WO2024066941A1 - Composition for detecting bladder cancer, kit, and use thereof - Google Patents

Composition for detecting bladder cancer, kit, and use thereof Download PDF

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WO2024066941A1
WO2024066941A1 PCT/CN2023/116826 CN2023116826W WO2024066941A1 WO 2024066941 A1 WO2024066941 A1 WO 2024066941A1 CN 2023116826 W CN2023116826 W CN 2023116826W WO 2024066941 A1 WO2024066941 A1 WO 2024066941A1
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methylation
detecting
bladder cancer
composition
kit
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PCT/CN2023/116826
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French (fr)
Chinese (zh)
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戴立忠
杨丽
彭磊
刘佳
洪梅
胡志红
陈明
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圣湘生物科技股份有限公司
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Priority claimed from CN202211216063.1A external-priority patent/CN117802225A/en
Priority claimed from CN202211216837.0A external-priority patent/CN117844922A/en
Priority claimed from CN202211212129.XA external-priority patent/CN117844921A/en
Application filed by 圣湘生物科技股份有限公司 filed Critical 圣湘生物科技股份有限公司
Publication of WO2024066941A1 publication Critical patent/WO2024066941A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present invention belongs to the field of molecular biological detection, specifically, to the field of bladder cancer detection, and more specifically, to the detection of the methylation level of bladder cancer gene markers.
  • Bladder cancer is the most common malignant tumor of the urinary system and one of the top ten common tumors in the body. It ranks first in the incidence of urogenital tumors in my country. There are about 550,000 new cases and 200,000 deaths each year in the world, and the incidence in China is increasing year by year. In my country, nearly 80% of bladder cancers are diagnosed as non-muscle-invasive bladder cancer (NMIBC). The prognosis is good after transurethral bladder tumor resection, but 70% of patients will experience tumor recurrence after surgery, and 15% of patients will progress in staging and grading; the remaining patients are diagnosed with muscle-invasive bladder cancer (MIBC), which is prone to distant metastasis and has a high risk of death.
  • MIBC muscle-invasive bladder cancer
  • bladder cancer patients usually need multiple follow-up examinations and bladder instillation treatment after surgery, which is expensive.
  • the preoperative diagnosis and postoperative recurrence monitoring of bladder cancer mainly rely on invasive cystoscopy, which not only causes pain to patients, but may also cause urinary tract damage and infection, etc., and patient compliance is poor.
  • Finding non-invasive, accurate, stable and effective molecular markers for bladder cancer is a key to the early diagnosis of bladder cancer.
  • Early screening, diagnosis and recurrence detection are of great significance.
  • Tumor marker detection is a method developed in recent years for detecting diseases, and finding accurate, stable and effective bladder cancer molecular markers is of great significance for early diagnosis and early treatment of bladder cancer.
  • DNA methylation, histone modification, and abnormal miRNA expression are all epigenetic changes, and the core link of tumor occurrence is also related to abnormal DNA methylation.
  • DNA methylation testing is stable, easy to detect, and its abnormality is often related to the progression of cancer. It is the marker with the greatest potential for early screening of tumors.
  • the present invention collected 413 cancer tissues, 231 adjacent tissues or normal control tissues and 656 healthy whole blood methylation data from the TCGA dataset (https://tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI).
  • the bladder cancer and control data were used for differential analysis. Through a large number of studies, the applicant found that the methylation levels of certain methylation sites are closely related to bladder cancer.
  • the present invention provides a composition for detecting bladder cancer, the composition comprising a detection reagent for detecting at least one of the following methylation sites: cg00206063, cg03278514, and cg13314394.
  • compositions of the present invention in tissue samples, all compositions can predict bladder cancer with a specificity of at least 0.8571, a sensitivity of 0.7203, and an area under the curve of 0.8585.
  • urine free DNA samples all compositions can predict bladder cancer with a specificity of at least 0.9048, a sensitivity of 0.6525, and an area under the curve of 0.8060.
  • the compositions of the present invention can detect bladder cancer with high sensitivity and good specificity in the clinic with fewer markers, saving both cost and time. In the clinic, it can effectively detect bladder cancer in the early stages of malignant transformation. Sensitive and specific detection.
  • the composition includes detection reagents for detecting at least two of the following methylation sites: cg00206063, cg03278514, and cg13314394.
  • the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg03278514.
  • the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg13314394.
  • the composition includes detection reagents for detecting the following methylation sites: cg03278514, cg13314394.
  • the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg03278514, and cg13314394.
  • the composition further comprises a detection reagent for detecting the methylation level of at least one of the following: SPATS2, PARP4.
  • the composition includes detection reagents for detecting the following methylation sites: cg00206063, SPATS2, PARP4.
  • the composition includes detection reagents for detecting the following methylation sites: SPATS2, PARP4, cg03278514.
  • the composition includes detection reagents for detecting the following methylation sites: SPATS2, PARP4, cg13314394.
  • the composition further comprises a detection reagent for detecting the methylation level of: SPATS2, PARP4.
  • the methylation level detection reagent can be a detection reagent for detecting the average methylation level of the entire gene.
  • the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene fragment.
  • the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene promoter region or a fragment thereof.
  • the methylation level detection reagent can also be a detection reagent for detecting one or more methylation sites of a gene.
  • the composition further comprises a detection reagent for detecting the methylation level of the methylation site cg06712013 of the SPATS2 gene.
  • the composition further comprises a detection reagent for detecting the methylation level of the PARP4 gene methylation site cg20765408.
  • the composition includes detection reagents for detecting the methylation levels of cg13314394, cg00206063, cg06712013, cg20765408, and cg03278514.
  • composition of the above scheme can detect bladder cancer with higher sensitivity and better specificity; clinically, bladder cancer can be detected sensitively and specifically in the early stages of malignant transformation.
  • the detection reagent of the present invention can be used to detect the methylation level of the corresponding gene present in the sample.
  • sample is a biological sample selected from an individual. Specifically, for example, selected from cell lines, tissue sections, biopsy tissues, paraffin-embedded tissues, body fluids, feces, colon effluent, urine, plasma, serum, whole blood, separated blood cells, cells separated from blood, or a combination thereof.
  • the "sample” of the present invention is urine, ie, free DNA in urine.
  • Free DNA in urine can be used to detect tumors, with the characteristics of little harm to patients and good specificity. However, due to its extremely low content in urine, it generally has the problem of low sensitivity when used for cancer detection.
  • free DNA in urine can be used as a sample for detection, with high sensitivity and specificity.
  • detection reagent refers to a reagent for detecting the methylation level of a gene in a sample, wherein the methylation level is measured by amplification-sequencing, chip, methylation fluorescence quantitative PCR, etc.
  • the detection reagents include, but are not limited to, nucleic acid primers and sequencing Tag sequences, for measuring methylation levels by amplification-sequencing.
  • the amplification-sequencing is performed by treating the nucleic acid in the sample with bisulfite, then constructing a pre-library, then constructing a final library, and finally performing sequencing evaluation.
  • the detection reagent includes but is not limited to a chip, and the chip is a methylation chip, and the methylation chip has a probe that specifically binds to the methylation region.
  • the chip can be, for example, but is not limited to, Agilent's Human CpG Island Microarrays and Human DNA Methylation Microarrays, Illumina's Infinium Human Methylation 27 Bead Chip, Infinium Human Methylation 450 Bead Chip and Golden Gate Methylation Assay, and Roche NimbleGen's Human DNA Methylation 2.1M Deluxe Promoter Array, Human DNA Methylation Array, etc., for measuring the methylation level through the chip.
  • the detection reagents include, but are not limited to, nucleic acid primers and nucleic acid probes for measuring methylation levels by methylation fluorescence quantitative PCR.
  • the detection reagent also includes an internal standard primer and an internal standard probe.
