JP6446151B1 - Inspection device and device - Google Patents

Abstract

A test device capable of evaluating the performance of an analytical test based on a genetic test including a nucleic acid amplification technique is provided. A test device has at least one well and has information on the uncertainty of the absolute number of amplifiable reagents and the absolute number of amplifiable reagents in the at least one well. An embodiment in which an amplifiable reagent is encapsulated in carrier particles is preferred, and an embodiment in which the amplifiable reagent is a nucleic acid is more preferred. The embodiment in which the nucleic acid is incorporated into the nucleic acid in the nucleus of the cell is particularly preferred. [Selection] Figure 2

Description

  The present invention relates to an inspection device and a device.

  In recent years, with the increased sensitivity of analytical technology, it has become possible to measure the target to be measured in copy number units, and the industrial use of gene detection technology for detecting trace nucleic acids is required for food, environmental testing, and medical care. ing. In particular, it is often confirmed that pathogens and unapproved genetically modified foods are not contained in specimens, and a high level of detection accuracy is required.

For example, the polymerase chain reaction (PCR) frequently used in the field of molecular biology research can theoretically be amplified even from one copy of nucleic acid because of its technical characteristics.
In such a small amount of gene detection, it is necessary to use a standard reagent when quantitative analysis is performed, for example, limiting the dilution of a DNA fragment having a specific base sequence, and the result of real-time PCR of the obtained diluted solution Therefore, a method for selecting a diluent having a target copy number has been proposed (see, for example, Patent Document 1).
In addition, a method for producing a standard reagent containing a DNA fragment having a desired copy number has been proposed by introducing a DNA fragment having a specific copy number into a cell by genetic recombination technology and isolating the cultured cell ( For example, see Patent Document 2).

  An object of the present invention is to provide a test device capable of performing performance evaluation of an analytical test based on a genetic test including a nucleic acid amplification technique.

  The test device of the present invention as a means for solving the above problems has at least one well, and the absolute number of amplifiable reagents in the at least one well and the uncertainty of the absolute number of the amplifiable reagent Information.

  ADVANTAGE OF THE INVENTION According to this invention, the test | inspection device which can perform the performance evaluation of the analytical test based on the gene test containing a nucleic acid amplification technique can be provided.

FIG. 1 is a perspective view showing an example of an inspection device of the present invention. FIG. 2 is a side view showing an example of the inspection device of the present invention. FIG. 3 is a diagram showing an example of the position of a well filled with an amplifiable reagent in the test device of the present invention. FIG. 4 is a diagram showing another example of the position of a well filled with an amplifiable reagent in the test device of the present invention. FIG. 5 is a graph showing an example of the relationship between the frequency of cells replicated with DNA and the fluorescence intensity. FIG. 6A is a schematic diagram illustrating an example of an electromagnetic valve type ejection head. FIG. 6B is a schematic diagram illustrating an example of a piezo-type ejection head. 6C is a schematic view of a modification of the piezo-type ejection head in FIG. 6B. FIG. 7A is a schematic diagram illustrating an example of a voltage applied to the piezoelectric element. FIG. 7B is a schematic diagram illustrating another example of the voltage applied to the piezoelectric element. FIG. 8A is a schematic diagram illustrating an example of a state of a droplet. FIG. 8B is a schematic diagram illustrating an example of a state of a droplet. FIG. 8C is a schematic diagram illustrating an example of a state of a droplet. FIG. 9 is a schematic view showing an example of a dispensing apparatus for sequentially landing droplets in a well. FIG. 10 is a schematic diagram illustrating an example of a droplet forming apparatus. FIG. 11 is a diagram illustrating hardware blocks of control means of the droplet forming apparatus of FIG. FIG. 12 is a diagram illustrating functional blocks of control means of the droplet forming apparatus of FIG. FIG. 13 is a flowchart showing an example of the operation of the droplet forming apparatus. FIG. 14 is a schematic view showing a modification of the droplet forming apparatus. FIG. 15 is a schematic view showing another modification of the droplet forming apparatus. FIG. 16A is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 16B is a diagram illustrating a case where two fluorescent particles are included in a flying droplet. FIG. 17 is a diagram illustrating the relationship between the luminance value Li when the particles do not overlap and the actually measured luminance value Le. FIG. 18 is a schematic view showing another modification of the droplet forming apparatus. FIG. 19 is a schematic diagram showing another example of the droplet forming apparatus. FIG. 20 is a schematic diagram illustrating an example of a method of counting cells that have passed through the microchannel. FIG. 21 is a schematic diagram illustrating an example of a method for acquiring an image near the nozzle portion of the ejection head. FIG. 22 is a graph showing the relationship between the probability P (> 2) and the average cell number.

(Inspection device)
The test device of the present invention has at least one well, has information on the absolute number of amplifiable reagents in the at least one well and the uncertainty of the absolute number of the amplifiable reagents, It preferably has a base material, and further has other members as necessary.

  In the present invention, when detecting a nucleic acid from a sample containing a nucleic acid having a low copy number, it is important for the accuracy control of the examination to grasp the detection sensitivity of the system, particularly the detection lower limit value. When collecting a specimen from a sample containing a low copy number nucleic acid, random variations according to the Poisson distribution occur according to the amount of nucleic acid contained, and it is difficult to improve the accuracy of the inspection apparatus itself. Based on knowledge.

  In addition, the low copy number nucleic acid standard used for conventional quality control does not show the uncertainty that arises when producing a low copy number nucleic acid standard, and the reliability of quality control cannot be guaranteed. It is based on the knowledge that there are things.

  When the test device of the present invention is used, the reliability of the measurement result can be evaluated when detecting a target reagent from a sample containing a low copy number amplifiable reagent. In the present invention, “low copy number” means “low copy number” of an amplifiable reagent.

The absolute number of amplifiable reagents means the known number of copies of the amplifiable reagent (eg, nucleic acid) contained in the well. Here, the number of molecules of the reagent that can be amplified may be associated with the copy number.
The absolute number of reagents that can be amplified is preferably 100 copies or less, and more preferably 10 copies or less.
The absolute number of amplifiable reagents is preferably two or more different integers.
Examples of combinations of absolute numbers of reagents that can be amplified include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 3, 5, 7, 9, 2, The case of 4, 6, 8, 10 is mentioned.
Also, the combination of absolute numbers of reagents that can be amplified may be, for example, four levels of 1, 10, 100, and 1,000. A calibration curve can be created using the inspection device of the present invention with a combination of a plurality of different absolute numbers.

Uncertainty is defined as “parameters that characterize the variability of values that can be reasonably linked to measurement quantities associated with measurement results” and ISO / IEC Guide 99: 2007 [international metrology terminology-basic and general concepts and related terms ( VIM)]. Here, “a value that can be reasonably linked to a measurement amount” means a candidate for a true value of the measurement amount. That is, uncertainty refers to information on variations in measurement results due to operations, equipment, etc. related to the manufacture of the measurement object. The greater the uncertainty, the greater the variation expected as a measurement result.
The uncertainty may be, for example, a standard deviation obtained from a measurement result, or may be a value that is half the confidence level expressed as a range of values in which a true value is included with a predetermined probability or more.
As a method of calculating the uncertainty, Guide to the Expression of Uncertainty in Measurement (GUM: ISO / IEC Guide 98-3), and the measurement uncertainty in the Japan Accumulation Board Note 10 test, which can be calculated based on the uncertainty in the measurement based on the Japan Accumulation Board Note 10 test, etc. . As a method of calculating uncertainty, for example, use type A evaluation method using statistics such as measured values, and uncertainty information obtained from calibration certificate, manufacturer's specifications, published information, etc. Uncertainty can be expressed with the same confidence level by converting all the uncertainties derived from factors such as operation and measurement into standard uncertainties. . Standard uncertainty refers to the variation in the average value obtained from the measured values.
As an example of a method for calculating the uncertainty, for example, a factor causing the uncertainty is extracted, and the uncertainty (standard deviation) of each factor is calculated. Further, the uncertainties of the calculated factors are combined by the sum of squares method to calculate the combined standard uncertainty. Since the sum of squares method is used in the calculation of the composite standard uncertainty, factors that cause sufficiently small uncertainty can be ignored.
There are a number of factors that can cause uncertainty. For example, when a target reagent is introduced into a cell, and the cell is counted and dispensed, the factor of uncertainty in the number of target reagents in each well The number of reagents that can be amplified in the cell, the means for placing the cells on the plate, the frequency with which the placed cells are placed at the appropriate location on the plate, and the cells being destroyed in the cell suspension Examples include contamination of the reagent by mixing an amplifiable reagent in the cell suspension.

<Well>
The shape, number, volume, material, color and the like of the well 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 nucleic acid or the like can be arranged, and can be appropriately selected according to the purpose. For example, a recess such as a flat bottom, a round bottom, a U bottom, and a V bottom, and a partition on the substrate Etc.
The number of wells is at least one, preferably two or more, more preferably 5 or more, and still more preferably 50 or more.
Examples of one having one well include a PCR tube.
As the number of wells being 2 or more, for example, a multi-well plate is preferably used.
Examples of the multiwell plate include 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. For example, in consideration of the amount of sample used in a general nucleic acid test apparatus, it is preferably 1 μL or more and 1,000 μL or less.
The material of the well is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate. .
Examples of the color of the well include transparent, translucent, coloring, and complete light shielding.

