WO2021072786A1 - 人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用 - Google Patents

人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用 Download PDF

Info

Publication number
WO2021072786A1
WO2021072786A1 PCT/CN2019/112439 CN2019112439W WO2021072786A1 WO 2021072786 A1 WO2021072786 A1 WO 2021072786A1 CN 2019112439 W CN2019112439 W CN 2019112439W WO 2021072786 A1 WO2021072786 A1 WO 2021072786A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
probe
methylation
colorectal cancer
gene
Prior art date
Application number
PCT/CN2019/112439
Other languages
English (en)
French (fr)
Inventor
颜宏利
杨璐平
宋立华
廖清华
Original Assignee
厦门信道生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门信道生物技术有限公司 filed Critical 厦门信道生物技术有限公司
Priority to US17/767,765 priority Critical patent/US20230242990A1/en
Priority to EP19949066.5A priority patent/EP4047101A4/en
Publication of WO2021072786A1 publication Critical patent/WO2021072786A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to the field of biotechnology, in particular to a primer set, reagents, kits and applications thereof for detecting methylation of specific regions of human colorectal cancer-related genes from stool, blood or tissues by using specific PCR, and are suitable for detecting humans Whether there is methylation in specific regions of colorectal cancer and precancerous adenoma-related genes in the genome.
  • Colorectal cancer (Colorectal cancer, colorectal cancer), also known as colorectal cancer and advanced adenoma, is a common malignant tumor of the digestive system.
  • the current research results believe that the occurrence of colorectal cancer is not only related to genetic factors, but also has obvious family inheritance. Tendency is also related to the role of various environmental factors.
  • Colorectal mucosal epithelial cells produce genetic and epigenetic changes under the combined action of many factors, acquire the ability of malignant proliferation, invasion, infiltration and metastasis, form masses in local areas, and form metastases in lymph nodes and other tissues and organs .
  • Stool testing includes fecal occult blood test (FOBT) and fecal DNA testing (stool DNA testing, sDNA); colon structural testing includes flexible sigmoidoscopy (FSIG), colonoscopy (CS), Double contrast barium enema (DCBE) and CT colonoscopy (CTC).
  • FOG flexible sigmoidoscopy
  • CS colonoscopy
  • DCBE Double contrast barium enema
  • CTC CT colonoscopy
  • Fecal occult blood detection FOBT is a simple and rapid screening method for colorectal cancer and advanced adenomas, used to detect trace hemoglobin in feces.
  • the FOBT test is simple, inexpensive, non-invasive, and can be used for large-scale screening studies, but it has many influencing factors.
  • Sigmoidoscopy is a method of directly inspecting the distal colon using endoscopic technology. It is relatively simple to operate, requires relatively low bowel preparation, and does not require intraoperative sedation. It can be used for biopsy and treatment of lesions. It is a relatively cheap, Simple and safe screening method; however, the examination site of FSIG is limited to the distal large intestine (with the splenic flexure as the boundary).
  • Colonoscopy is currently the gold standard for the diagnosis and treatment of colorectal cancer and advanced adenomas. It can conduct a complete observation of the entire colon, and can perform tissue biopsy and excision of polyps found.
  • CS has the advantages of good curative effect and high accuracy, it also has certain limitations: (1) CS is an invasive examination method, which has a higher risk of complications and may cause colon perforation (0.3%) or even death (1/5000); (2) It takes a long time and requires sufficient bowel preparation and sedation before the examination; (3) The price is more expensive, if a tissue biopsy is required to remove the polyps found, it will cost more; (4) Colon The missed diagnosis rate of colorectal cancer and advanced adenoma by microscope was 5%.
  • CT colonography CTC is a non-invasive examination method, which can perform tomography with a thickness of 1-2mm, and then examine intestinal lesions on two-dimensional and three-dimensional images.
  • CTC has the advantages of fast, accurate and low complications, and its screening efficiency for large polyps, colorectal cancer and advanced adenomas is close to that of CS.
  • CTC has obvious shortcomings: in order to obtain satisfactory images, it is still necessary to release 2-4 rads of X-rays to the patient’s abdomen during the CTC examination, which increases the risk of cancer; Injecting air or carbon dioxide into the intestinal cavity to expand the intestines will cause some discomfort during the examination. In addition, the high examination costs will bring a heavier economic burden to both individuals and society.
  • Stool exfoliated cells and genetic testing is currently the most cutting-edge technology for colorectal cancer and advanced adenoma screening. It is a non-invasive screening method. Compared with normal colorectal mucosa, colorectal cancer and advanced adenoma The renewal metabolism of adenoma cells increases significantly, and the number of exfoliated cells increases. Quantitative analysis of the DNA of fecal exfoliated cells can effectively screen some colorectal cancer and advanced adenomas.
  • the method generally includes two types of tests: one is fecal exfoliated cytology: the intestinal epithelial cells shed in the feces are subjected to routine pathological examinations in order to find out the tumor cells in them, so as to achieve a clear intestinal tumor. diagnosis.
  • the second category is the detection of fecal exfoliated cell DNA: because studies have shown that the mutated genes in the tumor cells shed from the patient's feces are highly consistent with the mutated genes in the tumor tissue itself.
  • the diagnosis of intestinal tumors can also be clear, and the method has high specificity, and a positive result can basically confirm the existence of the disease; no change is required Screen the eating habits of the population; there is no need to restrict the use of drugs, etc.
  • the PRDM12 gene (PRDI-BF1 and RIZ homology domain-containing protein 12) is located in human chromosome 9 9q34.12 (chr9:130,664,594-130,682,997,hg38), and belongs to a family of transcriptional regulators containing PR and SET protein domains ( PR/SET domain family).
  • the PRDM12 encoded protein contains the N-terminal PRDI-BF1 and RIZ homology domain (PR), one SET domain, and the C-terminal three C2H2 zinc finger DNA binding domains.
  • PRDM12 is an important transcriptional regulator that controls neural differentiation and formation, and is related to the occurrence of solid tumors and hematological malignancies. At present, there is no research report on the relationship between the degree of methylation in a specific region of the PRDM12 gene and tumorigenesis, nor has there been any report on the quantitative detection of the degree of methylation of PRDM12.
  • the FOXE1 gene (Forkhead Box E1) is located in human chromosome 9 9q22.33 (chr9:97,853,254-97,856,715, hg38). It is a member of the forkhead family of transcription factors and contains the forkhead domain shared by the genes of the family. (Forkhead domain), this domain is composed of more than 100 amino acids, which can recognize and bind to specific sequences of genomic DNA. Previous studies have shown that FOXE1 is an important transcription factor and a forkhead transcription factor specifically expressed by the thyroid. It is essential for thyroid morphogenesis and differentiation, and is also involved in the transcriptional regulation induced by thyroid hormone.
  • FOXE1 is directly related to the occurrence of thyroid cancer, pancreatic cancer, and skin squamous cell carcinoma.
  • FOXE1 gene hypermethylation has also been reported in skin squamous cell carcinoma and breast cancer.
  • FOXE1 gene hypermethylation has also been reported in skin squamous cell carcinoma and breast cancer.
  • the B3GAT2 gene (Beta-1,3-Glucuronyltransferase 2) is located on human chromosome 6 6q13 (chr6:70,856,679-70,957,189, hg38). It belongs to the glucuronyltransferase family member and encodes a transmembrane protein. The function is mainly It catalyzes the transfer of Beta-1,3-linked glucuronic acid to the galactosyl group at the end of Gal-Beta-1-4 GLcNAc or Gal-Beta-1-3 GLcNAc residues contained in various glycoproteins or glycolipids.
  • B3GAT2 participates in the synthesis of HNK-1 sugar chain epitopes, is related to cell recognition, adhesion, and migration in the nervous system, and has a regulatory effect on the growth and development of the nervous system, neuronal regeneration, and synaptic plasticity.
