Primer probe combination for rapidly identifying leukemia cells and normal hematopoietic cells, DNA methylation detection method and kit thereof

Abstract

The invention discloses a primer probe combination for rapidly identifying leukemia cells and normal hematopoietic cells, a DNA methylation detection method and a kit thereof. The primer and probe combination disclosed by the invention comprises a forward primer beta-actin-F, a reverse primer beta-actin-R, a probe beta-actin-Probe, a forward primer 182-MSP-F, a reverse primer 182-MSP-R, and a probe 182-MSP-Probe. The method comprises the following steps: extracting bone marrow mononuclear cell DNA (Deoxyribose Nucleic Acid); transforming and purifying the DNA by a hydrosulfite method; the primer probe combination is adopted to detect the methylation level of the miR-182 promoter and the methylation level of the reference gene beta-actin respectively through a methylation specific fluorescent quantitative PCR method, and respective Ct values are obtained; and calculating a difference value as a distinguishing standard. The kit provided by the invention comprises the primer probe combination. According to the invention, early screening and early diagnosis of diseases can be realized, the method is simple, convenient and feasible, the specificity is high, batch detection can be realized, the result interpretation is objective and accurate, a methylation detection kit is easy to prepare, and the application prospect is good.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a primer-probe combination for rapidly distinguishing leukemia cells from normal hematopoietic cells, a DNA methylation detection method and a kit thereof. Background Art

[0002] Acute Myeloid Leukemia (AML) is a highly malignant, heterogeneous malignant clonal disease of hematopoietic stem cells. Its main cause is that hematopoietic stem / progenitor cells (HSPC) mutate under the stimulation of various factors, lose the ability to further differentiate and mature, and cause hematopoiesis to be blocked at different stages. At the same time, the malignant hematopoietic stem cells acquire the ability to clone and proliferate indefinitely, and proliferate in large quantities, consuming various nutrients and inhibiting the production of normal hematopoietic cells, ultimately leading to the occurrence of various leukemias. The main clinical manifestations are infection, bleeding, anemia and infiltration of extramedullary tissues and organs. The disease progresses rapidly, and the natural course is only a few weeks to a few months.

[0003] Since hematopoietic stem cells of different lineages have the ability to transform into leukemia cells, the morphology of leukemia cells varies and the heterogeneity is high, making it difficult to distinguish leukemia cells from normal hematopoietic cells using morphological methods such as Wright staining. At the same time, leukemia cells of different lineages express different leukemia differentiation antigens, making it difficult for flow cytometry to accurately distinguish leukemia cells from normal hematopoietic cells (Canali A, Vergnolle I, Bertoli S, et al. Prognostic Impact of Unsupervised Early Assessment of Bulk and Leukemic Stem Cell Measurable Residual Disease in Acute Myeloid Leukemia. Clin Cancer Res. 2023; 29 (1): 134-142.). In order to quickly and effectively identify and distinguish leukemia cells from normal hematopoietic cells, the present methylation detection method is specially invented.

[0004] DNA methylation is an important epigenetic modification. DNA methylation refers to the transfer of the methyl group (CH3) on S-adenosylmethionine to the 5th carbon atom of cytosine in the double-stranded CpG dinucleotide of DNA under the action of DNA methyltransferases (DNMTs), forming 5'-methylcytosine. The vast majority of DNA methylation modifications occur in CpG islands, and 60-90% of CpG islands in the human genome are methylated. Because CpG islands are usually located in the promoter region of genes, they play an important role in regulating gene expression. Abnormal DNA methylation is a powerful epigenetic marker of leukemia and has a strong predictive ability in the prognosis and treatment decision-making of leukemia (Li ZG, Jiao Y, Li WJ, et al. Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia. Leuk Res. 2013; 37(10): 1287-1293.). Abnormal DNA methylation, including overall DNA hypomethylation and hypermethylation of specific tumor suppressor gene promoter sites, is one of the key indicators that distinguish leukemia cells from normal hematopoietic cells.

[0005] The methods for distinguishing leukemia cells from hematopoietic cells are as follows:

[0006] 1. Wright-Giemsa staining: Leukemia cells and normal hematopoietic cells are stained with Wright-Giemsa to distinguish malignant cells from normal cells morphologically.

