CN115851941A - Marker, nucleic acid product and kit for screening esophageal cancer and application of marker and kit - Google Patents

Abstract

The application relates to a marker, a nucleic acid product, a kit and application thereof for screening esophageal cancer, wherein the marker comprises one or more of VWC2, THBD and FAM19A4, and esophageal cancer is diagnosed by detecting the methylation level of the marker. According to the application, VWC2, THBD and FAM19A4 can be used as biomarkers to carry out high-sensitivity and high-specificity screening diagnosis on early esophageal cancer.

Description

Marker, nucleic acid product and kit for screening esophageal cancer and application of marker and kit

Technical Field

The application relates to the technical field of molecular biology, in particular to a marker, a nucleic acid product, a kit and application thereof for screening esophageal cancer.

Background

Esophageal cancer is one of the most common malignant tumors, seriously threatening human life and health. In 2020, the number of new cases of esophageal cancer worldwide exceeds 60 ten thousand.

There are two types of esophageal cancer, esophageal adenocarcinoma and esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma occurs predominantly in the upper and middle portions of the esophagus. Most of the esophageal cancer is in the late stage when obvious symptoms appear, so the death rate is high, the prognosis is poor, and the five-year survival rate is less than 20%. The earlier the esophageal cancer treatment, the better the treatment effect. Early detection is a key factor in determining the survival rate of esophageal cancer. Currently, the upper gastrointestinal endoscopy is the most common and popular examination means for EC, but the large-scale clinical application is limited due to the high price and certain discomfort.

Disclosure of Invention

The research of the application finds that the methylation of VWC2, THBD and FAM19A4 genes has great correlation with esophageal cancer. Based on the marker, the nucleic acid product, the kit and the application thereof are provided for screening esophageal cancer with high sensitivity and high specificity.

According to one aspect of the present application, there is provided a marker for screening for esophageal cancer, the marker comprising one or more of VWC2, THBD, FAM19A4, the esophageal cancer being diagnosed by detecting the methylation level of the marker.

In some of these embodiments, esophageal cancer is screened for by detecting the methylation level of one or more of the following regions, with reference to grch38. P14:

region 1: and (2) Chr7:49772138-49774638;

and (4) area 2: and (2) Chr20:23047672-23052672;

region 3: and (2) Chr3:68931547-68933547.

The application provides an application of the reagent for detecting the methylation level of the marker in preparing products for screening and diagnosing esophageal cancer.

The present application also provides a nucleic acid product for screening for esophageal cancer, comprising a primer pair for detecting the methylation level of the above marker.

In some of these embodiments, the nucleic acid product further comprises a nucleic acid probe corresponding to the primer pair.

In some of these embodiments, the primer pairs include one or more of:

a first primer pair with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 and used for detecting the methylation level of the VWC2 gene;

a second primer pair with nucleotide sequences shown as SEQ ID No.4 and SEQ ID No.5 and used for detecting the methylation level of the THBD gene; and

and the nucleotide sequences are shown as SEQ ID No.7 and SEQ ID No.8, and the third primer pair is used for detecting the methylation level of the FAM19A4 gene.

In some of these embodiments, the nucleotide sequence of the detection probe corresponding to the first primer pair is set forth in SEQ ID No. 3;

and/or the nucleotide sequence of the detection probe corresponding to the second primer pair is shown as SEQ ID No. 6;

and/or the nucleotide sequence of the detection probe corresponding to the third primer pair is shown as SEQ ID No. 9.

In some embodiments, the kit further comprises an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair.

In some embodiments, the nucleotide sequences of the reference primer pair are shown as SEQ ID No.10 and SEQ ID No.11, and the nucleotide sequence of the reference probe is shown as SEQ ID No. 12.

According to another aspect of the present application, there is provided a kit for screening esophageal cancer, comprising a reagent for detecting the methylation level of the esophageal cancer marker.

In some of these embodiments, the reagent detects the methylation level of the marker by one or more of the following methods:

methylation specificity PCR, bisulfite sequencing, methylation specificity high performance liquid chromatography, methylation specificity microarray, whole genome methylation sequencing, direct sequencing, methylation specificity high resolution solubility curve, methylation sensitivity restriction endonuclease and methylation fluorescence quantitative PCR.

In some embodiments, the kit comprises one or more of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents, and sequencing reagents, as well as the nucleic acid products described above.

