CN116162703A

CN116162703A - Reagent for methylation detection of esophageal cancer genes, kit and application thereof

Info

Publication number: CN116162703A
Application number: CN202210969834.8A
Authority: CN
Inventors: 李红乐; 李俊; 徐斌杰; 李香梅; 周长悦; 陈颖琪
Original assignee: Anhui Dajian Medical Technology Co ltd
Current assignee: Anhui Dajian Medical Technology Co ltd
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2023-05-26

Abstract

The invention belongs to the technical field of biology, discloses a reagent for detecting esophageal cancer gene methylation, a kit and application thereof, and particularly discloses a detection region for detecting esophageal cancer gene methylation. The detection region for detecting the methylation of the esophageal cancer gene provided by the invention can be used as an important detection index for early screening, process monitoring and prognosis evaluation of esophageal cancer, DNA methylation abnormality is taken as a detection object, the DNA methylation abnormality usually occurs in early cancer and penetrates through the occurrence and development processes of cancer, the methylation state of the detection region is changed once the methylation state is formed and needs to be continuously stimulated by the external environment for a long time, and therefore, the detection of the DNA methylation region can be used as an important biological index for early screening, process monitoring and prognosis evaluation of esophageal cancer.

Description

Reagent for methylation detection of esophageal cancer genes, kit and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a reagent for detecting esophageal cancer gene methylation, a kit and application thereof.

Background

Esophageal cancer refers to cancer of esophageal epithelium origin from the initial part of hypopharyngeal esophagus to the joint part of esophageal stomach, and is mainly classified into esophageal squamous cell cancer, esophageal adenocarcinoma and undifferentiated cancer (less frequently, high in malignancy), and is one of the most common ten malignant tumors. The surgical excision rate of patients with early esophageal cancer reaches 100%, the surgical death rate is below 2.5%, and the survival rate of patients with early esophageal cancer reaches 92.6% in 5 years after surgery; early symptoms of esophageal cancer are not obvious, and if symptoms such as dysphagia, burning sensation, stagnation sensation or satiety after eating appear, the early symptoms generally belong to middle and late stages, and the survival rate of patients with middle and late stages of esophageal cancer after operation is only about 30%. The auxiliary inspection means of the esophageal cancer at the present stage mainly comprise technologies such as imaging inspection (CT, upper gastrointestinal radiography, MRI, PET-CT, ultrasonic inspection and the like), endoscopy, tumor marker inspection and the like, and the inspection technologies or the detection cost is high; or is invasive; or low sensitivity, and is not suitable for being used as a means for early screening of patients with esophageal cancer. Therefore, the biomarker for early esophageal cancer is urgently needed to be searched, sensitive, specific and rapid detection technology and detection method are developed, the early detection rate of esophageal cancer is improved, more esophageal cancer patients can find out early cancers, timely intervention treatment is given, and the death rate of esophageal cancer patients is reduced.

DNA methylation can regulate cell proliferation, apoptosis and differentiation, and is one of the most recently discovered epigenetic regulatory mechanisms. The necessity of DNA methylation abnormalities and the occurrence of tumors has been one of the hot spots of research in the medical community. The only form of mammalian DNA methylation that is currently found is the methylation process that occurs at the 5-position carbon atom on cytosine in CpG dinucleotides. The abnormal methylation of genes is found in most human tumor tissues, the disorder of apparent genetic codes in cancer cells is also first shown by the disorder of DNA methylation level, and most abnormal methylation is hypermethylation of cancer suppressor genes, and the local hypermethylation of CpG islands of the cancer suppressor genes often leads to transcriptional silencing of the cancer suppressor genes, can be earlier than malignant proliferation of the cancer cells and penetrates through the whole development process of the cancer. The canceration process of the esophageal cancer is a complex process of multi-gene variation accumulation, and involves abnormal methylation of various oncogenes and cancer suppressor genes, so that the detection of DNA methylation indexes can be used for early screening, diagnosis and grading of tumors and monitoring of curative effects of anticancer drug treatment stages and prognosis.

Current methods for DNA methylation for tumor detection are largely divided into two categories, whole genome methylation analysis and specific site methylation detection. Genome-wide methylation assays are often used as a means for high throughput screening to find target genes due to their high detection costs. Specific site methylation detection methods can be further classified into restriction enzyme analysis method (COBRA) combined with sodium bisulphite, methylation specific PCR Method (MSP), methylation sensitive high resolution melting curve analysis method, methylation fluorescent quantitation method (methyl light) and the like. The restriction enzyme analysis method can only aim at the methylation condition of the obtained special enzyme cutting site, and has small application range; the methylation specific PCR method is based on common PCR and electrophoresis analysis, has complex operation and is easy to cause sample pollution; the methylation-sensitive high-resolution melting curve analysis method needs to use a fluorescence quantitative PCR instrument with a high-resolution melting (HRM) module for detection, and has higher requirements on instruments and equipment; the methylation fluorescent quantitative method is based on higher flux and sensitivity, does not need operations such as electrophoresis, hybridization and the like after PCR, reduces sample pollution and operation errors, and is widely applied to DNA methylation detection. However, when fluorescence quantitative PCR is used in the methylation fluorescent quantitative method, it is necessary to measure the amount of nucleic acid by means of a standard curve or a reference gene, and errors in and between batches are likely to occur; and the sensitivity of the detection of the low-concentration nucleic acid sample is insufficient, so that the false negative is easy to delay the illness state of the patient. Compared with fluorescent quantitative PCR, digital PCR has higher detection sensitivity and accuracy by absolute quantitative counting of nucleic acid molecules. The digital PCR is to dilute the nucleic acid sample, follow poisson distribution rule, complete PCR amplification of target nucleic acid segment in micro reaction unit effectively and sensitively, obtain fluorescent signal, directly give out copy number of target sequence, and perform statistical analysis.