<Base material>
The inspection device is preferably in the form of a plate in which wells are provided on a base material, but may be a connection type well tube such as an 8-unit tube.
There is no restriction | limiting in particular about the material, a shape, a magnitude | size, a structure etc. as a base material, According to the objective, it can select suitably.
There is no restriction | limiting in particular as a material of a base material, According to the objective, it can select suitably, For example, a semiconductor, ceramics, a metal, glass, quartz glass, plastics etc. are mentioned. Among these, plastics are preferable.
Examples of the plastics include polystyrene, polypropylene, polyethylene, fluorine resin, acrylic resin, polycarbonate, polyurethane, polyvinyl chloride, and polyethylene terephthalate.
There is no restriction | limiting in particular as a shape of a base material, Although it can select suitably according to the objective, For example, plate shape, plate shape, etc. are preferable.
There is no restriction | limiting in particular as a structure of a base material, According to the objective, it can select suitably, For example, a single layer structure or a multilayer structure may be sufficient.

<Identification means>
The test device preferably has an identification means capable of identifying the absolute number of amplifiable reagents and their uncertainty information.
The identification means is not particularly limited and can be appropriately selected according to the purpose. For example, it can be selected as a memory, an IC chip, a barcode, a QR code (registered trademark), a radio frequency identifier (hereinafter also referred to as “RFID”). Color coding, printing, etc.
There is no restriction | limiting in particular as a position which provides an identification means, and the number of identification means, According to the objective, it can select suitably.
As information to be stored in the identification means, in addition to the absolute number of amplifiable reagents and their uncertainty information, for example, analysis results (activity value, luminescence intensity, etc.), the number of amplifiable reagents (for example, the number of cells) Number), cell viability, number of copies of a specific base sequence, which well of a plurality of wells is filled with amplifiable reagent, type of amplifiable reagent, measurement date and time, name of the measurer, etc. It is done.
The 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 for writing information to the identification means is not particularly limited and can be appropriately selected according to the purpose.For example, the number of amplifiable reagents can be selected manually or when dispensing the amplifiable reagent into the well. Examples include a method of directly writing data from a droplet forming apparatus to count, transfer of data stored in a server, transfer of data stored in a cloud, and the like.

<Other members>
There is no restriction | limiting in particular as another member, According to the objective, it can select suitably, For example, a sealing member etc. are mentioned.

-Sealing member-
The inspection device preferably has a sealing member in order to prevent foreign matter from entering the well from flowing out the filler.
The sealing member is preferably configured to be separable by a tear line so that at least one well can be sealed and each well can be individually sealed or opened.
The shape of the sealing member is preferably a cap shape that matches the well inner wall diameter or a film shape that covers the well opening.
Examples of the material for the sealing member include polyolefin resin, polyester resin, polystyrene resin, polyamide resin, and the like.
The sealing member is preferably a film that can seal all wells at once. Moreover, it is preferable that the adhesive strength between the wells that need to be resealed and the unnecessary wells is different so that user misuse can be reduced.

The well preferably contains at least one of a primer and an amplification reagent.
The primer is a synthetic oligonucleotide having a complementary base sequence of 18 to 30 bases specific to the template DNA in the polymerase chain reaction (PCR). Two primers, a forward primer and a reverse primer, sandwich the region to be amplified. Place (pair) is set.
As an amplification reagent, in polymerase chain reaction (PCR), for example, a DNA polymerase as an enzyme, four types of bases (dGTP, dCTP, dATP, dTTP), Mg ²⁺ (2 mM magnesium chloride), an optimum pH (pH 7. 5 to 9.5) and the like

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

Here, FIG. 1 is a perspective view showing an example of the inspection device 1 of the present invention. FIG. 2 is a side view of the inspection device 1 of FIG. In the inspection device 1, a plurality of wells 3 are provided on a base material 2, and a nucleic acid 4 as a reagent that can be amplified in the well 3 (an inner space region surrounded by the well wall surface that constitutes the well) has a specific copy number Filled with. The test device 1 is associated with information on the absolute number of amplifiable reagents and the uncertainty of the absolute number of the amplifiable reagents. 1 and FIG. 2 show an example in which the inspection device 1 is covered with the opening of the well 3 by the sealing member 5.
For example, as shown in FIGS. 1 and 2, an IC chip or bar that stores information on the number of reagents filled in each well 3 and the uncertainty (certainty) of the number, or information associated with the information. A code (identification means 6) is disposed between the sealing member 5 and the base material 2 at a position other than the well opening. This is suitable for preventing unintended modification of the identification means.
Further, since the inspection device has the identification means, it can be distinguished from a general well plate not having the identification means. For this reason, it is possible to prevent confusion.

FIG. 3 is a diagram showing an example of the position of a well filled with an amplifiable reagent of the test device of the present invention. The numbers in the wells in FIG. 3 represent the absolute number of reagents that can be amplified. Wells not indicated by numbers in FIG. 3 are samples and control measurement wells.
FIG. 4 is a diagram showing another example of the position of the well filled with the amplifiable reagent of the test device of the present invention. The numbers in the wells in FIG. 4 represent the absolute number of reagents that can be amplified. Wells not indicated by numerals in FIG. 4 are samples and control measurement wells.

  The amplifiable reagent is preferably a nucleic acid. It is preferred that the nucleic acid is incorporated into the cell nucleus.

-Nucleic acid-
Nucleic acid means a macromolecular organic compound in which nitrogen-containing bases derived from purines or pyrimidines, sugars, and phosphates are regularly bonded, and also includes fragments of nucleic acids or analogs of these nucleic acids or fragments thereof. .
There is no restriction | limiting in particular as a nucleic acid, According to the objective, it can select suitably, For example, DNA, RNA, cDNA etc. are mentioned. Moreover, a plasmid can also be used as a nucleic acid. The nucleic acid may be modified or mutated.
Examples of nucleic acid or nucleic acid fragment analogs include non-nucleic acid components bound to nucleic acids or nucleic acid fragments, nucleic acids or nucleic acid fragments labeled with a labeling agent such as a fluorescent dye or an isotope (for example, fluorescent dyes or radioisotopes) And a primer or probe labeled with a nucleic acid or a nucleic acid or a fragment of a nucleotide constituting a nucleic acid fragment (for example, a peptide nucleic acid). These may be natural products obtained from living organisms or processed products thereof, or may be manufactured using genetic recombination techniques, or may be chemically synthesized. .

The nucleic acid is preferably a carrier that is preferably handled in a state of being supported on a carrier, and can be appropriately selected depending on the purpose, and examples thereof include cells, resins, liposomes, and microcapsules. The nucleic acid as an amplifiable reagent is preferably carried on a particle-shaped carrier (carrier particle) (more preferably encapsulated).
The cell is not particularly limited as long as it is a cell into which gene transfer can be performed, can be appropriately selected according to the purpose, and can be used regardless of the aforementioned cell types.
The nucleic acid preferably has a specific base sequence. Specific means that it is specifically defined.
The specific base sequence is not particularly limited and may be appropriately selected depending on the purpose. For example, base sequences used for infectious disease tests, non-natural base sequences that do not exist in nature, bases derived from animal cells Examples thereof include sequences and base sequences derived from plant cells. These may be used alone or in combination of two or more.
When a non-natural base sequence is used, the GC content is preferably 30% or more and 70% or less of a specific base sequence (see, for example, SEQ ID NO: 1).
There is no restriction | limiting in particular as base length of a specific base sequence, According to the objective, it can select suitably, For example, 20 or more and 10,000 or less base length etc. are mentioned.
When using a base sequence used for an infectious disease test, as long as it contains a base sequence peculiar to the infectious disease, there is no particular limitation, and it can be appropriately selected according to the purpose. It is preferable that the nucleotide sequence is included (see, for example, SEQ ID NOs: 2 and 3).

The nucleic acid may be a cell-derived nucleic acid to be used, or a nucleic acid introduced by gene introduction. When using a nucleic acid introduced by gene introduction and a plasmid as the nucleic acid, it is preferable to confirm that one copy of the nucleic acid is 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 depending on the purpose. For example, confirmation can be performed using a sequencer, PCR method, Southern blot method, or the like. it can.
The number of nucleic acids introduced by gene introduction may be one, or two or more. Even when the number of nucleic acids introduced by gene introduction is one, the same base sequence may be introduced in tandem according to the purpose.

  The method for gene transfer is not particularly limited as long as a specific nucleic acid sequence can be introduced into a target location and can be appropriately selected according to the purpose. For example, homologous recombination, CRISPR / Cas9, TALEN, Zinc finger nuclease, Flip-in, Jump-in, etc. are mentioned. Among these, in the case of yeast, homologous recombination is preferable from the viewpoint of high efficiency and ease of control.

-Cell-
A cell refers to a structural and functional unit that has nucleic acids and forms an organism.
There is no restriction | limiting in particular as a cell, According to the objective, it can select suitably, For example, it can use about all cells regardless of a eukaryotic cell, a prokaryotic cell, a multicellular biological cell, and a unicellular biological cell. . These may be used alone or in combination of two or more.

  The eukaryotic cell is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include animal cells, insect cells, plant cells, fungi, algae, protozoa and the like. These may be used alone or in combination of two or more. Among these, animal cells and fungi are preferable.

