JP2018157812A - Detection method of cells included in sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which, for target cells included in a sample, both (1) optically detecting the target cells and analyzing the number or characteristics of the target cells and (2) analyzing genes of the target cells can be easily executed.SOLUTION: A cell detection method includes: a process of concentrating target cells contained in a sample; a process of dividing target cell concentrated liquid obtained in the concentration process into 2 or more fractions; a process of optically detecting target cells by using at least one out of fractions obtained in the dividing process; a process of analyzing genes of the target cells by using at least one out of the fractions obtained in the dividing process.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a method for detecting cells contained in a sample. In particular, the present invention relates to a method for performing optical detection and genetic analysis of specific cells contained in a biological sample such as blood or urine together.

  In recent years, cells have been selectively separated and collected from samples such as body fluids such as blood, tissue suspensions obtained by suspending or dispersing tissues such as organs, and cell culture solutions. Research that applies to basic research, clinical diagnosis, and treatment is underway. For example, a tumor cell (Circulating Tumor Cell, hereinafter referred to as CTC) is detected from blood collected from a cancer patient, and morphological analysis, tissue type analysis, or gene analysis is performed on the cell. Research to determine treatment policy based on this is underway. However, CTC has a very low probability of existence (in the case where the sample is whole blood derived from a cancer patient), and requires high-sensitivity detection.

  As a method for detecting target cells contained in a sample, a detection method by flow cytometry is known (Non-patent Document 1). In the flow cytometry method, cells previously labeled with a fluorescent substance or a luminescent substrate are flowed through a flow channel in a state of being aligned in a line, and the labeled cells flowing in the flow channel are scanned using a laser beam and an optical detector. This is a method for detection and analysis, and the target cells contained in a sample can be counted with high accuracy. As another method, a method is also known in which cell groups containing target cells are arranged on a substrate such as a glass slide, labeled with a fluorescent substance or a luminescent substrate, and then the light derived from the label is detected by a microscope and counted. (Non-Patent Documents 2 and 3).

  As a label target used in these methods, biomolecules such as proteins and nucleic acids (for example, DNA and mRNA) present in target cells are often used. For example, in the detection of the aforementioned CTC, immunostaining using an antibody labeled with a fluorescent substance against cytokeratin, an intracellular protein characteristic of epithelial cells, as a target cell marker, or DNA in target cells In general, staining with DAPI (4 ′, 6-diamidino-2-phenyllinole), which is a fluorescent dye that binds strongly and strongly, is performed.

  However, in order to label a biomolecule in a target cell, the cell must be fixed or membrane permeabilized, and the biomolecule such as mRNA is eluted and / or denatured by the treatment. For this reason, it has been difficult to continuously detect biomolecules contained in target cells. In addition, since the detection method using immunostaining described above requires a relatively long time until detection, there is a possibility that biomolecules such as mRNA may be decomposed even when immobilization treatment or membrane permeation treatment is not performed. This is unsuitable for continuously detecting biomolecules contained in target cells.

  As another method for detecting target cells contained in a sample, after carrying out cell fixation treatment or membrane permeabilization treatment, intracellular biomolecules are labeled with an antibody labeled with a fluorescent substance against the protein of the target cells, A method of performing mutation analysis of genomic DNA after collecting labeled target cells has also been reported (Non-patent Document 3). If you target a stable large molecule such as genomic DNA, you can detect the target cell by detecting the target cell by immunostaining, but it is necessary to recover the target cell one by one. The complexity of operation was a problem.

  As a method for analyzing a gene contained in a target cell contained in a sample, a method for analyzing the gene such as mRNA by collecting the target cell contained in the sample using magnetic beads has been reported (Patent Document 1). However, this method collects and analyzes target cells as a group, and cannot detect individual target cells or analyze the number of target cells and their characteristics.

WO2016 / 033114

W. Jeffrey Allard, et al. , Clin. Cancer Res. 10, 6897-6904 (2004). Howard I. Scher, et al. , JAMA Oncology, 2 (11), 1441-1449 (2016) A. Morimoto, et al. , PLOS ONE, 10 (6), e0130418 (2015)

  An object of the present invention is to provide a method capable of easily performing both optical detection of a target cell contained in a sample and analysis of a gene possessed by the target cell.