  • the above composition may further include other reagents, specifically, for example, various reagents required for pre-treatment or pre-processing of the sample, such as a sample release agent for extracting sample nucleic acid, a purification agent for purifying sample nucleic acid, bisulfite or bisulfite used for conversion, etc.
  • reagents specifically, for example, various reagents required for pre-treatment or pre-processing of the sample, such as a sample release agent for extracting sample nucleic acid, a purification agent for purifying sample nucleic acid, bisulfite or bisulfite used for conversion, etc.
  • the above composition also includes a reagent for extracting free DNA in urine.
  • the present invention provides a kit for preparing a kit for detecting bladder cancer using the above composition. Uses in.
  • the above composition is used in the preparation of a kit for detecting bladder cancer, wherein the composition is a detection reagent for the following methylation site: cg13314394.
  • the present invention provides use of the above composition in preparing a kit for detecting bladder cancer using urine free DNA.
  • the present invention provides a kit for detecting bladder cancer, the kit comprising the composition as described above.
  • the kit also includes, but is not limited to, at least one of a reagent for extracting nucleic acid, a reagent for purifying nucleic acid, bisulfite, T4 polynucleotide kinase, and T4 ligase.
  • the reagents for extracting nucleic acid are reagents for extracting tissue DNA and reagents for extracting urine free DNA.
  • the reagent for extracting nucleic acid is a reagent for extracting free DNA in urine.
  • FIG1 is a ROC diagram of the methylation level of a single gene in distinguishing cancer from non-cancer in tissues
  • FIG2 is a ROC diagram of the methylation levels of two genes in differentiating cancer from non-cancer tissues
  • FIG3 is a ROC diagram of the methylation levels of five genes in differentiating cancer from non-cancerous tissues
  • FIG4 is a ROC diagram of the methylation level of a single gene in distinguishing cancer from non-cancer in urine;
  • FIG5 is a ROC diagram of the methylation levels of two genes in distinguishing cancer from non-cancer in urine
  • FIG. 6 is a ROC diagram of the methylation levels of five genes in distinguishing cancer from non-cancer in urine.
  • the present invention collected 413 cancer tissues, 231 paracancerous tissues or normal control tissues and 656 healthy whole blood methylation data from the TCGA data set (https://tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI).
  • the bladder cancer and control data were used for differential analysis, and the physical location and gene information of the differential sites were annotated.
  • the screening of methylated gene fragments needs to meet the following requirements at the same time: 1) The selected gene fragments are required to have no less than 2 adjacent sites with consistent methylation levels; 2) The bladder cancer and paracancerous tissues or normal control tissues were used for differential analysis, and the gene fragments with high consistency and high differential methylation in the bladder cancer samples were selected as candidate target genes; 3) The whole blood methylation detection data of bladder cancer and healthy samples were used for differential analysis, and the gene fragments with high differential methylation in bladder cancer were selected; 4) Finally, the methylation sites were analyzed one by one to obtain the candidate methylation sites.
  • the sample preparation of the present invention is to extract 4 ml of urine supernatant using VAHTS Free-Circulating DNA Maxi Kit and elute with 45 ⁇ L of elution buffer.
  • the extracted free nucleic acid must meet the following quality control conditions: the total amount of extracted nucleic acid is greater than 20 ng.
  • all free nucleic acids that have passed the quality control are treated with bisulfite using the EZ DNA Methylation-Lightning TM Kit. Subsequently, the sample DNA treated with bisulfite is used to construct a pre-library using a single-stranded library construction method. After the pre-library passes the quality inspection, the target region is captured and enriched by liquid chip hybridization to complete the construction of the final library.
  • Prelibrary construction steps 1) Phosphorylation: T4 polynucleotide kinase phosphorylates the 5 end of the bisulfite treated DNA; 2) SS1 ligation: T4 DNA Ligase (Rapid) ligates SS1 The adapter was connected to the 5 end of the phosphorylated DNA; 3) Nucleic acid purification: Use 2 volumes of Agencourt AMPure XP system to remove the remaining adapter; 4) SS2 connection: T4 DNA Ligase (Rapid) connected the SS2 adapter to the 3 end of the phosphorylated DNA; 5) Nucleic acid purification: Use 2 volumes of Agencourt AMPure XP system to remove the remaining adapter; 6) Amplification: Use NEBNext Q5U Master Mix, primer1.0 (universal primer) and Bacard sequence to amplify the nucleic acid in the previous step; 7) Nucleic acid purification: Use 1.2 volumes of Agencourt AMPure XP system to remove primer dimers and excess primers
  • Chip hybridization capture steps 1) Chip hybridization: vacuum concentrate 1.5ug of the qualified mixed library into powder in advance and then mix it with Panel, Hybridization Mix, Blocker Solution, Universal Blockers, and Hybridization Enhancer reagents, and place it in a PCR instrument and incubate it at 70 degrees for 16 hours overnight (the hot cover temperature is 85 degrees); 2) Magnetic bead capture: wash the captured magnetic beads 3 times with Streptavidin Binding Buffer in advance, add the hybridization product to the captured magnetic beads, incubate for 30 minutes, wash once with Wash Buffer I, wash 3 times with Wash Buffer 2, and finally elute with 42 ⁇ l ultrapure water; 3) Amplification: use KAPA HiFi HotStart ReadyMix and universal primers to amplify the captured library; 4) Purification: use 1 volume of Agencourt AMPure XP system to remove primer dimers and excess primers.
  • the purified library was used The dsDNA HS Assay Kit and LabChip GXII Touch were used to perform quality checks on the total nucleic acid content, fragment distribution, and primer dimer ratio in the library.
  • the libraries to be tested that have passed the quality inspection of the total amount of the library, the fragment size distribution of the amplified product, and the primer dimer ratio are mixed at a 1:1 ratio and used
  • the dsDNA HS Asay Kit was used to accurately quantify the mixed library, and the library was denatured and diluted before sequencing using the NextSeq500 desktop sequencer using PE75.
  • the original fastq data obtained by sequencing was filtered and then the methylation analysis of the chip captured fragments was performed using the bismark methylation analysis software to obtain the methylation level of the candidate gene.
  • the methylation level of the candidate gene fragment was used to perform differential analysis and model construction for bladder cancer and control samples.
  • Example 3 Test results of the test samples of the composition of the present invention
  • Tissue samples from 63 patients with primary bladder cancer, 23 patients with cystitis, 10 ml of urine from 118 patients with bladder cancer, and 10 ml of urine from 60 patients with bladder disease and healthy people were collected to detect and analyze the methylation levels of candidate gene fragment methylation markers in urine samples.
  • the test results are shown in Table 1 and Figures 1 to 6.
  • Table 1 1 is cg13314394, 2 is cg00206063, 3 is cg06712013, 4 is cg20765408 and 5 is cg03278514.
  • the compositions in tissue samples, all the compositions can predict bladder cancer with a specificity of at least 0.8571, a sensitivity of 0.7203, and an area under the curve of 0.8585.
  • all the compositions can predict bladder cancer with a specificity of at least 0.9048, a sensitivity of 0.6525, and an area under the curve of 0.8060. Therefore, the composition of the present invention has a good predictive effect on bladder cancer, especially, when urine free DNA is used as a sample, it has excellent specificity and sensitivity.
  • methylation sites upstream and downstream of these sites were selected. The detection was also performed according to the method of the above embodiment, and the detection results are shown in Tables 2 to 4 below.
  • the highest AUC of the upstream and downstream methylation sites of the cg13314394 site in tissue samples is 0.760, and the highest AUC in free DNA samples is 0.522, which is lower than the AUC of the cg13314394 site of the present invention.
  • the highest AUC of the upstream and downstream methylation sites of the cg03278514 site in tissue samples is 0.857, and the highest AUC in free DNA samples is 0.878, which are lower than the AUC of the cg03278514 site of the present invention.
  • the methylation sites cg16732616 and cg04974290 which are closely related to bladder cancer and are known in the art, were further selected.
  • the detection was also performed according to the method of the above embodiment, and the detection results are shown in Table 3 below.
  • the highest AUC of a single comparison site in tissue samples is 0.848, and the highest AUC of a single site in free DNA samples is 0.751; the highest AUC of the comparison site combination in tissue samples is 0.851, and the highest AUC of the combination in free DNA samples is 0.862, which are lower than the AUCs of the cg00206063, cg03278514, and cg13314394 sites of the present invention.

