CN111363812A - Lung cancer diagnostic agent and kit based on DMRTA2 gene - Google Patents
Lung cancer diagnostic agent and kit based on DMRTA2 gene Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention belongs to the field of biological medicine, and relates to a lung cancer detection/diagnosis reagent and a kit, wherein the reagent or the kit comprises a detection reagent aiming at DMRTA2 gene methylation, and is used for detecting a sequence of a DMRTA2 gene modified by bisulfite or hydrazine salt. The test proves that the reagent of the invention can detect and diagnose the lung cancer with high sensitivity and high specificity, and has extremely high clinical application value.
Description
Technical Field
The invention belongs to the field of gene diagnosis, and particularly relates to a human DMRTA2 gene methylation detection/diagnosis reagent for lung cancer detection and a kit containing the reagent.
Background
Lung cancer is a malignant tumor of the lung that originates in the bronchial mucosa, glands or alveolar epithelium. The classification can be made according to the type of pathology: 1) small Cell Lung Cancer (SCLC): lung cancer, a pathological type of special, has a clear tendency to distant metastasis with a poor prognosis, but most patients are sensitive to radiotherapy and chemotherapy; 2) non-small cell lung cancer (non-small cell lung cancer, NSCLC): other pathological types of lung cancer besides small cell lung cancer include squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and the like. There are certain differences in biological behavior and clinical course. According to the occurrence position, the method can be divided into the following steps: 1) central lung cancer (central lung cancer): lung cancer that grows in and above the segmental bronchiectasis; 2) peripheral lung cancer (periheral lung cancer): lung cancer that grows beyond the bronchial opening of the segment.
In recent years, the incidence and mortality of the lung cancer in China are gradually increased year by year due to the influence of factors such as aging population, air pollution, smoking and the like, and according to the annual report of 2017 Chinese tumor registration issued by the national cancer center, about 7 people per minute are diagnosed with the cancer nationally, wherein the incidence and mortality of the lung cancer are the first. China has become the world with the largest number of lung cancers, and experts predict that the number of lung cancers in China will reach 100 ten thousand in 2025. And according to epidemiological studies show that: smoking is an important factor causing lung cancer. About 80% -90% of lung cancers worldwide can be attributed to smoking. Compared with non-smokers, 1-19 cigarettes and more than 20 cigarettes smoked per day in the age of 45-64 years have relative risk of lung cancer of 4.27 and 8.61 respectively, and compared with non-smokers, 1-19 cigarettes and more than 20 cigarettes smoked per day for a long time have relative risk of lung cancer death of 6.14 and 10.73 respectively. Although the treatment technology of the lung cancer is changed day by day, the 5-year survival rate is only increased from 4% to about 12%, the existing antitumor drugs still only have the function of relieving the disease condition, the non-progress survival time of the patient is only prolonged by 3 months to 5 months on average, and for the first-stage lung cancer patient, the 5-year survival rate after the operation is as high as about 60% to 70%. Therefore, early diagnosis and early surgery of lung cancer are one of the most effective methods for improving 5-year survival rate and reducing mortality rate of lung cancer.
The current clinical auxiliary diagnosis of lung cancer mainly comprises the following diagnosis methods, but the diagnosis methods cannot completely realize early detection and early diagnosis:
(1) biochemical examination of blood: for primary lung cancer, there is currently no specific blood biochemical examination. The increase of blood alkaline phosphatase or blood calcium in lung cancer patients takes into account the possibility of bone metastasis, and the increase of blood alkaline phosphatase, glutamic-oxalacetic transaminase, lactate dehydrogenase or bilirubin takes into account the possibility of liver metastasis.
(2) Tumor marker examination: 1) CEA: abnormally high levels of CEA are found in the serum of 30-70% of lung cancer patients, but are found mainly in later stage lung cancer patients. The current examination of CEA in serum is mainly used to estimate lung cancer prognosis and to monitor the course of treatment. 2) NSE: the kit is a preferred marker for small cell lung cancer, is used for diagnosing the small cell lung cancer and monitoring the treatment response, and has different reference values according to different detection methods and used reagents. 3) CYFRA 21-1: the first choice marker of the non-small cell lung cancer has the sensitivity of 60 percent on the diagnosis of the squamous cell lung cancer, and the reference value is different according to different detection methods and used reagents.
(3) Imaging examination: 1) chest X-ray examination: chest orthoses and lateral pieces should be included. In primary hospitals, the positive chest radiograph is still the most basic and preferred image diagnosis method for the initial diagnosis of lung cancer. Once lung cancer is diagnosed or suspected, a chest CT examination is performed. 2) And (3) CT examination: chest CT is the most common and important examination method for lung cancer, and is used for diagnosis and differential diagnosis, staging and follow-up after treatment of lung cancer. CT guided lung biopsy is an important diagnostic technique for lung cancer, and a conditional hospital can be used for the diagnosis of lung lesions which are difficult to characterize and the clinical diagnosis of lung cancer needs cytological and histological verification and other methods are difficult to obtain materials. In recent years, multi-slice helical CT and Low Dose CT (LDCT) have been effective screening tools for early lung cancer and reduced mortality, and national lung cancer screening studies (NLST) in the united states have shown that LDCT can reduce lung cancer mortality by 20% compared to chest X-ray screening. Low dose helical CT is recommended as an important tool for early stage lung cancer screening, but human influence factors are more, and the false positive rate is very high. 3) Ultrasonic examination: the kit is mainly used for finding whether the vital organs of the abdomen, the abdominal cavity and the retroperitoneal lymph nodes are transferred or not, and is also used for detecting the cervical lymph nodes. For lung lesions or chest wall lesions close to the chest wall, the cyst solidity can be identified and puncture biopsy can be carried out under ultrasonic guidance; ultrasound is also commonly used for pleural effusion extraction positioning. 4) Bone scanning: the sensitivity to the detection of the bone metastasis of the lung cancer is high, but the false positive rate is certain. The following can be used: preoperative examination of lung cancer; patients with local symptoms.
(4) Other checks: 1) sputum cytology examination: the lung cancer is a simple and convenient noninvasive diagnosis method at present, the positive rate can be improved by about 60 percent through continuous smear examination, and the method is a routine diagnosis method for suspicious lung cancer cases. 2) Fiberbronchoscopy: one of the most important means in lung cancer diagnosis plays an important role in the qualitative and localized diagnosis of lung cancer and the selection of surgical schemes. Is a necessary routine examination item for a patient to be treated by surgery. And the bronchoscopy biopsy (TBNA) is beneficial to staging before treatment, but the technical difficulty and risk are higher, so that a person in need should go to a higher hospital for further examination. 3) And others: such as percutaneous lung puncture biopsy, thoracoscope biopsy, mediastinoscopic biopsy, hydrothorax cytology examination, etc., under the condition of an adaptation, the diagnosis can be assisted according to the existing conditions.
In clinical practice work, the success or failure of any lung cancer screening project is proved to depend on the identification of high risk groups, and a risk prediction model fusing multiple high risk factors is universally accepted as one of the methods for identifying the high risk groups of lung cancer. With the rapid development of the technology, the tumor marker detection becomes a new field of tumor diagnosis and treatment after the imaging diagnosis and the pathological diagnosis, and can have great influence on the diagnosis, the detection and the treatment of tumors. The tumor marker can be detected in body fluid or tissues and can reflect the existence, differentiation degree, prognosis estimation, personalized medicine, treatment effect and the like of tumors. Early lung cancer patients have no obvious symptoms and are difficult to detect by doctors and patients, and in addition, the early lung cancer patients have no obvious specific markers on blood or biochemical projects, so that early detection and early diagnosis are difficult to perform through a conventional diagnosis method, and the early lung cancer diagnosis, especially the screening of large-scale application population is difficult.
