG. 15C has a piezoelectric element 13c, a liquid chamber 11c, a cell suspension 300c, and a nozzle 111c.
In the ejection head of FIG. 15C, 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, by controlling the ejection direction of the droplets in flight by applying a voltage, it is possible to select whether the droplets should land in the well or be collected in the collection unit. Such a method is used in a cell sorter or a flow cytometer, and for example, a device name: Cell Sorter SH800Z manufactured by Sony Corporation can be used.

FIG. 16A is a schematic diagram showing an example of the voltage applied to the piezoelectric element. Also, FIG. 16B is a schematic diagram showing another example of the voltage applied to the piezoelectric element. FIG. 16A shows drive voltages for droplet formation. Droplets can be formed by varying the strength of the voltages (V _A , V _B , V _C ). FIG. 16B 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 the upper and lower electrodes formed on the piezoelectric element. 17A to 17C are schematic diagrams showing states of droplets at respective timings.
In FIG. 17A, 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. 17B, the droplet continues to be extruded from the nozzle until the pressure relaxes upward, and the droplet grows.
Finally, as shown in FIG. 17C, 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 wells are formed is fixed on a movable stage, and droplets are sequentially landed on concave portions by combining driving of the stage and formation of droplets from an ejection head. 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 wells generally used in the biotechnology field can be used.
The number of wells in the plate is not particularly limited and can be appropriately selected depending on the purpose, and may be singular or plural.

FIG. 18 is a schematic diagram showing an example of a dispensing device 400 for depositing droplets sequentially in wells of a plate.
As shown in FIG. 18 , 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 wells 710 (recesses) on 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 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 respective 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 the droplets to be ejected, it is preferable to land the droplets in the wells so as to obtain a plurality of levels when the cell suspension is deposited in the 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 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-well microtube, 8-well tube, 96-well, 384-well well plate, etc. When there are a plurality of wells, the wells of these plates should contain the same number of cells. It is also possible to dispense and to enter different levels of numbers. There may also be wells containing no cells. In particular, when preparing a plate used for evaluating a real-time PCR device or a digital PCR device that quantitatively evaluates the amount of nucleic acids, it is preferable to use a plate in which multiple levels of nucleic acids are dispensed. For example, it is conceivable to prepare plates in which cells (or nucleic acids) are dispensed at 7 levels of approximately 1, 2, 4, 8, 16, 32, and 64 cells. By using such a plate, it is possible to examine the quantification, linearity, evaluation lower limit, etc. of a real-time PCR device or a digital PCR device.

<<Cell count step>>
The cell counting step is a step of counting the number of cells contained in the droplet with a sensor after the droplet is ejected and before the droplet lands on the 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 count 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 well, and may be appropriately selected according to the purpose. It may include a process of observing cells before discharge and a process of counting cells after landing.

After the droplet is ejected and before the droplet lands on the well, the number of cells contained in the droplet is counted immediately above the well opening where the droplet is expected to reliably enter the well of the plate. It is preferable to observe the cells in the droplet at certain timings.

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. 19, 23 and 24. FIG.
FIG. 19 is a schematic diagram showing an example of the droplet forming device 401. As shown in FIG. 23 and 24 are schematic diagrams showing other examples of droplet forming devices 401A and 401B. As shown in FIG. 19 , 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. 19, a liquid obtained by fluorescently staining cells with a specific dye and dispersing them in a predetermined solution is used as a cell suspension, and light having a specific wavelength emitted from a light source is applied to droplets formed from an 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 that are 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 lateral direction of the paper surface, and the membrane 12 can be vibrated in the vertical direction of the paper surface. 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. 20 to 22. FIG.

FIG. 20 is a diagram illustrating hardware blocks of control means of the droplet forming apparatus of FIG. 21 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. 19. FIG. FIG. 22 is a flow chart showing an example of the operation of the droplet forming device.

As shown in FIG. 20 , 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 equipment or the like. The droplet forming device 401 may be connected to an external network or the like via the I/F 74 .

As shown in FIG. 21, 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 counting of the droplet forming device 401 will be described with reference to FIGS. 21 and 22. FIG.
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 (including the case where it is zero) based on the information from the light receiving element 60. 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、ＴｕｒｂｏＦＰ６０２、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.

That is, 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 the conventional one. 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. 23 is a schematic diagram showing a modification of the droplet forming device 401 of FIG. As shown in FIG. 23, the droplet forming device 401A is different from the droplet forming device 401 (see FIG. 19) 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. 23, it is possible to avoid the occurrence of interference.

