Composition and kit for polygene methylation detection of early prostate cancer
Through the EFEMP1 and MIR-193B promoter dual methylation test kit, urine specimens were used for methylation treatment and fluorescence quantitative PCR detection, which solved the problem of insufficient sensitivity and specificity of early screening for prostate cancer in the prior art, and achieved efficient and fast early screening effect.
Patent Information
- Application Number
- CN202311497395.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-10
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art has problems of insufficient sensitivity and specificity in early screening of prostate cancer, and traditional screening methods such as rectal digital examination, PSA examination, ultrasound and magnetic resonance examination have problems such as high missed detection rate and high cost.
Using the EFEMP1 and MIR-193B promoter dual methylation test kit, the premature screening and early diagnosis of prostate cancer is achieved by collecting urine samples and using methylation treatment and fluorescence quantitative PCR detection technology.
This method can significantly improve the sensitivity and specificity of early screening of prostate cancer, reduce the rate of missed detection, and is more convenient and fast than traditional methods, suitable for large-scale popularization.
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Figure CN119979705A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a composition for multi-gene methylation detection of early prostate cancer. Background Art
[0002] Prostate cancer is one of the tumor types with a higher incidence in my country, ranking in the top ten. It has a high cure rate in the early stage, which can reach more than 90%, but in the late stage it can only be relieved by treatment. Therefore, early screening and early diagnosis of prostate cancer are particularly critical. At present, the main screening methods for prostate cancer in clinical practice are rectal examination, prostate-specific antigen test (PSA), ultrasound, and magnetic resonance imaging. The gold standard for diagnosis requires a prostate tissue puncture biopsy. As a traditional screening method, rectal examination depends on the level of clinicians and has a high missed detection rate. PSA examination has low specificity in distinguishing prostate cancer from prostatitis. Ultrasound and magnetic resonance imaging are expensive and not convenient for popularization. Therefore, there is a need for an early screening method that is both sensitive and specific, convenient and fast. Summary of the invention
[0003] The purpose of the present invention is to provide a composition and a kit for multi-gene methylation detection of early prostate cancer, which can achieve early screening and diagnosis of prostate cancer by collecting urine specimens.
[0004] To achieve the above object, a technical solution adopted by the present invention is: a composition for multi-gene methylation detection of early prostate cancer, wherein the composition is a combined gene of EFEMP1 and MIR-193B promoters.
[0005] Furthermore, the base sequence of the EFEMP1 gene is SEQ ID No: 1 or SEQ ID No: 2; the base sequence of the MIR-193B promoter is SEQ ID No: 3 or SEQ ID No: 4.
[0006] Further, the base sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 5 or SEQ ID No: 6;
[0007] The base sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 7 or SEQ ID No: 8.
[0008] Preferably, the primer sequence of the methylated EFEMP1 gene is SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 14 or SEQ ID No: 15, and the probe primer sequence is SEQ ID No: 12 or SEQ ID No: 16;
[0009] The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 22 or SEQ ID No: 23, and the probe primer sequence is SEQ ID No: 20 or SEQ ID No: 24.
[0010] Preferably, the primer sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 11 or SEQ ID No: 13;
[0011] The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 18 or SEQ ID No: 21.
[0012] Most preferably, the primer sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 13;
[0013] The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 18.
[0014] Furthermore, the internal reference for the fluorescence quantitative PCR detection in step (3) is the ACTB gene, the primer sequence of the ACTB gene is SEQ ID No: 25, and the probe primer sequence is SEQ ID No: 26.
[0015] The sequences of the above primers and probes are shown in Table 1 below:
[0016] Table 1
[0017]
[0018]
[0019] A kit for detecting early colorectal cancer comprises EFEMP1 gene primers and probes, MIR-193B promoter primers and probes, ACTB internal reference gene primers and probes and PCR amplification enzyme reaction solution.
[0020] Beneficial effects of the present invention:
[0021] The present invention provides a combined detection kit for EFEMP1 and MIR-193B promoter dual methylation, which can realize early screening and early diagnosis of prostate cancer by collecting urine specimens. The detection kit provided by the present invention comprises amplification primers and probes for EFEMP1 and MIR-193B promoters and internal reference gene ACTB, and PCR amplification enzyme reaction solution, etc. The preparation principle of the detection kit provided by the present invention is as follows:
[0022] First, we established a methylation amplification system for the CG-rich segments of the EFEMP1 and MIR-193B promoters using methylation standards, and screened out probe primer combinations with high specificity and sensitivity for different segments of the two targets;
[0023] Secondly, the performance of different segments of the two targets was determined by target amplification of DNA from urine samples of prostate cancer patients and healthy subjects;
[0024] Next, a dual-target joint detection system and diagnostic method with optimal specificity and sensitivity were initially established;
[0025] Finally, the performance of the kit was verified through a large number of clinical sample tests. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is a ROC curve diagram of the PCR system detection of samples using the third pair of primers of EFEMP1 segment 1, the first pair of primers of EFEMP1 segment 2, the second pair of primers of MIR-193B promoter segment 1 and the first pair of primers of MIR-193B promoter segment 2 of the present invention. DETAILED DESCRIPTION
[0027] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.