More and more studies have shown that two broad classes of mechanisms are involved in the process of tumor formation. One is the formation of mutations by changes in the nucleotide sequence of the DNA, a genetic mechanism. Tumors have been identified in the field of molecular biology as a genetic disease. Another is the epigenetic (epigenetics) mechanism, i.e., the change of gene expression level independent of DNA sequence change, and the role of it in the process of tumor formation is increasingly emphasized. The two mechanisms of genetics and epigenetics exist in a mutual crossing way, and the formation of tumors is promoted together. Aberrant methylation of genes occurs early in tumorigenesis and increases in the course of tumor progression. Analysis of the genome of 98 common primary human tumors revealed at least 600 abnormally methylated CpG islands per tumor.
Many studies have shown that promoter abnormal methylation is a frequent early event in the development of many tumors, and thus the methylation status of tumor-associated genes is an early sensitive indicator of tumorigenesis and is considered to be a promising molecular biomarker (biomarker). More importantly, the cancerous cells can release DNA into the peripheral blood. Free DNA is present in normal human peripheral blood on the nanogram scale. The research finds that abnormal methylation of the promoter of the tumor-related gene existing in the tumor tissue can be detected in peripheral blood plasma/serum and tumor-involved organ-related body fluid (such as saliva, sputum and the like). The biological samples are easy to obtain, and DNA in the biological samples can be sensitively detected after being massively amplified by a PCR technology, so that the methylation state of the promoter regions of certain tumor-related genes can be detected, and very valuable information can be provided for early diagnosis of tumors. There are many advantages to detecting promoter abnormal methylation compared to other types of tumor molecular markers. The abnormal methylation regions of the promoter of a certain gene in different types of tumors are the same, so that the detection is more convenient; in addition, compared to markers such as allelic deletion, aberrant methylation is a positive signal and is readily distinguishable from the negative background in normal tissue. Esteller et al examined the abnormal methylation state of the promoter regions of genes such as p16, DAPK, GSTP1 and MGM T in 22 cases of non-small cell lung cancer (NSCLC) tumor tissues and serum, and found that 68% (15/22) tumor tissues have promoter methylation of at least one gene; in 15 cases of tissue positivity, the presence of abnormal promoter methylation was also detected in the serum in 11 cases. In addition, many researchers have also detected the methylation of the promoters of some tumor-related genes from tumor tissues and sera of patients with liver cancer, head and neck cancer, esophageal cancer and colon cancer, respectively. Palmisano et al examined p16 and MGMT promoter abnormal methylation in tumor tissues and sputum of 21 patients with lung squamous carcinoma, and found that abnormal methylation of promoter regions of one or two genes existed in all sputum samples. 10 of these sputum samples were collected after tumor diagnosis; another 11 sputum samples were from a high risk population with a history of smoking or other exposures, and these 11 subjects were confirmed to be lung cancer within the following 5-35 months. The 21 sputum samples were positive by sputum cell morphology and only 4 sputum samples were positive. Therefore, the detection of abnormal methylation of the promoter region of the gene is a very sensitive indicator. The results of these studies show that: detection of DNA methylation can be used as a means for early diagnosis and risk assessment of cancer.
Early lung cancer patients often have no obvious symptoms and signs, are easily ignored by the patients and are rarely diagnosed due to the symptoms. Clinical routine chest X-ray and sputum shedding cytology examination is far from meeting the requirement of screening early lung cancer, and the examination has not been proved to reduce the death rate. The screening omission factor of chest X-ray can reach 54-90%, although the cost of sputum shedding cytology examination is low, expensive equipment is not needed, the omission factor is high, the interference of result judgment human factors is more, and multiple times of submission are needed. Low dose helical CT is recommended as an important tool for early stage lung cancer screening, but human influence factors are more, and the false positive rate is very high.
Currently, there are many studies to detect the cell or DNA methylation state in blood, sputum, alveolar lavage fluid in order to find markers for early diagnosis of lung cancer. Although the prior art has found that DNA methylation of some genes is related to lung cancer, there is still a need in the art to further study related genes for lung cancer diagnosis, which can be applied practically, and to develop a detection reagent with higher detection accuracy.
Disclosure of Invention
The invention aims to provide application of a nucleic acid fragment of the DMRTA2 gene in preparing a tumor detection/diagnosis reagent or kit.
Another objective of the invention is to provide an application of the primer pair in preparing a tumor detection/diagnosis reagent or kit.
Another object of the present invention is to provide a use of the probe in preparing a tumor detection/diagnosis reagent or kit.
It is a further object of the invention to provide a reagent, kit and method for diagnosing methylation of the human DMRTA2 gene.
The present invention further aims to provide a lung cancer detection/diagnosis reagent and a kit with strong specificity and high sensitivity.
The invention further aims to provide a lung cancer detection/diagnosis reagent and a kit with wide application range on lung cancer.
It is a further object of the present invention to provide a lung cancer detection/diagnosis reagent and kit which are convenient to use.
The above object of the present invention is achieved by the following technical means:
in one aspect, the invention provides an application of a nucleic acid fragment in preparing a tumor detection/diagnosis reagent or kit. Wherein the nucleic acid fragment is derived from the DMRTA2 gene, in particular from the DMRTA2 gene located between the 50,883,222-50,889,172 (according to GRCh37 reference) positions on chromosome 1.
Specifically, the nucleic acid fragment is selected from SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23 or SEQ ID NO: 25. as a preferred embodiment of the present invention, said nucleic acid fragment is selected from the group consisting of SEQ ID NO: 19.
in another aspect, the present invention also provides a primer pair, wherein the primer pair is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 68 and SEQ ID NO: 69; preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 62 and SEQ ID NO: 63; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ id no: 39, or a primer set shown in the specification; most preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer set shown in (2).
In another aspect, the present invention also provides a probe selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70 is shown in any one of the figures; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 55, SEQ ID NO: 64 is shown in any one of the figures; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ id no: 37, SEQ ID NO: 40 is shown in any one of the figures. As a preferred embodiment of the present invention, said nucleic acid probe is selected from the group consisting of seq id NO: 3.
on the other hand, the invention also provides application of the primer pair or the probe in preparing a tumor detection/diagnosis reagent or kit.
In another aspect, the present invention provides a tumor detection/diagnosis reagent comprising a methylation detection reagent for DMRTA2 gene.
Wherein, the DMRTA2 gene (DMRT-like family A2) belongs to the DMRT gene family, encodes a protein binding to DM-binding motif (DM-domain), and contains DM-domain (DNA sequence binding to the protein encoded by the Doublesex and mab-3 genes) shared by the Drosophila doubesex gene and the nematode mab-3 gene. The DMRT gene is a transcription regulation factor, and the product coded by the gene is combined with a specific DNA sequence through a zinc finger structure to regulate the gene expression.
Methylation is caused by adding one more methyl group on cytosine, and cytosine can be changed into uracil after being treated by bisulfite or hydrazinate, because uracil is similar to thymine and can be identified as thymine when PCR amplification is carried out, namely cytosine which is not methylated is changed into thymine (C is changed into T) on a PCR amplification sequence, and methylated cytosine (C) is not changed. The technique for detecting methylated genes by PCR is usually Methylation Specific PCR (MSP), wherein primers are designed for the treated methylated fragments (i.e., unchanged C in the fragments), PCR amplification is carried out, if amplification exists, methylation occurs, and if amplification does not occur, methylation does not occur.
Further, the methylation detection reagent of the DMRTA2 gene detects the sequence of the DMRTA2 gene modified by bisulfite or hydrazine salt.
As an exemplary embodiment, the sequence of the DMRTA2 gene was determined after bisulfite modification.
In a preferred embodiment, the detection region of the DMRTA2 gene targeted by the reagent is specifically derived from the DMRTA2 gene located between the 50,883,222-50,889,172 (according to GRCh37 reference) positions on chromosome 1. More preferably, the detection region is selected from the group consisting of SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. or SEQ ID NO: 25 is shown; more preferably as set forth in SEQ ID NO: 19 as a detection area.
The inventor experimentally finds that the selection of the detection region of the DMRTA2 gene has an influence on the detection efficiency of the tumor. The inventors carried out RRBS methylation sequencing on the DMRTA2 gene according to the present invention, and obtained the DMRTA2 gene itself and the methylation of the base within 5000bp upstream of the gene. Preliminary analysis can obviously find that the methylation conditions of different regions of the gene are obviously different in lung cancer and non-lung cancer control groups, so that the selection of different region design primers and probes has important influence on the diagnosis of lung cancer and non-lung cancer. As shown in FIG. 1, the region in the left box is clearly better for primer and probe design than the region on the right.
