WO2023083348A1 - 用于食管癌诊断的甲基化检测试剂及试剂盒 - Google Patents

用于食管癌诊断的甲基化检测试剂及试剂盒 Download PDF

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WO2023083348A1
WO2023083348A1 PCT/CN2022/131724 CN2022131724W WO2023083348A1 WO 2023083348 A1 WO2023083348 A1 WO 2023083348A1 CN 2022131724 W CN2022131724 W CN 2022131724W WO 2023083348 A1 WO2023083348 A1 WO 2023083348A1
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seq
region
nucleic acid
target region
methylation
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PCT/CN2022/131724
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English (en)
French (fr)
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周俊
张良禄
董兰兰
熊杨辉
吴志诚
郑义慧
万康康
李婷婷
赵巴丽
黄越
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武汉艾米森生命科技有限公司
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Priority claimed from CN202111344786.5A external-priority patent/CN113789388B/zh
Priority claimed from CN202211009579.9A external-priority patent/CN117604095A/zh
Application filed by 武汉艾米森生命科技有限公司 filed Critical 武汉艾米森生命科技有限公司
Publication of WO2023083348A1 publication Critical patent/WO2023083348A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the disclosure belongs to the technical field of medical biological detection, and relates to a diagnostic marker for esophageal cancer, in particular to a methylation detection reagent and a kit for diagnosing esophageal cancer.
  • Esophageal cancer is one of the most common malignant tumors in the world, and it is also the most common malignant tumor of the digestive tract. Esophageal cancer is highly aggressive and often leads to a poor prognosis. According to statistics, in 2018, there were about 570,000 new cases of esophageal cancer worldwide, and about 500,000 deaths. Its morbidity and mortality rates ranked seventh and sixth among all malignant tumors, respectively. According to the pathological classification of tumors, esophageal cancer mainly includes two histological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC).
  • ESCC esophageal squamous cell carcinoma
  • EAC esophageal adenocarcinoma
  • DNA methylation is a common form of epigenetic modification, which plays an important role in the occurrence of human malignant tumors.
  • Abnormal methylation in the promoter region of tumor suppressor genes may lead to down-regulation or silencing of gene transcription, leading to the occurrence of cancer. Therefore, changes in DNA methylation patterns have become reliable potential biomarkers for early detection and diagnosis of cancer.
  • liquid biopsy technology based on circulating cell-free DNA (cfDNA) has shown great potential in the early diagnosis of cancer because it can detect early solid tumors with minimal invasiveness.
  • Tumor cells undergo genetic changes (such as mutations, copy number variations, or epigenetic changes, etc.), and when tumor cells undergo apoptosis or necrosis, their nucleic acids are released into the blood, allowing tumor DNA to be detected by liquid biopsy techniques.
  • the present disclosure provides the application of a reagent for detecting the methylation level of a target region in a gene in the preparation of a diagnostic reagent or kit for esophageal cancer, where the target region is the OTOP2 gene.
  • the target region is a full-length region or a partial region in the Chr17:74922901-74924924 CpG island region in the OTOP2 gene.
  • the methylation level of the target region is the methylation level of the full-length region or a partial region in at least one of the following CpG island regions in the OTOP2 gene: region 1, region 2, region 3, region 4, Region 5 and region 6; wherein, said region 1 is selected from Chr17:74923881-7492401 5 positive strands, said region 2 is selected from Chr17:74924159-74924335 positive strands, and said region 3 is selected from Chr17:74924422-74924581 positive strands , the region 4 is selected from Chrl7: 74924603-74924449 negative strand, the region 5 is selected from Chrl7: 74924425-74924297 negative strand, and the region 6 is selected from Chrl7: 74924080-74923981 negative strand.
  • the reagent or kit includes a nucleic acid combination for detecting the methylation level of the CpG island of the OTOP2 gene.
  • the nucleic acid combination is selected from at least one of the following nucleic acid combinations: nucleic acid combination 1 for detecting the region 1, nucleic acid combination 2 for detecting the region 2, and nucleic acid combination 2 for detecting the region 3
  • the nucleic acid combination 3 for detecting the region 4 the nucleic acid combination 4 for detecting the region 5, the nucleic acid combination 5 for detecting the region 5, and the nucleic acid combination 6 for detecting the region 6;
  • the base sequence of the nucleic acid combination 1 has at least 90% identity with the base sequence shown in SEQ ID NO.67-69, and the base sequence of the nucleic acid combination 2 is shown in SEQ ID NO.70-72
  • the base sequence has at least 90% identity
  • the base sequence of the nucleic acid combination 3 has at least 90% identity with the base sequence shown in SEQ ID NO.73-75
  • the base sequence of the nucleic acid combination 4 The base sequence has at least 90% identity with the base sequence shown in SEQ ID NO.76-78
  • the base sequence of the nucleic acid combination 5 has at least 90% identity with the base sequence shown in SEQ ID NO.79-81 % identity
  • the base sequence of the nucleic acid combination 6 has at least 90% identity with the base sequence shown in SEQ ID NO.82-84.
  • the present disclosure also provides the application of a reagent for detecting the methylation level of a target region in a gene in the preparation of a diagnostic reagent or kit for esophageal cancer, the target region includes a first target region belonging to the KCNA3 gene and a second target region belonging to the OTOP2 gene target area.
  • the first target region includes a full-length selected from at least one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 or partial sequence; and/or
  • the second target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.12.
  • the first target region includes a full-length selected from at least one of the nucleotide sequences shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 or partial sequence; and/or
  • the second target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.14.
  • the first target region includes a full-length or partial sequence whose nucleotide sequence is shown in SEQ ID NO.3; the second target region includes a nucleotide sequence whose nucleotide sequence is shown in SEQ ID NO.12. full-length or partial sequence.
  • the present disclosure also provides a reagent, the reagent comprising at least one of the following nucleic acid combinations: nucleic acid combination 1, nucleic acid combination 2, nucleic acid combination 3, nucleic acid combination 4, nucleic acid combination 5 and nucleic acid combination 6; the nucleic acid combination 1
  • the base sequence of the nucleic acid combination 2 has at least 90% identity with the base sequence shown in SEQ ID NO.67-69, and the base sequence of the nucleic acid combination 2 has the same At least 90% identity, the base sequence of the nucleic acid combination 3 has at least 90% identity with the base sequence shown in SEQ ID NO.73-75, the base sequence of the nucleic acid combination 4 is consistent with SEQ ID
  • the base sequence shown in NO.76-78 has at least 90% identity, and the base sequence of the nucleic acid combination 5 has at least 90% identity with the base sequence shown in SEQ ID NO.79-81, The base sequence of the nucleic acid combination 6 has at least 90% identity with the base sequence shown in SEQ ID NO.82-
  • the present disclosure also provides a detection reagent for esophageal cancer diagnosis, the detection reagent includes a reagent for detecting methylation levels of a first target region belonging to the KCNA3 gene and a second target region belonging to the OTOP2 gene.
  • the first target region includes a full-length selected from at least one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 or partial sequence; and/or
  • the second target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.12.
  • the first target region includes a full-length selected from at least one of the nucleotide sequences shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 or a partial sequence; and/or the second target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.14 .
  • the detection reagent includes detecting the nucleotide sequence selected from the first target region as shown in SEQ ID NO.3 and the nucleotide sequence selected from the second target region as shown in SEQ ID NO.12 Reagents for methylation levels of full-length or partial sequences.
  • the detection reagent comprises a nucleic acid molecule comprising a primer pair for detecting the methylation levels of the first target region and the second target region.
  • the detection reagent further includes a detection probe corresponding to the primer pair, and a fluorescent group and a fluorescence quenching group are connected to the detection probe.
  • the primer pair for detecting the methylation levels of the first target region and the second target region is shown in Table 1 below:
  • the combination of the primer pair for detecting the methylation level of the first target region and the second target region and the detection probe is shown in Table 2 below:
  • the present disclosure also provides a kit for diagnosing esophageal cancer, comprising the above-mentioned reagent or any one of the above-mentioned detection reagents.
  • the kit also includes at least one of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents.
  • the technical solution of "A, B, C, and/or, D" includes any one of A, B, C, and D (that is, the technical solutions connected by "logic or"), and also includes A , B, C, and any and all combinations of D, that is, including any two or any three of A, B, C, and D, and also including four combinations of A, B, C, and D (that is, All use the technical scheme of "logic and" connection).
  • the present disclosure refers to "plurality”, “multiple”, “multiple times”, “plurality”, etc., unless otherwise specified, means that the number is greater than 2 or equal to 2. For example, “one or more” means one or more than two. “Above” includes the original number, such as “more than two” includes two, three or more.
  • first”, “second”, “third”, “fourth”, etc. are used for descriptive purposes only, and shall not be construed as indicating or implying relative importance or quantity, nor shall they be construed as implying The importance or quantity of the indicated technical characteristics.
  • first, “second”, “third”, “fourth” and so on are only for the purpose of non-exhaustive enumeration and description or for distinguishing descriptions, and it should be understood that they do not constitute a closed limitation on quantity. Neither should be read as indicating or implying relative importance.
  • the technical features described in open form include closed technical solutions composed of the listed features, and open technical solutions including the listed features.
  • Esophageal cancer is a malignant tumor of the digestive tract derived from the esophageal epithelium between the hypopharynx and the esophagogastric junction.
  • Esophageal cancer mainly includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.
  • diagnosis refers to a single factor used to determine, verify or confirm the clinical status of a patient, including auxiliary diagnosis, assessment of recurrence risk, assessment of cancer risk and degree of cancer, and prognosis.
  • aided diagnosis is used to provide various information to assist in judgment during the determination or verification of a patient's clinical status, and is not intended to be the only indicator for determination.
  • the term "gene” refers to a DNA segment that codes to produce an amino acid polypeptide chain, which includes sequences located in the coding region and non-coding region, as well as exon and intron sequences involved in gene transcription/translation and transcription/translation regulation. It can be understood that detecting the methylation level of a gene includes detecting the methylation level of CpG sites in the coding region and non-coding region of the gene, such as the methylation level of the CpG sites in the promoter region of the gene . Therefore, the chromosomal coordinate position of the detection site may be located upstream or downstream of the coding region of the gene. In this disclosure, the chromosomal coordinate position of the gene takes GRCh38.p14 as the reference genome.
  • oligonucleotide or “polynucleotide” or “nucleotide” or “nucleic acid” refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, optionally multiple Molecules of more than three deoxyribonucleotides or ribonucleotides, and usually more than ten molecules of deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors, which in turn will depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any number of ways, including chemical synthesis, DNA replication, reverse transcription, or combinations thereof.
  • Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine.
  • Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.
  • DNA methylation is a form of chemical modification of DNA that alters genetic expression without altering the DNA sequence.
  • DNA methylation refers to the covalent binding of a methyl group at the 5th carbon position of cytosine of a genomic CpG dinucleotide under the action of DNA methyltransferase.
  • DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.
  • methylation level refers to whether cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/ratio/percentage of methylation occurs, which represents a qualitative concept It also represents the concept of quantification. In practical applications, different detection indicators can be used to compare the DNA methylation level according to the actual situation.
  • the comparison can be made based on the Ct value detected by the sample; in some cases, the ratio of gene methylation in the sample can be calculated, that is, the number of methylated molecules/(number of methylated molecules+non-methylated number of molecules) ⁇ 100%, and then compare; in some cases, it is necessary to analyze and integrate each index statistically to obtain the final judgment index.
  • the target region of the gene to be detected herein is a DNA sequence including at least one CpG dinucleotide (CG).
  • CpG island refers to a region of DNA rich in abundant cytosines and guanines linked by phosphate bonds. CpG dinucleotides are usually concentrated in the promoter regions and exons of human genes. In the normal human genome, the CpG sites outside the CpG islands are usually methylated, while the CpG sites in the CpG islands are usually in an unmethylated state. . When tumors occur, the degree of unmethylation of CpG sites outside the CpG islands of tumor suppressor genes increases, while the CpG sites in CpG islands are hypermethylated, resulting in increased chromosome helical degrees, transcriptional inhibition, and loss of gene expression.
  • CpG island region methylation level refers to the methylation level of cytosine in one or more CpG dinucleotides within a CpG island.
  • Methodylation site or “CpG site” refers to at least one CpG dinucleotide site in a region, especially cytosine in at least one CpG dinucleotide site in a region.
  • primer refers to an oligonucleotide that can be used in an amplification method such as polymerase chain reaction PCR to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a partial region thereof.
  • amplification method such as polymerase chain reaction PCR to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a partial region thereof.
  • at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence.
  • the exact length of a primer depends on many factors, including temperature, source of primer, and method used.
  • oligonucleotide primers typically contain at least 10, 15, 20, 25 or more nucleotides, but may contain fewer nucleotides, depending on the complexity of the target sequence.
  • primers refers to a pair of primers capable of hybridizing to the double strand of a target DNA molecule or to regions of the target DNA molecule flanking the nucleotide sequence to be amplified.
  • Taqman probe refers to an oligonucleotide sequence comprising a 5' fluorescent group and a 3' quencher group.
  • the probe When the probe binds to the corresponding site on the DNA, the probe does not fluoresce due to the presence of a quencher group near the fluorophore.
  • DNA polymerase such as Taq enzyme
  • the 5'-3' exonuclease activity of DNA polymerase such as Taq enzyme
  • the fluorescent signal After each cycle of PCR, the fluorescent signal also has a synchronous exponential growth process like the target fragment.
  • ddNTPs deoxyribonucleotides
  • ddNTPs dideoxyribonucleotides
  • the extended oligonucleotides are selectively terminated at G, A, T or C, and the four light wavelength signals are converted into computer-recognizable signals by photoexcitation
  • the electrical signal of the target DNA sequence was judged according to the fluorescence signal of the last ddNTP incorporated in the reaction tube.
  • reagent and “detection reagent” have the same meaning and can be used interchangeably, and refer to any reagent required for the detection of DNA methylation level in a sample, especially including primers capable of amplifying target regions Right, a probe capable of specifically recognizing a target region, or a combination thereof.
  • DNA methylation is an important chemical modification on the gene, which affects the regulation process of gene transcription and the structure of the nucleus.
  • the change of DNA methylation is an early event and accompanying event of cancer development, mainly reflected in the hypermethylation of tumor suppressor genes and the hypomethylation of proto-oncogenes in tumor tissues.
  • DNA methylation is relatively stable. If tumor-specific blood DNA methylation molecular markers can be found, it will have great clinical application value.
  • the present disclosure provides an application of a substance used to detect the methylation level of OTOP2 gene CpG island (English full name: otopetrin 2) in the preparation of products, and the use of the product is at least one of the following:
  • the product is selected from at least one of the following products: reagents, kits, chips and sequencing libraries.
  • the present disclosure provides the application of a substance for detecting the methylation level of a target region in a gene in the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, and/or in distinguishing esophageal cancer samples from non-cancer samples,
  • the target region is the OTOP2 gene.
  • the present disclosure provides the application of a reagent for detecting the methylation level of a target region in a gene in the preparation of a diagnostic reagent or kit for esophageal cancer, where the target region is the OTOP2 gene.
  • the present disclosure provides the application of the reagent for detecting the methylation level of the CpG island of OTOP2 gene in the preparation of diagnostic reagents or kits for esophageal cancer.
