Molecular marker methylation level detection reagent for primary hepatocellular carcinoma and its application

Abstract

The present invention discloses the application of a molecular marker methylation level detection reagent for primary hepatocellular carcinoma in the preparation of a detection reagent product, and belongs to the field of gene detection technology. By screening the methylation level of the CpG site in the detected region and its reverse complementary sequence, it is identified whether the target sample is from a primary hepatocellular carcinoma patient. The methylation level of the CpG site in the above region has good sensitivity and specificity for the screening or auxiliary diagnosis of primary hepatocellular carcinoma, and can be used to distinguish samples from patients with primary hepatocellular carcinoma, samples from patients with benign liver diseases (cirrhosis, hepatitis, etc.) and samples from healthy people. The detection reagent and application of the methylation level of the molecular marker can improve the detection rate of early primary hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and specifically relates to a molecular marker methylation level detection reagent for primary hepatocellular carcinoma and an application of the same in preparing a detection reagent product. Background Art

[0002] Liver cancer mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular cholangiocarcinoma (cHCC-CCA). The three types differ greatly in pathogenesis, biological behavior, pathological histology, treatment methods and prognosis, among which HCC accounts for 75% to 85% and ICC accounts for 10% to 15%.

[0003] Primary liver cancer is a malignant tumor that originates in the liver, of which hepatocellular carcinoma (HCC) is the most common type. For example, in 2022, 9.515 million people were diagnosed with primary hepatocellular carcinoma worldwide, and 8.757 million people died from primary hepatocellular carcinoma. In addition, the 5-year survival rate of 43% of patients diagnosed with localized primary hepatocellular carcinoma was 35%. The number of people recommended for early screening of primary hepatocellular carcinoma increased from 507.7 million in 2018 to 526.9 million in 2022, and is expected to further increase to 571.2 million in 2032.

[0004] At present, the hematological molecular markers for primary hepatocellular carcinoma recommended by the "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022)" are mainly serum AFP (Alpha-fetoprotein), but the sensitivity and specificity of AFP are only 58.2% and 85.3%, which is difficult to meet the clinical needs of early screening and early diagnosis of hepatocellular carcinoma. The American Association for the Study of Liver Diseases (AASLD) no longer uses AFP as a necessary indicator for screening and diagnosis of primary hepatocellular carcinoma. Abnormal prothrombin (Protein induced by vitamin Kabsence / antagonist-Ⅱ, PIVKAⅡ; Des-gamma carboxyprothrombin, DCP), plasma free microRNA (microRNA, miRNA) and serum alpha-fetoprotein isomers (Lens culinaris agglutinin-reactive fraction of AFP, AFP-L3) can also be used as early diagnostic markers for primary hepatocellular carcinoma. Des-γ-carboxyprothrombin (DCP) as a supplement to AFP has a certain diagnostic value for AFP-negative primary hepatocellular carcinoma. As a new marker for primary hepatocellular carcinoma, ctDNA is a specific mutant DNA fragment released by the tumor into the peripheral blood, which can reflect the genomic information of the tumor and can be used for early diagnosis, monitoring tumor progression and response to treatment. The sensitivity and specificity of ctDNA methylation detection technology for early diagnosis of primary hepatocellular carcinoma are better than serum AFP, and it can also reflect the dynamic changes of primary hepatocellular carcinoma after surgery.

[0005] Currently, there is a lack of a blood test with high sensitivity and specificity for primary hepatocellular carcinoma. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker methylation level detection reagent for primary hepatocellular carcinoma for use in the preparation of a detection reagent product. The molecular marker methylation level detection reagent has good sensitivity and specificity for the screening and auxiliary diagnosis of primary hepatocellular carcinoma, and can be used to distinguish samples of primary hepatocellular carcinoma patients from samples of patients with benign liver diseases (cirrhosis, hepatitis, etc.) and healthy people. The liver cancer referred to above refers only to hepatocellular carcinoma "HCC".

