Method and kit for screening colorectal tumor

Abstract

The present application relates to methods of diagnosing, screening for or assessing the progression or prognosis of colorectal tumor in an individual, methods of monitoring the response of an individual to a treatment in an individual receiving a treatment of colorectal tumor, and kits for use in the methods.

Description

Technical Field

[0001] The present application generally relates to the field of biomedicine. Specifically, the present application relates to a method for diagnosing colorectal tumors in an individual, screening for colorectal tumor formation or the risk of formation, or evaluating the progression or prognosis of colorectal tumors, a method for monitoring the response of the individual to treatment in an individual receiving colorectal tumor treatment, and a kit used in the method. Background Art

[0002] Early detection of colorectal cancer in the late stage of precancerous adenoma or early stage of cancer has been shown to significantly reduce mortality. Current colorectal cancer screening methods include colonoscopy or molecular testing of stool or blood samples, which are invasive or have very few markers, limiting patient compliance or detection sensitivity.

[0003] Therefore, there is an urgent need to develop a method and / or kit that can efficiently read epigenetic information from the extremely limited amount of extracellular free DNA in biological samples, and that can be easily configured and reliably applied in hospital laboratories. Brief description of the invention

[0005] In one aspect, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0006] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0007] (II) quantitatively analyzing the methylation levels of a group of target markers in the treated DNA of step (I), respectively, wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5;

[0008] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0009] In another aspect, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0010] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0011] (II) respectively quantitatively analyzing the methylation levels of a group of target markers in the treated DNA of step (I), wherein at least two target markers are selected from the group consisting of Septin9, BCAT1,

[0012] IKZF1, BCAN, PKNOX2, VAV3, NDRG4 and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12,

[0013] KCTD8, HMX1, MARCH11, and CRHBP, and

[0014] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0015] In some embodiments, a panel of target markers described herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more target markers.

[0016] In some embodiments, step (II) described in the present application comprises:

[0017] (i) pre-amplifying at least a portion of at least one target marker from a group of target markers in the treated DNA obtained from step (I) using a pre-amplification primer library, and the group of target markers is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3,

[0018] IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2,

[0019] SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0020] (ii) quantitatively analyzing the methylation levels of the group of target markers in the DNA obtained from the sub-step (i).

[0021] In some embodiments, the method described herein further comprises obtaining DNA from a biological sample from the individual prior to step (I).

[0022] In another aspect, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0023] (a) obtaining a biological sample containing DNA from the individual;

[0024] (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0025] (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4,

[0026] POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2,

[0027] NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8,

[0028] HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5; wherein step (c) is present or absent;

[0029] (d) If step (c) exists, the methylation level of at least one target marker is quantitatively analyzed based on the DNA obtained from step (c); if step (c) does not exist, the methylation level of at least one target marker in the treated DNA obtained from step (b) is quantitatively analyzed, wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3,

[0030] IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2,

[0031] SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12,

[0032] KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0033] (e) comparing the methylation level of at least one target marker in step (d) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0034] In some embodiments, the at least one target marker in step (c) or step (d) of the above method comprises a plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1 and IKZF1.

[0035] In another aspect, the present application provides a method for monitoring the response of an individual to treatment for colorectal tumors, comprising the steps of:

[0036] (a) obtaining a biological sample containing DNA from the individual;

[0037] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing unmethylated sites from methylated sites in the DNA, thereby obtaining treated DNA;

[0038] (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one (e.g., each) target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0039] (d) if step (c) exists, then based on the DNA obtained from step (c) respectively quantitatively analyzing the methylation level of at least one target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0040] (e) comparing the methylation level of the at least one target marker in step (d) with the corresponding methylation levels of one or more target markers obtained from the same individual before treatment, respectively, wherein the corresponding methylation levels are quantitatively analyzed by repeating steps (a), (b), optionally (c), and (d) on a biological sample containing DNA obtained from the individual before treatment, wherein a lower methylation level of the one or more target markers compared to its corresponding methylation level before treatment indicates that the individual has responded to the treatment.

[0041] In some embodiments, the at least one target marker in step (c) or step (d) of the above method comprises a plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1 and IKZF1.

[0042] In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0043] In some embodiments, each target marker comprises or is: a) each region defined by Hg19 coordinates as shown below:

[0044]

[0045]

[0046] and 5 kb upstream of each start site and 5 kb downstream of each end site of each of the above regions; or b) the corresponding region of a) after bisulfite conversion; or c) the corresponding region of a) after methylation sensitive restriction enzyme (MSRE) treatment.

[0047] In some embodiments, the DNA in the biological sample obtained from step (a) includes genomic DNA or extracellular free DNA. In some embodiments, the extracellular free DNA includes circulating tumor DNA. In some embodiments, the amount of the target marker in the extracellular free DNA in the biological sample does not exceed 1ng, 0.8ng, 0.6ng, 0.4ng, 0.2ng, 0.1ng, 0.08ng or does not exceed 0.04ng. In some embodiments, the concentration of the target marker in the extracellular free DNA in the biological sample is lower than the sensitivity level of the detection analysis for the target marker.

[0048] In some embodiments, the DNA obtained in sub-step (i) or step (c) is diluted with a diluent before sub-step (ii) or step (d).

[0049] In some embodiments, the biological sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, colon effluents, surgical resection specimens, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, urine, mucus, saliva, peritoneal fluid, pleural fluid, pleural effusions, synovial fluid, cerebrospinal fluid, thoracentesis fluid, peritoneal effusions, and any combination thereof. In some embodiments, the biological sample is obtained from the plasma of the individual. In some embodiments, the colon effluent is selected from the group consisting of fecal samples and enema wash samples.

[0050] In some embodiments, the reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA selectively modifies unmethylated cytosine residues at CpG sites to produce modified residues, but does not significantly modify methylated cytosine residues. In some embodiments, the reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA comprises a bisulfite reagent. In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof.

[0051] In some embodiments, the reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA selectively cuts unmethylated residues but not methylated residues, or selectively cuts methylated residues but not unmethylated residues. In some embodiments, the reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA is a methylation sensitive restriction enzyme (MSRE). In some embodiments, the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0052] In some embodiments, the preamplification primer library comprises at least one methylation-specific primer pair. In some embodiments, wherein the at least one methylation-specific primer pair comprises a forward primer and a reverse primer, wherein the primers each comprise an oligonucleotide sequence, wherein the oligonucleotide sequence hybridizes to at least 9 consecutive nucleotides of one of the target markers under stringent conditions, moderately stringent conditions, or highly stringent conditions, wherein the at least 9 consecutive nucleotides of one of the target markers comprise at least one CpG site.

[0053] In some embodiments, the pre-amplification primer library further comprises a control primer pair for amplifying a control marker. In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0054] In some embodiments, the at least one methylation-specific primer pair comprises one or more pairs of nucleotide sequences selected from the group consisting of SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171.

[0055] In some embodiments, in step (c), the at least one target marker is amplified in the presence of one or more blocking oligonucleotides.

[0056] In some embodiments, the quantitative analysis is performed by polymerase chain reaction (e.g., real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, mass-based separation (e.g., electrophoresis, mass spectrometry) or target capture (e.g., hybridization, microarray). In some embodiments, the quantitative analysis is performed by real-time polymerase chain reaction, optionally the real-time polymerase chain reaction is a multiplex real-time polymerase chain reaction.

[0057] In some embodiments, if step (c) exists, the quantitative analysis of step (d) includes amplifying the DNA obtained in step (c) using a quantitative primer pair and a DNA polymerase; if step (c) does not exist, the quantitative analysis of step (d) includes amplifying at least one target marker in the treated DNA obtained in step (b) using a quantitative primer pair and a DNA polymerase.

[0058] In some embodiments, if step (c) exists, the quantitative primer pair used in step (d) can hybridize with at least 9 consecutive nucleotides of the DNA obtained in step (c) under stringent conditions, moderately stringent conditions, or highly stringent conditions; if step (c) does not exist, the quantitative primer pair used in step (d) can hybridize with at least 9 consecutive nucleotides of at least one target marker in the treated DNA obtained in step (b) under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0059] In some embodiments, the step (d) is performed in the presence of a detection reagent. In some embodiments, the detection reagent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 57-85 and 172. In some embodiments, the 5' end of the fluorescent probe is labeled with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5), and the 3' end is labeled with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA).

[0060] In some embodiments, step (e) includes comparing the Ct value of the target marker in step (d) with a reference Ct value, wherein the Ct value of at least one target marker is the same as or lower than its corresponding reference Ct value, indicating that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or risk of formation, or the individual has colorectal tumor development or increased likelihood of development, or the individual has a poor prognosis or risk of poor prognosis of colorectal tumor; or the Ct value of at least one target marker is higher than its corresponding Ct value before treatment, indicating that the individual being treated for colorectal tumor has responded to the treatment.

[0061] In some embodiments, the pre-amplification comprises 5 to 30 reaction cycles, wherein each cycle comprises a reaction at 40-80° C. for 5 seconds to 5 minutes, followed by a reaction at 85-99° C. for 5 seconds to 5 minutes.

[0062] In some embodiments, if step (c) is present, the quantitative analysis of step (d) includes determining the methylation level based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides or CpA dinucleotides in the DNA obtained in step (c); in some embodiments, if step (c) is not present, the quantitative analysis of step (d) includes determining the methylation level of at least one target marker based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides or CpA dinucleotides in at least one target marker in the treated DNA obtained in step (b). In some embodiments, if step (c) is present, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in the DNA obtained in step (c); in some embodiments, if step (c) is not present, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in at least one target marker in the treated DNA obtained in step (b).

[0063] In some embodiments, the reference level in step (e) is determined based on clinical samples obtained from a group of individuals having or at risk of colorectal tumor and clinical samples obtained from a group of individuals who do not have or are not at risk of colorectal tumor.

[0064] In some embodiments, the colorectal tumor is a colorectal cancer, a colorectal adenoma, and / or a sessile serrated polyp. In some embodiments, the colorectal tumor is precancerous. In some embodiments, the individual is a human.

[0065] In another aspect, the present application provides a kit for diagnosing colorectal tumors, screening for colorectal tumor formation or risk of formation, or evaluating the progression or prognosis of colorectal tumors, comprising:

[0066] (a) a first reagent for treating DNA, wherein the first reagent is capable of distinguishing between unmethylated sites and methylated sites in the DNA;

[0067] (b) optionally, a first primer library comprising at least one primer pair for preamplifying at least one target sequence of at least one target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5, wherein the at least one primer pair can hybridize to at least 9 consecutive nucleotides of the at least one target sequence after treatment with the first reagent under stringent conditions, moderately stringent conditions, or highly stringent conditions, wherein the target sequence comprises at least one CpG site; and

[0068] (c) a second reagent, wherein if the first primer library exists, the second reagent is used to quantitatively analyze the methylation level in the at least one target marker pre-amplified by the first primer library; if the first primer library does not exist, the second reagent is used to quantitatively analyze the methylation level of at least one target marker in the DNA treated by the first reagent, wherein the at least one target marker comprises one or more markers selected from the following group:

[0069] Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5.

[0070] In some embodiments, the at least one target marker comprises a plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0071] In some embodiments, if the first primer library exists, the second reagent comprises a second primer library, the second primer library comprises a plurality of quantitative primer pairs, and the quantitative primer pairs can hybridize with at least 9 consecutive nucleotides of the at least one target sequence pre-amplified by the first primer library under stringent conditions, moderately stringent conditions, or highly stringent conditions; in some embodiments, if the first primer library does not exist, the second reagent includes a third primer library, the third primer library comprises a plurality of quantitative primer pairs, and the quantitative primer pairs can hybridize with at least 9 consecutive nucleotides of at least one target sequence in at least one target marker in the DNA treated with the first reagent under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0072] In some embodiments, at least one quantitative primer pair in the second primer library is the same as at least one primer pair in the first primer library. In some embodiments, if the first primer library exists, the quantitative primer pair in the second primer library is designed to amplify at least a portion of the at least one target sequence pre-amplified by the first primer library; in some embodiments, if the first primer library does not exist, the quantitative primer pair of the third primer library is designed to amplify at least a portion of at least one target sequence of at least one target marker in the DNA treated with the first reagent.

[0073] In some embodiments, the first primer pool, the second primer pool, or the third primer pool comprises at least one methylation-specific primer pair.

[0074] In some embodiments, the first primer pool and the second primer pool are packaged in a single container or in separate containers. In some embodiments, the kit further comprises one or more blocking oligonucleotides.

[0075] In some embodiments, the kit further comprises a detection reagent. In some embodiments, the detection reagent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 57-85 and 172. In some embodiments, the 5' end of the fluorescent probe is labeled with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5), and the 3' end is labeled with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, TAMRA, or lowa Black Dark Quenchers).

[0076] In some embodiments, the kit further comprises a DNA polymerase and / or a container suitable for storing the biological sample obtained from the individual. In some embodiments, the kit further comprises instructions for use and / or an explanation of the test results of the kit.

[0077] In some embodiments, the first reagent comprises a bisulfite reagent or a methylation sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof. In some embodiments, the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0078] In some embodiments, if the first primer library exists, the first primer library comprises a plurality of primer pairs for pre-amplifying at least one target sequence in a plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1 and IKZF1, and further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4 and IRF4. In some embodiments, if the first primer library does not exist, the third primer library comprises a plurality of primer pairs for amplifying at least one target sequence in a plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1 and IKZF1, and further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4 and IRF4. In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0079] In some embodiments, each target marker comprises or is: a) each region defined by Hg19 coordinates as shown below:

[0080]

[0081]

[0082] and 5 kb upstream of each start site and 5 kb downstream of each end site of each of the above regions; or b) the corresponding region of a) after bisulfite conversion; or c) the corresponding region of a) after MSRE treatment.

[0083] In some embodiments, if the first primer library exists, the first primer library comprises at least one primer pair, and the at least one primer pair comprises or consists of at least one pair of nucleotide sequences selected from the following group as shown in Table 2: SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171, optionally, wherein the second primer library comprises at least one primer pair identical to at least one primer pair in the first primer library. In some embodiments, if the first primer library does not exist, the third primer library comprises at least one primer pair, and the at least one primer pair comprises or consists of at least one pair of nucleotide sequences selected from the following group as shown in Table 2: SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171.

[0084] In some embodiments, the first primer library, the second primer library or, optionally, the third primer library further comprises a primer pair for amplifying a control marker. In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29.

[0085] In some embodiments, the kit further comprises a plurality of containers, each container being used to receive a component of the second primer library.

[0086] On the other hand, the present application provides the use of the kit described in the present application in the manufacture of a diagnostic kit for diagnosing colorectal tumors in individuals, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors, or monitoring the response of individuals receiving colorectal tumor treatment to treatment.

[0087] In another aspect, the present application provides the use of a reagent for quantitatively analyzing the methylation level of a target marker in the manufacture of a kit, wherein the kit is used in a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, wherein the method comprises the following steps:

[0088] (a) obtaining a biological sample containing DNA from the individual;

[0089] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG sites in the DNA, thereby obtaining treated DNA;

[0090] (c) pre-amplifying at least a portion of at least one (e.g., each) target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0091] (d) if step (c) exists, then based on the DNA obtained from step (c), respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0092] (e) comparing the methylation level of at least one (e.g., each) target marker in step (d) with the corresponding reference level, respectively, wherein the same or higher methylation level of one or more target markers relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased likelihood of colorectal tumor development or development, or the individual has a poor prognosis or a risk of poor prognosis of colorectal tumor.

[0093] In another aspect, the present application provides use of a reagent for quantitatively analyzing the methylation level of a target marker in the manufacture of a kit, wherein the kit is used in a method for monitoring the response of an individual to treatment in an individual receiving colorectal tumor treatment, wherein the method comprises the following steps:

[0094] (a) obtaining a biological sample containing DNA from the individual;

[0095] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG sites in the DNA, thereby obtaining treated DNA;

[0096] (c) pre-amplifying at least a portion of at least one (e.g., each) target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one (e.g., each) target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0097] (d) if step (c) exists, then based on the DNA obtained from step (c), respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0098] (e) comparing the methylation level of at least one (e.g., each) target marker in step (d) with the corresponding methylation level of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation level is quantitatively analyzed by repeating steps (a), (b), optionally (c), and (d) on a biological sample containing DNA obtained from the individual before treatment, wherein a lower methylation level of one or more target markers compared to its corresponding methylation level before treatment indicates that the individual has responded to the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 The target marker PKNOX2 ( Figure 1 A) and control marker ACTB ( Figure 1B) Validation of methylation-specific primers. The ordinate represents the ΔRn value, which is obtained by subtracting the fluorescence intensity baseline value from the fluorescence intensity value detected at a specific cycle number. The abscissa represents the cycle number. Figure 1 As shown in A, the Ct value decreased as the percentage of converted methylated DNA in the DNA mixture increased, indicating that the primers used for preamplification of PKNOX2 were methylation-specific. Figure 1 As shown in B, the curves for each DNA composition overlap, indicating that despite the increase in the percentage of converted methylated DNA, the Ct value remains unchanged, which is consistent with the fact that the primers used for preamplification of the control marker ACTB are non-methylation-specific primers.

[0100] Figure 2 The methylation abundance of the control marker ACTB and the target marker SALL1 and target marker PKNOX2 in leukocytes (indicated by solid circles "●"), paracancerous tissues (indicated by solid squares "■"), high-grade adenoma tissues (indicated by solid equilateral triangles "▲"), and colorectal cancer tissues (indicated by solid inverted triangles "▼") are shown respectively. The ordinate represents the Ct value, and the abscissa represents the names of the control marker and the target marker. The higher the Ct value, the lower the methylation abundance of the marker. Therefore, Figure 2 It can be seen that the methylation abundance of the target marker in leukocytes is much lower than that in tissue samples, especially the methylation abundance of the target marker in adjacent cancer tissues is lower than that in high-grade adenoma tissues and colorectal cancer tissues.