  • the present disclosure finds that the methylation level of the CpG island region of the OTOP2 gene is significantly higher in esophageal cancer samples/precancerous lesions than in normal samples, and the methylation of the CpG island region of the OTOP2 gene is used as a detection marker. It can effectively distinguish between cancer/precancerous lesion samples and normal samples in the samples to be tested, with a detection sensitivity of over 50% for cancer, a sensitivity of over 30% for precancerous lesions, and a specificity of over 95%. Moreover, using the methylation of the CpG island region of the OTOP2 gene as a detection marker can effectively distinguish esophageal cancer samples from paracancerous samples (non-cancer samples). The present disclosure provides a new idea for the non-invasive detection of esophageal cancer.
  • esophageal cancer includes but not limited to esophageal squamous cell carcinoma and esophageal adenocarcinoma
  • precancerous lesions include but not limited to esophageal squamous intraepithelial neoplasia, Barrett's esophagus-related heterogeneous hyperplasia, chronic esophagitis, esophageal epithelial hyperplasia, esophageal polyps , Esophageal ulcer, esophageal leukoplakia.
  • the methylation level of the OTOP2 gene is the methylation level of the full-length region or a partial region in the Chr17:74922901-74924924 CpG island region of the OTOP2 gene.
  • the methylation level of the OTOP2 gene is the methylation level of the full-length region or a partial region in at least one of the following CpG island regions in the OTOP2 gene: region 1, region 2, region 3, region 4, Area 5 and Area 6.
  • the partial region is the methylation level of cytosine in at least one CpG dinucleotide site in regions 1-6.
  • region 1 is selected from Chrl7:74923881-74924015 plus strand
  • region 2 is selected from Chrl7:74924159-74924335 plus strand
  • region 3 is selected from Chrl7:74924422-74924581 plus strand
  • region 4 is selected from Chrl7:74924603- 74924449 negative strand
  • region 5 is selected from Chr17:74924425-74924297 negative strand
  • region 6 is selected from Chr17:74924080-74923981 negative strand.
  • the methylation level of the OTOP2 gene is the methylation level of the full-length region or a partial region of at least one of the following CpG island regions in the OTOP2 gene: region 2 and region 4.
  • region 2 and region 4 The inventors found that compared with the detection sensitivity and specificity of other regions for samples, the detection sensitivity and specificity of regions 2 and 4 are further improved.
  • the above-mentioned substances include a nucleic acid combination for detecting the methylation level of the CpG island of the OTOP2 gene.
  • the reagent or kit comprises a nucleic acid combination for detecting the methylation level of a CpG island of the OTOP2 gene.
  • the CpG island of the OTOP2 gene includes the Chr17:74922901-74924924 region, the base sequence is as shown in SEQ ID NO.88, and the sequence (5'-3') is as follows:
  • the above CpG island region also includes a reverse complementary sequence to the above sequence.
  • the nucleic acid combination is selected from at least one of the following nucleic acid combinations: nucleic acid combination 1 for detection area 1, nucleic acid combination 2 for detection area 2, nucleic acid combination 3 for detection area 3, Nucleic acid combination 4 for detection area 4, nucleic acid combination 5 for detection area 5 and nucleic acid combination 6 for detection area 6;
  • the base sequence of nucleic acid combination 1 has at least 90% identity with the base sequence shown in SEQ ID NO.67-69, and the base sequence of nucleic acid combination 2 is consistent with the base sequence shown in SEQ ID NO.70-72 Has at least 90% identity, the base sequence of nucleic acid combination 3 has at least 90% identity with the base sequence shown in SEQ ID NO.73-75, and the base sequence of nucleic acid combination 4 is consistent with SEQ ID NO.76
  • the base sequence shown in -78 has at least 90% identity
  • the base sequence of nucleic acid combination 5 has at least 90% identity with the base sequence shown in SEQ ID NO.79-81
  • the base of nucleic acid combination 6 The base sequence has at least 90% identity with the base sequence shown in SEQ ID NO.82-84.
  • the base sequence of nucleic acid combination 1 has 90%, 92%, 95%, 98%, 99% or 100% identity with the base sequence shown in SEQ ID NO.67-69, and the base sequence of nucleic acid combination 2
  • the sequence has 90%, 92%, 95%, 98%, 99% or 100% identity with the base sequence shown in SEQ ID NO.70-72, and the base sequence of nucleic acid combination 3 is identical to that of SEQ ID NO.73
  • the base sequence shown in -75 has 90%, 92%, 95%, 98%, 99% or 100% identity
  • the base sequence of nucleic acid combination 4 is the base sequence shown in SEQ ID NO.76-78
  • the sequence has 90%, 92%, 95%, 98%, 99% or 100% identity
  • the base sequence of nucleic acid combination 5 has 90%, 92% with the base sequence shown in SEQ ID NO.79-81 , 95%, 98%, 99% or 100% identity
  • the above-mentioned products are at least one selected from the following products: reagents, kits, chips and sequencing libraries.
  • the above-mentioned products can be any form of in vitro diagnostic products, and are not limited to the above-mentioned product types of reagents, kits, chips and sequencing libraries, as long as they can meet the requirements of diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions. All requirements are within the protection scope of the present disclosure.
  • the above-mentioned OTOP2 gene methylation level is detected by at least one of the following methods: methylation-specific PCR method, sequencing method, methylation-specific high performance liquid chromatography, digital PCR method, Methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction endonuclease method and flap endonuclease (flap endonuclease) method (see patent documents US8715937, US8361720).
  • the aforementioned sequencing method is selected from methylation-specific PCR method, bisulfite sequencing method, genome-wide methylation sequencing method or pyrosequencing method.
  • the present disclosure also provides a reagent, which includes at least one of the following nucleic acid combinations: nucleic acid combination 1, nucleic acid combination 2, nucleic acid combination 3, nucleic acid combination 4, nucleic acid combination 5, and nucleic acid combination 6;
  • the base sequence of nucleic acid combination 1 has at least 90% identity with the base sequence shown in SEQ ID NO.67-69, and the base sequence of nucleic acid combination 2 is consistent with the base sequence shown in SEQ ID NO.70-72 Has at least 90% identity, the base sequence of nucleic acid combination 3 has at least 90% identity with the base sequence shown in SEQ ID NO.73-75, and the base sequence of nucleic acid combination 4 is consistent with SEQ ID NO.76
  • the base sequence shown in -78 has at least 90% identity
  • the base sequence of nucleic acid combination 5 has at least 90% identity with the base sequence shown in SEQ ID NO.79-81
  • the base of nucleic acid combination 6 The base sequence has at least 90% identity with the base sequence shown in SEQ ID NO.82-84.
  • the reagent comprises at least one nucleic acid combination of nucleic acid combination 2 and nucleic acid combination 4.
  • the above-mentioned reagents also include reagents capable of differentially modifying methylated DNA and unmethylated DNA (such as bisulfite and other substances), methylation-specific PCR technology or other commonly used digital PCR Conventional reagents (including but not limited to PCR buffer, dNTPs (deoxyribonucleotide triphosphates), Taq enzyme, water) and other conventional reagents used for time-of-flight mass spectrometry.
  • the above-mentioned reagents can be powders, granules, water-dispersible granules, liquids, emulsions or suspensions.
  • the bacterial powder prepared by vacuum freeze-drying or the powder prepared by spray-drying.
  • the nucleic acid assembly is dissolved in ultrapure water or buffer.
  • the present disclosure also provides a kit comprising the above-mentioned reagents.
  • the kit further includes at least one of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents.
  • the detection samples of the above kit include but are not limited to tissue samples, blood samples, saliva samples or cell samples derived from esophagus;
  • the blood sample is selected from a plasma sample, a serum sample, a whole blood sample or a blood cell sample.
  • An embodiment of the present disclosure also provides the application of a reagent for detecting the methylation level of a target region in a gene in the preparation of a diagnostic reagent or kit for esophageal cancer.
  • the detected gene includes the KCNA3 gene (English full name: Potassium voltage-gated channel subfamily A member 3) and OTOP2 gene.
  • KCNA3 gene and OTOP2 gene can be obtained from biological information databases known in the art. If GRCh38.p14 is used as the reference genome, the KCNA3 gene is located at Chr1:110653560-110674940; the OTOP2 gene is located at Chr17:74924273-74933912.
  • the nucleotide sequence information of the gene can be obtained by referring to the above information, but the present disclosure is not limited to relying on the detection of KCNA3 gene and OTOP2 gene that are completely identical to the nucleotide sequence in the reference genome.
  • KCNA3 and OTOP2 genes are used as target genes to detect the methylation level of the target region in the two genes. According to the methylation level of the target region in the two genes, it can be diagnosed or assisted in judging whether the subject has esophageal cancer or the occurrence of esophageal cancer. Precancerous lesions of the esophagus. Furthermore, the reagent for detecting the methylation level of the target region in the above two genes can be applied to the preparation of diagnostic reagents or kits for esophageal cancer.
  • the target regions of KCNA3 and OTOP2 genes are regions including at least one methylation site.
  • the target region can be a CpG island of a gene, or any other region including a methylation site.
  • the target region can be located in a promoter region or a coding region.
  • the methylation levels of the target regions of the two genes in the present disclosure can be detected by any detection method known in the art capable of detecting DNA methylation levels, including but not limited to one or more of the following methods to achieve Detection of the methylation level of the target region: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, genome-wide methylation sequencing, pyrosequencing, methylation Specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method and methylation-specific fluorescent quantitative PCR method.
  • the target region includes a first target region and a second target region, the first target region belongs to the KCNA3 gene, and the second target region belongs to the KCNA3 gene.
  • Reagents for detecting methylation levels of target regions in a gene include reagents for detecting methylation levels of a first target region and a second target region.
  • the first target region may include one continuous nucleotide sequence, or two or more non-connected nucleotide sequences.
  • the second target region may include one continuous nucleotide sequence, or two or more non-connected nucleotide sequences.
  • the first target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 and/or the second target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12.
  • the first target region listed above can be combined with any second target region derived from the OTOP2 gene.
  • the second target region can be shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12
  • the full-length or partial sequence of any of them may also be other regions including CpG sites in the OTOP2 gene.
  • the second target region listed above can be combined with any first target region derived from the KCNA3 gene.
  • the first target SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 The full-length or partial sequence of any one shown may also be other regions including CpG sites in the KCNA3 gene.
  • the first target region includes a full-length or partial sequence whose nucleotide sequence is shown in SEQ ID NO.3; the second target region includes a full-length or partial sequence whose nucleotide sequence is shown in SEQ ID NO.12. partial sequence.
  • the first target region is selected from any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4; the second target region is selected from SEQ ID NO .6. Any one of SEQ ID NO.7, SEQ ID NO.12, a combination of the two, or a combination of the three.
  • the first target region is selected from any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, a combination of the two, or a combination of the three A combination, or a combination of all four;
  • the second target region is selected from any one of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12.
  • the first target region includes a combination of two or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, and the second The two target regions also include a combination of two or more of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.12.
  • the first target region includes a complete sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11. Long or partial sequence; And/or the second target region includes a full-length or partial sequence selected from at least one of the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.14.
  • the first target region listed above can be combined with any second target region derived from the OTOP2 gene.
  • the second target region can be SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO.
  • the full-length or partial sequence of any one shown in 14 may also be other regions including CpG sites in the OTOP2 gene.
  • the second target region listed above can be combined with any first target region derived from the KCNA3 gene.
  • the first target SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 The full-length or partial sequence of any one shown may also be other regions including CpG sites in the KCNA3 gene.
  • the first target region and the second target region can also be a combination of one or more nucleotide sequences respectively.
  • One embodiment of the present disclosure also provides a detection reagent for diagnosing esophageal cancer, the detection reagent includes a reagent for detecting the methylation level of a target region in a gene, and the detected genes include KCNA3 gene and OTOP2 gene.
  • the gene and its target region detected by the detection reagent have the meanings as described above, and will not be repeated here.
  • the reagent for detecting the methylation level of the target region in the gene includes a nucleic acid molecule including a pair of primers for detecting the methylation level of the first target region and the second target region.
  • the reagents also include detection probes corresponding to the detection primer pairs.
  • the detection probe is connected with a fluorescent group and a fluorescent quenching group.
  • the 5' end of the fluorescent probe contains a fluorescent reporter group such as any one of FAM, HEX, VIC, CY5, ROX, Texsa Red, JOE and Quasar 705, and the 3' end contains a fluorescent quencher group , such as any one of MGB, BHQ-1, BHQ-2, BHQ-3.
  • a fluorescent reporter group such as any one of FAM, HEX, VIC, CY5, ROX, Texsa Red, JOE and Quasar 705
  • the 3' end contains a fluorescent quencher group , such as any one of MGB, BHQ-1, BHQ-2, BHQ-3.
  • the primer pair for detecting the methylation level of the first target region and the second target region is as follows:
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.1 in the first target region are respectively SEQ ID NO.15 and SEQ ID NO.16
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.1 are SEQ ID NO.17 and SEQ ID NO.18 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.2 in the first target region are SEQ ID NO.19 and SEQ ID NO.20, respectively
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.2 are SEQ ID NO.21 and SEQ ID NO.22 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.3 in the first target region are respectively SEQ ID NO.23 and SEQ ID NO.24
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.3 are SEQ ID NO.25 and SEQ ID NO.26 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.4 in the first target region are respectively SEQ ID NO.27 and SEQ ID NO.28
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.4 are SEQ ID NO.29 and SEQ ID NO.30 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.12 in the second target region are SEQ ID NO.31 and SEQ ID NO.32, respectively
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.12 are SEQ ID NO.33 and SEQ ID NO.34 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.6 in the second target region are SEQ ID NO.35 and SEQ ID NO.36, respectively
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.6 are SEQ ID NO.37 and SEQ ID NO.38 respectively;
  • the methylation upstream primer (5'-3') and the methylation downstream primer (5'-3') of SEQ ID NO.7 in the second target region are SEQ ID NO.39 and SEQ ID NO.40, respectively.
  • the unmethylated upstream primer (5'-3') and unmethylated downstream primer (5'-3') of SEQ ID NO.7 are SEQ ID NO.41 and SEQ ID NO.42 respectively.
  • the combination of the primer pair and the detection probe for detecting the methylation level of the first target region and the second target region is as follows:
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.8 of the first target region are respectively SEQ ID NO.43, SEQ ID NO.43, SEQ ID NO. ID NO.44 and SEQ ID NO.45;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.9 of the first target region are respectively SEQ ID NO.46, SEQ ID NO.46, SEQ ID NO. ID NO.47 and SEQ ID NO.48;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.10 of the first target region are respectively SEQ ID NO.49, SEQ ID NO.49, SEQ ID NO. ID NO.50 and SEQ ID NO.51;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.11 of the first target region are respectively SEQ ID NO.52, SEQ ID NO.52, SEQ ID NO. ID NO.53 and SEQ ID NO.54;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.5 of the second target region are respectively SEQ ID NO.55, SEQ ID NO.55, SEQ ID NO.5 ID NO.56 and SEQ ID NO.57;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.13 of the second target region are respectively SEQ ID NO.58, SEQ ID NO.58, SEQ ID NO. ID NO.59 and SEQ ID NO.60;
  • the upstream primer (5'-3'), downstream primer (5'-3') and detection probe (5'-3') of the SEQ ID NO.14 of the second target region are respectively SEQ ID NO.61, SEQ ID NO.61, SEQ ID NO. ID NO.62 and SEQ ID NO.63.
