CN117604095A

CN117604095A - Methylation detection reagent and kit for esophageal cancer diagnosis

Info

Publication number: CN117604095A
Application number: CN202211009579.9A
Authority: CN
Inventors: 周俊; 董兰兰; 张良禄; 熊杨辉; 郑义慧; 赵巴丽; 黄越
Original assignee: Wuhan Aimisen Life Technology Co ltd
Current assignee: Wuhan Aimisen Life Technology Co ltd
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2024-02-27

Abstract

The application relates to a methylation detection reagent and a kit for diagnosing esophageal cancer. By detecting the methylation level of the gene target region, the esophageal cancer pre-lesion and esophageal cancer patients can be effectively distinguished, the detection sensitivity is high, the specificity is good, and a novel technical scheme is provided for noninvasive detection of esophageal cancer or precancerous lesions.

Description

Methylation detection reagent and kit for esophageal cancer diagnosis

Technical Field

The application belongs to the technical field of medical biological detection, relates to a diagnosis marker of esophageal cancer, and in particular relates to a methylation detection reagent and a kit for esophageal cancer diagnosis.

Background

Esophageal cancer is one of the most common malignant tumors in the world, and is also the most common malignant tumor of the digestive tract. Esophageal cancer is highly invasive and often results in a poor prognosis. The statistics show that about 57 ten thousand new cases of esophageal cancer and about 50 ten thousand dead cases are seen in 2018 worldwide, and the morbidity and the mortality of the new cases are respectively located at the 7 th and the 6 th positions in all malignant tumors. Depending on the pathological typing of the tumor, esophageal cancer mainly comprises two histological subtypes: esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC). Esophageal squamous cell carcinoma is the most common cancer species in china, accounting for about 90% of the total number of esophageal cancer cases. China is one of the areas of high incidence of esophageal cancer and has more than half of esophageal cancer patients worldwide. Since symptoms of esophageal cancer are not apparent in the early stages of cancer occurrence and a convenient screening means is lacking, most patients have already developed middle and advanced esophageal cancer at the time of diagnosis. According to statistics, the overall survival rate of 5 years of esophageal cancer patients in China is about 30%, the survival rate of 5 years of middle and late stage esophageal cancer patients is lower than 10%, and the survival rate of 5 years of early stage esophageal cancer patients is higher than 90%. Therefore, the early diagnosis rate of patients with esophagus cancer or high-risk groups is improved, and the method has important significance for improving the life quality of patients and prolonging the life cycle.

At present, the screening strategy of the high-incidence area of the esophageal cancer in China is a biopsy tissue pathological diagnosis technology combination method based on endoscopic iodine staining, and is a gold standard for diagnosing the esophageal cancer. However, since the operation difficulty of the endoscopy is relatively high, the technical level requirement on doctors is high, and the patients have a certain fear of the endoscopy, and the compliance of the patients is poor. Therefore, the search of biomarkers with high sensitivity and high specificity in the development and metastasis process of esophageal cancer at the gene level can be helpful for early noninvasive diagnosis of esophageal cancer.

The pathogenesis of esophageal cancer involves complex interactions between genetics, epigenetic and environmental factors. DNA methylation is a common form of epigenetic modification that plays an important role in the development of human malignancies. Abnormal methylation of the promoter region of an oncogene may lead to down-regulation or silencing of transcription of the gene, leading to the occurrence of cancer. Thus, alterations in DNA methylation patterns have become a reliable potential biomarker for early detection and diagnosis of cancer. In recent years, liquid biopsy techniques based on circulating cell-free DNA (cfDNA) have shown great potential in early diagnosis of cancer, as they can detect early solid tumors in a minimally invasive manner. Genetic alterations (e.g., mutations, copy number variations, or epigenetic changes) that occur in tumor cells, release their nucleic acids into the blood upon apoptosis or necrosis of the tumor cells, allowing tumor DNA to be detected by liquid biopsy techniques. In view of this, diagnosing early esophageal cancer by detecting methylation of cfDNA in the blood is a viable method, and urgent need is to find highly sensitive, highly specific DNA methylation biomarkers that can distinguish cancer patients from non-cancer patients.

Disclosure of Invention

In view of the above, the application provides application of KCNA3 and OTOP2 gene methylation as esophageal cancer diagnosis markers, and also provides a reagent for detecting methylation levels of KCNA3 and OTOP2 gene target areas and a diagnosis kit, wherein the reagent and the kit have high sensitivity and good specificity in diagnosing esophageal cancer, and provide a new thought for noninvasive diagnosis of esophageal cancer and early esophageal cancer.

In a first aspect of the present application, there is provided the use of a reagent for detecting the methylation level of a target region in genes including the KCNA3 gene and OTOP2 gene in the preparation of a diagnostic esophageal cancer reagent or kit.

In some embodiments, the target region comprises a first target region that belongs to a KCNA3 gene and a second target region that belongs to an OTOP2 gene: the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, 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 as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO. 12.

Further, the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO. 11; and/or the second target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO. 14.

Further, the first target region comprises the full-length or partial sequence shown in SEQ ID NO. 3; the second target region comprises the full-length or partial sequence shown in SEQ ID NO. 12.