  Adhesive cells may be primary cells collected directly from tissues or organs, or may be passaged from some primary cells directly collected from tissues or organs, and can be appropriately selected according to the purpose. Examples thereof include differentiated cells and undifferentiated cells.

  The differentiated cell is not particularly limited and may be appropriately selected depending on the intended purpose. For example, hepatocytes that are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; Endothelial cells such as cells; fibroblasts; osteoblasts; osteoclasts; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; Such as epithelial cells; mammary cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; kidney cells; pancreatic Langerhans islet cells; peripheral nerve cells, nerve cells such as optic nerve cells; chondrocytes; .

  The undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. For example, pluripotent stem cells such as embryonic stem cells which are undifferentiated cells, mesenchymal stem cells having multipotency; Examples include unipotent stem cells such as vascular endothelial progenitor cells having unipotency; iPS cells and the like.

There is no restriction | limiting in particular as fungi, According to the objective, it can select suitably, For example, a mold, yeast, etc. are mentioned. These may be used alone or in combination of two or more. Among these, yeast is preferable because the cell cycle can be regulated and a haploid can be used.
The cell cycle means a process in which cell division occurs when cells increase, and a cell (daughter cell) generated by cell division again becomes a cell (mother cell) that undergoes cell division to generate a new daughter cell.

  There is no restriction | limiting in particular as yeast, According to the objective, it can select suitably, For example, the Bar-1 deficient yeast with which the sensitivity of the pheromone (sex hormone) which controls a cell cycle to G1 phase increased is preferable. If the yeast is a Bar-1-deficient yeast, the abundance ratio of yeasts whose cell cycle cannot be controlled can be lowered, so that an increase in the number of specific nucleic acids in the cells accommodated in the wells is prevented. be able to.

  The prokaryotic cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include eubacteria and archaea. These may be used alone or in combination of two or more.

As the cell, a dead cell is preferable. A dead cell can prevent cell division from occurring after sorting.
The cell is preferably a cell that can emit light when receiving light. If the cells can emit light when receiving light, the number of cells can be controlled with high accuracy and landed in the well.
Light reception means receiving light.
An optical sensor is a cell that collects visible light that can be seen by the human eye and light from the near-infrared, short-wavelength infrared, and thermal infrared regions with longer wavelengths. This means a passive sensor that acquires the shape of the image as image data.

-Cells that can emit light when receiving light-
The 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. Examples include cells that express proteins, cells that are labeled with fluorescently labeled antibodies, and the like.
There are no particular limitations on the site of staining with a fluorescent dye, the site of expression of a fluorescent protein, or the site of labeling with a fluorescently labeled antibody in cells, and examples include whole cells, cell nuclei, and cell membranes.

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

  Commercially available products can be used as the fluorescent dye. Examples of commercially available products include trade name: EosinY (Wako Pure Chemical Industries, Ltd.), trade name: Evans Blue (Wako Pure Chemical Industries, Ltd.), product 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 ( Wako Pure Chemical Industries, Ltd.).

--Fluorescent protein--
Examples of the fluorescent protein include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidorishiCyan, CFP, TurboGFP, AcGFP, TagGFP, AGFP-G, P, Gs, P. , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanaLa, KusiraOrange, mOrange, TurboRFP, DsRed-Express2, DsRed2, TagRFP, DsRed-MrR d, mCherry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and the like KikumeGR. These may be used individually by 1 type and may use 2 or more types together.

--Fluorescently labeled antibody--
The fluorescently labeled antibody is not particularly limited as long as it is fluorescently labeled, and can be appropriately selected according to the purpose. Examples thereof 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 diameter of the cells is preferably 100 μm or less, more preferably 50 μm or less, and particularly preferably 20 μm or less in the free state. If the volume average particle diameter is 100 μm or less, it can be suitably used for the ink jet method.

As a volume average particle diameter of a cell, it can measure with the following measuring method, for example.
A volume average particle diameter can be measured by taking out 10 μL from the prepared dyed yeast dispersion and placing it on a plastic slide made of PMMA and using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen). The number of cells can also be determined by the same measurement method.

There is no restriction | limiting in particular as a density | concentration of the cell in a cell suspension, Although it can select suitably according to the objective, 5 * 10 < ⁴ > / mL or more and 5 * 10 < ⁸ > / mL or less are preferable, and 5 * 10 More preferably, ⁴ pieces / mL or more and 5 × 10 ⁷ pieces / mL or less. When the number of cells is 5 × 10 ⁴ cells / mL or more and 5 × 10 ⁸ cells / mL or less, the discharged droplets can surely contain cells. 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 diameter measurement method.

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

<Inspection device manufacturing method>
Hereinafter, a method for producing a test device using cells having a specific nucleic acid as an amplifiable reagent will be described.
The method for producing a test device of the present invention includes a cell suspension generation step for generating a cell suspension containing a plurality of cells having a specific nucleic acid and a solvent, and discharging the cell suspension as droplets. A droplet landing process for sequentially landing droplets in the wells of the plate, and a cell number counting in which the number of cells contained in the droplets is counted by a sensor after the droplets are discharged and before the droplets land on the wells. And a nucleic acid extraction step for extracting nucleic acid from the cells in the well, preferably including a step of calculating the uncertainty of each step, an output step, and a recording step, and if necessary, other steps. Including.

<< Cell suspension production process >>
The cell suspension generation step is a step of generating a cell suspension containing a plurality of cells having a specific nucleic acid and a solvent.
The solvent means a liquid used for dispersing cells.
Suspension in a cell suspension means a state where cells are dispersed in a solvent.
Generating means creating.

-Cell suspension-
The cell suspension contains a plurality of cells having a specific nucleic acid and a solvent, preferably contains an additive, and further contains other components as necessary.
The plurality of cells having a specific nucleic acid are as described above.

--Solvent--
The solvent is not particularly limited and may be appropriately selected depending on the intended purpose. For example, water, culture solution, separation solution, dilution solution, buffer solution, organic solution solution, organic solvent, polymer gel solution, colloidal dispersion Liquid, electrolyte aqueous solution, inorganic salt aqueous solution, metal aqueous solution, and mixed liquids thereof. These may be used individually by 1 type and may use 2 or more types together. Among these, water and a buffer solution are preferable, and water, phosphate buffered saline (PBS), and Tris-EDTA buffer (TE) are more preferable.

--Additives--
There is no restriction | limiting in particular as an additive, According to the objective, it can select suitably, For example, surfactant, a nucleic acid, resin etc. are mentioned. These may be used alone or in combination of two or more.

  The surfactant can prevent aggregation between cells and improve the continuous ejection stability.

  There is no restriction | limiting in particular as surfactant, According to the objective, it can select suitably, For example, an ionic surfactant, a nonionic surfactant, etc. are mentioned. These may be used individually by 1 type and may use 2 or more types together. Among these, although it depends on the amount added, a nonionic surfactant is preferable from the viewpoint of not denaturing and deactivating the protein.

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

  Nonionic surfactants include, for example, alkyl glycosides, alkyl polyoxyethylene ethers (such as Brij series), octylphenol ethoxylates (Triton X series, Igepal CA series, Nonidet P series, Nikol OP series, etc.), polysorbates ( Tween series such as Tween 20), sorbitan fatty acid ester, polyoxyethylene fatty acid ester, alkyl maltoside, 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 preferable.

  There is no restriction | limiting in particular as content of surfactant, Although it can select suitably according to the objective, 0.001 to 30 mass% is preferable with respect to the whole cell suspension. When the content is 0.001% by mass or more, the effect of adding a surfactant can be obtained, and when the content is 30% by mass or less, cell aggregation can be suppressed. It is possible to strictly control the copy number of the nucleic acid therein.

  The nucleic acid is not particularly limited as long as it does not affect the detection of the nucleic acid to be detected, and can be appropriately selected according to the purpose. Examples thereof include ColE1 DNA. When it is a nucleic acid, it is possible to prevent a nucleic acid having a specific base sequence from adhering to the wall surface of a well.

  There is no restriction | limiting in particular as resin, According to the objective, it can select suitably, For example, a polyethyleneimide etc. are mentioned.

-Other materials-
Other materials are not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include a crosslinking agent, a pH adjuster, an antiseptic, an antioxidant, an osmotic pressure adjuster, a wetting agent, and a dispersant. Can be mentioned.

[Method of dispersing cells]
The method for dispersing cells is not particularly limited and can be appropriately selected according to the purpose. For example, a media method such as a bead mill, an ultrasonic method such as an ultrasonic homogenizer, or a pressure difference such as a French press is used. Examples include methods. These may be used individually by 1 type and may use 2 or more types together. Among these, the ultrasonic method is more preferable because of less damage to cells. In the media method, the crushing ability is strong, and the possibility of destroying the cell membrane and cell wall and media may be mixed as contamination.

[Cell screening method]
There is no restriction | limiting in particular as a screening method of a cell, According to the objective, it can select suitably, For example, the screening by wet classification, a cell sorter, a filter, etc. are mentioned. These may be used individually by 1 type and may use 2 or more types together. Among these, screening with a cell sorter or a filter is preferable because damage to cells is small.

The cell preferably estimates the number of nucleic acids having a specific 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 by cell division.
Estimating the number of nucleic acids means obtaining the copy number of nucleic acids from the number of cells.

