WO2022105774A1 - 一种用于检测肝癌的试剂组合，试剂盒及其用途 - Google Patents

一种用于检测肝癌的试剂组合，试剂盒及其用途 Download PDF

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WO2022105774A1
WO2022105774A1 PCT/CN2021/131103 CN2021131103W WO2022105774A1 WO 2022105774 A1 WO2022105774 A1 WO 2022105774A1 CN 2021131103 W CN2021131103 W CN 2021131103W WO 2022105774 A1 WO2022105774 A1 WO 2022105774A1
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reagent
reagent combination
methylation
detecting
gene
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PCT/CN2021/131103
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English (en)
French (fr)
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戴立忠
陈明
郭鑫武
洪梅
刘佳
刘让蛟
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圣湘生物科技股份有限公司
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Priority claimed from CN202011288235.7A external-priority patent/CN112501293B/zh
Priority claimed from CN202011290484.XA external-priority patent/CN112280865B/zh
Priority claimed from CN202011290799.4A external-priority patent/CN112322736B/zh
Application filed by 圣湘生物科技股份有限公司 filed Critical 圣湘生物科技股份有限公司
Priority to EP21893921.3A priority Critical patent/EP4134454A1/en
Publication of WO2022105774A1 publication Critical patent/WO2022105774A1/zh
Priority to US17/935,281 priority patent/US20230102121A1/en

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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention belongs to the field of molecular biology detection, in particular, to the field of liver cancer detection, and more specifically, to the detection of the methylation level of liver cancer gene markers.
  • Hepatocellular carcinoma is one of the most common and deadly diseases in the world, and it is a very harmful malignant tumor.
  • AFP serum alpha fetal protein
  • ultrasonography ultrasonography
  • the sensitivity of AFP screening can only reach 40-60%, and the AFP level of many patients with early-stage liver cancer is always maintained at a normal level; a considerable number of patients with liver disease also have abnormally elevated AFP; ultrasound detection is very dependent on Due to equipment and manual operations, it is affected by the distribution of medical resources and the experience of doctors; contrast-enhanced ultrasonography and puncture have disadvantages such as complicated operations and trauma, and are not suitable for early screening and early diagnosis. Finding accurate, stable and effective molecular markers of liver cancer is of great significance for early diagnosis and early treatment of liver cancer.
  • the detection of tumor markers is a method developed in recent years to detect diseases, and finding accurate, stable and effective molecular markers of liver cancer is of great significance for the early diagnosis and early treatment of liver cancer.
  • DNA methylation, histone modification and abnormal expression of miRNA are all epigenetic changes, and the core link of tumorigenesis is also related to abnormal DNA methylation.
  • DNA methylation detection has good stability, is easy to detect, and its abnormality is often related to the progress of cancer. It is the most potential marker for early tumor screening. At present, there are colorectal cancer, gastric cancer and other tumors based on DNA methylation detection.
  • Auxiliary diagnostic products have been certified by the FDA or CFDA, but no screening products for liver cancer have been approved for marketing.
  • the present invention collects and constructs liver cancer-related methylation big data sets in TCGA and GEO databases, and uses bioinformatics methods to screen out liver cancer DNA methylation markers with potential for application and development.
  • the methylation level of the methylation site is closely related to liver cancer.
  • ZNF397OS was generally considered to be a zinc finger protein in the art. Studies have shown that it has the function of regulating the activity of DNA-binding transcription factors. There is no literature report that it is associated with liver cancer.
  • the applicant also found that the methylation levels of the methylation sites of the OTX1 gene and the PAK1 gene are closely related to liver cancer.
  • the present invention provides a reagent combination for detecting liver cancer, the reagent combination comprising any one of the following detection reagents:
  • the reagent combination when the above-mentioned reagent combination includes 1), further, the reagent combination includes a detection reagent for detecting the methylation level of at least two of the following methylation sites of the PAK1 gene: cg17202086, cg26996201, cg18309286. Further, the reagent combination includes detection reagents for detecting the following three methylation sites of the PAK1 gene: cg17202086, cg26996201, and cg18309286.
  • the combination of reagents detects methylation levels in the following regions:
  • liver cancer can be predicted with a specificity of at least 0.360 and a sensitivity of 0.843 and an area under the curve of 0.8194 in a tissue sample, and a specificity of at least 0.8511 and a specificity of 0.7460 in a free DNA sample of plasma
  • the sensitivity and the area under the curve of 0.8286 are used to predict liver cancer.
  • the reagent combination of the present invention can detect liver cancer clinically with high sensitivity and good specificity with less markers, and the cost and time are saved; , it can be sensitively and specifically detected in the early stage of liver malignancy.
  • the reagent combination when the above-mentioned reagent combination includes 1), the reagent combination further includes a detection reagent for detecting the methylation level of at least one of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least two of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least three of the following genes:
  • the reagent combination further includes detection reagents for detecting the methylation levels of the following four genes:
  • the reagent combination when the above reagent combination includes 2), further, the reagent combination includes detection reagents for detecting the methylation levels of the following two methylation sites of the OTX1 gene: cg23229261 and cg10122865. Further, the reagent combination also includes a detection reagent for detecting the methylation level of the methylation site cg21472506 of the OTX1 gene.
  • the combination of reagents detects methylation levels in the following regions:
  • liver cancer can be predicted with a specificity of at least 0.846, a sensitivity of 0.860 and an area under the curve of 0.914 in tissue, and a specificity of at least 0.8298 and a sensitivity of 0.778 in free DNA in plasma and the area under the curve of 0.8823 to predict liver cancer, the reagent combination of the present invention can detect liver cancer clinically with high sensitivity and good specificity with less markers, and both cost and time are saved; The early stage of liver malignancy can be detected sensitively and specifically.
  • the reagent combination when the above reagent combination includes 2), the reagent combination further includes a detection reagent for detecting the methylation level of at least one of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least two of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least three of the following genes:
  • the reagent combination further includes detection reagents for detecting the methylation levels of the following four genes:
  • the reagent combination detects the methylation level of the following region: CGCCCCACTCACCCTTCGCTCTACCGGCGGCGGCGGGAACCCACCCCCGGGAGCGAACAATGCCCGGCCGCACGCGCCGGAAGTGGGAGAGTGCCCCTCTAGGAGCCCGGAGGACCGCAGCTCTGTGGCAGGCGCGGGTCGTGTCTCGCAGGAGGGGCGCGGGTCGCGCAGACCTGGCGGAGGAGCGCGCAGACCTGGAGGGGAGGCGCGCAGACCTGGAGGGGAGGCGCGCAGACCTGGAGGGGAGGCGCGCAGACCAGGAGGGGCGGCGCGCAGACCAGGAGGGGAGGCGCAGCA).
  • a sensitivity of 0.910, a specificity of 0.950, and an area under the curve of 0.941 in clinical tissue samples, and a sensitivity of 0.7619, 0.9574 in cell-free DNA samples, with fewer markers The specificity of the gene and the area under the curve of 0.8948 are used to detect liver cancer.
  • liver cancer can be detected sensitively and specifically, and the cost and time are saved; On the other hand, it can be sensitively and specifically detected in the early stage of liver malignancy.
  • the reagent combination when the above-mentioned reagent combination includes 3), the reagent combination further includes a detection reagent for detecting the methylation level of at least one of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least two of the following genes:
  • the reagent combination further includes a detection reagent for detecting the methylation level of any at least three of the following genes:
  • the reagent combination further includes detection reagents for detecting the methylation levels of the following four genes:
  • the methylation level detection reagent can be a detection reagent that detects the average methylation level of the entire gene.
  • the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene segment.
  • the methylation level detection reagent can also be a detection reagent for detecting the average methylation level of a gene promoter region or a fragment thereof.
