A marker composition and kit for liver cancer detection or auxiliary diagnosis

Abstract

The present invention discloses a marker composition and a kit for liver cancer detection or auxiliary diagnosis. The three markers and combinations used in the present invention are obtained by screening from a hybrid capture panel with a methylation site coverage that is 10 times higher than that of a traditional methylation chip. In the sample set used in the present invention, the screening markers and the detection performance of the combination are optimal; the methylation detection kit of the screening marker combination can achieve higher sensitivity and specificity to distinguish liver cancer, liver disease and healthy people, and has the advantages of being simple, convenient, sensitive, non-invasive and having diversified samples. It can be used as an early screening for liver cancer, and the sensitivity and specificity of liver cancer detection are greatly improved, which is more beneficial for realizing early screening and diagnosis of liver cancer and real-time monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a marker composition and a kit thereof for liver cancer detection or auxiliary diagnosis. Background Art

[0002] Liver cancer is a type of malignant tumor with a high incidence rate, and hepatocellular carcinoma is its main histopathological type. Most liver cancer patients have a history of hepatitis B or cirrhosis, but because there are no obvious symptoms in the early stages of liver cancer and screening awareness is not popular among high-risk groups, most of them are in the middle and late stages when they seek medical treatment. The 5-year survival rate of patients with early liver cancer is over 50%, while the 5-year survival rate of patients with advanced liver cancer is less than 20%. Therefore, early detection and early diagnosis and treatment are a key factor in improving the survival rate of liver cancer patients.

[0003] At present, ultrasound combined with serum alpha-fetoprotein (AFP) testing is the most widely used liver cancer screening technology. According to actual clinical practice and domestic and foreign reports, the sensitivity of AFP in diagnosing liver cancer is 25%~65%, and the specificity is 80%~94%, especially in the diagnosis of small liver cancer and early liver cancer. The false negative rate is high. The sensitivity of ultrasound in diagnosing liver cancer is 84%, but the sensitivity and specificity for early liver cancer are only 63% and 45%. Summary of the invention

[0004] Based on peripheral blood free DNA, genetic and epigenetic changes (somatic mutations, copy number variations and methylation changes) of tumor cells can be detected, and abnormal methylation is better than mutation and copy number variation in distinguishing tissue specificity. The purpose of the present invention is to provide a marker composition for liver cancer detection or auxiliary diagnosis, by using a large panel capture sequencing for liver cancer, paired adjacent cancer and liver disease tissue samples, the large panel covers 10 times the methylation sites of traditional methylation chips, and screens out 30,008 methylation change sites of genes related to liver cancer, forming a capture panel of about 480k in size, using the capture panel to screen out three methylation regions with obvious methylation differences in liver cancer, liver disease and healthy plasma samples, and after combination, higher sensitivity and specificity are obtained to distinguish liver cancer, liver disease and healthy people.

[0005] The present invention is achieved through the following technical solutions:

[0006] A marker composition for liver cancer detection or auxiliary diagnosis, the marker composition is composed of three markers;

[0007] The positions of each marker in the marker composition on the reference genome are as follows:

[0008] Marker 1, located in the entire region or part of chr17:30970348-30970741;

[0009] Marker 2, located in the entire region or a portion of chr2:63054088-63054326;

[0010] Marker 3, located in the entire region or part of chr6:26250469-26250942;

[0011] The reference genome is the hg38 genome.

[0012] Preferably, the marker 1 is selected from the whole or partial region of the following region containing at least one CpG site: region 1 is located at chr17:30970348-30970428; region 2 is located at chr17:30970452-30970550; region 3 is located at chr17:30970629-30970741.

[0013] Preferably, the marker 2 is selected from the whole or partial region of the following region containing at least one CpG site: region 4 is located at chr2:63054088-63054212; region 5 is located at chr2:63054112-63054305; region 6 is located at chr2:63054194-63054326.

[0014] Preferably, the marker 3 is selected from the entire region or a partial region of the following region containing at least one CpG site: region 7 is located at chr6:26250469-26250577; region 8 is located at chr6:26250563-26250692; region 9 is located at chr6:26250594-26250753; region 10 is located at chr6:26250594-26250753.

[0015] A marker composition for liver cancer detection or auxiliary diagnosis is used in the preparation of liver cancer detection or auxiliary diagnosis products.