The reagent of the present invention contains an amplification primer.
In a preferred embodiment, the primer is as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ id no: 37 and SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 55 and SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 59, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 64 and SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 68; preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 28 and SEQ ID NO: 29, seq id NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 52 and SEQ ID NO: 53, SEQ ID NO: 61 and SEQ ID NO: 62, and (b) a primer pair shown in the specification; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38, or a primer pair shown in figure 38. In a more preferred embodiment, the primer is as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The primers are used for amplifying a specific region of the DMRTA2 gene. It is well known in the art that successful design of primers is crucial for PCR. Compared with general PCR, in the methylation detection of genes, the design influence of primers is more critical, because the methylation reaction promotes the conversion of 'C' in a DNA chain into 'U', the GC content is reduced, long continuous 'T' appears in the sequence after the PCR reaction, the DNA chain is easy to break, and the selection of primers with proper Tm value and stability is difficult; on the other hand, in order to distinguish between DNA that is treated with and without sulfurization and not treated completely, a sufficient number of "C" s are required for the primers, which all increase the difficulty in selecting stable primers. Therefore, in the detection of DNA methylation, the selection of the amplified fragment to which the primer is directed, such as the length and position of the amplified fragment, the selection of the primer, and the like, all influence the sensitivity and specificity of the detection. The inventor also finds that different amplified target fragments and primer pairs have different detection effects through experiments. Many times, some genes or nucleic acid fragments are found to have expression difference between tumor and non-tumor, however, the distance is converted into a tumor marker, and the application in clinic still has a long distance. The main reason is that the detection sensitivity and specificity of the potential tumor marker cannot meet the detection requirement due to the limitation of detection reagents, or the detection method is complex in operation and high in cost, and is difficult to apply in large scale in clinic.
In an alternative embodiment, the kit of the present invention further comprises a probe. In a preferred embodiment, the probe is as set forth in SEQ ID NO: 3. SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 69; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 54, SEQ ID NO: 63; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39; as a most preferred embodiment, the probe is as set forth in SEQ ID NO: 3, respectively. As a preferred embodiment, the kit of the present invention comprises a primer and a probe, preferably, the primer is as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the probe is shown as SEQ ID NO: 3, respectively.
As a preferred embodiment, the reference gene is β -actin, COL2A 1.
In a preferred embodiment, the detection reagent for the reference gene is a primer and a probe for the reference gene. In a more preferred embodiment, the detection reagent for the reference gene is SEQ ID NO: 16. SEQ ID NO: 17 and the primer pair shown in SEQ ID NO: 18, or a probe as shown in figure 18. In another more preferred embodiment, the detection reagent for the reference gene is SEQ ID NO: 70. SEQ ID NO: 71 and the primer set shown in SEQ ID NO: 72, and (b) a probe of (e).
In a preferred embodiment, the reagent further comprises bisulfite, or hydrazonium salts to modify the DMRTA2 gene, although it may not be included.
In a preferred embodiment, the reagent comprises DNA polymerase, dNTPs, Mg2+One or more of ions and buffer solution, preferably DNA polymerase, dNTPs, Mg2+And the PCR reaction system of ions and buffer is used for amplifying the modified DMRTA2 gene.
The sample to be tested by the detection/diagnostic reagent of the present invention may be selected from alveolar lavage fluid, tissue, pleural fluid, sputum, blood, serum, plasma, urine, prostatic fluid, or stool. In a preferred embodiment, the sample is selected from the group consisting of alveolar lavage fluid, tissue, sputum; more preferably, the sample is selected from alveolar lavage fluid or sputum.
The detection/diagnostic reagent of the present invention is directed to a tumor selected from lung cancer tissue and paracancerous normal tissue (or benign lung disease tissue).
In another aspect, the present invention also provides a kit comprising the above-described detection/diagnostic reagent.
In another aspect, the present invention provides a method for detecting DNA methylation of DMRTA2 gene, comprising the steps of:
(1) processing a sample to be detected by bisulfite or hydrazine to obtain a modified sample to be detected;
(2) and (3) carrying out DMRTA2 gene methylation detection on the modified sample to be detected in the step (1) by using the reagent or the kit.
In a preferred embodiment, in step (2), the detection is performed by real-time fluorescence quantitative methylation-specific polymerase chain reaction.
In another aspect, the present invention also provides a system for detecting/diagnosing lung cancer. The system comprises:
(1) a DNA methylation detection module of the DMRTA2 gene, and,
(2) a result judgment system;
preferably, the DNA methylation detection means of DMRTA2 gene comprises the reagent or kit of any one of claims 5 to 14;
preferably, the result judging component is used for outputting the lung cancer disease risk and/or the lung cancer type according to the DNA methylation result of the DMRTA2 gene detected by the detection system;
more preferably, the disease risk is determined by comparing the methylation results of the test sample and the normal sample by the result determination component, and when the methylation of the test sample and the methylation of the normal sample have a significant difference or a very significant difference, the result determination component outputs that the disease risk of the test sample is high.
In a preferred embodiment, if the DMRTA2 gene is positive in DNA methylation, it indicates that the provider of the test sample is a lung cancer high-risk or lung cancer patient. In a preferred embodiment, the positive result is obtained by comparing the test result with the test result of a normal sample, and the donor of the test sample is positive when the amplification result of the test sample is significantly or very significantly different from the amplification result of the normal sample.
The invention has the beneficial effects that:
although, in the prior art, DMRTA2 gene methylation has been reported as one of the tumor markers for lung cancer. However, there are many reports on tumor markers of lung cancer, and the reports are really clinically applicable, but few of the reports are used as markers for lung cancer detection. The detection reagent for the DMRTA2 gene has high sensitivity and specificity on lung cancer, and is very hopeful to be used as a tumor marker for clinical diagnosis of the lung cancer.
The DMRTA2 gene has high sensitivity and specificity to lung cancer, and the detection rate of the lung cancer with the specificity of 100 percent and the sensitivity of 89.3 percent in a tissue specimen can be achieved based on the optimized methylation detection region and detection reagent of the DMRTA2 gene. Wherein squamous carcinoma can be detected completely. In the most difficult adenocarcinoma to detect, the specificity reaches 100.0%, and the sensitivity reaches 86.4%.
Lung cancer detection kits based on the SHOX2 gene are currently on the market. In one embodiment of the present invention, the detection of the sputum specimen, whether the lung cancer is comparatively analyzed as a whole or according to the subtype of the lung cancer, is superior to the detection of the SHOX2 gene in DMRTA 2. Particularly, the detection effect on adenocarcinoma is that the detection rate of DMRTA2 is 33.3%, and the detection rate of SHOX2 gene is 0%. In another embodiment of the present invention, when lung cancer is detected and compared as a whole, the detection rate of DMRTA2 is 61.9% which is much higher than 47.6% of SHOX2, and when lung cancer subtypes are compared, the detection result of DMRTA2 in squamous cell carcinoma group is 16.7% higher than that of SHOX 2. Particularly, the sensitivity of the kit reaches 54.5 percent and is far higher than 36.4 percent of that of SHOX2 in the detection effect on adenocarcinoma.
In addition, the detection marker of the invention has high specificity and sensitivity for different types of lung cancer, including squamous cell carcinoma and adenocarcinoma in small cell lung cancer and non-small cell lung cancer, has wide application range, and can be used as tumor markers of all lung cancers basically. The existing lung cancer markers for clinical use can only be generally applicable to detection of one type of lung cancer, such as NSE used for diagnosis of small cell lung cancer and monitoring of treatment response, while CYFRA21-1 is the first choice marker for non-small cell lung cancer.
The detection reagent containing the DMRTA2 gene and the method can conveniently and accurately judge the lung cancer and benign lung disease patients, and the gene detection method is expected to be converted into a gene detection kit and is used for screening, clinical detection and prognosis monitoring of the lung cancer.