[0007] 2. Flow cytometer analysis: Leukemia cells and normal hematopoietic cells are distinguished by detecting differentiation antigens on the cell membrane surface of leukemia cells and normal hematopoietic cells.

[0008] 3. Peroxidase (POX) staining: By intracellular staining of leukemia cells and hematopoietic cells, the intensity of staining can be detected to distinguish malignant leukemia cells from normal hematopoietic cells.

[0009] Currently, there is no mature method to distinguish leukemia cells from normal hematopoietic cells. Leukemia cells and normal hematopoietic cells are stained with Wright-Giemsa and differentially diagnosed by morphological methods. Since both leukemia cells and hematopoietic cells show primitive cell morphology such as large nuclei and less cytoplasm, it is easy to confuse the diagnosis. The main disadvantage of this method is that it is highly subjective, and qualified bone marrow morphology technicians need long-term training.

[0010] Peroxidase (POX) staining is a cytochemical staining method mainly used for the classification of acute leukemia. Strong positive POX staining indicates acute myeloid leukemia, but acute monocytic leukemia can also be negative or weakly positive, and POX staining of normal hematopoietic stem cells can also be negative or weakly positive. Therefore, peroxidase staining cannot distinguish leukemia stem cells from hematopoietic stem cells very well.

[0011] Current studies have shown that there is no unified leukocyte surface differentiation antigen that can accurately and quickly distinguish leukemia cells from normal hematopoietic cells. Therefore, traditional flow cytometry detection methods are not easy to distinguish leukemia cells from normal hematopoietic cells.

[0012] Some studies have reported that there are differentially expressed DNA methylation modifications in leukemia and normal hematopoietic cells. For example, SPRED1 (Sun J, Zhang J, Wang Y, et al. A Pilot Study of Aberrant CpG Island Hypermethylation of SPRED1 in Acute Myeloloid Leukemia. Int J Med Sci. 2019; 16 (2): 324-330.), RASSF6 (Younesian S, Shahkarami S, Ghaffari P, et al. DNA hypermethylation of tumor suppressor genes RASSF6 and RASSF10 as independent prognostic factors in adult acute lymphoblastic leukemia. Leuk Res. 2017; 61: 33-38.), DLX4 (Zhou JD, Zhang TJ, Wang YX, et al. DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia. Tumour Biol. 2016; 37(7): 8951-8960.) The DNA methylation modification level of gene promoters in AML cells is significantly higher than that in normal hematopoietic cells. However, the above papers did not detect the DNA methylation level in leukemia cells and normal hematopoietic cells by methylation-specific PCR method, and could not make good use of the differential DNA methylation level to distinguish leukemia cells from hematopoietic cells. Therefore, in order to effectively distinguish leukemia cells from normal hematopoietic cells, this patent preliminarily uses bioinformatics to analyze the existing DNA methylation data and finds that there are 4 CpG islands in the promoter region of chromosome 7 microRNA (miR-182). Preliminary analysis of big data shows that the CpG island of the miR-182 promoter may have a higher methylation modification. Summary of the invention

[0013] The present invention aims to solve the problem that it is not easy to distinguish leukemia cells from normal hematopoietic cells in the prior art, and to propose a primer-probe combination, a DNA methylation detection method and a kit for rapidly distinguishing leukemia cells from normal hematopoietic cells.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] As a first aspect, the present invention provides a primer-probe combination for rapidly distinguishing leukemia cells from normal hematopoietic cells, including a methylation-specific primer-probe combination of an internal reference gene β-actin and a methylation-specific primer-probe combination of a miR-182 promoter;

[0016] The methylation-specific primer-probe combination of the internal reference gene β-actin includes: a forward primer β-actin-F, whose sequence is shown in SEQ ID NO.1, a reverse primer β-actin-R, whose sequence is shown in SEQ ID NO.2, and a probe β-actin-Probe, whose sequence is shown in SEQ ID NO.3;

[0017] The miR-182 promoter methylation-specific primer-probe combination includes: a forward primer 182-MSP-F, whose sequence is shown in SEQ ID NO.4, a reverse primer 182-MSP-R, whose sequence is shown in SEQ ID NO.5, and a probe 182-MSP-Probe, whose sequence is shown in SEQ ID NO.6;

[0018] Wherein, the sequence is 5'-3'.

