Analysis method of chimerism

Abstract

To provide an analysis method of chimerism in which a ratio between cells of a recipient and cells of a donor can be easily measured.SOLUTION: An analysis method of chimerism for a plurality of cells derived from a subject includes the steps of: distributing one each of the plurality of cells to a plurality of first reaction sections; amplifying a first DNA marker specifically present in cells of a recipient and a second DNA marker specifically present in cells of a donor by PCR in each of the plurality of first reaction sections; and detecting an amplicon of the first DNA marker and an amplicon of the second DNA marker in each of the plurality of first reaction sections.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for analyzing chimerism. [Background technology]

[0002] Hematopoietic stem cell transplantation is a treatment aimed at completely curing blood cancers that are difficult to treat with chemotherapy or radiation therapy. In hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantation), hematopoietic stem cells collected from a donor are transplanted into the recipient (patient) after pre-transplant treatment such as chemotherapy and radiation therapy.

[0003] In hematopoietic stem cell transplantation, it is important to confirm the engraftment of donor-derived cells to confirm that the recipient's blood cells have been replaced by donor-derived blood cells. In particular, the proportion of umbilical cord blood transplants, which have a relatively low engraftment ability compared to bone marrow stem cells and require many days to engraft, is increasing, so it is increasingly important to accurately confirm engraftment. After transplantation, a state called chimerism, in which donor-derived blood cells and recipient-derived blood cells exist simultaneously, may occur in the recipient's body. Analysis of chimerism is clinically useful as a method to monitor the engraftment of donor-derived cells and the recurrence of blood cancer.

[0004] Chimerism analysis in hematopoietic stem cell transplantation is clinically useful as a method for monitoring graft survival or rejection, and recurrence of hematopoietic tumors (Non-Patent Document 1). One method for analyzing chimerism is the XY-FISH method, which detects sex chromosomes by fluorescence in situ hybridization (FISH). However, this method is only applicable to heterosexual transplants. On the other hand, one method for analyzing chimerism that can also be applied to homosexual transplants is the STR-PCR method, which analyzes short tandem repeats (STRs) of different lengths between individuals by PCR. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pamela Tozzo, et al., "Chimerism Monitoring Techniques after Hematopoietic Stem Cell Transplantation: An Overview of the Last 15 Years of Innovations, Diagnostics, Vol. 11, pp. 621. Summary of the Invention [Problem to be solved by the invention]

[0006] The above-mentioned XY-FISH and STR-PCR methods for chimerism analysis have problems in that the procedures are complicated and time-consuming, and the STR-PCR method also has problems in that it is difficult to measure the ratio of recipient cells to donor cells.

[0007] An object of the present invention is to provide a method for analyzing chimerism that can easily measure the ratio of recipient cells to donor cells. [Means for solving the problem]

[0008] The present invention relates to the following chimerism analysis method. [1] A method for analyzing chimerism in a plurality of cells derived from a subject, comprising: distributing said plurality of cells into a plurality of first reaction compartments, one each; amplifying a first DNA marker specifically present in recipient cells and a second DNA marker specifically present in donor cells by PCR in each of the plurality of first reaction compartments; detecting an amplicon of the first DNA marker and an amplicon of the second DNA marker in each of the plurality of first reaction compartments; A method for analyzing chimerism, comprising: [2] The method for analyzing chimerism described in [1], wherein in the step of detecting the amplicon of the first DNA marker and the amplicon of the second DNA marker, in each of the plurality of first reaction compartments, the amplicon of the first DNA marker is detected with a fluorescent probe of a first wavelength, and the amplicon of the second DNA marker is detected with a fluorescent probe of a second wavelength. [3] The method for analyzing chimerism described in [1] or [2], further comprising a step of confirming the presence of multiple candidate DNA markers in the recipient cells and the donor cells to determine the first DNA marker and the second DNA marker. [4] The step of determining the first DNA marker and the second DNA marker comprises: distributing fragments of genomic DNA from the recipient cells or the donor cells into a plurality of second reaction compartments; amplifying the plurality of candidate DNA markers by PCR in each of the plurality of second reaction compartments; detecting amplicons of the plurality of candidate DNA markers using a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength in each of the plurality of second reaction compartments; A method for analyzing chimerism described in [3], comprising: [5] In the step of detecting the amplicons of the multiple candidate DNA markers, a fluorescent probe having a third wavelength and a fluorescent probe having a fourth wavelength that specifically bind to the amplicon of a first candidate DNA marker among the multiple candidate DNA markers, and a fluorescent probe having a third wavelength and a fluorescent probe having a fourth wavelength that specifically bind to the amplicon of a second candidate DNA marker among the multiple candidate DNA markers are used; a ratio of the fluorescent probe at the third wavelength to the fluorescent probe at the fourth wavelength that specifically binds to the amplicon of the first candidate DNA marker is different from a ratio of the fluorescent probe at the third wavelength to the fluorescent probe at the fourth wavelength that specifically binds to the amplicon of the second candidate DNA marker; [4] The method for analyzing chimerism is described. [6] The method for analyzing chimerism described in any one of [1] to [5], wherein the first DNA marker and the second DNA marker are indel markers or CNV markers. [7] The method for analyzing chimerism described in any one of [1] to [6], wherein the plurality of cells are cells derived from peripheral blood of the subject. [8] The method for analyzing chimerism described in any one of [1] to [7], wherein the plurality of first reaction compartments are droplets. Effect of the Invention