Drawings
FIG. 1 comparison of the detection effects of primers and probes designed for different regions of DMRTA2
FIG. 2 shows ROC curves of five candidate genes DMRTA2, SIX3, PCDHGA12, HOXD8 and GATA3 for detecting lung cancer
FIG. 3 methylation degree of DMRTA2 gene in control group and lung cancer group
FIG. 4 shows a ROC curve for DMRTA2 detection of lung cancer in clinical tissue specimens;
FIG. 5 ROC curves of DMRTA2 and SHOX2 genes detected in sputum samples
FIG. 6 ROC curves for DMRTA2 and SHOX2 genes detected in lavage fluid samples
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Example 1: detection of target Gene selection
In order to complete the present invention, the inventors screened hundreds of genes, and selected better DMRTA2, SIX3, PCDHGA12, HOXD8, GATA3 as candidate detection genes, β -actin gene as reference gene, studied the distribution of methylation sites of each gene, designed primer probes for detection respectively as follows:
the detection primers and probes for DMRTA2 were:
SEQ ID NO: 1 DMRTA2 primer MF 1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO: 2 DMRTA2 primer MR 1: TACGACCTAACCGCGCTCTCA
SEQ ID NO: 3 d DMRTA2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
Detection primers and probes for SIX3 were:
SEQ ID NO: 4 SIX3 primer F: CGTTTTATATTTTTGGCGAGTAGC
SEQ ID NO: 5 SIX3 primer R: ACTCCGCCAACACCG
SEQ ID NO: 6 SIX3 probe: FAM-CGGCGGCGGCGCGGGAGGCGG-BQ1
The detection primers and probes of the PCDHGA12 are as follows:
SEQ ID NO: 7 PCDHGA12 primer F: TTGGTTTTTACGGTTTTCGAC
SEQ ID NO: 8 PCDHGA12 primer R: AAATTCTCCGAAACGCTCG
SEQ ID NO: 9 PCDHGA12 probe: FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1
The detection primers and probes for HOXD8 were:
SEQ ID NO: 10 HOXD8 primer F: TTAGTTTCGGCGCGTAGC
SEQ ID NO: 11 HOXD8 primer R: CCTAAAACCGACGCGATCTA
SEQ ID NO: 12 HOXD8 probe:
FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
the detection primers and probes of GATA3 are:
SEQ ID NO: 13 GATA3 primer F: TTTCGGTAGCGGGTATTGC
SEQ ID NO: 14 GATA3 primer R: AAAATAACGACGAACCAACCG
SEQ ID NO: 15 GATA3 Probe:
FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1
β -actin comprises the following detection primers and probes:
SEQ ID NO 16 β -actin primer F TTTTGGATTGTGAATTTGTG
SEQ ID NO 17 β -actin primer R AAAACCTACTCCTCCCTTAAA
18 β -actin probe of SEQ ID NO. FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
Sample information: the lung paraffin tissue samples count 36 cases, wherein the lung tissue samples used as controls count 11 cases, and comprise 4 cases of paracancer normal tissues and 7 cases of benign lung disease tissues; the cancer tissue samples comprise 25 cases, including 4 cases of squamous carcinoma and 21 cases of adenocarcinoma.
The test process comprises the following steps:
a. collecting operation excision specimen of lung cancer or benign lung disease, embedding with paraffin, staining pathological tissue section, and identifying tissue type and purity. Tissue sections DNA was extracted using the DNA extraction Kit from magenta (HiPureFFPE DNA Kit, D3126-03).
b. Bisulfite modification of DNA was performed using the DNA conversion Kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Biopsis.
c. The amplification detection system and the detection system are shown in tables 1-2:
TABLE 1 compounding System
TABLE 2 PCR reaction procedure
d. Calculating the methylation copy number of each gene in a specimen by using a standard curve, judging the methylation degree of two groups of tissues by adopting a ratio of the methylation copy number to the ACTB copy number 100, finally selecting a threshold value as a standard for judging a cancer group and a control group, judging the methylation degree of the two groups of tissues as positive when the converted ratio exceeds the set threshold value, and judging the methylation degree of the two groups of tissues as negative when the converted ratio is equal to or less than the set threshold value. The results of 36 tissue specimens tested according to this standard are shown in tables 3-4:
TABLE 3 results of the assays in the organization
Note: "+" indicates that the test result is a positive sample; "-" indicates that the test result was a negative sample.
TABLE 4 statistical results
In 36 cases of lung paraffin specimens, when the specificity is 100.0%, the detection sensitivity of the DMRTA2 gene is 96%, only 1 case of lung adenocarcinoma is judged to be negative, and the positive detection rate is far higher than that of the other 4 genes. Meanwhile, the detection results of benign lung disease tissue samples and paranormal lung cancer are compared, and the DMRTA2 genes show consistency, namely the DMRTA2 gene has better specificity. The DMRTA2 is suggested to have important significance in clinical detection and diagnosis of lung cancer.
Therefore, the inventors selected DMRTA2 as a candidate gene and optimized the detection conditions, as shown in example 2.
The ROC curve of five candidate genes DMRTA2, SIX3, PCDHGA12, HOXD8 and GATA3 for detecting lung cancer is shown in figure 2, and the area under the ROC curve of DMRTA2 gene is 0.996.
Example 2: detection of DMRTA2 gene in clinical specimens
a. The detection primer probes are as follows:
the detection primers and probes for DMRTA2 were:
SEQ ID NO: 1 DMRTA2 primer MF 1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO: 2 DMRTA2 primer MR 1: TACGACCTAACCGCGCTCTCA
SEQ ID NO: 3 d DMRTA2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
β -actin comprises the following detection primers and probes:
SEQ ID NO 16 β -actin primer F TTTTGGATTGTGAATTTGTG
SEQ ID NO 17 β -actin primer R AAAACCTACTCCTCCCTTAAA
18 β -actin probe of SEQ ID NO. FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
b. Sample information: a total of 56 lung paraffin tissue specimens were prepared by using 28 lung cancer tissue specimens and their corresponding paracancerous tissues as non-lung cancer controls. These included 6 squamous carcinomas and 22 adenocarcinomas.
c. Collecting the operation excision specimen for lung cancer, separating the cancer tissue and the tissue beside the cancer, embedding the cancer tissue and the tissue beside the cancer with paraffin respectively, staining the pathological tissue section, and identifying the tissue type and purity. Tissue sections DNA was extracted using the DNA extraction Kit from magenta (HiPure FFPE DNA Kit, D3126-03).
d. Bisulfite modification of DNA was performed using the DNA conversion Kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Biopsis.
e. The amplification detection system and the detection system are as follows:
TABLE 5 liquid formulation system
| DMRTA2 | β-actin | |
| Reaction component | Addition amount (μ l) | Addition amount (μ l) |
| Upstream primer (100uM) | 0.125 | 0.125 |
| Downstream primer (100uM) | 0.125 | 0.125 |
| Probe (100uM) | 0.05 | 0.05 |
| Magnesium ion (25mM) | 5 | 5 |
| dNTPs(10mM) | 1 | 1 |
| Taq polymerase (5unit/ul) | 0.5 | 0.5 |
| 5 Xbuffer solution | 5 | 5 |
| Sterilized water | 12.2 | 12.2 |
| |
1 | 1 |
| Total volume | 25 | 25 |
TABLE 6 PCR reaction procedure
f. The result of the detection
Calculating the methylation copy number of the DMRTA2 gene in a specimen by using a standard curve, judging the methylation degree of two groups of tissues by adopting a ratio of DMRTA2 copy number/ACTB copy number 100, finally selecting a numerical value of '4.3' as a standard for judging a cancer group and a control group, judging the cancer group to be positive when the converted ratio exceeds '4.3', and judging the cancer group to be negative when the converted ratio is equal to or less than '4.3'. According to this standard, the results of 56 specimens were as follows:
TABLE 7 test results
Note: "+" indicates that the test result is a positive sample; "-" indicates that the test result was negative
TABLE 8 statistical results
The above results indicate that 28 of the 28 samples were lung cancer samples, and that 25 of the 28 samples were positive for methylated DNA with a specificity of 100.0%, and the sensitivity was 89.3%, and among them, squamous cell carcinoma was detected in all cases and adenocarcinoma was missed in 3 cases. From the box plot and scatter plot (fig. 3), DNA methylation detected by the DMRTA2 gene was clearly differentiated between lung cancer and non-lung cancer. The DMRTA2 gene is proved to be a molecular marker for clinical diagnosis of lung cancer.