[0101] Figure 3 The distribution of the control marker ACTB and the target marker SALL1 and the target marker PKNOX2 of the biological samples obtained from the group with colorectal cancer (the plasma of colorectal cancer individuals, represented by the solid circle "●") and the biological samples obtained from the group with negative colonoscopy (the plasma of healthy individuals, represented by the solid equilateral triangle "▲") are shown. The vertical axis represents the Ct value, and the horizontal axis represents the name of the control marker and the target marker. The lower the Ct value, the higher the methylation level of the marker. Therefore, the Figure 3 It can be seen that the methylation level of each target marker in the group with colorectal cancer is significantly higher than that in the group with negative colonoscopy.

[0102] Figure 4The AUC values ​​of all 13 target markers tested are shown. The ordinate represents the number of occurrences within the same AUC value range, and the abscissa represents the AUC value. The AUC value is between 0 and 1, and the higher the AUC value, the better the classification ability. As shown in the figure, all the tested markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and intergenic region 1) have the ability to distinguish CRC (colorectal cancer) from controls, with AUC values ​​ranging from 0.8 to 0.9.

[0103] Figure 5 The ROC curve of the combination of markers SALL1, BCAT1 and Septin9 is shown. The ordinate represents the true positive rate (i.e., sensitivity), and the abscissa represents the false positive rate (i.e., 1-specificity). The solid line represents the ROC curve, and the dotted line represents the 45-degree diagonal line. The points above the diagonal line represent better classification results (i.e., better than random), while the points below the line represent poorer results (i.e., worse than random). Therefore, the combination of target markers SALL1, BCAT1 and Septin9 has high sensitivity and high specificity when classifying colorectal tumors. DETAILED DESCRIPTION OF THE INVENTION

[0105] Although the present application discloses various aspects and various embodiments of the present application, those skilled in the art can make various equivalent changes or modifications without departing from the spirit and scope of the present application. The various aspects and various embodiments disclosed in the present application are exemplary and are not intended to limit the scope of the present application. The actual scope of protection of the present application is subject to the claims. Unless otherwise stated, all technical and scientific terms used in the present application are the meanings commonly understood by those skilled in the art. All references, patents and patent applications cited in this application are incorporated into the present application by reference.

[0106] It should be noted that, in the specification and claims of this application, the singular forms "a", "an", and "the" include plural forms thereof, unless the context indicates otherwise. Thus, for example, "an agent" includes a plurality of agents.

[0107] In the specification and claims of the present application, unless otherwise stated, the terms "comprise", "include" or "contain" mean that the listed values, steps or components are included, but other values, steps or components are not excluded.

[0108] Traditionally, the diagnosis of cancer relies on the detection of a single marker (e.g., a gene mutation), but unfortunately, it is usually difficult to detect cancer by detecting a single marker, or it is difficult to distinguish multiple types of cancer by detecting a single marker. In addition, the level of a single marker in a biological sample is usually extremely limited, which further reduces the diagnostic specificity and / or diagnostic sensitivity of cancer. Therefore, analytical methods that only identify a single marker have been shown to have limited predictive value.

[0109] In one aspect, the present application preamplifies at least a portion of at least one target marker so that at least a portion of the at least one target marker is preamplified, and then quantitatively analyzes the methylation level of at least one (e.g., each) target marker based on the DNA obtained from the preamplification. Such a preamplification step can increase the number / level of the target marker, and can significantly improve the diagnostic specificity and / or diagnostic sensitivity of colorectal tumors. On the other hand, the present application simultaneously quantitatively analyzes the methylation levels of multiple target markers in a biological sample to improve the diagnostic specificity and / or diagnostic sensitivity of colorectal tumors. In some embodiments, the multiple target markers are not preamplified before quantitative analysis. In some embodiments, the multiple target markers are preamplified before quantitative analysis. In particular, the inventors of the present application unexpectedly found that the methylation levels of multiple target markers in a biological sample are quantitatively analyzed simultaneously, or the preamplification step and the quantitative analysis step are combined together to significantly improve the diagnostic specificity and / or diagnostic sensitivity of colorectal tumors, making it possible to detect colorectal tumors in the early stage, such as in the adenoma stage before canceration or in the early stage of cancer. It will be understood by those skilled in the art that the diagnostic "sensitivity" in this context defines the proportion of correctly identified positive results, that is, the percentage of individuals correctly identified as having the disease, while "specificity" defines the proportion of correctly identified negative results, that is, the percentage of individuals correctly identified as not having the disease.

[0110] 1. Methods

[0111] In one aspect, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0112] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0113] (II) quantitatively analyzing the methylation levels of a group of target markers in the treated DNA of step (I), respectively, wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5;

[0114] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0115] In another aspect, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0116] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0117] (II) quantitatively analyzing the methylation levels of a group of target markers in the treated DNA described in step (I), respectively, wherein at least two target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, PKNOX2, VAV3, NDRG4, and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP, and

[0118] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0119] In another aspect, the present application provides a method for monitoring the response of an individual to treatment for colorectal tumors, comprising the steps of:

[0120] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0121] (II) quantitatively analyzing the methylation levels of a group of target markers in the treated DNA of step (I), respectively, wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5;

[0122] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding methylation levels of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation levels are quantitatively analyzed by repeating steps (I) and (II) on a biological sample containing DNA obtained from the individual before treatment, wherein a methylation level of one or more target markers that is lower than its corresponding methylation level before treatment indicates that the individual has responded to the treatment.

[0123] In another aspect, the present application provides a method for monitoring the response of an individual to treatment for colorectal tumors, comprising the steps of:

[0124] (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0125] (II) quantitatively analyzing the methylation levels of a group of target markers in the treated DNA described in step (I), respectively, wherein at least two target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, PKNOX2, VAV3, NDRG4, and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP, and

[0126] (III) comparing the methylation level of at least one target marker in the group of target markers quantitatively analyzed in step (II) with the corresponding methylation levels of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation levels are quantitatively analyzed by repeating steps (I) and (II) on a biological sample containing DNA obtained from the individual before treatment, wherein a methylation level of one or more target markers that is lower than its corresponding methylation level before treatment indicates that the individual has responded to the treatment.

[0127] In some embodiments, a panel of target markers described herein includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more target markers.

[0128] In some embodiments, step (II) described in the present application comprises:

[0129] (i) pre-amplifying at least a portion of at least one target marker from a group of target markers in the treated DNA obtained from step (I) using a pre-amplification primer library, and the group of target markers is selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5; and

[0130] (ii) quantitatively analyzing the methylation levels of the group of target markers in the DNA obtained from the sub-step (i).

[0131] In some embodiments, there is a sub-step (i) of step (II). In some embodiments, there is no sub-step (i) of step (II). In some embodiments, the above method further comprises obtaining DNA from a biological sample from an individual before step (I).

[0132] In some embodiments, the present application provides a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, the method comprising the following steps:

[0133] (a) obtaining a biological sample containing DNA from the individual;

[0134] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between unmethylated sites and methylated sites in the DNA, thereby obtaining treated DNA;

[0135] (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0136] (d) if step (c) exists, then based on the DNA obtained from step (c) respectively quantitatively analyzing the methylation level of at least one target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0137] (e) comparing the methylation level of at least one target marker in step (d) with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has colorectal tumor formation or the risk of formation, or the individual has colorectal tumor development or the likelihood of development is increased, or the individual has a poor prognosis or the risk of a poor prognosis of colorectal tumor.

[0138] In another aspect, the present application provides a method for monitoring the response of an individual to treatment in an individual receiving colorectal tumor treatment, comprising the steps of:

[0139] (a) obtaining a biological sample containing DNA from the individual;

[0140] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing unmethylated sites from methylated sites in the DNA, thereby obtaining treated DNA;

[0141] (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0142] (d) if step (c) exists, then based on the DNA obtained from step (c) respectively quantitatively analyzing the methylation level of at least one target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0143] (e) respectively comparing the methylation level of the at least one target marker in step (d) with the corresponding methylation levels of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation levels are quantitatively analyzed by repeating steps (a), (b), optionally (c), and (d) on a biological sample containing DNA obtained from the individual before treatment, wherein a lower methylation level of the one or more target markers than the corresponding methylation level before treatment indicates that the individual has responded to the treatment.

[0144] As used herein, the term "screening" refers to the identification of a pathological state, disease or condition, such as the identification of colorectal tumors, or the identification of individuals who suffer from colorectal tumors but may benefit from a specific treatment regimen. In this application, the term "screening" and the term "diagnosis" may be used interchangeably. As used herein, the term "tumor" or "tumor" should be understood to refer to a lesion, tumor or other encapsulated or unencapsulated mass or other form of growth containing tumor cells. "Tumor cells" should be understood to refer to cells showing abnormal growth. The term "growth" should be understood in the broadest sense and include proliferation. In this regard, an example of abnormal cell growth is the uncontrolled proliferation of cells. Another example is that cells extend their usual lifespan due to failure of apoptosis. Tumor cells can be benign cells or malignant cells. In some embodiments, the tumor is an adenoma or adenocarcinoma. Without limiting the present invention to any theory or mode of action, adenoma is generally a benign tumor derived from epithelium, which originates from epithelial tissue or shows a clearly defined epithelial structure. These structures can have a glandular appearance. It may contain a malignant cell population within the adenoma, for example, as a benign adenoma or benign tumor lesion progresses to a malignant adenocarcinoma. In some embodiments, the tumor is malignant, such as a carcinoma. In some embodiments, the tumor is not malignant, such as an adenoma.

[0145] The term "colorectal tumor" as used herein refers to a tumor present in the colon, rectum and / or appendix. In some embodiments, the colorectal tumor is a colorectal cancer, a colorectal adenoma and / or a sessile serrated polyp. In some embodiments, the colorectal tumor is precancerous.

[0146] As used herein, the term "precancerous" refers to a neoplasm that exhibits some histological changes associated with an increased risk of progression to cancer. With respect to colorectal cell proliferative disorders, examples of such conditions include high-grade dysplastic cell proliferative diseases, such as adenomatous polyps of the colon.

[0147] The term "formation" as used herein in describing a tumor (e.g., an adenoma or adenocarcinoma) is understood to refer to one or more cells of an individual that exhibit abnormal development. In this regard, an adenoma or adenocarcinoma may have developed because an abnormally growing mass of cells has formed, or it may be an adenoma or adenocarcinoma in a very early stage, with only a relatively small number of cells showing abnormal division at the time of diagnosis. The present application also extends to assessing the risk of colorectal tumor (e.g., colorectal cancer) development in an individual.

[0148] The term "assessment" as used herein refers to the ability to distinguish samples from individuals with colorectal tumor progression and individuals without colorectal tumor progression, or the ability to distinguish samples from individuals at different stages of colorectal tumor progression. In some embodiments, the assessment involves determining whether the tumor of an individual has entered the progression stage or has a higher probability of entering the progression stage. In some embodiments, the assessment involves classifying the tumor of an individual, such as Phase I, Phase II, Phase III, Phase IV, etc. In some embodiments, the assessment involves determining whether the tumor of an individual is alleviated or aggravated. In some embodiments, the assessment can assist in evaluating the possibility that a treatment has a clinical benefit. In some embodiments, the assessment may involve whether the patient improves and / or improves after receiving treatment (e.g., treatment with a specific drug). By selecting the most appropriate treatment for any particular patient, the assessment method of the present application can be used to make a treatment decision clinically. After administering a treatment regimen (e.g., a given treatment regimen, including, for example, the administration of a given therapeutic agent or combination, surgical intervention, steroid therapy, etc.), the assessment method of the present application is a valuable tool in evaluating the possibility of long-term survival of the patient.

[0149] "Differentiation" as understood by those skilled in the art cannot achieve 100% correctness for the analyzed samples. However, it is still required that the samples with statistically significant quantities can be correctly classified. The statistically significant quantity can be determined by those skilled in the art by using different statistical tools, such as but not limited to determining confidence intervals, determining p values, Student's T test or Fisher differentiation equation. For more information, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. In some embodiments, the confidence interval is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In some embodiments, the p value is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.

[0150] As used herein, the term "progression" refers to the natural developmental process by which a cell's morphology and physiology change along a genetically determined pathway, for example, from an earlier, less advanced or early stage to a later, more complex or advanced stage of physiological maturation.

[0151] The term "prognosis" as used herein refers to the possibility of predicting the outcome of disease symptoms (including, for example, recurrence, aggravation, drug resistance) of a disease (e.g., cancer). The term also refers to the prediction of the possibility of clinical benefit of treatment. In some embodiments, a statistical algorithm is used to provide a prognosis of a disease for an individual. For example, the prognosis can be surgery, the progression of a clinical subtype of a cancer (e.g., solid tumors, such as colorectal cancer, melanoma, and renal cell carcinoma), the progression of one or more clinical factors, or recovery from the disease. The prognosis can be a poor prognosis (e.g., possible recurrence or drug resistance) or a good prognosis.

[0152] As used herein, the term "responsive" refers to a beneficial response of an individual to a treatment. The response of an individual to a treatment can be assessed by any endpoint that indicates a benefit to the individual, including, but not limited to: (1) inhibiting the progression of the disease to some extent, including slowing down and completely stopping the progression of the disease; (2) reducing the number of disease attacks and / or symptoms; (3) reducing the size of lesions; (4) inhibiting (i.e., reducing, slowing down, or completely stopping) the infiltration of disease cells into adjacent peripheral organs and / or tissues; (5) inhibiting (i.e., reducing, slowing down, or completely stopping) the spread of the disease; (6) alleviating to some extent one or more symptoms associated with the disease; (7) increasing the time without disease manifestation after treatment; (8) reducing the autoimmune response, which may but not necessarily lead to regression or ablation of disease lesions, such as progression-free survival; (9) increasing overall survival; (10) having a higher response rate; and / or (11) reducing mortality at a given time point after treatment. The terms "benefit", "beneficial", or "benefit" are used in the broadest sense to refer to any desired effect.

[0153] In the present application, the following detailed description of step (a), step (b), step (c) and step (d) is applicable to a method for diagnosing colorectal tumors in an individual, screening for the formation or risk of colorectal tumor formation, or assessing the progression or prognosis of colorectal tumors, and a method for monitoring the response of an individual to treatment for colorectal tumors. However, step (e) of these two methods will be described separately. In addition, in the present application, step (I) of the present application is the same or at least similar to step (b) of the present application. In addition, sub-step (i) of step (II) of the present application is the same or at least similar to step (c) of the present application. Sub-step (ii) of step (II) of the present application is the same or at least similar to step (d) of the present application. In addition, step (III) of the present application is the same or at least similar to step (e) of the present application. Therefore, step (I) and step (b) are collectively referred to as "step (b)" below, and sub-step (i) and step (c) of step (II) are collectively referred to as "step (c)" below. Sub-step (ii) of step (II) and step (d) are hereinafter collectively referred to as "step (d)", and step (III) and step (e) are hereinafter collectively referred to as "step (e)".

[0154] Step (a)

[0155] In step (a) of the method according to the present application, a biological sample containing DNA is obtained from the individual.

[0156] The term "biological sample" as used herein refers to a biological composition obtained or derived from a target individual, which contains cells and / or other molecular entities (e.g., DNA) to be characterized or identified based on physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of an individual obtained by any method known to those skilled in the art. In some embodiments, the biological sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissues, body fluids, colon effluents, surgical resection specimens, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, urine, mucus, saliva, peritoneal fluid, pleural fluid, pleural effusion, synovial fluid, cerebrospinal fluid, thoracentesis fluid, peritoneal effusion, and any combination thereof. In some embodiments, the colon effluent is selected from the group consisting of fecal samples and enema washing samples. The selection of the sample most suitable for detection according to the method of the present application will depend on the nature of the context. In some embodiments, the biological sample is obtained from the whole blood of an individual. In some embodiments, the biological sample is obtained from plasma of the individual. Those skilled in the art are aware of various methods for preparing plasma from whole blood. For example, in some embodiments, plasma is obtained by centrifuging whole blood from an individual once, twice, three times, four times, five times or more.

[0157] As used herein, the term "individual" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. An "individual" may also be a livestock, such as cattle, pigs, sheep, poultry, and horses; or a rodent, such as a rat, mouse; or a non-human primate, such as an ape, monkey, rhesus monkey; or a domesticated animal, such as a dog or a cat. In some embodiments, the individual is a human or a non-human primate. In some embodiments, the individual is a human. In this application, "individual" and "subject" may be used interchangeably.

[0158] In some embodiments, the DNA is separated from the biological sample. Separation and purification of DNA from biological samples can be implemented by using various methods known in the art, including the use of commercially available kits. For example, DNA is separated from cells and tissues in the following manner: cleavage of raw materials under highly denatured and reducing conditions, partial use of protein degrading enzymes, purification of nucleic acid components obtained by phenol / chloroform extraction processes, and recovery of nucleic acids from aqueous phases by dialysis or ethanol precipitation (see, for example, Sambrook, J., Fritsch, EFin T. Maniatis, CSH, Molecular Cloning, 1989). For another example, there are many reagent systems that are particularly suitable for purifying DNA fragments from agarose gels, separating plasmid DNA from bacterial lysates, and separating longer-chain nucleic acids (genomic DNA, total cell RNA) from blood, tissues or cell cultures. Many of these commercially available purification systems are based on fairly well-known principles, that is, nucleic acids are combined with mineral carriers in the presence of solutions of different chaotropic salts. In these systems, finely ground glass powder, diatomaceous earth or silica gel suspensions are used as carrier materials. Some other methods for isolating and purifying DNA from biological samples are described in, for example, US7888006B2 and EP1626085A1. The choice between methods will be influenced by several factors, including time, cost and the amount of DNA required.

[0159] In some embodiments, the DNA contained in the biological sample includes genomic DNA. The term "genomic DNA" used herein refers to the DNA containing the complete genome of a cell or organism and its fragments or parts. Genomic DNA is a large segment of DNA (e.g., longer than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 300 kb) derived from an individual and may have natural modifications, such as DNA methylation.

