Marker and probe composition for monitoring liver cancer and / or liver cancer relapse and application of marker and probe composition

Abstract

The invention provides a marker and a probe composition for monitoring liver cancer and / or liver cancer relapse and application of the marker and the probe composition. The marker comprises any one or two of an FJX1 gene and a ZNF135 gene. Meanwhile, the invention also provides a probe composition for monitoring liver cancer and / or liver cancer relapse. According to the application, the two genes are used as markers for monitoring liver cancer and / or liver cancer recurrence for the first time, and when the two genes are used as the markers and combined with the composition provided by the invention for detecting liver cancer and / or liver cancer recurrence, the detection result has relatively good sensitivity.

Description

Technical Field

[0001] This application relates to the technical field of cancer markers, and particularly relates to a marker, a probe composition and their applications for monitoring liver cancer and / or liver cancer recurrence. Background Art

[0002] Currently, the methods used clinically for diagnosing liver cancer include serum alpha-fetoprotein (AFP) and ultrasound examination. However, both of these detection methods lack sufficient sensitivity and specificity. Especially for the detection of early-stage liver cancer, the sensitivity of AFP is about 65%. In addition, the 5-year recurrence rate of liver cancer patients is as high as ~80%. Research reports indicate that minimal residual disease is closely related to early cancer recurrence. Liver cancer recurrence and metastasis are also one of the main factors inducing death. Therefore, it is urgent to develop a high-sensitivity and high-specificity liver cancer recurrence monitoring technology, which is an effective way to reduce the mortality rate of liver cancer and improve the survival rate.

[0003] Currently, a large number of studies have confirmed that changes in DNA methylation are closely related to the occurrence and development of cancer. Subsequently, the methylation profile of circulating tumor DNA (ctDNA) has become a very important type of molecule in liquid biopsy technology. The methylation signal of ctDNA in blood can achieve early cancer diagnosis, progression monitoring and efficacy evaluation earlier than clinical indicators and accurately.

[0004] For example, the related prior art discloses early recurrence markers for liver cancer, which include the methylation levels of BDNF, FOXL2, LMO7, NCAM1, MEIS3, PLA2G7 and LTB4R2. The detection method is:

[0005] MER = -0.941 - X1×A1 + X2×A2 + X3×A3 + X4×A4 + X5×A5 + X6×A6 - X7×A7, where the range of X1 is from -0.007 to 0.043, the range of X2 is from -0.019 to 0.025, the range of X3 is from -0.007 to 0.074, the range of X4 is from -0.046 to 0.079, the range of X5 is from 0.001 to 0.159, the range of X6 is from -0.002 to 0.006, and the range of X7 is from -0.026 to 0.006. And A1, A2, A3, A4, A5, A6 and A7 respectively represent the methylation degrees of BDNF, FOXL2, LMO7, NCAM1, MEIS3, PLA2G7 and LTB4R2. The sensitivity and specificity of this detection method are 85.7% and 73.7% respectively, and the AUC is 0.855.

[0006] In addition, currently clinically, the progress of liver cancer is monitored based on the patient's serum marker AFP and the ultrasound results. However, the sensitivity and specificity of these two technologies are not sufficient to meet the clinical needs. Summary of the Invention

[0007] This application provides a marker, a probe composition and their applications for the monitoring of liver cancer and / or recurrence of liver cancer. This study uses the changes in ctDNA methylation in patients' plasma to monitor the progression of liver cancer in patients, and provides a non-invasive, highly sensitive and highly specific methylation marker that can be dynamically monitored and its application in the monitoring of liver cancer recurrence.

[0008] This application relates to the following:

[0009] 1. A marker for the monitoring of liver cancer and / or recurrence of liver cancer, the marker comprising any one or two of the FJX1 gene and the ZNF135 gene;

[0010] Preferably, the marker is a combination of any one or two of the FJX1 gene and the ZNF135 gene.

[0011] 2. The marker according to item 1, wherein the target sequence of the FJX1 gene comprises any one, two, three or more of the following groups: the sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, SEQ ID NO.15-16;

[0012] Or, the target sequence of the FJX1 gene is any one, two, three or more selected from the following groups: the sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, SEQ ID NO.15-16;

[0013] And / or, wherein the target sequence of the ZNF135 gene comprises any one, two, three or more of the following groups: the sequences shown in SEQ ID NO.2, SEQ ID NO.13-14, SEQ ID NO.17-18;

[0014] Or, the target sequence of the ZNF135 gene is any one, two, three or more selected from the following groups: the sequences shown in SEQ ID NO.2, SEQ ID NO.13-14, SEQ ID NO.17-18.

[0015] 3. A probe composition for the monitoring of liver cancer and / or recurrence of liver cancer, the probe composition comprising a probe that specifically binds to the methylation product of the marker according to item 1 or 2.

[0016] 4. The probe composition according to item 3, the probe composition comprising a hypermethylated probe composition and a hypomethylated probe composition;

[0017] Among them, the hypermethylated probe composition comprises one, two or more hypermethylated probes that target and bind to the hypermethylated sequence of the biomarker;

[0018] Among them, the hypomethylated probe composition comprises x hypomethylated probes that target and bind to the hypomethylated sequence of the biomarker, where x is a natural number arbitrarily selected from 1 to 5, and there are a nucleic acid overlaps between any two probes, and a is a natural number arbitrarily selected from 0 to 100;

[0019] Among them, the hypermethylated probe composition comprises y hypermethylated probes that target and bind to the hypermethylated sequence of the biomarker, where y is a natural number arbitrarily selected from 1 to 5, and there are b nucleic acid overlaps between any two probes, and b is a natural number arbitrarily selected from 0 to 100.

[0020] 5. The probe composition according to item 4, wherein the hypermethylated probe composition comprises any one, two, three or four of the sequences shown in SEQ ID NO. 3-6; or, the hypermethylated probe composition is selected from any one, two, three or four of the sequences shown in SEQ ID NO. 3-6;

[0021] And / or, the hypomethylated probe composition comprises any one, two, three or four of the sequences shown in SEQ ID NO. 7-10; or, the hypomethylated probe composition is selected from any one, two, three or four of the sequences shown in SEQ ID NO. 7-10.

[0022] 6. A composition for monitoring liver cancer and / or recurrence of liver cancer, the composition comprising the probe composition according to any one of items 3-5.

[0023] 7. The composition according to item 6, wherein the composition further comprises primers;

[0024] The primers are fragments of at least 9 nucleotides in the target sequences of any one or two selected from the FJX1 gene and the ZNF135 gene, and the fragment of 9 nucleotides contains at least one CpG dinucleotide sequence;

[0025] Preferably, the composition further comprises a probe, and the probe is a fragment that targets and binds to at least 15 nucleotides in the target sequences of any one or two selected from the FJX1 gene and the ZNF135 gene, and the fragment of 15 nucleotides contains at least one CpG dinucleotide sequence;

[0026] More preferably, the probe further comprises a blocker that preferentially binds to the target sequence in the unmethylated state;

[0027] More preferably, the composition further comprises a reagent that converts the 5-position unmethylated cytosine base of the target sequence of any one or both of the FJX1 gene and the ZNF135 gene into uracil.