Particularly for adenocarcinoma, the specificity reaches 100.0%, the sensitivity reaches 86.4%, and the method has extremely high clinical application value. Lung adenocarcinoma is more likely to occur in women and non-smokers, no clinical symptoms are generally evident in early stages, and the adenocarcinoma peripheral type has higher omission rate due to the dendritic physiological structure of the bronchus, so that the detection of the lung adenocarcinoma peripheral type is more difficult and meaningful, and the ROC curve of the DMRTA2 in a tissue specimen is shown in fig. 4, and the AUC value is 0.953.
Example 3: detection of DMRTA2 gene in sputum specimen
A great deal of literature shows that SHOX2 can be used as a marker for detecting lung cancer, and SHOX2 has high detection rate in samples such as alveolar lavage fluid, lesion tissues, pleural fluid, sputum and the like. To verify the detection effect of DMRTA2, the inventors simultaneously detected the detection efficiency of DMRTA2 and SHOX2 genes in sputum. The gene detection primer probes are as follows:
the detection primers and probes for DMRTA2 were:
SEQ ID NO: 1 DMRTA2 primer MF 1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO: 2 DMRTA2 primer MR 1: TACGACCTAACCGCGCTCTCA
SEQ ID NO: 3 d DMRTA2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
The detection primers and probes for SHOX2 were:
SEQ ID NO: 74 SHOX2_ T _ MF3 primer F: TTTAAAGGGTTCGTCGTTTAAGTC
SEQ ID NO: 75 SHOX2_ T _ MR3 primer R: AAACGATTACTTTCGCCCG
SEQ ID NO: 76 SHOX2_ Taq _ P3_ probe: FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1
Sample information: the total number of sputum samples tested was 60, wherein 31 samples of the normal control group, 29 samples of the cancer group, and 9 samples of squamous carcinoma, 6 samples of small cell carcinoma, 9 samples of adenocarcinoma, 1 sample of large cell carcinoma, and 4 samples of lung cancer which is not classified clearly were selected from the 29 samples of cancer group.
The test process comprises the following steps:
a. sputum specimens of lung cancer patients and non-lung cancer patients were collected, and after being de-thickened with DTT, cells were separated by centrifugation and pelleted, and washed 2 times with PBS, and then DNA was extracted using the DNA extraction Kit of magenta (HiPure FFPE DNA Kit, D3126-03).
b. Bisulfite modification of DNA was performed using the DNA transformation Kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Biopsis.
c. The liquid preparation system is shown in Table 9:
TABLE 9 liquid formulation system
d. The amplification system is shown in Table 10:
TABLE 10 PCR reaction procedure
e. The detection results are as follows:
and calculating the methylation copy number of each gene in the specimen by using a standard curve, judging the methylation degree of two groups of tissues by adopting a ratio of the methylation copy number to the ACTB copy number of 100, finally selecting a threshold value of DMRTA2 of 25.9 and a threshold value of SHOX2 of 5.1 as standards for judging the cancer group and the control group, and judging the methylation copy number of the gene in the specimen to be positive when the converted ratio exceeds a set threshold value and judging the methylation copy number of the gene in the specimen to be negative when the converted ratio is equal to or less than the set threshold value. According to this standard, the results of 60 sputum specimens are shown in Table 11:
TABLE 11 sputum specimen test results
Note: "+" indicates that the test result is a positive sample; "-" indicates that the test result was a negative sample.
f. Analysis of results
TABLE 12 statistical results
From the above results, it can be seen that the detection effect of DMRTA2 is superior to that of SHOX2 gene regardless of whether lung cancer is comparatively analyzed as a whole or as subtypes of lung cancer. In particular, in the detection effect on adenocarcinoma, the detection rate of DMRTA2 is 33.3%, while the detection rate of SHOX2 gene is 0%, and adenocarcinoma is generally peripheral, and due to the dendritic physiological structure of bronchus, exfoliated cells in the deep lung are more difficult to be expectorated through sputum, so that the detection of the part is more difficult and meaningful. The ROC curves for DMRTA2 and SHOX2 in sputum specimens are shown in fig. 5, with the AUC value for DMRTA2 being 0.868 and the AUC value for SHOX2 being 0.847.
Example 4: detection of DMRTA2 gene in lavage fluid specimen
Sample information: the total number of alveolar lavage fluid samples tested was 79, 58 samples of the normal control group, 21 samples of the cancer group, 6 samples of squamous cell carcinoma, 4 samples of small cell carcinoma, and 11 samples of adenocarcinoma in the 21 cancer group.
The test process comprises the following steps:
a. alveolar lavage fluid specimens of patients diagnosed with lung cancer and non-lung cancer were collected, cells were centrifuged, and then DNA was extracted using a DNA extraction Kit from magenta (HiPure FFPE DNA Kit, D3126-03).
b. Bisulfite modification of DNA was performed using the DNA conversion Kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Biopsis.
c. The liquid preparation system is as in Table 9.
d. The amplification assay system is as in Table 10.
e. The detection results are as follows:
and calculating the methylation copy number of each gene in the specimen by using a standard curve, judging the methylation degrees of the two groups of tissues by adopting a ratio of the methylation copy number to the ACTB copy number 100, finally selecting a threshold value of DMRTA2 of 2.1 and a threshold value of SHOX2 of 0.6 as standards for judging the cancer group and the control group, and judging the methylation copy number of each gene in the specimen to be positive when the converted ratio exceeds a set threshold value and judging the methylation copy number of each gene to be negative when the converted ratio is equal to or less than the set threshold value. Results of detection of 79 lavage samples according to this standard are shown in Table 13:
TABLE 13 lavage sample test results
Note: "+" indicates that the test result is a positive sample; "-" indicates that the test result was a negative sample.
TABLE 14 statistical results
From the above results, it can be seen that, when DMRTA2 and SHOX2 are detected simultaneously and lung cancer is compared and analyzed as a whole, the detection rate of DMRTA2 is 61.9% and is much higher than 47.6% of SHOX2, and when compared and analyzed according to the subtype of lung cancer, the detection result of the DMRTA2 in the squamous cell carcinoma group is 16.7% higher than that of the SHOX 2. Particularly, the sensitivity of the kit reaches 54.5 percent and is far higher than 36.4 percent of that of SHOX2 in the detection effect on adenocarcinoma. Since adenocarcinomas are generally peripheral, alveolar lavage fluid does not readily reach the deep alveoli or cancerous tissues of the lungs due to the dendritic physiology of the bronchi, making detection of this fraction more difficult and meaningful. The ROC curves for DMRTA2 and SHOX2 in alveolar lavage fluid specimens are shown in fig. 6, with an AUC value of 0.870 for DMRTA2 and 0.784 for SHOX 2.
By combining the embodiments 1-4, it can be fully demonstrated that DMRTA2 has better detection effect on lung cancer detection and diagnosis, especially on biological samples such as sputum and alveolar lavage fluid. Can be more easily applied to large-scale population screening. Has more excellent social and economic values.
Example 5: selection of regions of the DMRTA2 Gene
Various research data show that the methylation state and distribution of the same gene are not uniform, so that for the same gene, methylation primers and probe detection systems designed by selecting different regions have different diagnostic detection efficiencies on the same sample, and even the selected regions are not suitable to cause no diagnostic effect on tumors at all. Table 15 lists various regions of the DMRTA2 gene selected during the course of the experiments of the present invention.
TABLE 15 sequences of different regions of the DMRTA2 gene
Designing different methylation primers and probes according to the region 1, the region 2, the region 3 and the region 4, wherein the information of each primer probe is shown in a table 16, wherein the group 1, the group 2, the group 3, the group 4 and the group 5 are the methylation primers and probes designed according to the region 1; group 6, group 7, group 8, group 9 are methylation primers and probes designed according to region 2; groups 10, 11, 12 are methylation primers and probes designed according to region 3; group 13, group 14, and group 15 are methylation primers and probes designed based on region 4. All primers and probes were synthesized by England Shafer (Shanghai) trade Limited.