[0019] Furthermore, the 5' ends of the probe β-actin-Probe and the probe 182-MSP-Probe are both connected to a fluorescent reporter group, and the 3' ends are both connected to a fluorescent quencher group.

[0020] As a second aspect, the present invention provides a methylation detection method for rapidly distinguishing leukemia cells from normal hematopoietic cells, comprising the following steps:

[0021] Extract DNA from bone marrow mononuclear cells;

[0022] DNA was converted and purified by the bisulfite method;

[0023] The methylation-specific primer-probe combination of the miR-182 promoter and the methylation-specific primer-probe combination of the internal reference gene β-actin are used to perform PCR amplification on the DNA treated by the bisulfite method, and the methylation levels of the miR-182 promoter and the internal reference gene β-actin are respectively detected by methylation-specific fluorescent quantitative PCR to obtain the Ct value of the miR-182 promoter and the Ct value of the internal reference gene β-actin;

[0024] ΔCt=miR-182 promoter Ct value-reference gene β-actin Ct value was calculated as the standard for distinguishing leukemia cells from normal hematopoietic cells.

[0025] Furthermore, the reaction system of the PCR amplification is: 10.0 μl 2×BioSmart MethyLight qPCRMix, 0.4 μl, 10 μg / μl forward primer, 0.4 μl, 10 μg / μl reverse primer, 0.2 μl, 10 μg / μl probe, 2 μl, 0.5 μg / μl DNA treated with bisulfite, and ultrapure water H2O is added to 20.0 μl; the reaction program of PCR amplification is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 50 cycles.

[0026] Furthermore, ΔCt+10 was calculated, ΔCt+10<15.82 was positive, and ΔCt+10≥15.82 was negative.

[0027] As a third aspect, the present invention also provides a kit for rapidly distinguishing leukemia cells from normal hematopoietic cells, comprising the primer-probe combination.

[0028] Preferably, the kit further comprises BioSmart MethyLight qPCR Mix reagent, a positive control and a negative control.

[0029] The present invention has the following beneficial effects:

[0030] (1) DNA samples are easy to obtain, and the chemical structure of DNA is stable. The storage and processing of samples do not require high conditions, making it easier to carry out clinical testing. DNA methylation is a stable biological indicator that will not change in the short term due to drug treatment or other changes in the patient's physical condition. This feature of DNA methylation makes the results of this method more stable and reliable.

[0031] (2) DNA methylation changes often occur in the early stages of tumors, enabling early screening and diagnosis of tumors.

[0032] (3) The method of the present invention is simple and easy to operate, has high specificity, can be used for batch detection, and the result interpretation is objective and accurate. It can be easily made into a methylation detection kit and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : is the methylation distribution map of the miR-182 promoter region, wherein (A) is the schematic diagram of the methylation distribution of the miR-182 promoter region; (B) is the methylation base distribution map of the miR-182 promoter region;

[0034] Figure 2The MethylTargetTM method was used to detect the methylation ratio of the miR-182 promoter region, wherein (A) the methylation levels of CpG1, CpG2 and CpG3 in leukemia cells were detected; (B) the methylation levels of CpG1, CpG2 and CpG3 in normal hematopoietic cells were detected; (C) the methylation levels of CpG3 in leukemia cells and normal hematopoietic cells were analyzed;

[0035] Figure 3 is a flow chart of the method of the present invention;

[0036] Figure 4 The figures are the test results of 36 normal hematopoietic cells and 110 acute myeloid leukemia cases, where (A) is the ROC curve, with ΔCT+10 as the Q-MSP result value, and (B) is a statistical graph drawn based on the test results. DETAILED DESCRIPTION

[0037] The present invention previously analyzed the methylation distribution of the miR-182 promoter region by bioinformatics and found that there were 4 CpG islands ( Figure 1 A and B). by MethylTarget TM Methods The methylation of miR-182 promoter was analyzed in 32 leukemia cells and 26 normal control cells. It was found that the methylation of malignant leukemia cells mainly existed in CpG3 and CpG1, but not CpG2. The methylation level of CpG3 was higher than that of CpG1. Figure 2 A). However, in normal hematopoietic cells, the promoter CpG1, CpG2, and CpG3 of miR-182 are all in a low methylation state ( Figure 2 B). At the same time, the methylation level of CpG3 in normal hematopoietic cells is significantly lower than that in malignant leukemia cells ( Figure 2 C). Therefore, the present invention finally selects the methylation site on CpG3 as the target for PCR amplification.