[0009] According to the present invention, a method for analyzing chimerism can be provided that can easily measure the ratio of recipient cells to donor cells. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a flowchart of a chimerism analysis method according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a scatter plot showing the results of detecting amplicons of 10 types of candidate DNA markers (PA1 to PA10) and two types of reference DNA markers. [Diagram 3] FIG. 3 is a scatter plot showing the results of detecting the amplicons of the other nine types of candidate DNA markers (PA11 to PA19) and two types of reference DNA markers. [Figure 4] FIG. 4 is a scatter plot showing the results of detecting the amplicons of the other nine types of candidate DNA markers (PA20 to PA28) and two types of reference DNA markers. [Diagram 5] FIG. 5A shows the results of ddPCR performed on recipient gDNA, and FIG. 5B shows the results of ddPCR performed on donor gDNA. [Figure 6] FIG. 6A shows the results of ddPCR performed on recipient gDNA, and FIG. 6B shows the results of ddPCR performed on donor gDNA. [Figure 7]FIG. 7A shows the results of ddPCR performed on recipient gDNA, and FIG. 7B shows the results of ddPCR performed on donor gDNA. [Figure 8] FIG. 8A is a graph showing the dot counts of candidate DNA markers and reference DNA markers for the recipient, and FIG. 8B is a graph showing the dot counts of candidate DNA markers and reference DNA markers for the donor. [Figure 9] FIG. 9A is a graph showing the dot counts of candidate and reference DNA markers for the recipient, and FIG. 9B is a graph showing the dot counts of candidate and reference DNA markers for the donor. [Figure 10] FIG. 10A is a graph showing the dot counts of candidate DNA markers and reference DNA markers for the recipient, and FIG. 10B is a graph showing the dot counts of candidate DNA markers and reference DNA markers for the donor. [Figure 11] Figure 11 shows the results of PCR performed on cells in the peripheral blood of a recipient 21 days after hematopoietic stem cell transplantation, using two types of first DNA markers, PA3 and PA11, and four types of second DNA markers, PA6, PA17, PA20, and PA21. [Figure 12] FIG. 12 shows the results of PCR performed on cells in the peripheral blood of a recipient 21 days after hematopoietic stem cell transplantation, using PA3 as the first DNA marker and PA6 as the second DNA marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The method for analyzing chimerism according to the present invention relates to a method for analyzing chimerism in a plurality of cells derived from a subject.

[0012] Fig. 1 is a flow chart of a method for analyzing chimerism according to an embodiment of the present invention. As shown in Fig. 1, the method for analyzing chimerism according to an embodiment of the present invention includes the steps of (1) distributing a plurality of cells derived from a subject into a plurality of first reaction compartments one by one, (2) amplifying a first DNA marker specifically present in a recipient cell and a second DNA marker specifically present in a donor cell by PCR in each of the plurality of first reaction compartments, and (3) detecting an amplicon of the first DNA marker and an amplicon of the second DNA marker in each of the plurality of first reaction compartments.

[0013] In the method for analyzing chimerism according to the present invention, the presence of multiple candidate DNA markers in the recipient's cells and the donor's cells may be confirmed, and the first DNA marker and the second DNA marker may be determined in advance. That is, the method for analyzing chimerism according to another embodiment of the present invention includes the above steps (1) to (3), and (4) a step of confirming the presence of multiple candidate DNA markers in the recipient's cells and the donor's cells, and determining the first DNA marker and the second DNA marker.

[0014] Each of the above steps (1) to (4) will be described below. In this specification, a numerical range indicated with "to" means a numerical range including the numerical values ​​before and after "to".

[0015] (1) Distribution of cells to the first reaction compartment First, a plurality of cells derived from a subject are distributed one by one into a plurality of first reaction compartments.

[0016] The type of the multiple cells derived from the subject is not particularly limited as long as they can be used in chimerism analysis. For example, the multiple cells are cells derived from the subject's peripheral blood (e.g., mononuclear cells) or cells derived from the subject's bone marrow.