The counting target may be not the number of cells but the number of specific DNA sequences. Usually, a specific DNA sequence is selected by selecting one containing one region per cell, or introduced by genetic recombination, so that the number of specific DNA sequences may be considered to be equal to the number of cells. However, in order for cells to undergo cell division at a specific cycle, nucleic acid replication occurs in the cells. Although the cell cycle varies depending on the cell type, by extracting a predetermined amount of solution from the cell suspension and measuring the cycle of multiple cells, the expected value and uncertainty for the number of specific nucleic acids contained in one cell can be determined. It is possible to calculate. This can be done, for example, by observing the nuclear-stained cells with a flow cytometer.
Uncertainty means information on variations in measurement results due to operations, equipment, etc. related to the manufacture of the measurement object.
Calculation means calculating a numerical value obtained by calculation.

FIG. 5 is a graph showing an example of the relationship between the frequency of cells replicated with DNA and the fluorescence intensity. As shown in FIG. 5, since two peaks appear depending on the presence or absence of DNA replication on the histogram, it is possible to calculate the proportion of cells that have undergone DNA replication. From this calculation result, the average number of DNA contained in one cell can be calculated, and the estimated number of nucleic acids can be calculated by multiplying the above-mentioned cell number counting result.
In addition, it is preferable to perform a process for controlling the cell cycle before preparing the cell suspension, and the number of specific nucleic acids can be determined from the number of cells by aligning with the state before or after the above-described replication occurs. It becomes possible to calculate more accurately.

  The number of nucleic acids to be estimated is preferably calculated as uncertainty. By calculating the uncertainty, it is possible to output the uncertainty as a variance or a standard deviation based on these numerical values. When summing up the effects of multiple factors, it is possible to use a square root sum of squares of standard deviation that is generally used. For example, the correct answer rate of the number of cells ejected as factors, the number of DNA in the cells, the landing rate at which the ejected cells land in the well, and the like can be used. It is also possible to select and calculate an item having a large influence among these.

<< Droplet landing process >>
The droplet landing step is a step of sequentially landing droplets in the wells of the device by discharging the cell suspension as droplets.
A droplet means a lump of liquid collected by surface tension.
Discharge means that the cell suspension is allowed to fly as droplets.
Sequential means to follow the order one after another.
Landing means that a droplet reaches the well.

  As the ejection means, a means for ejecting the cell suspension as droplets (hereinafter also referred to as “ejection head”) can be suitably used.

  Examples of the method for discharging the cell suspension as droplets include an on-demand method and a continuous method. Among these, in the case of the continuous method, for reasons such as empty ejection until reaching a stable ejection state, adjustment of the amount of droplets, and continuous droplet formation even when moving between wells, etc. The dead volume of the cell suspension used tends to increase. In the present invention, it is preferable to reduce the influence of dead volume from the viewpoint of adjusting the number of cells, and therefore the on-demand method is more preferable in the above two methods.

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

In the electrostatic method, it is necessary to install an electrode so as to face a discharge portion that holds a cell suspension and forms a droplet. In the plate generating method of the present invention, it is preferable that the plates for receiving the liquid droplets are arranged to face each other, and there is no electrode arrangement in order to increase the degree of freedom of the plate configuration.
In the thermal method, since local heating occurs, there is a concern about the influence on the cells that are biomaterials and the scorching (kogation) of the heater part. The influence of heat depends on the contents and the use of the plate, and therefore it is not necessary to exclude it in general. However, the pressure application method is preferable from the point that there is no fear of scorching on the heater part than the thermal method.

Examples of the pressure application method include a method of applying pressure to the liquid using a piezo element and a method of applying pressure using a valve such as an electromagnetic valve. 6A to 6C show configuration examples of a droplet generation device that can be used for discharging a cell suspension droplet.
FIG. 6A is a schematic diagram illustrating an example of an electromagnetic valve type ejection head. The electromagnetic valve type discharge head includes an electric motor 13a, an electromagnetic valve 112, a liquid chamber 11a, a cell suspension 300a, and a nozzle 111a.
As an electromagnetic valve type ejection head, for example, a dispenser manufactured by TechElan Co., Ltd. can be preferably used.
FIG. 6B is a schematic diagram illustrating an example of a piezo-type ejection head. The piezo-type ejection head includes a piezoelectric element 13b, a liquid chamber 11b, a cell suspension 300b, and a nozzle 111b.
As the piezo type ejection head, a Cytena single cell printer or the like can be suitably used.
Any of these discharge heads can be used, but the pressure application method using an electromagnetic valve cannot repeatedly form droplets at high speed, so the piezo method should be used to increase the plate generation throughput. Is preferred. In addition, in a piezo-type ejection head using a general piezoelectric element 13b, problems such as uneven cell concentration due to sedimentation and nozzle clogging may occur.
For this reason, the structure shown to FIG. 6C etc. is mentioned as a more preferable structure. FIG. 6C is a schematic diagram of a modification of the piezo-type ejection head using the piezoelectric element in FIG. 6B. The ejection head in FIG. 6C includes a piezoelectric element 13c, a liquid chamber 11c, a cell suspension 300c, and a nozzle 111c.
In the ejection head of FIG. 6C, by applying a voltage to the piezoelectric element 13c from a control device (not shown), a compressive stress is applied in the horizontal direction of the paper surface, and the membrane can be deformed in the vertical direction of the paper surface.

  As a method other than the on-demand method, for example, a continuous method in which droplets are continuously formed can be cited. In the continuous method, when liquid droplets are pressurized and pushed out from a nozzle, periodic fluctuations are given by a piezoelectric element or a heater, whereby fine droplets can be continuously produced. Furthermore, it is also possible to select whether to land on the well or to collect in the collection unit by controlling by applying a voltage in the ejection direction of the droplet during flight. Such a method is used in a cell sorter or a flow cytometer. For example, a device name: Cell Sorter SH800 manufactured by Sony Corporation can be used.

FIG. 7A is a schematic diagram illustrating an example of a voltage applied to the piezoelectric element. FIG. 7B is a schematic diagram illustrating another example of the voltage applied to the piezoelectric element. FIG. 7A shows a driving voltage for forming a droplet. _A droplet can be formed by the strength of the voltage (V _A , V _B , V _C ). FIG. 7B shows a voltage for stirring the cell suspension without discharging the droplet.

  By inputting multiple pulses that are not strong enough to eject droplets during periods when droplets are not ejected, it is possible to agitate the cell suspension in the liquid, and concentration distribution due to cell sedimentation Can be suppressed.

The droplet forming 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. FIG. 8A to FIG. 8C are schematic views showing the state of the liquid droplets at each timing.
FIG. 8A shows that a high pressure is generated between the cell suspension held in the liquid chamber 11c and the membrane 12c by applying a voltage to the piezoelectric element 13c to rapidly deform the membrane 12c. However, the liquid droplet is pushed out from the nozzle portion by this pressure.
Next, as shown in FIG. 8B, liquid extrusion from the nozzle portion continues for the time until the pressure is relaxed upward, and droplets grow.
Finally, as shown in FIG. 8C, when the membrane 12c returns to its original state, the fluid pressure near the interface between the cell suspension and the membrane 12c decreases, and a droplet 310 ′ is formed.

  In the manufacturing method of the inspection device, the plate on which the well is formed is fixed on a movable stage, and droplets are sequentially landed on the concave portions by combining the driving of the stage and the droplet formation from the ejection head. Here, the method of moving the plate as the movement of the stage has been described, but the ejection head may be moved as a matter of course.

There is no restriction | limiting in particular as a plate, It is possible to use what formed the well generally used in the bio field | area.
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. 9 is a schematic view showing an example of a dispensing apparatus 400 for sequentially landing droplets in the wells of the plate.
As shown in FIG. 9, the dispensing device 400 for landing droplets includes a droplet forming device 401, a plate 700, a stage 800, and a control device 900.

  In the dispensing apparatus 400, the plate 700 is disposed on a stage 800 configured to be movable. The plate 700 is formed with a plurality of wells 710 (concave portions) on which droplets 310 ejected from the ejection head of the droplet forming apparatus 401 are deposited. 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. Accordingly, the droplets 310 including the fluorescently stained cells 350 can be sequentially discharged from the discharge head of the droplet forming apparatus 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 the ROM or the like into the main memory and executing it by the CPU. However, part or all of the control device 900 may be realized only by hardware. The control device 900 may be physically configured by a plurality of devices.

As the liquid droplets to be discharged, it is preferable that the liquid droplets are landed in the well so as to obtain a plurality of levels when the cell suspension is landed in the well.
A plurality of levels means a plurality of standards serving as standards.
As a plurality of levels, it is preferable that a plurality of cells having a specific nucleic acid in a well have a predetermined concentration gradient. By having a concentration gradient, it can be suitably used as a calibration curve reagent. The plurality of levels can be controlled using values counted by the sensor.

