Multi-gene methylation detection kit and detection system for liver cancer and application of multi-gene methylation detection kit and detection system

Abstract

The invention relates to a polygene methylation detection kit for liver cancer, a detection system and application thereof, and aims to provide a marker combination for early liver cancer screening. The kit is used for detecting the methylation state of one or more of 114 genes of BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, and is combined with restriction enzyme digestion and fluorescent quantitative PCR (Polymerase Chain Reaction) technologies, so that the early diagnosis of the liver cancer is realized. The kit and the system provided by the invention have high accuracy, specificity and early diagnosis capability, and experiments prove that the detection method can effectively distinguish liver cancer tissues from para-carcinoma tissues, and has relatively high clinical application value. The method is not only suitable for early screening of liver cancer, but also can provide a scientific basis for early treatment of liver cancer patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a marker combination for early cancer screening, as well as the use of the marker combination for detecting cancer and the preparation of cancer diagnostic reagents. Background Art

[0002] Liver cancer is a common digestive system malignancy worldwide. According to GLOBOCAN2020 data, there are 906,000 new cases and 830,000 deaths worldwide each year. Liver cancer has an insidious onset, and most patients are diagnosed in the late stage. The development of liver cancer usually goes through a multi-step process from chronic hepatitis to cirrhosis, to atypical hyperplastic nodules, and finally to liver cancer. Early detection is crucial to improving treatment outcomes and reducing mortality, but currently 80% of liver cancer patients are already in the middle and late stages when they are discovered.

[0003] At present, the methods used in clinical practice for liver cancer detection have their own advantages and disadvantages: Although AFP is a commonly used serum marker for diagnosing liver cancer, it may be negative in about 30% of liver cancer patients, and it may also be elevated in patients with chronic hepatitis and cirrhosis, resulting in a high false negative rate for early liver cancer detection. Therefore, it is not an ideal indicator for early screening. Ultrasound (US) is a widely used liver cancer screening method. Although the diagnostic sensitivity for liver cancer at all stages can reach 84%, its sensitivity and specificity for early liver cancer are low and are affected by operator experience and patient obesity. Magnetic resonance imaging (MRI) is suitable for screening high-risk groups and early liver cancer diagnosis, but it is expensive and not suitable for large-scale screening. Histopathological biopsy, as the "gold standard" for tumor diagnosis, is also not suitable as a method for large-scale liver cancer screening due to its invasiveness, complexity and limitations on small tumors. Therefore, the medical community has been seeking more effective methods for early diagnosis of liver cancer.

[0004] In recent years, studies have shown that DNA methylation is closely related to the occurrence and development of liver cancer, and can also be used as an effective molecular marker for liver cancer screening. The combination of methylated DNA markers has a high accuracy in detecting HCC, including early lesions, which is better than AFP, and has high specificity in people with cirrhosis without HCC and healthy controls without known liver cancer.

[0005] Gene methylation is an important part of genetic epigenetics, affecting the regulation of gene expression without changing the DNA sequence. DNA methylation refers to the process of methylating the 5-carbon atom of the cytosine ring to form 5-methylcytosine under the action of DNA methyltransferases (DNMTs), which generally occurs at the CpG island position in the promoter region. In normal people, the CpG sites in the CpG island are generally in an unmethylated state. When the CpG sequence in the CpG island, especially the CpG sequence in the CpG island of the tumor suppressor gene, is highly methylated, it can cause the silencing and expression loss of the corresponding gene, thereby leading to the inactivation of the tumor suppressor gene and causing tumor growth.

[0006] The restriction endonuclease is sensitive to CpG methylation. In the reaction process, when the cytosine in the restriction endonuclease site is not methylated, the restriction endonuclease acts and the sequence will be cut off; when the cytosine in the restriction endonuclease site is methylated, the restriction endonuclease does not act and the sequence will not be cut off because it is sensitive to CpG methylation. The present invention uses three restriction endonucleases, Hin6I, HpaII and Bsh1236I, and the restriction endonucleases are GCGC, CCGG and CGCG, respectively.

[0007] Genes such as BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1 play important roles in the human body. They are involved in a series of biological processes from cell differentiation, proliferation to apoptosis, involving tumor development, immune response, cell adhesion, signal transduction, cytoskeleton maintenance and many other aspects.

[0008] The present invention discovered a genome associated with liver cancer through database analysis and multi-analysis verification. Selecting one or several genes among them can achieve early and accurate prediction of liver cancer, lay the foundation for early treatment, and has a high application prospect. Summary of the invention

[0009] The purpose of the present invention is to provide a multi-gene methylation detection kit and a detection system for liver cancer, and applications of the detection system and the kit.

[0010] The first aspect of the present invention provides a liver cancer diagnosis kit, which comprises a reagent for detecting the methylation degree of one or more of the target genes BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1.

[0011] Furthermore, in the kit, the reagents for detecting the methylation degree of one or more of the target genes BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1 are selected from one or more of the following restriction primer groups:

[0012] BDH1 forward primer: 5'-CTGTTCCCGGGGTTTGGG-3',

[0013] BDH1 reverse primer: 5′-CCAGCCTGTCCCCGCGGC-3′;

[0014] CPAMD8 forward primer: 5'-CCACCAGCGAACAGCTG-3',

[0015] CPAMD8 reverse primer: 5′-AGGGATCCTTGACCCGTATGC-3′;

[0016] DAB2IP forward primer: 5'-TGGGGCCTCCAGCCTGCG-3',

[0017] DAB2IP reverse primer: 5′-GGAGGAGAAACAGGCCCCG-3′;

[0018] DLEC1 forward primer: 5'-CTGGGATGTGAGGAGCGCCTCT-3',

[0019] DLEC1 reverse primer: 5′-GGCGGCTGCCGGGCGGAGGGG-3′;

[0020] HOXA1 forward primer: 5'-ACCTGGGTCAACCCAACGCG-3',

[0021] HOXA1 reverse primer: 5′-CCCTGCGGGCGCGCGTCAGGTA-3′;