[0017] The configuration of the first reaction compartment is not particularly limited as long as it can store cells derived from a subject, a PCR reaction system, and a cell lysis reagent, and can perform PCR appropriately in the subsequent steps. Usually, the multiple first reaction compartments have substantially the same configuration except for the cells to be distributed. For example, the first reaction compartment is a droplet of a water-in-oil emulsion type, or a microwell. The volume of the first reaction compartment is not particularly limited, but is, for example, 1 pL to 10 nL, preferably 1 pL to 2 nL, and particularly preferably 10 pL to 1 nL. As will be described later, in the minute first reaction compartment, DNA markers (first DNA marker and second DNA marker) in the genomic DNA contained in the distributed cells are amplified by PCR, and the amplicons are detected, so that it is possible to detect whether the cells distributed to the first reaction compartment are recipient (subject) cells or donor cells.

[0018] The PCR reaction system includes elements necessary for PCR to amplify the DNA marker in the subsequent step (2). For example, the PCR reaction system includes a set of primers corresponding to the first DNA marker and the second DNA marker, a probe, a DNA polymerase, a deoxynucleoside triphosphate (dNTP) mixture, a buffer solution, a cofactor, and the like. The cofactor includes, for example, magnesium ions. For example, the PCR reaction system may be prepared using primers prepared by a known method and a known PCR premix reagent. The type of probe is not particularly limited. For example, the probe is a fluorescent probe that hybridizes to the DNA marker or an amplicon obtained from the DNA marker, or a fluorescent intercalator that binds to double-stranded DNA.

[0019] The cell lysis reagent lyses the cells distributed in the first reaction compartment. This allows the genomic DNA contained in the cells to come into contact with the PCR reaction system. Examples of cell lysis reagents include sodium dodecyl sulfate, TRITON X-100 (TRITON is a registered trademark), and NP-40.

[0020] The method of storing the subject-derived cells, the PCR reaction system, and the cell lysis reagent in each of the multiple first reaction compartments is not particularly limited. When the first reaction compartment is a droplet, a droplet containing the subject-derived cells, the PCR reaction system, and the cell lysis reagent may be generated using a known droplet generating device used in droplet digital PCR (ddPCR), for example. For example, a mixture of the subject-derived cells, the PCR reaction system, and the cell lysis reagent may be made into droplets. In this case, it is preferable to perform the process from mixing the cell lysis reagent and the cells to making the droplets quickly so that the cell lysis reagent does not react with the cells. When the reaction compartment is a microwell, the subject-derived cells, the PCR reaction system, and the cell lysis reagent may be provided to each microwell of a known microwell plate used in digital PCR (dPCR) or the like. At this time, the subject-derived cells, the PCR reaction system, and the cell lysis reagent may be provided separately to the microwell, or a mixture of the subject-derived cells, the PCR reaction system, and the cell lysis reagent may be provided to the microwell. From the viewpoint of distributing one cell each to each of the multiple first reaction compartments, it is preferable to distribute the cells to each first reaction compartment by limiting dilution (ideally diluting the cells so that each first reaction compartment contains one or zero cells) so that some of the first reaction compartments do not contain cells. It is acceptable for multiple cells to be distributed to some of the first reaction compartments. For example, if there is a reaction compartment with zero cells, it is possible to correct the cell number using Poisson distribution, so there is no problem even if multiple cells are distributed to some of the first reaction compartments.

[0021] (2) Amplification by PCR Next, in each of the first reaction compartments prepared in step (1), the first DNA marker specifically present in the recipient cells and the second DNA marker specifically present in the donor cells are amplified by PCR. That is, the first reaction compartments are subjected to thermal cycling. At this time, in each of the first reaction compartments, the cells are lysed by a cell lysis reagent. For example, the first reaction compartments may be subjected to thermal cycling using a known thermal cycler used in droplet digital PCR or a known thermal cycler for microwell plates. In PCR, the genomic DNA contained in the cells derived from the subject serves as a template, and the first DNA marker or the second DNA marker contained in the genomic DNA is amplified. The conditions of the thermal cycle (for example, temperature, time, number of cycles, etc.) can be appropriately adjusted by a person skilled in the art.

[0022] The first DNA marker is a DNA marker that is specifically present in the recipient's cells. The second DNA marker is a DNA marker that is specifically present in the donor's cells. In each of the first reaction compartments, the number of the first DNA marker and the second DNA marker that are amplified by PCR is not particularly limited. For example, one type of the first DNA marker and one type of the second DNA marker may be amplified by PCR, or multiple types of the first DNA markers and multiple types of the second DNA markers may be amplified by PCR.

[0023] The types of the first DNA marker and the second DNA marker are not particularly limited. For example, the first DNA marker and the second DNA marker are biallelic markers. When a biallelic marker is used as the first DNA marker and / or the second DNA marker, if the marker is present in a homozygous or heterozygous form, the marker is detected in the next step (3), and if the marker is not present, the marker is not detected in the next step (3). Examples of the first DNA marker and the second DNA marker include an indel marker and a CNV marker. The first DNA marker and the second DNA marker may be any sequence determined based on an analysis such as a sequence.

