CN112280865A

CN112280865A - Reagent combination for detecting liver cancer, kit and application thereof

Info

Publication number: CN112280865A
Application number: CN202011290484.XA
Authority: CN
Inventors: 郭鑫武; 陈明; 洪梅; 刘让蛟; 戴立忠
Original assignee: Sansure Biotech Inc
Current assignee: Sansure Biotech Inc
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2021-01-29
Anticipated expiration: 2040-11-17
Also published as: CN112280865B

Abstract

The invention provides a reagent combination for detecting liver cancer, which comprises a detection reagent for detecting the methylation level of cg16657538 methylation sites of ZNF397OS gene. Meanwhile, the invention also provides the application of the reagent combination and a kit comprising the reagent combination. By using the reagent combination, the liver cancer can be detected in clinical tissue samples with the sensitivity of 0.910, the specificity of 0.950 and the area under the curve of 0.941 and free DNA samples with the sensitivity of 0.7619, the specificity of 0.9574 and the area under the curve of 0.8948 by using fewer markers, the liver cancer can be detected sensitively and specifically by detecting one methylation site of the gene, and the cost and the time are saved; so that it can be detected sensitively and specifically in the early stage of liver degeneration in clinic.

Description

Reagent combination for detecting liver cancer, kit and application thereof

Technical Field

The invention belongs to the field of molecular biological detection, specifically belongs to the field of liver cancer detection, and more specifically relates to detection of methylation level of a liver cancer gene marker.

Background

Liver cancer is one of the most common and fatal diseases in the world, and is a highly harmful malignant tumor. As many as 50% of liver cancer patients are in China all, and the liver cancer patients are the 4 th common malignant tumor and the 2 nd tumor lethal cause in China. According to data of '2015 Chinese malignant tumor prevalence analysis' published in 2019, it is shown that about 37.0 ten thousand people have liver cancer in 2015; about 32.6 million people die due to liver cancer, and the lives and health of people in China are seriously threatened. The liver cancer is hidden, has no specific symptoms in the early stage, has low operative cure rate and short life cycle when most liver cancer patients are in the middle and late stages.

At present, serum Alpha Fetoprotein (AFP) and ultrasonic examination are the common and important methods for clinically diagnosing liver cancer at present, but the sensitivity and specificity are not ideal. The sensitivity of AFP screening can only reach 40-60%, and the AFP level of many early liver cancer patients is always maintained at a normal level; there is also an abnormal increase in AFP in a significant proportion of patients with liver disease; the ultrasonic detection is very dependent on instruments and manual operation and is influenced by the limitation of medical resource distribution and the experience of doctors; the examination of ultrasonic radiography, puncture and the like has the defects of complex operation, trauma and the like, and is not suitable for early screening and early diagnosis. The search for accurate, stable and effective liver cancer molecular markers has important significance for early diagnosis and early treatment of liver cancer.

Tumor marker detection is a method for detecting diseases developed in recent years, and the search for accurate, stable and effective liver cancer molecular markers has important significance for early diagnosis and early treatment of liver cancer. With the understanding of the science community on tumors, more and more researches prove that the change of the cell epigenetic level is a key event of the occurrence and development of the tumors. DNA methylation, histone modification, and miRNA expression abnormalities are epigenetic changes, and the core link of tumorigenesis is also related to DNA abnormal methylation. The DNA methylation detection has good stability and easy detection, the abnormal degree of the DNA methylation detection is often related to the progress of cancer, the DNA methylation detection is the marker with the most tumor early screening potential, tumor auxiliary diagnosis products such as colorectal cancer, gastric cancer and the like based on the DNA methylation detection are certified by FDA or CFDA at present, but no liver cancer screening products are approved to be on the market.

Many studies have sought markers for early diagnosis of liver cancer by detecting changes in tumor cells or DNA methylation in peripheral blood. Clinical studies of methylation detection-based early liver cancer screening by the company of Exact Sciences in the United states show that in 135 cases of HCC (hepatocellular carcinoma) and 308 controls participating in clinical tests, the blood detection achieves 71% of sensitivity and 90% of specificity when diagnosing patients with early HCC. The project obtains FDA breakthrough medical appliance identification, but the clinical research is mainly based on European and American people and has the defect of low sensitivity; the patent CN107164508A detects liver cancer by detecting the content of 5-hydroxymethylcytosine of 9 genes, has excellent detection performance, the sensitivity is 90 percent, and the specificity is 91.3 percent, but the sample of the patent does not contain liver cancer high-risk people such as cirrhosis, hepatitis and the like, so the defect of low specificity may exist.