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Abstract

Provided is a composition for detecting bladder cancer. The composition comprises a detection reagent for detecting at least one of the following methylation sites: cg00206063, cg03278514, and cg13314394. Also provided are use of the composition, and a kit comprising the composition. In a tissue sample, all compositions can predict bladder cancer with a specificity of at least 0.8571, a sensitivity of 0.7203, and an area under the curve of 0.8585. However, in a urine free DNA sample, all the compositions can predict the bladder cancer with a specificity of at least 0.9048, a sensitivity of 0.6525, and an area under the curve of 0.8060. The composition can detect the bladder cancer clinically with a high sensitivity and a good specificity using fewer markers, such that both the cost and time are saved. Clinically, bladder cancer malignant transformation can be sensitively and specifically detected at an early stage.

Description

一种用于检测膀胱癌的组合物，试剂盒及其用途Composition, kit and use thereof for detecting bladder cancer
相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请基于，申请号为202211212129.X、申请日为2022年09月30日，申请号为202211216063.1、申请日为2022年09月30日以及申请号为202211216837.0、申请日为2022年09月30日的中国专利申请提出，并要求该些中国专利申请的优先权，该些中国专利申请的全部内容在此以引入方式并入本申请。This application is based on the Chinese patent applications with application number 202211212129.X and application date September 30, 2022, application number 202211216063.1 and application date September 30, 2022, and application number 202211216837.0 and application date September 30, 2022, and claims the priority of these Chinese patent applications. The entire contents of these Chinese patent applications are hereby incorporated into this application by introduction.
技术领域Technical Field
本发明属于分子生物学检测领域，具体地，属于膀胱癌检测领域，更具体地，涉及膀胱癌基因标志物的甲基化水平的检测。The present invention belongs to the field of molecular biological detection, specifically, to the field of bladder cancer detection, and more specifically, to the detection of the methylation level of bladder cancer gene markers.
背景技术Background technique
膀胱癌是泌尿系统最常见的恶性肿瘤，也是全身十大常见肿瘤之一，占我国泌尿生殖系肿瘤发病率的第一位。全世界每年约有55万例新发病例和20万死亡病例，在中国的发病率逐年升高。在我国，近80％的膀胱癌被诊断为非肌层浸润性膀胱癌(non-muscle-invasive bladder cancer，NMIBC)，行经尿道膀胱肿瘤切除术后预后较好，但其中70％的患者术后会出现肿瘤复发，15％的患者分期和分级会发生进展；其余患者被诊断为肌层浸润性膀胱癌(muscle-invasive bladder cancer，MIBC)，易发生远处转移，具有较高的死亡风险。因此，膀胱癌患者术后通常需要多次复查和膀胱灌注治疗，费用昂贵。目前，膀胱癌的术前诊断和术后复发监测主要依赖有创的膀胱镜检查，不但给患者带来疼痛，还可能导致尿路损伤和感染等，患者依从性较差。寻找无创、准确、稳定、有效的膀胱癌分子标志物对膀胱癌进行早 期筛查、诊断和复发检测具有重要意义。肿瘤标志物检测是近年发展起来的用于检测疾病的方法，而寻找准确、稳定、有效的膀胱癌分子标志物对膀胱癌进行早期诊断、早期治疗具有重要意义。Bladder cancer is the most common malignant tumor of the urinary system and one of the top ten common tumors in the body. It ranks first in the incidence of urogenital tumors in my country. There are about 550,000 new cases and 200,000 deaths each year in the world, and the incidence in China is increasing year by year. In my country, nearly 80% of bladder cancers are diagnosed as non-muscle-invasive bladder cancer (NMIBC). The prognosis is good after transurethral bladder tumor resection, but 70% of patients will experience tumor recurrence after surgery, and 15% of patients will progress in staging and grading; the remaining patients are diagnosed with muscle-invasive bladder cancer (MIBC), which is prone to distant metastasis and has a high risk of death. Therefore, bladder cancer patients usually need multiple follow-up examinations and bladder instillation treatment after surgery, which is expensive. At present, the preoperative diagnosis and postoperative recurrence monitoring of bladder cancer mainly rely on invasive cystoscopy, which not only causes pain to patients, but may also cause urinary tract damage and infection, etc., and patient compliance is poor. Finding non-invasive, accurate, stable and effective molecular markers for bladder cancer is a key to the early diagnosis of bladder cancer. Early screening, diagnosis and recurrence detection are of great significance. Tumor marker detection is a method developed in recent years for detecting diseases, and finding accurate, stable and effective bladder cancer molecular markers is of great significance for early diagnosis and early treatment of bladder cancer.
而随着科学界对肿瘤的认识逐渐深入，越来越多的研究证实细胞表观遗传水平的改变是肿瘤发生与发展的关键事件。DNA甲基化、组蛋白修饰及miRNA表达异常均属于表观遗传学改变，而肿瘤发生的核心环节也与DNA异常甲基化相关。DNA甲基化检测稳定性好，易于检测且其异常程度常与癌症的进展相关，是最具肿瘤早筛潜力的标志物。As the scientific community's understanding of tumors deepens, more and more studies have confirmed that changes in cell epigenetic levels are key events in the occurrence and development of tumors. DNA methylation, histone modification, and abnormal miRNA expression are all epigenetic changes, and the core link of tumor occurrence is also related to abnormal DNA methylation. DNA methylation testing is stable, easy to detect, and its abnormality is often related to the progression of cancer. It is the marker with the greatest potential for early screening of tumors.
因此，本领域需求一种基于DNA甲基化检测的兼具高灵敏度高特异性的膀胱癌诊断产品，在膀胱癌的早期阶段即能进行检出。Therefore, there is a need in the art for a bladder cancer diagnostic product based on DNA methylation detection that has high sensitivity and specificity and can detect bladder cancer in its early stages.
发明内容Summary of the invention
本发明从UCSC Xena网站的TCGA数据集(https://tcga.xenahubs.net)和美国国立生物技术信息中心(NCBI)的GEO数据库中共收集到413例癌组织、231例癌旁组织或正常对照组织和656例健康全血甲基化数据。以膀胱癌和对照数据进行差异分析，通过大量研究，申请人发现某些甲基化位点的甲基化水平与膀胱癌有着密切的联系。The present invention collected 413 cancer tissues, 231 adjacent tissues or normal control tissues and 656 healthy whole blood methylation data from the TCGA dataset (https://tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI). The bladder cancer and control data were used for differential analysis. Through a large number of studies, the applicant found that the methylation levels of certain methylation sites are closely related to bladder cancer.
第一方面，本发明提供一种用于检测膀胱癌的组合物，所述组合物包括用于检测以下甲基化位点中至少一个的检测试剂：cg00206063、cg03278514，以及cg13314394。In a first aspect, the present invention provides a composition for detecting bladder cancer, the composition comprising a detection reagent for detecting at least one of the following methylation sites: cg00206063, cg03278514, and cg13314394.