[0028] 8. Use of the biomarker according to item 1 or 2 and / or the probe composition according to any one of items 3-5 in the preparation of a product for monitoring liver cancer and / or recurrence of liver cancer;

[0029] Preferably, the product for monitoring liver cancer and / or recurrence of liver cancer is a kit for monitoring liver cancer and / or recurrence of liver cancer, or a chip for monitoring liver cancer and / or recurrence of liver cancer.

[0030] 9. A kit for monitoring liver cancer and / or recurrence of liver cancer, comprising the probe composition according to any one of items 3-5, and / or the composition according to item 6 or 7.

[0031] 10. A chip for monitoring liver cancer and / or recurrence of liver cancer, comprising the probe composition according to any one of items 3-5, and / or the composition according to item 6 or 7.

[0032] Advantages of the Invention

[0033] In this application, it is first discovered that the FJX1 gene and / or the ZNF135 gene can be used as biomarkers for monitoring liver cancer and / or recurrence of liver cancer. At the same time, high-methylation probes and low-methylation probes for the FJX1 gene for detecting high-methylation sequences and low-methylation sequences of the FJX1 gene respectively, and high-methylation probes and low-methylation probes for the ZNF135 gene for detecting high-methylation sequences and low-methylation sequences of the ZNF135 gene respectively are also provided. Using this detection method for monitoring liver cancer and / or recurrence of liver cancer has good sensitivity and specificity. Detailed Embodiments

[0034] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. The specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of components as the criterion for distinction.

[0035] Unless specifically defined elsewhere in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this application belongs.

[0036] As used throughout the specification and claims, the term "comprising" or "including" is an open-ended term and should be interpreted as "including but not limited to". The following description in the specification is the preferred embodiment for implementing the present application, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be subject to what is defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0037] It should be understood that the embodiments of the present application described herein include embodiments of "consisting of" and / or "consisting essentially of". References herein to a "about" value or parameter include (and describe) variations that are specific to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0038] As used herein, a reference to a "not" value or parameter generally means and describes a value or parameter "other than". For example, the method is not for treating cancer type X means the method is for treating cancers other than type X.

[0039] As used herein, the term "about X - Y" has the same meaning as "about X to about Y".

[0040] As used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a basis for antecedent use in conjunction with the recitation of claim elements of exclusive terms such as "only", "solely", etc., or the use of a "negative" limitation.

[0041] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, as used herein, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0042] As used herein, the term "methylation" refers to the methylation process occurring at the 5th carbon atom of cytosine in CpG dinucleotides. As a stable modification state, under the action of DNA methyltransferase, it can be inherited to the newly generated daughter DNA during DNA replication. It is an important epigenetic mechanism. When DNA is methylated, the methylation of the gene promoter region can lead to transcriptional silencing of tumor suppressor genes. Therefore, it is closely related to the occurrence of tumors. Aberrant methylation includes hypermethylation of tumor suppressor genes and DNA repair genes, hypomethylation of repetitive sequence DNA, and loss of imprinting of certain genes, which is related to the occurrence of various tumors. The methylation described in this application can be methylation level, methylation degree, or methylation state. When analyzing the methylation of such target sequences, those skilled in the art can use quantitative determination methods to determine methylation.

[0043] As used herein, the term "target" when used in reference to nucleic acid capture, detection, or analysis methods refers to a nucleic acid having a property to be detected or analyzed, such as a specific nucleotide sequence, for example, in a sample suspected of containing the target nucleic acid. In some embodiments, the target is a nucleic acid having a specific sequence for which its methylation status is desired to be determined. For example, when detecting a target, the "target" generally refers to the region of the nucleic acid that binds to the probe. Therefore, the "target" is preferably sorted out from other nucleic acid sequences that may be present in the sample.

[0044] As used herein, the term "marker" refers to a substance (such as a nucleic acid or a region of a nucleic acid or a protein) that can be used to distinguish abnormal cells (such as cancer cells) from normal cells, for example, based on the presence, absence, or status (such as methylation status) of the marker substance.

[0045] As used herein, the term "primer" refers to the following oligonucleotide: whether the oligonucleotide exists naturally in a purified restriction enzyme digestion or is produced synthetically, it can be used as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (for example, in the presence of nucleotides and an inducer such as a biocatalyst (such as DNA polymerase, etc.)). To obtain maximum efficiency in amplification, the primer is usually single-stranded, but alternatively can be partially or completely double-stranded. The portion of the primer that hybridizes to the template nucleic acid must be long enough to initiate the synthesis of an extension product in the presence of an inducer. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. The primer can include a label, tag, capture moiety, etc.

[0046] As used herein, the term "CpG" or "CpG island" refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides relative to total genomic DNA. A CpG island can be at least 100, 200 or more base pairs in length, wherein the G:C content of the region is at least 50% and the ratio of the observed CpG frequency to the expected frequency is 0.6. The observed CpG frequency relative to the expected frequency can be calculated according to the method provided by Gardiner-Garden et al. (1987) J. Mol. Biol. 196:261–281. For example, the observed CpG frequency relative to the expected frequency can be calculated according to the formula R = (A × B) / (C × D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence.

[0047] In the present application, methods well-known to those skilled in the art can be used to detect DNA methylation. For example, methylation-sensitive restriction endonuclease (MSRE) assays using southern or polymerase chain reaction (PCR) analysis, methylation-specific or methylation-sensitive PCR (MS-PCR), methylation-sensitive single nucleotide primer extension (Ms-SnuPE), high-resolution melting (HRM) analysis, bisulfite sequencing, pyrosequencing, methylation-specific single-strand conformation analysis (MS-SSCA), combined bisulfite restriction analysis (COBRA), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE), methylation-specific melting curve analysis (MS-MCA), methylation-specific denaturing high performance liquid chromatography (MSDHPLC), methylation-specific microarray (MSO). These assays can be PCR analysis, quantitative analysis using fluorescence labeling, or southern blot analysis.

[0048] As used herein, the term "detection" refers to any process of observing a marker or a change in a marker (e.g., a change in the methylation status of a marker or the expression level of a nucleic acid or protein sequence) in a sample, regardless of whether the marker or the change in the marker is actually detected. In other words, the act of probing for a marker or a change in a marker in a sample is "detection", even if the marker is determined to be absent or below the sensitivity level. Detection can be quantitative, semi-quantitative or non-quantitative observation, and can be based on comparison with one or more control samples.

[0049] In the present application, the calculation formula for the methylation level of the target region can be:

[0050] Target region methylation level = (Sum of methylation reads of all CpG sites within the target interval) / (Sum of reads of all CpG sites within the target interval). Here, the term "read" refers to a sequenced sequence, also known as "read sequence". "Methylation read" refers to a methylated sequence.