[0160] In some embodiments, the DNA contained in the biological sample includes cellular DNA. The term "cellular DNA" as used herein refers to DNA present in cells, or DNA obtained from cells in vivo and isolated in vitro, or otherwise manipulated in vitro, as long as the DNA is not removed from cells in vivo.

[0161] In some embodiments, the DNA contained in the biological sample includes extracellular free DNA. The term "extracellular free DNA" used herein refers to DNA fragments present outside the cell in vivo. The term can also be used to refer to DNA fragments obtained from extracellular sources in vivo and separated or manipulated in vitro. The DNA fragments in the extracellular free DNA generally have a length of about 100 to 200bp, which is speculated to be related to the length of the DNA fragments wrapped in nucleosomes. Extracellular free DNA includes, for example, extracellular free fetal DNA and circulating tumor DNA. Extracellular free fetal DNA circulates in the body (e.g., blood) of a pregnant woman, representing the fetal genome, while circulating tumor DNA circulates in the body (e.g., blood) of a cancer patient. In some embodiments, extracellular free DNA may be substantially free of individual cell DNA. For example, the extracellular free DNA may include less than about 1,000ng / mL, less than about 100ng / mL, less than about 10ng / mL, less than about 1ng / mL of cell DNA.

[0162] Extracellular free DNA can be prepared by using conventional techniques known in the art. For example, the extracellular free DNA of a blood sample can be obtained by centrifuging a blood sample at a speed of about 200-20,000g, about 200-10,000g, about 200-5,000g, about 300-4000g, etc. for about 3-30 minutes, about 3-15 minutes, about 3-10 minutes, about 3-5 minutes. For example, in some embodiments, the extracellular free DNA of a blood sample can be obtained by centrifuging the plasma or serum of an individual one, two, three, four, five or more times. In some embodiments, in order to separate cells and fragments thereof from a cell-free component comprising soluble DNA, the biological sample can be obtained by microfiltration. Generally speaking, microfiltration can be performed by using a filter, for example, a membrane filter of 0.1 micron to 0.45 micron, such as a membrane filter of 0.22 micron.

[0163] In some embodiments, a commercially available DNA extraction product is used to extract extracellular free DNA from whole blood, serum or plasma for analysis. This extraction method is said to have a high recovery rate (>50%) for circulating DNA, and some products (e.g., the QIAamp Circulating Nucleic Acid Kit produced by Qiagen) are said to be able to extract small-sized DNA fragments. The typical sample volume used is 1-5 mL of serum or plasma.

[0164] In some embodiments, the extracellular free DNA includes circulating tumor DNA. Circulating tumor DNA ("ctDNA") is a fragmented DNA of tumor origin in body fluids (such as blood, urine, saliva, sputum, feces, pleural fluid, cerebrospinal fluid, etc.) that are not related to cells. Typically, ctDNA is highly fragmented, with an average length of about 150 base pairs. ctDNA typically includes a very small portion of extracellular free DNA in body fluids (such as plasma), for example, ctDNA may constitute less than about 10% of plasma DNA. Typically, the percentage is less than about 1%, such as less than about 0.5% or less than about 0.01%. In addition, the total amount of plasma DNA is usually very low, such as about 10ng / mL plasma. The amount of ctDNA varies from person to person and depends on the type and location of the tumor, and for cancerous tumors, it depends on the stage of cancer. However, ctDNA is usually very rare in body fluids and can only be detected by extremely sensitive and specific techniques. Detecting ctDNA may help detect and diagnose tumors, guide tumor-specific treatments, monitor treatments, and monitor the remission of cancer.

[0165] Step (b)

[0166] In step (b) of the method according to the present application, the DNA in the biological sample obtained in step (a) is treated with a reagent capable of distinguishing between unmethylated sites and methylated sites in DNA, thereby obtaining treated DNA.

[0167] DNA methylation is a biological process in which a methyl group is added to a DNA molecule (e.g., to one or more cytosine bases of a DNA molecule) (e.g., by the action of a DNA methyltransferase). In mammals, DNA methylation occurs at the 5' position of a cytosine-phosphate-guanine (CpG) dinucleotide (i.e., a "CpG site"), and when it occurs in a 5'-CpG-3' dinucleotide in the promoter or first exon of a gene, it results in epigenetic inactivation of the gene. It is well documented that DNA methylation plays an important role in regulating gene expression, tumorigenesis, and other genetic and epigenetic diseases.

[0168] As used herein, the term "methylated cytosine residue" refers to a derivative of a cytosine residue in which one methyl group is attached to a carbon atom of the cytosine ring (e.g., C5). The term "unmethylated cytosine residue" refers to an underivatized cytosine residue in which, in contrast to a "methylated cytosine residue," no methyl group is attached to a carbon atom of the cytosine ring (e.g., C5). A CpG site in which a cytosine residue is methylated is a methylated CpG site, while a CpG site in which a cytosine residue is not methylated is an unmethylated CpG site.

[0169] In some embodiments, the reagent used in step (b) can distinguish between unmethylated and methylated CpG sites in DNA, thereby obtaining treated DNA. The reagent can selectively act on unmethylated cytosine residues, but cannot significantly act on methylated cytosine residues. Or the reagent can selectively act on methylated cytosine residues, but not significantly act on unmethylated cytosine residues. Therefore, the original DNA is converted into treated DNA in a manner depending on whether it is methylated, so that the treated DNA can be distinguished from the original DNA by its hybridization behavior.

[0170] For example, some reagents can selectively convert unmethylated cytosine residues to uracil, thymine, or another base that is hybridized differently from cytosine, while methylated cytosine residues remain unconverted. For another example, some reagents can selectively cleave methylated residues, or selectively cleave unmethylated residues.

[0171] As used herein, "treated DNA" refers to DNA that has been treated with an agent that can distinguish between unmethylated sites and methylated sites in DNA, ie, the DNA methylation status in the DNA has been changed.

[0172] In some embodiments, the reagent of step (b) selectively modifies unmethylated cytosine residues at CpG sites to produce modified residues, but does not significantly modify methylated cytosine residues.

[0173] In some embodiments, the reagent of step (b) includes a bisulfite reagent. As used herein, the term "bisulfite reagent" refers to, for example, a reagent disclosed in the present application that can be used to distinguish between methylated and unmethylated CpG dinucleotide sequences, including bisulfite, bisulfite ions, or any combination thereof. In the present application, treating DNA with a bisulfite reagent is also described as a "bisulfite reaction" or "bisulfite treatment", which refers to a reaction that converts unmethylated cytosine residues, particularly in the presence of bisulfite ions, where unmethylated cytosine residues in nucleic acids are converted to uracil bases, thymine bases, or other bases that are different from cytosine in hybridization behavior, while methylated cytosine residues are not significantly converted. In other words, bisulfite treatment can be used to distinguish between methylated CpG dinucleotides and unmethylated CpG dinucleotides.

[0174] The bisulfite reaction for detecting methylated cytosine residues is described in detail in Frommer, M., et al., Proc Natl Acad Sci USA 89 (1992) 1827-31 and Grigg, G., Clark, S., Bioessays 16 (1994) 431-6. The bisulfite reaction includes a deamination step and a desulfonation step (see Grigg and Clark, supra). The statement “methylated cytosine residues are not significantly converted” does not exclude that a very small percentage (e.g., less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%) of the methylated cytosine residues are converted to uracil, thymine, or other bases that differ from cytosine in hybridization behavior, although it is intended that only unmethylated cytosine residues are converted.

[0175] The person skilled in the art knows how to perform the bisulfite treatment, in particular the deamination step and the desulfonation step, with reference to, for example, Frommer M., et al. (supra) or Grigg and Clark (supra), which disclose the basic parameters of the bisulfite treatment. The effects of incubation time and temperature on the efficiency of deamination, as well as the parameters that affect DNA degradation, are disclosed.

[0176] In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ion, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, the bisulfite reagent is commercially available, for example, MethylCode TM Bisulfite Conversion Kit, EpiMark TM BisulfiteConversion Kit、EpiJET TM Bisulfite Conversion Kit, EZ DNA Methylation-Gold TM Kit, etc. In some embodiments, the bisulfite reaction is performed according to the instructions of the kit.

[0177] In some embodiments, the reagent of step (b) selectively cleaves unmethylated residues but not methylated residues, or selectively cleaves methylated residues but not unmethylated residues.

[0178] In some embodiments, the reagent of step (b) is a methylation-sensitive restriction enzyme (MSRE).

[0179] The term "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids based on the methylation state of its recognition site. For restriction enzymes that specifically shear when the recognition site is not methylated or hemimethylated, shearing will not occur when the recognition site is methylated, or shearing will be significantly reduced in efficiency. For restriction enzymes that specifically shear when the recognition site is methylated, shearing will not occur when the recognition site is not methylated, or shearing will be significantly reduced in efficiency. In some embodiments, the recognition sequence of the methylation-sensitive restriction enzyme contains a CG dinucleotide (e.g., cgcg or cccggg). In some embodiments, when the cytosine in the CG dinucleotide is methylated at the C5 carbon atom, the methylation-sensitive restriction enzyme does not shear.

[0180] In some embodiments, the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0181] Methylation is determined using methods known in the art using a methylation-sensitive restriction enzyme or a panel of restriction enzyme reagents comprising a methylation-sensitive restriction enzyme that can distinguish between methylated and unmethylated CpG dinucleotides in the target region, such as, but not limited to, differential methylation hybridization ("DMH").

[0182] In some embodiments, the DNA of step (a) can be cut before being treated with a methylation-sensitive restriction enzyme. Such methods are known in the art and can include both physical means and enzymatic means. Particularly preferably, one or more restriction enzymes that are insensitive to methylation and whose recognition sites are rich in AT and do not comprise CG dinucleotides are used. Such enzymes are used to preserve the CpG sites and CpG-enriched regions in the DNA fragments. In some embodiments, such restriction enzymes are selected from MseI enzyme, BfaI enzyme, Csp6I enzyme, Tru1I enzyme, Tru9I enzyme, MaeI enzyme, XspI enzyme and any combination thereof.

[0183] Step (c)

[0184] In the step (c) according to the method of the present application, at least one target marker in the treated DNA obtained from step (b) is pre-amplified with a pre-amplification primer library, wherein at least a portion of at least one (e.g., each) target marker is pre-amplified. In the present application, step (c) may also be referred to as a pre-amplification step. Although it is not desired to be bound by any theory, it is believed that step (c) is not necessarily required to achieve the purpose of the present application. In some embodiments, step (c) of the method of the present application exists. In some embodiments, step (c) of the method of the present application does not exist.

[0185] One of the purposes of pre-amplification of target markers is to increase the quantity of target markers in treated DNA. As used herein, the term "amplification" refers generally to any process that can cause the copy number of a molecule or a group of related molecules to increase. When "amplification" is used for polynucleotide molecules, it refers to multiple copies of a polynucleotide molecule or a part of a polynucleotide molecule that usually produces multiple copies from a small amount of polynucleotides, wherein the amplified material (amplicon, PCR amplicon) is usually detectable. The amplification of polynucleotides covers multiple chemical and enzymatic processes. The form of amplification includes by polymerase chain reaction (reverse transcription PCR, PCR), strand displacement amplification (SDA) reaction, transcription-mediated amplification (TMA) reaction, nucleic acid sequence-based amplification (NASBA) reaction or ligase chain reaction (LCR), one or more copies of template RNA or DNA molecules generate multiple DNA copies.

[0186] As used herein, the term "target marker" refers to a target nucleic acid or gene region whose methylation level indicates a colorectal tumor (e.g., colorectal cancer), or indicates the formation or risk of colorectal tumor (e.g., colorectal cancer), or indicates the progression or prognosis of colorectal tumor (e.g., colorectal cancer). In this application, the terms "marker" and "gene" can be used interchangeably. The term "marker" or "gene" should be considered to include all its transcriptional variants (e.g., the term "Septin9" should include, for example, its truncated transcript Q9HC74) and all its promoters and regulatory elements. As understood by those skilled in the art, certain genes are known to exhibit allelic variation or single nucleotide polymorphism ("SNP") between individuals. SNPs include insertions and deletions of simple repeat sequences (e.g., dinucleotide and trinucleotide repeats) of different lengths. Therefore, the present application should be understood to be extended to all forms of markers / genes produced by any other mutation, polymorphism or allelic variation. In addition, it should be understood that the terms "marker" and "gene" should include both the positive and negative strand sequences of the marker or gene.

[0187] The term "target marker" used herein is broadly interpreted as including both 1) the original marker found in a biological sample or genomic DNA (in a specific methylation state), and 2) its processed sequence (e.g., the corresponding region after bisulfite conversion or the corresponding region after MSRE treatment). The corresponding region after bisulfite conversion is different from the target marker in the genomic sequence in that one or more unmethylated cytosine residues are converted to uracil bases, thymine bases, or other bases that are different from cytosine in hybridization behavior. The corresponding region treated with MSRE is different from the target marker in the genomic sequence in that the sequence is cut at one or more MSRE cutting sites.

[0188] In some embodiments, the at least one target marker comprises one or more markers (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 markers) selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one target marker comprises 14 markers selected from the group consisting of NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and intergenic region 1. In some embodiments, the at least one target marker comprises 13 markers selected from the group consisting of NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and intergenic region 1. In some embodiments, the at least one target marker comprises 11 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and intergenic region 1. In some embodiments, the at least one target marker comprises 10 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and intergenic region 1. In some embodiments, the at least one target marker comprises 10 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2. In some embodiments, the at least one target marker comprises 9 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2. In some embodiments, the at least one target marker comprises 7 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, and NDRG4.In some embodiments, the at least one target marker comprises 6 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, and NDRG4. In some embodiments, the at least one target marker comprises 6 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, and BCAN. In some embodiments, the at least one target marker comprises 5 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and BCAN. In some embodiments, the at least one target marker comprises 5 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and IRF4. In some embodiments, the at least one target marker comprises 3 markers selected from the group consisting of SALL1, BCAT1, and Septin9.

[0189] In some embodiments, the at least one target marker may be at most one target marker (i.e., one marker and no more than one marker). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, the at least one target marker is intergenic region 1.

[0190] In some embodiments, the at least one target marker comprises a plurality of target markers. In some embodiments, the plurality of target markers comprises at least two or three markers selected from the group consisting of Septin9, BCAT1 and IKZF1. In some embodiments, the plurality of target markers described herein further comprises one, two, three, four or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4 and IRF4. In some embodiments, the plurality of target markers described herein further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0191] In some embodiments, the plurality of target markers described herein comprise Septin9 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers comprise BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers comprise NDRG4, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0192] In some embodiments, the plurality of target markers described herein comprises BCAT1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0193] In some embodiments, the plurality of target markers described herein comprises IKZF1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, or IRF4.

[0194] In some embodiments, the plurality of target markers described herein comprises BCAN and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, VAV3, and / or IRF4.

[0195] In some embodiments, the plurality of target markers described herein include VAV3 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BBCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, NDRG4, and / or IRF4.

[0196] In some embodiments, the plurality of target markers described herein comprises IRF4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or NDRG4.

[0197] In some embodiments, the plurality of target markers described herein include PKNOX2 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0198] In some embodiments, the plurality of target markers described herein comprise POU4F2 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0199] In some embodiments, the plurality of target markers described herein include SALL1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0200] In some embodiments, the plurality of target markers described herein include TMEFF2 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, ASCL4, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0201] In some embodiments, the plurality of target markers described herein include ASCL4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0202] In some embodiments, the multiple target markers described herein include FGF12 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27) additional target markers selected from the following group: Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0203] In some embodiments, the multiple target markers described herein include intergenic region 1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27) additional target markers selected from the following group: Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0204] In some embodiments, the plurality of target markers described herein include NDRG4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0205] In the present application, it should be understood that markers / genes are described both by citing their names and by their chromosome coordinates. The chromosome coordinates are consistent with the Hg19 version of the human genome database released in February 2009 (referred to herein as "Hg19 coordinates").

[0206] In the present application, it is understood that the target marker also includes intergenic regions, which are referred to as "intergenic region 1", "intergenic region 2", "intergenic region 3", "intergenic region 4", "intergenic region 5", and are defined by their respective chromosome coordinates. For example, in the present application, intergenic region 1 refers to the region defined by chr6:19679885-19693988; intergenic region 2 refers to the region defined by chr10:130082033-130087148; intergenic region 3 refers to the region defined by chr10:133107880-133113966; intergenic region 4 refers to the region defined by chr7:152620588-152624685; intergenic region 5 refers to the region defined by chr8:70945014-70949177.

[0207] In some embodiments, each target marker includes or is: a) each region defined by Hg19 coordinates as shown below:

[0208]

[0209]

[0210]

[0211] and 5 kb upstream of each starting site and 5 kb downstream of each end site in each region; or b) the corresponding region of a) after bisulfite conversion; or c) the corresponding region of a) after methylation sensitive restriction enzyme (MSRE) treatment. The specific nucleotide sequence of the Hg19 coordinates as described above, as well as 5 kb upstream of each starting site and 5 kb downstream of each end site in each region can be obtained in public databases (such as UCSC Genome Browser, Ensemble and NCBI websites).

[0212] In some embodiments, each target marker also includes all variants thereof. Variant includes a nucleotide sequence from the same region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (i.e., with one or more deletions, insertions, substitutions, reverse sequences, etc.) of markers / gene regions as described herein. Therefore, the present application content should be understood as extending to such variants that achieve the same result, despite the fact that the actual nucleotide sequence between individuals has slight genetic variation.

[0213] As used herein, the term "percentage (%) of sequence identity" refers to the same percentage of amino acid (or nucleic acid) residues of a candidate sequence and the amino acid (or nucleic acid) residues of a reference sequence after sequence alignment, and intervals may be introduced (if necessary) during alignment to maximize the number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered identical residues. The percentage of amino acid (or nucleic acid) sequence identity can be determined in the following manner, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute), see also Higgins D G et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. One skilled in the art can use the default parameters provided by the tool, or can customize parameters suitable for the alignment (eg, by selecting an appropriate algorithm).