  • Sanger sequencing can be used to assess the methylation level of target gene combinations.
  • a pair of methylated primers and a pair of unmethylated primers are respectively provided for the first target region and the second target region, and Sanger sequencing is used to detect the methylation level.
  • the primer pairs shown in Table 1 above can be used.
  • the methylation level of the target gene combination can also be assessed by using the methylation fluorescent quantitative PCR method.
  • primer pairs and detection probes may be provided for the first target region and the second target region respectively, to detect the methylation levels of the first target region and the second target region.
  • combinations of primer pairs and detection probes are shown in Table 2 above.
  • the reagent also includes a pair of primers for detecting internal reference genes and corresponding detection probes.
  • the internal reference gene includes the ACTB gene, and the ACTB gene corresponds to the (detection) primer pair shown in SEQ ID NO.64 and SEQ ID NO.65 and the detection probe shown in SEQ ID NO.66.
  • the biological sample to be tested is an esophageal tissue sample, an esophageal exfoliated cell sample or a blood sample; wherein the blood sample includes plasma, serum, whole blood, isolated blood cells or a combination thereof.
  • An embodiment of the present disclosure also provides a kit for diagnosing esophageal cancer, including the above detection reagent.
  • the kit further includes at least one of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents.
  • the methylation conversion reagent is bisulfite.
  • the PCR reaction reagents include one or more of amplification buffer, dNTPs, DNA polymerase, and Mg 2+ .
  • kit provided in the present disclosure may also include negative/positive reference materials, or other necessary reagents to achieve the purpose or effect of the present disclosure, such as some necessary sample preservation reagents.
  • One embodiment of the present disclosure also provides a method for detecting whether a subject has esophageal cancer or precancerous lesions, including:
  • Detect the methylation level of the KCNA3 gene and OTOP2 gene target region in the biological sample if the methylation level of at least one of the KCNA3 gene and OTOP2 gene increases, the subject has esophageal cancer or precancerous lesions Increased risk.
  • the method is implemented based on the foregoing solutions.
  • it can be realized by using the detection reagent or kit as mentioned above.
  • the biological sample is an ex vivo sample, including but not limited to an esophageal tissue sample, an esophageal exfoliated cell sample or a blood sample.
  • the blood sample includes plasma, serum, whole blood, isolated blood cells, or any combination thereof.
  • the methylation level of at least one of the KCNA3 gene and the OTOP2 gene is increased, which means that the methylation level of the KCNA3 gene and the OTOP2 gene is detected in the biological sample of the subject, and the result is the same as the average methylation level of the normal population. Compared with the level of culture, it is higher.
  • the ct value can be used to represent the relative level of gene methylation.
  • This disclosure provides the application of OTOP2 gene and the methylation of both KCNA3 and OTOP2 genes as diagnostic markers for esophageal cancer.
  • esophageal cancer can be specifically detected. diagnosis or auxiliary diagnosis.
  • the disclosure also provides reagents and diagnostic kits for detecting the methylation levels of the OTOP2 gene and the target regions of both KCNA3 and OTOP2 genes.
  • the reagents and the kits have high sensitivity and good specificity for diagnosing esophageal cancer. Improving the effect of early diagnosis and treatment of esophageal cancer and reducing mortality has important clinical application value and social significance, and provides a new idea for non-invasive diagnosis of esophageal cancer and early esophageal cancer.
  • the inventors found that by using the method of methylation-specific fluorescent quantitative PCR to detect the methylation levels of the target regions of KCNA3 gene and OTOP2 gene in blood samples, patients with esophageal cancer can be effectively distinguished from healthy people.
  • the sensitivity of esophageal low-grade tumors, high-grade tumors, early esophageal cancer, and advanced esophageal cancer can reach 76.67%, 89.19%, 93.33% and 96.0%, and its specificity in healthy people can reach 95%.
  • the technical solution provided by the present disclosure can realize non-invasive or minimally invasive diagnosis of esophageal cancer, has high diagnostic sensitivity and good specificity, and is conducive to improving the detection rate of esophageal precancerous lesions and esophageal cancer.
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 1.
  • Nucleic acid combination 1 comprises the nucleotides shown in SEQ ID NO.67-69.
  • the nucleic acid combination 1 can detect the methylation level of the Chr17:74923881-74924015 region positive strand (target region 1) on the OTOP2 gene.
  • the upstream primer sequence of region 1 methylation-specific PCR is (5'-3'):
  • TAATACCCACCGCGCCCTAAC SEQ ID NO. 68
  • the detection probe sequence for region 1 methylation-specific PCR is (5'-3'):
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 2.
  • Nucleic acid combination 2 comprises the nucleotides shown in SEQ ID NO.70-72.
  • the nucleic acid combination 2 can detect the methylation level of the Chr17:74924159-74924335 region positive strand (target region 2) on the OTOP2 gene.
  • the upstream primer sequence of region 2 methylation-specific PCR is (5'-3'):
  • the detection probe sequence for region 2 methylation-specific PCR is (5'-3'):
  • AAAGCGTTTGTTTATTTCGGCGTGG (SEQ ID NO. 72).
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 3.
  • Nucleic acid combination 3 comprises the nucleotides shown in SEQ ID NO.73-75.
  • the nucleic acid combination 3 can detect the methylation level of the Chr17:74924422-74924581 region positive strand (target region 3) on the OTOP2 gene.
  • the upstream primer sequence of region 3 methylation-specific PCR is (5'-3'):
  • CAAAACTCCAAATCAACCCTAACGA SEQ ID NO. 74
  • the detection probe sequence of region 3 methylation-specific PCR is (5'-3'):
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 4.
  • Nucleic acid combination 4 comprises the nucleotides shown in SEQ ID NO.76-78.
  • the nucleic acid combination 4 can detect the methylation level of the Chr17:74924603-74924449 region negative strand (target region 4) on the OTOP2 gene.
  • the upstream primer sequence of region 4 methylation-specific PCR is (5'-3'):
  • the detection probe sequence of region 4 methylation-specific PCR is (5'-3'):
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 5.
  • Nucleic acid combination 5 comprises the nucleotides shown in SEQ ID NO.79-81.
  • the nucleic acid combination 5 can detect the methylation level of the Chr17:74924425-74924297 region negative strand (target region 5) on the OTOP2 gene.
  • the upstream primer sequence of region 5 methylation-specific PCR is (5'-3'):
  • ATGGGAAGAAAGGAGAAGCG (SEQ ID NO. 79);
  • the detection probe sequence of region 5 methylation-specific PCR is (5'-3'):
  • TAAGTTCGATTGTTTTTCGGTTCGG (SEQ ID NO. 81).
  • This embodiment provides reagents for the diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which include nucleic acid combination 6.
  • Nucleic acid combination 6 comprises the nucleotides shown in SEQ ID NO.82-84.
  • the nucleic acid combination 6 can detect the methylation level of the Chr17:74924080-74923981 region negative strand (target region 6) on the OTOP2 gene.
  • the upstream primer sequence of region 6 methylation-specific PCR is (5'-3'):
  • the detection probe sequence of region 6 methylation-specific PCR is (5'-3'):
  • TTTCGTATTGCGGTATTTAGAGGGT SEQ ID NO. 84.
  • the methylation-specific PCR method was used to detect the methylation status of the 6-stage methylation region of the CpG island of the OTOP2 gene in esophageal cancer samples, high-grade esophageal neoplasia samples, and normal samples, and calculate the sensitivity and specificity .
  • Steps include:
  • Tissue DNA was extracted using QIAamp DNA FFPE Tissue Kit, see the kit instructions for operation.
  • Plasma samples Use Tiangen Biochemical Technology (Beijing) Co., Ltd. Magnetic Bead Method Serum/Plasma Free DNA Extraction Kit (DP709) for plasma cfDNA extraction, see the kit instructions for operation.
  • DP709 Magnetic Bead Method Serum/Plasma Free DNA Extraction Kit
  • the nucleic acid conversion kit used is the EZ DNA Methylation-Gold Kit of ZYMO RESEARCH.
  • kit manual unmethylated cytosine (C) is converted to uracil (U), methylated cytosine is unchanged, uracil is paired with adenine (A) in the subsequent PCR step, and cytosine Pyrimidines pair with guanine (G) to distinguish methylated from unmethylated sequences.
  • Methylation detection is performed on 6 regions within the CpG island, and the detection reagents for each region include a pair of methylated sequence-specific detection primers and a specific taqman probe.
  • the positions of the six regions on the chromosome and the sequences of PCR primers and probes are shown in Table 3.
  • step (2) The DNA after bisulfite conversion in step (2) was subjected to methylation-specific PCR reactions to detect the methylation status of OTOP2 gene regions 1-6, and each region was detected separately, that is, each time in a PCR tube Add detection primers and probes for only one region, and add detection probes for internal reference genes at the same time.
  • ACTB is used as an internal reference gene, wherein the upstream primer of ACTB is: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO.85); the downstream primer of ACTB is: AATAACACCCCCACCCCTGC (SEQ ID NO.86); the probe of ACTB is: GGAGTGGTTTTTGGGTTTG (SEQ ID NO.87).
  • Platinum II Taq hot-start DNA polymerase (product number 14966001) was used for PCR amplification, and the PCR reaction solution configuration system is shown in Table 4.
  • Negative control was purified water.
  • the preparation method of the positive control is as follows: the sequence corresponding to the amplified region of ACTB after bisulfite conversion (i.e., except the C in the CG dinucleotide in the amplified region remains unchanged, the C in the remaining positions are all converted into T, and the other three bases T, G and A remain unchanged) for artificial synthesis, and cloned into the vector pUC57 to form an artificially synthesized plasmid.
  • the bisulfite-converted sequences corresponding to the fully methylated regions 1-6 (that is, the C in the other positions in each region are kept unchanged except for the CG dinucleotides are converted into T , the other three bases T, G and A remain unchanged) were artificially synthesized, and cloned into the vector pUC57 to form an artificially synthesized plasmid.
  • the positive control of area 1-6 is 10 3 copies/ ⁇ l of ACTB artificially synthesized plasmid and 10 3 copies/ ⁇ l of area 1-6 artificially synthesized plasmid mixed at a volume ratio of 1:1, such as the positive control of area 1
  • the control is a mixture of 10 3 copies/ ⁇ l of ACTB artificially synthesized plasmid and 10 3 copies/ ⁇ l of region 1 artificially synthesized plasmid at a volume ratio of 1:1.
  • the negative control should have no amplification, the positive control should have a significant exponential growth period, and the Ct value of the internal reference gene of the sample to be tested should be ⁇ 35. After the negative control, positive control and internal reference genes all meet the above requirements, it indicates that the experiment is valid. The judgment of the next sample result can be carried out. Otherwise, the test is invalid and must be tested again.
  • Ct value reading After the PCR is completed, adjust the baseline, set the fluorescence value before the minimum Ct value of the sample in a PCR 1-2 cycles earlier as the baseline value, set the threshold at the inflection point of the S-type amplification curve, and use the PCR software The Ct value of each sample will be automatically given, and the Ct value of the target region to be detected in each sample and the Ct value of the internal reference gene ACTB will be read.
  • Result analysis and interpretation method If the Ct value of the region to be detected in a well is ⁇ 40, it is considered that the region is detected to be methylated in the well, and methylation is detected in at least two wells in the three multiple wells When it is methylated, it is determined that the region is methylation-positive in the sample, otherwise it is methylation-negative.
  • Sensitivity (%) number of positive methylation/(total number of esophageal precancerous lesions or esophageal cancer samples) ⁇ 100% (1)
  • the detection sensitivities of regions 1-6 to esophageal cancer tissue samples are all above 80%, and the specificities are all above 95%. It shows that these 6 regions can effectively distinguish esophageal cancer samples from paracancerous samples.
  • the detection performance of area 2 and area 4 is further improved, and the sensitivity of both is above 90%.
  • regions 1-6 are all negative for methylation in healthy plasma samples, and the detection specificity is 100%.
  • the detection sensitivity in esophageal cancer plasma samples is above 55%.
  • the detection sensitivity of the changed plasma sample is above 30%.
  • the detection sensitivity of areas 2 and 4 is further improved, and the detection sensitivity of both of them is greater than 65% for esophageal cancer samples, and the detection of high-grade esophageal neoplasia samples The sensitivities are all greater than 40%.
  • the dual-target esophageal cancer detection example of the present disclosure uses two different methods to verify the effect of the combination of methylated molecular markers in diagnosing esophageal cancer:
  • Example 8 demonstrates the use of Sanger sequencing to evaluate the combination of molecular markers to determine whether the tissue sample and blood sample are cancer samples;
  • Example 9 demonstrates the use of methylation fluorescent quantitative PCR method to evaluate the methylation level of the molecular marker combination, and then determine whether the tissue sample, blood sample Whether it is a cancer sample.
  • the methylation status of at least one sequence is used to analyze the sensitivity and specificity of the kit for esophageal cancer diagnosis or auxiliary diagnosis.
  • LGIN low-grade esophageal squamous intraepithelial neoplasia
  • HGIN high-grade esophageal
  • Blood samples from 60 patients with low-grade esophageal squamous intraepithelial neoplasia (LGIN) and 74 patients with high-grade esophageal squamous intraepithelial neoplasia (HGIN) were collected in a hospital in Zhengzhou.
  • the collection process of all samples was approved by the ethics committee, and all volunteers signed the informed consent. All samples were anonymized.
  • DNA was extracted using the QIAamp DNA FFPE Tissue Kit (56404), and the operation was performed according to the kit instructions.
  • plasma cfDNA extraction was performed using the magnetic bead method serum/plasma free DNA (cfDNA) extraction kit (DP709) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the operation was performed according to the kit instructions.
  • the extracted sample genome was subjected to bisulfite conversion.
  • the nucleic acid conversion kit used was the nucleic acid purification reagent of Wuhan Amison Life Science and Technology Co., Ltd. (Ehan Machinery Equipment 20200843).
  • Ehan Machinery Equipment 20200843 for the experimental operation, please refer to the kit manual.
  • nucleotide sequences SEQ ID NO.1 ⁇ 4 of the KCNA3 gene and the nucleotide sequences SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.12 of the OTOP2 gene respectively design methylated primer pairs and non-
  • the methylated primer pair amplifies the corresponding region, and the optimal ratio of the methylated primer pair and the unmethylated primer pair is screened to ensure that when there is greater than or equal to 1% of the methylated sequence in the template, that is Methylated products can be amplified, and only when the template is a non-methylated sequence, the amplified product is a non-methylated product.
  • the methylated primer pairs and unmethylated primer pairs used in PCR amplification are shown in Table 8. Configure the PCR reaction system according to the formula in Table 9, and the PCR amplification program is the same as Table 10. After the PCR amplification finishes, use mixed primers (comprising a methylated primer pair and a non-methylated primer pair) to perform mulberry on the amplified product. Geer sequencing (delivered to the sequencing company), simultaneously sequenced from the 5' end and the 3' end.
  • Table 8 The sequences of methylated primers and non-methylated primers in each target region
  • the methylation status of the CpG sites in each amplicon of each sample was analyzed according to the sequencing profile.