[0024] If there are DNA markers known in advance to be usable as the first and second DNA markers, they may be used as the first and second DNA markers. In addition, the first and second DNA markers may be determined by carrying out step (4) described below before step (1).

[0025] (3) Detection of amplicons Next, in step (3), the amplicons of the first DNA marker and the amplicons of the second DNA marker are detected in each of the first reaction compartments after the PCR is stopped. The cells distributed to the first reaction compartments in which the amplicons of the first DNA marker are detected are determined to be recipient cells, and the cells distributed to the first reaction compartments in which the amplicons of the second DNA marker are detected are determined to be donor cells.

[0026] The method of detecting the amplicon is not particularly limited. For example, the amount of the amplicon is measured by a fluorescent probe method. More specifically, in each of the multiple first reaction compartments, the amplicon of the first DNA marker is detected by a fluorescent probe of a first wavelength, and the amplicon of the second DNA marker is detected by a fluorescent probe of a second wavelength. The types of the 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 (fluorescein amidite), and the fluorescent substance of the second fluorescent wavelength is HEX (hexachloro-fluorescein).

[0027] By detecting the amplicon of the first DNA marker and the amplicon of the second DNA marker in each of the first reaction compartments, it is possible to determine whether each of the cells derived from the subject is a recipient cell or a donor cell, and also to determine the number of recipient cells and donor cells. That is, it is possible to perform chimerism analysis for the cells derived from the subject. In this case, the analysis of the number of cells can be performed by any known method.

[0028] (4) Determination of DNA markers If DNA markers that can be used as the first and second DNA markers are not known before carrying out steps (1) to (3), it is preferable to carry out a step of determining the first and second DNA markers before step (1). Specifically, the presence of multiple types of candidate DNA markers is confirmed for recipient cells and donor cells to determine the first and second DNA markers.

[0029] For example, the step of determining the DNA markers includes the steps of (4-1) distributing fragments of genomic DNA derived from recipient cells or donor cells into a plurality of second reaction zones, (4-2) amplifying a plurality of types of candidate DNA markers by PCR in each of the plurality of second reaction zones, and (4-3) detecting amplicons of the plurality of types of candidate DNA markers using a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength in each of the plurality of second reaction zones.

[0030] In the above step (4-1), fragments of genomic DNA derived from recipient cells or donor cells are distributed into a plurality of second reaction compartments.

[0031] The type of recipient or donor cells is not particularly limited. For example, recipient or donor cells may be peripheral blood-derived cells or bone marrow-derived cells.

[0032] The configuration of the second reaction compartment is not particularly limited as long as it can store the genomic DNA fragments and the PCR reaction system and can perform PCR appropriately in the subsequent step. Usually, the multiple second reaction compartments have substantially the same configuration except for the distributed genomic DNA fragments. For example, the second reaction compartment is a droplet of a water-in-oil emulsion type or a microwell. The volume of the second reaction compartment is not particularly limited, but is, for example, 1 pL to 10 nL, preferably 1 pL to 2 nL, and particularly preferably 10 pL to 1 nL. As will be described later, in the minute second reaction compartment, the candidate DNA markers in the distributed genomic DNA fragments are amplified by PCR and the amplicons are detected, thereby determining the first DNA marker specifically present in the recipient's cells and the second DNA marker specifically present in the donor's cells.

[0033] The PCR reaction system includes elements necessary for PCR to amplify the candidate DNA marker in the subsequent step (4-2). For example, the PCR reaction system includes a set of primers corresponding to the candidate DNA marker, a probe, a DNA polymerase, a mixture of deoxynucleoside triphosphates (dNTPs), a buffer solution, cofactors, and the like. The cofactors include, for example, magnesium ions. For example, the PCR reaction system may be prepared using primers prepared by a known method and a known premixed PCR reagent. The type of probe is not particularly limited. For example, the probe is a fluorescent probe that hybridizes to the candidate DNA marker or an amplicon obtained from the candidate DNA marker, or a fluorescent intercalator that binds to double-stranded DNA.

[0034] The method of storing the fragments of genomic DNA derived from the recipient's cells or the donor's cells and the PCR reaction system in each of the multiple second reaction compartments is not particularly limited. When the second reaction compartment is a droplet, a droplet containing the fragments of genomic DNA and the PCR reaction system may be generated using a known droplet generating device used in droplet digital PCR (ddPCR), for example. When the reaction compartment is a microwell, the fragments of genomic DNA and the PCR reaction system may be provided in each microwell of a known microwell plate used in digital PCR (dPCR) or the like. In this case, the fragments of genomic DNA and the PCR reaction system may be provided separately in the microwell, or a mixture of the fragments of genomic DNA and the PCR reaction system may be provided in the microwell. From the viewpoint of distributing one fragment of genomic DNA to each of the multiple second reaction compartments, it is preferable to distribute the fragments of genomic DNA to each second reaction compartment by limit diluting the fragments of genomic DNA (ideally, diluting so that each second reaction compartment contains one or zero fragments of genomic DNA) so that some second reaction compartments do not contain the fragments of genomic DNA.