In a second aspect of the present application, there is provided a detection reagent for diagnosis of esophageal cancer, the detection reagent comprising a reagent for detecting a methylation level of a target region in genes including a KCNA3 gene and an OTOP2 gene.

In some embodiments of the present application, the detection reagent comprises a reagent that detects 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 comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4;

the second target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 12.

In some embodiments of the present application, the first target region further comprises a full-length or partial sequence selected from at least one of the nucleotide sequences set forth in SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No. 11; the second target region further comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO. 14.

In some embodiments of the present application, the detection reagent comprises a reagent that detects the methylation level of a full or partial sequence shown in SEQ ID NO.3 for a nucleotide sequence selected from the first region of interest and SEQ ID NO.12 for a nucleotide sequence selected from the second region of interest.

In some embodiments of the present application, the reagent comprises a nucleic acid molecule comprising a primer pair that detects the methylation level of the first target region and the second target region.

In some embodiments of the present application, the reagent further comprises a detection probe corresponding to the detection primer, the detection probe having a fluorescent group and a fluorescence quenching group attached thereto.

In some embodiments of the present application, the 5 'end of the fluorescent probe contains a fluorescent reporter group such as any of FAM, HEX, VIC, CY, ROX, texsa Red, JOE, and Quasar 705, and the 3' end contains a fluorescent quenching group such as any of MGB, BHQ-1, BHQ-2, and BHQ-3.

In some embodiments of the present application, the primer pair for detecting methylation levels of the first target region and the second target region is as shown in table 1 below:

TABLE 1 primer pair summary for detecting methylation levels of first target region and second target region

In some embodiments of the present application, the combination of primer pairs and probes to detect methylation levels of the first and second target regions is shown in table 2 below:

TABLE 2 primer pair and probe combinations for detecting methylation levels of first target region and second target region

In some embodiments of the present application, the reagent further comprises a primer pair for detecting a reference gene and a corresponding detection probe.

In some embodiments of the present application, the reference gene comprises an ACTB gene corresponding to the pair of detection primers shown in SEQ ID No.64 and SEQ ID No.65 and the detection probe shown in SEQ ID No. 66.

In some embodiments of the present application, the reagent enables detection of the methylation level of the target region by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and methylation-specific fluorescent quantitative PCR.

In some embodiments of the present application, the biological sample to be tested is an esophageal tissue sample, an esophageal exfoliated cell sample, or a blood sample; wherein the blood sample comprises plasma, serum, whole blood, isolated blood cells, or a combination thereof.

The application adopts two different methods to verify the effect of the combination of methylation molecular markers on diagnosing esophageal cancer: the first example demonstrates that the methylation level of a molecular marker combination is evaluated by a method of sanger sequencing to determine whether a tissue sample, a blood sample, is a cancer sample; example two shows the method of using methylation fluorescence quantitative PCR to evaluate the methylation level of molecular marker combinations, and further to determine whether a tissue sample, blood sample, is a cancer sample.

In a third aspect of the present application there is provided a kit for diagnosing oesophageal cancer comprising a detection reagent as defined in the second aspect.

In some embodiments of the present application, the kit further comprises at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent, and a sequencing reagent.

In some embodiments of the present application, the methylation conversion reagent is bisulfite.

In some embodiments of the present application, the PCR reaction reagents include an amplification buffer, dNTPs, a DNA polymerase, and Mg ²⁺ One or more of the following.

The beneficial effects of this application are as follows:

the inventor finds that by using a methylation specific fluorescence quantitative PCR method, through detecting methylation levels of KCNA3 gene and OTOP2 gene target regions in blood samples, esophageal cancer patients and healthy people can be effectively distinguished, the sensitivity of diagnosing esophageal low-grade neoplasia, high-grade neoplasia, early-stage esophageal cancer and advanced-stage esophageal cancer can reach 76.67%, 89.19%, 93.33% and 96.0%, and the specificity of the kit in healthy people can reach 95%. The technical scheme provided by the application can realize noninvasive or minimally invasive diagnosis of the esophageal cancer, has high diagnosis sensitivity and good specificity, and is beneficial to improving the detection rate of lesion before the esophageal cancer and the esophageal cancer.

Detailed Description

The present application will now be described in detail with reference to examples, but the practice of the present application is not limited thereto.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or", "or" as used herein includes a selection of any one of two or more of the listed items and also includes any and all combinations of the listed items, including any two or more of the listed items, or all combinations of the listed items. It should be noted that, when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", it is understood that, in the present application, the technical solutions certainly include technical solutions all connected by "logical and", and also certainly include technical solutions all connected by "logical or". For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, B, C, and/or D" include any one of A, B, C, D (i.e., the technical schemes all connected by "logical or"), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and further include four combinations of A, B, C, D (i.e., the technical schemes all connected by "logical and").

The terms "plurality", "plural", "multiple", and the like are used herein, and refer to a number of 2 or more, unless otherwise specified. For example, "one or more" means one kind or two or more kinds. "above" includes the present number, for example "two or more" includes two, three or more.

