CN111363811A - Lung cancer diagnostic agent and kit based on FOXD3 gene - Google Patents
Lung cancer diagnostic agent and kit based on FOXD3 gene Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The invention belongs to the field of biological medicine, and relates to a lung cancer detection/diagnosis reagent and a reagent kit, wherein the reagent or the reagent kit comprises a detection reagent aiming at the methylation of FOXD3 gene, and is used for detecting a sequence of FOXD3 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 FOXD3 gene methylation detection/diagnosis reagent for lung cancer detection and a kit containing the same.
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 FOXD3 gene in preparation of 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 present invention to provide a reagent, a kit and a method for diagnosing human FOXD3 gene methylation.
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 FOXD3 gene, in particular from the fact that the FOXD3 gene is positioned on chromosome 1: 63788730-63790797 (GRCh 37 as reference).
Specifically, the nucleic acid fragment is selected from SEQ ID NO: 19. SEQ ID NO: 21 or SEQ ID NO: 23. 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: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and seq id NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO: 64; preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 51 and SEQ ID NO: 52; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, or a primer set shown in the specification. As a preferred embodiment of the present invention, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
In another aspect, the present invention also provides a probe selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 41. SEQ ID NO: 44. SEQ ID NO: 47. SEQ ID NO: 50. SEQ ID NO: 53. SEQ ID NO: 56. SEQ ID NO: 59. SEQ ID NO: 62. SEQ ID NO: 65; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 53; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. as a preferred embodiment of the present invention, the nucleic acid probe is selected from 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 of FOXD3 gene.
FOXD3 gene (forkhead box D3), forkhead box D3 gene, which belongs to the forkhead protein family transcription factor. It is reported that FOXD3 is mainly expressed in embryonic stem cells or pluripotent stem cells, has a close relationship with embryonic development and the pluripotency of stem cells, and is a marker member of tumor stem cells. In addition, FOXD3 has been reported to have cancer-inhibiting effects.
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.
Furthermore, the methylation detection reagent of the FOXD3 gene detects the sequence of the FOXD3 gene modified by bisulfite or hydrazine.
As an exemplary embodiment, the sequence of FOXD3 gene was determined after bisulfite modification.
In a preferred embodiment, the detection region of the FOXD3 gene targeted by the reagent is specifically derived from the fact that the FOXD3 gene is located on chromosome 1: 63788730-63790797 (GRCh 37 as reference). More preferably, the detection region is selected from the group consisting of SEQ ID NO: 19. SEQ ID NO: 21 or SEQ ID NO: 23 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 FOXD3 gene has an influence on the detection efficiency of the tumor. The inventors carried out RRBS methylation sequencing on the FOXD3 gene according to the present invention, and obtained the FOXD3 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 primers and probes designed in the left side of the box are better in detecting both adenocarcinoma and squamous carcinoma, while the right side of the box is better in detecting adenocarcinoma than squamous carcinoma. Although all were near the FOXD3 gene, it was clear that the left region was selected to design a primer probe more effectively.
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: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ id no: 36 and SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO: 64; preferably, the primer is selected from SEQ id no: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, seq id NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 51 and SEQ ID NO: 52; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37; most preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer set shown in (2).
The primer is used for amplifying a specific region of the FOXD3 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: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 41. SEQ ID NO: 44. SEQ ID NO: 47. SEQ ID NO: 50. SEQ ID NO: 53. SEQ ID NO: 56. SEQ ID NO: 59. SEQ ID NO: 62. SEQ ID NO: 65; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 53; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. any one of SEQ ID NOs: 38; most preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3. 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.
The detection reagent of the reference gene is β -actin and COL2A1, the detection reagent of the reference gene is a primer and a probe aiming at the reference gene, and as a more preferable embodiment, the detection reagent of the reference gene is a primer pair shown in SEQ ID NO. 16, SEQ ID NO. 17 and a probe shown in SEQ ID NO. 18.
