How to detect the copy number of a specific nucleic acid in a single cell

Abstract

The present invention detects the copy number of a specific nucleic acid per single cell in a cell population. This method comprises, in a reaction compartment containing a DNA sample, which is derived from nucleic acids in a single cell, and a PCR system, amplifying a target contained in the DNA sample by PCR. Then, PCR amplicons for each reaction compartment are quantified during the exponential amplification phase.

Description

[Technical field]

[0001] The present invention relates to a method for detecting the copy number of a specific nucleic acid in a single cell. [Background technology]

[0002] Non-Patent Document 1 discloses that genomic DNA extracted from cells is encapsulated in droplets and amplified by PCR, although this is not a single-cell analysis. In Non-Patent Document 1, a target sample and a reference sample are quantified independently in the same droplet. This is achieved by distinguishing the fluorescent signal of the intercalator to distinguish the length of the amplicon.

[0003] Non-Patent Document 2 discloses single-cell RT-PCR (reverse transcription PCR) targeting the mRNA of a specific gene. + The presence of HIV-1 in T cells is detected in a high-throughput manner using reverse transcription and single-cell-in-droplet (scd) PCR assays.

[0004] Non-Patent Document 4 discloses a single cell-droplet digital PCR (sc-ddPCR) method. In this method, a single cell is encapsulated in a droplet, and PCR is performed in the droplet using gene-specific primers and probes. One copy of the gene is artificially introduced into the cell.

[0005] Non-Patent Document 5 discloses dissolving a single cell within a droplet, and integrating the droplet in which the single cell is dissolved with a droplet of a RT-PCT reaction solution.

[0006] Patent Document 1 discloses, although not a single cell analysis, a method of determining the genetic status by analyzing the characteristics of DNA obtained from fetal cells concentrated from maternal blood. Patent Document 1 discloses amplifying the DNA of the concentrated fetal cells and analyzing the amplified DNA using digital PCR. Patent Document 1 also discloses detection of chromosome 21.

[0007] Patent Document 2 discloses a method for isolating blood cells containing potential fetal cells at the single-cell level, and then independently extracting chromosomal DNA from each of the isolated blood cells, and subsequently identifying the fraction containing fetal-derived chromosomal DNA.

[0008] Non-Patent Document 6 discloses that the above-mentioned sc-ddPCR method is used to detect the SRY gene on the genomic DNA of fetal cells circulating in maternal blood.

[0009] Non-Patent Document 7 discloses quantitative PCR using 1, 2, 4,... copies of yeast genome as a template, which are weighed out using the piezoelectric effect.

[0010] Non-patent documents 8 and 9 will be described later. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Special Publication No. 2014-533509 [Patent Document 2] JP 2018-102242 A [Non-patent literature]

[0012] [Non-Patent Document 1] Laura Miotke, et al., "High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR", Analytical chemistry, 86, 5, 2618-2624. [Non-Patent Document 2] Dennis J. Eastburn, et al., "Identification and genetic analysis of cancer cells with PCR-activated cell sorting", Nucleic Acids Research, 42, 16, e128. [Non-Patent Document 3] Robert W. Yucha, et al., "High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay", EBioMedicine, 20, 217-229. [Non-Patent Document 4] Yuka Igarashi, et al., "Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy", Molecular Therapy, Methods & Clinical Development, 6, 8-16. [Non-Patent Document 5] Samuel C. Kim, et al., "Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis", Analytical Chemistry, 90, 2, 1273-1279. [Non-Patent Document 6] Taisuke Sato, et al., "Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis", The Journal of Molecular Diagnostics, 22, 2, 2, 132-140. [Non-Patent Document 7] Unoh Ki, et al., "A Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA", Ricoh Technical Report, 2020, 44, 14-26. Retrieved from <https: / / jp.ricoh.com / technology / techreport / 44 / > [Non-Patent Document 8] Ichiro Hanamura, et al., "Frequent gain of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation", Blood, 108, 5, 1724-1732. [Non-Patent Document 9] Sadao Ishida, “Multiple myeloma: chromosomal abnormalities, clinical disease types, and prognosis” , [online], 2013-09-21, Clinical Hetology, 54, 10, 2, 1856-1866. Retrieved from<https: / / doi.org / 10.11406 / rinketsu.54.1856> Summary of the Invention [Problem to be solved by the invention]

[0013] Digital PCR qualitatively indicates whether or not a template is present in a microcompartment (e.g., a droplet). Therefore, digital PCR is not suitable for detecting discrete copy number variations (CNVs) of a specific nucleic acid in a microcompartment.

[0014] It is an object of the present invention to provide a method that can detect the copy number of a particular nucleic acid in every single cell in a population of cells. [Means for solving the problem]

[0015] The present invention relates to a method for detecting the copy number of a specific nucleic acid in each single cell as follows.

[0016] [1] A method for detecting the copy number of a specific nucleic acid in each single cell in a population of cells, comprising: amplifying a target contained in sample DNA by PCR in a reaction compartment containing sample DNA derived from a nucleic acid in the single cell and a PCR reaction system; and quantifying the PCR amplicon for each reaction compartment during the exponential amplification phase. [2] The detection method according to [1], wherein the sample DNA is a set of genomic DNA in a single cell, a reverse transcription product of RNA in a single cell, or a mitochondrial DNA in a single cell. [3] The detection method according to [1] or [2], wherein the amplicon is quantified using a fluorescent probe method. [4] The detection method described in [3], wherein the PCR reaction system includes a plurality of probes each having a fluorescent substance with a different fluorescent wavelength that is assigned to a different region on the sample DNA, the region containing one or more of the targets, and in the step of quantifying the amplicon, fluorescence of a plurality of wavelengths is detected from one of the reaction compartments to distinguish the reaction compartment in which the target contained in the sample DNA has been amplified from the reaction compartment in which only contaminating cell-free nucleic acid has been amplified. [5] The detection method described in [3] or [4], wherein the region includes a plurality of targets each assigned to a plurality of probes having fluorescent substances with the same fluorescent wavelengths, and in the step of quantifying the amplicon, the fluorescence from the plurality of probes is measured collectively for each region regardless of the differences in the targets, thereby quantifying the amplicon. [6] The method according to [3], wherein the sample DNA comprises a set of genomic DNA in the single cell and mitochondrial DNA in the single cell, the PCR reaction system comprises a probe having a fluorescent substance with a first fluorescent wavelength assigned to a region on the genomic DNA and a probe having a fluorescent substance with a second fluorescent wavelength assigned to the mitochondrial DNA, the region comprising one or more of the targets, and in the step of amplifying the target contained in the sample DNA by PCR, the target contained in the set of genomic DNA and the target contained in the mitochondrial DNA are amplified by PCR in the reaction compartments, and in the step of quantifying the amplicons, the amplicons of the genomic DNA and the mitochondrial DNA are quantified for each reaction compartment in a cycle in which amplification of the target contained in the mitochondrial DNA reaches a plateau and amplification of the target contained in the genomic DNA reaches the exponential amplification phase, and in the step of quantifying the amplicons, the fluorescence of the first and second fluorescent wavelengths is detected from one of the reaction compartments to distinguish the reaction compartment in which the target contained in the genomic DNA and the target contained in the mitochondrial DNA have been amplified from the reaction compartment in which only contaminating cell-free nucleic acid has been amplified. [7] The detection method described in any one of [1] to [6], further comprising a step of generating the reaction compartment by dissolving the single cell in a compartment containing a single cell, a cell lysis reagent, and a PCR premix. [8] The detection method described in any one of [1] to [6], further comprising the steps of: lysing a single cell in a compartment containing the single cell; and generating the reaction compartment by integrating the compartment containing the lysed single cell with a compartment containing a PCR premix. [9] The detection method described in any one of [1] to [6], further comprising the steps of: mixing a population of cell nuclei with a PCR premix in bulk; and generating the reaction compartment by individualizing each cell nucleus in the population of cell nuclei together with the premix.

[10] The detection method according to any one of [1] to [9], wherein the reaction compartment is a reaction droplet that contains the sample DNA and the PCR reaction system.

[11] The detection method according to any one of [1] to

[10] , wherein the sample DNA contains a complete set of genomic DNA in the single cell, and the detection method further comprises a step of detecting the presence of a single cell having at least one chromosomal mutation selected from the group consisting of full-length aneuploidy of a chromosome, partial aneuploidy of a chromosome, gene amplification, and gene deletion from the quantification result of the amplicon.

[12] The detection method according to

[11] , wherein the single cell having a chromosomal mutation contains genomic DNA having at least any one of the following chromosomal mutations: - Aneuploidy of chromosome 21 - Aneuploidy of chromosome 18 - Aneuploidy of chromosome 13 - Y chromosome aneuploidy - X chromosome aneuploidy - Deletion of the 22q11.2 region of the long arm of chromosome 22 - Deletion of the 5q region of the short arm of chromosome 5 - Deletion of the 15q11-q13 region of the long arm of chromosome 15 - Amplification of the long arm of chromosome 1 - Deletion of the short arm of chromosome 17 - Deletion of the long arm of chromosome 13 - Deletion of the long arm of chromosome 4 - Deletion of the long arm of chromosome 5 - Deletion of the long arm of chromosome 7 - Amplification of chromosome 8 - Deletion of chromosome 11 - Aneuploidy of chromosome 12 - Deletion of the long arm of chromosome 20 - Deletion of the long arm of chromosome 19 - Deletion of chromosome 1 - Deletion of the long arm of chromosome 18 - Deletion of the short arm of chromosome 8 - Deletion of chromosome 4 - Amplification of the long arm of chromosome 8 - Deletion of the long arm of chromosome 16 - Amplification of the short arm of chromosome 5 - Amplification of the long arm of chromosome 3 - Deletion of the short arm of chromosome 3 - Deletion of the short arm of chromosome 9 - Gene amplification of the MYCN gene - Genetic amplification of the HER2 gene - Gene amplification of the MET gene

[13] The method of

[11] , further comprising a step of generating data including information on whether a single cell having the chromosomal mutation has been detected, wherein the population of cells has been separated from amniotic fluid or maternal blood so as to include fetal cells, and the data is used to diagnose trisomy 13, trisomy 18, trisomy 21, Turner syndrome, triple X syndrome, XYY syndrome, Klinefelter syndrome, DiGeorge syndrome, Angelman syndrome, Prader-Willi syndrome, or cat's cry syndrome.

