WO2019105090A1 - 用于检测食道癌的组合物及其用途 - Google Patents

用于检测食道癌的组合物及其用途 Download PDF

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WO2019105090A1
WO2019105090A1 PCT/CN2018/104076 CN2018104076W WO2019105090A1 WO 2019105090 A1 WO2019105090 A1 WO 2019105090A1 CN 2018104076 W CN2018104076 W CN 2018104076W WO 2019105090 A1 WO2019105090 A1 WO 2019105090A1
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seq
sequence
target
esophageal cancer
gene
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PCT/CN2018/104076
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English (en)
French (fr)
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马竣
韩晓亮
王建铭
樊代明
聂勇战
胡思隽
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博尔诚（北京）科技有限公司
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Priority claimed from CN201711248825.5A external-priority patent/CN109868320B/zh
Priority claimed from CN201810989986.8A external-priority patent/CN110863046B/zh
Application filed by 博尔诚（北京）科技有限公司 filed Critical 博尔诚（北京）科技有限公司
Priority to KR1020207018984A priority Critical patent/KR102512282B1/ko
Priority to EP18882701.8A priority patent/EP3744858A4/en
Priority to JP2020545787A priority patent/JP6924335B2/ja
Publication of WO2019105090A1 publication Critical patent/WO2019105090A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is in the field of biotechnology, relates to compositions for detecting esophageal cancer and their use in disease detection, and in particular to compositions for detecting esophageal cancer and their corresponding kits and uses.
  • Esophageal cancer is a common digestive tract tumor. According to the National Cancer Prevention Office, in 2015, the incidence of esophageal cancer in China was 478 cases per 100,000 people, and the mortality rate was 375 cases per person, ranking fourth and third among common cancers. The mortality rate of esophageal cancer is close to 80%, which is a highly malignant cancer. About 300,000 people worldwide die from esophageal cancer each year, half of which comes from China.
  • esophageal cancer diagnosis technology has limited application in early detection and screening of esophageal cancer, mainly because: 1) tissue biopsy is invasive and not suitable for early cancer screening; 2) imaging detection technology (such as : Esophageal angiography and endoscopy: limitations in equipment cost, operating techniques, and invasiveness are also difficult to promote as a cancer screening technology; 3) traditional serum tumor markers (eg AFP, CEA) , CA125, and CA199, etc.) have low sensitivity to esophageal cancer detection and cannot fully meet the requirements of early cancer screening.
  • tissue biopsy is invasive and not suitable for early cancer screening
  • imaging detection technology such as : Esophageal angiography and endoscopy: limitations in equipment cost, operating techniques, and invasiveness are also difficult to promote as a cancer screening technology
  • 3) traditional serum tumor markers eg AFP, CEA
  • the present invention provides a composition for detecting esophageal cancer in view of the problems of inconvenient detection, low sensitivity, and high cost of existing esophageal cancer detection techniques.
  • the compositions provided by the present invention are capable of sensitively and specifically detecting esophageal cancer, and the present invention also provides kits comprising the compositions and their use in detecting esophageal cancer.
  • the kit provided by the invention has good sensitivity for detecting esophageal cancer, and can detect esophageal cancer conveniently, quickly and effectively.
  • the present invention provides compositions, kits and uses thereof for detecting esophageal cancer in vitro, and methods for performing detection based on the kit.
  • the invention relates to the following:
  • WHAT IS CLAIMED IS 1. A composition for detecting esophageal cancer in vitro, the composition comprising:
  • nucleic acid for detecting the methylation status of a target gene a nucleic acid for detecting the methylation status of a target gene
  • the target gene is one or both of an MT1A gene and an EPO gene.
  • composition according to item 1 wherein the target sequence of the MT1A gene is as shown in SEQ ID NO: 1.
  • composition according to item 1 wherein the target sequence of the EPO gene is as shown in SEQ ID NO: 3.
  • composition according to any one of items 1 to 3, wherein the nucleic acid for detecting a methylation status of a target gene comprises:
  • the fragment comprises at least one CpG dinucleotide sequence.
  • composition according to any one of items 1 to 4, wherein the nucleic acid for detecting the methylation status of the target gene further comprises:
  • Hybridizing at least 15 nucleotide fragments in the target gene target sequence under moderately stringent or stringent conditions at least 15 nucleotide fragments in the target gene target sequence under moderately stringent or stringent conditions
  • the fragment comprises at least one CpG dinucleotide sequence.
  • An agent that converts the 5-position unmethylated cytosine base of the target gene target sequence into uracil An agent that converts the 5-position unmethylated cytosine base of the target gene target sequence into uracil.
  • composition according to any one of items 1 to 6, wherein the nucleic acid for detecting the methylation status of the target gene further comprises:
  • a blocker that preferentially binds to a target sequence in an unmethylated state is a blocker that preferentially binds to a target sequence in an unmethylated state.
  • a blocker which is the sequence of SEQ ID NO: 8 or the sequence of SEQ ID NO: 12.
  • An oligonucleotide for detecting esophageal cancer in vitro comprising:
  • a blocker that preferentially binds to a target sequence in an unmethylated state is a blocker that preferentially binds to a target sequence in an unmethylated state.
  • An oligonucleotide for detecting esophageal cancer in vitro comprising:
  • An oligonucleotide for detecting esophageal cancer in vitro comprising:
  • a kit comprising the composition of any one of items 1 to 8 or the oligonucleotide of any one of items 9 to 17.
  • kit of clause 20 further comprising at least one other component selected from the group consisting of:
  • a nucleoside triphosphate A nucleoside triphosphate, a DNA polymerase, and a buffer required for the function of the DNA polymerase.
  • composition of any one of items 1 to 8 or the oligonucleotide of any one of clauses 9 to 17 in the preparation of a kit for the detection of esophageal cancer in vitro.
  • kit for detecting esophageal cancer in vitro detects esophageal cancer by a method comprising the following steps:
  • the extracted genomic DNA is treated with a reagent to convert 5 unmethylated cytosine bases into uracil or other bases;
  • the reagent-treated DNA sample is contacted with a DNA polymerase and a primer of the target gene target sequence, and the DNA polymerization reaction is carried out in the presence of a blocking agent preferentially bound to the target sequence in an unmethylated state;
  • a methylation state of at least one CpG dinucleotide of the target gene target sequence is determined based on the presence or absence of the amplification product.
  • a method of detecting esophageal cancer comprising the steps of:
  • the state of the biological sample is determined by the detection result of the methylation state of the target gene target sequence, thereby achieving in vitro detection of esophageal cancer.
  • a method of detecting esophageal cancer comprising the steps of:
  • the extracted genomic DNA is treated with a reagent to convert 5 unmethylated cytosine bases into uracil or other bases;
  • the reagent-treated DNA sample is contacted with a DNA polymerase and a primer of the target gene target sequence, and subjected to DNA polymerization in the presence of a blocking agent preferentially bound to the target sequence in an unmethylated state;
  • a methylation state of at least one CpG dinucleotide of the target gene target sequence is determined based on the presence or absence of the amplification product.
  • the target gene is one or both of the MT1A gene and the EPO gene.
  • the blocker is a blocker that preferentially binds to a target sequence in an unmethylated state.
  • primers are the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, or the sequences of SEQ ID NO: 9 and SEQ ID NO: 10.
  • probe is the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 11.
  • composition for detecting esophageal cancer in vitro comprising:
  • nucleic acid for detecting the methylation status of a target gene target sequence
  • the target gene is one or two of an MT1A gene and an EPO gene
  • the target protein is SNCG, ⁇ -synuclein.
  • composition of clause 38, wherein the target sequence of the MT1A gene is set forth in SEQ ID NO: 1.
  • composition according to any one of items 38 to 40, wherein the nucleic acid for detecting a methylation state of a target gene target sequence comprises:
  • the fragment comprises at least one CpG dinucleotide sequence.
  • composition of any one of items 38 to 41, wherein the nucleic acid for detecting a methylation status of a target gene further comprises:
  • Hybridizing at least 15 nucleotides in the target gene target sequence under moderately stringent or stringent conditions is provided.
  • the fragment comprises at least one CpG dinucleotide sequence.
  • composition of any one of items 38 to 42 further comprising:
  • An agent that converts the 5-position unmethylated cytosine base of the target gene target sequence into uracil An agent that converts the 5-position unmethylated cytosine base of the target gene target sequence into uracil.
  • composition according to any one of items 38 to 43 wherein the nucleic acid for detecting the methylation status of the target gene further comprises:
  • a blocker that preferentially binds to a target sequence in an unmethylated state is a blocker that preferentially binds to a target sequence in an unmethylated state.
  • a blocker which is the sequence of SEQ ID NO: 8 or the sequence of SEQ ID NO: 12.
  • composition of any one of items 38 to 45 further comprising:
  • the reagent being an enzyme-linked immunosorbent assay reagent
  • the reagent includes an antibody-coated reaction plate for detecting a target protein concentration, a SNCG protease conjugate, a substrate solution, and a wash Liquid and stop solution.
  • a kit comprising the composition of any one of items 38 to 46 or the oligonucleotide of any one of items 9 to 17.
  • kit of item 47 further comprising at least one other component selected from the group consisting of:
  • nucleoside triphosphate a nucleoside triphosphate
  • DNA polymerase a DNA polymerase
  • buffer required for the function of the DNA polymerase
  • An antibody-coated reaction plate for detecting a target protein concentration, a SNCG protease conjugate, a substrate solution, a washing solution, and a stop solution.
  • kit for detecting esophageal cancer in vitro detects esophageal cancer by a method comprising the following steps:
  • the in vitro detection of esophageal cancer is achieved by jointly determining whether the subject has esophageal cancer by jointly detecting the methylation status of the target gene target sequence and the target protein concentration.
  • the extracted free DNA is treated with a reagent to convert the 5-unmethylated cytosine base to uracil or other bases;
  • the reagent-treated DNA sample is contacted with a DNA polymerase and a primer of the target gene target sequence, and the DNA polymerization reaction is carried out in the presence of a blocking agent preferentially bound to the target sequence in an unmethylated state;
  • the amplification product is detected by a probe
  • the concentration of SNCG in plasma or serum was determined using an immunoreaction using an SNCG antibody.
  • a method of detecting esophageal cancer comprising the steps of:
  • the in vitro detection of esophageal cancer is achieved by jointly determining whether the subject has esophageal cancer by jointly detecting the methylation status of the target gene target sequence and the target protein concentration.
  • a method of detecting esophageal cancer comprising the steps of:
  • the reagent-treated DNA sample is contacted with a DNA polymerase and a primer of the target gene target sequence, and the DNA polymerization reaction is carried out in the presence of a blocking agent preferentially bound to the target sequence in an unmethylated state;
  • the amplification product is detected by a probe
  • the concentration of SNCG in plasma or serum was determined using an immunoreaction using an SNCG antibody.
  • the target gene is one or both of an MT1A gene and an EPO gene, and
  • the target protein is SNCG.
  • primers are the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, or the sequences of SEQ ID NO: 9 and SEQ ID NO: 10.
  • probe is the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 11.
  • the inventors of the present invention used epigenomics and bioinformatics techniques to analyze the genome-wide methylation data of esophageal cancer tissues and paracancerous control tissues, and found two methylation genes associated with esophageal cancer, and identified These two esophageal cancer methylation genes have a target sequence of methylation abnormality; further, the inventors of the present invention found that the target sequences of the two esophageal cancer methylation genes can be sensitively and specifically detected. The methylation status of the gene can be used for the detection of free DNA in peripheral blood.
  • compositions and assays described herein are capable of sensitively and specifically detecting esophageal cancer, including common esophageal cancers of two different cell types: squamous cell carcinoma and gland cancer. Therefore, the present invention provides a composition and a detection method which can be used for detecting esophageal cancer in vitro, and has important clinical application value.
  • the inventors of the present invention have found that in a plurality of tumor protein markers, the combined detection of SNCG protein and methylation of the target gene can significantly improve the detection of esophageal cancer. Therefore, the present invention provides a composition and a detection method which can be used for detecting esophageal cancer in vitro, and has important clinical application value.
  • Figure 1 shows a graph showing the results of screening the target genes of the present invention.
  • FIG. 2 is a diagram showing the composition and detection method of the present invention for leukocyte genomic DNA (negative reference product of methylation status of target gene target sequence) and DNA methyltransferase-treated leukocyte genomic DNA (target gene target sequence methyl group) Detection of positive reference in the state.
  • the results showed that the composition and the detection method provided by the present invention were negative for the detection of leukocyte genomic DNA, and the detection results of the leukocyte genomic DNA after DNA methyltransferase treatment were positive.
  • Fig. 3 is a graph showing the results of in vitro non-invasive detection of esophageal cancer by detecting the methylation status of a target gene target sequence by using the composition and the detection method.
  • Fig. 4 is a graph showing the results of in vitro non-invasive detection of esophageal cancer by detecting the methylation state of the target gene target sequence and the concentration of the target protein by using the composition and the detection method.
  • the present invention provides a composition for detecting esophageal cancer in vitro, the composition comprising a nucleic acid for detecting a methylation state in a target gene target sequence, and an antibody for detecting a target protein concentration;
  • the target gene is one or both of an MT1A gene and an EPO gene, and the target protein is SNCG.
