Liver cancer detection reagent based on OTX1

Abstract

The invention belongs to the field of gene diagnosis, and particularly relates to a liver cancer detection reagent based on OTX1. The reagent comprises a detection reagent for OTX1 gene methylation, and the detection reagent is used for detecting the modified sequence of the OTX1 gene. Experiments prove that the detection reagent disclosed by the invention can detect and diagnose liver cancer with high sensitivity and high specificity, and has extremely high clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of gene diagnostics, specifically, this invention relates to a liver cancer detection reagent based on OTX1. Background Technology

[0002] Primary hepatic carcinoma (PHC) is one of the most common malignant tumors worldwide. It mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA), with HCC being the most common. In 2020, there were 906,000 new cases of primary hepatic carcinoma globally, accounting for 4.7% of all new cancer cases, ranking sixth; and 830,000 deaths, accounting for 8.3% of all cancer deaths, ranking third. In 2020, China had 410,000 new cases of primary hepatic carcinoma, accounting for 45.27% of the global total; and 390,000 deaths, accounting for 47.12% of the global total. The incidence and mortality rates are higher in men than in women.

[0003] The main causative factors of primary liver cancer include chronic hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, alcohol consumption, aflatoxin B1 exposure, non-alcoholic fatty liver disease (NAFLD), and diabetes. Although effective control measures have been implemented in recent years to address factors such as hepatitis B virus infection and aflatoxin contamination in food, leading to a decline in the incidence and mortality rates of liver cancer, the 5-year survival rate for liver cancer patients remains low. This is because primary liver cancer has an insidious onset, complex etiologies, and is difficult to diagnose early. By the time it is discovered, it is often already in an advanced or late stage. Only 30% of patients have the opportunity for surgical resection, and the metastasis and recurrence rate within 5 years after resection is as high as 60%–70%, resulting in an overall low 5-year survival rate of only 7%–10%. Follow-up data from a study of patients with primary liver cancer showed that the 5-year survival rate after surgery was as high as 69.0%–86.2% for patients with early-stage BCLC (stage 0 or A), while the 5-year survival rate for patients with intermediate-to-late-stage BCLC was only 39.0%, highlighting the importance of early screening for high-risk groups of liver cancer. Therefore, achieving early screening and diagnosis is crucial for the effective treatment and improved prognosis of liver cancer.

[0004] Currently, clinical screening and diagnosis of liver cancer often combine imaging techniques with biomarker detection. Non-invasive screening methods primarily include routine abdominal ultrasound and alpha-fetoprotein (AFP) testing. However, the sensitivity of routine abdominal ultrasound for early-stage HCC is only 32%-63%. AFP, a special protein produced by embryonic stem cells, is expressed during the occurrence and development of liver cancer. It has high specificity and can be widely used as a serological marker for liver cancer screening and early diagnosis. However, the sensitivity and specificity of AFP in diagnosing liver cancer currently fall short of clinical needs. Approximately 30% of liver cancer patients have serum AFP levels within the normal range, and the sensitivity of AFP testing for early-stage HCC is only 45.3-62%. Even with a screening strategy combining abdominal ultrasound and serum AFP, it still cannot fully meet the need for "early intervention" in liver cancer specific prevention and control, and cannot effectively achieve early screening and diagnosis. Finding novel biomarkers to improve and supplement existing screening strategies is currently a key focus of research on liver cancer specific prevention and control.

[0005] In recent years, "liquid biopsy," including circulating cell-free microRNA, circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA), has demonstrated significant value in early tumor diagnosis and efficacy evaluation. Significant progress has been made in liver cancer "liquid biopsy," which may offer higher sensitivity and specificity compared to commonly used serum molecular markers such as serum AFP. CTC detection is considered a novel clinical tool for predicting liver cancer prognosis and evaluating treatment efficacy. Reports indicate that CTC detection has predictive value for liver cancer recurrence and progression after transcatheter arterial chemoembolization (TCA) and radiotherapy; CTCs in different locations can predict different metastatic types; and dynamic CTC detection can be used to monitor tumor recurrence after liver transplantation. ctDNA is a specific mutated DNA fragment released into the peripheral blood from tumor cell apoptosis or necrosis, carrying genomic information of the in situ tumor. Studies have shown that ctDNA has superior sensitivity and specificity compared to serum AFP for early liver cancer diagnosis, demonstrating good clinical application value. In addition, some studies have shown that epigenetic modification features of specific genes, such as methylation and 5-hmc, can also be used for the early diagnosis of liver cancer.

[0006] Currently, many studies are detecting cellular or DNA methylation status in blood, urine, tissues, and feces in an attempt to find biomarkers for the early diagnosis of primary liver cancer. Although existing technologies have identified some genes whose DNA methylation is associated with liver cancer, further research is needed in this field to identify genes that can be practically applied to liver cancer diagnosis, and to develop diagnostic reagents with high accuracy. Summary of the Invention

[0007] On one hand, the present invention provides an application of a nucleic acid fragment methylation detection reagent in the preparation of liver cancer detection reagents or kits, wherein the nucleic acid fragment is selected from the nucleic acid fragment shown in SEQ ID NO: 95.