[0016] Preferably, the detection product is any one of a reagent, a kit, a chip, a hybridization probe and a sequencing library.

[0017] A kit for liver cancer detection or auxiliary diagnosis comprises a primer probe set of a marker composition and a sequence having a similarity of 90% or more to any sequence in the primer probe set of the marker composition.

[0018] Preferably, the primers and probes for detecting methylation of marker 1 include primer-probe combination 1 for detecting region 1, primer-probe combination 2 for detecting region 2, and primer-probe combination 3 for detecting region 3;

[0019] wherein primer-probe combination 1 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.1-3, primer-probe combination 2 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.4-6, and primer-probe combination 3 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.7-9;

[0020] The primers and probes for detecting methylation of marker 2 include primer-probe combination 4 for detecting region 4, primer-probe combination 5 for detecting region 5, and primer-probe combination 6 for detecting region 6;

[0021] wherein primer-probe combination 4 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.10-12, primer-probe combination 5 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.13-15, and primer-probe combination 6 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.16-18;

[0022] The primers and probes for detecting methylation of marker 3 include a primer-probe combination 7 for detecting region 7, a primer-probe combination 8 for detecting region 8, a primer-probe combination 9 for detecting region 9, and a primer-probe combination 10 for detecting region 10; wherein primer-probe combination 7 is an upstream primer sequence, a downstream primer sequence, and a probe sequence as shown in SEQ ID NOs.19-21, primer-probe combination 8 is an upstream primer sequence, a downstream primer sequence, and a probe sequence as shown in SEQ ID NOs.22-24, primer-probe combination 9 is an upstream primer sequence, a downstream primer sequence, and a probe sequence as shown in SEQ ID NOs.25-27, and primer-probe combination 10 is an upstream primer sequence, a downstream primer sequence, and a probe sequence as shown in SEQ ID NOs.28-30.

[0023] Preferably, the kit further comprises a PCR primer-probe combination of an internal reference gene β-actin, such as an upstream primer sequence, a downstream primer sequence and a probe sequence as shown in SEQ ID NOs. 31-33.

[0024] The kit also includes a positive control, a negative control, a DNA polymerase and a PCR amplification buffer.

[0025] Preferably, the test samples of the kit include tissue samples, blood samples or ascites samples; tissue samples are from fresh ex vivo pathological tissues, paraffin-embedded tissues or liver cancer cells, and blood samples are from ex vivo plasma, ex vivo serum, ex vivo whole blood or ex vivo blood cells.

[0026] The 5' ends of the above-mentioned probe sequences are all labeled with a fluorescent reporter group, and the 3' ends of the probe sequences are all labeled with a fluorescent quencher group.

[0027] The fluorescent reporter group is any one of CY3, CY5, HEX, FAM, TET, CF532, JOE, TAMRA, ROX, Texas Red, NED, Alexa Flour, and VIC; the quencher group is any one of MGB, TAMRA, QSY, BHQ1, BHQ2, and BHQ3.

[0028] In other embodiments, the fluorescent reporter group and quencher group of the probe label can be adjusted according to the conditions and are not limited to the types described above.

[0029] On the other hand, the present invention also provides a liver cancer detection kit, which not only comprises the above-mentioned marker and the primer-probe combination of the internal reference gene β-actin, but also comprises a positive quality control product and a negative quality control product.

[0030] The positive quality control material for markers 1-3 is the DNA of liver cancer cell line, and the negative quality control material is the DNA of BJ cell line.

[0031] The above-mentioned methylation detection methods include pyrophosphate sequencing, methylation sequencing, methylation-specific microarray method, methylation-specific PCR method, digital PCR method, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, methylation-specific high-performance liquid chromatography method and FEN1 endonuclease method.

[0032] The methylation described in the present invention can be understood as the methylation level, degree of methylation or methylation state, which refers to whether the 5th carbon atom on the cytosine in the CpG dinucleotide on a DNA sequence is methylated, and the proportion / frequency / percentage of methylation in the sum of methylation and non-methylation, which can be a qualitative concept or a quantitative concept. Those skilled in the art can use qualitative or quantitative methods to determine methylation according to actual conditions.

