Single cell methylation detection method based on enriched cast-off cells and application

Abstract

The invention relates to the technical field of biology, in particular to a single cell methylation detection method based on enriched cast-off cells and application. The invention provides a single cell methylation detection method based on enriched cast-off cells. The single cell methylation detection method comprises the following steps: S1, separating, purifying and enriching cast-off cells to be analyzed; s2, carrying out DNA extraction on cast-off cells to be analyzed; s3, carrying out hydrosulfite conversion on the extracted DN A; s4, carrying out DNA multiple replacement amplification on the transformed DNA sample; and S5, carrying out methylation analysis on the amplified DNA. According to the method, DNA products are increased through a genome amplification technology, the DNA products can be increased to the level suitable for a conventional detection technology, and meanwhile non-specific loss in the capturing process is effectively avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a single cell methylation detection method based on exfoliated cell enrichment and application. Background Art

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, and its incidence ranks second among all urinary system tumors. The incidence and mortality of bladder cancer are on the rise, especially in elderly men. The high incidence and high recurrence rate of bladder cancer have a serious impact on the quality of life and survival rate of patients, and also bring a huge economic burden to the medical system.

[0003] Precision medicine is a medical model based on individual genomic information, environmental factors and lifestyle, which aims to achieve personalized disease prevention, diagnosis and treatment. With the rapid development of genomics, molecular biology and information technology, precision medicine has received widespread attention and rapid advancement around the world. The development of precision medicine is seen as an important way to improve the quality and efficiency of medical services, especially in the early diagnosis and personalized treatment of cancer.

[0004] At present, the diagnosis of bladder cancer mainly relies on cystoscopy, urine cytology and imaging examinations. However, these methods have some obvious limitations. Cystoscopy is invasive and requires the insertion of a cystoscope, which brings greater discomfort and infection risks to patients. Conventional urine cytology has low sensitivity, especially for early and low-grade tumors, which are prone to false negative interpretations and lead to missed diagnoses. Imaging examinations such as CT and MRI are expensive and are not suitable for large-scale screening and frequent monitoring. At the same time, the recurrence rate of bladder cancer is high, and frequent follow-up and examinations are required, which further increases the burden on patients.

[0005] Urine, as an easily accessible and non-invasive body fluid sample, can reflect a variety of physiological and pathological conditions of the whole body. In addition to metabolic waste in urine, there are also a certain number of exfoliated cells in urine. Exfoliated cells in urine can be used for the detection of a variety of projects. At present, early tumor screening can be achieved by detecting the DNA methylation status in urine exfoliated cells and identifying cancer-related epigenetic markers. It is also possible to assist in the diagnosis and prognosis of bladder cancer by analyzing mutations such as gene SNPs in exfoliated cells, such as TERT promoter mutations and FGFR3 mutations. Some technologies will also understand the molecular characteristics of tumors and guide personalized treatment by detecting the expression levels of mRNA and miRNA in urine exfoliated cells. Of course, protein marker detection will also be applied to urine exfoliated cells, such as detecting some specific proteins, such as NMP22 and BTA, as auxiliary diagnostic markers for bladder cancer.

[0006] Currently, the most advanced direct detection technology for bladder cancer is to directly extract DNA from urine exfoliated cells based on centrifugation for qPCR or NGS detection of DNA methylation. There are currently many problems with this test. First, the exfoliated cells in urine come from a wide range of sources, including normal cells and cancer cells, which causes the methylation signal to be diluted by normal cells, resulting in low detection sensitivity and large background noise. Second, the complexity of urine samples in actual clinical practice makes it difficult to separate and capture exfoliated cells, especially the presence of a large number of protein decomposition products and other cell fragments in the urine, which further increases the difficulty and complexity of detection. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a single cell methylation detection method and application based on exfoliated cell enrichment, which integrates advanced single cell separation and capture technologies, such as microfluidic chip technology and magnetic microbead sorting technology, to achieve efficient capture of cancer cells from urine samples. In addition, this method increases DNA products through genome amplification technology, which can increase DNA products to a level suitable for conventional detection technology, while effectively avoiding nonspecific losses during the capture process.

[0008] To this end, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides, in an optional embodiment, a single cell methylation detection method based on exfoliated cell enrichment, comprising the following steps:

[0010] S1: Isolate, purify and enrich the exfoliated cells to be analyzed;

[0011] S2: DNA extraction of the exfoliated cells to be analyzed;

[0012] S3: Bisulfite conversion of the extracted DNA;

[0013] S4: Perform DNA multiple displacement amplification on the converted DNA samples;

[0014] S5: Perform methylation analysis on the amplified DNA.