The application screens genes which are extremely relevant to esophageal cancer through a large amount of research by collecting and analyzing methylation data relevant to esophageal cancer in TCGA and GEO databases: VWC2, THBD and FAM19A4, and the nucleic acid product of the present application has proven >95% sensitivity for screening for esophageal cancer: 148 cases were detected in 155 samples of patients with esophageal cancer; specificity >98%: out of 230 healthy human samples, 4 samples were detected. Therefore, the VWC2, THBD and FAM19A4 genes can be used as markers for screening and diagnosing early esophageal cancer with high sensitivity and high specificity.

Drawings

FIG. 1 shows the detection result of a VWC2 gene methylation PCR reaction solution system for detecting a positive reference in example 3 of the present application;

FIG. 2 shows the result of detecting a positive reference substance in the methylated PCR reaction solution system of THBD gene in example 3 of the present application;

FIG. 3 shows the detection result of the FAM19A4 gene methylation PCR reaction solution system for detecting a positive reference substance in example 3 of the present application;

FIG. 4 shows the detection result of the positive reference substance detected by the VWC2/THBD/FAM19A4 gene methylation PCR reaction solution system in example 3 of the present application.

Detailed Description

The present invention is not limited to the above-described embodiments, but various modifications can be made without departing from the scope of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of embodiments in many different forms than those described herein and that modifications may be made by one skilled in the art without departing from the spirit and scope of the application and it is therefore not intended to be limited to the specific embodiments disclosed below.

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 present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or can be prepared by an existing method.

Methylation level refers to whether or not a cytosine in one or more CpG dinucleotides in a stretch of DNA is methylated or the frequency/ratio/percentage of methylation occurring, and represents both a qualitative and a quantitative concept. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

Circulating tumor DNA (ctDNA) is DNA from tumor cells that is free in the blood of a patient, and after necrosis or apoptosis of tumor cells, DNA in the cells is released into the circulatory system and is free in the blood. By detecting the methylation state of the related gene of ctDNA, the in-vivo tumor tissue information is convenient to obtain, thereby providing a basis for tumor diagnosis.

The inventor creatively proposes that by collecting and analyzing the methylation data related to esophageal cancer in TCGA and GEO databases, different methylation site analysis models are constructed by adopting a bioinformatics method, plasma free DNA methylation biomarkers with application values are screened out, and the methylation markers with high specificity and high sensitivity are further screened out by verifying clinical samples: VWC2, THBD and FAM19A4. In addition, the results of experiments show that compared with the method for screening the esophageal cancer by a single gene, the sensitivity and the specificity of screening the esophageal cancer by combining multiple genes are higher.

Accordingly, some embodiments of the present application provide a marker for screening esophageal cancer, including one or more of VWC2, THBD and FAM19A4, by detecting the methylation level of the above marker.

Esophageal cancer was screened by detecting the methylation level of one or more of the following regions, referenced grch38. P14:

region 1: and (2) Chr7:49772138-49774638;

region 2: and (2) Chr20:23047672-23052672;

region 3: and (2) Chr3:68931547-68933547.

In addition, an embodiment of the application also provides application of a reagent for detecting the methylation level of the marker in preparing a product for screening esophageal cancer.

In addition, an embodiment of the present application also provides a nucleic acid product for screening esophageal cancer, which comprises a primer pair for detecting the methylation level of the marker.

In one embodiment, the primer pair includes one or more of the following groups:

a first primer pair with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 and used for detecting the methylation level of the VWC2 gene; a second primer pair with nucleotide sequences shown as SEQ ID No.4 and SEQ ID No.5 and used for detecting the methylation level of the THBD gene; and a third primer pair with nucleotide sequences shown as SEQ ID No.7 and SEQ ID No.8 and used for detecting the methylation level of the FAM19A4 gene.

Specifically, the nucleotide sequence shown in SEQ ID No.1 is 5-; the nucleotide sequence shown as SEQ ID No.2 is 5-; the nucleotide sequence shown as SEQ ID No.4 is 5 'GGGTGTAAGAAGTATTATTT-3'; the nucleotide sequence shown as SEQ ID No.5 is CTCTACCCATAACTAACCA-3'; the nucleotide sequence shown as SEQ ID No.7 is 5-; the nucleotide sequence shown as SEQ ID No.8 is 5 'AACCTAAACTACTACTACT-doped 3'.

In one embodiment, the nucleic acid product further comprises a nucleic acid probe corresponding to the primer pair.