[0008] On one hand, the present invention provides a primer pair selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 20 and SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 3 ...6, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 39, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 40 and SEQ ID NO: 42, SEQ ID NO: 40 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 44 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 55, SEQ ID NO: 49 and SEQ ID NO: 56, SEQ ID NO: 50 and SEQ ID NO: 55, SEQ ID NO: 50 and SEQ ID NO: 56, SEQ ID NO: 51 and SEQ ID NO: 55, SEQ ID NO: 51 and SEQ ID NO: 56, SEQ ID NO: 11 and SEQ ID NO: 78, SEQ ID NO: 77 and SEQ ID SEQ ID NO: 12, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 80,Any one of the primer pairs shown in SEQ ID NO: 79 and SEQ ID NO: 14, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 40 and SEQ ID NO: 67, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 66, SEQ ID NO: 64 and SEQ ID NO: 67, SEQ ID NO: 40 and SEQ ID NO: 70, SEQ ID NO: 64 and SEQ ID NO: 43, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 44 and SEQ ID NO: 74, SEQ ID NO: 71 and SEQ ID NO: 46, SEQ ID NO: 71 and SEQ ID NO: 74, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 49 and SEQ ID NO: 55.

[0009] In some embodiments, the primer pairs are selected from SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 11 and SEQ ID NO: 78, SEQ ID NO: 77 and SEQ ID NO: 12, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 80, SEQ ID NO: 79 and SEQ ID NO: 14, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 40 and SEQ ID NO: 67, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 66, SEQ ID NO: 64 and SEQ ID NO: 67, SEQ ID NO: 40 and SEQ ID NO: 70, SEQ ID NO: 64 and SEQ ID NO: 43, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 44 and SEQ ID NO: 70. Any one of the primer pairs shown in NO:74, SEQ ID NO:71 and SEQ ID NO:46, SEQ ID NO:71 and SEQ ID NO:74, SEQ ID NO:19 and SEQ ID NO:22, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:49 and SEQ ID NO:55.

[0010] In some embodiments, the primer pairs are selected from any one of the primer pairs shown in SEQ ID NO: 77 and SEQ ID NO: 12, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 49 and SEQ ID NO: 55.

[0011] On the one hand, the present invention provides a nucleic acid probe, wherein the probe is selected from any one of the probes shown in SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99.

[0012] On the one hand, the present invention provides the application of the primer pairs and / or probes in the preparation of methylation detection reagents, or in the preparation of liver cancer detection reagents.

[0013] On the one hand, the present invention provides the application of the primer pairs, probes, and / or AFP detection reagents in the preparation of liver cancer detection reagents.

[0014] Although existing technologies have reported differential expression of some genes in liver cancer, in reality, the discovery of differential expression of certain genes or nucleic acid fragments in tumor and non-tumor cells cannot be simply interpreted as indicating a change in the methylation level of that gene or nucleic acid fragment. This is because the causes of differential expression of genes or nucleic acid fragments in tumor cells are diverse. According to Peter Laird's 2012 report in Genome Research, less than 8% of gene expression in tumors is significantly regulated by gene DNA methylation, while more than 90% of gene expression is regulated by various other intracellular mechanisms, including transcription factors, enhancers, nucleic acid conformation, histone modification, RNA modification, and RNA splicing. DNA methylation regulation is only one of the factors regulating gene expression (Hinoue, Toshinori et al. "Genome-scale analysis of aberrant DNA methylation in colorectal cancer." Genome Research vol. 22, 2(2012): 271-82.).

[0015] Methylation occurs when a methyl group is added to cytosine. After treatment with bisulfite, bisulfite, or hydrazine, cytosine is converted to uracil. Because uracil is similar to thymine during PCR amplification, it is recognized as thymine. This is reflected in the PCR amplified sequence as unmethylated cytosine becoming thymine (C becomes T), while methylated cytosine (C) remains unchanged. The PCR technique for detecting methylated genes typically uses MSP (methylated polystyrene). Primers are designed for the treated methylated fragment (i.e., the unaltered C group in the fragment), and PCR amplification is performed. If amplification occurs, methylation has occurred; if not, methylation has not occurred.

[0016] It is well known in the art that successful primer design is crucial for PCR. Compared to general PCR, primer design is even more critical in gene methylation detection. This is because methylation causes the "C" in the DNA chain to be converted to "U," leading to a decrease in GC content. This results in long, continuous "T" sequences in the PCR reaction, which easily cause DNA chain breaks, making it difficult to select primers with suitable Tm values ​​and stability. On the other hand, to distinguish between methylated and unmethylated DNA, as well as incompletely methylated DNA, primers need to have a sufficient number of "C" atoms, further increasing the difficulty of selecting stable primers. Therefore, in DNA methylation detection, the selection of the amplified fragment targeted by the primers, such as the length and location of the amplified fragment, as well as the primer selection itself, all affect the sensitivity and specificity of the detection.

[0017] In some implementation schemes, different amplified target fragments and primers result in varying detection effects. Often, while certain genes or nucleic acid fragments are found to express differently in tumor and non-tumor environments, there is still a long way to go before they can be translated into tumor biomarkers for clinical application. The primary reason for this is the limitation of detection reagents, which makes it difficult to meet detection requirements in terms of sensitivity and specificity, or the detection methods are complex and costly, hindering large-scale clinical application.

[0018] On one hand, the present invention provides a methylation detection reagent, comprising the primer pair and / or probe.

[0019] On one hand, the present invention provides a kit comprising the primer pair, or the probe, or the methylation detection reagent. In some embodiments, the kit further comprises an AFP detection reagent.

[0020] On the one hand, the present invention provides the application of the primer pair, or the probe, or the methylation detection reagent, or the kit in the preparation of a liver cancer detection / diagnostic kit.

[0021] In some embodiments, the methylation detection reagent, or the kit, is used to detect samples selected from blood, plasma, serum, saliva, tissue, or urine.

[0022] In some implementations, the test sample for the test reagent is selected from plasma or tissue.

[0023] In some implementation schemes, the liver cancer referred to is primary liver cancer.