Based on the defects existing in the prior art, the invention establishes the esophageal cancer gene methylation detection method based on digital PCR by screening related methylation genes of esophageal cancer, expects to obtain detection reagents with higher sensitivity, specificity and accuracy, and realizes early screening, diagnosis and grading of esophageal cancer patients and curative effect monitoring of anticancer drug treatment stages and prognosis.

Disclosure of Invention

It is an object of the first aspect of the present invention to provide a detection zone for methylation detection of esophageal cancer genes.

The object of the second aspect of the present invention is to provide a marker combination for esophageal cancer gene methylation detection.

In a third aspect of the present invention, there is provided a reagent.

The fourth aspect of the present invention is directed to a kit.

In a fifth aspect, the present invention provides a method for detecting methylation of esophageal cancer genes for the purpose of diagnosis of a non-disease.

The sixth aspect of the present invention is directed to providing the use of the methylation detection region for esophageal cancer gene of the first aspect of the present invention, the DNA methylation marker combination of the second aspect of the present invention, the reagent of the third aspect of the present invention or the kit of the fourth aspect of the present invention in a product for diagnosis and/or auxiliary diagnosis of esophageal cancer.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a gene methylation detection region for esophageal cancer comprising the gene methylation detection region shown in (a 1) and/or (a 2);

(a1) The IRF4 gene methylation detection region comprises a methylation detection region obtained by converting Chr6:391499-391683 into sulfite, wherein the nucleotide sequence of the Chr6:391499-391683 is shown as SEQ ID NO. 22;

(a2) The methylation detection region of the UNC5D gene comprises a methylation detection region obtained after the conversion of Chr8:35235143-35235452 by sulfite, and the nucleotide sequence of Chr8:35235143-35235452 is shown as SEQ ID NO. 23.

Preferably, the IRF4 gene methylation detection region described in (a 1) comprises the nucleotide sequence of Chr6:391506-391630 and/or the nucleotide sequence of Chr6:391531-391683, wherein the nucleotide sequence of Chr6:391506-391630 is shown as SEQ ID NO.24, and the nucleotide sequence of Chr6:391531-391683 is shown as SEQ ID NO. 26.

Preferably, the methylation detection region of the UNC5D gene described in (b 1) comprises a nucleotide sequence of Chr8:35235311-35235452 and/or a nucleotide sequence of Chr8:35235310-35235426, wherein the nucleotide sequence of Chr8:35235311-35235452 is shown as SEQ ID NO.27, and the nucleotide sequence of Chr8:35235310-35235426 is shown as SEQ ID NO. 28.

In a second aspect of the invention there is provided a DNA methylation marker combination for oesophageal cancer detection, said marker combination comprising CpG island regions in the sequence of SEQ ID No.22 and/or SEQ ID No. 23.

In a third aspect of the present invention, there is provided a reagent comprising a detection reagent capable of specifically detecting the methylation level of a CpG dinucleotide site in at least any one of the nucleotide sequences of interest in (b 1) to (b 3) in a biological sample;

(b1) The full length of the nucleotide sequence shown as SEQ ID No.22 and/or SEQ ID No.23 or any partial region thereof;

(b2) Full length or any partial region thereof complementary to the nucleotide sequence shown in SEQ ID No.22 and/or SEQ ID No. 23;

(b3) A nucleotide sequence having at least 90% identity to (b 1) or (b 2).

The inventor finds that human esophageal cancer is related to the DNA methylation level of the nucleotide sequence, and the methylation level in an esophageal cancer sample is obviously higher than that in a normal sample, and can provide reference for diagnosis or auxiliary diagnosis of esophageal cancer by detecting whether the subject has the risk of esophageal cancer or early lesions of esophageal cancer or has suffered from lesions of esophageal cancer or not.