[0035] In the above step (4-2), the multiple candidate DNA markers are amplified by PCR in each of the multiple second reaction compartments. That is, the multiple second reaction compartments are subjected to thermal cycling. For example, the multiple second reaction compartments may be subjected to thermal cycling using a known thermal cycler used in droplet digital PCR or a known thermal cycler for microwell plates. In PCR, genomic DNA serves as a template, and if the genomic DNA contains a specific candidate DNA marker among the multiple candidate DNA markers, the candidate DNA marker is amplified. The conditions of the thermal cycle (for example, temperature, time, number of cycles, etc.) can be appropriately adjusted by a person skilled in the art.

[0036] The type of candidate DNA marker is not particularly limited as long as it has the potential to distinguish between recipient cells (genomic DNA) and donor cells (genomic DNA). For example, the candidate DNA marker is a biallelic marker. When a biallelic marker is used as the candidate DNA marker, if the marker is present in a homozygous or heterozygous form, the marker is detected in the next step (4-3), and if the marker is not present, the marker is not detected in the next step (4-3). Examples of the candidate DNA marker include indel markers and CNV markers. The candidate DNA marker may be any sequence determined based on an analysis such as a sequence.

[0037] Candidate DNA markers can be appropriately selected from known DNA markers according to the occurrence rate, etc. The number of types of candidate DNA markers is not particularly limited and can be appropriately set according to the occurrence rate of each candidate DNA marker, the degree of blood relatedness between the donor and the recipient, etc., but from the viewpoint of reliably determining the first DNA marker specific to the recipient's cells and the second DNA marker specific to the donor's cells, it is preferable that there are 20 types or more, and more preferably 28 types or more. For example, in the following examples, 28 types of candidate DNA markers (PA1 to PA28; 18 types of indel markers and 10 types of CNV markers) are used. The upper limit of the number of types of candidate DNA markers is not particularly limited and is, for example, 50 types.

[0038] In the above step (4-3), the amplicons of the multiple candidate DNA markers are detected in each of the multiple second reaction compartments using a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength. In this step, it is preferable to use a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength that specifically bind to the amplicon of a first candidate DNA marker among the multiple candidate DNA markers, and a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength that specifically bind to the amplicon of a second candidate DNA marker among the multiple candidate DNA markers. In this case, it is preferable that the ratio of the fluorescent probe of the third wavelength and the fluorescent probe of the fourth wavelength that specifically bind to the amplicon of the first candidate DNA marker is different from the ratio of the fluorescent probe of the third wavelength and the fluorescent probe of the fourth wavelength that specifically bind to the amplicon of the second candidate DNA marker. Note that the third wavelength may be different from the fourth wavelength and may be the same as the first wavelength or the second wavelength. Similarly, the fourth wavelength may be different from the third wavelength and may be the same as the first wavelength or the second wavelength.

[0039] For example, when detecting amplicons of 10 types of candidate DNA markers, a fluorescent probe of the third wavelength and a fluorescent probe of the fourth wavelength that specifically bind to the amplicon are prepared for each of the amplicons of the 10 types of candidate DNA markers. Then, the ratio of the fluorescent probe of the third wavelength to the fluorescent probe of the fourth wavelength is changed for each of the amplicons of the 10 types of candidate DNA markers. In this way, the amplicons of the 10 types of candidate DNA markers can be detected at once using the two wavelengths of the third wavelength and the fourth wavelength.

[0040] FIG. 2 is a scatter diagram showing the results of detecting the amplicons of 10 types of candidate DNA markers (PA1 to PA10) and two types of reference DNA markers. The vertical axis shows the fluorescence intensity of FAM detected for each second reaction compartment, and the horizontal axis shows the fluorescence intensity of HEX detected for each second reaction compartment. In this example, the above steps (4-1) to (4-3) were performed using a mixture of genomic DNA from six males. For each of the amplicons of these 10 types of candidate DNA markers and two types of reference DNA markers, a FAM-labeled fluorescent probe and a HEX-labeled fluorescent probe that specifically bind to the amplicon were used. At this time, the ratio of the FAM-labeled fluorescent probe to the HEX-labeled fluorescent probe was changed for each of the amplicons of these 10 types of candidate DNA markers and two types of reference DNA markers. For example, looking at PA6 and PA8, the amount of HEX-labeled fluorescent probe relative to the FAM-labeled fluorescent probe was increased for PA6, so the dot (cluster) showing the amplicon of PA6 was located to the right of the dot (cluster) showing the amplicon of PA8. In this way, the amplicons of 10 types of candidate DNA markers could be detected simultaneously using two fluorescent substances, FAM and HEX.