[0214] In step (c) provided herein, at least a portion of at least one (eg, each) target marker is pre-amplified. In some embodiments, the pre-amplified portion of the target marker is within a sub-region of the target marker.

[0215] Without limiting the present application to any one theory or mode of action, it is believed that it is particularly useful to measure the methylation level within a subregion of the target marker, which contains a high density of CpG dinucleotides that are often hypermethylated in colorectal tumors (e.g., colorectal cancer). This discovery makes the subregion a particularly useful target for analysis because it can both simplify the screening process (because the DNA region to be analyzed is shorter and more clearly defined), and further, compared to the results obtained by analyzing the entire Hg19 region of the target marker, the results obtained from these subregions will provide significantly clearer results in terms of the presence or absence of hypermethylation. Therefore, this discovery simplifies the diagnosis, screening / monitoring process, and increases the sensitivity and specificity of colorectal tumor diagnosis. In some embodiments, the subregions of each target marker include or are: a) each region defined by the Hg19 coordinates as shown below:

[0216]

[0217]

[0218]

[0219] and 5 kb upstream of each start site and 5 kb downstream of each end site of each of the above regions; or b) the corresponding region of a) after bisulfite conversion; or c) the corresponding region of a) after MSRE treatment.

[0220] In some embodiments, the subregion of each target marker comprises or is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 86-112, 167, or a corresponding region thereof after bisulfite conversion, or a corresponding region thereof after MSRE treatment. In some embodiments, the corresponding region of the subregion of the target marker after bisulfite conversion comprises or is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 113-166, 168, 169. The SEQ ID NO of each subregion of the target marker is listed in Table 1 below.

[0221] Table 1. Exemplary subregions for various target markers

[0222]

[0223]

[0224] In some embodiments, the subregion of NDRG4 comprises a sequence selected from the group consisting of SEQ ID NOs: 86, 113, and 140; the subregion of BCAT1 comprises a sequence selected from the group consisting of SEQ ID NOs: 87, 114, and 141; the subregion of IKZF1 comprises a sequence selected from the group consisting of SEQ ID NOs: 88, 115, and 142; the subregion of Septin9 comprises a sequence selected from the group consisting of SEQ ID NOs: 89, 116, and 143; the subregion of SDC2 comprises a sequence selected from the group consisting of SEQ ID NOs: 90, 117, and 144; the subregion of VAV3 comprises a sequence selected from the group consisting of SEQ ID NOs: 91, 118, and 145; the subregion of TMEFF2 comprises a sequence selected from the group consisting of SEQ ID NOs: 92, 119, and 146; the subregion of SALL1 comprises a sequence selected from the group consisting of SEQ ID NOs: 93, 120, and 147; the subregion of BCAN comprises a sequence selected from the group consisting of SEQ ID NOs: NO:94, 121 and 148; the subregion of POU4F2 comprises a sequence selected from the group consisting of SEQ ID NO:95, 122 and 149; the subregion of PKNOX2 comprises a sequence selected from the group consisting of SEQ ID NO:96, 123 and 150; the subregion of ASCL4 comprises a sequence selected from the group consisting of SEQ ID NO:97, 124 and 151; the subregion of KCNA6 comprises a sequence selected from the group consisting of SEQ ID NO:98, 125 and 152; the subregion of SOX1 comprises a sequence selected from the group consisting of SEQ ID NO:99, 126 and 153; the subregion of HS3ST2 comprises a sequence selected from the group consisting of SEQ ID NO:100, 127 and 154; the subregion of FGF12 comprises a sequence selected from the group consisting of SEQ ID NO:101, 128 and 155; the subregion of KCTD8 comprises a sequence selected from the group consisting of SEQ ID NO: NO:102, 129 and 156; the subregion of HMX1 comprises a sequence selected from the group consisting of SEQ ID NO:103, 130 and 157; the subregion of MARCH11 comprises a sequence selected from the group consisting of SEQ ID NO:104, 131 and 158; the subregion of CRHBP comprises a sequence selected from the group consisting of SEQ ID NO:105, 132 and 159; the subregion of NKX2-6 comprises a sequence selected from the group consisting of SEQ ID NO:106, 133 and 160; the subregion of SLC24A2 comprises a sequence selected from the group consisting of SEQ ID NO:107, 134 and 161; the subregion of intergenic region 1 comprises a sequence selected from the group consisting of SEQ ID NO:108, 135 and 162; the subregion of intergenic region 2 comprises a sequence selected from the group consisting of SEQ ID NO:109, 136 and 163;The subregion of intergenic region 3 comprises a sequence selected from the group consisting of SEQ ID NOs: 110, 137 and 164; the subregion of intergenic region 4 comprises a sequence selected from the group consisting of SEQ ID NOs: 111, 138 and 165; the subregion of intergenic region 5 comprises a sequence selected from the group consisting of SEQ ID NOs: 112, 139 and 166; and / or the subregion of IRF4 comprises a sequence selected from the group consisting of SEQ ID NOs: 167, 168 and 169.;

[0225] In some embodiments, the target marker in the extracellular free DNA is present in the biological sample in an amount of no more than 1 ng, no more than 0.9 ng, no more than 0.8 ng, no more than 0.7 ng, no more than 0.6 ng, no more than 0.5 ng, no more than 0.4 ng, no more than 0.3 ng, no more than 0.2 ng, no more than 0.1 ng, no more than 0.09 ng, no more than 0.08 ng, no more than 0.07 ng, no more than 0.06 ng, no more than 0.05 ng, no more than 0.04 ng, no more than 0.03 ng, no more than 0.02 ng or no more than 0.01 ng. In some embodiments, the target marker in the extracellular free DNA is present in the biological sample in a percentage of no more than 0.1%, no more than 0.2%, no more than 0.3%, no more than 0.4%, no more than 0.5%, no more than 0.6%, no more than 0.7%, no more than 0.8%, no more than 0.9%, or no more than 1%. In some embodiments, the concentration of the target marker in the extracellular free DNA in the biological sample is lower than the sensitivity level of the detection analysis for the target marker." Sensitivity of detection analysis" is a measure of the ability of the detection analysis to distinguish between small differences in analytical concentration / amount. If the target marker in the extracellular free DNA present in the biological sample is lower than the sensitivity level of the detection analysis, conventional methods cannot be used to quantitatively analyze the methylation level of each target marker in the sample. On the contrary, the method disclosed in the present application is practical and superior in terms of a very small amount of target marker in the detection sample. In some embodiments, the target marker in the extracellular free DNA is present in the biological sample in an amount of no more than 0.08ng or no more than 0.04ng.

[0226] In some embodiments, the DNA obtained in step (c) is diluted with a diluent before the next step (i.e., step (d)). In some embodiments, the diluent is selected from the group consisting of nuclease-free water, Tris-EDTA buffer, and any other buffer without PCR inhibition. In some embodiments, the pre-amplified DNA of step (c) is directly added to the next step (i.e., step (d)) without prior dilution.

[0227] The at least one target marker in the treated DNA is pre-amplified with a pre-amplification primer library. As used herein, the term "primer" refers to a single-stranded oligonucleotide that can be used as a starting point for template-guided DNA synthesis under suitable conditions (e.g., buffer and temperature) in the presence of four different nucleoside triphosphates and reagents for polymerization (e.g., DNA polymerase). In any given case, the length of the primer depends on, for example, the intended use of the primer, and is generally within the range of 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. Primers do not have to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template. A primer site is a region on the template that hybridizes with a primer. A primer pair is a set of primers comprising a 5' forward primer that hybridizes with the 5' end of a sequence to be amplified and a 3' reverse primer that hybridizes with the complementary strand at the 3' end of a sequence to be amplified. Those skilled in the art can design primers according to the marker to be amplified based on the common knowledge in the art (see, for example, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995). In addition, some software packages for designing optimal probes and / or primers for use in various analyses are public, such as Primer3 available from the Center for Genome Research, Cambridge, Mass., USA. Obviously, its potential use should also be taken into account when designing probes or primers. For example, the primers designed for the purpose of the present invention may include at least one CpG site, or the amplification product obtained from the primers may include at least one CpG site. Tools for designing primers for detecting DNA methylation status are also known in the art, such as MethPrimer (Li LC and Dahiya R. Meth Primer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov; 18 (11): 1427-31). In the present application, any target marker (at least a portion of at least one (eg, each) target marker or a subregion of at least one target marker) in the treated DNA can be preamplified by using the preamplification primers as a primer library.

[0228] The term "oligonucleotide" as used herein is defined as a molecule comprising two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably at least about 10-15 nucleotides, more preferably at least about 15 to 30 nucleotides or longer (e.g., the length of an oligonucleotide is generally less than 200 residues (e.g., between 15 and 100 nucleotides), but, as used herein, the term is also intended to cover longer polynucleotide chains). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are usually referred to by their length. For example, an oligonucleotide with 24 residues is referred to as a "24-mer". Oligonucleotides can form secondary and tertiary structures by self-hybridization or hybridization with other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, crosses, bends, and triplexes. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or any combination thereof.

[0229] As used herein, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleic acids, for example, between the two strands of a double-stranded DNA molecule, or between a primer binding site and an oligonucleotide primer on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand are optimally aligned, compared, and with appropriate nucleotide insertions or deletions, and are paired with at least about 80% (usually at least about 90% to 95%, more preferably about 98% to 100%) of the nucleotides of the other strand. Alternatively, complementarity exists when an RNA strand or DNA strand hybridizes to its complementary sequence under selective hybridization conditions. Typically, selective hybridization will occur when there is at least about 65% (preferably at least about 75%, more preferably at least about 90%) complementarity over a stretch of at least 14 to 25 nucleotides. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

[0230] In some embodiments, the preamplification primer library comprises at least one methylation-specific primer pair. In some embodiments, the preamplification primer library comprises a plurality of methylation-specific primer pairs. In some embodiments, the preamplification step is performed by methylation-specific PCR ("MSP"), which is a PCR using methylation-specific primers. This technology (i.e., MSP) has been described in Herman et al., Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996 September 3; 93 (18): 9821-6 and United States Patent No. 6,265,171.

[0231] As used herein, the term "methylation-specific primer pair" refers to a primer pair specifically designed to identify CpG sites to utilize methylated differences to amplify specific target markers in treated DNA. Primers act only on molecules with a specific methylation state or without a specific methylation state. For example, a primer can be an oligonucleotide, which can hybridize specifically with a specific CpG site with methylation in a methylation-specific manner under stringent conditions, medium stringent conditions, or highly stringent conditions, but can not hybridize with a specific CpG site without methylation. Therefore, the primer will specifically amplify a target marker with methylation at a specific CpG site. For another example, a primer can be an oligonucleotide, which can hybridize specifically with a specific CpG site with unmethylation in a methylation-specific manner under stringent conditions, medium stringent conditions, or highly stringent conditions, but can not hybridize with a specific CpG site with methylation. Therefore, the primer will specifically amplify a target marker without methylation at a specific CpG site. Therefore, in the present application, methylation-specific primers are used in the preamplification of at least one target marker in the treated DNA to distinguish between methylated and unmethylated CpG sites. The methylation-specific primer pairs of the present application include at least one primer that hybridizes to a CpG dinucleotide treated with bisulfite. Therefore, the sequence of the primer specific for methylated DNA includes at least one CpG dinucleotide, and the sequence of the primer specific for unmethylated DNA includes "T" at the C position of CpG, and / or includes "A" at the G position in CpG.

[0232] In some embodiments, the at least one methylation-specific primer pair comprises a forward primer and a reverse primer, each of which comprises an oligonucleotide sequence that hybridizes to at least 9 consecutive nucleotides of one of the target markers (or a subregion of the target marker) under stringent conditions, moderately stringent conditions, or highly stringent conditions, wherein the at least 9 consecutive nucleotides of one of the target markers (or a subregion of the target marker) comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG site.

[0233] As used herein, the term "hybridization" may refer to a process in which two single-stranded polynucleotides are non-covalently bound to form a stable double-stranded polynucleotide. In one aspect, the resulting double-stranded polynucleotide may be a "hybrid" or "double-stranded". The salt concentration in the "hybridization conditions" is generally less than about 1 M, often less than about 500 mM and may be less than about 200 mM. "Hybridization buffer" includes a buffered salt solution, such as 5% SSPE, or other such buffers known in the art. The hybridization temperature may be as low as 5°C, but is generally higher than 22°C, and more generally higher than about 30°C, and generally exceeds 37°C. Hybridization is generally performed under stringent conditions, i.e., under which the sequence will hybridize with its target sequence but not with other non-complementary sequences. Stringent conditions are sequence-dependent and vary in different situations. For example, longer fragments may require higher hybridization temperatures than short fragments for specific hybridization. Since other factors may affect the stringency of hybridization, including base composition and the length of complementary chains, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one parameter alone. Generally, stringent conditions are selected to be about 5°C lower than the melting point (Tm) for a specific sequence at a defined ionic strength and pH.

[0234] Tm can be the temperature at which half of a population of double-stranded nucleic acid molecules is separated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As shown in standard references, when nucleic acids are in 1M NaCl aqueous solution, a simple estimated Tm value can be calculated by the formula Tm=81.5+0.41(%G+C) (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36:10581-94 (1997)) include alternative calculation methods that take into account structure and environment as well as sequence features when calculating Tm.

[0235] Typically, the stability of the hybrid is a function of ion concentration and temperature. Typically, the hybridization reaction is carried out under relatively low stringency conditions and then washed in a washing solution having different but higher stringency. Exemplary stringency conditions include pH about 7.0 to about 8.3, a temperature of at least 25°C, and a sodium ion (or other salt) concentration of at least 0.01M to no more than 1M. For example, 5xSSPE (750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH 7.4) and a temperature of about 30°C are suitable for allele-specific hybridization, although the appropriate temperature depends on the length and / or GC content of the hybridization region. In one aspect, the "hybridization stringency" for determining the percentage of mismatches can be as follows: 1) high stringency: 0.1x SSPE, 0.1% SDS, 65°C; 2) medium stringency (also referred to as moderate stringency): 0.2x SSPE, 0.1% SDS, 50°C; 3) low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It should be understood that the same stringency can be achieved using alternative buffers, salts and temperatures. For example, moderate stringency hybridization can refer to conditions that allow nucleic acid molecules (e.g., probes) to bind to complementary nucleic acid molecules. The hybridized nucleic acid molecules typically have at least 60% identity, including, for example, at least 70%, 75%, 80%, 85%, 90% or 95% identity. Moderate stringency conditions can be conditions that achieve the same effect as the following conditions: 42°C, 50% formamide, 5x Denhardt solution, 5x SSPE, 0.2% SDS hybridization, followed by washing with 42°C, 0.2x SSPE, 0.2% SDS. High stringency conditions can be provided by, for example, 42°C, 50% formamide, 5x Denhardt solution, 5x SSPE, 0.2% SDS hybridization, followed by washing at 65°C, 0.1x SSPE and 0.1% SDS. Low stringency hybridization can be conditions that achieve equivalent results to the following conditions: 22°C, 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS hybridization, followed by washing in 1x SSPE, 0.2% SDS at 37°C. Denhardt's solution contains 1% polysucrose, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA.Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, NY (1989) and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0236] In some embodiments, the preamplification primer library also includes a control primer pair for amplifying a control marker. Typically, a control marker is a nucleic acid with a known feature (e.g., a known sequence, a known copy number of each cell), for comparison with an experimental target (e.g., a nucleic acid with an unknown concentration). The control can be an endogenous, preferably an invariant gene, and the experimental nucleic acid or target nucleic acid in the analysis can be standardized. This type of control standardized because of differences between samples may occur in, for example, sample processing, analysis efficiency, etc., and allows accurate data comparison between samples, quantitative analysis of amplification efficiency and deviation.

[0237] In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29. In some embodiments, the sequence of the control primer pair is shown in SEQ ID NOs: 55 and 56 of Table 2 below.

[0238] In some embodiments, the at least one methylation-specific primer pair comprises one or more pairs of nucleotide sequences selected from the group consisting of SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2. The sequence numbers of the primer pairs used in this application are represented in the form of "SEQ ID NO: n / m". For example, SEQ ID NO: 1 / 2 refers to a primer pair having the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Table 2 below.

[0239] The primer pairs shown in SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171 were used to amplify the markers, respectively. The genes included NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, intergenic region 1, ASCL4, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 4, NKX2-6, SLC24A2, intergenic region 5, and IRF4.

[0240] In some embodiments, in step (c), at least one target marker is amplified in the presence of one or more blocking oligonucleotides. The purposes of such blocking oligonucleotides have been described in Yu et al., BioTechniques 23: 714-720, 1997. The blocking sequence and pre-amplification primer are simultaneously hybridized with the treated DNA. The pre-amplification of the target marker ends at the 5' position of the blocking sequence, so that the pre-amplification of the target marker is suppressed in the presence of a sequence complementary to the blocking sequence. The blocking sequence can be designed to hybridize with the treated DNA in a methylation state-specific manner. For example, in order to detect the methylated nucleic acid in the unmethylated nucleic acid colony, the amplification of the unmethylated nucleic acid in the position can be suppressed by using a blocking sequence comprising " CpA " or " TpA " in the relevant position, correspondingly, if it is necessary to suppress the amplification of the methylated nucleic acid, " CpG " is used.

[0241] For PCR methods using blocking oligonucleotides, effective interference with polymerase-mediated amplification requires that the blocking oligonucleotide cannot be extended by the polymerase. Preferably, this is achieved by using a blocker that is a 3'-deoxyoligonucleotide or an oligonucleotide other than a "free" hydroxyl group derivatized at the 3' position. For example, 3'-O-acetyl oligonucleotides are representatives of a preferred class of blocker molecules.