[0051] As used herein, the terms "identity" and "homology" are used interchangeably in this application and refer to sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 out of a total of 6 positions match). Generally, such comparisons are made when the two sequences are aligned to yield maximum identity. Such alignment can be achieved by methods such as those described in Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The PAM 120 weight residue table can also be used, with the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0). A gap length penalty of 12 and a gap penalty of 4 are used to determine the percentage identity between two amino acid sequences. In addition, the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm incorporated into the GAP program of the GCG software package (available from www.gcg.com) can be used, with the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percentage identity between two amino acid sequences.

[0052] As used herein, the term "probe" refers to single-stranded or double-stranded DNA with a length ranging from dozens to hundreds or even thousands of base pairs. Utilizing the denaturation and renaturation of molecules and the high precision of base complementary pairing, it can bind (hybridize) to complementary unlabeled single-stranded DNA or RNA in a test sample through hydrogen bonds to form a double-stranded complex (hybrid). After washing away the unpaired and bound probes, detection systems such as autoradiography or enzyme-linked reactions can be used to detect the results of the hybridization reaction. In the present application, the region that binds or hybridizes complementarily with the probe is the specific target region. Multiple probes are combined into a probe composition.

[0053] As used herein, the term "sensitivity", also known as sensitivity, represents the proportion of cancers detected from a certain cancer sample, and its calculation formula is: sensitivity = (detected cancers / all cancers), while "specificity", also known as specificity, represents the proportion of normals detected from a certain normal sample, and its calculation formula is specificity = (detected negatives / total negatives).

[0054] As used herein, the term "recurrence" refers to the situation where a tumor shows new progression after active treatment, with the condition improving or being controlled, and even shows spread and metastasis.

[0055] As used herein, the terms "subject", "individual", or "patient" are used interchangeably in the present application for treatment purposes and refer to any animal classified as a mammal, including humans, domestic animals, and farm animals, as well as zoo, farm, or pet animals such as dogs, horses, cats, cows, etc. In some embodiments, the individual is a human individual.

[0056] The present application provides a biomarker for the monitoring of liver cancer and / or liver cancer recurrence, and the biomarker comprises any one or both of the FJX1 gene and the ZNF135 gene.

[0057] In one embodiment, the biomarker is a combination of any one or both of the FJX1 gene and the ZNF135 gene. In one embodiment, the biomarker is the FJX1 gene and the ZNF135 gene.

[0058] In some embodiments, the target sequence of the FJX1 gene comprises any one, two, three or more of the following: the sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, SEQ ID NO.15-16. In some embodiments, the target sequence of the FJX1 gene comprises any one, two, three or more of the following: a sequence having at least 80% sequence identity with any of the sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, SEQ ID NO.15-16; for example, a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, SEQ ID NO.15-16. In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequences shown in SEQ ID NO.11 and SEQ ID NO.12. In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.11. In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.12. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequences shown in SEQ ID NO.15 and SEQ ID NO.16. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.15. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.16.

[0059] In some embodiments, the target sequence of the FJX1 gene is any one, two, three, or more selected from the group consisting of: the sequences shown in SEQ ID NO.1, SEQ ID NOs.11 - 12, SEQ ID NOs.15 - 16. In some embodiments, the target sequence of the FJX1 gene is any one, two, three, or more selected from the group consisting of: sequences having at least 80% sequence identity with any of the sequences shown in SEQ ID NO.1, SEQ ID NOs.11 - 12, SEQ ID NOs.15 - 16; for example, sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO.1, SEQ ID NOs.11 - 12, SEQ ID NOs.15 - 16.

[0060] In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequences shown in SEQ ID NO.11 and SEQ ID NO.12. In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.11. In some embodiments, the hypermethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.12. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequences shown in SEQ ID NO.15 and SEQ ID NO.16. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.15. In some embodiments, the hypomethylated target sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO.16.

[0061] In some embodiments, the target sequence of the ZNF135 gene comprises any one, two, three or more of the following: the sequences shown in SEQ ID NO.2, SEQ ID NOs.13-14, SEQ ID NOs.17-18. In some embodiments, the target sequence of the ZNF135 gene comprises any one, two, three or more of the following: sequences having at least 80% sequence identity with any of the sequences shown in SEQ ID NO.2, SEQ ID NOs.13-14, SEQ ID NOs.17-18; for example, sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the sequences shown in SEQ ID NO.2, SEQ ID NOs.13-14, SEQ ID NOs.17-18.

[0062] In some embodiments, the target sequence of the ZNF135 gene is any one, two, three or more selected from the following: the sequences shown in SEQ ID NO.2, SEQ ID NOs.13-14, SEQ ID NOs.17-18. In some embodiments, the target sequence of the ZNF135 gene is any one, two, three or more selected from the following: sequences having at least 80% sequence identity with any of the sequences shown in SEQID NO.1, SEQ ID NOs.13-14, SEQ ID NOs.17-18; for example, sequences having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the sequences shown in SEQ ID NO.2, SEQ ID NOs.13-14, SEQ ID NOs.17-18. The target sequences of the ZNF135 gene described above are all included in this application.

[0063] In some embodiments, the hypermethylated target sequences of the ZNF135 gene include the sequences shown in SEQ ID NO.13 and SEQ ID NO.14. In some embodiments, the hypermethylated target sequences of the ZNF135 gene include the sequence shown in SEQ ID NO.13. In some embodiments, the hypermethylated target sequences of the ZNF135 gene include the sequence shown in SEQ ID NO.14. In some embodiments, the hypomethylated target sequences of the ZNF135 gene include the sequences shown in SEQ ID NO.17 and SEQ ID NO.18. In some embodiments, the hypomethylated target sequences of the ZNF135 gene include the sequence shown in SEQ ID NO.17. In some embodiments, the hypomethylated target sequences of the ZNF135 gene include the sequence shown in SEQ ID NO.18. The target sequences of the ZNF135 gene described above are all included in this application.

[0064] Among them, hypermethylation means that after the biomarker is bisulfite-converted, base C becomes base T, but if it is base CG, base C remains unchanged; the hypomethylation means that after the biomarker is bisulfite-converted, all base CGs do not undergo methylation, and base C becomes base T.

[0065] This application provides a probe composition for monitoring liver cancer and / or recurrence of liver cancer. The probe composition includes probes that specifically bind to the methylation products of the above-mentioned biomarkers.

[0066] In some embodiments, the probe composition includes a hypermethylated probe composition and a hypomethylated probe composition.

[0067] In some embodiments, the hypermethylated probe composition includes one, two or more hypermethylated probes that specifically bind to the hypermethylated sequences of the biomarker. Among them, the hypomethylated probe composition includes x hypomethylated probes that specifically bind to the hypomethylated sequences of the biomarker, where x is a natural number arbitrarily selected from 1 to 5, and there are a nucleic acid overlaps between any two probes, and a is a natural number arbitrarily selected from 0 to 100. For example, x is 1, 2, 3, 4, or 5. For example, a is 0, 5, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, or 100. Among them, the hypermethylated probe composition includes y hypermethylated probes that specifically bind to the hypermethylated sequences of the biomarker, where y is a natural number arbitrarily selected from 1 to 5, and there are b nucleic acid overlaps between any two probes, and b is a natural number arbitrarily selected from 0 to 100. For example, y is 1, 2, 3, 4, or 5. For example, b is 0, 5, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, or 100.

