CN114032307A

CN114032307A - Composition for detecting esophageal cancer and use thereof

Info

Publication number: CN114032307A
Application number: CN202111355095.5A
Authority: CN
Inventors: 马竣; 韩晓亮; 王建铭
Original assignee: Biochain Beijing Science and Technology Inc
Current assignee: Fourth Military Medical University FMMU; Biochain Beijing Science and Technology Inc
Priority date: 2018-08-28
Filing date: 2018-08-28
Publication date: 2022-02-11
Also published as: CN110863046A; CN110863046B

Abstract

本发明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。本发明还提供了包括所述组合物的试剂盒。以及所述组合物在制备用于体外检测食管癌的试剂盒中的用途。The present invention provides a composition for detecting esophageal cancer, the composition comprising: a nucleic acid for detecting the methylation state of a target gene and an antibody for detecting the concentration of a target protein; wherein the target gene is MT1A One or both of the gene and the EPO gene; the target protein is SNCG. The present invention also provides kits comprising the compositions. And the use of the composition in the preparation of a kit for detecting esophageal cancer in vitro.

Description

Composition for detecting esophageal cancer and application thereof

The application is a divisional application of a patent application with Chinese application number of 201810989986.8, entitled composition for detecting esophageal cancer and application thereof and application date of 2018, 8 and 28.

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. National cancer prevention and treatment office data show: in 2015, the incidence rate of esophageal cancer is 478/10 ten thousand and the mortality rate is 375/ten thousand in China, and the esophageal cancer is respectively located at the fourth and third sites of common cancers. The death rate of esophageal cancer is close to 80%, and the cancer is high in malignancy degree. About 30 million people die of esophageal cancer each year worldwide, half of which are from china.

An important factor leading to high mortality of esophageal cancer is the low rate of definitive diagnosis of early esophageal cancer. Early esophageal cancer is much more cured than middle and advanced stages, but most subjects have progressed to middle and advanced 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 a subject 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 expected to be 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 has strong invasiveness 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 are also difficult to popularize on a large scale as a cancer screening technique; 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.

Disclosure of Invention

Based on the above, the invention provides a composition for detecting esophageal cancer, which aims at the problems of inconvenient detection, low sensitivity and high cost of the existing esophageal cancer detection technology. 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, and a method for performing detection based on the kit.

In particular, the invention relates to the following:

1. a composition for use in the in vitro detection of esophageal cancer, the composition comprising:

nucleic acid for detecting methylation state of target sequence of target gene, and

an antibody for detecting the concentration of a target protein,

wherein the target gene is one or two of MT1A gene and EPO gene,

the target protein is SNCG, namely gamma synuclein (gamma-synuclein).

2. The composition of item 1, wherein the target sequence of MT1A gene is shown in SEQ ID NO. 1.

3. The composition of item 1, wherein the target sequence of the EPO gene is shown in SEQ ID NO 3.

4. The composition according to any one of items 1 to 3, wherein the nucleic acid for detecting the methylation state of the target gene sequence comprises:

a fragment of at least 9 nucleotides of the target sequence of the target gene,

the fragment comprises at least one CpG dinucleotide sequence.

5. The composition according to any one of items 1 to 4, wherein the nucleic acid for detecting the methylation state of a target gene further comprises:

hybridizing 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 items 1 to 5, further comprising:

an agent that converts the 5-unmethylated cytosine base of the target sequence of the target gene to uracil.

7. The composition according to any one of items 1 to 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 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. The composition of any one of items 1 to 8, further comprising:

a reagent for detecting a concentration of a target protein, which is an enzyme-linked immunosorbent assay reagent, for example, the reagent comprises an antibody-coated reaction plate for detecting a concentration of a target protein, an SNCG protease conjugate, a substrate solution, a washing solution and a stop solution.

10. 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.

11. The oligonucleotide of item 10, 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.

12. The oligonucleotide of item 11, further comprising:

13. An oligonucleotide for detecting esophageal cancer in vitro, comprising:

the sequences of SEQ ID NO 5 and SEQ ID NO 6.

14. The oligonucleotide of item 13, further comprising:

the sequence of SEQ ID NO. 7.

15. The oligonucleotide of item 14, further comprising:

the sequence of SEQ ID NO 8.

