WO2019233448A1

WO2019233448A1 - Test kit for detecting early stage dna methylation markers in lung cancer and detection method

Info

Publication number: WO2019233448A1
Application number: PCT/CN2019/090190
Authority: WO
Inventors: 陈琦; 梁昊原; 谷东风
Original assignee: 深圳市圣必智科技开发有限公司
Priority date: 2018-06-07
Filing date: 2019-06-05
Publication date: 2019-12-12
Also published as: CN110577997A

Abstract

Disclosed by the present invention are a test kit for detecting early stage DNA methylation markers in lung cancer and a detection method, the method comprising: extracting a sample to be tested; detecting the sample to be tested by using the test kit for detecting early stage DNA methylation markers in lung cancer as provided by the present invention to obtain the degree of DNA methylation of an F2RL3 gene, the degree of DNA methylation of an SHOX2 gene, the degree of DNA methylation of an RASSF1A gene, the degree of DNA methylation of an ANKRD18B gene, and the degree of DNA methylation of an MPDZ gene; according to the degrees of DNA methylation of the five genes to be detected, determining whether a subject to be detected has a high risk of lung cancer or is a patient suffering from lung cancer.

Description

Lung cancer early DNA methylation marker detection test box and detection method

Technical field

The invention relates to the technical field of early detection of diseases, in particular to a test paper box and a detection method for detecting DNA methylation markers of lung cancer at an early stage.

Background technique

DNA methylation is the possibility that cytosines on specific sequences (continuous cytosines and guanines) on the DNA sequence can be methylated. The degree of methylation changes the expression of genes by changing the structure and stability of chromatin and changing the binding activity of transcription factors. Generally, low methylation is conducive to gene expression, and high methylation will inhibit gene expression. Studies have confirmed that the state of DNA methylation is highly correlated with the occurrence of certain tumors. DNA methylation can be used to evaluate whether a tumor has occurred and the efficacy of treatment measures for the tumor.

Lung cancer has become one of the leading causes of cancer death in humans. Lung cancer is the highest incidence cancer in China, and its morbidity and mortality are increasing rapidly. The incidence and mortality of lung cancer are the highest among all tumors, but lung cancer is not the most diagnosed tumor. In the United States, breast and prostate tumors have a higher diagnosis rate. The reason is that early diagnosis can lead to early treatment. The 5-year survival rate was improved (89 and 99%, respectively), while the 5-year survival rate of lung cancer was only 15%. In clinical practice, early diagnosis of lung cancer has always been difficult, and early detection of cancer is very important for effective treatment of cancer patients. At present, the diagnosis of cancer is mainly based on clinical symptoms, imaging tests, and histopathological examinations. However, many cancers have clinical symptoms that appear late, and biopsy and detection are difficult, which seriously affects the early diagnosis of cancer and the prognosis of patients.

If DNA methylation can be used to evaluate the risk of lung cancer, it is undoubtedly a simple and feasible method.

technical problem

The main purpose of the present invention is to provide a test box and a detection method for detecting DNA methylation markers in early stage of lung cancer, which aims to solve the technical problem that the prior art cannot use DNA methylation to evaluate the risk of lung cancer occurrence.

Technical solutions

To achieve the above object, the present invention provides a test kit for detecting early stage DNA methylation markers of lung cancer. The test kit for early stage DNA methylation markers of lung cancer includes a DNA methylation detection reagent of F2RL3 gene, SHOX2 DNA methylation detection reagent for genes, DNA methylation detection reagent for RASSF1A gene, DNA methylation detection reagent for ANKRD18B gene, and DNA methylation detection reagent for MPDZ gene, the early detection of DNA methylation markers of lung cancer The test box also contains PCR reaction solution, polymerase, positive control, negative control, and DNA modification solution.

Further, the PCR reaction solution includes a 0.2 uM F2RL3 gene amplification primer pair, a 0.06 uM F2RL3 gene detection probe, a 0.2 uM RASSF1A gene amplification primer pair, and a 0.06 uM RASSF1A gene detection probe. , 0.2uM SHOX2 gene amplification primer pair, 0.06uM SHOX2 gene detection probe, 0.2uM ANKRD18B gene amplification probe pair, 0.06uM ANKRD18B gene detection probe, 0.2uM MPDZ gene amplification Primer pair, 0.06uM MPDZ gene detection probe, and 0.67uM β-actin gene amplification primer pair and 0.02uM β-actin gene detection probe; 3.5mM MgCl2; 0.25mM dNTP.

Further, the polymerase is 200U of Taqman polymerase.