[0068] In some embodiments, the hypermethylated probe composition comprises any one, two, three, or four of the sequences shown in SEQ ID NOs. 3-6. In some embodiments, the hypermethylated probe composition is selected from any one, two, three, or four of the sequences shown in SEQ ID NOs. 3-6. In some embodiments, the hypomethylated probe composition comprises any one, two, three, or four of the sequences shown in SEQ ID NOs. 7-10. In some embodiments, the hypomethylated probe composition is selected from any one, two, three, or four of the sequences shown in SEQ ID NOs. 7-10. In some embodiments, the probe targeting the hypermethylated sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4; for example, the sequence shown in or comprising SEQ ID NO. 3 targets the sequence shown in or comprising SEQ ID NO. 11, and the sequence shown in or comprising SEQ ID NO. 4 targets the sequence shown in or comprising SEQ ID NO. 12. In some embodiments, the probe targeting the hypomethylated sequence of the FJX1 gene comprises the sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8; for example, the sequence shown in or comprising SEQ ID NO. 7 targets the sequence shown in or comprising SEQ ID NO. 15, and the sequence shown in or comprising SEQ ID NO. 8 targets the sequence shown in or comprising SEQ ID NO. 16. In some embodiments, the probe targeting the hypermethylated sequence of the ZNF135 gene comprises the sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6; for example, the sequence shown in or comprising SEQ ID NO. 5 targets the sequence shown in or comprising SEQ ID NO. 13, and the sequence shown in or comprising SEQ ID NO. 6 targets the sequence shown in or comprising SEQ ID NO. 14. In some embodiments, the probe targeting the hypomethylated sequence of the ZNF135 gene comprises the sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10; for example, the sequence shown in or comprising SEQ ID NO. 9 targets the sequence shown in or comprising SEQ ID NO. 17, and the sequence shown in or comprising SEQ ID NO. 10 targets the sequence shown in or comprising SEQ ID NO. 18.

[0069] The present application also provides a composition for monitoring liver cancer and / or recurrence of liver cancer, the composition comprising the above-mentioned probe composition.

[0070] In some embodiments, the composition further comprises primers.

[0071] In some embodiments, the primer is a fragment of at least 9 nucleotides in a target sequence selected from any one or both of the FJX1 gene and the ZNF135 gene, and the fragment of 9 nucleotides contains at least one CpG dinucleotide sequence.

[0072] In some preferred embodiments, the composition further comprises a probe, which is a fragment that specifically binds to at least 15 nucleotides in a target sequence selected from any one or both of the FJX1 gene and the ZNF135 gene, and the fragment of 15 nucleotides contains at least one CpG dinucleotide sequence.

[0073] In some embodiments, the probe further comprises a blocker that preferentially binds to a target sequence in an unmethylated state. The blocker is used to improve the amplification specificity of the PCR amplification primer. The 5' end of the blocker nucleotide sequence has an overlapping region of at least 5 nucleotides with the 3' end nucleotide sequence of the forward or reverse primer. The blocker is complementary to the forward or reverse primer and binds to the same strand of the target gene target sequence DNA. The melting temperature of the blocker is higher than that of the forward or reverse primer by more than (including) 5 °C. The nucleotide sequence of the blocker contains at least one CpG dinucleotide sequence and is complementary to the sequence of the unmethylated target gene target sequence DNA after bisulfite conversion. Therefore, when the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated states, especially when the DNA in the methylated state is far less than the DNA in the unmethylated state, the unmethylated DNA after bisulfite conversion will preferentially bind to the blocker, thereby preventing the binding of the DNA template to the PCR primer, and thus no PCR amplification occurs. However, the methylated DNA does not bind to the blocker, so it binds to the primer set and undergoes PCR amplification, and then the fragment obtained by amplification is detected directly or indirectly.

[0074] In some embodiments, the composition further comprises a reagent that converts the 5-position unmethylated cytosine base in the target sequence of any one or both of the FJX1 gene and the ZNF135 gene into uracil.

[0075] The present application also provides the use of the above-mentioned marker and / or the above-mentioned probe composition in the preparation of a product for monitoring liver cancer and / or liver cancer recurrence.

[0076] In some embodiments, the product for monitoring liver cancer and / or liver cancer recurrence is a kit for monitoring liver cancer and / or liver cancer recurrence; in some embodiments, the product for monitoring liver cancer and / or liver cancer recurrence is a chip for monitoring liver cancer and / or liver cancer recurrence.

[0077] The biological sample detected by the kit or chip can be peripheral whole blood, plasma or serum.

[0078] The present application also provides a kit for monitoring liver cancer and / or recurrence of liver cancer, which comprises the above-mentioned probe composition and / or the above-mentioned composition. Those skilled in the art should understand that the kit also comprises components necessary for storage, transportation, etc. of the kit, such as buffer solution, etc.

[0079] The present application also provides a chip for monitoring liver cancer and / or recurrence of liver cancer, which comprises the above-mentioned probe composition and / or the above-mentioned composition.

[0080] Examples

[0081] The specific embodiments of the present application will be described in more detail below with reference to specific examples. It should be understood that the technical solutions of the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0082] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0083] Example 1.

[0084] I. Markers and probes:

[0085] Markers:

[0086] The markers selected by the present application for monitoring liver cancer and / or recurrence of liver cancer are FJX1 gene and ZNF135 gene.

[0087] Specifically, the target sequence of the FJX1 gene is as shown in SEQ ID NO.1, the hypermethylated sequences of the FJX1 gene are as shown in SEQ ID NO.11 and SEQ ID NO.12, and the hypomethylated sequences of the FJX1 gene are as shown in SEQ ID NO.15 and SEQ ID NO.16;

[0088] The target sequence of the ZNF135 gene is as shown in SEQ ID NO.2, the hypermethylated sequences of the ZNF135 gene are as shown in SEQ ID NO.13 and SEQ ID NO.14, and the hypomethylated sequences of the ZNF135 gene are as shown in SEQ ID NO.17 and SEQ ID NO.18.

[0089] Probe composition:

[0090] The hypermethylation probes for the hypermethylated sequences of the FJX1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, and the hypomethylation probes for the hypomethylated sequences of the FJX1 gene are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0091] The hypermethylation probes for the hypermethylated sequences of the ZNF135 gene are shown in SEQ ID NO.5 and SEQ ID NO.6, and the hypomethylation probes for the hypomethylated sequences of the ZNF135 gene are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0092] II. Kit and monitoring method for monitoring liver cancer and / or recurrence of liver cancer

[0093] This method includes the following steps:

[0094] Collect samples from the subject;

[0095] Extract and purify cfDNA in the sample (using Qiagen Circulating Nucleic Acid Kit for cfDNA preparation, and the product number is 55114 for cfDNA extraction and purification);

[0096] Construct a cfDNA library for sequencing for the purified cfDNA sample (using Swift's ACCEL- METHYL-SEQ DNA LIBRARY KIT for library preparation, and the product number is 30096); Bisulfite convert the constructed cfDNA library;

[0097] Pre-PCR amplify the bisulfite-converted cfDNA library above;

[0098] Hybridization capture the pre-PCR amplified sample using the probe;

[0099] PCR amplify the product after hybridization capture;

[0100] Perform high-throughput next-generation sequencing on the product after hybridization capture and PCR amplification;

[0101] Analyze the sequencing data to determine the methylation level of the sample;

[0102] Calculate the threshold of each biomarker based on the methylation status of the existing samples, and judge the disease progression of the patient based on the biomarker methylation level of the sample. If it exceeds the threshold, it is cancer recurrence; if it is lower than the threshold, it is no recurrence.