DNA methylation is the covalent bonding of a methyl group at the cytosine carbon number 5 of a genomic CpG dinucleotide. The DNA methylation level refers to the proportion of CpG dinucleotide sites that are methylated in all CpG dinucleotide sites within a specific nucleotide sequence or a partial region thereof. In the present invention, DNA methylation detection means determining whether each CpG island is methylated or not by detecting, and calculating the methylation level in the nucleotide sequence of a specific region. In practical application, different detection indexes can be adopted to compare the methylation level of the DNA according to practical situations, for example, in some cases, the methylation proportion of the marker in the sample can be calculated according to the Ct value detected by the sample, namely, the methylation molecular number/(the methylation molecular number+the unmethylation molecular number) ×100% can be calculated in some cases, and then the comparison is carried out, and in some cases, statistical analysis and integration are required to be carried out on each index to obtain the final judgment index.

Preferably, the complementary sequence is a nucleotide sequence formed by complementary one-to-one correspondence to each base of the nucleotide sequence shown in SEQ ID NO.22 or SEQ ID NO. 23.

Preferably, the partial region depicted in (b 1) is the nucleotide sequence depicted at positions 8-132 in SEQ ID NO.22 (i.e., the nucleotide sequence depicted at positions 391506-391630,SEQ ID NO.24 of the IRF4 gene), the nucleotide sequence depicted at positions 32-185 in SEQ ID NO.22 (i.e., the nucleotide sequence depicted at positions 391531-391683,SEQ ID NO.26 of the IRF4 gene), the nucleotide sequence depicted at positions 192-310 in SEQ ID NO.23 (i.e., the nucleotide sequence depicted at positions 35235334-35235452,SEQ ID NO.27 of the UNC5D gene) and/or the nucleotide sequence depicted at positions 168-284 in SEQ ID NO.23 (i.e., the nucleotide sequence depicted at positions 35235310-35235426,SEQ ID NO.28 of the UNC5D gene).

Preferably, the nucleotide sequence of (b 3) is at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to (b 1) or (b 2).

Preferably, the sequence of identity still maintains unchanged the CpG dinucleotide site in the nucleotide sequence shown as SEQ ID NO.22 or SEQ ID NO.23, or the complement thereof.

Preferably, the reagent further comprises a nucleic acid molecule.

Preferably, the nucleic acid molecule comprises a primer pair which can amplify the nucleotide sequences shown in (b 1), (b 2) and/or (b 3).

Preferably, the primer pair comprises one or more of (c 1) - (c 4);

(c1) Nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2;

(c2) Nucleotide sequences shown in SEQ ID No.7 and SEQ ID No. 8;

(c3) Nucleotide sequences shown in SEQ ID No.10 and SEQ ID No. 11;

(c4) The nucleotide sequences shown as SEQ ID No.13 and SEQ ID No. 14.

Preferably, the nucleic acid molecule further comprises a probe capable of labelling the nucleotide sequences shown in (b 1), (b 2) and/or (b 3).

Preferably, the probe comprises one or more of (d 1) - (d 4);

(d1) A nucleotide sequence shown as SEQ ID NO. 3;

(d2) The nucleotide sequence shown in SEQ ID NO. 9;

(d3) A nucleotide sequence shown as SEQ ID NO. 12;

(d4) The nucleotide sequence shown as SEQ ID NO. 15.

Preferably, both ends of the sequence of the probe are marked with a modification group comprising a 5 'group and a 3' group.

Preferably, the 5' group comprises any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY 5.

Preferably, the 3' group includes any one of MGB, BHQ-1, BHQ-2, BHQ-3, MGB-NFQ; and further MGBs.

Preferably, the biological sample comprises at least one of a blood sample, a saliva sample, a tissue sample, and an esophageal-derived cell sample.

Preferably, the tissue sample comprises fresh pathological tissue, paraffin-embedded tissue, and the cell sample comprises esophageal exfoliated cells.

Preferably, the biological sample comprises an ex vivo biological sample derived from a mammal; further sources are humans.

In a fourth aspect of the invention there is provided a kit comprising the reagent of the third aspect of the invention.

Preferably, the kit further comprises a reagent capable of differentially modifying methylated DNA and unmethylated DNA.

Preferably, the reactant is bisulphite.

Preferably, the kit further comprises a primer pair of the reference gene, a probe, a quality control product and a buffer solution.

Preferably, the quality control comprises a positive quality control and a negative quality control.

Preferably, the positive quality control comprises esophageal cancer cell line DNA and the negative quality control comprises peripheral blood leukocyte DNA.

Preferably, the reference gene is GAPDH.

Preferably, the primer pair and the probe of the reference gene comprise the primer pair shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe shown in SEQ ID NO. 21.

Preferably, the esophageal cancer comprises esophageal cancer of different stages, such as high grade neoplasia, early stage esophageal cancer, advanced stage esophageal cancer.

The esophageal cancer of different stages referred to in the invention can be stage system defined by referring to the American cancer society (American Joint Committee onCancer, AJCC), such as esophageal squamous carcinoma, the 0-stage esophageal squamous carcinoma is TisN0M0, and the esophageal cancer is represented by severe dysplasia or high-grade neoplasia of the esophagus. That is, even if the patient is at stage 0 esophageal squamous carcinoma or earlier lesions. The kit can still keep higher sensitivity, has the sensitivity of more than 70 percent, can be applied to early screening of esophageal cancer, and has important significance for early therapeutic intervention and improvement of prognosis of patients.