[0007] A molecular marker methylation level detection reagent for primary hepatocellular carcinoma, wherein the molecular marker contained in the detection reagent is a CpG methylation site in the chr5:42992282-42992764 genomic region;

[0008] The molecular markers are located in the following regions containing at least one CpG site: region 1, region 2, region 3, region 4 and region 5;

[0009] The region 1 is Chr5: 42992282-42992514; the region 2 is Chr5: 42992299-42992414; the region 3 is Chr5: 42992451-42992557; the region 4 is Chr5: 42992522-42992764; the region 5 is Chr5: 42992565-42992764;

[0010] The genomic region is the hg38 human genome.

[0011] Experimental verification found that by detecting the methylation level of CpG sites in the chr5:42992282-42992764 region (with the hg38 genome as the reference genome), it is possible to identify whether the target sample is from a patient with primary hepatocellular carcinoma. If the target sample is from a patient with primary hepatocellular carcinoma, the CpG sites in the above region will show a high methylation level. If the target sample is from a patient with benign liver disease (cirrhosis, hepatitis, etc.) or a healthy person, the CpG sites in the above region will show a low methylation level.

[0012] Application of a molecular marker methylation level detection reagent for primary hepatocellular carcinoma in the preparation of a detection reagent product.

[0013] Preferably, the detection reagent product includes one or more combinations of nucleic acid combination 1 for detecting the area 1, nucleic acid combination 2 for detecting the area 2, nucleic acid combination 3 for detecting the area 3, nucleic acid combination 4 for detecting the area 4 and nucleic acid combination 5 for detecting the area 5.

[0014] Preferably, the nucleic acid combination 1 includes primer combination 1, the nucleic acid combination 2 includes primer combination 2, the nucleic acid combination 3 includes primer combination 3, the nucleic acid combination 4 includes primer combination 4, and the nucleic acid combination 5 includes primer combination 5;

[0015] The base sequence of the primer combination 1 has at least 90% identity with the base sequence shown in SEQ ID NO. 1-2;

[0016] The base sequence of the primer combination 2 has at least 90% identity with the base sequence shown in SEQ ID NO. 4-5;

[0017] The base sequence of the primer combination 3 has at least 90% identity with the base sequence shown in SEQ ID NO. 7-8;

[0018] The base sequence of the primer combination 4 has at least 90% identity with the base sequence shown in SEQ ID NO. 10-11;

[0019] The base sequence of the primer combination 5 has at least 90% consistency with the base sequence shown in SEQ ID NO. 13-14.

[0020] Preferably, the nucleic acid combination 1 further includes probe 1, the nucleic acid combination 2 further includes probe 2, the nucleic acid combination 3 further includes probe 3, the nucleic acid combination 4 further includes probe 4, and the nucleic acid combination 5 further includes probe 5;

[0021] The base sequence of the probe 1 has at least 90% identity with the base sequence shown in SEQ ID NO.3;

[0022] The base sequence of the probe 2 has at least 90% identity with the base sequence shown in SEQ ID NO.6;

[0023] The base sequence of the probe 3 has at least 90% identity with the base sequence shown in SEQ ID NO.9;

[0024] The base sequence of the probe 4 has at least 90% identity with the base sequence shown in SEQ ID NO. 12;

[0025] The base sequence of the probe 5 has at least 90% identity with the base sequence shown in SEQ ID NO.15.

[0026] Preferably, the detection reagent product includes any one of a detection reagent, a detection kit, a detection chip and a sequencing library.

[0027] In a preferred embodiment of the present invention, the above-mentioned substance is a nucleic acid combination for detecting the methylation level of molecular markers for screening or auxiliary diagnosis of primary hepatocellular carcinoma.

[0028] In a preferred embodiment of the present invention, a nucleic acid molecule containing at least one CpG site in any detection region can be selected, or a combination of nucleic acid molecules in several detection regions each containing at least one CpG site can be selected. As long as the molecular marker is from the above region, it is within the protection scope of the present invention.

[0029] In a preferred embodiment of the present invention, the 5' end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a fluorescent quenching group.

