WO2023082140A1 - 一种用于诊断肝癌的核酸检测试剂盒 - Google Patents

一种用于诊断肝癌的核酸检测试剂盒 Download PDF

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WO2023082140A1
WO2023082140A1 PCT/CN2021/130060 CN2021130060W WO2023082140A1 WO 2023082140 A1 WO2023082140 A1 WO 2023082140A1 CN 2021130060 W CN2021130060 W CN 2021130060W WO 2023082140 A1 WO2023082140 A1 WO 2023082140A1
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cancer
seq
gene
value
hist1h3g
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PCT/CN2021/130060
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English (en)
French (fr)
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周俭
樊嘉
杨欣荣
黄傲
郭德镇
王宇鹏
孙健泷
汪宇盈
蒋睿婧芳
李志隆
彭佳茜
朱师达
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华大数极生物科技(深圳)有限公司
深圳华大基因股份有限公司
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Priority to PCT/CN2021/130060 priority Critical patent/WO2023082140A1/zh
Publication of WO2023082140A1 publication Critical patent/WO2023082140A1/zh

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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Definitions

  • the invention relates to the field of biomedicine, in particular to a nucleic acid detection kit for diagnosing liver cancer.
  • Liver cancer is one of the cancers with high morbidity and mortality in the world.
  • the incidence of liver cancer in my country is particularly serious. More than 50% of liver cancers in the world occur in China.
  • the screening methods for liver cancer are mainly serum alpha-fetoprotein (AFP) examination and ultrasound imaging detection, but these methods have the problem of low sensitivity or insufficient specificity for early liver cancer, and imaging detection is more limited by examining doctors
  • AFP serum alpha-fetoprotein
  • ultrasound imaging detection is more limited by examining doctors
  • a large part of liver cancer is found at an advanced stage, and the treatment and prognosis of advanced liver cancer are poor, and the five-year survival rate of patients is poor. Therefore, it is of great significance to establish an accurate, simple and economical method for early screening of liver cancer.
  • DNA methylation is an important gene expression regulation mechanism, which can regulate gene expression and silencing, and has a significant impact on the occurrence and development of tumors. Abnormal methylation of cancer-related genes often occurs in the early stages of cancer, so DNA methylation signals are considered to be potential early screening markers for tumors.
  • the study first found gastric cancer-related candidate DNA methylation markers from the DNA methylome, and then used the methylation-specific PCR (MSP) method to analyze a large number of samples. After testing, a panel including 3 markers (ELMO1, ZNF569, C13orf18) was finally obtained, and the sensitivity of this method reached 86% (95% specificity, CI71-95%). More and more research reports have proved the great potential of DNA methylation markers in the field of tumor early screening. The process of transforming the early screening of cancer-based tumors into the clinical industry.
  • MSP methylation-specific PCR
  • the flow chart of the existing liver cancer screening program (the "Standards for Diagnosis and Treatment of Primary Liver Cancer (2019 Edition)” published by the National Health and Medical Commission) is shown in Figure 1.
  • the screening methods for liver cancer are mainly serum alpha-fetoprotein (AFP) examination and ultrasonography detection. These methods have the problem of low sensitivity or insufficient specificity for early liver cancer, and imaging detection is more limited by factors such as the experience of the examiner and the performance of the detection instrument.
  • the treatment and prognosis of advanced liver cancer are poor, and the five-year survival rate of patients is poor.
  • the OTX1 gene encodes a transcription factor protein that is involved in the development of human organs.
  • the HIST1H3G gene encodes a H3 histone, which plays an important role in transcription regulation, DNA repair, DNA replication and chromosome stability.
  • the purpose of the present invention is to provide a nucleic acid detection kit for diagnosing liver cancer.
  • the present invention claims to protect the use of the methylated OTX1 gene and the methylated HIST1H3G gene as markers in any of the following:
  • the methylated OTX1 gene is the methylation of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3.
  • the methylated HIST1H3G gene is the methylation of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.4.
  • the present invention claims the use of substances for detecting the methylation level of the OTX1 gene and the methylation level of the HIST1H3G gene in any of the following:
  • the methylation level of the OTX1 gene is the methylation level of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3.
  • the HIST1H3G gene methylation level is the methylation level of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.4.
  • the substance used to detect the methylation level of OTX1 gene can be a bisulfite reagent, a primer pair and a probe;
  • the primer pair is composed of any one of SEQ ID No.5-SEQ ID No.112.
  • the two single-stranded DNAs shown are composed;
  • the probe is any single-stranded DNA shown in SEQ ID No.113-SEQ ID No.115.
  • the primer pair is composed of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96; the probe is a single-stranded DNA shown in SEQ ID No.113.
  • the substance used to detect the methylation level of the HIST1H3G gene can be a bisulfite reagent, a primer pair and a probe; the primer pair is composed of any one of SEQ ID No.116-SEQ ID No.197.
  • the composition of the two single-stranded DNAs shown; the probe is any single-stranded DNA shown in SEQ ID No.198-SEQ ID No.202.
  • the primer pair consists of two single-stranded DNAs shown in SEQ ID No.140 and SEQ ID No.141; the probe is a single-stranded DNA shown in SEQ ID No.202.
  • the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
  • the cancer is liver cancer.
  • the benign lesion is a benign lesion of the liver or liver cirrhosis.
  • the benign lesions of the liver specifically include hepatic hemangioma, hepatic adenoma, hepatic abscess, hepatic cyst, focal nodular hyperplasia of the liver, idiopathic non-cirrhotic portal hypertension, or inflammatory pseudotumor.
  • Kit I a kit, which is designated as Kit I.
  • Said test kit 1 may comprise:
  • control nucleic acid 1 the sequence of said control nucleic acid 1 is shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3, and has a methylation status associated with non-cancer patients;
  • Control nucleic acid 2 the sequence of said control nucleic acid 2 is shown in SEQ ID No.4, and has the methylation status relevant to non-cancer patients.
  • the kit has any of the following purposes: diagnosing or screening cancer; early warning of cancer before clinical symptoms; distinguishing or assisting in distinguishing cancer from benign lesions.
  • the non-cancer patients may be healthy controls or patients with benign lesions.
  • Kit II a kit, which is designated as Kit II.
  • test kit II may comprise:
  • control nucleic acid 3 the sequence of the control nucleic acid 3 is shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3, and has a methylation status associated with cancer patients;
  • control nucleic acid 4 the sequence of the control nucleic acid 4 is shown in SEQ ID No.4, and has a methylation state relevant to cancer patients.
  • the kit has any of the following purposes: diagnosing or screening cancer; early warning of cancer before clinical symptoms; distinguishing or assisting in distinguishing cancer from benign lesions.
  • kit III a kit, which is designated as kit III.
  • Said kit III may comprise:
  • (D2) Primer pair A and probe A for detecting OTX1 gene methylation level; Described primer pair A is made up of two single-stranded DNAs shown in any group of SEQ ID No.5-SEQ ID No.112 ; The probe A is a single-stranded DNA shown in any one of SEQ ID No.113-SEQ ID No.115.
  • the primer pair A is composed of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96; the probe A is a single-stranded DNA shown in SEQ ID No.113.
  • the primer pair B consists of two single-stranded DNAs shown in SEQ ID No.140 and SEQ ID No.141; the probe B is a single-stranded DNA shown in SEQ ID No.202.
  • the kit has any of the following purposes: diagnosing or screening cancer; early warning of cancer before clinical symptoms; distinguishing or assisting in distinguishing cancer from benign lesions.
  • the kit can also contain a primer pair C and a probe C for amplifying the internal reference gene ACTB;
  • the primer pair C consists of two single-stranded DNAs shown in SEQ ID No.203 and SEQ ID No.204;
  • the probe C is a single-stranded DNA shown in SEQ ID No.205.
  • the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
  • the cancer is liver cancer
  • the benign lesion is a benign liver lesion or liver cirrhosis.
  • the benign lesions of the liver specifically include hepatic hemangioma, hepatic adenoma, hepatic abscess, hepatic cyst, focal nodular hyperplasia of the liver, idiopathic non-cirrhotic portal hypertension, or inflammatory pseudotumor.
  • the present invention claims a method for diagnosing or screening cancer.
  • the method for diagnosing or screening cancer claimed in the present invention may include the following steps: detecting the methylation levels of OTX1 gene and HIST1H3G gene in the sample from the test subject, so as to realize the diagnosis or screening of cancer.
  • the present invention claims a method for early warning of cancer before clinical symptoms.
  • the method for early warning of cancer before clinical symptoms claimed in the present invention may include the following steps: detecting the methylation levels of OTX1 gene and HIST1H3G gene in samples from the subject, so as to realize early warning of cancer before clinical symptoms.
  • the present invention claims a method for distinguishing or assisting in distinguishing cancer and benign lesions.
  • the method for distinguishing or assisting in distinguishing between cancer and benign lesions claimed in the present invention may include the following steps: detecting the methylation levels of OTX1 gene and HIST1H3G gene in samples from the subject, so as to realize or assist in distinguishing between cancer and benign lesions.
  • the methylation level of the OTX1 gene is the methylation level of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 basic level.
  • the methylation level of the HIST1H3G gene is the methylation level of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.4.
  • the methods for detecting the methylation levels of the OTX1 gene and the HIST1H3G gene in the sample from the subject include but are not limited to bisulfite conversion, PCR, methylation PCR-specific PCR (MS-PCR), pyrosequencing (pyrosequencing), high-throughput sequencing (High-throughput sequencing), three-generation sequencing or single-molecule sequencing (Third-generation sequencing), etc.
  • detecting the methylation level of the OTX1 gene and the HIST1H3G gene in the sample from the subject is carried out according to a method comprising the following steps:
  • E2 DNA (such as cfDNA) converted by bisulfite obtained through real-time fluorescent quantitative PCR amplification (E1); the primer pair for the OTX1 gene used when performing the real-time fluorescent quantitative PCR amplification is represented by SEQ ID No.