In a fifth aspect of the invention, there is provided a method for detecting esophageal cancer gene methylation for non-disease diagnostic purposes, comprising the step of employing the kit of the fourth aspect of the invention.

Preferably, the detection method specifically includes the following steps: preparing nucleic acid of a sample to be detected, and performing bisulphite conversion by using a reaction reagent to obtain converted DNA (deoxyribonucleic acid), namely Bis-DNA; the methylation status of Bis-DNA was detected using the kit of the third aspect of the present invention.

Preferably, the final concentration composition of the reaction system of the kit comprises: 0.1-1. Mu.M primer, 0.1-1. Mu.M probe; further preferably, the kit comprises 0.1 to 0.5. Mu.M primer and 0.1 to 0.5. Mu.M probe.

Preferably, the reaction conditions for detecting the methylation state of Bis-DNA by the kit are as follows: 90-95 ℃ for 10-15min; cycling at 90-94 deg.C for 25-30s,56-62 deg.C for 60-65s and 30-50 times; 95-98 ℃ for 8-10min; further at 95℃for 10min;94 ℃ for 30s,56 ℃ for 60s and 45 cycles; and 98 ℃ for 10min.

Preferably, the sample to be tested comprises at least one of a blood sample, a saliva sample, a tissue sample and an oesophageal derived cell sample.

Preferably, the esophageal cancer includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.

Preferably, the methylation status of Bis-DNA can be detected using methods known in the art, including, but not limited to, methylation-specific PCR, quantitative 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, fluorescent quantitation, and the like. The detection reagent can be determined and prepared by one skilled in the art based on reagents and tools required for known methods.

In a sixth aspect of the invention there is provided the use of a detection region according to the first aspect of the invention, a marker combination according to the second aspect of the invention, a reagent according to the third aspect of the invention or a kit according to the fourth aspect of the invention in the manufacture of a product for diagnosis and/or assistance in diagnosis of esophageal cancer.

The beneficial effects of the invention are as follows:

the detection area for detecting the methylation of the esophageal cancer gene provided by the invention can be used as an important detection index for early screening, process monitoring and prognosis evaluation of esophageal cancer, DNA methylation abnormality is taken as a detection object, the DNA methylation abnormality usually occurs in early cancer and penetrates through the occurrence and development processes of cancer, the methylation state of the DNA methylation abnormality can be changed once the methylation state is formed and needs to be continuously stimulated by the external environment for a long time, and therefore, the detection of the DNA methylation area can be used as an important biological index for early screening, process monitoring and prognosis evaluation of esophageal cancer.

The reagent for detecting DNA methylation and the kit for detecting esophageal cancer gene methylation, which are provided by the application, can be used for diagnosing and assisting diagnosis of patients with esophageal cancer on specific nucleotide sequences, and can be used for diagnosing patients with esophageal cancer in different stages, including high-grade neoplasia, early esophageal cancer and advanced esophageal cancer, so that the detection kit can be used for early screening of esophageal cancer, has sensitivity up to 70%, accuracy up to 92% or more and even 100%, shows good sensitivity and specificity, and has important significance for early therapeutic intervention and improvement of prognosis of patients.

Non-invasive detection is possible: the kit for detecting the methylation of the esophageal cancer genes can detect various samples, and can realize noninvasive detection by detecting the methylation state of the genes of esophageal exfoliated cells.

The accuracy is high: the esophageal cancer gene methylation detection kit provided by the invention is based on a digital PCR technology, can efficiently and sensitively complete PCR amplification of target nucleic acid fragments in a micro-reaction unit, acquires fluorescent signals for statistical analysis, thoroughly gets rid of dependence on a standard curve, directly gives out copy numbers of target sequences, improves the stability of experimental results in batches and among batches, realizes absolute quantification of initial samples, improves the sensitivity of a nucleic acid detection method, and effectively reduces the occurrence of false negative.

Drawings

FIG. 1 is a diagram of typical detection results of digital PCR detection of methylation of esophageal cancer genes provided by the implementation of the invention.

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be made clearly and completely in connection with the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.

The embodiment of the application provides a reagent for detecting esophageal cancer gene methylation, and a kit and application thereof. The following will describe in detail. The following description of the embodiments is not intended to limit the preferred embodiments. In addition, in the description of the present application, the term "comprising" means "including but not limited to".

Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified. Experimental methods, in which specific conditions are not specified, are generally performed under conventional conditions, or under conditions recommended by the manufacturer.

