Liver cancer related methylation gene combination in plasma and application thereof

Abstract

The invention discloses a methylation gene combination related to liver cancer in plasma and application thereof, and the methylation gene combination related to liver cancer in plasma comprises TSPYL5 and CLEC11A; the combination further comprises any one or more genes selected from the group consisting of LRRC4 and KCNA3. The invention also provides an application of the combination as a detection target in preparation of a liver cancer auxiliary diagnosis kit. According to the auxiliary diagnosis kit for the liver cancer, the detection sensitivity of the biomarker can be improved to 5-10 copies / microliter, and the methylation conditions of the provided four liver cancer related genes TSPYL5, CLEC11A, LRRC4 and KCNA3 are potential meaningful clinical indexes for diagnosis, prognosis evaluation and curative effect monitoring of the liver cancer (especially early liver cancer).

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and specifically to a plasma free DNA methylation gene panel for identifying liver cancer and its applications. Background Art

[0002] Liver cancer is currently a malignant tumor with high incidence and mortality rates. China is currently a country severely troubled by liver cancer in the world. In 2016, the incidence rate of liver cancer in China ranked fourth among all cancers, while the mortality rate ranked second among all cancers. Therefore, liver cancer seriously threatens the health and safety of people in China and even the world.

[0003] Primary liver cancer is clinically mainly divided into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Among them, HCC accounts for 75%-85%, and ICC accounts for 10%-15%. Since the molecular mechanisms, pathogenesis, biological behaviors, and pathological characteristics of these three cancers are different, the research on the three cancers is also different.

[0004] Currently, the overall 5-year survival rate of liver cancer is only 14.1%. The main reason for this phenomenon every year is that the onset of liver cancer is relatively hidden. Early liver cancer only shows atypical digestive tract symptoms such as abdominal distension, abdominal pain, nausea, vomiting, and loss of appetite, or is asymptomatic. Patients usually find it difficult to detect, delaying the best treatment opportunity. At the same time, the early screening of high-risk populations for liver cancer is insufficient, resulting in a low early diagnosis rate. If liver cancer can be detected as early as possible, through radical surgical operations such as hepatectomy and liver transplantation, the prognosis of liver cancer patients can be significantly improved.

[0005] Currently, the popular methods for early screening of liver cancer mainly include serological tumor marker examination, imaging examination, and pathological histology examination. Among them, the pathological histology examination samples are from percutaneous biopsy guided by ultrasound imaging (US) or computed tomography (CT), which is an invasive examination and not suitable for the screening of liver cancer. Imaging examinations mainly include US, CT, MRI (Magnetic Resonance Imaging), and nuclear medicine imaging examinations, etc. Among them, US has become a commonly used method for clinical liver cancer imaging examination due to its advantages such as low cost, non-invasiveness, and no radiation. If the US examination shows abnormalities, dynamic enhanced CT and multi-parametric MRI scans are the preferred imaging examination methods for clarifying the diagnosis of patients. Tumor markers can, to a certain extent, reflect the severity of the tumor condition and predict the development of the condition, such as alpha-fetoprotein (AFP), protein induced by vitamin K absence / antagonist-II (PIVKA-II, Des-gamma Carboxy Prothrombin, DCP), microRNA (miRNA), and lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), etc. However, the sensitivity and specificity of tumor marker examination are difficult to meet the clinical requirements.

[0006] Epigenetic alterations occur in the early stages of carcinogenesis and are prevalent in cancers. Late diagnosis is closely associated with poor prognosis and high mortality. Therefore, DNA methylation analysis, due to its advantages of high sensitivity, stability, and cost-effectiveness, is a biomarker that can be used for the early diagnosis of various cancers. By detecting abnormal DNA methylation, the early occurrence and development of liver cancer can be well monitored. DNA methylation tumor markers are expected to become an effective means for the early diagnosis of liver cancer, with broad prospects for clinical application.

[0007] Liquid biopsy is the most ideal method to avoid pain, and the biological samples for analysis can be obtained from blood, urine, saliva, sputum, hepatic alveolar lavage fluid, pleural effusion, or tissue samples. The applicable samples of this kit are mainly plasma cfDNA isolated from blood, which is not only the least invasive liquid biopsy sample but also can avoid the errors caused by tissue heterogeneity.

[0008] Currently, the main methods for DNA methylation detection are as follows: methylation-specific PCR, bisulfite sequencing, high-resolution melting curve method, fluorescence quantitative method, chip method, high-throughput sequencing, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. Among them, the real-time fluorescence quantitative PCR method has low cost, high popularity, is fast, and has excellent sensitivity and specificity. Therefore, this kit has greater clinical application value in choosing this method. Summary of the Invention

[0009] To solve the problems raised in the above background art, the purpose of the present invention is to provide a plasma-free DNA methylation gene combination for identifying liver cancer and its application. By detecting the relative methylation degree of liver cancer-related methylation genes and providing a plasma liver cancer-related methylation gene combination, it has great advantages of high clinical sensitivity and high clinical specificity in the diagnosis of early liver cancer.

[0010] To achieve the above purpose, the present invention provides the following technical solutions:

[0011] One object of the present invention is to provide a composition for detecting plasma liver cancer-related methylation genes, and the composition contains reagents for fluorescence quantitative PCR to detect any two or more of the plasma liver cancer-related methylation gene combinations of TSPYL5, CLEC11A, LRRC4, and KCNA3.