  In order to solve the above-mentioned problems, the present inventors have intensively studied to arrive at the present invention.

That is, the present invention includes the embodiments described in [1] to [5] below:
[1] A method for detecting a target cell contained in a sample,
(1) a step of concentrating target cells contained in the sample,
(2) dividing the target cell concentrate obtained in the step (1) into two or more fractions;
(3) a step of optically detecting a target cell using at least one of the fractions obtained in the step (2);
(4) A step of analyzing a gene possessed by the target cell using at least one of the fractions obtained in the step (2),
At least including the method.

  [2] The method according to [1], wherein the step (1) is performed by a specific gravity difference separation method.

  [3] The method according to [1], wherein the sample is a blood sample and the target cell is a tumor cell, and the step (1) is performed by a specific gravity difference method to which a leukocyte binding agent is added.

  [4] The method according to any one of [1] to [3], wherein the step (3) includes a step of detecting the target cell by fixing treatment and / or membrane permeation treatment.

  [5] The method according to any one of [1] to [4], wherein the step (4) is a step of analyzing mRNA contained in the target cell.

  Hereinafter, the present invention will be described in detail.

  Examples of samples containing target cells in the present invention include samples obtained from living bodies such as urine, whole blood, diluted blood, plasma, serum, saliva, semen, feces, sputum, spinal fluid, ascites, amniotic fluid, and cell aggregation Samples derived from organs or tissues such as tumors, tumors, lymph nodes, arteries, and culture samples such as cultures of cells and tissues and culture media thereof.

In the present invention, target cells contained in a sample are detected by at least the following steps (1) to (4).
(1) a step of concentrating target cells contained in the sample,
(2) dividing the target cell concentrate obtained in the step (1) into two or more fractions;
(3) a step of optically detecting a target cell using at least one of the fractions obtained in the step (2);
(4) A step of analyzing a gene possessed by the target cell using at least one of the fractions obtained in the step (2),
Hereafter, each said process is demonstrated in detail.

(1) Step of concentrating target cells contained in sample This step selects target cells more by reducing components (contaminated cells, contaminated biomolecules, liquid components, etc.) other than target cells contained in the sample. Recovery step. As an example of this process, a filter method that separates and concentrates using the difference in size between the target cell and components other than the target cell, and an antibody against a target cell-specific protein using a difference in the antibody expression profile on the cell surface There are a magnetic bead method in which target cells are separated and concentrated using magnetic particles bound to, and a specific gravity difference separation method using a specific gravity difference between target cells and components other than the target cells. Among these, the specific gravity difference separation method is particularly preferable as the method performed in this step because the target cells can be concentrated efficiently in a short time.

  The concentration of target cells by the specific gravity difference separation method (reduction of components other than the target cells) is not particularly limited as long as the target cells and components other than the target cells (such as contaminating cells) can be separated by the specific gravity difference. For example, by layering a sample containing target cells in a centrifuge tube containing a density gradient solution, and performing a centrifugation operation, a fraction containing target cells and a fraction containing components other than target cells (such as contaminated cells) And collecting the fraction containing the target cells to concentrate the target cells (reduce contaminating cells).

  When this step is performed by the specific gravity difference separation method, a substance that specifically binds to the target cell or a substance that specifically binds to a component other than the target cell (such as a contaminated cell) is added to the target cell or a component other than the target cell. The target cells can be more efficiently separated by combining the substances having different specific gravity to change the apparent specific gravity. Examples of the substance that specifically binds include biopolymers such as antibodies, antigens, peptides, polypeptides, growth factors, cytokines, and lectins that can specifically bind to target cells (or components other than target cells such as contaminated cells). It can be illustrated. Examples of substances having different specific gravities include synthetic polymers such as polypropylene and polystyrene, inorganic materials such as glass, silica and zirconia, natural polymers such as cellulose, dextran and agarose, and biological samples such as erythrocytes. As a specific example, when the sample is a blood sample such as whole blood or diluted blood and the target cell is a tumor cell such as CTC, a binding agent for leukocytes, which are contaminating cells, is added to change the apparent specific gravity of leukocytes. Thus, efficient leukocyte removal (ie, enrichment of tumor cells) can be performed by the specific gravity difference separation method. The leukocyte binding agent used here is not particularly limited as long as it is a substance that specifically binds to leukocytes and changes the apparent specific gravity, and a combination of a substance that specifically binds to leukocytes and a substance that changes the specific gravity. Illustrated. Among these, when using a leukocyte binding agent linked to an antibody capable of specifically binding to leukocytes represented by RosetteSep (manufactured by STEMCELL Technologies) and an antibody that specifically binds to red blood cells, a specific gravity difference separation method from a blood sample is used. This is preferable because the concentration of tumor cells (removal of leukocytes) can be performed particularly efficiently.