[0015] Preferably, in step S4, the method for DNA multiple displacement amplification comprises the following steps:

[0016] S41: Evenly mixing the bisulfite-converted DNA sample, 10× Reaction Buffer for Equ iPhi29 and 500 μM anti-exocrine random primers, centrifuging, preparing a denaturation system, centrifuging and denaturing the denaturation system, and obtaining a denatured product;

[0017] S42: the denatured product, 100 nM dithiothreitol, 2.5 nM dNTP premix solution, 10 U / μL EquiPhi29 DNA Polymerase and nuclease-free pure water are mixed and centrifuged to prepare an amplification system, and the amplification system is centrifuged and amplified to obtain an amplification product;

[0018] S43: mixing the amplified product, 10×Mung Bean Nuclease Buffer and mung bean nuclease and centrifuging to prepare an enzyme digestion system, centrifuging and digesting the enzyme digestion system, and then adding a terminator to obtain an enzyme digestion product;

[0019] S44: Purify and recover the enzyme cleavage product to obtain amplified DNA.

[0020] Preferably, in step S41, the volume of the DNA sample in the denaturation system is 7 μL, the volume of the 10×Reaction Buffer for EquiPhi29 is 1 μL, the volume of the 500 μM anti-exocrine random primer is 2 μL, and / or the 10×Reaction Buffer for EquiPhi29 includes 330-400 nM Tris-acetate, 100-150 nM magnesium acetate, 660-750 nM potassium acetate, 1-3% by mass of Tween 20, and 10-25 nM dithiothreitol.

[0021] Preferably, in step S42, the volume of the denatured product in the amplification system is 10 μL, the volume of 100 nM dithiothreitol is 0.5 μL, the volume of 2.5 nM dNTP premix solution is 20 μL, the volume of 10 U / μL EquiPhi29 DNA Polymerase is 2 μL, and the volume of nuclease-free pure water is 17.5 μL.

[0022] Preferably, the 10×Mung Bean Nuclease Buffer comprises 500-600 nM potassium acetate, 250-350 nM potassium chloride and 10-15 nM zinc sulfate.

[0023] Preferably, in step S41, the denaturation method is: denaturation at 95°C for 3 minutes; and / or, in step S42, the amplification method is: amplification at 30°C for 16 hours, and then maintaining at 65°C for 10 minutes to inactivate the enzyme; and / or, in step S43, the enzyme cleavage method is: incubation at 30°C for 30 minutes, and the terminator is 0.6 μL of a 1% sodium dodecyl sulfate solution.

[0024] Preferably, in step S3, the extracted DNA is subjected to bisulfite conversion, which converts unmethylated cytosine in the DNA sample into uracil, while methylated cytosine remains unchanged. In step S5, the methylation analysis method is real-time fluorescence PCR or high-throughput sequencing technology. In step S1, separation, purification and enrichment of exfoliated cells to be analyzed are performed using a fully automatic urine cell efficient enrichment system.

[0025] Specifically, in step S1, a fully automatic urine cell efficient enrichment system is used to achieve efficient separation and purification of exfoliated cells in urine:

[0026] (1) Ensure that all pipeline connection ports are properly connected and that a microporous filter membrane with a pore size of 8-10 μm is used in the module.

[0027] (2) Place 50 ml of urine sample into the sample module, set the flow rate of the liquid storage peristaltic pump to 2000 μL / min, the flow rate of the waste extraction peristaltic pump to 1000 μL / min, and start both peristaltic pumps at the same time.

[0028] (3) The enriched urine sample quickly fills the upper cover plate cross-flow layer chamber and the lower cover plate shear flow layer chamber of the urine cell separation module through the pipeline, and the system operates normally.

[0029] (4) After the enrichment is completed, first turn off the liquid storage peristaltic pump, and after observing that the residual liquid is completely discharged from the waste liquid port, turn off the waste extraction peristaltic pump.

[0030] (5) Take out the microporous filter membrane of the urine cell separation module, add 200 μl PBS and wash it twice to obtain a sample enriched with exfoliated cells.