  • B3GAT2 gene is related to tumorigenesis at home and abroad.
  • the methylation of a specific region of the B3GAT2 gene is related to the occurrence of Barrett's Esophagus, which is useful as a diagnostic biomarker for this disease.
  • Barrett’s esophagus may develop into esophageal cancer, the probability of canceration is very low.
  • Vimentin gene is located on chromosome 10 10P13 (chr10:17,229,548-17,229,607), which belongs to the type III intermediate filaggrin gene. It is a member of the intermediate fibroin family and exhibits complex gene expression patterns in many aspects. Mainly expressed in embryonic tissues and adult cells derived from mesenchymal tissues. More and more studies have shown that the expression of Vimentin is not a specific biomarker for mesenchymal origin cells and tumor cells that differentiate into mesenchymal cells.
  • Keratin and Vimentin have been found to be co-expressed in breast cancer, hepatocellular carcinoma, prostate cancer, endometrial cancer, gastric cancer, esophageal cancer, colon cancer, lung cancer and other epithelial malignant tumors, and they are highly invasive with cancer cells. It is closely related to Easy Transfer. Other studies have confirmed that the down-regulation of Vimentin expression related to tumors and precancerous lesions (such as adenomas) may be closely related to the methylation of its promoter. This suggests that DNA methylation can down-regulate the expression of Vimentin from the level of gene transcription, and it can promote tumor cell migration. Chen et al.
  • the SFRP2 gene is located in chromosome 4q31.3 (chr4:153,789,388-153,789,447).
  • the SFRP2 protein encoded by it contains a characteristic cysteine-rich region.
  • Many studies have shown that hypermethylation of the SFRP2 gene can cause the gene to increase Silent expression in this kind of tumor.
  • the SFRP2 gene contains two introns and three exons. There are high-density CpG islands around the first exon of the SFRP2 gene. This CpG island is closely related to DNA hypermethylation of tumor cells. . In recent years, many studies have found that SFRP2 gene hypermethylation is involved in the occurrence of colorectal cancer.
  • the first objective of the present invention is to provide a primer set for methylation of a specific region of a human colorectal cancer-related gene.
  • the primer set includes PRDM12, FOXE1, B3GAT2, Vimentin, and SFRP2 -1.
  • the detection primer set of the PRDM12 gene is: forward primer F: 5'-GTTAGATTAGAAGTATAAAATGTG-3'; hereinafter referred to as: primer 1; reverse primer R: 5'-ATCCCATTCCTCTCCTCC- 3'; hereinafter referred to as: primer 2; detection probe P: 5'-AGCGCGGTGGAGATTTG-3'; hereinafter referred to as: probe 1;
  • the detection primer set of the FOXE1 gene is: forward primer F: 5'-CGAGTttAAGtttTTGGtAGAGG-3'; hereinafter referred to as: primer 3; reverse primer R: 5'-CCGATCGACTaAaaCCCGa- 3'; hereinafter referred to as: primer 4; detection probe P: 5'-tCGAGTTCGGGCGtTGAGG-3'; hereinafter referred to as: probe 2;
  • the detection primer set of the B3GAT2 gene is: forward primer F: 5'-TtCGtCGGGTtTGGCG-3'; hereinafter referred to as: primer 5; reverse primer R: 5'-CAaaAaaTCGTaCAaCCCCG- 3'; hereinafter referred to as: primer 6; detection probe P: 5'-tCGCGAGtAAGtTCGGGAG-3'; hereinafter referred to as: probe 3;
  • the detection primer set of the Vimentin gene is: forward primer F: 5'-GtCGtAGttTtTACGttTCGT-3'; hereinafter referred to as: primer 7; reverse primer R: 5'-aaCGCACGaCAaAaaAaCG- 3'; hereinafter referred to as: primer 8; detection probe P: 5'-ttCGGGCGGCGTGTATG-3'; hereinafter referred to as: probe 4;
  • the detection primer set of the SFRP2-1 gene the specific primer probe set sequence is: forward primer F: 5'-CGGAtTGGGGtAAAAtAAGttC-3'; hereinafter referred to as: primer 9; reverse primer R: 5'- CTaaCGaaAACGCGCCTA-3'; hereafter called: primer 10; detection probe P: 5'-TAGGCGCGTTttCGttAGTAttTGG-3'; hereafter called: probe 5;
  • the detection primer set for the SFRP2-2 gene has a specific primer probe set sequence: forward primer F: 5'-AATGtAGtCGGCGCG-3'; hereinafter referred to as: primer 11; reverse primer R: 5'- CCTTaTTaaaAaTTCAAaaAaCCCG-3'; hereafter called: primer 12; detection probe P: 5'-AGttAtTTtCGGGCGTGCGGt-3'; hereafter called: probe 6;
  • primer set for internal reference ACTB gene.
  • the sequence of the detection primer probe set is: forward primer: 5'-GTGATGGAGGAGGTTTAGTAAG-3'; hereinafter referred to as: primer 13; reverse primer: 5'-CAATAAAACCTACTCCTCCCTT-3'; Hereinafter referred to as: primer 14; detection probe P: 5'-TGTGTTTGTTATTGTGTGTTGGGTGGT-3'; hereafter referred to as: probe 7.
  • the fluorescent reporter group at the 5'end of the detection probe includes: ALEX-350, FAM, VIC, TET, CAL Fluor Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, Cal Fluor Red 590, ROX, CAL Fluor 20 Red 610, TEXAS RED, CAL Floral Red 635, Quasar 670, Cy3, Cy5, Cy5.5 or Quasar 705; the quenching group at the 3'end includes TAMRA, BHQ1, BHQ2, MGB.
  • Another object of the present invention is to provide reagents for detecting methylation in specific regions of human colorectal cancer-related genes.
  • the reagents include 6 PCR systems, namely CRC PCR Mix-A/B/C/D/E/F; Each system includes the specific primer probe set for the methylation of the specific region of the corresponding gene, and the internal reference gene specific primer probe set.
  • the reagent system A includes the PRDM12 forward primer F (primer 1), PRDM12 reverse primer R (primer 2), PRDM12 detection probe P (probe 1);
  • the reagent system B includes FOXE1 forward primer F (primer 3), FOXE1 reverse primer R (primer 4), FOXE1 detection Probe P (probe 2);
  • the reagent system C includes B3GAT2 forward primer F (primer 5), B3GAT2 reverse primer R (primer 6), B3GAT2 detection probe P (probe 3);
  • Reagent system D includes Vimentin forward primer F (primer 7), Vimentin reverse primer R (primer 8), and Vimentin detection probe P (probe 4);
  • the reagent system E includes SFRP2-1 forward primer F ( Primer 9), SFRP2-1 reverse primer R (primer 10), SFRP2-1 detection probe P (probe 5);
  • the reagent system F includes SFRP2-2 forward primer F (primer 11), SFRP2- 2 Reverse primer R (primer
  • the six reagent systems CRC PCR Mix also include common components such as PCR buffer, dNTPs, and MgCl 2 .
  • the internal reference gene is the human housekeeping gene ACTB gene.
  • the primers and probes of the internal reference gene are used to monitor the quality of each sample and its amplification in each reaction system, so as to avoid false negatives or partial Suppress results.
  • the final volume of each system is between 20-40 ⁇ l, and the reaction system A/B/C/
  • the final concentration of the primers in D/E/F is: the forward primer F is between 0.3 and 0.8 ⁇ M, the reverse primer R is between 0.3 and 0.8 ⁇ M, and the probe P concentration is between 0.1 and 0.3 ⁇ M;
  • the final concentration of the common components in each of the 6 reaction systems is: dATP, dTTP, dCTP, and dGTP in the dNTPs are all 0.1-0.3 mM, and the MgCl2 solution is 1-2 mM.
  • the HS hot-start enzyme also includes a positive control, a negative control and a blank control;
  • the positive control is a mixture of the methylated plasmid DNA fragments of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 and the internal reference ACTB gene, Or it is a human whole genome DNA methylation positive control;
  • the negative control is a mixture of unmethylated plasmid DNA fragments of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 and the internal reference ACTB gene, or a human whole genome DNA methylation-free negative control;
  • Another object of the present invention is to provide an application of a methylation detection kit for a specific region of a human colorectal cancer-related gene.
  • DNA is directly extracted from human feces, blood or tissues, and the DNA is transformed by bisulfite treatment.
  • DNA as a template, amplified by PCR, and accumulated signals on the detection probe to achieve real-time detection of methylation in specific regions of human colorectal cancer related PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, and SFRP2-2 genes ;
  • ACTB gene is used as an internal reference gene; including the following PCR specific experimental steps:
  • Step 1 Collect a certain number of stool, blood or tissue samples from patients with colorectal cancer and patients with advanced adenoma;
  • Step 2 Extract human genomic DNA from stool, blood or tissue samples of the person to be tested;
  • Step 3 The extracted human genomic DNA is treated with bisulfite and transformed, and used as a template for PCR reaction;
  • Step 5 Dispense 30 ⁇ l per tube into PCR tubes, then add 10 ⁇ l of the transformed DNA template to be tested, the total volume is 40 ⁇ l;