As shown in FIG. 23, by changing the layout of the light-receiving element 60, it is 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. 24 is a schematic diagram showing another modification of the droplet forming device 401 of FIG. As shown in FIG. 24, 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. 19) 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).

25A and 25B are diagrams illustrating a case where a flying droplet contains two fluorescently stained cells. For example, as shown in FIG. 25A, fluorescently stained cells 350 ₁ and 350 ₂ overlap, and as shown in FIG. 25B, 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. 26 is a diagram illustrating the relationship between the luminance value Li when particles do not overlap and the luminance value Le that is actually measured. As shown in FIG. 26, 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 cells/droplet=n (n: natural number).

However, in practice, 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. 26). 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.

When a plurality of light-receiving elements are installed, the number of cells 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. 27 is a schematic diagram showing another modification of the droplet forming device 401 of FIG. As shown in FIG. 27, a droplet forming device 401C differs from the droplet forming device 401 (see FIG. 19) 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. For example, when the fluorescently stained cells 350 are animal cells, particularly human cells, the size of human cells is generally about 5 μm to 50 μm, so the diameter of the nozzle 121 is set to 10 μm or more according to the cells to be used. is preferred, and 100 µm or more is more preferred.

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 drive 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. coil 200 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 cells to move. 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 the microchannel 250 shown in FIG. 29, a method of acquiring an image of the vicinity of the nozzle portion of the ejection head shown in FIG. 30, and the like. is mentioned. FIG. 29 shows a method used in a cell sorter apparatus. For example, a cell sorter SH800Z manufactured by Sony Corporation can be used. In FIG. 29, the microchannel 250 is irradiated with laser light from a light source 260 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 type 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 given 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. 30, it is estimated that 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′ before ejection, and images before and after ejection. It is possible to estimate the number of cells that landed in a given well by estimating the number of cells that are considered to have been ejected from the difference from 1. Count the cells that have passed through the microchannel shown in FIG. While the method produces droplets continuously, FIG. 30 is more preferred as it allows for droplet formation on demand.

[Processing for counting cells after landing]
As a process for counting cells after landing, it is possible to adopt a method of detecting fluorescently stained cells by observing 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 I can't. 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 well, in addition to counting the number of cells contained in the droplet with a sensor and cell counting means, a process of observing the cells before ejection, It is preferable to perform a process of counting cells after impact.

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. 31 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 performing cell counting 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.

<<Step of calculating the likelihood of the number of nucleic acids to be estimated in the cell suspension generation step, the droplet landing step, and the cell number counting step>>
The step of calculating the likelihood of the number of nucleic acids to be estimated in the cell suspension generation step, the droplet landing step, and the cell counting step is performed in the cell suspension generation step, the droplet landing step, and the cell counting step, respectively. This is the step of calculating the certainty in the step of .
The likelihood of the number of nucleic acids to be estimated can be calculated in the same manner as the likelihood in the cell suspension generation step.
In addition, the calculation timing of the probability may be calculated collectively in the next step of the cell number counting process, or at the end of each step of the cell suspension generation process, the droplet landing process, and the cell number counting process may be calculated, and each uncertainty may be combined and calculated in the next step of the cell counting step. In other words, the certainty in each of the above steps may be appropriately calculated before the synthesis calculation.

<<Output process>>
The output step is a step of outputting the number of cells contained in the cell suspension that has landed in the well by the cell number counting means based on the detection result measured by the sensor.
The counted value means the number of cells contained in the well by cell 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 well of 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 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 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>>
The 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.

INDUSTRIAL APPLICABILITY The device of the present invention is widely used in the bio-related industry, the life science industry, the medical industry, and the like, and can be suitably used, for example, for device configuration, calibration curve preparation, accuracy control of inspection devices, 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.

(Inspection apparatus performance evaluation method, inspection apparatus performance evaluation apparatus, and inspection apparatus performance evaluation program)
A performance evaluation method for an inspection device according to the present invention is a performance evaluation method for an inspection device for evaluating performance of an inspection device that inspects an object to be inspected,
Using the device of the present invention, a Ct value information acquisition step of acquiring Ct value information in the device;
A performance evaluation step of evaluating the performance of the inspection apparatus based on the Ct value information, and further includes other steps as necessary.

A performance evaluation apparatus for an inspection apparatus of the present invention is a performance evaluation apparatus for an inspection apparatus that evaluates the performance of an inspection apparatus that inspects an object to be inspected,
Using the device of the present invention, a Ct value information acquisition unit that acquires Ct value information in the device;
a performance evaluation unit that evaluates the performance of the inspection apparatus based on information on the Ct value, and further has other means as necessary.