Further, the nucleotide sequence of the detection probe corresponding to the first primer pair is shown as SEQ ID No. 3; the nucleotide sequence of the detection probe corresponding to the second primer pair is shown as SEQ ID No. 6; the nucleotide sequence of the detection probe corresponding to the third primer pair is shown as SEQ ID No. 9. Specifically, the nucleotide sequence shown as SEQ ID No.3 is 5-; the nucleotide sequence shown as SEQ ID No.6 is 5 'TATTCGGCGTCGTAGTAATTTATTTG-3'; the nucleotide sequence shown as SEQ ID No.9 is 5 'TTGGGTTCGGTTTCGTATTGTTAG-3'.

In one embodiment, the nucleic acid product further comprises an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair.

In one embodiment, the reference gene is selected from one or more of ACTB, GAPDH, ALDOA and PGK 1.

Further, the reference gene is ACTB.

In one embodiment, the nucleotide sequences of the internal reference primer pair are shown as SEQ ID No.10 and SEQ ID No.11, and the nucleotide sequence of the internal reference probe is shown as SEQ ID No. 12.

Specifically, the nucleotide sequence shown in SEQ ID No.10 is 5-; the nucleotide sequence shown as SEQ ID No.11 is 5; the nucleotide sequence shown as SEQ ID No.12 is 5.

The nucleic acid product can be used for clinically screening early esophageal cancer with high sensitivity and high specificity.

In addition, an embodiment of the present application also provides a kit for screening esophageal cancer, comprising a reagent for detecting the methylation level of the above marker.

In one embodiment, the above reagent detects the methylation level of the marker by one or more of the following methods: methylation specificity PCR, bisulfite sequencing, methylation specificity high performance liquid chromatography, methylation specificity microarray method, whole genome methylation sequencing, direct sequencing, methylation specificity high resolution solubility curve method, methylation sensitivity restriction endonuclease method and methylation fluorescence quantitative PCR method.

In one embodiment, the kit comprises one or more of a nucleic acid extraction reagent, a methylation conversion reagent, a quality control reagent, a PCR reaction reagent and a sequencing reagent, and the nucleic acid product for screening esophageal cancer.

In one embodiment, the PCR reagent comprises the above-mentioned nucleic acid product, PCR buffer, dNTP, mgCl ₂ And TaqDNA polymerase.

In one embodiment, the concentration of the nucleic acid product is 0.1 to 1. Mu. Mol/L, the concentration of TaqDNA polymerase is 0.1 to 1U/μ L, the concentration of dNTP is 0.2 to 2mmol/L, and MgCl is added to the PCR reagent ₂ The concentration of (b) is 5 mmol/L-25 mmol/L, and the concentration of PCR buffer solution is 10 mmol/L-50 mmol/L.

Further, in the PCR reaction reagent, the concentration of the nucleic acid product is 0.1 to 0.4. Mu. Mol/L, the concentration of TaqDNA polymerase is 0.1 to 0.4U/μ L, the concentration of dNTP is 0.2 to 0.4mmol/L, and MgCl ₂ The concentration of the PCR buffer solution is 5mmol/L to 10mmol/L and the concentration of the PCR buffer solution is 20mmol/L to 30mmol/L.

It is understood that too low a concentration of nucleic acid product affects yield, that higher concentrations cause increased mismatch and non-specific products, and may increase the generation probability of dimers; among them, the enzyme amount of TaqDNA polymerase is increased to lower the reaction specificity, and the reaction yield is influenced by too small enzyme amount.

The gene quantitative detection technology for screening the esophageal cancer has the advantages of simplicity, convenience, sensitivity, accuracy and the like.

The present application also provides a method for detecting the methylation level of the above marker, comprising steps S10, S20 and S30. Specifically, the method comprises the following steps:

step S10: and extracting sample DNA.

In one embodiment, the sample is a biological sample selected from an individual, including but not limited to one or more of the following: cell lines, histological sections, tissue biopsies/paraffin-embedded tissues, body fluids, stool, colon effluents, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood.

Further, the sample is plasma. The free DNA in the plasma can be used for detecting tumors and has the characteristics of little harm to patients, high specificity and the like.

Step S20: and carrying out methylation conversion on the extracted DNA to obtain a conversion sample.

In one embodiment, the methylation is converted to one of a sulfite conversion or an enzymatic conversion.