  • the methylation level detection reagent can also be a detection reagent for detecting one or more methylation sites of a gene.
  • Those skilled in the art can select detection reagents to detect the methylation levels of GRASP, PAK1, PPFIA1, and OTX1.
  • GRASP is related to liver cancer
  • PPFIA1 is related to liver cancer
  • PAK1 gene is related to liver cancer.
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the entire gene of the GRASP gene.
  • the reagent combination further includes a detection reagent for detecting the average methylation level of the amplified fragment of the GRASP gene in Chinese patent CN110904225:
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the following gene segments of the GRASP gene:
  • the reagent combination further comprises a detection reagent for detecting the methylation level of one or more of the GRASP gene methylation sites cg04034767, cg00817367.
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the entire gene of the PPFIA1 gene.
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the following gene segments of the PPFIA1 gene:
  • the detection reagent for the methylation level of the PPFIA1 gene further includes a detection reagent for detecting the methylation level of one or more of the PPFIA1 gene methylation sites cg14999168, cg14088196, and cg25574765 reagents.
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the entire gene of the PAK1 gene.
  • the detection reagent for the methylation level of the PAK1 gene further comprises a methylation level for detecting one or more of the PAK1 gene methylation sites cg17202086, cg26996201, cg12269002, and cg18309286 detection reagents.
  • the reagent combination further comprises a detection reagent for detecting the average methylation level of the entire gene of the OTX1 gene.
  • the detection reagent for the methylation level of the OTX1 gene further includes a detection reagent for detecting the methylation level of one or more of the OTX1 gene methylation sites cg21472506, cg23229261, and cg10122865 reagents.
  • the detection reagent for the methylation level of the ZNF397OS gene further comprises a reagent for detecting the methylation level of one or more of the ZNF397OS gene methylation sites cg27249419, cg16657538, and cg00487232 detection reagents.
  • the detection reagent for the methylation level of the ZNF397OS gene further comprises a detection reagent for detecting the methylation level of one or more fragments covering the methylation site cg16657538 of the ZNF397OS gene.
  • liver cancer can be detected with higher sensitivity and better specificity; clinically, it can be detected sensitively and specifically in the early stage of liver malignant transformation.
  • the methylation level of the corresponding gene present in the sample can be detected using the detection reagents of the present invention.
  • a sample is a biological sample selected from an individual. Specifically, for example, selected from cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, feces, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood , or a combination thereof.
  • the "sample” of the present invention is plasma, ie cell-free DNA in plasma.
  • Cell-free DNA in plasma can be used to detect tumors, and has the characteristics of less harm to patients and good specificity. However, due to its extremely low content in plasma, it generally suffers from low sensitivity when used for cancer detection.
  • the detection reagent of the present invention the free DNA in the plasma can be used as a sample for detection, which has high sensitivity and specificity.
  • detection reagent refers to a reagent for detecting the methylation level of a gene in a sample.
  • the methylation level is measured by means of amplification-sequencing, chip, and methylation fluorescence quantitative PCR.
  • detection reagents include, but are not limited to, nucleic acid primers, sequencing Tag sequences, for measuring methylation levels by amplification-sequencing.
  • the amplification-sequencing is performed by subjecting the nucleic acid in the sample to bisulfite treatment, then constructing a pre-library, then constructing a final library, and finally performing sequencing evaluation.
  • detection reagents include, but are not limited to, a chip that is a methylation chip with probes that specifically bind to methylated regions.
  • the chips may be, for example, Human CpG Island Microarrays and Human DNA Methylation Microarrays from Agilent, Human DNA Methylation Microarrays from Illumina, Infinium HumanMethylation27 BeadChip, Infinium HumanMethylation 450 BeadChip and GoldenGate Methylation Assay and Human DNA Methylation 2.1M Deluxe Promoter Array from Roche NimbleGen, Human DNA Methylation Array et al., for measuring methylation levels by chip.
  • detection reagents include, but are not limited to, nucleic acid primers and nucleic acid probes for measuring methylation levels by quantitative PCR for methylation.
  • the detection reagent also includes an internal standard primer and an internal standard probe.
  • the above-mentioned reagent combination may also include other reagents, specifically, for example, various reagents required for pretreatment or pretreatment of the sample.
  • sample release agent for extracting sample nucleic acid for example, purifying agent for purifying sample nucleic acid, bisulfite or bisulfite used for transformation, etc.
  • the above reagent combination also includes a reagent for extracting plasma-free DNA.
  • the present invention provides the use of the above reagent combination in the preparation of a kit for detecting liver cancer.
  • the present invention provides the use of the above reagent combination in preparing a kit for detecting liver cancer using plasma free DNA.
  • the present invention provides a kit for detecting liver cancer, the kit comprising the reagent combination as described above.
  • the kit also includes, but is not limited to, at least one of nucleic acid extraction reagents, nucleic acid purification reagents, bisulfite, T4 polynucleotide kinase, and T4 ligase.
  • the reagent for extracting nucleic acid is a reagent for extracting tissue DNA and a reagent for plasma free DNA.
  • the reagent for extracting nucleic acid is a reagent for extracting plasma-free DNA.
  • Figure 1 shows the ROC diagram of ZNF397OS single gene in the identification of cancer and non-cancer in peripheral blood cell-free DNA samples
  • Figure 2 shows the methylation levels of ZNF397OS gene in different groups in peripheral blood cell-free DNA samples
  • Figure 3 shows the methylation levels of ZNF397OS gene methylation sites in different groups in tissue samples
  • Figure 4 shows the methylation levels of different groups of methylated sites outside the target region of ZNF397OS gene in different groups in tissue samples
  • Fig. 5 is the ROC diagram of PAK1 methylation site in differentiating cancer and non-cancer in cell-free DNA samples of peripheral blood;
  • Figure 6 shows the methylation levels of different groups of PAK1 gene methylation sites in peripheral blood cell-free DNA samples
  • Figure 7 shows the methylation levels of different groups of PAK1 gene methylation sites in tissue samples
  • Figure 8 shows the methylation levels of different groups of PAK1 gene comparative methylation sites in tissue samples
  • Fig. 9 is the ROC diagram of OTX1 gene methylation sites in cell-free DNA samples of peripheral blood to differentiate cancer and non-cancer;
  • Figure 10 shows the methylation levels of different groups of OTX1 gene methylation sites in peripheral blood cell-free DNA samples
  • Figure 11 is a comparison of methylation levels of different groups of OTX1 gene methylation sites in tissue samples
  • Figure 12 is a comparison of methylation levels of different groups of OTX1 gene methylation sites in tissue samples.
  • the present invention collected a total of 785 cancer tissues, 461 adjacent cancer tissues or normal controls from the TCGA dataset (https://tcga.xenahubs.net) of the UCSC Xena website and the GEO database of the National Center for Biotechnology Information (NCBI). Tissue and 656 healthy whole blood methylation data. Perform differential analysis with liver cancer and control data, and annotate the physical location and gene information of the differential sites.
  • the screening of methylated gene fragments needs to meet the following requirements at the same time: 1) The selected gene fragments are required to have no less than 2 adjacent sites with consistent methylation levels; 2) Perform differential analysis between liver cancer and adjacent tissue or normal control tissue, and select liver cancer samples with high consistency and differential hypermethylation 3) Perform differential analysis on the whole blood methylation detection data of liver cancer and healthy samples, and select the gene fragments that are differentially hypermethylated in liver cancer; 4) Finally, analyze the methylation sites one by one. Thus, candidate methylation sites are derived.
  • the sample preparation of the present invention is to extract 4ml of plasma by MagMAX TM Cell-Free DNA Isolation Kit and elute with 45 ⁇ L of eluent.
