CN115261467A - Composition for detecting esophageal cancer and application thereof - Google Patents
Composition for detecting esophageal cancer and application thereof Download PDFInfo
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- CN115261467A CN115261467A CN202210605952.0A CN202210605952A CN115261467A CN 115261467 A CN115261467 A CN 115261467A CN 202210605952 A CN202210605952 A CN 202210605952A CN 115261467 A CN115261467 A CN 115261467A
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- C—CHEMISTRY; METALLURGY
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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Abstract
The invention provides a composition for detecting esophageal cancer and application thereof, wherein the composition comprises the following components in parts by weight: a nucleic acid for detecting the methylation state of a target gene, wherein the target gene is one or both of the MT1A gene and the EPO gene. The invention also provides a kit comprising the composition. And the use of the composition in the preparation of a kit for the in vitro detection of esophageal cancer.
Description
The application is a divisional application of a patent application with the Chinese application number of 201711248825.5, the invention name of the patent application is 'composition for detecting esophageal cancer and application thereof', and the application date of the patent application is 2017, 12 months and 1 days.
Technical Field
The invention belongs to the technical field of biology, relates to a composition and application thereof in disease detection, and particularly relates to a composition for detecting esophageal cancer, a corresponding kit and application thereof.
Background
Esophageal cancer is a common tumor of the digestive tract, and about 30 million people die of esophageal cancer every year worldwide. China is one of the high incidence areas of esophageal cancer in the world, and half of the patients who die of esophageal cancer worldwide come from China. National cancer prevention and treatment office data show: in 2015, the incidence rate of esophageal cancer is 478/10 ten thousand in China, the mortality rate is 375/ten thousand in China, and the esophageal cancer is respectively the fourth and third place of common cancers in China. The death rate of esophageal cancer is close to 80%, and the cancer is high in malignancy degree. An important factor leading to high mortality of esophageal cancer is the low certainty of early esophageal cancer. Early esophageal cancer has a much higher cure rate than the middle and late stages, but most patients have progressed to the middle and late stages when diagnosed due to lack of obvious and specific symptoms of early esophageal cancer. Clinical studies have found that the progression of cancer from the onset of the lesion to the appearance of clinical symptoms in patients takes on average years; this provides an effective window period for the detection of early esophageal cancer and the improvement of the diagnosis rate of early esophageal cancer. The window period is fully utilized, so that the treatment effect of the esophageal cancer is hopefully improved, and the death rate of the esophageal cancer is reduced.
The current techniques for clinical diagnosis of esophageal cancer have limited applications in the detection and screening of early esophageal cancer, mainly because: 1) The tissue biopsy is highly invasive and is not suitable for early cancer screening; 2) Imaging detection techniques (e.g.: esophagography and endoscopy) in terms of equipment cost, operating techniques, invasiveness, and the like, and is also difficult to popularize as a cancer screening technique on a large scale; 3) Traditional serum tumor markers (e.g.: AFP, CEA, CA125, and CA199, etc.) has low sensitivity for esophageal cancer detection, and cannot sufficiently meet the requirement of early cancer screening.
Recent studies have shown that epigenetics plays an important role in the development and progression of cancer. As an important mechanism in epigenetics, DNA methylation regulation in a variety of cancers has been extensively studied. The study data showed that: regulation of gene methylation is related to biological mechanisms such as chromatin structure and gene expression regulation; changes in cellular gene methylation occur early in tumor formation and throughout the development and progression of cancer; methylation of cancer suppressor genes is an important molecular mechanism for transformation of precancerous lesion tissues into malignant tumor cells. However, detection technology, method and product for detecting esophageal cancer methylation genes are lacked at present. Therefore, there is a current need for methylation gene markers with high sensitivity and high specificity for esophageal cancer detection.
Disclosure of Invention
Based on the above, aiming at the problems of inconvenient detection, low sensitivity and high cost of the existing esophageal cancer detection technology, the invention provides a composition for detecting esophageal cancer, the composition provided by the invention can sensitively and specifically detect esophageal cancer, and the invention also provides a kit containing the composition and application thereof in detecting esophageal cancer. The kit provided by the invention has good esophageal cancer detection sensitivity, and can conveniently, quickly and effectively detect esophageal cancer.
The invention provides a composition for in vitro detection of esophageal cancer, a kit and application thereof, a method for performing detection based on the kit, and a method for detecting esophageal cancer.
In particular, the invention relates to the following:
1. a composition for use in the in vitro detection of esophageal cancer, the composition comprising:
a nucleic acid for detecting the methylation state of a target gene,
wherein the target gene is one or two of MT1A gene and EPO gene.
2. The composition of item 1, wherein the MT1A gene target sequence is shown in SEQ ID NO. 1.
3. The composition of item 1, wherein the target sequence of the EPO gene is set forth in SEQ ID NO. 3.
4. The composition of any one of items 1 to 3, wherein the nucleic acid for detecting the methylation state of a target gene comprises:
a fragment of at least 9 nucleotides in the target sequence of the gene of interest,
the fragment comprises at least one CpG dinucleotide sequence.
5. The composition of any one of items 1 to 4, wherein the nucleic acid for detecting the methylation state of a target gene further comprises:
hybridizes under moderately stringent or stringent conditions to a fragment of at least 15 nucleotides of the target sequence of the target gene,
the fragment comprises at least one CpG dinucleotide sequence.
6. The composition according to any one of items 1 to 5, further comprising:
an agent that converts an unmethylated cytosine base at position 5 of a target sequence of a gene of interest to uracil.
7. The composition of any one of items 1 to 6, wherein the nucleic acid for detecting the methylation state of a target gene further comprises:
a blocking agent that preferentially binds to a target sequence in a non-methylated state.
8. The composition according to item 7, wherein,
the fragment of at least 9 nucleotides is the sequence of SEQ ID NO. 5 and SEQ ID NO. 6, or the sequence of SEQ ID NO. 9 and SEQ ID NO. 10,
a fragment of said at least 15 nucleotides being the sequence of SEQ ID NO 7 or the sequence of SEQ ID NO 11,
a blocker which is the sequence of SEQ ID NO 8 or the sequence of SEQ ID NO 12.