[0038] Because MethylTarget TMIt is a high-throughput and high-cost detection method, and this method is not suitable for implementation in hospital laboratories. Therefore, the present invention uses efficient, rapid and low-cost real-time fluorescence quantitative methylation-specific PCR (real-time quantitative methylation-specific PCR, RQ-MSP) to detect miR-182 promoter methylation. The present invention separates 110 bone marrow mononuclear cells from acute myeloid leukemia, and simultaneously separates 36 bone marrow mononuclear cells from healthy donors as normal hematopoietic cells. Total cell DNA is extracted, and the DNA treated with bisulfite is used as a template to simultaneously detect the methylation levels of the internal reference gene β-actin and the miR-182 promoter by the RQ-MSP method. The results show that all 36 normal hematopoietic cells show hypomethylation of the miR-182 promoter, while the vast majority of malignant leukemia cells show hypermethylation of the miR-182 promoter.

[0039] The present invention has two key links, namely bisulfite conversion and qPCR after bisulfite conversion.

[0040] 1. Use Kit II to perform bisulfite conversion on the extracted genomic DNA. Unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged.

[0041] 2. Perform PCR amplification (qMSP) on bisulfite-converted DNA (Bis-DNA). The reagent contains miR-182 promoter methylation-specific primers and internal reference gene (β-actin) primers. Methylation-specific primers can specifically bind to the methylated miR-182 promoter sequence and can specifically detect the methylated sequence in the qMSP reaction. At the same time, the internal reference gene (β-actin) can monitor the extraction of each sample and the amplification of the PCR reaction tube, thereby avoiding the occurrence of false negative results. In addition, the kit also provides positive and negative controls, which need to be added at the same time in each test.

[0042] Among them, the present invention designs potential primers and probes through primer design software, and performs PCR experiments one by one, and finally finds a pair of primer and probe sequences with high specificity and sensitivity (Table 1).

[0043] Table 1: MiR-182 promoter methylation-specific primers and reference gene (β-actin) primer sequences

[0044]

[0045] The experimental results of the present invention are shown as (reference gene Ct value-miR-182 promoter Ct value) + 10. Figure 4As shown, (A) shows that the cutoff value corresponding to the maximum Youden index is 15.82, indicating that the optimal cutoff value of the detection method is 15.82. The detection specificity at this cutoff value is 0.97, indicating that the specificity of this method for effectively distinguishing leukemia cells from normal hematopoietic cells is 0.97, the area under the curve (AUC) is 0.97, and the sensitivity is 0.87. The experimental results have significant statistical differences (P<0.0001); (B) is to draw the test results into a statistical graph. According to the cutoff value suggested by the ROC curve, the true positive rate of the detection method is calculated to be 87.27%, the false positive rate is 2.87%, and the false negative rate is 12.73%.

[0046] In order to make the implementation of the method of the present invention more obvious and easy to understand, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the flow chart in the present invention. Obviously, the embodiment described below is only a part of the embodiment of the present invention, not all of the embodiments.

[0047] Example 1

[0048] The reagents and consumables that need to be prepared are as follows:

[0049] Kit I: Blood / Cell / Tissue Genomic DNA Extraction Kit (Spin Column Type)

[0050] Kit II: Bisulfite Conversion Kit (Spin Column Type)

[0051] Kit III: BioSmart MethyLight qPCR Mix

[0052] Others: anhydrous ethanol, ddH2O, 15ml centrifuge tube, 1.5ml EP tube, 200μl EP tube, etc.

[0053] The experimental process and detailed steps are as follows Figure 3 As shown:

[0054] 1. Isolation of mononuclear cells from blood samples

[0055] 1.1 Take all the anticoagulated bone marrow specimens, dilute them with an equal amount of PBS buffer, and mix them by gently pipetting.

[0056] 1.2 Take a 15ml centrifuge tube, add lymphocyte separation fluid, and then slowly add an equal amount of bone marrow cells diluted with PBS buffer to the top of the lymphocyte separation fluid along the wall of the tube. At this time, the upper layer of the centrifuge tube is the bone marrow specimen, and the lower layer is the lymphocyte separation fluid.