Therefore, there is a need in the art for a diagnostic product for liver cancer based on DNA methylation detection with high sensitivity and specificity, which can detect liver cancer at an early stage.

Disclosure of Invention

According to the invention, a large methylation data set related to liver cancer is collected and constructed in TCGA and GEO databases, a bioinformatics method is adopted to screen out liver cancer DNA methylation markers with application and development potential, and through a large amount of researches, the applicant discovers that the methylation level of a methylation site of ZNF397OS gene is closely related to liver cancer, and before that, ZNF397OS is generally considered to be a zinc finger protein in the field, and the researches show that the zinc finger protein has the function of regulating the activity of DNA binding transcription factors, and no literature reports that the zinc finger protein is related to liver cancer.

In a first aspect, the present invention provides a reagent combination for detecting liver cancer, the reagent combination comprising a detection reagent for detecting the methylation level of cg16657538 methylation site of ZNF397OS gene.

In a specific embodiment, the combination of reagents detects the level of methylation in the following regions: CGCCCCACTCACCCTTCGCTCTACCGGCGGCGGCGGGAACCCACCCCCGGGAGCGAGAACAATGCCCGGCCGCACGCGCGCCGGAAGTGGGAGAGTGCCCCTCTAGGAGCCCGGAGGACCGCAGCTCTGTGGCAGGCGCGGGTCGTGTCTCGCAGGAGGGGCGCGGGTCCGGCTCAGACCTGGCGGGGGCATCGCAGAGTACAAGCGGTTGACGCG (SEQ ID NO: 1).

By using the reagent combination, the liver cancer can be detected in clinical tissue samples with the sensitivity of 0.910, the specificity of 0.950 and the area under the curve of 0.941 as well as the sensitivity of 0.7619, the specificity of 0.9574 and the area under the curve of 0.8948 in free DNA samples by using fewer markers, and the liver cancer can be detected sensitively and specifically by detecting one methylation site of the gene, so that the cost and the time are saved; so that it can be detected sensitively and specifically in the early stage of liver degeneration in clinic.

In some specific embodiments, the combination of reagents further comprises a detection reagent for detecting the methylation level of any at least one of the following genes:

GRASP, PAK1, PPFIA1, and OTX 1.

The reagent combination further comprises detection reagents for detecting the methylation level of any at least two of the following genes:

GRASP, PAK1, PPFIA1, and OTX 1.

The reagent combination further comprises detection reagents for detecting the methylation level of any at least three of the following genes:

GRASP, PAK1, PPFIA1, and OTX 1.

The reagent combination further comprises detection reagents for detecting the methylation levels of the following four genes:

GRASP, PAK1, PPFIA1, and OTX 1.

In some embodiments, the detection reagent for methylation level can be a detection reagent for detecting an average methylation level of the entire gene.

In some embodiments, the detection reagent for methylation level can also be a detection reagent for detecting the average methylation level of a gene fragment.

In some embodiments, the methylation level detection reagent can also be a reagent that detects the average methylation level of a gene promoter region or a fragment thereof.

In some specific embodiments, the detection reagent for methylation level can also be a detection reagent for detecting one or more methylation sites of a gene.

One skilled in the art would be able to select detection reagents that detect methylation levels of GRASP, PAK1, PPFIA1, and OTX 1. For example, chinese patent CN110904225 mentioned GRASP related to liver cancer, chinese patent CN106947830 mentioned PPFIA1 related to liver cancer, and chinese patent CN1659287 mentioned PAK1 gene related to liver cancer.

In a specific embodiment, the combination of reagents further comprises a detection reagent for detecting the average methylation level of the entire gene of the GRASP gene.