[14] The method of detection described in

[11] , further comprising a step of generating data including information on whether a single cell having the chromosomal mutation has been detected, wherein the population of cells has been isolated from a patient, and the data is for use in diagnosing myelodysplastic syndrome, multiple myeloma, idiopathic eosinophilia, chronic eosinophilic leukemia, acute nonlymphocytic leukemia, myeloproliferative neoplasms, chronic lymphocytic leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, or head and neck cancer. Effect of the Invention

[0017] According to the present invention, the copy number of a specific nucleic acid in each single cell in a population of cells can be detected. For example, according to the present invention, the copy number variation (CNV) of a specific nucleic acid in each single cell can be detected. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a conceptual diagram of detection. [Diagram 2] FIG. 2 is a flow chart showing an example of detection. [Diagram 3] FIG. 3 is a diagram showing an outline of droplet preparation. [Figure 4] FIG. 4 is a diagram showing the assignment of probes. [Diagram 5] Figure 5 shows the scatter of detection events. [Figure 6] FIG. 6 is a diagram illustrating the allocation of multiple probes. [Figure 7] FIG. 7 shows the scatter of fluorescence enhanced detection events. [Figure 8] Figure 8 shows the scattering of droplets on the plateau. [Figure 9] FIG. 9 shows droplet scattering during the exponential amplification phase. [Figure 10]FIG. 10 shows droplet scattering with enhanced fluorescence. [Figure 11] FIG. 11 shows droplet scatter of a heterogeneous cell population. [Figure 12] FIG. 12 shows droplet scatter of a heterogeneous cell population. [Figure 13] Figure 13 is a scatter diagram that distinguishes between diploid and haploid plants. [Figure 14] Figure 14 is a scatter diagram that distinguishes between diploid and haploid plants. [Figure 15] Figure 15 is a scatter diagram that distinguishes between diploid and haploid plants. [Figure 16] FIG. 16 is a fluorescence enhanced scatter diagram distinguishing diploid from haploid. [Figure 17] FIG. 17 is a fluorescence enhanced scatter diagram distinguishing diploid from haploid. [Figure 18] FIG. 18 is a fluorescence enhanced scatter diagram distinguishing diploid from haploid. [Figure 19] FIG. 19 shows box plots corresponding to each scatter. [Figure 20] FIG. 20 shows the ROC curve when 23 types of probes were used. [Figure 21] FIG. 21 is a scatter plot including a line showing the cutoff value. [Figure 22] FIG. 22 shows the displacement of clusters for each measurement. [Diagram 23] FIG. 23 is a graph showing the variation in the corrected measurement values. [Figure 24] FIG. 24 is a diagram for explaining calculation of the cutoff value using the corrected measurement value. [Diagram 25] Figure 25 shows a scatter plot of droplets containing genomic DNA derived from human blood. [Figure 26] FIG. 26 shows a scatter of droplets containing CD45 cells. [Figure 27]FIG. 27 shows droplet scatter of a homogeneous cell population. [Figure 28] FIG. 28 shows droplet scatter of a heterogeneous cell population. [Figure 29] FIG. 29 shows the scattering of droplets from each cell population. [Diagram 30] FIG. 30 is a box plot showing the detection results of abnormal droplets for each cell population. [Diagram 31] FIG. 31 shows a scatter of droplets containing the products of reverse transcription of RNA. [Diagram 32] FIG. 32 shows a scatter plot of droplets containing mitochondrial DNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] <Summary> 1 is a conceptual diagram showing how to detect the copy number of a specific nucleic acid in each single cell in a population of cells, for example, CNV at a specific position on genomic DNA. In one embodiment, the cell is a human cell.

[0020] Hereinafter, in describing CNV, the term "haploid" includes the entire haploidization of a particular chromosome (i.e., monosomy) and a partial haploid state due to deletion. The entire or partial haploidization of a diploid chromosome is included in CNV.

[0021] The term "triploidy" includes the entire triploidization (i.e., trisomy) of a particular chromosome, and partial triploidy due to duplication. The entire or partial triploidization of a diploid chromosome is included in CNV. The entire or partial ploidization of a diploid chromosome is included in CNV.

[0022] CNVs also include changes in ploidy due to duplication of all or part of a male's sex chromosome, which is normally haploid. Hereinafter, the term "polyploidy" may refer to haploidy, diploidy, triploidy, and other ploidies.

[0023] CNVs include changes that result in the complete loss of all or part of a polyploid chromosome (biallelic deletion), as well as the creation of all or part of a chromosome that would not normally be present.

[0024] In the present invention, detecting cells having aneuploid chromosomes includes detecting that a population of cells containing cells having euploid chromosomes further contains cells having aneuploid chromosomes. In the present invention, detecting cells having aneuploid chromosomes includes detecting that a population of cells containing cells having euploid chromosomes is a population of cells not containing cells having aneuploid chromosomes, or contains aneuploid cells at a detection limit or lower. In the present invention, detecting cells having aneuploid chromosomes includes detecting that a population of cells having aneuploid chromosomes does not contain cells having euploid chromosomes.

[0025] <Overall flow> As shown on the left side of Fig. 1, a population of cells 17 is prepared. The population of cells 17 is a collection of single cells 18. The cells are eukaryotic cells or prokaryotic cells. In one embodiment, the cells are eukaryotic cells having one nucleus.

[0026] As shown in the center of FIG. 1, a reaction compartment containing a sample DNA derived from nucleic acid in a single cell 18 and a PCR reaction system is prepared. In FIG. 1, a droplet 20 is formed in oil 19 as a reaction compartment. The figure shows one embodiment of the droplet. The droplet 20 is a water-in-oil emulsion type droplet. In one embodiment, the volume of the reaction compartment (droplet 20) is 1 pL (picoliter) to 1 μL (microliter) or 1 pL (picoliter) to 10 nL (nanoliter). In the one embodiment, the volume of the reaction compartment (droplet 20) is 10 pL, 100 pL, 1 nL, 10 nL, or 100 nL. The configuration of the reaction compartment is not particularly limited as long as it is a minute compartment containing a sample DNA and a PCR reaction system, and may be, for example, a microwell.

[0027] The sample DNA is, for example, a set of genomic DNA in a single cell, a reverse transcription product of RNA in a single cell, or DNA of the mitochondrial genome in a single cell (hereinafter referred to as "mitochondrial DNA"). In FIG. 1, the droplet 20 contains a set of genomic DNA 21 in a single cell 18 as the sample DNA. The droplet 20 further contains a PCR reaction system. Hereinafter, the droplet 20 may be referred to as a reaction droplet.

[0028] In one embodiment, the sample DNA is a set of genomic DNA in a single cell. The "set of genomic DNA" may be interpreted as genomic DNA as extracted. In one embodiment, the set of genomic DNA is not subjected to whole genome amplification. This makes it possible to avoid allele dropout due to whole genome amplification. The "set of genomic DNA" includes DNA that has been unintentionally lost in part during cell lysis or other extraction procedures. In one embodiment, the "set of genomic DNA" does not include mitochondrial DNA. However, when a set of genomic DNA is used as the sample DNA, mitochondrial DNA may be mixed into the reaction compartment.

[0029] In one embodiment, the sample DNA is a reverse transcription product of RNA in a single cell.

[0030] In one embodiment, the sample DNA is mitochondrial DNA in a single cell.

[0031] In one embodiment, the sample DNA is a set of genomic DNA in a single cell and mitochondrial DNA in the same single cell. In this case, the mitochondrial DNA may be used as a control for detecting the copy number of chromosomal genomic DNA.

[0032] In other embodiments, the sample DNA is the nucleic acid of a microorganism or virus that has invaded the cell, or a nucleic acid that has been introduced into the cell.

[0033] As shown on the right side of FIG. 1, the reaction compartment is subjected to a thermal cycle. That is, in the reaction compartment including the sample DNA derived from the nucleic acid in the single cell and the PCR reaction system, the target contained in the sample DNA is amplified by PCR. In FIG. 1, the target contained in the set of genomic DNA 21 in the droplet 20 is amplified by PCR. In PCR, the sample DNA (e.g., genomic DNA) serves as a template. In FIG. 1, the droplets 22a and 22b are reaction droplets in the exponential amplification phase. At this stage, the amplicons are quantified for each reaction droplet. That is, the PCR amplicons are quantified for each reaction compartment (e.g., reaction droplet) in the exponential amplification phase. In one embodiment, the quantification of the amplicons is performed by a fluorescent probe method.

[0034] In Fig. 1, droplet 22b exhibits a stronger signal than droplet 22a. Therefore, the copy number of the target genome region of the single cell corresponding to droplet 22b is greater than the copy number of the target genome region of the single cell corresponding to droplet 22a. Therefore, the copy number and CNV of genomic DNA for a specific position (target position) on the genome can be detected.

[0035] <Detection flow> 2 is a flowchart showing an example of detection of the copy number of a nucleic acid. Here, an example will be described in which droplets are used as reaction compartments and a set of genomic DNA in a single cell is used as sample DNA.

[0036] In step 25 after initiation, a PCR premix is ​​prepared. The premix includes primers, DNA polymerase, deoxynucleoside triphosphates (dNTPs), a buffer, and necessary cofactors. In one embodiment, the cofactors include magnesium ions. In one embodiment, a cell lysis reagent is premixed with the premix. Examples of cell lysis reagents include sodium dodecyl sulfate, TRITONX-100 (TRITON is a registered trademark), and NP-40 (trademark). The premix includes necessary probes.

[0037] In step 26 shown in Fig. 2, the cell population 17 is suspended in a premix 31, as shown in the upper part of Fig. 3. The steps from suspension to droplet formation are carried out quickly so as not to cause a reaction between the cell lysis reagent and the cells.

[0038] In step 27 shown in Fig. 2, each single cell 18 is converted into a droplet together with the premix 31, as shown in the lower part of Fig. 3. This produces a droplet 33a containing the single cell 18.

[0039] As a system for forming droplets from single cells and a premix, for example, a QX200 Droplet Generator (BioRad) can be used. The system for forming single cells is not limited to this.

[0040] In one embodiment shown in FIG. 3, a cell lysis reagent is contained in the droplet 33a. In one embodiment, a single cell 18 in the droplet 33a is lysed within the droplet 33a with the above-mentioned cell lysis reagent. A reaction droplet is obtained by cell lysis. That is, a reaction compartment (reaction droplet) is generated by lysing the single cell 18 in a compartment (droplet 33a) containing the single cell 18, the cell lysis reagent, and a PCR premix. The reaction of the cell lysis reagent proceeds efficiently by heating.

[0041] In another embodiment of step 27 shown in FIG. 2, single cells are lysed in a compartment (droplet) containing single cells before mixing with the premix. The compartment (droplet) containing the lysed single cells is then merged with a compartment (droplet) containing a PCR premix. The merger can result in a reaction compartment (reaction droplet).