  • the extracted free nucleic acid must meet the following quality control conditions: the total amount of extracted nucleic acid is greater than 20ng.
  • all free nucleic acids qualified for quality control are treated with bisulfite using EZ DNA Methylation-LightningTM Kit (Zymo Research, Irvine, CA, USA). Subsequently, the sample DNA after bisulfite treatment is used to construct a pre-library by the single-strand library construction method. After passing the quality inspection of the pre-library, the target region is captured and enriched by liquid chip hybridization to complete the construction of the final library.
  • Pre-library construction steps 1) Phosphorylation: T4 polynucleotide kinase phosphorylates the 5-terminal of the bisulfite-treated DNA; 2) SS1 ligation: T4 DNA Ligase (Rapid) ligates the SS1 linker on the phosphorylated 3) Nucleic acid purification: use 2 volumes of Agencourt AMPure XP system (Beckman CouLter, CA, USA) to remove the remaining linker; 4) SS2 ligation: T4 DNA Ligase (Rapid) ligates the SS2 linker in phosphate 5) Nucleic acid purification: use 2 volumes of Agencourt AMPure XP system (Beckman CouLter, CA, USA) to remove the remaining linkers; 6) Amplification: use NEBNext Q5U Master Mix and primer1.0 (generic Primers) and Bacard sequences to amplify the nucleic acid in the previous step; 7) Nucleic acid purification: use 1.2 times the volume
  • Chip hybridization capture steps 1) Chip hybridization: Pre-concentrate 1.5 ⁇ g of the library that has passed the quality inspection into powder, and then mix with Panel, Hybridization Mix, Blocker Solution, Universal Blockers, and Hybridization Enhancer reagents ( The reagents used for chip hybridization were all provided by Twist Bioscience), placed in the PCR machine and incubated at 70 degrees for 16 hours overnight (the temperature of the hot lid was 85 degrees); 2) Magnetic bead capture: Use Streptavidin Binding Buffer to wash the capture magnetic beads 3 times in advance , the hybridized product was added to the capture magnetic beads, incubated for 30 minutes, washed once with Wash Buffer I, washed with Wash Buffer 2 three times, and finally eluted with 42 ⁇ l of ultrapure water; 3) Amplification: using KAPA HiFi HotStart ReadyMix and universal primers Amplify the captured library; 4) Purification: Use 1 volume of Agencourt AMPure XP system (Beckman Coutad DNA sample, and
  • the purified library was dsDNA HS Assay Kit (Life Technologies, CA, USA) and LabChip GXII Touch were used for quality control of the total amount of nucleic acid, fragment distribution and primer-dimer ratio in the library.
  • the total amount of the library, the fragment size distribution of the amplified products, and the primer-dimer ratio of the libraries to be tested that passed the quality inspection were mixed according to the amount of 1:1 substances, and then used
  • the dsDNA HS Assay Kit (Life Technologies, CA, USA) was used to accurately quantify the mixed library.
  • the library was denatured and diluted and then sequenced on a NextSeq500 desktop sequencer using PE75.
  • the bismark methylation analysis software For the raw fastq data obtained by sequencing, after filtering the raw data, use the bismark methylation analysis software to perform methylation analysis on the fragments captured by the chip, and obtain the methylation level of a single site of the candidate gene and the methylation level of the gene fragment.
  • methylation level of a single site of candidate genes and the methylation level of gene fragments differential analysis and model construction were performed for liver cancer and control samples.
  • the present invention constructs and evaluates the liver cancer classification model by adopting the logistic regression analysis to the data.
  • Samples from 63 patients with primary liver cancer, 25 patients with cirrhosis, 15 patients with hepatitis and 7 healthy people were collected, and the methylation level of the cg16657538 methylation site of ZNF397OS gene was detected and analyzed according to the method described in Example 2. , and the methylation levels of the combination of the ZNF397OS gene and the rest of the genes in the samples to verify its effect on detecting liver cancer.
  • the test results are shown in Table 1, Figure 1 and Figure 2.
  • Table 1 Predictive performance of the combination of ZNF397OS gene and other genes in the Logistics liver cancer classification model
  • 1 represents the GRASP gene (detects the average methylation level of the fragment shown in SEQ ID NO: 5), and 2 represents the PAK1 gene (detects the average methylation of fragments containing four methylation sites cg17202086, cg26996201, cg12269002, and cg18309286).
  • Cg21472506, cg23229261, cg10122865 The average methylation of the fragment containing the three methylation sites of cg21472506, cg23229261, cg10122865 methylation level)
  • 5 represents the ZNF397OS gene (the average methylation level of the cg16657538 methylation site fragment was detected).
  • the reagent combination of the present invention has a good predictive effect on liver cancer, and in particular, when plasma cell-free DNA is used as a sample, it has excellent specificity and sensitivity.
  • the genes PLAC8 and ATXN1 which are closely related to liver cancer known in the art, were further selected (see Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K, et al. al.Circulating tumor DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma. Nature Materials, 2017 and Chinese patent CN106947830B).
  • the detection is also carried out according to the method of the above-mentioned embodiment, and the detection results are shown in Table 3 below.
  • 6 represents the PLAC8 gene (detecting the methylation level of cg11606215 of this gene), and 7 representing the ATXN1 gene (detecting the methylation level of cg24067911 of this gene).
  • the highest area under the curve of a single comparative gene in tissue samples is 0.858, and the highest area under the curve in cell-free DNA samples is 0.75, which is lower than the area under the curve of ZNF397OS of the present invention.
  • the area under the curve of the combination is also lower than the value of the area under the curve of the combination of the agents of the present invention.
  • Samples from 63 patients with primary liver cancer, 25 patients with liver cirrhosis, 15 patients with hepatitis, and 7 healthy people were collected, and the methylation sites of PAK1 gene in the samples were detected and analyzed according to the method described in Example 2. Basalization level to verify its effect on detecting liver cancer.
  • the diagnosis of hepatocellular carcinoma, liver cirrhosis, hepatitis and healthy people is based on the final hospital pathological diagnosis. The test results are shown in Table 4, Figure 5, and Figure 6.
  • each methylation site of the PAK1 gene in tissues can predict liver cancer with a specificity of at least 0.360, a sensitivity of 0.843 and an area under the curve of 0.8194.
  • all reagent combinations were able to predict liver cancer with a specificity of at least 0.8511 and a sensitivity of 0.7460 and an area under the curve of 0.833.
  • 1 represents the GRASP gene (detects the average methylation level of the fragment shown in SEQ ID NO: 5)
  • 2 represents the PAK1 gene (detects the methylation levels of the three methylation sites cg17202086, cg26996201, and cg18309286)
  • 3 represents the PPFIA1 gene (detects the average methylation level of the fragment shown in SEQ ID NO: 6)
  • 4 represents the OTX1 gene (detects the methylation levels of three methylation sites cg21472506, cg23229261, and cg10122865)
  • 5 represents the ZNF397OS gene (Detect the methylation level of the cg16657538 methylation site).
  • the reagent combination of the present invention has a good predictive effect on liver cancer, and in particular, when plasma cell-free DNA is used as a sample, it has excellent specificity and sensitivity.
  • the PAK1 gene can be used as a marker for detecting liver cancer
  • PLAC8 and ATXN1 known in the art (see Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K, et al. al.Circulating tumor DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma. Nature Materials, 2017 and Chinese patent CN106947830B).
  • the detection is also carried out according to the method of the above-mentioned embodiment, and the detection results are shown in Table 6 below.
  • 6 represents the PLAC8 gene (detects the methylation level of cg11606215 of this gene), and 7 represents the ATXN1 gene (detects the methylation level of cg24067911 of this gene).
