WO2022170984A1 - 结直肠进展期腺瘤的筛查、风险评估及预后方法和试剂盒 - Google Patents

结直肠进展期腺瘤的筛查、风险评估及预后方法和试剂盒 Download PDF

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WO2022170984A1
WO2022170984A1 PCT/CN2022/074143 CN2022074143W WO2022170984A1 WO 2022170984 A1 WO2022170984 A1 WO 2022170984A1 CN 2022074143 W CN2022074143 W CN 2022074143W WO 2022170984 A1 WO2022170984 A1 WO 2022170984A1
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primer
enzymes
seq
pknox2
group
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French (fr)
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周平红
蔡明琰
朱博群
王辉
刘蕊
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复旦大学附属中山医院
上海鹍远生物科技股份有限公司
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Publication of WO2022170984A1 publication Critical patent/WO2022170984A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • This application relates generally to the field of biomedicine.
  • the present application relates to screening, risk assessment and prognosis methods and kits for advanced colorectal adenomas.
  • colorectal tumor screening methods include colonoscopy or molecular testing of stool and blood samples, which are either invasive or have few markers, limiting patient compliance or cancer screening. Detection sensitivity.
  • the invention improves the detection sensitivity of colorectal advanced adenoma by combining different detection targets and the method of hypermethylation and hypomethylation of plasma free DNA.
  • the present application provides a method of diagnosing colorectal advanced adenoma, screening for risk of colorectal advanced adenoma formation or formation, or assessing the progression or prognosis of colorectal advanced adenoma in an individual, wherein The method includes the following steps:
  • the target marker includes at least one target marker in each of the following two groups of target markers: (1) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A;
  • the methylation levels of the target markers and the corresponding reference levels are compared, respectively, wherein: the target markers of group (I) have higher methylation levels relative to their corresponding reference levels, and group (II) ) the target marker has a lower methylation level relative to its corresponding reference level indicating that the individual has advanced colorectal adenoma, or that the individual has advanced colorectal adenoma formation or formation of risk, or the individual has an increased likelihood of developing or developing advanced colorectal adenoma, or the individual has a poor prognosis or a risk of poor prognosis for advanced colorectal adenoma.
  • the method includes:
  • step (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing methylated and unmethylated CpG sites in the DNA, thereby obtaining a treated DNA ;
  • step (c) pre-amplifying at least one target in each of the following two sets of target markers in the treated DNA obtained from step (b) with a pool of pre-amplification primers Markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product;
  • step (d) quantitatively analyzing the methylation levels of the amplified target markers based on the pre-amplification products obtained in step (c);
  • step (e) respectively comparing the methylation level of the amplified target marker in step (d) with the corresponding reference level, wherein: the target marker described in group (I) is relative to its corresponding reference level Having higher methylation levels and lower methylation levels of the target markers described in group (II) relative to their corresponding reference levels indicates that the individual has advanced colorectal adenomas, or that all The individual is at risk for the formation or formation of advanced colorectal adenomas, or the individual has an increased likelihood of developing or developing advanced colorectal adenomas, or the individual has a poor prognosis or poor prognosis for advanced colorectal adenomas risks of.
  • step (c) of the above method at least 2 or all 3 markers of interest in said panel (I) are amplified.
  • step (c) of the above method Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in said panel (I) are amplified.
  • step (c) of the above method at least 2, at least 3 or all 4 markers of interest in said panel (II) are amplified.
  • step (c) of the above method in said group (II), PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and intergenic region Region A is amplified.
  • step (c) of the above method in said group (II), PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A is amplified.
  • step (c) of the above method the following target markers in the treated DNA obtained from step (b) are pre-amplified with a pool of pre-amplification primers: Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product.
  • the respective marker of interest comprises either:
  • the DNA in the biological sample obtained in step (a) comprises genomic DNA or extracellular DNA.
  • the extracellular cell-free DNA comprises circulating tumor DNA.
  • the amount of the marker of interest in the extracellular DNA does not exceed 1 ng, 0.8 ng, 0.6 ng, 0.4 ng, 0.2 ng, 0.1 ng, 0.08 ng or no more in the biological sample more than 0.04ng.
  • the concentration of the marker of interest in the extracellular free DNA in the biological sample is below the sensitivity level for the detection assay of the marker of interest.
  • the pre-amplification product in step (c) is diluted with a diluent prior to step (d).
  • the biological sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissues, bodily fluids, colonic effluents, surgically resected samples, isolated blood cells, cells isolated from blood, and the like random combination.
  • 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, abdominal cavity Effusion, and any combination thereof.
  • the biological sample is obtained from the plasma of the individual.
  • the colonic effluent is selected from the group consisting of a stool sample and an enema wash sample.
  • the agent of step (b) selectively modifies unmethylated cytosine residues at CpG sites to produce modified residues, but does not significantly modify methylated cytosines pyrimidine residues.
  • the reagent of step (b) comprises a bisulfite reagent.
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the reagent of step (b) selectively cleaves unmethylated residues but not methylated residues, or selectively cleaves methylated residues but not unmethylated residues methylated residues.
  • the reagent of step (b) is a methylation-sensitive restriction enzyme (MSRE).
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzymes, BbeI enzymes, NotI enzymes, SmaI enzymes, XmaI enzymes, MboI enzymes, BstBI enzymes, ClaI enzymes, MluI enzymes, NaeI enzymes, NarI enzymes, PvuI enzymes, SacII enzymes, Hhal enzymes, and any combination thereof.
  • the pool of pre-amplification primers comprises methylation-specific primer pairs.
  • the methylation-specific primer pair comprises a forward primer and a reverse primer
  • each of the primers comprises an oligonucleotide sequence that is associated with a corresponding target marker
  • At least 9 contiguous nucleotides of the substance hybridize under stringent, moderately stringent or highly stringent conditions, wherein the at least 9 contiguous nucleotides comprise at least one CpG site.
  • the pre-amplification primer pool further comprises a control primer pair for amplifying a control marker.
  • 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.
  • the primer pair for amplifying Septin9 is SEQ ID NO: 22/23; the primer pair for amplifying BCAT1 is SEQ ID NO: 24/25; the primer pair for amplifying IKZF1 is SEQ ID NO: 24/25 ID NO: 26/27; the primer pair for amplifying PKNOX2 is SEQ ID NO: 28/29; the primer pair for amplifying CRHBP is SEQ ID NO: 30/31; the primer pair for amplifying SOX1 is SEQ ID NO: 32/33;
  • the primer pair used to amplify the intergenic region A is SEQ ID NO: 34/35.
  • the methylation-specific primer pair comprises at least one or more primer pairs selected from the group consisting of: SEQ ID NOs: 22/23, 24/25, 26/27, 28/29 , 30/31, 32/33 and 34/35.
  • the methylation-specific primer pair comprises at least one, at least two or all three primer pairs selected from the group (I) below and at least one pair, at least two pairs, at least one pair from the group (II) below Three primer pairs: (I) SEQ ID NOs: 22/23, 24/25 and 26/27; (II) SEQ ID NOs: 28/29, 30/31, 32/33 and 34/35.
  • the methylation specific primer pair comprises SEQ ID NOs: 22/23, 24/25, 26/27, 28/29, 30/31, 32/33 and 34/35.
  • step (c) the target marker is amplified in the presence of one or more blocking oligonucleotides.
  • the quantitative analysis of step (d) is performed by polymerase chain reaction (eg, real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, mass-based separation (eg electrophoresis, mass spectrometry) or target capture (eg hybridization, microarray).
  • the quantitative analysis of step (d) is performed by real-time polymerase chain reaction, optionally the real-time polymerase chain reaction is a multiplex real-time polymerase chain reaction.
  • the quantitative analysis of step (d) comprises using a quantitative primer pair and a DNA polymerase to amplify the pre-amplification product of step (c), wherein the pre-amplification product is amplified by amplification increase product.
  • the quantitative primer pair used in step (d) is capable of at least 9 contiguous nucleotides of the pre-amplification product of step (c) under stringent conditions, moderately stringent conditions, or highly stringent conditions Hybridization conditions.
  • At least one of the quantitative primer pairs used in step (d) is the same as at least one of the methylation-specific primer pairs of the pre-amplification primer pool of step (c).
  • the quantitative primer pair used in step (d) is designed to amplify at least a portion within the at least one pre-amplification product of step (c).
  • the step (d) is performed in the presence of a detection reagent.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • the probe for detecting Septin9 is SEQ ID NO:36; the probe for detecting BCAT1 is SEQ ID NO:37; the probe for detecting IKZF1 is SEQ ID NO:38; the probe for detecting PKNOX2 It is SEQ ID NO: 39 and 40; the probe for detecting CRHBP is SEQ ID NO: 41; the probe for detecting SOX1 is SEQ ID NO: 42; the probe for detecting intergenic region A is SEQ ID NO: 43.
  • the probe comprises at least one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 36-43.
  • the probes comprise at least one, at least two or all three nucleotide sequences of SEQ ID NOs: 36-38 and SEQ ID NOs: 39 and/or 40 and SEQ ID NOs: 41-43 At least one, at least two or at least three nucleotide sequences.
  • the probes include the nucleotide sequences set forth in SEQ ID NOs: 36-38 and 41-43, and the nucleotide sequences set forth in SEQ ID NOs: 39 and/or 40.
  • 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).
  • a fluorescent dye eg, FAM, HEX/VIC, TAMRA, Texas Red, or Cy5
  • a quencher eg, BHQ1, BHQ2, BHQ3 , DABCYL or TAMRA
  • step (e) comprises comparing the Ct value of the target marker of step (d) with a reference Ct value, wherein the Ct value of the target marker of group (I) is higher than its corresponding reference A Ct value and a Ct value for the target marker of group (II) below its corresponding reference Ct value indicates that the individual has advanced colorectal adenoma, or that the individual has advanced colorectal adenoma formation or formation , or the individual has an increased likelihood of developing or developing advanced colorectal adenoma, or the individual has a poor prognosis or a risk of poor prognosis for advanced colorectal adenoma.
  • the pre-amplification of step (c) includes 5 to 30 reaction cycles, wherein each cycle includes a reaction at 40-80°C for 5 seconds-5 minutes, followed by a reaction at 85-99°C 5 seconds - 5 minutes.
  • the quantitative analysis of step (d) comprises determining the presence or level of a plurality of CpG dinucleotides, TpG dinucleotides or CpA dinucleotides in the pre-amplification product. methylation level. In some embodiments, the quantitative analysis of step (d) comprises determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in the pre-amplification product. In some embodiments, the quantitative analysis of step (d) is performed by dividing the pre-amplification product of step (c) into multiple components.
  • the reference level in step (e) is based on clinical samples obtained from a group of individuals with or at risk of developing colorectal tumors and having never had colorectal tumors or did not have colorectal tumors Clinical samples obtained from a group of individuals at risk of developing colorectal tumors.
  • the application provides a kit for diagnosing colorectal advanced adenoma, screening for risk of colorectal advanced adenoma formation or formation, or assessing the progression or prognosis of colorectal advanced adenoma, It contains:
  • a first primer pool comprising primer pairs for pre-amplifying at least one target marker in each of the following two groups of target markers: (1) Septin9, BCAT1 and IKZF1, and (II) PKNOX2, CRHBP, SOX1, and intergenic region A, wherein said primer pair is compatible with at least 9 contiguous nucleosides of the target sequence of said target marker after treatment with said first reagent
  • the acid hybridizes under stringent conditions, under moderately stringent conditions, or under highly stringent conditions, wherein the target sequence comprises at least one CpG site;
  • the second reagent comprises a second primer pool comprising a plurality of quantitative primer pairs capable of interacting with the quantitation primers pre-amplified by the first primer pool At least 9 contiguous nucleotides of the target sequence hybridize under stringent, moderately stringent or highly stringent conditions.
  • the quantitative primer pairs in the second primer pool are the same as the primer pairs in the first primer pool. In some embodiments, quantitative primer pairs in the second primer pool are designed to amplify at least a portion of the target sequence pre-amplified by the first primer pool.
  • the first primer pool comprises at least one methylation-specific primer pair.
  • the first primer pool and the second primer pool are packaged in a single container or in separate containers.
  • the kit further comprises one or more blocking oligonucleotides.
  • the kit further comprises detection reagents.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • the probe includes at least one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 36-43.
  • the probe comprises at least one, at least two or all three nucleotide sequences of the following group (I) and at least one, at least two or at least three nucleotide sequences of the following group (II): (I ) SEQ ID NOs: 36-38, (II) SEQ ID NOs: 39 or 40 and 41-43.
  • the probes include: the nucleotide sequences set forth in SEQ ID NOs: 36-38 and 41-43, and the nucleotide sequences set forth in SEQ ID NOs: 39 and/or 40.
  • 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, TAMRA or lowa Black Dark Quenchers).
  • a fluorescent dye eg, FAM, HEX/VIC, TAMRA, Texas Red, or Cy5
  • a quencher eg, BHQ1, BHQ2, BHQ3
  • the kit further comprises DNA polymerase and/or a container suitable for holding 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.
  • the first reagent comprises a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE).
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzymes, BbeI enzymes, NotI enzymes, SmaI enzymes, XmaI enzymes, MboI enzymes, BstBI enzymes, ClaI enzymes, MluI enzymes, NaeI enzymes, NarI enzymes, PvuI enzymes, SacII enzymes, Hhal enzymes, and any combination thereof.
  • the first primer pool includes primer pairs for amplifying at least 2 or all 3 markers of interest in the set (I).
  • the first primer pool includes primer pairs for amplifying Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in the set (I).
  • the first primer pool includes primer pairs for amplifying at least 2, at least 3, or all 4 markers of interest in the set (II).
  • the first primer pool includes PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and gene for amplification set (II) Primer pair for spacer A.
  • the first primer pool includes PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, Primer pair for SOX1 and intergenic region A.
  • the first primer pool includes primer pairs for amplifying Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and Intergenic Region A; wherein at least a portion of the target marker is preamplified to obtain pre-amplified products.
  • the primer pair for amplifying Septin9 is SEQ ID NO: 22/23; the primer pair for amplifying BCAT1 is SEQ ID NO: 24/25; the primer pair for amplifying IKZF1 is SEQ ID NO: 24/25 ID NO: 26/27; the primer pair for amplifying PKNOX2 is SEQ ID NO: 28/29; the primer pair for amplifying CRHBP is SEQ ID NO: 30/31; the primer pair for amplifying SOX1 is SEQ ID NO: 32/33;
  • the primer pair used to amplify the intergenic region A is SEQ ID NO: 34/35.
  • the respective marker of interest comprises either:
  • the first primer pool comprises primer pairs comprising or consisting of a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 22/23, 24/25 , 26/27, 28/29, 30/31, 32/33, and 34/35, optionally wherein the second primer pool comprises at least one primer that is identical to at least one primer pair in the first primer pool right.
  • the first primer pool and the second primer pool further comprise primer pairs for amplifying a control marker.
  • 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.
  • the kit further comprises a plurality of containers, each container for receiving components of the second primer pool.
  • the application provides a kit according to the application in the manufacture of a kit for diagnosing colorectal advanced adenoma, screening for colorectal advanced adenoma formation or risk of formation, or assessing colorectal progression in an individual Use in a diagnostic kit for the progression or prognosis of stage adenomas.
  • the application provides the use of a reagent for quantitative analysis of methylation levels of a marker of interest in the manufacture of a kit for use in diagnosing advanced colorectal adenoma, screening In a method of investigating the risk of formation or formation of advanced colorectal adenoma or assessing the progression or prognosis of advanced colorectal adenoma, wherein the method comprises the steps of:
  • step (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing methylated and unmethylated CpG sites in the DNA, thereby obtaining a treated DNA ;
  • step (c) pre-amplifying at least one target in each of the following two sets of target markers in the treated DNA obtained from step (b) with a pool of pre-amplification primers Markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product;
  • step (d) quantitatively analyzing the methylation levels of the amplified target markers based on the pre-amplification products obtained in step (c);
  • step (e) respectively comparing the methylation level of the amplified target marker in step (d) with the corresponding reference level, wherein: the target marker described in group (I) is relative to its corresponding reference level Having higher methylation levels and lower methylation levels of the target markers described in group (II) relative to their corresponding reference levels indicates that the individual has advanced colorectal adenomas, or that all The individual is at risk for the formation or formation of advanced colorectal adenomas, or the individual has an increased likelihood of developing or developing advanced colorectal adenomas, or the individual has a poor prognosis or poor prognosis for advanced colorectal adenomas risks of.
  • step (c) of the above method at least 2 or all 3 markers of interest in said panel (I) are amplified.
  • step (c) of the above method Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in said panel (I) are amplified.
  • step (c) of the above method at least 2, at least 3 or all 4 markers of interest in said panel (II) are amplified.
  • step (c) of the above method PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and intergenic region A are amplified.
  • step (c) of the above method PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A are amplified .
  • step (c) of the above method the following target markers in the treated DNA obtained from step (b) are pre-amplified with a pool of pre-amplification primers: Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product.
  • the respective marker of interest comprises either:
  • Figure 1 Detection of Septin9, BCAT1 and IKZF1 methylation in plasma cell-free DNA of patients with no abnormality and advanced adenoma detected by colonoscopy.
  • Figure 2 The results of methylation detection of PKNOX2, CRHBP, SOX1 and intergenic region A in plasma cell-free DNA of patients with no abnormality and advanced adenoma detected by colonoscopy.
  • Figure 3 shows the nucleotide sequence of an exemplary subregion of a marker of interest.
  • the present application pre-amplifies the target markers described herein to obtain pre-amplified products, and then quantifies the methylation level of each target marker separately based on its respective pre-amplified products.
  • a pre-amplification step can increase the quantity/level of the target marker and can significantly improve the diagnostic specificity and/or diagnostic sensitivity for colorectal advanced adenomas.
  • the present application simultaneously quantitatively analyzes the methylation levels of multiple target markers in biological samples to improve diagnostic specificity and/or diagnostic sensitivity for colorectal advanced adenomas.
  • the plurality of target markers are pre-amplified prior to quantitative analysis.
  • diagnostic specificity in this context is defined as the proportion of positive results that are correctly identified, ie the percentage of individuals who are correctly identified as having a disease. While “specificity” is defined as the proportion of correctly identified negative results, that is, the percentage of individuals correctly identified as not having the disease.
  • the present application provides a method of diagnosing colorectal advanced adenoma, screening for risk of colorectal advanced adenoma formation or formation, or assessing the progression or prognosis of colorectal advanced adenoma in an individual, wherein The method includes the following steps:
  • target markers include at least one target marker in each of the following two groups of target markers: (I ) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and Intergenic Region A;
  • the target marker of group (I) has a higher methylation level relative to its corresponding reference level
  • the target marker described in group (II) has a lower methylation level relative to its corresponding reference level indicating that the individual has advanced colorectal adenoma, or that the individual has advanced colorectal adenoma formation or the risk of developing, or that the individual has an increased likelihood of developing or developing advanced colorectal adenoma, or that the individual has a poor prognosis or a risk of poor prognosis of colorectal advanced adenoma;
  • the method comprises an optional step (c): pre-amplifying each of the following two groups of target markers in the treated DNA obtained from step (b) with a pool of pre-amplification primers At least one marker of interest in: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and Intergenic Region A; wherein at least a portion of the marker of interest is pre-amplified to obtain pre-amplification increase product.
  • the method includes:
  • step (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing methylated and unmethylated CpG sites in the DNA, thereby obtaining a treated DNA ;
  • step (c) pre-amplifying at least one target in each of the following two sets of target markers in the treated DNA obtained from step (b) with a pool of pre-amplification primers Markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product;
  • step (d) quantitatively analyzing the methylation levels of the amplified target markers based on the pre-amplification products obtained in step (c);
  • step (e) respectively comparing the methylation level of the amplified target marker in step (d) with the corresponding reference level, wherein: the target marker described in group (I) is relative to its corresponding reference level Having higher methylation levels and lower methylation levels of the target markers described in group (II) relative to their corresponding reference levels indicates that the individual has advanced colorectal adenomas, or that all The individual is at risk for the formation or formation of advanced colorectal adenomas, or the individual has an increased likelihood of developing or developing advanced colorectal adenomas, or the individual has a poor prognosis or poor prognosis for advanced colorectal adenomas risks of.
  • screening refers to the identification of a pathological state, disease, or condition, such as identification of advanced colorectal adenomas, or identification of patients with advanced colorectal adenomas that may be affected by a specific treatment regimen Individuals who benefit from it are identified.
  • screening and the term “diagnosis” may be used interchangeably.
  • tumor or tumor should be understood to refer to a foci, tumor or other encapsulated or unencapsulated mass or other form of growth comprising tumor cells.
  • Tuor cells should be understood to mean cells that exhibit abnormal growth.
  • growth should be understood in the broadest sense and includes proliferation. In this regard, one example of abnormal cell growth is uncontrolled proliferation of cells. Another instance is
  • Tumor cells can be benign or malignant cells.
  • the tumor is an adenoma or adenocarcinoma.
  • adenomas are generally benign tumors of epithelial origin that arise from epithelial tissue or display well-defined epithelial structures. These structures can have a glandular appearance. It may contain a population of malignant cells within the adenoma, eg, as a benign adenoma or benign tumor foci progresses into a malignant adenocarcinoma.
  • the tumor is malignant, such as cancer.
  • the tumor is not malignant, such as an adenoma.
  • colonal tumor refers to a tumor present in the colon, rectum and/or appendix.
  • the colorectal tumor is colorectal cancer, colorectal adenoma, and/or sessile serrated polyp.
  • the colorectal tumor is precancerous.
  • advanced colorectal adenoma refers to adenomas with tumors larger than 1 cm, or containing high-grade intraepithelial neoplasia, or containing a villous component.
  • precancerous refers to a neoplasm that exhibits some histological changes associated with an increased risk of cancer progression.
  • colorectal cell proliferative disorders examples of such conditions include highly dysplastic cell proliferative disorders such as adenomatous polyps of the colon.
  • adenoma or adenocarcinoma is understood to refer to one or more cells of an individual exhibiting dysplasia.
  • an adenoma or adenocarcinoma may have developed because of abnormally growing clumps of cells, or it may be a very early stage adenoma or adenocarcinoma with only a relatively small number at the time of diagnosis. Cells divide abnormally.
  • the present application also extends to assessing an individual's risk of developing colorectal advanced adenomas (eg, colorectal cancer).
  • the term "assessing" refers to the ability to distinguish between samples from individuals with advanced colorectal adenomas from individuals who do not have advanced colorectal adenomas, or to distinguish samples from individuals at different stages of colorectal tumor progression the capacity of a sample of individuals.
  • the assessment involves determining whether the individual's tumor has entered the advanced stage or has a high probability of entering the advanced stage.
  • the assessment involves classifying the individual's tumor, eg, stage I, II, III, IV, and the like.
  • the assessment involves determining whether the individual's tumor is reduced or worsened.
  • the assessment can assist in assessing the likelihood of a treatment having clinical benefit.
  • the assessment may relate to whether and/or the likelihood that the patient will get better after receiving treatment (eg, treatment with a particular drug).
  • treatment eg, treatment with a particular drug
  • the assessment methods of the present application can be used to make treatment decisions clinically by selecting the most appropriate treatment modality for any particular patient.
  • a treatment regimen eg, a given treatment regimen, including, for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.
  • the assessment methods of the present application are useful in assessing the likelihood of long-term survival of the patient is a valuable tool.
  • the confidence interval is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the p-value is less than 0.1, 0.05, 0.01, 0.005, or 0.0001.
  • progression refers to changes in the morphology and physiology of cells along genetically defined pathways, eg, natural developmental processes from previous, lower or early stages to later, more complex or advanced stages of physiological maturation.
  • prognosis refers to the likelihood of predicting the outcome of disease symptoms (including, eg, recurrence, exacerbation, drug resistance) of a disease (eg, cancer). The term also refers to a prediction of the likelihood of clinical benefit from treatment. In some embodiments, a statistical algorithm is used to provide an individual with a prognosis of the disease. For example, prognosis can be surgery, progression of a clinical subtype of cancer (eg, solid tumors such as colorectal cancer, melanoma, and renal cell carcinoma), progression of one or more clinical factors, or recovery from disease. The prognosis can be poor (eg, possible relapse or development of drug resistance) or good.
  • a clinical subtype of cancer eg, solid tumors such as colorectal cancer, melanoma, and renal cell carcinoma
  • the prognosis can be poor (eg, possible relapse or development of drug resistance) or good.
  • steps (a), (b), (c), (d), and (e) apply to the diagnosis of colorectal advanced adenomas, the screening of Risk of advanced rectal adenoma formation or formation or methods of assessing progression or prognosis of advanced colorectal adenoma.
  • step (a) of the method according to the present application a biological sample comprising DNA is obtained from the individual.
  • biological sample refers to a biological composition obtained or derived from a target individual comprising cells and/or other molecular entities to be characterized or identified based on physical, biochemical, chemical and/or physiological characteristics (e.g. DNA).
  • 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 of skill in the art.
  • the biological sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissues, bodily fluids, colonic effluents, surgically resected samples, isolated blood cells, cells isolated from blood, and the like random combination.
  • 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, abdominal cavity Effusion, and any combination thereof.
  • the colonic effluent is selected from the group consisting of a stool sample and an enema wash sample. Selection of the sample most suitable for detection according to the methods of the present application will depend on the nature of the situation.
  • the biological sample is obtained from whole blood of an individual.
  • the biological sample is obtained from the plasma of an 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 one, two, three, four, five or more times.
  • the term "individual” as used herein includes human and non-human animals.
  • Non-human animals include all vertebrates such as mammals and non-mammals.
  • An "individual” can also be a domestic animal, such as cattle, pigs, sheep, poultry, and horses; or rodents, such as rats, mice; or non-human primates, such as apes, monkeys, rhesus monkeys; or domesticated Animals such as dogs or cats.