  As the plate, it is preferable to use a 1-well microtube, 8-strip tube, 96-well, 384-well well plate or the like. However, when there are a plurality of wells, the same number of cells are placed in the wells of these plates. It can be dispensed or it can contain a number of different levels. There may also be wells that do not contain cells. In particular, when a plate used for evaluation of a real-time PCR apparatus or a digital PCR apparatus that quantitatively evaluates the amount of nucleic acid is prepared, it is preferable to use a plate in which a plurality of levels of nucleic acids are dispensed. For example, it is conceivable to produce a plate in which cells (or nucleic acids) are dispensed in seven levels of approximately 1, 2, 4, 8, 16, 32, and 64. By using such a plate, it is possible to examine the quantitativeness, linearity, evaluation lower limit value, etc. of the real-time PCR device or digital PCR device.

<< Cell count process >>
The cell number counting step is a step of counting the number of cells contained in the droplet by a sensor after the droplet is discharged and before the droplet reaches the well.
A sensor is a natural phenomenon or the mechanical / electromagnetic, thermal, acoustic, or chemical properties of artifacts, or spatial information / temporal information indicated by them, applying some scientific principle to human or It means a device that replaces the signal with another medium that is easy for the machine to handle.
Counting means counting numbers.

  The cell number counting step is not particularly limited as long as the number of cells contained in the droplet is counted by a sensor after the droplet is discharged and before the droplet reaches the well, and is appropriately selected according to the purpose. And may include a process of observing cells before discharge and a process of counting cells after landing.

  After discharging the droplet and before landing on the well of the droplet, the number of cells contained in the droplet is counted just above the well opening where the droplet is expected to enter the well of the plate. It is preferable to observe the cells in the droplets at the timing at the position.

  Examples of the method for observing cells in the droplet include an optical detection method and an electrical / magnetic detection method.

-Optical detection method-
The optical detection method will be described below with reference to FIGS. 10, 14, and 15. FIG.
FIG. 10 is a schematic diagram illustrating an example of the droplet forming apparatus 401. 14 and 15 are schematic views showing another example of the droplet forming apparatuses 401A and 401B. As shown in FIG. 10, the droplet forming apparatus 401 includes a discharge head (droplet discharge unit) 10, a drive unit 20, a light source 30, a light receiving element 60, and a control unit 70.

In FIG. 10, a cell suspension is used that is obtained by fluorescently staining cells with a specific dye and then dispersed in a predetermined solution. Light having a specific wavelength emitted from a light source is emitted to a droplet formed from an ejection head. Counting is performed by detecting the fluorescence emitted from the cells by irradiation with a light receiving element. At this time, in addition to the method of staining cells with a fluorescent dye, autofluorescence emitted from molecules originally contained in the cells may be used, or fluorescent proteins (for example, GFP (Green Fluorescent Protein)) are produced in the cells. Alternatively, a gene may be introduced in advance so that the cells emit fluorescence.
Irradiation means applying light.

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

  The liquid chamber 11 is a liquid holding unit that holds the cell suspension 300 in which the fluorescent staining 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 formed from, for example, metal, silicon, ceramic, or the like. Examples of the fluorescent dyed cells 350 include inorganic fine particles and organic polymer particles stained with a fluorescent dye.

  The membrane 12 is a film-like member fixed to the upper end portion of the liquid chamber 11. The planar shape of the membrane 12 can be a circle, for example, but may be an ellipse or a rectangle.

  The drive element 13 is provided on the upper surface side of the membrane 12. The shape of the drive 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 a circular driving element 13.

  The membrane 12 can be vibrated by supplying a drive signal from the drive means 20 to the drive element 13. Due to the vibration of the membrane 12, a droplet 310 containing fluorescently stained cells 350 can be discharged 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 to the upper surface and the lower surface of a piezoelectric material can be provided. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means 20, a compressive stress is applied in the horizontal direction on the paper surface, and the membrane 12 can be vibrated in the vertical direction on the paper surface. As the piezoelectric material, for example, lead zirconate titanate (PZT) can be used. In addition, various piezoelectric materials such as bismuth iron oxide, metal niobate, barium titanate, or a material obtained by adding a metal or a different oxide to these materials can be used.

  The light source 30 irradiates the flying droplet 310 with light L. Note that “in flight” means a state from when the droplet 310 is ejected from the droplet ejection means 10 until it has landed on the droplet deposition target. The flying droplet 310 is substantially spherical at the position where the light L is irradiated. Further, the beam shape of the light L is substantially circular.

  Here, the beam diameter of the light L is preferably about 10 to 100 times the diameter of the droplet 310. This is because the droplet 310 is surely irradiated with the light L from the light source 30 even when the position variation of the droplet 310 exists.

  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 applied to the droplet 310 is lowered, so that the amount of the fluorescence Lf emitted using the light L as excitation light is reduced and is difficult to detect by the light receiving element 60.

  The light L emitted from the light source 30 is preferably pulsed light. For example, a solid 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, and more preferably 1 μs or less. The energy per unit pulse largely depends on the optical system, such as the presence or absence of light collection, but is generally preferably 0.1 μJ or more, and more preferably 1 μJ or more.

  The light receiving element 60 receives the fluorescence Lf generated by the fluorescence stained cells 350 absorbing the light L as excitation light when the flying droplets 310 contain the fluorescence stained cells 350. Since the fluorescence Lf is emitted from the fluorescence-stained cells 350 in all directions, the light receiving element 60 can be disposed 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 is not directly incident.

  The light receiving element 60 is not particularly limited as long as it can receive the fluorescence Lf emitted from the fluorescence-stained cells 350 and can be appropriately selected according to the purpose. However, the light receiving element 60 irradiates the droplet with light having a specific wavelength. An optical sensor that receives fluorescence from the cells in the droplet is preferable. As the light receiving element 60, for example, a one-dimensional element such as a photodiode or a photosensor may be used. However, when 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.

  In addition, since the fluorescence Lf emitted from the fluorescent staining cell 350 is weaker than the light L emitted from the light source 30, a filter that attenuates the wavelength region of the light L may be installed in the front stage (light receiving surface side) of the light receiving element 60. . Thereby, in the light receiving element 60, it is possible to obtain an image of the fluorescence-stained cells 350 with very high contrast. As the filter, for example, a notch filter that attenuates a specific wavelength region including the wavelength of the light L can be used.

  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 to control the light receiving element 60 so that the light receiving element 60 can take in light at the timing when the continuously oscillating light is applied to the flying droplet 310, so that the light receiving element 60 receives the fluorescence Lf.

  The control unit 70 has a function of controlling the driving unit 20 and the light source 30. The control means 70 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 a case where it is zero). Yes. Hereinafter, the operation of the droplet forming apparatus 401 including the operation of the control means 70 will be described with reference to FIGS.

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

  As shown in FIG. 11, the control means 70 includes 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 connected to each other via a bus line 75.

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

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

  The cell number (particle number) counting of the droplet forming device 401 will be described with reference to FIGS. First, in step S <b> 11, the discharge control unit 701 of the control unit 70 issues a discharge command to the drive unit 20. The drive unit 20 that has received a discharge command from the discharge control unit 701 supplies a drive signal to the drive element 13 to vibrate the membrane 12. Due to the vibration of the membrane 12, a droplet 310 containing fluorescently stained cells 350 is discharged from the nozzle 111.

  Next, in step S <b> 12, the light source control unit 702 of the control unit 70 supplies the light source 30 in synchronization with the discharge of the droplet 310 (in synchronization with the drive signal supplied from the drive unit 20 to the droplet discharge unit 10). Give a lighting command. As a result, the light source 30 is turned on and the light L is irradiated to the flying droplet 310.

  Here, synchronizing means not emitting light at the same time as the droplet 310 is ejected by the droplet ejecting means 10 (simultaneously with the drive means 20 supplying a drive signal to the droplet ejecting means 10), but not the droplet. It means that the light source 30 emits light at the timing when the droplet 310 is irradiated with the light L when the 310 jumps 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 the ejection of the droplet 310 by the droplet ejection unit 10 (a driving signal supplied from the driving unit 20 to the droplet ejection unit 10). The light source 30 is controlled.

  For example, the velocity v of the droplet 310 ejected when a drive signal is supplied to the droplet ejection means 10 is measured in advance. Then, based on the measured velocity v, the time t to reach a predetermined position after the droplet 310 is ejected is calculated, and the light source 30 emits light at the timing when the drive signal is supplied to the droplet ejecting means 10. The timing to perform is delayed by t. Thereby, good light emission control is possible, and the light from the light source 30 can be reliably irradiated to the droplet 310.

  Next, in step S <b> 13, the cell number counting unit 703 of the control unit 70 determines the number of fluorescent stained cells 350 contained in the droplet 310 (including the case of zero) based on the information from the light receiving element 60. Count. Here, the information from the light receiving element 60 is a luminance value (light quantity) or an area value of the fluorescent staining cell 350.

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

  When a two-dimensional element is used as the light receiving element 60, the cell number counting unit 703 performs image processing for calculating the luminance value or area of the fluorescent stained cell 350 based on the two-dimensional image obtained from the light receiving element 60. The technique to perform may be used. In this case, the cell number counting means 703 calculates the luminance value or area value of the fluorescence-stained cells 350 by image processing, and compares the calculated luminance value or area value with a preset threshold value, thereby obtaining the fluorescence value. The number of stained cells 350 can be counted.