使用本发明的组合物，在组织样本中，所有组合物均能以至少0.8571的特异性和0.7203的灵敏度以及0.8585的曲线下面积对膀胱癌进行预测。而在尿液游离DNA样本中，所有组合物均能以至少0.9048的特异性和0.6525的灵敏度以及0.8060的曲线下面积对膀胱癌进行预测，本发明组合物以较少的标志物就能够在临床上以高灵敏度和好特异性对膀胱癌进行检测，成本和时间均得到节约；在临床上，在膀胱癌恶变的早期阶段就能灵 敏和特异性地检出。Using the compositions of the present invention, in tissue samples, all compositions can predict bladder cancer with a specificity of at least 0.8571, a sensitivity of 0.7203, and an area under the curve of 0.8585. In urine free DNA samples, all compositions can predict bladder cancer with a specificity of at least 0.9048, a sensitivity of 0.6525, and an area under the curve of 0.8060. The compositions of the present invention can detect bladder cancer with high sensitivity and good specificity in the clinic with fewer markers, saving both cost and time. In the clinic, it can effectively detect bladder cancer in the early stages of malignant transformation. Sensitive and specific detection.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点中至少两个的检测试剂：cg00206063、cg03278514，以及cg13314394。In some specific embodiments, the composition includes detection reagents for detecting at least two of the following methylation sites: cg00206063, cg03278514, and cg13314394.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg00206063、cg03278514。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg03278514.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg00206063、cg13314394。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg13314394.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg03278514、cg13314394。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: cg03278514, cg13314394.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg00206063、cg03278514和cg13314394。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: cg00206063, cg03278514, and cg13314394.
在一些具体的实施方案中，所述组合物进一步包括用于检测以下任意至少一个的甲基化水平的检测试剂：SPATS2、PARP4。In some specific embodiments, the composition further comprises a detection reagent for detecting the methylation level of at least one of the following: SPATS2, PARP4.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg00206063、SPATS2、PARP4。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: cg00206063, SPATS2, PARP4.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点的检测试剂：SPATS2、PARP4、cg03278514。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: SPATS2, PARP4, cg03278514.
在一些具体的实施方案中，所述组合物包括用于检测以下甲基化位点中的检测试剂：SPATS2、PARP4、cg13314394。In some specific embodiments, the composition includes detection reagents for detecting the following methylation sites: SPATS2, PARP4, cg13314394.
在一些具体的实施方案中，所述组合物进一步包括用于检测以下甲基化水平的检测试剂：SPATS2、PARP4。In some specific embodiments, the composition further comprises a detection reagent for detecting the methylation level of: SPATS2, PARP4.
在一些具体的实施方案中，所述甲基化水平的检测试剂可以是检测整个基因平均甲基化水平的检测试剂。In some specific embodiments, the methylation level detection reagent can be a detection reagent for detecting the average methylation level of the entire gene.
在一些具体的实施方案中，所述甲基化水平的检测试剂也可以是检测一个基因片段的平均甲基化水平的检测试剂。 In some specific embodiments, the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene fragment.
在一些具体的实施方案中，所述甲基化水平的检测试剂也可以是检测一个基因启动子区域或其片段的平均甲基化水平的检测试剂。In some specific embodiments, the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene promoter region or a fragment thereof.
在一些具体的实施方案中，所述甲基化水平的检测试剂还可以是检测基因的一个或多个甲基化位点的检测试剂。In some specific embodiments, the methylation level detection reagent can also be a detection reagent for detecting one or more methylation sites of a gene.
在一些具体的实施方案中，所述组合物进一步包括用于检测SPATS2基因甲基化位点cg06712013的甲基化水平的检测试剂。In some specific embodiments, the composition further comprises a detection reagent for detecting the methylation level of the methylation site cg06712013 of the SPATS2 gene.
在一些具体的实施方案中，所述组合物进一步包括用于检测PARP4基因甲基化位点cg20765408的甲基化水平的检测试剂。In some specific embodiments, the composition further comprises a detection reagent for detecting the methylation level of the PARP4 gene methylation site cg20765408.
在一个具体的实施方案中，所述组合物包括用于检测以下甲基化水平的检测试剂：cg13314394、cg00206063、cg06712013、cg20765408和cg03278514。In a specific embodiment, the composition includes detection reagents for detecting the methylation levels of cg13314394, cg00206063, cg06712013, cg20765408, and cg03278514.
使用上述方案的组合物，能够以更高的灵敏度和更好的特异性对膀胱癌进行检测；在临床上，在膀胱癌恶变的早期阶段就能灵敏和特异性地检出。The composition of the above scheme can detect bladder cancer with higher sensitivity and better specificity; clinically, bladder cancer can be detected sensitively and specifically in the early stages of malignant transformation.
在一些实施方案中，使用本发明的检测试剂，可以对样本中所存在的相应基因的甲基化水平进行检测。In some embodiments, the detection reagent of the present invention can be used to detect the methylation level of the corresponding gene present in the sample.
在本发明中，“样本”为选自个体的生物样本。具体地，例如，选自细胞系、组织切片、活检组织、石蜡包埋的组织、体液、粪便、结肠流出物、尿液、血浆、血清、全血、分离的血细胞、从血液中分离的细胞，或其组合。In the present invention, "sample" is a biological sample selected from an individual. Specifically, for example, selected from cell lines, tissue sections, biopsy tissues, paraffin-embedded tissues, body fluids, feces, colon effluent, urine, plasma, serum, whole blood, separated blood cells, cells separated from blood, or a combination thereof.
优选地，本发明的“样本”为尿液，即尿液中的游离DNA。Preferably, the "sample" of the present invention is urine, ie, free DNA in urine.
尿液中的游离DNA能用作检测肿瘤，具有对病人伤害小，特异性好等特点。但是由于其在尿液中的含量极低，因此，在用于癌症检测时普遍存在灵敏度较低的问题。使用本发明的检测试剂，能够使用尿液中的游离DNA作为样本进行检测，具有较高的灵敏度和特异性。 Free DNA in urine can be used to detect tumors, with the characteristics of little harm to patients and good specificity. However, due to its extremely low content in urine, it generally has the problem of low sensitivity when used for cancer detection. Using the detection reagent of the present invention, free DNA in urine can be used as a sample for detection, with high sensitivity and specificity.
在本发明中，“检测试剂”指的是对样本中的基因的甲基化水平进行检测的试剂。其中，所述甲基化水平是通过扩增-测序、芯片、甲基化荧光定量PCR等方式测量。In the present invention, "detection reagent" refers to a reagent for detecting the methylation level of a gene in a sample, wherein the methylation level is measured by amplification-sequencing, chip, methylation fluorescence quantitative PCR, etc.
在一些具体的实施方案中，检测试剂包括但不限于核酸引物、测序Tag序列，用于通过扩增-测序测量甲基化水平。In some specific embodiments, the detection reagents include, but are not limited to, nucleic acid primers and sequencing Tag sequences, for measuring methylation levels by amplification-sequencing.