  • the individual is a human or non-human primate.
  • the individual is a human.
  • "individual" and “subject” may be used interchangeably.
  • the DNA is isolated from the biological sample. Isolation and purification of DNA from biological samples can be performed using various methods known in the art, including the use of commercially available kits. For example, DNA is isolated from cells and tissues by lysing raw materials under highly denaturing and reducing conditions, partially using protein-degrading enzymes, purifying nucleic acid fractions obtained by a phenol/chloroform extraction process, and removing them from water by dialysis or ethanol precipitation Nucleic acids are recovered in phase (see, eg, Sambrook, J., Fritsch, E.F. in T. Maniatis, CS, Molecular Cloning, 1989).
  • the DNA contained in the biological sample includes genomic DNA.
  • genomic DNA refers to DNA comprising the entire genome of a cell or organism and fragments or portions thereof. Genomic DNA is a large stretch of DNA (eg, 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 .
  • the DNA contained in the biological sample includes cellular DNA.
  • cellular DNA refers to DNA present within a cell, or DNA obtained from cells in vivo and isolated in vitro, or otherwise manipulated in vitro, so long as the DNA is not removed from cells in vivo.
  • the DNA contained in the biological sample comprises extracellular cell-free DNA.
  • extracellular cell-free DNA refers to DNA fragments that exist outside of cells in vivo. The term can also be used to refer to DNA fragments obtained from extracellular sources in vivo and isolated, or manipulated in vitro. DNA fragments in cell-free DNA usually have a length of about 100 to 200 bp, presumably related to the length of DNA fragments encapsulated in nucleosomes.
  • Extracellular cell-free DNA includes, for example, extracellular cell-free fetal DNA and circulating tumor DNA.
  • Extracellular fetal DNA circulates in the body (e.g., blood) of pregnant women and represents the fetal genome, while circulating tumor DNA circulates in the body (e.g., blood) of cancer patients.
  • the extracellular cell-free DNA can be substantially free of the individual's cellular DNA.
  • the extracellular DNA can comprise less than about 1,000 ng/mL, less than about 100 ng/mL, less than about 10 ng/mL, less than about 1 ng/mL of cellular DNA.
  • Extracellular episomal DNA can be prepared by using conventional techniques known in the art.
  • the blood sample can be centrifuged at about 200-20,000 g, about 200-10,000 g, about 200-5,000 g, about 300-4000 g, etc. for about 3-30 minutes, about 3-15 minutes, about 3-10 minutes , about 3-5 minutes to obtain the extracellular DNA of the blood sample.
  • extracellular DNA from a blood sample can be obtained by centrifuging the individual's plasma or serum one, two, three, four, five or more times.
  • the biological sample can be obtained by microfiltration in order to separate cells and fragments thereof from a cell-free fraction comprising soluble DNA.
  • microfiltration can be performed by using a filter, eg, a 0.1 micron to 0.45 micron membrane filter, such as a 0.22 micron membrane filter.
  • extracellular cell-free DNA is extracted from whole blood, serum or plasma for analysis using commercially available DNA extraction products.
  • This extraction method is said to have a high recovery rate (>50%) of circulating DNA, and some products (such as the QIAamp Circulating Nucleic Acid Kit from Qiagen) are said to extract DNA fragments of small size.
  • Typical sample volumes used are 1-5 mL of serum or plasma.
  • the extracellular cell-free DNA includes circulating tumor DNA.
  • Circulating tumor DNA (“ctDNA”) is fragmented DNA of tumor origin in body fluids not associated with cells (eg, blood, urine, saliva, sputum, feces, pleural fluid, cerebrospinal fluid, etc.).
  • ctDNA is highly fragmented, with an average length of about 150 base pairs.
  • ctDNA typically comprises a very small fraction of extracellular free DNA in body fluids (eg, plasma), eg, 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%.
  • the total amount of plasma DNA is typically very low, eg, about 10 ng/mL plasma.
  • the amount of ctDNA varies from person to person and depends on the type of tumor, its location and, in the case of cancerous tumors, the stage of the cancer.
  • ctDNA is often very rare in body fluids and can only be detected by extremely sensitive and specific techniques. Detection of ctDNA may help detect and diagnose tumors, guide tumor-specific therapy, monitor treatment, and monitor cancer remission.
  • 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 and methylated CpG sites in the DNA, thereby obtaining a treated DNA.
  • DNA methylation is the biological process of adding methyl groups to a DNA molecule (eg, to one or more cytosine bases of a DNA molecule) (eg, by the action of DNA methyltransferases).
  • DNA methylation occurs at the 5' position of a cytosine-phosphate-guanine (CpG) dinucleotide (ie, a "CpG site") when it occurs at the promoter or the first 5'-CpG-3' dinucleotides in exons can cause 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.
  • methylated cytosine residue refers to a derivative of a cytosine residue in which a methyl group is attached to a carbon atom of the cytosine ring (eg, C5).
  • unmethylated cytosine residue refers to an underivatized cytosine residue in which, in contrast to "methylated cytosine residue,” there is no carbon atom (eg, C5) on the cytosine ring Methyl linkage.
  • CpG sites in which cytosine residues are methylated are methylated CpG sites, and CpG sites in which cytosine residues are not methylated are unmethylated CpG sites .
  • the reagents used in step (b) are capable of distinguishing between unmethylated and methylated CpG sites in DNA, thereby obtaining treated DNA.
  • the reagent can selectively act on unmethylated cytosine residues, but not significantly on methylated cytosine residues.
  • the agent may act selectively on methylated cytosine residues without significantly acting on unmethylated cytosine residues.
  • the original DNA is converted into the processed DNA in a manner that depends on whether it is methylated or not, so that the processed DNA can be distinguished from the original DNA by its hybridization behavior.
  • some reagents can selectively convert unmethylated cytosine residues to uracil, thymine, or another base hybridized to cytosine, while methylated cytosine residues remain unmethylated Conversion status.
  • some reagents can selectively cleave methylated residues, or selectively cleave unmethylated residues.
  • treated DNA refers to DNA that has been treated with an agent capable of distinguishing between unmethylated and methylated CpG sites in DNA, ie the DNA methylation status in the DNA has been altered.
  • the agent of step (b) selectively modifies unmethylated cytosine residues at CpG sites to produce modified residues, but does not significantly modify methylated cytosines pyrimidine residues.
  • the reagent of step (b) comprises a bisulfite reagent.
  • bisulfite reagent refers to, for example, a bisulfite, bisulfite, ions or any combination thereof.
  • treatment of DNA with a bisulfite reagent is also described as a "bisulfite reaction” or “bisulfite treatment” and refers to a reaction that converts unmethylated cytosine residues, particularly is the conversion of unmethylated cytosine residues in nucleic acids into uracil bases, thymine bases, or other bases that differ from cytosine in hybridization behavior in the presence of bisulfite ions, while where the methylated cytosine residues were not significantly converted.
  • bisulfite treatment can be used to distinguish methylated CpG dinucleotides from unmethylated CpG dinucleotides.
  • the bisulfite reaction includes a deamination step and a desulfonate step (see Grigg and Clark, supra).
  • methylated cytosine residues are not significantly converted does not exclude very small percentages (eg, 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%) methylated cells Pyrimidine residues are converted to uracil, thymine, or other bases that differ in hybridization behavior from cytosine, although it is intended to convert only unmethylated cytosine residues.
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the bisulfite reagent is sodium bisulfite.
  • bisulfite reagents are commercially available, eg, MethylCode TM Bisulfite Conversion Kit, EpiMark TM Bisulfite Conversion Kit, EpiJET TM Bisulfite Conversion Kit, EZDNAMethylation-Gold TM Kit, and the like.
  • the bisulfite reaction is performed according to the kit's instructions for use.
  • the reagent of step (b) selectively cleaves unmethylated residues but not methylated residues, or selectively cleaves methylated residues but not unmethylated residues methylated residues.
  • the reagent of step (b) is a methylation-sensitive restriction enzyme (MSRE).
  • MSRE methylation-sensitive restriction enzyme
  • methylation-sensitive restriction enzyme refers to an enzyme that selectively digests nucleic acids based on the methylation status of its recognition site. For restriction enzymes that specifically cleave when the recognition site is not methylated or hemimethylated, when the recognition site is methylated, no cleavage occurs, or cleavage occurs at a significantly reduced efficiency . For restriction enzymes that specifically cleave when the recognition site is methylated, when the recognition site is not methylated, no cleavage occurs, or cleavage occurs at a significantly reduced efficiency.
  • the recognition sequence of the methylation-sensitive restriction enzyme contains a CG dinucleotide (eg, cgcg or cccggg). In some embodiments, methylation-sensitive restriction enzymes do not cleave when the cytosine in the CG dinucleotide is methylated at the C5 carbon atom.
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzymes, BbeI enzymes, NotI enzymes, SmaI enzymes, XmaI enzymes, MboI enzymes, BstBI enzymes, ClaI enzymes, MluI enzymes, NaeI enzymes, NarI enzymes, PvuI enzymes, SacII enzymes, Hhal enzymes, and any combination thereof.
  • methylation-sensitive restriction enzyme capable of discriminating between methylated and unmethylated CpG dinucleotides within the target region using methods known in the art
  • a series of restriction enzyme reagents are used to determine methylation, such as, but not limited to, Differential Methylation Hybridization ("DMH").
  • DMH Differential Methylation Hybridization
  • the DNA of step (a) can be cleaved prior to treatment with a methylation-sensitive restriction enzyme.
  • a methylation-sensitive restriction enzyme Such methods are known in the art and can include both physical and enzymatic means. It is particularly preferred to use one or more restriction enzymes that are insensitive to methylation and whose recognition sites are AT-rich and do not contain CG dinucleotides. The use of such enzymes allows the preservation of CpG sites and CpG-rich regions in DNA fragments.
  • such restriction enzymes are selected from MseI enzymes, BfaI enzymes, Csp6I15 enzymes, Trull enzymes, Tru9I enzymes, MaeI enzymes, XspI enzymes, and any combination thereof.
  • step (c) of the method according to the present application the target markers in the treated DNA obtained from step (b) are pre-amplified with a pool of pre-amplification primers, wherein at least a portion of each target marker is used is pre-amplified to obtain at least one pre-amplified product.
  • step (c) may also be referred to as a pre-amplification step.
  • amplification generally refers to any process that results in an increase in the copy number of a molecule or group of related molecules.
  • amplifying refers to the production of multiple copies of a polynucleotide molecule or a portion of a polynucleotide molecule, usually starting from a small number of polynucleotides, wherein the amplified material ( amplicons, PCR amplicons) are usually detectable.
  • Amplification of polynucleotides encompasses multiple chemical and enzymatic processes.
  • Forms of amplification include 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 ligation
  • the enzyme chain reaction generates multiple copies of DNA from one or a few copies of a template RNA or DNA molecule.
  • the term "marker of interest” refers to a nucleic acid or gene region of interest whose methylation levels are indicative of colorectal tumors (eg, colorectal advanced adenomas), or colorectal tumors (eg, colorectal advanced adenomas) Colorectal advanced adenoma) formation or risk of formation, or indicative of the progression or prognosis of colorectal tumor (eg, colorectal advanced adenoma).
  • the terms “marker” and “gene” are used interchangeably.
  • the term “marker” or “gene” shall be considered to include all transcriptional variants thereof and all promoter and regulatory elements thereof.
  • SNPs single nucleotide polymorphisms
  • SNPs include insertions and deletions of simple repeat sequences (eg, dinucleotide and trinucleotide repeats) of varying lengths. Accordingly, this application should be understood to extend to all forms of markers/genes resulting from any other mutation, polymorphism or allelic variation.
  • the terms "marker” and “gene” shall include both the sense strand sequence of the marker or gene and the antisense strand sequence of the marker or gene.
  • the term "marker of interest” as used herein is to be interpreted broadly to include both 1) the original marker (in a specific methylation state) found in a biological sample or genomic DNA, and 2) its processed sequence ( For example, the corresponding region after bisulfite conversion or the corresponding region after MSRE treatment).
  • the corresponding region after bisulfite conversion differs from the target marker in the genomic sequence in that one or more unmethylated cytosine residues are converted to uracil bases, thymine bases or in hybridization. Other bases that behave differently from cytosine.
  • the MSRE-treated corresponding region differs from the marker of interest in the genomic sequence in that the sequence is cleaved at one or more MSRE cleavage sites.
  • the markers of interest are divided into the following two groups: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and Intergenic Region A. Wherein, at least one target marker in each group is pre-amplified or at least a portion of at least one target marker in each group is pre-amplified.
  • At least 2 or all 3 markers of interest in said group (I) are amplified, and/or, at least 2, at least 3 or all 4 in said group (II) target markers are amplified.
  • Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in said panel (I) are amplified.
  • PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and intergenic region A in group (II) are amplified.
  • PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A in group (II) are amplified.
  • the following markers of interest in the treated DNA obtained from step (b) are pre-amplified with a pool of pre-amplification primers: Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and intergenic regions A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product.
  • Hg19 coordinates Hg19 coordinates
  • the respective target markers include or:
  • Hg19 coordinates as described above are available in public databases (eg UCSC Genome Browser, Ensemble and the NCBI website), as well as 5 kb upstream of each start site and downstream of each end site for each region 5kb.
  • the respective marker of interest also includes all variants thereof.
  • Variants include those from the same region that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% from the marker/gene region described herein Nucleic acid sequences with sequence identity (ie, having one or more deletions, insertions, substitutions, reverse sequences, etc.). Accordingly, the present disclosure should be understood to extend to such variants that achieve the same result despite the fact that the actual nucleic acid sequence has minor genetic variation among individuals.
  • percent (%) sequence identity refers to the same percentage of amino acid (or nucleic acid) residues of a candidate sequence after sequence alignment with amino acid (or nucleic acid) residues of a reference sequence, when aligned Spacers can be introduced (if necessary) to maximize the number of identical amino acids (or nucleic acids).
  • the percent (%) sequence identity of an amino acid sequence can be calculated by dividing the number of amino acid residues (or bases) that are identical to the reference sequence by the number of amino acid residues (or bases) in the candidate or reference sequence. bases), whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Percent amino acid (or nucleic acid) sequence identity can be determined, for example, using published tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J. Mol. Biol., 215:403–410 (1990); Stephen F.
  • step c) at least a portion of each marker of interest is pre-amplified to obtain at least one pre-amplified product.
  • the pre-amplified portion of the marker of interest is within a subregion of the marker of interest.
  • each subregion of the marker of interest comprises or:
  • each sub-region of the marker of interest comprises or is selected from a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1-7, or their bisulfite-converted corresponding regions, or their MSRE-treated the corresponding area.
  • the bisulfite-converted corresponding region of each subregion of the marker of interest comprises or is selected from the group of polynucleotide sequences: SEQ ID NOs: 8-21.
  • SEQ ID NOs for the subregions of each target marker and the corresponding regions after bisulfite conversion are listed in Table 1 below, and the specific sequences are provided in Figure 3.
  • Table 1 Exemplary subregions for each marker of interest
  • the sub-region of Septin9 comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 8, 15;
  • the sub-region of BCAT1 comprises a sequence selected from the group consisting of: SEQ ID NO: 2, 9, 16;
  • Subregions of IKZF1 comprise sequences selected from the group consisting of SEQ ID NOs: 3, 10, 17.
  • the subregion of PKNOX2 comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 11, 18; the subregion of SOX1 comprises a sequence selected from the group consisting of SEQ ID NO: 5, 12, 19;
  • Subregions of CRHBP comprise sequences selected from the group consisting of SEQ ID NOs: 6, 13, 20; subregions of intergenic region A comprise sequences selected from the group consisting of SEQ ID NOs: 7, 14, 21.
  • the target marker in the extracellular cell-free DNA is 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 0.3ng, no more than 0.2ng, no more than 0.1ng, no more than 0.09ng, no more than 0.08ng, no more than 0.07ng, no more than 0.06ng, no more than 0.05ng, no more than 0.04ng, no more than 0.03ng, An amount of no more than 0.02 ng or no more than 0.01 ng is present in the biological sample.
  • the marker of interest in the extracellular DNA is 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%, no more than 1% are present in the biological sample.
  • the concentration of the marker of interest in the extracellular free DNA in the biological sample is below the sensitivity level of the assay for the detection of the marker of interest. "Sensitivity of an assay” is a measure of the ability of an assay to discriminate between small differences in assay concentration/amount.
  • the methods disclosed herein are practical and superior in detecting very small amounts of target markers in a sample.
  • the marker of interest in extracellular free DNA is present in the biological sample in an amount of no more than 0.08 ng or no more than 0.04 ng.
  • the at least one pre-amplification product of step (c) is diluted with a diluent prior to the next step (ie, step (d)).
  • the diluent is selected from the group consisting of nuclease-free water, Tris-EDTA buffer, and any other buffer that does not inhibit PCR.
  • the pre-amplified DNA of step (c) is added directly to the next step (ie, step (d)) without prior dilution.
  • the target marker in the treated DNA is pre-amplified with a pool of pre-amplification primers.
  • primer refers to a single-stranded oligonucleotide capable of reacting under suitable conditions (eg buffer and temperature) with four different nucleoside triphosphates and reagents for polymerization ( For example, DNA polymerase) as the initiation point for template-directed DNA synthesis.
  • suitable conditions eg buffer and temperature
  • reagents for polymerization For example, DNA polymerase
  • the length of the primer depends, for example, on the intended use of the primer, and typically ranges from 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 to the template.
  • the primer site is the region on the template to which the primer hybridizes.
  • a primer pair is a set of primers that includes a 5' forward primer that hybridizes to the 5' end of the sequence to be amplified and a 3' reverse primer that hybridizes to the complementary strand of the 3' end of the sequence to be amplified.
  • Those skilled in the art can design primers according to the markers to be amplified based on common knowledge in the art (see, for example, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).
  • several software packages for designing optimal probes and/or primers for use in a wide variety of assays are publicly available, for example, from the Center for Genome Research, Cambridge, MA, USA.
  • Primer 3 obtained.
  • a primer designed for the purposes of the present invention may include at least one CpG site, or an amplification product obtained from the primer may include at least one CpG site.
  • Tools for designing primers to detect DNA methylation status are also known in the art, such as MethPrimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov;18(11):1427-31) .
  • any marker of interest (each at least a portion of a marker of interest or a sub-region of a marker of interest) in the treated DNA can be pre-amplified by using the pre-amplification primers as a primer pool.
  • oligonucleotide as used herein is defined as a molecule comprising two or more nucleotides (eg, deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably is at least about 10-15 nucleotides, more preferably at least about 15 to 30 nucleotides or longer (eg, oligonucleotides are typically less than 200 residues in length (eg, between 15 and 100 nucleotides) between nucleotides), however, 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.
  • nucleotides eg, deoxyribonucleotides or ribonucleotides
  • Oligonucleotides are often 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 hybridizing to themselves or to other polynucleotides. Such structures may include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or any combination thereof.
  • complementary refers to hybridization or base pairing between nucleotides or nucleic acids, eg, between two strands of a double-stranded DNA molecule, or primers on a single-stranded nucleic acid to be sequenced or amplified between the binding site and the oligonucleotide primer.
  • Complementary nucleotides are usually A and T (or A and U), or C and G.
  • RNA or DNA molecules are said to be complementary if they are approximately 98% to 100%) of nucleotide pairing.
  • complementarity exists when an RNA or DNA strand hybridizes to its complementary sequence under selective hybridization conditions.
  • 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.
  • the pre-amplification primer pool comprises at least one methylation-specific primer pair. In some embodiments, the pre-amplification primer pool comprises a plurality of methylation-specific primer pairs. In some embodiments, the pre-amplification step is performed by methylation-specific PCR ("MSP"), which is PCR using methylation-specific primers.
  • MSP methylation-specific PCR
  • methylation-specific primer pair refers to a primer pair that is specifically designed to recognize CpG sites to take advantage of differences in methylation to amplify specific markers of interest in processed DNA. Primers only act on molecules with or without a specific methylation state.
  • primers can be oligonucleotides that can specifically hybridize to a specific CpG site with methylation in a methylation-specific manner, but not to Methylated specific CpG sites hybridize. Thus, the primers will specifically amplify target markers that are methylated at specific CpG sites.
  • a primer can be an oligonucleotide that can specifically hybridize to a specific unmethylated CpG site in a methylation-specific manner under stringent, moderately, or highly stringent conditions, but Cannot hybridize to specific CpG sites that are methylated.
  • the primers will specifically amplify target markers that are not methylated at specific CpG sites.
  • methylated and unmethylated CpG sites can be differentiated using methylation-specific primers in the pre-amplification of at least one marker of interest within the treated DNA.
  • the methylation-specific primer pairs of the present application comprise at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide.
  • sequence of the primer specific for methylated DNA comprises at least one CpG dinucleotide
  • sequence of the primer specific for unmethylated DNA comprises a "T" at the C position of the CpG, and/or contain "A” at the G position in the CpG.
  • the at least one methylation-specific primer pair comprises a forward primer and a reverse primer, the primers each comprising an oligonucleotide sequence that is identical to the target
  • At least 9 contiguous nucleotides of one of the markers (or a subregion of the marker of interest) hybridize under stringent conditions, under conditions of moderate stringency, or under conditions of high stringency, wherein one of the markers of interest (or the marker of interest) A subregion of at least 9 consecutive nucleotides comprising at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites.
  • hybridization can refer to a process in which two single-stranded polynucleotides are joined in non-covalent form to form a stable double-stranded polynucleotide.
  • the resulting double-stranded polynucleotide can be a "hybrid” or "double-stranded.”
  • the salt concentration in “hybridization conditions” is usually less than about 1 M, often less than about 500 mM and can be less than about 200 mM.
  • “Hybridization buffer” includes buffered saline solutions, such as 5% SSPE, or other such buffers known in the art.
  • Hybridization temperatures can be as low as 5°C, but are typically above 22°C, and more typically above about 30°C, and often in excess of 37°C.
  • Hybridization is generally carried out under stringent conditions, ie, conditions under which a sequence will hybridize to its target sequence but not to other non-complementary sequences.
  • Stringent conditions are sequence-dependent and will vary in different circumstances. For example, longer fragments may require higher hybridization temperatures than shorter fragments for specific hybridization. Since other factors may affect the stringency of hybridization, including base composition and length of complementary strands, the presence of organic solvents, and the extent of base mismatches, the combination of parameters is more important than absolute measurements of any one parameter alone.
  • Stringent conditions are generally selected to be about 5°C lower than the melting temperature (Tm) of a particular sequence at a particular ionic strength and pH.
  • Tm can be the temperature at which half of the population of double-stranded nucleic acid molecules are separated into single strands.
  • the stability of hybrids is a function of ion concentration and temperature.
  • hybridization reactions are performed under conditions of lower stringency, followed by washing in washes of different but higher stringency.
  • Exemplary stringent conditions include a pH of 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.
  • 5x SSPE 750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4
  • 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 hybridized region.
  • the "stringency of hybridization" to determine 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): 0.2 x SSPE, 0.1% SDS, 50°C; 3) Low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It will be appreciated that the same stringency can be achieved using alternative buffers, salts and temperatures.
  • moderately stringent hybridization can refer to conditions that allow a nucleic acid molecule (eg, a probe) to bind a complementary nucleic acid molecule.
  • Hybridized nucleic acid molecules typically are at least 60% identical, including, for example, at least 70%, 75%, 80%, 85%, 90%, or 95% identical.
  • Moderately stringent conditions may be equivalent to: 42°C, 50% formamide, 5x Denhardt's 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, hybridization at 42°C, 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C.
  • Hybridization of low stringency may be equivalent to the following conditions: hybridization at 22°C, 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS, followed by washing in 1x SSPE, 0.2% SDS at 37°C.
  • Denhardt's solution contained 1% Ficoll, 1% Polyvinylpyrrolidone and 1% Bovine Serum Albumin (BSA).
  • BSA Bovine Serum Albumin
  • 20x SSPE Sodium Chloride, Sodium Phosphate, EDTA
  • the pre-amplification primer pool further comprises a control primer pair for amplifying a control marker.
  • a control marker is a nucleic acid of known characteristics (eg, sequence is known, copy number per cell known) for comparison to an experimental target (eg, nucleic acid of unknown concentration).
  • a control can be an endogenous, preferably invariant, gene against which the experimental nucleic acid or target nucleic acid under analysis can be normalized. Such controls normalized for differences between samples may occur, for example, in sample processing, analytical efficiency, etc., and allow accurate data comparison between samples, quantitative analysis of amplification efficiency and bias.
  • 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.
  • sequences of the control primer pairs are set forth in SEQ ID NOs: 44 and 45 of Table 2 below.
  • the methylation-specific primer pair for Septin9 is SEQ ID NO:22/23; the methylation-specific primer pair for BCAT1 is SEQ ID NO:24/25; the methylation-specific primer pair for IKZF1 The pair is SEQ ID NO:26/27; the methylation-specific primer pair for PKNOX2 is SEQ ID NO:28/29; the methylation-specific primer pair for CRHBP is SEQ ID NO:30/31; the methylation-specific primer pair for SOX1 The methylation-specific primer pair is SEQ ID NO: 32/33; the methylation-specific primer pair for the intergenic region A is SEQ ID NO: 34/35.
  • the methylation-specific primer pair comprises at least one pair of nucleotide sequences selected from the group consisting of: SEQ ID NOs: 22/23, 24/25, 26/27 as shown in Table 2 below , 28/29, 30/31, 32/33 and 34/35.
  • the methylation-specific primer pair comprises at least one of SEQ ID NO: 22/23, 24/25 and 26/27, and SEQ ID NO: 28/29, 30/31, 32 At least one primer pair in /33 and 34/35.
  • the methylation-specific primer pair comprises: SEQ ID NOs: 22/23, 24/25, 26/27, 28/29, 30/31, 32/33 and 34/35.