  In addition, when performing this process by the specific gravity difference separation method, you may add operation which removes a contaminated cell actively. For example, when performing a concentration operation of tumor cells (target cells) such as CTC from a blood sample by a specific gravity difference separation method, a hemolysis operation that positively removes red blood cells that are contaminated cells may be added. The operation of actively removing the contaminating cells may be performed before the specific gravity difference separation operation (centrifugation operation) or after the specific gravity difference separation operation. In the case where the operation of positively removing contaminant cells is performed before the specific gravity difference separation operation, it is preferable to add a centrifugal operation after the removal operation.

(2) Step of dividing the target cell concentrate into two or more fractions In this step, the target cell concentrate (contaminant cell removal solution) obtained in the step (1) is divided into two or more fractions. . As for the number of divisions, one or more of the fraction for the step of optically detecting the target cell, which will be described later, and the fraction for the step of analyzing the gene of the target cell are secured, and the detection of the target cell As long as the necessary amount is secured, it may be divided into a large number. The amount of each divided fraction is not limited, and may be divided by equal amounts or may be divided with a bias.

(3) Step of optically detecting target cells In this step, for example, the target cells may be detected using an optical detector such as a flow cytometer, a fluorescence microscope, a confocal microscope, an optical microscope, or a spectrophotometer. As an example of the case where this step is performed using a microscope, one of the fractions of the target cell concentrate obtained in the step (2) above is applied to a slide or applied to a substrate having a holding part and applied. And a method of detecting a target cell held in a holding part using a microscope. Of the detection methods described above, the method of developing a sample on a substrate having a holding unit and detecting the cells held in the holding unit can observe / analyze each cell with high sensitivity and high accuracy, And after that, when the cells are relabeled and detected, the possibility that the retained cells are detached is preferable.

  In this step, the target cell may be directly detected by a bright-field image, but a signal that can be optically detected in advance by a recognition substance that can specifically bind to a substance that the target cell (or contaminating cell) specifically has. When the target cell (or the contaminating cell) is labeled using a labeling substance obtained by binding a label capable of emitting light, and the target cell is detected based on the label, the target cell is detected with high sensitivity and ease. This is preferable because it is possible. When the target cell is a tumor cell, the target cell (or contaminating cell) specifically has cytokeratin (CK), androgen receptor (AR) and its splice variant (AR-Vs), vimentin, etc. Intracellular biomolecules, and membrane proteins such as CD45, epidermal growth factor receptor (EGFR), EpCAM (Epithelial Cell Adhesion Molecule), PD-L1 (Programmed Death-Land 1). Examples of the recognition substance capable of specifically binding include biopolymers such as antibodies, antigens, peptides, polypeptides, growth factors, cytokines, and lectins. In particular, an immunostaining method using an antibody as a specifically binding recognizable substance is preferable from the viewpoint of high specificity and strong binding ability. Nucleation using a nuclear staining reagent such as DAPI (4 ′, 6-DiAmidino-2-PhenylIndole), hematoxylin, Hoechst 33342 (trade name), in addition to substances specifically possessed by target cells (or contaminating cells) Cell labeling or cell labeling using a cytoplasmic staining reagent such as orange G, light green, or eosin may be additionally performed.