[0012] Furthermore, the composition contains reagents for fluorescence quantitative PCR to detect plasma liver cancer-related methylation gene combinations selected from any one or more of the following:

[0013] Combination 1: LRRC4 and KCNA3;

[0014] Combination 2: CLEC11A and LRRC4;

[0015] Combination 3: CLEC11A and KCNA3;

[0016] Combination 4: TSPYL5 and LRRC4;

[0017] Combination 5: TSPYL5 and KCNA3;

[0018] Combination 6: TSPYL5 and CLEC11A;

[0019] Combination 7: CLEC11A, LRRC4, and KCNA3;

[0020] Combination 8: TSPYL5, LRRC4, and KCNA3;

[0021] Combination 9: TSPYL5, CLEC11A, and LRRC4;

[0022] Combination 10: TSPYL5, CLEC11A, and KCNA3;

[0023] Combination 11: TSPYL5, CLEC11A, LRRC4, and KCNA3.

[0024] The Genebank numbers of the above genes in the NCBI (National Center for Biotechnology Information) website are TSPYL5: NC_000008.11(97273488..97277928), CLEC11A: NC_000019.10(50723364..50725708), LRRC4: NC_000007.14(128027071..128032128), and KCNA3: NC_000001.11(110653560..110674940).

[0025] Furthermore, the reagent contains primers and probes for detecting the methylation status of the TSPYL5, CLEC11A, LRRC4, and KCNA3 genes.

[0026] The primers and probes for detecting the methylation status of the TSPYL5 gene are shown in sequences SEQ ID NO.1 - 3, or in sequences SEQ ID NO.4 - 6;

[0027] The primers and probes for detecting the methylation status of the CLEC11A gene are shown in sequences SEQ ID NO.7 - 9, or in sequences SEQ ID NO.10 - 12;

[0028] The primers and probes for detecting the methylation status of the LRRC4 gene are shown in sequences SEQ ID NO.13 - 15, or in sequences SEQ ID NO.16 - 18;

[0029] The primers and probes for detecting the methylation status of the KCNA3 gene are shown in sequences SEQ ID NO.19 - 21, or in sequences SEQ ID NO.22 - 24.

[0030] Furthermore, the reagent also contains primers and probes for detecting the internal reference gene GAPDH, and the primers and probes for detecting the internal reference gene GAPDH are shown in sequences SEQ ID NO.25 - 27.

[0031] Further, the above-mentioned specific probe is modified with a fluorescent dye. The fluorescent group labeled at the 5' end of the probe is any one of organic fluorescent dyes or inorganic dyes, and the quenching group labeled at the 3' end is any one of organic dyes. Preferably, the above-mentioned organic fluorescent dyes include FAM, VIC, ROX, TRT, Cy3, Cy5, HEX, and JOE, and the above-mentioned quenching groups include MGB, BHQ-1, BHQ-2, and BHQ-3.

[0032] The lengths of the above-mentioned specific primers are all between 15-30 nt, and they hybridize with the DNA of the methylated target gene region after bisulfite treatment under stringent conditions.

[0033] The above-mentioned specific probe is designed based on TaqManTM, with a length between 15-30 nt, and it hybridizes with the DNA of the methylated target gene region after bisulfite treatment under stringent conditions.

[0034] The second object of the present invention is to provide the application of the above-mentioned composition in the preparation of a kit for the auxiliary diagnosis of liver cancer.

[0035] The third object of the present invention is to provide a kit for the auxiliary diagnosis of liver cancer, which contains the above-mentioned composition.

[0036] Further, the kit also contains: a PCR reaction system, a negative control product, and a positive control product.

[0037] The PCR reaction system includes: each reaction volume is 10-50 μL, and each detection system contains a premixed solution of DNA polymerase and PCR reaction buffer at 5-25 μL / reaction; upstream and downstream primers and probe sets for the DNA of each liver cancer-related gene and the internal reference GAPDH after bisulfite conversion, where the concentrations of the upstream and downstream primers for each gene and the internal reference GAPDH are both 0.2 μΜ - 0.6 μΜ / reaction, and the probes for each gene and the internal reference GAPDH are both 0.2 μΜ - 0.6 μΜ / reaction; DNA template of the test sample: 3-10 μL; positive control: 3-10 μL; negative control product: 3-10 μL.

[0038] Preferably, the PCR reaction system includes: each reaction volume is 20 μL, and it contains a premixed solution of DNA polymerase and PCR reaction buffer at 10 μL / reaction; upstream and downstream primers and TaqMan probe sets for the DNA of each liver cancer-related gene and the internal reference GAPDH after bisulfite conversion, where the concentrations of the upstream and downstream primers for each gene and the internal reference GAPDH are both 0.2 μΜ - 0.6 μΜ / reaction, and the probes for each gene and the internal reference GAPDH are both 0.2 μΜ - 0.6 μΜ / reaction; DNA template of the test sample: 3-10 μL; positive control product: 3-10 μL; negative control product: 3-10 μL.

[0039] Further, the negative control product is DNase-free water or human genomic DNA after bisulfite conversion, and the positive control product is Huh7 genomic DNA after bisulfite conversion diluted to a certain concentration (1×10^3 copies / μL).