  • SEQ ID NO: 22/23 refers to a primer pair having the nucleic acid sequences shown in SEQ ID NO: 22 and SEQ ID NO: 23 shown in Table 2 below, respectively.
  • the target marker is amplified in the presence of one or more blocking oligonucleotides.
  • the use of such blocking oligonucleotides has been described in Yu et al., BioTechniques 23:714-720, 1997.
  • the blocking sequence and the preamplification primer pair hybridize to the treated DNA simultaneously.
  • Pre-amplification of the target marker terminates 5' of the blocking sequence such that pre-amplification of the target marker is inhibited in the presence of a sequence complementary to the blocking sequence.
  • Blocking sequences can be designed to hybridize to the treated DNA in a methylation state-specific manner.
  • amplification of unmethylated nucleic acids at the relevant positions can be inhibited by the use of blocking sequences comprising "CpA” or “TpA” at the relevant positions,
  • CpG is used if inhibition of methylated nucleic acid amplification is desired.
  • blocking oligonucleotides For PCR methods using blocking oligonucleotides, effective interference with polymerase-mediated amplification requires that the blocking oligonucleotides cannot be extended by the polymerase.
  • a blocker which is a 3'-deoxyoligonucleotide or an oligonucleotide derivatized at the 3' position with a "free" hydroxyl group other than that.
  • 3'-O-acetyl oligonucleotides are representative of a preferred class of blocker molecules.
  • polymerase-mediated cleavage of the blocking oligonucleotide should be prevented.
  • blocking involves the use of polymerases that lack 5'-3' exonuclease activity, or the use of, for example, modified blocking oligonucleotides having a thiolate bridge at their 5'-terminus, allowing the blocking molecule to Nuclease resistant.
  • Certain applications may not require 5' modification of the blocker. For example, if the blocker binding site and the primer binding site overlap, thereby preventing primer binding (eg, using excess blocker), degradation of the blocking oligonucleotide will be substantially prevented. This is because the polymerase does not extend the primer to and beyond the blocker (in the 5'-3' direction), a process that typically results in degradation of the hybridized blocking oligonucleotide.
  • particularly preferred blocker/PCR embodiments include the use of peptide nucleic acid (PNA) oligomers as blocking oligonucleotides.
  • PNA peptide nucleic acid
  • Such PNA-blocked oligomers are ideal because they are neither cleaved nor extended by polymerases.
  • the at least one marker of interest is pre-amplified with DNA polymerase.
  • DNA polymerase refers to an enzyme that catalyzes the synthesis of polydeoxyribonucleotides from monodeoxyribonucleotide triphosphates (dNTPs), which accomplishes the functions of DNA replication, repair and, in some cases, cell differentiation the most basic functions in .
  • dNTPs monodeoxyribonucleotide triphosphates
  • DNA polymerases in prokaryotes examples include DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, and DNA polymerase V.
  • DNA polymerases I, II and III are known in 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 ends of growing strands.
  • Retroviruses have a unique DNA polymerase, reverse transcriptase, which synthesizes DNA using an RNA template.
  • DNA polymerases are the polymerases alpha, beta, lambda, gamma, sigma, mu, delta, epsilon, n, iota, kappa, zeta, theta, and Rev1.
  • the DNA polymerase of animal cells is responsible for the replication of DNA in the nucleus and mitochondria.
  • the PCR reagents used in the pre-amplification step can be any commercially available PCR mix (e.g. KAPA2G Fast Multiplex PCR Kit, Universal Probe qPCR Master Mix, EpiTect MethyLightPCRKit, etc.), which are used to amplify processed DNA.
  • PCR reagents including Mg2+ , dNTPs, DNA polymerase, etc. in the laboratory.
  • Those skilled in the art can also select a suitable PCR reaction system and PCR reaction conditions according to actual needs.
  • the pre-amplification of step (c) comprises 5 to 30 reaction cycles, wherein each cycle comprises a reaction at 85-99°C for 5 seconds to 5 minutes, followed by a reaction at 40-80°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 a reaction at 90-99°C for 15 seconds to 2 minutes, followed by a reaction at 45-60°C for 30 seconds to 3 minutes. In some embodiments, the pre-amplification of step (c) includes 15 reaction cycles, wherein each cycle includes a reaction at 95°C for 30 seconds, followed by a reaction at 56°C for 60 seconds.
  • step (d) of the method according to the present application the methylation level of each target marker is quantitatively analyzed based on its respective pre-amplification product obtained in step (c), respectively.
  • steps may also be named as quantitative analysis steps.
  • methylation status refers to the presence, absence and/or amount of methylation of a particular nucleotide or nucleotides within a region of DNA.
  • the methylation status of a particular DNA sequence can indicate the methylation status of each base in the sequence, or can indicate the methylation status of a subset of base pairs in the sequence (for example, the methylation status of cytosine residues or the methylation status of one or more specific restriction enzyme recognition sequences), or may indicate information on the methylation density of a region in the sequence, although it does not provide information on the occurrence of methylation Precise information on where in the sequence.
  • Methylation status can optionally be represented or indicated by "methylation level”. Methylation levels can be determined, for example, by quantitative analysis of the amount of intact DNA present after restriction digestion with methylation-sensitive restriction enzymes. In this example, if quantitative PCR is used to quantify a specific sequence in DNA, the amount of template DNA is approximately equal to the mock-treated control indicating that the sequence is not hypermethylated, while the amount of template is significantly less than in the mock-treated sample The amount of template indicated the presence of methylated DNA in the sequence. Therefore, the methylation level as in the above example represents the methylation status and can therefore be used as a quantitative indicator of the methylation status. This is especially useful when the methylation status of sequences in a sample needs to be compared to a threshold level.
  • 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 " Hemimethylation".
  • the term “hemimethylation” refers to the methylation state of double-stranded DNA in which only one of its strands is methylated.
  • the term “hypermethylation” refers to the presence of one or more CpG dinucleotides in the DNA sequence of a tested DNA sample relative to the amount of 5-methylcytosine at the corresponding CpG dinucleotide in a normal control DNA sample. Average methylation status corresponding to an increase in the number of 5-methylcytosines at nucleotides.
  • the methylation status of a residue can be a qualitative read or a quantitative read, eg, expressed by methylation level.
  • methylation status and “methylation level” are used interchangeably. According to the present application, more than one different methylation level can be determined simultaneously.
  • the methylation level of each target marker is quantitatively analyzed based on its respective pre-amplification product obtained in step (c).
  • the methylation level/status of one or more CpG dinucleotide sequences within a DNA sequence can be determined by various analytical methods known in the art.
  • the quantitative analysis of step (d) is performed by polymerase chain reaction (eg, real-time polymerase chain reaction, digital polymerase chain reaction), nucleic acid sequencing, mass-based separation (eg electrophoresis, mass spectrometry) or target capture (eg hybridization, microarray).
  • polymerase chain reaction eg, real-time polymerase chain reaction, digital polymerase chain reaction
  • nucleic acid sequencing e.g, nucleic acid sequencing
  • mass-based separation eg electrophoresis, mass spectrometry
  • target capture eg hybridization, microarray
  • methylation levels of each marker of interest are individually quantified using MSP (see Herman, supra) based on pre-amplified DNA. For example, by using one or more primers that hybridize specifically to the unconverted sequence under moderate and/or high stringency conditions, amplification products are produced only when the template contains methylated cytosines at the CpG site.
  • the quantitative analysis of step (d) is performed by real-time PCR.
  • real-time PCR include HeavyMethyl TM PCR described by Cottrell et al., Nucl. Acids Res. 32:e10, 2003; MethyLight TM PCR described by Eads et al., Cancer Res. 59:2302-2306, 1999; Headloop PCR as described by Rand et al., Nucl. Acids Res. 33:e 127, 2005.
  • HeavyMethyl TM PCR refers to an art-recognized real-time PCR technique in which one or more non-extensible nucleic acid (eg, oligonucleotide) blockers are conjugated to sub-blocks in a methylation-specific manner.
  • Bisulfate-treated nucleic acid binding ie, the block specifically binds to unmutated DNA under moderate to high stringency conditions.
  • the amplification reaction is performed using one or more primers, which may optionally be methylation specific, but flanked by one or more blockers.
  • the blocker binds and no PCR product is produced.
  • the level of methylation of nucleic acids in a sample is determined using a TaqMan TM analytical method substantially as described, eg, by Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991.
  • Methods of Methods of Methods of Methods of Methods of Methods of Methods of Methods refers to an art-recognized fluorescence-based real-time PCR technique that employs dual-labeled fluorescent oligonucleotide probes called TaqMan TM probes and is designed to CpG-rich sequences that can be co-located between the forward and reverse amplification primers.
  • the TaqMan TM probes comprise a fluorescent "reporter moiety” and "quencher moiety” covalently bound to a linker moiety (eg, a phosphoramidite) linked to the nucleotides of the TaqMan TM oligonucleotide.
  • TaqMan TM probes hybridized to CpG-rich sequences are cleaved by the 5' nuclease activity of Taq polymerase, resulting in a signal that is detected in real-time during the PCR reaction.
  • 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).
  • Headloop PCR refers to an art-recognized type of real-time PCR that selectively amplifies a target nucleic acid, but suppresses non-specific amplification by extending the 3' stem-loop into a hairpin structure that cannot further provide template for amplification Amplification of Amplification Target Variants.
  • the real-time PCR is multiplex real-time PCR.
  • the term "multiplex" may refer to the use of more than one label, each label having at least one distinct detection characteristic, such as a fluorescence characteristic (eg, excitation wavelength, emission wavelength, emission intensity, FWHM (half peak) (full width at height) or fluorescence lifetime) or unique nucleic acid or protein sequence features, assays or other analytical methods that can simultaneously determine the presence and/or amount of multiple markers (eg, multiple nucleic acid sequences).
  • a fluorescence characteristic eg, excitation wavelength, emission wavelength, emission intensity, FWHM (half peak) (full width at height) or fluorescence lifetime
  • unique nucleic acid or protein sequence features eg, assays or other analytical methods that can simultaneously determine the presence and/or amount of multiple markers (eg, multiple nucleic acid sequences).
  • the quantitative analysis of step (d) is performed by nucleic acid sequencing.
  • nucleic acid sequencing Exemplary methods of nucleic acid sequencing are known in the art, see, eg, Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992; Clark et al., Nucl. Acids Res. 22: 2990-2997, 1994.
  • the identification of methyl groups in DNA sequences can be facilitated by comparing sequences obtained from samples not treated with bisulfite or known nucleotide sequences of target regions to sequences obtained from samples treated with bisulfite Cytosine.
  • a thymine residue detected at any cytosine site in a bisulfite-treated sample compared to an untreated sample can be considered a mutation caused by bisulfite treatment, i.e., the presence of methylated cytosines.
  • Methods for sequencing DNA 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).
  • the quantitative analysis of step (d) is performed by mass-based separation (eg, electrophoresis, mass spectrometry).
  • mass-based separation eg, electrophoresis, mass spectrometry
  • 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.
  • COBRA bisulfite restriction analysis
  • This method utilizes restriction enzymes between methylated and unmethylated nucleic acids following treatment with compounds that can selectively mutate unmethylated cytosine residues (eg, bisulfite) Identify differences in loci.
  • the restriction endonuclease Taq1 cleaves the sequence TCGA, which would be TTGA after bisulfite treatment of unmethylated nucleic acid and thus would not be cleaved.
  • Digested and/or undigested nucleic acids are then detected using detection means known in the art such as electrophoresis and/or mass spectrometry.
  • nucleic acid differences in amplified products based on differences in nucleotide sequence and/or secondary structure, such as methyl methacrylate.
  • MS-SSCA Methylation-specific single-strand conformation analysis
  • MS-DGGE methylation-specific denaturing gradient gel electrophoresis
  • MS-DHPLC methylation-specific denaturing high performance liquid chromatography
  • the quantitative analysis of step (d) is performed by target capture (eg, hybridization, microarray).
  • target capture eg, hybridization, microarray
  • probes for hybridization assays are detectably labeled.
  • nucleic acid-based probes for hybridization analysis are unlabeled. Such unlabeled probes can be immobilized on solid supports, such as microarrays, and can hybridize to detectably labeled target nucleic acid molecules.
  • microarray is a methylation-specific microarray, which can be used to distinguish sequences with converted cytosine residues from sequences with unconverted cytosine residues (see Adorjan et al., Nucl. Acids Res. , 30:e21, 2002).
  • Hybridization-based assays can also be used for nucleic acids after treatment with methylation-sensitive restriction enzymes.
  • the methylation status of CpG dinucleotide sequences within a DNA sequence can be determined by oligonucleotide probes that hybridize to bisulfite-treated DNA simultaneously with PCR amplification primers (wherein the primers may be methylation specific primers or standard primers).
  • step (d) is performed in the presence of a detection reagent.
  • detection reagent is a reagent used in a quantitative analysis step to detect the presence, absence or amount of nucleic acid.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • the probe for detecting Septin9 is SEQ ID NO:36; the probe for detecting BCAT1 is SEQ ID NO:37; the probe for detecting IKZF1 is SEQ ID NO:38; the probe for detecting PKNOX2 It is SEQ ID NO: 39 and 40; the probe for detecting CRHBP is SEQ ID NO: 41; the probe for detecting SOX1 is SEQ ID NO: 42; the probe for detecting intergenic region A is SEQ ID NO: 43.
  • the probe comprises at least one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 36-38, SEQ ID NOs: 41-43, and SEQ ID NOs: 39 and/or or SEQ ID NO:40.
  • the probes comprise at least one, at least two or all three nucleotide sequences of SEQ ID NOs: 36-38 and SEQ ID NOs: 39 and/or 40 and SEQ ID NOs: 41-43 At least one, at least two or at least three nucleotide sequences.
  • the probes include the nucleotide sequences set forth in SEQ ID NOs: 36-38 and 41-43, and the nucleotide sequences set forth in SEQ ID NOs: 39 and/or 40.
  • control marker is ACTB
  • nucleotide sequence of an exemplary probe is set forth in SEQ ID NO:58.
  • 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).
  • a fluorescent dye eg, FAM, HEX/VIC, TAMRA, Texas Red, or Cy5
  • a quencher eg, BHQ1, BHQ2, BHQ3, DABCYL
  • Marking can be done by direct or indirect methods.
  • Direct labeling involves direct (covalent or non-covalent) coupling of the label to the reagent.
  • Indirect labeling involves the binding (covalent or non-covalent) of the second reagent to the first reagent.
  • the second reagent should specifically bind to the first reagent.
  • the second reagent can be conjugated to a suitable label and/or the second reagent is the target (receptor) of the third reagent to which the second reagent can bind.
  • the use of secondary, tertiary, or even higher-order reagents often increases signal intensity.
  • Suitable secondary and advanced reagents can include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.).
  • the reagent or substrate can also be "labeled" with one or more tags known in the art.
  • the quantitative analysis of step (d) comprises using a quantitative primer pair and a DNA polymerase to amplify the pre-amplification product of step (c), wherein the pre-amplification product is amplified by amplification increase product.
  • quantitative primer pair refers to one or more primer pairs used in a quantitative analysis step.
  • the quantitative primer pair used in step (d) is capable of at least 9 contiguous nucleotides of the pre-amplification product of step (c) under stringent conditions, moderately stringent conditions, or highly stringent conditions Hybridization conditions. In some embodiments, the quantitative primer pair used in step (d) is the same as the methylation-specific primer pair in the preamplification primer pool of step (c).
  • the quantitative primer pair used in step (d) is designed to amplify at least a portion of the pre-amplified product of step (c), ie steps (c) and (d) are designed to Nested PCR.
  • Nested PCR is a modification of PCR designed 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 generate the first amplicon, and a second round of amplification is performed using a primer pair in which one or both primers anneal to a site within the region bounded by the initial primer pair, the second Primer pairs are considered "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 not further amplified in the second PCR reaction.
  • the quantitative analysis of step (d) comprises determining each of the plurality of CpG dinucleotides, TpG dinucleotides or CpA dinucleotides based on the presence or level of Methylation levels of target markers. In some embodiments, the quantitative analysis of step (d) comprises determining the methylation level of cytosine residues based on the presence or level of one or more CpG dinucleotides in the pre-amplification product. In some embodiments, the quantitative analysis of step (d) comprises determining the methylation level of cytosine residues based on the presence or level of one or more TpG dinucleotides in the pre-amplification product. In some embodiments, the quantitative analysis of step (d) comprises determining the methylation level of cytosine residues based on the presence of CpA dinucleotides in the pre-amplification product.
  • the quantitative analysis step is performed by dividing the pre-amplification product of step (c) into multiple components.
  • a plurality of different quantification experiments are performed on a plurality of components, wherein the difference in the pre-amplification product (if present in the component) is quantified in one of the plurality of components combination.
  • the control marker in each fraction is quantified.
  • step (e) of the method of diagnosing colorectal advanced adenoma, screening for risk of colorectal advanced adenoma formation or formation, or assessing the progression or prognosis of colorectal advanced adenoma in an individual step (d) The methylation levels of the obtained target markers were compared with the corresponding reference levels, wherein the target markers described in group (I) had higher methylation levels relative to their corresponding reference levels, and group (I) had higher methylation levels.
  • the target marker has a lower methylation level relative to its corresponding reference level indicating that the individual has advanced colorectal adenoma, or the individual has advanced colorectal adenoma formation or or the individual has an increased likelihood of developing or developing advanced colorectal adenoma, or the individual has a poor prognosis or a risk of a poor prognosis for colorectal advanced adenoma.
  • Step (e) of the method according to the present application may also be referred to as a comparison step.
  • comparing refers to comparing the methylation level of the target marker obtained by the quantitative analysis step contained in the detected biological sample with its corresponding reference level, respectively. It should be understood that the terms used herein refer to the comparison of corresponding parameters or values, for example, comparing an absolute amount to an absolute reference amount, comparing a concentration to a reference concentration, or an intensity signal obtained from a tested sample to that of a reference sample Compare the intensity signals of the same type. Comparisons can be made manually or computer-assisted. For computer-assisted comparisons, the value of the determined quantity may be compared to the value of a suitable reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison and automatically provide the desired evaluation in an appropriate output format.
  • the term "reference level” refers to a threshold level at which an individual is included in or excluded from colorectal advanced adenoma or the development or risk of developing colorectal advanced adenoma.
  • methylation level of the target marker detected in panel (I) is at least 1.1, 1.2, 1.3 of its corresponding reference level , 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.
  • Having a lower methylation level relative to its corresponding reference level means that the methylation level of the target marker detected in panel (II) is 98%, 95%, 93% of its corresponding reference level %, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or even lower.
  • no group (I) The methylation level of each target marker was higher than its corresponding reference level and the methylation level of each target marker of panel (II) was lower than its corresponding reference level. Precisely, it is sufficient if the methylation level of at least one target marker in each group to be quantitatively analyzed in the quantitative analysis step is equal to or higher than its corresponding reference level.
  • the reference level of methylation of the marker of interest can be derived from one or more reference samples, wherein the reference level is obtained from an experiment performed in parallel with the experiment to detect the sample of interest.
  • reference levels may be obtained in a database comprising a collection of data, standards or levels from one or more reference samples or disease reference samples.
  • sets of data, criteria or levels are normalized so as to be usable for the purpose of comparison with data from one or more samples.
  • "Standardization" is the process of converting measurement raw data into data that can be directly compared with other standardized data. Normalization is used to overcome assay-specific errors caused by different factors in different assays, such as differences in sample loading, differences in binding efficiencies, differences in detection sensitivity, and other types of errors.
  • the reference database includes methylation levels of target markers and/or other laboratory and clinical data from one or more reference samples.
  • the reference database includes the methylation levels of the marker of interest, each normalized to a percentage of the methylation level of the control marker detected under the same conditions as the reference sample. To compare with such normalized methylation levels of the target marker, the methylation level of the target marker of the test sample was also measured and calculated as the methylation level of the control marker detected under the same conditions as the test sample percentage.
  • the reference database is established by aggregating reference level data from reference samples obtained from healthy individuals and/or non-tumor individuals (ie, individuals known to be free of tumors). In some embodiments, the reference database is established by aggregating reference level data from reference samples obtained from individuals undergoing treatment for colorectal tumors. In some embodiments, the reference database is established by aggregating data obtained from reference samples of individuals with different stages of colorectal tumor evidenced by, for example, different methylation levels of a marker of interest .
  • the reference level can be selected by one skilled in the art according to the desired sensitivity and specificity. Means of determining suitable reference levels are known to those skilled in the art, eg reference levels may be determined from data collected in clinical studies.
  • the reference level of step (e) is based on clinical samples obtained from a group of individuals with or at risk of developing colorectal advanced adenoma and never having colorectal advanced adenoma It was determined from clinical samples obtained from a group of individuals with advanced colorectal adenomas or not at risk for developing colorectal advanced adenomas.
  • One of skill in the art can determine whether an individual has or is at risk for developing colorectal advanced adenoma based on various factors, such as age, gender, medical history, family history, symptoms, and the like.
  • a cycle threshold (ie, Ct value) is used to represent the methylation level of the marker of interest and the reference level.
  • Ct value refers to the number of cycles at which fluorescence of a PCR product can be detected above background signal. The Ct value is inversely proportional to the amount of target marker in the sample, that is, the lower the Ct value, the greater the amount of target marker in the sample.
  • step (e) of the method of diagnosing colorectal advanced adenoma screening for the formation or risk of colorectal advanced adenoma, or assessing the progression or prognosis of colorectal advanced adenoma in an individual
  • group (I ) at least one of said target markers has a low Ct value relative to its corresponding reference Ct value
  • group (II) at least one of said target markers has a Ct value relative to its corresponding reference
  • a high Ct value indicates that the individual has advanced colorectal adenoma, or that the individual is at risk of developing or developing advanced colorectal adenoma, or that the individual is at risk of developing or developing advanced colorectal adenoma increased sex, or the individual is at or at risk of poor prognosis or poor prognosis in advanced colorectal adenomas.
  • the individual is determined to have advanced colorectal adenoma, or the individual is at risk for the formation or formation of advanced colorectal adenoma, or the individual There is an increased likelihood of developing or developing advanced colorectal adenoma, or the individual is at or at risk of having a poor prognosis or a poor prognosis for colorectal advanced adenoma.
  • the term "increased likelihood” refers to an overall increase in the level of likelihood of an individual developing colorectal advanced adenoma or having a poor prognosis with colorectal advanced adenoma as compared to the individual from which the reference sample was obtained5 %, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.
  • the present application also provides a kit for diagnosing colorectal advanced adenoma, screening for the formation or risk of colorectal advanced adenoma, or assessing the progression or prognosis of colorectal advanced adenoma , which contains:
  • target markers include each of the following two groups of target markers at least one of the markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and Intergenic Region A; and optionally
  • a first primer pool comprising primer pairs for pre-amplifying at least one target marker in each of the following two groups of target markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A, wherein the primer pair can be linked to at least 9 consecutive nucleotides of the target sequence of the target marker after treatment with the first reagent Hybridizes under stringent, moderately stringent or highly stringent conditions, wherein the target sequence comprises at least one CpG site.
  • the kit comprises:
  • a first primer pool comprising primer pairs for pre-amplifying at least one target marker in each of the following two groups of target markers: (1) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A, wherein the primer pair can be linked to at least 9 consecutive nucleotides of the target sequence of the target marker after treatment with the first reagent hybridizes under stringent conditions, moderately stringent conditions or highly stringent conditions, wherein the target sequence comprises at least one CpG site;
  • the second reagent comprises a second primer pool comprising a plurality of quantitative primer pairs capable of interacting with the quantitation primers pre-amplified by the first primer pool At least 9 contiguous nucleotides of the target sequence hybridize under stringent, moderately stringent or highly stringent conditions.
  • At least one quantitative primer pair in the second primer pool is the same as at least one primer pair in the first primer pool. In some embodiments, quantitative primer pairs in the second primer pool are designed to amplify at least a portion of the target sequence pre-amplified by the first primer pool. In some embodiments, the first primer pool comprises at least one methylation-specific primer pair.
  • the first primer pool and the second primer pool are packaged in a single container or in separate containers.
  • the kit further comprises one or more blocking oligonucleotides.
  • the kit further comprises detection reagents.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • the probe comprises at least one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 36-38, SEQ ID NOs: 41-43, and SEQ ID NOs: 39 and/or or SEQ ID NO:40.
  • the probes comprise at least one, at least two or all three nucleotide sequences of SEQ ID NOs: 36-38 and SEQ ID NOs: 39 and/or 40 and SEQ ID NOs: 41-43 At least one, at least two or at least three nucleotide sequences.
  • the probes include the nucleotide sequences set forth in SEQ ID NOs: 36-38 and 41-43, and the nucleotide sequences set forth in SEQ ID NOs: 39 and/or 40.
  • 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, TAMRA or lowa Black Dark Quenchers).
  • a fluorescent dye eg, FAM, HEX/VIC, TAMRA, Texas Red, or Cy5
  • a quencher eg, BHQ1, BHQ2, BHQ3
  • 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 contains instructions for use and/or an explanation of the test results of the kit.
  • the kit may comprise a reaction buffer optimized for polymerase-mediated primer extension (eg, PCR) packaged in a separate container.
  • a kit which further comprises a container adapted to contain at least one (eg 2, 3, 4 or 5) target markers of the lower group (I) in a biological sample from an individual and means for the methylation of at least one (eg 2, 3 or 4) target markers in the following group (II): (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic regions A.
  • the first reagent comprises a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE).
  • the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, sulfurous acid Hydrogen ions, and any combination thereof.
  • the bisulfite reagent is sodium bisulfite.
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzymes, BbeI enzymes, NotI enzymes, SmaI enzymes, XmaI enzymes, MboI enzymes, BstBI enzymes, ClaI enzymes, MluI enzymes, NaeI enzymes, NarI enzymes, PvuI enzymes, SacII enzymes, Hhal enzymes, and any combination thereof.