  • the methylation status of cytosine in a CpG dinucleotide is divided into two types, that is, non-methylation and methylation, and methylation is further divided into complete methylation and partial methylation. If cytosine sequencing results in a thymine in a CpG dinucleotide, it is unmethylated. If the cytosine sequence in the CpG dinucleotide is still cytosine, it is fully methylated. If sequencing of cytosines in a CpG dinucleotide results in both cytosine and thymine (doublet), it is partially methylated.
  • a sample was considered positive for methylation in a gene region if more than 95% of the cytosines in the CpG dinucleotides in an amplicon were methylated. Calculate the number of methylation positives and methylation negatives in each region for each type of sample.
  • a sample is positive for methylation in at least one region of the KCNA3 gene or at least one region of the OTOP2 gene, the sample is considered to be positive for precancerous lesions or cancer; when a sample is positive for at least one region of the KCNA3 gene and A sample that was negative for methylation in at least one region of the OTOP2 gene was considered a precancer-negative and cancer-negative sample.
  • the nucleotide sequence in the KCNA3 gene is at least one region shown in SEQ ID NO.1-4 and at least one of the nucleotide sequences in the OTOP2 gene is shown in SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.12
  • SEQ ID NO.6 The methylation status, detection sensitivity and specificity of the region in tissue samples from patients with esophageal precancerous lesions and esophageal cancer are shown in Table 11; its methylation status, detection Sensitivity and specificity are as shown in Table 12
  • (+" in the sequence number of the composition hereinafter means to detect the methylation level of the two sequences before and after "+” simultaneously, for example SEQ ID NO.1+12 means to detect SEQ ID simultaneously NO.1 and the methylation level of SEQ ID NO.12).
  • the methylation level of the composition of at least one region shown in SEQ ID NO.12 has a good effect in detecting precancerous lesions of the esophagus and esophageal cancer.
  • the sensitivity for detecting low-grade neoplasia of the esophagus was the lowest at 40.63%, and the highest sensitivity for detecting low-grade neoplasia of the esophagus was 87.50%.
  • the lowest sensitivity was 54.17%, and the highest sensitivity for detecting high-grade esophageal neoplasia was 95.83%; the lowest sensitivity for detecting early esophageal cancer was 72.06%, and the highest sensitivity for early detection was 97.06%; the lowest sensitivity for detecting advanced esophageal cancer was The highest sensitivity for detecting advanced esophageal cancer was 98.75%; the lowest specificity for detecting adjacent tissues was 70.89%, and the highest specificity for detecting adjacent tissues was 94.94%.
  • the sensitivity for detecting low-grade neoplasia of the esophagus is the lowest at 38.33%, and the highest sensitivity for detecting low-grade neoplasia of the esophagus is 83.33%.
  • the lowest sensitivity was 63.51%, and the highest sensitivity for detecting high-grade esophageal neoplasia was 94.59%; the lowest sensitivity for detecting early esophageal cancer was 71.67%, and the highest sensitivity for detecting early esophageal cancer was 95.83%; the lowest sensitivity for detecting advanced esophageal cancer was 76.67%, the highest sensitivity for detecting advanced esophageal cancer is 97.33%; the lowest specificity for detecting blood samples from healthy people is 70%, and the highest specificity for detecting blood samples from healthy people is 96%.
  • the detection sensitivity of the composition tends to increase, but its specificity also tends to decrease.
  • both the sensitivity and specificity can reach a relatively high level, for example, the combination of SEQ ID NO.3 and SEQ ID NO.12 is used as a methylation marker.
  • Example 9 Methylation-specific fluorescent quantitative PCR method to assess the methylation level of the target gene
  • the main process of this embodiment includes: 1) Obtain tissue samples and blood samples from esophageal precancerous lesions or cancer patients or healthy people, and extract, transform, and purify template DNA according to different sample types; 2) use detection primer pairs and probes to detect the methylation status of each composition in the sample by methylation fluorescent quantitative PCR method, and then judge whether the sample is a cancer sample; 3) calculate and use the composition as a methylation marker to diagnose tissue samples, blood samples sensitivity and specificity. Considering that the combination of more than two marker sequences will reduce the specificity of detection, only the combination of two marker sequences was used in Example 9. Proceed as follows:
  • the DNA of each sample converted by bisulfite was subjected to methylation fluorescent quantitative PCR reaction to detect any one of the nucleotide sequences in the KCNA3 gene such as SEQ ID NO.
  • a detection primer pair, probe, and SEQ ID NO.5 in any region of SEQ ID NO.8-11 need to be added to a PCR tube , SEQ ID NO.13, SEQ ID NO.14 detection primer pair and probe for any region; at the same time, the detection primer pair and probe for the internal reference gene ACTB also need to be added.
  • the probe for detecting the target region is a Taqman probe, and the reporter group at the 5' end of the detection probe of SEQ ID NO.8 ⁇ 11 is FAM, and the quenching group at the 3' end is MGB; SEQ ID NO.5, SEQ ID NO.
  • the nucleotide sequences in the KCNA3 gene such as SEQ ID NO.8 ⁇ 11 and the nucleotide sequences in the OTOP2 gene such as SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO.14 and the upstream and downstream detection primers and probes of the internal reference gene ACTB
  • the needle sequence is shown in Table 13.
  • Negative control and positive control When testing samples in different combinations, the negative control and positive control should also be tested at the same time.
  • the DNA template of the negative control tube is TE buffer.
  • the preparation method of the DNA template of the positive control tube is as follows: artificially synthesize the sequence corresponding to the amplified region of the ACTB gene after complete bisulfite conversion, and clone it into the vector pUC57 to form an artificially synthesized plasmid; the target region such as SEQ The sequences of ID NO.8 ⁇ 11, SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.14 respectively after bisulfite conversion were artificially synthesized, and cloned into the vector pUC57 respectively to form an artificially synthesized plasmid.
  • the positive control DNA template is 10 3 copies/microliter of the artificially synthesized plasmid containing transformed ACTB, 10 3 copies/microliter of an artificially synthesized plasmid containing a sequence in SEQ ID NO.8-11 after transformation, and 10 3 copies
  • the artificially synthesized plasmid containing one of the sequences in SEQ ID NO.5, SEQ ID NO.13, and SEQ ID NO.14 after transformation is mixed in equal proportions per microliter.
  • Ct value reading After PCR is completed, adjust the baseline, set the fluorescence value before the minimum Ct value of the sample in one PCR 1-2 cycles earlier as the baseline value, set the threshold at the inflection point of the S-type amplification curve, and obtain the sample Ct values of individual genes.
  • the negative control should have no amplification, the positive control should have a significant exponential growth period, and the Ct value of each gene in the positive control should be between 26-30.
  • the Ct value of the internal reference gene of the sample to be tested should be ⁇ 35. After the negative control, positive control, and internal reference gene all meet the above requirements, it indicates that the experiment is valid, and the next step of sample results can be judged. Otherwise, the test is invalid and must be tested again.
  • the methylation level of the sample to be tested is judged.
  • tissue samples if the Ct value of the amplified gene region is ⁇ 38, the gene in the sample is considered positive for methylation; if the Ct value of the amplified gene region is > 38, the gene in the sample is considered positive. Because of methylation negative.
  • the Ct value of amplified gene region is less than or equal to 45, the gene in the sample is considered positive for methylation; if the Ct value of amplified gene region is > 45, the gene in the sample is considered positive Because of methylation negative.
  • the sample is a precancerous lesion-positive or cancer-positive sample, and only if both genes in the sample to be tested are methylation-negative, the sample is a precancerous lesion Negative and cancer negative samples. Judgment criteria are shown in Table 17.