[0040] Further, the kit further includes: The premixed solution of DNA polymerase and PCR reaction buffer in the above kit includes hot-start Taq DNA polymerase. The PCR buffer contains dNTP, Tris-HCl, potassium chloride, ammonium sulfate, magnesium chloride, etc.

[0041] The fourth object of the present invention is to provide a method for detecting methylation of multiple liver cancer-related genes in plasma, including: detecting the methylation content of target genes in a sample using the composition described in any one of claims 1 to 5; the target genes include any two or more genes among TSPYL5, CLEC11A, LRRC4, and KCNA3; the detection is not for the purpose of disease diagnosis or treatment.

[0042] A method for detecting methylation of multiple liver cancer-related genes in plasma specifically includes the following steps:

[0043] (1) Plasma free DNA extraction: Extract and purify free DNA from the plasma sample of the subject using a plasma free DNA extraction kit (other liquid biopsy samples can be extracted and purified using the corresponding extraction kit).

[0044] (2) Plasma free DNA methylation conversion: Treat the extracted plasma free DNA with bisulfite reagent using a plasma free DNA methylation conversion kit and then purify it (other liquid biopsy samples can be converted and purified using the corresponding bisulfite conversion kit).

[0045] (3) qPCR amplification: Perform PCR amplification on the bisulfite-treated plasma free DNA. Using the Taqman analysis method, three replicates are performed for the PCR amplification of each template; qPCR amplification is performed using the detection target primer-probe set and the internal reference primer and internal reference probe of the internal reference gene. The qPCR program is:

[0046] 95°C for 5 min;

[0047] 50 cycles: 95°C for 10 sec, 60°C for 30 sec;

[0048] 37°C for 30 sec;

[0049] After the PCR reaction ends, set the Ct threshold in the linear amplification range.

[0050] (4) Data analysis: First, using the CT value of the GAPDH gene as an internal reference, the ΔCT mean of three repeated detections is taken; Second, when the detection result of the internal reference site GAPDH is within the normal range, and the ΔCT mean value of any one or more target detections in the target gene is substituted into the standard curve equation to obtain the relative methylation degree value of each target, and then substituted into different combinations of regression equations. According to the cut-off value of the ROC curve, it is determined whether the sample is positive. Further, binomial regression fitting is performed on the target gene combination, and the performance indicators of the combined detection include: clinical sensitivity, clinical specificity, and the area under the receiver operating characteristic curve (AUC value).

[0051] The samples detected by the kit of the present invention are derived from human plasma but are not limited to plasma. The DNA of the test sample is a complete genome, cell-free DNA, or circulating tumor DNA that has been treated with a bisulfite conversion kit.

[0052] Further, multiple nucleic acid fragment detections are performed in one reaction tube, and multiple band fluorescence signal detections are simultaneously performed in one reaction tube to distinguish different DNA fragments by different fluorescence signals; alternatively, different nucleic acid fragment detections can be performed in different reaction tubes, and single band fluorescence signal detections are simultaneously performed in one reaction tube to distinguish different DNA fragments by fluorescence signals; alternatively, multiple nucleic acid fragment detections can be performed in different reaction tubes, and multiple band fluorescence signal detections are simultaneously performed in one reaction tube to distinguish different DNA fragments by fluorescence signals.

[0053] Compared with the prior art, the beneficial effects of the present invention are:

[0054] After experimental testing, the kit of the present invention can increase the detection sensitivity of biomarkers to 5-10 copies / μL. The improvement of detection sensitivity and clinical sensitivity is achieved by optimizing specific nucleotide sequence primers and DNA probes, screening suitable target gene detection sites, and optimizing each target combination. In the technical solution of the present invention, various gene combinations of 4 liver cancer-related genes, TSPYL5, CLEC11A, LRRC4, and KCNA3, can be used to verify liver cancer patients and normal people, and ROC curve fitting and multiple regression can be performed. Experiments show that when using the plasma cfDNA of liver cancer patients to verify the methylation detection of the above-mentioned multiple genes, the methylation levels of the above-mentioned liver cancer-related methylated genes are generally high; while in the negative verification experiment, when using the cfDNA in the plasma of normal people to detect the methylation of the above-mentioned multiple genes, the results show no methylation or low methylation. Therefore, compared with methylation qualitative detection, this kit uses a relative methylation quantification method, which can reduce the false positive rate. The methylation relative quantification standard curve is processed for the above detection results, and the methylation degree values of each detected gene are substituted into the multiple regression equation, and the risk of liver cancer occurrence is determined through the Cut-off value. In short, the methylation status of 4 liver cancer-related genes is a meaningful clinical indicator for the diagnosis, prognosis evaluation, and efficacy monitoring of potential liver cancer (especially early liver cancer). The present invention has important value for deeply understanding the pathogenesis of early liver cancer, understanding its occurrence and development laws, and improving the diagnosis and treatment level of early liver cancer in China, and also lays a foundation for seeking new targeted treatment strategies for early liver cancer. The kit provided by the present invention can be applied to the auxiliary diagnosis of liver cancer and the monitoring of liver cancer progression, and at the same time provides a new, fast, reliable, and accurate way for the diagnosis of multiple liver cancers, provides a basis for efficacy observation and dynamic observation of minimal residual disease, and the present invention will play an important role in the field of medical testing.