[0022] MIXL1 forward primer: 5'-CTCAAAGTGCTGGGATTA-3',

[0023] MIXL1 reverse primer: 5′-AGGCTTCTCCTTACCAACT-3′;

[0024] OTX1 forward primer: 5'-CTGGGCAGAGAGAAGGCCGG-3',

[0025] OTX1 reverse primer: 5′-GGGCGACCCCAAGATGCGGCC-3′;

[0026] PCDH8 forward primer: 5'-GCTGGACGTGCGGGCGCAGGA-3',

[0027] PCDH8 reverse primer: 5′-TTGTCATTGACGTCTCGGATGC-3′;

[0028] RNF135 forward primer: 5'-TAGGGGCGGCCGGGCAGAGG-3',

[0029] RNF135 reverse primer: 5′-CCCCTCTGCCCGGCCAGC-3′;

[0030] SIX2 forward primer: 5'-TTCAAGAACCGGCGGCAGCG-3',

[0031] SIX2 reverse primer: 5′-TTGGGCCCCGGGGAGCTGGCGCC-3′;

[0032] SPINT2 forward primer: 5'-AAAATTAGCCGGGCCTGGTGG-3',

[0033] SPINT2 reverse primer: 5′-CTGGCGCGATCTCAGCTCACT-3′;

[0034] TACSTD2 forward primer: 5'-CTCATTGACCTGCGCCACC-3',

[0035] TACSTD2 reverse primer: 5′-AGCCGATAGCGCTCGCGGAAGAG-3′;

[0036] VIM forward primer: 5'-CACCGTGATTGCCCCTCTGCG-3',

[0037] VIM reverse primer: 5′-GAGCAGCTGCGCCACCTC-3′;

[0038] ZAR1 forward primer: 5'-CTGTAAACCTCGCGCAGT-3',

[0039] ZAR1 reverse primer: 5′-ACTCTCCAGCCGGCAAGTGC-3′.

[0040] Furthermore, in the kit, the target genes detected are combination 1 consisting of genes BDH1, DAB2IP and OTX1, and / or combination 2 consisting of genes DLEC1, HOXA1, RNF135, SIX2 and SPINT2.

[0041] Furthermore, in the kit, the target gene detected is a combination of combination 1 and / or combination 2 and one or more of the genes CPAMD8, TACSTD2, VIM and ZAR1.

[0042] Furthermore, the kit also includes reagents for detecting the restriction quality control gene GAPDH_ED and / or the internal reference quality control gene GAPDH_PCR;

[0043] Preferably, the reagent for detecting the restriction endonuclease quality control gene GAPDH_ED and / or the internal reference quality control gene GAPDH_PCR is selected from the following restriction endonuclease primer groups:

[0044] GAPDH_ED forward primer: 5'-CTTTCTTTCCTTTCGCGCTCT-3',

[0045] GAPDH_ED reverse primer: 5'-CTACCGCAGGCGCAGCATCCGGA-3'; and / or

[0046] GAPDH_PCR forward primer: 5'-TTGGCAAATCAAAGCCCTG-3',

[0047] GAPDH_PCR reverse primer: 5′-CCCACCCCTTCTCTAAGTCCC-3′.

[0048] Furthermore, the kit also includes restriction endonuclease Hin6I, restriction endonuclease HpaII, restriction endonuclease Bsh1236I, 10XFastDigestBuffer, primer mixture, PowerSYBRGreenPCRMasterMix and nuclease-free water.

[0049] The second aspect of the present invention provides a device for diagnosing liver cancer, the device comprising 1) a sample processing module, 2) a target cause detection module, and 3) a determination module.

[0050] The 1) sample processing module comprises reagents for extracting genomic DNA of the liver tissue to be tested;

[0051] The 2) target gene detection module comprises a restriction endonuclease reaction system and the above-mentioned kit; the restriction endonuclease reaction system comprises: restriction endonuclease Hin6I, restriction endonuclease HpaII, restriction endonuclease Bsh1236I and reagents required for the restriction reaction;

[0052] The 3) determination module includes a step of calculating the fluorescence signal Ct value of each target gene generated by the 2) target gene detection module to obtain a relative fluorescence Ct value (dCt value), and the target gene relative fluorescence Ct value (dCt) is less than or equal to 6, and it is determined to be positive, and the relative fluorescence Ct value (dCt) of the enzyme digestion quality control gene GAPDH_ED is greater than or equal to 6, and the enzyme digestion reaction quality control is determined to be qualified;

[0053] The dCt value calculation formula is: dCt value (target gene) = Ct value (target gene) - Ct value (internal reference gene GAPDH_PCR).

[0054] Preferably, the target gene detection module 2) comprises a) a step of performing an enzyme cleavage reaction using the enzyme cleavage reaction system, and b) a step of performing a fluorescent quantitative PCR amplification reaction using the above-mentioned kit;

[0055] The enzymatic reaction procedure of the step a) of using the enzymatic reaction system to perform an enzymatic reaction is: 37° C. for 10 min, then 80° C. for 10 min;

[0056] The amplification reaction procedure of the step b) using the above kit to perform fluorescent quantitative PCR amplification reaction is: pre-denaturation at 95° C. for 10 min; denaturation at 95° C. for 15 sec, annealing and extension at 60° C. for 30 sec, for a total of 50 cycles.

[0057] The third aspect of the present invention provides the use of the kit and the device for diagnosing liver cancer in preparing a liver cancer diagnosis product.

[0058] A fourth aspect of the present invention provides an application of a detection reagent in the preparation of a liver cancer diagnostic reagent, wherein the detection reagent is a reagent for detecting one or more genes of BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1.

[0059] Furthermore, the detection reagent also includes a reagent for detecting an internal reference gene.

[0060] Furthermore, the reagent is selected from primers and probes.