Step S30: the transformed sample obtained in step S20 is used as a template, and the transformed template is detected by using the nucleic acid product.

Sulfite treatment converts all unmethylated cytosines to uracils, which are converted to thymines in subsequent PCR amplifications. Methylated cytosines remain unchanged during this process. The step can convert epigenetic information which cannot be detected at ordinary times into sequence information which is easy to detect, the resolution reaches a single base, and the sensitivity and specificity of marker detection are improved, so that the accuracy of esophageal cancer screening detection is ensured.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present application is further described below with reference to the following examples and drawings, but the present application shall not be construed as being limited to the scope of the present application.

Example 1 screening of methylated Gene and design of primer set and Probe

The method comprises the steps of mining data in a TCGA (TCGA) database, constructing a large methylation data set related to esophageal cancer by high-throughput methylation sequencing of clinical samples, constructing analysis models of different methylation sites by a bioinformatics method, screening free plasma DNA methylation biomarkers with application values, and further screening methylation markers with high specificity and high sensitivity by verifying the clinical samples.

Specifically, the application screens candidate targets suitable for plasma free DNA methylation by: performing differential analysis on the methylation data of esophageal cancer patients and healthy people in the TCGA database and clinical sample high-throughput methylation sequencing data, and screening to obtain methylation differential sites with hypermethylation of esophageal cancer patients and hypomethylation of healthy people; further, methylation difference analysis is carried out on the obtained methylation difference sites in other 18 cancers in the TCGA database, and methylation difference sites which are hypomethylated in other cancers are extracted; furthermore, clinical verification is carried out on the screened methylation difference sites, so that the optimal detection target of VWC2, THBD and FAM19A4 gene methylation is finally determined, and a nucleic acid product for early screening and diagnosing esophageal cancer is designed.

Primer pairs and probes were designed according to the human whole genome sequence published by the National Center for Biotechnology Information (NCBI) using Primer Express 3.0 software and Methyl Primer Express v1.0 software. Comprises a primer pair and a probe for detecting the methylation of VWC2 (Chr 7: 49772138-49774638), THBD (Chr 20: 23047672-23052672) and FAM19A4 (Chr 3: 68931547-68933547) genes and a primer pair and a probe for detecting the methylation of an internal reference gene ACTB; wherein the sequences of a primer pair for detecting the methylation of the VWC2 gene are shown as SEQ ID No.1 and SEQ ID No.2, and the sequence of a probe is shown as SEQ ID No. 3; the sequences of a primer pair for detecting the methylation state of the THBD gene are shown as SEQ ID No.4 and SEQ ID No.5, and the sequence of a probe is shown as SEQ ID No. 6; the sequences of the primer pair for detecting the methylation state of the FAM19A4 gene are shown as SEQ ID No.7 and SEQ ID No.8, and the sequence of the probe is shown as SEQ ID No. 9. The primer pair and the probe for detecting the methylation of the VWC2, THBD and FAM19A4 genes and the primer pair and the probe for detecting the ACTB gene are respectively included, and the nucleotide sequences of the primer pair and the probe are shown in the table 1.

TABLE 1

Wherein, the 5 'end of the probe SEQ ID No.3 for detecting the VWC2 is marked with a report fluorescent group FAM, and the 3' end is marked with a quenching fluorescent group BHQ; the probe SEQ ID No.6 for detecting the THBD is marked with a report fluorescent group FAM at the 5 'end and a quenching fluorescent group BHQ at the 3' end; a probe SEQ ID No.9 for detecting FAM19A4, wherein the 5 'end of the probe is marked with a report fluorescent group FAM, and the 3' end of the probe is marked with a quenching fluorescent group BHQ; the probe SEQ ID No.12 for detecting ACTB is labeled with a reporter fluorophore JOE at the 5 'end and a quenching fluorophore BHQ at the 3' end.

Example 2 kit for screening and diagnosing esophageal cancer

The kit for screening and diagnosing the esophageal cancer comprises a primer pair and a probe shown in the table 1. The kit also comprises at least one of a nucleic acid extraction reagent, a methylation conversion reagent, a PCR reaction solution, a positive quality control product and a negative quality control product; wherein, the PCR reaction solution comprises primers and probes shown as SEQ ID No. 1-SEQ ID No.3, primers and probes shown as SEQ ID No. 4-SEQ ID No.6, primers and probes shown as SEQ ID No. 7-SEQ ID No.9, primers and probes shown as SEQ ID No. 10-SEQ ID No.12, PCR buffer solution, dNTP, mgCl ₂ And TaqDNA polymerase, wherein the nucleic acid product, PCR buffer, dNTP, mgCl ₂ And the concentration ratio of TaqDNA polymerase is 0.1 mu mol/L:20mmol/L:0.2mmol/L:5mmol/L: 0.1U/. Mu.L, and the correspondence between the primers and the probes is specifically shown in Table 1 above; yang propertyThe control material adopts human methylated genomic DNA, and the negative quality control material adopts human non-methylated genomic DNA.