TABLE 16 primers and probes designed based on different regions of the DMRTA2 Gene
In 36 lung tissue samples, 15 primer probe sets in table 16 were tested, wherein 11 normal tissue samples, 25 cancer tissue samples, 4 squamous carcinomas and 21 adenocarcinoma samples were selected from the 25 cancer group samples. The results are shown in Table 17.
The sample treatment, the detection result judgment and the statistical mode are the same as the embodiment 1; the PCR solution preparation system and the reaction process are conventional operations in the field.
TABLE 17 results of detection of different combinations of primer probes in tissues
The results show that group 1, group 2, group 3, group 4, group 5, group 10, and group 13 all have better detectable rates. However, no matter what kind of primers and probes designed by the present invention are adopted, the detection sensitivity of the region 1 can reach 72% at the lowest, 96% at the highest, the detection rate is much higher than that of several pairs of primers designed for the region 2, and most of the primers of the region 1 have higher detection sensitivity than those of the primers of the regions 3 and 4, so that the detection rate of the region 1 is obviously higher than that of the other regions (see table 17).
EXAMPLE 6 selection of primer and Probe combinations
In order to further verify the detection rate of different combinations of primers and probes in sputum, the inventors selected 22 sputum samples and verified the primers and probes in table 16, wherein the samples include 7 normal controls, 15 lung cancer controls, 7 squamous cell carcinomas, 7 adenocarcinoma and 1 large cell carcinoma among 15 lung carcinomas, and the detection results are shown in table 18.
The sample treatment, the detection result judgment and the statistical mode are the same as the embodiment 3; the PCR solution preparation system and the reaction process are conventional operations in the field.
TABLE 18 test results in sputum
| Group of | Primer probe combination | Specificity of | Sensitivity of the |
| Group | |||
| 1 | DM2-F1,DM2-R1,DM2-P1 | 100％ | 80％ |
| Group 2 | DM2-F2,DM2-R2,DM2-P2 | 100％ | 60％ |
| Group 3 | DM2-F3,DM2-R3,DM2-P3 | 100％ | 66.7％ |
| Group 4 | DM2-F4,DM2-R4,DM2-P4 | 100％ | 46.7％ |
| Group 5 | DM2-F5,DM2-R5,DM2-P5 | 100％ | 66.7％ |
| Group 10 | DM2-F10,DM2-R10,DM2-P10 | 100％ | 60％ |
| Group 13 | DM2-F13,DM2-R13,DM2-P13 | 100％ | 46.7％ |
From the results of 22 sputum specimens, group 1: the detection rates of DM2-F1, DM2-R1 and DM2-P1 are the highest and reach 80 percent.
Although the sensitivity of group 1 is 96% and the sensitivity of group 2 is 88% in the tissue sample, the sensitivity of group 2 is greatly reduced to 60% for the sputum sample, which proves that it is particularly difficult to design a detection reagent with high sensitivity for the sputum sample.
Finally, based on the detection results of each set of primer probes, the most preferred primer probe sequence is the combination of set 1: DM2-F1, DM2-R1 and DM 2-P1.
Sequence listing
<110> Congliming Biotechnology, Inc. of Guangzhou City
<120> lung cancer diagnostic agent and kit based on DMRTA2 gene
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<170>SIPOSequenceListing 1.0
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ttcggtttag tgtgcgtcgt c 21
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tacgacctaa ccgcgctctc a 21
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cggcggcggc gcgggaggcg g 21
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aaattctccg aaacgctcg 19
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ttgtgtgttg ggtggtggtt 20
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gcgtggtgtc ggccctcaag ggccacaaac gctactgtcg ctggaaggac tgcctgtgcg 180
ccaagtgcac gctcatcgcg gagcgccagc gtgtcatggc ggcgcaggtg gcgctgcgca 240
ggcagcaggc gcaggaggag aacgaggcgc gcgagctgca gctgctctac ggcactgccg 300
aggggctggc gctggccgcc gccaacggca tcatcccccc gaggcccgcc tacgaggtct 360
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gcgtggtgtc ggtttttaag ggttataaac gttattgtcg ttggaaggat tgtttgtgcg 180
ttaagtgtac gtttatcgcg gagcgttagc gtgttatggc ggcgtaggtg gcgttgcgta 240
ggtagtaggc gtaggaggag aacgaggcgc gcgagttgta gttgttttac ggtattgtcg 300
aggggttggc gttggtcgtc gttaacggta ttattttttc gaggttcgtt tacgaggttt 360
tcggtttagt gtgcgtcgtc gacggcgggg gatttggagc gggagcgttc gcggggatcg 420
gaggcggagt agttggcgta gggggtttag gtgagagcgc ggttaggtcg taggtagggg 480
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<212>DNA
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accgcagtaa ttgaattgtc acctgccccc catcccgcct cagggctcca gagccacttg 180
ggaagctcgg ctttgccaag cgccccaggc cagtaagagc ggtgcagccg agccggcgcc 240
ccagaagccg agaggttaca caagtctacg cagagaggcg caccggacac ggtaattagg 300
agcaattcac acgctctcgg gcgcacggaa actacttgtc aggaaatttt caacgctcct 360
cctcgcccac ttttttccac tcctttcctg gcgcgccccc tcccctccca gaggcacgca 420
gtggagcttt tggagtttcg aatctttcgg acactgcaga atgcggggct ccccgggcgc 480
gggcccggcc aatcagagcg ccgtctgcct cccctggcca atggcaggcg ccacattgtt 540
gtccaattct gtctaatgga gccctaagga ccaacaatag agcgggcgag cggctcattg 600
agagtctggc agcgccgggc aactgggccc cggctgcgcg cagcgtccgg cgcgcaccgc 660
gaggggaccc ggcacggcga gagtcaggtc gcgggttcca accacgcggc ccgcagacga 720
gcctccgacc cggatttctg aagggagggt tggactgcgc tgcgttctga ggggtccgga 780
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<213> Artificial Sequence (Artificial Sequence)
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atcgtagtaa ttgaattgtt atttgttttt tatttcgttt tagggtttta gagttatttg 180
ggaagttcgg ttttgttaag cgttttaggt tagtaagagc ggtgtagtcg agtcggcgtt 240
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gggttcggtt aattagagcg tcgtttgttt tttttggtta atggtaggcg ttatattgtt 540
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caggccgccc gggcagcccg ctgccgccgc cggtgaagcc cttatcaccc gacggcgcag 180
actcggggcc cgggacgtcg tccccagagg tgcggcccgg ctcaggctcg gagaacggcg 240
atggcgagtc cttttctggt tcgcccctag ctcgggcctc caaagaggca ggtggcagct 300
gcccaggcag cgctggccct ggcggcggcg gcgaggagga cagcccgggc tccgctagcc 360
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<213> Artificial Sequence (Artificial Sequence)
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taggtcgttc gggtagttcg ttgtcgtcgt cggtgaagtt tttattattc gacggcgtag 180
attcggggtt cgggacgtcg tttttagagg tgcggttcgg tttaggttcg gagaacggcg 240
atggcgagtt tttttttggt tcgtttttag ttcgggtttt taaagaggta ggtggtagtt 300
gtttaggtag cgttggtttt ggcggcggcg gcgaggagga tagttcgggt ttcgttagtt 360
ttttgggttt tgaattcggt ttagaggttg ataaagaaga gggtgaggtc gcgtcggcgt 420
tagggttggg cggaggttcg ggtttacggt agcggacgtc gttggatatt ttgatacgcg 480
tgtttttagg ttatcggcga ggcgttttgg agttggtgtt gtagggttgc ggcggcgacg 540
tggtgtaggt tatcgagtag gtgttgaatt attatcgtgg gggtttggcg gtcggtttgg 600
gttttgcggc gtttttagat aaggtcgtcg tgggtgttgt agtagttgta gacgacgcgt 660
ggtttagtcg cgtcgacgtc gtcgtcgtcg tcgtcgtcgt cgtcgggggg tttgggttgt 720
ttgcgtcgtt gtaggcgggg ttcgtcgtat tttcgtatta tagatttttg ttggtcggcg 780
ttatggcgtt tggggcgttg ggttcgttga gtagtcgttc ggttttttcg tcgttgtagt 840
ttaacgttag ttatttcggt gtcgacgcgg gcgtttattc gttgggcgcg tcgttcggtt 900
ttagtttttt gcgtttggtt tatttcgcgg cggcggcgta tagtcgcggt ttggttttta 960
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gccaaactaa cggaatccgg cgcggccagg gaaggggtgg gtgcgggagg ggccattccg 180
tcccggagcc accggtttgg ggataattac ttttaatgtc agaaagattt agtttaatat 240
catctctatt aggctgccgg gagggtaatt aaacgggacg cgtcgcagcc ggcaaacaga 300
tggcgtctgc ttgctccgag gccgcgggca aacagcgcaa aagtcaatgc ctcccgggtc 360
ccccatcggc gcccctggct cttttactca gagctacact actcaccctc ggcccccttc 420
cccacgccgc gtctccagcc ggctctgcgc cctaggccct caaccccggc ctcaccccaa 480
cgcagtccct ttggacgcca ccagttcctg ggcgtgggcg tgggggatgg ggccgggggc 540