  • the nucleotide sequence in the KCNA3 gene is any one of the regions shown in SEQ ID NO.8 to 11 and the nucleotide sequence in the OTOP2 gene is any one shown in SEQ ID NO.5, SEQ ID NO.13, or SEQ ID NO.14
  • the methylation status, detection sensitivity and specificity of the composition of the region in the tissue samples of patients with esophageal precancerous lesions and esophageal cancer are shown in Table 18, and its methylation in the blood samples of patients with esophageal precancerous lesions and esophageal cancer Status, detection sensitivity and specificity are shown in Table 19.
  • the lowest sensitivity for diagnosing low-grade esophageal neoplasia was 43.75%, and the highest sensitivity for diagnosing low-grade esophageal neoplasia was 81.25%; for diagnosing high-grade esophageal neoplasia
  • the lowest sensitivity was 52.08%, and the highest sensitivity for diagnosing high-grade esophageal neoplasia was 91.67%; the lowest sensitivity for diagnosing early esophageal cancer was 72.06%, and the highest sensitivity for diagnosing early esophageal cancer was 94.12%;
  • the highest sensitivity for diagnosing advanced esophageal cancer was 96.25%; the lowest specificity for detecting adjacent tissues was 77.22%, and the highest specificity for detecting adjacent tissues was 93.67%.
  • the sensitivity for diagnosing low-grade neoplasia of the esophagus is the lowest at 46.67%, and the highest sensitivity for detecting low-grade neoplasia of the esophagus is 76.67%.
  • the lowest sensitivity was 51.35%, and the highest sensitivity for detecting high-grade esophageal neoplasia was 89.19%; the lowest sensitivity for detecting early esophageal cancer was 73.33%, and the highest sensitivity for detecting early esophageal cancer was 93.33%; the lowest sensitivity for detecting advanced esophageal cancer was The highest sensitivity for detecting advanced esophageal cancer is 96.0%; the lowest specificity for detecting blood samples from healthy people is 81.00%, and the highest specificity for detecting blood samples from healthy people is 95%.
  • nucleotide sequence of the KCNA3 gene is SEQ ID NO.10 and the nucleotide sequence of the OTOP2 gene is SEQ ID NO.5 as the combination of methylation markers, the sensitivity and specificity of detecting tissue samples and blood samples performance can be further improved.
  • the present disclosure provides the application of the OTOP2 gene or the methylation of both KCNA3 and OTOP2 genes as a diagnostic marker for esophageal cancer, and also provides a method for detecting the methylation level of the target region of the OTOP2 gene or both KCNA3 and OTOP2 genes
  • Reagents and diagnostic kits the reagents and kits have high sensitivity and specificity for diagnosing esophageal cancer, are conducive to improving the detection rate of esophageal precancerous lesions and esophageal cancer, and provide a new method for the non-invasive diagnosis of esophageal cancer and early esophageal cancer. Therefore, it has excellent practical performance and broad market prospects.

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Abstract

本公开涉及用于食管癌诊断的甲基化检测试剂及试剂盒。通过检测基因目标区域的甲基化水平，能够有效区分食管癌前病变和食管癌患者，且检测灵敏度高、特异性好，为食管癌或癌前病变的无创检测提供了新的技术方案。

Description

用于食管癌诊断的甲基化检测试剂及试剂盒
相关申请的交叉引用
本公开要求于2021年11月15日提交中国专利局的申请号为CN202111344786.5、名称为“一种食管癌基因甲基化水平检测试剂及其应用”以及于2022年08月22日提交中国专利局的申请号为CN202211009579.9、名称为“用于食管癌诊断的甲基化检测试剂及试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于医学生物检测技术领域，涉及食管癌的诊断标志物，具体涉及用于食管癌诊断的甲基化检测试剂及试剂盒。
背景技术
食管癌是世界上最常见的恶性肿瘤之一，同时也是消化道最常见的恶性肿瘤。食管癌具有高度的侵袭性，往往导致不良预后。据统计，2018年全球范围内食管癌的新发病例约57万例，死亡病例约50万例，其发病率和死亡率在所有恶性肿瘤中分别位居第7位和第6位。根据肿瘤的病理分型，食管癌主要包括两种组织学亚型：食管鳞状细胞癌(ESCC)和食管腺癌(EAC)。中国是食管癌高发地区之一，拥有全球一半以上的食管癌患者。由于食管癌的症状在癌症发生的早期并不明显，且缺乏方便的筛查手段，大多数患者在确诊时已经发展为中晚期食管癌。
在基因层面寻找食管癌发生发展和转移过程中高灵敏度、高特异性的生物标志物将有助于食管癌的早期无创诊断。食管癌的发病机制涉及遗传学、表观遗传学和环境因素之间复杂的相互作用。DNA甲基化是一种常见的表观遗传修饰形式，其在人类恶性肿瘤的发生中发挥了重要的作用。抑癌基因启动子区域的异常甲基化可能会导致基因的转录下调或沉默，从而导致癌症的发生。因此，DNA甲基化模式的改变已经成为癌症早期检测和诊断的可靠的潜在生物标志物。近年来，基于循环的无细胞DNA(circulating cell-free DNA,cfDNA)的液体活检技术在癌症的早期诊断方面展现出了巨大的潜力，因为其可以以最小的微创方式对早期实体瘤进行检测。肿瘤细胞发生遗传变异(如突变、拷贝数变异或表观遗传改变等)，在肿瘤细胞凋亡或坏死时，其核酸释放进入血液中，从而允许肿瘤DNA通过液体活检技术被检测到。鉴于此，通过检测血液中cfDNA的甲基化来诊断早期食管癌是一种可行的方法，而当务之急是寻找可以区分癌症患者和非癌患者的高灵敏度、高特异性的DNA甲基化生物标志物。
发明内容
本公开提供了检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，所述目标区域为OTOP2基因。
可选地，所述目标区域为OTOP2基因中Chr17:74922901-74924924CpG岛区域中的全长区域或部分区域。
可选地，所述目标区域的甲基化水平为OTOP2基因中如下至少一种CpG岛区域中的全长区域或部分区域的甲基化水平：区域1、区域2、区域3、区域4、区域5和区域6；其中，所述区域1选自Chr17:74923881-7492401 5正链，所述区域2选自Chr17:74924159-74924335正链，所述区域3选自Chr17:74924422-74924581正链，所述区域4选自Chr17:74924603-74924449负链，所述区域5选自Chr17:74924425-74924297负链，所述区域6选自Chr17:74924080-74923981负链。
可选地，所述试剂或试剂盒包括用于检测OTOP2基因的CpG岛的甲基化水平的核酸组合。
可选地，所述核酸组合选自如下核酸组合中的至少一种：用于检测所述区域1的核酸组合1、用于检测所述区域2的核酸组合2、用于检测所述区域3的核酸组合3、用于检测所述区域4的核酸组合4、用于检测所述区域5的核酸组合5和用于检测所述区域6的核酸组合6；
所述核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，所述核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，所述核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，所述核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，所述核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，所述核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
本公开还提供了检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，所述目标区域包括属于KCNA3基因的第一目标区域和属于OTOP2基因的第二目标区域。
可选地，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的至少一者的全长或部分序列；和/或
所述第二目标区域包括选自核苷酸序列为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一者的全长或部分序列。
可选地，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的至少一者的全长或部分序列；和/或
所述第二目标区域包括选自核苷酸序列为SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的至少一者的全长或部分序列。
可选地，所述第一目标区域包括核苷酸序列为SEQ ID NO.3所示的全长或部分序列；所述第二目标区域包括核苷酸序列为SEQ ID NO.12所示的全长或部分序列。
本公开还提供了一种试剂，所述试剂包括如下核酸组合的至少一种：核酸组合1、核酸组合2、核酸组合3、核酸组合4、核酸组合5和核酸组合6；所述核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，所述核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，所述核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，所述核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，所述核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，所述核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
本公开还提供了一种用于食管癌诊断的检测试剂，所述检测试剂包括检测属于KCNA3基因的第一目标区域和属于OTOP2基因的第二目标区域的甲基化水平的试剂。
可选地，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的至少一者的全长或部分序列；和/或
所述第二目标区域包括选自核苷酸序列为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一者的全长或部分序列。
可选地，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的至少一者的全长或部分序列；和/或所述第二目标区域包括选自核苷酸序列为SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的至少一者的全长或部分序列。
可选地，所述检测试剂包括检测选自第一目标区域的核苷酸序列为SEQ ID NO.3所示和选自第二目标区域的核苷酸序列为SEQ ID NO.12所示的全长或部分序列的甲基化水平的试剂。
可选地，所述检测试剂包括核酸分子，所述核酸分子包括检测第一目标区域和第二目标区域的甲基化水平的引物对。
可选地，所述检测试剂还包括与所述引物对对应的检测探针，所述检测探针上连接有荧光基团和荧光淬灭基团。
可选地，所述的检测第一目标区域和第二目标区域的甲基化水平的引物对如下表1所示：
表1
Figure PCTCN2022131724-appb-000001
可选地，所述的检测第一目标区域和第二目标区域的甲基化水平的引物对和所述检测探针的组合如下表2所示：
表2
Figure PCTCN2022131724-appb-000002
本公开还提供了一种诊断食管癌的试剂盒，包括上文所述的试剂或者上文任一项所述的检测试剂。
可选地，所述试剂盒还包括核酸提取试剂、甲基化转化试剂、质控试剂、PCR反应试剂和测序试剂中的至少一种。
具体实施方式
现结合实施方式，对本公开作详细描述，但本公开的实施不仅限于此。
除非另有定义，本文所使用的所有的技术和科学术语与属于本公开的技术领域的技术人员通常理解的含义相同。本文中在本公开的说明书中所使用的术语只是为了描述实施方式和实施例的目的，不是旨在于限制本公开。
除非另外说明或存在矛盾之处，本文中使用的术语或短语具有以下含义：
本公开所使用的术语“和/或”、“或/和”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”的连词组合连接至少三个项目时，应当理解，在本公开中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A和/或B”包括A、B和A+B三种并列方案。又比如，“A，B，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本公开中涉及“多个”、“多种”、“多次”、“多元”等，如无特别限定，指在数量上大于2或等于2。例如，“一种或多种”表示一种或大于等于两种。“以上”包括本数，比如“两种以上”包括两种、三种或更多种。
本公开中，“至少一者”、“至少一种”是指可以是所列项目中的任一项，或者是其中任意两种以上的组合。
本公开中所使用的“其组合”、“其任意组合”、“其任意组合方式”等中包括所列项目中任两个或任两个以上项目的所有合适的组合方式。
本公开中，“合适的组合方式”、“合适的方式”、“任意合适的方式”等中所述“合适”，以能够实施本公开的技术方案、解决本公开的技术问题、实现本公开预期的技术效果为准。
本公开中，“进一步”、“更进一步”、“特别”等用于描述目的，表示内容上的差异，但并不应理解为对本公开保护范围的限制。
本公开中，术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，不能理解为指示或暗示相对重要性或数量，也不能理解为隐含指明所指示的技术特征的重要性或数量。而且“第一”、“第二”、“第三”、“第四”等仅起到非穷举式的列举描述目的或用于区分描述，应当理解并不构成对数量的封闭式限定，同时不应理解为指示或暗示相对重要性。
本公开中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本公开中，“食管癌”是一种自下咽到食管胃交界部之间食管上皮来源的消化道恶性肿瘤。食管癌主要包括食管鳞状细胞癌和食管腺癌。
本公开中，术语“诊断”是指作为单一因素用于确定、验证或确认患者的临床状态，包括辅助诊断、复发风险评估、癌变风险和癌变程度的评估、预后判断等方面。术语“辅助诊断”用于在患者临床状态确定或验证过程中提供各种信息辅助判断，不作为唯一确定指标。
术语“基因”是指编码产生氨基酸多肽链的DNA区段，它包括参与基因转录/翻译和转录/翻译调控的位于编码区和非编码区的序列，以及外显子和内含子序列。可以理解，检测某一基因的甲基化水平，包括检测该基因编码区和非编码区 的CpG位点的甲基化水平，例如该基因的启动子区域中的CpG位点的甲基化水平。因此，检测位点的染色体坐标位置可能位于该基因编码区的上游或下游。本公开中，基因的染色体坐标位置以GRCh38.p14为参考基因组。
术语“寡核苷酸”或“多核苷酸”或“核苷酸”或“核酸”是指具有两个或更多个脱氧核糖核苷酸或核糖核苷酸的分子，可选地，多于三个脱氧核糖核苷酸或核糖核苷酸的分子，并且通常多于十个脱氧核糖核苷酸或核糖核苷酸的分子。确切的大小将取决于许多因素，而所述因素又取决于寡核苷酸的最终功能或用途。寡核苷酸可以任何方式产生，包括化学合成、DNA复制、逆转录或其组合。DNA的典型脱氧核糖核苷酸是胸腺嘧啶、腺嘌呤、胞嘧啶和鸟嘌呤。RNA的典型核糖核苷酸是尿嘧啶、腺嘌呤、胞嘧啶和鸟嘌呤。
术语“甲基化”为DNA化学修饰的一种形式，能够在不改变DNA序列的前提下，改变遗传表现。DNA甲基化是指在DNA甲基转移酶的作用下，在基因组CpG二核苷酸的胞嘧啶第5号碳位共价结合一个甲基基团。DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变，从而控制基因表达。