  In this step, target cells and / or contaminated cells may be fixed and / or membrane permeabilized. In particular, when labeling intracellular biomolecules, it is preferable to perform immobilization treatment and / or membrane permeation treatment for the purpose of preventing elution / degradation of the biomolecules and allowing the labeling reagent to penetrate into the cells. The fixation treatment may be any method as long as it maintains the intracellular position of the biomolecule and / or suppresses degradation, and the membrane permeabilization method may be any method that improves the permeability of the cell membrane, such as formaldehyde, paraformaldehyde, Examples include methods using ethanol, methanol, acetone, surfactants and the like.

(4) Step of analyzing gene of target cell The gene to be analyzed in this step may be DNA such as genomic DNA or RNA such as mRNA. Particularly, mRNA is suitable for the detection method of the present invention because it is a substance that is easily degraded and may be denatured and eluted by immobilization or membrane permeation that can be performed in the step (3). Gene of interest. If the target gene is DNA, this step is performed by, for example, sequence analysis by the Sanger method, sequence analysis by a next-generation sequencer, single nucleotide polymorphism (SNPs) analysis using gene amplification reaction (PCR method, etc.) When the gene is RNA, for example, RNA-seq method, detection method by electrophoresis after amplification of nucleic acid by RT-PCR method, real-time RT-PCR method, TRC (Transplication Reverse-translation Concerted) method, NASBA (Nucleic Acid Sequence- It can be performed by expression analysis using a method of directly amplifying and detecting RNA, such as the Base Amplification (TMA) method and the TMA (Transscription-Mediated Amplification) method. Good. Among them, a method of detecting a gene sequence or mRNA expression that is not present in a contaminated cell is preferable in that the influence of a contaminated cell (a cell other than the target cell) can be avoided. As an example, when the target cell is a prostate cancer-derived tumor cell, it is preferable to detect androgen receptor variant 7 (AR-V7) mRNA that, when expressed, contributes to acquisition of resistance to an anticancer drug, and the target cell is lung cancer. In the case of derived tumor cells, mutation analysis of genes (EGFR (Epidmal Growth Factor Receptor), etc.) that lead to an increase in anticancer drug resistance and tumor malignancy is preferable.

  The method for detecting a target cell contained in the sample of the present invention comprises a step of concentrating the target cell contained in the sample, and a step of dividing the target cell concentrate obtained in the concentration step into two or more fractions. , A step of optically detecting a target cell using at least one of the fractions obtained in the dividing step, and an analysis of a gene possessed by the target cell using at least one of the fractions in the dividing step This includes at least a step of performing analysis of the target cells by optical detection, and analysis of gene expression status and gene mutation status in the target cells.

It is a figure which shows the centrifuge tube used for concentration of a target cell (cancer cell) in the Example. It is a figure explaining concentration of a target cell (cancer cell) using the centrifuge tube shown in FIG. It is a figure explaining concentration of a target cell (cancer cell) using the centrifuge tube shown in FIG. It is a figure which shows the cell holding | maintenance apparatus (board | substrate) used for the optical detection of the target cell in the Example. It is the figure which showed the holding | maintenance and optical detection of a cell using the cell holding | maintenance apparatus (board | substrate) shown in FIG. It is a figure which shows the result of Example 3. It is a figure which shows the result of Example 4.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to an Example.

Example 1 Concentration of cancer cells contained in sample and division of the concentrated solution (1) About 40000 human prostate cancer cells (22Rv1 strain or PC3 strain, DS in 3 mL of healthy human blood obtained by obtaining informed consent) Pharma Biomedical) was added and suspended to prepare a blood sample used in this example.