[0031] In step S2, DNA nucleic acid extraction was performed using QIAamp DNABlood Mini Kit (purchased from QIAGEN, catalog number: 51104). The steps are as follows:

[0032] (1) Preparation: Add the corresponding buffer according to the instructions on the bottles of Buffer AW1 and Buffer AW2, and keep records;

[0033] (2) Add 200 μL Buffer AL and 20 μL Protease to a 1.5 mL centrifuge tube;

[0034] (3) Pipette 200 μL of the cell-enriched sample into a 1.5 mL centrifuge tube and mark it;

[0035] (4) Vortex to mix and centrifuge briefly;

[0036] (5) Place the sample centrifuge tube in a constant temperature metal bath and incubate at 56°C for 10 min;

[0037] (6) After the incubation, remove the sample tube and centrifuge briefly;

[0038] (7) Add 200 μl of pure alcohol (96%-100%) to the sample tube, vortex to mix, and centrifuge briefly;

[0039] (8) Add the liquid in the sample tube of step (7) into the corresponding labeled QIAamp Mini spin column, cap the tube tightly, centrifuge at 6000g for 1 min, discard the supernatant and collection tube, and replace with a new collection tube;

[0040] (9) Add 500 μL of Buffer AW1 to the QIAamp Mini spin column, close the lid, centrifuge at 6000 g for 1 min, discard the supernatant and collection tube, and replace with a new collection tube.

[0041] (10) Add 500 μL of Buffer AW2 to the QIAamp Mini spin column, close the lid, and centrifuge at 20,000 g for 1 min.

[0042] (11) Prepare a new 1.5 ml centrifuge tube (cut off the tube cap), place the QIAamp Mini spin column in it, discard the supernatant and the old tube, and centrifuge at 20,000 g for 3 min.

[0043] (12) Prepare a corresponding number of labeled 1.5 ml centrifuge tubes, place the centrifuged QIAamp Mini spincolumn into each tube, open the lid and let it sit at room temperature for 5 min to dry.

[0044] (13) Add 25 μL of buffer AE, cap the tube tightly, and incubate at room temperature for 5 min;

[0045] (14) After incubation, centrifuge at 11000g for 2 min.

[0046] (15) Repeat step (14) once.

[0047] (16) Remove the column, cover tightly, vortex to mix, and centrifuge briefly.

[0048] Take 1 μL of sample and use Thermo Fisher's Qubit TM The concentration of extracted DNA was detected by dsDNA HS Assay Kit.

[0049] In step S3, The Ultrafast Bisulfite Kit performs bisulfite (BS) conversion on sample DNA, converting unmethylated cytosine (C) in the DNA sample into uracil (U), while methylated cytosine (5-mC) remains unchanged, thereby distinguishing methylated and unmethylated cytosine sites and obtaining bisulfite-converted DNA. The specific steps are as follows:

[0050] (1) Add 22 ml of anhydrous ethanol to the Wash Buffer used for the first time, mix well, and cover the bottle tightly to prevent ethanol evaporation.

[0051] (2) Prepare the following reaction system in a PCR tube: 20 μL input DNA, 180 μL CT Conversion Buffer,

[0052] Mix well by pipetting and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0053] (3) Take 100 μL of the liquid in each tube and transfer it to new PCR tubes, marking them one by one with the original tubes.

[0054] (4) After brief centrifugation to collect the reaction solution at the bottom of the tube, place the PCR tube in a PCR instrument and keep it at 95°C for 5 min;

[0055] (5) Centrifuge the sample for 10 seconds.

[0056] (6) Transfer the sample to the Spin IC Columns installed in the collection tubes (the liquid in the two reaction tubes formed by aliquoting one sample in the first step of transformation should be added to the same Spin IC Columns).

[0057] (7) Centrifuge at 13000g for 1 min and discard the filtrate.

[0058] (8) Add 100 μL Wash Buffer (anhydrous ethanol has been added in advance) to the Spin IC Columns.

[0059] (9) Centrifuge at 13000 g for 1 min and discard the filtrate.

[0060] (10) Add 200 μL of Desulphonation Buffer to the Spin IC Columns and incubate at room temperature for 20 min.

[0061] (11) Centrifuge at 13000 g for 1 min and discard the filtrate.

[0062] (12) Add 200 μL Wash Buffer (with anhydrous ethanol added in advance) to the Spin IC Columns and centrifuge at 13,000 g for 1 min.

[0063] (13) Prepare a corresponding number of 1.5 mL centrifuge tubes and label them. Place the Spin IC Columns after centrifugation into them. Discard the supernatant and collection tubes.

[0064] (14) Open the lid and leave the column at room temperature for 5 minutes to dry.