[0103] The specific method is:

[0104] 1.1 cfDNA Extraction and Purification:

[0105] 1.1.1 Plasma Sample Preparation:

[0106] Centrifuge the blood sample at 2000g for 10 min at 4°C, and transfer the plasma to a new centrifuge tube. Centrifuge the plasma sample at 16000g for 10 min at 4°C. Depending on the type of collection tube used, perform the next step. The type of collection tube used in this experiment is other.

[0107] 1.1.2 Lysis and Binding:

[0108] 1.1.2.1 Pipette 100 μl, 200 μl, 300 μl, 400 μl, 500 μl of QIAGEN Proteinase K into a 50 ml centrifuge tube.

[0109] 1.1.2.2 Add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml of plasma or serum to the above 50 ml centrifuge tube.

[0110] 1.1.2.3 Add 0.8 ml, 1.6 ml, 2.4 ml, 3.2 ml, 4.0 ml of Buffer ACL (containing 1.0 μg carrier RNA), cover the lid and vortex for 30 s; Note: Mix well to ensure sufficient lysis; proceed to the next step immediately.

[0111] 1.1.2.4 Incubate at 60°C for 30 min.

[0112] 1.1.2.5 Take out the centrifuge tube and place it on the test bench, and unscrew the tube cap.

[0113] 1.1.2.6 Add 1.8 ml, 3.6 ml, 5.4 ml, 7.2 ml, 9.0 ml of Buffer ACB to the 50 ml centrifuge tube; cover the lid and mix for 15 - 30 s.

[0114] 1.1.2.7 Incubate the lysis mixture on ice for 5 min.

[0115] 1.1.2.8 Insert the QIAamp Mini column into the vacuum pump adapter, and insert the 20 ml tube extender onto the column; Note: Ensure that the tube extender is firmly inserted onto the QIAamp Mini column to avoid sample leakage.

[0116] 1.1.2.9. Carefully add the lysate-buffer ACB mixture from step 7 to the tube extender of the QIAamp Mini column, and turn on the vacuum pump; when all the lysate has been completely drawn out of the Mini column, turn off the vacuum pump and release the pressure to 0 mbar; carefully remove and discard the tube extender.

[0117] 1.1.3. Washing:

[0118] 1.1.3.1. Add 600 μl of Buffer ACW1 to the Mini column, leave the lid open, and turn on the vacuum pump; after all the liquid has passed through the column membrane, turn off the vacuum pump and release the pressure to 0 mbar.

[0119] 1.1.3.2. Add 750 μl of Buffer ACW2 to the Mini column, leave the lid open, and turn on the vacuum pump; after all the liquid has passed through the column membrane, turn off the vacuum pump and release the pressure to 0 mbar.

[0120] 1.1.3.3. Add 750 μl of ethanol (96 - 100%) to the Mini column, leave the lid open, and turn on the vacuum pump; after all the liquid has passed through the column membrane, turn off the vacuum pump and release the pressure to 0 mbar.

[0121] 1.1.3.4. Close the lid of the QIAamp Mini column, remove it from the adapter, and discard the VacConnector. Place the QIAamp Mini column into a clean 2 ml collection tube and centrifuge at high speed (20000 g; 14000 rpm) for 3 min.

[0122] 1.1.3.5. Place the QIAamp Mini column into a new 2 ml collection tube. Open the tube lid and incubate at room temperature for 5 min to completely dry the membrane.

[0123] 1.1.4. Eluting cfDNA:

[0124] 1.1.4.1. Place the QIAamp Mini column into a 1.5 ml elution tube and discard the 2 ml collection tube from step 14. Add 20 - 150 μl of Buffer AVE to the center of the Mini membrane; close the tube lid and incubate at room temperature for 3 min.

[0125] 1.1.4.2. Centrifuge at full speed (20000 g; 14000 rpm) for 1 min in the centrifuge to elute the nucleic acids.

[0126] For cfDNA samples, the Agilent 2100 is used for fragment detection, and Qubit is directly used for subsequent experiments.

[0127] 1.2. Bisulfite conversion and purification:

[0128] 1.2.1. Prepare CT Conversion Reagent:

[0129] 1.2.1.1. Add 700 μl of NF water, 300 μl of M-Dilution Buffer, and 50 μl of M-Dissolving Buffer to a tube of CT conversion reagent, mix well at room temperature, and vortex or shake frequently for 10 min.

[0130] 1.2.1.2. After mixing, aliquot the reagent to prepare enough for 10 reactions at a time.

[0131] 1.2.2. Perform bisulfite conversion on the DNA library and prepare the reaction system according to Table 1 below.

[0132] Table 1

[0133] Component Volume of high-concentration sample (1 ng - 2 μg) Sample at the end of the previous reaction 40 μl CT Conversion Reagent 110 μl Total volume 150 μl

[0134] 1.2.3. Set the pipette to 100 μl, gently pipette up and down 6 times to mix well, then divide the mixture into two tubes and place them on the PCR instrument.

[0135] 1.2.4. Set the following program on the PCR instrument for the reaction: The hot lid temperature is 105 °C.

[0136] Table 2

[0137] Temperature Time 98℃ 10 min 64℃ 2.5h 4℃ ∞

[0138] 1.2.5. Take a new 1.5 ml centrifuge tube and add 600 μl of M-Binding Buffer.

[0139] 1.2.6. After the PCR is completed, briefly centrifuge and transfer the two tubes of the same sample to the corresponding 1.5 ml centrifuge tubes respectively, and mix well.

[0140] 1.2.7. Add the above mixed sample to the Zymo-Spin TM IC Column, invert to mix well, and centrifuge at 10,000 x g for 30 s.

[0141] 1.2.8. Add 100 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s.

[0142] 1.2.9. Add 200 μl of M-Desulphonation Buffer to the column, let it stand at room temperature for 15 - 20 min, and centrifuge at 10,000 x g for 30 s.

[0143] 1.2.10. Add 200 μl of M-Wash Buffer to the column and centrifuge at 10,000 x g for 30 s.

[0144] 1.2.11. Repeat the previous step once.

[0145] 1.2.12. Place the column in a new collection tube and centrifuge at 10,000 x g for another 30 s.

[0146] 1.2.13. Place the recovery column in a new 1.5 ml EP tube, add 15 μl of LOW EDTA buffer to the center of the column membrane, and centrifuge at 10,000 x g for 30 s.

[0147] 1.3. Denaturation:

[0148] 1.3.1. Preheat the PCR instrument to 95 °C.