As a preferred embodiment, the reagent also includes bisulfite, bisulfite or hydrazonium salts to modify the FOXD3 gene, which of course 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 solution is used for amplifying the modified FOXD3 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 FOXD3 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 FOXD3 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 means for the FOXD3 gene, and,
(2) a result judgment system;
preferably, the DNA methylation detection means of FOXD3 gene comprises the reagent or the kit according to any one of claims 5 to 14;
preferably, the result judging means is used for outputting the risk of lung cancer and/or the type of lung cancer according to the DNA methylation result of the FOXD3 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 DNA methylation of the FOXD3 gene is positive, it indicates that the provider of the sample to be tested 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, it has been reported that FOXD3 gene methylation can be one of the tumor markers of 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 FOXD3 gene has high sensitivity and specificity to lung cancer, and is very hopeful to be used as a tumor marker for clinical diagnosis of lung cancer.
The FOXD3 gene has high sensitivity and specificity to lung cancer, and the detection rate of lung cancer with specificity of 95% and sensitivity of 77% in a tissue specimen can be reached only based on the optimized methylation detection region and detection reagent of the FOXD3 gene. Wherein the detection rate of squamous cell carcinoma reaches 91.3%; among the most difficult adenocarcinomas to detect, the detection rate of adenocarcinomas was 73.1%, and large cell carcinomas were all detectable.
Lung cancer detection kits based on the SHOX2 gene are currently on the market. In one embodiment of the invention, the FOXD3 detection effect is superior to that of SHOX2 gene in detecting sputum samples, whether lung cancer is compared as a whole or according to lung cancer subtypes. Particularly, the detection effect on adenocarcinoma is that the detection rate of FOXD3 is 22.2%, 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 FOXD3 is 61.9% which is much higher than 47.6% of SHOX2, and according to the lung cancer subtype, the detection result of FOXD3 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, adenocarcinoma and large cell carcinoma in small cell lung cancer and non-small cell lung cancer, has wide application range, and can be used as a tumor marker 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 FOXD3 gene and the method can conveniently and accurately judge the lung cancer and lung benign disease patients, and the gene detection method is expected to be converted into a gene detection kit and is used for lung cancer screening, clinical detection and prognosis monitoring.
Drawings
FIG. 1 comparison of detection effects of primers and probes designed for different regions of FOXD3
FIG. 2 ROC curves of five candidate genes FOXD3, SIX3, PCDHGA12, HOXD8 and GATA3 for detecting lung cancer
FIG. 3 methylation degree of FOXD3 gene in control group and lung cancer group
FIG. 4 ROC curve of FOXD3 gene in clinical tissue specimens for detection of lung cancer
FIG. 5 ROC curves for FOXD3 and SHOX2 genes detected in sputum samples
FIG. 6 ROC curves for detection of FOXD3 and SHOX2 genes 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 FOXD3, SIX3, PCDHGA12, HOXD8, GATA3 as candidate detection genes, β -actin as reference genes, studied the distribution of methylation sites of each gene, designed primer probes for detection respectively as follows:
the detection primers and probes of FOXD3 were:
SEQ ID NO: 1 FOXD3 primer F: CGTCGGGATCGGATTTTTTC
SEQ ID NO: 2 FOXD3 primer R: TCTCGACTCAAAAACCGACCG
SEQ ID NO: 3 FOXD3 probe: FAM-CGGTTTTTTGCGTTAAGGTTAG-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 the gene in the specimen by using a standard curve, judging the methylation degrees 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 the cancer group and the 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 negative
TABLE 4 statistical results
All samples of the control group were judged to be negative for FOXD3 methylation, while 21 of the 25 lung cancer samples were judged to be positive, and only 3 lung adenocarcinoma and 1 lung squamous carcinoma were judged to be negative. The detection sensitivity of the FOXD3 gene is 84%, the specificity is 100%, and the positive detection rate is far higher than that of the other 4 genes. The FOXD3 gene is suggested to have important significance in clinical detection and diagnosis of lung cancer.