[0033] Compared with the prior art, the present invention has at least the following technical effects:

[0034] (i) The three markers and combinations used in the present invention are obtained by screening from a hybridization capture panel with a methylation site coverage that is 10 times higher than that of a traditional methylation chip. In the sample set used in the present invention, the detection performance of the screened markers and combinations is the best;

[0035] (ii) The present invention uses an enzymatic conversion method to convert cytosine into uracil in both tissue and plasma screening markers and combined hybridization capture sequencing experiments. Compared with traditional bisulfite conversion, it causes less damage to DNA and will not cause cfDNA breakage, thereby preserving the true methylation status of cfDNA;

[0036] (III) The methylation detection kit of the marker combination screened by the present invention can achieve higher sensitivity and specificity to distinguish liver cancer, liver disease and healthy people. It has the advantages of being simple, convenient, sensitive, non-invasive and having diverse samples. It can be used as an important auxiliary means for early screening and real-time monitoring of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a research and development flow chart for the analysis and screening of methylation markers for liver cancer detection;

[0038] Figure 2 It is the detection performance result of the screened methylation marker combination in distinguishing in vitro liver cancer plasma from non-cancer plasma. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1: This example discloses the process and experimental method for screening and detecting liver cancer markers. The specific process is as follows Figure 1 shown.

[0041] By performing large-panel capture sequencing on liver cancer tissue, paired adjacent cancer tissue and liver disease tissue, as well as liver cancer plasma, liver disease plasma and healthy human plasma, the hybridization capture panel covered 3.98 million CpG methylation sites in the human genome, including the vast majority of CpG island regions and genes related to cancer occurrence. The coverage is 10 times that of traditional DNA methylation chips. 30,008 differentially methylated sites of genes related to liver cancer were screened out to form a capture panel of about 480k in size. The capture panel was used to perform a second round of targeted methylation high-depth sequencing on liver cancer, liver disease and healthy plasma samples. The average sequencing depth of this round of sequencing was 500X. The LASSO and Boruta machine learning methods were used for screening, and cross-validation and random forest models were used to determine that the methylation differences were obvious, and after combination, three methylation regions that could clearly distinguish liver cancer, liver disease and healthy people were found, including marker 1 located at chr17:30970348-30970741; marker 2 located at chr2:63054088-63054326; marker 3 located at chr6:26250469-26250942.

[0042] The clinical samples that underwent methylation capture sequencing using the large panel and 480k capture panel were as follows:

[0043]

[0044] The specific experimental steps and analysis steps for screening markers in the above liver cancer tissue samples and plasma samples are as follows:

[0045] 1. Extract DNA from samples:

[0046] 1.1 Genomic DNA was extracted from frozen tissue using QIAGEN's DNeasy® Blood & Tissue Kit #69506. For specific operations, refer to the kit instructions. Genomic DNA was sheared using a Covaris M220 ultrasonic shearing instrument. After shearing, the main peak detected was about 150bp-200bp.

[0047] 1.2 Plasma was used to extract cfDNA using Guangzhou Youze Biotechnology's "Free DNA Extraction Kit (Filtration Method) #C02-1". For specific operations, please refer to the kit instructions;

[0048] 2. Use SPRIselect magnetic beads to screen the fragment size of the extracted DNA sample according to the instructions, and filter out DNA molecules with fragments larger than 200 bp;

[0049] 3. Add 10 µL of 0.1 ng / µL CpG methylated pUC19 and 10 µL of 2 ng / µL non-methylated λDNA to each sample as methylation quality control. Both quality control products are DNA that has been sheared using a Covaris M220 ultrasonic shearer and the fragments have been screened to about 200 bp.

[0050] 4. Use NEBNext Enzymatic Methyl-seq kit to perform enzymatic conversion and construct a methylated DNA library for sequencing;

[0051] 4.1 DNA end repair and 3' end addition of "A"

[0052] 4.1.1 Take 50 ng of the sample to be tested, dilute it to 50 μL with nuclease-free water, add the following reagents and place it in a PCR instrument for reaction.

[0053]

[0054] 4.1.2 The PCR procedure is as follows:

[0055]

[0056] 4.2 Connector connection

[0057] 4.2.1 Add the following reagents to the end-repair product and place it in a PCR instrument for reaction.

[0058]

[0059] 4.2.2 The PCR program was set to: 60°C, 60 min, with the heated lid closed.