[0055] In addition, in the technical solution of the present invention, the relative methylation quantification method is mainly based on GAPDH as an internal reference, and a relative methylation reference product is constructed, and then the relative methylation standard curve is made. The methylation degree of liver cancer-related genes can be obtained through the standard curve, which is more sensitive than methylation quantitative detection, improves the detection accuracy, reduces the false positive rate, and has more clinical value for liver cancer diagnosis. Brief Description of the Drawings

[0056] Figure 1 It is the qPCR amplification curve graph of the TSPYL5 methylation negative / positive control product (A); the qPCR amplification curve graph of the CLEC11A methylation negative / positive control product (B); the qPCR amplification curve graph of the internal reference GAPDH methylation negative / positive control product (C)

[0057] Figure 2: qPCR amplification curve of TSPYL5 methylation positive gradient dilution reference (A); Standard curve of TSPYL5 methylation (B); qPCR amplification curve of CLEC11A methylation positive gradient dilution reference (C); Standard curve of CLEC11A methylation (D).

[0058] Figure 3 : ROC curves of 4 combinations of liver cancer methylation-related genes 1 - 11. Detailed implementation mode

[0059] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0060] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the scientific research technology in this field. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The reagents and equipment used in the following embodiments can be obtained from commercial channels or are commonly used in this industry unless otherwise specified.

[0061] To enable those skilled in the art to understand the effects and characteristics of the present invention, the terms mentioned herein are defined: Primary liver cancer: Generally refers to malignant tumors originating from hepatocytes and intrahepatic bile duct epithelial cells, which are mainly divided into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) clinically. Among them, HCC accounts for 75% - 85%, and ICC accounts for 10% - 15%. The "liver cancer" used in this article refers to HCC, which means malignant tumors occurring in hepatocytes.

[0062] The "early liver cancer" used in this article refers to cells in the early stage of transformation into cancer cells or cells prone to transform into cancer cells. Or stage I and II of liver cancer. Such cells can exhibit one or more phenotypic traits characteristic of cancer cells.

[0063] As used herein, "nucleic acid", "nucleic acid sequence", etc. refer to polynucleotides, which can be gDNA, plasmid DNA, cfDNA. "gDNA" refers to genomic DNA, plasmid DNA is in vitro recombinant DNA, and cfDNA refers to cell-free DNA circulating in human blood. It can also be a combination of these substances (i.e., a recombinant nucleic acid that is partially gDNA and partially plasmid DNA).

[0064] As used herein, "CpG island" means that the distribution of CpG dinucleotides in the human genome is very uneven, while in certain regions of the genome, CpG is maintained or higher than the normal probability. CpG islands are mainly located in the promoter and the first exon region of genes, and the promoters of more than about 60% of genes contain CpG islands. The GC content of CpG islands is greater than 50%, and the length is 500 - 1000 bp.

[0065] As used herein, "methylation" refers to the methylation of cytosine at the C5 position of the cytosine in the DNA double strand. DNA amplified in vitro is usually unmethylated because typical in vitro DNA amplification methods do not retain the methylation pattern of the amplification template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template is unmethylated or methylated, respectively.

[0066] As used herein, "hypermethylated" refers to a nucleic acid in which a specific locus (e.g., a CpG dinucleotide or a group of dinucleotides or a CpG-rich region) is methylated at a certain rate in a specific sample type or tissue type, and the rate is measurably greater than the rate observed for a comparable locus in the same DNA in another tissue or sample type. "Highly methylated" can refer to the average methylation rate within a single specific C residue or multiple Cs in a region, as a fraction of the copies of the locus in the measured sample. In some embodiments, without limiting the term to any specific methylation level, hypermethylated sites can be >10% methylated, preferably >20% to 40%, more preferably >50% to 75%, still more preferably between 75% and 100%.

[0067] As used herein, "primer-probe set" refers to the primers and probes required in the detection. A primer refers to an oligonucleotide (whether naturally occurring in a purified restriction digestion or produced synthetically) that can serve as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (e.g., in the presence of nucleotides and an inducer such as a biocatalyst). For maximum amplification efficiency, primers are usually single-stranded, but can alternatively be partially or fully double-stranded. The primer portion hybridizing to the template nucleic acid is long enough to initiate the synthesis of an extension product in the presence of an inducer. The exact length of the primer will depend on many factors, including temperature, primer source, and method of use.

[0068] Primers may include labels, tags, capture moieties, etc. A probe refers to an oligonucleotide (e.g., a nucleotide sequence) that is capable of hybridizing to another target oligonucleotide, whether naturally occurring when in a purified restriction digest or produced synthetically, recombinantly, or by PCR amplification. A probe can be single-stranded or double-stranded. Probes can be used for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that in some embodiments, any probe used in the present invention can be labeled with any "reporter molecule" such that it can be detected in any detection system, including but not limited to enzymes (e.g., ELISA and enzyme-based histochemical assays), fluorescence, radioactivity, and luminescence systems. The present invention is not intended to be limited to any specific detection system or label.

[0069] As used herein, "kit" refers to the reagents required for the detection, including primers, probes, enzymes, dNTPs, buffers, positive control products, and negative control products.

[0070] As used herein, "sensitivity" refers to the lowest analytical concentration that can be detected, or the limit of detection. The "specificity" of the detection method of the present kit refers to the ability to detect non-targets and unmethylated targets as negative. "Clinical sensitivity" refers to the proportion of the number of diseased people detected by the test among the total number of patients (true positive rate), and "clinical specificity" refers to the proportion of the number of healthy controls detected by the test among the total number of patients (true negative rate).