[0028] Embodiment 1
[0029] 1. Urine DNA Extraction
[0030] The present invention uses the phenol / chloroform method to extract urine DNA, and the process is as follows:
[0031] 1. Take 5 ml of urine sample, centrifuge at 10000 rpm for 5 min, remove the supernatant and collect the precipitate;
[0032] 2. Add 2 ml of 1x PBS buffer and shake to mix, resuspend the precipitate, centrifuge at 10000 rpm for 5 min, remove the supernatant and collect the precipitate;
[0033] 3. Add 0.5 ml of lysis buffer (0.7 M NaCl, 1% SDS, 0.1% Tween 20), shake and mix, incubate at 70°C for 30 min, invert and mix every 10 min;
[0034] 4. After incubation, centrifuge at 12000 rpm for 5 min, aspirate the supernatant, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), gently invert to mix, and centrifuge at 12000 rpm for 15 min;
[0035] 5. Transfer the supernatant to another new centrifuge tube, add 0.7 times the volume of isopropanol, gently invert to mix, and place at -20℃ for 30 minutes to precipitate nucleic acids;
[0036] 6. Centrifuge at 12000 rpm for 10 min, remove the supernatant and collect the precipitate;
[0037] 7. Discard the supernatant, add 1 ml of 80% ethanol for washing, centrifuge at 12000 rpm for 1 min, and discard the supernatant;
[0038] 8. Repeat step 7 once;
[0039] 9. Open the lid and let it dry for 10 minutes, add 50ul TE buffer, incubate in 56℃ water bath for 10 minutes, and then store at -20℃ for later use.
[0040] 2. DNA methylation treatment
[0041] The DNA methylation treatment of the present invention uses a commercial kit, EZ DNA Methylation Kit (Cat. No. D5005) produced by ZYMO Research, and the specific process is as follows:
[0042] 1. Prepare the conversion solution:
[0043] 1) Add 900 μl water, 300 μl M-Dilution Buffer, and 50 μl M-Dissolving Buffer to CT Conversion Reagent;
[0044] 2) Vortex mix at room temperature for 10 min.
[0045] 2. Conversion:
[0046] 1) Add 130μl CT Conversion Reagent and 20ul DNA to a PCR tube. If the amount is less than 20ul, add water to make up the amount.
[0047] 2) Reaction procedure:
[0048] a.98℃ 10min
[0049] b.64℃ 2.5h
[0050] c. Can be stored at 4℃ for 20h
[0051] 3) Add 600 μl M-Binding Buffer to the adsorption column and place the adsorption column into the collection tube;
[0052] 4. Add the conversion solution to the adsorption column containing M-Binding Buffer and mix by inverting;
[0053] 5. Centrifuge at 12000 rpm for 30 seconds and discard the waste liquid;
[0054] 6. Add 100 μl M-Wash Buffer to the column and centrifuge at 12000 rpm for 30 seconds;
[0055] 7. Add 200 μl M-Desulphonation Buffer to the column and place at room temperature for 15-20 min. After incubation, centrifuge at 12,000 rpm for 30 s.
[0056] 8. Add 200 μl M-Wash Buffer to the column and centrifuge at 12000 rpm for 30 seconds. Repeat this step once;
[0057] 9. Place the adsorption column in a 1.5 ml centrifuge tube, add 10 μl M-Elution Buffer to the membrane, and centrifuge at 12000 rpm for 30 seconds.
[0058] 3. Methylation qPCR detection
[0059] Use the selected kit primers and probes to perform qPCR detection. The specific process is as follows:
[0060] 1. Preparation of PCR reaction solution (PCR reaction system preparation area)
[0061] a. In each PCR reaction, the negative control, positive control and the sample to be tested are tested simultaneously;
[0062] b. Take out the qPCR reaction enzyme mixture, EFEMP1 & MIR-193B promoter & β-actin reaction solution, negative control and positive control, thaw, shake for 30 seconds, and centrifuge for 30 seconds to prevent the reagents from remaining in the tube cap;
[0063] c. Preparation of reaction solution:
[0064] Calculate the number of reaction solution tubes that need to be dispensed based on the number of samples for amplification, as shown below:
[0065] Number of reaction tubes (N) = number of samples (n) + positive control (1 tube) + negative control (1 tube)
[0066] Prepare the following reaction mixture Mix:
[0067] Reaction solution Mix: N x (15μL qPCR reaction enzyme mixture + 5μL EFEMP1 & MIR-193b promoter & β-actin reaction solution) x 1.1
[0068] 2. Packaging and adding samples (performed in the sample processing area)
[0069] a. Aliquot: Aliquot the prepared reaction solution Mix into reaction tubes / plates at 20 μL / tube;
[0070] b. Add samples: Take 10 μL of the treated sample DNA solution, positive control and negative control and add them to the reaction tube containing the reaction solution Mix;
[0071] c. Cover the PCR reaction tube tightly, mix well, and centrifuge to the bottom of the tube to avoid bubbles;
[0072] d. The prepared PCR reaction system should be placed on the machine as soon as possible. The PCR reaction tube with template added should not be left at 2-8℃ for more than 30 minutes, otherwise the experiment may fail.