[0061] Further, the primers provided by the present invention are detection primers for detecting 14 target genes including liver cancer-related genes BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, as well as restriction endonuclease quality control gene GAPDH_ED and internal reference quality control gene GAPDH_PCR, and the detection primer sequences are as follows:

[0062] BDH1 forward primer: 5'-CTGTTCCCGGGGTTTGGG-3'

[0063] BDH1 reverse primer: 5'-CCAGCCTGTCCCCGCGGC-3'

[0064] CPAMD8 forward primer: 5'-CCACCAGCGAACAGCTG-3'

[0065] CPAMD8 reverse primer: 5'-AGGGATCCTTGACCCGTATGC-3'

[0066] DAB2IP forward primer: 5'-TGGGGCCTCCAGCCTGCG-3'

[0067] DAB2IP reverse primer: 5'-GGAGGAGAAACAGGCCCCG-3'

[0068] DLEC1 forward primer: 5'-CTGGGATGTGAGGAGCGCCTCT-3'

[0069] DLEC1 reverse primer: 5'-GGCGGCTGCCGGGCGGAGGGG-3'

[0070] HOXA1 forward primer: 5'-ACCTGGGTCAACCCAACGCG-3'

[0071] HOXA1 reverse primer: 5'-CCCTGCGGGCGCGCGTCAGGTA-3'

[0072] MIXL1 forward primer: 5'-CTCAAAGTGCTGGGATTA-3'

[0073] MIXL1 reverse primer: 5'-AGGCTTCTCCTTACCAACT-3'

[0074] OTX1 forward primer: 5'-CTGGGCAGAGAGAAGGCCGG-3'

[0075] OTX1 reverse primer: 5'-GGGCGACCCCAAGATGCGGCC-3'

[0076] PCDH8 forward primer: 5'-GCTGGACGTGCGGGCGCAGGA-3'

[0077] PCDH8 reverse primer: 5'-TTGTCATTGACGTCTCGGATGC-3'

[0078] RNF135 forward primer: 5'-TAGGGGCGGCCGGGCAGAGG-3'

[0079] RNF135 reverse primer: 5'-CCCCTCTGCCCGGCCAGC-3'

[0080] SIX2 forward primer: 5'-TTCAAGAACCGGCGGCAGCG-3'

[0081] SIX2 reverse primer: 5'-TTGGGCCCCGGGGAGCTGGCGCC-3'

[0082] SPINT2 forward primer: 5'-AAAATTAGCCGGGCCTGGTGG-3'

[0083] SPINT2 reverse primer: 5'-CTGGCGCGATCTCAGCTCACT-3'

[0084] TACSTD2 forward primer: 5'-CTCATTGACCTGCGCCACC-3'

[0085] TACSTD2 reverse primer: 5'-AGCCGATAGCGCTCGCGGAAGAG-3'

[0086] VIM forward primer: 5'-CACCGTGATTGCCCCTCTGCG-3'

[0087] VIM reverse primer: 5'-GAGCAGCTGCGCCACCTC-3'

[0088] ZAR1 forward primer: 5'-CTGTAAACCTCGCGCAGT-3'

[0089] ZAR1 reverse primer: 5'-ACTCTCCAGCCGGCAAGTGC-3'

[0090] GAPDH_ED forward primer: 5'-CTTTCTTTCCTTTCGCGCTCT-3'

[0091] GAPDH_ED reverse primer: 5'-CTACCGCAGGCGCAGCATCCGGA-3'

[0092] GAPDH_PCR forward primer: 5'-TTGGCAAATCAAAGCCCTG-3'

[0093] GAPDH_PCR reverse primer: 5′-CCCACCCCTTCTCTAAGTCCC-3′.

[0094] A fifth aspect of the present invention provides a method for diagnosing liver cancer in a subject, the method comprising the following steps:

[0095] 1) DNA extracted from patients is digested with restriction endonucleases to distinguish methylated sequences from non-methylated sequences

[0096] 2) Detect the Ct values ​​of one or more of the 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, the restriction enzyme quality control gene GAPDH_ED (containing 3 restriction enzyme sites, used to control whether the restriction enzyme reaction is successful) and the internal reference quality control gene GAPDH_PCR (the sequence does not contain restriction enzyme sites, used for quality control DNA and dCt value calculation) by fluorescent quantitative PCR method;

[0097] 3) Obtain the fluorescence signal Ct value of each gene through step 2), and calculate the relative fluorescence Ct value of the target gene and the internal reference quality control gene GAPDH respectively, and the calculation formula is dCt value (target gene) = Ct value (target gene) - Ct value (internal reference gene GAPDH_PCR).

[0098] 4) According to the dCt values ​​of different clinical diagnostic samples, the threshold is divided to distinguish clinical samples with lesions and non-lesions.

[0099] In a preferred embodiment of the present invention, the judgment criteria for early diagnosis of liver cancer tissue are formulated as follows:

[0100] A) If the Ct value of the internal reference quality control gene GAPDH_PCR in the detection reaction is less than or equal to 35, the quality control is considered to be qualified; if the Ct value of the internal reference quality control gene GAPDH_PCR is greater than 35 or Undetermined, the quality control is considered to be unqualified, and if the quality control is unqualified, the experiment needs to be repeated.

[0101] B) If the relative fluorescence Ct value (dCt) of the restriction digestion quality control gene GAPDH_ED in the detection reaction is greater than or equal to 6, the restriction digestion reaction quality control is judged to be qualified; if the relative fluorescence Ct value (dCt) of the restriction digestion quality control gene GAPDH_ED is less than 6, the restriction digestion reaction quality control is judged to be unqualified, and if the quality control is unqualified, the experiment needs to be repeated.

[0102] C) Combined with the dCt experimental data of the restriction quality control gene GAPDH_ED of T1-T10 and P1-P10, the minimum value is 7.353 of sample P8, so the threshold setting of 6 is a good way to perform quality control. The relative fluorescence Ct values ​​(dCt) of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, are less than or equal to 6, and are judged as positive; the relative fluorescence Ct values ​​(dCt) of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, are greater than 6 or Undetermined, and are judged as negative.