  • the first primer pool comprises at least one methylation-specific primer pair for pre-amplifying at least one target sequence from at least one target marker selected from the following group (I): (I ) Septin9, BCAT1 and IKZF1. In some embodiments, the first primer pool comprises at least one methylation-specific primer pair for pre-amplifying at least one target sequence from at least one target marker selected from the group (II): PKNOX2, SOX1, CRHBP and intergenic region A.
  • the first primer pool comprises at least two methylation-specific primer pairs, one of which is used to pre-amplify at least one target sequence selected from at least one target marker selected from group (I) below : (I) Septin9, BCAT1 and IKZF1, another for pre-amplification of at least one target sequence selected from at least one target marker selected from group (II): PKNOX2, SOX1, CRHBP and Intergenic Region A.
  • the first primer pool comprises primer pairs for pre-amplifying two sets of markers of interest: at least 2 or all 3 markers of interest in the set (I), and the At least 2, at least 3 or all 4 markers of interest from group (II).
  • the first primer pool comprises primer pairs for pre-amplifying set (I) of the following markers of interest: Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 are amplified.
  • the first primer pool comprises primer pairs for the following markers of interest for pre-amplification set (II): PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and gene Spacer A, or SOX1 and intergenic region A.
  • the first primer pool comprises primer pairs for the following markers of interest for pre-amplification set (II): PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and Intergenic Region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A.
  • the first primer pool comprises primer pairs for pre-amplifying the following markers of interest: Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and Intergenic Region A; wherein the markers of interest At least a portion of is pre-amplified to obtain a pre-amplified product.
  • the respective target markers include or:
  • the first primer pool comprises at least one or more primer pairs selected from the group consisting of: SEQ ID NOs: 22/23, 24/25, 26/27, 28/29, 30/ 31, 32/33 and 34/35.
  • the first primer pool comprises at least one pair, at least two pairs or all three primer pairs selected from the group (I) below and at least one pair, at least two pairs, at least three pairs of primers from the group (II) below Pairs: (I) SEQ ID NOs: 22/23, 24/25, 26/27; (II) SEQ ID NOs: 28/29, 30/31, 32/33 and 34/35.
  • the first primer pool comprises the following primer pairs: SEQ ID NOs: 22/23, 24/25, 26/27, 28/29, 30/31, 32/33, and 34/35, any of Optionally wherein the second primer pool comprises at least one primer pair that is identical to at least one primer pair in the first primer pool. In some embodiments, the primer pairs in the second primer pool are identical to the primer pairs in the first primer pool.
  • the first primer pool and the second primer pool further comprise primer pairs for amplifying a control marker.
  • 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.
  • the kit further comprises a plurality of containers, each container for receiving components of the second primer pool.
  • the kit further comprises standard reagents useful for performing CpG position-specific methylation analysis, wherein the analysis includes one or more of the following techniques: MS-SNuPE, MSP, MethyLight TM , HeavyMethyl TM , COBRA and nucleic acid sequencing.
  • the kit may comprise additional reagents selected from the group consisting of buffers (eg, restriction enzymes, PCR, storage or washing buffers), DNA recovery reagents or kits (eg, precipitation, ultrafiltration, Affinity column) and DNA recovery components.
  • buffers eg, restriction enzymes, PCR, storage or washing buffers
  • DNA recovery reagents or kits eg, precipitation, ultrafiltration, Affinity column
  • kits of the present application may comprise:
  • a first primer pool comprising a plurality of methylation-specific primer pairs for pre-amplifying at least one target marker in lower group (I) and at least one target in lower group (II) Markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and Intergenic Region A, wherein the methylation specific primer pair comprises or consists of the following primer pair: SEQ ID NO: 22/23, 24/25, 26/27, 28/29, 30/31, 32/33 and 34/35;
  • a second reagent for quantitatively analyzing the methylation level of each of the target markers pre-amplified by the first primer pool, wherein the second reagent comprises a second primer pool,
  • the second primer pool comprises a plurality of quantitative primer pairs, the quantitative primer pairs capable of being pre-amplified by the first primer pool with at least 9 consecutive nucleotides of the target sequence under stringent conditions, medium stringency Hybridization conditions or high stringency conditions.
  • kits of the present application may also contain other components packaged in separate containers, such as buffers or solutions suitable for blocking, washing or coating.
  • the kit of the present application may further comprise one or more of the following components known in the field of DNA enrichment: a protein component that selectively binds methylated DNA; a triple-strand-forming nucleic acid component , one or more linkers, optionally in a suitable solution; substances or solutions for performing ligation, such as ligases, buffers; substances or solutions for performing column chromatography; for performing immunologically based A substance or solution for enrichment (e.g. immunoprecipitation); a substance or solution for nucleic acid amplification, e.g. PCR; a dye or dyes, if applicable to a coupling agent, if applicable in solution; for The substance or solution used to perform the hybridization; and/or the substance or solution used to perform the washing step.
  • a protein component that selectively binds methylated DNA such as ligases, buffers
  • substances or solutions for performing column chromatography for performing immunologically based
  • a substance or solution for enrichment e.g. immunoprecipitation
  • the application provides a kit of the application in the manufacture of a kit for diagnosing colorectal advanced adenoma, screening for colorectal advanced adenoma formation or risk of formation, or assessing colorectal advanced adenoma in an individual Use of a diagnostic kit for the progression or prognosis of or monitoring the response to therapy in an individual receiving treatment for a colorectal tumor.
  • the present application provides the use of a reagent for quantitative analysis of methylation levels of a marker of interest in the manufacture of a kit for use in diagnosing advanced colorectal adenomas, screening for Risk of colorectal advanced adenoma formation or formation or in a method of assessing progression or prognosis of colorectal advanced adenoma.
  • each step in the method and each reagent used in each step are as described in the "1. Method” section herein, therefore, the kit of the present invention can include the required for each step of the method according to the method. each reagent.
  • kit is described in the section "2. Kit” herein.
  • the target markers comprise: at least 2 or all 3 target markers in said group (I), and at least 2, at least 3 or all 4 targets in said group (II) Mark.
  • the target markers include Septin9, BCAT1, IKZF1, PKNOX2, CRHBP, SOX1 and intergenic region A.
  • the reagents include amplification primers, primer pairs for amplifying the target marker.
  • the reagents comprise primer pairs for amplifying: Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in the group (I); or Septin9, BCAT1 and IKZF1 in the group (I) primer pair.
  • the reagents comprise for amplifying: PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and intergenic region A in the group (II) primer pairs; primer pairs of PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A in said group (II); or said Primer pairs for PKNOX2, CRHBP, SOX1 and Intergenic Region A in panel (II).
  • the primer pair for amplifying Septin9 is SEQ ID NO: 22/23; the primer pair for amplifying BCAT1 is SEQ ID NO: 24/25; the primer pair for amplifying IKZF1 is SEQ ID NO: 26/27; the primer pair for amplifying PKNOX2 is SEQ ID NO: 28/29; the primer pair for amplifying CRHBP is SEQ ID NO: 30/31; the primer pair for amplifying SOX1 is SEQ ID NO: : 32/33; the primer pair used to amplify the intergenic region A is SEQ ID NO: 34/35.
  • the reagent further comprises a quantitative primer pair designed to amplify at least a portion of the target sequence that has been pre-amplified with the amplification primer, capable of interacting with the first At least 9 contiguous nucleotides of the target sequence pre-amplified by a primer pool hybridize under stringent conditions, under moderately stringent conditions or under highly stringent conditions.
  • a quantitative primer pair designed to amplify at least a portion of the target sequence that has been pre-amplified with the amplification primer, capable of interacting with the first At least 9 contiguous nucleotides of the target sequence pre-amplified by a primer pool hybridize under stringent conditions, under moderately stringent conditions or under highly stringent conditions.
  • the quantitative primer pair is the same as the amplification primer pair.
  • the reagent further comprises one or more blocking oligonucleotides.
  • the reagent further comprises a detection reagent.
  • the detection reagent is selected from the group consisting of fluorescent probes, intercalating dyes, chromophore-labeled probes, radioisotope-labeled probes, and biotin-labeled probes.
  • the probe comprises at least one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 36-43.
  • the probe comprises at least one, at least two or all three nucleotide sequences set forth in SEQ ID NOs: 36-38, and at least one of the nucleotide sequences set forth in SEQ ID NOs: 39 or 40 and 41-43 One, at least two, or at least three nucleotide sequences.
  • the probe comprises: the nucleotide sequences shown in SEQ ID NOs: 36-38 and 41-43, and the nucleotide sequences shown in SEQ ID NO: 39 and/or 40.
  • the 5' end of the fluorescent probe is labeled with a fluorescent dye
  • the 3' end is labeled with a quencher
  • the fluorescent dye is selected from FAM, HEX/VIC, TAMRA, Texas Red or Cy5
  • the The quencher is selected from BHQ1, BHQ2, BHQ3, DABCYL, TAMRA or lowa Black Dark Quenchers.
  • the reagent further includes a bisulfite reagent or a methylation-sensitive restriction enzyme.
  • 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.
  • the MSRE is selected from the group consisting of HpaII enzyme, SalI enzyme, Enzymes, ScrFI enzymes, BbeI enzymes, NotI enzymes, SmaI enzymes, XmaI enzymes, MboI enzymes, BstBI enzymes, ClaI enzymes, MluI enzymes, NaeI enzymes, NarI enzymes, PvuI enzymes, SacII enzymes, Hhal enzymes, and any combination thereof.
  • the reagents comprise primer pairs for amplifying a control marker.
  • 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.
  • the target marker comprises or:
  • the method comprises the steps of:
  • step (b) treating the DNA in the biological sample obtained in step (a) with a reagent capable of distinguishing methylated and unmethylated CpG sites in the DNA, thereby obtaining a treated DNA ;
  • step (c) pre-amplifying at least one target in each of the following two sets of target markers in the treated DNA obtained from step (b) with a pool of pre-amplification primers Markers: (I) Septin9, BCAT1 and IKZF1, (II) PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product;
  • step (d) quantitatively analyzing the methylation levels of the amplified target markers based on the pre-amplification products obtained in step (c);
  • step (e) respectively comparing the methylation level of the amplified target marker in step (d) with the corresponding reference level, wherein: the target marker described in group (I) is relative to its corresponding reference level Having higher methylation levels and lower methylation levels of the target markers described in group (II) relative to their corresponding reference levels indicates that the individual has advanced colorectal adenomas, or that all The individual is at risk for the formation or formation of advanced colorectal adenomas, or the individual has an increased likelihood of developing or developing advanced colorectal adenomas, or the individual has a poor prognosis or poor prognosis for advanced colorectal adenomas risks of.
  • step (c) of the above method at least 2 or all 3 markers of interest in said panel (I) are amplified.
  • step (c) of the above method Septin9 and BCAT1, BCAT1 and IKZF1, or Septin9 and IKZF1 in said panel (I) are amplified.
  • step (c) of the above method at least 2, at least 3 or all 4 markers of interest in said panel (II) are amplified.
  • step (c) of the above method PKNOX2 and CRHBP, PKNOX2 and SOX1, PKNOX2 and intergenic region A, CRHBP and intergenic region A, or SOX1 and intergenic region A are amplified.
  • step (c) of the above method PKNOX2, CRHBP and SOX1, PKNOX2, CRHBP and intergenic region A, PKNOX2, SOX1 and intergenic region A or CRHBP, SOX1 and intergenic region A are amplified .
  • step (c) of the above method the following target markers in the treated DNA obtained from step (b) are pre-amplified with a pool of pre-amplification primers: Septin9, BCAT1 and IKZF1, PKNOX2, CRHBP, SOX1 and intergenic region A; wherein at least a portion of the target marker is pre-amplified to obtain a pre-amplified product.
  • the respective marker of interest comprises either:
  • advanced colorectal adenomas were screened by detecting the hypermethylation of Septin9, BCAT1, IKZF1 and the hypomethylation of PKNOX2, CRHBP, SOX1, and intergenic region (hg19, chr10: 130085033-130085148).
  • the preamplification method includes the following steps:
  • the transformed cfDNA sample was pre-amplified by PCR reaction in the presence of methylation-specific primer pairs specific for Septin9, BCAT1, IKZF1, PKNOX2, CRHBP, SOX1 and the intergenic region (hg19) located at chr10:130085033-130085148.
  • the final concentration of each primer was 200 nM.