Further, the positive quality control substance is 500 cells / μL, which is DNA extracted from the non-small cell lung cancer HCC827 cell line after counting.

Further, the negative control is sterilized water.

Further, the DNA modification solution is 3M sodium bisulfite, pH = 5.0.

The invention also provides a method for detecting early stage DNA methylation markers of lung cancer. The method includes the following steps:

Extract the sample to be tested;

The aforementioned test sample for detecting DNA methylation markers for early stage lung cancer was used to detect the test sample to obtain the degree of DNA methylation of the F2RL3 gene, the degree of DNA methylation of the SHOX2 gene, and the DNA methylation of the RASSF1A gene. The degree of DNA methylation of the ANKRD18B gene and the degree of DNA methylation of the MPDZ gene;

According to the degree of DNA methylation of the five genes to be tested, determine whether the test object is a high-risk or lung cancer patient.

Further, the test sample is derived from a nucleic acid extracted from a cell-containing sample or a sample containing a cell-derived nucleic acid.

Further, the sample to be tested refers to a nucleic acid sample to be detected, which contains one nucleic acid or multiple nucleic acids, and it is necessary to detect whether a target nucleic acid exists therein.

Further, the probe refers to a single-stranded nucleic acid having a known nucleotide sequence, the single-stranded nucleic acid has a nucleotide sequence structure that is complementary to the target nucleic acid, and the single-stranded nucleic acid forms a double-strand with the target nucleic acid.

Beneficial effect

Compared with the prior art, the test box and method for detecting early stage DNA methylation markers of lung cancer according to the present invention adopt the above technical scheme to achieve the following technical effects: The early stage DNA methylation marker detection of lung cancer disclosed by the present invention The test box contains five lung cancer-related gene methylation detection reagents, which can jointly detect multiple lung cancer-related DNA methylation degrees, can significantly improve the early detection rate of lung cancer, and is suitable for large-scale popularization and application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flowchart of a method for detecting DNA methylation markers at an early stage of lung cancer according to the present invention.

The purpose, function characteristics and advantages of the present invention will be further described with reference to the embodiments and the accompanying drawings.

Embodiments of the invention

In order to further explain the technical means and effects adopted by the present invention to achieve the above-mentioned object, the specific implementation, structure, features, and effects of the present invention are described in detail below with reference to the drawings and preferred embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

The lung cancer early-stage DNA methylation marker detection test box provided by the present invention includes a DNA methylation detection reagent for the F2RL3 gene, a DNA methylation detection reagent for the SHOX2 gene, a DNA methylation detection reagent for the RASSF1A gene, and an ANKRD18B gene. DNA methylation detection reagent and DNA methylation detection reagent for MPDZ gene. The test kit for detecting DNA methylation markers in early stage of lung cancer further includes a PCR reaction solution, a polymerase, a positive control, a negative control, and a DNA modification solution.

The PCR reaction solution includes an amplification primer pair of F2RL3 gene of 0.2uM, a detection probe of F2RL3 gene of 0.06uM, an amplification primer pair of RASSF1A gene of 0.2uM, a detection probe of RASSF1A gene of 0.06uM, 0.2uM SHOX2 gene amplification primer pair, 0.06uM SHOX2 gene detection probe, 0.2uM ANKRD18B gene amplification primer pair, 0.06uM ANKRD18B gene detection probe, 0.2uM MPDZ gene amplification primer pair , 0.06uM of the MPDZ gene detection probe, and 0.67uM of the β-actin gene amplification primer pair and 0.02uM of the β-actin gene detection probe; 3.5mM MgCl2; 0.25mM dNTP;

The polymerase is 200U of Taqman polymerase;

The positive control was 500 cells / μL, which was the DNA extracted from the non-small cell lung cancer HCC827 cell line after counting;

The negative control is sterilized water;

The DNA modification solution is 3M sodium bisulfite, pH = 5.0.

The reagent for detecting DNA methylation of the F2RL3 gene is a primer and a probe against the human F2RL3 gene; the reagent for detecting the DNA methylation of the SHOX2 gene is a primer and a probe against the human SHOX2 gene; or The detection reagent for DNA methylation of the RASSF1A gene is a primer and probe for the human RASSF1A gene; or the detection reagent for DNA methylation of the ANKRD18B gene is a primer and a probe for the human ANKRD18B gene; or The detection reagent for DNA methylation of the MPDZ gene is a primer and a probe for the human MPDZ gene.