[0024] On one hand, the present invention provides a liver cancer detection system, the system comprising:

[0025] (1) A methylation detection component for a nucleic acid fragment as shown in SEQ ID NO: 95;

[0026] (2) Result judgment component;

[0027] The methylation detection component of the nucleic acid fragment shown in SEQ ID NO: 95 contains the primer pair, or the probe, or the methylation detection reagent, or the kit.

[0028] In some implementations, the result determination component is used to output the risk of liver cancer and / or the type of liver cancer based on the methylation result of the nucleic acid fragment as shown in SEQ ID NO: 95 detected by the detection component.

[0029] In some implementations, the disease risk is determined based on a comparison of the methylation results of the test sample and the methylation results of a normal sample; when the comparison of the methylation results of the test sample and the normal sample shows a significant or highly significant difference, the test sample is judged to have a high disease risk.

[0030] In this invention, "detection" is synonymous with diagnosis. In addition to the early diagnosis of liver cancer, it also includes the diagnosis of intermediate and late-stage liver cancer, as well as liver cancer screening, risk assessment, prognosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets.

[0031] Currently, there are many biomarkers associated with liver cancer, but accurate diagnosis through a single biomarker is still not possible. Although liver cancer biomarkers, such as alpha-fetoprotein (AFP), are simple and easy to use, especially suitable for HBV-related liver cancer, AFP still has shortcomings in sensitivity and specificity, which can easily lead to missed diagnoses and misdiagnoses. In addition, existing technologies have reported the combined use of various biomarkers for liver cancer screening. For example, Chang-Yi Lu et al. found that the sensitivity of the combination of four biomarkers used for liver cancer detection was 84.2%, and the specificity was 83% (Chang-Yi Lu et al. "Cell-free methylationmarkers with diagnostic and prognostic potential in hepatocellular carcinoma". Oncotarget. 2017 8(4):6406-6418). Lu CY et al. found that the AUCs of four biomarkers used to detect hepatocellular carcinoma were 0.644, 0.758, 0.666, and 0.55, respectively (Lu CY et al. "Cell-free methylation markers with diagnostic and prognostic potential in hepatocellular carcinoma". Oncotarget. 2017 Jan24; 8(4):6406-641). Zhang PJ et al. found that the specificity of THY1 gene methylation for detecting hepatocellular carcinoma was 80.65% (Zhang PJ et al. Methylation profiling of serum DNA from hepatocellular carcinoma patients using an infinium human methylation 450 bead chip". Hepatology International. 2013; 7:893–900.).Han LY et al. found that the sensitivity of TGR5 gene methylation for detecting liver cancer was 48.13% (Han LY et al. Aberrant DNA methylation of g-protein-coupled bile acid receptor gpbar1 (TGR5) is a potential biomarker for hepatitis B virus associated hepatocellular carcinoma. International Journal of Medical Sciences. 2014; 11:164–71). Dong X et al. found that the sensitivity of RASSF1A gene methylation for detecting liver cancer was 64.2% (Dong X et al.).

[0032] Combination of serum RASSF1A methylation and AFP is a promising non-invasive biomarker for HCC patients with chronic HBV infection. DiagnPathol. 2015 Aug 4; 10:133.). Kuo CC et al. found that the sensitivity of IRAK3 and GLOXD1 gene methylation in detecting hepatocellular carcinoma in tissues was 46.9% and 63.8%, respectively (Kuo CC et al. Methylation of IRAK3 is a novel prognostic marker in hepatocellular carcinoma. World J Gastroenterol. 2015 Apr 7; 21(13):3960-9.). Lin SY et al. found that the AUC of 0.908 for detecting hepatocellular carcinoma in ucfDNA using a combination of six markers: GRASP, HOXA9, BMP4, ECE1, GSTP1, and RASSF1A (Lin SY et al. Novel urine cell-free DNA methylation markers for hepatocellular carcinoma. Sci Rep. 2023 Dec 7; 13(1):21585.). Lewin J et al. found that the NGS panel had a sensitivity of 76.7% and a specificity of 64.1% for detecting hepatocellular carcinoma in blood (Lewin J et al. Plasma cell-free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study. BMC Gastroenterol. 2021 Mar 25; 21(1):136.). Kim SC et al. found that the sensitivity of the combination of RNF135 and LDHB in detecting liver cancer was 57% (Kim SC et al. A circulating cell-free DNA methylation signature for the detection of hepatocellular carcinoma. Mol Cancer. 2023 Oct 6; 22(1):164.).When existing biomarkers are used for liver cancer detection, the following problems exist: (1) the detection reagents have too many target genes and are mostly combinations of biomarkers; (2) they lack specific characteristics of tissues and tumors; and (3) the detection sensitivity and specificity are not high.

[0033] This invention proposes a convenient and accurate method to distinguish between liver cancer patients and non-liver cancer patients by detecting the methylation level of a single gene. The detection method for this gene is expected to be transformed into a gene detection kit and serve the screening, clinical testing, and prognostic monitoring of liver cancer. Attached Figure Description

[0034] Figure 1 The ROC curve for combination 3 in Example 4 distinguishes between high-risk and low-risk primary liver cancer in plasma samples.

[0035] Figure 2 The ROC curves for combining 9 in Example 4 distinguishing between high-risk and low-risk primary liver cancer in plasma samples.

[0036] Figure 3 The ROC curves for combining 12 in Example 4 are used to distinguish between high-risk and low-risk primary liver cancer in plasma samples.

[0037] Figure 4 The ROC curves for combining 19 in Example 4 are used to distinguish between high-risk and low-risk primary liver cancer in plasma samples.