A performance evaluation program for an inspection device of the present invention is a performance evaluation program for an inspection device for evaluating the performance of an inspection device that inspects an object to be inspected,
Using the device of the present invention, obtaining information on the Ct value in the device,
A computer is caused to execute a process of evaluating the performance of the inspection apparatus based on the information of the Ct value.

Since the control performed by the control unit and the like in the performance evaluation apparatus for inspection apparatus of the present invention is synonymous with implementing the performance evaluation method for inspection apparatus of the present invention, the performance evaluation apparatus for inspection apparatus of the present invention will be described. The details of the performance evaluation method of the inspection device of the invention will also be clarified. Further, since the performance evaluation program for the inspection apparatus of the present invention is realized as the performance evaluation apparatus for the inspection apparatus of the present invention by using a computer or the like as a hardware resource, the performance evaluation apparatus for the inspection apparatus of the present invention is Details of the performance evaluation program for the inspection apparatus of the present invention will also be clarified through the explanation.

<Ct value information acquisition step and Ct value information acquisition unit>
The Ct value information acquisition step is a step of acquiring information on the Ct value in the device using the device of the present invention, and is performed by the Ct value information acquisition unit.
Ct values can be determined by performing real-time PCR using the device of the present invention.
As the information of the Ct value, for example, the average Ct value, the standard deviation of the Ct value, the CV value [(standard deviation / average Ct value) × 100], [(Ct value (Max) - Ct value (min)) / 2 average Ct value]×100.
Ct value information can be determined for two or more groups with different specific copy numbers of amplifiable reagents in the device.

<Performance evaluation process and performance evaluation unit>
The performance evaluation process is a process of evaluating the performance of the inspection device based on the information on the Ct value, and is performed by the performance evaluation section.

For qualitative evaluation, the device of the present invention is used to perform real-time PCR, measure Ct values, and calculate mean Ct values. If the Ct value of each well is within 10% of the average Ct value, "○", and if the Ct value of each well is greater than 10% of the average Ct value, "X" can be used to evaluate the in-plane characteristics. .
In addition, the change in Ct value over time can be obtained by performing measurements for a certain period of time using the device of the present invention. As a result, similar to the in-plane characteristics, when the Ct value of each well exceeds 10% of the average Ct value, it is possible to calibrate the inspection device or not to use the measurement location. In addition, since the specific number of copies arranged is an absolute value, it is possible to compare the performance between inspection apparatuses by using devices in which the same specific number of copies are arranged.

In quantitative evaluation, the device of the present invention can be used to measure the Ct value over time by performing measurements for a certain period of time. As with the in-plane characteristics, if a numerical value deviating from the quality control value is obtained, it is possible to calibrate the inspection device or not to use the measurement location. In addition, since the number of arranged copies is an absolute value, performance can be compared between inspection apparatuses by using devices in which the same number of copies are arranged.
In the case of quantitative evaluation, the copy number (copy number or concentration) corresponding to the Ct value can be obtained from the calibration curve and PCR efficiency instead of the Ct value itself. ), or CV value converted to copy number (copy number or density), copy number (copy number or density conversion) (Max-Min) / 2 average value × 100, etc. may be evaluated.

<Other processes and other departments>
Other processes and other units are not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a display process and a display unit.

The processing by the performance evaluation program for the inspection apparatus of the present invention can be executed using a computer having a control unit that constitutes the performance evaluation apparatus for the inspection apparatus.
The hardware configuration and functional configuration of the performance evaluation device for the inspection device will be described below.

<Hardware Configuration of Performance Evaluation Device for Inspection Device>
FIG. 32 is a block diagram showing an example of the hardware configuration of the performance evaluation device 100 for inspection devices.
As shown in FIG. 32, a performance evaluation apparatus 100 for an inspection apparatus includes a CPU (Central Processing Unit) 101, a main storage device 102, an auxiliary storage device 103, an output device 104, an input device 105, and a communication interface (communication I/F). It has 106 parts. These units are connected via a bus 107 .
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 unit 130 of the performance evaluation apparatus 100 of the inspection apparatus by executing the performance evaluation program of the inspection apparatus.
Also, the CPU 101 controls the operation of the entire performance evaluation apparatus 100 of the inspection apparatus. In this embodiment, the CPU 101 is used as the device that controls the operation of the entire performance evaluation device 100 of the inspection device, but it is not limited to this, and may be, for example, an FPGA (Field Programmable Gate Array).