[0065] (15) Add 13 μL of Elution Buffer to the center of the Spin IC Columns, cap the tube tightly, and incubate at room temperature for 3 min.

[0066] (16) Centrifuge at 13000g for 1 min.

[0067] (17) Check whether there is residual liquid in the columns. If so, centrifuge again at 13,000 g for 1 min.

[0068] Take out the column, cover the 1.5mL centrifuge tube containing the sample DNA, mark it, and proceed to the next step of reaction.

[0069] In a second aspect, the present invention provides, in an optional embodiment, an application of the above-mentioned single-cell methylation detection method based on exfoliated cell enrichment in the detection of bladder cancer.

[0070] Compared with the prior art, the present invention has one of the following beneficial effects:

[0071] 1. The present invention uses a fully automatic urine cell efficient enrichment system and laminar filtration. After filtration, not only a large number of non-target cells such as inflammatory cells can be removed, but also the enrichment efficiency of urothelial cancer cells can be greatly improved, thereby achieving the removal of impurities and non-target cells while obtaining efficient enrichment of urothelial cancer cells. The enriched single cells are assumed to be used for conventional detection methods such as qPCR. The yield of DNA extraction may be too low due to problems with filtration chromatography. Therefore, we use BS conversion to preserve methylation signals, and then increase the amount of DNA to meet subsequent detection needs through self-developed DNA multiple displacement amplification technology (MDA). Finally, the methylation state of the target DNA is detected by qPCR or NGS method to avoid nonspecific losses during capture. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a schematic diagram of the detection results of the total amount of DNA in Example 1 of the present invention;

[0073] Figure 2This is a graph showing the results of qPCR detection of β-actin gene in Example 1 of the present invention;

[0074] Figure 3 This is a diagram showing the detection results of the total amount of DNA before and after multiple displacement amplification after the DNA is converted by BS in Example 1 of the present invention;

[0075] Figure 4 This is a schematic diagram of the data results of subtracting the ΔCt value of the sample processed by the cell enrichment method provided by the present invention from the ΔCt value of the sample processed by the traditional centrifugation method in Example 1 of the present invention. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0078] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0079] Traditional urine-based DNA methylation tumor detection technology faces the following major problems:

[0080] (1) Methylation signal dilution and background noise issues: Urine is used as a detection target for urinary system tumors. It is different from conventional blood ctDNA methylation detection in that its biggest advantage is that the signal comes directly from exfoliated cells. However, the genomic information contained in normal epithelial cells and white blood cells in urine will cause the methylation characteristics of tumor cells to be diluted during DNA methylation detection, resulting in low detection sensitivity and large background noise, which limits the accuracy and reliability of the results.

[0081] (2) The current DNA methylation detection kits for urothelial carcinoma or bladder cancer are often subject to significant limitations in clinical use. Samples with hematuria, late-stage tumors, inflammation, or medication that results in a high level of protein in the urine cannot be tested. These samples are precisely the ones that are at the highest risk and need to be tested. The reason is that the existing methylation detection methods use urine sediment to extract DNA. The protein decomposition products and other cell fragments in the urine interfere with the capture and separation of exfoliated cells, resulting in a reduction in DNA products in subsequent molecular experiments or even the inability to effectively extract DNA, affecting the subsequent methylation analysis process.

[0082] In order to solve the above technical problems, the present invention proposes a single-cell methylation detection method for exfoliated cells. The method utilizes advanced single-cell separation and capture technologies, such as microfluidics and magnetic bead sorting technology, to efficiently capture cancer cells from urine samples, increase DNA products to a point that can be used by conventional detection technologies through genome amplification, and avoid nonspecific losses during the capture process.

[0083] The technical solution of the present invention will be described below in conjunction with specific implementation methods.

[0084] Example 1

[0085] A total of 342 urine samples were selected, including 155 positive samples and 187 negative samples. The specific information of sample status is shown in Table 1.