[0057] 1.3 Adjust the centrifuge's lifting acceleration to the lowest level and centrifuge at 2000rpm for 20min (to separate mononuclear cells based on the different sedimentation coefficients of blood cells).

[0058] 1.4 Aspirate and discard the top layer of plasma, then transfer the mononuclear cell layer to a new 15 ml centrifuge tube, add PBS buffer to 10 ml, pipette and mix, and centrifuge at 1000 rpm for 5 min.

[0059] 1.5 Discard the supernatant and the remaining precipitate is the mononuclear cells.

[0060] 2 Extraction of DNA from bone marrow mononuclear cells

[0061] Use the TIANamp Genomic DNA Kit Blood / Cell / Tissue Genomic DNA Extraction Kit (centrifugal column type) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. (Medical Device Registration Certificate Number: Version Number: DP201831). The operation process is shown in the kit manual. The operation steps are summarized as follows:

[0062] 2.1 Take 10 μl of negative control, positive control and CD34 + To the cells, add 200 μl buffer GA and 20 μl proteinase K respectively, mix thoroughly, then add 200 μl buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0063] 2.2 Add 200 μl of anhydrous ethanol and shake thoroughly for 15 seconds.

[0064] 2.3 Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0065] 2.4 Add 500 μl of buffer GD to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0066] 2.5 Add 600 μl of rinse solution PW to the adsorption column CB3 (check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube. Repeat this step once.

[0067] 2.6 Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0068] 2.7 Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of elution buffer TE to the middle part of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 2 minutes, and collect the solution (i.e., genomic DNA) into the centrifuge tube.

[0069] 3. Bisulfite conversion

[0070] EpiArt DNA Methylation Bisulfite Kit (EM101) produced by Nanjing Novozyme Medical Technology Co., Ltd. (Medical Device Registration Certificate Number: Version Number: DP201831) was used. The operation process is shown in the kit manual. The operation steps are summarized as follows:

[0071] 3.1 Reagent preparation

[0072] 3.1.1 Before using E-Wash Buffer for the first time, a specified volume of anhydrous ethanol must be added.

[0073] 3.1.2 Prepare CT Conversion Mix: Add 1ml ddH2O, 200μl CT Conversion Diluent, and 100μl CT conversion Buffer to a tube of CT Conversion Powder, and vortex at room temperature for about 1 minute until dissolved. Each tube of mixed Mix can be used for about 10 reactions.

[0074] 3.2 Bisulfite conversion

[0075] 3.2.1 Equilibrate the CT Conversion Mix to room temperature and prepare the following reaction in a 200 μl sterile PCR tube (Table 2):

[0076] Table 2: Bisulfite Conversion Reagents

[0077]

[0078] 3.2.2 Mix by inverting or pipetting, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0079] 3.2.3 Place the PCR tube in a PCR instrument and perform the following reaction (Table 3):

[0080] Table 3: Bisulfite Conversion Reaction Temperatures

[0081]

[0082]

[0083] 3.3 Purification of transformation products

[0084] 3.3.1 Place the EpiArt DNA Columns into the Collection Tubes.

[0085] 3.3.2 Add 600 μl E-Binding Buffer to the adsorption column, and then add the converted reaction product to the adsorption column. Gently invert up and down 8-10 times to mix the reaction solution and E-Binding Buffer completely.

[0086] 3.3.3 Centrifuge at 12,000 rpm (13,400 × g) for 30-60 seconds. Discard the filtrate and return the adsorption column to the collection tube.

[0087] 3.3.4 Add 500 μl E-Wash Buffer (with ethanol) to the adsorption column and centrifuge at 12,000 rpm (13,400 × g) for 30–60 seconds. Discard the filtrate and put the adsorption column back into the collection tube.

[0088] 3.3.5 Add 500 μl E-Desulphonation Buffer to the adsorption column and let it react for 15 min at room temperature (15-25°C). Centrifuge at 12,000 rpm (13,400 × g) for 30-60 sec. Discard the filtrate and put the adsorption column back into the collection tube.

[0089] 3.3.6 Add 500 μl E-Wash Buffer (with ethanol) to the adsorption column and centrifuge at 12,000 rpm (13,400 × g) for 60 seconds. Discard the filtrate and put the adsorption column back into the collection tube. Repeat this step.