[0103] D) When both the internal reference quality control gene GAPDH_PCR and the restriction endonuclease quality control gene GAPDH_ED are qualified, only one or more target genes need to be judged as positive in rule 3, and the final test result of the sample to be tested will be judged as positive.

[0104] Furthermore, the step 1) comprises a detection kit comprising restriction endonuclease Hin6I, restriction endonuclease HpaII, restriction endonuclease Bsh1236I, 10X FastDigest Buffer, primer mixture, Power SYBR Green PCRMaster Mix and nuclease-free water.

[0105] Preferably, in the restriction endonuclease digestion experiment of step 1), the reaction system is 20 μL: including 1 μL restriction endonuclease Hin6I, 1 μL restriction endonuclease HpaII, 1 μL restriction endonuclease Bsh1236I, 2 μL 10X FastDigestBuffer and 5 μL DNA sample to be tested (1-100 ng), and the rest is supplemented to 20 μL with nuclease-free water. The digestion reaction program is: 37°C 10 min; 80°C 10 min.

[0106] Furthermore, in the fluorescent quantitative PCR experiment of step 2), the reaction system is 20 μL: including 10 μL PowerSYBR Green PCR Master Mix, 0.7 μL primer mixture and 1 μL product after enzyme digestion reaction, and the rest is supplemented to 20 μL with nuclease-free water.

[0107] Preferably, the amplification reaction procedure is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec, annealing and extension at 60°C for 30 sec (collecting fluorescence), for a total of 50 cycles.

[0108] Technical effects of the present invention:

[0109] High accuracy and specificity: According to the data provided in Example 2, the detection method can distinguish liver cancer tissue from adjacent tissue with 100% sensitivity and specificity. This means that the method can very accurately identify liver cancer samples, while correctly judging non-liver cancer samples (adjacent tissue) as negative.

[0110] Early diagnostic capability: By detecting the methylation status of 14 genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, the kit is able to detect liver cancer at an early stage, providing the possibility for early intervention and treatment.

[0111] High-throughput detection: The kit is able to detect the methylation status of multiple genes simultaneously, which enables high-throughput liver cancer screening and significantly improves detection efficiency.

[0112] The operation is simple and fast: compared with the traditional bisulfite method, the present invention adopts the restriction endonuclease digestion reaction principle, the experimental operation is relatively simple, the experimental time is less, and the difficulty of primer design is greatly reduced, making the detection method more efficient and easy to implement.

[0113] Saving medical resources: Since this method can accurately diagnose liver cancer in the early stage, it helps to reduce medical costs and save social resources. Early detection and treatment of liver cancer can reduce treatment costs and improve patient survival rate and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 It is a ROC curve diagram of Example 3 of the present invention.

[0115] Figure 2 It is the dCt box plot of genes BDH1, CPAMD8, DAB2IP, DLEC1, and HOXA1 of Example 3 of the present invention.

[0116] Figure 3 It is a dCt box plot of genes MIXL1, OTX1, PCDH8, RNF135 and SIX2 of Example 3 of the present invention.

[0117] Figure 4 It is a dCt box plot of the genes SPINT2, TACSTD2, VIM and ZAR1 of Example 3 of the present invention and the restriction enzyme digestion quality control gene GAPDH_ED.

[0118] Figure 5 This is a diagram showing the determination results of the sample according to the third embodiment of the present invention. DETAILED DESCRIPTION

[0119] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0120] Example 1 Target Screening

[0121] 1.1 Marker screening:

[0122] By analyzing the liver cancer methylation dataset, expression dataset and clinical samples in the Cancer Genome Atlas (TCGA) dataset, the methylation differences between liver cancer and healthy people were studied, and methylation regions (DMRs) with significant differences were screened out.

[0123] Step 1. Screen out differentially methylated genes from the methylation dataset: A total of 353 liver cancer tissues (177 in stage I, 88 in stage II, 86 in stage III, and 2 in stage IV) and 50 adjacent tissue methylation data were collected from the TCGA dataset. The position information of the human reference genome sequence and the high-frequency SNP sites of the population were used to clean the data to be analyzed and annotate the genomic position and gene information of the methylation sites. Methylation difference analysis was performed on liver cancer and adjacent tissue data, and the genes were sorted according to the differences and the methylation levels of the genes were checked according to the cancer stage. In order to ensure that the screened genes have consistent methylation levels, the screening of methylation genes needs to meet the following two requirements at the same time: The first requirement is that the selected genes have no less than 10 methylation sites. The second requirement is that the degree of difference in methylation sites is greater than 0.15.

[0124] Step 2. Screen out differentially expressed genes from the expression dataset: A total of 374 liver cancer tissues and 50 adjacent tissues were collected from the TCGA dataset. The data to be analyzed were cleaned using the location information of the human reference genome sequence and the high-frequency SNP sites of the population. DESeq2 was used to perform expression differential analysis on liver cancer and adjacent tissues, and the data were sorted according to expression differences. Genes that intersected with genes with high differential high methylation were screened out as candidate target genes.

[0125] Step 3. Combine differentially methylated genes and differentially expressed genes, divide the regions and screen out the methylation regions with significant differences.