According to the invention, bisulphite conversion is carried out on a nucleic acid sample of the esophageal cancer to be detected by adopting a bisulphite modification method, a digital PCR technology is combined, a candidate gene with obvious methylation difference of the esophageal cancer is screened by a TCGA database, a specific esophageal cancer gene methylation detection primer and a specific probe are designed, a DNA sample to be detected which is modified by the bisulphite is amplified, the methylation condition of a target gene in the sample to be detected is determined according to the number of positive microdroplets amplified by PCR, and auxiliary diagnosis is provided for the esophageal cancer.

In an exemplary embodiment of the present invention, digital PCR is used to detect the methylation status of Bis-DNA, and the digital PCR is performed using a droplet PCR technique (droplet digital PCR) by adding a digital PCR mixture to a droplet generator to generate 10000-100000 micro-reaction droplets for PCR amplification. After PCR amplification reaction, judging whether the sample to be detected contains methylated DNA target molecules according to the type of fluorescent signals, and setting internal reference genes simultaneously according to the formula to determine the quantity and the content of the methylated DNA target molecules: methylation ratio = methylation copy number/reference gene copy number x 100%, determined by experimental study results: the methylation proportion of the target gene is more than or equal to 3%, and the interpretation result is positive; the methylation proportion of the target gene is less than 3%, and the result is negative.

Example 1 sample DNA extraction and bisulfite conversion

1. Sample DNA extraction

The DNA of esophageal desquamation cells (which are provided by Henan tumor Hospital) was extracted using a nucleic acid extraction or purification reagent (general type) (product number: GE 100) produced by Anhuida medical science and technology Co., ltd., specific steps are as follows:

a) Taking a proper amount of cell samples, adding 500 mu L of lysate and 30 mu L of proteinase K, and performing pyrolysis at 70 ℃ for 40min after fully mixing;

b) Centrifuging briefly, adding 200 μL of isopropanol, mixing thoroughly, transferring to adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid;

d) Adding 600 mu L of rinsing liquid I for rinsing, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

e) Adding 600 mu L of rinsing liquid II for rinsing, centrifuging at 12000rpm for 30s, and discarding the waste liquid;

f) Adding 600 mu L of rinsing liquid II again for rinsing, centrifuging at 12000rpm for 30s, discarding the waste liquid, and centrifuging at 12000rpm for 3min;

g) Uncapping, and airing in a fume hood for 2min;

h) 60 mu L of eluent is added into the adsorption column, the mixture is kept stand for 3min at room temperature (20 ℃ to 30 ℃ and the same shall apply below), and the mixture is centrifuged at 12000rpm for 2min;

i) Repeating the step h), collecting DNA into a centrifuge tube, and preserving at-20 ℃ for standby.

2. Bisulphite conversion

The genomic DNA obtained above was subjected to bisulfite conversion using a nucleic acid extraction or purification reagent (centrifugal column) (cat# ME 100) produced by Anhuida medical science, inc., with the following steps:

a) Taking 45 mu L of DNA sample to be detected (45 ng/. Mu.L) in a new 1.5mL centrifuge tube, adding 5 mu L of conversion buffer solution, and placing in a metal bath for incubation at a constant temperature of 37 ℃ for 15min;

b) After the incubation is completed, 100 mu L of a pre-prepared conversion solution is added into each sample, the mixture is uniformly mixed and centrifuged for a short time, and the metal bath is incubated for 12 to 16 hours at 50 ℃ in a dark place;

c) Incubating the sample on ice (0-4deg.C) for 10min;

d) Placing the adsorption column in a collecting pipe, and adding 400 mu L of binding solution into the adsorption column;

e) C, adding the sample in the step into an adsorption column (containing a binding solution), covering a tube cover, uniformly mixing the sample with the binding solution in an upside down manner for a plurality of times, centrifuging the sample at full speed (14000 rpm) for 30s, and discarding waste liquid;

f) Adding 100 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, and discarding waste liquid;

g) Adding 200 mu L of desulfonation liquid into an adsorption column, incubating for 20min at room temperature (20-30 ℃), centrifuging for 30s at full speed, and discarding waste liquid;

h) Adding 200 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, repeatedly adding 200 mu L of rinsing liquid, centrifuging at full speed for 30s, discarding waste liquid and collecting the tube;

i) Placing the adsorption column into a 1.5mL sterile centrifuge tube, suspending and dripping 30 mu L of eluent into the middle part of the adsorption film, eluting and transforming DNA, centrifuging at full speed for 1min, collecting Bis-DNA, and preserving at-20 ℃ for later use.

Example 2 methylation differential Gene of esophageal cancer tissue and specific primer and Probe screening

1. Screening of methylation differential Gene of esophageal cancer tissue

The methylation chip data related to the esophageal cancer and the corresponding transcriptome sequencing data are obtained through a TCGA database (http:// cancerationname. Gov /), and analyzed, and methylation sites with obvious differences are screened, so that the result shows that IRF4 and UNC5D are methylation sites with obvious differences, namely IRF4 and UNC5D are taken as methylation difference genes of esophageal cancer tissues.