[0038] Figure 5 The ROC curves for combining 23 in Example 4 are used to distinguish between high-risk and low-risk primary liver cancer in plasma samples.

[0039] Figure 6 The ROC curves for combining 24 in Example 4 distinguishing between high-risk and low-risk primary liver cancer in plasma samples.

[0040] Figure 7 The ROC curves for combining 25 in Example 4 distinguishing between high-risk and low-risk primary liver cancer in plasma samples. Detailed Implementation

[0041] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0042] Example 1: Detection region of the OTX1 gene and sequence information of primers and probes

[0043] Through extensive experimentation, the inventors discovered that by detecting the methylation levels at certain sites on the OTX1 gene, liver cancer and benign lesions can be detected with extremely high sensitivity and specificity. The methylation sites on the OTX1 gene are widely distributed. The inventors designed multiple sets of primers and probes targeting regions 1, 2, 3, 4, and 5 of the OTX1 gene. The sequence information of the primers, probes, and regions 1-5 is shown in Table 1, the primer pair combinations are shown in Table 2-1, and the primer-probe combinations are shown in Table 2-2.

[0044] Table 1. Sequence information of primers, probes, and regions 1-5

[0045]

[0046]

[0047]

[0048]

[0049]

[0050] Note: (1) R8-1, R8-2, and R8-3 are R primers, all of which can be combined with F8 to form primer pairs; F9-1, F9-2, and F9-3 are F primers, all of which can be combined with R9 to form primer pairs; F17-1 and F17-2 are F primers, all of which can be combined with R17 to form primer pairs; R18-1, R18-2, and R18-3 are R primers, all of which can be combined with F18 to form primer pairs; R19-1 and R19-2 are R primers, all of which can be combined with F19 to form primer pairs; F21-1, F21- 2. F21-3, F21-4, F21-5, and F21-6 are F primers, and R21-1, R21-2, R21-3, R21-4, R21-5, R21-6, and R21-7 are R primers. Any one of the F primers can be combined with any one of the R primers to form a primer pair; (2) F18-T1, F18-T2, and F18-T3 are sequences in which F18 introduces a mismatch, and the mismatch position is the penultimate base at the 3' end of F18; R18- R18-1-T1, R18-1-T2, and R18-1-T3 are sequences where a mismatch is introduced in R18-1, with the mismatch located at the penultimate base from the 3' end of R18-1; R18-3-T1, R18-3-T2, and R18-3-T3 are sequences where a mismatch is introduced in R18-3, with the mismatch located at the penultimate base from the 3' end of R18-3; F19-T1, F19-T2, and F19-T3 are sequences where a mismatch is introduced in F19, with the mismatch located at the 3' end of F19. The penultimate base; R19-2-T1, R19-2-T2, and R19-2-T3 are sequences where a mismatch is introduced in F19-2, with the mismatch located at the penultimate base at the 3' end of F19-2; F6-T and R6-T are sequences where a mismatch is introduced in F6 and R6, respectively, with the mismatch located at the penultimate base at the 3' end of F6 and R6; F7-T and R7-T are sequences where a mismatch is introduced in F7 and R7, respectively, with the mismatch located at the penultimate base at the 3' end of F7 and R7.

[0051] Table 2-1 Primer Pair Combinations

[0052] Primer pair numbering primer pairs Remark Primer pair numbering primer pairs Remark Primer pair 1 F1+R1 Area 1 Primer pair 27 F20+R20 Area 5 Primer pair 2 F2+R2 Area 2 Primer pair 28 F21-1+R21-1 Area 5 Primer pair 3 F3+R3 Area 3 Primer pair 29 F21-1+R21-2 Area 5 Primer pair 4 F4+R4 Area 3 Primer pair 30 F21-2+R21-1 Area 5 Primer pair 5 F5+R5 Area 4 Primer pair 31 F21-2+R21-2 Area 5 Primer pair 6 F6+R6 Area 5 Primer pair 32 F21-3+R21-1 Area 5 Primer pair 7 F8+R8-1 Area 5 Primer pair 33 F21-3+R21-2 Area 5 Primer pair 8 F8+R8-2 Area 5 Primer pair 34 F6+R6-T Area 5 Primer pair 9 F8+R8-3 Area 5 Primer pair 35 F6-T+R6 Area 5 Primer pair 10 F9-1+R9 Area 5 Primer pair 36 F6-T+R6-T Area 5 Primer pair 11 F9-2+R9 Area 5 Primer pair 37 F7+R7 Area 5 Primer pair 12 F9-3+R9 Area 5 Primer pair 38 F7+R7-T Area 5 Primer pair 13 F10+R10 Area 5 Primer pair 39 F7-T+R7 Area 5 Primer pair 14 F11+R11 Area 5 Primer pair 40 F7-T+R7-T Area 5 Primer pair 15 F12+R12 Area 5 Primer pair 41 F18+R18-1-T1 Area 5 Primer pair 16 F13+R13 Area 5 Primer pair 42 F18+R18-1-T3 Area 5 Primer pair 17 F14+R14 Area 5 Primer pair 43 F18-T3+R18-1-T1 Area 5 Primer pair 18 F15+R15 Area 5 Primer pair 44 F18-T3+R18-1-T2 Area 5 Primer pair 19 F16+R16 Area 5 Primer pair 45 F18-T3+R18-1-T3 Area 5 Primer pair 20 F17-1+R17 Area 5 Primer pair 46 F18+R18-3-T3 Area 5 Primer pair 21 F17-2+R17 Area 5 Primer pair 47 F18-T3+R18-3 Area 5 Primer pair 22 F18+R18-1 Area 5 Primer pair 48 F18-T3+R18-3-T3 Area 5 Primer pair 23 F18+R18-2 Area 5 Primer pair 49 F19+R19-2-T1 Area 5 Primer pair 24 F18+R18-3 Area 5 Primer pairs 50 F19-T1+R19-2 Area 5 Primer pair 25 F19+R19-1 Area 5 Primer pair 51 F19-T1+R19-2-T1 Area 5 Primer pair 26 F19+R19-2 Area 5 Primer pair 52 F9-1+R9 Area 5 / / / Primer pair 53 F16+R16 Area 5 / / / Primer pair 54 F21-1+R21-1 Area 5