[0126] Step 4. Design restriction primers for each marker region. Combine the above methylation dataset screening and expression dataset screening to obtain gene regions with high methylation differences. Then use the Primer3 primer design tool to write a script for automated primer design. The designed primer region should contain three restriction sites: GCGC, CCGG, and CGCG.By comprehensively analyzing the methylation and expression data of liver cancer in the Cancer Genome Atlas (TCGA) database and combining it with the detection of clinical samples, we successfully screened out the restriction primers of 137 candidate genes, including ACTN3, CACNA1C, ELTD1, HOXD8, OTX2, SATB2, TLX2, AGRN, CCDC37, FAM110B, HOXD9, OXTR, SCRN1, TM6SF1, AKR1E2, CDX2, FAR1, HS3ST2, P2RX2, SFRP2, TMEM155, ALX3, CHST2, FGF19, KCNQ1DN, PCDH8, SH2B2, TRABD, AMN, CIDEA, FOXI2, KCNQ4, PCDHGA4, SIPA1, TRH, AMPD3, CLDN5, FZD7, KCNS1, PCDHGB5, SIX2, TXNRD1, ANKRD29, CNTNAP1, GATA3, LEP, PEX14, SIX3, VA SH2, ARL9, CPAMD8, GDAP1L1, LHX8, PFKP, SIX6, VIM, ASCL2, CTHRC1, GDF6, LMX1B, PITX1, SLC5A7, WDR8, BDH1, CYBA, GFI1, LOC100130274, PITX2, SLC9 A3R2, ZAR1, BNC1, CYP26C1, GPR25, LOC254559, POU4F3, SOX17, ZFP3, BNIP3, DAB2IP, GPR85, LOC494141, PPFIA3, SPINT2, ZIC1, C12orf42, DBX1, GRI N2C, MAP1D, PTCH2, TBR1, ZNF132, C14orf162, DLEC1, GRM6, MAPK8IP2, PRDM14, TACSTD2, ZIC5, C1orf114, DLX1, HCG9, MIR663, PTGDR, TBX1, ZNF274, C The methylation status of 20orf177, DLX5, HIST1H1D, MIXL1, PTCH2, TBX15, ZNF578, C2orf39, DMRTA2, HOXA1, NR2E1, RAX, TERT, ZSCAN1, C2orf40, DPP6, HOXA6, NTSR2, RNF135, TFAP2A, C6orf186, ECE1, HOXA7, NXPH1, RYR2, TLX1, C8orf84, EFNB2, HOXD10, OTX1, RYR2, and TLX1 is preliminarily speculated to be closely related to the occurrence and development of liver cancer, and is expected to serve as molecular markers for the early diagnosis of liver cancer.

[0127] Step 5. Use clinical sample testing and analysis to obtain a marker combination that can screen for liver cancer through methylation levels. Genomic DNA of cancer tissue samples (as the positive group) and adjacent tissue samples (as the negative group) of liver cancer patients were extracted for comparison. Restriction endonuclease Hin6I (ThermoFisherScientific, Catalog No.: FD0484), restriction endonuclease HpaII (ThermoFisherScientific, Catalog No.: FD0514) and restriction endonuclease Bsh1236I (ThermoFisher Scientific, Catalog No.: FD0924) were used for enzyme digestion experiments. Fluorescence quantitative PCR reaction was performed using the designed primers, and the methylation degree differences between cancer tissue samples (positive) and adjacent tissue samples (negative) of the same liver cancer patient were analyzed. Finally, 14 genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM, and ZAR1, were screened out, and the detection effects were relatively good, and the methylation degree differences were relatively large.

[0128] 1.2 Preparation of methylation detection kit

[0129] The present embodiment aims to prepare a methylation detection kit with liver cancer diagnosis effect, the kit includes forward and reverse primers for 14 genes including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM, and ZAR1, restriction endonuclease Hin6I (ThermoFisherScientific, Catalog No.: FD0484), restriction endonuclease HpaII (ThermoFisherScientific, Catalog No.: FD0514), restriction endonuclease Bsh1236I (ThermoFisherScientific, Catalog No.: FD0924), 10XFastDigestBuffer, BDH1 forward primer, BDH1 reverse primer, CPAMD8 forward primer, CPAMD8 reverse primer, DAB2IP forward primer, DAB 2IP reverse primer, DLEC1 forward primer, DLEC1 reverse primer, HOXA1 forward primer, HOXA1 reverse primer, MIXL1 forward primer, MIXL1 reverse primer, OTX1 forward primer, OTX1 reverse primer, PCDH8 forward primer, PCDH8 reverse primer, RNF135 forward primer, RNF135 reverse primer, SIX2 forward primer, SIX2 reverse primer, SPINT2 forward primer, SPINT2 reverse primer, TACSTD 2 forward primer, TACSTD2 reverse primer, VIM forward primer, VIM reverse primer, ZAR1 forward primer, ZAR1 reverse primer, GAPDH_ED forward primer, GAPDH_ED reverse primer, GAPDH_PCR forward primer, GAPDH_PCR reverse primer, Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, catalog number: 4368706) and nuclease-free water.

[0130] The sequences of the detection primers used above are shown in Table 1 below. They were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0131] Table 1 Detection primer sequence list

[0132]

[0133]

[0134] Example 2: Construction of diagnostic kit detection method

[0135] 2 Methods:

[0136] 2.1 Extraction of genomic DNA from liver tissue: Liver tissue samples were extracted using a conventional tissue genomic extraction kit (universal DNA kit from Guangzhou Meiji Biotechnology Co., Ltd.), and the experimental method was performed in accordance with the product instructions of the corresponding kit; the liver tissue samples used in this step were compared with the cancer tissue samples and adjacent tissue samples of the same liver cancer patient.

[0137] 2.2 Restriction endonuclease digestion reaction experiment: The digestion reaction system and digestion reaction procedure are shown in Tables 2 and 3.

[0138] Table 2. Restriction enzyme digestion reaction system

[0139]

[0140]

[0141] Table 3. Enzyme digestion reaction program

[0142] temperature time 37℃ 10min 80℃ 10min

[0143] 2.3 Fluorescence quantitative PCR detection: The product after restriction endonuclease digestion was used as a template, and the Thermo Fisher ABI7500 real-time fluorescence quantitative PCR instrument was used to detect the expression of BDH1 forward primer, BDH1 reverse primer, CPAMD8 forward primer, CPAMD8 reverse primer, DAB2IP forward primer, DAB2IP reverse primer, DLEC1 forward primer, DLEC1 reverse primer, HOXA1 forward primer, HOXA1 reverse primer, MIXL1 forward primer, MIXL1 reverse primer, OTX1 forward primer, OTX1 reverse primer, PCDH8 forward primer, PCDH8 reverse primer. Primer, RNF135 forward primer, RNF135 reverse primer, SIX2 forward primer, SIX2 reverse primer, SPINT2 forward primer, SPINT2 reverse primer, TACSTD2 forward primer, TACSTD2 reverse primer, VIM forward primer, VIM reverse primer, ZAR1 forward primer, ZAR1 reverse primer, GAPDH_ED forward primer, GAPDH_ED reverse primer, GAPDH_PCR forward primer, GAPDH_PCR reverse primer and PowerSYBR Green PCR Master Mix (ThermoFisher Scientific, Cat. No.: 4368706) were used for fluorescence quantitative PCR amplification. The fluorescence quantitative PCR reaction system and amplification reaction procedure are shown in Tables 4 and 5.