  • the present invention provides a target gene target sequence for aberrant methylation in esophageal cancer, including a target sequence of the MT1A gene and the EPO gene, the target sequence of the MT1A gene is shown in SEQ ID NO: 1-2, and the EPO gene is The target sequence is set forth in SEQ ID NO: 3-4.
  • the target sequence of the MT1A gene is shown in SEQ ID NO: 1.
  • SEQ ID NO: 2 The complement of SEQ ID NO: 1 is set forth in SEQ ID NO: 2.
  • sequence of the target sequence of the EPO gene is as shown in SEQ ID NO: 3.
  • SEQ ID NO: 3 The complement of SEQ ID NO: 3.
  • the nucleic acid for detecting the methylation status of the target gene comprises a fragment of at least 9 nucleotides in the target gene target sequence, wherein the fragment comprises at least one CpG dinucleotide sequence.
  • the nucleic acid for detecting the methylation status of the target gene comprises at least at least a bisulfite converted sequence of the target gene target sequence.
  • the nucleic acid for detecting the methylation status of the target gene comprises a fragment of at least 15 nucleotides that hybridizes to the target gene target sequence under moderately stringent or stringent conditions, wherein the fragment of the nucleotide Containing at least one CpG dinucleotide sequence.
  • the nucleic acid for detecting the methylation status of the target gene includes hybridization to the target gene target sequence under moderately tight or stringent conditions.
  • the composition further comprises an agent that converts the 5-position unmethylated cytosine base of the target gene target sequence into uracil. More preferably, the reagent is bisulfite.
  • the nucleic acid for detecting the methylation status of the target gene further comprises a blocking agent that preferentially binds to DNA in an unmethylated state.
  • the composition comprises one or more of the following primers, probes and/or blockers:
  • the invention provides an oligonucleotide for detecting esophageal cancer in vitro comprising: at least 9 nucleotides of SEQ ID NO: 1 or its complement and comprising at least one CpG dinucleotide sequence a fragment; and/or a fragment of at least 9 nucleotides of SEQ ID NO: 3 or its complement and comprising at least one CpG dinucleotide sequence.
  • the oligonucleotide for detecting esophageal cancer in vitro comprises: a fragment of at least 9 nucleotides in a sequence after bisulfite conversion of SEQ ID NO: 1 or its complement; and/or SEQ ID NO: 3 or a sequence thereof comprising at least 9 nucleotides in the bisulfite converted sequence and comprising at least one CpG dinucleotide sequence.
  • An oligonucleotide for detecting esophageal cancer in vitro of the present invention which further comprises: hybridizing to at least 15 nucleotides of said SEQ ID NO: 1 or its complement under moderately stringent or stringent conditions and comprising at least a fragment of a CpG dinucleotide sequence; and/or hybridizes to at least 15 nucleotides of said SEQ ID NO: 3 or its complement and comprises at least one CpG dinucleotide under moderately stringent or stringent conditions A fragment of the sequence.
  • the oligonucleotide for detecting esophageal cancer in vitro comprises: hybridizing under moderately stringent or stringent conditions to at least 15 nuclei in a sequence following bisulfite conversion of SEQ ID NO: 1 or its complement. And a fragment comprising at least one CpG dinucleotide sequence; and/or hybridizing under moderately stringent or stringent conditions to at least 15 of the sequences following bisulfite conversion of SEQ ID NO: 3 or its complement. Fragments of nucleotides and comprising at least one CpG dinucleotide sequence.
  • the oligonucleotide for detecting esophageal cancer in vitro of the present invention further comprises: a blocking agent which preferentially binds to DNA in an unmethylated state.
  • the oligonucleotide for detecting esophageal cancer in vitro comprises: the sequences of SEQ ID NO: 5 and SEQ ID NO: 6. It also includes the sequence of SEQ ID NO: 7. It also includes the sequence of SEQ ID NO: 8.
  • an oligonucleotide for detecting esophageal cancer in vitro comprises: the sequences of SEQ ID NO: 9 and SEQ ID NO: 10. It also includes the sequence of SEQ ID NO:11. It also includes the sequence of SEQ ID NO: 12.
  • the invention provides a kit comprising the composition.
  • the kit further comprises at least one other component selected from the group consisting of a nucleoside triphosphate, a DNA polymerase, and a buffer required for the function of the DNA polymerase.
  • composition for detecting esophageal cancer in vitro of the present invention further comprising an antibody for detecting a concentration of a target protein which is ⁇ synuclein (SNCG).
  • SNCG ⁇ synuclein
  • the invention also relates to the use of the MT1A gene, the EPO gene, and the SNCG protein in the preparation of a kit for detecting esophageal cancer in vitro.
  • the MT1A is a metallothionein 1A, the English name metallothionein 1A, located in the q13 region of human chromosome 16, belonging to the metallothionein gene family.
  • Metallothionein is a small molecule protein rich in cysteine, lacking amino acids containing aromatic groups and capable of binding bivalent heavy metal ions.
  • Metallothionein is an antioxidant that protects cells from free radicals containing hydroxyl groups, maintains the balance of metal ions in cells, and acts to remove heavy ions. Loss of metallothionein gene function leads to pathological phenomena such as the occurrence of cancer.
  • the EPO gene is an erythropoietin gene, the English name erythropoietin, located in the q22.1 region of human chromosome 7.
  • the protein encoded by this gene is a glycosylated cytokine secreted by the cell. When erythropoietin binds to the corresponding receptor, it can promote the synthesis of red blood cells.
  • the SNCG protein is synuclein- ⁇ and is a product of the SNCG gene.
  • the English name for the SNCG gene is synuclein gamma, located in the q23.2 region of human chromosome 10. This gene is a member of the synuclein gene family and its protein sequence is as follows :).
  • amino acid sequence of the protein encoded by the SNCG gene is:
  • the invention provides a method of detecting esophageal cancer in vitro, the method comprising the steps of:
  • the method further comprises the steps of:
  • step 2) using the reagent to process the DNA sample obtained in step 1), converting the 5 unmethylated cytosine base into uracil or other base, ie, the 5 unmethylated cytosine base of the target gene target sequence
  • the base is converted to uracil or other bases, and the converted base differs from the 5 unmethylated cytosine base in hybridization performance and is detectable;
  • step 2) contacting the DNA sample treated in step 2) with a DNA polymerase and a primer of the target gene target sequence, such that the processed target gene target sequence is amplified to produce an amplification product or not amplified.
  • the treated target gene target sequence generates an amplification product if DNA polymerization occurs; the treated target gene target sequence is not amplified if DNA polymerization does not occur;
  • the invention provides a method of detecting esophageal cancer in vitro, the method comprising the steps of:
  • the in vitro detection of esophageal cancer is achieved by jointly determining whether the subject has esophageal cancer by jointly detecting the methylation status of the target gene target sequence and the target protein concentration.
  • the biological sample is plasma or serum separated from the peripheral blood of the subject.
  • the method further comprises the steps of:
  • step 3 using the reagent to process the free DNA obtained in step 2), converting the 5 unmethylated cytosine base into uracil or other base, ie, the 5 unmethylated cytosine base of the target gene target sequence
  • the base is converted to uracil or other bases, and the converted base differs from the 5 unmethylated cytosine base in hybridization performance and is detectable;
  • step 4) contacting the free DNA treated in step 3) with a DNA polymerase and a primer of the target gene target sequence, such that the treated target gene target sequence is amplified to produce an amplification product or not amplified.
  • the treated target gene target sequence generates an amplification product if DNA polymerization occurs; the treated target gene target sequence is not amplified if DNA polymerization does not occur;
  • a typical primer comprises a fragment of the target gene target sequence, the fragment of the target gene target sequence comprising, respectively, identical to, complementary to, or hybridized under medium stringency or stringency conditions selected from the group consisting of SEQ ID NO: 1-2 And a fragment of at least 9 nucleotides of SEQ ID NO: 3-4.
  • a probe of the target gene target sequence is typically probed, and the fragment of the target gene target sequence comprises, respectively, is complementary to, complementary to, or hybridized under medium stringency or stringency conditions selected from the group consisting of SEQ ID NO: 1-2 And a fragment of at least 15 nucleotides of SEQ ID NO: 3-4.
  • a typical blocker is a blocking agent that preferentially binds to DNA in an unmethylated state.
  • one or more of the primers, probes and/or blockers are as follows:
  • the contacting or amplifying comprises using at least one of the following methods: using a thermostable DNA polymerase as the amplification enzyme, using a polymerase lacking 5-3' exonuclease activity, using a polymerase chain reaction (PCR), producing an amplification product nucleic acid molecule with a detectable label.
  • a thermostable DNA polymerase as the amplification enzyme
  • a polymerase lacking 5-3' exonuclease activity using a polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • the methylation status of at least one CpG dinucleotide in the target gene target sequence is determined by the cycle threshold Ct value of the PCR reaction.
  • the biological sample is selected from the group consisting of a cell line, histological section, tissue biopsy/paraffin-embedded tissue, body fluid, feces, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood. , or a combination thereof.
  • the biological sample is selected from the group consisting of peripheral blood whole blood, plasma, or serum.
  • kits comprising the compositions.
  • the kit includes a container for holding a biological sample of a subject.
  • the kit also includes instructions for using and interpreting the results of the assay.
  • the present invention provides a method for non-invasive detection of esophageal cancer in vitro by detecting the methylation status and target protein concentration of a target gene target sequence.
  • the present inventors have found that there is a significant difference in the methylation status of the MT1A gene and the EPO gene target sequence and the methylation status of the gene target sequence of normal esophageal tissue in esophageal cancer tissues: in the esophageal cancer tissue, the MT1A gene Methylation occurs with the EPO gene target sequence, whereas in normal esophageal tissues, the MT1A gene and the EPO gene target sequence are not methylated.
  • the present application provides a method for detecting esophageal cancer in vitro by detecting the methylation status of the MT1A gene and the EPO gene target sequence in the sample, and the method provided by the present invention can detect esophageal cancer non-invasively and rapidly.
  • the present inventors further found that the combined detection of the target gene target methylation state and target protein concentration can significantly increase the detection rate of esophageal cancer and the specificity of detection compared with the detection of the methylation status of the target gene target sequence alone. Sex has no significant effect. Therefore, the present application provides a method for detecting esophageal cancer in vitro by detecting the methylation status of the MT1A gene and the EPO gene target sequence and the SNCG protein concentration in the sample, and the method provided by the present invention can detect esophageal cancer non-invasively and rapidly. .
  • the present invention also provides a composition capable of sensitively and specifically detecting a target gene target methylation state and a target protein concentration; and a method and kit for non-invasive detection of esophageal cancer in vitro.
  • a first set of embodiments discloses a target gene and a target gene target sequence
  • a second set of embodiments discloses a composition for detecting a methylation status of a target gene target sequence, including for detecting a target gene target sequence methylation status Nucleic acid
  • a third set of embodiments discloses a method for non-invasive detection of esophageal cancer in vitro by detecting the methylation status and target protein concentration of a target gene target sequence.
  • the nucleic acid detected sequence comprises a fragment of at least 9 nucleotides in the target gene target sequence, wherein the fragment of the nucleotide comprises at least one CpG dinucleotide sequence; in certain preferred embodiments, for example, the sample DNA to be tested is converted using bisulfite, and the sequence detected by the nucleic acid comprises a fragment of at least 9 nucleotides in the sequence after bisulfite conversion of the target gene target sequence, wherein A fragment of the nucleotide comprises at least one CpG dinucleotide sequence;
  • the nucleic acid detected sequences comprise a fragment of at least 15 nucleotides that hybridizes to the target gene target sequence under moderately stringent or stringent conditions, wherein the fragment of the nucleotide comprises at least one CpG a dinucleotide sequence; in certain preferred embodiments, such as: transforming a test sample DNA using bisulfite, the nucleic acid detected sequence comprising hybridizing to a target gene target sequence under moderately stringent or stringent conditions A fragment of at least 15 nucleotides in the sequence after bisulfite conversion, wherein the fragment of the nucleotide comprises at least one CpG dinucleotide sequence.
  • the composition further comprises an agent that converts the 5-position unmethylated cytosine base of the gene to uracil.
  • the reagent is bisulfite.
  • the bisulfite modification of DNA is a known tool for assessing the methylation status of CpG.
  • 5-methylcytosine is the most common covalent base modification in the DNA of eukaryotic cells. 5-methylcytosine cannot be identified by sequencing because 5-methylcytosine has the same base pairing behavior as cytosine. In addition, epigenetic information carried by 5-methylcytosine was completely lost during PCR amplification.
  • the most commonly used method for the analysis of the presence of 5-methylcytosine in DNA is based on the specific reaction of bisulfite with cytosine; after subsequent alkaline hydrolysis, no methylated cytosine is converted to pairing behavior. Uracil corresponding to thymine; but under these conditions 5-methylcytosine remained unmodified.
  • the original DNA is transformed in such a way that the 5-methylcytosine originally unable to distinguish it from the cytosine in its hybridization behavior can now be detected as a remaining cytosine by conventional known molecular biology techniques. , for example, by amplification and hybridization. All of these techniques are based on different base pairing characteristics and can now be fully utilized.
  • the present application typically provides a combination of bisulfite technology in combination with one or more methylation assays for determining the methylation status of a CpG dinucleotide sequence within a target gene target sequence.
  • the method of the invention is suitable for the analysis of heterogeneous biological samples, such as low concentrations of tumor cells in blood or feces.