In the present application, "at least one" and "at least one" mean any one of the listed items, or a combination of any two or more thereof.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

In the present application, "suitable" is described in "suitable combination mode", "suitable mode", "any suitable mode", etc., so as to implement the technical scheme of the present application, solve the technical problem of the present application, and achieve the technical effect expected in the present application.

In this application, "preferred," "better," "preferred," and "preferred" are merely examples of better performing implementations or examples, and it should be understood that they are not limiting the scope of the application.

In this application, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the present application.

In this application, the terms "first," "second," "third," "fourth," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

As used herein, an "esophageal cancer" is a malignancy of the digestive tract from the hypopharynx to the esophageal epithelium between the esophageal and gastric junctions. Esophageal cancer mainly includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.

In this application, the term "diagnosis" includes auxiliary diagnosis, recurrence risk assessment, assessment of risk and extent of cancerous lesions, prognosis, and the like.

The term "gene" refers to a segment of DNA encoding a polypeptide chain that produces amino acids, and includes sequences located in coding and non-coding regions, as well as exon and intron sequences involved in gene transcription/translation and transcriptional/translational regulation.

The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and typically more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotides may be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine and guanine.

The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.

The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration of each index is also required to obtain a final decision index. It is understood that the target region of the gene to be detected herein is a DNA sequence comprising at least one CpG dinucleotide (CG).

The term "CpG island" refers to a region on DNA that is rich in a large number of cytosines and guanines linked by phosphoester linkages. CpG dinucleotides are typically concentrated in the promoter region and exons of human genes. In normal human genomes, cpG sites outside CpG islands are typically methylated, whereas CpG sites in CpG islands are typically in an unmethylated state, a form of methylation that is inherited stably with cell division. When the tumor occurs, the unmethylation degree of CpG sites outside the cancer suppressor gene CpG island is increased, and the CpG sites in the CpG island are in a hypermethylation state, so that the chromosome helix degree is increased, the transcription is inhibited, and the gene expression is deleted.

The term "methylation level of a CpG island region" refers to the methylation level of cytosine in one or more CpG dinucleotides within a CpG island. "methylation site" or "CpG site" refers to at least one CpG dinucleotide site in a region, and in particular to a cytosine in at least one CpG dinucleotide site in a region.

The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" refers to a pair of primers that hybridize to the double strand of a target DNA molecule or to regions of the target DNA molecule that flank the nucleotide sequence to be amplified.

The term "Taqman probe" refers to a stretch of oligonucleotide sequences comprising a 5 'fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.

The term "sanger sequencing", i.e., a generation of sequencing, the reaction system comprises: target fragment, four deoxyribonucleotides (dNTPs), DNA polymerase, primer, etc., and 4 kinds of dideoxyribonucleotides (ddNTPs) marked by different fluorophores are required. Because ddNTP lacks 3' -OH group required for extension, the extended oligonucleotide is selectively terminated at G, A, T or C, and the four optical wavelength signals are converted into computer recognizable electric signals through optical excitation, and the target DNA sequence is judged according to the fluorescence signal of the ddNTP finally doped in the reaction tube.

Various aspects of the present application are described in detail below.

In a first aspect of the present application, there is provided the use of a reagent for detecting the methylation level of a target region in genes, including the KCNA3 gene and OTOP2 gene, in the preparation of a diagnostic esophageal cancer reagent or kit.

The relevant information for the KCNA3 gene and OTOP2 gene can be obtained from bioinformatic databases known in the art. If GRCh38.p14 is used as the reference genome, KCNA3 gene is located in Ch1: 110653560-110674940; 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 application is not limited to relying on detection of KCNA3 gene and OTOP2 gene which are identical to the nucleotide sequence in the reference genome. It will be appreciated that there may be some differences in the nucleotide sequences of KCNA3 and OTOP2 genes obtained from different sets or different samples of human genomic information, but these differences do not affect the practice of the present application. The present application is applicable to different subject individuals.

The KCNA3 and OTOP2 genes are used as target genes, the methylation level of a target region in the two genes is detected, and whether the subject suffers from esophageal cancer or suffers from esophageal precancerous lesions can be diagnosed or assisted according to the methylation level of the target region in the two genes. Furthermore, the reagent for detecting the methylation level of the target region in the two genes can be used for preparing an esophageal cancer diagnostic reagent or a kit.

It is understood that the target region of the KCNA3 and OTOP2 genes is a region including at least one methylation site. In some embodiments, the region of interest may be a CpG island of a gene, or any other region that includes a methylation site. In some embodiments, the target region may be located in a promoter region or a coding region.

The methylation level of a target region of two genes in the present application can be detected by any detection method known in the art capable of detecting DNA methylation levels, including, but not limited to, detection of methylation levels of the target region by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and methylation-specific fluorescent quantitative PCR.

In some embodiments, the target region comprises a first target region and a second target region, the first target region being of a KCNA3 gene and the second target region being of a KCNA3 gene. Reagents for detecting the methylation level of a target region in a gene include reagents for detecting the methylation levels of a first target region and a second target region.

The first target region may comprise one contiguous nucleotide sequence or may comprise two or more non-contiguous nucleotide sequences. The second target region may comprise one contiguous nucleotide sequence or may comprise two or more non-contiguous nucleotide sequences.