In a specific embodiment, the reagent combination further comprises a detection reagent for detecting the average methylation level of the amplified fragment of the GRASP gene in Chinese patent CN 110904225:

CGCAGCCGCCACCCCTGGGCCCCCAGCGGACGAGCTGTACGCGGCGCTGGAGGACTATCACCCTGCCGAGCTGTACCGCGC(SEQ ID NO:2)。

in a specific embodiment, the combination of reagents further comprises detection reagents for detecting the average methylation level of the following gene segments of the GRASP gene:

CGGTCCCGACCCCGGGACCCCCTGCCGCAGCCGCCACCCCTGGGCCCCCAGCGGACGAGCTGTACGCGGCGCTGGAGGACTATCACCCTGCCGAGCTGTACCGCGCGCTCGCCGTGTCCGGGGGCACCCTGCCCCGCCGAAAGGTGCGTCCCCCGCCCGCCTTCAGGATCTGCTCAGCCCCTCTCCGACTCCCTACAGGGCCTGCTGACTCCGCAGTGCCCTCTCCTCGGCGTCCGCGGAGTCCCCCACCTTCTTCCCCGGCCCGCTGGGTGCCTCGACTCCCCGCGTTCCCCGCTGCTGCGAAGGCCGTGGCCCTCGCCTGCACACCGCGCCCAGGCTCG(SEQ ID NO:3)。

in some specific embodiments, the combination of agents further comprises a detection agent for detecting the level of methylation of one or more of the methylation sites cg04034767, cg00817367 of the GRASP gene.

In a specific embodiment, the reagent combination further comprises a detection reagent for detecting the average methylation level of the whole gene of the PPFIA1 gene.

In a specific embodiment, the reagent combination further comprises detection reagents for detecting the average methylation level of the following gene fragments of the PPFIA1 gene:

CGACCCAGTGTTAACAGGGAATGGTTATTCTGTACGGGCATCTGAACTGAAAAGTGAGAAGAGCGAACTTTGCCTCCTCGGCCCCTTCTCTGTGCCTGTGGCTTATGCGTGTGCCCCTCTCCTCTTTGTCACTGCTTCCCTTGCCCTGGATGTGGTTGGTGCACTGGGGTCACCTTAGACCACAGGAAATGTCTGGTTAACACACGAAGAGATGGAAACGCTCGCAGCCACG(SEQ ID NO:4)。

in some specific embodiments, the detection reagent for the methylation level of the PPFIA1 gene further comprises a detection reagent for detecting the methylation level of one or more of the methylation sites cg14999168, cg14088196 and cg25574765 of the PPFIA1 gene.

In some specific embodiments, the reagent combination further comprises a detection reagent for detecting the average methylation level of the entire gene of PAK1 gene.

In some specific embodiments, the detection reagent for the methylation level of the PAK1 gene further comprises a detection reagent for detecting the methylation level of one or more of the methylation sites cg17202086, cg26996201, cg12269002 and cg18309286 of the PAK1 gene.

In some specific embodiments, the reagent combination further comprises a detection reagent for detecting the average methylation level of the whole gene of OTX1 gene.

In some specific embodiments, the detection reagent for the methylation level of OTX1 gene further comprises a detection reagent for detecting the methylation level of one or more of OTX1 gene methylation sites cg21472506, cg23229261, cg 10122865.

By using the reagent combination of the scheme, the liver cancer can be detected with higher sensitivity and better specificity; clinically, it can be detected sensitively and specifically in the early stage of liver degeneration.

In some embodiments, the methylation level of a corresponding gene present in a sample can be detected using the detection reagents of the invention.

In the present invention, a "sample" is a biological sample selected from an individual. Specifically, for example, selected from the group consisting of cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, stool, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or combinations thereof.

Preferably, the "sample" of the invention is plasma, i.e. free DNA in plasma.

The free DNA in the plasma can be used for detecting tumors and has the characteristics of little harm to patients, good specificity and the like. However, the content of the compound in blood plasma is extremely low, so that the compound has the problem of low sensitivity when being used for cancer detection. The detection reagent can be used for detecting free DNA in plasma as a sample, and has higher sensitivity and specificity.

In the present invention, the "detection reagent" refers to a reagent for detecting the methylation level of a gene in a sample. Wherein, the methylation level is measured by means of amplification-sequencing, chip and methylation fluorescent quantitative PCR.

In some specific embodiments, detection reagents include, but are not limited to, nucleic acid primers, sequencing Tag sequences, for measuring methylation levels by amplification-sequencing.

In a specific embodiment, the amplification-sequencing is performed by bisulfite treating the nucleic acids in the sample, followed by construction of a pre-library, followed by construction of a final library, and finally by sequencing evaluation.