gaatagggat ggggtggatt ttcttggctg ggccctctca cccgggcccc cagcccctgg 600
cacagcagag gcgtcagcaa atgattctga gctaagagta cgcgtgtgga tgtgtgagga 660
agaccctctg ctctcagccg cctcatttgt cccggagggt tttggctttt tctgcccggg 720
gcccaactgt agaagtaaga ctggagctcc gcgcataaac cagggcatac ctgcgtaaga 780
atgtgctcct gtcggcgtgt gctatcaccg aggtggaaat ctgtgggagt ctgggaatgt 840
cacacaccaa gtggatggcc caggatgggg acatgcttat aagaacactg tgaggtcaaa 900
ctcactgaag gggtcccctt gagccggagg gtagcatcag agttgagcct gggcccggtg 960
gtgctctctg aatgctggcg tgagagtgtg tcccaaactt t 1001
<210>26
<211>1001
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
aggaatagga gagtttgtgg ttgtgcggga aaacgcgtgt agggtaattc gtggagattt 60
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gttaaattaa cggaattcgg cgcggttagg gaaggggtgg gtgcgggagg ggttatttcg 180
tttcggagtt atcggtttgg ggataattat ttttaatgtt agaaagattt agtttaatat 240
tatttttatt aggttgtcgg gagggtaatt aaacgggacg cgtcgtagtc ggtaaataga 300
tggcgtttgt ttgtttcgag gtcgcgggta aatagcgtaa aagttaatgt ttttcgggtt 360
ttttatcggc gtttttggtt tttttattta gagttatatt atttattttc ggtttttttt 420
tttacgtcgc gtttttagtc ggttttgcgt tttaggtttt taatttcggt tttattttaa 480
cgtagttttt ttggacgtta ttagtttttg ggcgtgggcg tgggggatgg ggtcgggggc 540
gaatagggat ggggtggatt tttttggttg ggttttttta ttcgggtttt tagtttttgg 600
tatagtagag gcgttagtaa atgattttga gttaagagta cgcgtgtgga tgtgtgagga 660
agattttttg tttttagtcg ttttatttgt ttcggagggt tttggttttt tttgttcggg 720
gtttaattgt agaagtaaga ttggagtttc gcgtataaat tagggtatat ttgcgtaaga 780
atgtgttttt gtcggcgtgt gttattatcg aggtggaaat ttgtgggagt ttgggaatgt 840
tatatattaa gtggatggtt taggatgggg atatgtttat aagaatattg tgaggttaaa 900
tttattgaag gggttttttt gagtcggagg gtagtattag agttgagttt gggttcggtg 960
gtgttttttg aatgttggcg tgagagtgtg ttttaaattt t 1001
<210>27
<211>972
<212>DNA
<213>Homo sapiens
<400>27
agcccggggc ggggtggggc tggagctcct gtctcttggc cagctgaatg gaggcccagt60
ggcaacacag gtcctgcctg gggatcaggt ctgctctgca ccccaccttg ctgcctggag 120
ccgcccacct gacaacctct catccctgct ctgcagatcc ggtcccatcc ccactgccca 180
ccccaccccc ccagcactcc acccagttca acgttccacg aacccccaga accagccctc 240
atcaacaggc agcaagaagg gccccccgcc catcgcccca caacgccagc cgggtgaacg 300
ttggcaggtc ctgaggcagc tggcaagacg cctgcagctg aaagatacaa ggccagggac 360
aggacagtcc catccccagg aggcagggag tatacaggct ggggaagttt gcccttgcgt 420
ggggtggtga tggaggaggc tcagcaagtc ttctggactg tgaacctgtg tctgccactg 480
tgtgctgggt ggtggtcatc tttcccacca ggctgtggcc tctgcaacct tcaagggagg 540
agcaggtccc attggctgag cacagccttg taccgtgaac tggaacaagc agcctccttc 600
ctggccacag gttccatgtc cttatatgga ctcatctttg cctattgcga cacacactca 660
gtgaacacct actacgcgct gcaaagagcc ccgcaggcct gaggtgcccc cacctcacca 720
ctcttcctat ttttgtgtaa aaatccagct tcttgtcacc acctccaagg agggggagga 780
ggaggaaggc aggttcctct aggctgagcc gaatgcccct ctgtggtccc acgccactga 840
tcgctgcatg cccaccacct gggtacacac agtctgtgat tcccggagca gaacggaccc 900
tgcccacccg gtcttgtgtg ctactcagtg gacagaccca aggcaagaaa gggtgacaag 960
gacagggtct tc 972
<210>28
<211>972
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
agttcggggc ggggtggggt tggagttttt gttttttggt tagttgaatg gaggtttagt 60
ggtaatatag gttttgtttg gggattaggt ttgttttgta ttttattttg ttgtttggag 120
tcgtttattt gataattttt tatttttgtt ttgtagattc ggttttattt ttattgttta 180
ttttattttt ttagtatttt atttagttta acgttttacg aatttttaga attagttttt 240
attaataggt agtaagaagg gtttttcgtt tatcgtttta taacgttagt cgggtgaacg 300
ttggtaggtt ttgaggtagt tggtaagacg tttgtagttg aaagatataa ggttagggat 360
aggatagttt tatttttagg aggtagggag tatataggtt ggggaagttt gtttttgcgt 420
ggggtggtga tggaggaggt ttagtaagtt ttttggattg tgaatttgtg tttgttattg 480
tgtgttgggt ggtggttatt ttttttatta ggttgtggtt tttgtaattt ttaagggagg 540
agtaggtttt attggttgag tatagttttg tatcgtgaat tggaataagt agtttttttt 600
ttggttatag gttttatgtt tttatatgga tttatttttg tttattgcga tatatattta 660
gtgaatattt attacgcgtt gtaaagagtt tcgtaggttt gaggtgtttt tattttatta 720
ttttttttat ttttgtgtaa aaatttagtt ttttgttatt atttttaagg agggggagga 780
ggaggaaggt aggttttttt aggttgagtc gaatgttttt ttgtggtttt acgttattga 840
tcgttgtatg tttattattt gggtatatat agtttgtgat tttcggagta gaacggattt 900
tgtttattcg gttttgtgtg ttatttagtg gatagattta aggtaagaaa gggtgataag 960
gatagggttt tt 972
<210>29
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
ttaagtgtac gtttatcgcg gag 23
<210>30
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
cgcacactaa accgaaaacc tc 22
<210>31
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
cgttaacgac gaccaacgcc aa 22
<210>32
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
gtcgttgtat cgttatcggt gag 23
<210>33
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gacaccacgc cataattacg aca 23
<210>34
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
ccgcaacaaa ccgcctacca c 21
<210>35
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
cgttgtatcg ttatcggtga gc 22
<210>36
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
gcacaaacaa tccttccaac gac 23
<210>37
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
acttctcgac tacccgcaac aaca 24
<210>38
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
cggtcgttgt atcgttatcg 20
<210>39
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
ctacccgcaa caacaataac g 21
<210>40
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
cgtggtaggc ggtttgttgc g 21
<210>41
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
cgggttttaa ttacgcggtt c 21
<210>42
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
cgcctcccga ctccactaac 20
<210>43
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
ccccgacccc ttatctaacc ac 22
<210>44
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
taagagcggt gtagtcgagt c 21
<210>45
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
aatttccgta cgcccgaaa 19
<210>46
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
cgttttagaa gtcgagaggt tat 23
<210>47
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
ggttaattag agcgtcgttt 20
<210>48
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
gcaaccgaaa cccaattacc c 21
<210>49
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
gacgctacca aactctcaat aaaccg 26
<210>50
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
ttttcggata ttgtagaatg c 21
<210>51
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gaaacccaat tacccgac 18
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
acgctctaat taaccgaacc 20
<210>53
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
ggggttcgtc gtattttcg 19
<210>54
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
aaccgaacga ctactcaacg 20
<210>55
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
cgttatggcg tttggggcgt tg 22
<210>56
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
tattcgacgg cgtagattcg 20
<210>57
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
gttctccgaa cctaaaccg 19
<210>58
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
cgtcgttttt agaggtgcgg tt 22
<210>59
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
cgtcgttgga tattttgata cg 22
<210>60
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
ctacaacacc aactccaaaa cg 22
<210>61
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
cgtgttttta ggttatcggc gagg 24
<210>62
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
gtcgggaggg taattaaacg 20
<210>63
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
cgacctcgaa acaaacaaac g 21
<210>64
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
cgcgtcgtag tcggtaaata gatgg 25
<210>65
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
tggttgtgcg ggaaaacg 18
<210>66
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
ctccgcccga aaataaaacg 20
<210>67
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
cgtgtagggt aattcgtgga gattt 25
<210>68
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
gtaaatgatt ttgagttaag agtacg 26
