Composition and kit for polygene methylation detection of early colorectal cancer

Abstract

The invention discloses a composition for polygene methylation detection of early colorectal cancer, the composition is an SDC2 and EYA4 combined gene, and the base sequence of the SDC2 gene is SEQ ID No: 1 or SEQ ID No: 2; the base sequence of the EYA4 gene is SEQ ID No: 3 or SEQ ID No: 4; meanwhile, the invention further provides a kit for detecting SDC2 and EYA4 gene methylation, the sensitivity of colorectal cancer patient detection reaches 96% or above, and the specificity is 96% or above.

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 colorectal cancer. Background Art

[0002] Colorectal cancer is one of the types of tumors with high morbidity and mortality in my country. Due to the strong concealment of colorectal cancer, many patients are already in the late stage when they are discovered, resulting in the inability to obtain timely and appropriate treatment and poor prognosis. Therefore, early screening and early diagnosis of colorectal cancer are particularly critical. At present, clinical colorectal cancer tumor screening is mainly carried out through two technologies: fecal occult blood test and colonoscopy. As a traditional screening method, the fecal occult blood test has a high false positive rate, poor stability, and is easily affected by personal physical condition and diet. Colonoscopy is an invasive examination method that requires advance preparation and is prone to complications, especially for middle-aged and elderly people with weak constitutions, who cannot tolerate it. Therefore, there is an urgent need for an early screening and early diagnosis technology with high compliance, convenient sampling, and good accuracy to improve the applicability of colorectal cancer screening. 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 colorectal cancer to solve the problems raised in the above background technology.

[0004] To achieve the above object, a technical solution adopted by the present invention is: a composition for multi-gene methylation detection of early colorectal cancer, wherein the composition is a combined gene of SDC2 and EYA4.

[0005] Furthermore, the base sequence of the SDC2 gene is SEQ ID No: 1 or SEQ ID No: 2; the base sequence of the EYA4 gene is SEQ ID No: 3 or SEQ ID No: 4.

[0006] Further, the base sequence of the SDC2 gene after methylation treatment is SEQ ID No: 5 or SEQ ID No: 6; The base sequence of the EYA4 gene after methylation treatment is SEQ ID No: 7 or SEQ ID No: 8.

[0007] Preferably, the primer sequence of the SDC2 gene after methylation treatment 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 EYA4 gene 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.

[0008] Preferably, the primer sequence of the SDC2 gene after methylation treatment is SEQ ID No: 9 or SEQ ID No: 14; The primer sequence of the EYA4 gene after methylation treatment is SEQ ID No: 17 or SEQ ID No: 23.

[0009] Preferably, the primer sequence of the SDC2 gene after methylation treatment is SEQ ID No: 9; The primer sequence of the EYA4 gene after methylation treatment is SEQ ID No: 17.

[0010] Furthermore, the internal reference of the fluorescence quantitative PCR detection 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.

[0011] The sequences of the above primers and probes are shown in Table 1 below: Table 1

[0012] A kit for detecting early colorectal cancer comprises SDC2 gene primers and probes, EYA4 gene primers and probes, ACTB internal reference gene primers and probes and PCR amplification enzyme reaction solution.

[0013] Beneficial effects of the present invention: The present invention provides a combined detection kit for SDC2 and EYA4 gene dual methylation, which can be used to achieve early screening and early diagnosis of colorectal cancer through the detection of fecal DNA. The detection kit provided by the present invention comprises amplification primers and probes for SDC2 and EYA4 genes and internal reference gene ACTB, as well as PCR amplification enzyme reaction solution, etc. The preparation principle of the detection kit provided by the present invention is as follows: First, we established a methylation amplification system for the CG-rich segments of the SDC2 and EYA4 genes using methylation standards, and screened out probe primer combinations with high specificity and sensitivity for different segments of the two targets; Secondly, the clinical performance of different segments and sites of each target was preliminarily evaluated and screened through actual detection of stool samples of colorectal cancer patients and healthy subjects (including single-target detection and dual-target combined detection); Next, a dual-target joint detection system and diagnostic method with optimal specificity and sensitivity were initially established; Finally, the performance of the kit was verified through a large number of clinical sample tests, including a comprehensive test of adenoma and polyp samples in the precancerous lesion stage, as well as other gastrointestinal tumor samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a ROC curve diagram of the PCR system detection of samples using the first pair of primers in SDC2 segment 1, the second pair of primers in SDC2 segment 2, the first pair of primers in EYA4 segment 1 and the third pair of primers in EYA4 segment 2 of the present invention. DETAILED DESCRIPTION

[0015] 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.