术语“甲基化水平”指的是一段DNA序列中一个或多个CpG二核苷酸中的胞嘧啶是否发生甲基化、或发生甲基化的频率/比例/百分数，既代表定性的概念又代表定量的概念。在实际应用中，可根据实际情况采用不同的检测指标比较DNA甲基化水平。如在一些情况下，可根据样本检测的Ct值进行比较；在一些情况下，可计算样本中基因甲基化的比例，即甲基化分子数/(甲基化分子数+非甲基化分子数)×100％，然后再进行比较；在一些情况下，还需要对各个指标进行统计学上的分析整合，得出最终的判定指标。可以理解，本文中被检测基因的目标区域是包括至少一个CpG二核苷酸(CG)的DNA序列。
术语“CpG岛”指DNA上的一个区域，此区域富含大量的用磷酸酯键相连的胞嘧啶和鸟嘌呤。CpG二核苷酸通常集中在人类基因的启动子区域和外显子中。在正常人基因组中，CpG岛外的CpG位点通常是甲基化的，而CpG岛中的CpG位点通常处于非甲基化状态，这种甲基化的形式在随细胞分裂稳定的遗传。当肿瘤发生时，抑癌基因CpG岛以外的CpG位点非甲基化程度增加,而CpG岛中的CpG位点呈高甲基化状态，导致染色体螺旋程度增加，转录抑制，基因表达缺失。
术语“CpG岛区域甲基化水平”指的是CpG岛内一个或多个CpG二核苷酸中胞嘧啶的甲基化水平。“甲基化位点”或“CpG位点”指的是区域中至少一个CpG二核苷酸位点，尤其指区域中至少一个CpG二核苷酸位点中的胞嘧啶。
术语“引物”是指可以用在扩增方法(例如聚合酶链式反应PCR)中，基于与目标基因或其一部分区域相对应的多核苷酸序列来扩增目的序列的寡核苷酸。通常，用于扩增多核苷酸序列的PCR引物中的至少一个对于该多核苷酸序列是序列特异性的。引物的确切长度取决于很多因素，包括温度、引物来源以及所用方法等。例如，对于诊断和预后应用，根据靶序列的复杂度，寡核苷酸引物通常含有至少10、15、20、25或更多个核苷酸，但是也可以含有更少的核苷酸。在本公开内容中，术语“引物”是指能与目标DNA分子的双链杂交或能与目标DNA分子中位于待扩增核苷酸序列两翼的区域杂交的一对引物。
术语“Taqman探针”是指包含5’荧光基团和3’淬灭基团的一段寡核苷酸序列。当探针与DNA上的相应位点结合时，因为荧光基团附近存在淬灭基团，探针不会发出荧光。在扩增过程中，如果探针与被扩增的链结合，DNA聚合酶(如Taq酶)的5’-3’核酸外切酶活性会消化探针，荧光基团远离淬灭基团，其能量不被吸收，即产生荧光信号。每经过一个PCR循环，荧光信号也和目的片段一样，有一个同步的指数增长的过程。
术语“桑格尔测序(Sanger测序)”即一代测序，其反应系统包含：目标片段、四种脱氧核糖核苷酸(dNTP)、DNA聚合酶，引物等，还需要加入不同荧光基团标记的4种双脱氧核糖核苷酸(ddNTP)。由于ddNTP缺乏延伸所需要的3’-OH基团，使延长的寡聚核苷酸选择性地在G、A、T或C处终止，通过光激发将四种光波长信号转化为电脑可识别的电信号，根据反应管中最后掺入的ddNTP的荧光信号判断目的DNA序列。
本公开中，术语“试剂”与“检测试剂”具有相同含义，可互换使用，是指用于实现样本中DNA甲基化水平检测所需的任意试剂，尤其包括能够扩增目标区域的引物对、能够特异性识别目标区域的探针或者它们的组合。
以下对本公开的各个方面进行详细说明。
(一)单靶标的食管癌检测
DNA甲基化是基因上重要的一种化学修饰，影响着基因转录的调控过程和细胞核结构。DNA甲基化的改变是癌症发展的早期事件和伴随事件，主要体现在肿瘤组织上抑癌基因的高甲基化和原癌基因的低甲基化等。而且DNA甲基化较稳定，如果可以发现肿瘤特异的血液DNA甲基化分子标志物则有巨大的临床应用价值。
本公开提供了一种用于检测OTOP2基因CpG岛(英文全称：otopetrin 2)甲基化水平的物质在制备产品中的应用，产品的用途为如下中的至少一种：
诊断或辅助诊断食管癌或癌前病变；
区分食管癌样本和非癌样本；该产品选自如下产品中的至少一种：试剂、试剂盒、芯片和测序文库。
在一些实施方式中，本公开提供了检测基因中目标区域的甲基化水平的物质在诊断或辅助诊断食管癌或癌前病变，和/或区分食管癌样本和非癌样本的应用，
该目标区域为OTOP2基因。
在一些实施方式中，本公开提供了检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，目标区域为OTOP2基因。
在一些实施方式中，本公开提供了检测OTOP2基因CpG岛甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用。
本公开发现，OTOP2基因CpG岛区域的甲基化水平在食管癌样本/食管癌前病变中的甲基化水平显著高于正常样本，用OTOP2基因CpG岛区域的甲基化作为检测标记物，能在待测样本中有效区分癌症/癌前病变样本和正常样本，对癌症的检测灵敏性超过50％，对癌前病变的检测灵敏性超过30％，特异性高于95％。而且用OTOP2基因CpG岛区域的甲基化作为检测标记物能够有效的区分食管癌样本和癌旁样本(非癌样本)。本公开为食管癌的无创检测提供了新思路。
在一些实施方式中，食管癌包括但不限于食管鳞癌和食管腺癌，癌前病变包括不限于食管鳞状上皮内瘤变、Barrett食管相关异性增生、慢性食管炎、食管上皮增生、食管息肉、食管溃疡、食管白斑。
在一些实施方式中，OTOP2基因甲基化水平为OTOP2基因中Chr17:74922901-74924924CpG岛区域中的全长区域或部分区域的甲基化水平。
在一些实施方式中，OTOP2基因的甲基化水平为OTOP2基因中如下至少一种CpG岛区域中的全长区域或部分区域的甲基化水平：区域1、区域2、区域3、区域4、区域5和区域6。
在一些实施方式中，部分区域为区域1-6中至少一个CpG二核苷酸位点中胞嘧啶的甲基化水平。
在一些实施方式中，区域1选自Chr17:74923881-74924015正链，区域2选自Chr17:74924159-74924335正链，区域3选自Chr17:74924422-74924581正链，区域4选自Chr17:74924603-74924449负链，区域5选自Chr17:74924425-74924297负链，区域6选自Chr17:74924080-74923981负链。
在一些实施方式中，OTOP2基因甲基化水平为OTOP2基因中如下至少一种CpG岛区域中的全长区域或部分区域的甲基化水平：区域2和区域4。发明人发现相比于其他区域对于样本的检测灵敏度和特异性，区域2和区域4对于样本的检测灵敏度和特异性进一步提高。
在一些实施方式中，上述物质包括用于检测OTOP2基因的CpG岛的甲基化水平的核酸组合。
在一些实施方式中，该试剂或试剂盒包括用于检测OTOP2基因的CpG岛的甲基化水平的核酸组合。
在一些实施方式中，OTOP2基因的CpG岛包括Chr17:74922901-74924924区域，碱基序列如SEQ ID NO.88所示，序列(5’-3’)如下：
Figure PCTCN2022131724-appb-000003
需要说明的是，上述CpG岛区域还包括与上述序列反向互补的序列。
在一些实施方式中，核酸组合选自如下核酸组合中的至少一种：用于检测区域1的核酸组合1、用于检测区域2的核酸组合2、用于检测区域3的核酸组合3、用于检测区域4的核酸组合4、用于检测区域5的核酸组合5和用于检测区域6的核酸组合6；
核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
例如核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性，核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性，核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性，核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性，核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性，核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有90％、92％、95％、98％、99％或100％的一致性。
在一些实施方式中，上述产品选自如下产品中的至少一种：试剂、试剂盒、芯片和测序文库。
需要说明的是，上述产品可以是任意一种的体外诊断产品形式，并不限于上述的试剂、试剂盒、芯片和测序文库的产品类型，只要能满足食管癌或癌前病变的诊断或辅助诊断的需求均在本公开的保护范围内。
在一些实施方式中，上述OTOP2基因甲基化水平通过如下至少一种的方法进行检测：甲基化特异性PCR法、测序法、甲基化特异性高效液相层析法、数字PCR法、甲基化特异性高分辨率溶解曲线法、甲基化特异性微阵列法、甲基化敏感性限制性内切酶法和flap endonuclease(瓣状核酸内切酶)法(参见专利文献US8715937、US8361720)。在一些实施方式中，上述测序法选自甲基化特异性PCR法、亚硫酸氢盐测序法、全基因组甲基化测序法或焦磷酸测序法。
本公开还提供一种试剂，其包括如下至少一种的核酸组合：核酸组合1、核酸组合2、核酸组合3、核酸组合4、核酸组合5和核酸组合6；
核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
在一些实施方式，该试剂包括如下至少一种的核酸组合：核酸组合2和核酸组合4。
在一些实施方式中，上述试剂还包括能差异化修饰甲基化DNA和非甲基化DNA的试剂(如亚硫酸氢盐等物质)、采用甲基化特异性PCR技术或数字PCR常用的其他常规试剂(包括不限于PCR缓冲液、dNTPs(脱氧核糖核苷酸三磷酸)、Taq酶、水)和进行飞行时间质谱所用的其他常规试剂。
在一些实施方式中，上述的试剂可以是粉剂、颗粒剂、水分散粒剂、液剂、乳剂或悬浮剂。可选的，真空冷冻干燥制备的菌粉或者喷雾干燥制备的粉剂。在使用时，将核酸组合溶解于超纯水或缓冲液中。
本公开还提供一种试剂盒，其包括上述的试剂。
在一些实施方式中，该试剂盒还包括核酸提取试剂、甲基化转化试剂、质控试剂、PCR反应试剂和测序试剂中的至少一种。
在一些实施方式中，上述试剂盒的检测样本包括不限于组织样本、血液样本、唾液样本或食管来源的细胞样本；
血液样本选自血浆样本、血清样本、全血样本或血细胞样本。
(二)双靶标的食管癌检测
本公开一实施方式还提供了检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，所检测的基因包括KCNA3基因(英文全称：Potassium voltage-gated channel subfamily A member 3)和OTOP2基因。
KCNA3基因和OTOP2基因的相关信息可由本领域已知的生物信息数据库获得。若以GRCh38.p14为参考基因组，KCNA3基因位于Chr1:110653560-110674940；OTOP2基因位于Chr17：74924273-74933912。参照上述信息可以获得基因的核苷酸序列信息，但本公开并不局限于依赖于检测与该参考基因组中核苷酸序列完全相同的KCNA3基因和OTOP2基因。可以理解，从不同集合或不同样本的人类基因组信息所得到的KCNA3与OTOP2基因的核苷酸序列可能存在一些区别，但这些区别并不影响本公开的实施。本公开可适用于不同的受试者个体。
以KCNA3和OTOP2基因作为靶基因，检测两个基因中目标区域的甲基化水平，根据该两个基因中目标区域的甲基化水平可以诊断或辅助判断受试者是否患有食管癌或者发生食管癌前病变。进而，检测上述两个基因中目标区域的甲基化水平的试剂可以应用于制备食管癌诊断试剂或试剂盒。
可以理解，KCNA3和OTOP2基因的目标区域是至少包括一个甲基化位点的区域。在一些实施方式中，该目标区域可以是基因的CpG岛，或者是其他任何包括甲基化位点的区域。在一些实施方式中，该目标区域可以位于启动子区域或者编码区域。
可以通过本领域已知的任何能够检测DNA甲基化水平的检测方法来检测本公开中两个基因目标区域的甲基化水平，包括但不限于通过如下方法中的一种或多种实现对该目标区域的甲基化水平的检测：甲基化特异性PCR、亚硫酸氢盐测序法、甲基化特异性微阵列法、全基因组甲基化测序法、焦磷酸测序法、甲基化特异性高效液相层析法、数字PCR法、甲基化特异性高分辨率溶解曲线法、甲基化敏感性限制性内切酶法和甲基化特异性荧光定量PCR法。
可选地，该目标区域包括第一目标区域和第二目标区域，第一目标区域是属于KCNA3基因的，第二目标区域是属于KCNA3基因的。检测基因中目标区域的甲基化水平的试剂包括检测第一目标区域和第二目标区域的甲基化水平的试剂。
第一目标区域可以包括一个连续的核苷酸序列，也可以包括两个或更多个不相连的核苷酸序列。第二目标区域可以包括一个连续的核苷酸序列，也可以包括两个或更多个不相连的核苷酸序列。
作为示例，第一目标区域包括选自核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的至少一者的全长或部分序列；和/或第二目标区域包括选自核苷酸序列为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一者的全长或部分序列。前述所列的第一目标区域可以和任何来源于OTOP2基因的第二目标区域组合，此时第二目标区域可以是SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的任一者的全长或部分序列，也可以是OTOP2基因中其他包括CpG位点的区域。前述所列的第二目标区域可以和任何来源于KCNA3基因的第一目标区域组合，此时第一目标SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的任一者的全长或部分序列，也可以是KCNA3基因中其他包括CpG位点的区域。作为其中一个示例，第一目标区域包括核苷酸序列为SEQ ID NO.3所示的全长或部分序列；第二目标区域包括核苷酸序列为SEQ ID NO.12所示的全长或部分序列。
在一些实施方式中，第一目标区域选自SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4中的任一者；第二目标区域选自SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12中的任一者，二者的组合，或者三者的组合。
在另一些实施方式中，第一目标区域选自SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4中的任一者，二者的组合，三者的组合，或者全部四者的组合；第二目标区域选自SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12中的任一者。在又一些实施方式中，也可以是第一目标区域包括SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4中两者或更多个的组合，并且第二目标区域也包括SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12中两者或更多个的组合。
在另一些实施方式中，第一目标区域包括选自核苷酸序列为SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的至少一者的全长或部分序列；和/或第二目标区域包括选自核苷酸序列为SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的至少一者的全长或部分序列。可选地，前述所列的第一目标区域可以和任何来源于OTOP2基因的第二目标区域组合，此时第二目标区域可以是SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的任一者的全长或部分序列，也可以是OTOP2基因中其他包括CpG位点的区域。前述所列的第二目标区域可以和任何来源于KCNA3基因的第一目标区域组合，此时第一目标SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的任一者的全长或部分序列，也可以是KCNA3基因中其他包括CpG位点的区域。在该实施方式中，第一目标区域和第二目标区域也可以分别是一个或多个核苷酸序列的组合。
本公开一实施方式还提供一种用于食管癌诊断的检测试剂，该检测试剂包括检测基因中目标区域的甲基化水平的试剂，所检测的基因包括KCNA3基因和OTOP2基因。
该检测试剂所检测的基因及其目标区域具有如前所述的含义，在此不再赘述。
可选地，检测基因中目标区域的甲基化水平的试剂包括核酸分子，该核酸分子包括检测第一目标区域和第二目标区域的甲基化水平的引物对。
在一些实施方式中，试剂还包括与检测引物对对应的检测探针。该检测检测探针上连接有荧光基团和荧光淬灭基团。
在一些实施方式中，荧光探针的5'端含有荧光报告基团如FAM、HEX、VIC、CY5、ROX、Texsa Red、JOE及Quasar 705中任意一种，3'端含有荧光淬灭基团，如MGB、BHQ-1、BHQ-2、BHQ-3中的任意一种。
在一些实施方式中，检测第一目标区域和第二目标区域的甲基化水平的引物对如下：
第一目标区域的SEQ ID NO.1的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.15和SEQ ID NO.16，SEQ ID NO.1的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.17和SEQ ID NO.18；
第一目标区域的SEQ ID NO.2的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.19和SEQ ID NO.20，SEQ ID NO.2的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.21和SEQ ID NO.22；
第一目标区域的SEQ ID NO.3的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.23和SEQ ID NO.24，SEQ ID NO.3的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.25和SEQ ID NO.26；
第一目标区域的SEQ ID NO.4的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.27和SEQ ID NO.28，SEQ ID NO.4的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.29和SEQ ID NO.30；
第二目标区域的SEQ ID NO.12的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.31和SEQ ID NO.32，SEQ ID NO.12的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.33和SEQ ID NO.34；
第二目标区域的SEQ ID NO.6的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.35和SEQ ID NO.36，SEQ ID NO.6的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.37和SEQ ID NO.38；
第二目标区域的SEQ ID NO.7的甲基化上游引物(5’-3’)和甲基化下游引物(5’-3’)分别为SEQ ID NO.39和SEQ ID NO.40，SEQ ID NO.7的非甲基化上游引物(5’-3’)和非甲基化下游引物(5’-3’)分别为SEQ ID NO.41和SEQ ID NO.42。
在一些实施方式中，检测第一目标区域和第二目标区域的甲基化水平的引物对和所述检测探针的组合如下所示：
第一目标区域的SEQ ID NO.8的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.43、SEQ ID NO.44和SEQ ID NO.45；
第一目标区域的SEQ ID NO.9的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.46、SEQ ID NO.47和SEQ ID NO.48；
