A colorectal cancer multi-gene methylation detection kit

Abstract

The application discloses a colorectal cancer multi-gene methylation detection kit, which can detect the methylation of FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5 and Septin9, and has high detection accuracy. DNA methylation abnormalities usually occur in the early stage of cancer, so the detection of the DNA methylation index can be used as an important index for early screening, diagnosis and prognosis evaluation of colorectal cancer. Furthermore, the detection accuracy can be further improved by combining NGS sequencing.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a detection kit for multi-gene methylation in colorectal cancer. Background Technology

[0002] DNA methylation ( DNA methylation DNA methylation, a form of chemical modification, can alter genetic expression without changing the DNA sequence. It plays a crucial regulatory role in individual growth, development, gene expression patterns, and genome stability, and this modification is stably transmitted during development and cell proliferation. Recent studies have shown a close link between abnormal DNA methylation and tumor development, progression, and carcinogenesis. Changes in tumor DNA methylation manifest as a decrease in overall genome methylation levels and an increase in CpG island methylation levels in certain gene promoter regions. Methylation of tumor suppressor genes and DNA repair genes silences tumor suppressor genes and inactivates repair genes, thus losing their inhibitory effect on tumors and increasing gene damage. Conversely, a decrease in overall genome methylation levels activates proto-oncogenes and retrotransposons, leading to decreased chromosome stability.

[0003] Colorectal cancer is one of the most common malignant tumors worldwide, and its incidence rate in my country is increasing year by year. According to data from the U.S. National Cancer Institute, the 5-year survival rate for colorectal cancer is as high as 90.6%, while the 5-year survival rate for metastatic colorectal cancer is only 14.7%, highlighting the importance of early screening for colorectal cancer. It not only relates to individual interests but also reduces the national healthcare and disease burden. However, due to technological limitations, my country has not yet established a complete and stable early screening technology for colorectal cancer.

[0004] Currently, the strategy for colorectal cancer screening in hospitals is based on chemical methods (…). guaiac FOBT, gFOBT ), Immunization method ( fecal immunochemical test, FIT Screening methods include colonoscopy, circulating tumor cells (CTC), circulating tumor DNA (ctDNA) markers such as Septin9 gene methylation, RNA and protein markers in blood and feces, fecal microbiota markers, and urine markers.

[0005] Among these methods: The gFOBT (glucose-free biochemical assay) for screening tumors and advanced adenomas has low sensitivity, is easily affected by dietary components, and has high false positive and false negative rates. The FIT (fibrillation-based immunoassay) method has high sensitivity but low specificity; it also has high sensitivity for detecting precancerous lesions, but the cost is relatively high. Colonoscopy is the gold standard for colorectal cancer screening and plays an irreplaceable role in the early diagnosis and treatment of precancerous lesions. However, as an invasive procedure, colonoscopy is costly, requires cumbersome preparation, and carries certain risks of perforation (3.1 / 10,000) and massive bleeding (14.6 / 10,000), resulting in poor patient compliance. Circulating tumor cell (CTC) markers, circulating tumor DNA (ctDNA) markers such as Septin9 gene methylation, RNA and protein markers from blood and feces, fecal microbiota markers, and urine markers all have unavoidable problems in terms of sensitivity, specificity, and accuracy. In addition, although some genes are currently being tested for methylation, this is mainly done on single genes, which may lead to false positives and reduce the accuracy, specificity, and sensitivity of patient prediction. Secondly, most methylation detection techniques still use the medip-qPCR method, which has a relatively large error.

[0006] Therefore, it is urgent for professionals in this field to develop a colorectal cancer detection method with high sensitivity, specificity and accuracy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a detection kit for multi-gene methylation in colorectal cancer, which has high sensitivity, specificity and accuracy.