  • 25 ⁇ L PCR mix consists of 10 ⁇ L transformed cfDNA, 2.5 ⁇ L master mix containing the above primers and 12.5 ⁇ L PCR reagent ( Universal Probe qPCR Master Mix (NEB)).
  • the PCR reaction conditions were as follows: 95°C for 3 minutes; 95°C for 30 seconds, 56°C for 60 seconds, 10 cycles. ProFlexTM PCR System (Thermo Fisher).
  • PCR reaction system the final concentration of each primer was 500 nM, and the final concentration of each detection probe was 200 nM.
  • PCR reaction conditions were as follows: 95°C for 5 minutes; 95°C for 15 seconds, 56°C for 40 seconds (to collect fluorescence), 50 cycles. Different fluorescence was detected in the corresponding fluorescence channel using the ABI 7500 Real-Time PCR System.
  • the Ct value of samples without amplified signal was set to 50.
  • the Ct averages of Septin9, BCAT1, and IKZF1, and the Ct averages of PKNOX2, CRHBP, SOX1, and intergenic region A were calculated respectively.
  • Figure 1 shows the Ct value distribution of target markers Septin9, BCAT1 and IKZF1 in the group with advanced adenoma and the group with negative colonoscopy.
  • the overall Ct value of the group with advanced adenoma is smaller than that of the negative group, suggesting that the Higher levels of basement.
  • the detected Ct of the target markers PKNOX2, CRHBP, SOX1 and the intergenic region (hg19) located at chr10:130085033-130085148 in the population with advanced adenoma were greater than those of the negative control, suggesting a lower methylation level.
  • Table 3 shows the detection results of Septin9, BCAT1, IKZF1, PKNOX2, CRHBP, SOX1 and the intergenic region (hg19) target markers located at chr10:130085033-130085148 in advanced adenomas in the preamplification method.
  • ROC curve of the average Ct value of Septin9, BCAT1 and IKZF1 it was determined that the average Ct value was less than 44 as a positive test; according to the ROC curve of the average Ct value of PKNOX2, CRHBP, SOX1 and the intergenic region (hg19) located in chr10:130085033-130085148, draw A positive test was defined as a mean Ct greater than 38.
  • the final result of the sample was positive for either the hypermethylation of Septin9, BCAT1, IKZF1 or the hypomethylation of PKNOX2, CRHBP, SOX1 and the intergenic region A (hg19) located at chr10:130085033-130085148.
  • the preamplification method showed ultrahigh sensitivity (54.7%) for advanced adenomas and high specificity (82.7%) for the colonoscopy-negative population.
  • the detection rate for advanced adenomas is much higher than that of currently available blood tests.
  • Colonoscopy results Accuracy positive sample negative sample total colorectal advanced adenoma 54.7% 64 53 117 negative colonoscopy 86.0% 14 86 100

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Abstract

本申请涉及结直肠进展期腺瘤的筛查、风险评估及预后方法和试剂盒。具体而言，本申请提供一种用于诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的试剂盒，包含处理DNA的第一试剂、第一引物池和第二试剂，该第一引物池含用于预扩增以下两组目标标记物中每一组目标标记物中的至少一种目标标记物的引物对：(I)Septin9、BCAT1和IKZF1，和(II)PKNOX2、CRHBP、SOX1和基因间隔区A，所述引物对可与被第一试剂处理后的目标标记物的目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，所述目标序列包含至少一个CpG位点。

Description

结直肠进展期腺瘤的筛查、风险评估及预后方法和试剂盒 技术领域
本申请总体上涉及生物医学领域。具体来说，本申请涉及结直肠进展期腺瘤的筛查、风险评估及预后方法和试剂盒。
背景技术
在癌变前的腺瘤晚期或癌症早期对结直肠瘤进行早期检测表明可以显著降低患者的死亡率。当前的结直肠瘤筛查手段包括结肠镜检查或对粪便、血液样品的分子检查，这些筛查手段都是侵入性的或只有极少的标记物，限制了患者对癌症筛查的配合度或检测灵敏度。
然而，虽然通过检测血浆游离DNA的高甲基化已被证实可用于结直肠肿瘤的筛查，但现有检测方法对进展期腺瘤的检出率较低。
因此，亟需开发一种方法和/或试剂盒，其可以从生物样品中数量极为有限的细胞外游离DNA高效地读取表观遗传学信息，而且可以在医院检验科里很容易地配置并可以可靠地应用。
发明内容
本发明通过不同检测靶点，联合血浆游离DNA高甲基化和低甲基化的方法，提高对结直肠进展期腺瘤的检测灵敏度。
在一个方面，本申请提供了一种在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法，所述方法包括如下步骤：
从所述个体获取含有DNA的生物样品；
用试剂处理所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
定量分析目标标记物(target marker)的甲基化水平；其中，所述目标标记物包括以下两组目标标记物中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；
分别比较所述目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
在一些实施方式中，所述方法包括：
(a)从所述个体获取含有DNA的生物样品；
(b)用试剂处理步骤(a)中获取的所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
(c)用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物(target marker)中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物；
(d)基于步骤(c)获取的预扩增产物来分别定量分析所述被扩增的目标标记物的甲基化水平；
(e)分别比较步骤(d)中的所述被扩增的目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的至少2个或全部3个目标标记物被扩增。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1被扩增。
在一些实施方案中，上述方法的步骤(c)中，所述组(II)中的至少2个、至少3个或全部4个目标标记物被扩增。
在一些实施方案中，上述方法的步骤(c)中，所述组(II)中，PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，所述组(II)中，PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下目标标记物：Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在一些实施方式中，所述各个目标标记物包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
在一些实施方式中，从步骤(a)中获取的所述生物样本中的所述DNA包括基因组DNA或细胞外游离DNA。在一些实施方式中，所述细胞外游离DNA包括循环肿瘤DNA。在一些实施方式中，所述细胞外游离DNA中的所述目标标记物在所述生物样品中的数量不超过1ng、0.8ng、0.6ng、0.4ng、0.2ng、0.1ng、0.08ng或不超过0.04ng。在一些实施方式中，所述细胞外游离DNA中的所述目标标记物在所述生物样品中的浓度低于用于所述目标标记物的检测分析的灵敏度水平。
在一些实施方式中，步骤(c)中所述预扩增产物在步骤(d)之前使用稀释剂稀释。
在一些实施方式中，所述生物样品选自下组：组织学切片、组织活检、石蜡包埋的组织、体液、结肠流出物、手术切除样本、分离的血细胞、分离自血液的细胞，及其任意组合。在一些实施方式中，所述体液选自下组：全血、血清、血浆、尿液、粘液、唾液、腹膜液、胸腔液、胸膜积液、滑液、脑脊髓液、胸腔穿刺液、腹腔积液，及其任意组合。在一些实施方式中，从所述个体的血浆中获得所述生物样品。在一些实施方式中，所述结肠流出物选自下组：粪便样品和灌肠洗涤样品。
在一些实施方式中，步骤(b)的所述试剂在CpG位点选择性地修饰未甲基化的胞嘧啶残基以产生修饰的残基，但并不显著性地修饰甲基化的胞嘧啶残基。在一些实施方式 中，步骤(b)的所述试剂包括亚硫酸氢盐试剂。在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。
在一些实施方式中，步骤(b)的所述试剂选择性地切割未甲基化的残基但不切割甲基化的残基，或者选择性地切割甲基化的残基但不切割未甲基化的残基。在一些实施方式中，步骤(b)的所述试剂是甲基化敏感限制酶(MSRE)。在一些实施方式中，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022074143-appb-000001
酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
在一些实施方式中，所述预扩增引物池包含甲基化特异性引物对。在一些实施方式中，其中所述甲基化特异性引物对包含一个正向引物和一个反向引物，所述引物均包含寡核苷酸序列，所述寡核苷酸序列与相应的目标标记物的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，其中所述至少9个连续核苷酸包含至少一个CpG位点。
在一些实施方式中，所述预扩增引物池进一步包含用于扩增对照标记物的对照引物对。在一些实施方式中，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白(tubulin)、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
在一些实施方案中，用于扩增Septin9的引物对为SEQ ID NO:22/23；用于扩增BCAT1的引物对为SEQ ID NO:24/25；用于扩增IKZF1的引物对为SEQ ID NO:26/27；用于扩增PKNOX2的引物对为SEQ ID NO:28/29；用于扩增CRHBP的引物对为SEQ ID NO:30/31；用于扩增SOX1的引物对为SEQ ID NO:32/33；用于扩增基因间隔区A的引物对为SEQ ID NO:34/35。
在一些实施方式中，所述甲基化特异性引物对至少包含选自以下的引物对中的一对或多对：SEQ ID NO：22/23、24/25、26/27、28/29、30/31、32/33和34/35。优选地，所述甲基化特异性引物对包含选自下组(I)的至少一对、至少两对或全部三对引物对和下组(II)的至少一对、至少两对、至少三对引物对：(I)SEQ ID NO：22/23、24/25和26/27；(II)SEQ ID NO：28/29、30/31、32/33和34/35。优选地，所述甲基化特异性引物对包含SEQ ID NO：22/23、24/25、26/27、28/29、30/31、32/33和34/35。
在一些实施方式中，在步骤(c)中，所述目标标记物在一个或多个封闭寡核苷酸存 在的情况下被扩增。
在一些实施方式中，步骤(d)的所述定量分析是通过以下方式进行：聚合酶链式反应(例如实时聚合酶链式反应、数字聚合酶链式反应)、核酸测序、基于质量的分离(例如电泳法、质谱法)或靶标捕获(例如杂交、微阵列)。在一些实施方式中，步骤(d)的所述定量分析是通过实时聚合酶链式反应进行的，任选地所述实时聚合酶链式反应是多重实时聚合酶链式反应。
在一些实施方式中，步骤(d)的所述定量分析包含使用定量引物对和DNA聚合酶对步骤(c)的预扩增产物进行扩增，其中所述预扩增产物通过扩增产生扩增产物。
在一些实施方式中，步骤(d)使用的所述定量引物对能够与步骤(c)的所述预扩增产物的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
在一些实施方式中，步骤(d)使用的至少一个所述定量引物对和步骤(c)的所述预扩增引物池的至少一个所述甲基化特异性引物对相同。
在一些实施方式中，步骤(d)使用的所述定量引物对被设计为用于扩增步骤(c)的所述至少一个预扩增产物内的至少一部分。
在一些实施方式中，所述步骤(d)在检测试剂存在的情况下进行。在一些实施方式中，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。优选地，用于检测Septin9的探针为SEQ ID NO:36；检测BCAT1的探针为SEQ ID NO:37；用于检测IKZF1的探针为SEQ ID NO:38；用于检测PKNOX2的探针为SEQ ID NO:39和40；用于检测CRHBP的探针为SEQ ID NO:41；用于检测SOX1的探针为SEQ ID NO:42；用于检测基因间隔区A的探针为SEQ ID NO:43。在一些实施方式中，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-43。优选地，所述探针包括SEQ ID NO:36-38中的至少一条、至少两条或全部三条核苷酸序列和SEQ ID NO:39和/或40以及SEQ ID NO:41-43中的至少一条、至少两条或至少三条核苷酸序列。在一些实施方式中，所述探针包括SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。
在一些实施方式中，所述荧光探针的5’端标记有荧光染料(例如FAM、HEX/VIC、TAMRA、Texas Red或Cy5)，3’端标记有猝灭剂(例如BHQ1、BHQ2、BHQ3、DABCYL或TAMRA)。
在一些实施方式中，步骤(e)包括比较步骤(d)的所述目标标记物的Ct值和参考Ct值，其中所述组(I)的目标标记物的Ct值高于其相应的参考Ct值、且组(II)的目标标记物的Ct值低于其相应的参考Ct值表明所述个体患有结直肠进展期腺瘤，或者所述 个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
在一些实施方式中，步骤(c)的所述预扩增包括5到30个反应循环，其中每个循环包括在40～80℃下反应5秒-5分钟，之后在85～99℃下反应5秒-5分钟。
在一些实施方式中，步骤(d)的所述定量分析包括基于所述预扩增产物中多个CpG二核苷酸、TpG二核苷酸或CpA二核苷酸的存在或水平来确定其甲基化水平。在一些实施方式中，步骤(d)的所述定量分析包括基于所述预扩增产物中一个或多个CpG二核苷酸的存在或水平来确定胞嘧啶残基的甲基化水平。在一些实施方式中，步骤(d)的所述定量分析是通过将步骤(c)所述预扩增产物分割为多个组分来进行的。
在一些实施方式中，步骤(e)中的所述参考水平是基于从患有结直肠瘤或具有患结直肠瘤风险的一组个体中获取的临床样本和从未患结直肠瘤或不具有患结直肠瘤风险的一组个体中获取的临床样本来确定的。
在另一方面，本申请提供了一种用于诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的试剂盒，其包含：
(a)处理DNA的第一试剂，其中所述第一试剂能够区分DNA中的甲基化和未甲基化的CpG位点；
(b)第一引物池，所述第一引物池包含用于预扩增以下两组目标标记物中每一组目标标记物中的至少一种目标标记物的引物对：(I)Septin9、BCAT1和IKZF1，和(II)PKNOX2、CRHBP、SOX1和基因间隔区A，其中所述引物对可与被所述第一试剂处理后的所述目标标记物的目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，其中所述目标序列包含至少一个CpG位点；
(c)第二试剂，所述第二试剂用于定量分析被所述第一引物池预扩增的所述目标标记物中每个目标标记物的甲基化水平。
在一些实施方式中，所述第二试剂包含第二引物池，所述第二引物池包含多个定量引物对，所述定量引物对能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
在一些实施方式中，所述第二引物池中的定量引物对和所述第一引物池中的引物对相同。在一些实施方式中，所述第二引物池中的定量引物对被设计为用于扩增被所述第一引物池预扩增的所述目标序列内的至少一部分。
在一些实施方式中，所述第一引物池包含至少一个甲基化特异性引物对。
在一些实施方式中，所述第一引物池和所述第二引物池被包装在单一容器内或被包 装在独立容器内。
在一些实施方式中，所述试剂盒进一步包含一个或多个封闭寡核苷酸。
在一些实施方式中，所述试剂盒进一步包含检测试剂。在一些实施方式中，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-43。优选地，所述探针包括下组(I)的至少一条、至少两条或全部三条核苷酸序列和下组(II)的至少一条、至少两条或至少三条核苷酸序列：(I)SEQ ID NO:36-38，(II)SEQ ID NO:39或40以及41-43。在一些实施方式中，所述探针包括：SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。在一些实施方式中，所述荧光探针的5’端标记有荧光染料(例如FAM、HEX/VIC、TAMRA、Texas Red或Cy5)，3’端标记有猝灭剂(例如BHQ1、BHQ2、BHQ3、DABCYL、TAMRA或lowa Black Dark Quenchers)。
在一些实施方式中，所述试剂盒进一步包含DNA聚合酶和/或一个适合存放从所述个体中获取的所述生物样品的容器。在一些实施方式中，所述试剂盒进一步包含使用说明书和/或对试剂盒检测结果的解释。
在一些实施方式中，所述第一试剂包括亚硫酸氢盐试剂或甲基化敏感限制酶(MSRE)。在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。在一些实施方式中，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022074143-appb-000002
酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
在一些实施方式中，所述第一引物池中包括用于扩增所述组(I)中的至少2个或全部3个目标标记物的引物对。
在一些实施方式中，所述第一引物池中包括用于扩增所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1的引物对。
在一些实施方案中，所述第一引物池中包括用于扩增所述组(II)中的至少2个、至少3个或全部4个目标标记物的引物对。
在一些实施方案中，所述第一引物池中包括用于扩增组(II)中的PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A的引物对。
在一些实施方案中，所述第一引物池中包括用于扩增组(II)中的PKNOX2、 CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A的引物对。
在一些实施方案中，所述第一引物池中包括用于扩增Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A的引物对；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在一些实施方案中，用于扩增Septin9的引物对为SEQ ID NO:22/23；用于扩增BCAT1的引物对为SEQ ID NO:24/25；用于扩增IKZF1的引物对为SEQ ID NO:26/27；用于扩增PKNOX2的引物对为SEQ ID NO:28/29；用于扩增CRHBP的引物对为SEQ ID NO:30/31；用于扩增SOX1的引物对为SEQ ID NO:32/33；用于扩增基因间隔区A的引物对为SEQ ID NO:34/35。
在一些实施方式中，所述各个目标标记物包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
在一些实施方式中，所述第一引物池包含的引物对包含选自下组的核苷酸序列，或由选自下组的苷酸序列组成：SEQ ID NO:22/23、24/25、26/27、28/29、30/31、32/33和34/35，任选地其中所述第二引物池包含至少一个与所述第一引物池中的至少一个引物对相同的引物对。
在一些实施方式中，所述第一引物池和所述第二引物池进一步包含用于扩增对照标记物的引物对。在一些实施方式中，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
在一些实施方式中，所述试剂盒进一步包含多个容器，每个容器均用于接收所述第二引物池的组分。
在另一方面，本申请提供了根据本申请所述的试剂盒在制造用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进 展或预后的诊断试剂盒中的用途。
在另一方面，本申请提供了用于定量分析目标标记物的甲基化水平的试剂在制造试剂盒中的用途，所述试剂盒被用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法中，其中所述方法包括如下步骤：
(a)从所述个体获取含有DNA的生物样品；
(b)用试剂处理步骤(a)中获取的所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
(c)用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物(target marker)中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物；
(d)基于步骤(c)获取的预扩增产物来分别定量分析所述被扩增的目标标记物的甲基化水平；
(e)分别比较步骤(d)中的所述被扩增的目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的至少2个或全部3个目标标记物被扩增。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1被扩增。
在一些实施方案中，上述方法的步骤(c)中，所述组(II)中的至少2个、至少3个或全部4个目标标记物被扩增。
在一些实施方案中，上述方法的步骤(c)中，PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1 和基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下目标标记物：Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在一些实施方式中，所述各个目标标记物包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
附图说明
图1：肠镜未发现异常与进展期腺瘤患者血浆游离DNA中Septin9、BCAT1和IKZF1甲基化检测结果。
图2：肠镜未发现异常与进展期腺瘤患者血浆游离DNA中PKNOX2、CRHBP、SOX1、基因间隔区A的甲基化检测结果。
图3显示了目标标记物的示例性亚区域(subregion)的核苷酸序列。
具体实施方式
虽然本申请公开了本申请的各个方面和各种实施方式，但是本领域技术人员
可以在不脱离本申请的精神和范围的前提下做出各种等同改变或修改。本申请公开的各个方面和各种实施方式均是示例性的，并不旨在限制本申请的范围，本申请的实际保护范围以权利要求书为准。除非另有说明，否则本申请中使用的所有技术和科学术语均是本领域技术人员通常理解的含义。本申请引用的所有参考文献、专利和专利申请均通过引用并入本申请。
需注意的是，在本申请的说明书和权利要求书中，单数形式的“一个”、“一种”和“所述”均包括其复数形式，除非上下文另有说明。因此，例如，“一种试剂”包括多种试剂。
在本申请的说明书和权利要求书，除非另有说明，否则术语“包含”、“包括”或“含有 ”是指含有所列出的数值、步骤或成分，但也不排除还含有其他数值、步骤或成分。
传统上，对癌症的诊断依赖于对单个标记物(例如，基因突变)的检测，但是很可惜的是，通常很难通过检测单个标记物来检测癌症，或者很难通过检测单个标记物来区分多种类型的癌症。此外，在生物样品中单个标记物的水平通常是极为有限的，这进一步降低了对癌症的诊断特异性和/或诊断灵敏度。因此，仅识别单个标记物的分析法被证明具有有限的预测价值。
在一个方面，本申请预扩增本文所述的目标标记物来获得预扩增产物，之后基于其各自的预扩增产物来分别定量分析每个目标标记物的甲基化水平。这样的预扩增步骤可以提高目标标记物的数量/水平，并可以显著提高对结直肠进展期腺瘤的诊断特异性和/或诊断灵敏度。在另一方面，本申请同时定量分析生物样品中的多个目标标记物的甲基化水平，以提高对结直肠进展期腺瘤的诊断特异性和/或诊断灵敏度。在一些实施方式中，所述多个目标标记物在定量分析之前先预扩增。特别地，本申请的发明人出人意料地发现将预扩增步骤和定量分析步骤结合在一起可以显著提高对结直肠进展期腺瘤的诊断特异性和/或诊断灵敏度，使得对结直肠进展期腺瘤的早期检测成为可能，例如在癌变前的腺瘤期或癌症早期。本领域技术人员可以理解的是，在上下文中的诊断“灵敏度”定义的是被正确鉴定为阳性结果的比例，也就是被正确鉴定出患病的个体的百分比。而“特异性”定义的是被正确鉴定为阴性结果的比例，也就是被正确鉴定出不患病的个体的百分比。
1.  方法
在一个方面，本申请提供了一种在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法，所述方法包括如下步骤：
(a)从所述个体获取含有DNA的生物样品；
(b)用试剂处理所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
(d)定量分析目标标记物(target marker)的甲基化水平；其中，所述目标标记物包括以下两组目标标记物中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；
(e)分别比较所述目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目 标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险；
其中，所述方法包括任选的步骤(c)：用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
优选地，所述方法包括：
(a)从所述个体获取含有DNA的生物样品；
(b)用试剂处理步骤(a)中获取的所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
(c)用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物(target marker)中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物；
(d)基于步骤(c)获取的预扩增产物来分别定量分析所述被扩增的目标标记物的甲基化水平；
(e)分别比较步骤(d)中的所述被扩增的目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
本文中所使用的术语“筛查”是指对病理状态、疾病或病况进行鉴定，例如对结直肠进展期腺瘤的鉴定，或对患有结直肠进展期腺瘤但可能从特定的治疗方案中获益的个体进行鉴定。在本申请中，术语“筛查”和术语“诊断”可能互换使用。
本文中所使用的术语“瘤”或“肿瘤”应当理解为是指包含肿瘤细胞的病灶、肿瘤或其他包囊化或未包囊化的块体或其他形式的生长物。“肿瘤细胞”应当理解为是指显示出异常生长的细胞。术语“生长”应当以最广泛的意义理解并且包括增殖。于此而言，细胞异常生长的一个实例是细胞失控的增殖。另一个实例是
细胞因凋亡失败而延长其通常的寿命。肿瘤细胞可以是良性细胞或恶性细胞。在一些实施方式中，肿瘤是腺瘤或腺癌。不使本发明限于任一理论或作用模式，腺瘤通常是源自上皮的良性肿瘤，其源自上皮组织或显示出清晰界定的上皮结构。这些结构物可以具有腺状外观。它可能在腺瘤内部包含恶性细胞群体，例如，随着良性腺瘤或良性肿瘤病灶进展成恶性腺癌而发生。在一些实施方式中，瘤是恶性的，例如癌。在一些实施方式中，瘤不是恶性的，例如腺瘤。
本文中所使用的术语“结直肠瘤”是指存在于结肠、直肠和/或阑尾的瘤。在一些实施方式中，结直肠瘤是结直肠癌、结直肠腺瘤和/或无蒂锯齿状息肉。在一些实施方式中，结直肠瘤是癌前的。
本文所述术语“结直肠进展期腺瘤”是指肿瘤大于1cm、或含高级别上皮内瘤变、或含绒毛成分的腺瘤。
本文中所使用的术语“癌前”指的是展示出与癌症进展风险升高相关的一些组织学变化的瘤。就结直肠细胞增殖性病症来说，这类状况的实例包括高度发育异常的细胞增殖性疾病，例如结肠的腺瘤状息肉。
本文中在描述瘤(例如，腺瘤或腺癌)时所用的术语“形成”被理解为是指表现出发育异常的个体的一个或多个细胞。对这一点而言，腺瘤或腺癌可能已形成发展，因为已形成非正常增长的细胞团块，也可以是腺瘤或腺癌处在极早期，在诊断时只有相比数量极少的细胞出现非正常分裂。本申请也延伸至评估个体的结直肠进展期腺瘤(例如，结直肠癌)形成风险。