[0016] Embodiment 1 1. Targeted capture and extraction of fecal DNA The present invention uses the nucleic acid extraction or purification reagent (Lulin Xiebei No. 20210113) produced and registered by Lixin Nuokang (Shandong) Biomedical Technology Co., Ltd. to extract fecal DNA, and the process is as follows: 1. Sample pretreatment: fully shake and mix the sample in the feces collection tube, take 5mL of feces suspension into a clean 15mL centrifuge tube, centrifuge at 12000rpm for 5min, gently remove the centrifuge tube from the centrifuge, carefully pipette 2mL of supernatant into a clean 15mL centrifuge tube (note: avoid floating impurities on the upper layer and sediment on the lower layer when pipetting, add PBS to 2mL if the supernatant is less than 2mL), add 30μL proteinase K and 1.34mL lysis buffer, shake and mix, and place the centrifuge tube in a 70℃ water bath for 15min; 2. Nucleic acid denaturation: After the water bath is finished, place the centrifuge tube in a 92°C water bath for 15 minutes; 3. Capture agent activation (the capture agent needs to be activated before use, which can be performed during the waiting process in sample processing step 2): Take out the capture agent stored at 2-8°C, shake and mix for 30 seconds, take the required volume V (V = number of processed samples × 60μL) of capture agent into a 2mL centrifuge tube, place it on a magnetic rack for adsorption for 2 minutes, discard the supernatant (remove the remaining liquid as much as possible), add 1mL of activation solution and shake and mix for 30 seconds, centrifuge for 2 seconds, place it on a magnetic rack for adsorption for 2 minutes, discard the supernatant and add V volume of activation solution and shake and mix for 30 seconds for use; 4. Hybridization capture: quickly add 60 μL of activated capture agent to the denatured sample, shake and mix for 30 seconds, and incubate at room temperature for 30 minutes (shake and mix every 10 minutes during the incubation period, each time for 5 seconds); 5. Magnetic separation: Place the above 15 mL centrifuge tube in a magnetic rack for 10 minutes and discard the supernatant (try not to move the centrifuge tube when absorbing the supernatant); 6. Rinse 1: Add 1 mL of rinse solution 1 to the 15 mL centrifuge tube after absorbing the supernatant, shake and mix for 10 seconds to elute the magnetic beads, transfer the magnetic beads to a clean 2 mL centrifuge tube, place it on a magnetic stand and absorb it for 2 minutes, slowly discard the supernatant, and absorb all the liquid to avoid absorbing the magnetic beads; 7. Rinse 2: Add 1 mL of rinse solution 2 to the 2 mL centrifuge tube after absorbing the supernatant, shake and mix for 10 seconds, place on a magnetic stand for 2 minutes, slowly discard the supernatant, and absorb all the liquid to avoid absorbing the magnetic beads; 8. Repeat step 7 above once, and after absorbing the supernatant, open the lid and let it stand at room temperature for 3 minutes; 9. Elution: Add 70 μL of extraction and elution buffer to a 2 mL centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 seconds, place in a 92°C water bath for 5 minutes, then take out the centrifuge tube, centrifuge for 5 seconds, and magnetically absorb for 3 minutes. Transfer the supernatant to a clean 500 μL nucleic acid storage tube to obtain the required DNA solution.

[0017] 2. Bisulfite Conversion The present invention uses the nucleic acid extraction or purification reagent (Lulin Xiebei No. 20210113) produced and registered by Lixin Nuokang (Shandong) Biomedical Technology Co., Ltd. to perform bisulfite conversion of fecal DNA. The specific process is as follows: Add 95μL of conversion solution, 50μL of DNA solution (total amount not more than 2μg) and 15μL of protection solution to a clean 0.2mL PCR tube, and shake to mix. Centrifuge briefly and place in PCR amplification instrument, set the amplification program according to Table 1-1. After the instrument runs, take out the product, centrifuge briefly and set aside; Table 1-1

[0018] 2. Take out a clean 2mL centrifuge tube, add 1mL of binding solution, 160μL of transformation product and 30μL of magnetic bead suspension (magnetic beads should be fully shaken and mixed before use), shake and mix for 10s, place in a rotary mixer and mix by inversion for 30min; 3. After the incubation, centrifuge for 5 seconds, place on a magnetic stand for 2 minutes, and discard the supernatant (try to absorb all the remaining liquid); 4. Add 500 μL of washing solution 1 to the centrifuge tube (please check whether anhydrous ethanol has been added before use), shake and mix for 10 seconds, centrifuge for 2 seconds, place on a magnetic stand for 2 minutes, and discard the supernatant; 5. Add 500 μL of washing solution 2 to the centrifuge tube (please check whether anhydrous ethanol has been added before use), shake and mix for 10 seconds, place it in a rotary mixer and mix it upside down for 10 minutes. After the time is up, place it on a magnetic stand and absorb it for 2 minutes, and discard the supernatant; 6. Add 500 μL of washing solution 3 to the centrifuge tube (please check whether anhydrous ethanol has been added before use), shake and mix for 10 seconds, centrifuge for 2 seconds, place on a magnetic stand for 2 minutes, and discard the supernatant; 7. Add 500 μL of washing solution 1 to the centrifuge tube (please check whether anhydrous ethanol has been added before use), shake and mix for 10 seconds, centrifuge for 2 seconds, place on a magnetic stand for 2 minutes, and discard the supernatant.