[0060] 4.2.3 Purify the adapter ligation product. The specific steps of purification are as follows:

[0061] a) Prepare 80% ethanol by mixing anhydrous ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0062] b) The purified magnetic beads were equilibrated at room temperature for 30 minutes and mixed thoroughly on a vortex machine;

[0063] c) Add 55 μL of purified magnetic beads to the ligation product and mix by pipetting. Incubate at room temperature for 5 min, place on a magnetic rack and wait for the solution to become clear, then discard the supernatant.

[0064] d) Wash with 80% ethanol and discard the supernatant;

[0065] e) Repeat step d once, washing twice in total;

[0066] f) Dry, elute with 15 μL elution buffer, and take 14 μL of supernatant for the next reaction.

[0067] 4.3 Methyl oxidation reaction

[0068] 4.3.1 Buffer preparation, the specific steps are as follows:

[0069] a) Add 400 μL of E1 reaction buffer to 100 μL of E1 reaction buffer supplement, shake to mix and mark the preparation date;

[0070] b) Add 1249 μL of nuclease-free water to 10 μL of 500 mM Fe (II) for dilution. The diluted solution should be used immediately and cannot be stored.

[0071] c) Dilute the stop buffer with nuclease-free water at a ratio of 1:10.

[0072] 4.3.2 Add the following reagents to 14 μL of purified ligation product and mix well.

[0073]

[0074] 4.3.3 Add 10 μL of diluted Fe(II) to the purified ligation product to which oxidase has been added, mix well, centrifuge, and place on a PCR instrument for reaction. The PCR program is: 37°C, 1h, hot cover temperature ≥ 45°C;

[0075] 4.3.4 Add 1 μL of diluted reaction stop solution to the completed product and place it on a PCR instrument for reaction. The PCR program is: 37°C, 30 min, and the heating cover temperature is ≥ 45°C.

[0076] 4.3.5 Purify the oxidation reaction product. The specific steps are as follows:

[0077] a) Prepare 80% ethanol by adding 100 μL of anhydrous ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0078] b) The purified magnetic beads were equilibrated at room temperature for 30 minutes and mixed thoroughly on a vortex machine;

[0079] c) Add 45 μL of purified magnetic beads to the reaction product and mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic stand and wait for the solution to become clear, and discard the supernatant;

[0080] d) Wash with 80% ethanol and discard the supernatant;

[0081] e) Repeat step d once, washing twice in total;

[0082] f) Dry, elute with 9.5 μL elution buffer, and take 8 μL of supernatant for the next reaction.

[0083] 4.4 Methyl deamination reaction

[0084] 4.4.1 DNA denaturation: Add 2 μL of formamide to 8 μL of purified oxidation reaction product, vortex to mix, and centrifuge briefly. Place on ice immediately after denaturation;

[0085] 4.4.2 Perform the operation on ice and add the following reagents to the denatured product:

[0086]

[0087] 4.4.3 Preheat the PCR instrument to 85°C in advance, open the hot cover, and place the instrument in the PCR instrument for reaction. The PCR program is: 37°C, 3h, hot cover temperature ≥ 45°C;

[0088] 4.4.4 Purify the cytosine deamination reaction product. The specific steps are as follows:

[0089] a) Prepare 80% ethanol by adding 100uL of anhydrous ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0090] b) The purified magnetic beads were equilibrated at room temperature for 30 minutes and mixed thoroughly on a vortex machine;

[0091] c) Add 50 μL of purified magnetic beads to the reaction product and mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic stand and wait for the solution to become clear, and discard the supernatant;

[0092] d) Wash with 80% ethanol and discard the supernatant;

[0093] e) Repeat step d once, washing twice in total;

[0094] f) Dry, elute with 11 μL elution buffer, and take 10 μL of supernatant for the next reaction.

[0095] 4.4.5 PCR amplification and purification

[0096] 4.4.5.1 Add the following reagents to the purified reaction product:

[0097]

[0098] 4.4.5.2 Place on PCR instrument and react according to the following conditions

[0099]

[0100] 4.4.5.3 Purify the PCR amplification product. The specific steps are as follows:

[0101] 4.4.5.4 Purify the cytosine deamination reaction product. The specific steps are as follows:

[0102] a) Prepare 80% ethanol by adding 100uL of anhydrous ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0103] b) The purified magnetic beads were equilibrated at room temperature for 30 minutes and mixed thoroughly on a vortex machine;

[0104] c) Add 22.5 μL of purified magnetic beads to the reaction product and mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack and wait for the solution to become clear, and discard the supernatant;

[0105] d) Wash with 80% ethanol and discard the supernatant;

[0106] e) Repeat step d once, washing twice in total;

[0107] f) Dry, elute with 15 μL elution buffer, and aspirate 14 μL of supernatant for quality control.