  • 5-SEQ ID No.112 is composed of two single-stranded DNAs shown in any group, and the probe is a single-stranded DNA shown in any one of SEQ ID No.113-SEQ ID No.115; carry out the real-time fluorescent quantitative PCR
  • the primer pair for the HIST1H3G gene used in the amplification consists of two single-stranded DNAs shown in any group of SEQ ID No.116-SEQ ID No.197, and the probe is SEQ ID No.198-SEQ ID No.202 Single-stranded DNA as indicated in either.
  • the primer pair for the OTX1 gene used when performing the real-time fluorescent quantitative PCR amplification is composed of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96, and the probe is SEQ ID No.113 Single-stranded DNA indicated.
  • the primer pair for the HIST1H3G gene used in the real-time fluorescent quantitative PCR amplification is composed of two single-stranded DNAs shown in SEQ ID No.140 and SEQ ID No.141, and the probe is shown in SEQ ID No.202 single-stranded DNA.
  • step (E2) the internal reference used when performing the real-time fluorescent quantitative PCR amplification is the ACTB gene, and the primer pair for amplifying the ACTB gene is shown in SEQ ID No.203 and SEQ ID No.204 Composed of two single-stranded DNAs, the probe is the single-stranded DNA shown in SEQ ID No.205.
  • step (E2) the following steps may also be included:
  • ⁇ Ct value (OTX1) ⁇ 5 and/or ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the subject is or is a candidate for cancer patients, or the subject is a high-risk cancer patient;
  • ⁇ Ct value (OTX1) >5 and ⁇ Ct value (HIST1H3G)>9 it is determined that the subject is or is a candidate for a non-cancer patient, or the subject is a low-risk cancer patient;
  • the ⁇ Ct value (OTX1) ⁇ 5 it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when the ⁇ Ct value (OTX1)>5, it is determined that the subject is or is a candidate Is a non-cancer patient, or the test subject is a low-risk cancer patient;
  • the selected ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when the ⁇ Ct value (HIST1H3G)>9, it is determined that the subject is or is a candidate It is a non-cancer patient, or the test subject is a low-risk cancer patient.
  • the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
  • the cancer is liver cancer;
  • the benign lesion is benign liver lesion or liver cirrhosis.
  • the benign lesions of the liver specifically include hepatic hemangioma, hepatic adenoma, hepatic abscess, hepatic cyst, focal nodular hyperplasia of the liver, idiopathic non-cirrhotic portal hypertension, or inflammatory pseudotumor.
  • the sample can be a sample from which DNA (preferably cfDNA) can be extracted, including but not limited to plasma, serum, blood, tissue, saliva, urine, feces and the like.
  • DNA preferably cfDNA
  • the present invention claims a system.
  • the system claimed in the present invention may include:
  • F2 A device, which includes a data collection module, a threshold value storage module, a data comparison module, a data processing and conclusion output module.
  • the data collection module is configured to collect (F1) the detected real-time fluorescent quantitative PCR amplification result data of the sample from the subject.
  • the threshold storage module is configured to store threshold A, threshold B and threshold C; the threshold A is the threshold of the Ct value of the ACTB gene; the threshold B is the threshold of the Ct value of the OTX1 gene; the threshold C is HIST1H3G Threshold for the Ct value of the gene.
  • the data comparison module is configured to receive the real-time fluorescent quantitative PCR amplification result data of the sample of the subject sent from the data collection module, and call the threshold A stored in the threshold storage module , the threshold B and the threshold C, then compare the Ct value of the ACTB gene of the subject with the threshold A, and compare the Ct value of the OTX1 gene of the subject with the threshold B Compare, and compare the Ct value of the HIST1H3G gene of the test subject with the threshold value C.
  • the data processing and conclusion output module is configured to receive the comparison result sent from the data comparison module, and then output the conclusion as follows:
  • the Ct value of the OTX1 gene of the subject is greater than the threshold B, and the Ct value of the HIST1H3G gene of the subject is greater than the threshold C, then it is determined that the subject is or is a candidate for a non-cancer patient, or The test subject is a low-risk cancer patient; if the Ct value of the OTX1 gene of the test subject is less than or equal to the threshold B, and/or the Ct value of the HIST1H3G gene of the test subject is less than or equal to the threshold C, according to (1)-(3) are further judged as follows:
  • ⁇ Ct value (OTX1) ⁇ 5 and/or ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the subject is or is a candidate for cancer patients, or the subject is a high-risk cancer patient;
  • ⁇ Ct value (OTX1) >5 and ⁇ Ct value (HIST1H3G)>9 it is determined that the subject is or is a candidate for a non-cancer patient, or the subject is a low-risk cancer patient;
  • the selected ⁇ Ct value (OTX1) ⁇ 5 it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when the ⁇ Ct value (OTX1)>5, it is determined that the subject is a cancer patient. Or the candidate is a non-cancer patient, or the test subject is a low-risk cancer patient;
  • the selected ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when the ⁇ Ct value (HIST1H3G)>9, it is determined that the subject is Either the candidate is a non-cancer patient, or the test subject is a low-risk cancer patient.
  • the threshold A, the threshold B, and the threshold C are all 37.
  • the present invention claims the application of the kit described in the third to fifth aspects above or the system described in the ninth aspect above in any of the following:
  • the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer and the like.
  • the cancer is liver cancer;
  • the benign lesion is benign liver lesion or liver cirrhosis.
  • test subjects are selected from: patients with liver cancer, patients with benign liver lesions, and patients with liver cirrhosis after healthy people.
  • the liver cancer may be primary hepatocellular carcinoma, intrahepatic cholangiocarcinoma or mixed liver cancer.
  • the benign lesions of the liver specifically include hepatic hemangioma, hepatic adenoma, hepatic abscess, hepatic cyst, focal nodular hyperplasia of the liver, idiopathic non-cirrhotic portal hypertension, or inflammatory pseudotumor.
  • Figure 1 shows that the current screening methods for liver cancer are mainly serum alpha-fetoprotein (AFP) examination and ultrasound imaging.
  • AFP serum alpha-fetoprotein
  • Figure 2 shows 2588 differentially methylated regions (DMRs) discovered based on 44 pairs of liver cancer tumor tissues and adjacent tissues, of which 333 are hypermethylated (Hyper) DMRs and 2255 are hypomethylated (Hypo) DMRs.
  • DMRs differentially methylated regions
  • Figure 3 is a heat map of the methylation rate of DMRs based on 51 models in the validation set I.
  • Figure 4 shows the effectiveness of the liver cancer methylation model based on 51 DMRs in the validation set I.
  • Figure 5 shows the performance of OTX1 and/or HIST1 H3G methylation in independent validation set II.
  • the present invention finds out 2588 differentially methylated regions (Differentially methylated region, DMR).
  • the present invention carries out targeted methylation high-throughput sequencing of a total of 529 plasma free cell DNA (plasma cfDNA) from 295 patients with liver cancer, 180 healthy people, 34 patients with benign liver lesions, and 21 patients with liver cirrhosis. , collected data on the methylation levels of these DMRs in the cfDNA of liver cancer patients and healthy individuals.
  • plasma cfDNA 529 plasma free cell DNA
  • the present invention builds a liver cancer risk prediction model through screening of certain conditions and machine learning, and selects 51 DMRs that can be used for liver cancer screening.
  • the OTX1 gene and the HIST1H3G gene with better performance were selected as methylation markers for detecting liver cancer.
  • the present invention uses OTX1 gene and HIST1H3G gene methylation as markers for liver cancer screening.
  • OTX1 gene and HIST1H3G gene methylation are markers for liver cancer screening.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
  • DMRs differentially methylated regions
  • DNeasy Blood & Tissue Kit (Qiagen, #69506) was used to extract DNA from 44 pairs of liver cancer and liver cancer paracancerous tissue samples. Quantification was performed with Qubit3.0 system (Invitrogen, USA).
  • KAPA HyperPlus Library Preparation Kit (KAPA, #KK8510) was used for library construction.
  • Kit instructions in which the adapters used in the adapter ligation step and the PCR primers used in the PCR step were replaced with adapters and primers suitable for the MGISEQ platform.
  • Hybridization, capture and elution were performed using Seq Cap EZ Hybridization and Wash Kit (ROCHE, 5634253001) and SeqCap Epi CpGiant Enrichment Kit (ROCHE, 7138911001). Since the sequencing instrument of the MGI platform is used, the Block used in the hybridization process must use the corresponding Block of the MGI platform.
  • PE100 sequencing was performed using MGISEQ-2000 (MGI).
  • FIG. 2 shows a heat map of 2588 DMRs discovered based on 44 pairs of HCC tissues and paracancerous tissues.
  • the liver cancer tissue is on the left, and the paracancerous tissue is on the right.
  • Hypermethylated DMRs are located above the heatmap and hypomethylated DMRs are located below the heatmap.
  • Each cell represents the DMR methylation rate of the corresponding sample at this site, and its range is 0-1. The closer the methylation rate is to 0, the darker the color.
  • Plasma cfDNA plasma cfDNA
  • 140 liver cancer patients and 84 healthy individuals from independent sample set I machine learning was used to construct a liver cancer risk model to verify these DMRs.
  • cfDNA was extracted from liver cancer and healthy human plasma samples using the MagPure Circulating DNA Maxi Kit (MAGEN, #12917PJ-100). Quantification was performed with Qubit3.0 system (Invitrogen, USA). Take 10ng of cfDNA, and add 0.05ng of unmethylated lambda DNA that was interrupted and screened to about 160bp for subsequent C-U transformation quality control.
  • KAPA HyperPlus Library Preparation Kit (KAPA, #KK8510) was used for library construction.