9. An oligonucleotide for detecting esophageal cancer in vitro, comprising:
1 or the complement thereof and comprises at least one fragment of a CpG dinucleotide sequence; and/or
3 or the complement thereof and comprises at least one fragment of a CpG dinucleotide sequence.
10. The oligonucleotide of item 9, further comprising:
a fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID No. 1 or the complement thereof and comprises at least one CpG dinucleotide sequence; and/or
A fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID NO 3 or the complement thereof and comprises at least one CpG dinucleotide sequence.
11. The oligonucleotide of item 10, further comprising:
a blocking agent that preferentially binds to a target sequence in a non-methylated state.
12. An oligonucleotide for detecting esophageal cancer in vitro, comprising:
the sequences of SEQ ID NO 5 and SEQ ID NO 6.
13. The oligonucleotide of item 12, further comprising:
the sequence of SEQ ID NO. 7.
14. The oligonucleotide of item 13, further comprising:
the sequence of SEQ ID NO 8.
15. An oligonucleotide for detecting esophageal cancer in vitro, comprising:
the sequences of SEQ ID NO 9 and SEQ ID NO 10.
16. The oligonucleotide of item 15, further comprising:
the sequence of SEQ ID NO. 11.
17. The oligonucleotide of item 16, further comprising:
12, SEQ ID NO.
Use of the MT1A gene in the preparation of a kit for detecting esophageal cancer in vitro.
Use of EPO gene in the preparation of a kit for the in vitro detection of esophageal cancer.
20. A kit comprising the composition of any one of claims 1 to 8 or comprising the oligonucleotide of any one of claims 9 to 17.
21. The kit of item 20, further comprising at least one additional component selected from the group consisting of:
nucleoside triphosphates, a DNA polymerase and buffers required for the function of the DNA polymerase.
22. The kit of item 20 or 21, further comprising: and (6) instructions.
23. Use of a composition according to any one of claims 1 to 8 or an oligonucleotide according to any one of claims 9 to 17 for the preparation of a kit for the in vitro detection of esophageal cancer.
24. The use according to any one of claims 18, 19 and 23, wherein the kit for in vitro detection of esophageal cancer detects esophageal cancer by a method comprising the steps of:
1) Separating a DNA sample comprising a target sequence of a target gene or a fragment thereof in a biological sample to be tested;
2) Determining the methylation state of the target sequence of the target gene;
3) And judging the state of the biological sample according to the detection result of the methylation state of the target gene target sequence, thereby realizing the in-vitro detection of the esophageal cancer.
25. The use according to item 24, wherein the method comprises the steps of:
extracting genome DNA of a biological sample to be detected;
treating the extracted genomic DNA with a reagent that converts the 5-unmethylated cytosine base to uracil or other bases;
contacting the reagent-treated DNA sample with a DNA polymerase and a primer for a target sequence of the target gene, and carrying out a DNA polymerization reaction in the presence of a blocker that preferentially binds to the target sequence in a non-methylated state;
detecting the amplification product with a probe; and
determining the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest based on the presence or absence of the amplification product.
26. The use of item 25, wherein the reagent is a bisulfite reagent.
27. A method of detecting esophageal cancer comprising the steps of:
separating a DNA sample comprising a target sequence of a target gene or a fragment thereof in a biological sample to be tested;
determining the methylation state of the target sequence of the target gene; and
and judging the state of the biological sample according to the detection result of the methylation state of the target gene target sequence, thereby realizing the in-vitro detection of the esophageal cancer.
28. A method of detecting esophageal cancer, comprising the steps of:
extracting the genome DNA of a biological sample to be detected;
treating the extracted genomic DNA with a reagent that converts the 5-unmethylated cytosine base to uracil or another base;
contacting the reagent-treated DNA sample with a DNA polymerase and a primer for a target sequence of a target gene, and performing a DNA polymerization reaction in the presence of a blocker that preferentially binds to the target sequence in a non-methylated state;
detecting the amplification product with a probe; and
determining the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest based on the presence or absence of the amplification product.
29. The method of clauses 27 or 28, wherein,
the target gene is one or two of MT1A gene and EPO gene.
30. The method of claim 29, wherein the MT1A gene target sequence is set forth in SEQ ID NO 1.
31. The method of item 29, wherein the target sequence of the EPO gene is set forth in SEQ ID No. 3.
32. The method of item 28, wherein the reagent is a bisulfite reagent.
33. The method of item 28, wherein the primers are:
1 or the complement thereof and comprises at least one fragment of a CpG dinucleotide sequence; and/or
3 or the complement thereof and comprises at least one fragment of a CpG dinucleotide sequence.
33. The method of item 28, wherein the blocking agent is one that preferentially binds to a target sequence in a non-methylated state.
34. The method of item 28, wherein the probe is:
a fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID No. 1 or the complement thereof and comprises at least one CpG dinucleotide sequence; and/or
A fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID NO 3 or the complement thereof and comprises at least one CpG dinucleotide sequence.
35. The method of item 33, wherein the primers are the sequences of SEQ ID NO 5 and SEQ ID NO 6, or they are the sequences of SEQ ID NO 9 and SEQ ID NO 10.
36. The method of item 33, wherein the blocking agent is the sequence of SEQ ID NO 8 or the sequence of SEQ ID NO 12
37. The method of claim 34, wherein the probe is the sequence of SEQ ID NO 7 or the sequence of SEQ ID NO 11.
The inventor of the invention utilizes epigenomics and bioinformatics technology, discovers two methylation genes related to esophageal cancer by analyzing the whole genome methylation data of esophageal cancer tissues and paracancer control tissues, and determines the target sequences of the two methylation genes of esophageal cancer with methylation abnormality; furthermore, the inventors of the present invention found that the methylation states of the two esophageal cancer methylation genes can be sensitively and specifically detected through the target sequences of the two genes, so that the methylation states can be used for detecting free DNA in peripheral blood. The detection of peripheral blood samples of esophageal cancer patients and normal control individuals shows that: the compositions and detection methods described herein are capable of sensitively and specifically detecting esophageal cancer, including common esophageal cancers of two different cell types: squamous carcinoma and adenocarcinoma. Therefore, the composition and the detection method for detecting esophageal cancer in vitro provided by the invention have important clinical application values.