SHOX2 (short stature homeobox 2) gene is a member of the dwarf homeobox gene family, and its nucleotide sequence may be substantially the same as the sequence of GenBank accession number: NC_000003. Its gene expression regulation is closely related to organ development. The study found that the SHOX2 gene is expressed in the mesoderm and ectoderm during the embryonic period and plays an important role in the development of the bone, heart, and nervous system. SHOX2 gene is abnormally expressed in a variety of solid tumors, such as lung cancer, breast cancer, and kidney cancer.

RASSF1A (Ras-association domain family 1A (RASSF1A) gene is a Ras-related region family 1A gene, and its nucleotide sequence may be substantially the same as the sequence of GenBank accession number: DQ444319. The target genes regulated by RASSF1A involve gene transcription, signal transduction, cytoskeleton, cell cycle, cell adhesion and apoptosis, etc., and have a wide range of biological functions. As a novel tumor suppressor gene, RASSF1A plays a particularly important role in tumorigenesis and development. At present, it is believed that the inactivation of RASSF1A gene expression is mainly related to abnormal hypermethylation of the promoter region, loss of heterozygosity and chromosome deletion.

ANKRDl8B (Ankyrin The repeat domain18B) gene contains a typical ANK structure region (ANK domain) with 13 CpG island sites. Studies have found that ANKRD18B gene is significantly hypermethylated during the development of lung cancer, suggesting that abnormal gene methylation may be related to the occurrence of lung cancer.

MPDZ (multiple PDZ domain protein) gene contains 47 exons and encodes a protein of 2070 amino acids. The protein contains 13 typical PDZ domains. The main role of this domain is to anchor membrane proteins to the cytoskeleton and form protein complexes. Signal transmission. MPDZ, as a tight junction related protein, participates in the formation of tight junctions such as epithelial cells and endothelial cells, and is related to cell polarity and cell osmotic pressure regulation. In terms of the mechanism of MPDZ and tumorigenesis, MPDZ protein can bind to coxsackie virus and adenovirus receptors, as well as proteins such as the tumor suppressor gene FAT4. It is speculated that it may be involved in the suppression of the carcinogenic effect of the virus and the implementation of FAT4 tumor suppressor effect. Studies have confirmed that the MPDZ gene is methylated at a higher frequency in lung cancer tissues, but no methylation is detected in distant cancerous tissues, suggesting that the MPDZ gene may be involved in the occurrence of lung cancer and may be a candidate Tumor suppressor gene.

The present invention provides a method for detecting early stage DNA methylation markers of lung cancer. Referring to FIG. 1, FIG. 1 is a schematic flowchart of a method for detecting early stage DNA methylation markers of lung cancer according to the present invention. The method uses the above five lung cancer-related DNA methylation markers as a basis for detecting early-stage DNA methylation markers of lung cancer. The method for detecting early-stage DNA methylation markers of lung cancer according to the present invention includes the following steps:

Step S1: extract a sample to be tested;

Step S2: detecting the sample to be tested using the early DNA methylation marker detection test box of the lung cancer of the present invention to obtain the DNA methylation degree of the F2RL3 gene, the DNA methylation degree of the SHOX2 gene, and the DNA of the RASSF1A gene The degree of methylation, the degree of DNA methylation of the ANKRD18B gene, and the degree of DNA methylation of the MPDZ gene.

Specifically, the sample to be tested is processed by using a DNA modification solution to obtain a modified sample to be tested;

Using the primer pair of the PCR reaction solution to amplify the modified test sample to obtain an amplified product;

Using a polymerase to digest the amplified product;

Perform transcription and enzyme digestion on the digested amplification product;

The mass spectrometry method was used to detect the transcription and digestion products to obtain the DNA methylation degree of the F2RL3 gene, the DNA methylation degree of the SHOX2 gene, the DNA methylation degree of the RASSF1A gene, and the ANKRD18B gene in the test sample. The degree of DNA methylation and the degree of DNA methylation of the MPDZ gene.

Step S3: Determine whether the test object is a high-risk lung cancer patient or a lung cancer patient according to the degree of DNA methylation of the five genes to be tested. Specifically, if one or more of the five genes to be tested are positive for DNA methylation, it is determined that the test subject may be a high-risk lung cancer or a lung cancer patient.

The test sample is derived from: nucleic acid extracted from a sample containing cells, such as alveolar lavage fluid, tissue from a diseased site, such as pleural fluid, sputum, etc .; or a sample containing nucleic acid derived from cells, such as plasma, serum Wait.

The test sample refers to a nucleic acid sample to be detected, which contains one nucleic acid or multiple nucleic acids, and it is necessary to know whether a target nucleic acid exists therein.