在一个具体的实施方案中，所述扩增-测序是通过将样本中的核酸进行重亚硫酸盐处理，随后构建预文库，再构建终文库，最后进行测序评价的方法进行的。In a specific embodiment, the amplification-sequencing is performed by treating the nucleic acid in the sample with bisulfite, then constructing a pre-library, then constructing a final library, and finally performing sequencing evaluation.
在一些具体的实施方案中，检测试剂包括但不限于芯片，所述芯片是甲基化芯片，所述甲基化芯片具有与甲基化区域特异性结合的探针。所述芯片可以是包括但不限于例如安捷伦的Human CpG Island Microarrays和Human DNA Methylation Microarrays、Illumina的Infinium HumanMethylation27BeadChip、Infinium HumanMethylation450BeadChip和GoldenGate Methylation Assay以及Roche NimbleGen的Human DNA Methylation 2.1M Deluxe Promoter Array、Human DNA Methylation Array等，用于通过芯片测量甲基化水平。In some specific embodiments, the detection reagent includes but is not limited to a chip, and the chip is a methylation chip, and the methylation chip has a probe that specifically binds to the methylation region. The chip can be, for example, but is not limited to, Agilent's Human CpG Island Microarrays and Human DNA Methylation Microarrays, Illumina's Infinium Human Methylation 27 Bead Chip, Infinium Human Methylation 450 Bead Chip and Golden Gate Methylation Assay, and Roche NimbleGen's Human DNA Methylation 2.1M Deluxe Promoter Array, Human DNA Methylation Array, etc., for measuring the methylation level through the chip.
在一些具体的实施方案中，检测试剂包括但不限于核酸引物以及核酸探针，用于通过甲基化荧光定量PCR测量甲基化水平。In some specific embodiments, the detection reagents include, but are not limited to, nucleic acid primers and nucleic acid probes for measuring methylation levels by methylation fluorescence quantitative PCR.
进一步地，所述检测试剂还包括内标引物以及内标探针。Furthermore, the detection reagent also includes an internal standard primer and an internal standard probe.
在一些具体的实施方案中，上述组合物还可以包括其余的试剂，具体地，例如，各种对样本进行前处理或者预处理所需要的试剂。例如，提取样本核酸的样本释放剂，纯化样本核酸的纯化剂，转化所用的重亚硫酸盐或亚硫酸氢盐等。In some specific embodiments, the above composition may further include other reagents, specifically, for example, various reagents required for pre-treatment or pre-processing of the sample, such as a sample release agent for extracting sample nucleic acid, a purification agent for purifying sample nucleic acid, bisulfite or bisulfite used for conversion, etc.
进一步地，上述组合物还包括提取尿液游离DNA的试剂。Furthermore, the above composition also includes a reagent for extracting free DNA in urine.
第二方面，本发明提供了上述组合物在制备用于检测膀胱癌的试剂盒 中的用途。In a second aspect, the present invention provides a kit for preparing a kit for detecting bladder cancer using the above composition. Uses in.
在一个具体的实施方案中，上述组合物在制备用于检测膀胱癌的试剂盒中的用途，其中，所述组合物为以下甲基化位点的检测试剂：cg13314394。In a specific embodiment, the above composition is used in the preparation of a kit for detecting bladder cancer, wherein the composition is a detection reagent for the following methylation site: cg13314394.
进一步地，本发明提供了上述组合物在制备使用尿液游离DNA检测膀胱癌的试剂盒中的用途。Furthermore, the present invention provides use of the above composition in preparing a kit for detecting bladder cancer using urine free DNA.
第三方面，本发明提供了一种用于检测膀胱癌的试剂盒，所述试剂盒包括如上所述的组合物。In a third aspect, the present invention provides a kit for detecting bladder cancer, the kit comprising the composition as described above.
进一步地，所述试剂盒还包括但不限于提取核酸的试剂、纯化核酸的试剂、重亚硫酸盐、T4多聚核苷酸激酶、T4连接酶中的至少一种。Furthermore, the kit also includes, but is not limited to, at least one of a reagent for extracting nucleic acid, a reagent for purifying nucleic acid, bisulfite, T4 polynucleotide kinase, and T4 ligase.
进一步地，所述提取核酸的试剂是提取组织DNA的试剂和尿液游离DNA的试剂。Furthermore, the reagents for extracting nucleic acid are reagents for extracting tissue DNA and reagents for extracting urine free DNA.
进一步地，所述提取核酸的试剂是提取尿液游离DNA的试剂。Furthermore, the reagent for extracting nucleic acid is a reagent for extracting free DNA in urine.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为单基因的甲基化水平在组织中鉴别癌与非癌的ROC图；FIG1 is a ROC diagram of the methylation level of a single gene in distinguishing cancer from non-cancer in tissues;
图2为双基因的甲基化水平在组织中鉴别癌与非癌的ROC图；FIG2 is a ROC diagram of the methylation levels of two genes in differentiating cancer from non-cancer tissues;
图3为五基因的甲基化水平在组织中鉴别癌与非癌的ROC图；FIG3 is a ROC diagram of the methylation levels of five genes in differentiating cancer from non-cancerous tissues;
图4为单基因的甲基化水平在尿液中鉴别癌与非癌的ROC图；FIG4 is a ROC diagram of the methylation level of a single gene in distinguishing cancer from non-cancer in urine;
图5为双基因的甲基化水平在尿液中鉴别癌与非癌的ROC图；FIG5 is a ROC diagram of the methylation levels of two genes in distinguishing cancer from non-cancer in urine;
图6为五基因的甲基化水平在尿液中鉴别癌与非癌的ROC图。FIG. 6 is a ROC diagram of the methylation levels of five genes in distinguishing cancer from non-cancer in urine.
具体实施方式Detailed ways
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.
实施例1、甲基化基因的筛选 Example 1: Screening of methylation genes
本发明从UCSC Xena网站的TCGA数据集(https://tcga.xenahubs.net)和美国国立生物技术信息中心(NCBI)的GEO数据库中共收集到413例癌组织、231例癌旁组织或正常对照组织和656例健康全血甲基化数据。以膀胱癌和对照数据进行差异分析，对差异位点进行物理位置和基因信息注释，为保证筛选片段具有一致的甲基化水平，甲基化基因片段的筛选需要同时符合以下几点要求：1)要求所选基因片段具有不少于2个的相邻位点具有一致的甲基化水平；2)以膀胱癌和癌旁组织或正常对照组织进行差异分析，挑选膀胱癌样品中一致性高差异高甲基化的基因片段作为候选靶基因；3)以膀胱癌和健康样本全血甲基化检测数据进行差异分析，挑选膀胱癌差异高甲基化的基因片段；4)最后再从中进行逐个甲基化位点的分析，从而得出候选甲基化位点。The present invention collected 413 cancer tissues, 231 paracancerous tissues or normal control tissues and 656 healthy whole blood methylation data from the TCGA data set (https://tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI). The bladder cancer and control data were used for differential analysis, and the physical location and gene information of the differential sites were annotated. To ensure that the screened fragments have consistent methylation levels, the screening of methylated gene fragments needs to meet the following requirements at the same time: 1) The selected gene fragments are required to have no less than 2 adjacent sites with consistent methylation levels; 2) The bladder cancer and paracancerous tissues or normal control tissues were used for differential analysis, and the gene fragments with high consistency and high differential methylation in the bladder cancer samples were selected as candidate target genes; 3) The whole blood methylation detection data of bladder cancer and healthy samples were used for differential analysis, and the gene fragments with high differential methylation in bladder cancer were selected; 4) Finally, the methylation sites were analyzed one by one to obtain the candidate methylation sites.