[0071] The present invention adopts a relative methylation quantification method. Using GAPDH as an internal reference gene (non-methylated), a standard curve equation for relative methylation amount relative to GAPDH is established. Substituting the detected value ΔCt mean into the equation can obtain the relative methylation degree of the liver cancer-related gene.

[0072] In the present invention, in some embodiments, a diagnostic model based on the methylation level of biomarker genes is constructed using statistical methods, and the statistical methods are selected from the following methods: multiple linear regression, logistic regression, cluster analysis, Bayesian and non-Bayesian methods, etc.

[0073] In the present invention, the kit of the present invention is used to assist a physician in grading the risk of a patient having liver cancer and planning the next diagnostic steps to be taken. Similarly, the method provided by the present invention can also be used to evaluate the risk of liver cancer in asymptomatic high-risk patients, and as a screening tool for the general population. It is contemplated that the method of the present invention can be used by a clinician as part of a comprehensive assessment of other predictive and diagnostic indicators.

[0074] The combination of biomarker genes in the present invention provides a sensitive, specific, and accurate means for predicting the presence of liver cancer or detecting liver cancer at different stages of liver cancer progression. The evaluation of the methylation level in plasma may also be correlated with the presence of pre-malignant or pre-clinical conditions in patients. Therefore, the disclosed method can be used to predict or detect the presence of liver cancer in a sample, the stage of liver cancer, the subtype of liver cancer, the benign or malignant nature of liver cancer, the likelihood of metastasis of liver cancer, and other liver cancer characteristics related to the prevention, diagnosis, and treatment of liver cancer in patients.

[0075] In addition, in the technical solution of the present invention, a relative methylation quantification method is adopted. Mainly using GAPDH as an internal reference, a relative methylation reference product is constructed, and then a relative methylation standard curve is made. The methylation degree of liver cancer-related genes can be obtained through the standard curve, which is more sensitive than methylation quantitative detection, improves the detection accuracy, reduces the false positive rate, and has more clinical value for the diagnosis of liver cancer and early liver cancer.

[0076] The 4 abnormal methylation genes related to liver malignancies involved in the present invention mainly include TSPYL5 (Cysteine dioxygenase type 1), CLEC11A (C-type lectin domain containing 11A), LRRC4 (leucine rich repeat containing 4), and KCNA3 (potassium voltage-gated channel subfamily A member 3).

[0077] The following uses specific implementation cases to elaborate on the present invention in detail and further explain the technical solution.

[0078] Example 1

[0079] Real-time fluorescence PCR detection of DNA methylation, primer-probe set design, and verification

[0080] In this example, a method and process for designing a primer-probe set for 4 liver cancer-related genes, TSPYL5, CLEC11A, LRRC4, and KCNA3, and the internal reference GAPDH are provided.

[0081] This example also provides a method and kit for detecting the methylation of liver cancer-related genes in plasma, which can screen and assist in the diagnosis of liver cancer. The detection results are verified through negative reference products and positive reference products, and the designed primer-probe set is verified.

[0082] The specific steps are as follows:

[0083] Through NCBI (National Center for Biotechnology Information), we searched for liver malignancy-related genes TSPYL5, CLEC11A, LRRC4, KCNA3, and internal reference GAPDH, and through the UCSC database, we entered the gene name in the "HumanGRChg38 / hg38" interface, and selected "show" in "CpG Island". We can see the CpG island situation on the resulting page and obtain the CpG nucleic acid sequence. We found the location of the CpG island 50 bp upstream and downstream, and downloaded and saved the standard sulfurized sequence, that is, the cytosine except the CpG dinucleotide is converted into uracil.

[0084] Design upstream and downstream primers and probes for the sequences of liver cancer-related genes TSPYL5, CLEC11A, LRRC4 and KCNA3 and the internal reference GAPDH: Use Methyl Primer Express v1.0 or Meth Primer or PrimerPremier5 or Oligo7.5 to design upstream and downstream primers and probes for the sequences of four liver cancer-related genes and the internal reference GAPDH. Use NCBI to verify the specificity of primers and probes.

[0085] The design of upstream and downstream primers must meet the following conditions:

[0086] (1) Cover 2-4 CpG sites; (2) Avoid complementary DNA sequences between two primers, especially at the 3' end, and avoid complementary sequences at the 3' end of the same primer itself, so that they cannot form primer dimers or hairpin structures; (3) The primer Tm value is usually 55-65°C, and the Tm values ​​of the upstream and downstream primers cannot exceed 2°C; (4) The length of the primer is generally 18-30bp and cannot be too long, because it will cause its extension temperature to be greater than the optimal temperature of the Taq enzyme; (5) The GC content of the primer is generally 40-60%;

[0087] (6) The length of the PCR amplification product is 50-150 bp.

[0088] The designed probes must meet the following conditions: (1) cover 2-4 CpG sites; (2) avoid the appearance of repeated sequences, especially 4 consecutive G sequences or 6 consecutive A sequences; (3) the probe Tm value is usually 61-67°C, which is 5°C higher than the primer Tm value, ensuring that the probe binds to the target fragment before the primer during annealing; (4) the probe length is generally 18-30bp to ensure binding specificity; (5) the GC content of the probe is generally 40-60%; (6) the 5' end of the probe should not contain G as much as possible, as the G base has a quenching effect on the fluorescence of the 5' end to a certain extent; (7) the first 4 sequences of the 3' end of the probe should not contain 3 or more Gs.