  • the area under the curve of the highest tissue of the control gene is 0.858, and the highest area under the curve of the cell-free DNA is 0.75, which is lower than the area under the curve of the PAK1 gene of the present invention.
  • the area under the curve of the combination is also lower than the value of the area under the curve of the combination of the agents of the present invention.
  • Samples from 63 patients with primary liver cancer, 25 patients with liver cirrhosis, 15 patients with hepatitis and 7 healthy people were collected, and the methylation level of OTX1 gene in the samples was detected and analyzed according to the method described in Example 2 to verify the It tests the effect of liver cancer. Diagnosis of hepatocellular carcinoma, cirrhosis, hepatitis and healthy people is based on the final hospital pathological diagnosis. The test results are shown in Table 7, Figure 9 and Figure 10.
  • each methylation site of the OTX1 gene in tissues can predict liver cancer with a specificity of at least 0.846, a sensitivity of 0.860 and an area under the curve of 0.914.
  • all reagent combinations were able to predict liver cancer with a specificity of at least 0.8298 and a sensitivity of 0.778 and an area under the curve of 0.8823.
  • Example 7 Detection of methylation level of clinical samples by the combination of reagents of the present invention
  • 1 represents the GRASP gene (detects the average methylation level of the fragment shown in SEQ ID NO: 5)
  • 2 represents the PAK1 gene (detects the methylation levels of four methylation sites cg17202086, cg26996201, cg12269002, and cg18309286)
  • 3 represents the PPFIA1 gene (detects the average methylation level of the fragment shown in SEQ ID NO: 6)
  • 4 represents the OTX1 gene (detects the average methylation level of the three methylation sites cg21472506, cg23229261, cg10122865)
  • 5 represents the ZNF397OS gene (detecting the methylation level of the cg16657538 methylation site).
  • the reagent combination of the present invention has a good predictive effect on liver cancer, and in particular, when plasma cell-free DNA is used as a sample, it has excellent specificity and sensitivity.
  • OTX1 gene can be used as a marker for detecting liver cancer
  • PLAC8 and ATXN1 known in the art (see Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K, et al. al.Circulating tumor DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma. Nature Materials, 2017 and Chinese patent CN106947830B).
  • the detection is also carried out according to the method of the above-mentioned embodiment, and the detection results are shown in Table 9 below.
  • 6 represents the PLAC8 gene (detects the methylation level of cg11606215 of this gene), and 7 represents the ATXN1 gene (detects the methylation level of cg24067911 of this gene).
  • the highest area under the curve of the control gene in the tissue sample is 0.858, and the highest area under the curve in the cell-free DNA sample is 0.75, which is lower than the area under the curve of the OTX1 gene of the present invention.
  • the area under the curve of the gene combination model is also lower than the value of the area under the curve of the reagent-related combination model of the present invention.

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Abstract

一种用于检测肝癌的试剂组合，该试剂组合包括以下检测试剂中的任意一种：1)用于检测PAK1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg17202086、cg26996201、cg18309286；2)用于检测OTX1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg23229261、cg10122865；以及3)用于检测ZNF397OS基因的cg16657538甲基化位点的甲基化水平的检测试剂。还提供了该试剂组合的用途，以及包括该试剂组合的试剂盒。

Description

一种用于检测肝癌的试剂组合，试剂盒及其用途
相关申请的交叉引用
本申请基于申请号为202011288235.7、申请日为2020年11月17日，申请号为202011290484.X、申请日为2020年11月17日，以及申请号为202011290799.4，申请日为2020年11月17日的中国专利申请提出，并要求该些中国专利申请的优先权，该些中国专利申请的全部内容在此以引入方式并入本申请。
技术领域
本发明属于分子生物学检测领域，具体地，属于肝癌检测领域，更具体地，涉及肝癌基因标志物的甲基化水平的检测。
背景技术
肝癌是世界上最常见的和最致命的疾病之一，危害极大的恶性肿瘤。全球高达50％肝癌患者均在中国，是我国第4位常见恶性肿瘤及第2位肿瘤致死病因。据2019年发布的《2015年中国恶性肿瘤流行情况分析》数据显示，2015年我国肝癌发病约37.0万人；因肝癌死亡约32.6万人，正严重威胁我国人民的生命和健康。肝癌起病隐匿，早期无特异性症状，大多数肝癌患者在就诊时已是中晚期，手术治愈率低，生存期短。
目前，血清甲胎蛋白(alpha fetal protein，AFP)和超声检查是当前临床上诊断肝癌常用而又重要的方法，然而其灵敏度和特异度并不理想。AFP筛查的灵敏度只能达到40-60％，许多早期肝癌患者的AFP水平总是维持在一个正常水平；而在相当一部分肝病患者中也存在着AFP异常升高的情况；超声检测则非常依赖于仪器和人为操作，受医疗资源分布的限制和医生的 经验影响；超声造影和穿刺等检查则存在操作复杂、创伤等缺点，不适用于早筛早诊。寻找准确、稳定、有效的肝癌分子标志物对肝癌进行早期诊断、早期治疗具有重要意义。