[0008] This invention solves the above-mentioned technical problems through the following technical solution: a detection kit for multi-gene methylation in colorectal cancer, wherein the colorectal cancer gene methylation detection sites detected by the kit include FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5, and Septin9; and the kit includes primer combinations shown in 1)-7) below:

[0009] 1) The primer pairs for FBXO5 methylation detection include primer pair 1, primer pair 2, primer pair 3, and primer pair 4 as shown in SEQ ID NO. 1-8;

[0010] 2) Primer pairs for eIF6 methylation detection include primer pairs 5, 6, 7, 8, and 9 as shown in SEQ ID NO. 9-18;

[0011] 3) The primer pairs for KIF2C methylation detection include primer pair 10, primer pair 11, primer pair 12, primer pair 13, and primer pair 14 as shown in SEQ ID NO.19-28;

[0012] 4) Primer pairs for IGF2BP3 methylation detection include primer pairs 15, 16, 17, and 18 as shown in SEQ ID NO.29-36;

[0013] 5) Primer pairs for CBX3 methylation detection include primer pair 19, primer pair 20, and primer pair 21 as shown in SEQ ID NO.37-42;

[0014] 6) Primer pairs for ALKBH5 methylation detection include primer pairs 22, 23, 24, and 25 as shown in SEQ ID NO.43-50;

[0015] 7) Primer pairs for Septin9 methylation detection include primer pair 26 and primer pair 27 as shown in SEQ ID NO. 51-54.

[0016] Furthermore, the test sample type of the kit is an ex vivo blood sample.

[0017] Furthermore, the kit detects the methylation status of the detection site using NGS.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The kit of the present invention has high sensitivity and specificity; and it can perform multi-gene methylation detection for colorectal cancer, which can make the detection results highly accurate. DNA methylation abnormalities usually occur in the early stage of cancer. Therefore, the detection of DNA methylation index can be used as an important indicator for early screening, diagnosis and prognosis assessment of colorectal cancer.

[0020] (2) The gene methylation status of the seven gene detection sites in this invention is obtained by NGS sequencing, which can further improve the accuracy of detection. Attached Figure Description

[0021] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0022] Figure 1 The ROC curves for predicting the methylation levels of seven genes in colorectal cancer in Example 4 of this invention are shown.

[0023] Figure 2 This is the ROC curve for predicting the degree of Septin9 methylation in colorectal cancer in Example 4 of the present invention. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can easily implement the invention based on the disclosure in the specification. The embodiments described in this invention are merely exemplary and are not intended to limit the invention. In addition, unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field, and experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturer; and the samples used in this invention are all ex vivo samples.

[0025] Example 1: Screening of methylation detection sites

[0026] The data used for screening methylation detection sites in this invention comes from the public database TCGA methylation data and corresponding clinical information of the samples. Logistic regression, ridge regression (LASSO), RFECV-SVM (SVM), and RFECV-RF (RF) algorithms are used for gene screening and evaluation. Logistic regression and ridge regression use hierarchical cross-validation for parameter optimization, setting the feature selection threshold to the average feature weights; features with weights greater than the average are retained. The RFECV algorithm uses different learning models, SVM and RF, for screening. The specific screening steps are as follows:

[0027] (1) Download rectal cancer methylation data and patient clinical information from the TCGA website;

[0028] (2) Due to expression profile matrix integration: The data is organized into gene and patient matrices. To facilitate comparison between different sequencing platforms, standardized data is selected in this embodiment.

[0029] (3) Survival prognosis analysis: The number of samples with an expression level of 0 is less than pct% of the total number of samples, and the expression level variance is greater than 1 of genes are used to form an expression matrix;

[0030] (4) Divide the original dataset into k equal parts using stratified sampling, and the current feature gene contains all genes;

[0031] (5) Select k-1 equal parts as the training set and the remaining equal part as the validation set, and train on the learning models SVM and RF;

[0032] (6) Run the classifier on the test set and calculate the evaluation index of the classification result; sort the genes in the training set according to their importance and delete the last m features; repeat (2), (3), and (4) until the remaining genes in the training set are empty;

[0033] (7) Select the iteration with the highest average evaluation index on the k test sets, and perform RFE iteration screening on the entire dataset again based on the number of genes N* retained in this iteration until the number of genes remaining is N*. The gene methylation detection sites finally selected are FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5, and Septin9.