  • a quantitative assay to determine the methylation level (eg, percentage, number of copies) of a particular CpG dinucleotide sequence. , ratio, ratio or degree), not methylation status.
  • the term methylation status or methylation status should also be taken to mean a value that reflects the methylation status of the CpG dinucleotide sequence.
  • the method of the present application specifically comprises: 1) extracting genomic DNA of a biological sample to be tested; 2) treating the DNA sample obtained in step 1) with a reagent to make 5 unmethylated cytosine bases Conversion to uracil or other bases, ie, the 5 unmethylated cytosine bases of the target gene target sequence are converted to uracil or other bases, and the transformed base differs from the 5 positions in the hybridization performance.
  • a cytosine base and is detectable; 3) contacting the DNA sample treated in step 2) with a DNA polymerase and a primer of the target gene target sequence, such that the treated target gene target
  • the sequence is amplified to produce an amplification product or not amplified; if the processed target gene target sequence undergoes DNA polymerization, an amplification product is produced; if the processed target gene target sequence does not undergo DNA polymerization The reaction is not amplified; 4) detecting the amplification product with a probe; 5) and determining the methylation status of at least one CpG dinucleotide of the target gene target sequence based on the presence or absence of the amplification product .
  • the methods of the present application specifically comprise: 1) extracting peripheral blood of a subject; 2) extracting free DNA in plasma or serum; 3) treating the free DNA obtained in step 2) with a reagent to make 5 positions Unmethylated cytosine bases are converted to uracil or other bases, ie, the 5 unmethylated cytosine bases of the target gene target sequence are converted to uracil or other bases, and the transformed bases are The hybridization performance is different from the 5-unmethylated cytosine base and is detectable; 4) contacting the DNA sample treated in step 3) with the DNA polymerase and the primer of the target gene target sequence, Causing the treated target gene target sequence to be amplified to produce an amplification product or not to be amplified; if the processed target gene target sequence undergoes DNA polymerization, an amplification product is produced; the processed product The target gene target sequence is not amplified if DNA polymerization does not occur; 5) detecting the amplification product with a probe; 6) determining at least one
  • the contacting or amplifying comprises using at least one of the following methods: using a thermostable DNA polymerase as the amplification enzyme; using a polymerase lacking 5-3' exonuclease activity; using PCR; generating a band An amplification product nucleic acid molecule having a detectable label.
  • the methylation status is determined by PCR, such as "fluorescence-based real-time PCR technology", methylation-sensitive single nucleotide primer extension reaction (Ms-SNuPE), methylation-specific PCR (MSP) Assays such as methylated CpG island amplification (MCA) are used to determine the methylation status of at least one CpG dinucleotide of the target gene target sequence.
  • MCA methylated CpG island amplification
  • the "fluorescence-based real-time PCR” assay is a high-throughput quantitative methylation assay using fluorescence-based real-time PCR (TaqMan) technology that does not require further manipulation after the PCR step.
  • the "fluorescence-based real-time PCR” method begins with a mixed sample of genomic DNA that is converted to a mixing pool of methylation-dependent sequence differences in a sodium bisulfite reaction according to standard procedures. Fluorescence-based PCR is then performed in a "biased” reaction (using PCR primers that overlap known CpG dinucleotides). Sequence differences can be made at the level of amplification as well as at the level of fluorescence detection amplification.
  • the "Fluorescence-Based Real-Time PCR” assay can be used as a quantitative test for methylation status in genomic DNA samples where sequence discrimination occurs at the level of probe hybridization.
  • the PCR reaction provides methylation-specific amplification in the presence of fluorescent probes that overlap specific CpG dinucleotides.
  • a non-offset control for the amount of starting DNA is provided by the reaction in which neither the primer nor the probe covers any CpG dinucleotide.
  • the "fluorescence-based real-time PCR” method can be used with any suitable probe, such as “TaqMan", "Lightcycler", and the like.
  • the TaqMan probe is double labeled with a fluorescent reporter (Reporter) and a quencher (Quencher) and is designed to be specific to a relatively high GC content region such that it is higher than the forward or reverse primer in the PCR cycle. Melting at a temperature of 10 ° C.
  • TaqMan probe This allows the TaqMan probe to remain sufficiently hybridized during the PCR annealing/extension step.
  • Taq polymerase enzymatically synthesizes a new strand in PCR, it eventually encounters an annealed TaqMan probe.
  • the Taq polymerase 5 to 3' endonuclease activity will then be displaced by digestion of the TaqMan probe, thereby releasing the fluorescent reporter molecule for quantitative detection of its now unrefined signal using a real-time fluorescence detection system.
  • Typical reagents for "fluorescence-based real-time PCR” analysis can include, but are not limited to, PCR primers for target gene target sequences; non-specific amplification blockers; TaqMan or Lightcycler probes; optimized PCR buffers and deoxygenation Nucleotide; and Taq polymerase, and the like.
  • the SNCG antibody is used to determine the concentration of SNCG in plasma or serum by immunological reaction, and the expression level of SNCG protein in serum is detected by enzyme-linked immunosorbent assay (ELISA);
  • the ELISA is a "sandwich method", in particular the method comprises the following steps:
  • HRP horseradish peroxidase
  • esophageal cancer and normal threshold values relative to SNCG gene serum or plasma protein levels are determined based on serum or plasma protein levels of the SNCG gene of a certain number of esophageal cancer samples and normal samples.
  • the inventors By analyzing data of 233 cases of esophageal cancer tissues and 171 cases of normal esophageal tissue genome-wide methylation chips (Illumina's HumanMethylation450k chip), the inventors found that the methylation levels of MT1A gene and EPO gene in esophageal cancer tissues were significantly higher. In normal esophageal tissue (analysis results are shown in Figure 1). Further, the present inventors have found that the two target genes are in esophageal cancer tissues and normal esophageal tissues by analyzing the probe sequences of the MT1A gene and the EPO gene on the whole genome methylation chip and the corresponding methylation rate data. The sequence fragments with the most significant differences in methylation were determined to be the target sequences of the two target genes.
  • the target sequence of the MT1A gene is shown in SEQ ID NO: 1.
  • the complement of the target sequence of the MT1A gene is set forth in SEQ ID NO: 2.
  • the target sequence of the EPO gene is shown in SEQ ID NO: 3.
  • the complement of the target sequence of the EPO gene is set forth in SEQ ID NO:4.
  • First step obtaining the DNA of the biological sample to be analyzed.
  • the source may be any suitable source, such as cell lines, histological sections, biopsy tissue, paraffin embedded tissue, body fluids, feces, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood and All possible combinations.
  • the DNA is then isolated from the sample by separation by any standard means in the prior art. Briefly, when DNA is encapsulated in a cell membrane, the biological sample must be broken up and cleaved by enzymatic, chemical or mechanical means. Proteins and other contaminants are subsequently removed, for example by digestion of protein kinase K. The DNA is then recovered from the solution. This can be accomplished by a variety of methods including salting out, organic extraction or binding DNA to a solid support.
  • the choice of method is influenced by a number of factors, including time, cost, and amount of DNA required.
  • standard methods of isolating and/or purifying DNA in the prior art can be used. These methods include the use of protein degradation agents such as chaotropic salts such as guanidine hydrochloride or urea; or detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide.
  • protein degradation agents such as chaotropic salts such as guanidine hydrochloride or urea
  • detergents such as sodium dodecyl sulfate (SDS), cyanogen bromide.
  • Other methods include, but are not limited to, ethanol precipitation or propanol precipitation, vacuum concentration by centrifugation, and the like.
  • Those skilled in the art can also utilize devices such as filters such as ultrafiltration, silicon surfaces or membranes, magnetic particles, polystyrene particles, polystyrene surfaces, positively charged surfaces, and positively charged membranes, charged membranes, charged Surface, charged conversion film, charged conversion surface.
  • filters such as ultrafiltration, silicon surfaces or membranes, magnetic particles, polystyrene particles, polystyrene surfaces, positively charged surfaces, and positively charged membranes, charged membranes, charged Surface, charged conversion film, charged conversion surface.
  • the biological sample DNA is leukocyte genomic DNA and leukocyte genomic DNA after DNA methyltransferase treatment.
  • the target gene target sequence of the leukocyte genomic DNA is unmethylated, and thus the leukocyte genomic DNA is a negative reference for the methylation status of the target gene target sequence.
  • the target gene target sequence of the leukocyte genomic DNA after DNA methyltransferase treatment is methylated, and thus the leukocyte genomic DNA after DNA methyltransferase treatment is a positive reference for the methylation state of the target gene target sequence.
  • Second step The above two DNA samples are separately treated such that the unmethylated cytosine base at position 5 is converted to uracil, thymine or another base that is not used for cytosine in hybridization behavior. Preferably, this is achieved by treatment with a bisulfite reagent.
  • bisulfite reagent refers to an agent comprising bisulfite, acid sulfite or a combination thereof, as disclosed herein, which can be used to distinguish between methylated and unmethylated CpG dinucleotide sequences.
  • the bisulfite treatment is carried out in the presence of a denaturing solvent such as, but not limited to, a normal alkyl diol, especially diethylene glycol dimethyl ether (DME), or in dioxane or
  • a denaturing solvent such as, but not limited to, a normal alkyl diol, especially diethylene glycol dimethyl ether (DME), or in dioxane or
  • DME diethylene glycol dimethyl ether
  • the denaturation solvent is used at a concentration of from 1% to 35% (v/v).
  • a scavenger such as, but not limited to, a chroman derivative such as 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid or Trihydroxybenzoic acid and its derivatives, such as gallic acid.
  • the bisulfite conversion is preferably carried out at a reaction temperature of from 30 ° C to 70 ° C, wherein the temperature is increased to over 85 ° C for a short period of time during the reaction.
  • the bisulfite treated DNA is preferably purified prior to quantification. This can be done by any method known in the art, such as, but not limited to, ultrafiltration.
  • the third step amplification of the fragment of the treated DNA using the primer of the present invention and the amplification enzyme.
  • Amplification of several DNA fragments can be performed simultaneously in the same reaction vessel.
  • the amplification product is from 100 to 2,000 base pairs in length.
  • the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated states, especially in the case where the DNA in the methylated state is much smaller than the DNA in the unmethylated state,
  • the present invention employs a blocking agent specific for the target gene target sequence in the PCR reaction system.
  • the 5' end of the blocker nucleotide sequence has an overlap region of 5' nucleotides greater than or equal to 5' to the forward (F) or reverse (R) primer; blocker and forward (F) or the reverse (R) primer is complementary to the same strand of the target gene target DNA; the blocking agent has a melting temperature higher than the forward (F) or reverse (R) primer exceeding (including) 5 ° C;
  • the nucleotide sequence of the blocker comprises at least one CpG dinucleotide sequence and is complementary to the sequence of the target gene target sequence DNA which has not been methylated after bisulfite conversion.
  • the genomic DNA of the biological sample to be detected is a mixture of methylated and unmethylated states, especially when the DNA in the methylated state is much smaller than the DNA in the unmethylated state.
  • the DNA in the unmethylated state is converted by bisulfite, it preferentially binds to the blocking agent, thereby inhibiting the binding of the DNA template to the PCR primer, thereby preventing PCR amplification and being methylated.
  • the DNA does not bind to the blocker and thus binds to the primer, and PCR amplification occurs. Thereafter, the fragment obtained by amplification is directly or indirectly detected.
  • the label is in the form of a fluorescent label, a radionuclide or an attachable molecular fragment.
  • Target gene target sequences SEQ ID NO: 1-2 and SEQ ID NO: 3-4, primers, probes and probes for detecting methylation status of target genes of two target genes, MT1A and EPO, are designed in the present invention.
  • Blocker sequence SEQ ID NO: 5-12:
  • one or more of the primers, probes and/or blockers are as follows:
  • real-time PCR detection can be performed using standard commercial operations on various commercial real-time PCR instrumentation devices according to the prior art.
  • Real-time PCR detection was performed on a Life Technologies instrument (7500 Fast) according to some embodiments.
  • the PCR reaction mixture consists of a bisulfite converted DNA template 25-40 ng and 300-600 nM primers and blockers, 150-300 nM probes, 1 UTaq polymerase, 50-400 ⁇ M of each dNTP, 1 to 10 mM of MgCl 2 and 2XPCR buffered to a final volume of 2 ⁇ l to 100 ⁇ l.
  • the ⁇ -actin gene can be used as an internal reference for PCR to create a ⁇ -actin gene amplicon by using a primer complementary to the ⁇ -actin gene sequence, and A specific probe detects the beta actin gene amplicon.
  • Each sample is subjected to at least one real-time PCR, and in some embodiments, two or three real-time PCR assays are performed.
  • the experimental results are shown in Figure 2: using the composition and detection method provided by the present invention, the detection of leukocyte genomic DNA (negative reference product of methylation status of target gene target sequence) was not PCR-amplified, and the detection result was negative. That is, the target gene target sequence of the test DNA sample is not methylated; the DNA methyltransferase-treated leukocyte genomic DNA (the target gene target sequence methylation status is positive using the composition and the detection method provided by the present invention)
  • the detection of the reference product occurs by PCR amplification, and the detection result is positive, that is, the target gene target sequence of the DNA sample to be tested is methylated. From this, it is judged that the composition and the detection method provided by the present invention are capable of specifically detecting the methylation state of the target gene target sequence.