  Note that the fluorescence-stained cells 350 may be cells or stained cells. The 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 may be appropriately selected depending on the intended purpose. Examples thereof include fluoresceins, rhodamines, coumarins, pyrenes, cyanines and azos. 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 preferable.
Examples of the fluorescent protein include Sirius, EBFP, ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidorishiCyan, CFP, TurboGFP, AcGFP, TagGFP, AGFP-G, P, Gs, P. , YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanaLa, KusiraOrange, mOrange, TurboRFP, DsRed-Express2, DsRed2, TagRFP, DsRed-MrR d, mCherry, mPlum, PS-CFP, Dendra2, Kaede, EosFP, and the like KikumeGR. 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, the drive signal is supplied from the driving unit 20 to the droplet discharge unit 10 that holds the cell suspension 300 in which the fluorescent stained cells 350 are suspended. The contained droplet 310 is ejected, and light L is irradiated from the light source 30 to the droplet 310 in flight. 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. Further, based on information from the light receiving element 60, the cell number counting means 703 counts (counts) the number of fluorescent stained cells 350 contained in the flying droplet 310.

  In other words, since the droplet forming apparatus 401 actually observes the number of fluorescent stained cells 350 contained in the flying droplet 310 on the spot, the counting accuracy of the number of fluorescent stained cells 350 can be improved as compared with the conventional case. Is possible. Further, since the fluorescence stained cells 350 contained in the flying droplets 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 is obtained with high contrast. Thus, the frequency of erroneous counting of the number of fluorescently stained cells 350 can be reduced.

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

  As described above, in the droplet forming apparatus 401A, the mirror 40 is arranged in front of the light receiving element 60, so that the degree of freedom of the layout of the light receiving element 60 can be improved.

  For example, when the nozzle 111 and the droplet deposition object are brought close to each other, in the layout of FIG. 10, there is a possibility that interference between the droplet deposition object and the optical system (particularly, the light receiving element 60) of the droplet forming apparatus 401 occurs. The layout shown in FIG. 14 can avoid the occurrence of interference.

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

FIG. 22 is a schematic diagram showing another modification of the droplet forming apparatus 401 of FIG. As shown in FIG. 22, the droplet forming apparatus 401B includes a light receiving element 61 that receives the fluorescence Lf ₂ emitted from the fluorescence stained cell 350 in addition to the light receiving element 60 that receives the fluorescence Lf ₁ emitted from the fluorescence stained cell 350. The provided point is different from the droplet forming apparatus 401 (see FIG. 10). Note that description of the same components as those of the above-described embodiment may be omitted.

Here, the fluorescences Lf ₁ and Lf ₂ indicate a part of the fluorescence emitted from the fluorescence-stained cells 350 in all directions. The light receiving elements 60 and 61 can be arranged at arbitrary positions where the fluorescence emitted from the fluorescent stained cells 350 in different directions can be received. Note that three or more light receiving elements may be arranged at positions where fluorescence emitted from the fluorescently stained cells 350 in different directions can be received. Each light receiving element may have the same specifications or different specifications.

  When the number of the fluorescence-stained cells 350 is included in the flying droplet 310 when the number of the light-receiving elements is one, the cell-counting unit 703 causes the droplet 310 to overlap. There is a risk of erroneously counting the number of fluorescently stained cells 350 contained in (count error occurs).

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

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

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

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

  However, in practice, when n is 2 or more, particles may overlap each other, and thus the actually measured luminance value is Lu ≦ Le ≦ nLu (shaded portion in FIG. 16A). Therefore, when the number of cells / droplet = n, for example, the threshold can be set as (nLu−Lu / 2) ≦ threshold <(nLu + Lu / 2). When a plurality of light receiving elements are installed, it is possible to suppress the occurrence of a count error 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 particles may be determined by an algorithm for estimating the number of cells based on a plurality of obtained shape data.
As described above, the droplet forming apparatus 401B has a plurality of light receiving elements that receive fluorescence emitted in different directions by the fluorescence stained cells 350, and therefore the frequency of erroneous counting of the number of fluorescence stained cells 350 is further increased. Can be reduced.

  FIG. 18 is a schematic diagram showing another modification of the droplet forming apparatus 401 of FIG. As shown in FIG. 18, the droplet forming apparatus 401C is different from the droplet forming apparatus 401 (see FIG. 10) in that the droplet discharging unit 10 is replaced with the droplet discharging unit 10C. Note that description of the same components as those of the above-described embodiment may be omitted.

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

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

  There is no particular limitation on the material of the membrane 12C, but if it is too soft, the membrane 12C will vibrate easily, and it is difficult to suppress vibration immediately when it is not ejected. . As a 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 a cell is used as the fluorescence-stained cell 350, a material having low adhesion to cells and proteins is preferable. Cell adhesion is generally said to be dependent on the contact angle of the material with water. When the material is highly hydrophilic or highly hydrophobic, the cell adhesion is low. Various metal materials and ceramics (metal oxides) can be used as the highly hydrophilic material, and fluorine resin or the like can be used as the highly hydrophobic material.

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

  The nozzle 121 is preferably formed as a substantially circular through hole substantially at the center of the membrane 12C. In this case, the diameter of the nozzle 121 is not particularly limited, but is preferably at least twice the size of the fluorescent stained cell 350 in order to avoid the fluorescent stained cell 350 from clogging the nozzle 121. When the fluorescence-stained cell 350 is, for example, an animal cell, particularly a human cell, the size of the human cell is generally about 5 μm to 50 μm. Therefore, the diameter of the nozzle 121 is 10 μm or more according to the cell to be used. Preferably, 100 μm or more is more preferable.

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

  The drive element 13C is formed on 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 12 </ b> C is a circle, it is preferable to form a driving element 13 </ b> C having an annular shape (ring shape) around the nozzle 121. The drive method of the drive element 13C can be the same as that of the drive element 13.

  The drive means 20 selectively (for example, alternately) the ejection waveform that forms the droplet 310 by vibrating the membrane 12C and the stirring waveform that vibrates the membrane 12C within a range where the droplet 310 is not formed. ) Can be granted.

  For example, the discharge waveform and the stirring waveform are both rectangular waves, and the driving voltage of the stirring waveform is lower than the driving voltage of the discharging waveform, so that the droplet 310 can be prevented from being formed by applying the stirring waveform. That is, the vibration state (degree of vibration) of the membrane 12C can be controlled by the level of the drive voltage.

  In the droplet discharge means 10C, since the driving element 13C is formed on the lower surface side of the membrane 12C, when the membrane 12 vibrates by the driving element 13C, a flow from the lower direction to the upper direction of the liquid chamber 11C may be generated. Is possible.

  At this time, the movement of the fluorescence-stained cells 350 is a movement from the bottom to the top, and convection is generated in the liquid chamber 11C, and the cell suspension 300 containing the fluorescence-stained cells 350 is stirred. Due to the flow from the lower direction to the upper direction of the liquid chamber 11C, the sedimented and aggregated fluorescent stained cells 350 are uniformly dispersed inside the liquid chamber 11C.

  That is, the drive unit 20 applies the discharge waveform to the drive element 13C and controls the vibration state of the membrane 12C, thereby causing the cell suspension 300 held in the liquid chamber 11C to be discharged from the nozzle 121 as the droplet 310. Can do. 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. In addition, the droplet 310 is not discharged from the nozzle 121 at the time of stirring.

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

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

  That is, when bubbles are mixed in the vicinity of the nozzle 121 or when a large number of bubbles are mixed on the membrane 12C, the discharge state is affected. It is necessary to discharge the mixed bubbles. Normally, air bubbles mixed on the membrane 12C move upward naturally or by vibration of the membrane 12C. However, since the liquid chamber 11C is provided with an air release portion 115, the air bubbles mixed from the air release portion 115 are removed. It becomes possible to discharge. Therefore, it is possible to prevent non-ejection from occurring even if bubbles are mixed in the liquid chamber 11C, and the droplets 310 can be formed continuously and stably.

  It should be noted that the membrane 12C may be vibrated within a range where droplets are not formed at the timing when droplets are not formed, and the bubbles may be positively moved above the liquid chamber 11C.

-Electrical or magnetic detection method-
As a method for electrical or magnetic detection, as shown in FIG. 19, for cell counting, a cell suspension is discharged from a liquid chamber 11 ′ as a droplet 310 ′ onto a plate 700 ′ immediately below a discharge head. The coil 200 is installed as a sensor. The cells are covered with magnetic beads that are modified by specific proteins and can adhere to the cells, so that the induced current generated when the cells with the magnetic beads attached pass through the coil. The presence or absence can be detected. In general, cells have cell-specific proteins on their surfaces, and it is possible to attach magnetic beads to cells by modifying antibodies that can adhere to this protein to magnetic beads. . As such magnetic beads, ready-made products can be used. For example, Dynabeads (registered trademark) manufactured by Veritas Co., Ltd. can be used.