16. An oligonucleotide for detecting esophageal cancer in vitro, comprising:

the sequences of SEQ ID NO 9 and SEQ ID NO 10.

17. The oligonucleotide of item 16, further comprising:

the sequence of SEQ ID NO. 11.

18. The oligonucleotide of item 17, further comprising:

12, SEQ ID NO.

Use of MT1A gene in the preparation of a kit for the in vitro detection of esophageal cancer.

Use of the EPO gene in the preparation of a kit for the in vitro detection of esophageal cancer.

21. A kit comprising the composition of any one of claims 1 to 9 or comprising the oligonucleotide of any one of claims 10 to 18.

22. The kit of item 21, further comprising at least one additional component selected from the group consisting of:

nucleoside triphosphates, DNA polymerase and buffers required for the function of said DNA polymerase, and

antibody-coated reaction plates for detecting the concentration of a target protein, SNCG protease conjugate, substrate solution, washing solution and stop solution.

23. The kit of item 21 or 22, further comprising: and (6) instructions.

24. Use of a composition according to any one of claims 1 to 9 or an oligonucleotide according to any one of claims 10 to 18 for the preparation of a kit for the in vitro detection of esophageal cancer.

25. The use according to any one of items 19, 20 and 24, wherein the kit for in vitro detection of esophageal cancer detects esophageal cancer by a method comprising the steps of:

1) determining the methylation state of the target sequence of the gene of interest in a biological sample from the subject;

2) determining the concentration of the protein of interest in a biological sample from the subject;

3) and judging whether the subject suffers from esophageal cancer or not by combining the methylation state of the target gene target sequence and the detection result of the target protein concentration, thereby realizing the in-vitro detection of the esophageal cancer.

26. The use according to item 25, wherein the method comprises the steps of:

drawing peripheral blood from the subject, separating plasma or serum;

extracting free DNA in blood plasma or blood serum;

treating the extracted free 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;

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; and

using SNCG antibodies, immune responses were used to determine the concentration of SNCG in plasma or serum.

27. The use of item 26, wherein the agent is a bisulfite agent.

28. A method of detecting esophageal cancer, comprising the steps of:

drawing a biological sample from a subject;

determining the methylation state of the gene target sequence of interest in a biological sample from the subject;

determining the concentration of the protein of interest in a biological sample of a subject; and

and judging whether the subject suffers from esophageal cancer or not by combining the methylation state of the target gene target sequence and the detection result of the target protein concentration, thereby realizing the in-vitro detection of the esophageal cancer.

29. A method of detecting esophageal cancer, comprising the steps of:

drawing peripheral blood from the subject, and separating plasma or serum;

extracting free DNA in blood plasma or blood serum;

converting the 5-unmethylated cytosine base to uracil or another base;

detecting the amplification product with a probe;

30. The method of item 28 or 29, wherein,

the target gene is one or both of MT1A gene and EPO gene, an

The target protein is SNCG.

31. The method of item 30, wherein the target sequence of the MT1A gene is set forth in SEQ ID NO. 1.

32. The method of item 30, wherein the target sequence of the EPO gene is set forth in SEQ ID NO 3.

33. The method of claim 29, wherein the reagent is a bisulfite reagent.

34. The method of item 29, wherein the primers are:

35. The method of item 29, wherein the blocking agent is one that preferentially binds to the target sequence in the unmethylated state.

36. The method of item 29, wherein the probe is:

37. The method of item 34, wherein the primer is the sequence of SEQ ID NO 5 and SEQ ID NO 6, or it is the sequence of SEQ ID NO 9 and SEQ ID NO 10.

38. The method of claim 35, wherein the blocking agent is the sequence of SEQ ID NO 8 or the sequence of SEQ ID NO 12

39. The method of claim 36, 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 subjects and normal control individuals shows that: the compositions and detection methods described herein are capable of sensitively and specifically detecting esophageal cancer; furthermore, the inventor of the invention finds that the detection of the esophageal cancer can be obviously improved by the combined detection of the SNCG protein and the methylation of the target gene in a plurality of tumor protein markers. 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 is a graph showing the results of screening for a gene of interest according to the present invention.

