WO2023082139A1 - Nucleic acid for diagnosing liver cancer and protein detection kit - Google Patents

Abstract

Disclosed is a nucleic acid detection kit for diagnosing liver cancer. The present invention provides a use of the combination of "methylated OTX1 gene, methylated HIST1H3G gene, alpha-fetoprotein and abnormal prothrombin" as combined markers in the following: diagnosing or screening cancer, warning about cancer before clinical symptoms, and distinguishing or assisting in distinguishing cancer from benign lesions. In the present invention, the methylation of OTX1 gene and HIST1H3G gene and the contents of alpha-fetoprotein and abnormal prothrombin are used as combined markers for liver cancer screening. By detecting the methylation levels of OTX1 gene and HIST1H3G gene and the contents of alpha-fetoprotein and abnormal prothrombin and analyzing obtained data, the possibility of a subject suffering from liver cancer can be predicted, and thus the purpose of screening liver cancer in the general population or in groups at high risk of liver cancer can be achieved.

Description

A nucleic acid and protein detection kit for diagnosing liver cancer technical field

The invention relates to the field of biomedicine, in particular to a nucleic acid and protein detection kit for diagnosing liver cancer.

Background technique

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. At present, 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 At present, 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.

When cells in the human body rupture or die, their DNA will be released into the circulation system, which is cell-free DNA (cfDNA). Similarly, when tumor cells rupture or die, circulating tumor DNA (circulating tumor DNA, ctDNA) is also released, which carries the genetic information of tumor cells. By detecting ctDNA mixed in cfDNA and analyzing the mutations and epigenetic information it carries, the possibility of a subject suffering from cancer can be inferred.

Scientists have developed many high-throughput sequencing technologies with high molecular utilization, which makes it possible to detect a small amount of mutation signals in plasma cell-free DNA, and also promotes the development of precise tumor early screening. Scientists first searched for biomarkers suitable for early screening of tumors from gene mutations, but studies have shown that the single use of mutation signals for early screening of tumors has limited effect. Therefore, scientists began to explore early tumor screening from the epigenetic level. 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.

From the perspective of domestic and foreign companies focusing on the research of early tumor screening technology: Grail currently mainly adopts cfDNA targeted sequencing, WGS and WGBS. Mutation and methylation molecular markers. This strategy can conduct a more comprehensive study on the tumor genome map, but the huge cost of high-depth whole-genome sequencing cannot be afforded by general research institutions. Guardant Health focuses on liquid biopsy technology and uses high-sensitivity detection technology for early tumor screening research. However, there are still many limitations in liquid biopsy technology for early tumor screening, such as: early tumor mutation signals are extremely weak, and some gene mutations are in multiple The existence and clonal hematopoiesis in different cancer types will bring huge interference to ctDNA detection. Therefore, the single use of mutations as molecular markers has limited effects. Genetron Health combined mutation and protein markers for detection. This study also showed that the application of multi-omics for detection can effectively improve detection performance. Kunyuan Genetics and Benchmark Medicine focus on the detection of methylation. Changes in DNA methylation often occur at multiple sites at the same time, so they have higher sensitivity than gene mutations at a single site, and DNA methylation The tissue specificity of the methylation signal makes it possible for early screening of pan-cancer tumors, so methylation is an ideal molecular marker for early screening of tumors.

At present, there are many studies on early screening of tumors based on DNA methylation at home and abroad. For example, Lo Yuk-ming, a well-known clinical application expert in molecular biology in Hong Kong, who is known as the "founder of non-invasive DNA prenatal testing", published in 2019 the use of low-depth WGBS sequencing detects methylation and copy number variation (CNA) in urine cfDNA. This method has a sensitivity of 93.5% (95.8% specificity) for bladder cancer detection; as published by Anderson, B.W. et al. in 2018 Regarding the clinical validation of gastric cancer methylation markers, 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.

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. At present, 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 equipment. 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. Alpha-fetoprotein (AFP) is currently the most commonly used clinical protein marker for liver cancer detection. Serum abnormal prothrombin (des-Y-carboxy-prothrombin, DCP) or vitamin K deficiency or antagonist-induced protein II (protein induced by vitamin K absence or antagonist-II, PIVKA-II), due to vitamin K deficiency Or in the presence of vitamin K antagonists, liver cancer cells cannot rely on vitamin K to synthesize normal coagulation factors, resulting in insufficient carboxylation of prothrombin precursors. In recent years, scholars such as FujitaK and KinukawaH have reported that PIVKA-II is elevated in the early stage of HCC, which can be used as a serological index for early diagnosis and screening of HCC.

invention disclosure

The purpose of the present invention is to provide a nucleic acid detection kit for diagnosing liver cancer.

In the first aspect, the present invention claims to protect the application of the combination of "methylated OTX1 gene, methylated HIST1H3G gene, alpha-fetoprotein and abnormal prothrombin" as a joint marker in any of the following:

(A1) Manufacture of products for the diagnosis or screening of cancer;

(A2) diagnosis or screening for cancer;

(A3) Preparation of products for early warning of cancer before clinical symptoms;

(A4) Early warning of cancer before clinical symptoms;

(A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

(A6) Distinguishing or assisting in differentiating between cancer and benign lesions.

Wherein, 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.

In the second aspect, the present invention claims to protect the application of substances for detecting "the methylation level of the OTX1 gene, the methylation level of the HIST1H3G gene, the content of alpha-fetoprotein and the content of abnormal prothrombin" in any of the following:

(A1) Manufacture of products for the diagnosis or screening of cancer;

(A2) diagnosis or screening for cancer;

(A3) Preparation of products for early warning of cancer before clinical symptoms;

(A4) Early warning of cancer before clinical symptoms;

(A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

(A6) Distinguishing or assisting in differentiating between cancer and benign lesions.

Wherein, 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.

Further, the substance used to detect the methylation level of the 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. Preferably, 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.

Further, 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. Preferably, 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.

Further, the substance used to detect the content of alpha-fetoprotein is a substance capable of specifically binding to alpha-fetoprotein, such as an anti-alpha-fetoprotein antibody.

Further, the substance used to detect the content of abnormal prothrombin is a substance capable of specifically binding to abnormal prothrombin, such as an antibody against abnormal prothrombin.

In the application of the above two aspects, the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.

In a specific embodiment of the present invention, the cancer is liver cancer. Correspondingly, 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.

In the third aspect, the present invention claims a kit, which is designated as Kit I.

Said test kit 1 may comprise:

(B1) bisulfite reagent; and

(B2) 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;

(B3) control nucleic acid 2, the sequence of the control nucleic acid 2 is as shown in SEQ ID No.4, and has a methylation state associated with non-cancer patients;

(B4) Substances used to detect the content of alpha-fetoprotein;

(B5) Substances for detection of abnormal prothrombin content.