  • Kit instructions in which the adapters used in the adapter ligation step and the PCR primers used in the PCR step were replaced with adapters and primers suitable for the MGISEQ platform.
  • Hybridization, capture and elution were performed using Seq Cap EZ Hybridization and Wash Kit (ROCHE, 5634253001) and SeqCap Epi CpGiant Enrichment Kit (ROCHE, 7138911001). Since the sequencing instrument of the MGI platform is used, the Block used in the hybridization process must use the corresponding Block of the MGI platform.
  • PE100 sequencing was performed using MGISEQ-2000 (MGI).
  • Figure 3 shows the heat map of the methylation rate of 51 DMRs in 140 HCC patients and 84 healthy people.
  • the horizontal axis is samples, liver cancer patients are on the far left, followed by healthy people. It can be seen from the figure that the screened 51 DMRs have a clear distinction between liver cancer patients and healthy samples.
  • Fig. 4 shows the performance of the liver cancer methylation model based on 51 DMRs in the verification set I of this embodiment.
  • the horizontal axis represents the false positive rate (1-specificity), and the vertical axis represents the sensitivity. It can be seen from the figure that the methylation model has a good judgment ability for the sample of this example.
  • the present invention selects OTX1 gene and HIST1H3G gene with better performance as methylation markers for detecting liver cancer (the methylation level of these two genes increases in liver cancer) .
  • the nucleotide sequence of the OTX1 gene liver cancer-specific methylated region is shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3.
  • the nucleotide sequence of the HIST1H3G gene liver cancer-specific methylation region is shown in SEQ ID No.4. See Table 1 for information about the specific methylated regions of the OTX1 gene in liver cancer. See Table 2 for information about HIST1H3G gene liver cancer-specific methylated regions.
  • the main purpose of this example is to verify the performance of OTX1 and HIST1H3G gene methylation screening for liver cancer in an independent verification set II, including 293 liver cancer patients, 33 patients with benign liver lesions, 19 healthy people and 105 liver cancer patients. Plasma cell-free DNA in patients with cirrhosis.
  • the inclusion criteria for liver cancer samples were: pathologically confirmed primary hepatocellular carcinoma, intrahepatic cholangiocarcinoma or mixed liver cancer, no previous history of malignant tumors, and no anti-tumor treatment in any form before surgery.
  • the benign liver lesions specifically include hepatic hemangioma, hepatic adenoma, hepatic abscess, hepatic cyst, focal nodular hyperplasia of the liver, idiopathic noncirrhotic portal hypertension, or inflammatory pseudotumor.
  • cfDNA was extracted from liver cancer and healthy human plasma samples using the MagPure Circulating DNA Maxi Kit (MAGEN, #12917PJ-100). Quantification was performed with Qubit 3.0 system (Invitrogen, USA).
  • bisulfite-converted cfDNA was amplified by qPCR.
  • the entire reaction system contains 5 ⁇ l 10 ⁇ PCR buffer (Novizumab, China), 2 units of Taq polymerase (Novizumab, China), 2.5mM dNTP (Novizumab, China), 2 ⁇ l (10pmol/ ⁇ l) PCR primers , 1 ⁇ l (10 pmol/ ⁇ l) probe (Table 3 and Table 4), and bisulfite-converted cfDNA.
  • the PCR reaction was carried out under the following conditions: pre-denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds.
  • the Hongshi 96S qPCR instrument was used for detection. The baseline and threshold were set by default. Fluorescent signals were collected at the end of each cycle, and finally extended at 72°C for 5 minutes.
  • the primer pair (SEQ ID No.95 and SEQ ID No.96) used to amplify SEQ ID No.3 in OTX1 the probe is SEQ ID No.113 (can detect the following CpG sites: chr2:63284080, chr2 :63284082, chr2:63284092, chr2:63284099, chr2:63284104, chr2:63284111, chr2:63284140, chr2:63284146), and the primer pair (SEQ ID No.14 for amplifying SEQ ID No.4 in HIST1H3G 0 and SEQ ID No.141), the probe is SEQ ID No.202 (the following CpG sites can be detected: chr6:26271462, chr6:26271464, chr6:26271474, chr6:26271510, chr6:26271513, chr6:26271520, chr6:
  • the threshold value of Ct value for each gene was 37.
  • ⁇ Ct value (OTX1) ⁇ 5 and/or ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the subject is a liver cancer patient; when ⁇ Ct value (OTX1)>5 and ⁇ Ct value (HIST1H3G)>9, It is determined that the test subject is a non-liver cancer patient;
  • the ⁇ Ct values of the two genes were integrated for analysis, and the AUC could reach 0.909.
  • the ⁇ Ct value (OTX1) ⁇ 5 and the ⁇ Ct value (HIST1H3G) ⁇ 9 it is determined that the test is positive.
  • the sample is determined to be positive.
  • the specificity including healthy people, When cirrhosis and benign liver lesions
  • the sensitivity liver cancer
  • the present invention finds that the hypermethylation of OTX1 gene and HIST1H3G gene is related to liver cancer, and can be used as a marker for liver cancer detection.
  • the present invention provides an accurate, simple and economical means for early screening of liver cancer, which can increase the detection rate of liver cancer, especially early liver cancer, among high-risk groups of liver cancer and general physical examination groups, thereby improving the survival rate of liver cancer patients and saving a lot of money. medical expenditure and reduce medical burden.

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Abstract