Other features and advantages of the invention will be described in detail in the following detailed description and claims.
Drawings
The above and other features of the present invention will be further explained by the following detailed description thereof taken in conjunction with the accompanying drawings. It is appreciated that these drawings depict only several exemplary embodiments in accordance with the invention and are therefore not to be considered limiting of its scope. The drawings are not necessarily to scale and wherein like reference numerals refer to like parts, unless otherwise specified.
FIG. 1 shows a graph of the results of screening for the target gene of the present invention.
FIG. 2 is a diagram showing the detection of leukocyte genomic DNA (negative reference of methylation state of target gene target sequence) and leukocyte genomic DNA (positive reference of methylation state of target gene target sequence) treated with DNA methyltransferase using the compositions and detection methods provided by the present invention. The results show that: the composition and the detection method provided by the invention have negative detection result on the leucocyte genome DNA and positive detection result on the leucocyte genome DNA treated by DNA methyltransferase.
FIG. 3 is a diagram showing the results of in vitro non-invasive detection of esophageal cancer by detecting the methylation state of a target gene target sequence using the composition and the detection method.
Detailed Description
In one aspect, the present invention provides a composition for detecting esophageal cancer in vitro, the composition comprising a nucleic acid for detecting methylation status within a target sequence of a target gene, wherein the target gene is one or both of MT1A gene and EPO gene.
The invention provides a group of target gene target sequences emitting abnormal methylation in esophageal cancer, which comprise MT1A gene target sequences and EPO gene target sequences, wherein the MT1A gene target sequences are shown in SEQ ID NO. 1-2, and the EPO gene target sequences are shown in SEQ ID NO. 3-4.
The target sequence of MT1A gene is shown in SEQ ID NO. 1.
SEQ ID NO:1
CACCCAGGGGAGCTCAGTGGACTGTGCGCCTTGCCTTTCTGCTGCGCAAAGCCCAGTCCAGGTCATCACCTCGGGCGGGGCGGACTCGGCTGGGCGGACTCAGCGGGGCGGGCGCAGGCGCAGGGCGGGTCCTTTGCGTCCGGCCCTCTTTCCCCTGACCATAAAAGCAGC
The amino acid sequence of SEQ ID NO:1 is as shown in SEQ ID NO:2, respectively.
SEQ ID NO:2
GCTGCTTTTATGGTCAGGGGAAAGAGGGCCGGACGCAAAGGACCCGCCCTGCGCCTGCGCCCGCCCCGCTGAGTCCGCCCAGCCGAGTCCGCCCCGCCCGAGGTGATGACCTGGACTGGGCTTTGCGCAGCAGAAAGGCAAGGCGCACAGTCCACTGAGCTCCCCTGGGTG
Preferably, the sequence of the target sequence of the EPO gene is shown in SEQ ID NO. 3.
SEQ ID NO:3
CGCGCACGCACACATGCAGATAACAGCCCCGACCCCCGGCCAGAGCCGCAGAGTCCCTGGGCCACCCCGGCCGCTCGCTGCGCTGCGCCGCACCGCGCTGTCCTCCCGGAGCCGGACCGGGGCCACCGCGCCCGCTCTGCTCCGACACCGCGCCCCCTGGACAGCCGCCCTCTCCTCCAGGCCCGTGGGGCTGGCCCTGCACCGCCGAGCTTCCCGGGATGAGGGCCCCCGGTGTGGTCACCCGGCGCGCCCCAGGTCG
The amino acid sequence of SEQ ID NO:3 is as shown in SEQ ID NO:4, respectively.
CGACCTGGGGCGCGCCGGGTGACCACACCGGGGGCCCTCATCCCGGGAAGCTCGGCGGTGCAGGGCCAGCCCCACGGGCCTGGAGGAGAGGGCGGCTGTCCAGGGGGCGCGGTGTCGGAGCAGAGCGGGCGCGGTGGCCCCGGTCCGGCTCCGGGAGGACAGCGCGGTGCGGCGCAGCGCAGCGAGCGGCCGGGGTGGCCCAGGGACTCTGCGGCTCTGGCCGGGGGTCGGGGCTGTTATCTGCATGTGTGCGTGCGCG
Preferably, the nucleic acid for detecting the methylation state of a target gene comprises a fragment of at least 9 nucleotides of the target sequence of the target gene, wherein the fragment comprises at least one CpG dinucleotide sequence. In certain preferred embodiments, such as bisulfite conversion of test sample DNA, the nucleic acid used to detect the methylation state of a target gene comprises a fragment of at least 9 nucleotides of the sequence after bisulfite conversion of the target sequence of the target gene, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence.
More preferably, the nucleic acid for detecting the methylation state of a target gene comprises a fragment of at least 15 nucleotides that hybridizes under moderate stringency or stringent conditions to the target sequence of said target gene, wherein said fragment of nucleotides comprises at least one CpG dinucleotide sequence. In certain preferred embodiments, such as using bisulfite conversion of test sample DNA, the nucleic acid used to detect the methylation state of a target gene comprises a fragment of at least 15 nucleotides that hybridizes under moderately stringent or stringent conditions to the target sequence of the target gene after bisulfite conversion, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence.
Preferably, the composition further comprises an agent that converts the unmethylated cytosine base at position 5 of the target gene sequence to uracil. More preferably, the agent is bisulfite.
Preferably, the nucleic acid for detecting the methylation state of a target gene further comprises a blocker that preferentially binds to DNA in a non-methylated state.
Preferably, the composition comprises one or more of the following primers, probes and/or blockers:
MT1A primer F
SEQ ID NO:5
CGGACGTAAAGGATTC
MT1A primer R
SEQ ID NO:6
GAAACGAACTCGACTAAACG
MT1A probe
SEQ ID NO:7
TGCGTTTGCGTTCGTTTCG
MT1A blocking agent
SEQ ID NO:8
CAAACTCAACTAAACAAACTCAACAAAACAAAC
EPO primer F
SEQ ID NO:9
AGTCGTAGAGTTTTTGGGTT
EPO primer R
SEQ ID NO:10
CAACGCGATACGACG
EPO probes
SEQ ID NO:11
CGCAACGAACGACCGA
EPO blockers
SEQ ID NO:12
GAGTTTTTGGGTTATTTTGGTTGTTTGTTG
In another aspect, the present invention provides an oligonucleotide for detecting esophageal cancer in vitro, comprising: 1 or the complement thereof and comprises at least one fragment of a CpG dinucleotide sequence; and/or a fragment of at least 9 nucleotides of SEQ ID NO 3 or the complement thereof and comprising at least one CpG dinucleotide sequence.