[0242] In addition, the decomposition of the polymerase-mediated blocking oligonucleotide should be prevented. Preferably, such prevention includes the use of a polymerase lacking 5'-3' exonuclease activity, or the use of a modified blocking oligonucleotide having a thiolate bridge at its 5'-end, making the blocker molecule nuclease-resistant. Specific applications may not require 5' modifications to the blocker. For example, if the blocker binding site overlaps with the primer binding site, thereby preventing the binding of the primer (e.g., using an excessive blocker), the degradation of the blocking oligonucleotide will be substantially prevented. This is because the polymerase will not extend the primer to and beyond the blocker (along the 5'-3' direction), and the process will usually result in the degradation of the hybridized blocking oligonucleotide.

[0243] For the purposes of this application and also as implemented herein, particularly preferred blocker / PCR embodiments include the use of peptide nucleic acid (PNA) oligomers as blocking oligonucleotides. Such PNA blocking oligomers are ideal because they are neither degraded nor extended by polymerases.

[0244] In some embodiments, the at least one target marker is pre-amplified using a DNA polymerase. As used herein, the term "DNA polymerase" refers to an enzyme that catalyzes the synthesis of polydeoxyribonucleotides from triphosphate monodeoxyribonucleotides (dNTPs), which performs the most basic functions in DNA replication, repair, and in some cases cell differentiation.

[0245] Examples of DNA polymerases in prokaryotes include DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, and DNA polymerase V. It is known that there are DNA polymerases I, II, and III in Escherichia coli (E. coli). DNA polymerase III appears to be the most important in genome replication. The importance of DNA polymerase I is that it can delete unpaired bases at the end of the growing chain. Retroviruses have a unique DNA polymerase, i.e., reverse transcriptase, which uses an RNA template to synthesize DNA. For eukaryotes, examples of DNA polymerases are polymerases α, β, λ, γ, σ, μ, δ, ε, η, ι, κ, ζ, θ, and Rev1. The DNA polymerases of animal cells are responsible for the replication of DNA in the nucleus and mitochondria.

[0246] The PCR reagents used in the preamplification step can be any commercially available PCR mixture (e.g., KAPA2GFast Multiplex PCR Kit, Universal Probe qPCR Master Mix, EpiTectMethyLight PCR Kit, etc.), which is used to amplify the treated DNA. Alternatively, those skilled in the art can prepare in the laboratory 2+ , dNTP, DNA polymerase and other PCR reagents. Those skilled in the art can also select suitable PCR reaction system and PCR reaction conditions according to actual needs. In some embodiments, the pre-amplification of step (c) includes 5 to 30 reaction cycles, wherein each cycle includes reacting at 85 to 99°C for 5 seconds to 5 minutes, and then reacting at 40 to 80°C for 5 seconds to 5 minutes. In some embodiments, the pre-amplification of step (c) includes 10 to 20 reaction cycles, wherein each cycle includes reacting at 90 to 99°C for 15 seconds to 2 minutes, and then reacting at 45 to 60°C for 30 seconds to 3 minutes. In some embodiments, the pre-amplification of step (c) includes 15 reaction cycles, wherein each cycle includes reacting at 95°C for 30 seconds, and then reacting at 56°C for 60 seconds.

[0247] Step (d)

[0248] In step (d) of the method according to the present application, if step (c) exists, the methylation level of at least one target marker is quantitatively analyzed based on the DNA obtained from step (c); if step (c) does not exist, the methylation level of at least one target marker in the treated DNA obtained from step (b) is quantitatively analyzed. In the present application, step (d) can also be named as a quantitative analysis step.

[0249] As used herein, the term "methylation state" refers to the presence, absence and / or methylation quantity of a specific nucleotide or multiple nucleotides in the DNA region. The methylation state of a specific DNA sequence (for example, target marker as described herein) can indicate the methylation state of each base in the sequence, or can indicate the methylation state of a subset of the base pair in the sequence (for example, the methylation state of cytosine residues or the methylation state of one or more specific restriction enzyme recognition sequences), or can indicate the information of regional methylation density in the sequence, although accurate information on where methylation occurs in the sequence can not be provided. Methylation state can optionally be represented or indicated by "methylation level". Methylation level can be determined by, for example, quantitative analysis of the amount of the complete DNA present after restrictive digestion with a methylation-sensitive restriction enzyme. In this example, if quantitative PCR is used to perform quantitative analysis on the specific sequence in the DNA, the amount of template DNA approximately equals the control of the simulation treatment, which shows that the sequence is not highly methylated, and the amount of template is significantly less than the amount of template in the sample of the simulation treatment, which shows that methylated DNA exists in the sequence. Therefore, the methylation level as in the above example represents the methylation state and can therefore be used as a quantitative indicator of the methylation state. This is particularly useful when the methylation state of a sequence in a sample needs to be compared to a threshold level.

[0250] The methylation status of one or more specific CpG methylation sites (each having two CpG dinucleotide sequences) within a DNA sequence includes "unmethylated", "fully methylated" and "hemimethylated". The term "hemimethylation" refers to the methylation state of double-stranded DNA in which only one strand is methylated. The term "hypermethylation" refers to the average methylation state corresponding to the increase in the number of 5-methylcytosines at one or more CpG dinucleotides in the DNA sequence of the detected DNA sample relative to the number of 5-methylcytosines at the corresponding CpG dinucleotides in the normal control DNA sample. The methylation state of a residue can be a qualitative reading or a quantitative reading, for example, represented by a methylation level. In the present application, the terms "methylation state" and "methylation level" can be used interchangeably. According to the present application, more than one different methylation levels can be determined simultaneously.

[0251] As described herein, if step (c) exists, the methylation level of at least one (e.g., each) target marker is quantitatively analyzed separately based on the DNA obtained from step (c); if step (c) does not exist, the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b) is quantitatively analyzed separately, wherein the at least one target marker comprises one or more markers selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5. In some embodiments, the at least one target marker comprises 5 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3 and BCAN. In some embodiments, the at least one target marker comprises 5 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3 and IRF4. In some embodiments, the at least one target marker comprises at least two, three, four, five, six or seven target markers selected from the group consisting of Septin9, BCAT1, IKZF1, NDRG4, BCAN, VAV3, IRF4 or any combination thereof. The detailed description of "target marker" in the subsection of step (c) above (including but not limited to the definition of target markers, specific combinations of target markers, etc.) also applies to "at least one target marker in the treated DNA obtained from step (b)" recorded in step (d) (for the absence of step (c)). The methylation level / state of one or more CpG dinucleotide sequences within a DNA sequence (e.g., a target marker) can be determined by various analytical methods known in the art.

[0252] In some embodiments, the quantitative analysis of step (d) is performed by polymerase chain reaction (e.g., real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, mass-based separation (e.g., electrophoresis, mass spectrometry), or target capture (e.g., hybridization, microarray).

[0253] In some embodiments, if step (c) is present, the methylation level of at least one target marker is separately and quantitatively analyzed based on the DNA obtained from step (c) by using MSP (see Herman, supra). For example, by using one or more primers that specifically hybridize to unconverted sequences under medium and / or high stringency conditions, an amplification product is generated only when the template contains a methylated cytosine at a CpG site.

[0254] In some embodiments, the quantitative analysis of step (d) is performed by real-time PCR. Non-limiting examples of real-time PCR include Heavy Methyl PCR described by Cottrell et al., Nucl. Acids Res. 32: e10, 2003. TM PCR; MethyLight described by Eads et al., Cancer Res. 59:2302-2306, 1999 TM PCR; Headloop PCR described by Rand et al., Nucl. Acids Res. 33:e 127, 2005.

[0255] As used herein, the term "Heavy Methyl TM "PCR" refers to a real-time PCR technique recognized in the art in which one or more non-extendable nucleic acid (e.g., oligonucleotide) blocks bind to bisulfite-treated nucleic acid in a methylation-specific manner (i.e., the blockers bind specifically to non-mutated DNA under moderate to high stringency conditions). An amplification reaction is performed using one or more primers, which may optionally be methylation-specific but are flanked by one or more blockers. In the presence of non-methylated nucleic acid (i.e., non-mutated DNA), the blockers bind and no PCR product is produced. Using TaqMan® essentially as described, for example, in Holland et al., Proc. Natl. Acad. Sci. USA, 88: 7276-7280, 1991 TM Analytical method, the methylation level of nucleic acid in the sample is determined.

[0256] As used herein, the term "MethyLight TM PCR refers to a fluorescence-based real-time PCR technique recognized in the art, which uses a technique called TaqMan TM The TaqMan probe is a dual-labeled fluorescent oligonucleotide probe designed to hybridize to a CpG-rich sequence located between the forward and reverse amplification primers. TM The probe consists of a fluorescent "reporter moiety" and a "quencher moiety" covalently bound to the TaqMan TMThe linker portion (e.g., phosphoramidite) to which the nucleotides of an oligonucleotide are attached. During PCR amplification, TaqMan TM The probe is cleaved by the 5' nuclease activity of Taq polymerase, thereby generating a signal that is detected in real time during the PCR reaction. In this method, molecular beacons can be used as detectable probes, and the system is independent of the 5'-3' exonuclease activity of the DNA polymerase used (see Mhlanga and Malmberg, Methods 25:463-471, 2001).

[0257] As used herein, the term "Headloop PCR" refers to an art-recognized real-time PCR that selectively amplifies a target nucleic acid but suppresses the amplification of non-amplified target variants by extending the 3' stem loop to form a hairpin structure that can no longer provide amplification template.

[0258] In some embodiments, the real-time PCR is multiplex real-time PCR.

[0259] As used herein, the term "multiplex" may refer to an assay or other analytical method that can simultaneously determine the presence and / or amount of multiple markers (e.g., multiple nucleic acid sequences) by using more than one marker, each marker having at least one different detection characteristic, such as a fluorescence characteristic (e.g., excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum) or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.

[0260] In some embodiments, the quantitative analysis of step (d) is performed by nucleic acid sequencing. Exemplary methods of nucleic acid sequencing are known in the art, see, for example, Frommer et al., Proc. Natl. Acad. Sci. USA 89: 1827-1831, 1992; Clark et al., Nucl. Acids Res. 22: 2990-2997, 1994. For example, by comparing the sequence obtained from a sample not treated with bisulfite or the known nucleotide sequence of the target region with the sequence obtained from a sample treated with bisulfite, it is helpful to identify methylated cytosine in the DNA sequence. Compared with the untreated sample, the thymine residue detected at any cytosine site in the bisulfite-treated sample can be considered as a mutation caused by bisulfite treatment, that is, there is a methylated cytosine at the site.

[0261] Methods for sequencing DNA are known in the art and include, for example, the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2 nd Ed., CSHP, New York 1989)), pyrosequencing (see Uhlmann et al., Electrophoresis, 23:4072-4079, 2002), solid phase pyrosequencing (see Landegren et al., Genome Res., 8(8):769-776, 1998), solid phase minisequencing (see, for example, Southern et al., Genomics, 13:1008-1017, 1992), minisequencing using FRET (see, for example, Chen and Kwok, Nucleic Acids Res. 25:347-353, 1997), ligation sequencing or ultra-deep sequencing (see Marguiles et al., Nature 437(7057):376-80 (2005)).

[0262] In some embodiments, the quantitative analysis of step (d) is performed by mass-based separation (eg, electrophoresis, mass spectrometry).

[0263] For example, the presence of methylated cytosine residues can be detected by combined bisulfite restriction analysis (COBRA), substantially as described by Xiong and Laird, Nucl. Acids Res., 25:2532-2534, 2001. This method takes advantage of the difference in restriction enzyme recognition sites between methylated and unmethylated nucleic acids after treatment with a compound (e.g., bisulfite) that can selectively mutate unmethylated cytosine residues. For example, the restriction endonuclease Taq1 cuts the sequence TCGA, which will be TTGA after bisulfite treatment of unmethylated nucleic acids and will therefore not be cut. The digested and / or undigested nucleic acids are then detected using detection means known in the art, such as electrophoresis and / or mass spectrometry.

[0264] As another example, after treatment with a compound that selectively mutates unmethylated cytosine residues, different techniques are used to detect nucleic acid differences in amplification products based on differences in nucleotide sequence and / or secondary structure, such as methylation-specific single-stranded conformation analysis (MS-SSCA) (Bianco et al., Hum. Mutat., 14:289-293, 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton, Methods Enzymol., 212:71-74, 1992) and methylation-specific denaturing high performance liquid chromatography (MS-DHPLC) (Deng et al., Chin. J. Cancer Res., 12:171-191, 2000).

[0265] In some embodiments, the quantitative analysis of step (d) is performed by target capture (eg, hybridization, microarray).

[0266] Suitable detection methods by hybridization are known in the art, such as Southern, dot blot, slot blot or other nucleic acid hybridization methods (Kawai et al., Mol. Cell. Biol. 14:7421-7427, 1994; Gonzalgo et al., Cancer Res. 57:594-599, 1997). In some embodiments, the probes used for hybridization analysis are detectably labeled. In some embodiments, nucleic acid-based probes used for hybridization analysis are unlabeled. Such unlabeled probes can be fixed on a solid support such as a microarray and can hybridize with a detectably labeled target nucleic acid molecule.

[0267] An example of a microarray is a methylation-specific microarray, which can be used to distinguish between sequences with converted cytosine residues and sequences with unconverted cytosine residues (see Adorjan et al., Nucl. Acids Res., 30: e21, 2002). Hybridization-based analysis can also be used for nucleic acids treated with methylation-sensitive restriction enzymes.

[0268] As another example, the methylation status of CpG dinucleotide sequences within a DNA sequence can be determined by oligonucleotide probes that hybridize with PCR amplification primers simultaneously with bisulfite-treated DNA (wherein the primers can be methylation-specific primers or standard primers).

[0269] In some embodiments, step (d) is performed in the presence of a detection reagent. As used herein, the term "detection reagent" is a reagent used to detect the presence, absence or amount of nucleic acid in a quantitative analysis step.

[0270] Various detection agents known in the art can be used in the present application. In some embodiments, the detection agent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.

[0271] In some embodiments, the fluorescent probe is selected from SEQ ID NOs: 57-85, 172 shown in Table 2 below.

[0272] In some embodiments, the 5' end of the fluorescent probe is labeled with a fluorescent dye (eg, FAM, HEX / VIC, TAMRA, Texas Red, or Cy5), and the 3' end is labeled with a quencher (eg, BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA).

[0273] Labeling can be accomplished by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the reagent. Indirect labeling involves the binding (covalently or non-covalently) of a second reagent to a first reagent. The second reagent should specifically bind to the first reagent. The second reagent can be coupled to a suitable label and / or the second reagent is the target (receptor) of a third reagent that can bind to the second reagent. The use of secondary, tertiary or even higher order reagents will usually increase signal intensity. Suitable secondary and higher order reagents can include antibodies, secondary antibodies and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). Reagents or substrates can also be "labeled" by one or more tags known in the art.

[0274] In some embodiments, if step (c) is present, the quantitative analysis of step (d) comprises amplifying the DNA obtained in step (c) using a quantitative primer pair and a DNA polymerase, wherein at least a portion of the obtained DNA is amplified. In some embodiments, if step (c) is absent, the quantitative analysis of step (d) comprises amplifying at least one target marker in the treated DNA obtained in step (b) using a quantitative primer pair and a DNA polymerase.

[0275] As used herein, the term "quantification primer pair" refers to one or more primer pairs used in the quantitative analysis step.

[0276] In some embodiments, if step (c) is present, the quantitative primer pair used in step (d) can hybridize with at least 9 consecutive nucleotides of the DNA obtained in step (c) under stringent conditions, medium stringent conditions, or highly stringent conditions. In some embodiments, if step (c) is not present, the quantitative primer pair used in step (d) can hybridize with at least 9 consecutive nucleotides of at least one target marker in the treated DNA obtained in step (b) under stringent conditions, medium stringent conditions, or highly stringent conditions. In some embodiments, if step (c) is present, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28 or more) methylation-specific primer pair used in step (d) is the same as at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28 or more) methylation-specific primer pair in the pre-amplification primer pool of step (c).

[0277] In some embodiments, if step (c) is absent, the quantitative primer pair used in step (d) is designed to amplify at least a portion of at least one target marker in the treated DNA obtained in step (b). In some embodiments, if step (c) is present, the quantitative primer pair used in step (d) is designed to amplify at least a portion of the DNA obtained in step (c), that is, step (c) and step (d) are designed as nested PCR.

[0278] Nested PCR is a modification of PCR that aims to increase sensitivity and specificity. Nested PCR involves the use of two primer sets and two consecutive PCR reactions. A first round of amplification is performed to produce a first amplicon, and a second round of amplification is performed using a primer pair in which one or both primers anneal to sites within the region defined by the initial primer pair, i.e., the second primer pair is considered to be "nested" within the first pair of primers. In this way, background amplification products from the first PCR reaction that do not contain the correct internal sequence are no longer amplified further in the second PCR reaction.

[0279] In some embodiments, if step (c) exists, the quantitative analysis of step (d) includes determining the methylation level of at least one (e.g., each) target marker based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in the DNA obtained in step (c). In some embodiments, if step (c) does not exist, the quantitative analysis of step (d) includes determining the methylation level of at least one (e.g., each) target marker based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in at least one target marker in the treated DNA obtained in step (b). In some embodiments, if step (c) exists, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in the DNA obtained in step (c). In some embodiments, if step (c) does not exist, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in at least one target marker in the treated DNA obtained in step (b). In some embodiments, if step (c) exists, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more TpG dinucleotides in the DNA obtained in step (c). In some embodiments, if step (c) does not exist, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more TpG dinucleotides in at least one target marker in the treated DNA obtained in step (b). In some embodiments, if step (c) exists, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpA dinucleotides in the DNA obtained in step (c). In some embodiments, if step (c) does not exist, the quantitative analysis of step (d) includes determining the methylation level of cytosine residues based on the presence or level of one or more CpA dinucleotides in at least one target marker in the treated DNA obtained in step (b).