第一目标区域的SEQ ID NO.10的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.49、SEQ ID NO.50和SEQ ID NO.51；
第一目标区域的SEQ ID NO.11的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.52、SEQ ID NO.53和SEQ ID NO.54；
第二目标区域的SEQ ID NO.5的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.55、SEQ ID NO.56和SEQ ID NO.57；
第二目标区域的SEQ ID NO.13的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.58、SEQ ID NO.59和SEQ ID NO.60；
第二目标区域的SEQ ID NO.14的上游引物(5’-3’)、下游引物(5’-3’)和检测探针(5’-3’)分别为SEQ ID NO.61、SEQ ID NO.62和SEQ ID NO.63。
在本公开的一些实施方式中，可以采用利用桑格尔测序的方法评估靶基因组合的甲基化水平。例如，分别针对第一目标区域和第二目标区域提供甲基化引物对和非甲基化引物对，采用桑格尔测序法进行甲基化水平检测。在一些实施方式中，可以采用如上表1所示的引物对。
在另一些实施方式中，还可以利用甲基化荧光定量PCR的方法评估靶基因组合的甲基化水平。此时，可以分别针对第一目标区域和第二目标区域提供引物对和检测探针，对第一目标区域和第二目标区域的甲基化水平进行检测。作为示例，引物对和检测探针的组合如上表2所示。
在一些实施方式中，该试剂还包括检测内参基因的引物对和对应的检测探针。
在一些实施方式中，内参基因包括ACTB基因，ACTB基因对应SEQ ID NO.64和SEQ ID NO.65所示的(检测)引物对和SEQ ID NO.66所示的检测探针。
在一些实施方式中，待测的生物样本为食管组织样本、食管脱落细胞样本或血液样本；其中血液样本包括血浆、血清、全血、分离的血细胞或它们的组合。
本公开一实施方式还提供了一种诊断食管癌的试剂盒，包括上述检测试剂。
在一些实施方式中，试剂盒还包括核酸提取试剂、甲基化转化试剂、质控试剂、PCR反应试剂和测序试剂中的至少一种。可选地，甲基化转化试剂为亚硫酸氢盐。
在一些实施方式中，PCR反应试剂包括扩增缓冲液、dNTPs、DNA聚合酶和Mg 2+中的一种或者多种。
可以理解，本公开所提供的试剂盒还可以包括阴性/阳性参考品，或其他实现本公开目的或效果必要的试剂，例如一些必要的样本保存试剂。
本公开一实施方式还提供一种检测受试者是否患有食管癌或癌前病变的方法，包括：
提供源于受试者的生物样本；
检测该生物样本中KCNA3基因和OTOP2基因目标区域的甲基化水平，如果KCNA3基因和OTOP2基因中至少一者的甲基化水平升高，则该受试者患有食管癌或癌前病变的风险增加。
可选地，该方法基于前述方案实现。例如可以用如前所述的检测试剂或者试剂盒实现。
可选地，该生物样本为离体样本，包括但不限于食管组织样本、食管脱落细胞样本或血液样本。可选地，血液样本包括血浆、血清、全血、分离的血细胞或它们中的任何组合。
可选地，KCNA3基因和OTOP2基因中至少一者的甲基化水平升高，是指对受试者的生物样本检测KCNA3基因和OTOP2基因的甲基化水平，结果与正常人群的平均甲基化水平相比，是更高的。作为示例，可以采用ct值表示基因甲基化的相对水平。
本公开提供了OTOP2基因以及KCNA3和OTOP2两者基因的甲基化作为食管癌诊断标志物的应用，通过检测OTOP2基因，或者KCNA3和OTOP2两者基因的甲基化水平，可以对食管癌进行特异性诊断或辅助诊断。
同时本公开还提供了检测OTOP2基因以及KCNA3和OTOP2两者基因目标区域的甲基化水平的试剂以及诊断试剂盒，该试剂和试剂盒诊断食管癌均具有灵敏度高、特异性好的特性，对于提高食管癌早期诊疗效果和降低死亡率均有重要的临床应用价值和社会意义，为食管癌和早期食管癌的无创诊断提供了新的思路。
发明人在本研究中发现，使用甲基化特异性荧光定量PCR的方法，通过检测血液样本中KCNA3基因和OTOP2基因目标区域的甲基化水平，可以有效区分食管癌患者和健康人，其诊断食管低级别瘤变、高级别瘤变、早期食管癌、进展期食管癌的灵敏度可达到76.67％、89.19％、93.33％和96.0％，其在健康人群中的特异性可达到95％。本公开提供的技术方案可实现食管癌的无创或微创诊断，且诊断灵敏度高、特异性好，有利于提高食管癌前病变和食管癌的检出率。
实施例
以下，结合实施例对本公开的方案进行进一步说明。
(一)单靶标的食管癌检测实施例
实施例1
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合1。
核酸组合1包括SEQ ID NO.67-69所示的核苷酸。该核酸组合1可检测OTOP2基因上Chr17:74923881-74924015区域正链(目的区域1)的甲基化水平。
区域1甲基化特异性PCR的上游引物序列为(5’-3’)：
TAGGAGTATAGGATTTAGGGTGCGT(SEQ ID NO.67)；
区域1甲基化特异性PCR的下游引物序列为(5’-3’)：
TAATACCCACCGCGCCCTAAC(SEQ ID NO.68)；
区域1甲基化特异性PCR的检测探针序列为(5’-3’)：
TTGGAGGGCGTTAGGGTGTTCGT(SEQ ID NO.69)。
实施例2
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合2。
核酸组合2包括SEQ ID NO.70-72所示的核苷酸。该核酸组合2可检测OTOP2基因上Chr17:74924159-74924335区域正链(目的区域2)的甲基化水平。
区域2甲基化特异性PCR的上游引物序列为(5’-3’)：
TTGGAGAAAGATGGATTATGTGAGC(SEQ ID NO.70)；
区域2甲基化特异性PCR的下游引物序列为(5’-3’)：
ACCTAACTAAACTTAAAACCGACGC(SEQ ID NO.71)；
区域2甲基化特异性PCR的检测探针序列为(5’-3’)：
AAAGCGTTTGTTTATTTCGGCGTGG(SEQ ID NO.72)。
实施例3
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合3。
核酸组合3包括SEQ ID NO.73-75所示的核苷酸。该核酸组合3可检测OTOP2基因上Chr17:74924422-74924581区域正链(目的区域3)的甲基化水平。
区域3甲基化特异性PCR的上游引物序列为(5’-3’)：
TTATTTAGCGAGAGGGGTAGGTTTC(SEQ ID NO.73)；
区域3甲基化特异性PCR的下游引物序列为(5’-3’)：
CAAAACTCCAAATCAACCCTAACGA(SEQ ID NO.74)；
区域3甲基化特异性PCR的检测探针序列为(5’-3’)：
CGAAGTTTTAATTTTGCGGGTTAGG(SEQ ID NO.75)。
实施例4
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合4。
核酸组合4包括SEQ ID NO.76-78所示的核苷酸。该核酸组合4可检测OTOP2基因上Chr17:74924603-74924449区域负链(目的区域4)的甲基化水平。
区域4甲基化特异性PCR的上游引物序列为(5’-3’)：
ATTATTGTAGGGGAGGAGCG(SEQ ID NO.76)；
区域4甲基化特异性PCR的下游引物序列为(5’-3’)：
ATTTTCTCTTCCCCTTTCCCAACG(SEQ ID NO.77)；
区域4甲基化特异性PCR的检测探针序列为(5’-3’)：
TGGGGATTAGGGAGTTTTTGATTCG(SEQ ID NO.78)。
实施例5
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合5。
核酸组合5包括SEQ ID NO.79-81所示的核苷酸。该核酸组合5可检测OTOP2基因上Chr17:74924425-74924297区域负链(目的区域5)的甲基化水平。
区域5甲基化特异性PCR的上游引物序列为(5’-3’)：
ATGGGAAGAAAGGAGAAGCG(SEQ ID NO.79)；
区域5甲基化特异性PCR的下游引物序列为(5’-3’)：
TAACCCCCAACTCAACGCC(SEQ ID NO.80)；
区域5甲基化特异性PCR的检测探针序列为(5’-3’)：
TAAGTTCGATTGTTTTTCGGTTCGG(SEQ ID NO.81)。
实施例6
本实施例提供用于食管癌或癌前病变的诊断或辅助诊断的试剂，其包括核酸组合6。
核酸组合6包括SEQ ID NO.82-84所示的核苷酸。该核酸组合6可检测OTOP2基因上Chr17:74924080-74923981区域负链(目的区域6)的甲基化水平。
区域6甲基化特异性PCR的上游引物序列为(5’-3’)：
TTATTTCGGTGCGGTTAGGTTC(SEQ ID NO.82)；
区域6甲基化特异性PCR的下游引物序列为(5’-3’)：
CTAAAATACTACGCGCCAAAACG(SEQ ID NO.83)；
区域6甲基化特异性PCR的检测探针序列为(5’-3’)：
TTTCGTATTGCGGTATTTAGAGGGT(SEQ ID NO.84)。
实施例7
本实施例采用甲基化特异性PCR的方法，检测OTOP2基因的CpG岛的6段甲基化区域在食管癌样本、食管高级别瘤变样本和正常样本中的甲基化状态，计算灵敏度和特异性。步骤包括：
(1)样本DNA提取
福尔马林固定、石蜡包埋的组织样本：采用QIAamp DNA FFPE Tissue Kit提取组织DNA，操作参见试剂盒说明书。
血浆样本：采用天根生化科技(北京)有限公司的磁珠法血清/血浆游离DNA提取试剂盒(DP709)进行血浆cfDNA提取，操作参见试剂盒说明书。
(2)亚硫酸氢盐转化
所用核酸转化试剂盒为ZYMO RESEARCH的EZ DNA Methylation-Gold Kit，实验操作参见试剂盒说明书。在本过程中，未甲基化的胞嘧啶(C)被转化成尿嘧啶(U)，甲基化的胞嘧啶不变，尿嘧啶在后续的PCR步骤中与腺嘌呤(A)配对，胞嘧啶与鸟嘌呤(G)配对，以此实现甲基化与未甲基化序列的区分。
(3)甲基化特异性PCR
对CpG岛内的6个区域进行甲基化检测，各个区域的检测试剂包括一对甲基化序列特异性检测引物和一条特异性taqman探针。6个区域在染色体上的位置及PCR引物、探针序列如表3所示。
表3 OTOP2基因CpG岛内6个区域的甲基化引物探针序列表
Figure PCTCN2022131724-appb-000004
将步骤(2)亚硫酸氢盐转化后的DNA分别进行甲基化特异性PCR反应以检测OTOP2基因区域1-6的甲基化状态，每个区域单独进行检测，即一个PCR管中每次只加入一个区域的检测引物和探针，同时加入内参基因的检测探针。
ACTB作为内参基因，其中ACTB上游引物为：AAGGTGGTTGGGTGGTTGTTTTG(SEQ ID NO.85)；ACTB下游引物为：AATAACACCCCCACCCTGC(SEQ ID NO.86)；ACTB探针为：GGAGTGGTTTTTGGGTTTG(SEQ ID NO.87)。
采用Platinum II Taq热启动DNA聚合酶(货号14966001)进行PCR扩增，PCR反应液配置体系如表4所示。
表4 基于表3中序列的PCR反应体系
Figure PCTCN2022131724-appb-000005
如表4所示，在检测OTOP2区域1-区域6任一区域在样本中的甲基化状态时，只需将某一区域对应的引物探针、ACTB引物探针、缓冲液、dNTP、DNA酶和样本DNA等按照表中的体积加入到反应体系中。每个样本设置三个复孔。
PCR反应条件如下表5所示。
表5 基于表3中序列的PCR反应程序
Figure PCTCN2022131724-appb-000006
(4)质量控制：
在每次检测时对阴性对照和阳性对照进行同步检测。
阴性对照为纯化水。
阳性对照的制备方法为：将ACTB的扩增区域对应的经亚硫酸氢盐转化后的序列(即扩增区域中除CG二核苷酸中的C保持不变外，其余位置的C均转换成T，其他三种碱基T、G和A保持不变)进行人工合成，并克隆至载体pUC57上，形成人工合成质粒。将完全甲基化的区域1-6对应的经亚硫酸氢盐转化后的序列(即分别将各区域中除CG二核苷酸中的C保持不变外，其余位置的C均转换成T，其他三种碱基T、G和A保持不变)进行人工合成，并克隆至载体pUC57上，形成人工合成质粒。区域1-6的阳性对照为10 3拷贝/微升的ACTB人工合成质粒和10 3拷贝/微升的区域1-6的人工合成质粒按体积比1：1混合而成，如区域1的阳性对照为10 3拷贝/微升的ACTB人工合成质粒和10 3拷贝/微升的区域1的人工合成质粒按体积比1：1混合而成。
阴性对照要无扩增，阳性对照要有明显的指数增长期，待检样本的内参基因的Ct值应≤35，阴性对照、阳性对照及内参基因均满足上述要求后，表明本次实验有效，可进行下一步样本结果的判定。否则，当次实验无效，须重新进行检测。
(5)PCR数据分析
Ct值读取：PCR完成后，调整基线，将一次PCR中样本最小Ct值提前1-2个循环前的荧光值设置为基线值，将阈值设置在S型扩增曲线的拐点处，PCR软件会自动给出每个样本的Ct值，读取每个样本待检测目的区域的Ct值和内参基因ACTB的Ct值。
结果分析和判读方法：若一孔中待检区域的Ct值≤40时，则认为该区域在该孔中被检出甲基化，当三个复孔中至少有两孔被检出甲基化时，则判定该区域在该样本中为甲基化阳性，否则为甲基化阴性。计算区域1-6在食管癌前病变样本、食管癌样本中的灵敏性，计算各区域在癌旁样本或正常样本中的特异性，其中，灵敏性和特异性的计算公式如下：
灵敏性(％)＝甲基化阳性个数/(食管癌前病变或食管癌样本总数)×100％   (1)
特异性(％)＝甲基化阴性个数/(癌旁样本或正常样本总数)×100％   (2)
实验例1
于郑州市某医院收集24例食管癌及其癌旁组织样本，所有样本均为福尔马林固定、石蜡包埋的组织样本，采用实施例2提供的方法依次进行组织样本DNA提取、对提取到的DNA进行亚硫酸氢盐转化和甲基化特异性PCR，然后分别检测区域1-6在24例食管癌组织样本和24例癌旁样本中的甲基化状态，计算灵敏度和特异性，结果如表6所示。
表6 区域1-6在食管癌组织样本和癌旁样本中的检测结果
Figure PCTCN2022131724-appb-000007
如表6所示，区域1-6对食管癌组织样本的检测灵敏度均在80％以上，特异性均大于95％。表明这6个区域均能有效地区分食管癌样本和癌旁样本。此外，相比于区域1、3、5和6，区域2和区域4的检测性能进一步提高，二者的灵敏度均在90％以上。
实验例2
于郑州市某医院收集67例食管癌血浆样本、45例食管高级别瘤变血浆样本和54例健康人血浆样本。采用实施例1提供的方法依次进行血浆样本DNA提取、对提取到的DNA进行亚硫酸氢盐转化和甲基化特异性PCR，分别检测区域1-6在食管癌样本、食管高级别瘤变样本和健康样本的甲基化状态，计算灵敏度和特异性，结果如表7所示。
表7 区域1-6在血浆样本中的检测结果
Figure PCTCN2022131724-appb-000008
如表7所示，区域1-6在健康血浆样本中均为甲基化阴性，检测特异性均为100％，在食管癌血浆样本中的检测灵敏性在55％以上，对食管高级别瘤变血浆样本的检测灵敏性在30％以上。此外，相比于区域1、3、5和6，区域2和区域4的检测灵敏性进一步提高，二者对食管癌样本的检测灵敏性均大于65％，对食管高级别瘤变样本的检测灵敏性均大于40％。
(二)双靶标的食管癌检测实施例
本公开的双靶标的食管癌检测实施例采用两种不同的方法来验证甲基化分子标志物的组合诊断食管癌的效果：实施例8展示了利用桑格尔测序的方法评估分子标志物组合的甲基化水平，进而判断组织样本、血液样本是否为癌症样本；实施例9展示了利用甲基化荧光定量PCR的方法评估分子标志物组合的甲基化水平，进而判断组织样本、血液样本是否为癌症样本。
实施例8亚硫酸氢盐测序法评估目标基因的甲基化水平
通过检测KCNA3基因中核苷酸序列如SEQ ID NO.1～4所示的至少一个序列和OTOP2基因中核苷酸序列如SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一个序列的甲基化状态来分析该试剂盒用于食管癌诊断或辅助诊断的灵敏度及特异性。
本实施例的主要流程包括：
1)从食管癌前病变或癌症患者或健康人中获取组织样本及血液样本，并根据不同的样本类型提取、转化、纯化模板DNA；2)使用测序引物对分别扩增SEQ ID NO.1-4中至少一个区域和SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12中至少一个区域，扩增产物进行桑格尔测序，进而判断各区域的甲基化状态；3)根据各区域的甲基化状态，计算以组合物作为甲基化标志物来诊断组织样本、血液样本的灵敏度及特异性。步骤如下：
1、样本收集、处理
于武汉某医院收集经病理组织活检确诊为低级别食管鳞状上皮内瘤变(LGIN)的食管增生组织32例，经病理活检确诊为高级别食管鳞状上皮内瘤变(HGIN)的食管增生组织48例，早期食管鳞癌组织68例，进展期食管鳞癌组织80例及无病变的癌旁组织79例，所有样本均为福尔马林浸泡、石蜡包埋的组织样本。所有样本的收集过程经过伦理委员会的审批，所有志愿者都签署了知情同意书，所有样本均采用匿名化处理。
于郑州某医院收集经病理组织活检确诊为低级别食管鳞状上皮内瘤变(LGIN)患者的血液样本60例、高级别食管鳞状上皮内瘤变(HGIN)患者的血液样本74例，早期食管癌患者的血液样本120例、进展期食管鳞癌患者的血液样本150例以及健康人的血液样本100例，所有样本的收集过程经过伦理委员会的审批，所有志愿者都签署了知情同意书，所有样本均采用匿名化处理。
2、DNA模板的提取
对于组织样本，使用QIAamp DNA FFPE Tissue Kit(56404)提取DNA，操作按照试剂盒说明书进行。对于血液样本，使用天根生化科技(北京)有限公司的磁珠法血清/血浆游离DNA(cfDNA)提取试剂盒(DP709)进行血浆cfDNA提取，操作按照试剂盒说明书进行。
3、亚硫酸氢盐的转化
将提取好的样本基因组进行亚硫酸氢盐转化，所用核酸转化试剂盒为武汉艾米森生命科技有限公司核酸纯化试剂(鄂汉械备20200843)，实验操作参见试剂盒说明书。
4、PCR扩增及测序
对于KCNA3基因的核苷酸序列SEQ ID NO.1～4和OTOP2基因的核苷酸序列SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12，分别设计甲基化引物对和非甲基化引物对扩增相应的区域，并筛选最佳的甲基化引物对和非甲基化引物对的配比，以保证当模板中存在大于等于1％的甲基化序列时，即可扩增得到甲基化的产物，仅当模板为非甲基化序列时，扩增产物为非甲基化产物。
PCR扩增时使用的甲基化引物对和非甲基化引物对如表8所示。按照表9中的配方配置PCR反应体系，PCR扩增程序同表10，在PCR扩增结束后，使用混合引物(包含甲基化引物对和非甲基化引物对)对扩增产物进行桑格尔测序(送测序公司)，同时从5’端和3’端测序。
表8 各目标区域的甲基化引物和非甲基化引物序列
Figure PCTCN2022131724-appb-000009
Figure PCTCN2022131724-appb-000010
表9 基于表8中序列的PCR反应体系
组分 用量(μL)
10×Taq buffer(Mg 2+Free) 5
25mM Mg 2+ 4
dNTP Mix(10mM each) 1
甲基化上游引物(10μM) 1
甲基化下游引物(10μM) 1
非甲基化上游引物(10μM) 0.5
非甲基化下游引物(10μM) 0.5
热启动Taq DNA聚合酶 0.5
模板DNA 10
超纯水 补至50
表10 基于表8中序列的PCR反应程序
Figure PCTCN2022131724-appb-000011
5、结果分析