[0034] Example 2: Design of corresponding primer pairs

[0035] Based on the nucleic acid sequences of gene methylation detection sites FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5, and Septin9 obtained from Example 1, the applicant repeatedly designed and refined corresponding specific primer pairs, and sent the designed primer pairs to Sangon Biotech (Shanghai) Co., Ltd. for synthesis; the specific sequences are shown in the following tables (Tables 1-7).

[0036] Table 1 Primer pair sequences for FBXO5 methylation detection

[0037]

[0038] Table 2 Primer pair sequences for eIF6 methylation detection

[0039]

[0040] Table 3 Primer pair sequences for KIF2C methylation detection

[0041]

[0042] Table 4 Primer pair sequences for IGF2BP3 methylation detection

[0043]

[0044] Table 5 Primer pair sequences for CBX3 methylation detection

[0045]

[0046] Table 6 Primer pair sequences for ALKBH5 methylation detection

[0047]

[0048] Table 7 Primer pair sequences for Septin9 methylation detection

[0049]

[0050] Example 3: Verification of Sensitivity and Specificity

[0051] (1) Collecting samples

[0052] Blood samples were collected from 30 patients diagnosed with colorectal cancer and 30 healthy individuals undergoing routine physical examinations. Each blood sample was 10 ml. All samples were approved by the ethics committee, and all patients and individuals undergoing physical examinations signed informed consent forms. All collected samples were anonymized.

[0053] (2) Extracting DNA from blood

[0054] DNA was extracted from blood samples using a nucleic acid extraction kit from Nanjing Novizan Biotechnology Co., Ltd. The specific steps are as follows:

[0055] 21) Centrifuge the collected 10ml blood sample at 1400g for 10min, collect about 5ml of supernatant plasma, transfer it to a new 2ml enzyme-free and nucleic acid-free EP tube, centrifuge at 11000g for 1min, transfer the supernatant plasma to a new 2ml EP tube for storage and future use;

[0056] 22) Lysis and binding: Prepare a clean 10mL centrifuge tube, add 2mL of the supernatant plasma obtained in step 21), and add 3mL of lysis and binding buffer, 20μL of magnetic beads and 200μL of proteinase K in sequence. Mix well by inverting the tube and place it on a mixer at room temperature for 30min, while keeping the magnetic beads in suspension.

[0057] 23) Washing: After the lysis is complete, place the centrifuge tube on a magnetic rack to attract the magnetic beads. After the solution is clear, rinse the tube cap several times to remove any remaining magnetic beads until all the beads are attracted. Then carefully remove the waste liquid, add 1 mL of washing solution, vortex and mix for 1 min to completely disperse the magnetic beads, attract the magnetic beads for 2 min, and rinse the tube cap several times to remove any remaining magnetic beads until all the beads are attracted.

[0058] 24) Rinsing: After carefully removing the waste liquid, first add 1 ml of freshly prepared 80% ethanol to wash the magnetic beads to the bottom. Then transfer the magnetic bead suspension to a new 2 mL centrifuge tube. Next, add 1 ml of freshly prepared 80% ethanol to completely wash the remaining magnetic beads on the wall of the 10 mL centrifuge tube to the bottom. After a brief centrifugation, transfer all the magnetic bead suspension to a 2 mL centrifuge tube. Vortex to mix more than 10 times to completely disperse the magnetic beads. Magnetize for 2 minutes until the solution is clear. Then, invert the tube several times to rinse the remaining magnetic beads on the cap until all magnetic beads have been magnetized. Repeat the rinsing once.