FIG. 2 is a diagram showing the detection of leukocyte genomic DNA (a negative reference of the methylation state of a target gene target sequence) and leukocyte genomic DNA (a positive reference of the methylation state of a target gene target sequence) treated with DNA methyltransferase by using the composition and the detection method 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 noninvasive detection of esophageal cancer by detecting the methylation state of a target gene target sequence and the concentration of a target protein 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, and an antibody for detecting the concentration of a target protein; wherein the target gene is one or two of MT1A gene and EPO gene, and the target protein is SNCG.

The invention provides a group of target gene target sequences emitting abnormal methylation in esophageal cancer, which comprise MT1A gene and EPO gene target sequences, wherein the MT1A gene target sequence is shown as SEQ ID NO:1-2, and the EPO gene target sequence is shown as SEQ ID NO: 3-4.

The target sequence of MT1A gene is shown in SEQ ID NO. 1.

SEQ ID NO:1

CACCCAGGGGAGCTCAGTGGACTGTGCGCCTTGCCTTTCTGCTGCGCAAAGCCCAGTCCAGGTCATCACCTCGGGCGGGGCGGACTCGGCTGGGCGGACTCAGCGGGGCGGGCGCAGGCGCAGGGCGGGTCCTTTGCGTCCGGCCCTCTTTCCCCTGACCATAAAAGCAGC

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

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: bisulfite converting the DNA of a test sample, and the nucleic acid for detecting 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 esophageal 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 moderate stringency or stringent conditions to at least 15 nucleotides of a sequence after bisulfite conversion of SEQ ID NO. 1 or its complement 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 composition for detecting esophageal cancer in vitro further comprises an antibody for detecting the concentration of a target protein, wherein the target protein is gamma Synuclein (SNCG).

The invention also relates to application of MT1A gene, EPO gene and SNCG protein in preparing a kit for detecting esophageal cancer in vitro.

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.

The SNCG protein is synuclein-gamma, and is a product of the SNCG gene. The SNCG gene, known by the english name synuclein gamma, is located in the q23.2 region of chromosome 10 in humans. The gene is a member of synuclein gene family, and the protein sequence is as follows: ).

The amino acid sequence of the protein coded by the SNCG gene is as follows:

MDVFKKGFSIAKEGVVGAVEKTKQGVTEAAEKTKEGVMYVGAKTKENVVQSVTSVAEKTKEQANAVSEAVVSSVNTVATKTVEEAENIAVTSGVVRKEDLRPSAPQQEGEASKEKEEVAEEAQSGGD

in yet another aspect, the present invention provides a method for detecting esophageal cancer in vitro, comprising the steps of:

Wherein the biological sample is plasma or serum separated from peripheral blood of the subject.

According to certain preferred embodiments, the method further comprises the steps of:

1) drawing peripheral blood from the subject, and separating plasma or serum;

2) extracting free DNA in blood plasma or blood serum;

3) treating the free DNA obtained in step 2) 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;

4) contacting the free DNA treated in step 3) with a DNA polymerase and primers 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;

5) detecting the amplification product with a probe;

6) 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.

7) Using SNCG antibodies, immune responses were used to determine the concentration of SNCG in plasma or serum.

Preferably, a typical primer comprises a fragment of the target gene sequence comprising a fragment of at least 9 nucleotides selected from SEQ ID NO 1-2 and SEQ ID NO 3-4, which is identical to, complementary to, or hybridizes under moderate stringency or stringency conditions, respectively.

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 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 peripheral blood whole blood, plasma, or serum.

The invention also provides a kit comprising the composition. Typically, the kit comprises a container for holding a biological sample of a subject. 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 in a non-invasive manner by detecting the methylation state of a target gene target sequence and the concentration of target protein. The inventor finds that the methylation states of the MT1A gene and the EPO gene target sequence in esophageal cancer tissues are significantly different from the methylation state of the gene target sequence in normal esophageal tissues: in esophageal cancer tissues, the MT1A gene and EPO gene target sequences are methylated, while in normal esophageal tissues, the MT1A gene and EPO gene target sequences are not methylated. The inventor further finds that the detection rate of esophageal cancer can be remarkably improved by jointly detecting the methylation state of the target gene target sequence and the concentration of target protein, and the detection specificity is not remarkably influenced. Therefore, the application provides a method for detecting esophageal cancer in vitro by detecting the methylation state of MT1A gene and EPO gene target sequences and the concentration of SNCG protein in a sample.