本文中所用的术语“评估”指的是区分来自患有结直肠进展期腺瘤的个体和未患有结直肠进展期腺瘤的个体的样本的能力，或者区分来自处于结直肠瘤进展不同阶段的个体的样本的能力。在一些实施方式中，该评估涉及确定个体的肿瘤是否进入进展阶段或是否具有较高可能性进入进展阶段。在一些实施方式中，该评估涉及对个体的肿瘤进行分类，例如I期、II期、III期、IV期等。在一些实施方式中，该评估涉及确定个体的肿瘤是否减轻或加重。在一些实施方式中，该评估可以协助评价一种治疗具有临床受益的可能性。在一些实施方式中，该评估可能涉及患者在接受治疗(例如，用特定的药物进行治疗)后是否好转和/或好转的可能性。通过为任何特定患者选择最合适的治疗方式，本申请的评估方法可以被用于在临床上做出治疗决定。在施用治疗方案(例如，给定的治疗方案，包括例如给定的治疗药剂或组合的施用，手术干预、类固醇治疗等)后，本申请的评估方法在评价该患者能否长期存活的可能性上是有价值的工具。
本领域技术人员所理解的“区分”不能达到对所分析的样品100％正确。但是，依然要 求对具有统计学意义的数量的样品能够正确分类。具有统计学意义的数量可以由本领域技术人员通过使用不同的统计工具来确定，例如但不限于确定置信区间、确定p值，学生氏T检验或Fisher区分方程。有关详细信息，请参见Dowdy and Wearden,Statistics for Research,John Wiley&Sons,New York 1983。在一些实施方式中，置信区间为至少90％、至少95％、至少96％、至少97％、至少98％或至少99％。在一些实施方式中，p值小于0.1、0.05、0.01、0.005或0.0001。
本文所用的术语“进展”是指细胞的形态和生理沿着遗传确定的途径改变，例如，从先前、较低或早期到后来的、更复杂或更高级阶段的生理成熟中的自然发展过程。
本文所用的术语“预后”是指预测疾病(例如，癌症)的疾病症状(包括例如复发、加剧、耐药性)结果的可能性。该术语还指对治疗的临床获益可能性的预测。在一些实施方式中，使用统计算法为个体提供疾病的预后。例如，预后可以是手术、癌症(例如，实体瘤，例如结直肠癌、黑素瘤和肾细胞癌)的临床亚型的进展、一种或多种临床因素的进展或从疾病中恢复。预后可以是预后不良(例如可能复发或产生耐药性)或预后良好。
在本申请中，以下关于步骤(a)、步骤(b)、步骤(c)、步骤(d)和步骤(e)的详细描述适用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法。
步骤(a)
在根据本申请的方法的步骤(a)中，从所述个体中获取包含DNA的生物学样本。
本文所用的术语“生物样品”是指获自或衍生自目标个体的生物组合物，其包含基于物理、生化、化学和/或生理特征待表征或待识别的细胞和/或其他分子实体(例如DNA)。生物样品包括但不限于通过本领域技术人员已知的任何方法获得的个体的细胞、组织、器官和/或生物体液。在一些实施方式中，所述生物样品选自下组：组织学切片、组织活检、石蜡包埋的组织、体液、结肠流出物、手术切除样本、分离的血细胞、分离自血液的细胞，及其任意组合。在一些实施方式中，所述体液选自下组：全血、血清、血浆、尿液、粘液、唾液、腹膜液、胸腔液、胸膜积液、滑液、脑脊髓液、胸腔穿刺液、腹腔积液，及其任意组合。在一些实施方式中，所述结肠流出物选自下组：粪便样品和灌肠洗涤样品。选择最适合根据本文申请的方法的检测的样品将取决于情境的性质。在一些实施方式中，所述生物样品获自个体的全血。在一些实施方式中，所述生物样品获自个体的血浆。本领域技术人员知道从全血制备血浆的各种方法。例如，在一些实施方 式中，血浆通过将来自个体的全血离心一次、两次、三次、四次、五次或更多次来获得。
本文所用的术语“个体”包括人类和非人类的动物。非人类动物包括所有脊椎动物，例如哺乳动物和非哺乳动物。“个体”也可以是家畜，例如牛、猪、绵羊、家禽和马；或啮齿动物，例如大鼠、小鼠；或非人类灵长类动物，例如猿、猴、恒河猴；或家养的动物，例如狗或猫。在一些实施方式中，个体是人类或非人类灵长类动物。在一些实施方式中，个体是人类。在本申请中，“个体”和“受试者”可能互换使用。
在一些实施方式中，所述DNA是从所述生物样本中分离的。从生物样品中分离和纯化DNA可以通过使用本领域已知的各种方法来实施，包括使用可商购的试剂盒。例如，通过以下方式从细胞和组织中分离DNA：在高度变性和还原条件下裂解原材料、部分使用蛋白质降解酶、纯化通过苯酚/氯仿提取工艺获得的核酸组分，并通过渗析或乙醇沉淀从水相中回收核酸(参见例如Sambrook,J.,Fritsch,E.F.in T.Maniatis,C S H,Molecular Cloning,1989)。又例如，现在有许多试剂体系特别适用于从琼脂糖凝胶中纯化DNA片段、从细菌裂解物中分离质粒DNA，以及从血液、组织或细胞培养物中分离较长链的核酸(基因组DNA、总细胞RNA)。许多这些可商购的纯化体系中是基于相当众所周知的原理，即，在不同离液盐的溶液的存在下将核酸与矿物载体相结合。在这些体系中，细磨的玻璃粉、硅藻土或硅胶的悬浮液被用作载体材料。在例如US7888006B2和EP1626085A1中描述了从生物样品中分离和纯化DNA的一些其他方法。在方法之间进行选择将受到几个因素的影响，包括时间、费用和所需的DNA数量。
在一些实施方式中，生物样品中包含的DNA包括基因组DNA。本文所用的术语“基因组DNA”是指包含细胞或生物体的完整基因组及其片段或部分的DNA。基因组DNA是来源于个体的大段DNA(例如长于大约10、20、30、40、50、60、70、80、90、100、200或300kb)，并且可以具有天然修饰，例如DNA甲基化。
在一些实施方式中，生物样品中包含的DNA包括细胞DNA。本文所用的术语“细胞DNA”是指存在于细胞内的DNA，或从体内细胞中获取DNA并在体外分离、或以其他方式在体外操作，只要该DNA未从体内细胞中移除。
在一些实施方式中，生物样品中包含的DNA包括细胞外游离DNA。本文所用的术语“细胞外游离DNA”是指在体内的细胞外存在的DNA片段。该术语也可以被用于指代获取自体内的细胞外来源并在体外分离、或操作的DNA片段。细胞外游离DNA中的DNA片段通常具有约100到200bp的长度，推测与被包裹于核小体的DNA片段的长度有关。细胞外游离DNA包括例如细胞外游离胎儿DNA和循环肿瘤DNA。细胞外游离胎儿DNA在孕 妇的体内(例如血液)中循环，代表胎儿基因组，而循环肿瘤DNA在癌症患者的体内(例如血液)中循环。在一些实施方式中，细胞外游离DNA可基本上不含个体的细胞DNA。例如，所述细胞外游离DNA可包含小于约1,000ng/mL、小于约100ng/mL、小于约10ng/mL、小于约1ng/mL的细胞DNA。
可以通过使用本领域已知的常规技术来制备细胞外游离DNA。例如，可以通过以约200-20,000g、约200-10,000g、约200-5,000g、约300-4000g等的速度离心血液样品约3-30分钟、约3-15分钟、约3-10分钟、约3-5分钟来获得血液样品的细胞外游离DNA。例如，在一些实施方式中，可以通过将个体的血浆或血清离心一、二、三、四、五次或更多次来获得血液样本的细胞外游离DNA。在一些实施方式中，为了从包含可溶性DNA的无细胞组分中分离细胞及其片段，可以通过微滤来获得所述生物样品。通常来说，微滤可以通过使用过滤器来进行，例如，0.1微米～0.45微米的膜过滤器，诸如0.22微米的膜过滤器。
在一些实施方式中，使用商购的DNA提取产品从全血、血清或血浆中提取细胞外游离DNA用于分析。这种提取方法据称对循环DNA的回收率高(>50％)，某些产品(例如Qiagen生产的QIAamp Circulating Nucleic Acid Kit)据称可提取小尺寸的DNA片段。所使用的典型样品量为1-5mL血清或血浆。
在一些实施方式中，细胞外游离DNA包括循环肿瘤DNA。循环肿瘤DNA(“ctDNA”)是与细胞无关的体液(例如血液、尿液、唾液、痰、粪便、胸膜液、脑脊液等)中肿瘤来源的片段化DNA。通常，ctDNA高度片段化，平均长度约为150个碱基对。ctDNA通常包括体液(例如血浆)中细胞外游离DNA的极小部分，例如ctDNA可能构成血浆DNA的不到约10％。通常，该百分比小于约1％，例如小于约0.5％或小于约0.01％。另外，血浆DNA的总量通常非常低，例如约10ng/mL血浆。ctDNA的数量因人而异，并且取决于肿瘤的类型、位置，对于癌性肿瘤，则取决于癌症的阶段。但是，ctDNA通常在体液中非常罕见，只能通过极其敏感和特异性的技术进行检测。检测ctDNA可能有助于检测和诊断肿瘤、指导肿瘤特异性治疗、监测治疗以及监测癌症的缓解。
步骤(b)
在根据本申请的方法的步骤(b)中，用能够区分DNA中的未甲基化和甲基化CpG位点的试剂处理步骤(a)中获取的生物样品中的DNA，从而获得经处理的DNA。
DNA甲基化是(例如，通过DNA甲基转移酶的作用)将甲基添加到DNA分子上(例如，添加至DNA分子的一个或多个胞嘧啶碱基)的生物学过程。在哺乳动物中，DNA 甲基化出现于胞嘧啶-磷酸-鸟嘌呤(CpG)二核苷酸(即“CpG位点”)的5’位置，当其出现在基因的启动子或第一个外显子中的5’-CpG-3’二核苷酸中时，会导致基因的表观遗传失活。充分证明DNA甲基化在调节基因表达、肿瘤发生、以及其他遗传和表观遗传疾病中起重要作用。
如本文所用，术语“甲基化的胞嘧啶残基”是指胞嘧啶残基的衍生物，其中一个甲基连接至胞嘧啶环的碳原子上(例如C5)。术语“未甲基化的胞嘧啶残基”是指未衍生化的胞嘧啶残基，其中与“甲基化的胞嘧啶残基”相反，在胞嘧啶环的碳原子(例如C5)上没有甲基连接。其内的胞嘧啶残基被甲基化的CpG位点就是甲基化的CpG位点，而其内的胞嘧啶残基未被甲基化的CpG位点是未甲基化的CpG位点。
在一些实施方式中，步骤(b)中使用的试剂能够区分DNA中的未甲基化和甲基化的CpG位点，从而获得经处理的DNA。该试剂可以选择性地作用于未甲基化的胞嘧啶残基，但不能显著地作用于甲基化的胞嘧啶残基。或者该试剂可以选择性地作用于甲基化的胞嘧啶残基，而不显著地作用于未甲基化的胞嘧啶残基。因此，原始DNA以取决于是否被甲基化的方式转化为经处理的DNA，从而可以通过其杂交行为将经处理的DNA与原始DNA区分开。
例如，一些试剂可以选择性地将未甲基化的胞嘧啶残基转化为尿嘧啶、胸腺嘧啶或杂交上与胞嘧啶不同的另一碱基，而甲基化的胞嘧啶残基依然处于未转化状态。又例如，一些试剂可以选择性地切割甲基化的残基，或者选择性地切割未甲基化的残基。
如本文所用，“经处理的DNA”是指已经用能够区分DNA中的未甲基化和甲基化的CpG位点的试剂处理后的DNA，即DNA中的DNA甲基化状态已经改变。
在一些实施方式中，步骤(b)的所述试剂在CpG位点选择性地修饰未甲基化的胞嘧啶残基以产生修饰的残基，但并不显著性地修饰甲基化的胞嘧啶残基。
在一些实施方式中，步骤(b)的所述试剂包括亚硫酸氢盐试剂。如本文所用，术语“亚硫酸氢盐试剂”是指，例如本申请所公开的可用于区分甲基化和未甲基化的CpG二核苷酸序列的包括亚硫酸氢盐、亚硫酸氢根离子或其任意组合的试剂。在本申请中，用亚硫酸氢盐试剂处理DNA也被描述为“亚硫酸氢盐反应”或“亚硫酸氢盐处理”，指的是转化未甲基化的胞嘧啶残基的反应，特别是在亚硫酸氢根离子存在的情况下，核酸中未甲基化的胞嘧啶残基被转化为尿嘧啶碱基、胸腺嘧啶碱基或在杂交行为上与胞嘧啶不同的其他碱基，而其中甲基化的胞嘧啶残基未被显著地转化。换言之，亚硫酸氢盐处理可用于区分甲基化的CpG二核苷酸和未甲基化的CpG二核苷酸。Frommer,M.,et al.,Proc Natl Acad Sci USA 89(1992)1827-31和Grigg,G.,Clark,S.,Bioessays 16(1994)431-6中详细描 述了用于检测甲基化的胞嘧啶残基的亚硫酸氢盐反应。亚硫酸氢盐反应包括脱氨基步骤和脱磺酸基步骤(参见Grigg and Clark,同上)。“甲基化的胞嘧啶残基未被显著地转化”这一陈述，不排除非常小的百分比(例如，小于0.1％、小于0.2％、小于0.3％、小于0.4％、小于0.5％、小于0.6％、小于0.7％、小于0.8％、小于0.9％、小于1％、小于2％、小于3％、小于4％、小于5％、小于6％、小于7％、小于8％、小于9％、小于10％、小于11％、小于12％、小于13％、小于14％、小于15％、小于16％、小于17％、小于18％、小于19％、小于20％)的甲基化的胞嘧啶残基被转化为尿嘧啶、胸腺嘧啶或在杂交行为上与胞嘧啶不同的其他碱基，尽管其意在仅仅转化未甲基化的胞嘧啶残基。
在例如参考Frommer M.,et al.(同上)或Grigg and Clark(同上)的情况下(它们公开了亚硫酸氢盐处理的基本参数)，本领域技术人员知道如何进行亚硫酸氢盐处理，特别是脱氨基步骤和脱磺酸基步骤。孵育时间和温度对脱氨基效率的影响、以及影响DNA降解的参数都已公开。
在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。在一些实施方式中，所述亚硫酸氢盐试剂是亚硫酸氢钠。在一些实施方式中，亚硫酸氢盐试剂是可商购的，例如，MethylCode TM Bisulfite Conversion Kit、EpiMark TM Bisulfite Conversion Kit、EpiJET TM Bisulfite Conversion Kit、EZDNAMethylation-Gold TM Kit等。在一些实施方式中，根据试剂盒的使用说明书进行亚硫酸氢盐反应。
在一些实施方式中，步骤(b)的所述试剂选择性地切割未甲基化的残基但不切割甲基化的残基，或者选择性地切割甲基化的残基但不切割未甲基化的残基。
在一些实施方式中，步骤(b)的所述试剂是甲基化敏感限制酶(MSRE)。
术语“甲基化敏感限制酶”是指根据其识别位点的甲基化状态而选择性地消化核酸的酶。对于当识别位点未被甲基化或半甲基化时才特异剪切的限制酶来说，当识别位点被甲基化时，不会发生剪切，或以显著降低的效率剪切。对于当识别位点被甲基化时才特异剪切的限制酶来说，当识别位点未被甲基化时，不会发生剪切，或以显著降低的效率剪切。在一些实施方式中，甲基化敏感限制酶的识别序列含有CG二核苷酸(例如cgcg或cccggg)。在一些实施方式中，当该CG二核苷酸中的胞嘧啶在C5碳原子处被甲基化时，甲基化敏感限制酶不进行剪切。
在一些实施方式中，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022074143-appb-000003
酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
使用本领域已知的方法，使用能区分目标区域内的甲基化的CpG二核苷酸和未甲基化的CpG二核苷酸的甲基化敏感限制酶或包含甲基化敏感限制酶的一系列限制酶试剂来确定甲基化，例如但不限于，差异性甲基化杂交(“DMH”)。
在一些实施方式中，步骤(a)的DNA可以在用甲基化敏感限制酶处理之前被切割。这样的方法是本领域已知的，并且可以既包括物理方式也包括酶促方式。特别优选的是使用一种或多种对甲基化不敏感的并且其识别位点富含AT并且不包含CG二核苷酸的限制酶。使用此类酶使得DNA片段中的CpG位点和CpG富集区域得以保存。在一些实施方式中，此类限制酶选自MseI酶、BfaI酶、Csp6I15酶、Tru1I酶、Tru9I酶、MaeI酶、XspI酶及其任意组合。
步骤(c)
在根据本申请的方法的步骤(c)中，用预扩增引物池对从步骤(b)获得的经处理的DNA中的目标标记物进行预扩增，其中每个目标标记物的至少一部分被预扩增以获得至少一个预扩增产物。在本申请中，步骤(c)也可以被称为预扩增步骤。
对目标标记物进行预扩增的目的之一是增加经处理的DNA中的目标标记物的数量。如本文所用，术语“扩增”大体上是指任何能够导致分子或一组相关分子的拷贝数增加的过程。当“扩增”被用于多核苷酸分子时，是指通常从少量多核苷酸开始产生多拷贝的多核苷酸分子或多核苷酸分子的一部分的多份拷贝，其中被扩增的物质(扩增子，PCR扩增子)通常是可被检测到的。多核苷酸的扩增涵盖多个化学和酶促过程。扩增的形式包括通过聚合酶链式反应(逆转录PCR、PCR)、链置换扩增(SDA)反应、转录介导扩增(TMA)反应、基于核酸序列的扩增(NASBA)反应或连接酶链反应(LCR)，从一个或几个拷贝的模板RNA或DNA分子生成多个DNA拷贝。
如本文所用，术语“目标标记物”是指这样的目的核酸或基因区域：其甲基化水平指示着结直肠瘤(例如，结直肠进展期腺瘤)，或指示着结直肠瘤(例如，结直肠进展期腺瘤)形成或形成的风险，或指示着结直肠瘤(例如，结直肠进展期腺瘤)的进展或预后。在本申请中，术语“标记物”和“基因”可以互换使用。术语“标记物”或“基因”应被认为包括其所有转录变体及其所有启动子和调控元件。如本领域技术人员所理解的，已知某些基因在个体之间表现出等位基因变异或单核苷酸多态性(“SNP”)。SNP包括不同长度的简单的重复序列(例如二核苷酸和三核苷酸重复)的插入和缺失。因此，本申请应被理解为扩展到由任何其他突变、多态性或等位基因变异产生的标记物/基因的所有形式。另外，应当理解，术语“标记物”和“基因”应既包括标记物或基因的正义链序列，也 包括标记物或基因的反义链序列。
本文所用的术语“目标标记物”被宽泛地解释为既包括1)在生物样品或基因组DNA中发现的原始标记物(处于特定的甲基化状态)，也包括2)其经过处理的序列(例如亚硫酸氢盐转化后的对应区域或MSRE处理后的对应区域)。亚硫酸氢盐转化后的对应区域与基因组序列中的目标标记物不同之处在于，一个或多个未甲基化的胞嘧啶残基被转化为尿嘧啶碱基、胸腺嘧啶碱基或在杂交行为上与胞嘧啶不同的其他碱基。经MSRE处理的对应区域与基因组序列中的目标标记物不同之处在于，该序列在一个或多个MSRE切割位点处被切割。
在本文所述的方法和试剂盒中，所述目标标记物分为以下两组：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A。其中，每一组中至少预扩增一种目标标记物或每组中至少一种目标标记物的至少一部分被预扩增。
在一些实施方式中，所述组(I)中的至少2个或全部3个目标标记物被扩增，和/或，所述组(II)中的至少2个、至少3个或全部4个目标标记物被扩增。
在一些实施方式中，所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1被扩增。
在一些实施方案中，组(II)中的PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A被扩增。在一些实施方案中，组(II)中的PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A被扩增。
在一些实施方中，用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下目标标记物：Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在本申请中，应该理解的是，标记物/基因既通过引用其名称又通过其染色体坐标来进行描述。所述染色体坐标与2009年2月发布的人类基因组数据库Hg19版本一致(在本文中称为“Hg19坐标”)。
在一些实施方式中，所述的各个目标标记物包括或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
在公共数据库(例如UCSC Genome Browser、Ensemble和NCBI网站)中可以获得如上所述的Hg19坐标的特定核苷酸序列，以及每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb。
在一些实施方式中，所述各个目标标记物也包括其所有变体。变体包括来自相同区域的、与本文所述的标记物/基因区域具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的序列同一性(即，具有一个或多个缺失、插入、取代、反向序列等)的核酸序列。因此，本申请内容应理解为延伸至实现相同结果的此类变体，尽管事实上个体间的实际核酸序列具有微小的遗传变异。
如本文所用，术语“序列同一性的百分比(％)”是指候选序列的氨基酸(或核酸)残基和参考序列的氨基酸(或核酸)残基进行序列比对后的相同百分比，比对时可以引入间隔(如有必要)以使得相同的氨基酸(或核酸)数目达到最多。
换句话说，氨基酸序列(或核酸序列)的序列同一性百分比(％)可以通过用与参考序列相同的氨基酸残基(或碱基)的数目除以候选序列或参考序列中氨基酸残基(或碱基)的总数(以较短者为准)来计算。氨基酸残基的保守取代可以被认为或可以不被认为是相同的残基。可以通过以下方式来确定氨基酸(或核酸)序列同一性的百分比，例如，可以使用公开的工具如BLASTN、BLASTp(可在美国国家生物技术信息中心(NCBI)的网站上获得，也可参见Altschul S.F.et al.,J.Mol.Biol.,215:403–410(1990)；Stephen F.et al.,Nucleic Acids Res.,25:3389–3402(1997))、ClustalW2(可在欧洲生物信息研究所的网站上找到)，也可参见Higgins D.G.et al.,Methods in Enzymology,266:383-402(1996)；Larkin M.A.et al.,Bioinformatics(Oxford,England),23(21):2947-8(2007))和ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以使用所述工具提供的默认参数，或者可以(例如，通过选择合适的算法)定制适合比对的参数。
在本文提供的步骤c)中，每个目标标记物的至少一部分被预扩增以获得至少一个预扩增产物。在一些实施方式中，目标标记物的预扩增部分在目标标记物的亚区域内。
在不使本申请限于任一理论或作用模式的情况下，据信测量目标标记物的亚区域内的甲基化水平尤其有用，所述亚区域包含在结直肠瘤(例如，结直肠进展期腺瘤)中经常被超甲基化(hypermethylated)的高密度CpG二核苷酸。这一发现使得亚区域成为分析的一个特别有用的目标，因为它既可以简化筛查过程(因为需要分析的DNA区域更短且界定更为清晰)，而且进一步地，相比于对目标标记物的整个Hg19区域进行分析所 获得的结果，从这些亚区域所获得的结果会在超甲基化的存在或不存在这方面提供显著地更为明确的结果。因此，该发现既简化了诊断、筛查/监测过程，又增加了结直肠进展期腺瘤诊断的敏感度和特异性。在一些实施方式中，各个目标标记物的亚区域包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：
目标标记物 Hg19坐标的亚区域
Septin9 chr17:75369603-75369693
BCAT1 chr12:25102016-25102110
IKZF1 chr7:50343793-50343896
PKNOX2 chr11:125036431-125036547
CRHBP chr5:76249633-76249729
SOX1 chr13:112758808-112758890
基因间隔区A chr10:130085033-130085148
以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或b)亚硫酸氢盐转化后的a)的对应区域；或c)MSRE处理后的a)的对应区域。
在一些实施方式中，各个目标标记物的亚区域包含或是选自下组的多核苷酸序列：SEQ ID NO：1-7、或其亚硫酸氢盐转化后的对应区域、或其MSRE处理的对应区域。在一些实施方式中，各目标标记物的亚区域的经亚硫酸氢盐转化后的对应区域包含或是选自下组的多核苷酸序列：SEQ ID NO：8-21。每个目标标记物的亚区域及经亚硫酸氢盐转化后的对应区域的SEQ ID NO列在下表1中，图3中提供了具体序列。
表1：各个目标标记物的示例性亚区域
Figure PCTCN2022074143-appb-000004
Figure PCTCN2022074143-appb-000005
在一些实施方式中，Septin9的亚区域包含选自下组的序列：SEQ ID NO:1、8、15；BCAT1的亚区域包含选自下组的序列：SEQ ID NO:2、9、16；IKZF1的亚区域包含选自下组的序列：SEQ ID NO:3、10、17。
在一些实施方式中，PKNOX2的亚区域包含选自下组的序列：SEQ ID NO:4、11、18；SOX1的亚区域包含选自下组的序列：SEQ ID NO:5、12、19；CRHBP的亚区域包含选自下组的序列：SEQ ID NO:6、13、20；基因间隔区A的亚区域包含选自下组的序列：SEQ ID NO:7、14、21。
在一些实施方式中，细胞外游离DNA中的目标标记物以不超过1ng、不超过0.9ng、不超过0.8ng、不超过0.7ng、不超过0.6ng、不超过0.5ng、不超过0.4ng、不超过0.3ng、不超过0.2ng、不超过0.1ng、不超过0.09ng、不超过0.08ng、不超过0.07ng、不超过0.06ng、不超过0.05ng、不超过0.04ng、不超过0.03ng、不超过0.02ng或不超过0.01ng的量存在于生物样品中。在一些实施方式中，细胞外游离DNA中的目标标记物以不超过0.1％、不超过0.2％、不超过0.3％、不超过0.4％、不超过0.5％、不超过0.6％、不超过0.7％、不超过0.8％、不超过0.9％、不超过1％的百分比存在于生物样品中。在一些实施方式中，细胞外游离DNA中的所述目标标记物在生物样品中的浓度低于用于目标标记物的检测分析的灵敏度水平。“检测分析的灵敏度”是对检测分析在分析浓度/量的微小差异之间进行区分的能力的度量。如果存在于生物样品中的细胞外游离DNA中的目标标记物低于检测分析的灵敏度水平，那么将无法使用常规方法来定量分析样品中每个目标标记物的甲基化水平。相反，本申请公开的方法在检测样品中极少量的目标标记物方面是实用并优越的。在一些实施方式中，细胞外游离DNA中的目标标记物以不超过0.08ng或不超过0.04ng的量存在于生物样品中。
在一些实施方式中，步骤(c)的所述至少一个预扩增产物在下一步骤(即步骤(d))之前用稀释剂稀释。在一些实施方式中，稀释剂选自下组：不含核酸酶的水、Tris-EDTA缓冲液和没有PCR抑制作用的任何其他缓冲液。在一些实施方式中，将步骤(c)的所述预扩增的DNA直接添加至下一步骤(即步骤(d))，而无需事先稀释。
用预扩增引物池预扩增经处理的DNA中的所述目标标记物。如本文所用，术语“引物”是指这样的单链寡核苷酸，其能够在合适的条件(例如缓冲液和温度)下，在四种不同的三磷酸核苷和用于聚合的试剂(例如DNA聚合酶)的存在下，作为模板指导的DNA 合成的起始点。在任何给定的情况下，引物的长度取决于例如引物的预期用途，并且通常在15至30个核苷酸的范围内。短的引物分子通常需要较低的温度才能与模板形成足够稳定的杂交复合物。引物不必反映模板的确切序列，但必须足够互补以能与该模板杂交。引物位点是模板上与引物杂交的区域。引物对是一组引物，其包括与待扩增的序列的5’末端杂交的5’正向引物和与待扩增的序列的3’末端的互补链杂交的3’反向引物。本领域技术人员可以基于本领域的公知常识根据待扩增的标记物设计引物(参见，例如PCR Primer:A Laboratory Manual,Cold Spring Harbor Laboratories,NY,1995)。此外，一些用于设计在各种各样分析中使用的最佳探针和/或引物的软件包是公开的，例如可从美国马萨诸塞州剑桥市的基因组研究中心(the Center for Genome Research,Cambridge,Mass.,USA)获得的Primer 3。显然，在设计探针或引物时其潜在用途也应考虑在内。例如，设计用于本发明目的的引物可以包括至少一个CpG位点，或者从该引物获得的扩增产物可以包括至少一个CpG位点。用于设计检测DNA甲基化状态的引物的工具也是本领域已知的，例如MethPrimer(Li LC and Dahiya R.MethPrimer:designing primers for methylation PCRs.Bioinformatics.2002Nov；18(11):1427-31)。在本申请中，通过将预扩增引物作为引物池，经处理的DNA中的任何目标标记物(目标标记物的每至少一部分或目标标记物的一个亚区域)均可以被预扩增。
本文所用的术语“寡核苷酸”定义为包含两个或更多个核苷酸(例如，脱氧核糖核苷酸或核糖核苷酸)的分子，优选为至少5个核苷酸，更优选为至少约10-15个核苷酸，更优选为至少约15至30个核苷酸或更长(例如，寡核苷酸的长度通常少于200个残基(例如，在15至100个核苷酸之间)，但是，如本文所用，该术语也意在覆盖更长的多核苷酸链)。确切的大小将取决于许多因素，而这些因素又取决于寡核苷酸的最终功能或用途。寡核苷酸通常用其长度来指代。例如，具有24个残基的寡核苷酸被称为“24聚体”(24-mer)。寡核苷酸可通过自身杂交或与其他多核苷酸杂交形成二级和三级结构。这样的结构可以包括但不限于双链体、发夹、十字形、弯折和三链体。可以以任何方式产生寡核苷酸，包括化学合成、DNA复制、反转录、PCR或其任意组合。
如本文所用，术语“互补”是指核苷酸或核酸之间的杂交或碱基配对，例如，双链DNA分子的两条链之间，或待测序或扩增的单链核酸上的引物结合位点和寡核苷酸引物之间。互补核苷酸通常是A和T(或A和U)，或C和G。当一条链的核苷酸以最佳的方式对齐、并比较、并有适当的核苷酸插入或缺失后，与另一链的至少约80％(通常至少约90％至95％，更优选地为约98％至100％)的核苷酸配对，两条单链RNA或DNA分子就被称为是互补的。或者，当RNA链或DNA链在选择性杂交条件下与其互补序列 杂交时，互补存在。通常，当在至少14至25个核苷酸的一段上具有至少约65％(优选至少约75％、更优选至少约90％)的互补性时，将发生选择性杂交。参见M.Kanehisa,Nucleic Acids Res.12:203(1984)，作为参考并入本文。
在一些实施方式中，预扩增引物池包含至少一个甲基化特异性引物对。在一些实施方式中，预扩增引物池包含多个甲基化特异性引物对。在一些实施方式中，预扩增步骤通过甲基化特异性PCR(“MSP”)进行，甲基化特异性PCR是使用甲基化特异性引物的PCR。Herman et al.,Methylation-specific PCR:a novelPCRassay for methylation status ofCpGislands.Proc Natl Acad Sci USA.1996September 3；93(18):9821-6和United States Patent No.6,265,171中已描述了该技术(即MSP)。
如本文所用，术语“甲基化特异性引物对”是指经特异性设计以识别CpG位点以利用甲基化的差异来扩增经处理的DNA中的特定目标标记物的引物对。引物仅作用于具有特定甲基化状态或没有特定甲基化状态的分子。例如，引物可以是寡核苷酸，在严紧条件、中等严紧条件或高度严紧条件下，其可以以甲基化特异性方式与具有甲基化的特定CpG位点特异性杂交，但不能与没有甲基化的特定CpG位点杂交。因此，引物将特异性扩增在特定CpG位点具有甲基化的目标标记物。又例如，引物可以是寡核苷酸，在严紧条件、中等严紧条件或高度严紧条件下，其可以以甲基化特异性的方式与未甲基化的特定的CpG位点特异性杂交，但是不能与甲基化的特定的CpG位点杂交。因此，引物将特异性扩增在特定CpG位点没有甲基化的目标标记物。因此，在本申请中，对在经处理的DNA内的至少一个目标标记物的预扩增中使用甲基化特异性引物，可以区分甲基化的和未甲基化的CpG位点。本申请的甲基化特异性引物对包含至少一个与亚硫酸氢盐处理的CpG二核苷酸杂交的引物。因此，所述特异性针对甲基化DNA的引物的序列包含至少一个CpG二核苷酸，并且所述特异性针对未甲基化DNA的引物的序列在CpG的C位置上包含“T”，和/或在CpG中G位置上包含“A”。
在一些实施方式中，所述至少一个甲基化特异性引物对包含一个正向引物和一个反向引物，所述引物均包含寡核苷酸序列，所述寡核苷酸序列与所述目标标记物之一(或目标标记物的亚区域)的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，其中所述目标标记物之一(或目标标记物的亚区域)的至少9个连续核苷酸包含至少一个(例如1、2、3、4、5、6、7、8、9、10或更多个)CpG位点。
如本文所用，术语“杂交”可以指其中两条单链多核苷酸非共价形式结合以形成稳定的双链多核苷酸的过程。在一个方面，所得的双链多核苷酸可以是“杂交物”或“双链”。“杂交条件”中的盐浓度通常约小于1M，经常小于约500mM并且可以小于约200mM。“杂 交缓冲液”包括缓冲盐溶液，例如5％SSPE，或本领域已知的其他此类缓冲液。杂交温度可以低至5℃，但是通常高于22℃，并且更为通常地高于约30℃，并且通常超过37℃。杂交通常在严紧条件下进行，即，在该条件下序列将与其目标序列杂交但不与其他非互补序列杂交。严紧条件是取决于序列的，并且在不同情况下有所不同。例如，更长的片段可能需要比短片段更高的杂交温度才能进行特异性杂交。由于其他因素可能会影响杂交的严紧性，包括碱基组成和互补链的长度，有机溶剂的存在以及碱基错配的程度，因此参数组合比单独使用任何一个参数的绝对测量更为重要。通常严紧条件被选定为比特定序列在特定的离子强度和pH下的解链温度(Tm)低约5℃。Tm可以是双链核酸分子群体中的一半被分离成单链的温度。用于计算核酸的Tm的几个方程式是本领域众所周知的。如标准参考文献所示，当核酸在1M NaCl水溶液中时，可以通过公式Tm＝81.5+0.41(％G+C)计算出简单估算的Tm值(参见例如Anderson and Young,Quantitative Filter Hybridization,in Nucleic Acid Hybridization(1985))。其他参考文献(例如Allawi and SantaLucia,Jr.,Biochemistry,36:10581-94(1997))包括替代的计算方法，其计算Tm时将结构和环境以及序列特征等考虑在内。