In some specific embodiments, the detection reagents include, but are not limited to, a chip that is a methylation chip having probes that specifically bind to a methylation region. The chip may be, for example, but not limited to, a chip including, for example, Human CpG Island Microarrays and Human DNA Methylation Microarrays of Agilent, Infinium Humanmethylation27 BeadChip of Illumina, Infinium Humanmethylation450 BeadChip and golden Gate Methylation Assay, and Human DNA Methylation 2.1M Deluxe Promoter Array, Human DNA Methylation Array, etc. of Roche NimbleGen for measuring Methylation levels by the chip.

In some specific embodiments, detection reagents include, but are not limited to, nucleic acid primers and nucleic acid probes for measuring methylation levels by methylation fluorescence quantitative PCR.

Further, the detection reagent further comprises an internal standard primer and an internal standard probe.

In some specific embodiments, the above-described reagent combination may further include remaining reagents, specifically, for example, various reagents required for pretreatment or pretreatment of a sample. For example, a sample releasing agent for extracting a sample nucleic acid, a purifying agent for purifying a sample nucleic acid, bisulfite or bisulfite for conversion, and the like.

Further, the reagent combination also comprises a reagent for extracting free DNA of plasma.

In a second aspect, the invention provides the use of the above reagent combination in the preparation of a kit for detecting liver cancer.

Further, the invention provides application of the reagent combination in preparing a kit for detecting liver cancer by using free plasma DNA.

In a third aspect, the present invention provides a kit for detecting liver cancer, the kit comprising the combination of reagents as described above.

Further, the kit also comprises at least one of a reagent for extracting nucleic acid, a reagent for purifying nucleic acid, bisulfite, T4 polynucleotide kinase and T4 ligase.

Further, the reagent for extracting nucleic acid is a reagent for extracting tissue DNA and a reagent for plasma-free DNA.

Further, the reagent for extracting nucleic acid is a reagent for extracting plasma-free DNA.

Drawings

FIG. 1 is a ROC diagram of ZNF397OS single gene for identifying cancer and non-cancer in peripheral blood free DNA samples;

FIG. 2 shows the methylation levels of ZNF397OS gene in different groups in peripheral blood free DNA samples;

FIG. 3 is the methylation levels of ZNF397OS gene methylation sites in different groupings of tissue samples;

FIG. 4 shows the methylation levels of different groupings of the ZNF397OS gene target regions versus the methylation sites.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Example 1 screening of methylated genes

785 cancer tissues, 461 paracancer tissues or normal control tissues and 656 healthy whole blood methylation data are collected from a TCGA data set (https:// TCGA. xenahubs. net) of a UCSC Xena website and a GEO database of the National Center for Biotechnology Information (NCBI). Carrying out difference analysis on liver cancer and control data, carrying out physical position and gene information annotation on difference sites, and ensuring that screening fragments have consistent methylation level, wherein the screening requirements of methylated gene fragments simultaneously meet the following requirements: 1) the selected gene fragment is required to have not less than 2 adjacent sites with consistent methylation level; 2) carrying out differential analysis on liver cancer and a tissue beside the cancer or a normal control tissue, and selecting a gene segment with high consistency and high differential hypermethylation in a liver cancer sample as a candidate target gene; 3) carrying out differential analysis on the liver cancer and healthy sample whole blood methylation detection data, and selecting liver cancer differential hypermethylated gene segments; 4) and finally, analyzing the methylation sites one by one to obtain candidate methylation sites.

Example 2 detection of Gene methylation levels in clinical samples

10ml of each of the peripheral blood of the sample was collected for detection of the methylation level of the analyte DNA methylation marker in the sample. The experimental procedure is as follows:

1. sample preparation

The sample preparation of the invention is through MagMAX^TM4ml of plasma was extracted with the Cell-Free DNA Isolation Kit, and 45. mu.L of the eluate was eluted. The extracted free nucleic acid needs to meet the following quality control conditions: the total amount of extracted nucleic acid is greater than 20 ng.

2. Library preparation

In the present invention, all free nucleic acids that are qualified for quality control are bisulfite-treated with EZ DNA Methylation-Lightning TM Kit (Zymo Research, Irvine, Calif., USA). And then, constructing a pre-library by using a single-strand library construction method for the sample DNA treated by the bisulfite, and performing hybridization capture on an enriched target area through a liquid chip after the quality of the pre-library is qualified so as to complete construction of a final library.