[0073] Embodiment 2
[0074] The specific preparation method of the detection kit provided by the present invention is as follows:
[0075] Establish a target segment amplification system. We screened two CpG-rich segments from each of the EFEMP1 and MIR-193B promoters, designed three pairs of primers for each segment, and screened out the best amplification system through whole genome methylation quality control products.
[0076] The amplification reaction system is as shown in Table 2-1:
[0077] Table 2-1
[0078]
[0079]
[0080] The sequences of the internal reference upstream primer and downstream primer are SEQ ID No: 25, and the sequence of the probe primer is SEQ ID No: 26.
[0081] The amplification reaction procedure is as shown in Table 2-2:
[0082] Table 2-2
[0083]
[0084] The sequence of EFEMP1 gene segment 1 is as shown in SEQ ID No: 1, and the sequence after methylation-positive sulfurization is as shown in SEQ ID No: 5.
[0085] The designed primer sequences and probe sequences are shown in Table 2-3, and the corresponding methylation positive standard test results are shown in Table 2-4:
[0086] Table 2-3
[0087]
[0088]
[0089] Table 2-4
[0090]
[0091] The sequence of EFEMP1 gene segment 2 is as shown in SEQ ID No: 2, and the sequence after methylation-positive sulfurization is as shown in SEQ ID No: 6.
[0092] The designed primer sequences and probe sequences are shown in Table 2-5, and the corresponding methylation positive standard test results are shown in Table 2-6:
[0093] Table 2-5
[0094]
[0095]
[0096] Table 2-6
[0097]
[0098] The sequence of MIR-193B promoter segment 1 is as shown in SEQ ID No: 3, and the sequence after methylation-positive sulfurization is as shown in SEQ ID No: 7.
[0099] The designed primer sequences and probe sequences are shown in Table 2-7, and the corresponding methylation positive standard test results are shown in Table 2-8:
[0100] Table 2-7
[0101]
[0102]
[0103] Table 2-8
[0104]
[0105] The sequence of MIR-193B promoter segment 2 is SEQ ID No: 4, and the sequence after methylation-positive sulfurization is SEQ ID No: 8.
[0106] The designed primer sequences and probe sequences are shown in Table 2-9, and the corresponding methylation positive standard test results are shown in Table 2-10:
[0107] Table 2-9
[0108]
[0109] Table 2-10
[0110]
[0111] Through the above experiments, we selected the third pair of probe primers combination for EFEMP1 segment 1, the first pair of probe primers combination for EFEMP1 segment 2, the second pair of probe primers combination for MIR-193B promoter segment 1, and the first pair of probe primers combination for MIR-193B promoter segment 2.
[0112] Embodiment 3
[0113] In order to screen a dual-target methylation detection system for colorectal cancer, we collected urine samples from 30 prostate cancer patients and 28 healthy subjects for testing.
[0114] We first extracted the exfoliated cell DNA in the urine sample. After the extraction, the extracted DNA was methylated. The DNA methylation treatment used a commercial kit, the EZ DNA Methylation Kit (Cat. No. D5005) produced by ZYMO Research. The specific kit conversion process was carried out according to the kit instructions. The converted DNA was tested by methylation PCR.
[0115] After being treated with the methylation treatment kit, the methylated EFEMP1 gene or mir-193b gene will generate a fluorescent signal during the fluorescence quantitative PCR detection process, while the unmethylated gene will not generate a fluorescent signal.