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.

Further, the substance used to detect the content of alpha-fetoprotein is a substance capable of specifically binding to alpha-fetoprotein, such as an anti-alpha-fetoprotein antibody.

Further, the substance used to detect the content of abnormal prothrombin is a substance capable of specifically binding to abnormal prothrombin, such as an antibody against abnormal prothrombin.

Wherein, the non-cancer patients may be healthy controls or patients with benign lesions.

In the fourth aspect, the present invention claims a kit, which is designated as kit II.

Said test kit II may comprise:

(C1) bisulfite reagent; and

(C2) control nucleic acid 3, the sequence of said 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 state relevant to cancer patients;

(C3) control nucleic acid 4, the sequence of the control nucleic acid 4 is as shown in SEQ ID No.4, and has a methylation state relevant to cancer patients;

(C4) Substances used to detect the content of alpha-fetoprotein;

(C5) Substances for detection of abnormal prothrombin content.

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.

Further, the substance used to detect the content of alpha-fetoprotein is a substance capable of specifically binding to alpha-fetoprotein, such as an anti-alpha-fetoprotein antibody.

Further, the substance used to detect the content of abnormal prothrombin is a substance capable of specifically binding to abnormal prothrombin, such as an antibody against abnormal prothrombin.

In the fifth aspect, the present invention claims a kit, which is designated as kit III.

Said kit III may comprise:

(D1) bisulfite reagent; and

(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.

Preferably, 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.

(D3) Primer pair B and probe B for detecting the methylation level of the HIST1H3G gene; the primer pair B consists of two single-stranded DNAs shown in any group of SEQ ID No.116-SEQ ID No.197 ; The probe B is a single-stranded DNA shown in any one of SEQ ID No.198-SEQ ID No.202.

Preferably, 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.

(D4) Substances used to detect the content of alpha-fetoprotein;

(D5) Substances for detection of abnormal prothrombin content.

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.

Further, the substance used to detect the content of alpha-fetoprotein is a substance capable of specifically binding to alpha-fetoprotein, such as an anti-alpha-fetoprotein antibody.

Further, the substance used to detect the content of abnormal prothrombin is a substance capable of specifically binding to abnormal prothrombin, such as an antibody against abnormal prothrombin.

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.

In the kit described in the third to fifth aspects above, the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.

In a specific embodiment of the present invention, the cancer is liver cancer, and correspondingly, the benign lesion is a benign liver lesion or liver cirrhosis. 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.

In the sixth aspect, 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 level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in the sample from the test subject, so as to realize diagnosis or Screen for cancer.

In the seventh aspect, 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 level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in the sample from the subject, so as to achieve Early warning of cancer before clinical symptoms.

In the eighth aspect, the present invention claims a method for distinguishing or assisting in distinguishing cancer and benign lesions.

The method for distinguishing or assisting in distinguishing cancer and benign lesions claimed in the present invention may include the following steps: detecting the methylation level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in the sample from the test subject, thereby Realize or assist in distinguishing between cancer and benign lesions.

In the sixth to eighth aspects above, 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.

In the sixth to eighth aspects above, 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.

In the above sixth to eighth aspects, 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.

In a specific embodiment of the present invention, 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:

(E1) extracting DNA (such as cfDNA) from the sample, and then performing bisulfite conversion;

(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 Any of the indicated single-stranded DNA.

Preferably, 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 The single-stranded DNA shown; the primer pair for the HIST1H3G gene used when carrying out the real-time fluorescence 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 SEQ ID No.141 Single-stranded DNA indicated by ID No.202.

Further, in 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.

In a specific embodiment of the present invention, immunofluorescence is used to detect the content of alpha-fetoprotein and abnormal prothrombin in the sample from the subject.

Further, after detecting the methylation level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in the sample from the subject, the following steps may also be included:

When the Ct value of the ACTB gene is greater than 37, it is judged that the detection results of the methylation level of the OTX1 gene and the HIST1H3G gene are not reliable; credible.

If the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible, further judge the results as follows:

When the following (a) and/or (b) and/or (c) are met at the same time, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk cancer patient; when neither of the following (a) , (b) and (c), it is determined that the test subject is or is a candidate for a non-cancer patient, or the test subject is a low-risk cancer patient:

(a) The Ct value of OTX1 gene and/or HIST1H3G gene is ≤37, calculate the difference between the amplified Ct value of OTX1 gene and/or HIST1H3G gene and the amplified Ct value of the internal reference ACTB gene, and record it as △Ct according to the target gene value (OTX1) and/or ΔCt value (HIST1H3G); ΔCt value (OTX1)≤5 and/or ΔCt value (HIST1H3G)≤9;

(b) Alpha-fetoprotein ≥ 100ng/ml;

(c) Abnormal prothrombin ≥ 40mAU/ml.

If the test results of OTX1 gene and HIST1H3G gene methylation level are not reliable, only the test results of alpha-fetoprotein and abnormal prothrombin content are considered, when alpha-fetoprotein≥100ng/ml and/or abnormal prothrombin≥40mAU /ml, it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when alpha-fetoprotein <100ng/ml and abnormal prothrombin <40mAU/ml, it is determined that the subject is or a candidate is Non-cancer patients, or patients with low risk of cancer.

In the sixth to eighth aspects above, the cancer includes but not limited to liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.

In a specific embodiment of the present invention, 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.

In the sixth to eighth aspects above, 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.

In a ninth aspect, the present invention claims a system.

The system claimed in the present invention may include:

(F1) the kit and real-time fluorescent quantitative PCR instrument described in the fifth aspect above;

(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 real-time fluorescence quantitative PCR amplification result data of the OTX1 gene and the HIST1H3G gene detected from the sample from the subject, and the detection of alpha-fetoprotein and abnormal blood coagulation The content detection result data of zymogen.

The threshold storage module is configured to store threshold A, threshold B, threshold C, threshold D and threshold E; 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 the threshold of the Ct value of the HIST1H3G gene; the threshold D is the threshold of the content of alpha-fetoprotein; the threshold E is the threshold of the content of abnormal prothrombin.

The data comparison module is configured to receive the real-time fluorescence quantitative PCR amplification result data for the OTX1 gene and the HIST1H3G gene of the sample of the subject sent from the data acquisition module, and the data for the alpha-fetoprotein and abnormal content detection result data of prothrombin, and call the threshold value A, the threshold value B, the threshold value C, the threshold value D and the threshold value E stored in the threshold value storage module, and then the The Ct value of the ACTB gene of the subject is compared with the threshold A, the Ct value of the OTX1 gene of the subject is compared with the threshold B, and the Ct value of the HIST1H3G gene of the subject is compared with the threshold A. The threshold C is compared, and the content value of alpha-fetoprotein of the subject is compared with the threshold D, and the content value of abnormal prothrombin of the subject is compared with the threshold E.