[0041] Similarly, FIG. 3 is a scatter plot showing the results of detecting the amplicons of the other nine candidate DNA markers (PA11 to PA19) and two reference DNA markers. FIG. 4 is a scatter plot showing the results of detecting the amplicons of the other nine candidate DNA markers (PA20 to PA28) and two reference DNA markers. In these examples, the above steps (4-1) to (4-3) were performed using a mixture of genomic DNA from six males.

[0042] In the following examples, these 28 types of candidate DNA markers (PA1 to PA28) were used to determine the first DNA marker specifically present in the recipient's cells and the second DNA marker specifically present in the donor's cells. For example, FIG. 5A is a scatter plot showing the results of detecting the amplicons of 10 types of candidate DNA markers (PA1 to PA10) and two types of reference DNA markers in the recipient's genomic DNA, and FIG. 5B is a scatter plot showing the results of detecting the amplicons of 10 types of candidate DNA markers (PA1 to PA10) and two types of reference DNA markers in the donor's genomic DNA. Comparing these figures, it can be seen that PA3 is present in the recipient's cells but not in the donor's cells, and therefore can be used as the first DNA marker specifically present in the recipient's cells. Similarly, it can be seen that PA6 is present in the donor's cells but not in the recipient's cells, and therefore can be used as the second DNA marker specifically present in the donor's cells.

[0043] By using the above procedure, the ratio of recipient cells to donor cells in multiple cells derived from a subject can be easily measured.

[0044] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0045] [Example 1] 1. Deciding which DNA markers to use (genotyping) Prior to hematopoietic stem cell transplantation, peripheral blood was collected from a leukemia patient (recipient) using a vacuum blood collection tube (Streck, Inc.) and hemolyzed. gDNA was extracted from the hemolyzed blood using NucleoSpin (registered trademark) Tissue XS (Takara Bio Inc.). Similarly, peripheral blood collected from a donor was hemolyzed, and gDNA was extracted from the hemolyzed blood.

[0046] ddPCR was performed using a QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol with some modifications. All preparations and reactions were performed in a dedicated chamber under a hood. The 20 μl ddPCR reaction solution constituting each droplet contained 5 μl of 4× ddPCR Multiplex Super Mix (Bio-Rad Laboratories, Inc.), template DNA (recipient gDNA or donor gDNA), forward primer, reverse primer, 6-carboxyfluorescein (FAM)-labeled probe, and hexachlorofluorescein (HEX)-labeled probe. The primer and probe sequences were designed to correspond to 28 candidate DNA markers (18 indel markers and 10 CNV markers). In addition, the 28 candidate DNA markers (PA1 to PA28) were divided into three groups (Group 1 to Group 3), and the probe concentrations were designed to have different ratios of FAM-labeled probes and HEX-labeled probes for each candidate DNA marker in each group. The names of each candidate DNA marker (PA1 to PA28) are arbitrary names, not common names. Droplets were generated by a QX200 Droplet Generator (Bio-Rad Laboratories, Inc.). The plate was sealed with a pierceable foil heat seal (MSF1001; Bio-Rad Laboratories, Inc.), and PCR was performed using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Inc.) under the specified conditions (one cycle at 95°C for 10 min, followed by 40 cycles of a set of 94°C for 30 s and 62.0°C for 2 min, and finally one cycle at 98°C for 10 min), and then maintained at 10°C. Fluorescent signals were measured using a QX200 Droplet Reader (Bio-Rad Laboratories, Inc.).

[0047] 5A, 6A, and 7A show the results of ddPCR performed on the recipient's gDNA, and 5B, 6B, and 7B show the results of ddPCR performed on the donor's gDNA. 5A and 5B show the results of 10 types of candidate DNA markers (PA1 to PA10) and two types of reference DNA markers (ref1 and ref2) in the first group, 6A and 6B show the results of 9 types of candidate DNA markers (PA11 to PA19) and two types of reference DNA markers (ref1 and ref2) in the second group, and 7A and 7B show the results of 9 types of candidate DNA markers (PA20 to PA28) and two types of reference DNA markers (ref1 and ref2) in the third group. In each figure, the vertical axis shows the fluorescence intensity derived from FAM, and the horizontal axis shows the fluorescence intensity derived from HEX. 5A to 7B, PA3 (indel marker) and PA11 (indel marker) are present in recipient cells but not in donor cells, and therefore can be used as a first DNA marker that is specifically present in recipient cells. Similarly, PA6 (indel marker), PA17 (indel marker), PA20 (CNV marker) and PA21 (CNV marker) are present in donor cells but not in recipient cells, and therefore can be used as a second DNA marker that is specifically present in donor cells.