[0042] In another embodiment of step 27 shown in Figure 2, the bulk cells are lysed to obtain a bulk population of cell nuclei prior to mixing the single cells with the premix. The bulk population of cell nuclei is mixed with a PCR premix. Individual cell nuclei in the population of cell nuclei are singulated with the premix to generate reaction compartments (reaction droplets).

[0043] In step 28 shown in FIG. 2, PCR proceeds in the reaction droplet by thermal cycling. Thermal cycling stops at the exponential amplification phase of PCR. In one embodiment, "exponential amplification phase" refers to the time when dNTPs, primer sets, probe sets, and active enzymes have not been exhausted and therefore template-dependent DNA synthesis is not limiting. In one embodiment, "exponential amplification phase" refers to the time when the chain reaction plateau has not been reached.

[0044] In step 29 shown in Fig. 2, the copy number of the target in the reaction droplet is quantified by analyzing the signal from the reaction compartment (reaction droplet).

[0045] In one embodiment of step 25 shown in Figure 2, the premix further comprises a fluorescent probe that hybridizes to the target or an amplicon derived from the target. In another embodiment, the premix further comprises a fluorescent intercalator that binds to DNA.

[0046] In one embodiment, the target of the probe contained in the amplicon is one. In another embodiment, the target of the probe contained in the amplicon is two or more. The multiple probes that bind to one of these amplicons have fluorescent substances that emit the same color of fluorescence, so that the fluorescence emitted from the amplicon can be enhanced.

[0047] <Two-color probe method> FIG. 4 shows an example of the assignment of two-color probes. A probe 35a (1st color) having a fluorescent substance with a first fluorescent wavelength is assigned to any target on chromosome 21. A probe 35b (2nd color) having a second fluorescent wavelength is assigned to any target on chromosome 18. These types of chromosomes are merely examples. These probes are mixed together in the reaction compartment (reaction droplet).

[0048] In this example, the target to which the probe of the first fluorescent wavelength is assigned or the region containing the target is the target for copy number (CNV).In this example, the target to which the probe of the second fluorescent wavelength is assigned or the region containing the target is the control.In one embodiment, the control is a region with relatively small CNV.

[0049] That is, as shown in Fig. 4, the PCR reaction system includes a plurality of probes each having a fluorescent substance with a different fluorescent wavelength, which is assigned to a different region on the sample DNA. The region in this example is the entire length of each chromosome. The region may be a part of each chromosome, or may span across chromosomes.

[0050] In the embodiment shown in Figure 4, each region contains one target. In an embodiment different from the figure, the region may contain two or more targets. In either case, fluorescence of each fluorescence wavelength assigned to each region is detected from one reaction compartment (reaction droplet).

[0051] In the ideal reaction conditions of the embodiment shown in Figure 4, if chromosome 21 is triploid in its entirety or in the portion to be detected, the intensity of the first fluorescent wavelength is 1.5 times the intensity of the first fluorescent wavelength when chromosome 21 is diploid in its entirety or in the portion to be detected. If chromosome 21 is haploid in its entirety or in the portion to be detected, the intensity of the first fluorescent wavelength is 0.5 times the intensity of the first fluorescent wavelength when chromosome 21 is diploid in its entirety or in the portion to be detected. This is shown in Figure 5.

[0052] FIG. 5 shows a scatter plot of detection events of fluorescence emitted from a reaction compartment (reaction droplet). The vertical axis represents the first fluorescence wavelength assigned to chromosome 21, and the horizontal axis represents the second fluorescence wavelength assigned to chromosome 18. Each circle represents the center of the fluorescence intensity distribution of the detection events on the plot. Since most of the events are accumulated within this circle, the accumulated events are hereinafter referred to as a "cluster." Cluster 39 represents events whose fluorescence intensity was not different from the background at any of the fluorescence wavelengths. Clusters 40a to 40c represent events whose fluorescence intensity was different from the background at any of the fluorescence wavelengths.

[0053] 5, chromosome 21 is diploid either entirely or in a portion to be detected, as indicated by the intensity of the first fluorescent wavelength, and chromosome 18 is diploid either entirely or in a portion to be detected, as indicated by the intensity of the second fluorescent wavelength.

[0054] 5, chromosome 21 is triploid in its entirety or in the portion to be detected, as indicated by the intensity of the first fluorescent wavelength, and chromosome 18 is diploid in its entirety or in the portion to be detected, as indicated by the intensity of the second fluorescent wavelength.

[0055] 5, chromosome 21 is shown to be monoploid in its entirety or in a portion to be detected by the intensity of the first fluorescent wavelength, and chromosome 18 is shown to be diploid in its entirety or in a portion to be detected by the intensity of the second fluorescent wavelength.

[0056] In one embodiment shown in Figure 5, a shift of the dots from the location of cluster 40a to the location of cluster 40b is detected. In such a shift, the intensity of the first fluorescent wavelength increases. In such a shift, the intensity of the second fluorescent wavelength remains unchanged. Indicative of the presence of a single cell in the population of cells in which chromosome 21 is triploid in its entirety or in the portion being detected.

[0057] In another embodiment shown in Figure 5, a shift of the dots from the location of cluster 40a to the location of cluster 40c is detected. In such a shift, the intensity of the first fluorescent wavelength is decreased. In such a shift, the intensity of the second fluorescent wavelength is unchanged. Indicative of the presence of a single cell in the population of cells in which chromosome 21 is haploid in its entirety or in the portion being detected.

[0058] The magnitude of the shift on these scatter plots may be estimated by preliminary experiments. The ploidy of cells in each cluster on these scatter plots may be estimated by preliminary experiments.

[0059] To explain the clusters 44a and 44b shown in Fig. 5, let us return to Fig. 3. When the droplet 33a is generated, cell-free nucleic acids may be entrained within the droplet. The cell-free nucleic acids are, for example, fragments of genomic DNA released from broken cells. Amplicons may also be generated by PCR from the droplet 33b containing such cell-free genomic DNA.

[0060] In the scatter plot shown in Figure 5, the amplicons derived from such cell-free molecules produce clusters 44a and 44b. The signals of the amplicons derived from cell-free molecules are unlikely to overlap with cluster 40a. This is because it is unlikely that two or more targets on different regions are simultaneously amplified in a droplet derived from a cell-free molecule. In addition, the number of copies of the target in a droplet containing a cell-free molecule is usually one.

[0061] Returning to Figure 4, fluorescent substances with different fluorescent wavelengths are used in each of the different regions as shown in Figure 4. In other words, a probe with a second fluorescent wavelength is used as a control. This allows reaction droplets in which genomic DNA has been amplified to be distinguished from droplets in which only contaminating cell-free nucleic acids have been amplified, as shown in Figure 5.

[0062] That is, in the example of Figure 4, the PCR reaction system includes a plurality of probes each having a fluorescent substance with a different fluorescent wavelength, which are assigned to different regions on the sample DNA. The regions also include one or more of the targets. In the step of quantifying the amplicons, fluorescence of multiple wavelengths is detected from one reaction compartment to distinguish a reaction compartment in which the target contained in the sample DNA has been amplified from a reaction compartment in which only contaminating cell-free nucleic acid has been amplified.

[0063] The types of fluorescent substance (fluorescent dye) and quencher added to the probe are not particularly limited. For example, the fluorescent substance of the first fluorescent wavelength is FAM (trademark, Fluorescein amidite), and the fluorescent substance of the second fluorescent wavelength is HEX (trademark, Hexachloro-fluorescein).

[0064] <Increased target and enhanced signal> FIG. 6 shows an example in which multiple types of probes are assigned to each color. The differences from FIG. 4 are explained below. Three types of probes (1st color) having fluorescent substances with a first fluorescent wavelength are assigned to three types of targets on chromosome 21. Three types of probes (2nd color) having fluorescent substances with a second fluorescent wavelength are assigned to three types of targets on chromosome 18. These types of chromosomes are merely examples. The PCR reaction system includes multiple probes, each having fluorescent substances with different fluorescent wavelengths, assigned to different regions on the sample DNA. These have different target sequences. These probes are mixed together in the droplet.

[0065] Figure 7 shows a scatter plot of detection events of fluorescence emitted from the reaction compartment (reaction droplet). Clusters 41a to 41c show events in which the fluorescence intensity was different from the background at all fluorescence wavelengths. A shift similar to that in Figure 5 is observed.

[0066] 7, chromosome 21 is diploid either entirely or in a portion to be detected, as indicated by the intensity of the first fluorescent wavelength, and chromosome 18 is diploid either entirely or in a portion to be detected, as indicated by the intensity of the second fluorescent wavelength.

[0067] 7, chromosome 21 is triploid in its entirety or in a portion to be detected, as indicated by the intensity of the first fluorescent wavelength, and chromosome 18 is diploid in its entirety or in a portion to be detected, as indicated by the intensity of the second fluorescent wavelength.

[0068] 7, the intensity of the first fluorescent wavelength indicates that chromosome 21 is monoploid in its entirety or in a portion to be detected, and the intensity of the second fluorescent wavelength indicates that chromosome 18 is diploid in its entirety or in a portion to be detected.

[0069] In one embodiment shown in Figure 7, a shift of the dots from the location of cluster 41a to the location of cluster 41b is detected. In such a shift, the intensity of the first fluorescent wavelength increases. In such a shift, the intensity of the second fluorescent wavelength remains unchanged. Indicative of the presence of a single cell in the population of cells that is triploid in the entire or detected portion of chromosome 21.

[0070] In another embodiment shown in Figure 7, a shift of the dots from the location of cluster 41a to the location of cluster 41c is detected. In such a shift, the intensity of the first fluorescent wavelength increases. In such a shift, the intensity of the second fluorescent wavelength does not change. Indicative of the presence of a single cell in the population of cells in which chromosome 21 is haploid in its entirety or in the portion being detected.

[0071] The magnitude of these shifts on the scatter plots may be estimated by preliminary experiments.

[0072] Clusters 44a and 44b shown in Fig. 7 are similar to those shown in Fig. 5. These clusters can be distinguished from clusters 41a to 41c by using a technique for detecting probes with different fluorescent wavelengths within the same droplet. This is also similar to Fig. 5.

[0073] Returning to Figure 6, three probes with a fluorescent substance of the first fluorescent wavelength are assigned to each chromosome 21. Similarly, three probes with a fluorescent substance of the second fluorescent wavelength are assigned to each chromosome 18. Therefore, under ideal reaction conditions, the fluorescence intensity of each color in the reaction droplet increases in proportion to the number of targets in the region. In addition, by increasing the number of targets in one reaction droplet, the variation in PCR efficiency is averaged out, and the variation in fluorescence intensity between reaction droplets is reduced.

[0074] Explaining this with reference to FIG. 7, clusters 41a to 41c are more likely to be separated from cluster 39, cluster 44a, and cluster 44b.