[0030] The fluorescent reporter group is HEX, FAM, TET, CF532, JOE, TAMRA, ROX, CY3, CY5, Texas Red, NED, Alexa Flour or VIC, and the quencher group is MGB, TAMRA, BHQ1, BHQ2, BHQ3 or QSY.

[0031] In the reagent product, the reagent can be in the form of freeze-dried powder, solution, suspension, emulsion, etc.

[0032] The methylation level of the molecular marker is detected by at least one of the following methods: methylation-specific PCR method, sequencing method, methylation-specific high-performance liquid chromatography method, digital PCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction endonuclease method and flap endonuclease method.

[0033] The sequencing method is bisulfite sequencing, whole genome methylation sequencing or pyrosequencing.

[0034] The kit of the present invention also comprises a positive control, a negative control, a detection primer of an internal reference gene, a detection probe of an internal reference gene, a DNA polymerase and a buffer.

[0035] The detection samples of the above kit are tissue samples, blood samples or ascites samples, and other body fluid samples such as saliva, urine, etc.; the blood samples are ex vivo plasma samples, ex vivo serum samples, ex vivo whole blood samples or ex vivo blood cell samples.

[0036] A primary hepatocellular carcinoma detection kit, comprising the nucleic acid combination.

[0037] Preferably, the detection sample of the kit is an in vitro blood sample or tissue sample.

[0038] Compared with the prior art, the present invention has at least the following technical effects:

[0039] (I) The present invention provides a detection reagent and application for detecting the methylation level of molecular markers for screening and auxiliary diagnosis of primary hepatocellular carcinoma. The present invention identifies whether the target sample is from a primary hepatocellular carcinoma patient by detecting the methylation level of CpG sites in the chr5:42992282-42992764 region and its reverse complementary sequence. The methylation level of the CpG sites in the above region has good sensitivity and specificity for screening or auxiliary diagnosis of primary hepatocellular carcinoma, and can be used to distinguish samples from patients with primary hepatocellular carcinoma, samples from patients with benign liver diseases (cirrhosis, hepatitis, etc.) and samples from healthy people.

[0040] (ii) The present invention provides a method for early screening and early diagnosis of primary hepatocellular carcinoma that is highly accurate, simple, non-invasive, safe, fast and economical.

[0041] (III) The detection reagents and applications of the methylation levels of molecular markers provided by the present invention can improve the detection rate of primary hepatocellular carcinoma in people at high risk of hepatocellular carcinoma and the general population, especially the detection rate of early primary hepatocellular carcinoma, so as to realize early detection, early diagnosis and early treatment, improve the survival rate of patients with primary hepatocellular carcinoma, reduce the mortality rate of patients with hepatocellular carcinoma, and reduce medical expenses.

[0042] (IV) The methylation level of the CpG sites in the detection region disclosed in the present invention has good sensitivity and specificity for the screening and auxiliary diagnosis of primary hepatocellular carcinoma, and can be used to distinguish samples from patients with primary hepatocellular carcinoma from patients with benign liver diseases (cirrhosis, hepatitis, etc.) and healthy people.

[0043] (V) The present invention provides excellent molecular markers for the screening and auxiliary diagnosis of primary hepatocellular carcinoma, and provides a reagent for detecting the methylation level of molecular markers with high accuracy, non-invasiveness, convenience, safety and rapidity for the screening and auxiliary diagnosis of primary hepatocellular carcinoma.

[0044] (VI) The DNA methylation markers and compositions used in the detection kit provided by the present invention are screened from genomic regions with a coverage rate 10 times that of the 450K methylation chip. In the sample data set used in the present invention, the detection performance of the screened markers and compositions is optimal. The methylation sequencing used in the marker screening process is based on the enzymatic conversion method to convert cytosine to uracil. Compared with the traditional bisulfite conversion, it can avoid damage to DNA, thereby solving the problem of low free DNA content in blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a flow chart of screening methylation markers for detecting liver cancer in Example 1;