  • Step 6 Perform amplification reaction.
  • the reaction procedure is:
  • Step 7 PCR result determination: In the process of sample detection, the baseline is determined as: take 3-15 cycles of fluorescence signal; threshold setting: make the threshold line exceed the highest point of the normal negative control amplification curve, and The Ct value is Undet; according to the reported Ct value, that is, the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold, the positive or negative result of the sample is judged.
  • the present invention identifies a new gene specific region methylation as a tumor marker for human colorectal cancer, which can be used for early patient screening and auxiliary detection.
  • the kit of the present invention selects suitable regions of the target gene and designs highly specific primers and probes.
  • the kit of the present invention can directly use feces to extract DNA, which is non-invasive and reduces the suffering of patients.
  • the present invention discloses for the first time a kit that can be used to detect PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, and SFRP2-2 gene methylation related to colorectal cancer and advanced adenoma and its application.
  • the detection kit contains the methylation-specific amplification forward primers, reverse primers and fluorescent probes of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 genes, PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2 -1.
  • the methylation level of SFRP2-2 gene leads to the low expression of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 genes in patients with colorectal cancer and advanced adenoma, leading to colorectal cancer and advanced adenoma Cells have a stronger ability to invade. Therefore, the methylation of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 genes can be used as molecular biology for evaluating the malignancy and prognosis of colorectal cancer and advanced adenomas mark.
  • Diagnostic kits based on the detection of colorectal cancer and advanced adenomas PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 gene methylation levels can quickly and easily achieve the molecular level of colorectal cancer and The detection of advanced adenomas can judge the malignancy and prognosis of colorectal cancer and advanced adenomas.
  • the detection efficiency is high, the target is strong, and it has the advantages of accuracy, reliability, flexibility, rapidity and economy, which is beneficial to colorectal cancer and advanced stage. Early detection and timely treatment of adenomas.
  • this kit uses methylation-specific PCR method to detect colorectal cancer by detecting the DNA of fecal exfoliated cells And advanced adenomas have the following advantages:
  • test kit 5 The operation is convenient. After obtaining the test kit, the user can complete the sample collection work at home, and then send it by express delivery to a professional for testing.
  • This method uses Real-time PCR, which is developed on the basis of ordinary polymerase chain reaction technology, and uses fluorescent dyes or electrophoresis detection in the ordinary polymerase chain reaction amplification process For quantification, the application of the probe method makes the technology more accurate, sensitive and high-throughput, and does not require operations such as electrophoresis and hybridization after PCR, reducing pollution and operating errors.
  • Figure 1 shows the results of PRDM12 methylation detection
  • Figure 2 shows the result of FOXE1 methylation detection
  • Figure 3 is a graph of B3GAT2 methylation detection results
  • Figure 4 shows the result of Vimentin methylation detection
  • Figure 5 shows the result of methylation detection of SFRP2-1
  • Figure 6 shows the result of methylation detection of SFRP2-2.
  • the feces of 31 patients with colorectal cancer from Shanghai Changhai Hospital and the feces of corresponding patients were collected as the research objects.
  • the diagnosis of the cases used is determined based on the patient's clinical data, imaging studies, colonoscopy and postoperative pathology.
  • the collected stool samples are stored in a collection tube containing stool DNA preservation solution and placed in a refrigerator at -20 degrees. Take an appropriate amount of fecal samples to extract genomic DNA.
  • the tissue samples were extracted using QIAamp DNA Mini Kit tissue whole-genome DNA extraction kit (Qiagen, Germany) to extract genomic DNA from cancerous tissues and adjacent tissues.
  • Stool samples use QIAamp DNA Stool Mini Kit stool genomic DNA extraction kit.
  • concentration and quality of genomic DNA were detected by NanoDrop2000 micro spectrophotometer (Thermo Fisher Scientific, USA) for subsequent PCR detection of methylation of specific regions of genes.
  • the specific extraction steps are carried out according to the extraction kit operating instructions.
  • the positive control is a mixture of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 methylated plasmid DNA fragments and the internal reference ACTB gene (or a human whole genome DNA methylation positive control);
  • the negative control It is a mixture of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 unmethylated plasmid DNA fragments and internal reference ACTB gene (or human whole genome DNA non-methylation negative control);
  • the blank control is TE buffer Liquid or nuclease-free water.
  • the obtained genomic DNA of stool, blood or tissues use ZYMO EZ DNA Methylation Gold Kit (ZYMO Company, the United States) DNA sulfite conversion kit, operate according to the instructions, after sulfonation, hydrolysis, deamination, and desulfonation
  • the three main steps are to convert unmethylated cytosine (C) into uracil (U), while methylated cytosine (5mC) cannot be converted.
  • the genomic DNA of the sample to be tested is transformed by sulfite and used as a template for methylation PCR detection.
  • the positive control and the negative control are genetically engineered synthetic plasmids.
  • the present invention simultaneously designs and verifies multiple pairs of primer and probe combination sequences for the methylated nucleic acid sequences of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, SFRP2-2 target gene fragments related to colorectal cancer and advanced adenoma
  • Table 1 a set of primer probe sequences for detecting the methylation of PRDM12, FOXE1, B3GAT2, Vimentin, SFRP2-1, and SFRP2-2 gene fragments were selected.
  • the specific primers and probe sequences are shown in Table 1:
  • the PCR reaction system includes the above-mentioned specific primers, and is divided into 6 reaction systems (A/B/C/D/E/F), of which:
  • Reaction system A includes PRDM12 forward primer (primer 1), PRDM12 reverse primer (primer 2), PRDM12 detection probe (probe 1);
  • reaction system B includes FOXE1 forward primer (primer 3), FOXE1 reverse primer ( Primer 4), FOXE1 detection probe (probe 2);
  • reaction system C includes B3GAT2 forward primer (primer 5), B3GAT2 reverse primer (primer 6), B3GAT2 detection probe (probe 3);
  • reaction system D includes Vimentin forward primer (primer 7), Vimentin reverse primer (primer 8), Vimentin detection probe (probe 4);
  • reaction system E includes SFRP2-1 forward primer (primer 9), SFRP2-1 reverse primer ( Primer 10), SFRP2-1 detection probe (probe 5);
  • reaction system F includes SFRP2-2 forward primer (primer 11), SFRP2-2 reverse primer (primer 12), SFRP2-2 detection probe (probe Pin 6).
  • the PCR reaction system also includes internal reference gene ACTB forward primer (primer 13), ACTB reverse primer (primer 14), ACTB detection probe (probe 7), PCR buffer, dNTPs, Taq HS enzyme, and MgCl 2 .
  • the use of the kit includes the following steps:
  • Genomic DNA is extracted from feces, blood or tissue samples and transformed by bisulfite treatment as a template for PCR reaction.
  • the baseline is determined as follows: take 3-15 cycles of fluorescence signal; threshold setting: make the threshold line exceed the highest point of the normal negative control amplification curve (irregular noise line), and The Ct value is Undet. Determine the positive or negative result of the sample based on the reported Ct value (the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold).
  • kits of the present invention can quickly and accurately detect the mutant gene and the normal gene in the fecal exfoliated cells of colorectal cancer and advanced adenoma. It can be seen that the kit of the present invention is not only fast and efficient, but also the interpretation of the results is very clear and intuitive, and the results are reliable and specific.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