Preferably the oligonucleotide for detecting oesophageal cancer in vitro comprises: a fragment of at least 9 nucleotides of the sequence after bisulfite conversion of SEQ ID NO. 1 or its complement; and/or a fragment of at least 9 nucleotides of the sequence after bisulfite conversion of SEQ ID NO 3 or its complement and comprising at least one CpG dinucleotide sequence.
The oligonucleotide for detecting esophageal cancer in vitro of the invention further comprises: a fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID No. 1 or the complement thereof and comprises at least one CpG dinucleotide sequence; and/or a fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of said SEQ ID NO 3 or the complement thereof and comprises at least one CpG dinucleotide sequence.
Preferably the oligonucleotide for detecting esophageal cancer in vitro comprises: a fragment that hybridizes under moderately stringent or stringent conditions to at least 15 nucleotides of a sequence after bisulfite conversion of SEQ ID NO. 1 or a complementary sequence thereof and comprises at least one CpG dinucleotide sequence; and/or a fragment that hybridizes under moderate stringency or stringent conditions to at least 15 nucleotides of the bisulfite converted sequence of SEQ ID NO. 3 or its complement and comprises at least one CpG dinucleotide sequence.
The oligonucleotide for detecting esophageal cancer in vitro of the invention further comprises: a blocker that preferentially binds to DNA in an unmethylated state.
In a specific embodiment, an oligonucleotide for detecting esophageal cancer in vitro, comprising: the sequences of SEQ ID NO 5 and SEQ ID NO 6. It still includes: the sequence of SEQ ID NO. 7. It still includes: the sequence of SEQ ID NO 8.
In another specific embodiment, an oligonucleotide for detecting esophageal cancer in vitro, comprising: the sequences of SEQ ID NO 9 and SEQ ID NO 10. It still includes: the sequence of SEQ ID NO. 11. It still includes: 12, SEQ ID NO.
In another aspect, the invention provides a kit comprising the composition. The kit further comprises at least one additional component selected from the group consisting of: nucleoside triphosphates, a DNA polymerase and buffers required for the function of said DNA polymerase.
The invention also relates to application of the MT1A gene and/or the EPO gene in preparation of a kit for in vitro detection of esophageal cancer.
Wherein MT1A is metallothionein 1A, english name metallothionein 1A, is located in the q13 region of human chromosome 16, and belongs to the metallothionein gene family. Metallothionein is a small molecule protein rich in cysteine, lacking amino acid containing aromatic group, and capable of binding divalent heavy metal ions. Metallothionein is an antioxidant that protects cells from hydroxyl-containing free radicals, maintains the balance of metal ions in cells, and simultaneously exerts a heavy ion toxicity removing effect. The loss of the function of the metallothionein gene can cause pathological phenomena such as cancer and the like.
The EPO gene is an erythropoietin gene, the English name erythropoetin, located in the q22.1 region of human chromosome 7. The protein encoded by the gene is a glycosylated cytokine (cytokine) secreted by the cell. Erythropoietin, when bound to the corresponding receptor, promotes the synthesis of red blood cells.
In yet another aspect, the present invention provides a method for detecting esophageal cancer in vitro, comprising the steps of:
1) Separating target gene sequences or fragments thereof of the target genes in the biological sample to be detected;
2) Determining the methylation state of the target sequence of the target gene;
3) And judging the state of the biological sample according to the detection result of the methylation state of the target gene target sequence, thereby realizing the in-vitro detection of the esophageal cancer.
According to certain preferred embodiments, the method further comprises the steps of:
1) Extracting the genome DNA of a biological sample to be detected;
2) Treating the DNA sample obtained in step 1) with a reagent to convert the 5-unmethylated cytosine base to uracil or another base, i.e., the 5-unmethylated cytosine base of the target sequence of the target gene is converted to uracil or another base, and the converted base is different from the 5-unmethylated cytosine base in hybridization properties and is detectable;
3) Contacting the DNA sample treated in step 2) with a DNA polymerase and a primer for the target gene sequence such that the treated target gene sequence is amplified to produce an amplification product or is not amplified; the processed target gene sequence of the target gene generates an amplification product if a DNA polymerization reaction occurs; the treated target gene sequence is not amplified if no DNA polymerization reaction occurs;
4) Detecting the amplification product with a probe; and
5) Determining the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest based on the presence or absence of the amplification product.
Preferably, a typical primer comprises a fragment of the gene target sequence of interest comprising a fragment of at least 9 nucleotides selected from SEQ ID NOS: 1-2 and SEQ ID NOS: 3-4 that is identical to, complementary to, or hybridizes under moderate or stringent conditions, respectively.
Preferably, a typical probe is a fragment of the gene target sequence of interest comprising a fragment of at least 15 nucleotides selected from SEQ ID NO 1-2 and SEQ ID NO 3-4 that is identical to, complementary to or hybridizes under moderate or stringent conditions, respectively.
Preferably, a typical blocker is one that preferentially binds to DNA in the unmethylated state.
Preferably, one or more of the primers, probes and/or blockers are as follows:
MT1A primer F
SEQ ID NO:5
CGGACGTAAAGGATTC
MT1A primer R
SEQ ID NO:6
GAAACGAACTCGACTAAACG
MT1A probe
SEQ ID NO:7
TGCGTTTGCGTTCGTTTCG
MT1A blocking agent
SEQ ID NO:8
CAAACTCAACTAAACAAACTCAACAAAACAAAC
EPO primer F
SEQ ID NO:9
AGTCGTAGAGTTTTTGGGTT
EPO primer R
SEQ ID NO:10
CAACGCGATACGACG
EPO probes
SEQ ID NO:11
CGCAACGAACGACCGA
EPO blockers
SEQ ID NO:12
GAGTTTTTGGGTTATTTTGGTTGTTTGTTG
And, 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 Polymerase Chain Reaction (PCR), producing an amplification product nucleic acid molecule with a detectable label.