[0046] Figure 2 This is the ROC diagram of the detection of the methylation marker in Example 3 in the ex vivo blood sample of liver cancer. DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0048] The term "methylation level" is the same as the general understanding, which refers to whether the cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency / proportion / percentage of methylation. It represents both qualitative and quantitative concepts. For example, if the cytosine (C) residue in the nucleic acid sequence is methylated, it can be called "highly methylated" or have "increased methylation". In practical applications, different detection indicators can be used to compare DNA methylation levels according to actual conditions. For example, in some cases, the comparison can be based on the Ct value of the sample detection. In some cases, the methylation ratio of the marker in the sample can be calculated, that is, the number of methylated molecules / (the number of methylated molecules + the number of non-methylated molecules) × 100, and then compared. In some cases, it is also necessary to statistically analyze and integrate the various indicators to obtain the final judgment indicator.

[0049] Example 1: Screening process for liver cancer detection methylation markers, such as Figure 1 As shown,

[0050] This embodiment discloses a methylation marker composition for detecting early liver cancer and a screening process thereof.

[0051] In order to discover and screen methylation markers with excellent detection performance, this example first used a customized hybridization capture large panel for targeted methylation sequencing.

[0052] This hybridization capture panel covers 3.98 million CpG methylation sites in the human genome, including most of the CpG island regions and genes related to cancer occurrence, with a coverage range 10 times that of traditional DNA methylation chips.

[0053] By performing targeted sequencing on 82 clinical frozen tissue samples and 115 ex vivo blood samples and comparing the changes in methylation patterns between cancer and non-cancer groups, this example preliminarily identified 30,008 methylation sites that can clearly distinguish cancer and non-cancer samples in both tissue and blood samples as potential cancer markers.

[0054] Furthermore, a hybrid capture panel was designed based on the 30,008 methylation sites initially screened above for the second round of targeted methylation sequencing. This round of sequencing adopted a deep sequencing strategy, with an average sequencing depth of 500X, which significantly improved the accuracy of methylation level calculation and facilitated the accurate screening of markers in the second round.

[0055] By performing targeted methylation sequencing on ex vivo plasma samples from 218 clinical liver cancer patients and 196 ex vivo plasma samples from non-cancer groups, univariate statistical analysis and machine learning methods were used to accurately screen out 12 DNA methylation markers with the best performance for liver cancer detection.

[0056] Finally, primer and probe nucleic acid combinations of the qPCR detection platform were designed for the genomic regions of the 12 screened methylation markers, and the performance of liver cancer detection was verified on the qPCR platform using ex vivo plasma samples from 32 clinical liver cancer patients and 47 ex vivo plasma samples from non-cancer groups.

[0057] Finally, the DNA methylation marker with the best performance for liver cancer detection was determined through univariate performance analysis, namely the methylation level of the chr5:42992282-42992764 genomic region.

[0058] The primer combination for detecting the methylation level of the marker includes any one of SEQ ID NO.1-2, SEQ ID NO.4-5, SEQ ID NO.7-8, SEQ ID NO.10-11 and SEQ ID NO.13-14.

[0059] The nucleic acid probe used for detection includes any one of SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO.15.

[0060] Example 2: Detection performance of the screened markers in liver cancer tissue samples

[0061] 33 clinical samples of liver cancer tissue and 49 non-cancer control tissue samples were collected. The tissue samples were subjected to genome extraction, enzyme-based methylation conversion, and targeted methylation sequencing. The specific experimental steps are as follows:

[0062] 1. Extraction of DNA samples: When the sample is a frozen fresh ex vivo tissue sample, use QIAGEN's "DNeasy® Blood&Tissue Kit #69506" to extract the genome. For specific operations, please refer to the kit instructions.

[0063] Genomic DNA was sheared using a Covaris M220 ultrasonic shearer. When cfDNA was extracted from ex vivo plasma samples, the Guangzhou Youze Biotechnology "Free DNA Extraction Kit (Filtration Method) #C02-1" was used. For specific operations, please refer to the kit instructions.

[0064] 2. Use SPRIselect magnetic beads to screen the extracted DNA samples according to the recommended steps in the instructions, and filter out DNA molecules larger than 200 bp.