[0053] Table 2-2 Primer-Probe Combinations

[0054]

[0055]

[0056] Example 2: Screening of primer pairs and primer-probe combinations

[0057] The sample information comes from standard CpGenome Universal Methylated DNA, HuH-7 (human liver cancer cell) DNA, and leukocyte DNA. The specific detection steps are as follows:

[0058] 1. Bisulfite conversion:

[0059] DNA was converted to bisulfite using the EZ DNA Methylation Kit (Zymo Research, ZRC008984).

[0060] 2. Quantitative methylation specific PCR (q-MSP):

[0061] qMSP amplification system: 18.1 μl nuclease-free water, 6 μl 5× buffer, 1 μl dNTPs (10 mM), 0.5 μl FTaq HotStart polymerase, 0.9 μl upstream primer (10 μM), 0.9 μl downstream primer (10 μM), 0.6 μl probe (10 μM), 2 μl template DNA, total volume of qMSP amplification system: 30 μl. Reaction program: 95℃ for 5 min, (95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s) × 45 Cycles, 40℃ for 30 s.

[0062] 3. Results Analysis:

[0063] The PCR amplification results of primer pairs 1-33 are shown in Tables 3-1 and 3-2. The PCR amplification results of primer selection are shown in Table 3-3. The PCR amplification results of combination 1-22 are shown in Table 3-4.

[0064] Table 3-1 Screening results of primer pairs 1-6

[0065]

[0066] Note: In CpGenome Universal Methylated DNA, a smaller Ct value indicates higher amplification efficiency; in HuH-7 (human liver cancer cell) DNA, a smaller Ct value indicates a higher methylation rate; in leukocyte DNA, a larger amplified Ct value or no amplification indicates good specificity and low background. A larger ΔCt leukocyte-Huh-7 cell value or a ΔCt leukocyte-Huh-7 cell value showing "none" indicates better differentiation between HuH-7 cells and leukocytes.

[0067] The results showed that methylation amplification primer pairs designed for different regions of the OTX1 gene (such as region 1, region 2, region 3, region 4, and region 5) exhibited good amplification efficiency. However, primer pairs 1, 2, 3, 4, and 5 had low ΔCt values ​​for leukocyte-Huh-7 cells, failing to effectively distinguish between HuH-7 cells and leukocyte DNA. Primer pair 6, on the other hand, showed no amplification in leukocyte DNA and effectively distinguished between HuH-7 cells and leukocytes. This indicates that there are significant differences in methylation and specificity among different regions of the OTX1 gene.

[0068] To screen for more suitable primer pairs, the inventors designed multiple primer pairs in region 5 (such as primer pair 7-33 shown in Table 2-1) for quantitative methylation-specific PCR. Based on the amplification curves and Ct values ​​(as shown in Table 3-2), it was found that different primer pairs designed in region 5 had different amplification effects.

[0069] Table 3-2 Screening results of primer pair 7-33

[0070]

[0071]

[0072] To further investigate the impact of individual base differences in primers on amplification efficiency, the inventors conducted experiments on primer pairs 22, 24, and 26. The specific procedures were as follows:

[0073] (1) Introduce a mismatch in primer pair 22(F18+R18-1) at the penultimate base of the 3' end of primer F and / or primer R to obtain primer pairs numbered 22-1 to 22-15.

[0074] (2) Introduce a mismatch in primer pair 24(F18+R18-3) at the penultimate base of the 3' end of primer F and / or primer R to obtain primer pairs numbered 24-1 to 24-15.

[0075] (3) Introduce a mismatch in primer pair 26(F19+R19-2) at the penultimate base of the 3' end of primer F and / or primer R to obtain primer pairs numbered 26-1 to 26-15.

[0076] Quantitative methylation-specific PCR was performed on the above primer pairs, and the results are shown in Table 3-3.

[0077] Table 3-3 Selection of primer pair 22, primer pair 24, and primer pair 26

[0078]

[0079]

[0080] The results showed that introducing a mismatch site into primer pair 22 significantly improved the specificity of most primer pairs. Introducing a mismatch site into primer pair 24 also significantly improved the specificity of most primer pairs. Introducing a mismatch site into primer pair 26 affected amplification efficiency to varying degrees; for example, primer pairs 26-1, 26-5, 26-6, 26-9, 26-10, and 26-11 showed no amplification. This indicates that introducing a mismatch site into a primer pair, such as creating a difference of a few bases from the original primer pair, has an unpredictable impact on amplification performance; it may improve primer pair specificity or result in no amplification.

[0081] In summary, based on the amplification curves, Ct values, and ΔCt values, suitable primer pairs were screened. Simultaneously, probes were designed for further validation in cellular DNA, tissue DNA, and plasma DNA.

[0082] Table 3-4 PCR amplification results of primer-probe combinations 1-25 in CpGenome Universal Methylated DNA and leukocyte DNA

[0083]

[0084]

[0085] In CpGenome Universal Methylated DNA, a smaller Ct value indicates higher amplification efficiency. In leukocyte DNA, a larger amplification Ct value or no amplification indicates good specificity and low background. A larger or no ΔCt value indicates greater variability, good specificity, and low background. Based on the amplification curves and Ct and ΔCt values, combinations 3, 9, 10, 11, 12, 19, 23, 24, and 25 were selected as having relatively high specificity and relatively good amplification efficiency.