[0086] Table 1 Sample status details

[0087] Sample Type Quantity (example) Bladder Cancer Stage Ta 33 Bladder Cancer T1 / T2 54 Bladder Cancer T3 / T4 68 Negative 187 total 342

[0088] The samples were treated with the exfoliated cell enrichment method of the present invention and the traditional centrifugation method, and then DNA was extracted, and then all were subjected to BS conversion (bisulfite conversion). The concentration of the BS conversion (bisulfite conversion) product treated with the exfoliated cell enrichment method of the present invention was determined, and then the DNA multiple displacement amplification technology (MDA) independently developed by the laboratory was used for constant temperature amplification to increase the amount of detectable DNA. The concentration of the amplified product was measured again, and the total amount of DNA before MDA amplification after BS conversion (bisulfite conversion) was compared with the total amount of DNA after DNA amplification. Finally, the amplified DNA and the DNA extracted and BS converted (bisulfite converted) after the traditional centrifugation method were analyzed using a qPCR methylation detection kit for bladder cancer, and the ΔCt values ​​of the target gene and the internal standard gene, as well as the final test results of the sample, were compared to evaluate the effects of the two different sample processing methods on the extraction efficiency of exfoliated cell DNA in urine and its test results.

[0089] S1: 342 urine samples were divided into two tubes of 50 ml each, and then evenly divided into two groups, each containing 342 samples, each 50 ml. The first group of samples was processed using the fully automatic urine cell efficient enrichment system developed by the present invention, and the steps were as follows:

[0090] (1) Ensure that all pipeline connection ports are properly connected and that a microporous filter membrane with a pore size of 8-10 μm is used in the module.

[0091] (2) Place the urine sample in the sample module, set the flow rates of the liquid storage peristaltic pump and the waste extraction peristaltic pump respectively, and start the two peristaltic pumps at the same time.

[0092] (3) The urine sample fills the upper and lower chambers of the separation module and operates normally.

[0093] (4) After the enrichment is completed, first turn off the liquid storage peristaltic pump. After the residual liquid is completely discharged, turn off the waste peristaltic pump.

[0094] (5) Remove the microporous filter membrane, add 200 μl PBS and wash it twice to obtain a sample enriched with exfoliated cells.

[0095] The second group was processed by traditional centrifugation. The steps of traditional centrifugation were as follows:

[0096] (1) Centrifuge a centrifuge tube containing 50 mL of urine sample at 2800-3000g for 10 min.

[0097] (2) Take out the centrifuge tube and pour it in the opposite direction of the mark, carefully remove the supernatant and retain the urine sediment cells. If the sediment is not visible, reserve 0.3 mL of liquid.

[0098] (3) Resuspend the sediment in 0.5 mL of 1x PBS on the marked side and pipette up and down five times.

[0099] (4) Transfer all the sediment and resuspension into a 2 mL EP tube, and place the liquid in the two centrifuge tubes with the same sample number into the same 2 mL EP tube (make sure the sample numbers are consistent).

[0100] (5) Close the cap of the 2 mL EP tube and centrifuge at 3000 g for 10 min in a small centrifuge.

[0101] Use a 1 mL pipette to carefully remove the supernatant liquid and leave the sediment. Be careful to keep the urine sediment intact. If the sediment is not visible, reserve 0.2 mL of liquid.

[0102] S2: After treatment, both groups of samples were extracted using the QIAamp DNA Blood Mini Kit (purchased from QIAGEN, Cat. No. 51104) according to the product instructions. The test results of the total amount of DNA obtained can be found in Figure 1 .

[0103] according to Figure 1 From the data analysis in, it can be observed that the total amount of DNA extracted from the samples obtained by the cell enrichment method used in the present invention is between 15ng and 25ng, with an average of about 20ng; in contrast, the total amount of DNA extracted from the samples processed by the traditional centrifugation method is between 120ng and 260ng, with no obvious aggregation range. Next, a qPCR verification experiment was performed to further compare the human DNA content in the two groups of DNA samples that can be used for methylation analysis.

[0104] The two sets of DNA extracted above were used as templates, and each sample was repeated 3 times. TM Taq DNA polymerase (purchased from Thermo Company, catalog number: 10966083) was used to design primer probes to detect β-actin, and the human DNA content in the extracted DNA was compared by comparing the Ct values ​​of the two sets of data.

[0105] Concentration of each component in the qPCR reaction system: Template 5ng, Platinum TM Taq DNA polymerase 2U (optional 1-3U), 1X PCR Buffer (Platinum TM Taq DNA polymerase supporting reagent), magnesium chloride 150nM (optional 100-200nM), dNTPs 200μM (optional 100-300μM), upstream primer 200μM (optional 150-400μM), downstream primer 200μM, probe 100μM (optional 50-200μM), 50nM ROX (optional 25-60μM), add enzyme-free nucleic acid water to the volume of 25μL. (β-actin gene primer probe is: upstream primer 5'-CCTGGCACCCAGCACAAT-3', downstream primer primer 5'-AAGGAAGGCTGGAAGAGTGC-3', probe 5'-VIC-CCGAGCGTGGCCATCCGCAAAG-TAMRA-3')

[0106] The reaction conditions were: 94°C for 2 min; 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 40 cycles; 72°C for 2 min, 4°C Hold.