[0089] Four pairs of upstream and downstream primers and probes for liver cancer methylation-related genes and the internal reference gene GAPDH are shown in Table 1. The primer and probe sequences shown in Table 1 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0090] Table 1 Specific primer and probe sets included in the kit of the present invention

[0091]

[0092]

[0093] Example 2

[0094] Construct the target positive and negative reference products of the kit and construct the standard linear equation for the target methylation degree.

[0095] The preparation raw material of the positive reference product is the human hepatocellular carcinoma Huh7 cell line with a methylation ratio of 100% for the TSPYL5, CLEC11A, LRRC4, and KCNA3 genes; the cell line is prepared into the positive reference product mother liquor through processes such as enlarged culture, gDNA extraction, bisulfite conversion, concentration determination, and gradient dilution. The preparation raw material of the negative reference product is healthy human genomic DNA; the gDNA is prepared into the negative reference product mother liquor through processes such as bisulfite conversion, concentration determination, and gradient dilution. The positive reference product mother liquor and the negative reference product mother liquor are verified by methylation-specific PCR and sequencing methods.

[0096] Culture of hepatocellular carcinoma Huh7 cells: (1) Check the STR (short tandem repeat) genotype detection report of the received Huh7 cells; (2) After confirming that the detection report is correct, place the cells in an incubator at 37°C for 2 h of buffering; (3) Observe under an inverted microscope. If the cells do not grow to 85%, spray the T25 cell culture flask with 75% alcohol and place it on the biological operation table, add 6 - 8 mL of newly prepared complete medium, and continue to culture in the cell incubator (37°C, 5% CO2); if the cell growth density is higher than 85%, the cells can be passaged; (4) After multiple cell passages, take a culture flask filled with Huh7 cells, digest with 1 mL of trypsin for 1 - 2 min, add 2 volumes of complete medium to terminate digestion, gently pipette to make the cells fall off and become a single-cell suspension, then centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of PBS buffer to resuspend the cells, and collect them into a 1.5 mL centrifuge tube for subsequent genomic DNA extraction.

[0097] Extraction of genomic DNA from hepatocellular carcinoma Huh7: Use a Blood / Cells / Tissues Genomic DNA Extraction Kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) for extraction, and refer to the instruction manual for the extraction steps.

[0098] Bisulfite conversion of genomic DNA from hepatocellular carcinoma Huh7: Use the EZ DNA Methylation-Lighting TM KIT Bisulfite Conversion Kit (purchased from ZYMO RESEARCH) to perform bisulfite conversion on the extracted Huh7 genomic DNA, and refer to the instruction manual for the conversion steps.

[0099] Use Nano Drop to quantitatively detect the converted genomic DNA to obtain a high-concentration positive reference. The calculation method for the copy number of the target sequence is as follows:

[0100] Copy number of the target sequence = Concentration (ng / μL) × 6.023 × 10 23 ÷ 330 ÷ (2.91 × 10 9 )

[0101] The detection of sensitivity and specificity using this kit is as follows (taking TSPYL5 / CLEC11A as an example):

[0102] At the same time, this kit also provides positive and negative controls, as follows: The positive control (PC) is the positive reference stock solution diluted to a concentration of 1 × 10 3 copies / μL; the negative control (NC) is the negative reference stock solution diluted to a concentration of 1 × 10 3 copies / μL or DNase-free water.

[0103] Using the above DNA template, perform qPCR experiments. The relative methylation reaction system for TSPYL5 / CLEC11A is as follows:

[0104] Table 2 Methylation Detection Reaction System

[0105]

[0106] TOROIVD 5G qPCRPremix consists of dNTP, Mg 2+, Taq DNA polymerase, Tris-HCl and KCl, etc., purchased from Tiansie (Shanghai) Technology Co., Ltd. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. after providing the sequences. The two sets of probes for TSPYL5 were labeled with FAM at the 5' end and MGB at the 3' end; the two sets of probes for CLEC11A were labeled with ROX at the 5' end and MGB at the 3' end; the probes for GAPDH were labeled with VIC at the 5' end and MGB at the 3' end; triple detection was achieved.

[0107] Three replicate wells were set for each detection group. The reaction conditions for TSPYL5 / CLEC11A methylation and GAPDH detection were as follows:

[0108] Table 3 Methylation detection amplification procedure

[0109] Reaction step Number of cycles Temperature Time Pre-denaturation 1 95℃ 5 min Cyclic reaction 50 95℃ 10 sec 60 °C (collecting signal) 30 sec Cooling 1 37℃ 30 sec

[0110] The results obtained are attached Figure 1 .

[0111] The test results show that the detection sensitivity can be as low as 10 copies per microliter. The positive quality control product tested by parallel experiments showed positive results, and the negative quality control product tested by the template of healthy human genomic DNA converted by bisulfite showed negative results. This shows that the primer probe of this kit has good specificity.

[0112] The methylation degree reference was prepared with the positive reference stock solution and diluted 5-fold, mainly including 10×5 5 copies / μL, 10×5 4 copies / μL, 10×5 3 copies / μL, 10×5 2 copies / μL, 10×5 1 The linearity reference of the gradient concentration of 100 copies / μL.