[0065] 3. Quality control preparation: 10 µL of 0.1 ng / µL CpG methylated pUC19 and 10 µL of 2 ng / µL unmethylated λ DNA should be added to each sample;

[0066] 4. Library construction for methylation sequencing: Use the NEBNext Enzymatic Methyl-Seq kit to construct the library.

[0067] 4.1 End repair and 3' end addition of "A"

[0068] 4.1.1 Take 50 ng of the sample to be tested, dilute it to 50 μL with NF water, and then add the following reagents for reaction.

[0069]

[0070] 4.1.2 Place in PCR instrument and perform reaction according to the following procedure

[0071]

[0072] 4.2 Connector connection

[0073] 4.2.1 Add the following reagents to the end repair product for reaction

[0074]

[0075] 4.2.2 Place in a PCR instrument and set the PCR reaction program according to the following conditions: 60°C, 60 min, with the heated lid closed.

[0076] 4.2.3 Purification of adapter ligation products

[0077] The purified magnetic beads were equilibrated at room temperature for 30 min and fully mixed on a vortexer.

[0078] Prepare 80% ethanol according to the ratio of anhydrous ethanol: nuclease-free water = 8:2 and set aside.

[0079] Add 55 μL of purified magnetic beads to the ligation products respectively, mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack, discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, repeat this step once; dry, elute with 15 μL elution buffer, and aspirate 14 μL of supernatant for the next reaction.

[0080] 4.3 Methylation: Oxidation reaction

[0081] 4.3.1 Buffer configuration:

[0082] Add 400 μL of E1 reaction buffer to 100 μL of E1 reaction buffer supplement, shake and mix, and mark the preparation date.

[0083] Take 10 μL of 500 mM Fe (II) and add it to 1249 μL of nuclease-free water. The diluted solution should be used immediately and should not be stored.

[0084] Dilute the Stop Buffer 1:10 with nuclease-free water.

[0085] 4.3.2 Add the following reagents to 14 μL of the purified ligation product and mix well. Then add 10 μL of diluted Fe(II) to the purified ligation product to which the oxidase has been added, mix well and centrifuge.

[0086]

[0087] 4.3.3 Place the tube in a PCR instrument and react under the following conditions: 37°C, 1h, with the cover temperature ≥ 45°C.

[0088] 4.3.4 After the reaction is completed, add 1 μL of the diluted reaction stop solution to the product and place it on the PCR instrument for reaction under the following conditions: 37°C, 30 min, and the temperature of the heated cover ≥ 45°C.

[0089] 4.3.5 Purification of oxidation reaction products

[0090] The purified magnetic beads were equilibrated at room temperature for 30 min and fully mixed on a vortexer;

[0091] Prepare 80% ethanol in a ratio of 8:2 for use;

[0092] Add 45 μL of NEB Next Sample purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 9.5 μL of elution buffer, and aspirate 8 μL of supernatant for the next reaction.

[0093] 4.4 Methylation: Cytosine Deamination

[0094] 4.4.1 Denaturation:

[0095] Preheat the PCR instrument to 85°C in advance and open the heated lid;

[0096] Add 2 μL of formamide to 8 μL of the purified oxidation reaction product, vortex to mix, and centrifuge briefly.

[0097] 4.4.2 Add the following reagents to the denatured product for reaction

[0098]

[0099] 4.4.3 Place in PCR instrument and react under the following conditions: 37℃, 3h, heating cover temperature ≥45℃.

[0100] 4.4.4 Purification of Cytosine Deamination Reaction Products

[0101] The purified magnetic beads were equilibrated at room temperature for 30 min and fully mixed on a vortexer;

[0102] Prepare 80% ethanol in a ratio of 8:2 for use;

[0103] Add 50 μL of NEB Next Sample purified magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack, and discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, and repeat this step once; dry, elute with 11 μL of elution buffer, and aspirate 10 μL of supernatant for the next reaction.