The ROC curve of five candidate genes including FOXD3, SIX3, PCDHGA12, HOXD8 and GATA3 for detecting lung cancer is shown in FIG. 2, and the area under the ROC curve of FOXD3 gene detection is 0.916.
Therefore, the inventor selects FOXD3 as a candidate gene, optimizes the detection condition, expands the detection range, and collects 122 lung cancer tissue samples and the corresponding paracarcinoma tissues as non-lung cancer controls for a total of 244 lung paraffin tissue samples. These included 23 squamous carcinomas, 93 adenocarcinomas, 3 large cell carcinomas, 1 mixed carcinoma, and 2 not clearly diagnosed lung cancer types. See example 2 for details.
Example 2: detection of FOXD3 gene in clinical specimens
a. The detection primer probes are as follows:
the detection primers and probes of FOXD3 were:
SEQ ID NO: 1 FOXD3 primer F: CGTCGGGATCGGATTTTTTC
SEQ ID NO: 2 FOXD3 primer R: TCTCGACTCAAAAACCGACCG
SEQ ID NO: 3 FOXD3 probe: FAM-CGGTTTTTTGCGTTAAGGTTAG-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: 122 lung cancer tissue samples and their corresponding paracancerous tissues were used as non-lung cancer controls, and a total of 244 lung paraffin tissue specimens were collected. These included 23 squamous carcinomas, 93 adenocarcinomas, 3 large cell carcinomas, 1 mixed carcinoma, and 2 not clearly diagnosed lung cancer types.
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 detection system are shown in tables 5-6:
TABLE 5 liquid formulation system
TABLE 6 PCR reaction procedure
f. The result of the detection
Calculating methylation copy number of FOXD3 gene in a specimen by using a standard curve, judging the methylation degree of two groups of tissues by adopting a ratio of FOXD3 copy number/ACTB copy number 100, finally selecting a value of '13.8' as a standard for judging a cancer group and a control group, judging the cancer group to be positive when the converted ratio exceeds '13.8', and judging the cancer group to be negative when the converted ratio is equal to or less than '13.8'. According to this standard, the results of the test on 244 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 among the 122 samples, 122 samples were lung cancer samples, and 94 positive methylated DNA samples were detected with a specificity of 95.0%, and the sensitivity was 77.0%. Wherein the detection rate of squamous cell carcinoma reaches 91.3 percent, and 2 cases are missed; the adenocarcinoma detection rate is 73.1%, and large cell carcinoma can be completely detected. From the box plot and scatter plot (FIG. 3), the FOXD3 gene detected DNA methylation with more distinct discrimination between lung cancer group and non-lung cancer group. The FOXD3 gene is proved to be a molecular marker for clinical diagnosis of lung cancer.
Particularly for adenocarcinoma, the specificity reaches 95.0%, the sensitivity reaches 73.1%, and the method has extremely high clinical application value. Lung adenocarcinoma is relatively easy to occur in women and non-smokers, no obvious clinical symptoms are generally found in early stage, and the detection of the adenocarcinoma peripheral type is more difficult and meaningful due to the higher omission rate of the bronchial tree-shaped physiological structure. And the ROC curve of FOXD3 detected in the tissue specimen is shown in FIG. 4, and the AUC value is 0.937.
Example 3: detection of FOXD3 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. In order to verify the detection effect of FOXD3, the present inventors simultaneously detected the detection efficiency of FOXD3 and SHOX2 genes in sputum.