[0280] In some embodiments, if step (c) exists, the quantitative analysis of step (d) is carried out by dividing the DNA obtained in step (c) into multiple parts. In some embodiments, if step (c) does not exist, the quantitative analysis of step (d) is carried out by dividing at least one target marker in the treated DNA obtained in step (b) into multiple parts. In some embodiments, multiple parts are subjected to multiple different quantitative analysis experiments, wherein the DNA obtained from step (c) (or at least one target marker in the treated DNA obtained from step (b)) is quantitatively analyzed in one of the multiple parts, if present in a part, it is quantified in one of the multiple parts. In some embodiments, the control marker in each part is quantitatively analyzed.

[0281] Step (e)

[0282] In step (e) of the method for diagnosing colorectal tumor, screening for colorectal tumor formation or risk of formation, or assessing the progression or prognosis of colorectal tumor in an individual, the methylation level of at least one (e.g., each) target marker of step (d) is compared with the corresponding reference level, respectively, wherein one or more target markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual has colorectal tumor, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased likelihood of developing or developing colorectal tumor, or the individual has a poor prognosis or a risk of poor prognosis of colorectal tumor.

[0283] In step (e) of the method for monitoring the response of an individual who is being treated for colorectal tumor to treatment, the methylation level of at least one (e.g., each) target marker in step (d) is compared with the corresponding methylation level of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation levels are quantitatively analyzed by repeating steps (a), (b), optionally (c), and (d) on a biological sample containing DNA obtained from the individual before treatment, wherein a lower methylation level of one or more target markers than its corresponding methylation level before treatment indicates that the individual has responded to treatment.

[0284] Step (e) of the method according to the present application may also be referred to as a comparison step.

[0285] The term "comparison" as used herein refers to the comparison of the methylation level of at least one (e.g., each) target marker contained in the detected biological sample obtained by the quantitative analysis step with its corresponding reference level. It should be understood that the terms used herein refer to the comparison of corresponding parameters or values, for example, comparing the absolute amount with the absolute reference amount, comparing the concentration with the reference concentration, or comparing the intensity signal obtained from the detected sample with the intensity signal of the same type of the reference sample. The comparison can be performed manually or computer-assisted. For the comparison performed by computer-assisted, the value of the determined amount can be compared with the value of the appropriate reference stored in the database by the computer program. The computer program can further evaluate the results of the comparison and automatically provide the desired evaluation in a suitable output format. Based on the comparison of the methylation level of at least one (e.g., each) target marker in the quantitative analysis step with the corresponding reference level, individuals with colorectal tumors, or with the risk of colorectal tumor formation or formation, or with the possibility of colorectal tumor development or development, or with poor prognosis or poor prognosis of colorectal tumors can be identified; the response of the individual to treatment can also be monitored in individuals receiving colorectal tumor treatment.

[0286] As used herein, the term "reference level" refers to a threshold level that includes or excludes an individual from colorectal tumor or the development or risk of developing a colorectal tumor, or refers to a threshold level used to monitor the individual's response to treatment in an individual receiving treatment for a colorectal tumor.

[0287] For example, for a method of diagnosing colorectal tumors in an individual, screening for the risk of colorectal tumor formation or formation, or assessing the progress or prognosis of colorectal tumors, if the methylation level of one or more target markers in the test sample is equal to or higher than its corresponding reference level, the individual may be considered to have colorectal tumors, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased possibility of colorectal tumor development or development, or the individual has a poor prognosis or a poor prognosis of colorectal tumors. In some embodiments, the methylation level of one or more target markers in the test sample is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more of its corresponding reference level. In the present application, in order to diagnose colorectal tumors in an individual, screen for the risk of colorectal tumor formation or formation, or assess the progress or prognosis of colorectal tumors, it is not necessary for the methylation level of each target marker to be equal to or higher than its corresponding reference level. To be precise, it is sufficient if the methylation level of at least one target marker quantitatively analyzed in the quantitative analysis step is equal to or higher than its corresponding reference level.

[0288] For another example, for a method of monitoring the response of the individual to treatment in an individual receiving colorectal tumor treatment, if the methylation level of one or more target markers of the test sample is lower than the corresponding methylation level before treatment, the individual can be considered to have responded to the treatment. In some embodiments, the methylation level of one or more target markers in the biological sample obtained after colorectal tumor treatment is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% lower than the corresponding methylation level before colorectal tumor treatment. In the present application, in order to monitor the response of the individual to treatment in an individual receiving colorectal tumor treatment, it is not necessary for the methylation level of each target marker to be lower than the corresponding reference level before the individual receives colorectal tumor treatment. To be precise, it is sufficient if the methylation level of at least one target marker quantitatively analyzed in the quantitative analysis step is lower than the corresponding reference level before the individual receives colorectal tumor treatment.

[0289] The reference level of methylation of the target marker can be derived from one or more reference samples, wherein the reference level is obtained from an experiment performed in parallel with the experiment of detecting the target sample. Alternatively, the reference level can be obtained in a database, which includes a collection of data, standards or levels from one or more reference samples or disease reference samples. In some embodiments, the collection of such data, standards or levels is standardized so that it can be used for the purpose of comparing with the data from one or more samples. "Standardization" is the process of converting the measured raw data into data that can be directly compared with other standardized data. Standardization is used to overcome analytical method-specific errors caused by different factors in different analytical methods, such as differences in sample loading, differences in binding efficiency, differences in detection sensitivity and other types of errors.

[0290] In some embodiments, the reference database includes methylation levels of target markers and / or other laboratory and clinical data from one or more reference samples. In some embodiments, the reference database includes methylation levels of target markers, each of which is standardized as a percentage of the methylation level of a control marker detected under the same conditions as the reference sample. In order to compare with such standardized methylation levels of the target marker, the methylation level of the target marker of the test sample is also measured and calculated as a percentage of the methylation level of the control marker detected under the same conditions as the test sample.

[0291] In some embodiments, a reference database is established by aggregating reference level data of reference samples obtained from healthy individuals and / or non-tumor individuals (i.e., individuals known to be tumor-free). In some embodiments, a reference database is established by aggregating reference level data of reference samples obtained from individuals being treated for colorectal tumors. In some embodiments, a reference database is established by aggregating data of reference samples obtained from individuals at different stages of colorectal tumors, where the different stages of colorectal tumors are demonstrated by, for example, different methylation levels of target markers.

[0292] Those skilled in the art can select the reference level according to the desired sensitivity and specificity. The means for determining a suitable reference level are known to those skilled in the art, for example, the reference level can be determined from data collected in clinical studies.

[0293] In some embodiments, the reference level of step (e) is determined based on clinical samples obtained from a group of individuals having or at risk of colorectal tumor and clinical samples obtained from a group of individuals who do not have or are not at risk of colorectal tumor.

[0294] A person skilled in the art can determine whether an individual suffers from colorectal tumor or has a risk of suffering from colorectal tumor based on various factors, such as age, gender, medical history, family history, symptoms, etc.

[0295] In some embodiments, the methylation level and reference level of the target marker are represented by a cycle threshold value (i.e., Ct value). As used herein, the term "Ct value" refers to the number of cycles at which the fluorescence of the PCR product can be detected above the background signal. The Ct value is inversely proportional to the number of target markers in the sample, i.e., the lower the Ct value, the greater the number of target markers in the sample.

[0296] For example, in step (e) of a method for diagnosing colorectal tumors, screening for the formation or risk of colorectal tumor formation, or assessing the progression or prognosis of colorectal tumors in an individual, the Ct value of at least one target marker is the same or lower relative to its corresponding reference Ct value, indicating that the individual suffers from colorectal tumors, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased likelihood of colorectal tumor development or development, or the individual has a poor prognosis or risk of poor prognosis of colorectal tumors. In some embodiments, if the Ct value of at least one of the multiple target markers of step (d) is 2-10 cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles) lower than its corresponding reference Ct value, it is determined that the individual suffers from colorectal tumors, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased likelihood of colorectal tumor development or development, or the individual has a poor prognosis or risk of poor prognosis of colorectal tumors.

[0297] As used herein, the term "increased likelihood" refers to an overall increase of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of likelihood that an individual will develop colorectal tumor or have a poor prognosis for colorectal tumor compared to the individual from whom the reference sample was obtained.

[0298] For another example, in step (e) of the method for monitoring the response of the individual to treatment in an individual receiving colorectal tumor treatment, the Ct value of the target marker in step (d) is compared with the reference Ct value, wherein the Ct value of at least one target marker is higher than the corresponding Ct value before treatment, indicating that the individual has responded to the treatment. In some embodiments, if the Ct value of at least one of the multiple target markers in step (d) is 2-10 cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles) higher than the corresponding reference Ct value before treatment, it is determined that the individual has responded to the treatment of colorectal tumor.

[0299] 2. Kit

[0300] In another aspect, the present application also provides a kit for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or evaluating the progression or prognosis of colorectal tumors, comprising:

[0301] (a) a first reagent for treating DNA, wherein the first reagent is capable of distinguishing between unmethylated sites and methylated sites in the DNA;

[0302] (b) optionally, a first primer library comprising at least one primer pair for preamplifying at least one target sequence of at least one target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5, wherein the at least one primer pair can hybridize to at least 9 consecutive nucleotides of the at least one target sequence after treatment with the first reagent under stringent conditions, moderately stringent conditions, or highly stringent conditions, wherein the target sequence comprises at least one CpG site; and

[0303] (c) a second reagent, wherein if the first primer library exists, the second reagent is used to quantitatively analyze the methylation level in the at least one (e.g., each) target marker pre-amplified by the first primer library; if the first primer library does not exist, the second reagent is used to quantitatively analyze the methylation level of at least one (e.g., each) target marker in the DNA treated with the first reagent, wherein the at least one target marker comprises one or more markers selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5.

[0304] In some embodiments, the at least one target marker comprises a plurality of target markers, wherein the plurality of target markers comprises at least two (eg, two, three) markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0305] In some embodiments, if the first primer library exists, the second reagent comprises a second primer library, wherein the second primer library comprises a plurality of quantitative primer pairs capable of hybridizing with at least 9 consecutive nucleotides of the at least one target sequence pre-amplified by the first primer library under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0306] In some embodiments, if the first primer library does not exist, the second reagent includes a third primer library, which contains a plurality of quantitative primer pairs capable of hybridizing with at least 9 consecutive nucleotides of at least one target sequence in at least one target marker in the DNA treated with the first reagent under stringent conditions, moderately stringent conditions, or highly stringent conditions.

[0307] In some embodiments, if the first primer library exists, at least one quantitative primer pair in the second primer library is the same as at least one primer pair in the first primer library. In some embodiments, if the first primer library exists, the quantitative primer pair in the second primer library is designed to be used to amplify at least a portion of the at least one target sequence pre-amplified by the first primer library. In some embodiments, if the first primer library does not exist, the quantitative primer pair of the third primer library is designed to be used to amplify at least a portion of at least one target sequence of at least one target marker in the DNA treated with the first reagent. In some embodiments, the first, second or third primer library contains at least one methylation-specific primer pair.

[0308] In some embodiments, the first primer pool and the second primer pool are packaged in a single container or in separate containers. In some embodiments, the kit further comprises one or more blocking oligonucleotides.

[0309] In some embodiments, the kit further comprises a detection reagent. In some embodiments, the detection reagent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NO: 57-85, 172. In some embodiments, the 5' end of the fluorescent probe is labeled with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5), and the 3' end is labeled with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, TAMRA, or lowa Black Dark Quenchers).

[0310] In some embodiments, the kit further comprises a DNA polymerase and / or a container suitable for storing the biological sample obtained from the individual. In some embodiments, the kit further comprises instructions for use and / or an explanation of the test results of the kit.

[0311] In some embodiments, the kit may include a reaction buffer packaged in a separate container, the reaction buffer being optimized for primer extension mediated by a polymerase (e.g., PCR). Preferred is a kit that further includes a container suitable for containing a device for determining the methylation of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) target marker selected from the group consisting of Septin9, BCAT1, IK ZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, intergenic spacer 1, TMEFF2, intergenic spacer 4, NKX2-6, intergenic spacer 5, SLC24A2, NDRG4, intergenic spacer 2, intergenic spacer 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0312] In some embodiments, the first reagent comprises a bisulfite reagent or a methylation sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite ions, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, enzyme, ScrFI enzyme, BbeI enzyme, NotI enzyme, SmaI enzyme, XmaI enzyme, MboI enzyme, BstBI enzyme, ClaI enzyme, MluI enzyme, NaeI enzyme, NarI enzyme, PvuI enzyme, SacII enzyme, HhaI enzyme, and any combination thereof.

[0313] In some embodiments, the first primer library comprises at least one methylation-specific primer pair for pre-amplifying at least one target sequence of at least one target marker selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5.

[0314] In some embodiments, the at least one target marker comprises one or more markers (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 markers) selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5.

[0315] In some embodiments, the at least one target marker may be at most one target marker (i.e., one marker and no more than one marker). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, the at least one target marker is intergenic region 1.

[0316] In some embodiments, the at least one target marker comprises a plurality of target markers. In some embodiments, the plurality of target markers comprises at least two or three markers selected from the group consisting of Septin9, BCAT1 and IKZF1. In some embodiments, the plurality of target markers of the present application further comprises one, two, three, four or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4 and IRF4. In some embodiments, the multiple target markers of the present application further include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the following group: POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11 and CRHBP.

[0317] In some embodiments, the plurality of target markers described herein comprise Septin9 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0318] In some embodiments, the plurality of target markers described herein comprises BCAT1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0319] In some embodiments, the plurality of target markers described herein comprises IKZF1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0320] In some embodiments, the plurality of target markers described herein comprises BCAN and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, VAV3, and / or IRF4.

[0321] In some embodiments, the plurality of target markers described herein comprise VAV3 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, NDRG4, and / or IRF4.

[0322] In some embodiments, the plurality of target markers described herein comprises IRF4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or NDRG4.

[0323] In some embodiments, the plurality of target markers described herein comprises PKNOX2 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0324] In some embodiments, the plurality of target markers described herein comprises NDRG4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0325] In some embodiments, each target marker includes or is: a) each region defined by Hg19 coordinates as shown below:

[0326]

[0327]

[0328] and 5 kb upstream of each start site and 5 kb downstream of each end site of each of the above regions; or b) the corresponding region of a) after bisulfite conversion; or c) the corresponding region of a) after methylation sensitive restriction enzyme (MSRE) treatment.

[0329] In some embodiments, if the first primer library exists, the first primer library comprises at least one primer pair, and the at least one primer pair comprises or consists of at least one pair of nucleotide sequences shown in Table 2 below selected from the group consisting of SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171, optionally wherein the second primer library comprises at least one primer pair that is identical to at least one primer pair in the first primer library.

[0330] In some embodiments, if the first primer library does not exist, the third primer library comprises at least one primer pair, and the at least one primer pair comprises or consists of at least one pair of nucleotide sequences shown in Table 2 below selected from the group consisting of SEQ ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171.

[0331] In some embodiments, the first primer library, the second primer library, or optionally the third primer library further comprises a primer pair for amplifying a control marker. In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0332] In some embodiments, the kit further comprises a plurality of containers, each container being used to receive a component of the second primer library.

[0333] In some embodiments, the kit further comprises standard reagents that can be used to perform CpG position-specific methylation analysis, wherein the analysis comprises one or more of the following techniques: MS-SNuPE, MSP, MethyLight TM 、HeavyMethylTM , COBRA and nucleic acid sequencing.

[0334] In some embodiments, the kit may comprise additional reagents selected from the group consisting of buffers (eg, restriction enzyme, PCR, storage or wash buffers), DNA recovery reagents or kits (eg, precipitation, ultrafiltration, affinity columns), and DNA recovery components.

[0335] In some embodiments, the kit of the present application may include:

[0336] a) bisulfite reagent;

[0337] b) optionally, a first primer library comprising a plurality of methylation-specific primer pairs for preamplifying at least two target sequences in a plurality of target markers comprising at least 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, PO U4F2, SALL1, PKNOX2, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, NDRG4, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11 and CRHBP, wherein the methylation-specific primer pair comprises or consists of at least two pairs of nucleotide sequences selected from the group consisting of: SEQ ID NO: 1 ID NO: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54 and 170 / 171;

[0338] c) a second reagent, wherein if the first primer library exists, the second reagent is used to quantitatively analyze the methylation level of at least one (e.g., each) of the plurality of target markers pre-amplified by the first primer library, wherein the second reagent comprises a second primer library, the second primer library comprises a plurality of quantitative primer pairs, and the quantitative primer pairs are capable of hybridizing with at least 9 consecutive nucleotides of the plurality of target sequences pre-amplified by the first primer library under stringent conditions, medium stringent conditions, or highly stringent conditions; if the first primer library does not exist, the second reagent is used to quantitatively analyze the methylation level of at least one (e.g., each) target marker in the DNA treated with the first reagent, wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, , IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5, wherein the second reagent comprises a third primer library, the third primer library comprises a plurality of quantitative primer pairs, and the quantitative primer pairs are capable of hybridizing to at least 9 consecutive nucleotides of at least one target sequence in at least one target marker in the DNA treated with the first reagent under stringent conditions, moderately stringent conditions or highly stringent conditions.

[0339] The kit of the present application may also include other components packaged in separate containers, such as buffers or solutions suitable for blocking, washing or coating.

[0340] The kit of the present application may further include one or more of the following components known in the field of DNA enrichment: a protein component that selectively binds to methylated DNA; a triplex-forming nucleic acid component, one or more linkers, optionally in a suitable solution; a substance or solution for ligation, such as a ligase, a buffer; a substance or solution for column chromatography; a substance or solution for immunologically-based enrichment (such as immunoprecipitation); a substance or solution for nucleic acid amplification, such as PCR; a dye or several dyes, if suitable for a coupling agent, if suitable in a solution; a substance or solution for hybridization; and / or a substance or solution for a washing step.