本发明公开了一种用于诊断肝癌的核酸检测试剂盒。本发明提供了甲基化OTX1基因和甲基化HIST1H3G基因作为标记物在如下中的应用：诊断或筛查癌症、在临床症状之前预警癌症、区分或辅助区分癌症和良性病变。本发明用OTX1基因和HIST1H3G基因甲基化作为肝癌筛查的标志物。通过检测OTX1基因和HIST1H3G基因的甲基化水平，并对所得数据进行分析，可以预测受检者罹患肝癌的可能性，进而实现在普通人群或肝癌高危人群中筛查肝癌的目的。

Description

一种用于诊断肝癌的核酸检测试剂盒 技术领域
本发明涉及生物医学领域，具体涉及一种用于诊断肝癌的核酸检测试剂盒。
背景技术
肝癌是世界上发病、死亡率很高的癌症之一，我国的肝癌发病情况尤为严重，全球超过50％的肝癌发生在中国。目前肝癌的筛查手段主要是血清甲胎蛋白(AFP)检查和超声显像检测，但这些方法存在对早期肝癌的灵敏性低或特异性不足的问题，且影像学检测更受限于检查医生的经验、检测仪器的性能等因素，目前有很大一部分肝癌发现时已经是晚期，而晚期肝癌的治疗、预后较差，患者的五年生存率较差。因此，建立一种准确、简便、经济的肝癌早期筛查方法具有重大意义。
当人体内的细胞破裂或死亡时，会释放其DNA进入循环系统内，即为游离DNA(cell-freeDNA，cfDNA)。同样的，当肿瘤细胞破裂或死亡时，也会释放出循环肿瘤DNA(circulatingtumorDNA，ctDNA)，这些DNA携带有肿瘤细胞的遗传学信息。通过检测混杂在cfDNA里的ctDNA，分析其携带的突变和表观遗传学等信息，即可推断受检者罹患癌症的可能性。
科学家们开发出了许多高分子利用率的高通量测序技术，使得在血浆游离DNA中检测到微量的突变信号成为了可能，也推动着精准肿瘤早筛的发展。科学家们先从基因突变中寻找适用于肿瘤早筛的生物标志物，但是研究表明，单一采用突变信号进行肿瘤早筛效果有限。因此，科学家们开始从表观遗传学层面进行肿瘤早筛探索。DNA甲基化是一种重要的基因表达调控机制，能够调节基因的表达和沉默，在肿瘤的发生发展中具有重大的影响。癌症相关基因的异常甲基化常出现于癌症发生的早期，因此DNA甲基化信号被认为是有潜力的肿瘤早期筛查标志物。
综合国内外专注于肿瘤早筛技术研究的公司来看：目前Grail主要采用的研究技术是cfDNA靶向测序、WGS和WGBS，通过对大量癌症样本和非癌对照样本进行全基因组测序，挖掘肿瘤特异的突变和甲基化分子标志物。此策略能够较全面地对肿瘤基因组图谱进行研究，但高深度的全基因组测序带来的巨额成本并非一般研究单位能够承担。Guardant Health则专注于液体活检技术，采用高灵敏度的检测技术进行肿瘤早筛研究，但液体活检技术用于肿瘤早筛依然存在许多局限，如：早期肿瘤突变信号极弱、部分基因突变在多个不同癌种中存在和克隆性造血会对ctDNA检测带来巨大的干扰等。因此单一采用突变作为分子标志物效果受限较大。泛生子则联合突变和蛋白标志物进行检测，该研究也显示了应用多组学进行检测能够有效提高检测性能。鹍远基因和基准医疗则专注于甲基化的检测，DNA甲基化的变化往往是多个位点同时发生，因此相比单个位点的基因突变具有更高的灵敏性，而且DNA甲基化信号的组织特异性，使得泛癌种肿 瘤早筛成为可能，因此甲基化是肿瘤早筛非常理想的分子标志物。
目前国内外有许多基于DNA甲基化的肿瘤早筛研究，如被誉为“无创DNA产前检测的奠基人”的香港著名分子生物学临床应用专家卢煜明，在2019年时发表了使用低深度WGBS测序检测尿液cfDNA中的甲基化和拷贝数变异(CNA)情况，此方法用于膀胱癌检测灵敏性达到93.5％(特异性95.8％)；如Anderson,B.W.等人在2018年发表的关于胃癌甲基化标志物临床验证的研究，该研究首先从DNA甲基化组中找到了胃癌相关的候选DNA甲基化标志物，然后采用甲基化特异性PCR(MSP)方法对大量样本进行测试，最终获得一个包括3个marker的panel(ELMO1,ZNF569,C13orf18)，该方法的灵敏性达到86％(特异性95％，CI71-95％)。越来越多的研究报道证明了DNA甲基化标志物在肿瘤早筛领域的巨大潜力，在大量的研究基础上，开发一种基于甲基化的、“轻便”的检测方法将会加速甲基化肿瘤早筛向临床产业转化的进程。
现有的肝癌筛查方案(卫健委公布《原发性肝癌诊疗规范(2019年版)》)流程图如图1。目前肝癌的筛查手段主要是血清甲胎蛋白(AFP)检查和超声显像检测。这些方法存在对早期肝癌的灵敏性低或特异性不足的问题，且影像学检测更受限于检查医生的经验、检测仪器的性能等因素，目前有很大一部分肝癌发现时已经是晚期，而晚期肝癌的治疗、预后较差，患者的五年生存率较差。
OTX1基因编码一种转录因子蛋白，参与人体器官发育。HIST1H3G基因编码一种H3组蛋白，组蛋白在转录调控、DNA修复、DNA复制及染色体的稳定性等方面都有重要的作用。
发明公开
本发明的目的是提供一种用于诊断肝癌的核酸检测试剂盒。
第一方面，本发明要求保护甲基化OTX1基因和甲基化HIST1H3G基因作为标记物在如下任一中的应用：
(A1)制备用于诊断或筛查癌症的产品；
(A2)诊断或筛查癌症；
(A3)制备用于在临床症状之前预警癌症的产品；
(A4)在临床症状之前预警癌症；
(A5)制备用于区分或辅助区分癌症和良性病变的产品；
(A6)区分或辅助区分癌症和良性病变。
其中，所述甲基化OTX1基因为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化。所述甲基化HIST1H3G基因为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化。
第二方面，本发明要求保护用于检测OTX1基因甲基化水平和HIST1H3G基因甲基化水平的物质在如下任一中的应用：
(A1)制备用于诊断或筛查癌症的产品；
(A2)诊断或筛查癌症；
(A3)制备用于在临床症状之前预警癌症的产品；
(A4)在临床症状之前预警癌症；
(A5)制备用于区分或辅助区分癌症和良性病变的产品；
(A6)区分或辅助区分癌症和良性病变。
其中，所述OTX1基因甲基化水平为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化水平。所述HIST1H3G基因甲基化水平为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化水平。
进一步地，所述用于检测OTX1基因甲基化水平的物质可为重亚硫酸盐试剂以及引物对和探针；所述引物对由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成；所述探针为SEQ ID No.113-SEQ ID No.115中任一所示单链DNA。优选地，所述引物对由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成；所述探针为SEQ ID No.113所示单链DNA。
进一步地，所述用于检测HIST1H3G基因甲基化水平的物质可为重亚硫酸盐试剂以及引物对和探针；所述引物对由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成；所述探针为SEQ ID No.198-SEQ ID No.202中任一所示单链DNA。优选地，所述引物对由SEQ ID No.140和SEQ ID No.141所示两条单链DNA组成；所述探针为SEQ ID No.202所示单链DNA。
在上述两方面的应用中，所述癌症包括但不限于肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌等。
在本发明的具体实施方式中，所述癌症为肝癌。相应的，所述良性病变为肝脏良性病变或肝硬化。所述肝脏良性病变具体为肝血管瘤，肝腺瘤，肝脓肿，肝囊肿，肝局灶性结节性增生，特发性非肝硬化性门静脉高压或炎性假瘤等。
第三方面，本发明要求保护一种试剂盒，记为试剂盒I。
所述试剂盒I可包含：
(B1)重亚硫酸盐试剂；以及
(B2)对照核酸1，所述对照核酸1的序列如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示，并且具有与非癌症患者相关的甲基化状态；
(B3)对照核酸2，所述对照核酸2的序列如SEQ ID No.4所示，并且具有与非癌症患者相关的甲基化状态。
所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
其中，所述非癌症患者可为健康对照或良性病变患者。
第四方面，本发明要求保护一种试剂盒，记为试剂盒II。
所述试剂盒II可包含：
(C1)重亚硫酸盐试剂；以及
(C2)对照核酸3，所述对照核酸3的序列如SEQ ID No.1或SEQ ID No.2 或SEQ ID No.3所示，并且具有与癌症患者相关的甲基化状态；
(C3)对照核酸4，所述对照核酸4的序列如SEQ ID No.4所示，并且具有与癌症患者相关的甲基化状态。
所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
第五方面，本发明要求保护一种试剂盒，记为试剂盒III。
所述试剂盒III可包含：
(D1)重亚硫酸盐试剂；以及
(D2)用于检测OTX1基因甲基化水平的引物对A和探针A；所述引物对A由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成；所述探针A为SEQ ID No.113-SEQ ID No.115中任一所示单链DNA。
优选地，所述引物对A由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成；所述探针A为SEQ ID No.113所示单链DNA。
(D3)用于检测HIST1H3G基因甲基化水平的引物对B和探针B；所述引物对B由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成；所述探针B为SEQ ID No.198-SEQ ID No.202中任一所示单链DNA。
优选地，所述引物对B由SEQ ID No.140和SEQ ID No.141所示两条单链DNA组成；所述探针B为SEQ ID No.202所示单链DNA。
所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
所述试剂盒中还可含有用于扩增内参基因ACTB的引物对C和探针C；所述引物对C由SEQ ID No.203和SEQ ID No.204所示两条单链DNA组成；所述探针C为SEQ ID No.205所示单链DNA。
在上述第三到第五方面所述试剂盒中，所述癌症包括但不限于肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌等。
在本发明的具体实施方式中，所述癌症为肝癌，相应的，所述良性病变为肝脏良性病变或肝硬化。所述肝脏良性病变具体为肝血管瘤，肝腺瘤，肝脓肿，肝囊肿，肝局灶性结节性增生，特发性非肝硬化性门静脉高压或炎性假瘤等。
第六方面，本发明要求保护一种诊断或筛查癌症的方法。
本发明要求保护的诊断或筛查癌症的方法，可包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现诊断或筛查癌症。
第七方面，本发明要求保护一种在临床症状之前预警癌症的方法。
本发明要求保护的在临床症状之前预警癌症的方法，可包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现在临床症状之前预警癌症。
第八方面，本发明要求保护一种区分或辅助区分癌症和良性病变的方法。
本发明要求保护的区分或辅助区分癌症和良性病变的方法，可包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现区分或辅助区分癌症和良性病变。
在上述第六至第八方面中，所述OTX1基因甲基化水平为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化水平。
在上述第六至第八方面中，所述HIST1H3G基因甲基化水平为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化水平。
在上述第六至第八方面中，检测来自所述待测者的所述样本中OTX1基因和HIST1H3G基因甲基化水平的方法包括但不限于重硫酸盐转化(bisulfite conversion)、PCR、甲基化特异性PCR(MS-PCR)、焦磷酸测序(pyrosequencing)、高通量测序(High-throughput sequencing)、三代测序或单分子测序(Third-generation sequencing)等。