[0113] The qPCR experiment was performed using the above gradient diluted DNA template. The reaction system is shown in Table 2.

[0114] Three replicate wells were set for each detection group, and the relative methylation reaction conditions of TSPYL5 / CLEC11A are shown in Table 3.

[0115] The results obtained are attached Figure 2 .

[0116] The test results show that within the concentration range of 10 copies / μL to 10×5^5 copies / μL, both TSPYL5 and CLEC11A targets showed good linearity, R 2 >0.99.

[0117] Example 3

[0118] Verification of Clinical Sensitivity and Clinical Specificity of a Kit for the Combined Detection of 4 Different Hepatocellular Carcinoma Methylation Genes in the Plasma of Hepatocellular Carcinoma Patients and Normal Individuals

[0119] In this example, the clinical sensitivity and clinical specificity of the gene combinations selected in this kit were determined by verifying the DNA methylation status of different combinations of 4 hepatocellular carcinoma-related methylation genes in hepatocellular carcinoma plasma and the methylation status of cell-free DNA in normal plasma.

[0120] Extraction of cell-free DNA: Use Serum / Plasma Circulating DNA KIT (purchased from Nanjing Novoprotein Biotechnology Co., Ltd.), and operate according to the kit instructions.

[0121] Bisulfite conversion of cell-free DNA: Use the EZ DNA Methylation-Lighting TM KIT bisulfite conversion kit (purchased from ZYMO RESEARCH) to perform bisulfite conversion on cell-free DNA, and refer to the instruction part of the kit for the conversion steps.

[0122] Amplification and detection: Using real-time fluorescence PCR assay, the relative methylation levels of different combinations of the above 4 marker genes were detected in the plasma of 30 patients pathologically diagnosed with hepatocellular carcinoma and 30 normal individuals.

[0123] The purified nucleic acid was subjected to qPCR amplification, with three replicates set. The internal reference GAPDH was detected in each reaction. The reaction system and conditions were the same as those in the verification test in Example 1. After the reaction, the data was analyzed, the ΔCT mean value of the relevant methylation genes of each sample was read, the relative methylation degree was calculated according to the target relative methylation standard linear equation, and then the target combination detection was carried out.

[0124] In this invention, commercially available software packages (IBM SPSS Statistics 24 purchased from IBM, GraphPad Prism 8.0.2 purchased from GraphPad) were used to perform normal distribution tests on the ΔCT mean values of hepatocellular carcinoma patients and normal individuals respectively. Both cohorts had to pass the normal distribution test to use the unpaired t-test for significance analysis; if one or both of the two cohorts did not pass the normal distribution test, the Mann-Whitney test had to be used for significance analysis. After significance analysis, the P value was obtained. P < 0.05 was considered a significant statistical difference.

[0125] Four marker genes were combined using logistic regression, and the resulting AUC was 0.9556 (95% CI: 0.8881 - 1.000; P value: <0.0001) (attached Figure 3 ). To make the monitoring and analysis method simpler, multiple combinations of different markers were made and a logistic regression model was established. The obtained AUC values and P values are shown in Table 4, and the ROC curve graph is shown in the attached Figure 3 .

[0126] Table 4 AUC values and P values of the ROC curves for different marker combinations

[0127] Marker combination AUC P Combination 1 LRRC4 and KCNA3 0.6756 0.0195 Combination 2 CLEC11A and LRRC4 0.7122 0.0047 Combination 3 CLEC11A and KCNA3 0.7533 0.0007 Combination 4 TSPYL5 and LRRC4 0.9022 ＜0.0001 Combination 5 TSPYL5 and KCNA3 0.9044 ＜0.0001 Combination 6 TSPYL5 and CLEC11A 0.9556 ＜0.0001 Combination 7 CLEC11A, LRRC4 and KCNA3 0.7811 0.0002 Combination 8 TSPYL5, LRRC4 and KCNA3 0.9400 ＜0.0001 Combination 9 TSPYL5, CLEC11A and LRRC4 0.9600 ＜0.0001 Combination 10 TSPYL5, CLEC11A and KCNA3 0.9556 ＜0.0001 Combination 11 TSPYL5, CLEC11A, LRRC4 and KCNA3 0.9556 ＜0.0001

[0128] Generally speaking, the clinical sensitivity and clinical specificity of the logistic regression model of the four markers are slightly better. However, considering the operation analysis procedure and cost, several other marker combinations are also good choices, and combination 6 is the best.

[0129] According to the regression equation, the ΔCt values of each gene were substituted into the equation, and the cut-off value was obtained according to the ROC curve. The combinations of the detected biomarkers, as well as the clinical sensitivity and clinical specificity results, are shown in Table 5.