[0116] The results of EFEMP1 gene methylation detection in urine samples of 64 prostate cancer patients and 56 healthy subjects are shown in Table 3-1:
[0117] Table 3-1
[0118]
[0119]
[0120]
[0121]
[0122] The results of MIR-193b promoter methylation detection in urine samples of 64 prostate cancer patients and 56 healthy subjects are shown in Table 3-2:
[0123] Table 3-2
[0124]
[0125]
[0126]
[0127] refer to Figure 1 The ROC curves of the data of the four PCR system test samples are shown in Table 3-3:
[0128] Table 3-3
[0129]
[0130]
[0131] Using dual-target combined detection of urine samples, the analysis results are summarized in the following Tables 3-4.1, 3-4.2, 3-4.3, and 3-4.4 based on the above data:
[0132] Table 3-4.1
[0133]
[0134] Table 3-4.2
[0135]
[0136] Table 3-4.3
[0137]
[0138]
[0139] Table 3-4.4
[0140]
[0141] Calculations showed that the best effect was achieved by combined detection of dual methylation of EFEMP1 segment 2 and MIR-193B promoter segment 1. When the positive and negative judgment method was as follows, 62 of the 64 prostate cancer samples were positive for methylation detection, with a sensitivity of 96.8%; 3 of the 56 healthy samples were positive for methylation detection, with a specificity of 94.6% and an overall compliance rate of 95.8%.
[0142] 1. If the CT value of EFEMP1 gene is ≤37, it is judged as EFEMP1 methylation positive; if the CT value is greater than 37 or Unde, it is judged as EFEMP1 methylation negative;
[0143] 2. MIR-193B promoter CT value ≤ 37 was determined as MIR-193B promoter methylation positive; CT value > 37 or Unde was determined as MIR-193B promoter methylation negative;
[0144] 3. The method for determining the sample test results is shown in Table 3-4 below:
[0145] Table 3-4
[0146]
[0147] Embodiment 4
[0148] In order to verify the clinical performance of the dual-gene methylation joint detection kit, we collected urine samples from several patients diagnosed with prostate cancer by tissue puncture biopsy, healthy people, and patients with prostate intraepithelial neoplasia and prostate hyperplasia, and used the EFEMP1 and MIR-193b gene dual-gene methylation system for detection and verification.
[0149] A total of 72 urine samples from prostate cancer patients, 80 urine samples from healthy people, 22 urine samples from prostate intraepithelial neoplasia patients, and 28 urine samples from benign prostatic hyperplasia patients were tested. The reaction system is shown in Table 4-1:
[0150] Table 4-1
[0151]
[0152] The reaction procedure is shown in Table 4-2:
[0153] Table 4-2
[0154]
[0155] The test results of urine samples from prostate cancer patients, healthy people, prostatic intraepithelial neoplasia patients, and benign prostatic hyperplasia patients are shown in Table 4-3, where the detection sensitivity of prostate cancer is 95.8%, the detection specificity of healthy people is 97.5%, the detection sensitivity of prostatic intraepithelial neoplasia is 68.1%, and the detection specificity of prostatic hyperplasia is 92.8%.
[0156] Table 4-3
[0157] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A composition for multi-gene methylation detection of early prostate cancer, characterized in that: The composition is a combination of EFEMP1 and MIR-193B promoter genes.
2. A composition for multi-gene methylation detection of early prostate cancer according to claim 1, characterized in that: The base sequence of the EFEMP1 gene is SEQ ID No: 1 or SEQ ID No: 2; the base sequence of the MIR-193B promoter is SEQ ID No: 3 or SEQ ID No:
4.
3. A composition for multi-gene methylation detection of early prostate cancer according to claim 2, characterized in that: The base sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 5 or SEQ ID No: 6; The base sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 7 or SEQ ID No:
8.
4. A composition for multi-gene methylation detection of early prostate cancer according to claim 3, characterized in that: The primer sequence of the methylated EFEMP1 gene is SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 14 or SEQ ID No: 15, and the probe primer sequence is SEQ ID No: 12 or SEQ ID No: 16; The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 21, SEQ ID No: 22 or SEQ ID No: 23, and the probe primer sequence is SEQ ID No: 20 or SEQ ID No:
24.
5. A composition for multi-gene methylation detection of early prostate cancer according to claim 4, characterized in that: The primer sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 11 or SEQ ID No: 13; The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No: 18 or SEQ ID No:
21.
6. A composition for multi-gene methylation detection of early prostate cancer according to claim 5, characterized in that: The primer sequence of the EFEMP1 gene after methylation treatment is SEQ ID No: 13; The primer sequence of the MIR-193B promoter after methylation treatment is SEQ ID No:
18.
7. A composition for multi-gene methylation detection of early prostate cancer according to claim 1, characterized in that: The internal reference for the fluorescence quantitative PCR detection in step (3) is the ACTB gene, the primer sequence of the ACTB gene is SEQ ID No: 25, and the probe primer sequence is SEQ ID No:
26.
8. A kit for detecting early colorectal cancer, characterized in that: It includes EFEMP1 gene primers and probes, MIR-193B promoter primers and probes, ACTB internal reference gene primers and probes, and PCR amplification enzyme reaction solution.