According to certain preferred embodiments, the methylation state of at least one CpG dinucleotide in the target sequence of the target gene of interest is determined from the cycle threshold Ct value of the PCR reaction. The method for analyzing the DNA in the biological sample by utilizing the PCR reaction can conveniently realize the detection aiming at the methylation state of the target gene target sequence, and can quickly and conveniently judge whether the detected sample is positive according to the cycle threshold value of the PCR reaction, thereby providing a noninvasive and quick in-vitro detection method for the esophageal cancer.
The biological sample is selected from the group consisting of a cell line, a histological section, a tissue biopsy/paraffin embedded tissue, a bodily fluid, a stool, a colonic exudate, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, or a combination thereof.
A preferred biological sample is plasma.
The invention also provides a kit comprising the composition. Typically, the kit includes a container for holding a biological sample from a patient. Also, the kit may include instructions for using and interpreting the results of the assay.
The invention provides a method for detecting esophageal cancer in vitro and noninvasively by detecting the methylation state of a target gene target sequence. The inventor finds that the methylation states of the MT1A gene and the EPO gene target sequence in esophageal cancer tissues and the methylation state of the gene target sequence in normal esophageal tissues have significant difference: in esophageal cancer tissues, the MT1A gene and the EPO gene target sequence are methylated, while in normal esophageal tissues, the MT1A gene and the EPO gene target sequence are not methylated. Therefore, the method for detecting the esophageal cancer in vitro by detecting the methylation states of the MT1A gene and the EPO gene target sequences in the sample can detect the esophageal cancer noninvasively and quickly.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or experimental applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention also provides a composition capable of sensitively and specifically detecting the methylation state of the target sequence of the target gene; and a method and a kit for non-invasive detection of esophageal cancer in vitro.
The following are described as examples of the compositions, kits, nucleic acid sequences, and methods of detection of the invention. A first set of embodiments disclose a gene of interest and a gene target sequence of interest; a second set of embodiments discloses a composition for detecting the methylation state of a target sequence of a gene of interest, comprising a nucleic acid for detecting the methylation state of a target sequence of a gene of interest; the third group of embodiments discloses a method for non-invasive in vitro detection of esophageal cancer by detecting the methylation state of a target gene target sequence.
In certain embodiments, the composition further comprises an agent that converts an unmethylated cytosine base at position 5 of the gene to uracil. Preferably, the agent is a bisulfite. Bisulfite modification of DNA is a known tool for assessing CpG methylation status. Among eukaryotic DNA, 5-methylcytosine is the most common covalent base modification. 5-methylcytosine cannot be identified by sequencing because 5-methylcytosine has the same base-pairing behavior as cytosine. In addition, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification. The most commonly used method for analyzing the presence of 5-methylcytosine in DNA is based on the specific reaction of bisulfite with cytosine; following subsequent alkaline hydrolysis, unmethylated cytosines are converted to uracils which correspond in pairing behavior to thymines; however, under these conditions 5-methylcytosine remains unmodified. The original DNA is thereby converted in such a way that the 5-methylcytosines, which were originally indistinguishable from cytosine in their hybridization behavior, can now be detected as the only remaining cytosines by the customary known molecular biological techniques, for example by amplification and hybridization. All of these techniques are based on different base pairing properties and can now be fully exploited. Thus, typically, the present application provides for the use of bisulfite techniques in combination with one or more methylation assays for determining the methylation state of a CpG dinucleotide sequence within a target sequence of a gene of interest. Furthermore, the method of the invention is suitable for analyzing low concentrations of tumor cells in heterogeneous biological samples, such as blood or faeces. Thus, when analyzing the methylation status of a CpG dinucleotide sequence in such a sample, one skilled in the art can use a quantitative assay to determine the methylation level (e.g., percentage, fraction, ratio, proportion, or degree) of a particular CpG dinucleotide sequence, rather than the methylation status. Correspondingly, the term methylation status or methylation state shall also be taken to mean a value which reflects the methylation state of a CpG dinucleotide sequence.
In certain embodiments, the methods of the present application specifically comprise: 1) Extracting the genome DNA of a biological sample to be detected; 2) Treating the DNA sample obtained in step 1) with a reagent to convert the 5-unmethylated cytosine base to uracil or another base, i.e., the 5-unmethylated cytosine base of the target sequence of the target gene is converted to uracil or another base, and the converted base is different from the 5-unmethylated cytosine base in hybridization properties and is detectable; 3) Contacting the DNA sample treated in step 2) with a DNA polymerase and a primer for the target gene sequence such that the treated target gene sequence is amplified to produce an amplification product or is not amplified; the processed target gene sequence of the target gene generates an amplification product if a DNA polymerization reaction occurs; the processed target gene sequence of interest is not amplified if no DNA polymerization reaction occurs; 4) Detecting the amplification product with a probe; 5) And determining the methylation status of at least one CpG dinucleotide of the target sequence of the target gene based on the presence or absence of the amplification product.
Typically, 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; producing an amplification product nucleic acid molecule with a detectable label. Preferably, the methylation status is determined by means of PCR, and determination methods such as "fluorescence-based real-time PCR technique", methylation-sensitive single nucleotide primer extension reaction (Ms-SNuPE), methylation-specific PCR (MSP), and methylated CpG island amplification (MCA) are used to determine the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest. Among these, "fluorescence-based real-time PCR" assays are high-throughput quantitative methylation assays that use fluorescence-based real-time PCR (TaqMan) technology, requiring no further manipulation after the PCR step. Briefly, the "fluorescence-based real-time PCR" method starts with a mixed sample of genomic DNA that is converted to a mixed pool of methylation-dependent sequence differences in a sodium bisulfite reaction according to standard procedures. Fluorescence-based PCR was then performed in a "biased" reaction (using PCR primers that overlap known CpG dinucleotides). Sequence differences can be generated at the level of amplification as well as at the level of fluorescence detection amplification. "fluorescence-based real-time PCR" assays can be used as quantitative tests of methylation status in genomic DNA samples, where sequence discrimination occurs at the probe hybridization level. In this quantitative format, the PCR reaction provides methylation specific amplification in the presence of a fluorescent probe that overlaps a specific CpG dinucleotide. Unbiased controls for the starting DNA amounts are provided by the following reactions: wherein neither the primer nor the probe covers any CpG dinucleotides. 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 dual-labeled with a fluorescent Reporter (Reporter) and a Quencher molecule (Quencher) and is designed to be specific to a region of relatively high GC content, so that it melts at a temperature about 10 ℃ higher than the forward or reverse primer during the PCR cycle. This allows the TaqMan probe to remain fully hybridized during the PCR annealing/extension step. Taq polymerase eventually encounters an annealed TaqMan probe when it enzymatically synthesizes a new strand in PCR. The Taq polymerase 5-to 3' endonuclease activity will then displace the TaqMan probe by digesting it, releasing the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system. Typical reagents for "fluorescence-based real-time PCR" assays may include, but are not limited to: PCR primers for target sequences of the target genes; a non-specific amplification blocker; taqMan or Lightcycler probes; optimized PCR buffer solution and deoxynucleotide; and Taq polymerase, etc.