  • the Ct value capable of effectively distinguishing between esophageal cancer and a normal target gene is determined.
  • the methylation status of at least one CpG dinucleotide of the target gene target sequence is determined by the cycle threshold Ct value of the polymerase chain reaction, and the target is determined by comparing the Ct value of the sample to be measured with a predetermined threshold value.
  • the analysis of the gene is negative (normal) or positive (esophageal cancer).
  • plasma samples from 20 patients with esophageal cancer and 22 normal subjects were obtained. All samples were obtained from Borcheng.
  • the peripheral blood free DNA of the test sample is then extracted, and the DNA sample is pretreated such that the unmethylated cytosine base at position 5 is converted to uracil, thymine or not used for cytosine in hybridization behavior. Another base.
  • the pretreatment is achieved by treatment with a bisulfite reagent.
  • the extraction and processing of the DNA can be carried out by any standard means in the prior art. Specifically, in this embodiment, all sample DNA extraction and bisulfite DNA modification are performed by using Borcheng Corporation. Extracted from the plasma processing kit.
  • the above-mentioned target gene primers, probes, and blockers were added to the DNA samples of 20 treated esophageal cancer patients and 22 normal humans, and the methylation status of the target gene target sequence was detected by PCR.
  • the PCR taken in this experimental example was carried out on a Life Technologies instrument (7500).
  • the PCR reaction mixture was buffered from bisulfite converted DNA template 35 ng, 450 nM primer and blocker, 225 nM probe, 1 UTaq polymerase, 200 ⁇ M of each dNTP, 4.5 mM MgCl 2 and 2X PCR to a final volume of 50 ⁇ l.
  • the sample was amplified at 94 ° C for 20 minutes to pre-cycle the sample, followed by 45 cycles of annealing at 62 ° C for 5 seconds, annealing at 55.5 ° C for 35 seconds, and denaturation at 93 ° C for 30 seconds.
  • the detection of normal human target shows that the specificity of target gene target methylation is 95% (MT1A) and 95% (EPO) for normal human detection; 3) combined with MT1A gene And the EPO gene methylation test results, the sensitivity of detecting esophageal cancer can be increased to 60%, and the specific inconvenience is still 95%.
  • MT1A target gene target methylation
  • EPO 95%
  • plasma samples of 63 subjects with esophageal cancer were additionally obtained.
  • the free DNA in the plasma is then extracted and the genomic DNA sample is pretreated such that the cytosine base that is unmethylated at the 5' position is converted to uracil, thymine or not used for cytosine in hybridization behavior.
  • Another base is another base.
  • the pretreatment is achieved by treatment with a bisulfite reagent.
  • the extraction and processing of the DNA can be carried out by any standard means in the prior art. Specifically, in this embodiment, all sample DNA extraction and bisulfite DNA modification are performed by using Borcheng Corporation. Extracted from the plasma processing kit.
  • PCR amplification conditions employed in this experimental example were: real-time PCR was performed on a Life Technologies instrument (7500).
  • the PCR reaction mixture consisted of bisulfite converted DNA template 35 ng and 450 nM primers, 225 nM probe, 1 UTaq polymerase, 200 u ⁇ m of each dNTP, 4.5 mM MgCl 2 and 2X PCR buffer to a final volume of 30 u ⁇ l.
  • the sample was amplified by pre-circulation at 94 ° C for 20 minutes, followed by 45 cycles of annealing at 62 ° C for 5 seconds, annealing at 55.5 ° C for 35 seconds, and denaturation at 93 ° C for 30 seconds.
  • ELISA detection technique is to detect the serum protein level of the SNCG gene by a "sandwich method": firstly, a 96-well plate is coated with a SNCG monoclonal antibody (mouse) (source Borcheng) (1 ⁇ g/well); Then add 10-fold diluted clinical serum samples and serial dilutions (2.5 ng/mL to 0.04 ng/mL) of human SNCG protein solution (50 ⁇ l/well); then add 50 ⁇ l of 0.47 ⁇ g/mL horseradish to each tube.
  • Roche's electrochemiluminescence immunoassay system was used to measure the levels of commonly used cancer markers in 63 esophageal cancer subjects, including CEA, CA199, CA125, and AFP.
  • MT1A and EPO gene DNA methylation and SNCG protein combined detection have significant complementarity in the detection of esophageal cancer, and the combined detection of the two greatly improved the detection rate of esophageal cancer.
  • the complementarity of DNA methylation detection and SNCG protein detection of MT1A and EPO genes is unique, and common cancer markers (such as CEA, CA199, CA125, and AFP) do not have this property.
  • the above experimental results indicate that the combined detection of the target gene target methylation status and target protein concentration can detect esophageal cancer sensitively and specifically.
  • detecting the methylation DNA of the target gene target sequence of the present invention non-invasive detection of esophageal cancer can be achieved in vitro, and the detection rate of esophageal cancer can be improved.
  • the present application utilizes the above-described composition, nucleic acid sequence, kit and use thereof, and the above detection method to achieve target gene target by detecting methylation status and target protein concentration of target gene target sequence.
  • the methylation status and target protein concentration were used to detect esophageal cancer in vitro, which effectively improved the sensitivity and specificity of esophageal cancer in vitro detection.
  • By analyzing the free protein in plasma samples by real-time PCR and analyzing the target protein concentration in plasma it is convenient to achieve the combined detection of the methylation status and target protein concentration of the target gene target sequence, and can be based on the CT value of real-time PCR.
  • the detection concentration of ELISA to quickly and conveniently determine whether the sample is positive, providing a non-invasive and convenient method for in vitro detection of esophageal cancer.

Abstract

提供了一种用于检测食管癌的组合物及其用途，该组合物包括：用于检测目标基因甲基化状态的核酸，其中，所述目标基因为MT1A基因和EPO基因中的一种或两种。还提供了一种用于检测食管癌的组合物，所述组合物包括：用于检测目标基因甲基化状态的核酸和用于检测目标蛋白浓度的抗体；其中，所述目标基因为MT1A基因和EPO基因中的一种或两种；所述目标蛋白为SNCG。还提供了包括上述组合物的试剂盒，以及上述组合物在制备用于体外检测食管癌的试剂盒中的用途。

Description

用于检测食道癌的组合物及其用途 技术领域
本发明属于生物技术领域，涉及用于检测食道癌的组合物及其在疾病检测中的用途，具体地涉及用于检测食管癌的组合物及其相应的试剂盒和用途。
背景技术
食管癌是一种常见消化道肿瘤。全国肿瘤防治办公室数据显示：2015年，中国食管癌发病率为478例/10万人，死亡率为375例/万人，分别位居常见癌症中的第四位和第三位。食管癌的病死率接近80％，是一种恶性程度很高的癌症。全球每年约有30万人死于食管癌，其中一半来自中国。
导致食管癌高死亡率的重要因素是早期食管癌的确诊率低。早期食管癌的治愈率远高于中晚期，但是由于早期食管癌缺乏明显和特异的症状，绝大部分受试者确诊时已经发展进入中晚期。临床研究发现，癌症从病灶开始形成到受试者出现临床症状的过程平均需要数年时间；这为发现早期食管癌、提高早期食管癌的确诊率提供了一个有效的窗口期。充分利用这个窗口期，有望提高食管癌治疗效果、降低食道癌死亡率。