一种利用特异性PCR从粪便、血液或组织中检测人结直肠癌相关基因特定区域甲基化的引物组、试剂、试剂盒及其应用，所述引物组包括基因标志物PRDM12、FOXE1、B3GAT2基因特定区域甲基化区域的特异性扩增引物探针序列，所述引物探针组还包含Vimentin、SFRP2-1、SFRP2-2基因特定区域甲基化区域的特异性扩增引物探针序列和内参基因ACTB的特异性扩增引物探针组，检测效率高，针对性强。

Description

人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用 技术领域：
本发明涉及生物技术领域，特别涉及一种利用特异性PCR从粪便、血液或组织中检测人结直肠癌相关基因特定区域甲基化的引物组、试剂、试剂盒及其应用，适用于检测人类基因组中是否存在结直肠癌及癌前腺瘤相关基因特定区域甲基化情况。
背景技术：
结直肠癌(Colorectal cancer,结直肠癌)又称结直肠癌及进展期腺瘤，是常见的消化系统恶性肿瘤，目前研究结果认为结直肠癌的发生既与遗传因素有关，有明显的家族遗传倾向，又与各种环境因素的作用有关。结直肠粘膜上皮细胞在多种因素的共同作用下，产生遗传和表观遗传的变化，获得恶性增殖、侵袭、浸润和转移的能力，在局部形成肿块，在淋巴结和其他组织器官中形成转移灶。近年来，随着我国人民生活水平提高，饮食结构和习惯改变，预期寿命延长，人口老龄化趋势等的影响，我国结直肠癌的发病率和死亡率呈直线上升的趋势，而且发病年龄提前的趋势。根据发表在《CA:A Cancer Journal for Clinicians》杂志上《2015年中国癌症统计数据》显示：结直肠癌在男性肿瘤发病率中排第5位，在女性肿瘤发病率中排第4位；结直肠癌在男性和女性肿瘤死亡率中都排在第5位。尽管目前的手术方式和辅助治疗手段不断改进，结直肠癌患者的术后生存情况有很大改善，但是患者的五年总体生存率依旧不够理想，主要因为结直肠癌患者的生存率与肿瘤分期密切相关，早期结直肠癌患者术后5年生存率超过90％，但大多数患者出现症状或发现疾病时已发展到肿瘤中、晚期。由于93％的结直肠癌源于腺瘤，早期多无特征性症状，而从腺瘤发展到癌症需要3～17年，这就为早期筛查提供了时间基础。因此，对结直肠癌高危 人群的筛查，对结直肠癌患者的早发现、早诊断，从而实现结直肠癌患者早治疗，是提高患者总体生存率的重要方面。
目前结直肠癌及进展期腺瘤筛查方法主要分为粪便检测和结肠结构性检测两大类。粪便检测包括粪便潜血检测(fecal occult blood test，FOBT)及粪便DNA检测(stool DNA testing，sDNA)；结肠结构性检测包括乙状结肠镜检查(flexible sigmoidoscopy，FSIG)、结肠镜检查(colonoscopy，CS)、结肠气钡双重造影(double contrast barium enema，DCBE)及CT结肠成像(CT colonoscopy，CTC)。粪便潜血检测FOBT是一种简单、快速的结直肠癌及进展期腺瘤筛查方法，用于检测粪便中的微量血红蛋白。FOBT检测简便、廉价、无创，可用于大规模筛查研究，但其影响因素较多，需在肿瘤形成后破溃出血的情况下才能检测到阳性结果，而息肉或癌组织的出血常呈间歇性，不可能被任意粪便标本所包含，因此，其特异性不高，容易出现漏诊。乙状结肠镜检查(FSIG)是运用内镜技术直接检查远端结肠的方法，操作较简单，肠道准备要求相对较低，无需术中镇静，可对病变进行活检和治疗，是一种相对廉价、简便又安全的筛查方法；然而FSIG的检查部位仅限于远端大肠(以脾曲为界)，如果FSIG发现远端大肠肿瘤后才进行结肠镜检查，将会造成72.0％的近端大肠肿瘤漏诊。结肠镜检查(CS)是目前结直肠癌及进展期腺瘤诊断与治疗的金标准，可以对整个结肠进行完整的观察，并能进行组织活检以及切除发现的息肉。尽管CS具有疗效好、准确性高等优点，但也有一定的局限性：(1)CS是一种侵入性的检查方法，发生并发症的风险更高，可能会引起结肠穿孔(0.3％)甚至死亡(1/5000)；(2)耗时长，在检查前需要充分的肠道准备和镇静处理；(3)价格较贵，如果需要组织活检并切除发现的息肉则花费更多；(4)结肠镜对结直肠癌及进展期腺瘤的漏诊率为5％。CT结肠成像(CTC)属于非侵入性检查方法，能以1-2mm 的厚度进行断层扫描，进而在二维和三维图像上检查肠道病变。CTC具有快速、准确及并发症低等优点，对较大息肉及结直肠癌及进展期腺瘤的筛查效能接近于CS。但作为一种常规的筛查方法，CTC存在明显的不足：为了获得令人满意的影像，CTC检查过程中仍需释放2-4 rads X射线到患者腹部，增加患癌的风险；通常会向肠腔内注入空气或二氧化碳以扩张肠道，患者检查时会有一定的不适；此外，高额的检查费用，不管对个人还是社会都会带来更重的经济负担。
粪便脱落细胞及基因检测(sDNA)是目前结直肠癌及进展期腺瘤筛查的最前沿技术，是一种非侵入性的筛查方法，与正常大肠黏膜相比，结直肠癌及进展期腺瘤细胞更新代谢速度明显增快，脱落细胞随之增加，通过对粪便脱落细胞DNA定量分析，可有效筛查出部分结直肠癌及进展期腺瘤。该法总体包括两类的检测：一类是粪便脱落细胞学的检测：就是将粪便当中脱落的肠道上皮细胞进行常规的病理学检查以期找出其中的肿瘤细胞，从而达到明确肠道肿瘤的诊断。第二类是粪便脱落细胞DNA的检测：因为有研究表明，患者粪便中脱落的肿瘤细胞中的突变基因和肿瘤组织本身的突变基因有很高的一致性。如果能从粪便中脱落的肿瘤细胞中检测出该类基因，那么肠道肿瘤的诊断也可明确，而且该法还具有有很高的特异性，阳性结果基本能确诊病变的存在；不需要改变筛查人群的饮食习惯；不需要限制药物的使用等。
PRDM12基因(PRDI-BF1 and RIZ homology domain-containing protein 12)位于人第9号染色体9q34.12中(chr9:130,664,594-130,682,997,hg38)，属于一个含有PR和SET蛋白结构域的转录调控因子家族(PR/SET domain family)。PRDM12编码蛋白含有N端的PRDI-BF1和RIZ同源结构域(PR)、1个SET结构域、C端的3个C2H2锌指DNA结合域。国内外研究表明，PRDM12是重要的转录调节因子，控制神经分化和形成，与实体肿瘤和血液恶性肿瘤发生相 关。目前，国内外还未见PRDM12基因特定区域甲基化程度与肿瘤发生之间关系的研究报道，也未见任何关于定量检测PRDM12甲基化程度的检测试剂盒相关研究报道。
FOXE1基因(Forkhead Box E1)位于人第9号染色体9q22.33中(chr9:97,853,254-97,856,715，hg38)，是叉头转录因子家族的成员(Forkhead family)，含有该家族基因共有的叉头结构域(Forkhead domain)，此结构域由100多个氨基酸构成，可以识别并结合基因组DNA特定序列。既往研究表明，FOXE1是重要转录因子，是甲状腺特异性表达的叉头转录因子，对甲状腺形态发生、分化至关重要，还参与甲状腺激素诱导的转录调控。FOXE1与甲状腺癌、胰腺癌、皮肤鳞状细胞癌的发生直接相关，FOXE1基因高甲基化在皮肤鳞状细胞癌、乳腺癌中也有报道。尽管FOXE1在结直肠癌中的作用尚不清楚，但最新的研究发现，FOXE1基因异常高甲基化造成基因低表达，与结直肠癌转移和患者预后相关，有作为结直肠癌检测的生物标志物(biomarker)的潜质。尽管上述研究中有检测FOXE1基因特定区域甲基化的报道，但是目前国内外未见有定量检测FOXE1基因特定区域甲基化程度的检测试剂盒的报道。
B3GAT2基因(Beta-1,3-Glucuronyltransferase 2)位于人第6号染色体6q13中(chr6:70,856,679-70,957,189，hg38)，属于葡糖醛酸基转移酶家族成员，编码一个跨膜蛋白，功能主要是催化Beta-1,3连接的葡萄糖醛酸转移到多种糖蛋白或糖脂含有的Gal-Beta-1-4 GLcNAc或Gal-Beta-1-3 GLcNAc残基末端的半乳糖基上。已有研究发现，B3GAT2参与HNK-1糖链表位的合成，与神经系统的细胞识别、黏附、迁移有关,对神经系统生长发育、神经元再生、突触可塑性均有调节作用。目前国内外未见B3GAT2基因与肿瘤发生相关的研究报道，仅有一例研究发现B3GAT2基因特定区域甲基化与巴雷特氏食管(Barrett's  Esophagus)发生有关，有作为此疾病诊断生物学标志物的潜质，而巴雷特氏食管虽然有发展为食道癌的可能，但是癌变几率很低。目前，国内外未见有定量检测B3GAT2基因特定区域甲基化程度的检测试剂盒的报道。
Vimentin基因位于10号染色体10P13(chr10:17,229,548-17,229,607),属于第Ⅲ型中间丝纤维蛋白基因。是中间丝纤维蛋白家族的一员，在很多方面表现出复杂的基因表达模式。主要表达于胚胎组织和由间叶组织来源的成体细胞。越来越多的研究已经表明，Vimentin的表达并非是间充质起源细胞及向间叶分化的肿瘤细胞的特异性生物标志。已经发现角蛋白和Vimentin在乳腺癌、肝细胞肝癌、前列腺癌、子宫内膜癌、胃癌、食管癌、结肠癌、肺癌等上皮源性恶性肿瘤中的共同表达，而且与癌细胞的侵袭性强和易转移密切相关。另有研究证实，与肿瘤、癌前病变(如腺瘤)产生相关的Vimentin的表达下调可能与其启动子的甲基化密切相关。这提示DNA甲基化可以从基因转录水平下调Vimentin的表达，其对于肿瘤细胞迁移具有促进作用。Chen等研究显示未发生转移的早期结直肠癌及进展期腺瘤组织和晚期结直肠癌及进展期腺瘤的Vimentin基因的甲基化率是相等的，还有研究表明Vimentin基因特定区域甲基化与肿瘤大小和Duke's分期无关，这些都说明Vimentin基因特定区域甲基化检测对结直肠癌及进展期腺瘤早期诊断的重要性。