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:

If the Ct value of the ACTB gene of the subject to be tested is greater than the threshold A, it is determined that the detection results for the methylation level of the OTX1 gene and the HIST1H3G gene are unreliable; if the Ct value of the ACTB gene is less than or equal to the threshold A, then It is determined that the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible;

If the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible, further judge the results as follows:

If the following (a) and/or (b) and/or (c) are met at the same time, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk cancer patient; if neither of the following (a) , (b) and (c), it is determined that the test subject is or is a candidate for a non-cancer patient, or the test subject is a low-risk cancer patient:

(a) The Ct value of the OTX1 gene is less than or equal to the threshold value B, and/or the Ct value of the HIST1H3G gene is less than or equal to the threshold value C, and the amplification Ct value of the OTX1 gene and the HIST1H3G gene and the amplification Ct value of the internal reference ACTB gene are calculated According to different target genes, it is recorded as △Ct value (OTX1) and/or △Ct value (HIST1H3G); △Ct value (OTX1)≤5 and/or △Ct value (HIST1H3G)≤9;

(b) alpha-fetoprotein ≥ the threshold D;

(c) Abnormal prothrombin ≥ said threshold E.

If the test results of OTX1 gene and HIST1H3G gene methylation level are not reliable, only the test results of alpha-fetoprotein and abnormal prothrombin content are considered, if alpha-fetoprotein ≥ the threshold D and/or abnormal prothrombin ≥ If the threshold E is used, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk patient for cancer; The test subject is or a candidate is a non-cancer patient, or the test subject is a low-risk cancer patient.

In a specific embodiment of the present invention, the threshold A, the threshold B and the threshold C are all 37; the threshold D is 100 ng/ml; the threshold E is 40 mAU/ml.

In the tenth aspect, 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:

(A1) Manufacture of products for the diagnosis or screening of cancer;

(A2) diagnosis or screening for cancer;

(A3) Preparation of products for early warning of cancer before clinical symptoms;

(A4) Early warning of cancer before clinical symptoms;

(A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

(A6) Distinguishing or assisting in differentiating between cancer and benign lesions.

Wherein, 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.

In a specific embodiment of the present invention, the cancer is liver cancer; the benign lesion is benign liver lesion or liver cirrhosis.

In a specific embodiment of the present invention, the test subjects are selected from: patients with liver cancer, patients with benign liver lesions, and patients with liver cirrhosis after healthy people.

In the present invention, 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.

Description of drawings

Figure 1 shows that the current screening methods for liver cancer are mainly serum alpha-fetoprotein (AFP) examination and ultrasound imaging.

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.

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 each marker in the independent validation set II.

The best way to practice the invention

Firstly, the present invention finds out 2588 differentially methylated regions (Differentially methylated region, DMR).

Then 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.

Combined with the collected data, 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.

Finally, from the 51 DMRs, the OTX1 gene and the HIST1H3G gene with better performance were selected as methylation markers for detecting liver cancer.

At the same time, it was found that when the clinically commonly used detection of alpha-fetoprotein (AFP) and abnormal prothrombin (PIVKA-II, also known as DCP) is incorporated into the liver cancer diagnostic model of the present invention, the detection performance will be improved.

The invention uses methylation of OTX1 gene and HIST1H3G gene, alpha-fetoprotein and abnormal prothrombin as markers for liver cancer screening. By detecting the methylation level of the OTX1 gene and HIST1H3G gene, as well as the content of alpha-fetoprotein and abnormal prothrombin, and analyzing the obtained data, the possibility of the subject suffering from liver cancer can be predicted, and then realized in the general population or high-risk groups of liver cancer for the purpose of screening for liver cancer.

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.

Example 1. Discovery of Liver Cancer-Specific Methylated Genes

In order to screen biomarkers of specific methylation in liver cancer, whole genome methylation sequencing (wGBS) was performed on the genomic DNA of liver cancer tissues and liver cancer paracancerous tissues from 44 liver cancer patients. Through the analysis and calculation of the data, 2588 differentially methylated regions (Differentially methylated regions, DMRs) that may be related to the occurrence and development of liver cancer were found.

1. Tissue sample extraction

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).

2. DNA fragmentation

Take 200ng of extracted DNA, and add 1ng Unmethylated lambda DNA (PROMEGA, #D1521) at the same time for subsequent C-U conversion quality control, use an ultrasonic breaker to break the DNA, and then use AMPure XP (AGENCOURT, #A63882) to analyze the DNA Fragment selection, so that the DNA fragment size is concentrated at about 160bp.

3. Library construction

KAPA HyperPlus Library Preparation Kit (KAPA, #KK8510) was used for library construction. For specific operations, refer to the 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.

4. Bisulfite conversion

Use the EZ DNA Metlylation-Gold Kit (Zymo Research, #D5006) to perform bisulfite conversion on the constructed DNA library.

5. Library hybrid capture

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.

6. High-throughput sequencing

PE100 sequencing was performed using MGISEQ-2000 (MGI).

7. Data Analysis

Perform quality control, filtering, comparison, and calculation on the sequencing data, and calculate the methylation rate of each CpG site. By comparing the similarities and differences of the methylation rates of each CpG site in liver cancer tissues and adjacent tissues, 2588 Differentially methylated regions (DMRs) in liver cancer tissues and adjacent tissues were identified by hierarchical Bayesian method. ), which is the HCC-specific methylated region.

Figure 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 grid 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.

Example 2. Verification of Liver Cancer-Specific Methylated Genes

Targeted methylation high-throughput sequencing was performed on plasma cfDNA (plasma cfDNA) from 140 liver cancer patients and 84 healthy individuals from independent sample set I, and machine learning was used to construct a liver cancer risk model to verify these DMRs.

1. Plasma sample extraction

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.

2. Library construction

KAPA HyperPlus Library Preparation Kit (KAPA, #KK8510) was used for library construction. For specific operations, refer to the 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.

3. Bisulfite conversion

Use the EZ DNA Metlylation-Gold Kit (Zymo Research, #D5006) to perform bisulfite conversion on the constructed DNA library.

4. Library hybrid capture

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.

5. High-throughput sequencing

PE100 sequencing was performed using MGISEQ-2000 (MGI).