[0019] 8. Repeat rinsing step 7 once; 9. After the repeated operation is completed, centrifuge for 5 seconds immediately, magnetically absorb for 1 minute, absorb the remaining liquid, open the centrifuge tube cover, and dry it at room temperature for 5 minutes; 10. Add 35 μL of conversion eluent to the centrifuge tube, shake and mix for 10 seconds, centrifuge for 2 seconds, and place the centrifuge tube in a 56°C water bath for 5 minutes; 11 After the water bath is over, place the centrifuge tube on the magnetic rack. After magnetic absorption for 2 minutes, transfer the supernatant to a clean 500μL nucleic acid storage tube and store it at -20±5℃ for later use.

[0020] 3. Methylation qPCR detection Use the selected kit primers and probes to perform qPCR detection. The specific process is as follows: 1. Preparation of PCR reaction solution (PCR reaction system preparation area) a. In each PCR reaction, the negative control, positive control and the sample to be tested are tested simultaneously; b. Take out the qPCR reaction enzyme mixture, SDC2&EYA4&β-actin reaction solution, negative control and positive control, thaw them, shake for 30 seconds, and centrifuge for 30 seconds to prevent the reagents from remaining in the tube cap; c. Preparation of reaction solution: Calculate the number of reaction solution tubes that need to be dispensed based on the number of samples for amplification, as shown below: Number of reaction tubes (N) = number of samples (n) + positive control (1 tube) + negative control (1 tube) Prepare the following reaction mixture Mix: Reaction solution Mix: N x (15μL qPCR reaction enzyme mixture + 5μL SDC2&EYA4&β-actin reaction solution) x1.1 2. Packaging and sample addition (performed in the sample processing area) a. Aliquot: Aliquot the prepared reaction solution Mix into reaction tubes / plates at 20 μL / tube; 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; c. Cover the PCR reaction tube tightly, mix well, and centrifuge to the bottom of the tube to avoid bubbles; d. The prepared PCR reaction system should be loaded onto 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. Example

[0021] The specific preparation method of the detection kit provided by the present invention is as follows: Establish a target segment amplification system. For the SDC2 and EYA4 genes, we screened two CpG-rich segments from each target, designed three pairs of primers for each segment, and screened out the best amplification system through whole genome methylation quality control products.

[0022] The amplification reaction system is as shown in Table 2-1: Table 2-1

[0023] 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.

[0024] The amplification reaction procedure is as shown in Table 2-2: Table 2-2

[0025] The sequence of SDC2 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.

[0026] 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: Table 2-3

[0027] Table 2-4

[0028] The sequence of SDC2 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.

[0029] 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: Table 2-5

[0030] Table 2-6

[0031] The sequence of EYA4 gene 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.

[0032] 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: Table 2-7

[0033] Table 2-8

[0034] The sequence of EYA4 gene segment 2 is shown in SEQ ID No: 4, and the sequence after methylation-positive sulfurization is shown in SEQ ID No: 8.

[0035] 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: Table 2-9

[0036] Table 2-10

[0037] Through the above experiments, we selected the first pair of probe primers for SDC2 segment 1, the second pair of probe primers for SDC2 segment 2, the first pair of probe primers for EYA4 segment 1, and the third pair of probe primers for EYA4 segment 2. These four PCR systems were used to detect clinical samples (see Table 2-11).

[0038] Table 2-11

[0039] The above results show that SDC2-segment 1 capture probe 1 has the best efficiency and no non-specific capture; SDC2-segment 2 capture probe 1 has the best efficiency and no non-specific capture; EYA4-segment 1 capture probe 1 has the best efficiency and no non-specific capture. EYA4-segment 2 capture probe 1 has the best efficiency and no non-specific capture. The following experiments selected the selected probes to capture target DNA.

[0040] In order to screen the dual-target methylation detection system for colorectal cancer, we collected stool samples from 56 colorectal cancer patients (determined by clinical colonoscopy and pathological screening) and 81 healthy people for testing.