[0341] 3. Purpose

[0342] On the other hand, the present application provides the use of the kit of the present application in the manufacture of a diagnostic kit for diagnosing colorectal tumors in individuals, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors, or monitoring the response of individuals receiving colorectal tumor treatment to treatment.

[0343] In another aspect, the present application provides the use of a reagent for quantitatively analyzing the methylation level of a target marker in the manufacture of a kit, wherein the kit is used in a method for diagnosing colorectal tumors, screening for colorectal tumor formation or the risk of formation, or assessing the progression or prognosis of colorectal tumors in an individual, wherein the method comprises the following steps:

[0344] (a) obtaining a biological sample containing DNA from the individual;

[0345] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG sites in the DNA, thereby obtaining treated DNA;

[0346] (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one (e.g., each) target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0347] (d) if step (c) exists, then based on the DNA obtained from step (c), respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0348] (e) comparing the methylation level of at least one (e.g., each) target marker in step (d) with the corresponding reference level, respectively, wherein the same or higher methylation level of one or more target markers relative to their corresponding reference levels indicates that the individual suffers from colorectal tumor, or the individual has a risk of colorectal tumor formation or formation, or the individual has an increased likelihood of colorectal tumor development or development, or the individual has a poor prognosis or a risk of poor prognosis of colorectal tumor.

[0349] In another aspect, the present application provides use of a reagent for quantitatively analyzing the methylation level of a target marker in the manufacture of a kit, wherein the kit is used in a method for monitoring the response of an individual to treatment in an individual receiving colorectal tumor treatment, wherein the method comprises the following steps:

[0350] (a) obtaining a biological sample containing DNA from the individual;

[0351] (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG sites in the DNA, thereby obtaining treated DNA;

[0352] (c) pre-amplifying at least a portion of at least one (e.g., each) target marker in the treated DNA obtained from step (b) using a pre-amplification primer library, wherein at least a portion of at least one (e.g., each) target marker is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; wherein step (c) is present or absent;

[0353] (d) if step (c) exists, then based on the DNA obtained from step (c), respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker; if step (c) does not exist, then respectively quantitatively analyzing the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5; and

[0354] (e) comparing the methylation level of at least one (e.g., each) target marker in step (d) with the corresponding methylation level of one or more target markers obtained from the same individual before treatment, wherein the corresponding methylation level is quantitatively analyzed by repeating steps (a), (b), optionally (c), and (d) on a biological sample containing DNA obtained from the individual before treatment, wherein a lower methylation level of one or more target markers compared to its corresponding methylation level before treatment indicates that the individual has responded to the treatment.

[0355] In some embodiments, the at least one target marker of step (c) above comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5.

[0356] In some embodiments, the at least one target marker of step (c) above may be at most one target marker (i.e., one marker and no more than one marker). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, the at least one target marker is intergenic region 1.

[0357] In some embodiments, the at least one target marker of step (c) above comprises a plurality of target markers. In some embodiments, the plurality of target markers comprises at least two or three markers selected from the group consisting of Septin9, BCAT1 and IKZF1. In some embodiments, the plurality of target markers of the present application further comprises one, two, three, four or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4 and IRF4. In some embodiments, the plurality of target markers described herein further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, intergenic region 1, TMEFF2, intergenic region 4, NKX2-6, intergenic region 5, SLC24A2, intergenic region 2, intergenic region 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0358] In some embodiments, the plurality of target markers described herein comprise Septin9 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, BCAT1, and / or IKZF1.

[0359] In some embodiments, the at least one (eg, at least 1, 2, or 3) additional target markers comprise BCAN, VAV3, and / or IRF4.

[0360] In some embodiments, the plurality of target markers described herein comprises BCAT1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0361] In some embodiments, the plurality of target markers described herein comprises IKZF1 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0362] In some embodiments, the plurality of target markers described herein comprises BCAN and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, VAV3, NDRG4, IRF4, PKNOX2, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0363] In some embodiments, the plurality of target markers described herein comprise VAV3 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0364] In some embodiments, the plurality of target markers described herein comprises IRF4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0365] In some embodiments, the plurality of target markers described herein comprises PKNOX2 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0366] In some embodiments, the plurality of target markers described herein comprises NDRG4 and at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target marker selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, or 7) additional target markers include Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4. DETAILED DESCRIPTION

[0367] The biological materials used in all the examples, including various cloning and expression plasmids, culture media, enzymes, buffers, various culture methods, protein extraction and purification methods, and other molecular biology procedures, are well known to those skilled in the art. For more details, please refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY and Short Protocols in Molecular Biology by Frederick M. Ausubel et al. (translated by Yan Ziying et al., Science Press (Beijing), 1998).

[0368] Example 1: Validation of methylation-specific primers

[0369] For the initial proof of concept, the inventors selected bisulfite-converted reference DNA to evaluate primer / probe specificity. Custom primer / probe combinations were designed for 28 target markers (i.e., NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and 5 intergenic regions, including intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4, and intergenic region 5). In the proof of concept experiment, the inventors prepared a mixture of DNA fully methylated at all CpG sites and DNA fully unmethylated (ratios of 10%, 25%, 50%, 100%), with a total input of 4 ng. By using primers and probes (sequences are shown in Table 2), 28 target markers in the above mixture were evaluated (three replicates were set). The experimental method is described in detail as follows.

[0370] Bisulfite-converted fully methylated DNA and bisulfite-converted completely unmethylated DNA were purchased from Qiagen (EpiTect Control DNA) and mixed to provide DNA mixtures containing 100%, 50%, 25%, and 10% of fully methylated DNA in completely unmethylated DNA, respectively, with a total DNA amount of 4 ng in each DNA mixture.

[0371] The methylation-specific primer pairs (see Table 2) and primers for 28 target markers (i.e., NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and 5 intergenic regions (including intergenic region 1, In the presence of a detection probe (see Table 2) with specificity for intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5), the DNA mixture is amplified by PCR reaction. In the PCR reaction, the control marker ACTB is also amplified by methylated non-specific primers (see Table 2) and detection probes (see Table 2). In a separate detection analysis, each of the 28 target markers and a control marker are amplified separately. The detection probes of different markers are labeled with different fluorescent tags (FAM, HEX, VIC, TAMRA, Texas Red or Cy5) and corresponding quenchers (BHQ1, BHQ2, BHQ3, DABCYL or TAMRA). In the PCR reaction system, the final concentration of each primer is 500nM, and the final concentration of each detection probe is 200nM.

[0372] Table 2. Primer pair sequences and probe sequences for each target marker

[0373]

[0374]

[0375]

[0376]

[0377] Prepare a PCR reaction system, which includes: 10 μL of DNA mixture (4 ng DNA), 2.5 μL of the premix of the above primers and probes; PCR reagents ( Universal Probe qPCR Master Mix (NEB)) 12.5μL.

[0378] The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 56°C for 40 seconds (collecting fluorescence), and 50 cycles. Different fluorescence was detected in the corresponding fluorescence channels using the ABI 7500 Real-Time PCR System.

[0379] result

[0380] The Ct (cycle threshold) value was calculated for each PCR reaction and the Ct values ​​of the PCR reactions for each marker of the different DNA mixtures were analyzed. For each marker tested, it was found that the Ct values ​​provided by the methylation-specific primer pairs used in the PCR reaction decreased proportionally with the increase in the percentage of converted methylated DNA in the DNA mixture. For all tested markers, the percentage of methylated templates and the expected Ct values ​​were highly correlated (correlation coefficient R>0.9 for all tested markers) and linear, indicating that the primers used to preamplify the target markers are methylation-specific. Figure 1 The horizontal shift of the curve A (obtained using methylation-specific primers for PKNOX2) is Figure 1 The correlation was obtained by comparing the overlapping curves of B (obtained with non-methylation-specific primers for the control marker ACTB). Figure 1 A is similar and not shown here.

[0381] Example 2: Comparison of methylation abundance of target markers in different tissues

[0382] To demonstrate the feasibility and specificity of the selected target markers for tumor tissues, we tested 28 markers in colorectal cancer tissues (CRC-tissues), high-grade adenoma tissues (AA-tissues), and paracancerous tissues (para-tissues) of colorectal cancer patients, and white blood cells (WBC) of people with negative colonoscopy were used as controls. The experimental methods are described in detail below.

[0383] The methylation abundance of target markers is detected in DNA samples from different cells and tissues to explore the potential of these target markers in the diagnosis or screening of colorectal tumors. The target markers detected in this embodiment include NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2 and 5 intergenic regions, including intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5.

[0384] The process includes the following steps:

[0385] 1. DNA samples were obtained from leukocytes, paracancerous tissues, high-grade adenoma tissues, and colorectal cancer tissues, with 10 biological samples for each sample (i.e., a total of 40 samples). According to the instructions, leukocyte DNA was extracted using the Qiagen QIAamp DNAMini Kit, and tissue DNA was extracted using the Qiagen QIAamp DNA FFPE Tissue Kit.

[0386] 2. The DNA sample obtained in step 1 above was treated with bisulfite reagent (MethylCode TM The transformed DNA was obtained by using a Bisulfite Conversion Kit.

[0387] 3. Fluorescence PCR was performed on the transformed DNA. Briefly, methylation-specific primer pairs (see Table 2) and primers for 27 target markers (i.e., NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2 and 5 intergenic regions (including The converted DNA obtained in step 2 is amplified by PCR reaction in the presence of detection probes (see Table 2) specific to intergenic spacer 1, intergenic spacer 2, intergenic spacer 3, intergenic spacer 4 and intergenic spacer 5. In the PCR reaction, the control marker ACTB is also amplified by methylated non-specific primers (see Table 2) and detection probes (see Table 2). Different fluorescent labels are added to the detection probes of different markers. In the PCR reaction system, the final concentration of each primer is 500nM, and the final concentration of each detection probe is 200nM.

[0388] Prepare a PCR reaction system, which includes: 10 μL of converted DNA, 2.5 μL of the premix containing the above primers and probes; PCR reagents ( Universal Probe qPCR Master Mix (NEB)) 12.5μL.

[0389] The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 30 seconds, 56°C for 60 seconds (collecting fluorescence), 10 cycles. Different fluorescence was detected in the corresponding fluorescence channels using the ABI 7500 Real-Time PCR System.

[0390] 4. Calculate, summarize and compare the Ct values ​​of samples obtained from leukocytes, paracancerous tissues, high-grade adenoma tissues and colorectal cancer tissues. The Ct value of the wells not measured was set to 50.

[0391] result

[0392] The results showed that the methylation abundance of the target markers of the present application (NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2 and 5 intergenic regions, including intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5) in leukocytes of people with negative colonoscopy was significantly (p<0.01) lower than that in tissue samples of colon cancer patients (see Figure 2 ), taking SALL1 and PKNOX2 as examples. Significant differences were also observed in each of the other detected target markers (p<0.01), the results of which are not shown here. In particular, the methylation abundance of the target markers in paracancerous tissues was lower than that in advanced adenoma tissues and colorectal cancer tissues. This suggests that each of the detected target markers has potential application value in the diagnosis and screening of colorectal tumors by using leukocyte samples.

[0393] Example 3: Quantitative analysis of methylation of target markers using extracellular free DNA

[0394] To validate the clinical performance of methylation markers for CRC plasma samples, we used the method disclosed herein (also referred to as the pre-amplification method) to detect 13 markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and intergenic region 1) in 88 plasma samples clinically diagnosed with CRC and 107 colonoscopy-negative plasma control samples. Of the 88 plasma samples clinically diagnosed with CRC, 15 samples were from individuals diagnosed with CRC stage I, 26 samples were from individuals diagnosed with CRC stage II, 28 samples were from individuals diagnosed with CRC stage III, and 19 samples were from individuals diagnosed with CRC stage IV.

[0395] Pre-amplification methods

[0396] The pre-amplification method includes the following steps:

[0397] 1. Use QIAamp Circulating Nucleic Acid Kit (Qiagen) to obtain extracellular free DNA (cfDNA) samples from 1-4 ml plasma samples.

[0398] 2. Use bisulfite reagent (MethylCode TM 20 ng cfDNA was subjected to bisulfite conversion using a Bisulfite Conversion Kit to obtain converted cfDNA.

[0399] 3. Pre-amplify the converted cfDNA samples. In short, the converted cfDNA obtained from step 2 above was pre-amplified by PCR reaction in the presence of methylation-specific primer pairs (see Table 2), which were specifically designed for NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2 and intergenic region 1. In the PCR reaction system, the final concentration of each primer was 200 nM.

[0400] 25 μL PCR mixture consists of 10 μL converted cfDNA, 2.5 μL premix containing the above primers and 12.5 μL PCR reagent ( Universal Probe qPCR Master Mix (NEB)).

[0401] PCR reaction conditions were as follows: 95°C for 3 minutes; 95°C for 30 seconds, 56°C for 60 seconds, 8 cycles. TM PCR system (Thermo Fisher).

[0402] 4. The product obtained from step 3 above was diluted 10-fold and then used for multiplex fluorescence PCR detection specifically targeting NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2 and intergenic region 1.

[0403] The qPCR mixture consisted of 10 μL of diluted product from step 3, 2.5 μL of primer / probe pool, 12.5 μL of PCR reagent ( Universal Probe qPCR Master Mix (NEB)). The non-CpG ACTB region was used as the internal reference for each reaction well (see Table 2). The detection probes of different markers were labeled with different fluorescent markers. In the PCR reaction system, the final concentration of each primer was 500 nM, and the final concentration of each detection probe was 200 nM.

[0404] The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 56°C for 40 seconds (collecting fluorescence), and 50 cycles. Different fluorescence was detected in the corresponding fluorescence channels using the ABI 7500 Real-Time PCR System.

[0405] result

[0406] The Ct value of the sample without amplification signal is set to 50. A reference Ct value is set for each detection marker. If the Ct value of any detected marker is equal to or lower than its corresponding reference Ct value, the sample is classified as a positive sample. Figure 3 The Ct value distribution of the target markers SALL1 and BCAN in the group with CRC and the group with negative colonoscopy is shown. Figure 3 As shown, the methylation levels of the target markers SALL1 and BCAN in the group with CRC were significantly higher than those in the group with negative colonoscopy (for SALL1 and BCAN, p values ​​were 2.14E-4 and 1.07E-8, respectively). The results of other target markers were similar (p<0.01) and are not shown here.

[0407] Table 3 below shows the comparative results of using 5 target markers (i.e. Septin9, BCAT1, IKZF1, BCAN and VAV3) in the preamplification method. As shown in Table 3, the preamplification method showed ultra-high sensitivity (86.4%) for CRC and high specificity (90.7%) for the group with negative colonoscopy, which is much better than the existing commercial markers, such as Septin9, whose sensitivity for CRC in clinical trial samples is 48.2% (see TR Church et al., Gut.; 63: 317-325 (2014)). Other marker combinations of the 13 target markers were also analyzed (e.g., the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, and NDRG4; the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2; the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and intergenic region 1, etc.), and the results showed that the sensitivity for CRC was not less than 85%, and the specificity for the population with negative colonoscopy was not less than 90%.

[0408] Table 3. Comparison of preamplification method results and colonoscopy results

[0409] Colonoscopy results Accuracy Positive samples Negative samples total Colorectal cancer 86.4％ 76 12 88 Negative colonoscopy 90.7％ 10 97 107

[0410] The sensitivity of the preamplification method and the Septin 9 method alone in CRC classification was also compared. The Septin 9 method alone operates similarly to the preamplification method, except that the former only has Septin 9 as the target marker.

[0411] As shown in Table 4, in the preamplification method, the sensitivity of CRC stage I, stage II, stage III, stage IV was 73.3%, 80.8%, 89.3%, and 100%, respectively. In contrast, in the Septin9 method alone, the sensitivity of CRC stage I, stage II, stage III, and stage IV was 26.7%, 65.4%, 75.0%, and 79%, respectively. Therefore, compared with the Septin9 method alone, the preamplification method showed a significant improvement in sensitivity.

[0412] Table 4. Comparison of the results of the pre-amplification method and the results of the Septin9 method alone

[0413]

[0414] The Ct value of each detected target marker is quantitatively analyzed to determine whether there are methylated copies in the CRC sample. Alternatively, the relative Ct value of each detected target marker relative to the internal control ACTB can be calculated to represent its relative methylation level. Importantly, all detected markers have a classification ability to distinguish CRC from controls with an AUC ranging from 0.8 to 0.9 (e.g., Figure 4 For example, different algorithms such as linear discriminant analysis, support vector machine, random forest algorithm, linear regression, logistic regression, etc. have been used to establish classification tools for early cancer detection. Different combinations of markers have been used to achieve optimal performance. The ROC curve of one of the combinations (SALL1, BCAT1 and Septin9) is shown in FIG. Figure 5 The ROC curves of other combinations are shown in Figure 5 Similar, not shown here.

[0415] Example 4: Comparison of LOD between pre-amplification method and direct qPCR method

[0416] In order to compare the LOD of the pre-amplification method and the direct qPCR method, the inventors detected 13 target markers (i.e., VAV3, NDRG4, Septin9, BCAT1, IKZF1, BCAN, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2 and intergenic region 1) using both the pre-amplification method and the direct qPCR method. The operation of the direct qPCR method is the same as the pre-amplification method, except that there is no pre-amplification step. In each method, 13 target markers are amplified / pre-amplified simultaneously, but each target marker is quantitatively analyzed separately. The comparison of the LOD of the pre-amplification method and the direct qPCR method for the target marker VAV3 is shown below. The comparison of the LOD of the pre-amplification method and the direct qPCR method for the other 12 target markers adopts similar operations and is not shown here.