[Process to observe cells before discharge]
As processing for observing cells before discharge, a method of counting the cells 350 ′ that have passed through the microchannel 250 shown in FIG. 20, a method of acquiring an image near the nozzle portion of the discharge head shown in FIG. Is mentioned. FIG. 20 shows a method used in the cell sorter apparatus. For example, a cell sorter SH800 manufactured by Sony Corporation can be used. In FIG. 20, the presence or absence of a cell and the type of cell are identified by irradiating laser light from a light source 260 into a microchannel 250 and detecting scattered light and fluorescence with a detector 255 using a condenser lens 265. However, it is possible to form droplets. By using this method, it is possible to estimate the number of cells that have landed in a predetermined well from the number of cells that have passed through the microchannel 250.
Further, as the ejection head 10 ′ shown in FIG. 21, a single cell printer manufactured by Cytena can be used. In FIG. 21, it is estimated that the cells 350 ″ near the nozzle portion have been discharged from the result of image acquisition in the image acquisition portion 255 ′ via the lens 265 ′ near the nozzle portion before discharging, or images before and after discharging. It is possible to estimate the number of cells that have landed in a given well by estimating the number of cells that are considered to be ejected due to the difference from the number of cells that have passed through the microchannel shown in FIG. In the method, droplets are generated continuously, whereas FIG. 21 is more preferable because droplet formation is possible on demand.

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

The method of observing cells before and after droplet ejection has the following problems, but it is most preferable to observe cells in the droplet being ejected depending on the type of plate to be generated. In the method of observing cells before discharge, the number of cells that have landed is counted from the number of cells that have passed through the flow path and image observation before (and after) discharge. It has not been confirmed whether it was done, and unexpected errors may occur. For example, there are cases where the nozzle portion is dirty and the droplets are not ejected correctly, adhere to the nozzle plate, and the cells in the droplets do not land accordingly. In addition, there may be a problem that cells remain in a narrow region of the nozzle portion, or that the cells move more than expected due to the ejection operation and go out of the observation range.
There is also a problem in the method of detecting cells on the plate after landing. First, it is necessary to prepare a plate that can be observed with a microscope. As an observable plate, a plate with a transparent and flat bottom surface is generally used, particularly a plate with a glass bottom surface. However, since a special plate is used, a general well may be used. There is a problem that can not be done. In addition, when the number of cells is large, such as several tens of cells, there is a problem in that accurate counting cannot be performed because of overlapping of cells. Therefore, in addition to counting the number of cells contained in the droplet by the sensor and the particle number (cell number) counting means after discharging the droplet and before landing the droplet on the well, It is preferable to perform a process of observing and a process of counting cells after landing.

As the light receiving element, a light receiving element having one or a small number of light receiving portions, for example, a photodiode, an avalanche photodiode, or a photomultiplier tube can be used. In addition, the light receiving elements are provided in a two-dimensional array. 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 parts, it is conceivable to determine how many cells are contained from the fluorescence intensity using a calibration curve prepared in advance. A binary detection of the presence or absence of cells is performed. When discharging in a state where the cell concentration of the cell suspension is sufficiently low and only 1 or 0 cells are contained in the droplet, counting can be performed with sufficient accuracy by binary detection. 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 P (2) > 2) is represented by the following formula (1). FIG. 22 is a graph showing the relationship between the probability P (> 2) and the average cell number. Here, λ is the average number of cells in the droplet, and is obtained by multiplying the cell concentration in the cell suspension by the volume of the discharged droplet.
P (> 2) = 1− (1 + λ) × e ^−λ Formula (1)

  When performing cell counting by binary detection, it is preferable that the probability P (> 2) is a sufficiently small value to ensure accuracy, and that the probability P (> 2) is 1% or less λ < It is preferably 0.15. The light source is not particularly limited as long as it can excite the fluorescence of the cell, and can be appropriately selected according to the purpose, so that a general lamp such as a mercury lamp or a halogen lamp is irradiated with a specific wavelength. It is possible to use a filter, an LED (Light Emitting Diode), a laser, or the like. However, it is preferable to use a laser because it is necessary to irradiate a narrow region with high light intensity, particularly when forming a micro droplet of 1 nL or less. As the laser light source, it is possible to use a variety of generally known lasers such as a solid laser, a gas laser, and a semiconductor laser. Further, the excitation light source may be one that continuously irradiates the region through which the droplets pass, or at a timing with a predetermined time delay with respect to the droplet discharge operation in synchronization with the droplet discharge. Irradiation in a pulse manner may be used.

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

<< Output process >>
The output step is a step of outputting the value obtained by counting the number of cells contained in the cell suspension landed in the well by the particle number counting means based on the detection result measured by the sensor.
The counted value means the number of cells contained in the well by the particle number counting means from the detection result measured by the sensor.
Output refers to sending a value counted by a device such as a prime mover, a communication device, or a computer as electronic information to a server as an external counting result storage means, or printing the counted value as printed matter. Means that.

The output step observes or estimates the number of cells or the number of nucleic acids in each well in the plate at the time of generating the plate, and outputs the observed value or estimated value to an external storage unit.
The output may be performed simultaneously with the cell number counting step or may be performed after the cell number counting step.

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

<< Nucleic acid extraction process >>
The nucleic acid extraction step is a step of extracting nucleic acid from cells in the well.
Extraction means destroying a cell membrane, a cell wall, etc., and extracting nucleic acid.

  As a method for extracting nucleic acid from cells, a method of heat treatment at 90 ° C. to 100 ° C. is known. When heat treatment is performed at 90 ° C. or lower, DNA may not be extracted, and when heat treatment is performed at 100 ° C. or higher, DNA may be decomposed. At this time, it is preferable to add a surfactant and heat-treat.

  There is no restriction | limiting in particular as surfactant, According to the objective, it can select suitably, For example, an ionic surfactant, a nonionic surfactant, etc. are mentioned. These may be used individually by 1 type and may use 2 or more types together. Among these, although it depends on the amount added, nonionic surfactants are preferred from the viewpoint of not denaturing and deactivating proteins.

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

  Nonionic surfactants include, for example, alkyl glycosides, alkyl polyoxyethylene ethers (such as Brij series), octylphenol ethoxylates (Totonton X series, Igepal CA series, Nonidet P series, Nikol OP series, etc.), polysorbates ( Tween series such as Tween 20), sorbitan fatty acid ester, polyoxyethylene fatty acid ester, alkyl maltoside, 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 preferable.

The content of the surfactant is preferably 0.01% by mass or more and 5.00% by mass or less based on the total amount of the cell suspension in the well. If the content is 0.01% by mass or more, an effect can be exerted on DNA extraction, and if it is 5.00% by mass or less, inhibition of amplification can be prevented during PCR. The above-mentioned range of 0.01% by mass to 5.00% by mass is preferable for obtaining the above effect.
For cells having cell walls, DNA may not be sufficiently extracted by the above method. In this case, for example, osmotic shock method, freeze-thaw method, enzyme digestion method, use of DNA extraction kit, ultrasonic treatment method, French press method, homogenizer and the like can be mentioned. Among these, the enzymatic digestion method is preferable because the loss of extracted DNA is small.

<< Other processes >>
There is no restriction | limiting in particular as another process, According to the objective, it can select suitably, For example, an enzyme deactivation process etc. are mentioned.

-Enzyme deactivation process-
The enzyme deactivation step is a step of deactivating the enzyme.
Examples of the enzyme include DNase, RNase, and the enzyme used for nucleic acid extraction in the nucleic acid extraction step.
There is no restriction | limiting in particular as a method of inactivating an enzyme, According to the objective, it can select suitably, A well-known method can be used suitably.

The inspection 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, for example, apparatus calibration, calibration curve creation, and inspection apparatus accuracy management.
As an inspection device, when implemented against infectious diseases, it can be applied to methods stipulated in the official law or notification law,

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

<Production of inspection device>
An inspection device was produced as follows.
-Genetically modified yeast-
Saccharomyces cerevisiae w303-1a (ATCC, manufactured by ATCC 4001408) was used as a carrier cell for one copy of a specific nucleic acid sequence for production of a recombinant. The specific nucleic acid sequence is a concentrated nucleic acid sample DNA600-G (NMIJ CRM 6205-a, manufactured by National Institute of Advanced Industrial Science and Technology, see SEQ ID NO: 1), and a plasmid created so that URA3 as a selection marker is aligned in tandem As described above, a genetically modified yeast was prepared by introducing one copy of a specific nucleic acid sequence into yeast genomic DNA by homologous recombination targeting the BAR1 region of a carrier cell.

-Culture and cell cycle control-
In an Erlenmeyer flask in which 90 mL of genetically modified yeast cultured in a 50 g / L YPD medium (manufactured by Takara Bio Inc., CLN-630409) was collected, Α1-Mating Factor acetate salt (Sigma-Aldrich, T6901-5MG, hereinafter referred to as “α factor”) prepared to be 500 μg / mL using 14190-144, hereinafter referred to as “DPBS”) 900 μL was added.
Next, using a bioshaker (manufactured by Taitec Co., Ltd., BR-23FH), the mixture is incubated for 2 hours at a shaking speed of 250 rpm and a temperature of 28 ° C., and the yeast suspension is synchronized with the G0 / G1 phase. Obtained. The cell cycle of synchronized cells was confirmed by staining with a SYTOX Green Nucleic Acid Stain (device name: S7020, manufactured by Thermofisher) and using a flow cytometer (device name: SH800Z, manufactured by Sony Corporation). Thus, it was confirmed that the excitation wavelength was 488 nm and the G0 / G1 phase was synchronized. The ratio in the G1 period was 99.5%, and the ratio in the G2 period was 0.5%.