[0075] In this embodiment, each region includes a plurality of targets each assigned to a plurality of probes having fluorescent substances with the same fluorescence wavelength. In addition, when quantifying the amplicon, the fluorescence from the plurality of probes is measured collectively for each region by color regardless of the difference between the targets, thereby quantifying the amplicon. In this way, the fluorescence from the regions to which the targets are assigned is enhanced. Therefore, it becomes easier to separate the target signals from the background noise.

[0076] In one embodiment, the number of targets in one region is 1 to 1,000. In one embodiment, the number of targets in one region is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, The number of targets may be the same or different between the regions. In each region, the number of probes is the same as the number of targets. The same fluorescent wavelength is assigned to these probes.

[0077] In one embodiment, each combination of primer pair and probe specifies only one target on the genomic DNA, and n targets are detected in each region by using n types of probes in the same region.

[0078] <Allocating 3 or more colors to 3 or more areas> In the above embodiment, two fluorescent wavelengths were used. The number of regions may be further increased and other fluorescent wavelengths may be assigned to the additional regions. In one embodiment, a third fluorescent wavelength may be assigned to a chromosome other than chromosomes 18 and 21.

[0079] In one embodiment, the number of region and fluorescence wavelength pairs is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In another embodiment, there is one region and fluorescence wavelength pair.

[0080] The types of fluorescent substances (fluorescent dyes) and quenchers added to the probes are not particularly limited. Examples of fluorescent substances include FAM, HEX, VIC, TAMRA, ROX, Cy5, and Cy5.5 (all trademarks).

[0081] <Test Example 1. Quantitation at Plateau> Figure 8 shows the scattering of plateau droplets. One probe with a fluorescent substance of a first fluorescent wavelength (FAM) was assigned to one target on chromosome 21. Also, one probe with a fluorescent substance of a second fluorescent wavelength (HEX) was assigned to one target on chromosome 18. These probes were mixed together in one droplet.

[0082] The number of PCR cycles was 40. The PCR reaction passed the exponential amplification phase and reached a plateau. The reaction was saturated. Figure 8 shows the scatter after single cell-droplet PCR.

[0083] The top part of Figure 8 shows the results of single-cell droplet PCR performed on the human cultured cell line GM22948. In the human cultured cell line GM22948, chromosomes 21 and 18 are euploid, i.e., diploid (solid arrow). The solid arrow indicates the center of the cluster. The arrowhead is a cluster of cell-free DNA. The same applies to the following figures.

[0084] The middle part of Figure 8 shows the results of single-cell droplet PCR performed on the human cultured cell line AG17487. In the human cultured cell line AG17487, chromosome 21 is triploid (indicated by the dashed arrow) and chromosome 18 is euploid.

[0085] The bottom panel of Figure 8 shows the sum of the scatter plots for both strains. There was no difference in droplet signal intensity between diploid and triploid strains of chromosome 21.

[0086] <Test Example 2. Quantification during exponential amplification> Figure 9 shows droplet scattering during the exponential amplification phase. Unlike in Test Example 1, the PCR cycle number was 24. The PCR reaction was in the exponential amplification phase. Other points were the same as in Test Example 1.

[0087] The upper part of FIG. 9 shows the results of single-cell droplet PCR performed on the human cultured cell line GM22948.

[0088] The middle part of Figure 9 shows the results of single-cell droplet PCR performed on the human cultured cell line AG17487.

[0089] The lower part of Figure 9 shows the results of adding up the scatter of both strains. There was a difference in the signal intensity of the droplets between diploid and triploid chromosome 21. Diploid and triploid could be distinguished by quantifying the fluorescence of the droplets during the exponential amplification phase. Therefore, in the following test example, the fluorescence intensity of the droplets was measured during the exponential amplification phase.

[0090] In addition, in each scatter in Figure 9, the multiple events present in the upper right corner of the cluster in which chromosome 21 is diploid and in which chromosome 21 is diploid or triploid are thought to represent signals from droplets containing multiple cells.

[0091] <Test Example 3. Enhancement of fluorescence> FIG. 10 shows droplet scatter with enhanced fluorescence. Unlike Test Example 2, eight types of probes having a fluorescent substance with a first fluorescent wavelength were assigned to chromosome 21. One type of probe having a fluorescent substance with a first fluorescent wavelength was assigned to each of the eight targets on chromosome 21. Eight types of probes having a fluorescent substance with a second fluorescent wavelength were assigned to chromosome 18. One type of probe having a fluorescent substance with a second fluorescent wavelength was assigned to each of the eight targets on chromosome 18. Other points were the same as Test Example 2.

[0092] The upper part of FIG. 10 shows the results of single-cell droplet PCR performed on the human cultured cell line GM22948.

[0093] The middle part of Figure 10 shows the results of single-cell-droplet PCR performed on the human cultured cell line AG17487.

[0094] The bottom panel of Figure 10 shows the results of adding up the scatter of both strains. The diploid cluster and the triploid cluster on chromosome 21 are now more clearly separated. This makes it easier to detect the cluster shift itself. This is because increasing the number of targets in one area corresponding to a probe with the same fluorescent wavelength enhances the fluorescence of the droplets and reduces the variation in fluorescence intensity. In the following test example, the fluorescence was enhanced by assigning multiple targets to one area.

[0095] In addition, in each scatter in Figure 10, the multiple events present in the upper right corner of the cluster in which chromosome 21 is diploid and in which chromosome 21 is diploid or triploid are thought to represent signals from droplets containing multiple cells.

[0096] <Test Example 4. Detection of CNV in heterogeneous cell population> Figures 11 and 12 show droplet scatter of heterogeneous cell populations. Human cultured cell line GM22948 (diploidy) and human cultured cell line AG17487 (triploidy) were mixed to obtain heterogeneous cell populations. The mixing ratio was 99 / 1 to 75 / 25. Single cell-droplet PCR was performed on these cell populations. Other points were the same as in Test Example 3. The shift of the cluster from diploidy to triploidy reflected the ratio of the number of cells.

[0097] <Test Example 5. Shifting clusters from diploid to haploid> Figure 13 shows droplet scatter of a homozygous cell population. The vertical axis, Chr13-8p, indicates that eight types of probes are assigned to eight targets on chromosome 13. The horizontal axis, Chr18-8p, indicates that eight types of probes are assigned to eight targets on chromosome 18. These probes were mixed together in one droplet.

[0098] The upper part of Figure 13 shows the results of single-cell droplet PCR performed on the human cultured cell line GM22948. In the human cultured cell line GM22948, chromosomes 13 and 18 are euploid, i.e., diploidy (solid arrow).

[0099] The bottom part of Figure 13 shows the results of single-cell droplet PCR performed on the human cultured cell line NCI-H929. In the human cultured cell line NCI-H929, chromosome 13 is monoploid (dashed arrow) and chromosome 18 is euploid.

[0100] The top panel of Figure 14 shows the results of adding up the scatter of both strains. There was a difference in the signal intensity of the droplets between diploid and haploid chromosome 13. Diploid and haploid strains could be distinguished by quantifying the fluorescence of the droplets during the exponential amplification phase.

[0101] The lower part of Figure 14 shows droplet scatter of a heterogeneous cell population. A heterogeneous cell population was obtained by mixing human cultured cell line GM22948 (diploidy) and human cultured cell line NCI-H929 (monoploidy). The mixture ratio was 50 / 50. Single cell-droplet PCR was performed on these cell populations. The shift of the cluster from diploidy to haploidy reflected the ratio of the number of cells.

[0102] <Test Example 6. Enhancement of fluorescence> A PCR reaction solution was prepared by adding 2 μL of a cell suspension containing 10,000 cells (GM22948 or GM13721 strain) and PBS to a PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, etc. in the PCR reaction solution are as follows. ·1X ddPCR Multiplex Super Mix (BioRad) 500nM / 575nM Chromosome 13-FAM probe 1μM chromosome 13 primer 1000nM / 1250nM Chromosome 18-HEX probe 1μM chromosome 18 primer ·Distilled water (for filling up)

[0103] The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.

[0104] In this test example, 1, 5, 10 or 23 types of probes having a fluorescent substance (FAM) with a first fluorescent wavelength were assigned to chromosome 13. One type of probe having a fluorescent substance with a first fluorescent wavelength was assigned to each of 1, 5, 10 or 23 targets on chromosome 13. When multiple types of probes are used, the concentration of each probe is the same, and the above-mentioned probe concentration (1, 5 or 10 types: 500 nM, 23 types: 575 nM) is the total concentration of multiple types of probes. In addition, 1, 5, 10 or 25 types of probes having a fluorescent substance with a second fluorescent wavelength (HEX) were assigned to chromosome 18. One type of probe having a fluorescent substance with a second fluorescent wavelength was assigned to each of 1, 5, 10 or 25 targets on chromosome 18. When multiple types of probes were used, the concentration of each probe was the same, and the above probe concentrations (1, 5, or 10 types: 1000 nM, 25 types: 1250 nM) were the total concentrations of the multiple types of probes. These probes were mixed together in one droplet.

[0105] 15 to 18 show droplet scattering during the exponential amplification phase.

[0106] The upper rows of Figures 15 to 18 show the results of single-cell droplet PCR performed on the human cultured cell line GM22948. In the human cultured cell line GM22948, chromosomes 13 and 18 are euploid, i.e., diploidy (solid arrow). The solid arrow indicates the center of the cluster.

[0107] The lower panels of Figures 15 to 18 show the results of single-cell droplet PCR performed on the human cultured cell line GM13721. In the human cultured cell line GM13721, a partial region of one long arm of chromosome 13 is monoploid (broken arrow), and chromosome 18 is euploid. All of the probes having the fluorescent substance of the first fluorescent wavelength (FAM) are assigned to the missing region of chromosome 13.

[0108] FIG. 15 shows the results when one type of probe is assigned to each of chromosomes 13 and 18. FIG. 16 shows the results when five types of probes are assigned to each of chromosomes 13 and 18. FIG. 17 shows the results when ten types of probes are assigned to each of chromosomes 13 and 18. FIG. 18 shows the results when 23 types of probes are assigned to chromosome 13 and 25 types of probes are assigned to chromosome 18.

[0109] In addition, in each scatter in Figures 15 to 18, the multiple events present in the upper right corner of the cluster in which chromosome 18 is diploid and chromosome 13 is haploid or diploid are thought to represent signals from droplets containing multiple cells.

[0110] As can be seen by the different positions of the solid and dashed arrows in these scatters, by increasing the number of targets in one region that correspond to probes of the same fluorescent wavelength, differences in copy number can be more easily determined.