Example 3 application of kit in esophageal cancer screening and diagnosis

The present embodiment includes the following detection steps:

1. materials, reagents, apparatus

The nucleic acid extraction reagent and the methylation conversion kit are self-developed reagents of the company; PCR buffer, dNTP, taqDNA polymerase were purchased from Takara; mgCl ₂ Purchased from Sigma company; the primers and the probes were synthesized by Shanghai Biotech, inc.; the fluorescent quantitative PCR instrument is ABI7500.

2. Sample preparation

The positive reference substance is human methylated genomic DNA, the negative reference substance is human unmethylated genomic DNA, and the sensitivity reference substance is 10ng/mL human unmethylated genomic DNA containing 0.1% human methylated genomic DNA; the samples to be treated were 1 plasma sample from a patient diagnosed with esophageal cancer and 1 plasma sample from a normal healthy person.

3. DNA extraction

1. Adding 2mL of plasma sample, 5mL of nucleic acid cracking adsorption solution and 100 mu L of magnetic beads into a 15mL centrifuge tube, uniformly mixing by vortex, and placing the centrifuge tube at 56 ℃ for 10 minutes;

2. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 1mL of washing liquid A, and uniformly mixing to ensure that the magnetic beads are thoroughly resuspended;

3. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, sucking away the residual liquid as much as possible by using a 10-100 mu L gun head, moving the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 5min;

4. adding 40 μ L of eluent, covering the tube cover, vortex, mixing, resuspending magnetic beads, placing the centrifuge tube in 56 deg.C, and incubating for 5min;

5. the centrifuge tube was placed in a magnetic rack for magnetic attraction for 2min and the eluate was transferred to a new 0.2mL PCR tube.

4. Sulfite conversion

1. Adding 110 mu L of sulfite solution into a 0.2mL PCR tube containing 40 mu L of DNA, covering the centrifugal tube, whirling, uniformly mixing, centrifuging for a short time, placing the centrifugal tube in a common PCR instrument for reaction, wherein the reaction conditions are set to 95 ℃ for 5min, 60 ℃ for 10min, 95 ℃ for 5min and 60 ℃ for 10min;

2. transferring the DNA solution after the reaction to a new 1.5mL centrifuge tube, adding 600 μ L binding solution and 10 μ L magnetic bead, mixing by vortex, and standing at room temperature for 5min;

3. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid A, and performing vortex mixing to ensure that the magnetic beads are thoroughly resuspended;

4. placing the centrifuge tube in a magnetic frame, magnetically attracting for 2min, sucking away all the waste liquid, adding 200 μ L desulfonation liquid, vortex and mixing to ensure that the magnetic beads are thoroughly resuspended, and standing at room temperature for 15min;

5. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid B, and vortex mixing to ensure thorough resuspension of magnetic beads;

6. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid C, and vortex mixing to ensure thorough resuspension of magnetic beads;

7. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, sucking away all the waste liquid, adding 500 μ L of washing liquid D, and vortex mixing to ensure thorough resuspension of magnetic beads;

8. placing the centrifugal tube in a magnetic rack for magnetic attraction for 2min, sucking away all the waste liquid, removing the residual liquid as much as possible by using a 10-100 mu L gun head, transferring the centrifugal tube to a nonmagnetic test tube rack, opening a tube cover, and drying at room temperature for 5min;

9. adding 50 mu L of eluent, covering a tube cover, whirling, uniformly mixing and suspending magnetic beads, and placing a centrifugal tube in a 56 ℃ for incubation for 5min;

10. placing the centrifuge tube in a magnetic frame for magnetic attraction for 2min, and transferring all the eluates to a new centrifuge tube for later use.