根据测序峰图对每个样本各个扩增子中的CpG位点的甲基化情况进行分析。一个CpG二核苷酸中胞嘧啶的甲基化情况分为两种，即非甲基化和甲基化，其中甲基化又分为完全甲基化和部分甲基化。若CpG二核苷酸中胞嘧啶测序结果为胸腺嘧啶，则其为非甲基化的。若CpG二核苷酸中胞嘧啶测序结果仍然为胞嘧啶，则其为完全甲基化的。若CpG二核苷酸中胞嘧啶测序结果既有胞嘧啶也有胸腺嘧啶(双峰)，则其为部分甲基化的。
如果一个扩增子中95％以上的CpG二核苷酸中胞嘧啶是甲基化的，则认为此样本在该基因区域是甲基化阳性的。计算各类样本在每个区域中的甲基化阳性数目和甲基化阴性数目。
若某个样本在KCNA3基因的至少一个区域或OTOP2基因的至少一个区域为甲基化阳性，则认为该样本为癌前病变阳性或癌症阳性样本；当某个样本在KCNA3基因的至少一个区域且在OTOP2基因的至少一个区域为甲基化阴性，则认为该样本为癌前病变阴性且癌症阴性样本。
KCNA3基因中核苷酸序列如SEQ ID NO.1～4所示的至少一个区域和OTOP2基因中核苷酸序列如SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一个区域在食管癌前病变和食管癌患者组织样本中的甲基化状态、检测灵敏度和特异性如表11所示；其在食管癌前病变和食管癌患者血液样本中的甲基化状态、检测灵敏度和特异性如表12所示(下文中组合物序列编号中“+”表示同时检测“+”前后的两个序列的甲基化水平，例如SEQ ID NO.1+12表示同时检测SEQ ID NO.1和SEQ ID NO.12的甲基化水平)。
表11 KCNA3和OTOP2基因至少一个区域在组织样本中的检测结果
Figure PCTCN2022131724-appb-000012
Figure PCTCN2022131724-appb-000013
表12 KCNA3和OTOP2基因至少一个区域在血液样本中的检测结果
Figure PCTCN2022131724-appb-000014
Figure PCTCN2022131724-appb-000015
由表11和表12可以看出，利用KCNA3基因中核苷酸序列如SEQ ID NO.1-4所示的至少一个区域和OTOP2基因中核苷酸序列如SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一个区域的组合物的甲基化水平检测食管癌前病变和食管癌的效果良好。
在组织样本中，通过检测各类组合物的甲基化水平，检测食管低级别瘤变的灵敏度最低为40.63％，检测食管低级别瘤变的最高灵敏度为87.50％；检测食管高级别瘤变的灵敏度最低为54.17％，检测食管高级别瘤变的最高灵敏度为95.83％；检测早期食管癌的最低灵敏度为72.06％，检测早期食管癌的最高灵敏度为97.06％；检测进展期食管癌的最低灵敏度为78.75％，检测进展期食管癌的最高灵敏度为98.75％；其检测癌旁组织的特异性最低为70.89％，其检测癌旁组织的特异性最高为94.94％。
在血液样本中，通过检测各类组合物的甲基化水平，检测食管低级别瘤变的灵敏度最低为38.33％，检测食管低级别瘤变的最高灵敏度为83.33％；检测食管高级别瘤变的灵敏度最低为63.51％，检测食管高级别瘤变的最高灵敏度为94.59％；检测早期食管癌的最低灵敏度为71.67％，检测早期食管癌的最高灵敏度为95.83％；检测进展期食管癌的最低灵敏度为76.67％，检测进展期食管癌的最高灵敏度为97.33％；其检测健康人血液样本的特异性最低为70％，其检测健康人血液样本的特异性最高为96％。
综上可以看出，随着组合物中检测区域数量的增加，该组合物检测的灵敏度有增加的趋势，但是其特异性也有降低的趋势。综合考虑，当组合物中检测区域为2个时，其灵敏度和特异性可以均达到较高水平，如以SEQ ID NO.3与SEQ ID NO.12的组合作为甲基化标志物。
实施例9甲基化特异性荧光定量PCR法评估目标基因的甲基化水平
通过检测KCNA3基因中核苷酸序列如SEQ ID NO.8～11所示的任一个序列和OTOP2基因中核苷酸序列如SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的任一个序列的甲基化状态来分析该试剂盒用于食管癌诊断或辅助诊断的灵敏度及特异性。该实施例的主要流程包括：1)从食管癌癌前病变或癌症患者或健康人中获取组织样本及血液样本，并根据不同的样本类型提取、转化、纯化模板DNA；2)使用检测引物对及探针通过甲基化荧光定量PCR法检测样本中各组合物的甲基化状态，进而判断样本是否为癌症样本；3)计算以组合物作为甲基化标志物来诊断组织样本、血液样本的灵敏度及特异性。考虑到2种以上的标记物序列的组合会导致检测的特异性降低，因此在实施例9中仅使用两种标记物序列的组合方式。步骤如下：
1、样本收集、处理
临床样本的收集、模板DNA的提取、转化和纯化同实施例8。
2、甲基化荧光定量PCR反应
将各样本经亚硫酸氢盐转化后的DNA分别进行甲基化荧光定量PCR反应以检测各个样本中KCNA3基因中核苷酸序列如SEQ ID NO.8～11所示的任一个序列和OTOP2基因中核苷酸序列如SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的任一个序列的甲基化状态。
每种组合方式单独进行检测，即一个PCR管中除加入必须的反应成分、模板以外，需加入SEQ ID NO.8～11中任一个区域的检测引物对、探针，和SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14中任一个区域的检测引物对、探针；同时还需加入内参基因ACTB的检测引物对和探针。检测目标区域的探针为Taqman探针，SEQ ID NO.8～11等序列的检测探针5’端的报告基团为FAM，3’端猝灭基团为MGB；SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14等序列的检测探针5’端的报告基团为ROX，3’端猝灭基团为MGB；ACTB探针5’端的报告基团为VIC，3’端猝灭基团为BHQ1。KCNA3基因中核苷酸序列如SEQ ID NO.8～11和OTOP2基因中核苷酸序列如SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14及内参基因ACTB的上下游检测引物及探针序列如表13所示。
采用Invitrogen Platinum II Taq热启动DNA聚合酶进行PCR扩增，PCR反应液配置体系如表14所示，按照表15展示的扩增程序进行PCR扩增。利用表13中的引物对及对应的探针可以检测的SEQ ID NO.8～11、SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14等序列中CpG位点如表16所示。
表13 检测引物对及探针序列
Figure PCTCN2022131724-appb-000016
Figure PCTCN2022131724-appb-000017
表14 基于表13中序列的PCR反应体系
Figure PCTCN2022131724-appb-000018
表15 基于表13中序列的PCR反应程序
Figure PCTCN2022131724-appb-000019
Figure PCTCN2022131724-appb-000020
表16 检测引物对及探针可检测的CpG二核苷酸中胞嘧啶的甲基化位点
Figure PCTCN2022131724-appb-000021
阴性对照和阳性对照：在按照不同的组合方式检测样本时，阴性对照和阳性对照也应同时进行检测，阴性对照管的DNA模板为TE缓冲液。阳性对照管的DNA模板的制备方法为：将ACTB基因扩增区域对应的经亚硫酸氢盐完全转化后的序列进行人工合成，并克隆至载体pUC57上，形成人工合成质粒；将目标区域如SEQ ID NO.8～11、SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14各自对应的经亚硫酸氢盐转化后的序列进行人工合成，并分别克隆至载体pUC57，形成人工合成质粒。阳性对照DNA模板为10 3拷贝/微升的含转化后ACTB的人工合成质粒、10 3拷贝/微升的含转化后SEQ ID NO.8～11中一种序列的人工合成质粒和10 3拷贝/微升的含转化后SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14中一种序列的人工合成质粒等比例混合而成。
Ct值读取：PCR完成后，调整基线，将一次PCR中样本最小Ct值提前1-2个循环前的荧光值设置为基线值，将阈值设置在S型扩增曲线的拐点处，得到样本各个基因的Ct值。
质量控制：阴性对照要无扩增，阳性对照要有明显的指数增长期，且阳性对照各基因的Ct值应在26-30之间。待检样本的内参基因的Ct值应≤35，阴性对照、阳性对照及内参基因均满足上述要求后，表明本次实验有效，可进行下一步样本结果的判定。否则，当次实验无效，须重新进行检测。
3、PCR结果分析
根据KCNA3基因、OTOP2基因的Ct值来判断待测样本的甲基化水平。对于组织样本，若扩增某一基因区域的Ct值≤38，则认为该样本中此基因为甲基化阳性，若扩增某一基因区域的Ct值＞38，则认为该样本中此基因为甲基化阴性。对于血液样本，若扩增某一基因区域的Ct值≤45，则认为该样本中此基因为甲基化阳性，若扩增某一基因区域的Ct值＞45，则认为该样本中此基因为甲基化阴性。若待测样本中至少一个基因为甲基化阳性，则该样本为癌前病变阳性或癌症阳性样本，仅当待测样本中两个基因均为甲基化阴性，则该样本为癌前病变阴性且癌症阴性样本。判断标准如表17所示。
KCNA3基因中核苷酸序列如SEQ ID NO.8～11所示的任一个区域和OTOP2基因中核苷酸序列如SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的任一个区域的组合物在食管癌前病变和食管癌患者组织样本中的甲基化状态、检测灵敏度和特异性如表18所示，其在食管癌前病变和食管癌患者血液样本中的甲基化状态、检测灵敏度和特异性如表19所示。
表17 荧光定量法中样本的判断标准
Figure PCTCN2022131724-appb-000022
Figure PCTCN2022131724-appb-000023
表18 KCNA3和OTOP2基因组合物在癌前病变、癌变、癌旁组织样本的检测结果
Figure PCTCN2022131724-appb-000024
表19 KCNA3和OTOP2基因组合物在癌前病变、癌症、健康血液样本的检测结果
Figure PCTCN2022131724-appb-000025
Figure PCTCN2022131724-appb-000026
由表18和表19可以看出，利用甲基化荧光定量PCR法，通过检测KCNA3基因中核苷酸序列如SEQ ID NO.8～11所示的任一个区域和OTOP2基因中核苷酸序列如SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的任一个区域的组合物的甲基化水平诊断食管癌前病变、食管癌患者的效果良好。
在组织样本中，通过检测各类组合物的甲基化水平，其诊断食管低级别瘤变的灵敏度最低为43.75％，诊断食管低级别瘤变的最高灵敏度为81.25％；诊断食管高级别瘤变的灵敏度最低为52.08％，诊断食管高级别瘤变的最高灵敏度为91.67％；诊断早期食管癌的最低灵敏度为72.06％，诊断早期食管癌的最高灵敏度为94.12％；诊断进展期食管癌的最低灵敏度为80.00％，诊断进展期食管癌的最高灵敏度为96.25％；其检测癌旁组织的特异性最低为77.22％，其检测癌旁组织的特异性最高为93.67％。
在血液样本中，通过检测各类组合物的甲基化水平，诊断食管低级别瘤变的灵敏度最低为46.67％，检测食管低级别瘤变的最高灵敏度为76.67％；检测食管高级别瘤变的灵敏度最低为51.35％，检测食管高级别瘤变的最高灵敏度为89.19％；检测早期食管癌的最低灵敏度为73.33％，检测早期食管癌的最高灵敏度为93.33％；检测进展期食管癌的最低灵敏度为73.33％，检测进展期食管癌的最高灵敏度为96.0％；其检测健康人血液样本的特异性最低为81.00％，其检测健康人血液样本的特异性最高为95％。
综上，选择KCNA3基因中核苷酸序列为SEQ ID NO.10和OTOP2基因中核苷酸序列为SEQ ID NO.5作为甲基化标志物的组合时，其检测组织样本和血液样本的灵敏度和特异性可以达到进一步的提高。
以上已对本公开创造的较佳实施例进行了具体说明，但本公开创造并不限于所述实施例，熟悉本领域的技术人员在不违背本公开创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本公开权利要求所限定的范围内。
工业实用性
本公开提供了以OTOP2基因或者以KCNA3和OTOP2两者基因的甲基化作为食管癌诊断标志物的应用，还提供了检测OTOP2基因或者KCNA3和OTOP2两者基因的目标区域的甲基化水平的试剂以及诊断试剂盒，该试剂和试剂盒诊断食管癌的灵敏度高、特异性好，有利于提高食管癌前病变和食管癌的检出率，为食管癌和早期食管癌的无创诊断提供了新的思路，因此具有优异的实用性能和广阔的市场前景。

Claims (20)

  1. 检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，其特征在于，所述目标区域为OTOP2基因CpG岛。
  2. 根据权利要求1所述的应用，其特征在于，所述目标区域为OTOP2基因中Chr17:74922901-74924924 CpG岛区域中的全长区域或部分区域。
  3. 根据权利要求2所述的应用，其特征在于，所述目标区域的甲基化水平为OTOP2基因中如下至少一种CpG岛区域中的全长区域或部分区域的甲基化水平：区域1、区域2、区域3、区域4、区域5和区域6；其中，所述区域1选自Chr17:74923881-7492401 5正链，所述区域2选自Chr17:74924159-74924335正链，所述区域3选自Chr17:74924422-74924581正链，所述区域4选自Chr17:74924603-74924449负链，所述区域5选自Chr17:74924425-74924297负链，所述区域6选自Chr17:74924080-74923981负链。
  4. 根据权利要求3所述的应用，其特征在于，所述试剂或试剂盒包括用于检测OTOP2基因的CpG岛的甲基化水平的核酸组合。
  5. 根据权利要求4所述的应用，其特征在于，所述核酸组合选自如下核酸组合中的至少一种：用于检测所述区域1的核酸组合1、用于检测所述区域2的核酸组合2、用于检测所述区域3的核酸组合3、用于检测所述区域4的核酸组合4、用于检测所述区域5的核酸组合5和用于检测所述区域6的核酸组合6；
    所述核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，所述核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，所述核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，所述核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，所述核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，所述核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
  6. 检测基因中目标区域的甲基化水平的试剂在制备食管癌诊断试剂或试剂盒中的应用，其特征在于，所述目标区域包括属于KCNA3基因的第一目标区域和属于OTOP2基因的第二目标区域。
  7. 根据权利要求6所述的应用，其特征在于，
    所述第一目标区域包括选自核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的至少一者的全长或部分序列；和/或
    所述第二目标区域包括选自核苷酸序列为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一者的全长或部分序列。
  8. 根据权利要求6所述的应用，其特征在于，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的至少一者的全长或部分序列；和/或
    所述第二目标区域包括选自核苷酸序列为SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的至少一者的全长或部分序列。
  9. 根据权利要求7所述的应用，其特征在于，所述第一目标区域包括核苷酸序列为SEQ ID NO.3所示的全长或部分序列；所述第二目标区域包括核苷酸序列为SEQ ID NO.12所示的全长或部分序列。
  10. 一种试剂，其特征在于，其包括如下核酸组合的至少一种：核酸组合1、核酸组合2、核酸组合3、核酸组合4、核酸组合5和核酸组合6；所述核酸组合1的碱基序列与SEQ ID NO.67-69所示的碱基序列具有至少90％的一致性，所述核酸组合2的碱基序列与SEQ ID NO.70-72所示的碱基序列具有至少90％的一致性，所述核酸组合3的碱基序列与SEQ ID NO.73-75所示的碱基序列具有至少90％的一致性，所述核酸组合4的碱基序列与SEQ ID NO.76-78所示的碱基序列具有至少90％的一致性，所述核酸组合5的碱基序列与SEQ ID NO.79-81所示的碱基序列具有至少90％的一致性，所述核酸组合6的碱基序列与SEQ ID NO.82-84所示的碱基序列具有至少90％的一致性。
  11. 一种用于食管癌诊断的检测试剂，其特征在于，
    所述检测试剂包括检测属于KCNA3基因的第一目标区域和属于OTOP2基因的第二目标区域的甲基化水平的试剂。
  12. 根据权利要求11所述的检测试剂，其特征在于，
    所述第一目标区域包括选自核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4所示的至少一者的全长或部分序列；和/或
    所述第二目标区域包括选自核苷酸序列为SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.12所示的至少一者的全长或部分序列。
  13. 根据权利要求11所述的检测试剂，其特征在于，所述第一目标区域包括选自核苷酸序列为SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11所示的至少一者的全长或部分序列；和/或所述第二目标区域包括选自核苷酸序列为SEQ ID NO.5、SEQ ID NO.13、SEQ ID NO.14所示的至少一者的全长或部分序列。
  14. 根据权利要求12所述的检测试剂，其特征在于，所述检测试剂包括检测选自第一目标区域的核苷酸序列为SEQ ID NO.3所示和选自第二目标区域的核苷酸序列为SEQ ID NO.12所示的全长或部分序列的甲基化水平的试剂。
  15. 根据权利要求11-14任一项所述的检测试剂，其特征在于，所述检测试剂包括核酸分子，所述核酸分子包括检测所述第一目标区域和第二目标区域的甲基化水平的引物对。
  16. 根据权利要求15所述的检测试剂，其特征在于，所述检测试剂还包括与所述引物对对应的检测探针，所述检测探针上连接有荧光基团和荧光淬灭基团。
  17. 根据权利要求15所述的检测试剂，其特征在于，所述检测第一目标区域和第二目标区域的甲基化水平的引物对如下表所示，
    Figure PCTCN2022131724-appb-100001
    Figure PCTCN2022131724-appb-100002
  18. 根据权利要求16所述的检测试剂，其特征在于，所述检测第一目标区域和第二目标区域的甲基化水平的引物对和所述检测探针的组合如下表所示，
    Figure PCTCN2022131724-appb-100003
  19. 一种诊断食管癌的试剂盒，其特征在于，所述试剂盒包括权利要求10所述的试剂或者权利要求11-18任一项所述的检测试剂。
  20. 根据权利要求19所述的试剂盒，其特征在于，所述试剂盒还包括核酸提取试剂、甲基化转化试剂、质控试剂、PCR反应试剂和测序试剂中的至少一种。
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