[0086] Example 3: Detection of OTX1 gene primers and probes in tissue samples

[0087] The tissue specificity of hepatocellular carcinoma was detected using primer-probe combinations 3, 9, 10, 11, 12, and 19. The tissue samples included 16 independent hepatocellular carcinoma tissues (numbered AP) and 14 benign liver lesion tissues (numbered 1-14), all derived from hospital samples. The specific testing steps are as follows:

[0088] 1. DNA extraction from tissue samples:

[0089] DNA was extracted from paraffin-embedded tissue samples using the HiPure FFPEDNAKit (Medex, D3126-03).

[0090] 2. Bisulfite conversion:

[0091] 1000 ng of tissue DNA was taken and subjected to bisulfite conversion using the EZDNAMethylation Kit (Zymo Research, ZRC008984).

[0092] 3. Quantitative methylation-specific PCR (q-MSP):

[0093] The methylation of candidate genes in tissue DNA was detected using q-MSP, or methylation-specific quantitative PCR. Primers and probes used were combinations 3, 9, 10, 11, 12, and 19 from Table 2 of Example 1. ACTB gene amplification primers (SEQ ID NO: 86, SEQ ID NO: 87) and probe (SEQ ID NO: 88) were also designed to assess sample DNA quality. The qMSP amplification system consisted of: 18.1 μl nuclease-free water, 6 μl 5X buffer, 1 μl dNTPs (10 mM), 0.5 μl FTaq Hot Start polymerase, 0.9 μl upstream primer (10 μM), 0.9 μl downstream primer (10 μM), 0.6 μl probe (10 μM), and 2 μl template DNA. The total volume of the qMSP amplification system was 30 μl. Reaction program: 95℃ for 5 min, (95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s) × 45 Cycles, 40℃ for 30 s.

[0094] 4. Results Analysis:

[0095] The Ct value threshold for the target gene was set to 37. When the Ct value of the target gene was ≤37, the sample was considered to have methylated target gene. When the Ct value of the target gene was >37, the sample was considered to have very low or no methylation of the target gene. The specific detection data of combinations 3, 9, 10, 11, 12, and 19 in 16 independent liver cancer tissue samples and 14 benign liver lesion tissue samples are shown in Table 4, and the statistical results are shown in Table 5.

[0096] Table 4. Specific detection data of different primer-probe combinations in tissue samples.

[0097]

[0098]

[0099] Note: In Table 4, "+" indicates a positive sample and "-" indicates a negative sample.

[0100] The detection performance of combinations 3, 9, 10, 11, 12 and 19 is shown in Table 5 below.

[0101] Table 5. Statistical results of detection performance of different primer-probe combinations in tissue samples.

[0102]

[0103] The sensitivity and specificity of primer-probe combination 3 were 93.75% and 85.71%, respectively; the sensitivity and specificity of primer-probe combination 10 were 93.75% and 71.43%, respectively; the sensitivity and specificity of primer-probe combination 12 were 93.75% and 57.14%, respectively; and the sensitivity and specificity of primer-probe combination 19 were 87.50% and 92.86%, respectively. Although the detection sensitivity of primer-probe combinations 9 and 11 reached 100%, their specificity was much lower than that of combinations 3, 9, 10, 11, 12, and 19. Therefore, combinations 3, 12, and 19 were selected as the better combinations based on comprehensive screening.

[0104] Example 4: Detection of OTX1 gene primers and probes in plasma samples

[0105] Primer and probe combinations 3, 9, 12, 19, 23, 24, and 25 were used to detect the virus in 142 plasma samples (102 cases of primary liver cancer, 11 cases of cirrhosis, 15 cases of benign liver disease, and 14 cases of fatty liver). All plasma samples were obtained from hospital samples. The specific testing steps are as follows:

[0106] 1. Extraction of cfDNA from plasma samples:

[0107] Take approximately 2 mL of plasma sample and extract cfDNA using the MagPure Circulating DNA Kit (Meg, 12917PC-100).

[0108] 2. Bisulfite conversion:

[0109] All cfDNA extracted from plasma samples underwent bisulfite treatment. The EZ DNA Methylation Kit (Zymo Research, ZRC008984) was used for bisulfite conversion.

[0110] 3. Quantitative methylation specific PCR (q-MSP):

[0111] The methylation of candidate genes in plasma DNA was detected using q-MSP, or methylation-specific quantitative PCR. Primers and probes used were combinations 3, 9, 12, 19, 23, 24, and 25 from Table 3 of Example 1. ACTB gene amplification primers (SEQ ID NO: 86, SEQ ID NO: 87) and probe (SEQ ID NO: 88) were also designed to assess sample DNA quality.

[0112] qMSP amplification system: 8.7 μl nuclease-free water, 6 μl 5× Buffer, 5 μl MgCl2 (25 mM), 1 μl dNTPs (10 mM), 0.5 μl ZTaq HotStart polymerase, 0.9 μl each upstream primer (10 μM), 0.9 μl each downstream primer (10 μM), 0.6 μl each probe (10 μM), 4 μl template DNA, total volume of qMSP amplification system is 30 μl.

[0113] The reaction conditions for qMSP detection were: 95℃ for 5 min, (95℃ for 15 s, 58℃ for 30 s, 72℃ for 30 s) × 45 Cycles, and 40℃ for 30 s.