(2) Using the centrifuge tube 100 shown in FIG. 1, cancer cells were concentrated from the blood sample prepared in (1) by the method shown below. Of the members constituting the centrifuge tube 100 shown in FIG. 1, the lower cylindrical member 110 is a polypropylene member having a bottom with an inner diameter of 10 mm, a length of 54 mm, and a capacity of 3 mL. The upper cylindrical member 120 is a polypropylene member having a length of 70 mm and a capacity of 15 mL. The inner diameter is 18 mm on the upper surface side (lid 140 side) and 2 mm on the lower surface side (communication opening end 130 side). The inclination angle is 30 °.
(2-1) 3 mL of a density gradient solution having a density of 1.084 g / mL was injected into the lower cylindrical member 110. The liquid level height of the density gradient solution is about 1 mm from the lower surface side (communication opening end 130 side) of the upper cylindrical member 120 (therefore, the liquid level is positioned inside the upper cylindrical member 120). .
(2-2) On a density gradient solution, 3 mL of blood sample, 3 mL of physiological saline, 180 μL of 0.5 mg / mL tirofiban, 75 μL of leukocyte binding agent (trade name RosetteSep, manufactured by StemCell Technologies) The mixed liquid 150 was overlaid (in FIG. 2, the black coating portion is the portion of the mixed liquid 150 overlaid). The leukocyte binding agent to be added here is a reagent for binding contaminating cells (erythrocytes, leukocytes) other than cancer cells contained in the blood sample to each other, increasing the density of the contaminating cells, and density with cancer cells. By increasing the difference, the specific gravity difference separation using the centrifuge tube 100 can separate cancer cells with high recovery rate and selectivity.
(2-3) After sample stacking, the upper surface of the upper cylindrical member 120 was sealed with a lid 140 (made of polypropylene), and centrifuged at 2000 × g for 10 minutes at room temperature. By the centrifugation operation, the cancer cells were maintained at the position of the interface 160 between the density gradient solution and the sample (FIG. 3).
(2-4) After disconnecting the upper cylindrical member 120 from the lower cylindrical member 110 without removing the lid 140, the lid 140 is removed to open the seal, so that the upper cylindrical member 120 is connected to the communication opening end 130. A part of the density gradient solution and the cells maintained at the position of the interface 160 are allowed to flow out and collected by a 50 mL tube (not shown) installed below, and the inner wall of the upper cylindrical member 120 is mainly composed of ammonium chloride. The cells adhering to the wall of the upper cylindrical member 120 were also collected at the same time by washing with the erythrocyte disruption solution (FIG. 3).

  (3) A red blood cell crushing solution containing ammonium chloride as a main component was added to the collected cell suspension, and the volume was increased to 30 mL, followed by centrifugation at 300 × g for 10 minutes at room temperature. After centrifugation, the liquid at the top of the pellet was removed with a pipette, and the cells in the pellet were resuspended in 30 mL of a solution containing 300 mM mannitol and centrifuged at 300 × g for 5 minutes at room temperature. This operation is an operation for removing blood components and concentrating target cells.

  (4) After centrifugation, the liquid at the top of the pellet was removed with a pipette, and the liquid volume was adjusted to about 1.3 mL. The pellet is suspended by pipetting, half of the suspension (approx. 0.65 mL) is subjected to a cell number counting step by optical detection (Example 2 described later), and the other half (approx. 0.65 mL) is analyzed by mRNA. It used for the process (Example 3 mentioned later), respectively.

Example 2 Counting the number of cancer cells by optical detection After the cells collected in Example 1 were held in the cell holding device 200 shown in FIGS. 4 and 5 by the method shown below, the cancer cells were optically detected by the fluorescence microscope 300. Detected. The cell holding device 200 includes a flat insulating film 210 having a through hole 211, a flat light shielding film 220 having a through hole 221, a flat plate having an inlet 231, an outlet 232, and a through part 233. Spacer 230, electrodes 241 and 242 provided in close contact with the lower surface of light shielding member 220 and the upper surface of spacer 230, conductive wire 250 connecting electrodes 241 and 242, and AC power supply for applying a signal to electrodes 241 and 242 260, and the electrode 241 provided in close contact with the lower part of the through-hole 211, the through-hole 221 and the light-shielding member 211 constitutes a holding part 270 capable of holding cells, and the cell is introduced from the inlet 231 into the cell. When the sample containing 400 is introduced, the cell 400 is introduced into the holding part 270 through the penetration part 233. In the cell holding device 200 used in this embodiment, about 300,000 holding portions 270 having a diameter of 30 μm and a depth of 40 μm are provided.

  (1) After introducing the cell suspension recovered in Example 1 from the introduction unit 231, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) is applied to the electrodes 241, 242 from the AC power supply 260 for 3 minutes. Then, the cells were held in the holding unit 270 by the dielectrophoretic force 500.