6. Data analysis

Perform quality control, filtering, comparison, and calculation on the sequencing data, and calculate the methylation rate of each CpG site. By comparing the similarities and differences of the methylation rate of each CpG site in the plasma of liver cancer patients and the plasma of healthy people, 10-fold cross-validation based on random forest is used to screen according to the importance of features in hypermethylated DMR (feature importance > 0.15) , screened out 51 DMRs that can be used as markers for liver cancer screening. The performance of the model was evaluated using each half-way validation set, and the average sensitivity of the validation set was 0.929, the specificity was 0.894, and the AUC was 0.96.

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. The vertical axis is DMR. 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.

Example 3. Evaluation of the methylation status of OTX1 gene and HIST1H3G gene, as well as the content of alpha-fetoprotein and abnormal prothrombin in the diagnosis of liver cancer

From the 51 DMRs obtained in Example 2, 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.

Table 1. Specific methylation regions of OTX1 gene in liver cancer

Table 2. HIST1H3G gene liver cancer-specific methylated regions

The main purpose of this example is to verify the performance of OTX1 and HIST1 H3G gene methylation status and alpha-fetoprotein and abnormal prothrombin content screening for liver cancer in an independent validation set II, including 293 liver cancer patients, 33 liver benign Plasma cell-free DNA of lesion patients, 19 healthy people and 105 liver cirrhosis patients. The inclusion criteria for liver cancer samples were: primary hepatocellular carcinoma, intrahepatic cholangiocarcinoma or mixed liver cancer confirmed by pathology, no previous history of malignant tumors, and no anti-tumor treatment in any form before surgery. 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. Healthy subjects were medical samples with no reported abnormalities.

1. Plasma sample extraction

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).

2. Bisulfite conversion

Take 10ng of cfDNA, and use EZ DNA Metlylation-Gold Kit (Zymo Research, #D5006) for bisulfite conversion.

3. Real-time fluorescent quantitative PCR (qPCR)

Using the ACTB gene as an internal reference gene, bisulfite-converted cfDNA was amplified by qPCR. The entire reaction system contains 5 μl 10×PCR buffer (Novizumab, China), 2 units of Taq polymerase (Novizumab, China), 2.5mM dNTP (Novizumab, China), 2μl (10pmol/μl) PCR primers , 1 μl (10 pmol/μl) 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.

Table 3, real-time fluorescence quantitative PCR primer probe sequence (amplification OTX1 gene)

Table 4. Real-time fluorescence quantitative PCR primer probe sequence (amplification of HIST1H3G and ACTB internal reference)

serial number	Sequence (5'-3')	use
SEQ ID No.116	TTTACGTCGTCGGTGGTC	HIST1H3G forward primer 1
SEQ ID No.117	CGCAAATCCACCGATAACA	HIST1H3G reverse primer 1
SEQ ID No.118	TTTACGTCGTCGGTGGTC	HIST1H3G forward primer 2
SEQ ID No.119	CCGCACCAAACAAAACTACAC	HIST1H3G reverse primer 2
SEQ ID No.120	TTTGTTATCGGTGGATTTGC	HIST1H3G forward primer 3
SEQ ID No.121	AAACAATTTCCACTTCTACCAAATAACT	HIST1H3G reverse primer 3
SEQ ID No.122	GGACGGTAGCGATGAGGT	HIST1H3G forward primer 4
SEQ ID No.123	AACAACTAACCACTAAAACGACTCG	HIST1H3G reverse primer 4
SEQ ID No.124	GAGTCGTTTTAGTGGTTAGTTGTTTG	HIST1H3G forward primer 5
SEQ ID No.125	CGCACCAAACAAAACTACACG	HIST1H3G reverse primer 5
SEQ ID No.126	TGAGATGGTTCGTATTAAGTAGATTGTAC	HIST1H3G forward primer 6
SEQ ID No.127	AACCGCCTTAATAACCAACTACTTAC	HIST1H3G reverse primer 6
SEQ ID No.128	GTAAGTTTATCGGTGGTAAAGC	HIST1H3G forward primer 7
SEQ ID No.129	AAACGATAACGATAAAATTTCTTCAC	HIST1H3G reverse primer 7
SEQ ID No.130	TTTATCGGTGGTAAAGCGTC	HIST1H3G forward primer 8
SEQ ID No.131	AAACGATAACGATAAAATTTCTTCAC	HIST1H3G reverse primer 8
SEQ ID No.132	TTTTAGTGGTTAGTTGTTTGC	HIST1H3G forward primer 9
SEQ ID No.133	ATTTCCACTCTACCAAATAACTA	HIST1H3G reverse primer 9
SEQ ID No.134	TTTTAGTGGTTAGTTGTTTGC	HIST1H3G forward primer 10
SEQ ID No.135	ATTTCCACTCTACCAAATAACTA	HIST1H3G reverse primer 10
SEQ ID No.136	TTTTACGTCGTCGGTGGT	HIST1H3G forward primer 11
SEQ ID No.137	CGATAACAAAACGCCGCG	HIST1H3G reverse primer 11
SEQ ID No.138	GACGGTAGCGATGAGGTT	HIST1H3G forward primer 12