目前临床上应用于食管癌诊断的技术在早期食道癌的检测和筛查方面的应用有限，主要因为：1)组织活检侵入性强，不适于早癌筛查；2)影像学检测技术(如：食管造影检查和内镜检查)在设备成本、操作技术、和侵入性等方面上的限制，也难以作为癌症筛查技术而大范围推广；3)传统血清肿瘤标识物(如：AFP、CEA、CA125、和CA199等)对食管癌检测的灵敏度较低，无法充分满足早癌筛查的要求。
发明内容
基于此，针对现有食管癌检测技术存在的检测不便、灵敏度低、和成本高的问题，本发明提供了用于检测食道癌的组合物。本发明提供的组合物能够灵敏和特异地检测食管癌，本发明还提供了包含所述组合物的试剂盒以及 其在检测食管癌中的用途。本发明提供的试剂盒具有良好的食管癌检测灵敏性，能够方便、快捷、有效地检测食管癌。
本发明提供了用于体外检测食管癌的组合物、试剂盒及其用途，以及基于该试剂盒来执行检测的方法。
具体来说，本发明涉及以下内容：
1.一种用于体外检测食管癌的组合物，所述组合物包括：
用于检测目标基因甲基化状态的核酸，
其中，所述目标基因为MT1A基因和EPO基因中的一种或两种。
2.根据项1所述的组合物，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
3.根据项1所述的组合物，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
4.根据项1～3中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸包括：
所述目标基因的靶序列中的至少9个核苷酸的片段，
所述片段包含至少一个CpG二核苷酸序列。
5.根据项1～4中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸还包括：
在中等严紧或严紧条件下杂交于所述目标基因靶序列中的至少15个核苷酸的片段，
所述片段包含至少一个CpG二核苷酸序列。
6.根据项1～5中任一项所述的组合物，其还包括：
将目标基因靶序列的5位未甲基化胞嘧啶碱基转化为尿嘧啶的试剂。
7.根据项1～6中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸还包括：
优先与处于非甲基化状态的靶序列结合的阻断剂。
8.根据项7所述的组合物，其中，
所述至少9个核苷酸的片段，其为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列，
所述至少15个核苷酸的片段，其为SEQ ID NO:7的序列或SEQ ID NO:11的序列，
阻断剂，其为SEQ ID NO:8的序列或SEQ ID NO:12的序列。
9.一种用于体外检测食管癌的寡核苷酸，其包括：
所述SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
所述SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
10.根据项9所述的寡核苷酸，其还包括：
在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
11.根据项10所述的寡核苷酸，其还包括：
优先与处于非甲基化状态的靶序列结合的阻断剂。
12.一种用于体外检测食管癌的寡核苷酸，其包括：
SEQ ID NO:5和SEQ ID NO:6的序列。
13.根据项12所述的寡核苷酸，其还包括：
SEQ ID NO:7的序列。
14.根据项13所述的寡核苷酸，其还包括：
SEQ ID NO:8的序列。
15.一种用于体外检测食管癌的寡核苷酸，其包括：
SEQ ID NO:9和SEQ ID NO:10的序列。
16.根据项15所述的寡核苷酸，其还包括：
SEQ ID NO:11的序列。
17.根据项16所述的寡核苷酸，其还包括：
SEQ ID NO:12的序列。
18.MT1A基因在制备用于体外检测食管癌的试剂盒中的用途。
19.EPO基因在制备用于体外检测食管癌的试剂盒中的用途。
20.一种试剂盒，其包括项1～8中任一项所述的组合物或包括项9～17中任一项所述的寡核苷酸。
21.根据项20所述的试剂盒，其还包含选自下述的至少一种其它组分：
三磷酸核苷、DNA聚合酶和所述DNA聚合酶功能所需的缓冲液。
22.根据项20或21所述的试剂盒，其还包含：说明书。
23.根据项1～8中任一项所述的组合物或项9～项17中任一项所述的寡核苷酸在用于制备用于体外检测食管癌的试剂盒中的用途。
24.根据项18、19以及23中任一项所述的用途，其中，所述用于体外检测食管癌的试剂盒通过包括如下步骤的方法来检测食管癌：
1)分离待测生物样品中的包括目标基因靶序列或其片段的DNA样品；
2)确定所述目标基因靶序列的甲基化状态；
3)通过所述目标基因靶序列的甲基化状态的检测结果判断生物样品的状态，从而实现对食管癌的体外检测。
25.根据项24所述的用途，其中，所述方法包括如下步骤：
提取待测生物样品的基因组DNA；
使用试剂处理提取的基因组DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
用探针检测扩增产物；以及
基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
26.根据项25所述的用途，其中，所述试剂为亚硫酸氢盐试剂。
27.一种检测食管癌的方法，其包括如下步骤：
分离待测生物样品中的包括目标基因靶序列或其片段的DNA样品；
确定所述目标基因靶序列的甲基化状态；以及
通过所述目标基因靶序列的甲基化状态的检测结果判断生物样品的状态，从而实现对食管癌的体外检测。
28.一种检测食管癌的方法，其包括如下步骤：
提取待测生物样品的基因组DNA；
使用试剂处理提取的基因组DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA 聚合反应；
用探针检测扩增产物；以及
基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
29.根据项27或28所述的方法，其中，
所述目标基因为MT1A基因和EPO基因中的一种或两种。
30.根据项29所述的方法，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
31.根据项29所述的方法，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
32.根据项28所述的方法，其中，所述试剂为亚硫酸氢盐试剂。
33.根据项28所述的方法，其中，所述引物为：
所述SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
所述SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
33.根据项28所述的方法，其中，所述阻断剂为优先与处于非甲基化状态的靶序列结合的阻断剂。
34.根据项28所述的方法，其中，所述探针为：
在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
35.根据项33所述的方法，其中，所述引物为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列。
36.根据项33所述的方法，其中，所述阻断剂为SEQ ID NO:8的序列或SEQ ID NO:12的序列
37.根据项34所述的方法，其中，所述探针为SEQ ID NO:7的序列或SEQ ID NO:11的序列。
38.一种用于体外检测食管癌的组合物，所述组合物包括：
用于检测目标基因靶序列甲基化状态的核酸，以及
用于检测目标蛋白浓度的抗体，
其中，所述目标基因为MT1A基因和EPO基因中的一种或两种，
所述目标蛋白为SNCG，即γ突触核蛋白(γ-synuclein)。
39.根据项38所述的组合物，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
40.根据项38所述的组合物，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
41.根据项38～40中任一项所述的组合物，其中，所述用于检测目标基因靶序列甲基化状态的核酸包括：
所述目标基因靶序列中至少9个核苷酸的片段，
所述片段包含至少一个CpG二核苷酸序列。
42.根据项38～41中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸包括还包括：
在中等严紧或严紧条件下杂交于所述目标基因靶序列中至少15个核苷酸的片段，
所述片段包含至少一个CpG二核苷酸序列。
43.根据项38～42中任一项所述的组合物，其还包括：
将目标基因靶序列的5位未甲基化胞嘧啶碱基转化为尿嘧啶的试剂。
44.根据项38～43中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸还包括：
优先与处于非甲基化状态的靶序列结合的阻断剂。
45.根据项44所述的组合物，其中，
所述至少9个核苷酸的片段，其为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列，
所述至少15个核苷酸的片段，其为SEQ ID NO:7的序列或SEQ ID NO:11的序列，
阻断剂，其为SEQ ID NO:8的序列或SEQ ID NO:12的序列。
46.根据项38～45中任一项所述的组合物，所述组合物还包括：
用于检测目标蛋白浓度的试剂，所述试剂为酶联免疫吸附测定试剂，例如，所述试剂包括用于检测目标蛋白浓度的抗体包被的反应板，SNCG蛋白酶结合物，底物液，洗液和终止液。
47.一种试剂盒，其包括项38～46中任一项所述的组合物或包括项9～17中任一项所述的寡核苷酸。
48.根据项47所述的试剂盒，其还包含选自下述的至少一种其它组分：
三磷酸核苷、DNA聚合酶和所述DNA聚合酶功能所需的缓冲液，以及
用于检测目标蛋白浓度的抗体包被的反应板，SNCG蛋白酶结合物，底物液，洗液和终止液。
49.根据项47或48所述的试剂盒，其还包含：说明书。
50.根据项38～46中任一项所述的组合物或项9～项17中任一项所述的寡核苷酸在用于制备用于体外检测食管癌的试剂盒中的用途。
51.根据项18、19以及50中任一项所述的用途，其中，所述用于体外检测食管癌的试剂盒通过包括如下步骤的方法来检测食管癌：
1)确定受试者生物样品中所述目标基因靶序列的甲基化状态；
2)确定受试者生物样品中所述目标蛋白的浓度；
3)通过所述目标基因靶序列甲基化状态和目标蛋白浓度的检测结果联合判断受试者是否患有食管癌，从而实现对食管癌的体外检测。
52.根据项51所述的用途，其中，所述方法包括如下步骤：
抽取受试者的外周血，分离血浆或血清；
提取血浆或血清中的游离DNA；
使用试剂处理提取的游离DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
用探针检测扩增产物；
基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态；以及
使用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度。
53.根据项52所述的用途，其中，所述试剂为亚硫酸氢盐试剂。
54.一种检测食管癌的方法，其包括如下步骤：
抽取受试者的生物样品；
确定受试者生物样品中的所述目标基因靶序列的甲基化状态；
确定受试者生物样品中的所述目标蛋白的浓度；以及
通过所述目标基因靶序列甲基化状态和目标蛋白浓度的检测结果联合判断受试者是否患有食管癌，从而实现对食管癌的体外检测。
55.一种检测食管癌的方法，其包括如下步骤：
抽取受试者外周血，分离血浆或血清；
提取血浆或血清中的游离DNA；
使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
用探针检测扩增产物；
基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态；以及
使用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度。
56.根据项54或55所述的方法，其中，
所述目标基因为MT1A基因和EPO基因中的一种或两种，以及
所述目标蛋白为SNCG。
57.根据项56所述的方法，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
58.根据项56所述的方法，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
59.根据项55所述的方法，其中，所述试剂为亚硫酸氢盐试剂。
60.根据项55所述的方法，其中，所述引物为：
所述SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
所述SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
61.根据项55所述的方法，其中，所述阻断剂为优先与处于非甲基化状态的靶序列结合的阻断剂。
62.根据项55所述的方法，其中，所述探针为：
在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的 至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
63.根据项60所述的方法，其中，所述引物为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列。
64.根据项61所述的方法，其中，所述阻断剂为SEQ ID NO:8的序列或SEQ ID NO:12的序列
65.根据项62所述的方法，其中，所述探针为SEQ ID NO:7的序列或SEQ ID NO:11的序列。
本发明的发明人利用表观基因组学和生物信息学技术，通过分析食管癌组织和癌旁对照组织的全基因组甲基化数据，发现两个与食管癌相关的甲基化基因，并确定了这两个食管癌甲基化基因发生甲基化异常的靶序列；进一步，本发明的发明人发现，通过这两个食管癌甲基化基因的靶序列，能够灵敏和特异地检测这两个基因的甲基化状态，从而可以用于对外周血游离DNA的检测。通过对食管癌患者和正常对照个体的外周血样本的检测显示：本发明描述的组合物和检测方法能够灵敏和特异地检测食管癌，包括两种不同细胞类型的常见食管癌：鳞癌和腺癌。因此，本发明提供了一种可用于体外检测食管癌的组合物和检测方法，具有重要的临床应用价值。
进一步，本发明的发明人发现，在众多肿瘤蛋白标识物中，SNCG蛋白与所述目标基因甲基化的联合检测能够显著提高对食管癌的检出。因此，本发明提供了一种可用于体外检测食管癌的组合物和检测方法，具有重要的临床应用价值。
本发明的其他特点和优势将由下面的具体说明和权利要求书作详细的描述。
附图说明
本发明的上述及其它特征将通过下面结合附图及其详细描述作进一步说明。应当理解的是，这些附图仅示出了根据本发明的若干示例性的实施方式，因此不应被视为是对本发明保护范围的限制。除非特别说明，附图不必是成比例的，并且其中类似的标号表示类似的部件。
图1显示筛选本发明的目标基因的结果图。
图2为使用本发明提供的组合物和检测方法对白细胞基因组DNA(目标基因靶序列甲基化状态的阴性参考品)和DNA甲基转移酶处理后的白细胞基因组DNA(目标基因靶序列甲基化状态的阳性参考品)的检测。结果显示：本发明提供的组合物和检测方法对白细胞基因组DNA的检测结果为阴性，对DNA甲基转移酶处理后的白细胞基因组DNA的检测结果为阳性。
图3为利用所述的组合物和检测方法，通过检测目标基因靶序列的甲基化状态，实现对食管癌的体外无创检测的结果图。
图4为利用所述的组合物和检测方法，通过检测目标基因靶序列的甲基化状态和目标蛋白的浓度，实现对食管癌的体外无创检测的结果图。
具体实施方式
一方面，本发明提供了一种用于体外检测食管癌的组合物，所述组合物包括用于检测目标基因靶序列内甲基化状态的核酸，和用于检测目标蛋白浓度的抗体；其中，所述目标基因为MT1A基因和EPO基因中的一种或两种，所述目标蛋白为SNCG。