[0107] The data were sorted and the test results are shown in Table 2.

[0108] Table 2 Results of qPCR detection of β-actin gene

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] The samples treated by the two methods in Table 2 were compared one by one, and the results of qPCR detection of β-actin gene were summarized as follows: Figure 2 shown.

[0119] observe Figure 2 It can be clearly seen from Table 2 that, under the condition of equal amount of template DNA input, the Ct value obtained by qPCR detection of β-actin gene in samples treated by the method proposed in the present invention is generally lower than that in samples treated by traditional centrifugation. The smaller the Ct value, the higher the human DNA content in the template input. This phenomenon shows that although the traditional centrifugation method can extract a higher total amount of DNA from urine samples, it may contain a large amount of non-human DNA, such as bacteria and fungal DNA in urine. In contrast, urine samples treated by the exfoliated cell enrichment method provided by the present invention can extract DNA with higher purity, which is more conducive to subsequent methylation analysis.

[0120] S3: The extracted DNA is used Ultrafast Bisulfite Kit performs bisulfite (BS) conversion on sample DNA. The steps are as follows:

[0121] (1) Add 22 ml of anhydrous ethanol to the Wash Buffer used for the first time and mix well.

[0122] (2) Prepare the following reaction system in a PCR tube: 20 μL input DNA, 180 μL CT Conversion Buffer, mix well and centrifuge.

[0123] (3) Take 100 μL of the liquid in each tube and transfer it to a new PCR tube and mark it.

[0124] (4) After centrifugation, place the PCR tube in a PCR instrument and keep it at 95°C for 5 minutes;

[0125] (5) Centrifuge the sample briefly after removing it from the machine.

[0126] (6) Transfer the sample to the Spin IC Columns installed in the collection tubes (the liquid in the two reaction tubes divided from one sample in the first step of transformation should be added to the same Spin IC Columns).

[0127] (7) Centrifuge and discard the filtrate.

[0128] (8) Add 100 μL Wash Buffer (anhydrous ethanol has been added in advance) to the Spin IC Columns.

[0129] (9) Centrifuge and discard the filtrate.

[0130] (10) Add 200 μL Desulphonation Buffer and incubate at room temperature for 20 min.

[0131] (11) Centrifuge and discard the filtrate.

[0132] (12) Add 200 μL Wash Buffer (anhydrous ethanol has been added in advance) and centrifuge.

[0133] (13) Prepare a 1.5 mL centrifuge tube and label it. Place the Spin IC Columns after centrifugation into the tube. Discard the supernatant and the collection tube.

[0134] (14) Open the cover and let the column air dry at room temperature for 5 minutes.

[0135] (15) Add 13 μL of Elution Buffer to the center of the Spin IC Columns and incubate at room temperature for 3 min.

[0136] (16) Centrifugation.

[0137] (17) Check whether there is residual liquid in the columns. If so, centrifuge again.

[0138] (18) Remove the column, cap the 1.5 mL centrifuge tube containing the sample DNA, and label it.

[0139] (19) Using Qubit TM ssDNA Assay Kit (purchased from Thermo Company, catalog number: Q10212) was used to measure the concentration using Qubit4.0 instrument.

[0140] Record the measured DNA concentration and calculate the total amount. The remaining DNA will proceed to the next step of reaction.

[0141] S4: The transformed samples are then subjected to DNA multiple displacement amplification, and the specific steps are as follows:

[0142] S41: Evenly mix the bisulfite-converted DNA sample, 10×Reaction Buffer for EquiPhi29 and 500 μM anti-exocrine random primers, centrifuge, prepare a denaturation system, centrifuge and denature the denaturation system at 95° C. for 3 min to obtain a denatured product; the volume of the DNA sample in the denaturation system is 7 μL, the volume of 10×Reaction Buffer for EquiPhi29 is 1 μL, the volume of 500 μM anti-exocrine random primers is 2 μL, and / or,

[0143] 10×Reaction Buffer for EquiPhi29 includes 330-400nM Tris-acetate, 100-150nM magnesium acetate, 660-750nM potassium acetate, 1-3% by mass of Tween 20, and 10-25nM dithiothreitol.