The performance evaluation program of the inspection apparatus and various databases may not necessarily be stored in the main storage device 102, the auxiliary storage device 103, or the like. The performance evaluation program and various databases of the inspection apparatus are stored in another information processing apparatus or the like connected to the performance evaluation apparatus 100 of the inspection apparatus via the Internet, LAN (Local Area Network), WAN (Wide Area Network), or the like. may The inspection apparatus performance evaluation apparatus 100 may acquire and execute the inspection apparatus performance evaluation program and various databases from these other information processing apparatuses.
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 performance evaluation device 100 of the inspection device, and a known device can be used as appropriate. Examples include a keyboard, mouse, and touch panel.
The communication interface (communication I/F) 106 is not particularly limited, and a known one can be used as appropriate, such as a wireless or wired communication device.
With the hardware configuration as described above, the processing functions of the performance evaluation apparatus 100 of the inspection apparatus can be realized.

<Functional configuration of performance evaluation device for inspection device>
FIG. 33 is a diagram showing an example of the functional configuration of the performance evaluation device 100 for inspection devices.
As shown in FIG. 33, a performance evaluation apparatus 100 for an inspection apparatus has an input section 110, an output section 120, a control section 130, and a storage section 140. FIG.
The control unit 130 has a Ct value information acquisition unit 131 and a performance evaluation unit 132 . The control unit 130 controls the entire performance evaluation apparatus 100 of the inspection apparatus.
The storage unit 140 has a Ct value information database 141 and a performance evaluation result database 142 . Hereinafter, "database" may be referred to as "DB".

The Ct value information acquisition unit 131 acquires Ct value information using the Ct value information data stored in the Ct value information DB 141 of the storage unit 140 . The Ct value information DB 141 stores, for example, Ct value data obtained in advance by experiments as described above. Information on the Ct value associated with the device may be stored in the Ct value information DB 141 . The input to the DB may be performed from another information processing device connected to the performance evaluation device 100 of the inspection device, or may be performed by the operator.
The performance evaluation unit 132 evaluates the performance of the inspection device based on the Ct value information. A specific method for evaluating the performance of the inspection apparatus is as described above.
The performance evaluation result of the inspection apparatus obtained by the performance evaluation unit 132 is stored in the performance evaluation result DB 142 of the storage unit 140 .

Next, the processing procedure of the performance evaluation program for the inspection apparatus of the present invention will be described. FIG. 34 is a flow chart showing a processing procedure of an inspection apparatus performance evaluation program in the control unit 130 of the inspection apparatus performance evaluation apparatus 100. As shown in FIG.

In step S110, the Ct value information acquisition unit 131 of the control unit 130 of the performance evaluation apparatus 100 of the inspection apparatus acquires the information data of the Ct value stored in the Ct value information DB 141 of the storage unit 140, and the process proceeds to S111. do.
In step S111, the performance evaluation unit 132 of the control unit 130 of the inspection apparatus performance evaluation apparatus 100 evaluates the performance of the inspection apparatus based on the acquired Ct value information, and the process proceeds to S112.
In step S112, the control unit 130 of the inspection apparatus performance evaluation apparatus 100 saves the obtained inspection apparatus performance evaluation result in the performance evaluation result DB 142 of the storage unit 140, and ends this process.