本发明提供了一组在食管癌中发出异常甲基化的目标基因靶序列，包括MT1A基因和EPO基因的靶序列，MT1A基因的靶序列如SEQ ID NO:1-2所示，EPO基因的靶序列如SEQ ID NO:3-4所示。
MT1A基因的靶序列如SEQ ID NO:1所示。
SEQ ID NO:1
Figure PCTCN2018104076-appb-000001
SEQ ID NO：1的互补序列如SEQ ID NO：2所示。
SEQ ID NO:2
Figure PCTCN2018104076-appb-000002
优选地，EPO基因的靶序列的序列如SEQ ID NO:3所示。
SEQ ID NO:3
Figure PCTCN2018104076-appb-000003
SEQ ID NO：3的互补序列如SEQ ID NO：4所示。
Figure PCTCN2018104076-appb-000004
优选地，用于检测目标基因甲基化状态的核酸包括目标基因靶序列中的至少9个核苷酸的片段，其中所述片段包含至少一个CpG二核苷酸序列。在某些优选实施方式中，如使用亚硫酸氢盐对待测样本DNA进行转化，用于检测目标基因甲基化状态的核酸包括对目标基因靶序列进行亚硫酸氢盐转化后的序列中的至少9个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列。
更优选地，用于检测目标基因甲基化状态的核酸包括在中等严紧或严紧条件下杂交于所述目标基因靶序列中的至少15个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列。在某些优选实施方式中，如：使用亚硫酸氢盐对待测样本DNA进行转化，用于检测目标基因甲基化状态的核酸包括在中等严紧或严紧条件下，杂交于目标基因靶序列进行亚硫酸氢盐转化后序列中的至少15个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列。
优选地，所述组合物还包括将目标基因靶序列的5位未甲基化胞嘧啶碱基转化为尿嘧啶的试剂。更优选地，所述试剂为亚硫酸氢盐。
优选地，用于检测目标基因甲基化状态的核酸还包括优先与处于非甲基化状态的DNA结合的阻断剂。
优选地，所述组合物包括如下的引物、探针和/或阻断剂中的一种或多种：
MT1A引物F
SEQ ID NO:5
Figure PCTCN2018104076-appb-000005
MT1A引物R
SEQ ID NO:6
Figure PCTCN2018104076-appb-000006
MT1A探针
SEQ ID NO:7
Figure PCTCN2018104076-appb-000007
MT1A阻断剂
SEQ ID NO:8
Figure PCTCN2018104076-appb-000008
EPO引物F
SEQ ID NO:9
Figure PCTCN2018104076-appb-000009
EPO引物R
SEQ ID NO:10
Figure PCTCN2018104076-appb-000010
EPO探针
SEQ ID NO:11
Figure PCTCN2018104076-appb-000011
EPO阻断剂
SEQ ID NO:12
Figure PCTCN2018104076-appb-000012
另一方面，本发明提供用于体外检测食管癌的寡核苷酸，其包括：SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
优选地该用于体外检测食管癌的寡核苷酸包括：对SEQ ID NO:1或其互补序列进行亚硫酸氢盐转化后的序列中的至少9个核苷酸的片段；和/或对 SEQ ID NO:3或其互补序列进行亚硫酸氢盐转化后的序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
本发明的用于体外检测食管癌的寡核苷酸，其还包括：在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
优选地该用于体外检测食管癌的寡核苷酸包括：在中等严紧或严紧条件下杂交于对SEQ ID NO:1或其互补序列进行亚硫酸氢盐转化后的序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或在中等严紧或严紧条件下杂交于对SEQ ID NO:3或其互补序列进行亚硫酸氢盐转化后的序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
本发明的用于体外检测食管癌的寡核苷酸，其还包括：优先与处于非甲基化状态的DNA结合的阻断剂。
在一个具体的实施方式中，用于体外检测食管癌的寡核苷酸，其包括：SEQ ID NO:5和SEQ ID NO:6的序列。其还包括：SEQ ID NO:7的序列。其还包括：SEQ ID NO:8的序列。
在另一个具体的实施方式中，用于体外检测食管癌的寡核苷酸，其包括：SEQ ID NO:9和SEQ ID NO:10的序列。其还包括：SEQ ID NO:11的序列。其还包括：SEQ ID NO:12的序列。
另一方面，本发明提供了包括所述组合物的试剂盒。该试剂盒还包含选自下述的至少一种其它组分：三磷酸核苷、DNA聚合酶和所述DNA聚合酶功能所需的缓冲液。
本发明的用于体外检测食管癌的组合物，所述还包括用于检测目标蛋白浓度的抗体，所述目标蛋白为γ突触核蛋白(SNCG)。
本发明还涉及MT1A基因、EPO基因、SNCG蛋白在制备用于体外检测食管癌的试剂盒中的用途。
其中，所述MT1A为金属硫蛋白1A，英文名称metallothionein 1A，位于人类的第16号染色体的q13区域，属于金属硫蛋白基因家族。金属硫蛋白是一种小分子蛋白，富含半胱氨酸，缺乏含有芳香基的氨基酸，能够结合二价重金属离子。金属硫蛋白是一种抗氧化剂，能够保护细胞免受含有羟基 的自由基损害，维持细胞内金属离子的平衡，同时发挥去除重离子毒性的作用。金属硫蛋白基因功能的丧失会导致癌症的发生等病理现象。
所述EPO基因为红细胞生成素基因，英文名称erythropoietin，位于人类的第7号染色体的q22.1区域。该基因所编码的蛋白是一种由细胞分泌的糖基化的细胞活素(cytokine)。当红细胞生成素与相应受体结合后，能够促进红细胞的合成。
所述SNCG蛋白为突触核蛋白-γ，是SNCG基因的产物。SNCG基因的英文名称为synuclein gamma，位于人类的第10号染色体的q23.2区域。该基因是突触核蛋白基因家族的一员，其蛋白序列如下：)。
SNCG基因所编码蛋白的氨基酸序列为：
Figure PCTCN2018104076-appb-000013
再一方面，本发明提供了一种体外检测食管癌的方法，所述方法包括以下步骤：
1)分离待测生物样品中的目标基因靶序列或其片段；
2)确定所述目标基因靶序列的甲基化状态；
3)通过所述目标基因靶序列的甲基化状态的检测结果判断生物样品的状态，从而实现对食管癌的体外检测。
根据某些优选实施方式，所述方法还包括以下步骤：
1)提取待测生物样品的基因组DNA；
2)使用试剂处理步骤1)得到的DNA样品，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，即目标基因靶序列的5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，转化后的碱基在杂交性能方面不同于5位未甲基化的胞嘧啶碱基，并且是可检测的；
3)将经步骤2)处理过的DNA样品与DNA聚合酶和所述目标基因靶序列的引物接触，使得所述经处理的目标基因靶序列被扩增以产生扩增产物或不被扩增；所述经处理的目标基因靶序列如果发生DNA聚合反应，会产生扩增产物；所述经处理的目标基因靶序列如果不发生DNA聚合反应，则不被扩增；
4)用探针检测扩增产物；以及
5)基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
再一方面，本发明提供了一种体外检测食管癌的方法，所述方法包括以下步骤：
1)确定受试者生物样品中所述目标基因靶序列的甲基化状态；
2)确定受试者生物样品中所述目标蛋白的浓度；
3)通过所述目标基因靶序列甲基化状态和目标蛋白浓度的检测结果联合判断受试者是否患有食管癌，从而实现对食管癌的体外检测。
其中，生物样品是由受试者的外周血中分离的血浆或血清。
根据某些优选实施方式，所述方法还包括以下步骤：
1)抽取受试者外周血，分离血浆或血清；
2)提取血浆或血清中的游离DNA；
3)使用试剂处理步骤2)得到的游离DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，即目标基因靶序列的5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，转化后的碱基在杂交性能方面不同于5位未甲基化的胞嘧啶碱基，并且是可检测的；
4)将经步骤3)处理过的游离DNA与DNA聚合酶和所述目标基因靶序列的引物接触，使得所述经处理的目标基因靶序列被扩增以产生扩增产物或不被扩增；所述经处理的目标基因靶序列如果发生DNA聚合反应，会产生扩增产物；所述经处理的目标基因靶序列如果不发生DNA聚合反应，则不被扩增；
5)用探针检测扩增产物；
6)基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
7)使用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度。
优选地，典型的引物包括所述目标基因靶序列的片段，所述目标基因靶序列的片段包含分别等同于、互补于或在中等严紧或严紧条件下杂交于选自SEQ ID NO:1-2和SEQ ID NO:3-4中的至少9个核苷酸的片段。
优选地，典型的探针所述目标基因靶序列的片段，所述目标基因靶序列的片段包含分别等同于、互补于或在中等严紧或严紧条件下杂交于选自SEQ ID NO:1-2和SEQ ID NO:3-4中的至少15个核苷酸的片段。
优选地，典型的阻断剂其为优先与处于非甲基化状态的DNA结合的阻 断剂。
优选地，所述引物、探针和/或阻断剂中的一种或多种如下所示：
MT1A引物F
SEQ ID NO:5
Figure PCTCN2018104076-appb-000014
MT1A引物R
SEQ ID NO:6
Figure PCTCN2018104076-appb-000015
MT1A探针
SEQ ID NO:7
Figure PCTCN2018104076-appb-000016
MT1A阻断剂
SEQ ID NO:8
Figure PCTCN2018104076-appb-000017
EPO引物F
SEQ ID NO:9
Figure PCTCN2018104076-appb-000018
EPO引物R
SEQ ID NO:10
Figure PCTCN2018104076-appb-000019
EPO探针
SEQ ID NO:11
Figure PCTCN2018104076-appb-000020
EPO阻断剂
SEQ ID NO:12
Figure PCTCN2018104076-appb-000021
并且，所述接触或扩增包括使用至少一种如下的方法：使用耐热DNA聚合酶作为所述扩增酶、使用缺乏5-3’外切酶活性的聚合酶、使用聚合酶链式反应(PCR)、产生带有可检测标记的扩增产物核酸分子。
据某些优选实施方式，所述目标基因靶序列中至少一个CpG二核苷酸的甲基化状态是由PCR反应的循环阈值Ct值确定的。通过利用PCR反应分 析生物样本中DNA的方法，能方便地实现针对目标基因靶序列甲基化状态的检测，并且能根据PCR反应的循环阈值来快速、便捷地判断受检样本是否呈阳性，因而提供了一种无创、快速的食管癌体外检测方法。
所述的生物样品选自细胞系、组织学切片、组织活检/石蜡包埋的组织、体液、粪便、结肠流出物、尿、血浆、血清、全血、分离的血细胞、从血液中分离的细胞，或其组合。所述的生物样品选自外周血全血、血浆、或血清。
本发明还提供了包括所述组合物的试剂盒。典型地，所述的试剂盒包括用于容纳受试者生物样品的容器。并且，所述的试剂盒也包括使用和解释检测结果的说明。
本发明提供了一种通过检测目标基因靶序列甲基化状态和目标蛋白浓度，体外无创检测食管癌的方法。本发明人发现，在食管癌组织中MT1A基因和EPO基因靶序列的甲基化状态和正常食管组织的所述基因靶序列的甲基化状态存在显著性差异：在食管癌组织中，MT1A基因和EPO基因靶序列发生甲基化，而在正常食管组织中，MT1A基因和EPO基因靶序列不发生甲基化。因此本申请提供了一种通过检测样本中的MT1A基因和EPO基因靶序列甲基化状态对食管癌进行体外检测的方法，本发明提供的方法能够无创、快速地检测食管癌。
本发明人进一步发现，与单独检测目标基因靶序列甲基化状态相比，联合检测目标基因靶序列甲基化状态和目标蛋白浓度能够显著提高对食管癌的检出率，同时对检测的特异性没有显著影响。因此本申请提供了一种通过检测样本中的MT1A基因和EPO基因靶序列甲基化状态和SNCG蛋白浓度，对食管癌进行体外检测的方法，本发明提供的方法能够无创、快速地检测食管癌。
除非另外定义，本说明书中有关技术的和科学的术语与本领域内的技术人员所通常理解的意思相同。虽然在实验或实际应用中可以应用与此间所述相似或相同的方法和材料，本文还是在下文中对材料和方法做了描述。在相冲突的情况下，以本说明书包括其中定义为准，另外，材料、方法和例子仅供说明，而不具限制性。
本发明还提供一种能够灵敏和特异地检测目标基因靶序列甲基化状态和目标蛋白浓度的组合物；以及一种可用于体外无创检测食管癌的方法和试剂盒。
下述描述为本发明的组合物、试剂盒、核酸序列以及检测方法的实施例。第一组实施方案公开了目标基因和目标基因靶序列；第二组实施方案公开了用于检测目标基因靶序列甲基化状态的组合物，包括用于检测目标基因靶序列甲基化状态的核酸；第三组实施方案公开了一种通过检测目标基因靶序列甲基化状态和目标蛋白浓度进行体外无创检测食管癌的方法。
优选地，所述核酸检测的序列包括所述目标基因靶序列中的至少9个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列；在某些优选实施方式中，如：使用亚硫酸氢盐对待测样本DNA进行转化，所述核酸检测的序列包括对目标基因靶序列进行亚硫酸氢盐转化后的序列中的至少9个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列；
更优选地，所述核酸检测的序列分别包括在中等严紧或严紧条件下杂交于所述目标基因靶序列中的至少15个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列；在某些优选实施方式中，如：使用亚硫酸氢盐对待测样本DNA进行转化，所述核酸检测的序列包括在中等严紧或严紧条件下，杂交于目标基因靶序列进行亚硫酸氢盐转化后序列中的至少15个核苷酸的片段，其中所述核苷酸的片段包含至少一个CpG二核苷酸序列。
在某些实施方式中，所述组合物还包括将基因的5位未甲基化的胞嘧啶碱基转化为尿嘧啶的试剂。优选地，该试剂是亚硫酸氢盐。DNA的亚硫酸氢盐修饰为已知的用于评估CpG甲基化状态的工具。在真核细胞的DNA中，5-甲基胞嘧啶是最常见的共价碱基修饰。5-甲基胞嘧啶不能通过测序来鉴定，因为5-甲基胞嘧啶与胞嘧啶有相同的碱基配对行为。此外，在PCR扩增过程中，5-甲基胞嘧啶携带的表观遗传信息则完全丢失。最常用于分析DNA中5-甲基胞嘧啶存在的方法是基于亚硫酸氢盐与胞嘧啶的特异反应；在随后的碱性水解后，没有甲基化的胞嘧啶被转变为在配对行为上对应胸腺嘧啶的尿嘧啶；但在这些条件下5-甲基胞嘧啶保持不被修饰。由此原始的DNA以此方式被转变，使得原来在其杂交行为上不能与胞嘧啶区分开的5-甲基胞嘧啶现在可作为仅剩的胞嘧啶被常规的已知分子生物学技术检测到，例如通过扩增和杂交。所有这些技术都基于不同的碱基配对特性，现在可被充分利用了。因此，典型地，本申请提供了亚硫酸氢盐技术与一种或多种甲基化测定 的联合使用，用于确定目标基因靶序列内的CpG二核苷酸序列的甲基化状态。此外，本发明的方法适于分析异质的生物样品，例如血液或粪便中的低浓度肿瘤细胞。因此，当分析这种样品中CpG二核苷酸序列的甲基化状态时，本领域技术人员可以使用定量测定法来确定特定CpG二核苷酸序列的甲基化水平(例如百分比、份数、比率、比例或程度)，而不是甲基化状态。相应地，术语甲基化状况或甲基化状态还应被认为是指反映CpG二核苷酸序列甲基化状态的值。