As an example, the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, 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 as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO. 12. The first target region listed above may be combined with any second target region derived from the OTOP2 gene, in which case the second target region may be the full length or partial sequence shown in any one of SEQ ID No.6, SEQ ID No.7, SEQ ID No.12, or may be other regions of the OTOP2 gene including CpG sites. The second target region listed above may be combined with any of the first target regions derived from the KCNA3 gene, in which case the full or partial sequence of any of the first targets SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 may be used, as well as other regions of the KCNA3 gene including CpG sites. As a specific example thereof, the first target region includes a full-length or partial sequence represented by SEQ ID No. 3; the second target region comprises the full-length or partial sequence shown in SEQ ID NO. 12.

In some embodiments, 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 any one of SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12, a combination of the two or a combination of the three.

In other embodiments, 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, combinations of the two, combinations of the three, or combinations 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. In still other embodiments, it is also possible that the first target region comprises a combination of two or more of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and the second target region comprises a combination of two or more of SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO. 12.

In other embodiments, the first region of interest comprises a full length or partial sequence selected from at least one of the nucleotide sequences set forth in SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No. 11; and/or the second target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO. 14. Furthermore, the first target region listed above may be combined with any second target region derived from the OTOP2 gene, in which case the second target region may be the full length or partial sequence of any one of SEQ ID No.5, SEQ ID No.13, and SEQ ID No.14, or may be other CpG site-containing regions of the OTOP2 gene. The second target region listed above may be combined with any of the first target regions derived from the KCNA3 gene, in which case the full or partial sequence of any of the first targets SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 may be used, as well as other regions of the KCNA3 gene including CpG sites. In this embodiment, the first target region and the second target region may also be a combination of one or more nucleotide sequences, respectively.

In a second aspect of the present application, there is also provided a detection reagent for diagnosis of esophageal cancer, comprising a reagent for detecting methylation level of a target region in genes, the genes detected comprising a KCNA3 gene and an OTOP2 gene, based on the above-mentioned applications.

The genes detected by the detection reagent and the target region thereof have the meanings as described above and are not described in detail herein.

Further, the reagent for detecting the methylation level of a target region in a gene comprises a nucleic acid molecule comprising a primer pair for detecting the methylation level of a first target region and a second target region.

In some embodiments of the present application, the reagent further comprises a detection probe corresponding to the detection primer. The detection probe is connected with a fluorescent group and a fluorescence quenching group.

In some embodiments of the present application, methods utilizing Sanger sequencing may be employed to assess the methylation level of a target gene combination. For example, methylation level detection is performed using a Sanger sequencing method by providing a pair of methylated primers and a pair of unmethylated primers for the first target region and the second target region, respectively. In some embodiments, primer pairs as set forth in Table 1 above may be employed.

In other embodiments, methylation levels of target gene combinations can also be assessed using methylation fluorescent quantitative PCR methods. At this time, a primer pair and a detection probe may be provided for the first target region and the second target region, respectively, and methylation levels of the first target region and the second target region may be detected. As a specific example, the combinations of primer pairs and probes are shown in table 2 above.

In some embodiments of the present application, the kit further comprises at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent, and a sequencing reagent. The methylation converting reagent is bisulphite.

It will be appreciated that the kits provided herein may also include negative/positive references, or other reagents necessary to achieve the purposes or effects of the present application, such as some of the necessary sample preservation reagents.

The present application also provides a method of detecting whether a subject has esophageal cancer or a precancerous condition, comprising:

providing a biological sample derived from a subject;

detecting the methylation level of the KCNA3 gene and the OTOP2 gene target region in the biological sample, wherein the subject is at increased risk of having esophageal cancer or a precancerous lesion if the methylation level of at least one of the KCNA3 gene and the OTOP2 gene is increased.

In some embodiments, the method is implemented based on the foregoing scheme. This can be achieved, for example, with the detection reagents or kits as described above.

In some embodiments, 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; wherein the blood sample comprises plasma, serum, whole blood, isolated blood cells, or any combination thereof.

In some embodiments, an elevated methylation level of at least one of the KCNA3 gene and the OTOP2 gene refers to detecting the methylation level of the KCNA3 gene and the OTOP2 gene in a biological sample of the subject, with the result being higher compared to the average methylation level of the normal population. By way of example, the relative level of methylation of a gene may be expressed in terms of ct values, where in a tissue sample, ct.ltoreq.38 is considered to be elevated (positive, diseased); in the blood sample, ct.ltoreq.45, an elevated methylation level (positive, diseased) is considered.

The following is a further description of the aspects of the present application in connection with specific embodiments.

EXAMPLE 1 bisulfite sequencing method to assess methylation level of target genes

The sensitivity and specificity of the kit for diagnosis or auxiliary diagnosis of esophageal cancer are analyzed by detecting the methylation state of at least one sequence with the nucleotide sequences shown as SEQ ID NO.1-4 in KCNA3 gene and at least one sequence with the nucleotide sequences shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12 in OTOP2 gene.