SFRP2基因位于染色体4q31.3中(chr4:153,789,388-153,789,447),其编码的SFRP2蛋白质含有一特征性半胱氨酸富含区域，许多研究表明，SFRP2基因的超甲基化会引起该基因在多种肿瘤中表达沉默。SFRP2基因包含二个内含子和三个外显子，在SFRP2基因的第一个外显子周围有着高密度的CpG岛，这个CpG岛与肿瘤细胞DNA超甲基化(DNA methylation)密切相关。近年来，许多研究发现SFRP2基因超甲基化参与了结直肠癌的发生过程。大量研究证明SFRP2甲 基化的程度是诊断结直肠癌最灵敏的标记，因此，从粪便脱落细胞中检测SFRP2基因特定区域的甲基化情况，可作为筛查结直肠癌的依据之一。
发明内容：
针对现有技术中存在的缺陷，本发明的第一个目的是提供一种人结直肠癌相关基因特定区域甲基化的引物组，所述的引物组包括PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2六个特定基因甲基化区域的任意一种基因或任意几种基因组合的甲基化引物组。
进一步，所述PRDM12基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-GTTAGATTAGAAGTATAAAATGTG-3’；以下称：引物1；反向引物R：5’-ATCCCATTCCTCTCCTCC-3’；以下称：引物2；检测探针P：5’-AGCGCGGTGGAGATTTG-3’；以下称：探针1；
进一步，所述FOXE1基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-CGAGTttAAGtttTTGGtAGAGG-3’；以下称：引物3；反向引物R：5’-CCGATCGACTaAaaCCCGa-3’；以下称：引物4；检测探针P：5’-tCGAGTTCGGGCGtTGAGG-3’；以下称：探针2；
进一步，所述B3GAT2基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-TtCGtCGGGTtTGGCG-3’；以下称：引物5；反向引物R：5’-CAaaAaaTCGTaCAaCCCCG-3’；以下称：引物6；检测探针P：5’-tCGCGAGtAAGtTCGGGAG-3’；以下称：探针3；
进一步，所述Vimentin基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-GtCGtAGttTtTACGttTCGT-3’；以下称：引物7；反向引物R：5’-aaCGCACGaCAaAaaAaCG-3’；以下称：引物8；检测探针P：5’-ttCGGGCGGCGTGTATG-3’；以下称：探针4；
进一步，所述SFRP2-1基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-CGGAtTGGGGtAAAAtAAGttC-3’；以下称：引物9；反向引物R：5’-CTaaCGaaAACGCGCCTA-3’；以下称：引物10；检测探针P：5’-TAGGCGCGTTttCGttAGTAttTGG-3’；以下称：探针5；
进一步，所述SFRP2-2基因的检测引物组，其特异性引物探针组序列为：正向引物F：5’-AATGtAGtCGGCGCG-3’；以下称：引物11；反向引物R：5’-CCTTaTTaaaAaTTCAAaaAaCCCG-3’；以下称：引物12；检测探针P：5’-AGttAtTTtCGGGCGTGCGGt-3’；以下称：探针6；
进一步，还包括内参ACTB基因的引物组，其检测引物探针组序列为：正向引物：5’-GTGATGGAGGAGGTTTAGTAAG-3’；以下称：引物13；反向引物：5’-CAATAAAACCTACTCCTCCCTT-3’；以下称：引物14；检测探针P：5’-TGTGTTTGTTATTGTGTGTTGGGTGGT-3’；以下称：探针7。
进一步，所述的检测探针5’端的荧光报告基团包括：ALEX-350、FAM、VIC、TET、CAL Fluor Gold 540、JOE、HEX、CAL Flour Orange 560、TAMRA、Cal Fluor Red 590、ROX、CAL Fluor 20 Red 610、TEXAS RED、CAL Flour Red 635、Quasar  670、Cy3、Cy5、Cy5.5或Quasar 705；3’端的淬灭基团包括TAMRA、BHQ1、BHQ2、MGB。
本发明的另一个目的是提供人结直肠癌相关基因特定区域甲基化检测试剂，所述的试剂包括6个PCR体系，分别为CRC PCR Mix-A/B/C/D/E/F；每个体系中包括如上述相应的基因特定区域甲基化情况的特异性引物探针组，内参基因特异性引物探针组，所述的试剂体系A包含PRDM12正向引物F(引物1)、PRDM12反向引物R(引物2)、PRDM12检测探针P(探针1)；所述的试剂体系B包含FOXE1正向引物F(引物3)、FOXE1反向引物R(引物4)、FOXE1检测探针P(探针2)；所述的试剂体系C包含B3GAT2正向引物F(引物5)、B3GAT2反向引物R(引物6)、B3GAT2检测探针P(探针3)；所述的试剂体系D包含Vimentin正向引物F(引物7)、Vimentin反向引物R(引物8)、Vimentin检测探针P(探针4)；所述的试剂体系E包含SFRP2-1正向引物F(引物9)、SFRP2-1反向引物R(引物10)、SFRP2-1检测探针P(探针5)；所述的试剂体系F包含SFRP2-2正向引物F(引物11)、SFRP2-2反向引物R(引物12)、SFRP2-1检测探针P(探针6)。
进一步，所述6个试剂体系CRC PCR Mix(A/B/C/D/E/F)都还包括通用成分有PCR buffer、dNTPs、MgCl 2。
进一步，所述内参基因为人类管家基因ACTB基因，所述内参基因的引物及探针，用于监控每一个样本的质量及其在每一个反应体系中的扩增情况，从而避免假阴性或者部分抑制结果的产生。
进一步，所述的六个PCR反应体系CRC PCR Mix-A/B/C/D/E/F，每个体系终体积都在20～40μl之间，所述的反应体系A/B/C/D/E/F中引物终浓度为：正向引物F均在0.3～0.8μM之间，反向引物R均在0.3～0.8μM之间，探针P 浓度均在0.1～0.3μM之间；所述6个反应体系中每个体系的通用成分终浓度为：dNTPs中的dATP、dTTP、dCTP、dGTP都为0.1～0.3mM，MgCl2溶液为1～2mM。
本发明的再一个目的是提供人结直肠癌相关基因特定区域甲基化检测试剂盒，所述的试剂盒包括上述的6个PCR体系中的任意一个或者任意几个的组合，0.5～2U Taq HS热启动酶，还包括阳性对照、阴性对照和空白对照；所述阳性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2的甲基化质粒DNA片段和内参ACTB基因的混合物，或者为人全基因组DNA甲基化阳性对照；所述阴性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2的非甲基化质粒DNA片段和内参ACTB基因的混合物，或者为人全基因组DNA无甲基化阴性对照；所述空白对照为pH＝8.5±0.1的TE缓冲液或无核酸酶水。
本发明的又一个目的是提供人结直肠癌相关基因特定区域甲基化检测试剂盒的应用，从人的粪便、血液或组织中直接提取DNA，DNA经过亚硫酸氢盐处理转化，以转化后的DNA为模板，通过PCR方法扩增，并通过检测探针上信号累积从而实现人结直肠癌相关PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因特定区域甲基化的实时检测；同时ACTB基因作为内参基因；包括如下PCR具体实验步骤：
步骤一：收集一定数量的结直肠癌患者及进展期腺瘤患者的粪便、血液或组织样本；
步骤二：从待检测人的粪便、血液或组织样本中提取人基因组DNA；
步骤三：提取的人基因组DNA经亚硫酸氢盐处理，转化后，作为PCR反应的模板；
步骤四：从试剂盒中取出29.7μl×(n+1)，PCR Mix和0.3μl×(n+1)Taq HS热启动酶混合液，混匀20s，瞬时离心10s；n＝检测样本数量。
步骤五：按每管30μl分装至PCR管中，再加入待检测的转化后DNA模板10μl，总体积40μl；
步骤六：进行扩增反应。反应程序为：
Figure PCTCN2019112439-appb-000001
步骤七：PCR结果判定：在样本的检测过程中，基线的确定为：取3～15个循环的荧光信号；阈值设定：使阀值线超过正常阴性对照品扩增曲线的最高点，且Ct值为Undet；根据所报告的Ct值，即每个反应管内的荧光信号达到设定的阈值时所经历的循环数判读样本的阳性或阴性结果。
采用上述技术方案，本发明的有益效果：
1.本发明鉴定了新的基因特定区域甲基化作为人结直肠癌的肿瘤标志物，可用于患者早期筛查和辅助检测。
2、本发明试剂盒选取靶基因适宜的区域并设计了高特异性的引物和探针。3、
3、本发明试剂盒可直接采用粪便提取DNA，因其无创，减少病人的痛苦。
4、本发明首次公开了可用于检测与结直肠癌及进展期腺瘤相关的PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因甲基化的试剂盒及其应用， 所述的检测试剂盒包含PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因的甲基化特异性扩增正向引物、反向引物及其荧光探针，PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因甲基化水平导致结直肠癌及进展期腺瘤患者PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因低表达，从而导致结直肠癌及进展期腺瘤细胞具有更强的侵袭能力，因此，PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因甲基化情况能用作结直肠癌及进展期腺瘤恶性程度和预后评价的分子生物学标记。以检测结直肠癌及进展期腺瘤PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因甲基化水平为基础的诊断试剂盒可以方便快捷地在分子水平上实现对结直肠癌及进展期腺瘤的检测，对结直肠癌及进展期腺瘤恶性程度和预后判断，检测效率高，针对性强，具有准确可靠、灵活快速和经济节约的优点，有利于结直肠癌及进展期腺瘤的早期发现和及时治疗。