实施例2、临床样本的基因甲基化水平检测Example 2: Detection of gene methylation levels in clinical samples
收集样本的尿液各10ml，用于检测分析DNA甲基化标记物在样本中的甲基化水平。实验过程如下所述：10 ml of urine was collected from each sample to detect and analyze the methylation level of DNA methylation markers in the sample. The experimental process is as follows:
1、样本制备1. Sample preparation
本发明的样本制备是通过VAHTS Free-Circulating DNA Maxi Kit提取4ml尿液上清，45μL洗脱液洗脱。提取的游离核酸需满足以下质控条件：提取的核酸总量大于20ng。The sample preparation of the present invention is to extract 4 ml of urine supernatant using VAHTS Free-Circulating DNA Maxi Kit and elute with 45 μL of elution buffer. The extracted free nucleic acid must meet the following quality control conditions: the total amount of extracted nucleic acid is greater than 20 ng.
2、文库制备2. Library preparation
本发明将质控合格的全部游离核酸采用EZ DNA Methylation-Lightning TM Kit进行重亚硫酸盐处理。随后，重亚硫酸盐处理后的样本DNA采用单链建库方法构建预文库，预文库质检合格后通过液态芯片杂交捕获富集目标区域而完成终文库的构建。In the present invention, all free nucleic acids that have passed the quality control are treated with bisulfite using the EZ DNA Methylation-Lightning TM Kit. Subsequently, the sample DNA treated with bisulfite is used to construct a pre-library using a single-stranded library construction method. After the pre-library passes the quality inspection, the target region is captured and enriched by liquid chip hybridization to complete the construction of the final library.
预文库构建步骤：1)磷酸化：T4多聚核苷酸激酶将重亚硫酸盐处理后的DNA的5端磷酸化；2)SS1连接：T4DNA Ligase(Rapid)将SS1 接头连接在磷酸化的DNA的5端；3)核酸纯化：使用2倍体积的Agencourt AMPure XP system去除剩余的接头；4)SS2连接：T4DNA Ligase(Rapid)将SS2接头连接在磷酸化的DNA的3端；5)核酸纯化：使用2倍体积的Agencourt AMPure XP system去除剩余的接头；6)扩增：采用NEBNext Q5U Master Mix以及primer1.0(通用引物)和Bacard序列扩增上一步的核酸；7)核酸纯化：使用1.2倍体积的Agencourt AMPure XP system去引物二聚体和多余的引物；8)质检：纯化处理后的预文库采用dsDNA HS Assay Kit进行文库总量质检，采用LabChip GXII Touch进行文库片段分布质检。Prelibrary construction steps: 1) Phosphorylation: T4 polynucleotide kinase phosphorylates the 5 end of the bisulfite treated DNA; 2) SS1 ligation: T4 DNA Ligase (Rapid) ligates SS1 The adapter was connected to the 5 end of the phosphorylated DNA; 3) Nucleic acid purification: Use 2 volumes of Agencourt AMPure XP system to remove the remaining adapter; 4) SS2 connection: T4 DNA Ligase (Rapid) connected the SS2 adapter to the 3 end of the phosphorylated DNA; 5) Nucleic acid purification: Use 2 volumes of Agencourt AMPure XP system to remove the remaining adapter; 6) Amplification: Use NEBNext Q5U Master Mix, primer1.0 (universal primer) and Bacard sequence to amplify the nucleic acid in the previous step; 7) Nucleic acid purification: Use 1.2 volumes of Agencourt AMPure XP system to remove primer dimers and excess primers; 8) Quality inspection: The purified pre-library is used The dsDNA HS Assay Kit was used to check the total amount of the library, and the LabChip GXII Touch was used to check the fragment distribution of the library.
芯片(Twist Bioscience)杂交捕获步骤：1)芯片杂交：将质检合格混合好的1.5ug的文库预先真空浓缩成粉末状再和Panel、Hybridization Mix、Blocker Solution、Universal Blockers、Hybridization Enhancer试剂混匀，放置在PCR仪中70度孵育16小时过夜(热盖温度为85度)；2)磁珠捕获：预先使用Streptavidin Binding Buffer将捕获磁珠洗涤3次，将杂交完成的产物加入捕获磁珠中，孵育30分钟，Wash Buffer I清洗一次，Wash Buffer 2清洗3次，最后42μl超纯水洗脱；3)扩增：采用KAPA HiFi HotStart ReadyMix以及通用引物扩增捕获后的文库；4)纯化：采用1倍体积的Agencourt AMPure XP system去引物二聚体和多余的引物。Chip (Twist Bioscience) hybridization capture steps: 1) Chip hybridization: vacuum concentrate 1.5ug of the qualified mixed library into powder in advance and then mix it with Panel, Hybridization Mix, Blocker Solution, Universal Blockers, and Hybridization Enhancer reagents, and place it in a PCR instrument and incubate it at 70 degrees for 16 hours overnight (the hot cover temperature is 85 degrees); 2) Magnetic bead capture: wash the captured magnetic beads 3 times with Streptavidin Binding Buffer in advance, add the hybridization product to the captured magnetic beads, incubate for 30 minutes, wash once with Wash Buffer I, wash 3 times with Wash Buffer 2, and finally elute with 42μl ultrapure water; 3) Amplification: use KAPA HiFi HotStart ReadyMix and universal primers to amplify the captured library; 4) Purification: use 1 volume of Agencourt AMPure XP system to remove primer dimers and excess primers.
纯化处理后的文库采用dsDNA HS Assay Kit和LabChip GXII Touch进行文库核酸总量、片段分布和引物二聚体比例质检。The purified library was used The dsDNA HS Assay Kit and LabChip GXII Touch were used to perform quality checks on the total nucleic acid content, fragment distribution, and primer dimer ratio in the library.
3、测序3. Sequencing
将文库总量、扩增产物的片段大小分布和引物二聚体比例质检均合格的待测文库，按照1:1的物质的量进行混合，使用dsDNA HS Asay Kit对混合文库进行精确定量，将文库变性稀释后使用NextSeq500台式测序仪采用PE75进行上机测序。 The libraries to be tested that have passed the quality inspection of the total amount of the library, the fragment size distribution of the amplified product, and the primer dimer ratio are mixed at a 1:1 ratio and used The dsDNA HS Asay Kit was used to accurately quantify the mixed library, and the library was denatured and diluted before sequencing using the NextSeq500 desktop sequencer using PE75.
4、膀胱癌分类模型的建立和评价4. Establishment and evaluation of bladder cancer classification model
对于测序得到的原始fastq数据，对原始数据过滤后使用bismark甲基化分析软件对芯片捕获片段进行甲基化分析，获得候选基因甲基化水平。利用候选基因片段甲基化水平，针对膀胱癌和对照样品进行差异分析和模型构建。For the original fastq data obtained by sequencing, the original data was filtered and then the methylation analysis of the chip captured fragments was performed using the bismark methylation analysis software to obtain the methylation level of the candidate gene. The methylation level of the candidate gene fragment was used to perform differential analysis and model construction for bladder cancer and control samples.
实施例3、本发明组合物测试样本的检测结果Example 3: Test results of the test samples of the composition of the present invention
收集了63例原发性膀胱癌患者、23例膀胱炎患者组织样本和118例膀胱癌患者的尿液10ml以及60例膀胱患者和健康人尿液10ml，用于检测分析候选基因片段甲基化标记物在样本尿液中的甲基化水平。检测结果如表1和图1～6所示。Tissue samples from 63 patients with primary bladder cancer, 23 patients with cystitis, 10 ml of urine from 118 patients with bladder cancer, and 10 ml of urine from 60 patients with bladder disease and healthy people were collected to detect and analyze the methylation levels of candidate gene fragment methylation markers in urine samples. The test results are shown in Table 1 and Figures 1 to 6.
表1