  (2) A 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine is introduced from the introduction unit 231 while applying the AC voltage, and left to stand for 3 minutes, and then the application of the AC voltage is stopped and discharged. The aqueous solution was removed by suction from the part 232.

  (3) An aqueous solution containing 50% (v / v) ethanol and 1% (w / v) formaldehyde (hereinafter referred to as a cell membrane permeation reagent) is introduced from the introduction part 231 and allowed to stand for 10 minutes, thereby permeating the cell membrane. The cells introduced into the holding part were sampled. Thereafter, the cell membrane permeation reagent was aspirated and removed from the introduction part 231, and the remaining cell membrane permeation reagent was washed by introducing PBS (Phosphate buffered saline).

  (4) From introduction part 231, FITC (fluorescein isothiocyanate) labeled anti-cytokeratin antibody (Miltenyi Biotec) (hereinafter referred to as CK-FITC), PE (phycoerythrin) labeled anti-CD45 antibody (Beckman-Coulter) (Hereinafter referred to as CD45-PE) and DAPI (4 ′, 6-DiAmidino-2-PhenylIndole) (0.5 μg / mL) (manufactured by Dojindo Laboratories) were introduced, and 800 μL of a cell staining solution was introduced to label the cells. (25 ° C., 30 minutes) and then washed by introducing a 300 mM mannitol aqueous solution from the introduction part 231.

  (5) In order to observe all the cells 400 held in the holding unit 270, a fluorescence microscope 300 (IX71 manufactured by Olympus) equipped with a computer-controlled electric stage and an electron multiplying cooled CCD camera (ADT-100 manufactured by FLOVEL) ) Were used to take bright field images and fluorescent images of all the holding units 270.

(6) The image photographed in (5) was analyzed using analysis software LabVIEW (manufactured by National Instruments), and cells were discriminated based on the following conditions. The cells not stained with DAPI (having no cell nuclei) were excluded on the analysis software because they were contaminated cells having no cell nuclei such as erythrocytes and dead cell debris. In addition, although 100,000 to 500,000 leukocytes, which are contaminating cells, were held in the holding unit 270, the amount was not an obstacle when detecting human prostate cancer cells, which are target cells.
Cancer cells (target cells): cell leukocytes (contaminated cells) stained with DAPI (having cell nuclei), stained with CK-FITC (expressing cytokeratin) and not stained with CD45-PE (not expressing CD45) ): Cells stained with DAPI (having cell nuclei), not stained with CK-FITC (does not express cytokeratin), and stained with CD45-PE (expresses CD45). Table 1 shows. Target cancer cells added to the blood of healthy subjects could be detected optically.

実施例３ 癌細胞のｍＲＮＡ解析
以下に示す方法で、実施例１で回収した癌細胞濃縮液からアンドロゲン受容体スプライスバリアント７（ＡＲ−Ｖ７）のｍＲＮＡ解析を行なった。

Example 3 Cancer Cell mRNA Analysis Androgen receptor splice variant 7 (AR-V7) mRNA was analyzed from the cancer cell concentrate collected in Example 1 by the method described below.

  (1) The cancer cell concentrate collected in Example 1 was centrifuged at 300 × g for 5 minutes at room temperature, and the supernatant was removed. Total RNA was extracted from the collected cells using NucleoSpin RNA XS (manufactured by Machalai Nagel). 77 ng / μL RNA was obtained from the sample to which the 22Rv1 strain was added, and 56 ng / μL RNA was obtained from the sample to which the PC3 strain was added.

  (2) cDNA was synthesized by SuperScript IV First-Strand Synthesis System (manufactured by Thermo Fisher Scientific) using 400 ng of total RNA extracted in (1) as a template. A random hexamer was used as a primer.