[0144] S42: The denatured product, 100 nM dithiothreitol, 2.5 nM dNTP premix solution, 10 U / μL EquiPhi29 DNA Polymerase and nuclease-free pure water were mixed and centrifuged to prepare an amplification system. The amplification system was centrifuged and amplified at 30°C for 16 hours, and then kept at 65°C for 10 minutes to inactivate the enzyme to obtain an amplification product. The volume of the denatured product in the amplification system was 10 μL, the volume of 100 nM dithiothreitol was 0.5 μL, the volume of 2.5 nM dNTP premix solution was 20 μL, the volume of 10 U / μL EquiPhi29 DNA Polymerase was 2 μL, and the volume of nuclease-free pure water was 17.5 μL.

[0145] S43: The amplified product, 10×Mung Bean Nuclease Buffer and mung bean nuclease are mixed and centrifuged to prepare an enzyme digestion system, the enzyme digestion system is centrifuged and incubated at 30° C. for 30 minutes, and then a 1% sodium dodecyl sulfate solution is added to terminate the enzyme digestion to obtain an enzyme digestion product; in step S43, the volume of the amplified product in the enzyme digestion system is 50 μL, the volume of the 10×Mung Bean Nuclease Buffer is 5.9 μL, and the volume of the mung bean nuclease is 3 μL; the 10×Mung Bean Nuclease Buffer includes 500-600 nM potassium acetate, 250-350 nM potassium chloride and 10-15 nM zinc sulfate.

[0146] S44: Purify and recover the enzyme cleavage product to obtain amplified DNA.

[0147] Using Qubit TMThe dsDNA HS Assay Kit (purchased from Thermo, catalog number: Q32854) was used to determine the concentration of amplified DNA and record and calculate the total amount. The total amount of samples before and after DNA multiple displacement amplification (MDA) was compared. Figure 3 shown.

[0148] Figure 3 The results showed that the samples that had undergone DNA extraction and bisulfite (BS) conversion had a relatively serious DNA loss, resulting in a significant reduction in the amount of DNA remaining, which had an adverse effect on subsequent detection and analysis. After the bisulfite (BS) conversion product was amplified by DNA multiple displacement amplification (MDA), the amount of DNA was significantly increased, effectively making up for the lack of previous losses and providing a sufficient sample basis for subsequent conventional detection technologies, thereby ensuring the smooth progress of the entire methylation detection process and the accuracy of the results.

[0149] S5: Using the human Twist1 gene methylation detection kit (fluorescence PCR method) (purchased from Dajian Company), according to the instruction manual, a total of 684 DNA samples from two groups were tested for bladder cancer. The interpretation method of the kit is shown in Table 3:

[0150] Table 3. Judgment method of the kit

[0151]

[0152] After arranging the data, the final test results of this sample are shown in Table 4.

[0153] Table 4 Test results

[0154] Treatment True Positive False Positive True Negative False Negative Detection method of the present invention 138 15 172 17 Traditional centrifugation 131 23 164 24

[0155] In the two urine processing methods, the samples were grouped according to the sample type and compared with the samples processed by the traditional centrifugation method. It was found that the ΔCt value of the samples processed by the traditional centrifugation method was larger. The ΔCt value of the samples processed by the cell enrichment method provided by the present invention was subtracted from the ΔCt value of the samples processed by the traditional centrifugation method. For data collation, see Figure 4 .

[0156] Figure 4The results in Table 4 show that there are differences in the detection effects between the cell enrichment method provided by the present invention and the traditional centrifugation method (the detection results are better than the traditional centrifugation method), especially in the detection of early (Ta stage, T1 / T2 stage) samples. This phenomenon is mainly caused by changes in the volume of bladder cancer cells and tumor characteristics. The size of bladder cancer cells is generally between 20μm-100μm. By setting the pore size of the microporous filtration membrane to 8-10μm, bladder cancer cells can hardly pass through the microporous membrane and are effectively captured for subsequent nucleic acid extraction and detection. However, in muscle-invasive bladder cancer samples (T3 / T4 stage), although the cancer cells may be larger and more abnormal, because the tumor has invaded the bladder wall muscle layer and tissues or organs outside the bladder, the exfoliated cells in the urine may not be as many or obvious as in the early stages.

[0157] The results confirmed that the enrichment technology of exfoliated cells in urine samples used in the present invention significantly improved the accuracy of bladder cancer methylation detection. This method can efficiently extract cancer cells from urine samples and increase the amount of DNA through genome amplification technology, while preventing non-specific loss during the capture process, ensuring that tumor samples can be identified with high accuracy in the early stages of bladder cancer.