[0114] 4. Results Analysis: When ΔCt ≤ 6.660 for combination 3, it is considered positive (+); when ΔCt > 6.660 for combination 3, it is considered negative (-); when ΔCt ≤ 7.530 for combination 9, it is considered positive (+); when ΔCt > 7.530 for combination 9, it is considered negative (-); when ΔCt ≤ 6.880 for combination 12, it is considered positive (+); when ΔCt > 6.880 for combination 12, it is considered negative (-); when ΔCt ≤ 8.105 for combination 19, it is considered positive (+); when ΔCt > 8.105 for combination 19, it is considered negative (-); when ΔCt ≤ 5.405 for combination 23, it is considered positive (+); when ΔCt > 5.405 for combination 23, it is considered negative (-); when ΔCt ≤ 6.660 for combination 24, it is considered positive (+); when ΔCt > ...

[0115] A value of 6.160 is considered positive (+), and a ΔCt > 6.160 for combination 24 is considered negative (-); a ΔCt ≤ 6.495 for combination 24 is considered positive (+), and a ΔCt > 6.495 for combination 25 is considered negative (-); where positive indicates a high risk of primary liver cancer, and negative indicates a low risk of primary liver cancer.

[0116] The specific test data of 142 plasma samples are shown in Table 6, and the statistical results are shown in Table 7. The ROC curves of combinations 3, 9, 12, 19, 23, 24, and 25 are shown in Table 7. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 .

[0117] Table 6 shows the specific detection data of combinations 3, 9, 12, 19, 23, 24, and 25 in 142 plasma samples.

[0118]

[0119]

[0120]

[0121]

[0122] Note: "+" indicates a positive test result; "-" indicates a negative test result; "Benign - Eight Categories" indicates that the sample is not a liver cancer sample, but has one or more of the following eight diseases: gallstones, cholecystitis, bile duct stones, cholangitis, gallbladder polyps, liver abscess, liver cysts, or liver hemangioma; "Benign - Cirrhosis" indicates that the sample is not a liver cancer sample, but has cirrhosis; "Benign - Fatty Liver" indicates that the sample is not a liver cancer sample, but has fatty liver.

[0123] Table 7 shows the statistical results of combinations 3, 9, 12, 19, 23, 24, and 25 in 142 plasma samples.

[0124] Primer-probe combination AUC 95% confidence interval cutoff Sensitivity Specificity Combination 3 0.8598 0.8011-0.9186 ΔCt≤6.660 76.47％(78 / 102) 90％(4 / 40) Combination 9 0.8474 0.7861-0.9087 ΔCt≤7.530 71.57％(73 / 102) 90％(4 / 40) Combination 12 0.8810 0.8254-0.9366 ΔCt≤6.880 81.37％(83 / 102) 90％(4 / 40) Combination 19 0.8616 0.8037-0.9196 ΔCt≤8.105 73.53％(75 / 102) 90％(4 / 40) Combination 23 0.8890 0.8373-0.9407 ΔCt≤5.405 76.47％(78 / 102) 90％(4 / 40) Combination 24 0.8967 0.8458-0.9475 ΔCt≤6.160 82.35％(84 / 102) 90％(4 / 40) Combination 25 0.8816 0.8285 to 0.9348 ΔCt≤6.495 75.49％(77 / 102) 90％(4 / 40)

[0125] When the specificity of each primer-probe combination is 90%, the sensitivity of primer-probe combination 24 for detecting primary liver cancer is 82.35%, primer-probe combination 12 is 81.37%, primer-probe combination 23 is 76.47%, primer-probe combination 3 is 76.47%, primer-probe combination 25 is 75.49%, primer-probe combination 19 is 73.53%, and primer-probe combination 9 is 71.57%.

[0126] Example 5: Detection of OTX1 gene primers and probes combined with AFP protein in plasma samples.

[0127] The detection data of primer-probe combinations 3, 9, 12, 19, 23, 24, and 25 in 142 plasma samples (102 cases of primary liver cancer, 11 cases of cirrhosis, 15 cases of benign liver disease, and 14 cases of fatty liver) were analyzed in conjunction with AFP detection data. The detection data of primer-probe combinations 3, 9, 12, 19, 23, 24, and 25 in plasma samples are shown in Example 4. The AFP protein concentrations of these 142 plasma samples were obtained from the corresponding hospital case reports.

[0128] The combined statistical analysis of methylation and AFP results is shown in Table 8.

[0129] Table 8. Statistical results of different combinations of AFP in 142 plasma samples.

[0130]

[0131]

[0132] Note: AFP > 100 ng / mL is considered positive; ΔCt ≤ 6.660 for combination 3 is considered positive (+), and ΔCt > 6.660 for combination 3 is considered negative (-); ΔCt ≤ 7.530 for combination 9 is considered positive (+), and ΔCt > 7.530 for combination 9 is considered negative (-); ΔCt ≤ 6.880 for combination 12 is considered positive (+), and ΔCt > 6.880 for combination 12 is considered negative (-); ΔCt ≤ 8.105 for combination 19 is considered positive (+), and ΔCt > 8.105 for combination 19 is considered negative (-); ΔCt ≤ 5.405 for combination 23 is considered positive (+), and ΔCt > 5.405 for combination 23 is considered negative (-); ΔCt ≤ A value of 6.160 is considered positive (+), and a ΔCt > 6.160 for combination 24 is considered negative (-); a ΔCt ≤ 6.495 for combination 24 is considered positive (+), and a ΔCt > 6.495 for combination 25 is considered negative (-); a positive result indicates a high risk of liver cancer, and a negative result indicates a low risk of liver cancer.