1为cg13314394、2为cg00206063、3为cg06712013、4为cg20765408
和5为cg03278514。Table 1


1 is cg13314394, 2 is cg00206063, 3 is cg06712013, 4 is cg20765408
and 5 is cg03278514.
从表1中可以看出，在组织样本中，所有组合物均能以至少0.8571的特异性和0.7203的灵敏度以及0.8585的曲线下面积对膀胱癌进行预测。而在尿液游离DNA样本中，所有组合物均能以至少0.9048的特异性和0.6525的灵敏度以及0.8060的曲线下面积对膀胱癌进行预测。因此，本发明的组合物具有对膀胱癌很好的预测效果，特别地，在使用尿液游离DNA作为样本时，具有优异的特异性和灵敏度。As can be seen from Table 1, in tissue samples, all the compositions can predict bladder cancer with a specificity of at least 0.8571, a sensitivity of 0.7203, and an area under the curve of 0.8585. In urine free DNA samples, all the compositions can predict bladder cancer with a specificity of at least 0.9048, a sensitivity of 0.6525, and an area under the curve of 0.8060. Therefore, the composition of the present invention has a good predictive effect on bladder cancer, especially, when urine free DNA is used as a sample, it has excellent specificity and sensitivity.
对比例1Comparative Example 1
为了考察cg00206063、cg03278514、cg13314394位点附近其他甲基化位点能否统一作为检测膀胱癌的标志物，选取了这些位点上下游甲基化位点。同样按照上述实施例的方法进行检测，检测结果如下表2～4所示。In order to investigate whether other methylation sites near cg00206063, cg03278514, and cg13314394 sites can be used as markers for detecting bladder cancer, methylation sites upstream and downstream of these sites were selected. The detection was also performed according to the method of the above embodiment, and the detection results are shown in Tables 2 to 4 below.
表2、对比位点在膀胱癌分类模型中的预测性能

6为cg26644674、7为cg06048750。Table 2. Predictive performance of comparison sites in bladder cancer classification model