[0104] 4.5 PCR amplification and purification

[0105] 4.5.1 Add the following reagents to the above purified product for reaction

[0106]

[0107] 4.5.2 Place on PCR instrument and react according to the following conditions

[0108]

[0109] 4.5.3 Purification of PCR amplification products

[0110] The purified magnetic beads were equilibrated at room temperature for 30 min and fully mixed on a vortexer;

[0111] Prepare 80% ethanol in a ratio of 8:2 for use;

[0112] Add 22.5 μL of NEB purified magnetic beads to the ligation products respectively, mix by pipetting, incubate at room temperature for 5 min, place on a magnetic rack, discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, repeat this step once; dry, elute with 15 μL of elution buffer, aspirate 14 μL of supernatant, and conduct quality inspection.

[0113] 5. Hybridization and elution

[0114] 5.1 Hybridization:

[0115] Take 10 ng of each library to be hybridized, calculate the library volume required for combined hybridization, and mix the 12 libraries together;

[0116] Add the following reagents to the library mixture for reaction;

[0117] Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature;

[0118] Immediately take 20 μL of hybridization mixture and add it to the concentrated dry powder, mix well and let it stand at room temperature for 5 minutes, then add 30 μL of hybridization enhancement solution, mix well and centrifuge;

[0119] Place on a PCR instrument and perform the reaction under the following conditions.

[0120] Reagent composition table

[0121]

[0122] PCR reaction conditions table

[0123]

[0124] 5.2 Capture and elution

[0125] 5.2.1 Buffer preheating:

[0126] Preheat the rapid binding solution and rapid washing solution 2 at 48°C until the precipitate is dissolved, and preheat the rapid washing solution 1 at 63°C until the precipitate is dissolved. Equilibrate the streptavidin magnetic beads at room temperature for at least 30 minutes.

[0127] 5.2.2 Magnetic bead capture

[0128] Vortex to mix the streptavidin beads that have been equilibrated to room temperature;

[0129] Take 70 μL of streptavidin magnetic beads and add them to a 1.5 mL centrifuge tube;

[0130] Add 20 μL of binding buffer, mix by pipetting, centrifuge briefly, place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0131] Repeat steps b and c twice, performing streptavidin magnetic bead washing three times in total;

[0132] Add 20 μL of binding buffer to resuspend the streptavidin magnetic beads;

[0133] Transfer 20 μL of resuspended streptavidin magnetic beads to the hybridization reaction tube, and then transfer all the liquid to a 1.5 mL centrifuge tube.

[0134] At room temperature, mix on a mixer for 30 minutes without vortexing.

[0135] After mixing, centrifuge instantly, place on a magnetic stand, let stand for 1 min, and discard the supernatant.

[0136] 5.2.3 Elution

[0137] Add 200 μL of 54°C preheated rapid wash buffer 1, pipette to mix, and incubate in a thermostatic metal bath at 54°C for 5 min;

[0138] After the incubation is completed, centrifuge briefly and transfer all the liquid to a new 1.5 mL centrifuge tube to remove the non-specific capture fragments bound to the surface of the centrifuge tube;

[0139] Place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0140] Add 200 μL of 63°C preheated washing buffer 1, pipette to mix, and incubate in a thermostatic metal bath at 54°C for 5 min;

[0141] After incubation, centrifuge immediately, place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0142] Add 200 μL of 54°C preheated washing buffer 2, pipette to mix, and incubate in a thermostatic metal bath at 54°C for 5 min;

[0143] After incubation, centrifuge immediately, place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0144] Repeat steps a to g twice for a total of three washes;

[0145] Centrifuge briefly, discard the remaining supernatant with a 10 μL pipette tip, immediately add 45 μL of nuclease-free water, mix by pipetting, and incubate on ice.

[0146] 5.3 PCR enrichment of capture products

[0147] 5.3.1 Take 22.5 μL of the captured product (streptavidin magnetic bead suspension) and add the following reagents to react.