[0111] Next, for each scatterer, the ratio of the fluorescence intensity of the first fluorescence wavelength to the fluorescence intensity of the second fluorescence wavelength, "FAM / HEX," was calculated for each droplet containing one cell, and the results were summarized in a box plot. Figure 19 shows the box plot for each scatterer.

[0112] In Figure 19, the boxes representing the third and first quartiles of haploid and diploid cells for chromosome 13 do not overlap in any of the graphs, indicating that haploid and diploid cells can be distinguished. In particular, when 23 probes were used for chromosome 13 and 25 probes were used for chromosome 18, the whiskers did not overlap.

[0113] Figure 20 shows the results of ROC analysis of the performance of detecting GM13728 strain (cells with haploid chromosome 13) from a cell population containing GM22948 strain (cells with diploid chromosome 13) and GM13728 strain (cells with haploid chromosome 13) using 23 types of probes as explanatory variables with "FAM / HEX". The Yoden Index was 0.743. When the Yoden Index was cut off, the sensitivity was 0.997 (99.7%) and the specificity was 0.994 (99.4%). The AUC was 0.997 (95% CI: 0.996-0.999), which was an extremely high value.

[0114] <Internal standard> In one embodiment, the measurement of ploidy is performed with an internal standard. In one embodiment, the cell that is euploid in the region to be detected is the internal standard of CNV. For example, in the latter half of Test Example 4 and Test Example 5, the copy number of the region to be detected in a heterogeneous cell population is measured. In addition, for euploid clusters, whether they are triploid or haploid is determined based on whether or not there is a cluster with CNV.

[0115] In the heterogeneous cell population, the composition ratio of euploid cells in the assigned region of the target is greater than 0% and less than 100%. The ratio of euploid cells in the heterogeneous cell population is, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, 99.95, 99.96, 99.97, 99.98 or 99.99%.

[0116] In the heterogeneous cell population, the composition ratio of cells with the CNV of interest in the assigned region of the target is greater than 0% and less than 100%. The ratio of cells with the CNV in the heterogeneous cell population is, for example, 99.99, 99.98, 99.97, 99.96, 99.95, 99.9, 99.8, 99.7, 99.6, 99.5, 99, 98, 97, 96, 95, 90, 85, 80, 75, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.04, 0.03, 0.02 or 0.01%.

[0117] <External standard> In Test Examples 1 to 3 and the first half of Test Example 5, the copy number of the detection target region in a population of cells homozygous for ploidy was measured. CNV of these cells had been identified in advance. The following method can provide a standard copy number of the detection target region for cells whose CNV has not been identified in advance.

[0118] That is, in one embodiment, ploidy is measured by external standard.For example, in another emulsion system that is not mixed with sample, PCR is performed simultaneously or before and after using genomic DNA of a cell whose ploidy is known as a template, and the fluorescence intensity of the amplicon is measured.The coordinates of the cluster obtained from the fluorescence intensity are compared with the coordinates of the cluster of the sample.

[0119] Examples of such external standard tests are shown in the upper (diploidy) and middle (triploidy) rows of Figure 9, the upper (diploidy) and middle (triploidy) rows of Figure 10, and the upper (diploidy) and lower (haploidy) rows of Figure 13.

[0120] Take for example the measurement of the ploidy of one of the chromosomes in the tumor cells of a cancer patient. As the treatment progresses smoothly, the original chromosomal mutation disappears in all the cells being measured. In this case, the region on the chromosome being measured becomes euploid. Therefore, according to the above measurement method, the shift of the aneuploid cluster on the scatter disappears. In other words, only euploid clusters remain among the various clusters.

[0121] On the other hand, when the cancer patient's disease progresses significantly, it is assumed that all cells to be measured will have chromosomal mutations. Or, it is assumed that the number of aneuploid cells will be significantly greater than that of euploid cells, making it impossible to detect euploid cells. According to the above measurement method, the euploid clusters on the scatter will disappear. In other words, only aneuploid ones will be detected among various clusters.

[0122] Such aneuploid clusters can be homogeneous populations of cells with the particular ploidy of the region being measured, or can be heterogeneous populations of cells with a range of ploidy.

[0123] In one embodiment, the genome ploidy of tumor cells of cancer patients is measured by using an external standard of ploidy.By using an external standard, the ploidy of cells can be measured without relying on the internal standard of euploid cells (non-cancerous healthy cells) that should be originally included in the cell population.More accurate information on these ploidy is useful for determining the necessity of additional treatment in cancer treatment or for diagnosing complete remission.

[0124] Next, let us take the example of measuring fetal cells in amniotic fluid as part of prenatal diagnosis. If the fetal cells in amniotic fluid are not contaminated by maternal cells, the normal population of cells will contain only fetal cells. The population of cells is homogeneous in terms of genome ploidy.

[0125] In one embodiment, the ploidy of the genome of the fetal cells is measured using an external ploidy standard, which allows the ploidy of the cells to be measured without relying on an internal standard of euploid cells that should be present in the population of cells, in this case cells of maternal origin.

[0126] If it is possible to confirm in advance that fetal cells have been obtained from amniotic fluid with 100% purity, the above-mentioned measurement method can be used to determine whether the population of fetal cells is heterogeneous or homogeneous in terms of genome ploidy, i.e., whether or not there is mosaicism in the chromosome ploidy of fetal cells.

[0127] In this embodiment, the copy number of the genomic region to be measured and the center of the fluorescence intensity of the cluster have a very clear linear relationship. Therefore, it is not necessary to obtain the fluorescence intensity from the external standard cells every time a sample is measured. In other words, by comparing the previously obtained information on the fluorescence intensity of the external standard with the information on the fluorescence intensity of the sample, the ploidy of the region to be measured in the genome of the sample can be determined.

[0128] <Test Example 7. Setting the cutoff value as an external standard> 21 shows a scatter plot including a straight line corresponding to the cutoff value to be set in this test example. In this test example, as shown in this figure, a cutoff value that separates cells in which a portion of the long arm of one of chromosomes 13 is monoploid and cells in which chromosome 13 is diploid is set as an external standard.

[0129] A PCR reaction solution was prepared by adding 2 μL of a cell suspension containing 10,000 cells (GM22948 strain) and PBS to a PCR premix containing sodium dodecyl sulfate. The concentrations of primers, probes, etc. in the PCR reaction solution are as follows. ·1X ddPCR Multiplex Super Mix (BioRad) 25nM chromosome 13-FAM probe x 23 types (total concentration: 575nM) 1μM chromosome 13 primer x 23 types 50nM chromosome 18-HEX probe x 25 types (total concentration: 1250nM) 1μM chromosome 18 primer x 25 types ·Distilled water (for filling up)

[0130] The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and cells were lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. PCR was performed separately using the 23 PCR reaction solutions prepared separately.

[0131] FIG. 22 shows the displacement of clusters for each measurement. The upper and middle rows of FIG. 22 show the results of single-cell droplet PCR performed on the human cultured cell line GM22948. In the human cultured cell line GM22948, chromosomes 13 and 18 are euploid, i.e., diploidy (solid arrow). As shown in these figures, when the same cell line is measured multiple times using the same probe, the positions of the clusters may be displaced between different measurements. Here, the direction in which each cluster is displaced is the same for each measurement.

[0132] The lower part of Fig. 22 is a graph showing the variability of the measured values ​​when 23 measurements were performed. In this graph, FAM / HEX is used as the measured value. As can be seen from this graph, even when the same cell line is measured using the same probe, the measurement results vary. In order to set an appropriate cutoff value, it is preferable to correct this variability before setting the cutoff value.

[0133] Therefore, in this test example, the median value of the fluorescence intensity of the first fluorescence wavelength (FAM) (hereinafter also referred to as the "FAM measurement value") and the median value of the fluorescence intensity of the second fluorescence wavelength (HEX) (hereinafter also referred to as the "HEX measurement value") of the negative cluster (the cluster located at the lower left in the scatter) were calculated for each of the 23 measurement results. The average value of the 23 FAM measurement values ​​was set as the FAM reference value of the negative cluster, and the average value of the 23 HEX measurement values ​​was set as the HEX reference value of the negative cluster. Then, for each of the 23 measurement results, "(FAM reference value + α) / (FAM measurement value + α)" and "(HEX reference value + α) / (HEX measurement value + α)" were calculated as correction values ​​for correcting the cluster shift (variation in the measurement results). α is a value that is arbitrarily set to bring the "FAM reference value / FAM measurement value" and the "HEX reference value / HEX measurement value" closer to the true fluorescence intensity ratio, taking into account the detection limit of the fluorescence intensity of the measurement device. Here, α was set to 2000. The obtained FAM correction value and HEX correction value were multiplied to all measured values ​​to correct the variation in the measurement results.

[0134] Figure 23 is a graph showing the variability of the corrected measurements. The measurement numbers correspond to the graph in the lower part of Figure 22. It can be seen that the variability of the measurement results (cluster shift) is significantly reduced. The cutoff value was set using these corrected measurements.

[0135] FIG. 24 is a diagram for explaining the calculation of the cutoff value using the measured values ​​after correction. The upper part of FIG. 24 is a scatter showing 95341 measured values ​​after correction. When a linear function (y=ax+b) that approximates the distribution of all measured values ​​was obtained using a standard principal axis regression equation, the equation y=0.84511x-404.24 was obtained. Then, in order to integrate the fluorescence intensity of the first fluorescent wavelength (FAM) and the fluorescence intensity of the second fluorescent wavelength (HEX) of each measured value, the above linear equation was converted to the equation b=y-0.84511x, where x (fluorescence intensity of the second fluorescent wavelength (HEX)) and y (fluorescence intensity of the first fluorescent wavelength (FAM)) were substituted for each measured value to obtain the value of b for each measured value. The graph in the middle part of FIG. 24 shows the distribution of the calculated 95341 b values. In this study, with an emphasis on specificity, the value equivalent to -3SD (standard deviation) of the distribution shown in this graph was set as the cutoff value for separating cells with haploid chromosome 13 from cells with diploid chromosome 13. The graph in the lower part of Figure 24 is a scatter graph that includes a line (y=0.84511x-2121.17) indicating the cutoff value. 99.85% of the 95,341 measured values ​​for cells with diploid chromosome 13 are located above this line.

[0136] Table 1 shows the results of judging the measurement results using the determined cutoff value. When detecting the GM13721 strain (cells in which a portion of the long arm of one of the chromosomes 13 is haploid) from a cell population containing the GM22948 strain (cells in which chromosome 13 is diploid) and the GM13721 strain (cells in which chromosome 13 is haploid), the sensitivity was 84.1% and the specificity was 99.3%. As mentioned above, the cutoff value set this time places emphasis on specificity, and it is of course possible to set the cutoff value with emphasis on sensitivity. [Table 1]

[0137] The method for setting the cutoff value is not limited to the above example. For example, the cutoff value that maximizes the sensitivity and specificity may be calculated using ROC analysis.