Constructing a pre-library: 1) phosphorylation: t4 Polynucleotide kinase phosphorylates the 5-terminus of DNA after bisulfite treatment; 2) the SS1 is connected with: t4 DNA Ligase (Rapid) attaches a SS1 linker to the 5-terminus of phosphorylated DNA; 3) nucleic acid purification: the remaining linker was removed using 2 volumes of Agencourt XP system (Beckman Coulter, Calif., USA); 4) the SS2 is connected with: t4 DNA Ligase (Rapid) attaches a SS2 linker at the 3-terminus of phosphorylated DNA; 5) nucleic acid purification: the remaining linker was removed using 2 volumes of Agencourt XP system (Beckman Coulter, Calif., USA); 6) amplification: amplifying the nucleic acid of the previous step by using NEBNext Q5U Master Mix and primer1.0 (universal primer) and Bacard sequence; 7) nucleic acid purification: 1.2 volumes of Agencour AMPure XP system (Beckman Coulter, Calif., USA) were used to remove primer dimers and excess primers; 8) quality inspection: purification of the processed pre-library

The dsDNA HS Assay Kit (Life Technologies, CA, USA) was used for quality inspection of total library quantity, and LabChip GXII Touch was used for quality inspection of library fragment distribution.

Chip (Twist Bioscience) hybridization capture step: 1) chip hybridization: 1.5 mu g of the library which is qualified for quality inspection and mixed well is concentrated in vacuum in advance to be powdery and then is mixed with Panel, Hybridization Mix, Blocker Solution, Universal blocks and Hybridization Enhancer reagents (reagents used for chip Hybridization are all provided by Twist Bioscience), and the mixture is placed in a PCR instrument for incubation at 70 ℃ for 16hours overnight (the temperature of a hot cover is 85 ℃); 2) and (3) magnetic bead capture: washing the capture magnetic beads 3 times by using a Streptavidin Binding Buffer in advance, adding the hybridized products into the capture magnetic beads, incubating for 30 minutes, washing once by using Wash Buffer I, washing 3 times by using Wash Buffer 2, and finally eluting by using 42 mu l of ultrapure water; 3) amplification: amplifying the captured library by using KAPA HiFi HotStart ReadyMix and a universal primer; 4) and (3) purification: a1-fold volume of Agencour AMPure XP system (Beckman Coulter, Calif., USA) was used to remove primer dimers and excess primers.

Purified library application

The dsDNA HS Assay Kit (Life Technologies, CA, USA) and LabChip GXII Touch were used for quality control of total nucleic acid amount, fragment distribution and primer dimer ratio in the library.

3. Sequencing

Mixing the libraries to be tested with the total library amount, the fragment size distribution of the amplification products and the primer dimer proportion quality inspection qualified according to the amount of substances of 1:1, and using

The mixed library was quantitated accurately by the dsDNA HS Assay Kit (Life Technologies, CA, USA), and the library was denatured and diluted for on-machine sequencing using a NextSeq500 bench-top sequencer with PE 75.

4. Establishment and evaluation of liver cancer classification model

And for original fastq data obtained by sequencing, filtering the original data, and performing methylation analysis on the chip capture fragment by using bismark methylation analysis software to obtain the methylation level of a single site of the candidate gene and the methylation level of the gene fragment. And performing differential analysis and model construction on the liver cancer and the control sample by using the methylation level of a single site of the candidate gene and the methylation level of the gene fragment. According to the invention, the construction and evaluation of the liver cancer classification model are carried out by adopting Logitics regression analysis on data.

Example 3 detection of methylation levels in clinical samples by the combination of reagents of the invention

Samples of 63 primary liver cancer patients, 25 liver cirrhosis patients, 15 hepatitis patients and 7 healthy persons were collected and examined for methylation level of cg16657538 methylation site of ZNF397OS gene and methylation level of ZNF397OS gene in combination with the remaining genes in the samples according to the method described in example 2 to verify the effect of examining liver cancer. The results of the measurements are shown in table 1, fig. 1 and fig. 2.