SEQ ID No.139	CGATAACAAAACGCCGCG	HIST1H3G reverse primer 12
SEQ ID No.140	CGCGTAGAGTTACGGTGT	HIST1H3G forward primer 13
SEQ ID No.141	CGATAACAAAACGCCGCG	HIST1H3G reverse primer 13
SEQ ID No.142	TTTAGGTAGTTTTGAGATGGT	HIST1H3G forward primer 14
SEQ ID No.143	CCTTAATAACCAACTACTTACG	HIST1H3G reverse primer 14
SEQ ID No.144	TTATTTTGTTAAGTGGTTGAGT	HIST1H3G forward primer 15
SEQ ID No.145	CCTTAATAACCAACTACTTACG	HIST1H3G reverse primer 15
SEQ ID No.146	GTTAGTTGTTTGCGCGGC	HIST1H3G forward primer 16
SEQ ID No.147	TCAAATAATCTAAAATAACCCGCACCA	HIST1H3G reverse primer 16
SEQ ID No.148	GTTAGTTGTTTGCGCGGC	HIST1H3G forward primer 17
SEQ ID No.149	AACAATTTCCACTTCTACCAAATAACT	HIST1H3G reverse primer 17
SEQ ID No.150	TTTTACGTCGTCGGTGGT	HIST1H3G forward primer 18
SEQ ID No.151	CAAATCCACCGATAACAAAACG	HIST1H3G reverse primer 18
SEQ ID No.152	GGACGGTAGCGATGAGGT	HIST1H3G forward primer 19
SEQ ID No.153	GCAAATCCACCGATAACAAAACG	HIST1H3G reverse primer 19
SEQ ID No.154	GACGGTAGCGATGAGGTT	HIST1H3G forward primer 20
SEQ ID No.155	CAAATCCACCGATAACAAAACG	HIST1H3G reverse primer 20
SEQ ID No.156	GTAAGTTTATCGGTGGTAAAGC	HIST1H3G forward primer 21
SEQ ID No.157	ATAACCGACGCGCTTTTC	HIST1H3G reverse primer 21
SEQ ID No.158	TAAGTTTATCGGTGGTAAAGC	HIST1H3G forward primer 22
SEQ ID No.159	AAACGATAACGATAAAATTTCTTCA	HIST1H3G reverse primer 22
SEQ ID No.160	GTTTGAGATGGTTCGTATTAAGTAGA	HIST1H3G forward primer 23
SEQ ID No.161	CCAACTACTTACGCGACG	HIST1H3G reverse primer 23
SEQ ID No.162	GTAGTTTTTATTTTGTTAAGTGGTTGAGT	HIST1H3G forward primer 24
SEQ ID No.163	CCAACTACTTACGCGACG	HIST1H3G reverse primer 24
SEQ ID No.164	TTTAGTGGTTAGTTGTTTGCGC	HIST1H3G forward primer 25
SEQ ID No.165	CTTTCAAATAATCTAAAATAACCCG	HIST1H3G reverse primer 25
SEQ ID No.166	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 26
SEQ ID No.167	ACTAAAACGACTCGAAAAAACG	HIST1H3G reverse primer 26
SEQ ID No.168	GTTAGTTGTTTGCGCGGC	HIST1H3G forward primer 27
SEQ ID No.169	TTCAAATAATCTAAAATAACCCGCACCA	HIST1H3G reverse primer 27
SEQ ID No.170	GACGGTAGCGATGAGGTT	HIST1H3G forward primer 28
SEQ ID No.171	CAAATCCACCGATAACAAAACG	HIST1H3G reverse primer 28

SEQ ID No.172	TTTTTAGGTAGTTTGAGATGGTTCG	HIST1H3G forward primer 29
SEQ ID No.173	GCTTTACCACCGATAAACTTACGT	HIST1H3G reverse primer 29
SEQ ID No.174	AGGTAGTTTGAGATGGTTCGTA	HIST1H3G forward primer 30
SEQ ID No.175	CCAACTACTTACGCGACG	HIST1H3G reverse primer 30
SEQ ID No.176	TGATTCGTAAGTTGTTTTTTTAAC	HIST1H3G forward primer 31
SEQ ID No.177	CCTCCTACAAAACCATCA	HIST1H3G reverse primer 31
SEQ ID No.178	TGATTCGTAAGTTGTTTTTTTAAC	HIST1H3G forward primer 32
SEQ ID No.179	AAACTCTAAAAACGCAAATCT	HIST1H3G reverse primer 32
SEQ ID No.180	GGTAATTTGCGGATTAGTAGTTTAGTC	HIST1H3G forward primer 33
SEQ ID No.181	GACACCGTAACTCTACGCGA	HIST1H3G reverse primer 33
SEQ ID No.182	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 34
SEQ ID No.183	ACAACTAACCACTAAAACGACTCG	HIST1H3G reverse primer 34
SEQ ID No.184	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 35
SEQ ID No.185	ACTAAAACGACTCGAAAAAACG	HIST1H3G reverse primer 35
SEQ ID No.186	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 36
SEQ ID No.187	GCAAACAACTAACCACTAAAACGA	HIST1H3G reverse primer 36
SEQ ID No.188	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 37
SEQ ID No.189	AACTAACCACTAAAACGACTCGAA	HIST1H3G reverse primer 37
SEQ ID No.190	AGTAGTTGGTTATTAAGGCGGTTC	HIST1H3G forward primer 38
SEQ ID No.191	ATAACGATAAAATTTCTTCACGCCG	HIST1H3G reverse primer 38
SEQ ID No.192	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 39
SEQ ID No.193	ACTAAAACGACTCGAAAAAACGCG	HIST1H3G reverse primer 39
SEQ ID No.194	GTAGAGTTACGGTGTCGGGAC	HIST1H3G forward primer 40
SEQ ID No.195	ACTAAAACGACTCGAAAAAACG	HIST1H3G reverse primer 40
SEQ ID No.196	GTAATAAAATTATTGGTTGAAGAGTTTAAC	HIST1H3G forward primer 41
SEQ ID No.197	CTTAAATCGCAAACTAACCGAA	HIST1H3G reverse primer 41
SEQ ID No.198	ACCACTAAAACGACTCGAAAAAACGCGCCG	HIST1H3G probe 1
SEQ ID No.199	ACCGCCTTAATAACCAACTACTTACGCGAC	HIST1H3G probe 2
SEQ ID No.200	CAAACAACTAACCACTAAAACGACTC	HIST1H3G probe 3
SEQ ID No.201	AACCGCCTTAATAACCAACTACTTAC	HIST1H3G probe 4
SEQ ID No.202	AACGCGCCGACCACCGACGA	HIST1H3G probe 5
SEQ ID No.203	AAGGGGTTTATATGGTAATTGTG	ACTB forward primer sequence
SEQ ID No.204	GATTGAGTGTGGTAGGGATT	ACTB reverse primer sequence

SEQ ID No.205

CCCACCACACTAAATCTCCCCTCCT

ACTB probe sequence

4. Result analysis

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: 26271523, chr6:26271525, chr6:26271560, chr6:26271562, chr6:26271565, chr6:26271576) as an example, as follows:

Principle: Estimate the methylation rate based on the difference between the Ct value of the target gene and the amplified Ct value of the internal reference ACTB gene (△Ct value). The smaller the △Ct value, the higher the methylation rate of the target gene.

The threshold value of Ct value for each gene was 37.