[0133] The sensitivity and specificity of combination 24 combined with AFP for detecting primary liver cancer were 91.18% and 90.00%, respectively; the sensitivity and specificity of combination 23 combined with AFP for detecting primary liver cancer were 89.22% and 90.00%, respectively; the sensitivity and specificity of combination 12 combined with AFP for detecting primary liver cancer were 88.24% and 90.00%, respectively; the sensitivity and specificity of combination 19 combined with AFP for detecting primary liver cancer were 87.25% and 90.00%, respectively; the sensitivity and specificity of combination 3 combined with AFP for detecting primary liver cancer were 87.25% and 90.00%, respectively; and the sensitivity and specificity of combination 25 combined with AFP for detecting primary liver cancer were 85.29%, respectively.

[0134] The sensitivity and specificity of combination 9 in detecting primary liver cancer with AFP were 84.31% and 90.00%, respectively. The results indicate that the primers and probes provided by this invention can be effectively applied to the diagnosis of liver cancer with high detection accuracy. Moreover, when combined with conventional non-invasive screening methods in the field (such as AFP detection), they can achieve even better detection accuracy, which is of great significance in the clinical detection and diagnosis of liver cancer.

Claims

1. The application of a nucleic acid fragment methylation detection reagent in the preparation of liver cancer detection reagents or kits, wherein the nucleic acid fragment is selected from the nucleic acid fragment shown in SEQ ID NO:

95.

2. A primer pair, characterized in that, The primer pairs are selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 15 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 20 and SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 3 ... SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 39, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 40 and SEQ ID NO: 42, SEQ ID NO: 40 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 44 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 55, SEQ ID NO: 49 and SEQ ID NO: 56, SEQ ID NO: 50 and SEQ ID NO: 55, SEQ ID NO: 50 and SEQ ID NO: 56, SEQ ID NO: 51 and SEQ ID NO: 55, SEQ ID NO: 51 and SEQ ID NO: 56, SEQ ID NO: 11 and SEQ ID NO: 78, SEQ ID NO: 77 and SEQ ID NO: 12, SEQ ID SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 80,Any one of the primer pairs shown in SEQ ID NO: 79 and SEQ ID NO: 14, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 40 and SEQ ID NO: 67, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 66, SEQ ID NO: 64 and SEQ ID NO: 67, SEQ ID NO: 40 and SEQ ID NO: 70, SEQ ID NO: 64 and SEQ ID NO: 43, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 44 and SEQ ID NO: 74, SEQ ID NO: 71 and SEQ ID NO: 46, SEQ ID NO: 71 and SEQ ID NO: 74, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 49 and SEQ ID NO: 55; Preferably, the primer pairs are selected from SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 11 and SEQ ID NO: 78, SEQ ID NO: 77 and SEQ ID NO: 12, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 13 and SEQ ID NO: 80, SEQ ID NO: 79 and SEQ ID NO: 14, SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 40 and SEQ ID NO: 67, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 66, SEQ ID NO: 64 and SEQ ID NO: 67, SEQ ID NO: 40 and SEQ ID NO: 70, SEQ ID NO: 64 and SEQ ID NO: 43, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 44 and SEQ ID NO:

70. Any one of the primer pairs shown in NO: 74, SEQ ID NO: 71 and SEQ ID NO: 46, SEQ ID NO: 71 and SEQ ID NO: 74, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 49 and SEQ ID NO: 55; Preferably, the primer pair is selected from any one of the primer pairs shown in SEQ ID NO: 77 and SEQ ID NO: 12, SEQ ID NO: 40 and SEQ ID NO: 65, SEQ ID NO: 64 and SEQ ID NO: 70, SEQ ID NO: 19 and SEQ ID NO: 22, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 49 and SEQ ID NO:

55.

3. A nucleic acid probe, characterized in that, The probe is selected from any one of the probes shown in SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO:

99.

4. The application of the primer pair as described in claim 2, and / or the probe as described in claim 3, in the preparation of methylation detection reagents, or in the preparation of auxiliary diagnostic reagents for liver cancer.

5. The use of the primer pair as described in claim 2, the probe as described in claim 3, and / or the AFP detection reagent in the preparation of liver cancer detection reagents.

6. A methylation detection reagent, characterized in that, Includes the primer pair as described in claim 2, and / or the probe as described in claim 3.

7. A kit comprising the primer pair as described in claim 2, or the probe as described in claim 3, or the methylation detection reagent as described in claim 6; Preferably, the kit further includes an AFP detection reagent.

8. The use of the primer pair of claim 2, the probe of claim 3, the methylation detection reagent of claim 6, or the kit of claim 7 in the preparation of a liver cancer detection kit.

9. The methylation detection reagent as described in claim 6, or the kit as described in claim 7, characterized in that, The test samples for the aforementioned test reagents are selected from blood, plasma, serum, saliva, tissue, or urine; Preferably, the test sample for the test reagent is selected from plasma or tissue.

10. A liver cancer detection system, characterized in that, The system includes: (1) A methylation detection component for a nucleic acid fragment as shown in SEQ ID NO: 95; (2) Result judgment component; The methylation detection component comprises the primer pair as described in claim 2, the probe as described in claim 3, the methylation detection reagent as described in claim 6, or the kit as described in claim 7; Preferably, the result determination component is used to output the risk of liver cancer and / or the type of liver cancer based on the methylation result of the nucleic acid fragment as shown in SEQ ID NO: 95 detected by the detection component; Preferably, the disease risk is determined based on a comparison between the methylation results of the test sample and the methylation results of the normal sample; when the comparison between the methylation results of the test sample and the methylation results of the normal sample shows a significant or highly significant difference, the test sample is judged to have a high disease risk.

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