[0158] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. A single cell methylation detection method based on exfoliated cell enrichment, characterized in that: The following steps are involved: S1: Isolate, purify and enrich the exfoliated cells to be analyzed; S2: DNA extraction of the exfoliated cells to be analyzed; S3: Bisulfite conversion of the extracted DNA; S4: Perform DNA multiple displacement amplification on the converted DNA samples; S5: Perform methylation analysis on the amplified DNA.

2. The single cell methylation detection method based on exfoliated cell enrichment according to claim 1, characterized in that: In step S4, the method for DNA multiple displacement amplification comprises the following steps: S41: Evenly mixing the bisulfite-converted DNA sample, 10× Reaction Buffer for Equ iPhi29 and 500 μM anti-exocrine random primers, centrifuging, preparing a denaturation system, centrifuging and denaturing the denaturation system, and obtaining a denatured product; S42: the denatured product, 100 nM dithiothreitol, 2.5 nM dNTP premix solution, 10 U / μL EquiPhi29 DNA Polymerase and nuclease-free pure water are mixed and centrifuged to prepare an amplification system, and the amplification system is centrifuged and amplified to obtain an amplification product; S43: mixing the amplified product, 10×Mung Bean Nuclease Buffer and mung bean nuclease and centrifuging to prepare an enzyme digestion system, centrifuging and digesting the enzyme digestion system, and then adding a terminator to obtain an enzyme digestion product; S44: Purify and recover the enzyme cleavage product to obtain amplified DNA.

3. The single cell methylation detection method based on exfoliated cell enrichment according to claim 2, characterized in that: In step S41, the volume of the DNA sample in the denaturation system is 7 μL, the volume of 10×Reaction Buffer for EquiPhi29 is 1 μL, the volume of 500 μM anti-exocrine random primers is 2 μL, and / or, The 10×Reaction Buffer for EquiPhi29 comprises 330-400 nM Tris-acetate, 100-150 nM magnesium acetate, 660-750 nM potassium acetate, 1-3% Twee n 20 by mass fraction and 10-25 nM dithiothreitol.

4. The single cell methylation detection method based on exfoliated cell enrichment according to claim 2, characterized in that: In step S42, the volume of the denatured product in the amplification system is 10 μL, the volume of 100 nM dithiothreitol is 0.5 μL, the volume of 2.5 nM dNTP premix solution is 20 μL, the volume of 10 U / μL EquiPhi29 DNA Polymerase is 2 μL, and the volume of nuclease-free pure water is 17.5 μL.

5. The single cell methylation detection method based on exfoliated cell enrichment according to claim 2, characterized in that: In step S43, the volume of the amplified product in the enzyme digestion system is 50 μL, the volume of 10×Mung Bean Nuclease Buffer is 5.9 μL, and the volume of mung bean nuclease is 3 μL; and / or, The 10×Mung Bean Nuclease Buffer includes 500-600 nM potassium acetate, 250-350 nM potassium chloride and 10-15 nM zinc sulfate.

6. The single cell methylation detection method based on exfoliated cell enrichment according to claim 2, characterized in that: In step S41, the denaturation method is: denaturation at 95° C. for 3 min; and / or, In step S42, the amplification method is: amplification at 30°C for 16 hours, and then maintained at 65°C for 10 minutes to inactivate the enzyme; and / or, In step S43, the enzyme cleavage method is: incubating at 30°C for 30 minutes, and the terminator is 0.6 μL of a 1% sodium dodecyl sulfate solution.

7. The single cell methylation detection method based on exfoliated cell enrichment according to claim 1, characterized in that: In step S3, the extracted DNA is subjected to bisulfite conversion to convert unmethylated cytosine in the DNA sample into uracil, while the methylated cytosine remains unchanged.

8. The single cell methylation detection method based on exfoliated cell enrichment according to claim 1, characterized in that: In step S5, the methylation analysis method is real-time fluorescence PCR or high-throughput sequencing technology.

9. The single cell methylation detection method based on exfoliated cell enrichment according to claim 1, characterized in that: In step S1, the exfoliated cells to be analyzed are separated, purified and enriched using a fully automatic urine cell efficient enrichment system.

10. Use of the single cell methylation detection method based on exfoliated cell enrichment according to any one of claims 1 to 9 in detecting bladder cancer.

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