5、与传统的结肠镜检筛查结直肠癌及进展期腺瘤比较，及常规的粪便隐血试验比较，本试剂盒采用甲基化特异性PCR法通过检测粪便脱落细胞DNA筛查结直肠癌及进展期腺瘤具有以下优势：
1)完全无创，仅需2～5克粪便，无症状人群接受度高；
2)准确性高，在临床前研究中可以检测出绝对多数的结直肠癌及进展期腺瘤和大部分的癌前腺瘤；能检测到96.3％的早期结直肠癌，85.8％的癌前腺瘤(≥1厘米)，特异性为100％；
3)可以同时发现左右两侧结肠的肿瘤，减少检测盲区和漏诊；
4)不仅可以发现结直肠癌及进展期腺瘤，也可以发现癌前腺瘤，为通过摘除癌前腺瘤而预防结直肠癌及进展期腺瘤提供了可能性；
5)操作方便，在获得检测试剂盒后，用户可以居家完成样本的收集工作， 之后快递寄出由专业人员进行检测。
6、本方法采用实时荧光PCR法(Real-time PCR)，这种技术是在普通聚合酶链式反应技术上发展起来的，在普通聚合酶链式反应扩增过程中利用荧光染料或者电泳检测进行定量，探针法的应用使得该技术具有更高的精确性、高敏感性和较高通量，且无需在PCR后电泳、杂交等操作，减少了污染和操作误差。
附图说明：
此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为PRDM12甲基化检测结果图；
图2为FOXE1甲基化检测结果图；
图3为B3GAT2甲基化检测结果图；
图4为Vimentin甲基化检测结果图；
图5为SFRP2-1甲基化检测结果图；
图6为SFRP2-2甲基化检测结果图。
具体实施方式：
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚、明白，以下结合附图和实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
如图1－图6所示，相关基因甲基化检测结果图(FAM通道)。
下面结合实施例对本发明作进一步详细说明：
一、研究对象的样本收集：
本研究中，选取上海长海医院手术切除的31例结直肠癌患者标本为研究对象。所有病例诊断依据患者的临床资料、影像学和结肠镜检查等，都得到术后病理诊断确定。手术切除的结直肠癌组织和癌旁组织(距离癌组织≥5cm的正常结直肠粘膜组织)经液氮速冻后，保存于-80度冰箱中。取适量癌组织和癌旁组织提取基因组DNA。
本研究中，收集来自上海长海医院31例结直肠癌患者的粪便和对应病人的粪便作为研究对象。所用病例诊断依据患者的临床资料、影像学、结肠镜检查和术后病理等确定。收集的粪便样本，保存于含有粪便DNA保存液的收集管，置于-20度冰箱中。取适量粪便样本提取基因组DNA。
二、基因组DNA的提取
组织样本采用QIAamp DNA Mini Kit组织全基因组DNA提取试剂盒(Qiagen公司，德国)提取癌组织和癌旁组织的基因组DNA。粪便样本采用QIAamp DNA Stool Mini Kit粪便基因组DNA提取试剂盒。通过NanoDrop2000微量分光光度计(Thermo Fisher Scientific，美国)检测基因组DNA的浓度和质量，供后续基因特定区域甲基化PCR检测。具体提取步骤根据提取试剂盒操作说明书进行。
三、对照品选择
所述阳性性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2甲基化质粒DNA片段和内参ACTB基因的混合物(或者为人全基因组DNA甲基化阳性对照)；所述阴性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2非甲基化质粒DNA片段和内参ACTB基因的混合物(或者为人全基因组DNA无甲基化阴性对照)；所述空白对照为TE缓冲液或无核酸酶水。
四、DNA亚硫氢酸盐转化
获取的粪便、血液或组织的基因组DNA，使用ZYMO EZ DNA Methylation Gold Kit(ZYMO公司，美国)的DNA亚硫氢酸盐转化试剂盒，依照说明书操作，经过磺化、水解脱氨、去磺化三个主要步骤，将非甲基化的胞嘧啶(C)转化为尿嘧啶(U)，而甲基化的胞嘧啶(5mC)不能进行转化。待测样本的基因组DNA经亚硫氢酸盐转化后作为甲基化PCR检测的模板。阳性对照和阴性对照为基因工程合成质粒。
五、引物和探针的设计与合成
针对人基因组中PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因特定甲基化位点以及内参基因ACTB(序列参见NCBI数据库公开的人类全基因组序列)，使用Primer Premier 3.0及BeaconDesigner软件，设计特异性引物及探针。引物和探针由生工生物工程(上海)有限公司合成。
本发明针对结直肠癌及进展期腺瘤相关的PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2靶基因片段甲基化的核酸序列同时设计并验证了多对引物和探针组合序列，经多次反复优化实验，优选了以下(表1)一组检测PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因片段甲基化的引物探针序列。特异性引物及探针序列如表1所示：
表1检测基因特定区域甲基化的特异性引物探针序列
Figure PCTCN2019112439-appb-000002
Figure PCTCN2019112439-appb-000003
六、PCR反应体系组成
PCR反应体系包括含有上述特异性引物，分为包括6个反应体系(A/B/C/D/E/F)，其中：
反应体系A包含PRDM12正向引物(引物1)、PRDM12反向引物(引物2)、PRDM12检测探针(探针1)；反应体系B包含FOXE1正向引物(引物3)、FOXE1反向引物(引物4)、FOXE1检测探针(探针2)；反应体系C包含B3GAT2正向引物(引物5)、B3GAT2反向引物(引物6)、B3GAT2检测探针(探针3)；反应体系D包含Vimentin正向 引物(引物7)、Vimentin反向引物(引物8)、Vimentin检测探针(探针4)；反应体系E包含SFRP2-1正向引物(引物9)、SFRP2-1反向引物(引物10)、SFRP2-1检测探针(探针5)；反应体系F包含SFRP2-2正向引物(引物11)、SFRP2-2反向引物(引物12)、SFRP2-2检测探针(探针6)。所述PCR反应体系还包括内参基因ACTB正向引物(引物13)、ACTB反向引物(引物14)、ACTB检测探针(探针7)、PCR buffer、dNTPs、Taq HS酶、MgCl 2。
在各PCR反应体系总加入转化后的DNA，各成分终浓度如表2所示：
表2反应体系组分表
Figure PCTCN2019112439-appb-000004
七、试剂盒的使用，包括以下步骤：
(1)准备待测样品，粪便、血液或组织来源样本均适用于本发明。从粪便、 血液或组织样本提取基因组DNA，并经亚硫酸氢盐处理转化后作为模板，用于PCR反应。
(2)从试剂盒中取出PCR反应试剂，平衡至室温，29.7μl×(n+1)，PCR反应液和0.3μl×(n+1)酶混合液，充分混匀20s，瞬时离心；(n＝检测样本数量)，瞬时离心。
(3)按每管30μl分装于PCR管中。
(4)用微量移液器向每个PCR管中再加入10μL模版DNA溶液(转化后的待测样本DNA或转化后的阴性/阳性/空白对照品)，每个反应终体积为40ul。立即盖严管盖，充分混匀，瞬时离心，将PCR管移至实时荧光定量PCR扩增仪上。
(5)在实时荧光定量PCR仪上，设置一个扩增程序，连续完成PCR扩增以及扩增过程中实时荧光信号采集、分析。反应程序设定如表3所示：
表3：
Figure PCTCN2019112439-appb-000005
(6)结果的判定：
在样本的检测过程中，基线的确定为：取3～15个循环的荧光信号；阈值设定：使阀值线超过正常阴性对照品扩增曲线(无规则的噪音线)的最高点，且Ct值为Undet。根据所报告的Ct值(每个反应管内的荧光信号达到设定的阈 值时所经历的循环数)判读样本的阳性或阴性结果。
结论：(1)31例阳性样本FAM通道对应检测孔均有扩增曲线，检测结果为阳性，与病理诊断结果一致；10例阴性样本只有对照的内参基因ROX通道有扩增曲线，检测靶基因结果在FAM通道均为阴性，与病理诊断结果一致表明本试剂盒的检测结果具有极高的特异性和可靠性。本发明各样本的详细检测结果的Ct值见表3。
由样本的荧光定量PCR检测结果中可以看出，各样本PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因在结直肠癌及进展期腺瘤样本中高度扩增，而正常样本中扩增极少，由此证明本发明试剂盒能快速、准确的检测结直肠癌及进展期腺瘤粪便脱落细胞中突变基因和正常基因。由此看出本发明试剂盒不仅是快速、高效的，而且其结果的判读非常明确、直观，结果可靠、特异的。
表3各样本荧光定量PCR检测结果的Ct值(编号“1-N、1-P、1-F”中1代表样本号，N代表正常组织、P代表癌症或腺瘤组织、F代表粪便样本，以此类推)
Figure PCTCN2019112439-appb-000006
Figure PCTCN2019112439-appb-000007
Figure PCTCN2019112439-appb-000008
Figure PCTCN2019112439-appb-000009
Figure PCTCN2019112439-appb-000010
Figure PCTCN2019112439-appb-000011
Figure PCTCN2019112439-appb-000012
Figure PCTCN2019112439-appb-000013
上述说明示出并描述了本发明的优选实施例，如前所述，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。