通常，杂交物的稳定性是关于离子浓度和温度的函数。通常，杂交反应在较低严紧条件下进行，然后在具有不同但较高严紧性的洗涤液中洗涤。示例性的严紧条件包括pH约7.0至约8.3、温度至少25℃、钠离子(或其他盐)浓度为至少0.01M至不超过1M。例如，5x SSPE(750mM NaCl，50mM磷酸钠，5mM EDTA，pH 7.4)和约30℃的温度适合于等位基因特异性杂交，尽管合适的温度取决于杂交区域的长度和/或GC含量。在一个方面，确定错配百分比的“杂交严紧性”可以如下：1)高度严紧性：0.1x SSPE，0.1％SDS，65℃；2)中等严紧性(也称为中度严紧性)：0.2x SSPE，0.1％SDS，50℃；3)低严紧性：1.0x SSPE，0.1％SDS，50℃。应当理解，使用替代的缓冲剂、盐和温度可以达到相同的严紧性。例如，中等严紧杂交可以是指允许核酸分子(例如探针)结合互补核酸分子的条件。杂交的核酸分子通常具有至少60％的同一性，包括例如至少70％、75％、80％、85％、90％或95％的同一性。中等严紧条件可以是与下述条件达到同等效果的条件：42℃，50％甲酰胺，5x Denhardt溶液，5x SSPE，0.2％SDS杂交，然后用42℃，0.2x SSPE，0.2％SDS进行洗涤。高度严紧条件可以通过如下条件提供，例如，42℃，50％甲酰胺，5x Denhardt溶液，5x SSPE，0.2％SDS杂交，然后65℃，0.1x SSPE和0.1％SDS中洗涤。低严紧性杂交可以是与下述条件达到同等效果的条件：22℃，10％甲酰胺，5x Denhardt溶液，6x SSPE，0.2％SDS杂交，然后在1x SSPE，0.2％SDS中于37℃洗涤。Denhardt的溶液包含1％聚蔗糖，1％聚乙烯吡咯烷酮和1％ 牛血清白蛋白(BSA)。20x SSPE(氯化钠，磷酸钠，EDTA)包含3M氯化钠、0.2M磷酸钠和0.025M EDTA。其他合适的中等严紧性和高度严紧性杂交缓冲液和条件是本领域技术人员众所周知的，并且描述于例如Sambrook et al.,Molecular Cloning:A Laboratory Manual,2nd ed.,Cold Spring Harbor Press,Plainview,N.Y.(1989)和Ausubel et al.,Short Protocols in Molecular Biology,4th ed.,John Wiley&Sons(1999)。
在一些实施方式中，预扩增引物池还包含用于扩增对照标记物的对照引物对。通常，对照标记物是具有已知特征(例如，序列已知，每个细胞的拷贝数已知)的核酸，用于与实验目标(例如，浓度未知的核酸)进行比较。对照可以是内源的，优选为不变的基因，可以将分析中的实验核酸或目标核酸相对其进行标准化。此类因为样品间差异而标准化的对照可能发生在例如样品处理，分析效率等，并且允许精确的样品间数据比较，定量分析扩增效率和偏差。
在一些实施方式中，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。在一些实施方式中，对照引物对的序列如下表2的SEQ ID NO：44和45所示。
在一些实施方式中，Septin9的甲基化特异性引物对为SEQ ID NO:22/23；BCAT1的甲基化特异性引物对为SEQ ID NO:24/25；IKZF1的甲基化特异性引物对为SEQ ID NO:26/27；PKNOX2的甲基化特异性引物对为SEQ ID NO:28/29；CRHBP的甲基化特异性引物对为SEQ ID NO:30/31；SOX1的甲基化特异性引物对为SEQ ID NO:32/33；基因间隔区A的甲基化特异性引物对为SEQ ID NO:34/35。在一些实施方案中，所述甲基化特异性引物对包含如下表2所示的选自下组的至少一对核苷酸序列：SEQ ID NO：22/23，24/25，26/27，28/29，30/31，32/33和34/35。优选地，所述甲基化特异性引物对包括SEQ ID NO：22/23，24/25和26/27中的至少一对引物对，以及SEQ ID NO：28/29，30/31，32/33和34/35中的至少一对引物对。优选地，所述甲基化特异性引物对包含：SEQ ID NO：22/23，24/25，26/27，28/29，30/31，32/33和34/35。本申请中使用的引物对的序列号以“SEQ ID NO：n/m”的形式表示。例如，SEQ ID NO：22/23是指分别具有如下表2所示的SEQ ID NO：22和SEQ ID NO：23所示的核酸序列的引物对。
表2
Figure PCTCN2022074143-appb-000006
Figure PCTCN2022074143-appb-000007
在一些实施方式中，在步骤(c)中，所述目标标记物在一个或多个封闭寡核苷酸的存在下被扩增。此类封闭寡核苷酸的用途已经被描述于Yu et al.,BioTechniques 23:714-720,1997。封闭序列与预扩增引物对同时与经处理的DNA杂交。目标标记物的预扩增终止于封闭序列的5’位置，使得目标标记物的预扩增在与封闭序列互补的序列存在的情况下被抑制。封闭序列可以被设计为以甲基化状态特异性方式与经处理的DNA杂交。例如，为了检测未甲基化核酸群体中的甲基化核酸，可以通过使用在相关位置包含“CpA”或“TpA”的封闭序列来抑制在所述位置未甲基化的核酸的扩增，与之对应，如果需要抑制甲基化核酸扩增，则使用“CpG”。
对于使用了封闭寡核苷酸的PCR方法，对聚合酶介导的扩增的有效干扰要求封闭寡核苷酸不能被聚合酶延长。优选地，这是通过使用封闭物来实现的，所述封闭物为3'-脱氧寡核苷酸或在3'位置衍生的具有“游离”羟基以外的寡核苷酸。例如，3'-O-乙酰基寡核苷酸是优选的封闭物分子类别的代表。
另外，聚合酶介导的封闭寡核苷酸的分解应当被阻止。优选地，此类阻止包括使用缺乏5'-3'核酸外切酶活性的聚合酶，或使用例如在其5'-末端具有硫醇盐桥的修饰的封闭寡核苷酸，使封闭物分子抗核酸酶。特定的应用可能不需要对封闭物进行5'修饰。例如 ，如果封闭物结合位点和引物结合位点重叠，因而阻止了引物的结合(例如使用过量的封闭物)，封闭寡核苷酸的降解将被基本上阻止。这是因为聚合酶不会将引物延伸至、并超过封闭物(沿5'-3'方向)，该过程通常会导致杂交的封闭寡核苷酸降解。
出于本申请的目的并且也如本文所实施，特别优选的封闭物/PCR实施方式包括使用肽核酸(PNA)寡聚物作为封闭寡核苷酸。此类PNA封闭低聚物是理想的，因为它们既不会被分解也不会被聚合酶延伸。
在一些实施方式中，所述至少一个目标标记物是用DNA聚合酶预扩增的。
如本文所用，术语“DNA聚合酶”是指催化三磷酸单脱氧核糖核苷酸(dNTP)合成多聚脱氧核糖核苷酸的酶，其完成在DNA复制、修复以及在某些情况下细胞分化中的最基本功能。
原核生物中的DNA聚合酶的实例包括DNA聚合酶I、DNA聚合酶II、DNA聚合酶III、DNA聚合酶IV和DNA聚合酶V。已知在大肠杆菌(E.coli)中有DNA聚合酶I、II和III。DNA聚合酶III在基因组复制中似乎是最重要的。DNA聚合酶I的重要性在于其可以在增长的链的末端删除掉未配对的碱基。逆转录病毒具有独特的DNA聚合酶，即，逆转录酶，它使用RNA模板合成DNA。对于真核生物，DNA聚合酶的实例是聚合酶α、β、λ、γ、σ、μ、δ、ε、η、ι、κ、ζ、θ和Rev1。动物细胞的DNA聚合酶负责DNA在细胞核和线粒体中的复制。
在预扩增步骤中使用的PCR试剂可以是任何可商购的PCR混合物(例如KAPA2G Fast MultiplexPCRKit,
Figure PCTCN2022074143-appb-000008
Universal Probe qPCR Master Mix,EpiTect MethyLightPCRKit等)，其用于扩增经处理的DNA。或者，本领域技术人员可以在实验室中制备包括Mg 2+、dNTP、DNA聚合酶等的PCR试剂。本领域技术人员还可以根据实际需要选择合适的PCR反应体系和PCR反应条件。在一些实施方式中，步骤(c)的预扩增包括5至30个反应循环，其中每个循环包括在85～99℃下反应5秒到5分钟，然后在40～80℃下反应5秒到5分钟。在一些实施方式中，步骤(c)的预扩增包括10至20个反应循环，其中每个循环包括在90～99℃下反应15秒到2分钟，然后在45～60℃下反应30秒到3分钟。在一些实施方式中，步骤(c)的预扩增包括15个反应循环，其中每个循环包括在95℃下反应30秒，然后在56℃下反应60秒。
步骤(d)
在根据本申请的方法的步骤(d)中，每个目标标记物的甲基化水平分别基于其在步骤(c)中获得的各个预扩增产物而进行定量分析。在本申请中，步骤也可以被命名为定 量分析步骤。
如本文所用，术语“甲基化状态”指的是DNA区域内的一个特定的核苷酸或多个核苷酸的甲基化的存在、不存在和/或甲基化的数量。特定DNA序列的甲基化状态(例如，本文所述的目标标记物)可以指示序列中每个碱基的甲基化状态，或者可以指示序列中的碱基对的子集的甲基化状态(例如，胞嘧啶残基的甲基化状态或一个或多个特定的限制酶识别序列的甲基化状态)，或者可以指示序列中区域甲基化密度的信息，虽然不能提供甲基化发生在序列中何处的精确信息。甲基化状态可以任选地由“甲基化水平”来表示或指示。甲基化水平可以通过例如定量分析在用甲基化敏感性限制性酶进行限制性消化后存在的完整DNA的量来确定。在该例中，如果使用定量PCR对DNA中的特定序列进行定量分析，模板DNA的量大约等于模拟处理的对照则表明该序列未高度甲基化，而模板量明显少于模拟处理的样品中的模板量则表明该序列中存在甲基化DNA。因此，如上述例子中的甲基化水平代表着甲基化状态，并且因此可以用作甲基化状态的定量指标。当需要将样品中序列的甲基化状态与阈值水平进行比较时，这尤其有用。
在DNA序列内一个或多个特定的CpG甲基化位点(每个具有两个CpG二核苷酸序列)的甲基化状态包括“未甲基化”、“完全甲基化”和“半甲基化”。术语“半甲基化”是指双链DNA其中仅其一条链被甲基化的甲基化状态。术语“超甲基化”是指，相对于正常对照DNA样品中的相应的CpG二核苷酸处5-甲基胞嘧啶的数量，在检测的DNA样品的DNA序列中一个或多个CpG二核苷酸处5-甲基胞嘧啶的数量增加所对应的平均甲基化状态。一个残基的甲基化状态可以是定性读数或定量读数，例如通过甲基化水平来表示的。在本申请中，术语“甲基化状态”和“甲基化水平”可以互换使用。根据本申请，可以同时确定一个以上的不同甲基化水平。
如本文所述，每个目标标记物的甲基化水平分别基于步骤(c)获得的其各自的预扩增产物而进行定量分析。DNA序列(例如目标标记物)内的一个或多个CpG二核苷酸序列的甲基化水平/状态可以通过本领域中已知的各种分析方法来确定。
在一些实施方式中，步骤(d)的所述定量分析是通过以下方式进行：聚合酶链式反应(例如实时聚合酶链式反应、数字聚合酶链式反应)、核酸测序、基于质量的分离(例如电泳法、质谱法)或靶标捕获(例如杂交、微阵列)。
在一些实施方式中，基于预扩增的DNA通过使用MSP(参见Herman，同上)分别定量分析每个目标标记物的甲基化水平。例如，通过使用在中等和/或高度严紧条件下与未转化序列特异性杂交的一种或多种引物，仅当模板在CpG位点包含甲基化胞嘧啶时才产生扩增产物。
在一些实施方式中，步骤(d)的定量分析通过实时PCR进行。实时PCR的非限制性实例包括Cottrell et al.,Nucl.Acids Res.32:e10,2003描述的HeavyMethyl TMPCR；Eads et al.,Cancer Res.59:2302-2306,1999描述的MethyLight TM PCR；Rand et al.,Nucl.Acids Res.33:e 127,2005描述的Headloop PCR。
如本文所用，术语“HeavyMethyl TM PCR”是指本领域公认的一种实时PCR技术，其中一个或多个不可延伸性核酸(例如，寡核苷酸)封闭物以甲基化特异性方式与亚硫酸氢盐处理的核酸结合(即，封闭物在中等至高等严紧条件下与未突变的DNA特异性结合)。使用一种或多种引物进行扩增反应，所述引物可以任选地是甲基化特异性的，但旁侧分布一个或多个封闭物。在未甲基化的核酸(即突变的DNA)存在的情况下，封闭物结合并且无PCR产物产生。使用基本上像例如Holland et al.,Proc.Natl.Acad.Sci.USA,88:7276-7280,1991所述的TaqMan TM分析方法，样品中核酸的甲基化水平得以确定。
如本文所用，术语“MethyLight TM PCR”是指基于本领域公认的一种基于荧光的实时PCR技术，其中采用了称为TaqMan TM探针的双标记荧光寡核苷酸探针，并且被设计为可同位于正向和反向扩增引物之间的富含CpG的序列杂交。所述的TaqMan TM探针包含一个荧光“报告因子部分”和“淬灭剂部分”共价结合到与TaqMan TM寡核苷酸的核苷酸相连的接头部分(例如，亚磷酰胺)。在PCR扩增过程中，与富含CpG的序列杂交的TaqMan TM探针被Taq聚合酶的5’核酸酶活性切割，从而在PCR反应过程中产生以实时方式检测的信号。在该方法中，可以将分子信标用作可检测的探针，并且该系统不依赖于所使用的DNA聚合酶的5’-3’核酸外切酶活性(参见Mhlanga and Malmberg,Methods 25:463-471,2001)。
如本文所用，术语“Headloop PCR”是指本领域公认的一种实时PCR，其选择性地扩增目标核酸，但是通过将3’茎环延伸形成不能进一步提供扩增模板的发卡结构来抑制非扩增目标变体的扩增。
在一些实施方式中，所述实时PCR是多重实时PCR。
如本文所用，术语“多重”可指，通过使用一个以上的标记物，每个标记物具有至少一个不同的检测特征，例如荧光特征(例如，激发波长、发射波长、发射强度、FWHM(半峰高处的全宽度)或荧光寿命)或独特的核酸或蛋白序列特征，可以同时对多个标记物(例如多个核酸序列)的存在和/或量进行测定的分析或其他分析方法。
在一些实施方式中，步骤(d)的定量分析通过核酸测序进行。核酸测序的示例性方法是本领域已知的，参见，例如Frommer et al.,Proc.Natl.Acad.Sci.USA 89:1827-1831,1992；Clark et al.,Nucl.Acids Res.22:2990-2997,1994。例如，通过将未使用亚硫酸氢盐处 理的样品获得的序列或目标区域的已知核苷酸序列与使用亚硫酸氢盐处理的样品获得的序列进行比较，有助于鉴定DNA序列中甲基化胞嘧啶。与未处理的样品相比，在亚硫酸氢盐处理的样品中的任意胞嘧啶位点检测到的胸腺嘧啶残基都可以认为是由亚硫酸氢盐处理而引起的突变，即该位点存在甲基化的胞嘧啶。
用于测序DNA的方法是本领域已知的，并且包括例如双脱氧链终止法或Maxam-Gilbert法(参见Sambrook et al.,Molecular Cloning,A Laboratory Manual(2 nd Ed.,CSHP,New York 1989))、焦磷酸测序(参见Uhlmann et al.,Electrophoresis,23:4072-4079,2002)、固相焦磷酸测序(参见Landegren et al.,Genome Res.,8(8):769-776,1998)、固相微测序(参见例如，Southern et al.,Genomics,13:1008-1017,1992)、采用FRET的微测序(参见例如，Chen and Kwok,Nucleic Acids Res.25:347-353,1997)、连接法测序或超深度测序(参见Marguiles et al.,Nature 437(7057):376-80(2005))。
在一些实施方式中，步骤(d)的所述定量分析通过基于质量的分离(例如电泳、质谱法)进行。
例如，甲基化胞嘧啶残基的存在可以通过联合亚硫酸氢盐限制分析法(COBRA)进行检测，基本如Xiong and Laird,Nucl.Acids Res.,25:2532-2534,2001所述。这种方法利用了在使用可以选择性地突变未甲基化的胞嘧啶残基的化合物(例如，亚硫酸氢盐)处理之后，在甲基化和未甲基化的核酸之间的限制酶识别位点的差异。例如，限制性核酸内切酶Taq1切割序列TCGA，在对未甲基化核酸进行亚硫酸氢盐处理后该序列将是TTGA，因此将不被切割。然后使用本领域已知的检测手段例如电泳和/或质谱法，检测消化的和/或未消化的核酸。
又例如，在用选择性突变未甲基化胞嘧啶残基的化合物处理后，基于核苷酸序列和/或二级结构的差异，使用不同的技术来检测扩增产物中核酸差异，例如甲基化特异性单链构象分析(MS-SSCA)(Bianco et al.,Hum.Mutat.,14:289-293,1999)、甲基化特异性变性梯度凝胶电泳(MS-DGGE)(Abrams and Stanton,Methods Enzymol.,212:71-74,1992)和甲基化特异性变性高效液相色谱(MS-DHPLC)(Deng et al.,Chin.J.Cancer Res.,12:171-191,2000)。
在一些实施方式中，步骤(d)的定量分析是通过靶标捕获(例如杂交、微阵列)来进行的。
通过杂交的合适的检测方法是本领域已知的，例如Southern、斑点印迹、狭缝印迹或其他核酸杂交方式(Kawai et al.,Mol.Cell.Biol.14:7421-7427,1994；Gonzalgo et al.,Cancer Res.57:594-599,1997)。在一些实施方式中，用于杂交分析的探针被可检测地标 记。在一些实施方式中，用于杂交分析的基于核酸的探针是未标记的。这种未标记的探针可以固定在固体载体如微阵列上，并且可以与被可检测地标记的目标核酸分子杂交。
微阵列的一个实例是甲基化特异性微阵列，其可用于区分具有转化的胞嘧啶残基的序列和具有未转化的胞嘧啶残基的序列(参见Adorjan et al.,Nucl.Acids Res.,30:e21,2002)。基于杂交的分析还可被用于用甲基化敏感的限制酶处理后的核酸。
又例如，可通过寡核苷酸探针确定DNA序列内CpG二核苷酸序列的甲基化状态，所述寡核苷酸探针与PCR扩增引物同时与亚硫酸氢盐处理的DNA杂交(其中所述引物可以是甲基化特异性引物或标准引物)。
在一些实施方式中，步骤(d)在检测试剂的存在下进行。如本文所用，术语“检测试剂”是在定量分析步骤中用于检测核酸的存在、不存在或量的试剂。
本领域已知的各种检测试剂在本申请中都可使用。在一些实施方式中，检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。
优选地，用于检测Septin9的探针为SEQ ID NO:36；检测BCAT1的探针为SEQ ID NO:37；用于检测IKZF1的探针为SEQ ID NO:38；用于检测PKNOX2的探针为SEQ ID NO:39和40；用于检测CRHBP的探针为SEQ ID NO:41；用于检测SOX1的探针为SEQ ID NO:42；用于检测基因间隔区A的探针为SEQ ID NO:43。
在一些实施方式中，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-38、SEQ ID NO:41-43、以及SEQ ID NO:39和/或SEQ ID NO:40。优选地，所述探针包括SEQ ID NO:36-38中的至少一条、至少两条或全部三条核苷酸序列和SEQ ID NO:39和/或40以及SEQ ID NO:41-43中的至少一条、至少两条或至少三条核苷酸序列。在一些实施方式中，所述探针包括SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。
在一些实施方式中，对照标记物为ACTB，其示例性的探针的核苷酸序列如SEQ ID NO:58所述。
在一些实施方式中，荧光探针的5’端标记有荧光染料(例如FAM、HEX/VIC、TAMRA、Texas Red或Cy5)，3’端标记有猝灭剂(例如BHQ1、BHQ2、BHQ3、DABCYL或TAMRA)。
标记可以通过直接或间接方法来完成。直接标记涉及将标记直接(共价或非共价)偶联至试剂上。间接标记涉及第二试剂与第一试剂的结合(共价或非共价)。第二试剂应与第一试剂特异性结合。所述第二试剂可以与合适的标记偶联和/或第二试剂是可与第 二试剂结合的第三试剂的目标(受体)。使用二级、三级甚至更高阶的试剂通常会增加信号强度。合适的二级和高级试剂可以包括抗体、二级抗体和众所周知的链霉亲和素-生物素系统(Vector Laboratories，Inc.)。试剂或底物也可以被本领域中已知的一个或多个标签“标记”。
在一些实施方式中，步骤(d)的所述定量分析包含使用定量引物对和DNA聚合酶对步骤(c)的预扩增产物进行扩增，其中所述预扩增产物通过扩增产生扩增产物。
如本文所用，术语“定量引物对”是指在定量分析步骤中使用的一个或多个引物对。
在一些实施方式中，步骤(d)使用的所述定量引物对能够与步骤(c)的所述预扩增产物的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。在一些实施方式中，步骤(d)中所使用的定量引物对与在步骤(c)的预扩增引物池中的甲基化特异性引物对相同。
在一些实施方式中，步骤(d)中使用的定量引物对被设计为扩增步骤(c)的所述预扩增产物内的至少一部分，即步骤(c)和步骤(d)被设计为巢式PCR。
巢式PCR是PCR的一种改进，旨在提高灵敏度和特异性。巢式PCR涉及使用两个引物组和两个连续的PCR反应。进行第一轮扩增以产生第一扩增子，并使用一个引物对进行第二轮扩增，其中一个或两个引物与由初始引物对界定的区域内的位点退火，即第二个引物对被认为是“嵌套”在第一对引物中。以这种方式，不包含正确内部序列的来自第一次PCR反应的背景扩增产物在第二次PCR反应中不再被进一步扩增。
在一些实施方式中，步骤(d)的定量分析包括基于所述预扩增产物中的多个CpG二核苷酸、TpG二核苷酸或CpA二核苷酸的存在或水平，确定每个目标标记物的甲基化水平。在一些实施方式中，步骤(d)的所述定量分析包括基于所述预扩增产物中一个或多个CpG二核苷酸的存在或水平来确定胞嘧啶残基的甲基化水平。在一些实施方式中，步骤(d)的所述定量分析包括基于所述预扩增产物中一个或多个TpG二核苷酸的存在或水平来确定胞嘧啶残基的甲基化水平。在一些实施方式中，步骤(d)的所述定量分析包括基于所述预扩增产物中CpA二核苷酸的存在来确定胞嘧啶残基的甲基化水平。
在一些实施方式中，定量分析步骤是通过将步骤(c)的所述预扩增产物分为多个组分来进行的。在一些实施方式中，对多个组分进行多个不同的定量分析实验，其中在多个组分之一中定量分析所述预扩增产物(如果存在于所述组分中的话)的不同组合。在一些实施方式中，定量分析每个组分中的对照标记物。
步骤(e)
在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法的步骤(e)中，由步骤(d)的获得的目标标记物的甲基化水平分别与相应的参考水平进行比较，其中，组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平、且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
根据本申请的方法的步骤(e)也可以被称为比较步骤。
本文所用的术语“比较”是指分别对检测的生物样本所含有的通过定量分析步骤获取的目标标记物的甲基化水平与其相应的参考水平进行对比。应当理解，本文所用的术语是指相应参数或值的比较，例如，将绝对量与绝对参考量进行比较，将浓度与参考浓度进行比较，或从检测的样本中获得的强度信号同参考样本的同类型的强度信号进行比较。可以通过手动或计算机辅助进行比较。对于计算机辅助进行的比较，可以将所确定的量的值与通过计算机程序存储在数据库中的合适参考的值进行比较。该计算机程序可以进一步评估比较的结果，并以合适的输出格式自动提供期望的评估。基于定量分析步骤中每个目标标记物的甲基化水平与相应参考水平的比较，可以识别出患有结直肠进展期腺瘤、或者有结直肠进展期腺瘤形成或形成的风险、或者有结直肠进展期腺瘤发展或发展的可能性增加、或者有结直肠进展期腺瘤预后不良或预后不良的风险的个体。
如本文所用，术语“参考水平”是指将个体纳入或排除结肠直肠进展期腺瘤或结肠直肠进展期腺瘤的形成或形成的风险的阈值水平。
本文所述的“相对于其相应的参考水平具有更高的甲基化水平”指组(I)中所检测的目标标记物的甲基化水平至少是其相应参考水平的1.1、1.2、1.3、1.4、1.5、2、3、4、5、6、7、8、9、10倍或更多倍。本文所述的“相对于其相应的参考水平具有较低的甲基化水平”指组(II)所检测的目标标记物的甲基化水平是其相应参考水平的98％、95％、93％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％甚至更低。在本申请中，为了在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后，不需要组(I)的每个目标标记物的甲基化水平均高于其相应的参考水平以及组(II)的每个目标标记物的甲基化水平均低于其相应的参考水平。准确的说，如果在定量分析步骤中进行定量分析的每一组中至少有一种目标标记物的甲基化水平等于或高于其相应的参考水平就足够了。
目标标记物的甲基化的参考水平可以源自一个或多个参考样品，其中参考水平获自与检测目的样品的实验平行进行的实验。或者，可以在数据库中获得参考水平，该数据库包括来自一个或多个参考样品或疾病参考样品的数据、标准或水平的集合。在一些实施方式中，此类数据、标准或水平的集合被标准化，以便可用于与来自一个或多个样品的数据进行比较的目的。“标准化”是将测量原始数据转换为可以直接与其他标准化数据进行比较的数据的过程。标准化被用于克服因不同的分析方法里因素不同而导致的、分析方法特异性的误差，例如上样量的不同、结合效率的不同、检测灵敏度的不同和其他各类的误差。
在一些实施方式中，参考数据库包括来自一个或多个参考样品的目标标记物和/或其他实验室和临床数据的甲基化水平。在一些实施方式中，参考数据库包括目标标记物的甲基化水平，其各自被标准化为在与参考样品相同的条件下检测的对照标志物的甲基化水平的百分比。为了与目标标记物的如此标准化甲基化水平进行比较，测试样品的目标标记物的甲基化水平也被测量并计算为在与测试样品相同的条件下检测的对照标记物的甲基化水平的百分比。
在一些实施方式中，通过汇总获自健康个体和/或非肿瘤个体(即已知没有肿瘤的个体)的参考样品的参考水平数据来建立参考数据库。在一些实施方式中，通过汇总获自正在接受结直肠瘤治疗的个体的参考样品的参考水平数据来建立参考数据库。在一些实施方式中，通过汇总获自结直肠瘤不同阶段的个体的参考样品的数据来建立参考数据库，所述结直肠瘤不同阶段是通过例如目标标记物的不同的甲基化水平来证明的。
本领域技术人员可以根据期望的灵敏度和特异性来选择参考水平。确定合适的参考水平的手段是本领域技术人员已知的，例如参考水平可以从临床研究中收集的数据来确定。
在一些实施方式中，步骤(e)的参考水平是基于从患有结直肠进展期腺瘤或具有患结直肠进展期腺瘤风险的一组个体中获取的临床样本和从未患结直肠进展期腺瘤或不具有患结直肠进展期腺瘤风险的一组个体中获取的临床样本来确定的。
本领域技术人员可以基于各种因素，例如年龄、性别、病史、家族史、症状等，来确定个体是否患结直肠进展期腺瘤或具有患结直肠进展期腺瘤的风险。
在一些实施方式中，用循环阈值(即Ct值)来表示目标标记物的甲基化水平和参考水平。如本文所用，术语“Ct值”是指在背景信号以上可以检测到PCR产物的荧光的循环数。Ct值与样品中目标标记物的数量成反比，即Ct值越低，样品中目标标记物的数量就越大。
例如，在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法的步骤(e)中，组(I)所述目标标记物中至少一个目标标记物的Ct值相对于其相应的参考Ct值低，且组(II)所述目标标记物中至少一个目标标记物的Ct值相对于其相应的参考Ct值高，表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。在一些实施方式中，如果组(I)所述目标标记物中至少一个目标标记物Ct值比其对应的参考Ct值低2-10个循环(例如2、3、4、5、6、7、8、9、10个循环)且组(II)所述目标标记物中至少一个目标标记物的Ct值相对于其相应的参考Ct值高2-10个循环(例如2、3、4、5、6、7、8、9、10个循环)，则确定所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
如本文所用，术语“可能性增加”，是指与从中获得参考样品的个体相比，在个体发展结直肠进展期腺瘤或有结直肠进展期腺瘤预后不良的可能性水平方面总体增加5％、10％、15％、20％、25％、30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％或更高。
2.  试剂盒
在另一方面，本申请还提供了一种用于诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的试剂盒，其包含：
(a)处理DNA的第一试剂，其中所述第一试剂能够区分DNA中的甲基化和未甲基化的CpG位点；
(b)第二试剂，所述第二试剂用于定量分析目标标记物中每个目标标记物的甲基化水平；其中，所述目标标记物包括以下两组目标标记物中每一组目标标记物中的至少一种：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；和任选的
(c)第一引物池，所述第一引物池包含用于预扩增以下两组目标标记物中每一组目标标记物中的至少一种目标标记物的引物对：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A，其中所述引物对可与被所述第一试剂处理后的所述目标标记物的目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或 高度严紧条件下杂交，其中所述目标序列包含至少一个CpG位点。
优选地，所述试剂盒包含：
(a)处理DNA的第一试剂，其中所述第一试剂能够区分DNA中的甲基化和未甲基化的CpG位点；
(b)第一引物池，所述第一引物池包含用于预扩增以下两组目标标记物中每一组目标标记物中的至少一种目标标记物的引物对：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A，其中所述引物对可与被所述第一试剂处理后的所述目标标记物的目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，其中所述目标序列包含至少一个CpG位点；
(c)第二试剂，所述第二试剂用于定量分析被所述第一引物池预扩增的所述目标标记物中每个目标标记物的甲基化水平。
在一些实施方式中，所述第二试剂包含第二引物池，所述第二引物池包含多个定量引物对，所述定量引物对能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
在一些实施方式中，所述第二引物池中的至少一个定量引物对和所述第一引物池中的至少一个引物对相同。在一些实施方式中，所述第二引物池中的定量引物对被设计为用于扩增被所述第一引物池预扩增的所述目标序列内的至少一部分。在一些实施方式中，所述第一引物池包含至少一个甲基化特异性引物对。
在一些实施方式中，所述第一引物池和所述第二引物池被包装在单一容器内或被包装在独立容器内。在一些实施方式中，所述试剂盒进一步包含一个或多个封闭寡核苷酸。
在一些实施方式中，所述试剂盒进一步包含检测试剂。在一些实施方式中，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。在一些实施方式中，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-38、SEQ ID NO:41-43、以及SEQ ID NO:39和/或SEQ ID NO:40。优选地，所述探针包括SEQ ID NO:36-38中的至少一条、至少两条或全部三条核苷酸序列和SEQ ID NO:39和/或40以及SEQ ID NO:41-43中的至少一条、至少两条或至少三条核苷酸序列。在一些实施方式中，所述探针包括SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。在一些实施方式中，所述荧光探针的5’端标记有荧光染料(例如FAM、HEX/VIC、TAMRA、Texas Red或Cy5)，3’端标记有猝灭剂(例如BHQ1、BHQ2、BHQ3、DABCYL、TAMRA或lowa Black Dark  Quenchers)。
在一些实施方式中，所述试剂盒进一步包含DNA聚合酶和/或适合存放从所述个体中获取的所述生物样品的容器。在一些实施方式中，所述试剂盒进一步含使用说明书和/或对试剂盒检测结果的解释。