The target nucleic acid refers to a nucleic acid fragment of interest, which is a methylated DNA-specific fragment of the F2RL3 gene, the SHOX2 gene, the RASSF1A gene, the ANKRD18B gene, and the MPDZ gene.

The probe refers to a single-stranded nucleic acid having a known nucleotide sequence, which has a nucleotide sequence structure that is substantially complementary to a target nucleic acid, and can form a double strand with the target nucleic acid. The probe may carry a label. For example, the label can be attached to the 5 'end or 3' end of the probe.

The lung cancer early DNA methylation marker detection test box disclosed by the present invention includes five lung cancer related gene methylation detection reagents, which can jointly detect multiple lung cancer related DNA methylation degrees, and can significantly improve early detection of lung cancer. Rate, suitable for large-scale promotion and application.

The above are only preferred embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent functional transformation made by using the description and drawings of the present invention, or directly or indirectly used in other related technical fields All are included in the patent protection scope of the present invention.

Industrial applicability

Claims

An early-stage lung cancer DNA methylation marker detection test case, characterized in that the early-stage lung cancer DNA methylation marker detection test case includes a DNA methylation detection reagent of the F2RL3 gene and a DNA methylation of the SHOX2 gene. Detection reagent, DNA methylation detection reagent for RASSF1A gene, DNA methylation detection reagent for ANKRD18B gene, and DNA methylation detection reagent for MPDZ gene, the test kit for early detection of DNA methylation markers in lung cancer also includes PCR reaction solution, polymerase, positive control, negative control and DNA modification solution.
The test kit for detecting early stage DNA methylation markers of lung cancer according to claim 1, wherein the PCR reaction solution includes a primer pair for amplification of the F2RL3 gene at 0.2 uM, and a probe for detecting the F2RL3 gene at 0.06 uM. , 0.2uM RASSF1A gene amplification primer pair, 0.06uM RASSF1A gene detection probe, 0.2uM SHOX2 gene amplification probe pair, 0.06uM SHOX2 gene detection probe, 0.2uM ANKRD18B gene amplification Primer pair, 0.06uM ANKRD18B gene detection probe, 0.2uM MPDZ gene amplification primer pair, 0.06uM MPDZ gene detection probe, and 0.67uM β-actin gene amplification primer pair and 0.02 uM β-actin gene detection probe; 3.5 mM MgCl2; 0.25 mM dNTP.
The test kit for detecting DNA methylation markers at an early stage of lung cancer according to claim 1, wherein the polymerase is 200U Taqman polymerase.
The test kit for detecting early stage DNA methylation markers of lung cancer according to claim 1, wherein the positive quality control substance is 500 cells / μL, which is DNA extracted from the non-small cell lung cancer HCC827 cell line after counting. .
The test kit for detecting DNA methylation markers at an early stage of lung cancer according to claim 1, wherein the negative control is sterilized water.
The test kit for detecting DNA methylation markers at an early stage of lung cancer according to claim 1, wherein the DNA modification solution is 3M sodium bisulfite, and pH = 5.0.
A method for detecting DNA methylation markers in early stage of lung cancer, characterized in that the method includes the following steps:

Extract the sample to be tested;

The test sample is detected by using a lung cancer early stage DNA methylation marker detection test box according to any one of claims 1 to 6 to obtain a DNA methylation degree of the F2RL3 gene and a DNA methylation of the SHOX2 gene. Degree, DNA methylation degree of RASSF1A gene, DNA methylation degree of ANKRD18B gene, and DNA methylation degree of MPDZ gene;

According to the degree of DNA methylation of the five genes to be tested, determine whether the test object is a high-risk or lung cancer patient.
The method for detecting a DNA methylation marker at an early stage of lung cancer according to claim 7, wherein the test sample is derived from a nucleic acid extracted from a cell-containing sample or a sample containing a cell-derived nucleic acid.
The method for detecting early stage DNA methylation markers of lung cancer according to claim 7, wherein the sample to be detected refers to a nucleic acid sample to be detected, which contains one kind of nucleic acid or multiple kinds of nucleic acids and needs to be detected for its presence Target nucleic acid.
The method for detecting a DNA methylation marker at an early stage of lung cancer according to claim 9, wherein the probe refers to a single-stranded nucleic acid having a known nucleotide sequence, and the single-stranded nucleic acid has a target nucleic acid Complementary nucleotide sequence structure, the single-stranded nucleic acid forms a double strand with the target nucleic acid.