在某些实施方式中，本申请的方法具体包括：1)提取待测生物样品的基因组DNA；2)使用试剂处理步骤1)得到的DNA样品，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，即目标基因靶序列的5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，转化后的碱基在杂交性能方面不同于5位未甲基化的胞嘧啶碱基，并且是可检测的；3)将经步骤2)处理过的DNA样品与DNA聚合酶和所述目标基因靶序列的引物接触，使得所述经处理的目标基因靶序列被扩增以产生扩增产物或不被扩增；所述经处理的目标基因靶序列如果发生DNA聚合反应，会产生扩增产物；所述经处理的目标基因靶序列如果不发生DNA聚合反应，则不被扩增；4)用探针检测扩增产物；5)以及基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
在某些实施方式中，本申请的方法具体包括：1)抽取受试者外周血；2)提取血浆或血清中的游离DNA；3)使用试剂处理步骤2)得到的游离DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，即目标基因靶序列的5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基，转化后的碱基在杂交性能方面不同于5位未甲基化的胞嘧啶碱基，并且是可检测的；4)将经步骤3)处理过的DNA样品与DNA聚合酶和所述目标基因靶序列的引物接触，使得所述经处理的目标基因靶序列被扩增以产生扩增产物或不被扩增；所述经处理的目标基因靶序列如果发生DNA聚合反应，会产生扩增产物；所述经处理的目标基因靶序列如果不发生DNA聚合反应，则不被扩增；5)用探针检测扩增产物；6)基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态；7)利用SNCG抗体，通过免疫反应(优选地，酶联免疫吸附试验，ELISA)，检测血浆或血清中目标蛋白的浓度。
典型地，所述接触或扩增包括使用至少一种如下的方法：使用耐热DNA 聚合酶作为所述扩增酶；使用缺乏5-3’外切酶活性的聚合酶；使用PCR；产生带有可检测标记的扩增产物核酸分子。优选地，用PCR方式来测定甲基化状态，诸如“基于荧光的实时PCR技术”、甲基化敏感的单核苷酸引物延伸反应(Ms-SNuPE)、甲基化特异性PCR(MSP)、和甲基化CpG岛扩增(MCA)等测定方法被用于测定目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。其中，“基于荧光的实时PCR”测定为高通量定量甲基化测定，其使用基于荧光的实时PCR(TaqMan)技术，在PCR步骤后不需要进一步的操作。简言之，“基于荧光的实时PCR”方法以基因组DNA的混合样品开始，该混合样品根据标准操作在亚硫酸氢钠反应中被转变为甲基化依赖的序列差异的混合池。随后在“偏移的(biased)”反应(采用重叠已知CpG二核苷酸的PCR引物)中进行基于荧光的PCR。可在扩增水平以及在荧光检测扩增水平上产生序列差别。“基于荧光的实时PCR”测定可以用作基因组DNA样品中甲基化状态的定量测试，其中序列区分发生在探针杂交水平上。在该定量方式中，在重叠特定的CpG二核苷酸的荧光探针存在下，PCR反应提供了甲基化特异的扩增。用于起始DNA量的无偏移对照由以下反应提供：其中引物和探针都不覆盖任何CpG二核苷酸。“基于荧光的实时PCR”方法可与任何适合的探针一起使用，如“TaqMan”、“Lightcycler”等。TaqMan探针为荧光报道物(Reporter)和淬灭分子(Quencher)双标记的，并被设计为特异于相对高GC含量区，以至于其在PCR循环中以比正向或反向引物高约10℃的温度熔解。这使得TaqMan探针在PCR退火/延伸步骤中保持充分杂交。当Taq聚合酶在PCR中酶合成新链时，其最终会遇到退火的TaqMan探针。Taq聚合酶5至3’内切酶活性随后将通过消化TaqMan探针而顶替它，从而释放荧光报道物分子用于采用实时荧光检测系统定量检测其现在未被淬灭的信号。用于“基于荧光的实时PCR”分析的典型试剂可以包括，但不限于：用于目标基因靶序列PCR引物；非特异扩增阻断剂；TaqMan或Lightcycler探针；优化的PCR缓冲液以及脱氧核苷酸；以及Taq聚合酶等。
其中利用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度是通过酶联免疫吸附法(ELISA)检测SNCG蛋白在血清中的表达水平；
优选地，所述ELISA为“三明治法”，具体来说该方法包括以下步骤：
1)使用SNCG单克隆抗体(鼠)对96孔板进行包被；
2)加入10倍稀释的临床血清样本或血浆样本和系列稀释的人源SNCG 蛋白溶液；
3)在每孔中加入辣根过氧化酶(horseradish peroxidase，HRP)标记的鼠抗人源SNCG的多抗(IgG)；
4)装有以上混合物的免疫反应板在室温、震荡条件下，孵育，用洗液清洗个反应孔后，加入显色底物；
5)加入终止液终止显色反应后，使用读板仪对各反应孔的光谱进行检测；
6)利用系列稀释的人源SNCG蛋白溶液的检测值建立标准曲线，并基于标准曲线对临床血清样本进行定量。
在本发明的具体事实方式中，基于一定数量的食管癌样本和正常样本的SNCG基因的血清或血浆蛋白水平，确定相对于SNCG基因血清或血浆蛋白水平的食管癌和正常的临界值。
实施例
实施例1
通过分析233例食管癌组织及171例正常食道组织的全基因组甲基化芯片(Illumina公司的HumanMethylation450k芯片)数据，本发明人发现MT1A基因和EPO基因在食道癌组织中的甲基化水平显著高于正常食道组织(分析结果如图1所示)。进一步地，本发明人通过分析MT1A基因和EPO基因在全基因组甲基化芯片上的探针序列及相应的甲基化率数据，发现了这两个目标基因在食道癌组织和正常食道组织中甲基化差异最明显的序列片段，从而确定为这两个目标基因的靶序列。
MT1A基因的靶序列如SEQ ID NO:1所示。
SEQ ID NO:1
Figure PCTCN2018104076-appb-000022
MT1A基因的靶序列的互补序列如SEQ ID NO：2所示。
SEQ ID NO:2
Figure PCTCN2018104076-appb-000023
Figure PCTCN2018104076-appb-000024
EPO基因的靶序列如SEQ ID NO:3所示。
SEQ ID NO:3
Figure PCTCN2018104076-appb-000025
EPO基因的靶序列的互补序列如SEQ ID NO：4所示。
SEQ ID NO：4
Figure PCTCN2018104076-appb-000026
实施例2
第一步：获得待分析的生物样品的DNA。该来源可以是任何适合的来源，例如细胞系、组织学切片、活检组织、石蜡包埋的组织、体液、粪便、尿、血浆、血清、全血、分离的血细胞、从血液分离的细胞及其所有可能的组合。然后通过现有技术中的任何标准手段来分离，从所述样品分离DNA。简言之，当DNA被包裹在细胞膜中时，该生物样品必须被破碎并通过酶、化学或机械手段被裂解。随后例如通过蛋白激酶K的消化而清除蛋白和其它的污染物。接着从溶液回收DNA。这可以通过各种方法来实现，包括盐析、有机提取或将DNA结合到固相支持物。对方法的选择会受到多种因素的影响，包括时间、费用和所需的DNA的量。当所述样品DNA未被包裹在细胞膜中时(例如来自血液样品的循环DNA)，可以使用现有技术中分离和/或纯化DNA的标准方法。这些方法包括使用蛋白质降解试剂，例如离液盐， 如盐酸胍或脲；或去污剂，如十二烷基磺酸钠(SDS)、溴化氰。其它方法包括但不限于乙醇沉淀或丙醇沉淀、通过离心的真空浓缩等。本领域技术人员也可以利用装置，例如诸如超滤的滤器，硅表面或膜，磁性颗粒，聚苯乙烯颗粒，聚苯乙烯表面，带正电荷的表面以及带阳性电荷的膜，带电膜，带电表面，带电转换膜，带电转换表面。一旦核酸被提取，就将DNA用于分析。
在本实施方案中，生物样本DNA为白细胞基因组DNA和DNA甲基转移酶处理后的白细胞基因组DNA。白细胞基因组DNA的目标基因靶序列的为非甲基化状态，因此白细胞基因组DNA是目标基因靶序列甲基化状态的阴性参考品。DNA甲基转移酶处理后的白细胞基因组DNA的目标基因靶序列为甲基化状态，因此DNA甲基转移酶处理后的白细胞基因组DNA是目标基因靶序列甲基化状态的阳性参考品。
第二步：将上述两种DNA样品分别处理，以使得在5位未甲基化的胞嘧啶碱基被转变为尿嘧啶、胸腺嘧啶或在杂交行为上不用于胞嘧啶的另一碱基。优选地，通过亚硫酸氢盐试剂处理来实现。术语“亚硫酸氢盐试剂”指包括亚硫酸氢盐、酸式亚硫酸盐或其组合的试剂，如这里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。优选地，该亚硫酸氢盐处理在变性溶剂存在下进行，所述变性溶剂诸如但不限于正烷基二醇，尤其是二乙二醇二甲基醚(DME)，或者在二噁烷或二噁烷衍生物存在下进行。在优选的实施方案中，所述变性溶剂以1％至35％(v/v)的浓度使用。还优选该亚硫酸氢盐反应在清除剂存在下进行，例如但不限于色原烷衍生物，如6-羟基-2,5,7,8,-四甲基色原烷2-羧酸或三羟基苯甲酸及其衍生物，例如没食子酸。该亚硫酸氢盐转变优选在30℃至70℃的反应温度下进行，其中在反应期间温度短时间地增加至超过85℃。经亚硫酸氢盐处理的DNA优选在定量之前进行纯化。这可通过任何现有技术中已知的方法来进行，例如但不限于超滤。
第三步：采用本发明的引物以及扩增酶扩增经处理的DNA的片段。可在同一个反应容器中同时进行几种DNA片段的扩增。优选地，所述扩增产物的长度为100至2,000个碱基对。当所要检测的生物样本的基因组DNA是处于甲基化和非甲基化状态的混合物时，尤其是在处于甲基化状态的DNA远远少于处于非甲基化状态的DNA的情况下，如：癌症受试者外周血中游离DNA，为了提高PCR扩增引物的扩增特异性，本发明采用了在PCR反应体系中使用目标基因靶序列特异的阻断剂。阻断剂核苷酸序列的5端与正向 (F)或者反向(R)引物的3’端核苷酸序列有大于或等于5个核苷酸的重叠区域；阻断剂与正向(F)或者反向(R)引物互补于目标基因靶序列DNA的同一条链；阻断剂的解链温度高于正向(F)或者反向(R)引物超过(包括)5℃；阻断剂的核苷酸序列包含至少一个CpG二核苷酸序列，并与亚硫酸氢盐转化后的未发生甲基化的目标基因靶序列DNA的序列互补。因此，当所要检测的生物样本的基因组DNA是处于甲基化和非甲基化状态的混合物时，尤其是在处于甲基化状态的DNA远远少于处于非甲基化状态的DNA的情况下，处于非甲基化状态的DNA经亚硫酸氢盐转化后，会优先与阻断剂相结合，从而抑制DNA模板与PCR引物结合，因而不发生PCR扩增，而处于甲基化状态的DNA不与阻断剂相结合，因而与引物结合，发生PCR扩增。之后，直接或间接地检测通过扩增获得的片段。优选的是标记物为荧光标记物、放射性核素或可附着的分子片段的形式。
根据目标基因靶序列SEQ ID NO:1-2和SEQ ID NO:3-4，在本发明中设计了用于检测MT1A和EPO这两个目标基因靶序列甲基化状态的引物、探针和阻断剂序列(SEQ ID NO:5-12)：
优选地，所述引物、探针和/或阻断剂中的一种或多种如下所示：
MT1A引物F
SEQ ID NO:5
Figure PCTCN2018104076-appb-000027
MT1A引物R
SEQ ID NO:6
Figure PCTCN2018104076-appb-000028
MT1A探针
SEQ ID NO:7
Figure PCTCN2018104076-appb-000029
MT1A阻断剂
SEQ ID NO:8
Figure PCTCN2018104076-appb-000030
EPO引物F
SEQ ID NO:9
Figure PCTCN2018104076-appb-000031
EPO引物R
SEQ ID NO:10
Figure PCTCN2018104076-appb-000032
EPO探针
SEQ ID NO:11
Figure PCTCN2018104076-appb-000033
EPO阻断剂
SEQ ID NO:12
Figure PCTCN2018104076-appb-000034
在本发明中，可以利用各种商业用实时PCR仪器设备上根据现有技术的标准操作进行实时PCR的检测。根据某些具体实施方式，在Life Technologies仪器(7500Fast)上进行实时PCR的检测。PCR反应混合物由经亚硫酸氢盐转化的DNA模板25-40ng和300-600nM引物和阻断剂、150-300nM探针、1UTaq聚合酶、50-400μM的各个dNTP、1至10mM的MgCl 2和2XPCR缓冲至最终的2μl至100μl的体积。在85至99℃持续3-60分钟，以用预循环扩增样品，紧接着在50至72℃进行1至30秒的35-55个循环的退火，在45至80℃下退火及延伸5至90秒，在85至99℃下变性5至90秒。通过仅仅在甲基化的目标基因靶序列上观测扩增，用与含5-甲基胞嘧啶的目标基因靶序列的CpG岛区域的特异性的探针检测所述基因片段。并且，在某些具体实施方式中，可以以β肌动蛋白基因(ACTB)作为PCR的内参，通过使用与β肌动蛋白基因序列互补的引物来创建β肌动蛋白基因扩增子，并且用特定的探针检测β肌动蛋白基因扩增子。每个样品进行至少一次的实时PCR，在某些具体实施方式中，进行两次或三次实时PCR检测。
实验结果显示如图2所述：使用本发明提供的组合物和检测方法对白细胞基因组DNA(目标基因靶序列甲基化状态的阴性参考品)的检测没有发生PCR扩增，检测结果为阴性，即：受检DNA样本的目标基因靶序列没有发生甲基化；使用本发明提供的组合物和检测方法对DNA甲基转移酶处理后的白细胞基因组DNA(目标基因靶序列甲基化状态的阳性参考品)的检测发生PCR扩增，检测结果为阳性，即：受检DNA样本的目标基因靶序列发生甲基化。由此判断，本发明提供的组合物和检测方法能够特异性地检测目标 基因靶序列的甲基化状态。
实施例3
根据本申请的具体实施方式，基于一定数量的食管癌样本和正常样本的检测结果的平均Ct值，确定能够有效区分食管癌和正常的目标基因的Ct值，即：临界值。目标基因靶序列的至少一个CpG二核苷酸的甲基化状态是由聚合酶链式反应的循环阈值Ct值确定，通过比较所测样本的Ct值与预先设定的临界值，确定基于目标基因的分析结果是阴性(正常)、还是阳性(食管癌)。
本实施例包括以下步骤：
首先，得到20例食管癌患者和22例正常人的血浆样本。所有样品来源于博尔诚公司。然后提取受测样本的外周血游离DNA，并对所述DNA样品预处理以使得在5位未甲基化的胞嘧啶碱基被转变为尿嘧啶、胸腺嘧啶或在杂交行为上不用于胞嘧啶的另一碱基。在本实施例中，通过亚硫酸氢盐试剂处理来实现该预处理。所述DNA的提取和处理可以采用现有技术中的任何标准手段来进行，具体而言，在本实施例中，所有的样品DNA的提取和亚硫酸氢盐DNA修饰是通过使用博尔诚公司的血浆处理试剂盒提取的。
然后，上述经过处理的20例食道癌患者和22例正常人的DNA样本中加入上述的目标基因引物、探针和阻断剂组合，通过PCR检测目标基因靶序列的甲基化状态。其中，本实验例中采取的PCR在Life Technologies仪器(7500)上进行。PCR反应混合物由经亚硫酸氢盐转化的DNA模板35ng、450nM引物和阻断剂、225nM探针、1UTaq聚合酶、200μM的各个dNTP、4.5mM的MgCl2和2XPCR缓冲至最终的50μl的体积。在94℃持续20分钟，以用预循环扩增样品，紧接着在62℃进行5秒的45个循环的退火，在55.5℃下退火及延伸35秒，在93℃下变性30秒。