肿瘤标志物检测是近年发展起来的用于检测疾病的方法，而寻找准确、稳定、有效的肝癌分子标志物对肝癌进行早期诊断、早期治疗具有重要意义。而随着科学界对肿瘤的认识逐渐深入，越来越多的研究证实细胞表观遗传水平的改变是肿瘤发生与发展的关键事件。DNA甲基化、组蛋白修饰及miRNA表达异常均属于表观遗传学改变，而肿瘤发生的核心环节也与DNA异常甲基化相关。DNA甲基化检测稳定性好，易于检测且其异常程度常与癌症的进展相关，是最具肿瘤早筛潜力的标志物，目前已有基于DNA甲基化检测的结直肠癌、胃癌等肿瘤辅助诊断产品获得FDA或CFDA的认证，但尚无肝癌的筛查产品获批上市。
很多研究通过检测外周血中的肿瘤细胞或DNA甲基化变化，寻找用于肝癌早期诊断的标志物。美国Exact Sciences公司基于甲基化检测的早期肝癌筛查临床研究显示，在135例HCC(Hepatocellular Carcinoma)病例和308例对照参与的临床试验中，这一血液检测在诊断早期HCC患者时达到了71％的灵敏度度和90％的特异性。该项目获得了FDA突破性医疗器械认定，但其临床研究主要基于欧美人群且存在灵敏度偏低的缺陷；专利CN107164508A通过检测9个基因的5-羟甲基胞嘧啶含量检测肝癌，其具有较优良的检测性能，灵敏度为90％，特异性为91.3％，但其入组的样本并不包含肝硬化、肝炎等肝癌高风险人群，因此可能存在特异性偏低的不足。
因此，本领域需求一种基于DNA甲基化检测的兼具高灵敏度高特异性的肝癌诊断产品，在肝癌的早期阶段即能进行检出。
发明内容
本发明通过在TCGA和GEO数据库搜集和构建肝癌相关的甲基化大数据集，采用生物信息学方法筛选出有应用开发潜力的肝癌DNA甲基化标记物，通过大量研究，申请人发现ZNF397OS基因的甲基化位点的甲基化水平与肝癌有着密切的联系，而在此之前，本领域通常仅认为ZNF397OS是一种锌指蛋白，研究表明其具有调控DNA结合转录因子活性的功能，尚无文献报导其与肝癌存在关联。除此之外，申请人还发现OTX1基因和PAK1基因的甲基化位点的甲基化水平与肝癌有着密切的联系。
第一方面，本发明提供一种用于检测肝癌的试剂组合，所述试剂组合包括以下检测试剂中的任意一种：
1)用于检测PAK1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg17202086、cg26996201、cg18309286；
2)用于检测OTX1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg23229261、cg10122865；以及
3)用于检测ZNF397OS基因的cg16657538甲基化位点的甲基化水平的检测试剂。
在一些具体的实施方案中，当上述试剂组合包括1)时，进一步地，所述试剂组合包括用于检测PAK1基因的以下甲基化位点中至少两个的甲基化水平的检测试剂：cg17202086、cg26996201、cg18309286。更进一步地，所述试剂组合包括用于检测PAK1基因的以下三个甲基化位点的检测试剂：cg17202086、cg26996201、cg18309286。
在一些具体的实施方案中，当上述试剂组合包括1)时，所述试剂组合检测以下区域的甲基化水平：
Figure PCTCN2021131103-appb-000001
Figure PCTCN2021131103-appb-000002
使用本发明的试剂组合，在组织样本中能以至少0.360的特异性和0.843的灵敏度以及0.8194的曲线下面积对肝癌进行预测，在血浆的游离DNA样本中，能以至少0.8511的特异性和0.7460的灵敏度以及0.8286的曲线下面积对肝癌进行预测，本发明试剂组合以较少的标志物就能够在临床上以高灵敏度和好特异性对肝癌进行检测，成本和时间均得到节约；在临床上，在肝脏恶变的早期阶段就能灵敏和特异性地检出。
在一些具体的实施方案中，当上述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中任意至少一个的甲基化水平的检测试剂：
GRASP、ZNF397OS、PPFIA1，以及OTX1。
当上述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中任意至少两个的甲基化水平的检测试剂：
GRASP、ZNF397OS、PPFIA1，以及OTX1。
当上述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中任意至少三个的甲基化水平的检测试剂：
GRASP、ZNF397OS、PPFIA1，以及OTX1。
当上述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及OTX1。
在一些具体的实施方案中，当上述试剂组合包括2)时，进一步地，所述试剂组合包括用于检测OTX1基因的以下两个甲基化位点的甲基化水平的检测试剂：cg23229261和cg10122865。更进一步地，所述试剂组合还包 括用于检测OTX1基因的甲基化位点cg21472506的甲基化水平的检测试剂。
在一些具体的实施方案中，当上述试剂组合包括2)时，在一个具体的实施方案中，所述试剂组合检测以下区域的甲基化水平：
Figure PCTCN2021131103-appb-000003
使用本发明的试剂组合，在组织中能以至少0.846的特异性和0.860的灵敏度以及0.914的曲线下面积对肝癌进行预测，在血浆的游离DNA中，能以至少0.8298的特异性和0.778的灵敏度以及0.8823的曲线下面积对肝癌进行预测，本发明试剂组合以较少的标志物就能够在临床上以高灵敏度和好特异性对肝癌进行检测，成本和时间均得到节约；在临床上，在肝脏恶变的早期阶段就能灵敏和特异性地检出。
在一些具体的实施方案中，当上述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中任意至少一个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及ZNF397OS。
当上述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中任意至少两个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及ZNF397OS。
当上述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中任意至少三个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及ZNF397OS。
当上述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及ZNF397OS。
在一些具体的实施方案中，当上述试剂组合包括3)时，所述试剂组合检测以下区域的甲基化水平：CGCCCCACTCACCCTTCGCTCTACCGGCGGCGGCGGGAACCCACCCCCGGGAGCGAGAACAATGCCCGGCCGCACGCGCGCCGGAAGTGGGAGAGTGCCCCTCTAGGAGCCCGGAGGACCGCAGCTCTGTGGCAGGCGCGGGTCGTGTCTCGCAGGAGGGGCGCGGGTCCGGCTCAGACCTGGCGGGGGCATCGCAGAGTACAAGCGGTTGACGCG(SEQ ID NO:3)。
使用本发明的试剂组合，以较少的标志物就能够在临床上的组织样本中以0.910的灵敏度、0.950的特异性，和0.941的曲线下面积，以及在游离DNA样本中0.7619的灵敏度、0.9574的特异性和0.8948的曲线下面积对肝癌进行检测，通过检测该基因的一个甲基化位点就能够对肝癌灵敏和特异性地检出，其成本和时间均得到节约；而使得其在临床上，在肝脏恶变的早期阶段就能灵敏和特异性地检出。
在一些具体的实施方案中，当上述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下基因中任意至少一个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及OTX1。
当上述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下基因中任意至少两个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及OTX1。
当上述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下基因中任意至少三个的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及OTX1。
当上述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：
GRASP、PAK1、PPFIA1，以及OTX1。
在一些具体的实施方案中，所述甲基化水平的检测试剂可以是检测整个基因平均甲基化水平的检测试剂。
在一些具体的实施方案中，所述甲基化水平的检测试剂也可以是检测一个基因片段的平均甲基化水平的检测试剂。
在一些具体的实施方案中，所述甲基化水平的检测试剂也可以是检测一个基因启动子区域或其片段的平均甲基化水平的检测试剂。
在一些具体的实施方案中，所述甲基化水平的检测试剂还可以是检测基因的一个或多个甲基化位点的检测试剂。