[0138] Mutation detection In one embodiment, the CNV is detected to detect the presence of a single cell with a mutation in a population of cells. In one embodiment, the mutation is a somatic mutation or a germline mutation. In one embodiment, the mutation is a chromosomal mutation or a genetic mutation.

[0139] <Detection of chromosomal mutations> In one embodiment, when the sample DNA contains a complete set of genomic DNA in a single cell, the presence of a single cell having a chromosomal mutation is detected from a population of cells by detecting CNV from the quantitative result of PCR amplicon. In one embodiment, the chromosomal mutation is at least one of aneuploidy over the entire length of a chromosome, partial aneuploidy of a chromosome, gene amplification, and gene deletion. Figures 4 to 7 described above are one embodiment of detection of aneuploidy over the entire length of a chromosome.

[0140] Gene amplification is the multiplication of a gene by partial duplication of a chromosome. In one embodiment, there is no limit to the number of times duplication occurs. Gene deletion is the loss of a gene by partial deletion of a chromosome. In one embodiment, the gene deletion is haplo or null. In one embodiment, gene amplification and gene deletion are polymorphisms.

[0141] As described above, unless the cell population is dominated by cells with genomic aneuploidy, the cell population is contaminated with euploid cells. The cell population includes normal single cells that do not have chromosomal mutations at the target genomic location. Any of the single cells assigned to each of the above-mentioned reaction compartments (reaction droplets) has euploid chromosomes in the region that is the target of aneuploidy detection. On the scatterer, the cluster of single cells with chromosomal mutations shifts from the position of the cluster created by the normal single cells. The cluster 40a shown in FIG. 5 and the cluster 41a shown in FIG. 7 described above are examples of this shift. The cluster 40a functions as a kind of internal standard.

[0142] The external standard described above can also be used to absolutely quantify the ploidy of a cluster, even if the cell population is dominated by cells with genomic aneuploidy, and can also be applied to heterogeneous cell populations consisting of euploid and aneuploid cells.

[0143] A reaction droplet that has come to have an amplicon amount different from the amplicon amount of a reaction droplet of a single cell that does not have a chromosomal mutation at a position on the target genome is detected. Hereinafter, this is called an aneuploid droplet. Clusters 40b and 40c shown in FIG. 5 and clusters 41b and 41c shown in FIG. 7 described above are detection examples.

[0144] Examples of chromosomal mutations that result in aneuploid droplets are as follows: That is, a single cell having a detectable chromosomal mutation contains, for example, genomic DNA having any of the following chromosomal mutations. - Aneuploidy of chromosome 21 - Aneuploidy of chromosome 18 - Aneuploidy of chromosome 13 - Y chromosome aneuploidy - X chromosome aneuploidy - Deletion of the 22q11.2 region of the long arm of chromosome 22 - Deletion of the 5q region of the short arm of chromosome 5 - Deletion of the 15q11-q13 region of the long arm of chromosome 15 - Amplification of the long arm of chromosome 1 - Deletion of the short arm of chromosome 17 - Deletion of the long arm of chromosome 13 - Deletion of the long arm of chromosome 4 - Deletion of the long arm of chromosome 5 - Deletion of the long arm of chromosome 7 - Amplification of chromosome 8 - Deletion of chromosome 11 - Aneuploidy of chromosome 12 - Deletion of the long arm of chromosome 20 - Deletion of the long arm of chromosome 19 - Deletion of chromosome 1 - Deletion of the long arm of chromosome 18 - Deletion of the short arm of chromosome 8 - Deletion of chromosome 4 - Amplification of the long arm of chromosome 8 - Deletion of the long arm of chromosome 16 - Amplification of the short arm of chromosome 5 - Amplification of the long arm of chromosome 3 - Deletion of the short arm of chromosome 3 - Deletion of the short arm of chromosome 9 - Gene amplification of the MYCN gene - Genetic amplification of the HER2 gene - Gene amplification of the MET gene.

[0145] In one embodiment, the deletion excludes biallelic deletion, so-called null. In one embodiment, a pair of a chromosome with a deleted portion and a normal chromosome with the deleted portion is detected.

[0146] In one embodiment, data is further generated including information on whether an aneuploid droplet is detected, i.e., whether a single cell having a chromosomal mutation is detected. By selecting a population of cells that produces a single cell, data useful for specific diagnostic areas such as:

[0147] <Application to prenatal diagnosis> In one embodiment, single cells are obtained from either amniotic fluid or maternal blood. Specifically, a bulk population of cells is obtained from amniotic fluid or maternal blood. The population of cells includes fetal cells. In one embodiment, the population of cells is enriched for fetal cells. The population of cells includes maternal cells. In one embodiment, the maternal cells are euploid in the region of genomic DNA to be detected. In one embodiment, the fetal cells are fetal nucleated red blood cells (fetal nucleated erythroblasts, fNRBC) circulating in maternal blood, and the maternal cells are white blood cells circulating in maternal blood. In one embodiment, the fetal cells are fetal cells suspended in amniotic fluid, and the maternal cells are maternal cells suspended in amniotic fluid.

[0148] In one embodiment, the data provides for a diagnosis of trisomy 13, trisomy 18, trisomy 21, Turner syndrome, triple X syndrome, XYY syndrome, Klinefelter syndrome, DiGeorge syndrome, Angelman syndrome, Prader-Willi syndrome, or Cat's Bark syndrome.

[0149] <Application to cancer diagnosis> In one embodiment, single cells are obtained from the blood of a cancer patient. Specifically, a bulk cell population is isolated from the blood of a cancer patient. The cell population includes cancer cells consisting of at least one of circulating tumor cells (CTCs) and cancerous blood cells. In another embodiment, the cell population is obtained by biopsying a solid tumor and dispersing the tumor cells by a predetermined chemical and / or physical treatment. In one embodiment, the cancerous blood cells are cancerous plasma cells. In one embodiment, these cancer cells are enriched in the cell population. The cell population further includes non-cancer cells. In one embodiment, the non-cancer cells are euploid in the region on the genomic DNA to be detected. In one embodiment, the non-cancer cells are normal white blood cells circulating in the blood of a cancer patient. In one embodiment, the normal white blood cells are normal plasma cells.

[0150] In one aspect, the data is for the diagnosis of myelodysplastic syndrome, multiple myeloma, idiopathic eosinophilia, chronic eosinophilic leukemia, acute nonlymphocytic leukemia, myeloproliferative neoplasms, chronic lymphocytic leukemia, acute myeloid leukemia, brain cancer, neuroblastoma, colon cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer or head and neck cancer.

[0151] <Application to multiple myeloma> As described in Non-Patent Documents 8 and 9, multiple myeloma (MM) is a type of hematopoietic tumor, a disease in which plasma cells become tumorous. Chromosomal abnormalities are observed in the majority of MM cases. Trisomies have been reported frequently in chromosomes 3, 5, 7, 9, 11, 15, and 19. Monosomies are frequently observed in chromosomes 13, 14, 16, and 22. Among these, monosomy of chromosome 13 and deletion of the long arm of chromosome 13 (13q deletion) are the most frequent examples of chromosome mutations. These chromosomal mutations are observed in approximately 50% of new patients. Of these, 85% are monoploid in the entire body or in the part to be detected, and 15% are intraarm deletions including 13q14 deletion. Deletions of both alleles are rare. In addition, deletion of 17p13 in the short arm of chromosome 17 is observed in approximately 10%. These are one of the poor prognostic factors of MM. Another poor prognostic factor is amplification of the long arm of chromosome 1 at 1q21, which has been reported to be present in 43% of untreated MM patients.

[0152] In this embodiment, the amount of tumor remaining in a patient with these chromosomal abnormalities is monitored by periodically measuring the amount of plasma cells (cancerous plasma cells) that have these chromosomal mutations remaining in the body during the post-treatment period.

[0153] In this embodiment, the minimal residual disease (MRD) consisting of cells having the above-mentioned chromosomal mutation is measured. Among the residual diseases, MRD is particularly difficult to measure using existing technology. By measuring MRD, the effectiveness of treatment can be evaluated in depth. Based on this evaluation, the doctor decides whether to increase, decrease, discontinue, or resume the therapeutic drug.

[0154] An example of an actual test method is shown below. In one example, bone marrow fluid or blood taken from a patient is used as a sample. Red blood cells are removed from these fluids. Hemolysis, centrifugation, and a hydrodynamic classification chip are used to remove the red blood cells.

[0155] The sample is mixed with a predetermined probe-primer set and PCR premix, and the cells are lysed.The probe-primer set to be detected is selected to have a target in the 13q region.In one embodiment, the control probe-primer set is selected to have a target anywhere in the entire chromosome 18.

[0156] 13q deletion of chromosome 13, i.e., partial monoploidization, is detected by shifting of the cluster as shown in FIG. 5 and FIG. 7. Events in the cluster have a distribution of fluorescence intensity. By combining with necessary statistical analysis, cells with 13q deletion of chromosome 13 present in the range of 0 to 100% in the population of cells of the sample are detected. In one embodiment, the abundance ratio of cells with 13q deletion of chromosome 13 detected is any of 10, 1, 0.1, 0.05, 0.04, 0.03, 0.02, and 0.01% in the population of cells of the sample. As described above, cells with 13q deletion of chromosome 13 are expected to be cancerous plasma cells. In another embodiment, targets are assigned to regions where both 13q deletion and monosomy of chromosome 13 can be detected.

[0157] In one embodiment, plasma cells, including circulating tumor cells, are enriched using an antibody against an antigen specific to plasma cells prior to measurement, allowing for more sensitive detection of cancerous plasma cells, i.e., tumor-forming plasma cells, present in bone marrow fluid or blood.

[0158] In other embodiments, the cluster shift provides information about whether cells in a sample have 17p13 deletions and / or 1q21 amplifications, which information is useful for predicting the prognosis of MM.

[0159] <Applications to myelodysplastic syndromes and other hematopoietic tumors> Myelodysplastic syndromes (MDS) are a type of hematopoietic tumor. At least 50% of MDS cases are caused by chromosomal mutations (specifically, full-length aneuploidy, partial aneuploidy, gene amplification, and gene deletion) occurring in hematopoietic stem cells. Representative chromosomal mutations in MDS are deletion of the long arm of chromosome 5 (5q), monosomy of chromosome 7, and deletion of the long arm of chromosome 20 (20q). MRD is measured periodically by detecting cells with chromosomal mutations present in a sample.

[0160] Diseases with 5q deletion are independently defined as a type of MDS called 5q-syndrome. According to European and American studies, approximately 10% of MDS cases are 5q-syndrome. Lenalidomide, which is generically known as lenalidomide, is effective for 5q-syndrome. The therapeutic effect of lenalidomide is monitored by shifting the above clusters.