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 experimental or practical 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 gene target sequence and the concentration of the target protein; and a method and a kit for noninvasive 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 sequence of a target gene and the concentration of a target protein.

Preferably, the nucleic acid detection sequence comprises a fragment of at least 9 nucleotides in the target sequence of the target gene, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence; in certain preferred embodiments, such as: bisulfite converting the DNA of a sample to be detected, wherein the sequence detected by the nucleic acid comprises a fragment of at least 9 nucleotides in the sequence after bisulfite converting the target sequence of the target gene, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence;

more preferably, the nucleic acid detection sequences comprise a fragment of at least 15 nucleotides that hybridizes under moderate stringency or stringency conditions to the target sequence of the target gene, respectively, wherein the fragment of nucleotides comprises at least one CpG dinucleotide sequence; in certain preferred embodiments, such as: and (2) converting the DNA of the sample to be detected by using bisulfite, wherein the sequence detected by the nucleic acid comprises a fragment of at least 15 nucleotides in the sequence after the bisulfite conversion of the target gene target sequence under the medium or strict condition, wherein the fragment of the nucleotides comprises at least one CpG dinucleotide sequence.

In certain embodiments, the composition further comprises an agent that converts 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 thus converted in such a way that the 5-methylcytosine, which originally could not be distinguished from cytosine in its hybridization behavior, can now be detected as the only remaining cytosine 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 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 quantitative assays to determine the methylation level (e.g., percentage, fraction, ratio, proportion, or degree) of a particular CpG dinucleotide sequence, rather than the methylation status. Accordingly, the term methylation status or methylation state shall also be taken to mean a value reflecting the methylation state of a CpG dinucleotide sequence.

In certain embodiments, the methods of the present application specifically comprise: 1) drawing peripheral blood from the subject; 2) extracting free DNA in blood plasma or blood serum; 3) treating the free DNA obtained in step 2) 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; 4) contacting the DNA sample treated in step 3) 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; 5) detecting the amplification product with a probe; 6) 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; 7) the concentration of the target protein in plasma or serum is detected by an immune reaction (preferably, enzyme-linked immunosorbent assay, ELISA) using an SNCG antibody.

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 overlapping 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", etc. 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" analysis 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.

Wherein the SNCG antibody is used, and the concentration of the SNCG in blood plasma or blood serum is determined by immune reaction, and the expression level of the SNCG protein in the blood serum is detected by enzyme-linked immunosorbent assay (ELISA);

preferably, the ELISA is a "sandwich method", in particular the method comprises the steps of:

1) coating 96-well plates with SNCG monoclonal antibody (mouse);

2) adding clinical serum samples or plasma samples diluted by 10 times and human-derived SNCG protein solution diluted in series;

3) adding a Horse Radish Peroxidase (HRP) labeled mouse anti-human SNCG polyclonal antibody (IgG) to each well;

4) incubating the immunoreaction plate filled with the mixture under the conditions of room temperature and shaking, washing each reaction hole by using washing liquor, and adding a chromogenic substrate;

5) after the stop solution is added to stop the color reaction, detecting the spectrum of each reaction hole by using a plate reader;

6) and establishing a standard curve by using the detection values of the serially diluted human-derived SNCG protein solution, and quantifying a clinical serum sample based on the standard curve.

In a particular embodiment of the invention, the threshold values for esophageal cancer and normal relative to the serum or plasma protein level of the SNCG gene are determined based on the serum or plasma protein levels of the SNCG gene in a number of esophageal cancer samples and normal samples.

Examples

Example 1

By analyzing the data of 233 cases of esophageal cancer tissues and 171 cases of normal esophageal tissues (human methylation450k chip of Illumina), the present inventors found that the methylation levels of the MT1A gene and EPO gene in esophageal cancer tissues were significantly higher than that 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.

SEQ ID NO:1

The complementary sequence of the target sequence of the MT1A gene is shown as SEQ ID NO: 2, respectively.