Examples
Example 1
By analyzing the data of 233 cases of esophageal cancer tissues and 171 cases of normal esophageal tissues on a genome-wide methylation chip (human methylation450k chip from Illumina), the present inventors found that the methylation levels of the MT1A gene and EPO gene in esophageal cancer tissues were significantly higher than those in normal esophageal tissues (the analysis results are shown in fig. 1). Further, the inventors found sequence segments of the two target genes with the most obvious methylation difference in esophageal cancer tissues and normal esophageal tissues by analyzing probe sequences of MT1A gene and EPO gene on a whole genome methylation chip and corresponding methylation rate data, and thereby determined the target sequences of the two target genes. The target sequence of MT1A gene is shown in SEQ ID NO. 1. The complementary sequence of the target sequence of the MT1A gene is shown as SEQ ID NO:2, respectively. The target sequence of EPO gene is shown in SEQ ID NO. 3. The complementary sequence of the target sequence of the EPO gene is shown as SEQ ID NO:4, respectively.
Example 2
The first step is as follows: 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, stool, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and all possible combinations thereof. The DNA is then isolated from the sample by any standard means known in the art. Briefly, when DNA is encapsulated in a cell membrane, the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. Followed by removal of proteins and other contaminants, for example by digestion with protein kinase K. The DNA is then recovered from the solution. This can be achieved by various methods, including salting out, organic extraction, or binding of the DNA to a solid support. The choice of method can be influenced by a number of factors, including time, expense, and the amount of DNA required. When the sample DNA is not encapsulated in a cell membrane (e.g., circulating DNA from a blood sample), standard methods of isolating and/or purifying DNA in the prior art can be used. These methods include the use of protein degrading agents, for example chaotropic salts, such as guanidine hydrochloride or urea; or 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. The skilled person may also utilize devices such as filters such as ultrafiltration, silicon surfaces or membranes, magnetic particles, polystyrene surfaces, positively charged surfaces and positively charged membranes, charged surfaces, charged transfer membranes, charged transfer surfaces. Once the nucleic acids are extracted, the DNA is used for analysis.
In this embodiment, the biological sample DNA is leukocyte genomic DNA and leukocyte genomic DNA after DNA methyltransferase treatment. The target gene sequence of the leukocyte genomic DNA is in a non-methylated state, so the leukocyte genomic DNA is a negative reference for the methylation state of the target gene sequence. The target gene target sequence of the leukocyte genomic DNA treated by the DNA methyltransferase is in a methylated state, so the leukocyte genomic DNA treated by the DNA methyltransferase is a positive reference of the methylated state of the target gene target sequence.
The second step is that: the two DNA samples were treated separately so that the cytosine base that was unmethylated in the 5 position was converted to uracil, thymine or another base that was not used for cytosine in the hybridization behavior. Preferably, this is achieved by treatment with a bisulphite reagent. The term "bisulfite reagent" refers to a reagent comprising bisulfite, or a combination thereof, as disclosed herein, that can be used to distinguish between methylated and unmethylated CpG dinucleotide sequences. Preferably, the bisulfite treatment is carried out in the presence of a denaturing solvent such as, but not limited to, n-alkylene glycols, especially diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In a preferred embodiment, the denaturing solvent is used at a concentration of 1% to 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chromane derivative, such as 6-hydroxy-2, 5,7,8, -tetramethylchromane 2-carboxylic acid or trihydroxybenzoic acid and derivatives thereof, such as gallic acid. The bisulfite conversion is preferably carried out at a reaction temperature of from 30 ℃ to 70 ℃, wherein the temperature is increased to over 85 ℃ for a short time during the reaction. The bisulfite treated DNA is preferably purified prior to quantification. This may be done by any method known in the art, such as, but not limited to, ultrafiltration.
The third step: the primers and amplification enzymes of the invention are used to amplify fragments of the treated DNA. Amplification of several DNA fragments can be performed simultaneously in the same reaction vessel. Preferably, the amplification product is 100 to 2,000 base pairs in length. When the genomic DNA of the biological sample to be tested is a mixture of methylated and unmethylated states, this is particularly the case when the DNA in the methylated state is much less than the DNA in the unmethylated state, as follows: in order to improve the amplification specificity of PCR amplification primers, the invention adopts a blocker which is specific to a target sequence of a target gene and is used in a PCR reaction system. The 5 end of the blocker nucleotide sequence and the 3' end nucleotide sequence of the forward (F) or reverse (R) primer have an overlapping region of more than or equal to 5 nucleotides; the blocker and the forward (F) or reverse (R) primer are complementary to the same strand of the target sequence DNA of the target gene; the blocker melting temperature is more than (including) 5 ℃ above the forward (F) or reverse (R) primer; the nucleotide sequence of the blocker comprises at least one CpG dinucleotide sequence and is complementary to the sequence of the bisulfite converted unmethylated target gene sequence DNA. Thus, when the genomic DNA of the biological sample to be tested is a mixture of methylated and unmethylated DNA, particularly if the methylated DNA is much less abundant than the unmethylated DNA, after bisulfite conversion, will preferentially bind to the blocking agent, thereby inhibiting the binding of the DNA template to the PCR primers, and thus PCR amplification will not occur, whereas the methylated DNA will not bind to the blocking agent and thus will bind to the primers, and PCR amplification will occur. Thereafter, the fragments obtained by amplification are detected directly or indirectly. Preferably the label is in the form of a fluorescent label, radionuclide or attachable molecular fragment.