[0048] 8A, 9A, and 10A are graphs showing the number of dots of candidate DNA markers (PA1 to PA28) and one type of reference DNA marker (ref1) known to be homozygous for the recipient, and FIG. 8B, 9B, and 10B are graphs showing the number of dots of candidate DNA markers (PA1 to PA28) and reference marker (ref1) for the donor. From these graphs, the number of dots of PA3 and PA11 usable as the first DNA marker, and PA6, PA17, PA20, and PA21 usable as the second DNA marker, were about half the number of dots of the reference DNA marker (ref1) known to be homozygous. From this, it was found that PA3 and PA11 are both heterozygous in the recipient's cells, and that PA6, PA17, PA20, and PA21 are all heterozygous in the donor's cells.

[0049] 2. Chimerism Analysis Peripheral blood was collected from a leukemia patient (recipient) 21 days after hematopoietic stem cell transplantation using a vacuum blood collection tube (Streck, Inc.) and hemolyzed. The hemolyzed blood 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 (Bio-Rad Laboratories, Inc.), and cells were further lysed in each droplet. Then, PCR was performed for the first DNA marker (PA3 and PA11) and the second DNA marker (PA6, PA17, PA20, and PA21) in each droplet. Specifically, the plate was sealed with a pierceable foil heat seal (MSF1001), and PCR was performed using a C1000 Touch Thermal Cycler under the specified conditions (85°C for 60 minutes, followed by 95°C for 10 minutes, followed by 50 cycles of 94°C for 30 seconds and 62.0°C for 2 minutes, and finally 98°C for 10 minutes). A FAM-labeled probe was used for the first DNA marker and a HEX-labeled probe was used for the second DNA marker. Fluorescence signals were measured using a QX200 Droplet Reader.

[0050] FIG. 11 shows the results of PCR performed on cells in the peripheral blood of a recipient 21 days after hematopoietic stem cell transplantation using two types of first DNA markers, PA3 and PA11, and four types of second DNA markers, PA6, PA17, PA20, and PA21. The vertical axis shows the fluorescence intensity derived from FAM, and the horizontal axis shows the fluorescence intensity derived from HEX. In the figure, there is a cluster showing droplets containing recipient-derived cells in the upper left, a cluster showing droplets containing donor-derived cells in the lower right, and a cluster showing droplets containing both recipient-derived cells and donor-derived cells in the upper right. Here, the total number of recipient-derived cells was calculated by adding up the number of dots contained in the upper left cluster and the number of dots contained in the upper right cluster, and the total number of donor-derived cells was calculated by adding up the number of dots contained in the lower right cluster and the number of dots contained in the upper right cluster. In addition, since one droplet may contain multiple cells, the total number of recipient-derived cells and the total number of donor-derived cells were corrected according to the Poisson model. As a result, the number of recipient-derived cells was 1462.25 (12.83%) and the number of donor-derived cells was 9938.10 (87.17%). From this result, it was found that the recipient-derived cells accounted for 12-13% of the total number of cells in this recipient.

[0051] Table 1 shows the results of PCR performed on cells in the peripheral blood of a recipient 21 days after hematopoietic stem cell transplantation, using PA3 as the first DNA marker and PA6, PA17, PA20 or PA21 as the second DNA marker. Figure 12 shows the results of No. 1 in Table 1. These results also revealed that 10 to 14% of cells were derived from the recipient in this recipient. [Table 1]

[0052] [Example 2] 1. Deciding which DNA markers to use (genotyping) Prior to hematopoietic stem cell transplantation, peripheral blood was collected from a leukemia patient (recipient) using a vacuum blood collection tube (Streck, Inc.) and hemolyzed. gDNA was extracted from the hemolyzed blood using NucleoSpin (registered trademark) Tissue XS (Takara Bio Inc.). Similarly, peripheral blood collected from a donor was hemolyzed, and gDNA was extracted from the hemolyzed blood. Similarly, peripheral blood collected from a donor was hemolyzed, and gDNA was extracted from the hemolyzed blood.

[0053] In the same manner as in Example 1, ddPCR was performed for the same 28 types of candidate DNA markers as in Example 1 using the recipient gDNA or donor gDNA as a template.

[0054] As a result, it was found that PA3 (indel marker) is present in the recipient cells but not in the donor cells, and therefore can be used as a first DNA marker that is specifically present in the recipient cells. Similarly, PA6 (indel marker) and PA13 (indel marker) are present in the donor cells but not in the recipient cells, and therefore can be used as a second DNA marker that is specifically present in the donor cells.

[0055] 2. Chimerism Analysis 21 days after hematopoietic stem cell transplantation, peripheral blood was collected from a leukemia patient (recipient) using a vacuum blood collection tube (Streck, Inc.), and bone marrow aspirate was collected into a cell preservative tube (Cell Preservative, Streck, Inc.). The hemolyzed blood or bone marrow aspirate 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, and cells were lysed in each droplet. Then, PCR was performed in each droplet for the first DNA marker (PA3) and the second DNA marker (PA6 and PA13) under the same conditions as in Example 1. A FAM-labeled probe was used for the first DNA marker, and a HEX-labeled probe was used for the second DNA marker. Fluorescence signals were measured using a QX200 Droplet Reader.