[0059] 25) Elution and collection: Remove the centrifuge tube and briefly centrifuge to collect the residual liquid. Place it on a magnetic rack to attract the magnets. After magnetization, use a small pipette tip to remove the residual liquid. Open the centrifuge tube and let it stand for 5 minutes to make the surface of the magnetic beads dull. Then add 50 μL of elution buffer TE and gently shake to disperse the magnetic beads. Then place it at 56°C for elution for 10 minutes. Take it out every 3 minutes and gently shake it to keep the magnetic beads in suspension. Then remove the centrifuge tube, centrifuge to collect the liquid on the cap and tube wall, place it on a magnetic rack to attract the magnets for 1 minute, and carefully aspirate the supernatant to obtain the DNA solution.

[0060] (3) DNA transformation

[0061] Take 40 μL of the DNA solution obtained in step (2) above into a 200 μL PCR tube, then add 110 μL of transformation mixture, mix well and place in a PCR instrument for reaction (where the PCR instrument reaction program is set as follows: 95℃ for 10 min, 64℃ for 90 min, 4℃~8℃ for 1 h).

[0062] (4) DNA purification

[0063] 41) Transfer the reaction product obtained in step (3) into a 2 mL centrifuge tube, add 600 μL of binding solution and 10 μL of magnetic beads, mix well and let stand for 15 min, shaking and mixing for 5 s every 3 min to keep the magnetic beads in suspension; centrifuge briefly, place the centrifuge tube on a magnetic rack, and after the magnetic beads are completely adsorbed (about 1 min), carefully remove the supernatant.

[0064] 42) Add 600 μL of rinsing solution, vortex to mix and disperse the magnetic beads for 20 seconds; centrifuge briefly, place the centrifuge tube on a magnetic rack, and after the magnetic beads are completely adsorbed (about 1 min), carefully remove the supernatant.

[0065] 43) Add 800μL of desulfurizing agent, vortex mix for 20s to disperse the magnetic beads, let stand at room temperature for 15min to desulfurize, and shake and mix for 5s every 5min during this period to keep the magnetic beads in a suspended state.

[0066] 44) Add 800 μL of washing solution, vortex to disperse the magnetic beads for 20 s; briefly centrifuge, place the centrifuge tube on a magnetic rack; after the magnetic beads are completely adsorbed (about 1 min), carefully remove the supernatant;

[0067] 45) Repeat step d) once;

[0068] 46) Briefly centrifuge to collect the liquid to the bottom of the tube, place the centrifuge tube on a magnetic rack, and carefully aspirate the supernatant; open the cap and let it sit at room temperature for about 5 minutes until the surface of the magnetic beads is no longer shiny;

[0069] 47) Add 20-50 μL of elution buffer TE, vortex to fully suspend the magnetic beads in the elution buffer, and incubate at 56°C for 10 min, vortexing once every 3 min to promote complete elution of nucleic acids;

[0070] 48) Collection: Briefly centrifuge, place the centrifuge tube on a magnetic rack and let it stand for 2 minutes, then transfer the DNA solution to a new centrifuge tube to obtain sulfite-treated cfDNA for later use.

[0071] (5) PCR amplification and NGS sequencing

[0072] Using the purified DNA from step (4) as a template (i.e., sulfite-treated cfDNA), methylated and unmethylated DNA fragments were simultaneously amplified using the primer pairs listed in Tables 1 to 7 of Example 2 as multiplex panel primers (using MultipSeq multiplex amplicon sequencing technology). Specifically:

[0073] The first round of PCR amplification consisted of the following reaction mixture: PCR mix 15 μL, Panel 8 μL, and sulfite-treated cfDNA 7 μL. The reaction procedure is shown in the table below.

[0074]

[0075] 52) Recovery of first-round PCR amplification products using magnetic beads:

[0076] a. Take 25 μL of the PCR amplification product from step 51) into a PCR tube, add 1.0 times the volume of magnetic beads, shake to suspend fully, let stand for 5 min, place on a magnetic rack to adsorb until the liquid is clear, and carefully aspirate the supernatant with a pipette.

[0077] b. Add 90 μL of 80% ethanol, place the tube upside down on a magnetic rack so that the magnetic beads are adsorbed onto the other side of the PCR tube. Repeat this process 2-3 times until the liquid is clear and the supernatant is removed.