The main flow of this embodiment includes:

1) Obtaining a tissue sample and a blood sample from a patient with esophageal precancerous lesions or cancer or a healthy person, and extracting, converting and purifying template DNA according to different sample types; 2) Amplifying at least one region of SEQ ID NO.1-4 and at least one region of SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12 respectively by using a sequencing primer, and performing Sanger sequencing on the amplified products so as to judge the methylation state of each region; 3) Based on the methylation status of each region, the sensitivity and specificity of diagnosing tissue samples and blood samples using the composition as a methylation marker were calculated. The method comprises the following specific steps:

1. Sample collection, processing

32 cases of esophageal hyperplasia tissues confirmed by pathological tissue biopsy as low-level esophageal squamous intraepithelial neoplasia (LGIN) were collected in a certain Wuhan hospital, 48 cases of esophageal hyperplasia tissues confirmed by pathological biopsy as high-level esophageal squamous intraepithelial neoplasia (HGIN), 68 cases of early stage esophageal squamous carcinoma tissues, 80 cases of progressive stage esophageal squamous carcinoma tissues and 79 cases of lesion-free paracancerous tissues, and all samples were formalin-immersed paraffin-embedded tissue samples. All samples were approved by the ethics committee, all volunteers signed informed consent, and all samples were anonymized.

Blood samples of 60 cases of patients with low-level esophageal squamous intraepithelial neoplasia (LGIN), 74 cases of patients with high-level esophageal squamous intraepithelial neoplasia (HGIN), 120 cases of patients with early esophageal cancer, 150 cases of patients with advanced esophageal squamous carcinoma and 100 cases of blood samples of healthy people were collected in a certain hospital in zheng state, all the collection processes of the samples were approved by the ethical committee, all volunteers signed informed consent, and all the samples were anonymized.

2. Extraction of DNA templates

For tissue samples, QIAamp DNA FFPE Tissue Kit (56404) was used to extract DNA, and the specific procedure was performed according to the kit instructions. For blood samples, plasma cfDNA extraction was performed using a magnetic bead serum/plasma free DNA (cfDNA) extraction kit (DP 709) from the company of the biochemical technology of the root of the heaven limited company, the specific procedure was performed according to the kit instructions.

3. Conversion of bisulphite

The extracted sample genome is subjected to bisulphite conversion, and the nucleic acid conversion kit is a nucleic acid conversion reagent (Huhan mechanical preparation 20200843) of the life technology Co.Ltd.Wohan Ai Misen, and specific experimental operation is described in the specification of the kit.

4. PCR amplification and sequencing

For the nucleotide sequences SEQ ID NO. 1-4 of KCNA3 gene and the nucleotide sequences SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12 of OTOP2 gene, respectively designing a methylation primer pair and a non-methylation primer pair to amplify corresponding regions, and screening the optimal ratio of the methylation primer pair and the non-methylation primer pair so as to ensure that a methylated product can be obtained by amplification when 1% or more of the methylation sequence exists in the template, and the amplification product is a non-methylation product only when the template is the non-methylation sequence.

The methylation primer pairs and unmethylation primer pairs used in PCR amplification are shown in Table 3. The PCR reaction system was prepared according to the formulation shown in Table 4, and the PCR amplification procedure was the same as that of Table 5, and after the completion of PCR amplification, the amplified products were subjected to Sanger sequencing (sequencing Co.) using mixed primers (comprising a methylated primer pair and an unmethylated primer pair) while sequencing from the 5 'end and the 3' end.

TABLE 3 methylation primer and unmethylation primer sequences for each target region

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TABLE 4 PCR reaction System

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TABLE 5 PCR reaction procedure

5. Analysis of results

Methylation of CpG sites in each amplicon was analyzed according to the sequencing peak plots for each sample. Methylation of cytosine in a CpG dinucleotide is classified into two types, namely unmethylation and methylation, where methylation is classified into complete methylation and partial methylation. If thymine is the result of cytosine sequencing in a CpG dinucleotide, it is unmethylated. If the cytosine sequencing result in a CpG dinucleotide is still cytosine, it is fully methylated. If the cytosine sequencing results in a CpG dinucleotide with both cytosine and thymine (bimodal), it is partially methylated.

If more than 95% of the cytosine in a CpG dinucleotide in an amplicon is methylated, the sample is considered methylation positive in that region of the gene. The number of methylation positives and the number of methylation negatives in each region were calculated for each type of sample.

If a certain sample is methylation positive in at least one region of the KCNA3 gene or at least one region of the OTOP2 gene, the sample is considered to be a pre-cancerous lesion positive or a cancer positive sample; a sample is considered a pre-cancerous negative and cancer negative sample when it is methylation negative in at least one region of the KCNA3 gene and in at least one region of the OTOP2 gene.

At least one region with nucleotide sequences shown as SEQ ID NO.1-4 in KCNA3 gene and at least one region with nucleotide sequences shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.12 in OTOP2 gene show methylation state, detection sensitivity and specificity in esophageal cancer precancerous lesions and esophageal cancer patient tissue samples as shown in Table 6; the methylation status, detection sensitivity and specificity of the probe in the blood sample of patients with esophageal cancer and precancerous lesions are shown in Table 7.