[0149] 1.3.2. Set the following program on the PCR instrument according to Table 3: The hot lid temperature is 105 °C.

[0150] Table 3

[0151] Temperature Time 95℃ ∞ 95℃ 2 min 95℃ ∞

[0152] 1.3.3. Immediately place the test tube on ice for 2 min after incubation.

[0153] 1.4. Adapter ligation and purification:

[0154] 1.4.1. Prepare the reaction system according to Table 4 below:

[0155] Table 4

[0156] Component Volume Low EDTA TE 11.5 μl Buffer G1 4 μl Reagent G2 4 μl Reagent G3 2.5 μl Enzyme G4 1 μl Enzyme G5 1 μl Enzyme G6 1 μl DNA 15 μl Total Volume 40 μl

[0157] 1.4.2. Set the following program on the PCR instrument according to Table 5: The hot lid temperature is 105 °C.

[0158] Table 5

[0159] Temperature Time 37℃ ∞ 37℃ 15 min 95℃ 2 min 4℃ ∞

[0160] 1.5. Sample extension and purification:

[0161] 1.5.1. Prepare the reaction system according to Table 6 below:

[0162] Table 6

[0163] Component Volume Reagent Y1 2 μl Enzyme Y2 42 μl totalVolume 44 μl

[0164] 1.5.2. Set up the following program on a PCR instrument according to Table 7: The hot lid temperature is 105 °C.

[0165] Table 7

[0166] Temperature Time 98℃ ∞ 98℃ 1 min 62℃ 2 min 65℃ 5 min 4℃ ∞

[0167] 1.5.3. Add the DNA protection buffer to the liquid until it turns blue. Gently pipette and mix well, then divide it into two tubes and place them on the PCR instrument.

[0168] 1.5.4. Set up the following program and run: The hot lid is 105 °C.

[0169] Table 8

[0170] Temperature Time 95℃ 5 min 60℃ 10 min 95℃ 5 min 60℃ 10 min 4℃ ∞

[0171] 1.5.5. Prepare the purification system according to Table 9 below:

[0172] Table 9

[0173] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 84 μl 101 μl (ratio: 1.2) 15 μl

[0174] 1.5.6. Add the magnetic beads in the above ratio to each sample for recovery, shake and mix well, and centrifuge briefly.

[0175] 1.5.7. Incubate at room temperature for 5 min.

[0176] 1.5.8. Shake and mix well, centrifuge briefly, place on a magnetic stand for adsorption until the solution becomes clear (~2 min), and aspirate the supernatant after the solution is clear.

[0177] 1.5.9. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all the remaining ethanol on the inner wall of the pipette.

[0178] 1.5.10. Repeat the above steps.

[0179] 1.5.11. Add the optimal volume of low EDTA TE buffer recommended in the above table for elution, and then shake and mix well.

[0180] 1.5.12. Incubate at room temperature for 2 min.

[0181] 1.5.13. Place on a magnetic stand for adsorption until the solution becomes clear (~2 min), and aspirate the supernatant after the solution is clear.

[0182] 1.5.14. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0183] 1.6. Adapter Ligation and Purification:

[0184] 1.6.1. Prepare the library reaction system according to Table 10 below:

[0185] Table 10

[0186] Component Volume Buffer B1 3 μl Reagent B2 10 μl Enzyme B3 2 μl TotalVolume 15 μl

[0187] 1.6.2. Set the following program according to Table 11 and run: Hot lid 0°C:

[0188] Table 11

[0189] Temperature Time 25℃ ∞ 25℃ 15 min 4℃ ∞

[0190] 1.6.3. Prepare the purification system according to Table 12 below:

[0191] Table 12

[0192] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 30 μl 36 μl (ratio: 1.2) 20 μl

[0193] 1.6.4. Add the magnetic beads in the above ratio to each sample for recovery, shake well and centrifuge briefly.

[0194] 1.6.5. Incubate at room temperature for 5 min.

[0195] 1.6.6. Shake well and centrifuge briefly, then place on the magnetic stand for adsorption until the solution becomes clear (~2 min). After the solution is clear, aspirate the supernatant.

[0196] 1.6.7. Add 200 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all the remaining ethanol on the inner wall of the pipette.

[0197] 1.6.8. Repeat the above steps.

[0198] 1.6.9. Add the optimal volume of low EDTA TE buffer recommended in the above table for elution, then shake well.

[0199] 1.6.10. Incubate at room temperature for 2 min.

[0200] 1.6.11. Place on the magnetic stand for adsorption until the solution becomes clear (~2 min). After the solution is clear, aspirate the supernatant.

[0201] 1.6.12. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0202] 1.7. Library Amplification and Purification:

[0203] 1.7.1. Prepare the library reaction system according to Table 13 below:

[0204] Table 13

[0205]

[0206] 1.7.2. Set the following program according to Table 14 and run: Hot lid at 105 °C:

[0207] Table 14

[0208]

[0209] 1.7.3. The recommended number of cycles is as shown in Table 15 below:

[0210] Table 15

[0211] Input amount Recommended cycle number 20 ng cfDNA 10-11 100 ng gDNA 9-10 20 ng gDNA 11-12

[0212] 1.7.4. Prepare the purification system according to Table 16 below:

[0213] Table 16

[0214] Input amount Reaction volume Amount of magnetic beads Volume 200 bp (SeqCap Epi) 50 μl 60 μl (ratio: 1.2) 22 μl

[0215] 1.7.5. Transfer the PCR product into a 1.5 ml centrifuge tube.

[0216] 1.7.6. Add the magnetic beads in the above ratio to each sample for recovery, shake well and centrifuge briefly.

[0217] 1.7.7. Incubate at room temperature for 5 min.

[0218] 1.7.8. Shake well and centrifuge briefly, then place on a magnetic stand to adsorb until the solution becomes clear (~2 min). After the solution is clear, aspirate the supernatant.

[0219] 1.7.9. Add 500 μl of 80% ethanol to wash the magnetic beads for 30 s, discard the supernatant, and carefully remove all the remaining ethanol on the inner wall of the pipette.

[0220] 1.7.10. Repeat the above steps.

[0221] 1.7.11. Place on the magnetic stand for 5 - 10 minutes until the beads are dry (avoid over-drying as it may reduce the DNA recovery rate).

[0222] 1.7.12. Add the optimal volume of low EDTA TE buffer recommended in the above table for elution, then shake well.

[0223] 1.7.13. Incubate at room temperature for 2 min.

[0224] 1.7.14. Place it on the magnetic rack and adsorb until the solution becomes clear (~2 min). After the solution becomes clear, aspirate the supernatant.

[0225] 1.7.15. Transfer the entire eluate to a new 0.2 mL PCR tube, ensuring that the eluate does not contain magnetic beads.

[0226] 1.7.16. Aspirate 1 μl for qubit calibration and perform 2100 quality inspection.

[0227] 1.8. Sample hybridization with the probe:

[0228] 1.8.1. Mix the samples:

[0229] 1.8.1.1. Refer to Table 17 below for the DNA library usage. The total usage can exceed 1500 ng but not exceed 4 μg.