[0108] 4.4.5.5 Hybridization

[0109] a) Take 10 ng of each library to be hybridized, calculate the library volume required for hybridization, and mix the 12 libraries together;

[0110] b) Add the following reagents to the library mixture;

[0111]

[0112] c) Turn on the vacuum concentrator, set the V-AQ mode, and concentrate into dry powder at room temperature;

[0113] d) Immediately take 20 μL of hybridization mixture and add it to the concentrated dry powder, mix well and let stand at room temperature for 5 min, then add 30 μL of hybridization enhancement solution, mix well and centrifuge;

[0114] e) Place in PCR instrument, PCR program is as follows:

[0115]

[0116] 6. Capture and elution

[0117] 6.1 Buffer Preheating

[0118] 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 to room temperature for at least 30 min;

[0119] 6.2 Magnetic Bead Capture

[0120] 1) Vortex the streptavidin magnetic beads that have been equilibrated to room temperature to mix;

[0121] 2) Take 70 μL of streptavidin magnetic beads and add them to a 1.5 mL centrifuge tube;

[0122] 3) 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;

[0123] 4) Repeat steps 2) and 3) twice, performing streptavidin magnetic bead washing three times in total;

[0124] 5) Add 20 μL binding buffer to resuspend the streptavidin magnetic beads;

[0125] 6) 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;

[0126] 7) Mix on a mixer at room temperature for 30 minutes without vortexing.

[0127] 8) After mixing, centrifuge briefly, place on a magnetic rack, let stand for 1 minute, and discard the supernatant.

[0128] 6.3 Elution

[0129] 1) Add 200 μL of 54°C preheated rapid wash buffer 1, mix well by pipetting, and incubate in a thermostatic metal bath at 54°C for 5 min;

[0130] 2) After incubation, centrifuge briefly and transfer all liquid to a new 1.5 mL centrifuge tube to remove nonspecific capture fragments bound to the surface of the centrifuge tube;

[0131] 3) Place on a magnetic stand, let stand for 1 min, and discard the supernatant;

[0132] 4) Repeat step 1);

[0133] 5) After mixing, centrifuge immediately, place on a magnetic stand, let stand for 1 minute, and discard the supernatant;

[0134] 6) 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;

[0135] 7) Repeat step 5);

[0136] 8) Repeat steps 1) to 7) twice for a total of three washes;

[0137] 9) Centrifuge briefly, discard the remaining supernatant, immediately add 45 μL of nuclease-free water, pipette to mix, and incubate on ice.

[0138] 6.4 PCR enrichment of capture products

[0139] 1) Take 22.5 μL of the captured product (streptavidin magnetic bead suspension) and add the following reagents

[0140]

[0141] 2) Place on the PCR instrument, and the PCR program is as follows:

[0142]

[0143] 3) PCR product purification

[0144] a) Prepare 80% ethanol by adding 100 μL of anhydrous ethanol and nuclease-free water in a ratio of 8:2 for later use;

[0145] b) The purified magnetic beads were equilibrated at room temperature for 30 minutes and mixed thoroughly on a vortex machine;

[0146] c) Add 90 μL of purified magnetic beads to the reaction product and mix well by pipetting, incubate at room temperature for 5 min, place on a magnetic rack and wait for the solution to become clear, and discard the supernatant;

[0147] d) Wash with 80% ethanol and discard the supernatant;

[0148] e) Repeat step d once, washing twice in total;

[0149] f) Dry, elute with 32 μL elution buffer, aspirate 30 μL of supernatant for quality control, and sequence.

[0150] 7. Use fastQC and Trimgalore to perform quality control on the raw data from the sequencing machine, and process the adapter sequences and polyA / T sequences; use BSMAP to post-pair to the hg38 human reference genome, and screen out reads with higher post-pair quality; use picard to remove PCR duplicate sequences, and use samtools to select reads that post-pair to the target genomic region (the region corresponding to the preset capture of DNA methylation) and calculate the methylation level of the target CpG site in the captured region.