[0041] We first captured human DNA in feces, and then methylated the DNA extracted from feces. 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 manual, and the converted DNA was tested for methylation PCR.

[0042] After being treated with the methylation treatment kit, the methylated SDC2 gene or EYA4 gene will generate a fluorescent signal during the fluorescence quantitative PCR detection process, while the unmethylated gene will not generate a fluorescent signal.

[0043] The results of SDC2 gene methylation detection in 56 colorectal cancer patients and 81 healthy subjects are shown in Table 3-1: Table 3-1

[0044] The results of EYA4 gene methylation detection in 56 colorectal cancer patients and 81 healthy subjects are shown in Table 3-2: Table 3-2

[0045] refer to Figure 1 The ROC curves of the data of the four PCR system test samples are shown in Table 3-3: Table 3-3

[0046] Using dual-target combined detection of fecal 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: Table 3-4.1

[0047] Table 3-4.2

[0048] Table 3-4.3

[0049] Table 3-4.4

[0050] Calculations showed that the combined detection of dual methylation in SDC2 segment 1 and EYA4 segment 1 had the best effect. When the positive and negative judgment method was as follows, 54 of the 56 colorectal cancer samples were positive for methylation, with a sensitivity of 96.4%; 3 of the 81 healthy samples were positive for methylation, with a specificity of 96.2% and an overall compliance rate of 96.4%.

[0051] 1. SDC gene CT value ≤ 36, judged as SDC2 methylation positive; CT value > 36 or Unde, judged as SDC2 methylation negative; 2. EYA4 gene CT value ≤ 36 was determined as EYA4 methylation positive; CT value > 36 or Unde was determined as EYA4 gene methylation negative; 3. The method for determining the sample test results is shown in Table 3-4 below: Table 3-4

[0052] Embodiment 4 In order to verify the clinical performance of the dual-gene methylation joint detection kit, we collected stool samples from several patients diagnosed with colorectal cancer by colonoscopy, gastric cancer patients, normal people, patients with benign intestinal lesions, and some people with polyps and adenomas, and used the SDC2 and EYA4 gene dual-gene methylation system for detection and verification.

[0053] A total of 120 stool samples of colorectal cancer, 168 stool samples of healthy people, 26 stool samples of gastric cancer, 32 stool samples of benign intestinal lesions, and 24 polyp samples were tested. The reaction system is shown in Table 4-1: Table 4-1

[0054] The reaction procedure is shown in Table 4-2: Table 4-2

[0055] The test results of stool samples from patients with colorectal cancer, healthy persons, patients with gastric cancer, patients with enteritis and patients with adenomatous polyps are shown in Table 4-3, where the detection sensitivity of colorectal cancer is 95.8%, the detection specificity of healthy persons is 97%, the detection specificity of gastric cancer and enteritis is 100%, the detection sensitivity of adenoma is 63.2%, and the detection sensitivity of polyps is 75.6%.

[0056] Table 4-3

[0057] 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 colorectal cancer, characterized in that: The combination is a combination of SDC2 and EYA4 genes.

2. A composition for multi-gene methylation detection of early colorectal cancer according to claim 1, characterized in that: The base sequence of the SDC2 gene is SEQ ID No: 1 or SEQ ID No: 2; the base sequence of the EYA4 gene is SEQ ID No: 3 or SEQ ID No:

4.

3. A composition for multi-gene methylation detection of early colorectal cancer according to claim 2, characterized in that: The base sequence of the SDC2 gene after methylation treatment is SEQ ID No: 5 or SEQ ID No: 6; The base sequence of the EYA4 gene after methylation treatment is SEQ ID No: 7 or SEQ ID No:

8.

4. A composition for multi-gene methylation detection of early colorectal cancer according to claim 3, characterized in that: The primer sequence of the SDC2 gene after methylation treatment 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 EYA4 gene 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 colorectal cancer according to claim 4, characterized in that: The primer sequence of the SDC2 gene after methylation treatment is SEQ ID No: 9 or SEQ ID No: 14; The primer sequence of the EYA4 gene after methylation treatment is SEQ ID No: 17 or SEQ ID No:

23.

6. A composition for multi-gene methylation detection of early colorectal cancer according to claim 5, characterized in that: The primer sequence of the SDC2 gene after methylation treatment is SEQ ID No: 9; The primer sequence of the EYA4 gene after methylation treatment is SEQ ID No:

17.

7. A composition for multi-gene methylation detection of early colorectal cancer according to claim 1, characterized in that: The internal reference of the fluorescence quantitative PCR detection 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 SDC2 gene primers and probes, EYA4 gene primers and probes, ACTB internal reference gene primers and probes, and PCR amplification enzyme reaction solution.

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