<210>69
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
ccctccgaaa caaataaaac g 21
<210>70
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
cgtgtggatg tgtgaggaag attt 24
<210>71
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
ttttggattt aaggggaaga taaa 24
<210>72
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>72
tttttccttc tctacatctt tctacct 27
<210>73
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>73
aagggaaatt gagaaatgag agaaggga 28
<210>74
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>74
tttaaagggt tcgtcgttta agtc 24
<210>75
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>75
aaacgattac tttcgcccg 19
<210>76
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>76
ttagaaggta ggaggcggaa aattag 26
Claims (17)
1. The application of a nucleic acid fragment in preparing a tumor detection/diagnosis reagent or kit; the nucleic acid fragment is selected from SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23 or SEQ ID NO: 25; preferably, the nucleic acid fragment is selected from the group consisting of SEQ ID NO: 19.
2. a primer selected from the group consisting of SEQ ID NOs: 1, and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ id no: 38 and SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 63, SEQ ID NO: 65 and SEQ ID NO: 66, SEQ ID NO: 68 and SEQ ID NO: 69; preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, seq id NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 62 and SEQ ID NO: 63; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 39, or a primer set shown in the specification; most preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer set shown in (2).
3. A nucleic acid probe selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 31, SEQ id no: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70 is shown in any one of the figures; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 55, SEQ ID NO: 64 is shown in any one of the figures; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40; most preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3.
4. use of the primer pair or the nucleic acid probe according to claim 2 or 3 for preparing a tumor detection/diagnosis reagent or kit.
5. A tumor detection/diagnosis reagent, which comprises a detection reagent for methylation of DMRTA2 gene.
6. The detection/diagnostic reagent of claim 5, wherein the detection reagent for methylation of DMRTA2 gene detects the sequence of DMRTA2 gene modified with bisulfite or hydrazine;
preferably, it is bisulphite modified.
7. The detection/diagnostic reagent of claim 5, wherein the reagent is set forth as SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23 or SEQ ID NO: 25 is shown;
preferably, as shown in SEQ ID NO: 19, respectively.
8. The detection/diagnostic reagent according to claim 5, wherein the reagent comprises an amplification primer; preferably, the primer is shown as SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69; preferably, the primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 62 and SEQ ID NO: 63; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ id no: 38 and SEQ ID NO: 39, or a primer set shown in the specification; most preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer set shown in (2).
9. The detection/diagnostic reagent according to claim 5, wherein the reagent further comprises a probe; preferably, the probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70 is shown in any one of the figures; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 55, SEQ ID NO: 64 is shown in any one of the figures; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40; most preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3.
10. the detection/diagnostic reagent according to claim 5, wherein the reagent comprises a detection reagent comprising an internal reference gene;
preferably, the internal reference gene is β -actin, COL2A1
Preferably, the detection reagent of the reference gene is a primer and a probe aiming at the reference gene;
more preferably, the detection reagent for the reference gene is SEQ ID NO: 16. SEQ ID NO: 17 and the primer pair shown in SEQ ID NO: 18 with a probe;
or preferably, the detection reagent of the reference gene is SEQ ID NO: 71. SEQ ID NO: 72 and the primer pair shown in SEQ ID NO: 73 in the same manner as described above.
11. The detection/diagnostic reagent of claim 5, wherein said reagent further comprises bisulfite, bisulfite or hydrazonium.
12. The detection/diagnostic reagent according to claim 5, wherein the reagent further comprises DNA polymerase, dNTPs, Mg2+One or more of ions and buffer solution; preferably, DNA polymerase, dNTPs, Mg are included2+Ions and buffers.
13. The detection/diagnostic reagent of claim 5, wherein the test sample of the reagent is selected from the group consisting of alveolar lavage fluid, tissue, pleural fluid, sputum, blood, serum, plasma, urine, prostatic fluid, and stool;
preferably, the sample is selected from alveolar lavage fluid, tissue, sputum; more preferably, the sample is selected from alveolar lavage fluid or sputum.
14. A kit comprising the tumor detection/diagnosis reagent according to any one of claims 5 to 13.
15. The nucleic acid fragment or the use or the detection/diagnostic reagent or kit according to any one of claims 1, 5 to 14, characterized in that said tumor is selected from the group consisting of lung cancer;
preferably, the lung cancer is selected from small cell lung cancer and non-small cell lung cancer;
more preferably, the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma.
16. A method for detecting DNA methylation of a DMRTA2 gene, comprising the steps of:
(1) processing a sample to be detected by bisulfite or hydrazine to obtain a modified sample to be detected;
(2) carrying out DMRTA2 gene methylation detection on the modified sample to be tested in step (1) by using the reagent or kit according to any one of claims 5 to 14.
Preferably, in step (2), the detection is performed by real-time fluorescence quantitative methylation specific polymerase chain reaction.
17. A system for detecting/diagnosing lung cancer, comprising:
a DNA methylation detection means for the DMRTA2 gene, and,
b. a result judgment system;
preferably, the DNA methylation detection means of DMRTA2 gene comprises the reagent or kit of any one of claims 5 to 14;
preferably, the result judging component is used for outputting the lung cancer disease risk and/or the lung cancer type according to the DNA methylation result of the DMRTA2 gene detected by the detection system;
more preferably, the disease risk is determined by comparing the methylation results of the test sample and the normal sample according to the result, and when the methylation of the test sample and the methylation of the normal sample have a significant difference or a very significant difference, the disease risk of the test sample is determined to be high according to the result.
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| CN201811591956.8A CN111363812B (en) | 2018-12-25 | 2018-12-25 | Lung cancer diagnostic agent and reagent kit based on DMRTA2 gene |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811591956.8A CN111363812B (en) | 2018-12-25 | 2018-12-25 | Lung cancer diagnostic agent and reagent kit based on DMRTA2 gene |
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| Publication Number | Publication Date |
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| CN111363812B CN111363812B (en) | 2023-12-19 |
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| WO2023116593A1 (en) * | 2021-12-20 | 2023-06-29 | 江苏鹍远生物技术有限公司 | Tumor test method and application |
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