[0148]

[0149] Place on PCR instrument and react according to the following conditions

[0150]

[0151] 5.3.2 PCR product purification

[0152] The DNA purification magnetic beads were equilibrated at room temperature for 30 min and mixed thoroughly on a vortex machine;

[0153] Prepare 80% ethanol in a ratio of 8:2 for use;

[0154] Add 90 μL of DNA purification magnetic beads to each ligation product, mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack, discard the supernatant after the solution is clarified; wash with freshly prepared 80% ethanol, discard the supernatant, repeat this step once; dry, elute with 32 μL of elution buffer, aspirate 30 μL of supernatant for quality control, and sequence on the machine.

[0155] 6. Analysis of sequencing data:

[0156] The raw data was first filtered using fastQC to filter low-quality sequences (phred33 score ≤ 20) and adapter sequences and polyA / T sequences in the reads. The filtered high-quality reads were mapped to the hg38 human genome using BSMAP, and reads with high mapping quality were screened out. After removing PCR duplicates using picard, reads mapped to the target genomic region (the region corresponding to the DNA methylation marker) were selected using samtools and the methylation levels of each DNA methylation marker were calculated.

[0157] The detection performance of the determined methylation markers and the combination for distinguishing liver cancer tissues from non-cancer tissues is shown in the following table:

[0158]

[0159] Example 3: Detection performance of the screened markers in liver cancer blood samples

[0160] 414 clinical blood samples were collected, including 218 blood samples from patients with primary liver cancer, 173 blood samples from patients with benign liver diseases, and 23 blood samples from healthy people. The blood samples were subjected to free DNA extraction, methylation conversion, and targeted methylation sequencing according to the method described in Example 2, and the methylation level of each methylation marker was calculated.

[0161] Blood sample statistical results

[0162]

[0163] A univariate statistical analysis was performed on the single methylation markers to calculate the detection performance of the ex vivo blood samples for distinguishing liver cancer patients from the control group. The detection sensitivity and specificity of the determined methylation markers in liver cancer blood samples are shown in the following table:

[0164]

[0165] like Figure 2As shown, the results showed that the AUC value of the determined methylation marker for methylation sequencing detection of liver cancer ex vivo blood samples was 0.927.

[0166] Example 4: Performance of the identified methylation markers in a liver cancer ex vivo blood sample qPCR detection dataset

[0167] The primer probe combination required for the methylation qPCR detection platform was designed for the identified chr5:42992282-42992764 biomarker. The design results are as follows:

[0168]

[0169] A total of 79 clinical blood samples were collected, including 32 blood samples from patients with primary liver cancer, 32 blood samples from patients with benign liver diseases, and 15 blood samples from healthy people.

[0170]

[0171] The 79 blood samples collected above were subjected to quantitative methylation qPCR detection, and the qPCR detection Ct values ​​were recorded. The specific experimental steps are as follows:

[0172] 1. Separation of plasma: First, centrifuge the collected 10 mL clinical blood sample at 1600×g for 10 minutes to separate the plasma. Centrifuge the separated plasma at 16000×g for 10 minutes to separate impurities. The purified plasma sample should be stored in a -80℃ refrigerator for future use.

[0173] 2. Free DNA extraction: Use the "Free DNA Extraction Kit" (Cat. No.: C03-2) of Guangzhou Youze Biotechnology Co., Ltd. to extract plasma free DNA from the separated plasma samples.

[0174] 3. Free DNA conversion: Use ZYMO's "EZ-96 DNA Methvlation-Lightning MagPrep" to perform methylation C->T conversion on the extracted free DNA samples.

[0175] 4. Negative and positive controls for methylation quantitative PCR: The negative control was the genomic DNA of the cell line (cell line: QSG-7701) transformed by the ZYMO kit, with a concentration of 1 ng / μL. After transformation, the C in the negative control was changed to T. The positive control was the genomic DNA of the cell line (cell line: HepG2) transformed by bisulfite using the ZYMO kit, with a concentration of 1 ng / μL. After transformation, the C at the methylated CpG position did not change, and the C at other positions was changed to T.