Among them, when the Ct value of ACTB is greater than 37, the result is judged to be unreliable; when the Ct value of ACTB gene is less than or equal to 37, the result is judged to be credible, and further judgment is carried out as follows:

When the Ct values of the OTX1 gene and the HIST1H3G gene are both greater than 37, it is determined that the subject is not a liver cancer patient; when the Ct values of the OTX1 gene and the HIST1H3G gene are not equal to greater than 37 (that is, when either or both ≤ 37), the following (1)-(3) Further judgment:

(1) When the Ct values of OTX1 gene and HIST1H3G gene are both ≤37, calculate the difference between the amplified Ct value of OTX1 gene and HIST1H3G gene and the amplified Ct value of internal reference ACTB gene, and record them as △Ct value (OTX1) and △Ct value (HIST1H3G);

When the selected △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;

(2) When the Ct value of the OTX1 gene is ≤37, but the Ct value of the HIST1H3G gene is >37, calculate the difference between the amplified Ct value of the OTX1 gene and the amplified Ct value of the internal reference ACTB gene, and record it as the ΔCt value (OTX1 );

When the selected △Ct value (OTX1)≤5, it is determined that the subject is a liver cancer patient; when the △Ct value (OTX1)>5, it is determined that the subject is not a liver cancer patient;

(3) When the Ct value of the HIST1H3G gene is ≤37, but the Ct value of the OTX1 gene is >37, calculate the difference between the amplified Ct value of the HIST1H3G gene and the amplified Ct value of the internal reference ACTB gene, and record it as the △Ct value (HIST1H3G );

When the selected △Ct value (HIST1H3G)≤9, it is determined that the subject is a liver cancer patient; when the △Ct value (HIST1H3G)>9, it is determined that the subject is not a liver cancer patient.

Use SPSS software to make the ROC curve of the ΔCt of the two genes to be detected. The ROC curve is shown in Figure 5, and the performance and area under the curve (AUC) of each gene are shown in Table 5.

5. Immunochemiluminescence:

(1) Take 20 μL plasma sample and 250 μL antibody-conjugated particles (250 μg/mL alpha-fetoprotein/abnormal prothrombin monoclonal antibody (mouse)-bound magnetic particles containing protein stabilizer (bovine, calf or mouse) and chemical stabilizer 0.15M sodium chloride/trishydroxymethylaminomethane (Tirs) buffer) solution of the agent was mixed, incubated at 37°C for 10min, and washed 3 times in a magnetic environment.

(2) Add 250 μL alkaline phosphatase-labeled alpha-fetoprotein/abnormal prothrombin antibody and mix well. Incubate at 37°C for 10 minutes to form a luminescent immune complex including anti-abnormal prothrombin antibody coated with magnetic microspheres, abnormal prothrombin to be tested, anti-abnormal prothrombin antibody and alkaline phosphatase, and Magnetic microsphere-coated anti-alpha-fetoprotein antibody, alpha-fetoprotein to be tested, anti-alpha-fetoprotein antibody and alkaline phosphatase-coupled luminescent immune complex;

(3) Detection: Apply an external magnetic field to precipitate the above-mentioned luminescent immune complex, remove the supernatant, wash with the buffer solution of the automatic perfusion system, add 200 μL of substrate solution (AMPPD), and detect the relative light intensity emitted at a wavelength of 447nm (RLU). The contents of abnormal prothrombin and alpha-fetoprotein were converted, and the ROC curve of the two protein marker contents was made using SPSS software. The ROC curve is shown in Figure 5, and the performance of each gene and the area under the curve (AUC) are shown in Table 5 Show.

6. Combined use of four markers

In order to further improve the performance of the kit, four marker values were integrated for analysis, as follows:

When the Ct value of the ACTB gene is greater than 37, it is judged that the detection results of the methylation level of the OTX1 gene and the HIST1H3G gene are not reliable; believable.

If the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible, further judge the results as follows:

When the following (a) and/or (b) and/or (c) are met at the same time, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk cancer patient; when neither of the following (a) , (b) and (c), it is determined that the test subject is or is a candidate for a non-cancer patient, or the test subject is a low-risk cancer patient:

(a) The Ct value of OTX1 gene and/or HIST1H3G gene is ≤37, calculate the difference between the amplified Ct value of OTX1 gene and/or HIST1H3G gene and the amplified Ct value of the internal reference ACTB gene, and record it as △Ct according to the target gene value (OTX1) and/or ΔCt value (HIST1H3G); ΔCt value (OTX1)≤5 and/or ΔCt value (HIST1H3G)≤9;

(b) Alpha-fetoprotein ≥ 100ng/ml;

(c) Abnormal prothrombin ≥ 40mAU/ml.

If the test results of OTX1 gene and HIST1H3G gene methylation level are not reliable, only the test results of alpha-fetoprotein and abnormal prothrombin content are considered, when alpha-fetoprotein≥100ng/ml and/or abnormal prothrombin≥40mAU /ml, it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when alpha-fetoprotein <100ng/ml and abnormal prothrombin <40mAU/ml, it is determined that the subject is or a candidate is Non-cancer patients, or patients with low risk of cancer.

Using SPSS software to make ROC curve, the AUC can reach 0.992. It is finally determined that △Ct (OTX1) ≤ 5, △ Ct (HIST1H3G) ≤ 9, alpha-fetoprotein ≥ 100ng/ml, abnormal prothrombin ≥ 40mAU/ml when at least one condition is met, it is determined that the test is positive, that is, when any marker is positive Then it is judged that the sample is positive. Under the judgment conditions, when the specificity (including healthy people, liver cirrhosis and benign liver lesions) is 92.7%, the sensitivity (liver cancer) can reach 87.6%. See Table 5.

The above results show that the present invention can obtain better detection performance than single gene detection through joint detection of multiple genes.

Table 5. Performance, area under the curve (AUC), sensitivity and specificity of each marker

industrial application

Through DNA methylation sequencing of liver cancer tissue, normal tissue, liver cancer patient plasma, and control population plasma, the present invention found that the hypermethylation of OTX1 gene and HIST1H3G gene is related to liver cancer, which can be used as markers for liver cancer detection, and further Used in combination with alpha-fetoprotein and abnormal prothrombin. 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.

Claims (34)

1. Application of the combination of "methylated OTX1 gene, methylated HIST1H3G gene, alpha-fetoprotein and abnormal prothrombin" as a combined marker in any of the following:

   (A1) Manufacture of products for the diagnosis or screening of cancer;

   (A2) diagnosis or screening for cancer;

   (A3) Preparation of products for early warning of cancer before clinical symptoms;

   (A4) Early warning of cancer before clinical symptoms;

   (A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

   (A6) Distinguishing or assisting in differentiating between cancer and benign lesions.
2. The application according to claim 1, characterized in that: the methylated OTX1 gene is 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 methylation of the HIST1H3G gene is the methylation of all or part of the CpG sites in the DNA fragment shown in SEQ ID No.4.
3. The application of substances used to detect "OTX1 gene methylation level, HIST1H3G gene methylation level, alpha-fetoprotein content and abnormal prothrombin content" in any of the following:

   (A1) Manufacture of products for the diagnosis or screening of cancer;

   (A2) diagnosis or screening for cancer;

   (A3) Preparation of products for early warning of cancer before clinical symptoms;

   (A4) Early warning of cancer before clinical symptoms;