5. PCR procedure

1.preparation of PCR reaction solution

According to the experimental quantity, a VWC2 gene methylation PCR reaction solution system is shown in a table 2;

TABLE 2

Components of a Single PCR reaction	Amount of Individual PCR reactions
		SEQ ID No.1(10μM)	0.25μL
SEQ ID No.2(10μM)	0.25μL
		SEQ ID No.3(10μM)	0.25μL
SEQ ID No.10(10μM)	0.25μL
		SEQ ID No.11(10μM)	0.25μL
SEQ ID No.12(10μM)	0.25μL
		TaqDNA polymerase (5U/. Mu.L)	0.5μL
dNTP(10mM)	0.5μL
		MgCl 2 (25mM)	5μL
PCR buffer (5X)	5μL
		dd H 2 O	2.5μL
Total	15μL

The THBD gene methylation PCR reaction solution system is shown in Table 3;

TABLE 3

The FAM19A4 gene methylation PCR reaction solution system is shown in Table 4;

TABLE 4

Components of a Single PCR reaction	Amount of Individual PCR reactions
		SEQ ID No.7(10μM)	0.25μL
SEQ ID No.8(10μM)	0.25μL
		SEQ ID No.9(10μM)	0.25μL
SEQ ID No.10(10μM)	0.25μL
		SEQ ID No.11(10μM)	0.25μL
SEQ ID No.12(10μM)	0.25μL
		TaqDNA polymerase (5U/. Mu.L)	0.5μL
dNTP(10mM)	0.5μL
		MgCl 2 (25mM)	5μL
PCR buffer (5X)	5μL
		dd H 2 O	2.5μL
Total	15μL

The VWC2/THBD/FAM19A4 gene methylation PCR reaction solution system is shown in Table 5;

TABLE 5

2. Sample application

mu.L of the PCR reaction solution and 10. Mu.L of the sample DNA were added to the prepared PCR reaction tube, and then the tube cap was closed and centrifuged at low speed instantaneously. The sample adding of the negative quality control and the positive quality control is the same as that of the sample to be detected.

3. Fluorescent quantitative PCR detection

(1) Fluorescence channel selection: each sample selects 2 channels of FAM, JOE. The Reference fluorescence (Passive Reference) is set to none;

(2) The reaction conditions were set as shown in Table 6 (25. Mu.L of reaction system);

TABLE 6

6. Analysis of results

Analysis of PCR results

And (3) automatically storing the result after the reaction is finished, automatically analyzing the result by using instrument matched software, and if any channel in the PCR amplification has an amplification curve, calculating the Ct value (Cycle threshold) of the channel, wherein the Ct value reflects the relative content of the detected gene.

2. Determination of detection result

Taking the result when the Ct of the ACTB gene is less than or equal to 32 as a valid result, indicating that the DNA content in the sample is sufficient, otherwise, taking the result as a invalid result; the result is positive when the Ct of the FAM channel is less than 43, which indicates that the risk of esophageal cancer is high, and the result is negative when the Ct of the FAM channel is greater than 43 or N.D., which indicates that the risk of esophageal cancer is low, wherein the N.D. is an abbreviation of 'Not Detected', which means 'undetected'.

3. The result of the detection

FIG. 1 shows the result of detecting a positive reference substance by a VWC2 gene methylation PCR reaction solution system, FIG. 2 shows the result of detecting a positive reference substance by a THBD gene methylation PCR reaction solution system, FIG. 3 shows the result of detecting a positive reference substance by a FAM19A4 gene methylation PCR reaction solution system, and FIG. 4 shows the result of detecting a positive reference substance by a VWC2/THBD/FAM19A4 gene methylation PCR reaction solution system.

(1) The methylation of the VWC2 gene is not detected in a negative reference substance and a normal sample, the result is negative, and the methylation of the VWC2 gene is detected in a positive reference substance, a sensitivity reference substance and an esophageal cancer sample, and the result is positive. The results are shown in Table 7;

TABLE 7

(2) THBD gene methylation is not detected in a negative reference product and a normal sample, the result is negative, and the THBD gene methylation is detected in a positive reference product, a sensitivity reference product and an esophageal cancer sample, and the result is positive. The results are shown in Table 8;

TABLE 8

(3) FAM19A4 gene methylation is not detected in a negative reference substance and a normal sample, the result is negative, and the result is positive when the FAM19A4 gene methylation is detected in a positive reference substance, a sensitivity reference substance and an esophageal cancer sample. The results are shown in Table 9;