最后，分别测得20例食道癌患者和22例正常人的DNA样本对于目标基因靶序列的实时PCR的Ct值。检测结果如图3所示：1)选用特定的临界值，优选地临界值Ct＝37，通过使用本发明提供的组合物和检测方法对目标基因靶序列甲基化状态的检测能够有效检出食管癌患者；在该实施例中，当以Ct值37为临界值时，目标基因靶序列甲基化检测食管癌的灵敏度分别是55％(MT1A)和45％(EPO)；2)目标基因靶序列的甲基化具有良好的特异性，对正常人的检测显示目标基因靶序列甲基化对正常人检测的特异性为 95％(MT1A)和95％(EPO)；3)联合MT1A基因和EPO基因甲基化检测结果，检测食管癌的灵敏度可提高至60％，而特异性不便，仍为95％。
实施例4
然后，针对另外得到63例食道癌受试者的血浆样本。然后提取血浆中的游离DNA，并对所述基因组DNA样品预处理以使得在5’位未甲基化的胞嘧啶碱基被转变为尿嘧啶、胸腺嘧啶或在杂交行为上不用于胞嘧啶的另一碱基。在本实施例中，通过亚硫酸氢盐试剂处理来实现该预处理。所述DNA的提取和处理可以采用现有技术中的任何标准手段来进行，具体而言，在本实施例中，所有的样品DNA的提取和亚硫酸氢盐DNA修饰是通过使用博尔诚公司的血浆处理试剂盒提取的。然后，上述经过处理的63例食道癌受试者的DNA样本中加入上述的MT1A和EPO基因引物、探针组合和内参基因ACTB基因引物、探针组合，通过PCR检测MT1A和EPO基因的甲基化状态。其中，本实验例中采取的PCR扩增条件为：在Life Technologies仪器(7500)上进行实时PCR。PCR反应混合物由经亚硫酸氢盐转化的DNA模板35ng和450nM引物、225nM探针、1UTaq聚合酶、200uμm的各个dNTP、4.5mM的MgCl2和2XPCR缓冲液组成至最终的30uμl的体积。在94℃保持20分钟用预循环扩增样品，紧接着在62℃进行5秒的45个循环的退火，在55.5℃下退火35秒，在93℃下变性30秒。
同时，对这63例食道癌受试者的血浆样本进行SNCG基因血清蛋白水平的检测。通过酶联免疫吸附剂测定技术(ELISA)检测SNCG基因的血清蛋白水平。此ELISA检测技术是通过“三明治法”实现对SNCG基因的血清蛋白水平的检测：首先以SNCG单克隆抗体(鼠)(来源博尔诚公司)对96孔板进行包被(1μg/孔)；之后加入10倍稀释的临床血清样本和系列稀释(2.5ng/mL～0.04ng/mL)的人源SNCG蛋白溶液(50μl/孔)；然后在每管中加入50μl的0.47μg/mL的辣根过氧化酶(horseradish peroxidase，HRP)标记的鼠抗人源SNCG的多抗(IgG)(来源博尔诚公司)。装有以上混合物的免疫反应板在室温、震荡条件下，孵育3小时；用洗液清洗个反应孔后，加入显色底物。通过加入2M硫酸终止显色反应后，使用读板仪(如：Bio-RAD 550)在450nm光谱波段对各反应孔进行检测。最后，利用系列稀释的人源SNCG蛋白溶液的检测值建立标准曲线，并基于标准曲线对临床血清样本进行定量。SNCG 基因血清水平在食道癌和正常个体中的临界值为2.0ng/mL。
最后，利用罗氏公司(Roche)的电化学发光免疫检测系统分别测得63例食道癌受试者样本中的常用癌症标识物的水平，包括：CEA、CA199、CA125、和AFP。
检测结果显示(图4)：食道癌受试者中MT1A和EPO基因甲基化检测和SNCG蛋白检测食道癌的阳性率显著高于常用癌症标识物(如：CEA、CA199、CA125、和AFP)。同时，MT1A和EPO基因DNA甲基化和SNCG蛋白联合检测对食道癌的检测具有突出的互补性，二者联检大大提高了食道癌的检出率。另外，MT1A和EPO基因DNA甲基化检测和SNCG蛋白检测的互补性是独特的，常用癌症标识物(如：CEA、CA199、CA125、和AFP)并不具备这种特性。
上述实验结果表明了目标基因靶序列甲基化状态和目标蛋白浓度的联合检测能够灵敏、特异地检测食管癌。通过本发明的目标基因靶序列甲基化DNA检测，可以实现体外无创检测食管癌，并能够提高食管癌的检出率。
综上所述，本申请利用以上所述的组合物、核酸序列、试剂盒及其用途，以及上述检测方法，通过检测目标基因靶序列甲基化状态和目标蛋白浓度，实现了利用目标基因靶序列甲基化状态和目标蛋白浓度来对食管癌进行体外检测，从而有效提高了食管癌体外检测的灵敏度和特异性。通过利用实时PCR分析血浆样本游离DNA的方法和ELISA方法分析血浆中目标蛋白浓度，能够方便地实现针对目标基因靶序列甲基化状态和目标蛋白浓度的联合检测，并且能够根据实时PCR的CT值和ELISA的检测浓度来快速、便捷地判断样本是否呈阳性，提供了一种无创、便捷的食管癌体外检测方法。
尽管在此公开了本发明的各个方面和实施例，但其他方面和实施例对于本领域技术人员而言也是显而易见的。在此公开的各个方面和实施例仅用于说明目的，而非限制目的。本发明的保护范围和主旨仅通过后附的权利要求书来确定。

Claims (44)

  1. 一种用于体外检测食管癌的组合物，所述组合物包括：
    用于检测目标基因甲基化状态的核酸，
    其中，所述目标基因为MT1A基因和EPO基因中的一种或两种。
  2. 根据权利要求1所述的用于体外检测食管癌的组合物，所述组合物还包括：用于检测目标蛋白浓度的抗体，所述目标蛋白为SNCG，即γ突触核蛋白(γ-synuclein)。
  3. 根据权利要求1或2所述的组合物，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
  4. 根据权利要求1或2所述的组合物，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
  5. 根据权利要求1～4中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸包括：
    所述目标基因的靶序列中的至少9个核苷酸的片段，
    所述片段包含至少一个CpG二核苷酸序列。
  6. 根据权利要求1～5中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸包括：
    在中等严紧或严紧条件下杂交于所述目标基因靶序列中的至少15个核苷酸的片段，
    所述片段包含至少一个CpG二核苷酸序列。
  7. 根据权利要求1～6中任一项所述的组合物，其还包括：
    将目标基因靶序列的5位未甲基化胞嘧啶碱基转化为尿嘧啶的试剂。
  8. 根据权利要求1～7中任一项所述的组合物，其中，所述用于检测目标基因甲基化状态的核酸还包括：
    优先与处于非甲基化状态的靶序列结合的阻断剂。
  9. 根据权利要求8所述的组合物，其中，
    所述至少9个核苷酸的片段，其为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列，
    所述至少15个核苷酸的片段，其为SEQ ID NO:7的序列或SEQ ID NO:11的序列，
    阻断剂，其为SEQ ID NO:8的序列或SEQ ID NO:12的序列。
  10. 根据权利要求2～9中任一项所述的组合物，所述组合物还包括：
    用于检测目标蛋白浓度的试剂，所述试剂为酶联免疫吸附测定试剂，例如，所述试剂包括用于检测目标蛋白浓度的抗体包被的反应板，SNCG蛋白酶结合物，底物液，洗液和终止液。
  11. 一种用于体外检测食管癌的寡核苷酸，其包括：
    所述SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
    所述SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
  12. 根据权利要求11所述的寡核苷酸，其还包括：
    在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
    在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
  13. 根据权利要求12所述的寡核苷酸，其还包括：
    优先与处于非甲基化状态的靶序列结合的阻断剂。
  14. 一种用于体外检测食管癌的寡核苷酸，其包括：
    SEQ ID NO:5和SEQ ID NO:6的序列。
  15. 根据权利要求14所述的寡核苷酸，其还包括：
    SEQ ID NO:7的序列。
  16. 根据权利要求15所述的寡核苷酸，其还包括：
    SEQ ID NO:8的序列。
  17. 一种用于体外检测食管癌的寡核苷酸，其包括：
    SEQ ID NO:9和SEQ ID NO:10的序列。
  18. 根据权利要求17所述的寡核苷酸，其还包括：
    SEQ ID NO:11的序列。
  19. 根据权利要求18所述的寡核苷酸，其还包括：
    SEQ ID NO:12的序列。
  20. MT1A基因在制备用于体外检测食管癌的试剂盒中的用途。
  21. EPO基因在制备用于体外检测食管癌的试剂盒中的用途。
  22. 一种试剂盒，其包括权利要求1～10中任一项所述的组合物或包括权利要求11～19中任一项所述的寡核苷酸。
  23. 根据权利要求22所述的试剂盒，其还包含选自下述的至少一种其它组分：
    三磷酸核苷、DNA聚合酶和所述DNA聚合酶功能所需的缓冲液。
  24. 根据权利要求23所述的试剂盒，其还包含选自下述的至少一种其它组分：
    用于检测目标蛋白浓度的抗体包被的反应板，SNCG蛋白酶结合物，底物液，洗液和终止液。
  25. 根据权利要求22～24中任一项所述的试剂盒，其还包含：说明书。
  26. 根据权利要求1～10中任一项所述的组合物或权利要求11～19中任一项所述的寡核苷酸在用于制备用于体外检测食管癌的试剂盒中的用途。
  27. 根据权利要求20、21以及26中任一项所述的用途，其中，所述用于体外检测食管癌的试剂盒通过包括如下步骤的方法来检测食管癌：
    1)分离待测生物样品中的包括目标基因靶序列或其片段的DNA样品；
    2)确定所述目标基因靶序列的甲基化状态；
    3)通过所述目标基因靶序列的甲基化状态和/或目标蛋白浓度的检测结果判断生物样品的状态，从而实现对食管癌的体外检测。
  28. 根据权利要求27所述的用途，其中，所述方法包括如下步骤：
    提取待测生物样品的基因组DNA；
    使用试剂处理提取的基因组DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
    将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
    用探针检测扩增产物；以及
    基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
  29. 根据权利要求27所述的用途，其中，所述方法包括如下步骤：
    抽取受试者的外周血，分离血浆或血清；
    提取血浆或血清中的游离DNA；
    使用试剂处理提取的游离DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
    将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
    用探针检测扩增产物；
    基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态；以及
    使用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度。
  30. 根据权利要求28或29所述的用途，其中，所述试剂为亚硫酸氢盐试剂。
  31. 一种检测食管癌的方法，其包括如下步骤：
    分离待测生物样品中的包括目标基因靶序列或其片段的DNA样品；
    确定所述目标基因靶序列的甲基化状态；以及
    通过所述目标基因靶序列的甲基化状态的检测结果判断生物样品的状态，从而实现对食管癌的体外检测。
  32. 一种检测食管癌的方法，其包括如下步骤：
    提取待测生物样品的基因组DNA；
    使用试剂处理提取的基因组DNA，使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
    将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
    用探针检测扩增产物；以及
    基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态。
  33. 一种检测食管癌的方法，其包括如下步骤：
    抽取受试者的生物样品；
    确定受试者生物样品中的所述目标基因靶序列的甲基化状态；
    确定受试者生物样品中的所述目标蛋白的浓度；以及
    通过所述目标基因靶序列甲基化状态和目标蛋白浓度的检测结果联合 判断受试者是否患有食管癌，从而实现对食管癌的体外检测。
  34. 一种检测食管癌的方法，其包括如下步骤：
    抽取受试者外周血，分离血浆或血清；
    提取血浆或血清中的游离DNA；
    使5位未甲基化的胞嘧啶碱基转化为尿嘧啶或其它碱基；
    将试剂处理过的DNA样品与DNA聚合酶和目标基因靶序列的引物接触，并在优先与处于非甲基化状态的靶序列结合的阻断剂的存在下进行DNA聚合反应；
    用探针检测扩增产物；
    基于所述扩增产物是否存在，确定所述目标基因靶序列的至少一个CpG二核苷酸的甲基化状态；以及
    使用SNCG抗体，利用免疫反应确定血浆或血清中SNCG的浓度。
  35. 根据权利要求31～34中任一项所述的方法，其中，
    所述目标基因为MT1A基因和EPO基因中的一种或两种。
  36. 根据权利要求35所述的方法，其中，所述MT1A基因的靶序列如SEQ ID NO:1所示。
  37. 根据权利要求35所述的方法，其中，所述EPO基因的靶序列如SEQ ID NO:3所示。
  38. 根据权利要求32或34所述的方法，其中，所述试剂为亚硫酸氢盐试剂。
  39. 根据权利要求32或34所述的方法，其中，所述引物为：
    所述SEQ ID NO:1或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
    所述SEQ ID NO:3或其互补序列中的至少9个核苷酸且包含至少一个CpG二核苷酸序列的片段。
  40. 根据权利要求32或34所述的方法，其中，所述阻断剂为优先与处于非甲基化状态的靶序列结合的阻断剂。
  41. 根据权利要求32或34所述的方法，其中，所述探针为：
    在中等严紧或严紧条件下杂交于所述SEQ ID NO:1或其互补序列中的至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段；和/或
    在中等严紧或严紧条件下杂交于所述SEQ ID NO:3或其互补序列中的 至少15个核苷酸且包含至少一个CpG二核苷酸序列的片段。
  42. 根据权利要求39所述的方法，其中，所述引物为SEQ ID NO:5和SEQ ID NO:6的序列，或者其为SEQ ID NO:9和SEQ ID NO:10的序列。
  43. 根据权利要求40所述的方法，其中，所述阻断剂为SEQ ID NO:8的序列或SEQ ID NO:12的序列
  44. 根据权利要求41所述的方法，其中，所述探针为SEQ ID NO:7的序列或SEQ ID NO:11的序列。
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CN113999915A (zh) * 2021-12-01 2022-02-01 杭州求臻医学检验实验室有限公司 用于食管癌早期筛查的生物标志物组合、试剂盒及应用

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