本领域技术人员能够选择检测GRASP、PAK1、PPFIA1，以及OTX1的甲基化水平的检测试剂。例如，中国专利CN110904225提及了GRASP与肝癌相关、中国专利CN106947830提及了PPFIA1与肝癌相关、中国专利CN1659287提及了PAK1基因与肝癌相关。
在一个具体的实施方案中，所述试剂组合进一步包括用于检测GRASP基因整个基因平均甲基化水平的检测试剂。
在一个具体的实施方案中，所述试剂组合进一步包括用于检测中国专利CN110904225中GRASP基因扩增片段的平均甲基化水平的检测试剂：
Figure PCTCN2021131103-appb-000004
在一个具体的实施方案中，所述试剂组合进一步包括用于检测GRASP基因以下基因片段的平均甲基化水平的检测试剂：
Figure PCTCN2021131103-appb-000005
Figure PCTCN2021131103-appb-000006
在一些具体的实施方案中，所述试剂组合进一步包括用于检测GRASP基因甲基化位点cg04034767、cg00817367中的一个或多个的甲基化水平的检测试剂。
在一个具体的实施方案中，所述试剂组合进一步包括用于检测PPFIA1基因整个基因平均甲基化水平的检测试剂。
在一个具体的实施方案中，所述试剂组合进一步包括用于检测PPFIA1基因以下基因片段的平均甲基化水平的检测试剂：
Figure PCTCN2021131103-appb-000007
在一些具体的实施方案中，所述PPFIA1基因的甲基化水平的检测试剂进一步包括用于检测PPFIA1基因甲基化位点cg14999168、cg14088196、cg25574765中的一个或多个的甲基化水平的检测试剂。
在一些具体的实施方案中，所述试剂组合进一步包括用于检测PAK1基因整个基因平均甲基化水平的检测试剂。
在一些具体的实施方案中，所述PAK1基因的甲基化水平的检测试剂进一步包括用于检测PAK1基因甲基化位点cg17202086、cg26996201、cg12269002、cg18309286中的一个或多个的甲基化水平的检测试剂。
在一些具体的实施方案中，所述试剂组合进一步包括用于检测OTX1基因整个基因平均甲基化水平的检测试剂。
在一些具体的实施方案中，所述OTX1基因的甲基化水平的检测试剂 进一步包括用于检测OTX1基因甲基化位点cg21472506、cg23229261、cg10122865中的一个或多个的甲基化水平的检测试剂。
在一些具体的实施方案中，所述ZNF397OS基因的甲基化水平的检测试剂进一步包括用于检测ZNF397OS基因甲基化位点cg27249419、cg16657538，和cg00487232中的一个或多个的甲基化水平的检测试剂。
在一个优选的实施方案中，所述ZNF397OS基因的甲基化水平的检测试剂进一步包括用于检测覆盖ZNF397OS基因甲基化位点cg16657538片段中的一个或多个的甲基化水平的检测试剂。
使用上述方案的试剂组合，能够以更高的灵敏度和更好的特异性对肝癌进行检测；在临床上，在肝脏恶变的早期阶段就能灵敏和特异性地检出。
在一些实施方案中，使用本发明的检测试剂，可以对样本中所存在的相应基因的甲基化水平进行检测。
在本发明中，“样本”为选自个体的生物样本。具体地，例如，选自细胞系、组织学切片、组织活检/石蜡包埋的组织、体液、粪便、结肠流出物、尿、血浆、血清、全血、分离的血细胞、从血液中分离的细胞，或其组合。
优选地，本发明的“样本”为血浆，即血浆中的游离DNA。
血浆中的游离DNA能用作检测肿瘤，具有对病人伤害小，特异性好等特点。但是由于其在血浆中的含量极低，因此，在用于癌症检测时普遍存在灵敏度较低的问题。使用本发明的检测试剂，能够使用血浆中的游离DNA作为样本进行检测，具有较高的灵敏度和特异性。
在本发明中，“检测试剂”指的是对样本中的基因的甲基化水平进行检测的试剂。其中，所述甲基化水平是通过扩增-测序、芯片、甲基化荧光定量PCR的方式测量。
在一些具体的实施方案中，检测试剂包括但不限于核酸引物、测序Tag序列，用于通过扩增-测序测量甲基化水平。
在一个具体的实施方案中，所述扩增-测序是通过将样本中的核酸进行重亚硫酸盐处理，随后构建预文库，再构建终文库，最后进行测序评价的方法进行的。
在一些具体的实施方案中，检测试剂包括但不限于芯片，所述芯片是甲基化芯片，所述甲基化芯片具有与甲基化区域特异性结合的探针。所述芯片可以是包括但不限于例如安捷伦的Human CpG Island Microarrays和Human DNA Methylation Microarrays、Illumina的Infinium HumanMethylation27 BeadChip、Infinium HumanMethylation450 BeadChip和GoldenGate Methylation Assay以及Roche NimbleGen的Human DNA Methylation 2.1M Deluxe Promoter Array、Human DNA Methylation Array等，用于通过芯片测量甲基化水平。
在一些具体的实施方案中，检测试剂包括但不限于核酸引物以及核酸探针，用于通过甲基化荧光定量PCR测量甲基化水平。
进一步地，所述检测试剂还包括内标引物以及内标探针。
在一些具体的实施方案中，上述试剂组合还可以包括其余的试剂，具体地，例如，各种对样本进行前处理或者预处理所需要的试剂。例如，提取样本核酸的样本释放剂，纯化样本核酸的纯化剂，转化所用的重亚硫酸盐或亚硫酸氢盐等。
进一步地，上述试剂组合还包括提取血浆游离DNA的试剂。
第二方面，本发明提供了上述试剂组合在制备用于检测肝癌的试剂盒中的用途。
进一步地，本发明提供了上述试剂组合在制备使用血浆游离DNA检测肝癌的试剂盒中的用途。
第三方面，本发明提供了一种用于检测肝癌的试剂盒，所述试剂盒包括如上所述的试剂组合。
进一步地，所述试剂盒还包括但不限于提取核酸的试剂、纯化核酸的试剂、重亚硫酸盐、T4多聚核苷酸激酶、T4连接酶中的至少一种。
进一步地，所述提取核酸的试剂是提取组织DNA的试剂和血浆游离DNA的试剂。
进一步地，所述提取核酸的试剂是提取血浆游离DNA的试剂。
附图说明
图1为ZNF397OS单基因在外周血游离DNA样本中鉴别癌与非癌的ROC图；
图2为ZNF397OS基因在外周血游离DNA样本中不同分组的甲基化水平；
图3为ZNF397OS基因甲基化位点在组织样本中不同分组的甲基化水平；
图4为ZNF397OS基因靶区外对比例甲基化位点在组织样本中不同分组的甲基化水平；
图5为PAK1甲基化位点在外周血游离DNA样本中鉴别癌与非癌的ROC图；
图6为PAK1基因甲基化位点在外周血游离DNA样本中不同分组的甲基化水平；
图7为PAK1基因甲基化位点在组织样本中不同分组的甲基化水平；
图8为PAK1基因对比例甲基化位点在组织样本中不同分组的甲基化水平；
图9为OTX1基因甲基化位点在外周血游离DNA样本中鉴别癌与非癌的ROC图；
图10为OTX1基因甲基化位点在外周血游离DNA样本中不同分组的甲基化水平；
图11为OTX1基因甲基化位点在组织样本中不同分组的甲基化水平比较；
图12为OTX1基因对比例甲基化位点在组织样本中不同分组的甲基化水平比较。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1、甲基化基因的筛选
本发明从UCSC Xena网站的TCGA数据集(https://tcga.xenahubs.net)和美国国立生物技术信息中心(NCBI)的GEO数据库中共收集到785例癌组织、461例癌旁组织或正常对照组织和656例健康全血甲基化数据。以肝癌和对照数据进行差异分析，对差异位点进行物理位置和基因信息注释，为保证筛选片段具有一致的甲基化水平，甲基化基因片段的筛选需要同时符合以下几点要求：1)要求所选基因片段具有不少于2个的相邻位点具有一致的甲基化水平；2)以肝癌和癌旁组织或正常对照组织进行差异分析，挑选肝癌样品中一致性高差异高甲基化的基因片段作为候选靶基因；3)以肝癌和健康样本全血甲基化检测数据进行差异分析，挑选肝癌差异高甲基化的基因片段；4)最后再从中进行逐个甲基化位点的分析，从而得出候选甲基化位点。
实施例2、临床样本的基因甲基化水平检测
收集样本的外周血各10ml，用于检测分析DNA甲基化标记物在样本中的甲基化水平。实验过程如下所述：
1、样本制备
本发明的样本制备是通过MagMAX TM Cell-Free DNA Isolation Kit提取4ml血浆，45μL洗脱液洗脱。提取的游离核酸需满足以下质控条件：提取的核酸总量大于20ng。
2、文库制备
本发明将质控合格的全部游离核酸采用EZ DNA Methylation-Lightning TM Kit(Zymo Research,Irvine,CA,USA)进行重亚硫酸盐处理。随后，重亚硫酸盐处理后的样本DNA采用单链建库方法构建预文库，预文库质检合格后通过液态芯片杂交捕获富集目标区域而完成终文库的构建。
预文库构建步骤：1)磷酸化：T4多聚核苷酸激酶将重亚硫酸盐处理后的DNA的5端磷酸化；2)SS1连接：T4 DNA Ligase(Rapid)将SS1接头连接在磷酸化的DNA的5端；3)核酸纯化：使用2倍体积的Agencourt AMPure XP system(Beckman CouLter,CA,USA)去除剩余的接头；4)SS2连接：T4 DNA Ligase(Rapid)将SS2接头连接在磷酸化的DNA的3端；5)核酸纯化：使用2倍体积的Agencourt AMPure XP system(Beckman CouLter,CA,USA)去除剩余的接头；6)扩增：采用NEBNext Q5U Master Mix以及primer1.0(通用引物)和Bacard序列扩增上一步的核酸；7)核酸纯化：使用1.2倍体积的Agencourt AMPure XP system(Beckman CouLter,CA,USA)去引物二聚体和多余的引物；8)质检：纯化处理后的预文库采用