[0151] 8. Combining a large amount of public data and 450k chip data in TCGA, we analyzed and screened out regions with consistent methylation differences between liver cancer and adjacent tissues, liver cancer and liver disease tissues, liver cancer plasma and liver disease plasma, and liver cancer plasma and healthy human plasma, forming a liver cancer-specific capture panel with a size of 480k.

[0152] 9. 480k specific panel targeted capture and sequencing of plasma sample methylated cfDNA library.

[0153] 10. After quality control, preprocessing, alignment and methylation level calculation of sequencing data, plasma samples were divided into training set and test set in a 5:5 ratio according to sample groups and different stages, and randomly divided 20 times. Lasso and Boruta machine learning methods were used for feature screening, and marker 1, marker 2, marker 3 and their combination were screened out based on cross-validation and random forest models.

[0154] The performance of the markers in tissue sequencing data is as follows:

[0155]

[0156] The performance of the markers in plasma sequencing data is as follows:

[0157]

[0158] The results are as follows Figure 2 As shown, when the three methylation markers were combined for simultaneous detection, the sensitivity for liver cancer plasma samples was 94.5%, the specificity for non-cancer plasma samples was 90.3%, and the overall detection AUC value was 0.968.

[0159] Embodiment 2:

[0160] This embodiment discloses a primer-probe combination for liver cancer methylation detection, including primer-probe sequences of marker 1, marker 2 and marker 3 in each corresponding region. The specific sequences are shown in the following table:

[0161] Marker 1:

[0162] Primer probe combination 1 (region 1):

[0163] Upstream primer: SEQ ID NO.1: 5'-TTTGTTGGGAAACGCGTTTA-3'

[0164] Downstream primer: SEQ ID NO.2: 5'-TTTTAACTCGCAAATATAAT-3'

[0165] Probe: SEQ ID NO.3: 5'-AAGGCGGTAAGGATATAGCG-3'

[0166] Primer probe combination 2 (region 2):

[0167] Upstream primer: SEQ ID NO.4: 5'-ATGTTCGGTTTCGTTCG-3'

[0168] Downstream primer: SEQ ID NO.5: 5'-CTTAAATACGCGTCAAAATC-3'

[0169] Probe: SEQ ID NO.6: 5'-TGGAGTACGGGTATTTTGTAA-3'

[0170] Primer probe combination 3 (region 3):

[0171] Upstream primer: SEQ ID NO.7: 5'-TCGTTCGTTGGTTTAAAAAAA-3'

[0172] Downstream primer: SEQ ID NO.8: 5'-ACACTTCGTAAACTCATTC-3'

[0173] Probe: SEQ ID NO.9: 5'-AAGATGTAATATCGATAATTT-3'

[0174] Marker 2:

[0175] Primer probe combination 4 (region 4):

[0176] Upstream primer: SEQ ID NO.10: 5'-CGTTTATAGTTGGACGTGTT-3'

[0177] Downstream primer: SEQ ID NO.11: 5'-CGAAAAATACGCACCTAAAC-3'

[0178] Probe: SEQ ID NO.12: 5'-GGCGTTTTTCGTTAAGATTCGTTATTTTG-3'

[0179] Primer probe combination 5 (region 5):

[0180] Upstream primer: SEQ ID NO.13: 5'-CACCCCCACAAAATAATCTC-3'

[0181] Downstream primer: SEQ ID NO.14: 5'-GCGTTTTTCGTTAAGATTCG-3'

[0182] Probe: SEQ ID NO. 15: 5'-CCCTAAAACCCGAAAAATACGCAC-3'

[0183] Primer probe combination 6 (region 6):

[0184] Upstream primer: SEQ ID NO.16: 5'-TTTAGGTGCGTATTTTTCGG-3'

[0185] Downstream primer: SEQ ID NO.17: 5'-CGACTACCCAATCTATAAACC-3'

[0186] Probe: SEQ ID NO.18: 5'-GAGGTTGTTCGGGGAAGGTTTAG-3'

[0187] Marker 3:

[0188] Primer probe combination 7 (region 7):

[0189] Upstream primer: SEQ ID NO.19: 5'-AATCCACTAACGACAAAACC-3'

[0190] Downstream primer: SEQ ID NO.20: 5'-GTATTAGGTTTGTAGCGGTG-3'