[0144] Table 4. Fluorescence quantitative PCR reaction system

[0145]

[0146] Table 5. Fluorescence quantitative PCR amplification reaction program

[0147]

[0148] Result analysis: Read and record the fluorescence signal Ct values ​​of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, the restriction endonuclease quality control gene GAPDH_ED and the internal reference quality control gene GAPDH_PCR, and calculate the relative fluorescence Ct values ​​(dCt) of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, and the restriction endonuclease quality control gene GAPDH_ED. The calculation formula is: dCt value (target gene) = Ct value (target gene) - Ct value (internal reference gene GAPDH_PCR).

[0149] According to the Ct value and dCt value displayed by the fluorescence quantitative PCR instrument, the inventors have established the following criteria for early diagnosis of liver cancer tissue:

[0150] 1. If the Ct value of the internal reference quality control gene GAPDH_PCR in the detection reaction is less than or equal to 35, the quality control is considered qualified; if the Ct value of the internal reference quality control gene GAPDH_PCR is greater than 35 or Undetermined, the quality control is considered unqualified, and if the quality control is unqualified, the experiment needs to be repeated.

[0151] 2. If the relative fluorescence Ct value (dCt) of the restriction digestion quality control gene GAPDH_ED in the detection reaction is greater than or equal to 6, the restriction digestion reaction quality control is judged to be qualified; if the relative fluorescence Ct value (dCt) of the restriction digestion quality control gene GAPDH_ED is less than 6, the restriction digestion reaction quality control is judged to be unqualified, and if the quality control is unqualified, the experiment needs to be repeated.

[0152] 3. Combining the dCt experimental data of the restriction quality control gene GAPDH_ED of T1-T10 and P1-P10, the minimum value is 7.353 of sample P8, so the threshold setting of 6 is a good way to perform quality control. The relative fluorescence Ct values ​​(dCt) of 14 target genes including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1 are less than or equal to 6, which is considered positive; the relative fluorescence Ct values ​​(dCt) of 14 target genes including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1 are greater than 6 or Undetermined, which is considered negative.

[0153] 4. When both the internal reference quality control gene GAPDH_PCR and the restriction endonuclease quality control gene GAPDH_ED are qualified, it is only necessary to judge that one or more target genes in rule 3 are positive, and the final test result of the sample to be tested is positive.

[0154] Example 3: Verification of sensitivity and specificity of diagnostic target gene combination

[0155] The sensitivity and specificity were verified by using the diagnostic target gene combination constructed according to the method of Example 2, the fluorescent quantitative PCR method and the judgment criteria used in Example 2.

[0156] 3.1 Primer extension specificity verification

[0157] Genomic DNA was extracted from three normal human blood samples. After no restriction endonuclease digestion reaction, the extracted genomic DNA was directly used for fluorescence quantitative PCR detection to test whether the primers can amplify. The experimental results are shown in Table 6 below.

[0158] Table 6 Results of direct extraction of DNA fluorescence quantitative PCR

[0159]

[0160]

[0161]

[0162] As can be seen from Table 6, the 16 pairs of primers synthesized and tested in Example 2 can all achieve specific amplification of the target gene, which shows that the primers are specific for the amplification of the target gene.

[0163] 3.2 Example 2 Verification of specificity and sensitivity of gene combination diagnosis

[0164] Tissue samples were extracted from 10 patients with liver cancer. Liver cancer tissue samples (T1-T10) and paracancerous tissue samples (P1-P10) were extracted from the same patient with liver cancer, for a total of 10 groups of samples. The sample numbers are shown in Table 7. The extracted DNA was used as a template for restriction endonuclease digestion experiments, and fluorescence quantitative PCR was performed after the digestion reaction. A total of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, were read and recorded, and the fluorescence signal Ct value of the restriction quality control gene GAPDH_ED and the internal reference quality control gene GAPDH_PCR were calculated to calculate the relative fluorescence Ct value (dCt). The liver cancer tissue samples (T1-T10) are shown in Table 8, and the paracancerous tissue samples (P1-P10) are shown in Table 9.

[0165] Table 7 Sample No.

[0166]

[0167] Table 8. Liver cancer tissue samples (T1-T10)

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Table 9. Paracancerous tissue samples (P1-P10)

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] From the data in Tables 7 and 8, the internal reference quality control gene GAPDH_PCR was amplified in all 10 groups of samples, and the CT value was relatively low, indicating that there was no problem with DNA quality control, while the restriction digestion quality control gene GAPDH_ED was amplified, but the dCT was also relatively high, indicating that the restriction digestion experiment was successful. The detection results of 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, showed that the methylation levels of liver cancer samples and para-cancer samples were different. The methylation levels of the same gene in different liver cancer samples were also different. The methylation levels in some liver cancer samples were relatively high, but the methylation levels in other liver cancer samples were not so high, and the dCt values ​​in para-cancer samples were relatively high.