TABLE 1 predictive Performance of ZNF397OS Gene in combination with the remaining genes in Logitics liver cancer Classification model

Wherein, 1 represents GRASP gene (detecting the average methylation level of the fragments shown in SEQ ID NO: 3), 2 represents PAK1 gene (detecting the average methylation level of the fragments containing the four methylation sites of cg17202086, cg26996201, cg12269002 and cg 18309286), 3 represents PPFIA1 gene (detecting the average methylation level of the fragments shown in SEQ ID NO: 4), 4 represents OTX1 gene (detecting the average methylation level of the fragments containing cg21472506, cg23229261 and cg 10122865), and 5 represents ZNF397OS gene (detecting the average methylation level of the fragments containing cg16657538 methylation sites).

As can be seen from table 1, all reagent combinations in the tissue samples were able to predict liver cancer with a specificity of at least 0.9176 and a sensitivity of 0.8981 and an area under the curve of 0.9396. In the free DNA sample of plasma, all reagent combinations can predict liver cancer with specificity of at least 0.8936, sensitivity of 0.7619 and area under curve of 0.8845. Therefore, the reagent combination of the present invention has a good predictive effect on liver cancer, and particularly, has excellent specificity and sensitivity when plasma free DNA is used as a sample.

Comparative example 1

In order to examine whether other methylation sites on the ZNF397OS gene can be used as a marker for detecting liver cancer, other methylation sites in the ZNF397OS gene are selected as comparative examples (which are all located outside the selected target section of the current ZNF397OS gene), and the methylation difference of the comparative examples in cancer and non-cancer samples is compared, and the results are shown in FIG. 3 and FIG. 4. As can be seen from fig. 3 and 4, the methylation sites in ZNF397OS gene upstream and downstream of the selected target segment do not have significant differences in liver cancer and non-cancer tissues and are not suitable as markers for detecting liver cancer.

Comparative example 2

In order to examine whether ZNF397OS gene can be used as a marker for detecting liver cancer, the genes PLAC8 and ATXN1, which are known in the art and closely related to liver cancer, were further selected (see Xu RH, Wei W, Krawczyk M, Wang W, Luo H, flag K, et al. circulating tumor DNA methylation markers for diagnosis and diagnosis of liver cancer. Nature Materials,2017 and Chinese patent CN 106947830B). The same procedures as in the above examples were carried out, and the results are shown in Table 3 below.

TABLE 3 predicted Performance of comparison genes and their combinations in Logitics liver cancer Classification model

Wherein 6 represents the PLAC8 gene (tested for cg11606215 methylation level of the gene), and 7 represents the ATXN1 gene (tested for cg24067911 methylation level of the gene).

As can be seen from Table 3, the area under the highest curve in the tissue sample for the control gene alone was 0.858 and the area under the highest curve in the free DNA sample was 0.75, which is lower than the area under the curve for ZNF397OS of the present invention. The area under the curve of the combination is also lower than the value of the area under the curve of the combination of agents according to the invention.

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Claims

1. A reagent combination for detecting liver cancer, which comprises a detection reagent for detecting the methylation level of cg16657538 methylation sites of ZNF397OS gene.

2. The reagent combination of claim 1, wherein the reagent combination further comprises a detection reagent for detecting the methylation level of at least one of the following genes: GRASP, PAK1, PPFIA1, and OTX 1.

3. The reagent combination of claim 1, wherein the reagent combination further comprises detection reagents for detecting the methylation levels of at least two of the following genes: GRASP, PAK1, PPFIA1, and OTX 1.

4. The reagent combination of claim 1, wherein the reagent combination further comprises detection reagents for detecting the methylation level of at least three of the following genes: GRASP, PAK1, PPFIA1, and OTX 1.

5. The reagent combination of claim 1, wherein the reagent combination further comprises detection reagents for detecting the methylation levels of the following four genes: GRASP, PAK1, PPFIA1, and OTX 1.

6. The reagent combination of any one of claims 1 to 5, wherein the detection reagent is any one or more of a nucleic acid primer, a sequencing Tag sequence, a methylation chip, and a nucleic acid probe.

7. The reagent combination of any one of claims 1-5, wherein the reagent combination further comprises a reagent to extract plasma free DNA.

8. Use of a combination of reagents according to any one of claims 1-7 in the manufacture of a kit for the detection of liver cancer.

9. A kit for detecting liver cancer, the kit comprising the combination of reagents of any one of claims 1-7.

10. The kit according to claim 9, wherein the kit further comprises remaining reagents, such as reagents for extracting nucleic acids, preferably reagents for extracting plasma free DNA, reagents for purifying nucleic acids, bisulfite, T4 polynucleotide kinase, T4 ligase.