The gene detection primer probes are as follows:
the detection primers and probes of FOXD3 were:
SEQ ID NO: 1 FOXD3 primer F1: CGTCGGGATCGGATTTTTTC
SEQ ID NO: 2 FOXD3 primer R1: TCTCGACTCAAAAACCGACCG
SEQ ID NO: 3 FOXD3 probe P1: FAM-CGGTTTTTTGCGTTAAGGTTAG-BQ1
The detection primers and probes for SHOX2 were:
SHOX2_ T _ MF3 primer F: TTTAAAGGGTTCGTCGTTTAAGTC
SHOX2_ T _ MR3 primer R: AAACGATTACTTTCGCCCG
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 sample 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 FOXD3 threshold value of 8.9 and a SHOX2 threshold value of 5.1 as standards for judging the cancer group and the control group, and judging the methylation copy number of each gene in the sample to be positive when the converted ratio exceeds a set threshold value and judging the methylation copy number of the gene 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 FOXD3 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, the detection effect on adenocarcinoma is 22.2% for FOXD3 and 0% for SHOX2, which is a peripheral type adenocarcinoma, and the exfoliated cells in the deep lung are more difficult to be expectorated by sputum due to the dendritic physiological structure of the bronchus, so that the detection of the adenocarcinoma is more difficult and meaningful. The ROC curves of FOXD3 and SHOX2 detected in sputum samples are shown in FIG. 5, the AUC value of FOXD3 is 0.894, and the AUC value of SHOX2 is 0.847.
Example 4: detection of FOXD3 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 sample 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 FOXD3 threshold value of 2.6 and a SHOX2 threshold value of 0.6 as standards for judging a cancer group and a control group, and judging the methylation copy number of each gene in the sample to be positive when the converted ratio exceeds a set threshold value and judging the methylation copy number of the gene to be negative when the converted ratio is equal to or less than the set threshold value. The results of the assay 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 FOXD3 and SHOX2 are simultaneously detected and lung cancer is comparatively analyzed as a whole, the detection rate of FOXD3 is 61.9% which is much higher than 47.6% of SHOX2, and the detection result of FOXD3 in the squamous cell carcinoma group is 16.7% higher than that of SHOX2 by the comparative analysis according to the subtype of lung cancer. 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 of FOXD3 and SHOX2 in alveolar lavage fluid specimens are shown in FIG. 6, the AUC value of FOXD3 is 0.791, and the AUC value of SHOX2 is 0.784.
By combining the examples 1-4, it can be fully demonstrated that FOXD3 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, detection primers and probes for the FOXD3 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, the same tumor has different diagnostic detection efficiencies, even the selected regions are not suitable to cause no diagnostic effect on the tumor at all, and Table 15 lists different regions of the FOXD3 gene selected in the experimental process of the invention.
TABLE 15 sequences of different regions of the FOXD3 gene
Designing different methylation primers and probes according to the region 1, the region 2 and the region 3, 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, group 10 are methylation primers and probes designed according to region 2; group 11, group 12, group 13, group 14 are methylation primers and probes designed based on region 3. All primers and probes were synthesized by England Shafer (Shanghai) trade Limited.