(3) The following reaction solution was prepared, and AR-V7 and β-actin cDNA were amplified by PCR. The amplification reaction by the PCR method was performed under the following temperature conditions. In addition, amplification of AR-V7 was performed by Antonarakis ES, et al. , N.M. Engl. J. et al. Med. , 371, 1028-1038 (2014), β-actin amplification was performed according to Guo Z. , Et al. , Cancer Cell, 10, 309-319 (2006).
(Reaction solution composition)
TaKaRa Ex Taq (Takara Bio Inc.) 0.1 μL
10x ExTaq Buffer 2μL
dNTP Mixture 1.6μL
cDNA 2μL
Each 5 μM Forward / Reverse primer (Table 2) 2 μL each
Sterile distilled water 10.3 μL

（増幅反応の温度条件）
９８℃で１０秒間：１サイクル
９８℃で１０秒間、５５℃で３０秒間、７２℃で３０秒間：３０サイクル
７２℃で３０秒間：１サイクル
（４）ＤＮＡの増幅を、アガロースゲル電気泳動で確認した。

(Temperature conditions for amplification reaction)
98 ° C for 10 seconds: 1 cycle 98 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds: 30 cycles 72 ° C for 30 seconds: 1 cycle (4) DNA amplification confirmed by agarose gel electrophoresis did.

  A result (electrophoresis photograph) is shown in FIG. In the sample to which 22Rv1 strain known as an AR-V7 positive cell line was added, a band due to the amplification of AR-V7 was confirmed. On the other hand, AR-V7 amplification was not confirmed in the sample to which the PC3 strain known as AR-V7 negative cell line was added.

From the above results, it was possible to perform both optical detection of target cells in a sample and gene analysis of target cells by dividing the concentrated solution of target cells.
Example 4 Addition of leukocyte binding agent in cancer cell concentration step The effect of adding a leukocyte binding agent in the cancer cell concentration step was verified by the following method.

  (1) By the method shown in Example 1, the cancer cells contained in the blood sample to which the 22Rv1 strain was added were concentrated and the concentrated solution was divided. However, the amount of leukocyte binding agent added in the step of Example 1 (2-2) was 0 μL, 75 μL, and 150 μL, respectively.

  (2) By the method shown in Example 3, mRNA analysis of androgen receptor splice variant 7 (AR-V7) was performed from the cancer cell concentrate recovered in (1). However, the process of Example 3 (4) was performed using an Agilent 2100 bioanalyzer and an Agilent DNA1000 kit (manufactured by Agilent Technologies).

  The quantification result of the AR-V7 PCR product by the bioanalyzer is shown in FIG. It can be seen that the PCR product was efficiently amplified under the condition (75 μL) in which the leukocyte binding agent was added and the condition (150 μL) in which the amount was further increased compared to the condition in which the leukocyte binding agent was not added (0 μL). Based on the above results, the step of concentrating cancer cells (tumor cells) contained in the blood sample is carried out by the specific gravity difference method with the addition of a leukocyte binding agent, so that the number of contaminating leukocytes in the cancer cell concentrate is reduced. Since contaminating RNA derived from leukocytes is reduced, it can be seen that the gene of interest (AR-V7) present in the cancer cell (22Rv1 strain) can be efficiently amplified in the gene analysis step.

100: centrifuge tube 110/120: cylindrical member 121: tapered shape 130: communication open end 140: lid 150: sample containing target cells 160: fraction containing target cells 200: cell holding device 210: insulating film 211: penetration Hole 220: Light shielding film 221: Through hole 230: Spacer 231: Introduction port 232: Discharge port 233: Through portion 241, 242: Electrode 250: Conductor 260: AC power supply 270: Holding portion 300: Fluorescence microscope 400: Cell 500: Dielectric Migration force

Claims (5)

A method for detecting a target cell contained in a sample, comprising:
(1) a step of concentrating target cells contained in the sample,
(2) dividing the target cell concentrate obtained in the step (1) into two or more fractions;
(3) a step of optically detecting a target cell using at least one of the fractions obtained in the step (2);
(4) A step of analyzing a gene possessed by the target cell using at least one of the fractions obtained in the step (2),
At least including the method.   The method according to claim 1, wherein the step (1) is performed by a specific gravity difference separation method.   The method according to claim 1, wherein the sample is a blood sample, the target cells are tumor cells, and the step (1) is performed by a specific gravity difference separation method to which a leukocyte binding agent is added.   The method according to any one of claims 1 to 3, wherein the step (3) includes a step of detecting the target cell by fixing treatment and / or membrane permeation treatment.   The method according to any one of claims 1 to 4, wherein the step (4) is a step of analyzing mRNA possessed by the target cell.

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