[0161] In another embodiment, the detection of other hematopoietic tumors with chromosomal aneuploidies, particularly chromosomal deletions and amplifications, is performed by shifting the clusters.

[0162] <Detection of gene mutations> In one embodiment, the CNV is detected to detect the presence of a single cell having a genetic mutation, which may be at least one of a base substitution, a base insertion, and a base deletion.

[0163] In one embodiment, any of the single cells assigned to each of the above-mentioned reaction compartments (reaction droplets) is an internal standard that does not have a genetic mutation to be detected, at least at the target genomic position. A cluster of reaction droplets that has an amplicon amount different from the amplicon amount of such a reaction droplet will shift from the position of the cluster without a genetic mutation. These are detected as mutant droplets.

[0164] In another embodiment, the fluorescence intensity of the external standard is measured in advance in a separate well or at a separate time. The information on the fluorescence intensity is compared with the information on the fluorescence intensity at the center of each cluster. Based on the comparison, the copy number of the gene mutation in each cluster is measured. Even if the cell population is dominated by cells with gene mutations and cells having only wild-type genes cannot be used as an internal standard, the copy number of the gene mutation can be measured.

[0165] In one embodiment, the gene mutation is introduced into an unspecified region on the genome by a lentivirus vector or other vector. Examples of cells having a gene mutation caused by a vector include CAR-T cells (Chimeric antigen receptor T cells) and TCR-T cells. In the detection in this embodiment, the target may be DNA introduced by the vector. For example, the copy number of DNA introduced by the vector may be detected.

[0166] <Test Example 8. Detection of copy number in human samples> Peripheral blood from a healthy individual was hemolyzed and added to a PCR premix to prepare a PCR reaction solution. The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and cells were further lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.

[0167] In this test example, 23 types of probes having a fluorescent substance with a first fluorescent wavelength (FAM) were assigned to chromosome 13. In addition, 25 types of probes having a fluorescent substance with a second fluorescent wavelength (HEX) were assigned to chromosome 18. These probes were mixed together in one droplet.

[0168] Figure 25 shows the results of single-cell droplet PCR performed on human blood. The cluster in the center is the measurement result corresponding to a droplet containing one cell. The cluster to the upper right is the measurement result corresponding to a droplet containing two cells. As the clusters can be clearly distinguished, it is suggested that the copy number of nucleic acids can be detected even when human blood is used as a sample.

[0169] Next, peripheral blood from healthy subjects was hemolyzed, and CD45-positive cells were isolated using EasySep Human CD45 Depletion Kit II (STEMCELL Technologies). The obtained CD45 cells were added to a PCR premix to prepare a PCR reaction solution. The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were further lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.

[0170] In this test example, 23 types of probes having a fluorescent substance with a first fluorescent wavelength (FAM) were assigned to chromosome 13. In addition, 25 types of probes having a fluorescent substance with a second fluorescent wavelength (HEX) were assigned to chromosome 18. These probes were mixed together in one droplet.

[0171] Figure 26 shows the results of single-cell droplet PCR performed on CD45 cells derived from human blood. The cluster in the center is the measurement result corresponding to a droplet containing one cell. The cluster to the upper right is the measurement result corresponding to a droplet containing two cells. The clearly distinguishable clusters suggest that the copy number of nucleic acids can be detected even when cells enriched for a specific antigen are used.

[0172] Next, a cell population obtained by mixing the above CD45-positive cells and cells of the human cultured cell line GM13721 at a predetermined ratio was added to a PCR premix to prepare a PCR reaction solution. The above PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were further lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. In the CD45-positive cells, chromosomes 13 and 18 are euploid, i.e., diploidy. In the human cultured cell line GM13721, a portion of the long arm of chromosome 13 is monoploidy, and chromosome 18 is euploid.

[0173] In this test example, 23 types of probes having a fluorescent substance with a first fluorescent wavelength (FAM) were assigned to the above-mentioned missing region of chromosome 13. In addition, 25 types of probes having a fluorescent substance with a second fluorescent wavelength (HEX) were assigned to chromosome 18. These probes were mixed together in one droplet.

[0174] Figure 27 shows droplet scatter of a homozygous cell population. The upper part of Figure 27 shows the results of single-cell-droplet PCR performed on CD45-positive cells. The lower part of Figure 27 shows the results of single-cell-droplet PCR performed on human cultured cell line GM13721.

[0175] Figure 28 shows droplet scatter of a heterogeneous cell population. The upper part of Figure 28 shows the results of single-cell droplet PCR performed on a cell population in which the mixture ratio of CD45 positive cells and human cultured cell line GM13721 is 75 / 25. The lower part of Figure 28 shows the results of single-cell droplet PCR performed on a cell population in which the mixture ratio of CD45 positive cells and human cultured cell line GM13721 is 95 / 5.

[0176] These results clearly distinguished CD45-positive cells from the human GM13721 cell line, suggesting that cells with abnormal copy numbers can be detected even in cell populations derived from human blood.

[0177] <Test Example 9. Evaluation of detection sensitivity when using human samples> A cell population was prepared by mixing CD45-positive cells and human cultured cell line GM13721 cells at a predetermined ratio in the same manner as in Test Example 8 above. The ratio of GM13721 cells in the cell population was 0%, 0.1%, or 3%. Each cell population was added to a PCR premix to prepare a PCR reaction solution. The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were further lysed in each droplet. Then, PCR was performed in each droplet under general conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase. In CD45-positive cells, chromosomes 13 and 18 are euploid, i.e., diploidy. In human cultured cell line GM13721, a portion of one long arm of chromosome 13 is monoploidy, and chromosome 18 is euploid.

[0178] In this test example, 23 types of probes having a fluorescent substance with a first fluorescent wavelength (FAM) were assigned to the above-mentioned missing region of chromosome 13. In addition, 25 types of probes having a fluorescent substance with a second fluorescent wavelength (HEX) were assigned to chromosome 18. These probes were mixed together in one droplet.

[0179] Figure 29 shows droplet scatter for each cell population. The top of Figure 29 shows the results of single-cell-droplet PCR performed on a cell population with a GM13721 cell ratio of 0%. The middle of Figure 29 shows the results of single-cell-droplet PCR performed on a cell population with a GM13721 cell ratio of 0.1%. The bottom of Figure 29 shows the results of single-cell-droplet PCR performed on a cell population with a GM13721 cell ratio of 3%.

[0180] In this test example, dots with a fluorescence intensity of 5000 or more at the first fluorescence wavelength (FAM) or 9000 or more at the second fluorescence wavelength (HEX) were defined as positive droplets to be evaluated. In addition, the cutoff (y=0.84806x-1044) determined in the same manner as in Test Example 7 was used to calculate the ratio of abnormal droplets (droplets thought to be monoploid on chromosome 13) to positive droplets.

[0181] FIG. 30 is a box plot showing the detection results of abnormal droplets for each cell population. The asterisk indicates p<0.05 (result of Steel test compared to control (0%) after confirming that the results of Kruskal-Wallis test were significant). This graph shows that the detection results of cell populations with 3% or 0.1% GM13721 cells are significantly different from the detection results of cell populations with 0% GM13721 cells. Therefore, it can be seen that detection is possible down to at least 0.1%.

[0182] <Test Example 10. Detection of copy number in reverse transcription product of RNA> Using the reverse transcription product of RNA in a single cell as the sample DNA, the copy numbers of B2M gene mRNA and GAPDH gene mRNA were detected.

[0183] In this test example, single-cell droplet RT-PCR was performed on the human cultured cell line GM22948. The premix contained primers for the B2M gene, primers for the GAPDH gene, a probe for the B2M gene (FAM labeling), a probe for the GAPDH gene (HEX labeling), and reverse transcriptase (SuperScriptIV). In order to amplify the mRNA of the gene by PCR, rather than the gene in the genomic DNA, the primers for both genes were designed to flank an intron.

[0184] Specifically, 2 μL of a cell suspension containing 10,000 cells (GM22948 strain) and PBS was added to a PCR premix containing sodium dodecyl sulfate to prepare a PCR reaction solution. The concentrations of primers, probes, etc. in the PCR reaction solution were as follows. ·1X SuperScriptIV (Thermo Fisher Scientific) ·1X ddPCR Multiplex Super Mix (BioRad) 200nM B2M-FAM probe 400nM B2M primer 200nM GAPDH-HEX probe 400nM GAPDH primer ·Distilled water (for filling up)

[0185] The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were dissolved in each droplet. The droplets were then left to stand at 50°C for 10 minutes to perform reverse transcription, and the DNA was denatured at 95°C for 10 minutes, after which PCR was performed under standard conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.

[0186] Figure 31 shows the results of single-cell droplet RT-PCR performed on the human cultured cell line GM22948. This result indicates that the copy number of RNA can be detected even when the reverse transcription product of RNA is used as the sample DNA.

[0187] When the premix did not contain reverse transcriptase, no positive droplets were detected. In other words, because the primers were designed to flank the intron as described above, both genes in the genomic DNA were hardly amplified.

[0188] <Test Example 11. Detection of mitochondrial DNA copy number> Mitochondrial DNA was used as the sample DNA to detect the copy number of the ND1 gene.

[0189] In this test example, single-cell droplet PCR was performed on human cultured cell line GM22948. Each droplet contains not only mitochondrial DNA in a single cell but also genomic DNA. The premix contains primers for chromosome 13 (23 types), primers for the ND1 gene of mitochondrial DNA, probes for chromosome 13 (23 types, FAM-labeled), and probes for the ND1 gene (HEX-labeled).

[0190] Specifically, 2 μL of a cell suspension containing 10,000 cells (GM22948 strain) and PBS was added to a PCR premix containing sodium dodecyl sulfate to prepare a PCR reaction solution. The concentrations of primers, probes, etc. in the PCR reaction solution were as follows. ·1X ddPCR Multiplex Super Mix (BioRad) 25nM Chromosome 13-FAM probe 1μM chromosome 13 primer 500nM ND1-HEX probe 1 μM ND1 primer ·Distilled water (for filling up)

[0191] The PCR reaction solution was made into droplets using a QX200 Droplet Generator (BioRad), and the cells were lysed in each droplet. After denaturing the DNA at 95°C for 10 minutes, PCR was performed under standard conditions. The number of PCR cycles was 23. The PCR reaction was in the exponential amplification phase.