[0176] 5. Reaction system for methylation quantitative PCR:

[0177]

[0178] 6. Reaction procedure of methylation quantitative PCR:

[0179]

[0180] 7. On-machine detection: Mix the sample DNA to be tested, negative reference, and positive reference according to the reaction system of methylation quantitative PCR in step 5. Put the sample into Shanghai Hongshi SLAN-96S quantitative PCR instrument, and set up the quantitative PCR instrument according to the program conditions in step 6 for detection, and collect the fluorescence values ​​of each channel of HEX / ROX / FAM / CY5.

[0181] 8. Result recording: After the methylation quantitative PCR reaction is completed, the threshold is set at the inflection point of the S-type amplification curve to obtain the Ct value of the sample to be tested at the methylation site.

[0182] 9. Quality control: Negative and positive references are tested simultaneously in each test. The negative control is the transformed cell genomic DNA (cell line: QSG-7701) containing the β-actin gene and the low-methylation target gene sequence, and the positive control is the transformed cell cfDNA (cell line: HepG2) containing the β-actin gene and the high-methylation target gene sequence, both at a concentration of 1ng / μL. When the negative reference is not amplified, the Ct value of the positive reference is between 28-32, and the Ct value of the internal reference gene is less than 38, the experiment is valid and the next step of sample result determination can be carried out. Otherwise, the result is invalid and the test must be repeated.

[0183] 10. Result determination: If the Ct value of the target marker is less than 35, the test result is positive, otherwise the test result is negative.

[0184] The Ct values ​​of qPCR detection in ex vivo blood samples using the designed primer-probe combination were used to distinguish liver cancer and non-cancer samples. The detection performance is shown in the following table.

[0185]

[0186] From the blood qPCR test results, it can be seen that the liver cancer detection performance of different primer probe combinations designed for the chr5:42992282-42992764 marker is similar, with a specificity of 89.4-91.5% and a sensitivity of 78.1-84.4%. Compared with traditional liver cancer screening methods, this single methylation marker has ideal detection performance, is easy to use and has low cost, which is of great significance for the popularization and promotion of early screening and treatment of liver cancer.

[0187] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a molecular marker methylation level detection reagent in the preparation of a primary hepatocellular carcinoma detection reagent product, characterized in that: The molecular marker contained in the molecular marker methylation level detection reagent is a CpG methylation site in the chr5:42992282-42992764 genomic region; The molecular markers are located in the following regions containing a CpG site: region 1, region 2, region 3, region 4 and region 5; The region 1 is Chr5: 42992282-42992514; the region 2 is Chr5: 42992299-42992414; the region 3 is Chr5: 42992451-42992557; the region 4 is Chr5: 42992522-42992764; the region 5 is Chr5: 42992565-42992764; The genomic region is the hg38 human genome; The detection reagent includes a nucleic acid combination 1 for detecting the region 1, a nucleic acid combination 2 for detecting the region 2, a nucleic acid combination 3 for detecting the region 3, a nucleic acid combination 4 for detecting the region 4, and a nucleic acid combination 5 for detecting the region 5; The nucleic acid combination 1 includes a primer combination 1, the nucleic acid combination 2 includes a primer combination 2, the nucleic acid combination 3 includes a primer combination 3, the nucleic acid combination 4 includes a primer combination 4, and the nucleic acid combination 5 includes a primer combination 5; The base sequences of the primer combinations 1-5 are shown in SEQ ID NOs. 1-2, 4-5, 7-8, 10-11, and 13-14, respectively; The nucleic acid combination 1 further includes probe 1, the nucleic acid combination 2 further includes probe 2, the nucleic acid combination 3 further includes probe 3, the nucleic acid combination 4 further includes probe 4, and the nucleic acid combination 5 further includes probe 5; The base sequences of the probes 1-5 are shown in SEQ ID NOs. 3, 6, 9, 12, and 15, respectively.

2. An application according to claim 1, characterized in that: The detection reagent product includes any one of a detection reagent, a detection kit, a detection chip and a sequencing library.

3. An application according to claim 1, characterized in that: The test sample is an in vitro blood sample or tissue sample.

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