2. Specific primer and probe design and screening for methylation detection of esophageal cancer

1) Specific primer and probe screening

According to the above-mentioned IRF4, UNC5D gene and its promoter region nucleic acid sequence, through repeated design, push and screening, the inventor determines that the genomic position of IRF4 gene methylation detection region is Ch6:391499-391683 (GRCh 38/hg 38), i.e. the methylation detection region obtained by sulfite conversion of the nucleic acid sequence shown in SEQ ID NO.22, and the genomic position of UNC5D gene methylation detection region is Ch8: 35235143-35235452 (GRCh 38/hg 38), i.e. the methylation detection region obtained by sulfite conversion of the nucleic acid sequence shown in SEQ ID NO.23, on the basis of which digital PCR probes and primers for related gene methylation are designed, the specific sequences are shown in Table 1, and specific primers and probes for internal reference gene GAPDH are set at the same time, the specific sequences are shown in Table 2. Primers and probes were synthesized by Biotechnology Inc. of the family Bosch, beijing.

TABLE 1 sequence information of primers and probes for IRF4 and UNC5D

TABLE 2 primer and probe sequence information for reference gene GAPDH

The two ends of the probe sequence are marked with modification groups, including a 5 'group and a 3' group. Wherein the 5 'group is selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY5, and the 3' group is selected from any one of MGB, BHQ-1, BHQ-2, BHQ-3 and MGB-NFQ.

2) PCR amplification

The primer probe combinations (exemplified by the bure QX200 microdroplet digital PCR) were further screened using PCR amplification as follows:

the reaction system: 10. Mu.L of 2 Xdigital PCR reaction premix (Berle (Bio-Rad) Co., ltd., cat# 1863023), 0.1. Mu.L of each of 10. Mu.M GAPDH primer and probe, 0.5. Mu.L of each of 10. Mu.M primer and probe in the primer-probe combination, and 6. Mu.L of Bis-DNA (prepared in example 1) were added with water to 20. Mu.L.

Droplet preparation: the reaction system of 20. Mu.L was loaded with a droplet-generating card containing 70. Mu.L of a droplet-generating oil (Berle Co., ltd., cat# 1863005) and placed on a droplet generator to generate droplets, which were completed in generally 2 minutes, and the generated droplets were transferred to a 96-well plate, and then sealed with a preheated PX1 heat sealer, and the film was sealed and then subjected to PCR within 30 minutes. The PCR reaction procedure is shown in Table 3.

TABLE 3 PCR reaction procedure

The annealing temperature was selected at 56-62℃depending on the TM value of each primer combination.

Droplet reading and signal analysis: the PCR 96-well plate with the reaction completed was placed in a microdroplet reader and QuantaSoft was opened ^TM And the software establishes sample module information according to the sample quantity and the sample layout, and runs after the setting is completed. After the data reading is completed, the threshold is automatically adjusted for positive and negative droplet assignment for each detection channel. Sample adjustment partitioning threshold, methylation data collection and analysis are shown in QuantaSoft ^TM In a software interface.

The PCR amplification was performed simultaneously with a DNA sample of esophageal cast-off cells of healthy persons (DNA extraction and bisulfite conversion as in example 1) as a control group.

The methylation ratio was calculated according to the following formula: methylation ratio = methylation copy number/reference gene copy number x 100%.

The primer probe combination selected in 1) is selected by PCR amplification by taking DNA samples of esophageal cancer confirmed diagnosis patients and esophageal exfoliated cells of healthy people (provided by tumor hospitals in Henan province) as templates, and the result shows that the methylation proportion of IRF4 genes is less than 3% (0.59%, 0.55% and 0.56% respectively) no matter when the primer probe combination 1, 2 and 3 detect the esophageal exfoliated cell samples of the same normal person, the detection results are negative, and when the exfoliated cells of the same esophageal cancer patients are detected, the methylation copy number (1324) obtained by the detection of the primer probe combination 2 is obviously less than that of the primer probe combination 1 (2298) and the primer probe combination 3 (2021), the methylation proportion of the IRF4 genes obtained by the detection of the primer probe combination is obviously lower than that of the primer probe combination 1 (30.70%) and the primer probe combination 3 (25.62%) (Table 4), so that the subsequent experiments are finally carried out by using the primer probe combination 1 and the primer probe combination 3.

As can be seen from Table 5, when the primer probe combinations 4, 5 and 6 detect the same normal human endometrial cast-off cell sample, the methylation proportion of the UNC5D gene is less than 3% (0.93%, 0.84% and 0.85% respectively), the detection results are all negative, and when the cast-off cells of the same endometrial cancer patient are detected, the methylation copy number (3341) of the primer probe combination 6 is obviously less than that of the primer probe combination 4 (5282) and the primer probe combination 5 (4989), the methylation proportion of the detected UNC5D gene is 25.70% and is obviously lower than that of the primer probe combination 4 (44.41%) and the primer probe combination 5 (49.56%), so that the primer probe combination 4 and the primer probe combination 5 are finally selected for subsequent experiments for the target gene UNC 5D.