Aspects of the present invention are, for example, as follows.
<1> having a plurality of wells,
The device comprises two or more groups of wells in which a specific copy number of the amplifiable reagent is arranged, wherein the group of wells has a different specific copy number of the amplifiable reagent.
<2> The device according to <1>, wherein at least one of the groups of wells has a specific copy number of the amplifiable reagent near the detection limit.
<3> The device according to <1>, wherein at least one of the groups of wells is a group in which the specific copy number of the amplifiable reagent exceeds the lower limit of quantification.
<4> The device according to any one of <1> to <3>, wherein at least one of the groups of wells is a negative control group in which the specific copy number of the amplifiable reagent is 0. be.
<5> The device according to any one of <1> to <4>, wherein at least one of the groups of wells is a positive control group in which the amplifiable reagent has a specific copy number of 100 or more. is.
<6> At least one of the groups of wells is the group with the lowest copy number excluding the negative control group, and is arranged in at least a well approximately on the periphery of the device From <1> The device according to any one of <5>.
<7> The device according to any one of <1> to <6>, comprising identification means capable of identifying information on the specific copy number of the amplifiable reagent.
<8> The device according to any one of <1> to <7>, wherein the amplifiable reagent is a nucleic acid.
<9> The device according to any one of <1> to <8>, wherein the nucleic acid is incorporated into a nucleic acid in the nucleus of a cell.
<10> The device according to <9>, wherein the cells are yeast.
<11> The device according to any one of <1> to <10>, wherein the wells contain at least one of primers and amplification reagents.
<12> The device according to any one of <1> to <11>, wherein the specific copy number is the counted copy number of the amplifiable reagent.
<13> The device according to any one of <9> to <12>, wherein the cells are ejected by an inkjet method.
<14> An inspection device performance evaluation program for evaluating the performance of an inspection device that inspects an object to be inspected,
Using the device according to any one of <1> to <13>, obtaining Ct value information in the device,
The inspection apparatus performance evaluation program causes a computer to execute a process of evaluating the performance of the inspection apparatus based on the Ct value information.
<15> An inspection apparatus performance evaluation method for evaluating performance of an inspection apparatus that inspects an object to be inspected,
A Ct value information acquisition step of acquiring information of the Ct value in the device using the device according to any one of <1> to <13>;
a performance evaluation step of evaluating the performance of the inspection device based on the Ct value information;
A performance evaluation method for an inspection device, comprising:
<16> A performance evaluation device for an inspection device that evaluates the performance of an inspection device that inspects an object to be inspected,
A Ct value information acquisition unit that acquires information on the Ct value of the device using the device according to any one of <1> to <13>;
a performance evaluation unit that evaluates the performance of the inspection device based on the Ct value information;
It is a performance evaluation device for an inspection device characterized by having

The device according to any one of <1> to <13>, the inspection apparatus performance evaluation program according to <14>, the inspection apparatus performance evaluation method according to <15>, and <16>. The described performance evaluation apparatus for an inspection apparatus can solve the above-mentioned conventional problems and achieve the object of the present invention.

REFERENCE SIGNS LIST 1 device 2 substrate 3 well 4 amplifiable reagent 5 sealing member

JP 2014-33658 A JP 2015-195735 A

Claims (15)

having a plurality of wells,
A group of wells in which a specific number of molecules of a nucleic acid extracted in the well from a carrier placed in the well is arranged , wherein the specific number of molecules of the nucleic acid is different from each other. 2 or more. 2. The device according to claim 1, wherein at least one of the groups of wells is a group in which a specific number of molecules of the nucleic acid is near the detection limit. 2. The device according to claim 1, wherein at least one of the groups of wells is a group in which the specific number of molecules of the nucleic acid exceeds the lower limit of quantification. 4. The device according to any one of claims 1 to 3, wherein at least one of the groups of wells is a negative control group in which the number of specific molecules of the nucleic acid is zero. 5. The device according to any one of claims 1 to 4, wherein at least one of the groups of wells is a positive control group in which the number of specific molecules of the nucleic acid is 100 or more. 5. The device of claim 4 , wherein at least one of said groups of wells is the group with the lowest number of molecules excluding said negative control group, and is arranged in wells at least approximately around the perimeter of the device. 7. The device according to any one of claims 1 to 6, further comprising identification means capable of identifying information on a specific number of molecules of said nucleic acid . 8. The device of claim 7, wherein said nucleic acid is integrated into nucleic acid in the nucleus of a cell. 9. The device of claim 8, wherein said cells are yeast. 10. The device of any of claims 1-9, wherein the wells contain primers and/or amplification reagents. 11. The device according to any one of claims 1 to 10, wherein the specific number of molecules is the number of nucleic acid molecules counted. 9. The device according to claim 8, wherein the cells are ejected by an inkjet method. A performance evaluation program for an inspection apparatus for evaluating the performance of an inspection apparatus that inspects an object to be inspected, wherein the device according to any one of claims 1 to 12 is used to obtain information on the Ct value of the device,
Evaluating the performance of the inspection device based on the information of the Ct value
A performance evaluation program for an inspection device, characterized by causing a computer to execute processing. A performance evaluation method for an inspection device for evaluating the performance of an inspection device that inspects an object to be inspected,
A Ct value information acquisition step of acquiring information of the Ct value in the device using the device according to any one of claims 1 to 12;
a performance evaluation step of evaluating the performance of the inspection device based on the Ct value information;
A performance evaluation method for an inspection device, comprising: A performance evaluation device for an inspection device that evaluates the performance of an inspection device that inspects an object to be inspected,
A Ct value information acquisition unit that uses the device according to any one of claims 1 to 12 and acquires Ct value information in the device;
a performance evaluation unit that evaluates the performance of the inspection device based on the Ct value information;
A performance evaluation device for an inspection device, comprising:

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