[0056] In addition, gDNA was extracted from the hemolyzed blood or bone marrow fluid using NucleoSpin (registered trademark) Tissue XS (Takara Bio Inc.). The obtained gDNA 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, and cells were further lysed in each droplet. Then, in each droplet, PCR was performed for the first DNA marker (PA3) and the second DNA marker (PA6 and PA13) under the same conditions as in Example 1. A FAM-labeled probe was used for the first DNA marker, and a HEX-labeled probe was used for the second DNA marker. Fluorescence signals were measured using a QX200 Droplet Reader.

[0057] Table 2 shows the results of PCR performed on cells in the peripheral blood or bone marrow of recipients 21 days after hematopoietic stem cell transplantation, using PA3 as the first DNA marker and PA6 and PA13 as the second DNA markers. Nos. 1, 2, 5, 6, 9, and 10 are the results when the recipient's peripheral blood was used, and Nos. 3, 4, 7, 8, 11, and 12 are the results when the bone marrow was used. [Table 2]

[0058] Table 3 shows the results of PCR performed on gDNA derived from cells in the peripheral blood or bone marrow of recipients 21 days after hematopoietic stem cell transplantation, using PA3 as the first DNA marker and PA6 and PA13 as the second DNA markers. Nos. 1, 2, 5, and 6 are the results when the recipient's peripheral blood was used, and Nos. 3, 4, 7, and 8 are the results when the bone marrow was used. [Table 3]

[0059] These results indicate that the recipient cells accounted for approximately 0% of all recipient-derived cells. In addition, when PCR reaction mixtures containing cells were made into droplets, the results were similar to those when PCR reaction mixtures containing gDNA were made into droplets. [Industrial Applicability]

[0060] The method for analyzing chimerism of the present invention is clinically useful, for example, as a method for monitoring the engraftment of donor-derived cells and the recurrence of blood cancer after hematopoietic stem cell transplantation.

Claims

1. A method for analyzing chimerism in a plurality of cells derived from a subject, comprising: distributing said plurality of cells into a plurality of first reaction compartments, one each; amplifying a first DNA marker specifically present in recipient cells and a second DNA marker specifically present in donor cells by PCR in each of the plurality of first reaction compartments; detecting an amplicon of the first DNA marker and an amplicon of the second DNA marker in each of the plurality of first reaction compartments; A method for analyzing chimerism, comprising:

2. 2. The method for analyzing chimerism according to claim 1, wherein in the step of detecting the amplicon of the first DNA marker and the amplicon of the second DNA marker, in each of the plurality of first reaction compartments, the amplicon of the first DNA marker is detected with a fluorescent probe of a first wavelength, and the amplicon of the second DNA marker is detected with a fluorescent probe of a second wavelength.

3. The method for analyzing chimerism according to claim 1 , further comprising the step of confirming the presence of a plurality of candidate DNA markers in the recipient cells and the donor cells to determine the first DNA marker and the second DNA marker.

4. The step of determining the first DNA marker and the second DNA marker comprises: distributing fragments of genomic DNA from the recipient cells or the donor cells into a plurality of second reaction compartments; amplifying the plurality of candidate DNA markers by PCR in each of the plurality of second reaction compartments; detecting amplicons of the plurality of candidate DNA markers using a fluorescent probe of a third wavelength and a fluorescent probe of a fourth wavelength in each of the plurality of second reaction compartments; The method for analyzing chimerism according to claim 3, comprising:

5. In the step of detecting the amplicons of the plurality of candidate DNA markers, a fluorescent probe having a third wavelength and a fluorescent probe having a fourth wavelength that specifically bind to the amplicon of a first candidate DNA marker among the plurality of candidate DNA markers, and a fluorescent probe having a third wavelength and a fluorescent probe having a fourth wavelength that specifically bind to the amplicon of a second candidate DNA marker among the plurality of candidate DNA markers are used; a ratio of the fluorescent probe having the third wavelength to the fluorescent probe having the fourth wavelength that specifically binds to the amplicon of the first candidate DNA marker is different from a ratio of the fluorescent probe having the third wavelength to the fluorescent probe having the fourth wavelength that specifically binds to the amplicon of the second candidate DNA marker; The method for analyzing chimerism according to claim 4.

6. The method for analyzing chimerism according to claim 1 , wherein the first DNA marker and the second DNA marker are indel markers or CNV markers.

7. The method for analyzing chimerism according to claim 1 , wherein the plurality of cells are cells derived from peripheral blood of the subject.

8. The method for analyzing chimerism according to claim 1 , wherein the plurality of first reaction compartments are droplets.

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