6 is cg26644674 and 7 is cg06048750.
从表2中可以看出，cg00206063位点上下游甲基化位点在组织样本中的最高AUC为0.565，在游离DNA样本中的最高AUC为0.728，低于本发 明的cg00206063位点的AUC。As can be seen from Table 2, the highest AUC of the upstream and downstream methylation sites of cg00206063 in tissue samples was 0.565, and the highest AUC in free DNA samples was 0.728, which was lower than that of the present invention. The AUC of the cg00206063 locus was shown in Figure 2.
表3、对比位点在膀胱癌分类模型中的预测性能

8为cg07200122、9为cg05554320。Table 3. Predictive performance of comparison sites in bladder cancer classification model

8 is cg07200122 and 9 is cg05554320.
从表3中可以看出，cg13314394位点上下游甲基化位点在组织样本中的最高AUC为0.760，在游离DNA样本中的最高AUC为0.522，低于本发明的cg13314394位点的AUC。As can be seen from Table 3, the highest AUC of the upstream and downstream methylation sites of the cg13314394 site in tissue samples is 0.760, and the highest AUC in free DNA samples is 0.522, which is lower than the AUC of the cg13314394 site of the present invention.
表4、对比位点在膀胱癌分类模型中的预测性能

10为cg12737588、11为cg03578041。Table 4. Predictive performance of comparison sites in bladder cancer classification model

10 is cg12737588 and 11 is cg03578041.
从表4中可以看出，cg03278514位点上下游甲基化位点在组织样本中的最高AUC为0.857，在游离DNA样本中的最高AUC为0.878，低于本发明的cg03278514位点的AUC。As can be seen from Table 4, the highest AUC of the upstream and downstream methylation sites of the cg03278514 site in tissue samples is 0.857, and the highest AUC in free DNA samples is 0.878, which are lower than the AUC of the cg03278514 site of the present invention.
对比例2Comparative Example 2
为了考察cg00206063、cg03278514、cg13314394位点能否作为检测膀胱癌的标志物，进一步选取了本领域知晓的与膀胱癌密切相关的甲基化位点cg16732616和cg04974290。同样按照上述实施例的方法进行检测，检测结果如下表3所示。In order to investigate whether the cg00206063, cg03278514, and cg13314394 sites can be used as markers for detecting bladder cancer, the methylation sites cg16732616 and cg04974290, which are closely related to bladder cancer and are known in the art, were further selected. The detection was also performed according to the method of the above embodiment, and the detection results are shown in Table 3 below.
表5、对比位点及其组合膀胱癌分类模型中的预测性能


12为cg16732616、13为cg04974290。Table 5. Prediction performance of comparison sites and their combination in bladder cancer classification model


12 is cg16732616 and 13 is cg04974290.
从表5中可以看出，对比位点单个位点在组织样本中的最高AUC为0.848，单个位点在游离DNA样本中的最高AUC为0.751；对比位点组合在组织样本中的最高AUC为0.851，组合在游离DNA样本中的最高AUC为0.862，低于本发明的cg00206063、cg03278514、cg13314394位点的AUC。 As can be seen from Table 5, the highest AUC of a single comparison site in tissue samples is 0.848, and the highest AUC of a single site in free DNA samples is 0.751; the highest AUC of the comparison site combination in tissue samples is 0.851, and the highest AUC of the combination in free DNA samples is 0.862, which are lower than the AUCs of the cg00206063, cg03278514, and cg13314394 sites of the present invention.

Claims (10)

  1. 一种用于检测膀胱癌的组合物，所述组合物包括用于检测以下甲基化位点中至少一个的检测试剂：cg00206063、cg03278514，以及cg13314394。A composition for detecting bladder cancer, comprising a detection reagent for detecting at least one of the following methylation sites: cg00206063, cg03278514, and cg13314394.
  2. 根据权利要求1所述的组合物，其中，所述组合物包括用于检测以下甲基化位点中至少两个的检测试剂：cg00206063、cg03278514，以及cg13314394。The composition according to claim 1, wherein the composition comprises detection reagents for detecting at least two of the following methylation sites: cg00206063, cg03278514, and cg13314394.
  3. 根据权利要求2所述的组合物，其中，所述组合物包括用于检测以下甲基化位点的检测试剂：cg00206063、cg03278514和cg13314394。The composition according to claim 2, wherein the composition comprises detection reagents for detecting the following methylation sites: cg00206063, cg03278514 and cg13314394.
  4. 根据权利要求1～3中任一项所述的组合物，其中，所述组合物进一步包括用于检测以下任意至少一个的甲基化水平的检测试剂：SPATS2、PARP4。The composition according to any one of claims 1 to 3, wherein the composition further comprises a detection reagent for detecting the methylation level of any one of the following: SPATS2, PARP4.
  5. 根据权利要求4所述的组合物，其中，所述检测SPATS2基因甲基化水平的检测试剂为检测甲基化位点cg06712013的甲基化水平的检测试剂；所述检测PARP4基因甲基化水平的检测试剂为检测甲基化位点cg20765408的甲基化水平的检测试剂。The composition according to claim 4, wherein the detection reagent for detecting the methylation level of the SPATS2 gene is a detection reagent for detecting the methylation level of the methylation site cg06712013; the detection reagent for detecting the methylation level of the PARP4 gene is a detection reagent for detecting the methylation level of the methylation site cg20765408.
  6. 根据权利要求1-5中任一项所述的组合物，其中，所述检测试剂为核酸引物、测序Tag序列、甲基化芯片、核酸探针中的任意一种或多种。The composition according to any one of claims 1 to 5, wherein the detection reagent is any one or more of a nucleic acid primer, a sequencing tag sequence, a methylation chip, and a nucleic acid probe.
  7. 根据权利要求1-5中任一项所述的组合物，其中，所述组合物进一步包括提取尿液游离DNA的试剂。The composition according to any one of claims 1 to 5, wherein the composition further comprises a reagent for extracting urine free DNA.
  8. 如权利要求1-7中任一项所述的组合物在制备用于检测膀胱癌的试剂盒中的用途。Use of the composition according to any one of claims 1 to 7 in the preparation of a kit for detecting bladder cancer.
  9. 一种用于检测膀胱癌的试剂盒，所述试剂盒包括如权利要求1-7中任一项所述的组合物。A kit for detecting bladder cancer, comprising the composition according to any one of claims 1 to 7.
  10. 根据权利要求9所述的试剂盒，其中，所述试剂盒还包括提取核 酸的试剂，纯化核酸的试剂、重亚硫酸盐、T4多聚核苷酸激酶、T4连接酶中的至少一种。 The kit according to claim 9, wherein the kit further comprises a nucleic acid extraction At least one of a reagent for purifying nucleic acid, a reagent for purifying nucleic acid, bisulfite, T4 polynucleotide kinase, and T4 ligase.
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