TABLE 9

(4) Methylation of the VWC2/THBD/FAM19A4 gene is not detected in a negative reference substance and a normal sample, the result is negative, and the methylation of the VWC2/THBD/FAM19A4 gene is detected in a positive reference substance, a sensitivity reference substance and an esophageal cancer sample, and the result is positive. The results are shown in Table 10;

watch 10

/>

Example 4 use of primers and probes for detection of clinical specimens

Several clinical plasma samples were collected, 176 patients with primary esophageal cancer and 192 healthy human plasma samples. Clinical sample testing was performed using the kit shown in example 3. The specific steps are consistent with example 3, and the detection results of the VWC2 gene methylation clinical samples are shown in Table 11;

TABLE 11

The detection results of the clinical sample of the THBD gene methylation are shown in Table 12;

TABLE 12

The detection results of the FAM19A4 gene methylation clinical samples are shown in Table 13;

watch 13

/>

The detection results of the VWC2/THBD/FAM19A4 gene methylation clinical samples are shown in Table 14;

TABLE 14

According to the experimental result, the sensitivity of the primer and the probe for detecting the methylation state of any one gene of VWC2, THBD and FAM19A4 is more than 80%, and the specificity is more than 98%. And the sensitivity of the combined detection of methylation of three genes is >95%: 148 of 155 samples of patients with esophageal cancer were detected; specificity >98%: out of 230 healthy human samples, 4 were detected. The results show that VWC2, THBD and FAM19A4 can be used as markers to detect early esophageal cancer with high specificity and high sensitivity.

According to the embodiments, the primer, the probe and the kit can detect the methylation levels of VWC2, THBD and FAM19A4 genes in a sample with high specificity and high sensitivity, and provide reference for screening and diagnosing early esophageal cancer.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (12)

1. A marker for screening for esophageal cancer, wherein the marker comprises one or more of VWC2, THBD, FAM19A4, and wherein esophageal cancer is screened by detecting the methylation level of the marker.

2. The marker of claim 1, wherein esophageal cancer is screened for by detecting the level of methylation of one or more of the following regions, with reference to grch38. P14:

region 1: and (2) Chr7:49772138-49774638;

region 2: and (2) Chr20:23047672-23052672;

and (4) area 3: and (2) Chr3:68931547-68933547.

3. Use of a reagent for detecting the methylation level of a marker according to claim 1 for the manufacture of a product for screening for the diagnosis of esophageal cancer.

4. A nucleic acid product for screening for esophageal cancer, comprising a primer pair for detecting the level of methylation of the marker of claim 1.

5. The nucleic acid product of claim 4, further comprising a nucleic acid probe corresponding to the primer pair.

6. The nucleic acid product of claim 4 or 5, wherein the primer pair comprises one or more of:

a first primer pair with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2 and used for detecting the methylation level of the VWC2 gene;

a second primer pair with nucleotide sequences shown as SEQ ID No.4 and SEQ ID No.5 and used for detecting the methylation level of the THBD gene; and

and the nucleotide sequences are shown as SEQ ID No.7 and SEQ ID No.8, and the third primer pair is used for detecting the methylation level of the FAM19A4 gene.

7. The nucleic acid product of claim 6, wherein the nucleotide sequence of the detection probe corresponding to the first primer pair is shown as SEQ ID No. 3;

and/or the nucleotide sequence of the detection probe corresponding to the second primer pair is shown as SEQ ID No. 6;

and/or the nucleotide sequence of the detection probe corresponding to the third primer pair is shown as SEQ ID No. 9.

8. The nucleic acid product of claim 4 or 5, further comprising an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair.

9. The nucleic acid product of claim 8, wherein the nucleotide sequences of the internal reference primer pair are shown as SEQ ID No.10 and SEQ ID No.11, and the nucleotide sequence of the internal reference probe is shown as SEQ ID No. 12.

10. A kit for screening esophageal cancer, comprising a reagent for detecting the methylation level of the esophageal cancer marker of claim 1.

11. The kit of claim 10, wherein the reagents detect the methylation level of the marker by one or more of the following methods:

methylation specificity PCR, bisulfite sequencing, methylation specificity high performance liquid chromatography, methylation specificity microarray, whole genome methylation sequencing, direct sequencing, methylation specificity high resolution solubility curve, methylation sensitivity restriction endonuclease and methylation fluorescence quantitative PCR.

12. The kit of claim 11, wherein the kit comprises one or more of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents, and the nucleic acid product of any one of claims 4 to 9.

Images