SEQ ID NO:2

The target sequence of EPO gene is shown in SEQ ID NO. 3.

SEQ ID NO:3

The complementary sequence of the target sequence of the EPO gene is shown as SEQ ID NO: 4, respectively.

SEQ ID NO：4

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. In short, when the DNA is encapsulated in the 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 can 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 target 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 target 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 the 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 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 primer and blocker, 150-300nM probe, 1UTaq polymerase, 50-400. mu.M of each dNTP, 1-10 mM 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 detection of the leukocyte genome DNA (a positive reference product of the methylation state of a target gene target sequence) treated by the DNA methyltransferase, and the detection result is positive, namely: the target gene sequence of the tested DNA sample is methylated. Thus, 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 analysis result based on the target gene is negative (normal) or positive (esophageal cancer) is determined by comparing the Ct value of the tested 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 from the test sample is then extracted and the DNA sample is pretreated so that the unmethylated cytosine base at 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 can be performed by any standard means known in the art, and in particular, in this example, all of the sample DNA extraction and bisulfite DNA modification is performed by using the plasma processing kit from Boercheng.

Then, the above-mentioned 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, and a specific critical value is selected, preferably, the critical value Ct is 37.

Then, plasma samples were obtained for an additional 63 esophageal cancer subjects. Free DNA in plasma is then extracted and the genomic DNA sample is pretreated to convert the cytosine base that is unmethylated at the 5' position 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 can be performed by any standard means known in the art, and in particular, in this example, all of the sample DNA extraction and bisulfite DNA modification is performed by using the plasma processing kit from Boercheng. Then, the above-mentioned MT1A and EPO gene primer, probe combination and reference gene ACTB gene primer, probe combination were added to the DNA samples of 63 patients with esophageal cancer, and the methylation states of MT1A and EPO gene were detected by PCR. The PCR amplification conditions adopted 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 35ng and 450nM primers, 225nM probe, 1UTaq polymerase, 200u μm of each dNTP, 4.5mM MgCl2 and 2XPCR buffer to a final volume of 30u μ l. The samples were amplified with pre-cycling at 94 ℃ for 20 minutes, followed by 5 seconds of 45 cycles of annealing at 62 ℃, 35 seconds at 55.5 ℃ and 30 seconds of denaturation at 93 ℃.

Meanwhile, plasma samples of 63 esophageal cancer subjects were tested for serum protein level of SNCG gene. The serum protein level of the SNCG gene was detected by enzyme-linked immunosorbent assay (ELISA). The ELISA detection technology realizes the detection of the serum protein level of the SNCG gene by a 'three-Ming-Zhi-method': the 96-well plates were first coated (1 μ g/well) with SNCG monoclonal antibody (murine) (sourced borchenne corporation); then adding clinical serum samples with 10-fold dilution and human source SNCG protein solution (50 mu.l/hole) with serial dilution (2.5 ng/mL-0.04 ng/mL); then 50. mu.l of 0.47. mu.g/mL horseradish peroxidase (HRP) -labeled mouse anti-human SNCG polyclonal antibody (IgG) from Boehringer Corp.) was added to each tube. Incubating the immunoreaction plate with the mixture for 3 hours at room temperature under shaking conditions; after washing each reaction well with a washing solution, a chromogenic substrate is added. After the color reaction is terminated by adding 2M sulfuric acid, each reaction well is examined at 450nm spectral band using a plate reader (e.g., Bio-RAD 550). And finally, establishing a standard curve by using the detection values of the serially diluted human SNCG protein solution, and quantifying a clinical serum sample based on the standard curve. The threshold for SNCG gene serum levels in esophageal cancer and normal individuals was 2.0 ng/mL.

Finally, the levels of commonly used cancer markers were measured in samples of 63 esophageal cancer subjects using the electrochemiluminescence immunoassay system of Roche (Roche), respectively, and included: CEA, CA199, CA125, and AFP.