[0078] c. Heat the PCR tube in a 55°C dry bath for 5 minutes to allow the alcohol inside to evaporate completely;

[0079] d. Add 30 μL of Elution Buffer for elution;

[0080] e. Place the PCR tube on the adsorption rack for 5 minutes to allow for complete adsorption, then transfer the supernatant into a clean 1.5 mL centrifuge tube;

[0081] 53) The product recovered in step 52) was quantified and its concentration was determined using Qubit 3.0 and its matching kit.

[0082] 54) Second round of PCR amplification: The reaction system is as follows: PCR-mix2 10μL, i5-primer (10uM) 1μL, i7-primer (10uM) 1μL, PCR-DNA (previous round amplification product) 30ng, add water to 30μL; the reaction procedure is shown in the table below.

[0083]

[0084] 55) Recovering second-round PCR products using magnetic beads: The specific operating steps are the same as in step 52.

[0085] 56) After quantification of the recovered second-round PCR products using Qubit 3.0, sequencing was performed using an Illumina machine with a sequencing length of PE150 or longer.

[0086] (6) Results Analysis and Evaluation

[0087] 61) Sequencing data quality detection and quality control: The Cut_QC tool of the EQMS (Eruda NGS Laboratory Quality Monitoring System, abbreviated as EQMS; registration number: 2021SR2050233) was used to trim the sequences obtained from step (5) to remove low-quality reads and adapters (the specific command is as follows: Cut_QC -f file -o output.file, where -f is the directory where the original data is located and -o is the directory where the clean data is located); at the same time, the sequencing data quality was detected, and the quality of the trimmed reads was checked to ensure that QC30 was greater than 80%;

[0088] 62) Constructing a reference genome index and sequence alignment: The Somatools analysis software (Eruton next-generation sequencing data analysis platform, abbreviated as Somatools; registration number: 2021SR1783362) was used to construct a reference genome index with a custom fragment length. After the index was constructed, MS_Seeker was used to perform sequence alignment. After the alignment was completed, a bam file was generated, which is the alignment result file.

[0089] 63) Calculate the methylation level of each base site: Continue to use the analysis software Somatools to read in the alignment result file obtained in step 62), count the methylation site information in the file, and then generate a file in which each column is in turn the basic sequencing information, gene location information (including chromosome, start and end positions), methylation status (+ / present, - / absent), and methylation call (uppercase represents methylation).

[0090] 64) Result evaluation: The sequencing results of step 63) are evaluated by using a methylation prediction model constructed with machine learning to obtain the overall frequency of methylation of each gene, the frequency statistics of each site, and the probability that the sample may have colorectal cancer.

[0091] Furthermore, by analyzing blood samples from 30 patients diagnosed with colorectal cancer and 30 healthy individuals undergoing routine physical examinations in this embodiment, predictions were made for a single gene and all 7 genes screened in this invention in these 60 samples. At the same time, endoscopic gold standard tests were performed as a reference. The results are shown in Tables 8-15 below.

[0092] Table 8. Septin9 gene detection results

[0093]

[0094] Table 9 FBXO5 gene detection results

[0095]

[0096] Table 10 eIF6 gene detection results

[0097]

[0098] Table 11 KIF2C gene detection results

[0099]

[0100] Table 12 Results of IGF2BP3 gene detection

[0101]

[0102] Table 13 CBX3 gene detection results

[0103]

[0104] Table 14 ALKBH5 gene detection results

[0105]

[0106] Table 15 Results of 7 Gene Detection

[0107]

[0108] As shown in Tables 8 to 15, compared with using only one single gene among FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5, and Septin9 to jointly detect the methylation level of the 7 genes in this invention, the detection sensitivity of colorectal cancer is increased to 90.00%, and the detection specificity reaches 91.67%, which means that this invention has high sensitivity and specificity.