Claims (13)

  1. 人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述的引物组包括PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2六个特定基因甲基化区域的任意一种基因或任意几种基因组合的甲基化引物组。
  2. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述PRDM12基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-GTTAGATTAGAAGTATAAAATGTG-3’；以下称：引物1；
    反向引物R：5’-ATCCCATTCCTCTCCTCC-3’；以下称：引物2；
    检测探针P：5’-AGCGCGGTGGAGATTTG-3’；以下称：探针1；
  3. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述FOXE1基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-CGAGTttAAGtttTTGGtAGAGG-3’；以下称：引物3；
    反向引物R：5’-CCGATCGACTaAaaCCCGa-3’；以下称：引物4；
    检测探针P：5’-tCGAGTTCGGGCGtTGAGG-3’；以下称：探针2；
  4. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述B3GAT2基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-TtCGtCGGGTtTGGCG-3’；以下称：引物5；
    反向引物R：5’-CAaaAaaTCGTaCAaCCCCG-3’；以下称：引物6；
    检测探针P：5’-tCGCGAGtAAGtTCGGGAG-3’；以下称：探针3；
  5. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述Vimentin基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-GtCGtAGttTtTACGttTCGT-3’；以下称：引物7；
    反向引物R：5’-aaCGCACGaCAaAaaAaCG-3’；以下称：引物8；
    检测探针P：5’-ttCGGGCGGCGTGTATG-3’；以下称：探针4；
  6. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述SFRP2-1基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-CGGAtTGGGGtAAAAtAAGttC-3’；以下称：引物9；
    反向引物R：5’-CTaaCGaaAACGCGCCTA-3’；以下称：引物10；
    检测探针P：5’-TAGGCGCGTTttCGttAGTAttTGG-3’；以下称：探针5；
  7. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述SFRP2-2基因的检测引物组，其特异性引物探针组序列为：
    正向引物F：5’-AATGtAGtCGGCGCG-3’；以下称：引物11；
    反向引物R：5’-CCTTaTTaaaAaTTCAAaaAaCCCG-3’；以下称：引物12；
    检测探针P：5’-AGttAtTTtCGGGCGTGCGGt-3’；以下称：探针6；
  8. 如权利要求1所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：还包括内参ACTB基因的引物组，其检测引物探针组序列为：
    正向引物：5’-GTGATGGAGGAGGTTTAGTAAG-3’；以下称：引物13；
    反向引物：5’-CAATAAAACCTACTCCTCCCTT-3’；以下称：引物14；
    检测探针P：5’-TGTGTTTGTTATTGTGTGTTGGGTGGT-3’；以下称：探针7。
  9. 如权利要求2-8任意一项所述的人结直肠癌相关基因特定区域甲基化的引物组，其特征在于：所述的检测探针5’端的荧光报告基团包括：ALEX-350、FAM、VIC、TET、CAL Fluor Gold 540、JOE、HEX、CAL Flour Orange 560、TAMRA、Cal Fluor Red 590、ROX、CAL Fluor 20Red 610、TEXAS RED、CAL Flour Red 635、Quasar 670、Cy3、Cy5、Cy5.5或Quasar 705；3’端的淬灭基团包括TAMRA、BHQ1、BHQ2、MGB。
  10. 人结直肠癌相关基因特定区域甲基化检测试剂，其特征在于：所述的试剂包括6个PCR体系，分别为CRC PCR Mix-A/B/C/D/E/F；每个体系中包括如权利要求2-7任意一项相应的基因特定区域甲基化情况的特异性引物探针组，权利如权利要求8的内参基因特异性引物探针组，还包括通用成分有PCR buffer、dNTPs、MgCl 2。
  11. 如权利要求10所述的人结直肠癌相关基因特定区域甲基化检测试剂，其特征在于：所述的六个PCR反应体系CRC PCR Mix-A/B/C/D/E/F，每个体系终体积都在20～40μl之间，所述的反应体系A/B/C/D/E/F中引物终浓度为：正向引物F均在0.3～0.8μM之间，反向引物R均在0.3～0.8μM之间，探针P浓度均在0.1～0.3μM之间；所述6个反应体系中每个体系的通用成分终浓度为：dNTPs中的dATP、dTTP、dCTP、dGTP都为0.1～0.3mM，MgCl 2溶液为1～2mM。
  12. 人结直肠癌相关基因特定区域甲基化检测试剂盒，其特征在于：所述的试剂盒包括如权利要求10中的6个PCR体系中的任意一个或者任意几个的组合，0.5～2U Taq HS热启动酶，还包括阳性对照、阴性对照和空白对照；所述阳 性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2的甲基化质粒DNA片段和内参ACTB基因的混合物，或者为人全基因组DNA甲基化阳性对照；所述阴性对照为PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2的非甲基化质粒DNA片段和内参ACTB基因的混合物，或者为人全基因组DNA无甲基化阴性对照；所述空白对照为pH＝8.5±0.1的TE缓冲液或无核酸酶水。
  13. 如权利要求12所述的人结直肠癌相关基因特定区域甲基化检测试剂盒的应用，其特征在于：从人的粪便、血液或组织中直接提取DNA，DNA经过亚硫酸氢盐处理转化，以转化后的DNA为模板，通过PCR方法扩增，并通过检测探针上信号累积从而实现人结直肠癌相关PRDM12、FOXE1、B3GAT2、Vimentin、SFRP2-1、SFRP2-2基因特定区域甲基化的实时检测；同时ACTB基因作为内参基因；包括如下PCR具体实验步骤：
    步骤一：收集一定数量的结直肠癌患者及进展期腺瘤患者的粪便、血液或组织样本；
    步骤二：从待检测人的粪便、血液或组织样本中提取人基因组DNA；
    步骤三：提取的人基因组DNA经亚硫酸氢盐处理，转化后，作为PCR反应的模板；
    步骤四：从试剂盒中取出29.7μl×(n+1)，PCR Mix和0.3μl×(n+1)Taq HS热启动酶混合液，混匀20s，瞬时离心10s；n＝检测样本数量
    步骤五：按每管30μl分装至PCR管中，再加入待检测的转化后DNA模板10μl，总体积40μl；
    步骤六：进行扩增反应。反应程序为：
    Figure PCTCN2019112439-appb-100001
    Figure PCTCN2019112439-appb-100002
    步骤五：PCR结果判定：在样本的检测过程中，基线的确定为：取3～15个循环的荧光信号；阈值设定：使阀值线超过正常阴性对照品扩增曲线的最高点，且Ct值为Undet；根据所报告的Ct值，即每个反应管内的荧光信号达到设定的阈值时所经历的循环数判读样本的阳性或阴性结果。
PCT/CN2019/112439 2019-10-16 2019-10-22 人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用 WO2021072786A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/767,765 US20230242990A1 (en) 2019-10-16 2019-10-22 Primer set, reagent, and kit for methylation of specific regions of human colorectal cancer-related genes, and application of the kit
EP19949066.5A EP4047101A4 (en) 2019-10-16 2019-10-22 PRIMER SET FOR METHYLATION OF A SPECIFIC REGION OF A GENE LINKED TO HUMAN COLORECTAL CANCER, TEST, TEST KIT AND ASSOCIATED APPLICATION