TABLE 6 detection Properties of at least one region of KCNA3 and OTOP2 genes in tissue samples

TABLE 7 detection Performance of at least one region of KCNA3 and OTOP2 genes in blood samples

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As can be seen from tables 6 and 7, the detection of precancerous lesions and esophageal cancer is excellent by using the methylation level of a composition of at least one region of the KCNA3 gene having the nucleotide sequences shown as SEQ ID NO.1-4 and at least one region of the OTOP2 gene having the nucleotide sequences shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 12.

In the tissue sample, the sensitivity of detecting the lower grade esophageal neoplasia is at least 40.63 percent (SEQ ID NO. 2+7) and the highest sensitivity of detecting the lower grade esophageal neoplasia is at least 87.50 percent (SEQ ID NO. 1+3+12+6) by detecting the methylation level of various compositions; the sensitivity for detecting the esophageal high-grade neoplasia is 54.17 percent (SEQ ID NO. 2+7) at the lowest, and the highest sensitivity for detecting the esophageal high-grade neoplasia is 95.83 percent (SEQ ID NO. 1+3+12+6/3+4+12+6); the lowest sensitivity for detecting early-stage esophageal cancer is 72.06% (SEQ ID NO. 2+7), and the highest sensitivity for detecting early-stage esophageal cancer is 97.06% (SEQ ID NO. 3+4+12+6); the lowest sensitivity for detecting the progressive esophageal cancer is 78.75 percent (SEQ ID NO. 1+7), and the highest sensitivity for detecting the progressive esophageal cancer is 98.75 percent (SEQ ID NO. 3+4+12+6); the specificity of the tissue beside the cancer is 70.89 percent (SEQ ID NO. 3+4+12+6), and the specificity of the tissue beside the cancer is 94.94 percent (SEQ ID NO. 3+12).

In the blood sample, the sensitivity of detecting esophageal low-grade neoplasia is as low as 38.33 percent (SEQ ID NO. 2+7) and the highest sensitivity of detecting esophageal low-grade neoplasia is as high as 83.33 percent (SEQ ID NO. 3+12+6) by detecting the methylation level of various compositions; the lowest sensitivity of detecting the high-grade tumor of the esophagus is 63.51 percent (SEQ ID NO. 2+7), and the highest sensitivity of detecting the high-grade tumor of the esophagus is 94.59 percent (SEQ ID NO. 1+3+12+6); the lowest sensitivity for detecting early-stage esophageal cancer is 71.67% (SEQ ID NO. 2+7), and the highest sensitivity for detecting early-stage esophageal cancer is 95.83% (SEQ ID NO. 3+12+6/1+3+12+6); the lowest sensitivity for detecting the progressive esophageal cancer is 76.67% (SEQ ID NO. 2+7), and the highest sensitivity for detecting the progressive esophageal cancer is 97.33% (SEQ ID NO. 1+3+12+6); the specificity of the detection of the healthy human blood sample is 70% (SEQ ID NO. 1+4+12+6) at the minimum, and the specificity of the detection of the healthy human blood sample is 96% (SEQ ID NO. 3+12) at the maximum.

In summary, it can be seen that the sensitivity of the composition to detection tends to increase with increasing number of detection areas in the composition, but the detection tends to decrease. In combination, when the number of detection regions in the composition is 2, the combination with optimal sensitivity and specificity can be achieved, such as the combination with SEQ ID NO.3+12 as methylation marker.

Example two methylation-specific fluorescent quantitative PCR method for assessing methylation level of target Gene

The sensitivity and specificity of the kit for diagnosis or auxiliary diagnosis of esophageal cancer are analyzed by detecting the methylation state of any one of the nucleotide sequences shown as SEQ ID NO. 8-11 in the KCNA3 gene and any one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14 in the OTOP2 gene. The main flow of this embodiment includes: 1) Obtaining a tissue sample and a blood sample from a precancerous lesion of esophageal cancer or a cancer patient or a healthy person, and extracting, converting and purifying template DNA according to different sample types; 2) Detecting the methylation state of each composition in the sample by using a detection primer pair and a probe through a methylation fluorescent quantitative PCR method, and further judging whether the sample is a cancer sample or not; 3) The sensitivity and specificity of the composition as a methylation marker for diagnosing tissue samples and blood samples were calculated. Considering that a combination of 2 or more kinds of marker sequences results in a decrease in the specificity of detection, only a combination of two kinds of marker sequences was used in example 2. The method comprises the following specific steps:

1. Sample collection, processing

The collection of clinical samples, extraction of template DNA, transformation and purification were as in example 1.

2. Methylation fluorescent quantitative PCR reaction

And (3) respectively carrying out methylation fluorescent quantitative PCR reaction on the DNA of each sample after bisulfite conversion to detect the methylation state of any one of the sequences of which the nucleotide sequences are shown as SEQ ID NO. 8-11 and any one of the sequences of which the nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14 in the KCNA3 gene and the OTOP2 gene in each sample.