[0181] 3.3 ROC curve plotting to verify the diagnostic effect of the diagnostic genome of the present invention

[0182] Figure 1 is the ROC curve diagram of this embodiment. Figure 1From the displayed content, it can be seen that the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by BDH1 gene methylation level is 1.000 (95% CI: 1.000-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by CPAMD8 gene methylation level is 0.850 (95% CI: 0.647-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by DAB2IP gene methylation level is 1.000 (95% CI: 1.000-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by DLEC1 gene methylation level is 0.850 (95% CI: 0.647-1.000). The area under the ROC curve of adjacent samples was 0.930 (95% CI: 0.794-1.000); the area under the ROC curve of differential diagnosis of liver cancer and adjacent samples by HOXA1 gene methylation level was 0.990 (95% CI: 0.958-1.000); the area under the ROC curve of differential diagnosis of liver cancer and adjacent samples by MIXL1 gene methylation level was 0.770 (95% CI: 0.522-1.000); the area under the ROC curve of differential diagnosis of liver cancer and adjacent samples by OTX1 gene methylation level was 1.000 (95% CI: 1.000-1.000). 000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by PCDH8 gene methylation level was 0.700 (95% CI: 0.426-0.974); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by RNF135 gene methylation level was 0.880 (95% CI: 0.728-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by SIX2 gene methylation level was 0.880 (95% CI: 0.691-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by SPINT2 gene methylation level was 0.860 (95% CI: 0.912-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by SPINT2 gene methylation level was 0.850 (95% CI: 0.911-1.000). The area under the ROC curve was 0.920 (95% CI: 0.782-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by TACSTD2 gene methylation level was 0.800 (95% CI: 0.579-1.000); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by VIM gene methylation level was 0.740 (95% CI: 0.490-0.990); the area under the ROC curve for differential diagnosis of liver cancer and adjacent cancer samples by ZAR1 gene methylation level was 0.710 (95% CI: 0.459-0.961);

[0183] The above results show that the AUC values ​​of BDH1, DAB2IP, and OTX1 genes reached the perfect diagnosis threshold of 1.000, indicating that the methylation levels of these genes can distinguish liver cancer and adjacent cancer samples with extremely high accuracy. In addition, DLEC1, HOXA1, RNF135, SIX2, and SPINT2 genes also showed excellent diagnostic effects, with AUC values ​​above 0.880, while CPAMD8, TACSTD2, VIM, and ZAR1 genes, although with slightly lower diagnostic effects, also showed certain diagnostic value. These findings suggest that the methylation levels of specific genes can be used as a powerful tool for liver cancer diagnosis. In particular, the methylation status of BDH1, DAB2IP, and OTX1 genes, due to their excellent diagnostic accuracy, can alone become promising biomarkers for early detection and screening of liver cancer.

[0184] 3.4 Use target gene dCt box plot to show the difference in expression of target genes in cancer tissue and adjacent tissue

[0185] Figure 2-Figure 4 It is a dCt box plot of the target gene of Example 2 of the present invention. The box plot is a method for intuitively displaying data distribution and variability, which can help us understand the expression changes of the target gene in different samples.

[0186] Figure 2 The dCt box plots of BDH1, CPAMD8, DAB2IP, DLEC1 and HOXA1 genes are shown. The results show that the dCt of genes BDH1, CPAMD8, DAB2IP, DLEC1 and HOXA1 in liver cancer samples are generally lower than those in adjacent cancer samples, and the methylation levels are higher than those in adjacent cancer samples, indicating that the methylation levels of these genes in liver cancer tissues are high, and the detection of their methylation levels can be used to distinguish cancer tissues from adjacent cancer tissues.

[0187] Figure 3 The dCt box plots of MIXL1, OTX1, PCDH8, RNF135, and SIX2 genes are shown. Similarly, the median dCt values ​​of these genes in liver cancer samples were lower than those in adjacent normal samples, further confirming that liver cancer tissues have increased methylation levels in these genes and that their detection can distinguish cancer tissues from adjacent normal tissues. Figure 4The dCt box plots of the four target genes SPINT2, TACSTD2, VIM and ZAR1 and the dCt box plots of the restriction enzyme quality control gene GAPDH_ED are displayed. The methylation levels of the four genes SPINT2, TACSTD2, VIM and ZAR1 in liver cancer samples also show obvious differences between liver cancer samples and adjacent paracancerous samples. Moreover, since the GAPDH gene is relatively stable as a housekeeping gene, the probability of methylation is low, and the restriction enzyme quality control gene GAPDH_ED has little difference between liver cancer samples and adjacent paracancerous samples, indicating that there is no problem with the quality control of this specific experiment.

[0188] Combination Figure 2 , Figure 3 , Figure 4 The results showed that liver cancer samples and adjacent cancer samples could be distinguished by detecting the methylation degree of one or more of the 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1. Figure 2-4 The box plot results verified the differences in gene methylation modification between liver cancer tissue and adjacent tissue, providing a biomarker combination for early diagnosis and treatment of liver cancer. In addition, these data also emphasize the important role of gene methylation status as a molecular marker in cancer development.

[0189] 3.5 Use target genes alone or in combination to determine cancer tissues T1 to T10 and adjacent tissues P1 to P10

[0190] The results of these samples are recorded in Figure 5 In. By Figure 5 As a result, it can be seen that the sensitivity of the liver cancer samples of the present invention is as high as 100%, and no detection is made in the adjacent cancer tissues. Therefore, the specificity of the target gene combination of the present invention for liver cancer samples is as high as 100%. Therefore, using the restriction endonuclease method, detecting the methylation degree of one or more of the 14 target genes, including BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1, can be used as a biomarker for auxiliary diagnosis of liver cancer.

[0191] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A liver cancer diagnosis kit, characterized in that: The kit comprises reagents for detecting the methylation degree of one or more of the target genes BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1.