[0192] FIG. 32 shows the results of single-cell droplet PCR performed on the human cultured cell line GM22948. The cluster slightly to the right of center is the measurement result corresponding to a droplet containing one cell (containing approximately 1X mitochondrial DNA). The cluster to the upper right is the measurement result corresponding to a droplet containing two cells (containing approximately 2X mitochondrial DNA). As shown above, as the copy number of mitochondrial DNA increased, the cluster shifted to the right. This result shows that the copy number of genes in mitochondrial DNA can be detected even when mitochondrial DNA is used as sample DNA.

[0193] <Use of mitochondrial DNA as a control> To detect CNV in the assigned region of the target, a control is assigned to a region that is considered to be euploid with few CNVs. In cells where chromosomal abnormalities occur frequently, such as cancer cells, it may be difficult to find a suitable chromosome as a control. In such cases, mitochondrial DNA may be used as a control. That is, a fluorescent wavelength is selected, and a probe having a fluorescent substance with the selected fluorescent wavelength is assigned to mitochondrial DNA.

[0194] For example, the sample DNA includes a set of genomic DNA in a single cell and a mitochondrial DNA in a single cell. The PCR reaction system includes a probe having a fluorescent substance with a first fluorescent wavelength assigned to a region on the genomic DNA and a probe having a fluorescent substance with a second fluorescent wavelength assigned to the mitochondrial DNA. The region includes one or more targets. In the reaction compartment, the target included in the set of genomic DNA and the target included in the mitochondrial DNA are amplified by PCR. In a cycle in which the amplification of the target included in the mitochondrial DNA reaches a plateau and the amplification of the target included in the genomic DNA reaches an exponential amplification phase, the amplicons of the genomic DNA and the mitochondrial DNA are quantified for each reaction compartment. At this time, by detecting the fluorescence of the first and second fluorescent wavelengths from one reaction compartment, the reaction compartment in which the target included in the genomic DNA and the target included in the mitochondrial DNA are amplified can be distinguished from the reaction compartment in which only the contaminating cell-free nucleic acid is amplified.

[0195] During droplet PCR, the amplification using mitochondrial DNA as a template proceeds early. Therefore, primers or probes are depleted early. This is because the copy number of mitochondrial DNA per cell is greater than the copy number of genomic DNA per cell. Therefore, PCR is stopped at the cycle number at which the mitochondrial DNA reaches a plateau. However, at this cycle number, the amplification using chromosomal genomic DNA as a template is in the exponential amplification phase.

[0196] For example, in Figures 4 to 7, when a probe of the second fluorescent wavelength (2nd color) is assigned to a chromosome with a large CNV, the cluster is unlikely to converge with respect to the intensity of the second fluorescent wavelength. Alternatively, the cluster does not converge to the intensity corresponding to euploidy with respect to the intensity of the second fluorescent wavelength.

[0197] For example, in Figures 4 to 7, when the second fluorescent wavelength is assigned to mitochondrial DNA, the fluorescent intensity reaches a plateau in PCR at the second fluorescent wavelength. Therefore, the clusters of the intensity of the second fluorescent wavelength converge to the plateau intensity.

[0198] This application claims priority to Japanese Patent Application No. 2020-216149, filed December 25, 2020. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety. [Industrial Applicability]

[0199] The detection method of the present invention is useful, for example, for prenatal diagnosis and cancer diagnosis. [Explanation of symbols]

[0200] 17 Cell population 19 Oil 20 Droplets 21 Complete set of genomic DNA 22a, 22b Droplets 31 Premix 33a, 33b Droplets 35a, 35b Probe 39 Cluster Clusters 40a, 40b, and 40c Clusters 41a, 41b, and 41c Cluster 44a, 44b

Claims

1. 1. A method for detecting the copy number of a particular nucleic acid per single cell in a population of cells, comprising: amplifying a target contained in the sample DNA by PCR in a reaction compartment containing a sample DNA derived from a nucleic acid in a single cell and a PCR reaction system; quantitating the PCR amplicons by measuring the intensity of fluorescence in each reaction compartment during the exponential amplification phase to quantify the copy number of the specific nucleic acid; Including, In the step of quantifying the copy number of the specific nucleic acid, the amplicon is quantified by a fluorescent probe method; the PCR reaction system includes a plurality of probes each having a fluorescent substance with a different fluorescent wavelength, the probes being assigned to different regions on the sample DNA; the region includes a plurality of targets to which a plurality of probes each having a fluorescent substance with the same fluorescent wavelength are assigned, In the step of quantifying the amplicon, the intensities of fluorescence from the plurality of probes are collectively measured for each region, regardless of the differences in their targets, thereby quantifying the amplicon. Detection methods.

2. A method for detecting the copy number of a specific nucleic acid in each single cell in a population of cells, comprising: amplifying a target contained in the sample DNA by PCR in a reaction compartment containing a sample DNA derived from a nucleic acid in a single cell and a PCR reaction system; quantitating the PCR amplicons by measuring the intensity of fluorescence in each reaction compartment during the exponential amplification phase to quantify the copy number of the specific nucleic acid; Including, In the step of quantifying the copy number of the specific nucleic acid, the amplicon is quantified by a fluorescent probe method; the sample DNA includes a set of genomic DNA in the single cell and a set of mitochondrial DNA in the single cell; the PCR reaction system includes a probe having a fluorescent substance with a first fluorescent wavelength assigned to a region on genomic DNA, and a probe having a fluorescent substance with a second fluorescent wavelength assigned to a region on mitochondrial DNA; the region comprises one or more of the targets; In the step of amplifying a target contained in the sample DNA by PCR, a target contained in the set of genomic DNA and a target contained in the mitochondrial DNA are amplified by PCR in the reaction compartment; In the step of quantifying the amplicons, the amplicons of the genomic DNA and the mitochondrial DNA are quantified for each reaction compartment in a cycle in which the amplification of the target contained in the mitochondrial DNA reaches a plateau and the amplification of the target contained in the genomic DNA reaches the exponential amplification phase; In the step of quantifying the amplicons, the intensities of the fluorescence at the first and second fluorescent wavelengths are measured from one of the reaction compartments to distinguish the reaction compartment in which the target contained in the genomic DNA and the target contained in the mitochondrial DNA have been amplified from the reaction compartment in which only contaminating cell-free nucleic acid has been amplified. Detection methods.

3. The step of quantifying the amplicons comprises: stopping the PCR cycles during the exponential amplification phase and measuring the intensity of the fluorescence for each reaction compartment; quantitating the PCR amplicon from the intensity of the fluorescence; The detection method according to claim 1 or claim 2, comprising:

4. The detection method according to claim 1 , wherein the sample DNA is a set of genomic DNA in a single cell, a reverse transcription product of RNA in a single cell, or a mitochondrial DNA in a single cell.

5. In the step of quantifying the amplicons, the reaction compartment in which the target contained in the sample DNA has been amplified is distinguished from the reaction compartment in which only contaminating cell-free nucleic acid has been amplified by measuring the intensities of fluorescence at multiple wavelengths from one of the reaction compartments. The detection method according to claim 1 .

6. The detection method according to any one of claims 1 to 5, further comprising a step of generating the reaction compartment by lysing the single cell in a compartment containing a single cell, a cell lysis reagent, and a PCR premix.

7. lysing a single cell in a compartment containing said single cell; generating said reaction compartment by combining a compartment containing said lysed single cells with a compartment containing a PCR premix; The detection method according to any one of claims 1 to 5, further comprising:

8. mixing the population of cell nuclei with a PCR premix in bulk; singulating individual cell nuclei in the population of cell nuclei with the premix to generate the reaction compartment; The detection method according to any one of claims 1 to 5, further comprising:

9. The detection method according to any one of claims 1 to 8, wherein the reaction compartment is a reaction droplet that contains the sample DNA and the PCR reaction system.

10. In the step of quantifying the copy number of the specific nucleic acid, the copy number of the specific nucleic acid is quantified using a cutoff value as an external standard; The cutoff value is preset based on the quantitative results of the PCR amplicon for cells with a known copy number of the specific nucleic acid. The detection method according to any one of claims 1 to 9.

11. The detection method according to claim 10, wherein the quantitative results of the PCR amplicons are corrected using the quantitative results of negative clusters before being used to set the cutoff value.

12. the sample DNA comprises a complete set of genomic DNA in the single cell; The detection method further includes a step of detecting the presence of a single cell having at least one selected from the group consisting of aneuploidy over the entire length of a chromosome, partial aneuploidy of a chromosome, gene amplification, and gene deletion, based on the quantification result of the amplicon. The detection method according to any one of claims 1 to 11.

13. The detection method according to claim 12 , wherein the single cell contains genomic DNA having at least any one of the following chromosomal mutations: - Aneuploidy of chromosome 21 - Aneuploidy of chromosome 18 - Aneuploidy of chromosome 13 - Y chromosome aneuploidy - X chromosome aneuploidy - Deletion of the 22q11.2 region of the long arm of chromosome 22 - Deletion of the 5q region of the short arm of chromosome 5 - Deletion of the 15q11-q13 region of the long arm of chromosome 15 - Amplification of the long arm of chromosome 1 - Deletion of the short arm of chromosome 17 - Deletion of the long arm of chromosome 13 - Deletion of the long arm of chromosome 4 - Deletion of the long arm of chromosome 5 - Deletion of the long arm of chromosome 7 - Amplification of chromosome 8 - Deletion of chromosome 11 - Aneuploidy of chromosome 12 - Deletion of the long arm of chromosome 20 - Deletion of the long arm of chromosome 19 - Deletion of chromosome 1 - Deletion of the long arm of chromosome 18 - Deletion of the short arm of chromosome 8 - Deletion of chromosome 4 - Amplification of the long arm of chromosome 8 - Deletion of the long arm of chromosome 16 - Amplification of the short arm of chromosome 5 - Amplification of the long arm of chromosome 3 - Deletion of the short arm of chromosome 3 - Deletion of the short arm of chromosome 9 - Gene amplification of the MYCN gene - Gene amplification of the HER2 gene - Gene amplification of the MET gene

14. generating data including information on whether the presence of the single cell is detected; the population of cells is isolated from amniotic fluid or maternal blood so as to contain fetal cells; The data are diagnostic of trisomy 13, trisomy 18, trisomy 21, Turner syndrome, triple X syndrome, XYY syndrome, Klinefelter syndrome, Di George syndrome, Angelman syndrome, Prader-Willi syndrome, or cat cry syndrome; The detection method according to claim 12.

15. generating data including information on whether the presence of the single cell is detected; the population of cells is isolated from a patient; the data being useful for diagnosing myelodysplastic syndrome, multiple myeloma, idiopathic eosinophilia, chronic eosinophilic leukemia, acute nonlymphocytic leukemia, myeloproliferative neoplasms, chronic lymphocytic leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, colon cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, esophageal cancer, thyroid cancer, or head and neck cancer; The detection method according to claim 12.

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