[0109] Example 4: Accuracy Verification

[0110] Using colorectal cancer methylation data and corresponding clinical survival data from the TCGA database, the data were normalized; the methylation levels of Septin9 alone and the seven genes screened using this invention were compared with the probability of colorectal cancer occurrence, and ROC curves were plotted. The ROC curves for predicting colorectal cancer using the methylation levels of the seven genes in this invention are shown below. Figure 1 As shown, the ROC curve for predicting the degree of Septin9 methylation in colorectal cancer is as follows. Figure 2 As shown, combined with Figure 1 and Figure 2 This indicates that the accuracy of using the methylation levels of the seven genes in this invention to predict the probability of colorectal cancer is significantly higher than that of using only the Septin9 single gene.

[0111] In summary, this invention provides a multi-gene methylation detection kit for colorectal cancer. By jointly detecting methylation sites of the FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5, and Septin9 genes, it exhibits high sensitivity and specificity. Furthermore, its multi-gene methylation detection for colorectal cancer ensures high accuracy of the results. Since abnormal DNA methylation usually occurs in the early stages of cancer, the detection of DNA methylation indicators can serve as an important indicator for early screening, diagnosis, and prognostic assessment of colorectal cancer.

[0112] Furthermore, it should be noted that all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

Claims

1. A colorectal cancer multi-gene methylation detection kit, characterized in that: The colorectal cancer gene methylation detection sites detected by the kit include FBXO5, eIF6, KIF2C, IGF2BP3, CBX3, ALKBH5 and Septin9; and the kit comprises the primer combinations shown in 1) to 7) as follows: 1) The primer pairs for FBXO5 methylation detection include primer pair 1, primer pair 2, primer pair 3 and primer pair 4 shown in SEQ ID NO. 1 to 8; 2) The primer pairs for eIF6 methylation detection include primer pair 5, primer pair 6, primer pair 7, primer pair 8 and primer pair 9 shown in SEQ ID NO. 9 to 18; 3) The primer pairs for KIF2C methylation detection include primer pair 10, primer pair 11, primer pair 12, primer pair 13 and primer pair 14 shown in SEQ ID NO. 19 to 28; 4) The primer pairs for IGF2BP3 methylation detection include primer pair 15, primer pair 16, primer pair 17 and primer pair 18 shown in SEQ ID NO. 29 to 36; 5) The primer pairs for CBX3 methylation detection include primer pair 19, primer pair 20 and primer pair 21 shown in SEQ ID NO. 37 to 42; 6) The primer pairs for ALKBH5 methylation detection include primer pair 22, primer pair 23, primer pair 24 and primer pair 25 shown in SEQ ID NO. 43 to 50; 7) The primer pairs for Septin9 methylation detection include primer pair 26 and primer pair 27 shown in SEQ ID NO. 51 to 54.

2. The detection kit for colorectal cancer multi-gene methylation according to claim 1, characterized in that: The sample type detected by the kit is an ex vivo blood sample.

3. The kit of claim 1, wherein the kit comprises at least one primer or probe selected from the group consisting of SEQ ID NOs: 1- 12. The kit detects the methylation state of the detection sites by NGS detection.

4. The kit of claim 1, wherein the kit comprises at least one primer or probe selected from the group consisting of SEQ ID NOs: 1- 12. The first round of reaction system and reaction procedure of the kit are as follows: The first round of PCR amplification reaction system is: PCR mix1 15 μL, Panel 8 μL, and sulfite-treated cfDNA 7 μL; the reaction procedure is as shown in the following table: ； The Panel is the primer combination shown in 1) to 7) of claim 1.

5. The kit of claim 1, wherein the kit comprises at least one primer or probe selected from the group consisting of SEQ ID NOs: 1- 12. The second round of reaction system and reaction procedure of the kit are as follows: The second round of reaction system is: PCR-mix2 10 μL, i5-primer 1 μL with a concentration of 10 μM, i7-primer 1 μL with a concentration of 10 μM, 30 ng of the amplification product of the previous round, and water to 30 μL; the reaction procedure is as shown in the following table: 。

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