Figure PCTCN2021131103-appb-000008
dsDNA HS Assay Kit(Life Technologies,CA,USA)进行文库总量质检，采用LabChip GXII Touch进行文库片段分布质检。
芯片(Twist Bioscience)杂交捕获步骤：1)芯片杂交：将质检合格混合好的1.5μg的文库预先真空浓缩成粉末状再和Panel、Hybridization Mix、Blocker Solution、Universal Blockers、Hybridization Enhancer试剂混匀(芯片杂交用到的试剂均由Twist Bioscience提供)，放置在PCR仪中70度孵育16hours过夜(热盖温度为85度)；2)磁珠捕获：预先使用Streptavidin  Binding Buffer将捕获磁珠洗涤3次，将杂交完成的产物加入捕获磁珠中，孵育30分钟，Wash Buffer I清洗一次，Wash Buffer 2清洗3次，最后42μl超纯水洗脱；3)扩增：采用KAPA HiFi HotStart ReadyMix以及通用引物扩增捕获后的文库；4)纯化：采用1倍体积的Agencourt AMPure XP system(Beckman CouLter,CA,USA)去引物二聚体和多余的引物。
纯化处理后的文库采用
Figure PCTCN2021131103-appb-000009
dsDNA HS Assay Kit(Life Technologies,CA,USA)和LabChip GXII Touch进行文库核酸总量、片段分布和引物二聚体比例质检。
3、测序
将文库总量、扩增产物的片段大小分布和引物二聚体比例质检均合格的待测文库，按照1:1的物质的量进行混合，使用
Figure PCTCN2021131103-appb-000010
dsDNA HS Assay Kit(Life Technologies,CA,USA)对混合文库进行精确定量，将文库变性稀释后使用NextSeq500台式测序仪采用PE75进行上机测序。
4、肝癌分类模型的建立和评价
对于测序得到的原始fastq数据，对原始数据过滤后使用bismark甲基化分析软件对芯片捕获片段进行甲基化分析，获得候选基因单个位点的甲基化水平和基因片段的甲基化水平。利用候选基因单个位点的甲基化水平和基因片段的甲基化水平，针对肝癌和对照样品进行差异分析和模型构建。本发明通过对数据采用Logistics回归分析进行了肝癌分类模型的构建和评价。
实施例3、本发明试剂组合检测临床样本的甲基化水平
收集63例原发性肝癌患者、25例肝硬化患者、15例肝炎患者和7例健康人的样本，按照实施例2所述的方法检测分析ZNF397OS基因cg16657538甲基化位点的甲基化水平，以及ZNF397OS基因与其余基因的组合在样本中的甲基化水平，以验证其检验肝癌的效果。检测结果如表1、 图1和图2所示。
表1 ZNF397OS基因与其余基因的组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000011
其中，1代表GRASP基因(检测SEQ ID NO:5所示片段的平均甲基化水平)，2代表PAK1基因(检测包含cg17202086、cg26996201、cg12269002、cg18309286四个甲基化位点片段的平均甲基化水平)，3代表PPFIA1基因(检测SEQ ID NO:6所示片段的平均甲基化水平)，4代表OTX1基因(检测包含cg21472506、cg23229261、cg10122865三个甲基化位点片段的平均甲基化水平)，5代表ZNF397OS基因(检测cg16657538甲基化位点片段的平均甲基化水平)。
从表1中可以看出，在组织样本中，所有试剂组合均能以至少0.9176的特异性和0.8981的灵敏度以及0.9396的曲线下面积对肝癌进行预测。而在血浆的游离DNA样本中，所有试剂组合均能以至少0.8936的特异性和 0.7619的灵敏度以及0.8845的曲线下面积对肝癌进行预测。因此，本发明的试剂组合具有对肝癌很好的预测效果，特别地，在使用血浆游离DNA作为样本时，具有优异的特异性和灵敏度。
对比例1
为了考察ZNF397OS基因上其他甲基化位点能否统一作为检测肝癌的标志物，选取了ZNF397OS基因中另外的甲基化位点作为对比例(其均位于当前ZNF397OS基因所选靶区段外部)，比较其在癌与非癌样品中甲基化差异，结果如图3和图4所示。从图3和图4中可以看出，ZNF397OS基因中在所选靶区段上下游的甲基化位点在肝癌与非癌组织中不具有显著差异，不适合作为检测肝癌的标志物。
对比例2
为了考察ZNF397OS基因能否作为检测肝癌的标志物，进一步选取了本领域知晓的与肝癌密切相关的基因PLAC8和ATXN1(参见Xu RH,Wei W,Krawczyk M,Wang W,Luo H,Flagg K,et al.Circulating tumour DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma.Nature Materials,2017和中国专利CN106947830B)。同样按照上述实施例的方法进行检测，检测结果如下表3所示。
表3对比基因及其组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000012
其中，6代表PLAC8基因(检测该基因的cg11606215甲基化水平)，7 代表ATXN1基因(检测该基因的cg24067911甲基化水平)。
从表3中可以看出，对比基因单个在组织样本中的最高曲线下面积为0.858，在游离DNA样本中的最高曲线下面积为0.75，低于本发明的ZNF397OS的曲线下面积。而组合的曲线下面积也低于本发明试剂组合的曲线下面积的值。
实施例4、本发明试剂组合检测临床样本的甲基化水平
收集63例原发性肝癌患者、25例肝硬化患者、15例肝炎患者和7例健康人的样本，按照实施例2所述的方法检测分析PAK1基因各甲基化位点在样本中的甲基化水平，以验证其检验肝癌的效果。肝细胞癌、肝硬化、肝炎和健康人的诊断均以最终的医院病理诊断为依据，检测结果如表4、图5、图6所示。
表4 PAK1基因甲基化位点在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000013
从表4中可以看出，在组织中PAK1基因的各甲基化位点能以至少0.360的特异性和0.843的灵敏度以及0.8194的曲线下面积对肝癌进行预测。而 在血浆的游离DNA中，所有试剂组合均能以至少0.8511的特异性和0.7460的灵敏度以及0.833的曲线下面积对肝癌进行预测。
实施例5、本发明试剂组合检测临床样本的甲基化水平
收集63例原发性肝癌患者、25例肝硬化患者、15例肝炎患者和7例健康人的样本，按照实施例2所述的方法检测分析PAK1基因与其余基因的组合在样本中的甲基化水平，以验证其检验肝癌的效果。检测结果如下表5所示。
表5 PAK1基因与其余基因的组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000014
其中，1代表GRASP基因(检测SEQ ID NO:5所示片段的平均甲基化水平)，2代表PAK1基因(检测cg17202086、cg26996201、cg18309286三个甲基化位点的甲基化水平)，3代表PPFIA1基因(检测SEQ ID NO:6所示片段的平均甲基化水平)，4代表OTX1基因(检测cg21472506、 cg23229261、cg10122865三个甲基化位点的甲基化水平)，5代表ZNF397OS基因(检测cg16657538甲基化位点的甲基化水平)。
从表5中可以看出，在组织样本中，所有试剂组合均能以至少0.9176的特异性和0.8904的灵敏度以及0.8944的曲线下面积对肝癌进行预测。而在血浆游离DNA样本中，所有试剂组合均能以至少0.7937的特异性和0.8578的灵敏度以及0.8578的曲线下面积对肝癌进行预测。因此，本发明的试剂组合具有对肝癌很好的预测效果，特别地，在使用血浆游离DNA作为样本时，具有优异的特异性和灵敏度。
对比例3
为了考察PAK1基因上其他甲基化位点能否统一作为检测肝癌的标志物，选取了PAK1基因中所选靶区段上下游甲基化位点，比较其在癌与非癌样品中甲基化差异，结果如图7和图8所示。从图7和图8中可以看出，PAK1基因中在所选靶区段上下游的甲基化位点在癌与非癌组织中不具有显著差异，不适合作为检测肝癌的标志物。
对比例4
为了考察PAK1基因能否作为检测肝癌的标志物，进一步选取了本领域知晓的常见与肝癌相关的基因PLAC8和ATXN1(参见Xu RH,Wei W,Krawczyk M,Wang W,Luo H,Flagg K,et al.Circulating tumour DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma.Nature Materials,2017和中国专利CN106947830B)。同样按照上述实施例的方法进行检测，检测结果如下表6所示。
表6对比基因及其组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000015
Figure PCTCN2021131103-appb-000016
其中，6代表PLAC8基因(检测该基因的cg11606215甲基化水平)，7代表ATXN1基因(检测该基因的cg24067911甲基化水平)。
从表6中可以看出，对照基因单个的最高组织曲线下面积为0.858，游离DNA最高曲线下面积为0.75，低于本发明的PAK1基因的曲线下面积。而组合的曲线下面积也低于本发明试剂组合的曲线下面积的值。