[0191] Probe: SEQ ID NO. 21: 5'-CAACTAACCACTAAAACGACTCGC-3'

[0192] Primer probe combination 8 (region 8):

[0193] Upstream primer: SEQ ID NO.22: 5'-TACCGCCAATAAACTTACGA-3'

[0194] Downstream primer: SEQ ID NO.23: 5'-CGGATTAATTAGAAGGATTTTGTT-3'

[0195] Probe: SEQ ID NO.24: 5'-TTACTTCGTACGTACCATAAAAAT-3'

[0196] Primer probe combination 9 (region 9):

[0197] Upstream primer: SEQ ID NO.25: 5'-CCTAATAACTAATCACAAATCCTATC-3'

[0198] Downstream primer: SEQ ID NO.26: 5'-CGTGCGTGTTATAGGAATG-3'

[0199] Probe: SEQ ID NO. 27: 5'-CGCGAAACTAAAAAATTCGAACCAA-3'

[0200] Primer probe combination 10 (region 10):

[0201] Upstream primer: SEQ ID NO.28: 5'-CTTCCAATTTCGCGCC-3'

[0202] Downstream primer: SEQ ID NO.29: 5'-ACGCGGTATTTTTGAATCG-3'

[0203] Probe: SEQ ID NO.30: 5'-CAAACGAAAATAAACGTTCTACAATTCCG-3'

[0204] The primer and probe sequences of the internal reference gene β-actin are as follows:

[0205] The primer and probe sequences of the internal reference gene β-actin are as follows:

[0206] β-actin upstream primer: SEQ ID NO.31: 5'-ATTGTTGAGATTGTGTTTGTTTTGT-3'

[0207] β-actin downstream primer: SEQ ID NO.32: 5'-TCAACTAACCAAACTTACCTAACAA-3'

[0208] β-actin probe: SEQ ID NO.33: 5'-GTTTTTGTTGATTTGTTGTTTGTTT-3'

[0209] Embodiment 3:

[0210] This embodiment discloses the following specific experimental steps for detecting the methylation level of a marker in a blood sample by using fluorescent quantitative PCR technology:

[0211] 1. Use Guangzhou Youze Biological Free DNA Extraction Kit "Free DNA Extraction Kit (Filtration Method) #C02-1" to extract plasma cfDNA.

[0212] 2. Use ZYMO's "EZ-96 DNA Methylation-Lighting MagPrep" to perform bisulfite conversion on the extracted cfDNA.

[0213] 3. The negative control is QSG-7701 cell line DNA, and the positive control for markers 1-3 is HepG2 liver cancer cell line DNA. They were all converted with bisulfite using the ZYMO kit.

[0214] 4. The composition and concentration system of PCR reaction are as follows:

[0215]

[0216] 5. The PCR reaction program is set as follows:

[0217]

[0218] *Indicates fluorescence signal collection during annealing and extension

[0219] 6. Operate the converted sample DNA to be tested, negative quality control DNA, and positive quality control DNA according to the PCR reaction composition and concentration system, and perform PCR amplification reaction according to the PCR reaction procedure.

[0220] 7. After the PCR reaction is completed, if the amplification curve is S-shaped, set the threshold at the inflection point to obtain the Ct value of this marker. If the amplification curve is a straight line, record the Ct value of this marker as NA. The Ct value of the negative control should be NA, the Ct value of the positive control should be between 28 and 32, and the Ct value of the internal reference gene should be less than 38. When the positive control, negative control and internal reference gene meet the standards, the sample can be judged, otherwise the PCR test must be repeated.

[0221] 8. If the Ct value of a target marker in a sample is less than 35, the test result of the target marker in the sample is determined to be positive, otherwise the test result is negative. When the test result of at least one marker in the combined test of three markers is positive, the sample is determined to be positive, and when the test results of all three markers are negative, the sample is determined to be negative.