[0417] Briefly, CRC tissue DNA was spiked into blood cell DNA at ratios of 0.5% and 0.2%. TM 40 ng DNA was treated with a Bisulfite Conversion Kit, of which half of the converted DNA was used for preamplification and subsequent qPCR (i.e., preamplification method), and the remaining half of the converted DNA was directly used for qPCR (i.e., direct qPCR method). The final concentration of the primers in the preamplification step was 50 nM. The 25 μL PCR mixture contained 10 μL of converted DNA, 2.5 μL of the premix containing the above primers, and 12.5 μL of PCR reagents ( Universal Probe qPCR Master Mix (NEB). The PCR program was set to 95°C for 3 minutes; 95°C for 30 seconds, 56°C for 60 seconds, and 8 cycles. The product obtained after the pre-amplification step was diluted 10 times for qPCR. The qPCR mixture consisted of 10 μL template DNA, 2.5 μL primer / probe pool, and 12.5 μL LUNA master mix. qPCR was run on an ABI 7500 Real-Time PCR System and the program was set to 95°C for 5 minutes; 95°C for 15 seconds, 56°C for 40 seconds (collecting fluorescence), and 50 cycles. Four replicates were performed in parallel. The results are shown in Table 5 below.

[0418] Table 5. Comparison of the results of the preamplification method and the direct qPCR method

[0419]

[0420] As shown in Table 5, the preamplification method showed improved LOD (0.50% vs. 0.20% CRC DNA percentage), better stability, and higher detection sensitivity compared with the direct qPCR method. The preamplification method results of the other 12 target markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and intergenic region 1) were better or not worse than the direct qPCR method, and the results are not shown here.

[0421] Example 5: Quantitative analysis of methylation of target markers using extracellular free DNA and comparison with a method without pre-amplification.

[0422] To validate the clinical performance of the methylation markers for CRC plasma samples, we tested five markers (i.e., Septin9, BCAT1, IKZF1, BCAN, and VAV3) in 32 plasma samples with clinical diagnosis of CRC and 29 colonoscopy-negative plasma control samples using both preamplification and no preamplification methods. The no preamplification method was similar to the preamplification method, but did not include the preamplification step and the dilution step. Of the 32 plasma samples with clinical diagnosis of CRC, 2 samples were from individuals diagnosed with CRC stage I, 9 samples were from individuals diagnosed with CRC stage II, 13 samples were from individuals diagnosed with CRC stage III, 5 samples were from individuals diagnosed with CRC stage IV, and 3 samples were of unknown stage.

[0423] The experiment includes the following steps:

[0424] 1. Obtain cell-free DNA (cfDNA) samples from 3-5 ml plasma samples using QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0425] 2. If the DNA is less than 40 ng, the cfDNA is divided into two parts and used as a bisulfite reagent (MethylCode TM The substrate for bisulfite conversion was prepared by using a preamplification method with a 10 μL elution kit and a 20 μL elution kit with a 20 μL elution kit. If the DNA was more than 40 ng, 20 ng cfDNA was used in each reaction and the elution method was the same as above.

[0426] 3. For the pre-amplification method, the converted cfDNA sample in one reaction (10 μL eluate) was pre-amplified. In short, the converted cfDNA sample obtained from step 2 above was pre-amplified by PCR reaction in the presence of methylation-specific primer pairs specific for Septin9, BCAT1, IKZF1, BCAN, and VAV3 (see Table 2). In the PCR reaction system, the final concentration of each primer was 200 nM. The pre-amplification procedure, dilution, and qPCR analysis method were the same as in Example 3.

[0427] 4. For the no pre-amplification method, the converted cfDNA sample (20 μL eluate) from another reaction was used for qPCR analysis in two different wells, each containing 10 μL of converted DNA. The qPCR mixture and procedure were the same as for the pre-amplification method.

[0428] 5. The non-CpG ACTB region was used as an internal control for each reaction well (see Table 2). The detection probes for different markers were labeled with different fluorescence. In the PCR reaction system, the final concentration of each primer was 500 nM, and the final concentration of each detection probe was 200 nM.

[0429] result

[0430] The Ct value of the sample without amplification signal is set to 50. A reference Ct value is set for each detection marker. If the Ct value of any detected marker is equal to or lower than its corresponding reference Ct value, the sample is classified as a positive sample.

[0431] Table 6 below shows the comparative results of using 5 target markers (i.e., Septin9, BCAT1, IKZF1, BCAN, and VAV3) in the preamplification method and the non-preamplification method. As shown in Table 6, the preamplification method showed ultra-high sensitivity (96.9%) for CRC and high specificity (93.1%) for the colonoscopy-negative population, and the sensitivity and specificity of the non-preamplification method were 84.4% and 93.1%, respectively. The sensitivity of the non-preamplification method was also much higher than that of the Septin9 method alone.

[0432] Table 6. Comparison of the results of the pre-amplification method and the results of the method without pre-amplification

[0433]

[0434] To validate the clinical performance of the methylation markers for CRC plasma samples, we tested more target markers, including any combination of markers selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4 and IRF4, in plasma samples clinically diagnosed with CRC and plasma control samples with negative colonoscopy using the pre-amplification method and the no-preamplification method as described above. For example, any of the following combinations are tested: (1) Septin9; (2) Septin9 and BCAT1; (3) Septin9 and IKZF1; (4) Septin9 and NDRG4; (5) Septin9 and BCAN; (6) Septin9 and VAV3; (7) Septin9 and IRF4; (8) BCAT1 and IKZF1; (9) BCAT1 and NDRG4; (10) BCAT1 and BCAN; (11) BCAT1 and VAV3; (12) BCAT1 and IRF4; (13) IKZF1 and NDRG4; (14) IKZF1 and BCAN; (15) IKZF1 and VAV3; (16) IKZF1 and IRF4; (17) NDRG4 and BCAN; (18) NDRG4 and VAV3; (19) NDRG4 and IRF 4; (20) BCAN and VAV3; (21) BCAN and IRF4; (22) VAV3 and IRF4; (23) Septin9, BCAT1 and IKZF1; (24) BCAT1, IKZF1 and NDRG4; (25) IKZF1, NDRG4 and BCAN; (26) NDRG4, BCAN and VAV3; (27) BCAN, VAV3 and IRF4; (28) Septin9, BCAT1 and NDRG4; (29) Septin9, BCAT1 and BCAN; (30) Septin9, BCAT1 and VAV3; (31) Septin9, BCAT1 and IRF4; (32) BCAT1, IKZF1 and BCAN; (33) BCAT1, IKZF1 and VAV3; (34) BCAT1, IKZF1 and IRF4.

[0435] Example 6: CRC detection was performed by quantitatively analyzing CRC methylation target markers (Septin9, BCAT1, IKZF1, VAV3 and IRF4) using extracellular free DNA.

[0436] In order to evaluate the clinical performance of more marker combinations, we used the method disclosed herein (also referred to as the pre-amplification method) to detect 5 markers (i.e., Septin9, BCAT1, IKZF1, VAV3, and IRF4) in 286 plasma samples clinically diagnosed with CRC and 112 colonoscopy-negative plasma control samples. Among the 286 plasma samples clinically diagnosed with CRC, 48 samples were from individuals diagnosed with CRC stage I, 113 samples were from individuals diagnosed with CRC stage II, 107 samples were from individuals diagnosed with CRC stage III, and 18 samples were from individuals diagnosed with CRC stage IV.

[0437] The experimental method is similar to that of Example 3.

[0438] result

[0439] The Ct value of the sample without amplification signal is set to 50. A reference Ct value is set for each detection marker. If the Ct value of any detected marker is equal to or lower than its corresponding reference Ct value, the sample is classified as a positive sample.

[0440] As shown in Table 7, the pre-amplification method (quantitative analysis of CRC methylation markers Septin9, BCAT1, IKZF1, VAV3, and IRF4) showed ultra-high sensitivity for CRC (84.3%) and high specificity for the colonoscopy-negative population (90.3%).

[0441] Table 7. Comparison of results between the preamplification method (methylation markers of Septin9, BCAT1, IKZF1, VAV3, and IRF4) and colonoscopy

[0442]

[0443] As shown in Table 8, in the pre-amplification method (quantitative analysis of CRC methylation markers Septin9, BCAT1, IKZF1, VAV3 and IRF4), the sensitivities of CRC stages I, II, III and IV were 62.5%, 85.8%, 88.8% and 100%, respectively.

[0444] Table 8. CRC detection sensitivity of the preamplification method for quantitative analysis of Septin9, BCAT1, IKZF1, VAV3 and IRF4

[0445] Clinical classification Accuracy Positive number Negative number total Phase I 62.5％ 30 18 48 Phase II 85.8％ 97 16 113 Stage III 88.8％ 95 12 107 Stage IV 100.0％ 18 0 18

[0446] To validate the clinical performance of more combinations of markers on CRC plasma samples, we used the method described above to test more target markers in plasma samples clinically diagnosed with CRC and plasma control samples with negative colonoscopy, including any combination of markers selected from the following group: Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4 and IRF4. For example, any of the following combinations are tested: (1) Septin9; (2) Septin9 and BCAT1; (3) Septin9 and IKZF1; (4) Septin9 and NDRG4; (5) Septin9 and BCAN; (6) Septin9 and VAV3; (7) Septin9 and IRF4; (8) BCAT1 and IKZF1; (9) BCAT1 and NDRG4; (10) BCAT1 and BCAN; (11) BCAT1 and VAV3; (12) BCAT1 and IRF4; (13) IKZF1 and NDRG4; (14) IKZF1 and BCAN; (15) IKZF1 and VAV3; (16) IKZF1 and IRF4; (17) NDRG4 and BCAN; (18) NDRG4 and VAV3; (19) NDRG4 and IRF 4; (20) BCAN and VAV3; (21) BCAN and IRF4; (22) VAV3 and IRF4; (23) Septin9, BCAT1 and IKZF1; (24) BCAT1, IKZF1 and NDRG4; (25) IKZF1, NDRG4 and BCAN; (26) NDRG4, BCAN and VAV3; (27) BCAN, VAV3 and IRF4; (28) Septin9, BCAT1 and NDRG4; (29) Septin9, BCAT1 and BCAN; (30) Septin9, BCAT1 and VAV3; (31) Septin9, BCAT1 and IRF4; (32) BCAT1, IKZF1 and BCAN; (33) BCAT1, IKZF1 and VAV3; (34) BCAT1, IKZF1 and IRF4.

Claims

1. A methylation marker for diagnosing colorectal cancer or screening for the formation or risk of colorectal cancer, characterized in that: The methylation markers include Septin9, BCAT1, IKZF1 and fragment sequences within their regions.

2. The methylation marker according to claim 1, characterized in that The methylation markers also include BCAN and / or VAV3 and fragment sequences within their regions; preferably, the methylation markers also include IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, one or more sub-region sequences in intergenic region 1, intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5 and fragment sequences within their regions.

3. The methylation marker according to claim 2, characterized in that The Hg19 coordinates of the methylation marker Septin9 include chr17:75276651-75496678, the Hg19 coordinates of the methylation marker BCAT1 include chr12:24964295-25102393, the Hg19 coordinates of the methylation marker IKZF1 include chr7:50343720-50472799, the Hg19 coordinates of the methylation marker BCAN include chr1:156611182-156629324, the Hg19 coordinates of the methylation marker VAV3 include chr1:108113782-108507766, the methylation marker IR The Hg19 coordinates of F4 include chr6:391739-411447, the Hg19 coordinates of the methylation marker POU4F2 include chr4:147560045-147563626, the Hg19 coordinates of the methylation marker SALL1 include chr16:51169886-51185278, the Hg19 coordinates of the methylation marker PKNOX2 include chr11:125034583-125303285, the Hg19 coordinates of the methylation marker SDC2 include chr8:97505579-97624000, the Hg19 coordinates of the methylation marker ASCL4 include ch r12:108168162-108170421, the Hg19 coordinates of the methylation marker TMEFF2 include chr2:192813769-193060435, the Hg19 coordinates of the methylation marker SLC24A2 include chr9:19507450-19786926, the Hg19 coordinates of the methylation marker NDRG4 include chr16:58496750-58547532, the Hg19 coordinates of the methylation marker NKX2-6 include chr8:23559964-23564111, the Hg19 coordinates of the methylation marker KCNA6 include chr12:49 18342-4960277, the Hg19 coordinates of the methylation marker SOX1 include chr13:112721913-112726020, the Hg19 coordinates of the methylation marker HS3ST2 include chr16:22825498-22927659, the Hg19 coordinates of the methylation marker FGF12 include chr3:191857184-192485553, the Hg19 coordinates of the methylation marker KCTD8 include chr4:44175926-44450824, the Hg19 coordinates of the methylation marker HMX1 include chr4:8847802-8873543,The Hg19 coordinates of the methylation marker MARCH11 include chr5:16067248-16180871, the Hg19 coordinates of the methylation marker CRHBP include chr5:76248538-76276983, the Hg19 coordinates of the methylation marker intergenic region 1 include chr6:19679885-19693988, and the Hg19 coordinates of the methylation marker intergenic region 2 include chr1 0:130082033-130087148, the Hg19 coordinates of the methylation marker intergenic region 3 include chr10:133107880-133113966, the Hg19 coordinates of the methylation marker intergenic region 4 include chr7:152620588-152624685, and the Hg19 coordinates of the methylation marker intergenic region 5 include chr8:70945014-70949177. , 4. A methylation marker for diagnosing colorectal cancer or screening for the development or risk of colorectal cancer, characterized in that: The methylation markers include: VAV3 and fragment sequences within its region; preferably, the methylation markers also include BCAN and fragment sequences within its region; more preferably, the methylation markers also include one or more subregion sequences of Septin9, BCAT1, IKZF1 and fragment sequences within their regions; more preferably, the methylation markers also include IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, intergenic region 1 , one or more subregion sequences in intergenic region 2, intergenic region 3, intergenic region 4 and intergenic region 5 and fragment sequences within the regions; more preferably, the Hg19 coordinates of the methylation marker Septin9 include chr17:75276651-75496678, the Hg19 coordinates of the methylation marker BCAT1 include chr12:24964295-25102393, the Hg19 coordinates of the methylation marker IKZF1 include chr7:50343720-50472799, the Hg19 coordinates of the methylation marker BCAN include chr1:156611182-156629324, the methylation marker V The Hg19 coordinates of AV3 include chr1:108113782-108507766, the Hg19 coordinates of the methylation marker IRF4 include chr6:391739-411447, the Hg19 coordinates of the methylation marker POU4F2 include chr4:147560045-147563626, the Hg19 coordinates of the methylation marker SALL1 include chr16:51169886-51185278, the Hg19 coordinates of the methylation marker PKNOX2 include chr11:125034583-125303285, and the Hg19 coordinates of the methylation marker SDC2 include chr8:975 05579-97624000, the Hg19 coordinates of the methylation marker ASCL4 include chr12:108168162-108170421, the Hg19 coordinates of the methylation marker TMEFF2 include chr2:192813769-193060435, the Hg19 coordinates of the methylation marker SLC24A2 include chr9:19507450-19786926, the Hg19 coordinates of the methylation marker NDRG4 include chr16:58496750-58547532, the Hg19 coordinates of the methylation marker NKX2-6 include chr8:23559964-23564111,The Hg19 coordinates of the methylation marker KCNA6 include chr12:4918342-4960277, the Hg19 coordinates of the methylation marker SOX1 include chr13:112721913-112726020, the Hg19 coordinates of the methylation marker HS3ST2 include chr16:22825498-22927659, the Hg19 coordinates of the methylation marker FGF1 2 includes chr3:191857184-192485553, the Hg19 coordinates of the methylation marker KCTD8 include chr4:44175926-44450824, the Hg19 coordinates of the methylation marker HMX1 include chr4:8847802-8873543, and the Hg19 coordinates of the methylation marker MARCH11 include chr5: 16067248-16180871, the Hg19 coordinates of the methylation marker CRHBP include chr5:76248538-76276983, the Hg19 coordinates of the methylation marker intergenic region 1 include chr6:19679885-19693988, and the Hg19 coordinates of the methylation marker intergenic region 2 include chr10:130082033-13 0087148, the Hg19 coordinates of the methylation marker intergenic region 3 include chr10:133107880-133113966, the Hg19 coordinates of the methylation marker intergenic region 4 include chr7:152620588-152624685, and the Hg19 coordinates of the methylation marker intergenic region 5 include chr8:70945014-70949177.

5. A primer or probe for detecting a methylation marker according to any one of claims 1 to 4, characterized in that: The primer uses the nucleotide sequence where the methylation marker is located as the target sequence and is used for specific amplification of the target sequence; the probe specifically captures the nucleotide sequence where the methylation marker is located.

6. A kit for diagnosing colorectal cancer or screening for the formation or risk of colorectal cancer, characterized in that: The kit comprises a reagent for specifically detecting the presence and / or abundance of the methylation state of the methylation marker according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that The kit comprises the primer or probe of the methylation marker according to claim 3.

8. The kit according to claim 6, characterized in that The kit also includes a bisulfite reagent and / or a methylation-sensitive restriction enzyme.

9. Use of the methylation marker as described in any one of claims 1-4, or the primer or probe as described in claim 5, or the kit as described in any one of claims 6-8 in the preparation of a product for diagnosing colorectal cancer, a product for screening the formation of colorectal cancer, a product for screening the risk of colorectal cancer formation, a product for assessing the progression of colorectal cancer, a product for assessing the prognostic risk of colorectal cancer, and a product for assessing the response of an individual receiving treatment for colorectal cancer to treatment.

10. An information data processing terminal, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the following steps are implemented: (a) obtaining the methylation level of at least one region of the methylation marker according to any one of claims 1 to 4 in an individual test sample; (b) comparing the methylation level in step (a) with the corresponding reference level, respectively, wherein one or more methylation markers having the same or higher methylation level relative to their corresponding reference levels indicates that the individual suffers from colorectal cancer, or the individual has a risk of colorectal cancer formation or formation; preferably, (a) also includes treating the corresponding region of the methylation marker with a bisulfite reagent and / or a methylation-sensitive restriction enzyme.

Images