According to the target gene target sequences SEQ ID NO:1-2 and SEQ ID NO:3-4, primers, probes and blocker sequences (SEQ ID NO: 5-12) for detecting the methylation states of the target sequences of the MT1A and EPO are designed in the invention:
preferably, one or more of the primers, probes and/or blockers are as follows:
MT1A primer F
SEQ ID NO:5
CGGACGTAAAGGATTC
MT1A primer R
SEQ ID NO:6
GAAACGAACTCGACTAAACG
MT1A probe
SEQ ID NO:7
TGCGTTTGCGTTCGTTTCG
MT1A blocking agent
SEQ ID NO:8
CAAACTCAACTAAACAAACTCAACAAAACAAAC
EPO primer F
SEQ ID NO:9
AGTCGTAGAGTTTTTGGGTT
EPO primer R
SEQ ID NO:10
CAACGCGATACGACG
EPO probes
SEQ ID NO:11
CGCAACGAACGACCGA
EPO blockers
SEQ ID NO:12
GAGTTTTTGGGTTATTTTGGTTGTTTGTTG
In the present invention, detection of real-time PCR can be performed according to standard procedures of the prior art on various commercially available real-time PCR instrumentation. According to certain embodiments, detection of real-time PCR is performed on a Life Technologies instrument (7500 Fast). The PCR reaction mixture was composed of bisulfite converted DNA template 25-40ng and 300-600nM primers and blockers, 150-300nM probe, 1UTaq polymerase, 50-400. Mu.M of each dNTP, 1-10 mM of MgCl2And 2XPCR buffered to a final volume of 2. Mu.l to 100. Mu.l. The sample is amplified with pre-cycles at 85 to 99 ℃ for 3-60 minutes, followed by 35-55 cycles of annealing at 50 to 72 ℃ for 1 to 30 seconds, annealing and extension at 45 to 80 ℃ for 5 to 90 seconds, and denaturation at 85 to 99 ℃ for 5 to 90 seconds. The gene fragment is detected with a probe specific for the region of the CpG island of the target gene target sequence containing 5-methylcytosine by observing amplification only on the methylated target gene target sequence. Also, in certain embodiments, a beta actin gene (ACTB) may be used as an internal reference for PCR, a beta actin gene amplicon may be created by using a primer complementary to the beta actin gene sequence, and the beta actin gene amplicon may be detected with a specific probe. At least one real-time PCR is performed per sample, and in certain embodiments, two or three real-time PCR assays are performed.
The experimental results show that as shown in figure 2: the composition and the detection method provided by the invention have no PCR amplification in the detection of the leucocyte genome DNA (negative reference substance of the methylation state of the target gene target sequence), and the detection result is negative, namely: the target gene target sequence of the tested DNA sample is not methylated; the composition and the detection method provided by the invention are used for carrying out PCR amplification on the leucocyte genome DNA (a positive reference substance of the methylation state of a target gene target sequence) treated by DNA methyltransferase, and the detection result is positive, namely: the target sequence of the target gene in the tested DNA sample is methylated. Therefore, the composition and the detection method provided by the invention can specifically detect the methylation state of the target sequence of the target gene.
Example 3
According to the specific embodiment of the application, based on the average Ct values of the detection results of a certain number of esophageal cancer samples and normal samples, the Ct values of the target genes capable of effectively distinguishing esophageal cancer from normal are determined, that is: the critical value. The methylation state of at least one CpG dinucleotide in the target sequence of the target gene is determined by the cycle threshold Ct value of the polymerase chain reaction, and whether the result of the analysis based on the target gene is negative (normal) or positive (esophageal cancer) is determined by comparing the Ct value of the measured sample with a preset critical value.
The embodiment comprises the following steps:
first, plasma samples were obtained from 20 esophageal cancer patients and 22 normal persons. All samples were from the bor-cheng company. Peripheral blood free DNA of the test sample is then extracted and the DNA sample is pretreated so that the cytosine base that is unmethylated in the 5-position is converted to uracil, thymine or another base that is not used for cytosine in hybridization behavior. In this example, the pretreatment is achieved by bisulfite reagent treatment. The extraction and processing of the DNA may be performed by any standard means known in the art, in particular, in this example, all of the sample DNA extraction and bisulfite DNA modification are extracted by using plasma processing kit from Boerci.
Then, the above combination of the target gene primer, probe and blocker was added to the DNA samples of 20 patients with esophageal cancer and 22 normal persons, and the methylation state of the target sequence of the target gene was detected by PCR. In this example, PCR was performed on a Life Technologies apparatus (7500). The PCR reaction mixture was buffered to a final volume of 50. Mu.l by bisulfite converted DNA template 35ng, 450nM primer and blocker, 225nM probe, 1UTaq polymerase, 200. Mu.M of each dNTP, 4.5mM MgCl2 and 2 XPCR. The sample was amplified with pre-cycling at 94 ℃ for 20 minutes, followed by 5 seconds of 45 cycles of annealing at 62 ℃, annealing and extension at 55.5 ℃ for 35 seconds, and denaturation at 93 ℃ for 30 seconds.
Finally, ct values of the DNA samples of 20 esophageal cancer patients and 22 normal persons for the real-time PCR of the target gene target sequence are measured. The detection results are shown in fig. 3: 1) By selecting a specific critical value, preferably the critical value Ct =37, the esophageal cancer patient can be effectively detected by detecting the methylation state of the target gene target sequence by using the composition and the detection method provided by the invention; in this example, the sensitivity of methylation detection of esophageal cancer by the target gene target sequence was 55% (MT 1A) and 45% (EPO), respectively, when Ct value 37 was used as a cutoff value; 2) Methylation of the target gene sequence has good specificity, and the detection of a normal person shows that the methylation of the target gene sequence has the specificity of 95 percent (MT 1A) and 95 percent (EPO) for the detection of the normal person; 3) By combining the methylation detection results of MT1A gene and EPO gene, the sensitivity of esophageal cancer detection can be improved to 60%, and the specificity is still 95% due to inconvenient detection.