-Immobilization-
Transfer 45 mL of the synchronized yeast suspension to a centrifuge tube (manufactured by ASONE, VIO-50R), and centrifuge for 5 minutes at 3000 rpm with a centrifuge (F16RN, manufactured by Hitachi, Ltd.). The supernatant was removed to obtain a yeast pellet. By adding 4 mL of formalin (manufactured by Wako Pure Chemical Industries, Ltd., 062-1661) to the obtained yeast pellet, allowing to stand for 5 minutes, centrifuging to remove the supernatant, adding 10 mL of ethanol and suspending. An immobilized yeast suspension was obtained.

−Dyeing−
500 μL of the immobilized yeast suspension was transferred to a 1.5 mL light-shielding tube (Watson Co., Ltd., 131-915BR), centrifuged at 3,000 rpm for 5 minutes using a centrifuge, and the supernatant was removed. 400 μL of DPBS (1 mM EDTA) prepared to be 1 mM EDTA (TOCRIS, 200-449-4) was added and suspended well by pipetting, and then the rotational speed was 3 using a centrifuge. The yeast pellet was obtained by centrifuging at 1,000 rpm for 5 minutes and removing the supernatant. 1 mL of Evans Blue aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd., 054-04061) prepared to 1 mg / mL was added to the obtained pellets, stirred for 5 minutes using a vortex, and then rotated using a centrifuge. After centrifuging at 3,000 rpm for 5 minutes, the supernatant was removed, DPBS (1 mM EDTA) was added, and vortexing was performed to obtain a dyed yeast suspension.

-Dispersion-
The dyed yeast suspension is dispersed using an ultrasonic homogenizer (LUH150, manufactured by Yamato Kagaku Co., Ltd.), output: 30%, for 10 seconds, and rotated at a rotational speed of 3,000 rpm using a centrifuge at 5%. The supernatant was removed by centrifugation for 1 minute, and 1 mL of DPBS was added for washing. Centrifugation and removal of the supernatant were performed twice in total, and the suspension was again suspended in 1 mL of DPBS to obtain a yeast suspension ink.

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

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

  Next, in order to consider the reliability of the result obtained from the plate with a known number of cells, a plate with a known number of cells is manufactured, and the uncertainty in the number of cells is calculated. Note that the uncertainty at various copy numbers can be calculated by using the following method for each specific copy number.

-Calculation of uncertainty-
In this example, the number of cells in the droplet, the number of copies of the amplifiable reagent in the cell, the number of cells in the well, and contamination were used as factors of uncertainty.
The number of cells in the liquid droplets is the number of cells in the liquid droplets obtained by analyzing and counting the images of the liquid droplets ejected from the ejection means, and the number of the liquid droplets ejected by the ejection means on the slide glass and landing The number of cells obtained by microscopic observation was used.
The number of nucleic acid copies in the cell (cell cycle) was calculated using the ratio of cells corresponding to the G1 phase of the cell cycle (99.5%) and the ratio of cells corresponding to the G2 phase (0.5%). .
As for the number of cells in the well, the number of discharged droplets landed in the well was counted. However, since all the droplets landed in the well in the 96-sample counting, the factor of the number of cells in the well was uncertain. Excluded from the calculation.
Contamination was confirmed by performing three trials with 4 μL of the ink filtrate to determine whether nucleic acids other than the amplifiable reagent in the cells were mixed in the ink liquid by real-time PCR. As a result, the detection limit was reached in all three times, so contamination factors were also excluded from the uncertainty listing.
For uncertainty, the standard deviation is obtained from the measured value of each factor, and the combined standard uncertainty is obtained by the sum of squares standard uncertainties that are unified to the unit of measurement by multiplying the sensitivity coefficient. Since the synthetic standard uncertainty includes only a value in the range of about 68% of the normal distribution, the uncertainty considering the range of about 95% of the normal distribution is obtained by setting the expanded standard uncertainty twice the synthetic standard uncertainty. Can be obtained. The results are shown in the budget sheet of Table 1 below.

In Table 1, “symbol” means an arbitrary symbol associated with an uncertainty factor.
In Table 1, “value (±)” is the average experimental standard deviation, and is obtained by dividing the calculated experimental standard deviation by the value of the square root of the number of data.
In Table 1, “probability distribution” is a probability distribution of an uncertainty factor. In the case of A type uncertainty evaluation, it is blank, and for B type uncertainty evaluation, a normal distribution or a rectangular distribution is used. Enter one. In this embodiment, only the A-type uncertainty evaluation is performed, so the probability distribution column is blank.
In Table 1, “divisor” means a number that normalizes the uncertainty obtained from each factor.
In Table 1, “standard uncertainty” is a value obtained by dividing “value (±)” by “divisor”.
In Table 1, “sensitivity coefficient” means a value used to unify the unit of measurement.

  Based on the above results, the obtained expanded uncertainty is stored as data for each well as an index of measurement variation, so that the person using the experiment can determine the reliability index of the measurement results for each well. Can be used as a reference. By using the above criteria for determining reliability, the performance evaluation of analytical inspection can be performed with high accuracy.

Aspects of the present invention are as follows, for example.
<1> having at least one well,
A test device having information on the absolute number of amplifiable reagents in at least one well and uncertainty on the absolute number of amplifiable reagents.
<2> The inspection device according to <1>, wherein the amplifiable reagent is contained in a carrier.
<3> The inspection device according to any one of <1> to <2>, wherein the amplifiable reagent is a nucleic acid.
<4> The inspection device according to any one of <2> to <3>, wherein the nucleic acid is incorporated into a nucleic acid in a cell nucleus.
<5> The inspection device according to any one of <3> to <4>, wherein the nucleic acid has a base length of 20 or more and 10,000 or less.
<5> The inspection device according to any one of <3> to <4>, wherein the nucleic acid has a non-natural base sequence that does not exist in nature.
<7> The test device according to any one of <3> to <6>, wherein the GC content of the nucleic acid is 30% to 70% of the specific base sequence.
<8> The uncertainty information is at least one of a means for arranging the cell in a device, a cell cycle of the cell, a number of cells arranged in the well, and contamination in the well. The inspection device according to any one of 4> to <7>.
<9> The inspection device according to any one of <1> to <8>, further including identification means capable of identifying information on the absolute number of the amplifiable reagent and the uncertainty of the absolute number of the amplifiable reagent. is there.
<10> The inspection device according to any one of <4> to <9>, wherein the cell is yeast.
<11> The inspection device according to any one of <1> to <10>, wherein the well has at least one of a primer and an amplification reagent.
<12> A plurality of the wells,
The inspection device according to any one of <1> to <11>, wherein the absolute number of nucleic acids in each well and the uncertainty of the absolute number of nucleic acids are used as information for each well.
<13> A substrate on which the well is held, and a sealing unit capable of sealing the well of the substrate, wherein the identification unit is disposed between the sealing unit and the substrate. 9> to <12>.
<14> A device used for evaluation of a PCR apparatus capable of amplifying a nucleic acid,
Having at least one well,
A device having information associated with at least one absolute number of nucleic acids in the well and uncertainty of the absolute number of nucleic acids.
<15> The device according to <14>, which is used for the management of the PCR apparatus, the result of nucleic acid amplification by the device, the absolute number of the nucleic acid and the uncertainty of the absolute number of the nucleic acid using the associated information, and It is.

  The inspection device according to any one of <1> to <13> and the device according to any one of <14> to <15> solve the problems in the related art, and achieve the object of the present invention. Can be achieved.

DESCRIPTION OF SYMBOLS 1 Inspection device 2 Base material 3 Well 4 Reproducible reagent 5 Sealing member

JP 2014-33658 A JP-A-2015-195735

Claims (13)

Having at least one well,
Device comprising: the absolute number of information of nucleic acid extracted from the cells arranged in at least one of said wells, and information of the absolute number of uncertainty of the nucleic acid.   The device of claim 1, wherein the nucleic acid is incorporated into a nucleic acid in a cell nucleus.   The device according to claim 1, wherein the nucleic acid has a base length of 20 or more and 10,000 or less.   The device according to claim 1, wherein the nucleic acid has a non-natural base sequence that does not exist in nature.   The device according to any one of claims 1 to 4, wherein the nucleic acid has a specific base sequence, and the GC content of the nucleic acid is 30% to 70% of the specific base sequence.   The uncertainty factor in the uncertainty information is at least one of the number of cells in a droplet, the cell cycle of the cells, the number of cells arranged in the well, and contamination in the well. The device according to any one of 1 to 5.   The device according to any one of claims 1 to 6, further comprising identification means capable of identifying information on the absolute number of the nucleic acid and the uncertainty of the absolute number of the nucleic acid.   The device according to claim 1, wherein the cell is yeast.   The device according to claim 1, comprising at least one of a primer and an amplification reagent in the well. A plurality of the wells,
The device according to any one of claims 1 to 9, which has, as information for each well, the absolute number of nucleic acids in each well and the uncertainty of the absolute number of nucleic acids.   The base material on which the well is held and sealing means capable of sealing the well of the base material are provided, and the identification means is disposed between the sealing means and the base material. A device according to any of the above. A device used for evaluation of a PCR apparatus capable of amplifying a nucleic acid,
Having at least one well,
Devices having the absolute number of information of nucleic acid extracted from the cells arranged in at least one of said wells, and information of the absolute number of uncertainty of the nucleic acid.   The device according to claim 12, wherein the result of nucleic acid amplification by the device and information on the absolute number of the nucleic acid and the uncertainty of the absolute number of the nucleic acid are used for management of the PCR device.