[0090] 3.3.7 Centrifuge the empty column at 12,000×rpm for 2 min.

[0091] 3.3.8 Transfer the adsorption column to a new 1.5 ml centrifuge tube, open the cover for 2 minutes to dry thoroughly, add 10-20 μl E-Elution Buffer to the center of the adsorption column membrane. Let stand at room temperature for 1-2 minutes, centrifuge at 12,000 rpm (13,400 × g) for 2 minutes, and collect the DNA filtrate.

[0092] 3.3.9 Discard the adsorption column and store the DNA at -20℃. For long-term storage, place it at -70℃.

[0093] 4qMSP test samples

[0094] 4.1 Design of MSP primers.

[0095] 4.2 Configuring the Amplification System

[0096] Table 4: qMSP reaction reagent ratio

[0097]

[0098] Wherein, the concentration of the forward primer, the reverse primer and the probe is 10 μg / μl, and the concentration of the DNA after bisulfite treatment is 0.5 μg / μl.

[0099] 4.3 Amplification Procedure

[0100] Table 5: qMSP reaction temperature

[0101]

[0102] 5. Interpretation of test results

[0103] Table 6: qMSP result interpretation

[0104]

[0105] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A primer-probe combination for rapid identification of leukemia cells and normal hematopoietic cells, characterized in that: It includes a methylation-specific primer probe combination of an internal reference gene β-actin and a methylation-specific primer probe combination of a miR-182 promoter; The methylation-specific primer-probe combination of the internal reference gene β-actin includes: a forward primer β-actin-F, whose sequence is shown in SEQ ID NO.1, a reverse primer β-actin-R, whose sequence is shown in SEQ ID NO.2, and a probe β-actin-Probe, whose sequence is shown in SEQ ID NO.3; The miR-182 promoter methylation-specific primer-probe combination includes: a forward primer 182-MSP-F, whose sequence is shown in SEQ ID NO.4, a reverse primer 182-MSP-R, whose sequence is shown in SEQ ID NO.5, and a probe 182-MSP-Probe, whose sequence is shown in SEQ ID NO.6; Wherein, the sequence is 5'-3'.

2. The primer-probe combination according to claim 1, characterized in that: The 5' ends of the probe β-actin-Probe and the probe 182-MSP-Probe are both connected to a fluorescent reporter group, and the 3' ends are both connected to a fluorescent quencher group.

3. A methylation detection method for rapidly distinguishing leukemia cells from normal hematopoietic cells, characterized in that: The following steps are involved: Extract DNA from bone marrow mononuclear cells; DNA was converted and purified by the bisulfite method; The methylation-specific primer-probe combination of the miR-182 promoter and the methylation-specific primer-probe combination of the internal reference gene β-actin according to claim 1 or 2 are used to perform PCR amplification on the DNA treated with the bisulfite method, and the methylation levels of the miR-182 promoter and the internal reference gene β-actin are respectively detected by methylation-specific fluorescent quantitative PCR to obtain the Ct value of the miR-182 promoter and the Ct value of the internal reference gene β-actin; ΔCt=miR-182 promoter Ct value-reference gene β-actin Ct value was calculated as the standard for distinguishing leukemia cells from normal hematopoietic cells.

4. The methylation detection method according to claim 3, characterized in that The reaction system of the PCR amplification is: 10.0 μl 2×BioSmart MethyLight qPCR Mix, 0.4 μl, 10 μg / μl forward primer, 0.4 μl, 10 μg / μl reverse primer, 0.2 μl, 10 μg / μl probe, 2 μl, 0.5 μg / μl DNA treated by bisulfite method, and ultrapure water H2O is added to 20.0 μl; the reaction program of PCR amplification is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 50 cycles.

5. The methylation detection method according to claim 3, characterized in that: The ΔCt+10 was calculated, ΔCt+10<15.82 was positive, and ΔCt+10≥15.82 was negative.

6. A kit for rapidly distinguishing leukemia cells from normal hematopoietic cells, characterized in that: Comprising the primer-probe combination of claim 1 or 2.

7. The kit according to claim 6, characterized in that Also included are BioSmart MethyLight qPCR Mix reagents, positive controls, and negative controls.

Images