TABLE 4 primer probe combination detection results of target gene IRF4

TABLE 5 primer probe combination detection results for target gene UNC5D

Example 3

A kit for methylation detection of esophageal cancer genes, comprising the following components: the primer probe set (at least one set of primer probe set 1, primer probe set 3, primer probe set 4, or primer probe set 5) in example 2, the primer and probe of the internal reference gene GAPDH (same as in example 2), negative quality control (human peripheral blood leukocyte DNA), positive quality control (human esophageal cancer cell line DNA), 2 x digital PCR reaction premix, bisulfite.

Example 4 clinical sample detection

Clinical samples (142 samples of tissue of patients with esophageal cancer, 42 samples of high-grade neoplasia, 46 samples of early esophageal cancer and 54 samples of esophageal cancer in advanced stage, 40 samples of tissue of healthy people, all provided by Henan tumor Hospital and pathologically identified) were tested by using the kit of example 3 to verify the effect of the kit, pretreatment of the sample to be tested was the same as the sample DNA extraction and bisulfite conversion in example 1, and the procedure of the detection reaction system agent was the same as that of 2) PCR amplification part in example 2). The sum of the methylation copy number of the detected sample and the copy number of the reference gene is more than or equal to 100, so that the detection result of the sample is effective, and the analysis of the result can be continued; if less than 100, the sample detection is not effective and a re-detection is required. The methylation ratio of the sample is calculated from the following formula: methylation ratio = methylation copy number/reference gene copy number x 100%, determined by experimental study results: the methylation proportion of the target gene is more than or equal to 3%, and the interpretation result is positive; the methylation proportion of the target gene is less than 3%, and the result is negative; typical test results are shown in FIG. 1.

1. Single-nucleotide detection using a kit comprising a single primer probe combination

The detection sensitivity and the specificity of the kit only containing a single primer probe combination in the tissue sample are examined by respectively using the primer probe combination 1, the primer probe combination 3, the primer probe combination 4 or the primer probe combination 5 to detect 142 samples of esophageal cancer patients and 40 samples of healthy people.

The results are shown in Table 6, the primer probe sets 1, 3, 4 and 5 have the detection sensitivity of more than 92% for esophageal cancer in the progressive stage, the detection sensitivity of more than 73% for early stage esophageal cancer and the detection sensitivity of more than 71% for high-grade neoplasia, and the kit containing the single primer probe combination has higher detection sensitivity for tissues of patients with esophageal cancer. Meanwhile, the detection of the tissue sample of a normal person shows that the detection specificity of the kit containing the single primer probe combination is 95-100%, and the kit has higher detection specificity.

TABLE 6 detection sensitivity and specificity of Single primer probe combinations containing kits in tissue samples

2. Single-nucleotide detection using a kit comprising a combination of two primer probes

And (3) respectively using the primer probe combination 1, the primer probe combination 3, the primer probe combination 4 and the primer probe combination 5 to carry out single-nucleotide detection on the samples of 142 esophageal cancer patients and the samples of 40 healthy people, and superposing the single-nucleotide detection results of the two primer probe combinations on the statistical judgment detection junction, wherein when any detection result of the two primer probe combinations is positive, the detection sensitivity and the specificity of the kit containing the two primer probe combinations in the tissue samples are further examined.

As shown in Table 7, the detection sensitivity of the kit containing the two primer probe combinations for esophageal cancer in the progressive stage is 96% -100%, the detection sensitivity for early stage esophageal cancer is 80% -83%, the detection sensitivity for high-grade neoplasia samples is 73% -79%, and the detection specificity for normal human samples is 92% -98%, and when the detection results of the single-nucleic acid detection results of the kit containing the two primer probe combinations are overlapped and statistically judged, the detection sensitivity is higher than the single-nucleic acid detection results of the kit containing the single primer probe combinations, but the detection specificity is reduced, which indicates that the single-nucleic acid detection mode of the kit containing the two primer probe combinations can possibly lead to the reduction of the specificity while the sensitivity is improved.

TABLE 7 detection sensitivity and specificity of kit containing two primer probe combinations in tissue samples

3. Dual nucleic acid detection using a kit containing a combination of two primer probes

And (3) respectively carrying out double nucleic acid detection on the samples of 142 esophageal cancer patients and the samples of 40 healthy people by using the kit of any two combinations of the primer probe combination 1, the primer probe combination 3, the primer probe combination 4 and the primer probe combination 5, carrying out double nucleic acid detection of the double primer probe combination in a single tube, and when any detection result of the two primer probe combinations is positive, judging that the detection result is positive, and further examining the detection sensitivity and the specificity of the kit containing the two primer probe combinations in the tissue sample (table 8).