The assay results show (fig. 3): the positive rate of MT1A and EPO gene methylation detection and SNCG protein detection of esophageal cancer in esophageal cancer subjects is significantly higher than that of common cancer markers (such as CEA, CA199, CA125, and AFP). Meanwhile, the MT1A and EPO gene DNA methylation and SNCG protein combined detection has outstanding complementarity for the detection of the esophageal cancer, and the detection rate of the esophageal cancer is greatly improved by the combined detection of the MT1A and the EPO gene DNA methylation and the SNCG protein. In addition, the complementarity of MT1A and EPO gene DNA methylation detection and SNCG protein detection is unique, and common cancer markers (such as CEA, CA199, CA125, and AFP) do not have this property.

The experimental results show that the combined detection of the methylation state of the target gene target sequence and the concentration of the target protein can sensitively and specifically detect the 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 methylation state of the target gene target sequence and the concentration of the target protein, so that the in-vitro detection of the esophageal cancer by using the methylation state of the target gene target sequence and the concentration of the target protein is realized, and the sensitivity and the specificity of the in-vitro detection of the esophageal cancer are effectively improved. The method for analyzing the free DNA of the plasma sample by using the real-time PCR and the ELISA method are used for analyzing the concentration of the target protein in the plasma, so that the combined detection of the methylation state of the target sequence of the target gene and the concentration of the target protein 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 and the detection concentration of the ELISA, 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 for purposes of illustration only and are not intended to be limiting. 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

<130> PB00212D1

<160> 13

<170> PatentIn version 3.5

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<211> 171

<212> DNA

<213> Artificial sequence

<220>

<223> target sequence of MT1A gene

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cacccagggg agctcagtgg actgtgcgcc ttgcctttct gctgcgcaaa gcccagtcca 60

ggtcatcacc tcgggcgggg cggactcggc tgggcggact cagcggggcg ggcgcaggcg 120

cagggcgggt cctttgcgtc cggccctctt tcccctgacc ataaaagcag c 171

<210> 2

<211> 171

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO:1, the complement of

<400> 2

gctgctttta tggtcagggg aaagagggcc ggacgcaaag gacccgccct gcgcctgcgc 60

ccgccccgct gagtccgccc agccgagtcc gccccgcccg aggtgatgac ctggactggg 120

ctttgcgcag cagaaaggca aggcgcacag tccactgagc tcccctgggt g 171

<210> 3

<211> 259

<212> DNA

<213> Artificial sequence

<220>

<223> sequences of target sequences of EPO genes

<400> 3

cgcgcacgca cacatgcaga taacagcccc gacccccggc cagagccgca gagtccctgg 60

gccaccccgg ccgctcgctg cgctgcgccg caccgcgctg tcctcccgga gccggaccgg 120

ggccaccgcg cccgctctgc tccgacaccg cgccccctgg acagccgccc tctcctccag 180

gcccgtgggg ctggccctgc accgccgagc ttcccgggat gagggccccc ggtgtggtca 240

cccggcgcgc cccaggtcg 259

<210> 4

<211> 259

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO:3, and the complement of

<400> 4

cgacctgggg cgcgccgggt gaccacaccg ggggccctca tcccgggaag ctcggcggtg 60

cagggccagc cccacgggcc tggaggagag ggcggctgtc cagggggcgc ggtgtcggag 120

cagagcgggc gcggtggccc cggtccggct ccgggaggac agcgcggtgc ggcgcagcgc 180

agcgagcggc cggggtggcc cagggactct gcggctctgg ccgggggtcg gggctgttat 240

ctgcatgtgt gcgtgcgcg 259

<210> 5

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<213> Artificial sequence

<220>

<223> MT1A primer F

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cggacgtaaa ggattc 16

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<213> Artificial sequence

<220>

<223> MT1A primer R

<400> 6

gaaacgaact cgactaaacg 20

<210> 7

<211> 19

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<213> Artificial sequence

<220>

<223> MT1A Probe

<400> 7

tgcgtttgcg ttcgtttcg 19

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<213> Artificial sequence

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caaactcaac taaacaaact caacaaaaca aac 33

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agtcgtagag tttttgggtt 20

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<213> Artificial sequence

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caacgcgata cgacg 15

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cgcaacgaac gaccga 16

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gagtttttgg gttattttgg ttgtttgttg 30

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<213> Artificial sequence

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Met Asp Val Phe Lys Lys Gly Phe Ser Ile Ala Lys Glu Gly Val Val