Each combination mode is independently detected, namely, a PCR tube is added with necessary reaction components and templates, and detection primer pairs and probes of any one region of SEQ ID NO. 8-11, and detection primer pairs and probes of any one region of SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14 are required to be added; meanwhile, a detection primer pair and a probe of an internal reference gene ACTB are also required to be added. The probe of the detection target area is a Taqman probe, the reporter group at the 5 'end of the detection probe with the sequence of SEQ ID NO. 8-11 is FAM, and the quenching group at the 3' end is MGB; the reporter group at the 5 'end of the detection probe with the sequences of SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO.14 and the like is ROX, and the quenching group at the 3' end is MGB; the reporting group at the 5 'end of the ACTB probe is VIC, and the quenching group at the 3' end is BHQ1. The nucleotide sequences of the KCNA3 gene are shown in SEQ ID NO. 8-11, the nucleotide sequences of the OTOP2 gene are shown in SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO.14 and upstream and downstream detection primers and probe sequences of the reference gene ACTB are shown in Table 8.

PCR was performed using Invitrogen Platinum II Taq hot-start DNA polymerase, and the PCR reaction solution was prepared as shown in Table 9, and the PCR amplification was performed according to the amplification procedure shown in Table 10. The CpG sites in the sequences of SEQ ID NO. 8-11, SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO.14, etc. which can be detected by using the primer pair and the corresponding probe in Table 6 are shown in Table 11.

Table 8 detection primer pairs and probe sequences

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TABLE 9 PCR reaction System

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TABLE 10 PCR reaction procedure

TABLE 11 detection primer pairs and probe-detectable methylation sites for cytosine in CpG dinucleotides

Negative and positive controls: in detecting samples according to different combinations, negative control and positive control should be detected simultaneously, and the DNA template of the negative control tube is TE buffer. The preparation method of the DNA template of the positive control tube comprises the following steps: artificially synthesizing the sequence which corresponds to the ACTB gene amplified region and is subjected to complete conversion by bisulphite, and cloning the sequence onto a vector to form an artificially synthesized plasmid; the target areas are respectively corresponding to SEQ ID NO. 8-11, SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14, and the sequences are artificially synthesized after being converted by bisulphite and are respectively cloned to vectors to form artificially synthesized plasmids. Positive control DNA template 10 ³ Copy/microliter of synthetic plasmid containing post-transformation ACTB, 10 ³ Copy/microliter of synthetic plasmid and 10 containing one of the sequences SEQ ID No. 8-11 after transformation ³ Copy/microliter of artificially synthesized plasmid containing one sequence of SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14 after transformation is mixed in equal proportion.

Ct value reading: after the PCR is completed, a baseline is adjusted, a fluorescence value before a minimum Ct value of a sample in one PCR is advanced by 1-2 cycles is set as a baseline value, and a threshold value is set at the inflection point of an S-type amplification curve to obtain Ct values of all genes of the sample.

And (3) quality control: the negative control needs no amplification, the positive control needs obvious index increase period, and the Ct value of each gene of the positive control is between 26 and 30. The Ct value of the reference gene of the sample to be detected is less than or equal to 35, and after the negative control, the positive control and the reference gene meet the requirements, the experiment is effective, and the next sample result can be judged. Otherwise, when the experiment is invalid, the detection is needed again.

3. Analysis of PCR results

And judging the methylation level of the sample to be tested according to the Ct values of the KCNA3 gene and the OTOP2 gene. For a tissue sample, if the Ct value of a certain gene region is less than or equal to 38, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene region is greater than 38, the gene in the sample is considered to be methylation negative. For a blood sample, if the Ct value of a certain gene region is amplified to be less than or equal to 45, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene region is amplified to be more than 45, the gene in the sample is considered to be methylation negative. If at least one gene in the sample to be detected is methylation positive, the sample is a positive sample of precancerous lesions or a positive sample of cancers, and only if two genes in the sample to be detected are methylation negative, the sample is a negative sample of precancerous lesions and a negative sample of cancers. Specific criteria are shown in table 12.

The methylation state, detection sensitivity and specificity of the composition of any one of the regions of the KCNA3 gene with the nucleotide sequences shown in SEQ ID NO. 8-11 and any one of the regions of the OTOP2 gene with the nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14 in tissue samples of patients suffering from esophageal cancer are shown in Table 13, and the methylation state, detection sensitivity and specificity of the composition in blood samples of patients suffering from esophageal cancer are shown in Table 14.

Table 12 criteria for samples in fluorescent quantitation

TABLE 13 detection Properties of KCNA3 and OTOP2 Gene compositions in precancerous lesions, cancerations, paracancerous tissue samples

TABLE 14 detection Properties of KCNA3 and OTOP2 Gene compositions in precancerous lesions, cancer, healthy blood samples

As can be seen from tables 13 and 14, the methylation level of the composition for diagnosing precancerous lesions of esophagus and patients with esophageal cancer by detecting the methylation level of the composition in any one of the regions of the KCNA3 gene, the nucleotide sequences of which are shown as SEQ ID NO. 8-11, and the OTOP2 gene, the nucleotide sequences of which are shown as SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO.14, by using a methylation fluorescent quantitative PCR method.