The results show that the detection sensitivity of the dual nucleic acid detection of the kit containing the two primer probe combinations for esophageal cancer in the progressive stage is 96% -99%, the detection sensitivity of the kit containing the two primer probe combinations for early stage esophageal cancer is 80% -83%, the detection sensitivity of the kit containing the two primer probe combinations for high-level neoplasia samples is 73% -79%, and the detection specificity of the kit containing the two primer probe combinations for normal human samples is 95% -98%, compared with the single-nucleic acid detection result superposition statistical judgment detection of the kit containing the two primer probe combinations, the detection sensitivity of the dual nucleic acid detection of the kit containing the two primer probe combinations is slightly reduced, but the detection specificity of the kit containing the two primer probe combinations is improved to a certain extent; meanwhile, compared with the single-nucleic acid detection of the kit with the single primer probe combination, the detection specificity of the double-nucleic acid detection of the kit with the two primer probe combinations is slightly reduced, but the detection sensitivity is obviously improved. In addition, the dual nucleic acid detection of the kit containing the combination of the two primer probes can reduce reagent consumption to a great extent, reduce consumable cost, reduce the operation of experimenters and reduce labor cost.

Table 8 results of double nucleic acid detection in a kit containing two primer probe combinations

In conclusion, the kit for methylation detection of esophageal cancer genes provided in the embodiment 3 has higher detection sensitivity and detection specificity, and is ideal for diagnosis and early screening of esophageal cancer, so that early diagnosis and early treatment of esophageal cancer are assisted, and the kit has good clinical application prospects.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. A detection region for methylation detection of an esophageal cancer gene, comprising the detection region shown in (a 1) and/or (a 2);

(a1) The IRF4 gene methylation detection region comprises a detection region obtained by converting Chr6:391499-391683 into sulfite, wherein the nucleotide sequence of the Chr6:391499-391683 is shown as SEQ ID NO. 22;

(a2) The methylation detection region of the UNC5D gene comprises a detection region obtained by converting Chr8:35235143-35235452 into sulfite, and the nucleotide sequence of the Chr8:35235143-35235452 is shown as SEQ ID NO. 23.

2. A marker combination for methylation detection of esophageal cancer genes, characterized in that the marker combination comprises CpG island regions in the sequence of SEQ ID No.22 and/or SEQ ID No. 23.

3. A reagent comprising a detection reagent capable of specifically detecting the methylation level of a CpG dinucleotide site in at least any one of the nucleotide sequences of interest (b 1) to (b 3) in a biological sample;

(b1) The full length of the nucleotide sequence shown as SEQ ID No.22 and/or SEQ ID No.23 or any partial region thereof; (b2) Full length or any partial region thereof complementary to the nucleotide sequence shown in SEQ ID No.22 and/or SEQ ID No. 23;

(b3) A nucleotide sequence having at least 90% identity to (b 1) or (b 2).

4. The reagent of claim 3, wherein the reagent further comprises a nucleic acid molecule; preferably, the nucleic acid molecule comprises a primer pair which can amplify the nucleotide sequences shown in (b 1), (b 2) and/or (b 3); preferably, the nucleic acid molecule further comprises a probe capable of labelling the nucleotide sequences shown in (b 1), (b 2) and/or (b 3); preferably, the probe is labeled with a modifying group at both ends of its sequence.

5. The reagent of claim 4, wherein the primer pair comprises one or more of (c 1) - (c 4);

(c1) Nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2;

(c2) Nucleotide sequences shown in SEQ ID No.7 and SEQ ID No. 8;

(c3) Nucleotide sequences shown in SEQ ID No.10 and SEQ ID No. 11;

(c4) The nucleotide sequences shown as SEQ ID No.13 and SEQ ID No. 14.

6. The reagent of claim 5, wherein the probe comprises one or more of (d 1) - (d 4);

(d1) A nucleotide sequence shown as SEQ ID NO. 3;

(d2) The nucleotide sequence shown in SEQ ID NO. 9;

(d3) A nucleotide sequence shown as SEQ ID NO. 12;

(d4) The nucleotide sequence shown as SEQ ID NO. 15.

7. A kit comprising the reagent of any one of claims 3-6; preferably, the kit further comprises a reagent capable of differentially modifying methylated DNA and unmethylated DNA; preferably, the kit further comprises a primer pair of the reference gene, a probe, a quality control product and a buffer solution.

8. The kit according to claim 7, wherein the primer pair and the probe of the reference gene comprise primer pairs shown in SEQ ID NO.19 and SEQ ID NO.20, and a probe shown in SEQ ID NO. 21.

9. A method for detecting methylation of esophageal cancer genes for the purpose of non-disease diagnosis, comprising the step of using the kit according to claim 7 or 8; preferably, the detection method specifically includes the following steps: preparing nucleic acid of a sample to be detected, and performing bisulphite conversion by using a reaction reagent to obtain converted DNA (deoxyribonucleic acid), namely Bis-DNA; detecting the methylation status of Bis-DNA using the kit of claim 7 or 8.

10. Use of the detection region of claim 1, the marker combination of claim 2, the reagent of any one of claims 3-6 or the kit of claim 7 or 8 for the preparation of a product for diagnosis and/or auxiliary diagnosis of esophageal cancer.