在本发明的具体实施方式中，检测来自所述待测者的所述样本中OTX1基因和HIST1H3G基因甲基化水平按照包括如下步骤的方法进行：
(E1)从所述样本中提取DNA(如cfDNA)，然后进行重亚硫酸盐转化；
(E2)通过实时荧光定量PCR扩增(E1)所得经重亚硫酸盐转化的DNA(如cfDNA)；进行所述实时荧光定量PCR扩增时采用的针对OTX1基因的引物对由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成，探针为SEQ ID No.113-SEQ ID No.115中任一所示单链DNA；进行所述实时荧光定量PCR扩增时采用的针对HIST1H3G基因的引物对由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成，探针为SEQ ID No.198-SEQ ID No.202任一中所示单链DNA。
优选地，进行所述实时荧光定量PCR扩增时采用的针对OTX1基因的引物对由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成，探针为SEQ ID No.113所示单链DNA。进行所述实时荧光定量PCR扩增时采用的针对HIST1H3G基因的引物对由SEQ ID No.140和SEQ ID No.141所示两条单链DNA组成，探针为SEQ SEQ ID No.202所示单链DNA。
进一步地，在步骤(E2)中，进行所述实时荧光定量PCR扩增时采用的内参为ACTB基因，用于扩增ACTB基因的引物对由SEQ ID No.203和SEQ ID No.204所示两条单链DNA组成，探针为SEQ ID No.205所示单链DNA。
更进一步地，在步骤(E2)后，还可包括如下步骤：
(E3)根据(E2)中所述实时荧光定量PCR扩增的结果按照如下进行判定：
当ACTB基因的Ct值大于37时，判定为结果不可信；当ACTB基因的Ct值≤37时，判定为结果可信,按照如下进行进一步判定：
当OTX1基因和HIST1H3G基因的Ct值均大于37时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；当OTX1基因和HIST1H3G基因的Ct值不均大于37时(即均或之一≤37时)，按照如下(1)-(3)进一步判 定：
(1)当OTX1基因和HIST1H3G基因的Ct值均≤37时，计算OTX1基因和HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，分别记为△Ct值(OTX1)和△Ct值(HIST1H3G)；
选定△Ct值(OTX1)≤5和/或△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5且△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
(2)当OTX1基因的Ct值≤37，但是HIST1H3G基因的Ct值＞37时，计算OTX1基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(OTX1)；
选定△Ct值(OTX1)≤5时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
(3)当HIST1H3G基因的Ct值≤37，但是OTX1基因的Ct值＞37时，计算HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(HIST1H3G)；
选定△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者。
在上述第六至第八方面中，所述癌症包括但不限于肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌等。
在本发明的具体实施方式中，所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。所述肝脏良性病变具体为肝血管瘤，肝腺瘤，肝脓肿，肝囊肿，肝局灶性结节性增生，特发性非肝硬化性门静脉高压或炎性假瘤等。
在上述第六至第八方面中，所述样本可为能够提取DNA(优选cfDNA)的样本，包括但不限于血浆、血清、血液、组织、唾液、尿液、粪便等。
第九方面，本发明要求保护一种系统。
本发明要求保护的系统，可包括：
(F1)前文第五方面所述的试剂盒和实时荧光定量PCR仪；
(F2)装置，所述装置包括数据采集模块、阈值存储模块、数据比较模块、数据处理及结论输出模块。
所述数据采集模块被配置为采集(F1)检测得到的来自所述待测者的所述样本的实时荧光定量PCR扩增结果数据。
所述阈值存储模块被配置为存储阈值A、阈值B和阈值C；所述阈值A为ACTB基因的Ct值的阈值；所述阈值B为OTX1基因的Ct值的阈值；所述阈值C为HIST1H3G基因的Ct值的阈值。
所述数据比较模块被配置为接收来自所述数据采集模块发送的所述待测者 的所述样本的实时荧光定量PCR扩增结果数据，并调用所述阈值存储模块中存储的所述阈值A、所述阈值B和所述阈值C，然后将所述待测者的ACTB基因的Ct值与所述阈值A进行比较，将所述待测者的OTX1基因的Ct值与所述阈值B行比较，并将所述待测者的HIST1H3G基因的Ct值与所述阈值C行比较。
所述数据处理及结论输出模块被配置为接收来自所述数据比较模块发送的比较结果，然后按照如下输出结论：
若所述待测者的ACTB基因的Ct值大于所述阈值A，则判定为结果不可信；当ACTB基因的Ct值≤37时，判定为结果可信,按照如下进行进一步判定：
若所述待测者的OTX1基因的Ct值大于所述阈值B，且所述待测者的HIST1H3G基因的Ct值大于所述阈值C，则判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；若所述待测者的OTX1基因的Ct值小于等于所述阈值B，和/或所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，按照如下(1)-(3)进一步判定：
(1)若所述待测者的OTX1基因的Ct值小于等于所述阈值B，且所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，则计算OTX1基因和HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，分别记为△Ct值(OTX1)和△Ct值(HIST1H3G)；
选定△Ct值(OTX1)≤5和/或△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5且△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
(2)若所述待测者的OTX1基因的Ct值小于等于所述阈值B，但是所述待测者的HIST1H3G基因的Ct值大于所述阈值C，则计算OTX1基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(OTX1)；
选定△Ct值(OTX1)≤5时，判定为待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5时，判定为待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
(3)若所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，但是所述待测者的OTX1基因的Ct值大于所述阈值B，但是则计算HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(HIST1H3G)；
选定△Ct值(HIST1H3G)≤9时，判定为待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(HIST1H3G)>9时，判定为待测者为或候选为非癌症患者，或者待测者为癌症低风险患者。
在本发明的具体实施方式中，所述阈值A、所述阈值B和所述阈值C均为37。
第十方面，本发明要求保护前文第三到第五方面所述试剂盒或前文第九方面所述系统在如下任一中的应用：
(A1)制备用于诊断或筛查癌症的产品；
(A2)诊断或筛查癌症；
(A3)制备用于在临床症状之前预警癌症的产品；
(A4)在临床症状之前预警癌症；
(A5)制备用于区分或辅助区分癌症和良性病变的产品；
(A6)区分或辅助区分癌症和良性病变。
其中，所述癌症包括但不限于肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌等。
在本发明的具体实施方式中，所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。
在本发明的具体实施方式中，所述待测者选自：肝癌患者、肝良性病变患者、健康人后肝硬化患者。
在本发明中，所述肝癌可为原发肝细胞癌、肝内胆管癌或混合型肝癌。
所述肝脏良性病变具体为肝血管瘤，肝腺瘤，肝脓肿，肝囊肿，肝局灶性结节性增生，特发性非肝硬化性门静脉高压或炎性假瘤等。
附图说明
图1为目前肝癌的筛查手段主要是血清甲胎蛋白(AFP)检查和超声显像检测。
图2为基于44对肝癌肿瘤组织与癌旁组织发现的2588个差异甲基化区域(DMR)，其中333个为高甲基化(Hyper)DMR,2255个为低甲基化(Hypo)DMR。
图3为基于51个模型DMR的甲基化率在验证集I的热图。
图4为基于51个DMR的肝癌甲基化模型在验证集I中的效能。
图5为OTX1和/或HIST1H3G甲基化在独立验证集II中的性能。
实施发明的最佳方式
首先本发明通过对44对肝癌组织和肝癌癌旁组织进行甲基化高通量测序，通过对数据进行分析计算，找出了2588个可能与肝癌发生发展有关的差异化甲基化区域(Differentially methylated region，DMR)。
然后本发明通过来自对295个肝癌患者、180个健康人、34个肝良性病变患者、21个肝硬化患者的总计529个血浆游离细胞DNA(plasma cfDNA)进行靶向甲基化高通量测序，收集了这些DMR在肝癌病人和健康人cfDNA内的甲基化水平数据。
结合已经收集的数据，本发明通过一定条件的筛选及机器学习构建肝癌风险预测模型，选出了可用于肝癌筛查的51个DMR。
最后从这51个DMR中，选取了性能较好的OTX1基因和HIST1H3G基因作为用于检测肝癌的甲基化标志物。
本发明用OTX1基因和HIST1H3G基因甲基化作为肝癌筛查的标志物。通过检测OTX1基因和HIST1H3G基因的甲基化水平，并对所得数据进行分析，可以预测 受检者罹患肝癌的可能性，进而实现在普通人群或肝癌高危人群中筛查肝癌的目的。
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、肝癌特异性甲基化基因的发现
为了筛选在肝癌中特异性甲基化的生物标志物，对来自44个肝癌病人的肝癌组织和肝癌癌旁组织的基因组DNA进行全基因组甲基化测序(whole genome bisulfite sequencing,WGBS)。通过对数据进行分析计算，找出了2588个可能与肝癌发生发展有关的差异化甲基化区域(Differentially methylated region，DMR)。
1、组织样本提取
使用DNeasy Blood & Tissue Kit(Qiagen，#69506)对44对肝癌和肝癌癌旁组织样本进行DNA提取。用Qubit3.0系统(Invitrogen，美国)进行定量。
2、DNA片段化
取200ng提取好的DNA，同时加入1ng Unmethylated lambda DNA(PROMEGA，#D1521)用于后续C-U转化质控，使用超声打断仪将DNA打断，再使用AMPure XP(AGENCOURT，#A63882)对DNA进行片段选择，使DNA片段大小集中在160bp左右。
3、文库构建
使用KAPA HyperPlus Library Preparation Kit(KAPA，#KK8510)进行文库构建，具体操作参考试剂盒说明书，其中接头连接步骤所使用的接头以及PCR步骤所使用的PCR引物更换为适用于MGISEQ平台的接头和引物。
4、重亚硫酸盐转化
使用EZ DNA Metlylation-Gold Kit(Zymo Research，#D5006)对构建好的DNA文库进行重亚硫酸盐转化。
5、文库杂交捕获
使用Seq Cap EZ Hybridization and Wash Kit(ROCHE，5634253001)和SeqCap Epi CpGiant Enrichment Kit(ROCHE，7138911001)进行杂交、捕获和洗脱。由于使用了MGI平台的测序仪器，杂交过程中所用的Block须使用MGI平台对应的Block。