In the tissue sample, the sensitivity of diagnosing the lower grade esophageal neoplasia is at least 43.75 percent (SEQ ID NO. 9+14), and the highest sensitivity of diagnosing the lower grade esophageal neoplasia is at least 81.25 percent (SEQ ID NO. 10+5) by detecting the methylation level of various compositions; the sensitivity of diagnosing the high-grade tumor of the esophagus is at least 52.08 percent (SEQ ID NO. 11+14), and the highest sensitivity of diagnosing the high-grade tumor of the esophagus is at least 91.67 percent (SEQ ID NO. 10+13); the lowest sensitivity of diagnosing early-stage esophageal cancer is 72.06% (SEQ ID NO. 9+14), and the highest sensitivity of diagnosing early-stage esophageal cancer is 94.12% (SEQ ID NO. 10+5/10+13); the lowest sensitivity for diagnosing the progressive esophageal cancer is 80.00% (SEQ ID NO. 9+14), and the highest sensitivity for diagnosing the progressive esophageal cancer is 96.25% (SEQ ID NO. 10+5); the specificity of the tissue beside the cancer is 77.22 percent (SEQ ID NO. 9+14) at the minimum, and the specificity of the tissue beside the cancer is 93.67 percent (SEQ ID NO. 10+5/10+13) at the maximum.

In the blood sample, the sensitivity of diagnosing esophageal low-grade neoplasia is at least 46.67% (SEQ ID NO. 9+14) and the highest sensitivity of detecting esophageal low-grade neoplasia is at least 76.67% (SEQ ID NO. 10+5) by detecting methylation levels of various compositions; the lowest sensitivity of detecting the high-grade tumor of the esophagus is 51.35 percent (SEQ ID NO. 11+14), and the highest sensitivity of detecting the high-grade tumor of the esophagus is 89.19 percent (SEQ ID NO. 10+5); the lowest sensitivity for detecting early-stage esophageal cancer is 73.33% (SEQ ID NO. 8+14/11+14), and the highest sensitivity for detecting early-stage esophageal cancer is 93.33% (SEQ ID NO. 10+5); the lowest sensitivity for detecting the progressive esophageal cancer is 73.33 percent (SEQ ID NO. 9+14), and the highest sensitivity for detecting the progressive esophageal cancer is 96.0 percent (SEQ ID NO. 10+5); the specificity of the sample for detecting the healthy human blood is as low as 81.00 percent (SEQ ID NO. 9+14), and the specificity of the sample for detecting the healthy human blood is as high as 95 percent (SEQ ID NO. 10+5).

In conclusion, when the combination of the KCNA3 gene with the nucleotide sequence of SEQ ID NO.10 and the OTOP2 gene with the nucleotide sequence of SEQ ID NO.5 is selected as a methylation marker, the sensitivity and the specificity of detecting the tissue sample and the blood sample can be optimized.

While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these are intended to be included within the scope of the present invention as defined in the appended claims.

Claims

1. Use of a reagent for detecting the methylation level of a target region in a gene for the preparation of an esophageal cancer diagnostic reagent or kit, characterized in that the target region comprises a first target region belonging to the KCNA3 gene and a second target region belonging to the OTOP2 gene.

2. The use according to claim 1, wherein,

the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; and/or

3. The use according to claim 1, wherein the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences set forth in SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No. 11; and/or

The second target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13 and SEQ ID NO. 14.

4. The use according to claim 2, wherein the first target region comprises the full-length or partial sequence shown in SEQ ID No. 3; the second target region comprises the full-length or partial sequence shown in SEQ ID NO. 12.

5. A test agent for diagnosis of esophageal cancer, characterized in that the test agent comprises an agent for detecting a methylation level of a first target region belonging to KCNA3 gene and a second target region belonging to OTOP2 gene.

6. The detection reagent according to claim 5, wherein,

7. The detection reagent according to claim 5, wherein the first target region comprises a full-length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO. 11; and/or the second target region comprises a full length or partial sequence selected from at least one of the nucleotide sequences shown as SEQ ID NO.5, SEQ ID NO.13, SEQ ID NO. 14.

8. The detection reagent according to claim 6, wherein the detection reagent comprises a reagent for detecting a methylation level of a full-length or partial sequence represented by SEQ ID NO.3 as a nucleotide sequence selected from the first target region and SEQ ID NO.12 as a nucleotide sequence selected from the second target region.

9. The detection reagent of any one of claims 5 to 8, wherein the reagent comprises a nucleic acid molecule comprising a primer pair that detects the methylation levels of the first and second target regions.

10. The detection reagent according to claim 9, further comprising a detection probe corresponding to the detection primer, wherein the detection probe has a fluorescent group and a fluorescence quenching group attached thereto.

11. The detection reagent according to claim 9, wherein the primer pair for detecting methylation levels of the first target region and the second target region is as shown in the following table,

12. the detection reagent according to claim 10, wherein the combination of the primer pair and the probe for detecting methylation levels of the first target region and the second target region is as shown in the following table,

13. A kit for diagnosing esophageal cancer, comprising the detection reagent according to any one of claims 5 to 12.

14. The kit of claim 13, further comprising at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent, and a sequencing reagent.