1 5 10 15

Gly Ala Val Glu Lys Thr Lys Gln Gly Val Thr Glu Ala Ala Glu Lys

20 25 30

Thr Lys Glu Gly Val Met Tyr Val Gly Ala Lys Thr Lys Glu Asn Val

35 40 45

Val Gln Ser Val Thr Ser Val Ala Glu Lys Thr Lys Glu Gln Ala Asn

50 55 60

Ala Val Ser Glu Ala Val Val Ser Ser Val Asn Thr Val Ala Thr Lys

65 70 75 80

Thr Val Glu Glu Ala Glu Asn Ile Ala Val Thr Ser Gly Val Val Arg

85 90 95

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100 105 110

Lys Glu Lys Glu Glu Val Ala Glu Glu Ala Gln Ser Gly Gly Asp

115 120 125

Claims

1. a composition for in vitro detection of esophageal cancer, the composition comprising:

Nucleic acids used to detect the methylation status of target sequences of target genes, and

Antibodies to detect target protein concentration,

Wherein, the target gene is one or both of MT1A gene and EPO gene,

The target protein is gamma synuclein (SNCG).

2. The composition according to claim 1, wherein the target sequence of the MT1A gene is shown in SEQ ID NO:1.

3. The composition according to claim 1, wherein the target sequence of the EPO gene is shown in SEQ ID NO:3.

4. The composition according to any one of claims 1 to 3, wherein the nucleic acid for detecting the methylation state of a target sequence of a target gene comprises:

A fragment of at least 9 nucleotides in the target gene target sequence,

The fragment comprises at least one CpG dinucleotide sequence.

5. The composition according to any one of claims 1 to 4, wherein the nucleic acid for detecting the methylation state of a target sequence of a target gene comprises:

hybridizes to a fragment of at least 15 nucleotides in the target sequence of the gene of interest under moderate or stringent conditions,

The fragment comprises at least one CpG dinucleotide sequence.

6. The composition of any one of claims 1 to 5, further comprising:

A reagent that converts the unmethylated cytosine base at position 5 of the target sequence of the target gene to uracil.

7. The composition according to any one of claims 1 to 6, wherein the nucleic acid for detecting the methylation state of the target gene further comprises:

Blockers that bind preferentially to target sequences in their unmethylated state.

8. The composition of claim 7, wherein,

the fragment of at least 9 nucleotides, which is the sequence of SEQ ID NO:5 and SEQ ID NO:6, or it is the sequence of SEQ ID NO:9 and SEQ ID NO:10,

the fragment of at least 15 nucleotides, which is the sequence of SEQ ID NO:7 or the sequence of SEQ ID NO:11,

A blocking agent, which is the sequence of SEQ ID NO:8 or the sequence of SEQ ID NO:12.

9. The composition of any one of claims 1 to 8, further comprising:

A reagent for detecting the concentration of the target protein, the reagent is an enzyme-linked immunosorbent assay reagent, for example, the reagent includes an antibody-coated reaction plate for detecting the concentration of the target protein, SNCG protease conjugate, substrate solution, washing solution and stop solution.

10. Use of the composition according to any one of claims 1 to 9 in the preparation of a kit for in vitro detection of esophageal cancer.

11. purposes according to claim 10, wherein, described test kit for detecting esophageal cancer in vitro detects esophageal cancer by the method comprising the steps:

extracting biological samples from subjects,

Extracting DNA samples including target gene target sequences or fragments thereof in biological samples;

determining the methylation status of the target gene target sequence;

determining the target protein concentration in the biological sample; and

Whether the subject suffers from esophageal cancer is determined by combining the detection results of the target gene target sequence methylation state and the target protein concentration, thereby realizing the in vitro detection of esophageal cancer.

12. The use of claim 10, wherein the method comprises the steps of:

Draw the peripheral blood of the subject;

isolation of plasma or serum from peripheral blood samples;

Extraction of cell-free DNA from plasma or serum;

Treating the extracted free DNA with a reagent to convert the 5-position unmethylated cytosine base into uracil or other bases;

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 an unmethylated state;

Detection of amplified products with probes;

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 concentration of SNCG protein in plasma or serum is determined by immunoreaction using SNCG antibodies.

13. The use of claim 12, wherein the reagent is a bisulfite reagent.