6、高通量测序
使用MGISEQ-2000(MGI)进行PE100测序。
7、数据分析
对测序数据进行质控、过滤、比对、计算，计算出了每个CpG位点的甲基化率。通过比较各个CpG位点在肝癌组织与癌旁组织中甲基化率的异同，采用层次贝叶方法辨析出了2588个肝癌组织与肝癌癌旁组织差异化甲基化区域(Differentially methylated region，DMR)，即肝癌特异性甲基化区域。
图2展示了基于44对肝癌组织和癌旁组织发现的2588个DMR的热图。肝癌组织于左侧，癌旁组织于右侧。高甲基化DMR位于热图的上方，低甲基化DMR位于热图的下方。每格代表对应的该样本于该位点的DMR甲基化率,其范围为0-1。甲基化率越接近0，颜色越深。
实施例2、肝癌特异性甲基化基因的验证
对来自独立样本集合I的140例肝癌患者及84例健康人的血浆游离细胞DNA(plasma cfDNA)进行靶向甲基化高通量测序，利用机器学习构建肝癌风险模型，验证这些DMR。
1、血浆样本提取
使用MagPure Circulating DNA Maxi Kit(MAGEN，#12917PJ-100)对肝癌和健康人血浆样本进行cfDNA提取。用Qubit3.0系统(Invitrogen，美国)进行定量。取10ng cfDNA，并加入0.05ng打断并筛选至160bp左右的Unmethylated lambda DNA用于后续C-U转化质控。
2、文库构建
使用KAPA HyperPlus Library Preparation Kit(KAPA，#KK8510)进行文库构建，具体操作参考试剂盒说明书，其中接头连接步骤所使用的接头以及PCR步骤所使用的PCR引物更换为适用于MGISEQ平台的接头和引物。
3、重亚硫酸盐转化
使用EZ DNA Metlylation-Gold Kit(Zymo Research，#D5006)对构建好的DNA文库进行重亚硫酸盐转化。
4、文库杂交捕获
使用Seq Cap EZ Hybridization and Wash Kit(ROCHE，5634253001)和SeqCap Epi CpGiant Enrichment Kit(ROCHE，7138911001)进行杂交、捕获和洗脱。由于使用了MGI平台的测序仪器，杂交过程中所用的Block须使用MGI平台对应的Block。
5、高通量测序
使用MGISEQ-2000(MGI)进行PE100测序。
6、数据分析
对测序数据进行质控、过滤、比对、计算，计算出了每个CpG位点的甲基化率。通过比较各个CpG位点在肝癌病人血浆与健康人血浆中甲基化率的异同，采用基于随机森林的10折交叉验证，在高甲基化DMR中根据特征重要性进行筛选(特征重要性>0.15)，筛选出51个可作为肝癌筛查的标志物的DMR。利用每 折中验证集对模型性能进行评估，由此可得验证集的平均灵敏度为0.929，特异性为0.894，AUC为0.96。
图3展示了51个DMR在140个肝癌患者和84个健康人中区域内甲基化率的热图。横轴为样本，肝癌患者于最左侧，随后为健康人。由图可见，筛选的51个DMR对肝癌患者与健康人样本有明显的区分度。
图4展示了基于51个DMR的肝癌甲基化模型在该实施例的验证集I中的性能。横轴代表假阳性率(1-特异性)，纵轴代表灵敏度。由图可见，该甲基化模型对该实施例样本有良好的判断能力。
实施例3、对OTX1基因和HIST1H3G基因甲基化诊断肝癌能力的评估
本发明从实施例2获得的51个DMR中，选取了性能较好的OTX1基因和HIST1H3G基因作为用于检测肝癌的甲基化标志物(这两个基因在肝癌中甲基化水平升高)。OTX1基因肝癌特异性甲基化区域核苷酸序列如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示。HIST1H3G基因肝癌特异性甲基化区域核苷酸序列如SEQ ID No.4所示。OTX1基因肝癌特异性甲基化区域相关信息参见表1。HIST1H3G基因肝癌特异性甲基化区域相关信息参见表2。
表1、OTX1基因肝癌特异性甲基化区域
Figure PCTCN2021130060-appb-000001
表2、HIST1H3G基因肝癌特异性甲基化区域
Figure PCTCN2021130060-appb-000002
此实施例的主要目的是在独立的验证集II里验证OTX1和HIST1H3G基因甲基化筛查肝癌的性能，包括来自293个肝癌患者，33个肝良性病变患者，19个健康人及105个肝硬化患者的血浆游离DNA。肝癌样本的入组标准为：经病理确诊的原发肝细胞癌、肝内胆管癌或混合型肝癌，无既往恶性肿瘤史，术前未接收任何形式的抗肿瘤治疗。所述肝脏良性病变具体为肝血管瘤，肝腺瘤，肝脓肿，肝囊肿，肝局灶性结节性增生，特发性非肝硬化性门静脉高压或炎性假瘤等。
1、血浆样本提取
使用MagPure Circulating DNA Maxi Kit(MAGEN，#12917PJ-100)对肝癌和健康人血浆样本进行cfDNA提取。用Qubit 3.0系统(Invitrogen，美国) 进行定量。
2、重亚硫酸盐转化
取10ng cfDNA，使用EZ DNA Metlylation-Gold Kit(Zymo Research，#D5006)进行重亚硫酸盐转化。
3、实时荧光定量PCR(qPCR)
以ACTB基因为内参基因，通过qPCR扩增经重亚硫酸盐转化的cfDNA。整个反应体系包含5μl 10×PCR缓冲液(诺唯赞，中国)，2单位Taq聚合酶(诺唯赞，中国)，2.5mM dNTP(诺唯赞，中国)，2μl(10pmol/μl)PCR引物，1μl(10pmol/μl)探针(表3和表4)，以及经重亚硫酸盐转化的cfDNA。在如下条件下进行PCR反应：95℃预变性3分钟，然后进行95℃变性30秒、60℃退火30秒和72℃延伸30秒的循环40个。采用宏石96S qPCR仪进行检测，基线、阈值按照默认设置，在每个循环结束时收集荧光信号，最终进行72℃延伸5分钟。
表3、实时荧光定量PCR引物探针序列(扩增OTX1基因)
Figure PCTCN2021130060-appb-000003
Figure PCTCN2021130060-appb-000004
Figure PCTCN2021130060-appb-000005
Figure PCTCN2021130060-appb-000006
表4、实时荧光定量PCR引物探针序列(扩增HIST1H3G和ACTB内参)
编号 序列(5’-3’) 用途
SEQ ID No.116 TTTACGTCGTCGGTGGTC HIST1H3G正向引物1
SEQ ID No.117 CGCAAATCCACCGATAACA HIST1H3G反向引物1
SEQ ID No.118 TTTACGTCGTCGGTGGTC HIST1H3G正向引物2
SEQ ID No.119 CCGCACCAAACAAACTACAC HIST1H3G反向引物2
SEQ ID No.120 TTTGTTATCGGTGGATTTGC HIST1H3G正向引物3
SEQ ID No.121 AAACAATTTCCACTCTACCAAATAACT HIST1H3G反向引物3
SEQ ID No.122 GGACGGTAGCGATGAGGT HIST1H3G正向引物4
SEQ ID No.123 AACAACTAACCACTAAAACGACTCG HIST1H3G反向引物4
SEQ ID No.124 GAGTCGTTTTAGTGGTTAGTTGTTTG HIST1H3G正向引物5
SEQ ID No.125 CGCACCAAACAAACTACACG HIST1H3G反向引物5
SEQ ID No.126 TGAGATGGTTCGTATTAAGTAGATTGTAC HIST1H3G正向引物6
SEQ ID No.127 AACCGCCTTAATAACCAACTACTTAC HIST1H3G反向引物6
SEQ ID No.128 GTAAGTTTATCGGTGGTAAAGC HIST1H3G正向引物7
SEQ ID No.129 AAACGATAACGATAAAATTTCTTCAC HIST1H3G反向引物7
SEQ ID No.130 TTTATCGGTGGTAAAGCGTC HIST1H3G正向引物8
SEQ ID No.131 AAACGATAACGATAAAATTTCTTCAC HIST1H3G反向引物8
SEQ ID No.132 TTTTAGTGGTTAGTTGTTTGC HIST1H3G正向引物9
SEQ ID No.133 ATTTCCACTCTACCAAATAACTA HIST1H3G反向引物9
SEQ ID No.134 TTTTAGTGGTTAGTTGTTTGC HIST1H3G正向引物10
SEQ ID No.135 ATTTCCACTCTACCAAATAACTA HIST1H3G反向引物10
SEQ ID No.136 TTTTACGTCGTCGGTGGT HIST1H3G正向引物11
SEQ ID No.137 CGATAACAAAACGCCGCG HIST1H3G反向引物11
SEQ ID No.138 GACGGTAGCGATGAGGTT HIST1H3G正向引物12
SEQ ID No.139 CGATAACAAAACGCCGCG HIST1H3G反向引物12
SEQ ID No.140 CGCGTAGAGTTACGGTGT HIST1H3G正向引物13
SEQ ID No.141 CGATAACAAAACGCCGCG HIST1H3G反向引物13
SEQ ID No.142 TTTAGGTAGTTTGAGATGGT HIST1H3G正向引物14
SEQ ID No.143 CCTTAATAACCAACTACTTACG HIST1H3G反向引物14
SEQ ID No.144 TTATTTTGTTAAGTGGTTGAGT HIST1H3G正向引物15
SEQ ID No.145 CCTTAATAACCAACTACTTACG HIST1H3G反向引物15
SEQ ID No.146 GTTAGTTGTTTGCGCGGC HIST1H3G正向引物16
SEQ ID No.147 TCAAATAATCTAAAATAACCCGCACCA HIST1H3G反向引物16
SEQ ID No.148 GTTAGTTGTTTGCGCGGC HIST1H3G正向引物17
SEQ ID No.149 AACAATTTCCACTCTACCAAATAACT HIST1H3G反向引物17
SEQ ID No.150 TTTTACGTCGTCGGTGGT HIST1H3G正向引物18
SEQ ID No.151 CAAATCCACCGATAACAAAACG HIST1H3G反向引物18
SEQ ID No.152 GGACGGTAGCGATGAGGT HIST1H3G正向引物19
SEQ ID No.153 GCAAATCCACCGATAACAAAACG HIST1H3G反向引物19
SEQ ID No.154 GACGGTAGCGATGAGGTT HIST1H3G正向引物20
SEQ ID No.155 CAAATCCACCGATAACAAAACG HIST1H3G反向引物20
SEQ ID No.156 GTAAGTTTATCGGTGGTAAAGC HIST1H3G正向引物21
SEQ ID No.157 ATAACCGACGCGCTTTTC HIST1H3G反向引物21
SEQ ID No.158 TAAGTTTATCGGTGGTAAAGC HIST1H3G正向引物22
SEQ ID No.159 AAACGATAACGATAAAATTTCTTCA HIST1H3G反向引物22
SEQ ID No.160 GTTTGAGATGGTTCGTATTAAGTAGA HIST1H3G正向引物23
SEQ ID No.161 CCAACTACTTACGCGACG HIST1H3G反向引物23
SEQ ID No.162 GTAGTTTTTATTTTGTTAAGTGGTTGAGT HIST1H3G正向引物24
SEQ ID No.163 CCAACTACTTACGCGACG HIST1H3G反向引物24
SEQ ID No.164 TTTAGTGGTTAGTTGTTTGCGC HIST1H3G正向引物25
SEQ ID No.165 CTTTCAAATAATCTAAAATAACCCG HIST1H3G反向引物25
SEQ ID No.166 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物26
SEQ ID No.167 ACTAAAACGACTCGAAAAAACG HIST1H3G反向引物26
SEQ ID No.168 GTTAGTTGTTTGCGCGGC HIST1H3G正向引物27