The above experimental results indicate that the methylated DNA of the target sequence of the target gene is a marker of esophageal cancer. The methylation DNA detection of the target gene target sequence can realize the in-vitro noninvasive detection of the esophageal cancer and improve the detection rate of the esophageal cancer.
In summary, the composition, the nucleic acid sequence, the kit and the use thereof, and the detection method are used for detecting the methylated nucleic acid sequence of the target gene target sequence and the segment thereof, so that the in vitro detection of the esophageal cancer by using the methylated biomarker of the target gene target sequence is realized, and the sensitivity and the specificity of the in vitro detection of the esophageal cancer are effectively improved. By utilizing the method for analyzing the free DNA of the plasma sample by utilizing the real-time PCR, the detection aiming at the methylation state of the target gene target sequence can be conveniently realized, and whether the sample is positive or not can be quickly and conveniently judged according to the CT value of the real-time PCR, thereby providing a noninvasive and convenient in-vitro detection method for the esophageal cancer.
While various aspects and embodiments of the invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are presented for purposes of illustration and not limitation. The scope and spirit of the present invention are to be determined only by the appended claims.
Sequence listing
<110> Boercheng (Beijing) science and technology Limited
<120> composition for detecting esophageal cancer and use thereof
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Claims (12)
1. A composition for use in the in vitro detection of esophageal cancer, the composition comprising:
a nucleic acid for detecting the methylation state of a target gene,
wherein the target gene is one or two of MT1A gene and EPO gene.
2. The composition according to claim 1, wherein the target sequence of MT1A gene is shown in SEQ ID NO 1.
3. The composition of claim 1, wherein the EPO gene has a target sequence as set forth in SEQ ID NO 3.
4. The composition of any one of claims 1 to 3, wherein the nucleic acid for detecting the methylation state of a target gene comprises:
a fragment of at least 9 nucleotides in the target sequence of the gene of interest,
the fragment comprises at least one CpG dinucleotide sequence.
5. The composition of any one of claims 1 to 4, wherein the nucleic acid for detecting the methylation state of a target gene comprises:
hybridizes under moderately stringent or stringent conditions to a fragment of at least 15 nucleotides of the target sequence of the target gene,
the fragment comprises at least one CpG dinucleotide sequence.
6. The composition of any one of claims 1-5, further comprising:
an agent that converts an unmethylated cytosine base at position 5 of a target sequence of a gene of interest to uracil.
7. The composition of any one of claims 1-6, wherein the nucleic acid for detecting the methylation state of a target gene further comprises:
a blocker that preferentially binds to a target sequence in a non-methylated state.
8. The composition of claim 7, wherein,
the fragment of at least 9 nucleotides is the sequence of SEQ ID NO. 5 and SEQ ID NO. 6, or the sequence of SEQ ID NO. 9 and SEQ ID NO. 10,
the fragment of at least 15 nucleotides is a sequence of SEQ ID NO. 7 or a sequence of SEQ ID NO. 11,
a blocker which is the sequence of SEQ ID NO 8 or the sequence of SEQ ID NO 12.
9. Use of a composition according to any one of claims 1 to 8 for the preparation of a kit for the in vitro detection of esophageal cancer.
10. The use according to claim 9, wherein the kit for the in vitro detection of esophageal cancer detects esophageal cancer by a method comprising the steps of:
separating a DNA sample comprising a target sequence of a target gene or a fragment thereof in a biological sample to be tested;
determining the methylation state of the target sequence of the target gene; and
and judging the state of the biological sample according to the detection result of the methylation state of the target gene target sequence, thereby realizing the in-vitro detection of the esophageal cancer.
11. Use according to claim 9, wherein the method comprises the steps of:
extracting the genome DNA of a biological sample to be detected;
treating the extracted genomic DNA with a reagent that converts the 5-unmethylated cytosine base to uracil or another base;
contacting the reagent-treated DNA sample with a DNA polymerase and a primer for a target sequence of the target gene, and carrying out a DNA polymerization reaction in the presence of a blocker that preferentially binds to the target sequence in a non-methylated state;
detecting the amplification product with a probe; and
determining the methylation status of at least one CpG dinucleotide of the target sequence of the gene of interest based on the presence or absence of the amplification product.
12. Use according to claim 11, wherein the reagent is a bisulphite reagent.
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| CN201711248825.5A CN109868320B (en) | 2017-12-01 | 2017-12-01 | Composition for detecting esophageal cancer and application thereof |
| CN202210605952.0A CN115261467A (en) | 2017-12-01 | 2017-12-01 | Composition for detecting esophageal cancer and application thereof |
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| CN112662764A (en) * | 2020-03-17 | 2021-04-16 | 博尔诚（北京）科技有限公司 | Probe composition for detecting 11 cancers |
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| TWI408235B (en) * | 2010-06-03 | 2013-09-11 | Univ China Medical | Gene marker and method for detection of oral cancer |
| CN102851354B (en) * | 2012-01-16 | 2014-06-11 | 中国人民解放军总医院 | Primer pair and kit for detecting methylation state of DNA at cell DACH1 gene promoter region |
| CN103667344B (en) * | 2012-09-18 | 2018-03-27 | 上海吉凯基因化学技术有限公司 | A kind of method for activating erythropoietin gene expression |
| CN104513851B (en) * | 2013-10-08 | 2017-01-11 | 陈永恩 | Novel epigenetic biomarker for detecting bladder cancer and method thereof |
| WO2016097319A1 (en) * | 2014-12-19 | 2016-06-23 | Epigenomics Ag | METHODS FOR DETECTING CpG METHYLATION AND FOR DIAGNOSING CANCER |
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Effective date of registration: 20231019 Address after: 100176 No.18, Hongda South Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing Applicant after: BIOCHAIN (BEIJING) SCIENCE & TECHNOLOGY Co.,Ltd. Applicant after: Air Force Medical University Address before: 100176 No.18, Hongda South Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing Applicant before: BIOCHAIN (BEIJING) SCIENCE & TECHNOLOGY Co.,Ltd. |