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910982813 2019-10-16
CN201910982813.8 2019-10-16

Publications (1)

Publication Number Publication Date
WO2021072786A1 true WO2021072786A1 (zh) 2021-04-22

Family

ID=68852132

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/112439 WO2021072786A1 (zh) 2019-10-16 2019-10-22 人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用

Country Status (4)

Country Link
US (1) US20230242990A1 (zh)
EP (1) EP4047101A4 (zh)
CN (1) CN110592224B (zh)
WO (1) WO2021072786A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114395623A (zh) * 2021-12-16 2022-04-26 上海市杨浦区中心医院(同济大学附属杨浦医院) 一种基因甲基化检测引物组合物及其试剂盒和应用
CN116064788A (zh) * 2022-08-01 2023-05-05 山东大学 乳腺癌早期筛查的多重基因甲基化检测荧光定量pcr试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115896027A (zh) * 2022-04-07 2023-04-04 广州燃石医学检验所有限公司 一种生物组合物、其制备方法及应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250588A (zh) * 2008-04-09 2008-08-27 王道荣 一种利用粪便中sfrp2基因超甲基化筛查大肠癌的方法
WO2011126768A2 (en) * 2010-03-29 2011-10-13 Mayo Foundation For Medical Education And Research Methods and materials for detecting colorectal cancer and adenoma
WO2012167145A2 (en) * 2011-06-01 2012-12-06 University Of Southern California Genome-scale analysis of aberrant dna methylation in colorectal cancer
WO2018211404A1 (en) * 2017-05-17 2018-11-22 King Abdullah University Of Science And Technology Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer
CN109486955A (zh) * 2018-12-29 2019-03-19 厦门艾德生物医药科技股份有限公司 一种用于粪便检测进展期腺瘤、结直肠癌相关基因甲基化位点的试剂盒
CN109811056A (zh) * 2019-02-28 2019-05-28 苏州唯善生物科技有限公司 用于结直肠癌及其癌前病变早期诊断、检测或筛查的引物探针组和试剂盒
CN110168108A (zh) * 2016-12-21 2019-08-23 加利福尼亚大学董事会 血浆中稀少dna的去卷积和检测

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2541178T3 (es) * 2009-02-03 2015-07-16 Mdxhealth Sa Método de detección de cáncer colorrectal
US9546403B1 (en) * 2011-12-14 2017-01-17 University Of Utah Research Foundation Substrate for methylated DNA testing
CN102787174B (zh) * 2012-09-06 2013-08-07 南京大学 胃肠肿瘤发病相关抑癌基因表观突变检测试剂盒及其用途
CN103849680A (zh) * 2013-12-23 2014-06-11 浙江大学医学院附属邵逸夫医院 Foxe1基因和wnt3基因的应用及该基因的甲基化扩增引物和探针
CN104164516B (zh) * 2014-09-05 2016-08-24 中国科学院上海微系统与信息技术研究所 一种基于人结直肠癌特异性甲基化检测的引物及试剂盒
US20210032703A1 (en) * 2018-01-23 2021-02-04 Exellon Medical Technology Method and kit for identifying state of colorectal cancer

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250588A (zh) * 2008-04-09 2008-08-27 王道荣 一种利用粪便中sfrp2基因超甲基化筛查大肠癌的方法
WO2011126768A2 (en) * 2010-03-29 2011-10-13 Mayo Foundation For Medical Education And Research Methods and materials for detecting colorectal cancer and adenoma
WO2012167145A2 (en) * 2011-06-01 2012-12-06 University Of Southern California Genome-scale analysis of aberrant dna methylation in colorectal cancer
CN110168108A (zh) * 2016-12-21 2019-08-23 加利福尼亚大学董事会 血浆中稀少dna的去卷积和检测
WO2018211404A1 (en) * 2017-05-17 2018-11-22 King Abdullah University Of Science And Technology Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer
CN109486955A (zh) * 2018-12-29 2019-03-19 厦门艾德生物医药科技股份有限公司 一种用于粪便检测进展期腺瘤、结直肠癌相关基因甲基化位点的试剂盒
CN109811056A (zh) * 2019-02-28 2019-05-28 苏州唯善生物科技有限公司 用于结直肠癌及其癌前病变早期诊断、检测或筛查的引物探针组和试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"2015 China Cancer Statistics", CA: A CANCER JOURNAL FOR CLINICIANS
"EDITORIAL COMMENT", JOURNAL OF UROLOGY, LIPPINCOTT WILLIAMS & WILKINS, BALTIMORE, MD, US, vol. 155, no. 2, 1 February 1996 (1996-02-01), BALTIMORE, MD, US, pages 586, XP005580230, ISSN: 0022-5347, DOI: 10.1016/S0022-5347(01)66455-3 *
See also references of EP4047101A4

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114395623A (zh) * 2021-12-16 2022-04-26 上海市杨浦区中心医院(同济大学附属杨浦医院) 一种基因甲基化检测引物组合物及其试剂盒和应用
CN114395623B (zh) * 2021-12-16 2024-03-08 上海市杨浦区中心医院(同济大学附属杨浦医院) 一种基因甲基化检测引物组合物及其试剂盒和应用
CN116064788A (zh) * 2022-08-01 2023-05-05 山东大学 乳腺癌早期筛查的多重基因甲基化检测荧光定量pcr试剂盒
CN116064788B (zh) * 2022-08-01 2023-09-08 山东大学 乳腺癌早期筛查的多重基因甲基化检测荧光定量pcr试剂盒

Also Published As

Publication number Publication date
US20230242990A1 (en) 2023-08-03
EP4047101A4 (en) 2024-04-03
CN110592224A (zh) 2019-12-20
EP4047101A1 (en) 2022-08-24
CN110592224B (zh) 2023-02-28

Similar Documents

Publication Publication Date Title
CN108977543B (zh) 一种基于联合检测sdc2和sfrp2基因甲基化水平的结直肠癌早期诊断试剂
CN113755603B (zh) 子宫内膜癌早期筛查诊断用标志物、引物探针及试剂盒
WO2023071890A1 (zh) 胃癌淋巴结转移相关的甲基化生物标记物及其组合和检测试剂盒
WO2021072786A1 (zh) 人结直肠癌相关基因特定区域甲基化的引物组及试剂以及试剂盒及其应用
TWI730429B (zh) Hoxa7甲基化檢測試劑
US20240035096A1 (en) Composition, kit, and application for detection of colorectal cancer
CN111500731A (zh) 一种甲状腺癌早期诊断、检测或者筛查的试剂盒及其使用方法、应用
CN116144782A (zh) 一种用于肺癌检测的组合标志物及其应用
CN110964812B (zh) Hoxa9甲基化检测试剂在制备肺癌诊断试剂中的用途
CN111363811A (zh) 基于foxd3基因的肺癌诊断剂及试剂盒
CN110964813B (zh) Hoxa7甲基化检测试剂在制备肺癌诊断试剂中的用途
US20230076141A1 (en) Markers, primers, probes and kit for early screening and diagnosis of endometrial cancer
CN116121387A (zh) 一种用于结直肠癌检测的组合标志物及其应用
CN108796078B (zh) 一种用于消化道癌诊断、检测或筛查的引物和探针组
CN110964811B (zh) Hoxa9甲基化检测试剂
CN111549136A (zh) 一种用于甲状腺癌检测的试剂盒及其使用方法、应用
RU2791172C1 (ru) Набор праймеров, реагент и коммерческий набор для метилирования конкретных областей генов, связанных с колоректальным раком у человека, и применение коммерческого набора
CN116656810B (zh) 胰腺癌的生物标志物、核酸产品和试剂盒
WO2024001602A1 (zh) 一种用于检测胃癌的组合物，试剂盒及其用途
CN111363817B (zh) 基于hoxd12基因的肺癌诊断剂及试剂盒
WO2024045160A1 (zh) Oplah基因的差异性甲基化区域、试剂盒和用途
WO2024045162A1 (zh) Adhfe1基因的差异性甲基化区域、试剂盒和用途
CN114959024A (zh) 用于粪便脱落细胞dna甲基化检测的生物靶点、引物探针组合物和应用和试剂盒及其用法
CN117106918A (zh) 基因甲基化鉴别诊断肺良性结节和恶性肿瘤方法及其试剂盒
CN114959025A (zh) 一种用于血浆游离dna甲基化检测肠癌的引物探针组合物及其应用和试剂盒

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19949066

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2022111047

Country of ref document: RU

ENP Entry into the national phase

Ref document number: 2019949066

Country of ref document: EP

Effective date: 20220516