实施例6、本发明试剂组合检测临床样本的甲基化水平
收集63例原发性肝癌患者、25例肝硬化患者、15例肝炎患者和7例健康人的样本，按照实施例2所述的方法检测分析OTX1基因在样本中的甲基化水平，以验证其检验肝癌的效果。肝细胞癌、肝硬化、肝炎和健康人的诊断均以最终的医院病理诊断为依据。检测结果如表7、图9和图10所示。
表7 OTX1基因甲基化位点在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000017
从表7中可以看出，在组织中OTX1基因的各甲基化位点能以至少0.846的特异性和0.860的灵敏度以及0.914的曲线下面积对肝癌进行预测。而在血浆的游离DNA中，所有试剂组合均能以至少0.8298的特异性和0.778的灵敏度以及0.8823的曲线下面积对肝癌进行预测。
实施例7、本发明试剂组合检测临床样本的甲基化水平
收集63例原发性肝癌患者、25例肝硬化患者、15例肝炎患者和7例健康人的样本，按照实施例2所述的方法检测分析OTX1基因与其余基因的组合在样本中的甲基化水平，以验证其检验肝癌的效果。检测结果如下表8所示：
表8 OTX1基因与其余基因的组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000018
其中，1代表GRASP基因(检测SEQ ID NO:5所示片段的平均甲基化水平)，2代表PAK1基因(检测cg17202086、cg26996201、cg12269002、cg18309286四个甲基化位点的甲基化水平)，3代表PPFIA1基因(检测SEQ ID NO:6所示片段的平均甲基化水平)，4代表OTX1基因(检测cg21472506、cg23229261、cg10122865三个甲基化位点的平均甲基化水平)，5代表ZNF397OS基因(检测cg16657538甲基化位点的甲基化水平)。
从表中可以看出，在组织样本中，所有试剂组合均能以至少0.9024的 特异性和0.8904的灵敏度以及0.9398的曲线下面积对肝癌进行预测。而在血浆游离DNA样本中，所有试剂组合均能以至少0.8723的特异性和0.8571的灵敏度以及0.9132的曲线下面积对肝癌进行预测。因此，本发明的试剂组合具有对肝癌很好的预测效果，特别地，在使用血浆游离DNA作为样本时，具有优异的特异性和灵敏度。
对比例5
为了考察OTX1基因上其他甲基化位点能否统一作为检测肝癌的标志物，选取了OTX1基因中所选靶区段上游甲基化位点，比较其在癌与非癌样品中甲基化差异，结果如图11和图12所示。从图11和图12中可以看出，OTX1基因中在所选靶区段上游的甲基化位点在癌与非癌组织中不具有显著差异，不适合作为检测肝癌的标志物。
对比例6
为了考察OTX1基因能否作为检测肝癌的标志物，进一步选取了本领域知晓的常见与肝癌相关的基因PLAC8和ATXN1(参见Xu RH,Wei W,Krawczyk M,Wang W,Luo H,Flagg K,et al.Circulating tumour DNA methylation markers for diagnosis and prognosis for diagnosis and prognosis of hepatocellular carcinoma.Nature Materials,2017和中国专利CN106947830B)。同样按照上述实施例的方法进行检测，检测结果如下表9所示。
表9对比基因及其组合在Logistics肝癌分类模型中的预测性能
Figure PCTCN2021131103-appb-000019
其中，6代表PLAC8基因(检测该基因的cg11606215甲基化水平)，7代表ATXN1基因(检测该基因的cg24067911甲基化水平)。
从表9中可以看出，在组织样本中对照基因单个的最高曲线下面积为0.858，在游离DNA样本中最高曲线下面积为0.75，低于本发明的OTX1基因的曲线下面积。而基因组合模型的曲线下面积也低于本发明试剂相关组合模型的曲线下面积的值。

Claims (21)

  1. 一种用于检测肝癌的试剂组合，所述试剂组合包括以下检测试剂中的任意一种：
    1)用于检测PAK1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg17202086、cg26996201、cg18309286；
    2)用于检测OTX1基因的以下甲基化位点中至少一个的甲基化水平的检测试剂：cg23229261、cg10122865；以及
    3)用于检测ZNF397OS基因的cg16657538甲基化位点的甲基化水平的检测试剂。
  2. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中至少一个的甲基化水平的检测试剂：GRASP、ZNF397OS、PPFIA1，以及OTX1。
  3. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中至少两个的甲基化水平的检测试剂：GRASP、ZNF397OS、PPFIA1，以及OTX1。
  4. 根据权利要求1中任一项所述的试剂组合，其中，当所述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下基因中至少三个的甲基化水平的检测试剂：GRASP、ZNF397OS、PPFIA1，以及OTX1。
  5. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括1)时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：GRASP、ZNF397OS、PPFIA1，以及OTX1。
  6. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2)时，所述试剂组合包括用于检测OTX1基因的以下甲基化位点的甲基化水平的检测试剂：cg23229261、cg10122865。
  7. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2) 时，所述试剂组合还包括用于检测OTX1基因的甲基化位点cg21472506的甲基化水平的检测试剂。
  8. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中至少一个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及ZNF397OS。
  9. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中至少两个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及ZNF397OS。
  10. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下基因中至少三个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及ZNF397OS。
  11. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括2)时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及ZNF397OS。
  12. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括3)时，根据权利要求1所述的试剂组合，其中，所述试剂组合进一步包括用于检测以下基因中至少一个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及OTX1。
  13. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下基因中至少两个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及OTX1。
  14. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括3)时，所述试剂组合进一步包括用于检测以下基因中至少三个的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及OTX1。
  15. 根据权利要求1所述的试剂组合，其中，当所述试剂组合包括3) 时，所述试剂组合进一步包括用于检测以下四个基因的甲基化水平的检测试剂：GRASP、PAK1、PPFIA1，以及OTX1。
  16. 根据权利要求1-15中任一项所述的试剂组合，其中，所述检测试剂为核酸引物、测序Tag序列、甲基化芯片、核酸探针中的任意一种或多种。
  17. 根据权利要求1-15中任一项所述的试剂组合，其中，所述试剂组合进一步包括提取血浆游离DNA的试剂。
  18. 如权利要求1-17中任一项所述的试剂组合在制备用于检测肝癌的试剂盒中的用途。
  19. 如权利要求1-17中任一项所述的试剂组合在制备用于使用血浆游离DNA检测肝癌的试剂盒中的用途。
  20. 一种用于检测肝癌的试剂盒，所述试剂盒包括如权利要求1-17中任一项所述的试剂组合。
  21. 根据权利要求20所述的试剂盒，其中，所述试剂盒还包括其余的试剂，例如提取核酸的试剂，优选地提取血浆游离DNA的试剂、纯化核酸的试剂、重亚硫酸盐、T4多聚核苷酸激酶、T4连接酶中的至少一种。
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