   (A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

   (A6) Distinguishing or assisting in differentiating between cancer and benign lesions.
4. The application according to claim 3, characterized in that: the OTX1 gene methylation level is all or part of the CpG positions in the DNA fragment shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 The methylation level of the point; 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.
5. The application according to claim 3 or 4, characterized in that: the material used to detect the methylation level of the OTX1 gene is bisulfite reagent and primer pair A and probe A; the primer pair A consists of SEQ ID No.5-SEQ ID No.112 shown in any group of two single-stranded DNA composition; said probe A is shown in any one of SEQ ID No.113-SEQ ID No.115 single-stranded DNA A;

   The substance used to detect the methylation level of the HIST1H3G gene is a bisulfite reagent, a primer pair B and a probe B; the primer pair B is shown by any group of SEQ ID No.116-SEQ ID No.197 The two single-stranded DNA composition; The probe B is the single-stranded DNA shown in any one of SEQ ID No.198-SEQ ID No.202;

   The substance used to detect the content of alpha-fetoprotein is a substance that can specifically bind to alpha-fetoprotein;

   The substance used to detect the abnormal prothrombin content is a substance capable of specifically binding to abnormal prothrombin.
6. The application according to claim 5, characterized in that: said primer pair A consists of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96; said probe A is SEQ ID No.113 Single-stranded DNA as indicated;

   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.
7. The use according to any one of claims 1-6, wherein the cancer is liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
8. The application according to claim 7, characterized in that: the cancer is liver cancer; the benign lesion is a benign lesion of the liver or liver cirrhosis.
9. A test kit comprising:

   (B1) bisulfite reagent; and

   (B2) 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;

   (B3) control nucleic acid 2, the sequence of the control nucleic acid 2 is as shown in SEQ ID No.4, and has a methylation state associated with non-cancer patients;

   (B4) Substances used to detect the content of alpha-fetoprotein;

   (B5) Substances used to detect abnormal prothrombin levels;

   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.
10. A test kit comprising:

    (C1) bisulfite reagent; and

    (C2) control nucleic acid 3, the sequence of said 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 state relevant to cancer patients;

    (C3) control nucleic acid 4, the sequence of the control nucleic acid 4 is as shown in SEQ ID No.4, and has a methylation state relevant to cancer patients;

    (C4) Substances used to detect the content of alpha-fetoprotein;

    (C5) Substances used to detect abnormal prothrombin levels;

    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.
11. A test kit comprising:

    (D1) bisulfite reagent; and

    (D2) Primer pair A and probe A for detecting the methylation level of the OTX1 gene; two single-stranded DNAs shown in any group of SEQ ID No.5-SEQ ID No.112 form; the probe Needle A is a single-stranded DNA shown in any one of SEQ ID No.113-SEQ ID No.115;

    (D3) Primer pair B and probe B for detecting the methylation level of the HIST1H3G gene; the primer pair B consists of two single strands shown by any group of SEQ ID No.116-SEQ ID No.197 DNA composition; the probe B is single-stranded DNA shown in any one of SEQ ID No.198-SEQ ID No.202;

    (D4) Substances used to detect the content of alpha-fetoprotein;

    (D5) Substances used to detect abnormal prothrombin content;

    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.
12. The kit according to claim 11, wherein: said primer pair A consists of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96; said probe A is SEQ ID No. Single-stranded DNA shown in 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.
13. The kit according to claim 11 or 12, characterized in that: said kit also contains primer pair C and probe C for amplifying internal reference gene ACTB; said primer pair C consists of SEQ ID No.203 and two single-stranded DNAs shown in SEQ ID No.204; the probe C is the single-stranded DNA shown in SEQ ID No.205.
14. The kit according to any one of claims 11-13, wherein the cancer is liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
15. The kit according to claim 14, characterized in that: the cancer is liver cancer; the benign lesion is a benign liver lesion or liver cirrhosis.
16. A method for diagnosing or screening cancer, comprising the following steps: detecting the methylation level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in samples from a test subject, so as to realize the diagnosis or screening of cancer.
17. A method for early warning of cancer before clinical symptoms, comprising the following steps: detecting the methylation levels of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in samples from a subject, so as to realize early warning before clinical symptoms cancer.
18. A method for distinguishing or assisting in distinguishing cancer and benign lesions, comprising the following steps: detecting the methylation level of OTX1 gene and HIST1H3G gene and the content of alpha-fetoprotein and abnormal prothrombin in the sample from the subject, so as to realize the distinction or assist Differentiate between cancer and benign lesions.
19. According to the method described in any one of claims 16-18, it is characterized in that: the methylation level of the OTX1 gene is in the DNA fragment shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3 The methylation level of all or part of the CpG sites; 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.
20. The method according to any one of claims 16-19, characterized in that: the method for detecting the methylation levels of OTX1 gene and HIST1H3G gene in the sample from the subject is bisulfate conversion, PCR, formazan methylation-specific PCR, pyrosequencing, Sanger sequencing, high-throughput sequencing or three-generation sequencing or single-molecule sequencing.
21. The method according to any one of claims 16-20, characterized in that: detecting the methylation level of OTX1 gene and HIST1H3G gene in the sample from the subject is carried out according to the method comprising the following steps:

    (E1) extracting DNA from the sample, and then performing bisulfite conversion;

    (E2) DNA converted by bisulfite obtained through real-time fluorescence quantitative PCR amplification (E1); the primer pair for the OTX1 gene used when carrying out the real-time fluorescence 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 any single-stranded DNA shown in SEQ ID No.113-SEQ ID No.115; when performing the real-time fluorescence quantitative PCR amplification, it is used The primer pair for the HIST1H3G gene consists of two single-stranded DNAs shown in any one of SEQ ID No.116-SEQ ID No.197, and the probe is any one of SEQ ID No.198-SEQ ID No.202 Single-stranded DNA is shown.
22. The method according to claim 21, characterized in that: the primer pair for the OTX1 gene used when carrying out the real-time fluorescent quantitative PCR amplification consists of two single-stranded DNAs shown in SEQ ID No.95 and SEQ ID No.96 Composition, the probe is single-stranded DNA shown in SEQ ID No.113; The primer pair for HIST1H3G gene that adopts when carrying out described real-time fluorescence quantitative PCR amplification is shown in SEQ ID No.140 and SEQ ID No.141 two Composed of single-stranded DNA, the probe is the single-stranded DNA shown in SEQ ID No.202.
23. The method according to claim 21 or 22, characterized in that: in step (E2), the internal reference used when carrying out the real-time fluorescent quantitative PCR amplification is ACTB gene, and the primer pair for amplifying the ACTB gene consists of SEQ The two single-stranded DNAs shown in ID No.203 and SEQ ID No.204 consist of two single-stranded DNAs, and the probe is the single-stranded DNA shown in SEQ ID No.205.
24. The method according to any one of claims 16-23, characterized in that: immunofluorescence is used to detect the content of alpha-fetoprotein and abnormal prothrombin in the sample from the subject.
25. The method according to any one of claims 21-24, characterized in that: after detecting the methylation levels of the OTX1 gene and the HIST1H3G gene, alpha-fetoprotein and abnormal prothrombin in the sample from the subject After content, also include the following steps:

    When the Ct value of the ACTB gene is greater than 37, it is judged that the detection results of the methylation level of the OTX1 gene and the HIST1H3G gene are not reliable; Credible;

    If the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible, further judge the results as follows:

    When the following (a) and/or (b) and/or (c) are met at the same time, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk cancer patient; when neither of the following (a) , (b) and (c), it is determined that the test subject is or is a candidate for a non-cancer patient, or the test subject is a low-risk cancer patient:

    (a) The Ct value of OTX1 gene and/or HIST1H3G gene is ≤37, calculate the difference between the amplified Ct value of OTX1 gene and/or HIST1H3G gene and the amplified Ct value of the internal reference ACTB gene, and record it as △Ct according to the target gene value (OTX1) and/or ΔCt value (HIST1H3G); ΔCt value (OTX1)≤5 and/or ΔCt value (HIST1H3G)≤9;

    (b) Alpha-fetoprotein ≥ 100ng/ml;

    (c) Abnormal prothrombin ≥ 40mAU/ml;

    If the test results of OTX1 gene and HIST1H3G gene methylation level are not reliable, only the test results of alpha-fetoprotein and abnormal prothrombin content are considered, when alpha-fetoprotein≥100ng/ml and/or abnormal prothrombin≥40mAU /ml, it is determined that the subject is or is a candidate for cancer, or the subject is a high-risk cancer patient; when alpha-fetoprotein <100ng/ml and abnormal prothrombin <40mAU/ml, it is determined that the subject is or a candidate is Non-cancer patients, or patients with low risk of cancer.
26. The method according to any one of claims 16-25, characterized in that the cancer is liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
27. The method according to claim 26, characterized in that: the cancer is liver cancer; the benign lesion is a benign liver lesion or liver cirrhosis.
28. The method according to any one of claims 16-27, characterized in that: the sample is a sample from which DNA can be extracted.
29. The method according to claim 28, characterized in that: said sample is plasma, serum, blood, tissue, saliva, urine, feces.
30. systems, including:

    (F1) kit and real-time fluorescent quantitative PCR instrument according to claim 13;

    (F2) device, said device includes a data acquisition module, a threshold value storage module, a data comparison module, a data processing and a conclusion output module;

    The data collection module is configured to collect (F1) the real-time fluorescence quantitative PCR amplification result data of the OTX1 gene and the HIST1H3G gene detected from the sample from the subject, and the detection of alpha-fetoprotein and abnormal blood coagulation zymogen content test result data;

    The threshold storage module is configured to store threshold A, threshold B, threshold C, threshold D and threshold E; 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 the threshold of the Ct value of the HIST1H3G gene; the threshold D is the threshold of the content of alpha-fetoprotein; the threshold E is the threshold of the content of abnormal prothrombin;

    The data comparison module is configured to receive the real-time fluorescence quantitative PCR amplification result data for the OTX1 gene and the HIST1H3G gene of the sample of the subject sent from the data acquisition module, and the data for the alpha-fetoprotein and abnormal content detection result data of prothrombin, and call the threshold value A, the threshold value B, the threshold value C, the threshold value D and the threshold value E stored in the threshold value storage module, and then the The Ct value of the ACTB gene of the subject is compared with the threshold A, the Ct value of the OTX1 gene of the subject is compared with the threshold B, and the Ct value of the HIST1H3G gene of the subject is compared with the threshold A. Comparing the threshold C, comparing the alpha-fetoprotein content of the subject with the threshold D, and comparing the abnormal prothrombin content of the subject with the threshold E;

    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:

    If the Ct value of the ACTB gene of the subject to be tested is greater than the threshold A, it is determined that the detection results for the methylation level of the OTX1 gene and the HIST1H3G gene are unreliable; if the Ct value of the ACTB gene is less than or equal to the threshold A, then It is determined that the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible;

    If the detection results of the methylation levels of the OTX1 gene and the HIST1H3G gene are credible, further judge the results as follows:

    If the following (a) and/or (b) and/or (c) are met at the same time, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk cancer patient; if neither of the following (a) , (b) and (c), it is determined that the test subject is or is a candidate for a non-cancer patient, or the test subject is a low-risk cancer patient:

    (a) The Ct value of the OTX1 gene is less than or equal to the threshold value B, and/or the Ct value of the HIST1H3G gene is less than or equal to the threshold value C, and the amplification Ct value of the OTX1 gene and the HIST1H3G gene and the amplification Ct value of the internal reference ACTB gene are calculated According to different target genes, it is recorded as △Ct value (OTX1) and/or △Ct value (HIST1H3G); △Ct value (OTX1)≤5 and/or △Ct value (HIST1H3G)≤9;

    (b) alpha-fetoprotein ≥ the threshold D;

    (c) Abnormal prothrombin ≥ said threshold E.

    If the test results of OTX1 gene and HIST1H3G gene methylation level are not reliable, only the test results of alpha-fetoprotein and abnormal prothrombin content are considered, if alpha-fetoprotein ≥ the threshold D and/or abnormal prothrombin ≥ If the threshold E is used, it is determined that the subject is or is a candidate for a cancer patient, or the subject is a high-risk patient for cancer; The test subject is or a candidate is a non-cancer patient, or the test subject is a low-risk cancer patient.
31. The system according to claim 30, characterized in that: the threshold A, the threshold B and the threshold C are all 37; the threshold D is 100 ng/ml; the threshold E is 40 mAU/ml.
32. The application of the kit according to any one of claims 9-15 or the system according to claim 30 or 31 in any of the following:

    (A1) Manufacture of products for the diagnosis or screening of cancer;

    (A2) diagnosis or screening for cancer;

    (A3) Preparation of products for early warning of cancer before clinical symptoms;

    (A4) Early warning of cancer before clinical symptoms;

    (A5) Preparation of products for distinguishing or assisting in distinguishing between cancer and benign lesions;

    (A6) Distinguishing or assisting in differentiating between cancer and benign lesions.
33. The application according to claim 32, wherein the cancer is liver cancer, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer or urothelial cancer.
34. The application according to claim 33, characterized in that: the cancer is liver cancer; the benign lesion is a benign liver lesion or liver cirrhosis.

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