[0230] Table 17

[0231] Number of mixed samples Usage per library Total amount per reaction library 1 500 ng 500 ng 2 500 ng 1000 ng 3 500 ng 1500 ng 4 375 ng 1500 ng 8 187.5 ng 1500 ng

[0232] 1.8.1.2. Calculate the usage of different samples and mix them evenly in a centrifuge tube.

[0233] 1.8.1.3. Add the following pre-hybridization reagents in Table 18 to the mixed samples respectively, mix well, and try not to generate bubbles.

[0234] Table 18

[0235] Component Volume Twist probe panel 4 μl Universal blocker 8 μl Blocker solution 5 μl Methylation Enhancer 2 μl

[0236] 1.8.1.4. Dry the above mixed pre-hybridization reagents at room temperature (use low temperature if heating is required) in a vacuum concentrator.

[0237] 1.8.2. Hybridization:

[0238] 1.8.2.1. Incubate the Fast Hybridization Mix at 65 °C for 10 min or until all precipitates dissolve. Quickly vortex and add 20 μl to the sample dried in the previous step to resuspend the sample (do not let the hybridization solution return to room temperature). Gently flick with your fingertips to mix well and avoid generating bubbles.

[0239] 1.8.2.2. Centrifuge quickly to remove bubbles and add 30 μl of Hybridization Enhancer to the surface of the above reagent.

[0240] 1.8.2.3. Place the PCR tube in a preheated PCR instrument for hybridization.

[0241] 1.8.2.4. Set the following program according to Table 19 and run: Hot lid 85 °C.

[0242] Table 19

[0243] Temperature Time 95℃ ∞ 95℃ 5 min 60℃ 15 min - 4 h

[0244] 1.8.3. Binding:

[0245] 1.8.3.1. Vortex the streptavidin magnetic beads for pre - equilibration until completely mixed, and add 100 μl of magnetic beads into a 1.5 - ml centrifuge tube.

[0246] 1.8.3.2. Add 200 μl of binding buffer and pipette to mix well.

[0247] 1.8.3.3. Place the centrifuge tube on the magnetic stand for 1 min or until the solution becomes clear, discard the supernatant, and remove the centrifuge tube from the magnetic stand.

[0248] 1.8.3.4. Repeat the above washing steps 2 times, for a total of 3 times.

[0249] 1.8.3.5. After the last wash, add 200 μl of binding buffer, vortex to resuspend and mix well.

[0250] 1.8.3.6. After the hybridization is completed, open the lid of the PCR instrument and quickly transfer all the hybridization solution to the pre - equilibrated magnetic beads.

[0251] 1.8.3.7. Mix the magnetic beads with the hybridization solution thoroughly on a shaker, rocker or rotator at room temperature for 30 min.

[0252] 1.8.3.8. Remove the centrifuge tube from the mixer, centrifuge quickly, place it on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0253] 1.8.3.9. Add 200 μl of pre - heated Wash Buffer 1 and mix well.

[0254] 1.8.3.10. Incubate at 63 °C or 65 °C for 5 min.

[0255] 1.8.3.11. Place the centrifuge tube on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0256] 1.8.3.12. Repeat the above steps, add another 200 μl of pre - heated Wash Buffer 1 and mix well.

[0257] 1.8.3.13. Incubate at 63 °C or 65 °C for 5 min.

[0258] 1.8.3.14. Transfer the liquid to a new tube; place it on the magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0259] 1.8.3.15. Add 200 μl of pre-warmed wash buffer 2 and mix well with a pipette tip.

[0260] 1.8.3.16. Incubate at 48 °C for 5 min.

[0261] 1.8.3.17. Place on a magnetic stand for 1 min, discard the supernatant, and remove the tube.

[0262] 1.8.3.18. Repeat steps 3.15 - 3.17 two more times for a total of three washes.

[0263] 1.8.3.19. For the last wash, use a 10 μl pipette tip to completely aspirate the wash solution.

[0264] 1.8.3.20. Add 45 μl of water, mix well, and incubate the solution on ice.

[0265] 1.8.4. Post-capture PCR amplification, purification, and quality control:

[0266] 1.8.4.1. Set the following program according to Table 20 and run: Heat lid at 105 °C.

[0267] Table 20

[0268]

[0269] 1.8.4.2. Mix the magnetic bead mixture in 1.3, and pipette 22.5 μl into a 0.2 ml PCR tube.

[0270] 1.8.4.3. Add 2.5 μl of amplification primer and 25 μl of KAPA HiFi HotStart ReadyMix to the 0.2 ml PCR tube for a total reaction volume of 50 μl.

[0271] 1.8.4.4. Gently mix with a pipette tip, briefly centrifuge, and place in a PCR machine to start amplification.

[0272] 1.8.4.5. Vortex the pre-equilibrated DNA purification magnetic beads thoroughly.

[0273] 1.8.4.6. Add 90 μl (1.8*) of DNA purification magnetic beads to the amplified PCR product and vortex thoroughly to mix.

[0274] 1.8.4.7. Incubate at room temperature for 5 min.

[0275] 1.8.4.8. Place the centrifuge tube on a magnetic stand for 1 min. After the solution becomes clear, discard the supernatant.

[0276] 1.8.4.9. Without removing the centrifuge tube from the magnetic stand, directly add 200 μl of freshly prepared 80% ethanol, incubate for 1 min, and discard the supernatant; repeat the 80% ethanol wash once (for a total of 2 times), keeping the centrifuge tube on the magnetic stand.

[0277] 1.8.4.10. Carefully remove the residual ethanol with a 10 μl pipette tip and let it stand at room temperature for 5 - 10 min or until the magnetic beads are dry. Note that the magnetic beads should not be overdried.

[0278] 1.8.4.11. Remove the tube from the magnetic stand and add 32 μl of water. Mix well by pipetting and incubate at room temperature for 2 min.

[0279] 1.8.4.12. Place the centrifuge tube on the magnetic stand for 3 min or until the solution is clear.

[0280] 1.8.4.13. Transfer 30 μl of the supernatant to a clean 0.2 ml centrifuge tube.

[0281] 1.8.4.14. Take 1 μl of the library and quantify it using Qubit, and record the library concentration.

[0282] 1.8.4.15. Take 1 μl of the sample and measure the library fragment length using Agilent 2100.

[0283] 1.8.4.16. Perform sequencing using the Illumina high - throughput sequencing platform.

[0284] 1.9. Bioinformatics analysis process for methylation.

[0285] Roughly as follows: Use the fastp quality control software to view the sequencing quality, remove low - quality reads, then use the Bismark alignment software to align the quality - controlled clean data to the reference genome, and use the Bismar_methylation_extractor software to extract the corresponding methylation sites. Finally, use self - developed code to calculate the methylation level of the target region, and the calculation method is as follows. Use this value to calculate the score to judge cancer or normal.

[0286] Methylation level of the target region = (Sum of methylation reads of all CpG sites in the target interval) / (Sum of reads of all CpG sites in the target interval).