[0222] Embodiment 4:

[0223] In order to verify that the qPCR probe primer combination under the marker combination provided by the present invention can be used for liver cancer detection, the performance of the designed primer probe was verified using clinically collected blood samples. The following are the verified markers and marker combinations:

[0224]

[0225] A total of 79 samples, including 32 plasma samples from hepatocellular carcinoma, 32 plasma samples from benign liver diseases, and 15 plasma samples from healthy individuals, were tested for methylation by qPCR using the designed primer-probe combination. The sensitivity and specificity of the single marker and the three-marker combination were statistically analyzed. The specific results are as follows:

[0226]

[0227] From the results of plasma qPCR test, it can be seen that the detection sensitivity of ex vivo blood samples of liver cancer using chr17:30970348-30970741, chr2:63054088-63054326 and chr6:26250469-26250942 methylation markers is 81.3% to 87.5%, and the specificity is about 90%. When the three markers are combined, the detection sensitivity can be increased to 90.6~93.8%, and the specificity remains at about 90%.

[0228] 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. A marker composition for liver cancer detection or auxiliary diagnosis, characterized in that: The marker composition is composed of three markers; The positions of each marker in the marker composition on the reference genome are as follows: Marker 1, located in the entire region or part of chr17:30970348-30970741; Marker 2, located in the entire region or a portion of chr2:63054088-63054326; Marker 3, located in the entire region or part of chr6:26250469-26250942; The reference genome is the hg38 genome; The marker 1 is selected from the whole region or a part of the region containing at least one CpG site as follows: region 1 is located at chr17:30970348-30970428; region 2 is located at chr17:30970452-30970550; region 3 is located at chr17:30970629-30970741; The marker 2 is selected from the whole region or a part of the region containing at least one CpG site as follows: region 4 is located at chr2:63054088-63054212; region 5 is located at chr2:63054112-63054305; region 6 is located at chr2:63054194-63054326; The marker 3 is selected from the entire region or a partial region of the following region containing at least one CpG site: region 7 is located at chr6:26250469-26250577; region 8 is located at chr6:26250563-26250692; region 9 is located at chr6:26250594-26250753; region 10 is located at chr6:26250594-26250753.

2. Use of the marker composition for liver cancer detection or auxiliary diagnosis as claimed in claim 1 in the preparation of liver cancer detection or auxiliary diagnosis products.

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

4. A kit for liver cancer detection or auxiliary diagnosis, characterized in that: A primer probe set comprising detecting the marker composition as claimed in claim 1.

5. A kit for liver cancer detection or auxiliary diagnosis according to claim 4, characterized in that: The primer probe set includes a primer probe combination 1 for detecting region 1, a primer probe combination 2 for detecting region 2, and a primer probe combination 3 for detecting region 3; wherein primer-probe combination 1 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.1-3, primer-probe combination 2 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.4-6, and primer-probe combination 3 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.7-9; The primers and probes for detecting methylation of marker 2 include primer-probe combination 4 for detecting region 4, primer-probe combination 5 for detecting region 5, and primer-probe combination 6 for detecting region 6; wherein primer-probe combination 4 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.10-12, primer-probe combination 5 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.13-15, and primer-probe combination 6 comprises the upstream primer sequence, downstream primer sequence and probe sequence as shown in SEQ ID NOs.16-18; The primers and probes for detecting methylation of marker 3 include primer-probe combination 7 for detecting region 7, primer-probe combination 8 for detecting region 8, primer-probe combination 9 for detecting region 9, and primer-probe combination 10 for detecting region 10; Among them, primer probe combination 7 is an upstream primer sequence, a downstream primer sequence and a probe sequence as shown in SEQ ID NOs.19-21, primer probe combination 8 is an upstream primer sequence, a downstream primer sequence and a probe sequence as shown in SEQ ID NOs.22-24, primer probe combination 9 is an upstream primer sequence, a downstream primer sequence and a probe sequence as shown in SEQ ID NOs.25-27, and primer probe combination 10 is an upstream primer sequence, a downstream primer sequence and a probe sequence as shown in SEQ ID NOs.28-30.

6. A kit for liver cancer detection or auxiliary diagnosis according to claim 4, characterized in that: The kit also includes a PCR primer-probe combination of an internal reference gene β-actin, such as an upstream primer sequence, a downstream primer sequence and a probe sequence shown in SEQ ID NO.31-33.

7. A kit for liver cancer detection or auxiliary diagnosis according to claim 4, characterized in that: The test samples of the kit include tissue samples, blood samples or ascites samples; tissue samples come from fresh ex vivo pathological tissues, paraffin-embedded tissues or liver cancer cells, and blood samples come from ex vivo plasma, ex vivo serum, ex vivo whole blood or ex vivo blood cells.

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