[0130] Table 5 Combinations of the detected biomarkers, and clinical sensitivity and clinical specificity results

[0131] Marker combination Specificity Sensitivity Combination 1 LRRC4 and KCNA3 80.00％ 56.67％ Combination 2 CLEC11A and LRRC4 93.33％ 60.00％ Combination 3 CLEC11A and KCNA3 93.33％ 73.33％ Combination 4 TSPYL5 and LRRC4 96.67％ 80.00％ Combination 5 TSPYL5 and KCNA3 96.67％ 90.00％ Combination 6 TSPYL5 and CLEC11A 96.67％ 96.67％ Combination 7 CLEC11A, LRRC4 and KCNA3 96.67％ 73.33 Combination 8 TSPYL5, LRRC4 and KCNA3 96.67％ 90.00％ Combination 9 TSPYL5, CLEC11A and LRRC4 96.67％ 96.67％ Combination 10 TSPYL5, CLEC11A and KCNA3 96.67％ 96.67％ Combination 11 TSPYL5, CLEC11A, LRRC4 and KCNA3 96.67％ 96.67％

[0132] To achieve different intended uses and purposes, the detection kit provided by the present invention can achieve different clinical sensitivity and clinical specificity indexes by adjusting the cut-off value. In Example 3, in order to improve clinical sensitivity, setting the cut-off value does not mean that the present invention can only use this as the cut-off value. Different cut-off values can be selected as the determination criteria according to the ROC curve provided by the present invention.

[0133] The results in Table 4 and Table 5 show that by combining four methylation genes related to liver cancer in different ways, the combined detection performance indexes are good, indicating that the combined detection of relative methylation quantification of the above liver cancer-related genes can improve the accuracy.

[0134] It should be noted that, in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0135] It should be noted that the above content only illustrates the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements all fall within the protection scope of the claims of the present invention.

Claims

1. A composition for detecting liver cancer-related methylation genes in plasma, characterized in that: The composition comprises a reagent for detecting a combination of liver cancer-related methylation genes in plasma of any two or more of TSPYL5, CLEC11A, LRRC4 and KCNA3 by fluorescent quantitative PCR.

2. A composition for detecting liver cancer-related methylation genes in plasma according to claim 1, characterized in that: The composition comprises a reagent for fluorescent quantitative PCR detection of a combination of methylated genes associated with liver cancer in plasma selected from any one or more of the following: Combination 1: LRRC4 and KCNA3; Combination 2: CLEC11A and LRRC4; Combination 3: CLEC11A and KCNA3; Combination 4: TSPYL5 and LRRC4; Combination 5: TSPYL5 and KCNA3; Combination 6: TSPYL5 and CLEC11A; Combination 7: CLEC11A, LRRC4, and KCNA3; Combination 8: TSPYL5, LRRC4, and KCNA3; Combination 9: TSPYL5, CLEC11A, and LRRC4; Combination 10: TSPYL5, CLEC11A, and KCNA3; Combination 11: TSPYL5, CLEC11A, LRRC4 and KCNA3.

3. A composition for detecting liver cancer-related methylation genes in plasma according to claim 1, characterized in that: The reagent comprises primers and probes for detecting the methylation status of TSPYL5, CLEC11A, LRRC4, and KCNA3 genes, wherein the primers and probes for detecting the methylation status of the TSPYL5 gene are as shown in the sequences SEQ ID NO.1-3, or as shown in the sequences SEQ ID NO.4-6; The primers and probes for detecting the methylation status of the CLEC11A gene are shown in SEQ ID NOs.7-9, or in SEQ ID NOs.10-12; The primers and probes for detecting the methylation status of the LRRC4 gene are shown in the sequences of SEQ ID NO.13-15, or in the sequences of SEQ ID NO.16-18; The primers and probes for detecting the methylation status of the KCNA3 gene are shown in sequences SEQ ID NOs.19-21, or in sequences SEQ ID NOs.22-24.

4. A composition for detecting liver cancer-related methylated genes in plasma according to claim 3, characterized in that: The reagent also includes primers and probes for detecting the internal reference gene GAPDH, and the primers and probes for detecting the internal reference gene GAPDH are shown in sequences SEQ ID NO.25-27.

5. A composition for detecting liver cancer-related methylation genes in plasma according to claim 4, characterized in that: The fluorescent group labeled at the 5' end of the probe is any one of organic fluorescent dyes or inorganic dyes, and the quenching group labeled at the 3' end is any one of organic dyes.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a liver cancer auxiliary diagnosis kit.

7. A liver cancer auxiliary diagnosis kit, characterized in that: A composition comprising any one of claims 1 to 5.

8. A liver cancer auxiliary diagnosis kit according to claim 7, characterized in that: The kit also includes: a PCR reaction system; the PCR reaction system includes: each reaction volume is 10-50 μL, wherein the premix of DNA polymerase and PCR reaction buffer is 5-25 μL / reaction; upstream and downstream primers and TaqMan probe sets of DNA converted by bisulfite of each liver cancer-related gene and internal reference GAPDH, wherein the concentration of upstream and downstream primers of each gene and internal reference GAPDH is 0.2 μM-0.6 μM / reaction, and the concentration of each gene and internal reference GAPDH probe is 0.2 μΜ-0.6μΜ / reaction; test sample DNA template: 3-10μL; positive quality control: 3-10μL; negative quality control: 3-10μL.

9. A liver cancer auxiliary diagnosis kit according to claim 8, characterized in that: The negative quality control product is DNase-free water or human genomic DNA converted by bisulfite, and the positive quality control product is human hepatocellular carcinoma cell genomic DNA converted by bisulfite.

10. A method for detecting methylation of multiple liver cancer-related genes in plasma, characterized in that: include: Using the composition described in any one of claims 1 to 5 to detect the methylation content of the target gene in the sample; The target genes include Any two or more genes among TSPYL5, CLEC11A, LRRC4 and KCNA3; the detection is not for the purpose of diagnosis or treatment of the disease.

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