[0287] Example 2. Testing of follow - up plasma samples of recurrent and non - recurrent samples

[0288] Collect the follow-up plasma samples of 24 cases of recurrent liver cancer and 20 cases of non-recurrent liver cancer within 2 years, and use the method in Example 1 to detect the methylation level of the target segment. The calculation method of the methylation level threshold is as follows: draw an ROC curve based on the data set (including the type and methylation level of each sample), and the confusion matrix corresponding to the optimal threshold point on the ROC curve is the basis for calculating indicators such as sensitivity, specificity, and accuracy. Usually, we will select through the Youden index. The Youden index, also known as the correct index, refers to the sum of sensitivity and specificity minus 1: Youden index = Sensitivity + Specificity – 1. The range of the Youden index is between 0 and 1, representing the total ability of the classification model to identify true patients and non-patients. The larger the Youden index, the better the performance of the classification model. The threshold of FJX1 is set to 0.02357. If the detection result is greater than the threshold, it is judged as positive, otherwise it is judged as negative. The threshold of ZNF135 is set to 0.02638. If the detection result is greater than the threshold, it is judged as positive, otherwise it is judged as negative. Use these two target genes to discriminate the plasma samples to be tested. If any one of the target genes is detected as positive, it is judged that the patient has recurrent liver cancer. Otherwise, it is judged as non-recurrent.

[0289] The discrimination results are shown in Table 21:

[0290] Table 21

[0291]

[0292]

[0293] The test results of this implementation show that the sensitivity of this detection method is: 95.83%, the specificity is: 85%, and the accuracy is: 90.9%.

[0294] Example 3. Test of preoperative primary cancer plasma samples of recurrent samples and non-recurrent samples

[0295] Collect the preoperative primary cancer plasma samples of 21 cases of recurrent liver cancer and 20 cases of non-recurrent liver cancer, and use the method in Example 1 to detect the methylation level of the target segment. Use these two target genes to discriminate the preoperative primary cancer plasma samples, and the results are shown in Table 22:

[0296] Table 22

[0297]

[0298] The test results show that the sensitivity of this detection method is 76.19%, the specificity is 85%, and the accuracy is 80.5%.

[0299] Example 4. Preoperative Plasma Sample Test of Primary Cancer in Other Liver Cancer Samples

[0300] Collect preoperative plasma samples of primary cancer from 44 liver cancer patients, and detect the methylation level of the target region using the method in Example 1. Use these two target genes to discriminate the preoperative plasma samples of primary cancer, and the results are shown in Table 23 below:

[0301] Table 23

[0302]

[0303]

[0304] The sequences used in the above embodiments of the present application are shown in the following sequence listing. It should be understood that the following sequences are only exemplary sequences of the implementation schemes of the present application, and do not impose any limitations on the present application scheme.

[0305]

[0306] The description of the present disclosure is given for purposes of illustration and description, and is not intended to be exhaustive or to limit the present disclosure to the disclosed form. Many modifications and variations are obvious to those of ordinary skill in the art. The embodiments are chosen and described in order to best explain the principles of the present disclosure and its practical application, and to enable those of ordinary skill in the art to understand the present disclosure and design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A marker for monitoring liver cancer and / or liver cancer recurrence, the marker comprising any one or both of the FJX1 gene and the ZNF135 gene; Preferably, the marker is any one of the FJX1 gene and the ZNF135 gene or a combination of both.

2. The marker according to claim 1, wherein the target sequence of the FJX1 gene comprises any one, two, three or more of the following groups: sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, and SEQ ID NO.15-16; Or, the target sequence of the FJX1 gene is any one, two, three or more selected from the following group: sequences shown in SEQ ID NO.1, SEQ ID NO.11-12, and SEQ ID NO.15-16; And / or, wherein the target sequence of the ZNF135 gene comprises any one, two, three or more of the following groups: sequences shown in SEQ ID NO.2, SEQ ID NO.13-14, and SEQ ID NO.17-18; Or, the target sequence of the ZNF135 gene is any one, two, three or more selected from the following group: sequences shown in SEQ ID NO.2, SEQ ID NO.13-14, and SEQ ID NO.17-18.

3. A probe composition for monitoring liver cancer and / or liver cancer recurrence, the probe composition comprising a probe that targets and binds to a methylation product of the marker according to claim 1 or 2.

4. The probe composition according to claim 3, wherein the probe composition comprises a high methylation probe composition and a low methylation probe composition; in, The hypermethylation probe composition comprises one, two or more hypermethylation probes that bind to the hypermethylated sequence of the marker in a targeted manner; The hypomethylation probe composition comprises x hypomethylation probes that bind to the hypomethylated sequences of the markers in a targeted manner, x is a natural number selected from 1 to 5, and there are a nucleic acid overlaps between any two probes, and a is a natural number selected from 0 to 100; The hypermethylation probe composition comprises y hypermethylation probes that target and bind to the hypermethylated sequences of the marker, y is a natural number selected from 1-5, and there are b nucleic acid overlaps between any two probes, and b is a natural number selected from 0-100.

5. The probe composition according to claim 4, wherein the hypermethylated probe composition comprises any one, two, three or four of the sequences shown in SEQ ID NO.3-6; or, the hypermethylated probe composition is selected from any one, two, three or four of the sequences shown in SEQ ID NO.3-6; And / or, the hypomethylation probe composition comprises any one, two, three or four of the sequences shown in SEQ ID NO.7-10; or, the hypomethylation probe composition is selected from any one, two, three or four of the sequences shown in SEQ ID NO.7-10.

6. A composition for monitoring liver cancer and / or liver cancer recurrence, comprising the probe composition according to any one of claims 3 to 5.

7. The composition according to claim 6, further comprising a primer; The primer is a fragment of at least 9 nucleotides selected from the target sequence of any one or both of the FJX1 gene and the ZNF135 gene, and the fragment of 9 nucleotides contains at least one CpG dinucleotide sequence; Preferably, the composition further comprises a probe, wherein the probe is a fragment that targets and binds to at least 15 nucleotides in a target sequence selected from any one or both of the FJX1 gene and the ZNF135 gene, and the 15-nucleotide fragment comprises at least one CpG dinucleotide sequence; More preferably, the probe further comprises a blocker that preferentially binds to a target sequence in an unmethylated state; More preferably, the composition further comprises a reagent for converting the unmethylated cytosine base at position 5 of the target sequence of either or both of the FJX1 gene and the ZNF135 gene into uracil.

8. Use of the marker according to claim 1 or 2 and / or the probe combination according to any one of claims 3 to 5 in preparing a product for monitoring liver cancer and / or liver cancer recurrence; Preferably, the liver cancer and / or liver cancer recurrence monitoring product is a liver cancer and / or liver cancer recurrence monitoring kit, or a liver cancer and / or liver cancer recurrence monitoring chip.

9. A kit for monitoring liver cancer and / or liver cancer recurrence, comprising the probe composition according to any one of claims 3 to 5, and / or the composition according to claim 6 or 7.

10. A chip for monitoring liver cancer and / or liver cancer recurrence, comprising the probe composition according to any one of claims 3 to 5, and / or the composition according to claim 6 or 7.

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