SEQ ID No.169 TTCAAATAATCTAAAATAACCCGCACCA HIST1H3G反向引物27
SEQ ID No.170 GACGGTAGCGATGAGGTT HIST1H3G正向引物28
SEQ ID No.171 CAAATCCACCGATAACAAAACG HIST1H3G反向引物28
SEQ ID No.172 TTTTTAGGTAGTTTGAGATGGTTCG HIST1H3G正向引物29
SEQ ID No.173 GCTTTACCACCGATAAACTTACGT HIST1H3G反向引物29
SEQ ID No.174 AGGTAGTTTGAGATGGTTCGTA HIST1H3G正向引物30
SEQ ID No.175 CCAACTACTTACGCGACG HIST1H3G反向引物30
SEQ ID No.176 TGATTCGTAAGTTGTTTTTTTAAC HIST1H3G正向引物31
SEQ ID No.177 CCTCCTACAAAACCATCA HIST1H3G反向引物31
SEQ ID No.178 TGATTCGTAAGTTGTTTTTTTAAC HIST1H3G正向引物32
SEQ ID No.179 AAACTCTAAAAACGCAAATCT HIST1H3G反向引物32
SEQ ID No.180 GGTAATTTGCGGATTAGTAGTTTAGTC HIST1H3G正向引物33
SEQ ID No.181 GACACCGTAACTCTACGCGA HIST1H3G反向引物33
SEQ ID No.182 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物34
SEQ ID No.183 ACAACTAACCACTAAAACGACTCG HIST1H3G反向引物34
SEQ ID No.184 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物35
SEQ ID No.185 ACTAAAACGACTCGAAAAAACG HIST1H3G反向引物35
SEQ ID No.186 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物36
SEQ ID No.187 GCAAACAACTAACCACTAAAACGA HIST1H3G反向引物36
SEQ ID No.188 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物37
SEQ ID No.189 AACTAACCACTAAAACGACTCGAA HIST1H3G反向引物37
SEQ ID No.190 AGTAGTTGGTTATTAAGGCGGTTC HIST1H3G正向引物38
SEQ ID No.191 ATAACGATAAAATTTCTTCACGCCG HIST1H3G反向引物38
SEQ ID No.192 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物39
SEQ ID No.193 ACTAAAACGACTCGAAAAAACGCG HIST1H3G反向引物39
SEQ ID No.194 GTAGAGTTACGGTGTCGGGAC HIST1H3G正向引物40
SEQ ID No.195 ACTAAAACGACTCGAAAAAACG HIST1H3G反向引物40
SEQ ID No.196 GTAATAAAATTATTGGTTGAAGAGTTTAAC HIST1H3G正向引物41
SEQ ID No.197 CTTAAATCGCAAACTAACCGAA HIST1H3G反向引物41
SEQ ID No.198 ACCACTAAAACGACTCGAAAAAACGCGCCG HIST1H3G探针1
SEQ ID No.199 ACCGCCTTAATAACCAACTACTTACGCGAC HIST1H3G探针2
SEQ ID No.200 CAAACAACTAACCACTAAAACGACTC HIST1H3G探针3
SEQ ID No.201 AACCGCCTTAATAACCAACTACTTAC HIST1H3G探针4
SEQ ID No.202 AACGCGCCGACCACCGACGA HIST1H3G探针5
SEQ ID No.203 AAGGGGTTTATATGGTAATTGTG ACTB正向引物序列
SEQ ID No.204 GATTGAGTGTGGTAGGGATT ACTB反向引物序列
SEQ ID No.205 CCCACCACACTAAATCTCCCCTCCT ACTB探针序列
4.结果分析
以用于扩增OTX1中SEQ ID No.3的引物对(SEQ ID No.95和SEQ ID No.96)，探针为SEQ ID No.113(可检测如下CpG位点：chr2:63284080、chr2:63284082、chr2:63284092、chr2:63284099、chr2:63284104、chr2:63284111、chr2:63284140、chr2:63284146)，以及用于扩增HIST1H3G中SEQ ID No.4的引物对(SEQ ID No.140和SEQ ID No.141)，探针为SEQ ID No.202为(可检测如下CpG位点：chr6:26271462、chr6:26271464、chr6:26271474、chr6:26271510、chr6:26271513、chr6:26271520、chr6:26271523、chr6:26271525、chr6:26271560、chr6:26271562、chr6:26271565、chr6:26271576)为例进行说明，如下：
原理：根据目标基因的Ct值与内参ACTB基因的扩增Ct值的差值(△Ct值)估算甲基化率，△Ct越小，目标基因的甲基化率越高。
各基因Ct值的阈值为37。
其中，当ACTB的Ct值大于37时，判定为结果不可信；当ACTB基因的Ct值≤37时，判定为结果可信,按照如下进行进一步判定：
当OTX1基因和HIST1H3G基因的Ct值均大于37时，判定待测者为非肝癌患者；当OTX1基因和HIST1H3G基因的Ct值不均大于37时(即均或之一≤37时)，按照如下(1)-(3)进一步判定：
(1)当OTX1基因和HIST1H3G基因的Ct值均≤37时，计算OTX1基因和HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，分别记为△Ct值(OTX1)和△Ct值(HIST1H3G)；
选定△Ct值(OTX1)≤5和/或△Ct值(HIST1H3G)≤9时，判定待测者为肝癌患者；△Ct值(OTX1)>5且△Ct值(HIST1H3G)>9时，判定待测者为非肝癌患者；
(2)当OTX1基因的Ct值≤37，但是HIST1H3G基因的Ct值＞37时，计算OTX1基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(OTX1)；
选定△Ct值(OTX1)≤5时，判定待测者为肝癌患者；△Ct值(OTX1)>5时，判定待测者为非肝癌患者；
(3)当HIST1H3G基因的Ct值≤37，但是OTX1基因的Ct值＞37时，计算HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(HIST1H3G)；
选定△Ct值(HIST1H3G)≤9时，判定待测者为肝癌患者；△Ct值(HIST1H3G)>9时，判定待测者为非肝癌患者。
使用SPSS软件作两个待检测基因△Ct的ROC曲线，ROC曲线如图5所示，各基因的性能及曲线下面积(AUC)如表5所示。
为了进一步提高试剂盒性能，整合两个基因的△Ct值进行分析，AUC可达0.909。最终确定△Ct值(OTX1)≤5、△Ct值(HIST1H3G)≤9时判定检测阳性，当任意基因阳性时则判定该样本检测阳性，在此判定条件下，在特异性(包含健康人、肝硬化和肝良性病变)为92.4％时，灵敏性(肝癌)可达82.2％。
以上结果表明，本发明通过多个基因联合检测，可以获得比单一基因检测更好的检测性能。
表5、各基因的性能及曲线下面积(AUC)以及灵敏度和特异性
Figure PCTCN2021130060-appb-000007
工业应用
通过对肝癌组织、正常组织、肝癌病人血浆、对照人群血浆的DNA进行甲基化测序，本发明发现OTX1基因和HIST1H3G基因的高甲基化与肝癌相关，可作为肝癌检测的标志物。本发明提供了一种准确、简便、经济的肝癌早期筛查手段，可提高肝癌高危人群及普通体检人群中肝癌，尤其是早期肝癌的检出率，进而提高肝癌病人的生存率，并节约大量的医疗支出和降低医疗负担。

Claims (33)

  1. 甲基化OTX1基因和HIST1H3G基因作为标记物在如下任一中的应用：
    (A1)制备用于诊断或筛查癌症的产品；
    (A2)诊断或筛查癌症；
    (A3)制备用于在临床症状之前预警癌症的产品；
    (A4)在临床症状之前预警癌症；
    (A5)制备用于区分或辅助区分癌症和良性病变的产品；
    (A6)区分或辅助区分癌症和良性病变。
  2. 根据权利要求1所述的应用，其特征在于：所述甲基化OTX1基因为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化；所述甲基化HIST1H3G基因为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化。
  3. 用于检测OTX1基因和HIST1H3G基因甲基化水平的物质在如下任一中的应用：
    (A1)制备用于诊断或筛查癌症的产品；
    (A2)诊断或筛查癌症；
    (A3)制备用于在临床症状之前预警癌症的产品；
    (A4)在临床症状之前预警癌症；
    (A5)制备用于区分或辅助区分癌症和良性病变的产品；
    (A6)区分或辅助区分癌症和良性病变。
  4. 根据权利要求3所述的应用，其特征在于：所述OTX1基因甲基化水平为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化水平；所述HIST1H3G基因甲基化水平为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化水平。
  5. 根据权利要求3或4所述的应用，其特征在于：用于检测所述OTX1基因甲基化水平的物质为重亚硫酸盐试剂以及引物对A和探针A；所述引物对A由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成；所述探针A为SEQ ID No.113-SEQ ID No.115任一中所示单链DNA；
    用于检测所述HIST1H3G基因甲基化水平的物质为重亚硫酸盐试剂以及引物对B和探针B；所述引物对B由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成；所述探针B为SEQ ID No.198-SEQ ID No.202中任一所示单链DNA。
  6. 根据权利要求5所述的应用，其特征在于：所述引物对A由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成；所述探针A为SEQ ID No.113所示单链DNA；
    所述引物对B由SEQ ID No.140和SEQ ID No.141所示两条单链DNA组成； 所述探针B为SEQ ID No.202所示单链DNA。
  7. 根据权利要求1-6中任一所述的应用，其特征在于：所述癌症为肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌。
  8. 根据权利要求7所述的应用，其特征在于：所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。
  9. 一种试剂盒，包含：
    (B1)重亚硫酸盐试剂；以及
    (B2)对照核酸1，所述对照核酸1的序列如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示，并且具有与非癌症患者相关的甲基化状态；
    (B3)对照核酸2，所述对照核酸2的序列如SEQ ID No.4所示，并且具有与非癌症患者相关的甲基化状态；
    所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
  10. 一种试剂盒，包含：
    (C1)重亚硫酸盐试剂；以及
    (C2)对照核酸3，所述对照核酸3的序列如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示，并且具有与癌症患者相关的甲基化状态；