2. The kit according to claim 1, characterized in that The reagents for detecting the methylation degree of one or more of the target genes BDH1, CPAMD8, DAB2IP, DLEC1, HOXA1, MIXL1, OTX1, PCDH8, RNF135, SIX2, SPINT2, TACSTD2, VIM and ZAR1 are selected from one or more of the following restriction primer sets: BDH1 forward primer: 5'-CTGTTCCCGGGGTTTGGG-3', BDH1 reverse primer: 5′-CCAGCCTGTCCCCGCGGC-3′; CPAMD8 forward primer: 5'-CCACCAGCGAACAGCTG-3', CPAMD8 reverse primer: 5′-AGGGATCCTTGACCCGTATGC-3′; DAB2IP forward primer: 5'-TGGGGCCTCCAGCCTGCG-3', DAB2IP reverse primer: 5′-GGAGGAGAAACAGGCCCCG-3′; DLEC1 forward primer: 5'-CTGGGATGTGAGGAGCGCCTCT-3', DLEC1 reverse primer: 5′-GGCGGCTGCCGGGCGGAGGGG-3′; HOXA1 forward primer: 5'-ACCTGGGTCAACCCAACGCG-3', HOXA1 reverse primer: 5′-CCCTGCGGGCGCGCGTCAGGTA-3′; MIXL1 forward primer: 5'-CTCAAAGTGCTGGGATTA-3', MIXL1 reverse primer: 5′-AGGCTTCTCCTTACCAACT-3′; OTX1 forward primer: 5'-CTGGGCAGAGAGAAGGCCGG-3', OTX1 reverse primer: 5′-GGGCGACCCCAAGATGCGGCC-3′; PCDH8 forward primer: 5'-GCTGGACGTGCGGGCGCAGGA-3', PCDH8 reverse primer: 5′-TTGTCATTGACGTCTCGGATGC-3′; RNF135 forward primer: 5'-TAGGGGCGGCCGGGCAGAGG-3', RNF135 reverse primer: 5′-CCCCTCTGCCCGGCCAGC-3′; SIX2 forward primer: 5'-TTCAAGAACCGGCGGCAGCG-3', SIX2 reverse primer: 5′-TTGGGCCCCGGGGAGCTGGCGCC-3′; SPINT2 forward primer: 5'-AAAATTAGCCGGGCCTGGTGG-3', SPINT2 reverse primer: 5′-CTGGCGCGATCTCAGCTCACT-3′; TACSTD2 forward primer: 5'-CTCATTGACCTGCGCCACC-3', TACSTD2 reverse primer: 5′-AGCCGATAGCGCTCGCGGAAGAG-3′; VIM forward primer: 5'-CACCGTGATTGCCCCTCTGCG-3', VIM reverse primer: 5′-GAGCAGCTGCGCCACCTC-3′; ZAR1 forward primer: 5'-CTGTAAACCTCGCGCAGT-3', ZAR1 reverse primer: 5′-ACTCTCCAGCCGGCAAGTGC-3′.

3. The kit according to claim 1-2, characterized in that The detected target genes are combination 1 consisting of genes BDH1, DAB2IP and OTX1, and / or combination 2 consisting of genes DLEC1, HOXA1, RNF135, SIX2 and SPINT2.

4. The kit according to any one of claims 1 to 3, characterized in that The target gene detected is a combination of combination 1 and / or combination 2 and one or more of the genes CPAMD8, TACSTD2, VIM and ZAR1.

5. The kit according to any one of claims 1 to 4 further comprises a reagent for detecting the restriction quality control gene GAPDH_ED and / or the internal reference quality control gene GAPDH_PCR; Preferably, the reagent for detecting the restriction endonuclease quality control gene GAPDH_ED and / or the internal reference quality control gene GAPDH_PCR is selected from the following restriction endonuclease primer groups: GAPDH_ED forward primer: 5'-CTTTCTTTCCTTTCGCGCTCT-3', GAPDH_ED reverse primer: 5′-CTACCGCAGGCGCAGCATCCGGA-3′; and / or GAPDH_PCR forward primer: 5'-TTGGCAAATCAAAGCCCTG-3', GAPDH_PCR reverse primer: 5′-CCCACCCCTTCTCTAAGTCCC-3′.

6. The kit according to any one of claims 1 to 5, characterized in that Also included are restriction enzyme Hin6I, restriction enzyme HpaII, restriction enzyme Bsh1236I, 10X FastDigest Buffer, primer mix, PowerSYBR Green PCR Master Mix, and nuclease-free water.

7. A device for diagnosing liver cancer, characterized in that: The device comprises 1) a sample processing module, 2) a target factor detection module, and 3) a determination module. The 1) sample processing module comprises reagents for extracting genomic DNA of the liver tissue to be tested; 2) The target gene detection module comprises a restriction endonuclease reaction system and a kit according to any one of claims 1 to 5; The restriction endonuclease reaction system comprises: restriction endonuclease Hin6I, restriction endonuclease HpaII, restriction endonuclease Bsh1236I and reagents required for the restriction reaction; The 3) determination module includes a step of calculating the fluorescence signal Ct value of each target gene generated by the 2) target gene detection module to obtain a relative fluorescence Ct value (dCt value), and the target gene relative fluorescence Ct value (dCt) is less than or equal to 6, and it is determined to be positive, and the relative fluorescence Ct value (dCt) of the enzyme digestion quality control gene GAPDH_ED is greater than or equal to 6, and the enzyme digestion reaction quality control is determined to be qualified; The dCt value calculation formula is: dCt value (target gene) = Ct value (target gene) - Ct value (internal reference gene GAPDH_PCR).

8. The liver cancer diagnosis device according to claim 7, characterized in that: The target gene detection module 2) comprises a) a step of performing an enzyme cleavage reaction using the enzyme cleavage reaction system, and b) a step of performing a fluorescent quantitative PCR amplification reaction using the kit according to any one of claims 1 to 4; The enzymatic reaction procedure of the step a) of performing an enzymatic reaction using the enzymatic reaction system is: 37° C., 10 min, then 80° C., 10 min; The amplification reaction procedure of the step b) using the kit according to any one of claims 1 to 4 for fluorescent quantitative PCR amplification reaction is: pre-denaturation at 95° C. for 10 min; denaturation at 95° C. for 15 s, annealing and extension at 60° C. for 30 s, for a total of 50 cycles.

9. Use of the kit according to any one of claims 1 to 5 and the device for liver cancer diagnosis according to any one of claims 7 to 8 in the preparation of liver cancer diagnosis products.

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