在一些实施方式中，所述试剂盒可包含包装在独立的容器中的反应缓冲液，该反应缓冲液针对由聚合酶介导的引物延伸(例如PCR)进行了优化。优选的是这样的试剂盒：其进一步包括容器，所述容器适合容纳用于在个体的生物样品中确定下组(I)中的至少一个(例如2、3、4或5个)目标标记物以及下组(II)中的至少一个(例如2、3或4个)目标标记物的甲基化的装置：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A。
在一些实施方式中，所述第一试剂包括亚硫酸氢盐试剂或甲基化敏感限制酶(MSRE)。在一些实施方式中，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。在一些实施方式中，亚硫酸氢盐试剂是亚硫酸氢钠。在一些实施方式中，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022074143-appb-000009
酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
在一些实施方式中，所述第一引物池包含至少一个甲基化特异性引物对，用于预扩增选自下组(I)的至少一个目标标记物中的至少一个目标序列：(I)Septin9、BCAT1和IKZF1。在一些实施方式中，所述第一引物池包含至少一个甲基化特异性引物对，用于预扩增选自下组(II)的至少一个目标标记物中的至少一个目标序列：PKNOX2、SOX1、CRHBP和基因间隔区A。在一些实施方式中，所述第一引物池包含至少两个甲基化特异性引物对，其中一个用于预扩增选自下组(I)的至少一个目标标记物中的至少一个目标序列：(I)Septin9、BCAT1和IKZF1，另一个用于预扩增选自下组(II)的至少一个目标标记物中的至少一个目标序列：PKNOX2、SOX1、CRHBP和基因间隔区A。
在一些实施方式中，所述第一引物池包含用于预扩增以下两组目标标记物的引物对：所述组(I)中的至少2个或全部3个目标标记物，以及所述组(II)中的至少2个、至少3个或全部4个目标标记物。
在一些实施方式中，所述第一引物池包含用于预扩增组(I)的下述目标标记物的引物对：Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1被扩增。
在一些实施方案中，所述第一引物池包含用于预扩增组(II)的下述目标标记物的 引物对：PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A。在一些实施方案中，所述第一引物池包含用于预扩增组(II)的下述目标标记物的引物对：PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A。
在一些实施方中，所述第一引物池包含用于预扩增下述目标标记物的引物对：Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在一些实施方式中，所述的各个目标标记物包括或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
在一些实施方式中，所述第一引物池至少包含选自以下的引物对中的一对或多对：SEQ ID NO：22/23、24/25、26/27、28/29、30/31、32/33和34/35。优选地，所述第一引物池包含选自下组(I)的至少一对、至少两对或全部三对引物对和下组(II)的至少一对、至少两对、至少三对引物对：(I)SEQ ID NO：22/23、24/25、26/27；(II)SEQ ID NO：28/29、30/31、32/33和34/35。在一些实施方式中，所述第一引物池包含以下引物对：SEQ ID NO：22/23、24/25、26/27、28/29、30/31、32/33和34/35，任选地其中所述第二引物池包含至少一个与所述第一引物池中的至少一个引物对相同的引物对。在一些实施方式中，第二引物池中的引物对与第一引物池中的引物对完全相同。
在一些实施方式中，所述第一引物池和所述第二引物池进一步包含用于扩增对照标记物的引物对。在一些实施方式中，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
在一些实施方式中，所述试剂盒进一步包含多个容器，每个容器均用于接收所述第二引物池的组分。
在一些实施方式中，所述试剂盒还包含可用于进行CpG位置特异性甲基化分析的标准试剂，其中所述分析包括以下一种或多种技术：MS-SNuPE、MSP、MethyLight TM、HeavyMethyl TM、COBRA和核酸测序。
在一些实施方式中，所述试剂盒可包含选自下组的额外的试剂：缓冲液(例如限制酶、PCR、保存或洗涤缓冲液)、DNA回收试剂或试剂盒(例如沉淀、超滤、亲和柱)和DNA回收组件。
在一些实施方式中，本申请的试剂盒可包含：
a)亚硫酸氢盐试剂；
b)第一引物池，所述引物池包含多个甲基化特异性引物对，用于预扩增下组(I)中的至少一个目标标记物和下组(II)中的至少一个目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A，其中所述甲基化特异性引物对包含以下引物对，或由以下引物对组成：SEQ ID NO:22/23、24/25、26/27、28/29、30/31、32/33和34/35；
c)第二试剂，所述第二试剂用于定量分析被所述第一引物池预扩增的目标标记物的每一个的甲基化水平，其中所述第二试剂包含第二引物池，所述第二引物池包含多个定量引物对，所述定量引物对能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
本申请的试剂盒还可包含包装在独立容器中的其他组分，例如适用于封闭、洗涤或包被的缓冲液或溶液。
本申请的试剂盒可进一步包含在DNA富集领域中已知的以下组分的一种或几种：蛋白组分，所述蛋白选择性地结合甲基化的DNA；三链形成核酸组分，一个或多个接头，任选地在合适的溶液中；用于进行连接的物质或溶液，例如连接酶、缓冲液；用于进行柱层析的物质或溶液；用于进行免疫学为基础的富集(例如免疫沉淀)的物质或溶液；用于进行核酸扩增的物质或溶液，例如PCR；一种染料或几种染料，若适用于偶联剂，若适用于溶液中；用于进行杂交的物质或溶液；和/或用于进行洗涤步骤的物质或溶液。
3.  用途
在另一方面，本申请提供了本申请的试剂盒在制造用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后或监测接受结直肠瘤治疗的个体对治疗的应答的诊断试剂盒中的用途。
在另一方面，本申请提供用于定量分析目标标记物的甲基化水平的试剂在制造试剂 盒中的用途，所述试剂盒被用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法中。
优选地，所述方法中的各个步骤以及各步骤所用的各试剂如本文“1.方法”部分所述，因此，本发明的试剂盒可根据该方法而包括用于该方法各步骤所需的各试剂。
优选地，所述试剂盒为本文“2.试剂盒”部分所述。
优选地，所述目标标记物包括：所述组(I)中的至少2个或全部3个目标标记物，和所述组(II)中的至少2个、至少3个或全部4个目标标记物。
优选地，所述目标标记物包括Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A。
优选地，所述试剂包括扩增引物，用于扩增所述目标标记物的引物对。
优选地，所述试剂包括用于扩增：所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1的引物对；或所述组(I)的Septin9、BCAT1和IKZF1的引物对。
优选地，所述试剂包括用于扩增：所述组(II)中的PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A的引物对；所述组(II)中的PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A的引物对；或所述组(II)中的PKNOX2、CRHBP、SOX1和基因间隔区A的引物对。
优选地，用于扩增Septin9的引物对为SEQ ID NO:22/23；用于扩增BCAT1的引物对为SEQ ID NO:24/25；用于扩增IKZF1的引物对为SEQ ID NO:26/27；用于扩增PKNOX2的引物对为SEQ ID NO:28/29；用于扩增CRHBP的引物对为SEQ ID NO:30/31；用于扩增SOX1的引物对为SEQ ID NO:32/33；用于扩增基因间隔区A的引物对为SEQ ID NO:34/35。
优选地，所述试剂还包括定量引物对，所述定量引物对被设计为用于扩增已用所述扩增引物预扩增的所述目标序列内的至少一部分，能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
优选地，所述定量引物对与所述扩增引物对相同。
优选地，所述试剂进一步包含一个或多个封闭寡核苷酸。
优选地，所述试剂进一步包含检测试剂。
优选地，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射 性同位素标记的探针和生物素标记的探针。
优选地，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-43。
优选地，所述探针包括SEQ ID NO:36-38中所示的至少一条、至少两条或全部三条核苷酸序列，和SEQ ID NO:39或40以及41-43中所示的至少一条、至少两条或至少三条核苷酸序列。
优选地，所述探针包括：SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。
优选地，所述荧光探针的5’端标记有荧光染料，3’端标记有猝灭剂；优选地，所述荧光染料选自FAM、HEX/VIC、TAMRA、Texas Red或Cy5，所述淬灭剂选自BHQ1、BHQ2、BHQ3、DABCYL、TAMRA或lowa Black Dark Quenchers。
优选地，所述试剂还包括亚硫酸氢盐试剂或甲基化敏感限制酶。
优选地，所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合。
优选地，所述MSRE选自下组：HpaII酶、SalI酶、
Figure PCTCN2022074143-appb-000010
酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
优选地，所述试剂包含用于扩增对照标记物的引物对。
优选地，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
优选地，所述目标标记物包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
优选地，所述方法包括如下步骤：
(a)从所述个体获取含有DNA的生物样品；
(b)用试剂处理步骤(a)中获取的所述生物样品中的DNA，所述试剂能够区分所述 DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
(c)用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物(target marker)中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物；
(d)基于步骤(c)获取的预扩增产物来分别定量分析所述被扩增的目标标记物的甲基化水平；
(e)分别比较步骤(d)中的所述被扩增的目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的至少2个或全部3个目标标记物被扩增。
在一些实施方式中，上述方法的步骤(c)中，所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1被扩增。
在一些实施方案中，上述方法的步骤(c)中，所述组(II)中的至少2个、至少3个或全部4个目标标记物被扩增。
在一些实施方案中，上述方法的步骤(c)中，PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A被扩增。
在一些实施方案中，上述方法的步骤(c)中，用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下目标标记物：Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物。
在一些实施方式中，所述各个目标标记物包含或是：
a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678 ；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
b)亚硫酸氢盐转化后的a)的对应区域；或
c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
具体实施方式
所有实施例中使用的生物材料，包括各种克隆和表达质粒、培养基、酶、缓冲液、各种培养方法、蛋白质提取和纯化方法以及其他分子生物学操作方法，都是本领域技术人员所熟知的。更多细节请参照Sambrook et al.,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press,N.Y.和Frederick M.Ausubel等人编写的“Short Protocols in Molecular Biology”(Yan Ziying等译，科学出版社(北京)，1998)。
实施例
本实施例通过检测Septin9、BCAT1、IKZF1高甲基化及PKNOX2、CRHBP、SOX1、基因间隔区(hg19，chr10:130085033-130085148)低甲基化来筛查结直肠进展期腺瘤。
为评估甲基化标志物在结直肠进展期腺瘤检测中的临床性能，我们使用预扩增检测法，检测了117份经临床诊断为结直肠进展期腺瘤的血浆样品和100份结肠镜检查为阴性的血浆对照样品。检测位点为Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区(hg19)。
预扩增方法
预扩增方法包括以下步骤：
1、使用QIAamp Circulating Nucleic Acid Kit(Qiagen)从1-4ml血浆样品中获得细胞外游离DNA(cfDNA)样品。
2、使用亚硫酸氢盐试剂(MethylCodeTM Bisulfite Conversion Kit)对20ng cfDNA进行亚硫酸氢盐转化以获得转化的cfDNA。
3、将转化的cfDNA样品进行预扩增。简而言之，在甲基化特异性引物对的存在下，通过PCR反应对从上述步骤2获得的转化的cfDNA进行预扩增，所述甲基化特异性引物对专门针对Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和位于 chr10:130085033-130085148的基因间隔区(hg19)而设计。在PCR反应体系中，每个引物的终浓度均为200nM。25μL PCR混合物由10μL转化的cfDNA、2.5μL含有上述引物的预混液和12.5μL PCR试剂(
Figure PCTCN2022074143-appb-000011
Universal Probe qPCR Master Mix(NEB))组成。
PCR反应条件如下：95℃3分钟；95℃30秒，56℃60秒，10个循环。ProFlex TM PCR系统(Thermo Fisher)。
4、将从上述步骤3获得的预扩增产物稀释10倍，然后用于多重荧光PCR检测，专门针对Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区(hg19)。qPCR混合物由10μL稀释的预扩增产物、2.5μL引物/探针池、12.5μL PCR试剂(
Figure PCTCN2022074143-appb-000012
Universal Probe qPCR Master Mix(NEB))组成。非CpG的ACTB区域用作每个反应孔的内参。不同标记物的检测探针用不同的荧光进行标记。在PCR反应体系中，每个引物的终浓度为500nM，每个检测探针的终浓度为200nM。PCR反应条件如下：95℃5分钟；95℃15秒，56℃40秒(采集荧光)，50个循环。使用ABI 7500 Real-Time PCR System在相应的荧光通道检测不同的荧光。
结果
将没有扩增信号样品的Ct值设置为50。分别统计Septin9、BCAT1、IKZF1的Ct平均值及PKNOX2、CRHBP、SOX1、基因间隔区A(hg19，chr10:130085033-130085148)的Ct平均值。图1显示患有进展期腺瘤的群体和结肠镜检查为阴性的群体中目标标志物Septin9、BCAT1、IKZF1Ct值分布，患有进展期腺瘤的群体整体Ct值比阴性群体要小，提示甲基化水平更高。如图2所示，患有进展期腺瘤的群体中目标标志物PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区(hg19)的检测Ct较阴性对照要大，提示更低的甲基化水平。
表3显示了在预扩增方法中检测Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区(hg19)目标标记物对进展期腺瘤的检测结果。根据Septin9、BCAT1、IKZF1的平均Ct值ROC曲线，确定平均Ct小于44为检测阳性；根据PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区(hg19)的平均Ct值ROC曲线，划定平均Ct大于38为检测阳性。样本最终结果Septin9、BCAT1、IKZF1的高甲基化或PKNOX2、CRHBP、SOX1和位于chr10:130085033-130085148的基因间隔区A(hg19)的低甲基化任一为阳性即为检测阳性。如表3所示，预扩增方法显示出对进展期腺瘤具有超高灵敏度(54.7％)和对结肠镜 检查为阴性的群体的高特异性(82.7％)。对进展期腺瘤的检出率远高于目前已有的血液检测方法。
表3：预扩增方法结果和结肠镜检查结果的对比
结肠镜检查结果 准确率 阳性样本 阴性样本 总数
结直肠进展期腺瘤 54.7％ 64 53 117
结肠镜阴性 86.0％ 14 86 100

Claims (44)

  1. 一种用于诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的试剂盒，其包含：
    (a)处理DNA的第一试剂，其中所述第一试剂能够区分DNA中的甲基化和未甲基化的CpG位点；
    (b)第一引物池，所述第一引物池包含用于预扩增以下两组目标标记物中每一组目标标记物中的至少一种目标标记物的引物对：(I)Septin9、BCAT1和IKZF1，和(II)PKNOX2、CRHBP、SOX1和基因间隔区A，其中所述引物对可与被所述第一试剂处理后的所述目标标记物的目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交，其中所述目标序列包含至少一个CpG位点；
    (c)第二试剂，所述第二试剂用于定量分析被所述第一引物池预扩增的所述目标标记物中每个目标标记物的甲基化水平。
  2. 如权利要求1所述的试剂盒，其特征在于，所述目标标记物包含或是：
    a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
    b)亚硫酸氢盐转化后的a)的对应区域；或
    c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
  3. 如权利要求1或2所述的试剂盒，其特征在于，所述第一引物池中包括用于扩增所述组(I)中的至少2个或全部3个目标标记物的引物对；和/或所述第一引物池中包括用于扩增所述组(II)中的至少2个、至少3个或全部4个目标标记物的引物对。
  4. 如权利要求3所述的试剂盒，其特征在于，
    所述第一引物池中包括用于扩增所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1引物对；或所述第一引物池中包括用于扩增所述组(I)中的Septin9、BCAT1与IKZF1的引物对；和/或
    所述第一引物池中包括用于扩增组(II)中的PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A的引物对；或所述第一引物池中包括用于扩增组(II)中的PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间 隔区A的引物对。
  5. 如权利要求3所述的试剂盒，其特征在于，所述第一引物池中包括用于扩增Septin9、BCAT1和IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A的引物对。
  6. 如权利要求1所述的试剂盒，其特征在于，用于扩增Septin9的引物对为SEQ ID NO:22/23；用于扩增BCAT1的引物对为SEQ ID NO:24/25；用于扩增IKZF1的引物对为SEQ ID NO:26/27；用于扩增PKNOX2的引物对为SEQ ID NO:28/29；用于扩增CRHBP的引物对为SEQ ID NO:30/31；用于扩增SOX1的引物对为SEQ ID NO:32/33；用于扩增基因间隔区A的引物对为SEQ ID NO:34/35。
  7. 如权利要求1所述的试剂盒，其特征在于，所述第一引物池包含以下引物对，或由以下引物对组成：SEQ ID NO:22/23、24/25、26/27、28/29、30/31、32/33和34/35。
  8. 如权利要求1所述的试剂盒，其特征在于，所述第二试剂包含第二引物池，所述第二引物池包含多个定量引物对，所述定量引物对被设计为用于扩增被所述第一引物池预扩增的所述目标序列内的至少一部分，能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
  9. 如权利要求8所述的试剂盒，其特征在于，所述第二引物池中的定量引物对和所述第一引物池中的引物对相同。
  10. 如权利要求8或9所述的试剂盒，其特征在于，所述第一引物池和所述第二引物池被包装在单一容器内或被包装在独立容器内。
  11. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒进一步包含一个或多个封闭寡核苷酸。
  12. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒进一步包含检测试剂。
  13. 如权利要求12所述的试剂盒，其特征在于，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。
  14. 如权利要求13所述的试剂盒，其特征在于，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-43。
  15. 如权利要求14所述的试剂盒，其特征在于，所述探针包括SEQ ID NO:36-38中所示的至少一条、至少两条或全部三条核苷酸序列，和SEQ ID NO:39或40以及41-43中所示的至少一条、至少两条或至少三条核苷酸序列。
  16. 如权利要求15所述的试剂盒，其特征在于，所述探针包括：SEQ ID NO:36-38和41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。
  17. 如权利要求13所述的试剂盒，其特征在于，所述荧光探针的5’端标记有荧光染 料，3’端标记有猝灭剂；优选地，所述荧光染料选自FAM、HEX/VIC、TAMRA、Texas Red或Cy5，所述淬灭剂选自BHQ1、BHQ2、BHQ3、DABCYL、TAMRA或lowa Black Dark Quenchers。
  18. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒进一步包含DNA聚合酶和/或一个适合存放从所述个体中获取的所述生物样品的容器。
  19. 如权利要求1所述的试剂盒，其特征在于，所述第一试剂包括亚硫酸氢盐试剂或甲基化敏感限制酶。
  20. 如权利要求19所述的试剂盒，其特征在于，
    所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合；
    所述MSRE选自下组：HpaII酶、SalI酶、
    Figure PCTCN2022074143-appb-100001
    酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
  21. 如权利要求1所述的试剂盒，其特征在于，所述第一引物池和所述第二试剂包含的第二引物池进一步包含用于扩增对照标记物的引物对。
  22. 如权利要求21所述的试剂盒，其特征在于，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
  23. 用于定量分析目标标记物的甲基化水平的试剂在制造试剂盒中的用途，所述试剂盒用于在个体中诊断结直肠进展期腺瘤、筛查结直肠进展期腺瘤形成或形成的风险或评估结直肠进展期腺瘤的进展或预后的方法中；其中，所述目标标记物包括下组(I)中的至少一种目标标记物和下组(II)中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A。
  24. 如权利要求23所述的用途，其特征在于，所述目标标记物包括：所述组(I)中的至少2个或全部3个目标标记物，和所述组(II)中的至少2个、至少3个或全部4个目标标记物。
  25. 如权利要求23所述的用途，其特征在于，所述目标标记物为Septin9、BCAT1、IKZF1、PKNOX2、CRHBP、SOX1和基因间隔区A。
  26. 如权利要求23-25中任一项所述的用途，其特征在于，所述试剂包括扩增引物对，用于扩增所述目标标记物的引物对。
  27. 如权利要求27所述的用途，其特征在于，所述扩增引物对包括用于扩增：
    所述组(I)中的Septin9与BCAT1，BCAT1与IKZF1，或Septin9与IKZF1引物对；或
    所述组(I)的Septin9、BCAT1和IKZF1的引物对。
  28. 如权利要求23所述的用途，其特征在于，所述扩增引物对包括用于扩增：
    所述组(II)中的PKNOX2与CRHBP，PKNOX2与SOX1，PKNOX2与基因间隔区A，CRHBP与基因间隔区A，或SOX1与基因间隔区A的引物对；
    所述组(II)中的PKNOX2、CRHBP和SOX1，PKNOX2、CRHBP和基因间隔区A，PKNOX2、SOX1和基因间隔区A或CRHBP、SOX1和基因间隔区A的引物对；或
    所述组(II)中的PKNOX2、CRHBP、SOX1和基因间隔区A的引物对。
  29. 如权利要求26-28中任一项所述的用途，其特征在于，用于扩增Septin9的引物对为SEQ ID NO:22/23；用于扩增BCAT1的引物对为SEQ ID NO:24/25；用于扩增IKZF1的引物对为SEQ ID NO:26/27；用于扩增PKNOX2的引物对为SEQ ID NO:28/29；用于扩增CRHBP的引物对为SEQ ID NO:30/31；用于扩增SOX1的引物对为SEQ ID NO:32/33；用于扩增基因间隔区A的引物对为SEQ ID NO:34/35。
  30. 如权利要求23所述的用途，其特征在于，所述试剂还包括定量引物对，所述定量引物对被设计为用于扩增已被预扩增的所述目标序列内的至少一部分，能够与被所述第一引物池预扩增的所述目标序列的至少9个连续核苷酸在严紧条件下、中等严紧条件下或高度严紧条件下杂交。
  31. 如权利要求30所述的用途，其特征在于，所述定量引物对与权利要求28或29所述的引物对相同。
  32. 如权利要求23所述的用途，其特征在于，所述试剂进一步包含一个或多个封闭寡核苷酸。
  33. 如权利要求23所述的用途，其特征在于，所述试剂进一步包含检测试剂。
  34. 如权利要求33所述的用途，其特征在于，所述检测试剂选自下组：荧光探针、嵌入染料、生色团标记的探针、放射性同位素标记的探针和生物素标记的探针。
  35. 如权利要求34所述的用途，其特征在于，所述探针至少包括选自下组的一条或多条核苷酸序列：SEQ ID NO:36-43。
  36. 如权利要求35所述的用途，其特征在于，所述探针包括SEQ ID NO:36-38中所示的至少一条、至少两条或全部三条核苷酸序列，和SEQ ID NO:39或40以及41-43中所示的至少一条、至少两条或至少三条核苷酸序列。
  37. 如权利要求35所述的用途，其特征在于，所述探针包括：SEQ ID NO:36-38和 41-43所示的核苷酸序列，以及SEQ ID NO:39和/或40所示的核苷酸序列。
  38. 如权利要求34所述的用途，其特征在于，所述荧光探针的5’端标记有荧光染料，3’端标记有猝灭剂；优选地，所述荧光染料选自FAM、HEX/VIC、TAMRA、Texas Red或Cy5，所述淬灭剂选自BHQ1、BHQ2、BHQ3、DABCYL、TAMRA或lowa Black Dark Quenchers。
  39. 如权利要求23所述的用途，其特征在于，所述试剂还包括亚硫酸氢盐试剂或甲基化敏感限制酶。
  40. 如权利要求39所述的用途，其特征在于，
    所述亚硫酸氢盐试剂选自下组：亚硫酸氢铵、亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢钙、亚硫酸氢镁、亚硫酸氢铝、亚硫酸氢根离子，及其任意组合；
    所述MSRE选自下组：HpaII酶、SalI酶、
    Figure PCTCN2022074143-appb-100002
    酶、ScrFI酶、BbeI酶、NotI酶、SmaI酶、XmaI酶、MboI酶、BstBI酶、ClaI酶、MluI酶、NaeI酶、NarI酶、PvuI酶、SacII酶、HhaI酶及其任意组合。
  41. 如权利要求23所述的用途，其特征在于，所述试剂包含用于扩增对照标记物的引物对。
  42. 如权利要求41所述的用途，其特征在于，所述对照标记物选自下组：ACTB、GAPDH、微管蛋白、ALDOA、PGK1、LDHA、RPS27A、RPL19、RPL11、ARHGDIA、RPL32、C1orf43、CHMP2A、EMC7、GPI、PSMB2、PSMB4、RAB7A、REEP5、SNRPD3、VCP和VPS29。
  43. 如权利要求23所述的用途，其特征在于，所述目标标记物包含或是：
    a)如下所示的通过Hg19坐标定义的各个区域：Septin9，chr17:75276651-75496678；BCAT1，chr12:24964295-25102393；IKZF1，chr7:50343720-50472799；PKNOX2，chr11:125034583-125303285；CRHBP，chr5:76248538-76276983；SOX1，chr13:112721913-112726020；基因间隔区A，chr10:130082033-130087148；以及上述每个区域的各个起始位点的上游5kb和各个末端位点的下游5kb；或
    b)亚硫酸氢盐转化后的a)的对应区域；或
    c)甲基化敏感限制酶(MSRE)处理后的a)的对应区域。
  44. 如权利要求23所述的用途，其特征在于，所述方法包括如下步骤：
    (a)从所述个体获取含有DNA的生物样品；
    (b)用试剂处理步骤(a)中获取的所述生物样品中的DNA，所述试剂能够区分所述DNA中的甲基化和未甲基化的CpG位点，从而获得经处理的DNA；
    (c)用预扩增引物池预扩增从步骤(b)获取的所述经处理的DNA中的以下两组目标标记物(target marker)中每一组目标标记物中的至少一种目标标记物：(I)Septin9、BCAT1和IKZF1，(II)PKNOX2、CRHBP、SOX1和基因间隔区A；其中所述目标标记物的至少一部分被预扩增以获得预扩增产物；
    (d)基于步骤(c)获取的预扩增产物来分别定量分析所述被扩增的目标标记物的甲基化水平；
    (e)分别比较步骤(d)中的所述被扩增的目标标记物的甲基化水平和相应的参考水平，其中：组(I)所述的目标标记物相对于其相应的参考水平具有更高的甲基化水平，且组(II)所述的目标标记物相对于其相应的参考水平具有较低的甲基化水平表明所述个体患有结直肠进展期腺瘤，或者所述个体有结直肠进展期腺瘤形成或形成的风险，或者所述个体有结直肠进展期腺瘤发展或发展的可能性增加，或者所述个体有结直肠进展期腺瘤预后不良或预后不良的风险。
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