    (C3)对照核酸4，所述对照核酸4的序列如SEQ ID No.4所示，并且具有与癌症患者相关的甲基化状态；
    所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
  11. 一种试剂盒，包含：
    (D1)重亚硫酸盐试剂；以及
    (D2)用于检测所述OTX1基因甲基化水平的引物对A和探针A；所述引物对A由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成；所述探针A为SEQ ID No.113-SEQ ID No.115中任一所示单链DNA；
    (D3)用于检测所述HIST1H3G基因甲基化水平的引物对B和探针B；所述引物对B由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成；所述探针B为SEQ ID No.198-SEQ ID No.202中任一所示单链DNA；
    所述试剂盒具有如下任一用途：诊断或筛查癌症；在临床症状之前预警癌症；区分或辅助区分癌症和良性病变。
  12. 根据权利要求11所述的试剂盒，其特征在于：所述引物对A由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成；所述探针A为SEQ ID No.113所示单链DNA；
    所述引物对B由SEQ ID No.140和SEQ ID No.141所示两条单链DNA组成；所述探针B为SEQ ID No.202所示单链DNA。
  13. 根据权利要求11或12所述的试剂盒，其特征在于：所述试剂盒中还 含有用于扩增内参基因ACTB的引物对C和探针C；所述引物对C由SEQ ID No.203和SEQ ID No.204所示两条单链DNA组成；所述探针C为SEQ ID No.205所示单链DNA。
  14. 根据权利要求9-13中任一所述的试剂盒，其特征在于：所述癌症为肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌。
  15. 根据权利要求14所述的试剂盒，其特征在于：所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。
  16. 一种诊断或筛查癌症的方法，包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现诊断或筛查癌症。
  17. 一种在临床症状之前预警癌症的方法，包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现在临床症状之前预警癌症。
  18. 一种区分或辅助区分癌症和良性病变的方法，包括如下步骤：检测来自待测者的样本中OTX1基因和HIST1H3G基因甲基化水平，从而实现区分或辅助区分癌症和良性病变。
  19. 根据权利要求16-18中任一所述的方法，其特征在于：所述OTX1基因甲基化水平为SEQ ID No.1或SEQ ID No.2或SEQ ID No.3所示的DNA片段中全部或部分CpG位点的甲基化水平；所述HIST1H3G基因甲基化水平为SEQ ID No.4所示的DNA片段中全部或部分CpG位点的甲基化水平。
  20. 根据权利要求16-19中任一所述的方法，其特征在于：检测来自所述待测者的所述样本中OTX1基因和HIST1H3G基因甲基化水平的方法为重硫酸盐转化、PCR、甲基化特异性PCR、焦磷酸测序、桑格测序、高通量测序或三代测序或单分子测序。
  21. 根据权利要求16-20中任一所述的方法，其特征在于：检测来自所述待测者的所述样本中OTX1基因和HIST1H3G基因甲基化水平按照包括如下步骤的方法进行：
    (E1)从所述样本中提取DNA，然后进行重亚硫酸盐转化；
    (E2)通过实时荧光定量PCR扩增(E1)所得经重亚硫酸盐转化的DNA；进行所述实时荧光定量PCR扩增时采用的针对OTX1基因的引物对由SEQ ID No.5-SEQ ID No.112任一组所示的两条单链DNA组成，探针为SEQ ID No.113-SEQ ID No.115中任一所示单链DNA；进行所述实时荧光定量PCR扩增时采用的针对HIST1H3G基因的引物对由SEQ ID No.116-SEQ ID No.197任一组所示的两条单链DNA组成，探针为SEQ ID No.198-SEQ ID No.202中任一所示单链DNA。
  22. 根据权利要求21所述的方法，其特征在于：进行所述实时荧光定量PCR扩增时采用的针对OTX1基因的引物对由SEQ ID No.95和SEQ ID No.96所示两条单链DNA组成，探针为SEQ ID No.113所示单链DNA；进行所述实时荧光定量PCR扩增时采用的针对HIST1H3G基因的引物对由SEQ ID No.140和SEQ ID No.141 所示两条单链DNA组成，探针为SEQ SEQ ID No.202所示单链DNA。
  23. 根据权利要求21或22所述的方法，其特征在于：在步骤(E2)中，进行所述实时荧光定量PCR扩增时采用的内参为ACTB基因，用于扩增ACTB基因的引物对由SEQ ID No.203和SEQ ID No.204所示两条单链DNA组成，探针为SEQ ID No.205所示单链DNA。
  24. 根据权利要求21-23中任一所述的方法，其特征在于：在步骤(E2)后，还包括如下步骤：
    (E3)根据(E2)中所述实时荧光定量PCR扩增的结果按照如下进行判定：
    当ACTB基因的Ct值大于37时，判定为结果不可信；当ACTB基因的Ct值≤37时，判定为结果可信,按照如下进行进一步判定：
    当OTX1基因和HIST1H3G基因的Ct值均大于37时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；当OTX1基因和HIST1H3G基因的Ct值不均大于37时，按照如下(1)-(3)进一步判定：
    (1)当OTX1基因和HIST1H3G基因的Ct值均≤37时，计算OTX1基因和HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，分别记为△Ct值(OTX1)和△Ct值(HIST1H3G)；
    选定△Ct值(OTX1)≤5和/或△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5且△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
    (2)当OTX1基因的Ct值≤37，但是HIST1H3G基因的Ct值＞37时，计算OTX1基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(OTX1)；
    选定△Ct值(OTX1)≤5时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
    (3)当HIST1H3G基因的Ct值≤37，但是OTX1基因的Ct值＞37时，计算HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(HIST1H3G)；
    选定△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者。
  25. 根据权利要求16-24中任一所述的方法，其特征在于：所述癌症为肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌。
  26. 根据权利要求25所述的方法，其特征在于：所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。
  27. 根据权利要求16-26中任一所述的方法，其特征在于：所述样本为能够提取DNA的样本。
  28. 根据权利要求27所述的方法，其特征在于：所述样本为血浆、血清、血液、组织、唾液、尿液、粪便。
  29. 系统，包括：
    (F1)权利要求13所述的试剂盒和实时荧光定量PCR仪；
    (F2)装置，所述装置包括数据采集模块、阈值存储模块、数据比较模块、数据处理及结论输出模块；
    所述数据采集模块被配置为采集(F1)检测得到的来自所述待测者的所述样本的实时荧光定量PCR扩增结果数据；
    所述阈值存储模块被配置为存储阈值A、阈值B和阈值C；所述阈值A为ACTB基因的Ct值的阈值；所述阈值B为OTX1基因的Ct值的阈值；所述阈值C为HIST1H3G基因的Ct值的阈值。
    所述数据比较模块被配置为接收来自所述数据采集模块发送的所述待测者的所述样本的实时荧光定量PCR扩增结果数据，并调用所述阈值存储模块中存储的所述阈值A、所述阈值B和所述阈值C，然后将所述待测者的ACTB基因的Ct值与所述阈值A进行比较，将所述待测者的OTX1基因的Ct值与所述阈值B行比较，并将所述待测者的HIST1H3G基因的Ct值与所述阈值C行比较；
    所述数据处理及结论输出模块被配置为接收来自所述数据比较模块发送的比较结果，然后按照如下输出结论：
    若所述待测者的ACTB基因的Ct值大于所述阈值A，则判定为结果不可信；当ACTB基因的Ct值≤37时，判定为结果可信,按照如下进行进一步判定：
    若所述待测者的OTX1基因的Ct值大于所述阈值B，且所述待测者的HIST1H3G基因的Ct值大于所述阈值C，则判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；若所述待测者的OTX1基因的Ct值小于等于所述阈值B，和/或所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，按照如下(1)-(3)进一步判定：
    (1)若所述待测者的OTX1基因的Ct值小于等于所述阈值B，且所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，则计算OTX1基因和HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，分别记为△Ct值(OTX1)和△Ct值(HIST1H3G)；
    选定△Ct值(OTX1)≤5和/或△Ct值(HIST1H3G)≤9时，判定待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(OTX1)>5且△Ct值(HIST1H3G)>9时，判定待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
    (2)若所述待测者的OTX1基因的Ct值小于等于所述阈值B，但是所述待测者的HIST1H3G基因的Ct值大于所述阈值C，则计算OTX1基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(OTX1)；
    选定△Ct值(OTX1)≤5时，判定为待测者为或候选为癌症患者，或者待 测者为癌症高风险患者；△Ct值(OTX1)>5时，判定为待测者为或候选为非癌症患者，或者待测者为癌症低风险患者；
    (3)若所述待测者的HIST1H3G基因的Ct值小于等于所述阈值C，但是所述待测者的OTX1基因的Ct值大于所述阈值B，但是则计算HIST1H3G基因的扩增Ct值与内参ACTB基因的扩增Ct值的差值，记为△Ct值(HIST1H3G)；
    选定△Ct值(HIST1H3G)≤9时，判定为待测者为或候选为癌症患者，或者待测者为癌症高风险患者；△Ct值(HIST1H3G)>9时，判定为待测者为或候选为非癌症患者，或者待测者为癌症低风险患者。
  30. 根据权利要求29所述的系统，其特征在于：所述阈值A、所述阈值B和所述阈值C均为37。
  31. 权利要求9-15中任一所述试剂盒或权利要求29或30所述系统在如下任一中的应用：
    (A1)制备用于诊断或筛查癌症的产品；
    (A2)诊断或筛查癌症；
    (A3)制备用于在临床症状之前预警癌症的产品；
    (A4)在临床症状之前预警癌症；
    (A5)制备用于区分或辅助区分癌症和良性病变的产品；
    (A6)区分或辅助区分癌症和良性病变。
  32. 根据权利要求32所述的应用，其特征在于：所述癌症为肝癌、结直肠癌、肺癌、胃癌、胰腺癌、前列腺癌、食管癌或尿路上皮癌。
  33. 根据权利要求32所述的应用，其特征在于：所述癌症为肝癌；所述良性病变为肝脏良性病变或肝硬化。
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