TABLE 16 primers and probes designed from different regions of FOXD3 Gene
The 14 primer probe combinations in table 16 were tested in 36 lung tissue samples, wherein 11 samples of normal tissue, 25 samples of cancer tissue, 4 samples of squamous carcinoma and 21 samples of adenocarcinoma were tested in 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
| In the area | Group of | Primer probe combination | Specificity of | Sensitivity of the reaction |
| Region 1 | Group 1 | FOXD3-F1,FOXD3-R1,FOXD3-P1 | 100％ | 84％ |
| Region 1 | Group 2 | FOXD3-F2,FOXD3-R2,FOXD3-P2 | 100％ | 68％ |
| Region 1 | Group 3 | FOXD3-F3,FOXD3-R3,FOXD3-P3 | 100％ | 80％ |
| Region 1 | Group 4 | FOXD3-F4,FOXD3-R4,FOXD3-P4 | 100％ | 72％ |
| Region 1 | Group 5 | FOXD3-F5,FOXD3-R5,FOXD3-P5 | 100％ | 84％ |
| Region 2 | Group 6 | FOXD3-F6,FOXD3-R6,FOXD3-P6 | 100％ | 40％ |
| Region 2 | Group 7 | FOXD3-F7,FOXD3-R7,FOXD3-P7 | 100％ | 40％ |
| Region 2 | Group 8 | FOXD3-F8,FOXD3-R8,FOXD3-P8 | 100％ | 52％ |
| Region 2 | Group 9 | FOXD3-F9,FOXD3-R9,FOXD3-P9 | 100％ | 48％ |
| Region 2 | Group 10 | FOXD3-F10,FOXD3-R10,FOXD3-P10 | 100％ | 68％ |
| Region 3 | Group 11 | FOXD3-F11,FOXD3-R11,FOXD3-P11 | 100％ | 64％ |
| Region 3 | Group 12 | FOXD3-F12,FOXD3-R12,FOXD3-P12 | 100％ | 52％ |
| Region 3 | Group 13 | FOXD3-F13,FOXD3-R13,FOXD3-P13 | 100％ | 36％ |
| Region 3 | Group 14 | FOXD3-F14,FOXD3-R14,FOXD3-P14 | 100％ | 40％ |
The results show that the groups 1, 2, 3, 4, 5, and 10 all have higher detection rates. However, no matter what kind of primers and probes designed by the present invention are used, the detection sensitivity of the region 1 can reach 68% at the lowest, 84% at the highest, and the detection rate is higher than that of the pairs of primers designed for the regions 2 and 3, so that the detection rate of the region 1 is significantly 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
From the results of 22 sputum specimens, group 1: the highest detection rate of FOXD3-F1, FOXD3-R1 and FOXD3-P1 reaches 73.3 percent.
Although the sensitivity of both group 1 and group 5 reached 84% in the tissue samples, the sensitivity of group 5 decreased greatly to 53.3% for the sputum test samples, which confirms that it is particularly difficult to design a detection reagent with high sensitivity for the sputum samples.
Finally, based on the detection results of each set of primer probes, the most preferred primer probe sequence is the combination of set 1: FOXD3-F1, FOXD3-R1, FOXD 3-P1.
Sequence listing
<110> Congliming Biotechnology, Inc. of Guangzhou City
<120> lung cancer diagnostic agent and kit based on FOXD3 gene
<160>71
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
cgtcgggatc ggattttttc 20
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tctcgactca aaaaccgacc g 21
<210>3
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
cggttttttg cgttaaggtt ag 22
<210>4
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cgttttatat ttttggcgag tagc 24
<210>5
<211>15
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
actccgccaa caccg 15
<210>6
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
cggcggcggc gcgggaggcg g 21
<210>7
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
ttggttttta cggttttcga c 21
<210>8
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
aaattctccg aaacgctcg 19
<210>9
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
attcggtgcg tataggtatc gcgc 24
<210>10
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
ttagtttcgg cgcgtagc 18
<210>11
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
cctaaaaccg acgcgatcta 20
<210>12
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
aaaacttacg atcgtctacc ctccg 25
<210>13
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
tttcggtagc gggtattgc 19
<210>14
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
aaaataacga cgaaccaacc g 21
<210>15
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
cgcgtttatg taggagtggt tgaggttc 28
<210>16
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
ttttggattg tgaatttgtg 20
<210>17
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
aaaacctact cctcccttaa a 21
<210>18
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
ttgtgtgttg ggtggtggtt 20
<210>19
<211>348
<212>DNA
<213>Homo sapiens
<400>19
agcccgacat ctagccggtc tccggcagga ccctgcaccg cgtcgggatc ggacccttcc 60
gctggggcgg cctcctgcgt caaggccagc aggaaccttc ctgtcgccct ccccggccgc 120
cgcttcgcct ccttcccgcc cccggaggtt gtgcaggcgc tatggtccgc ctggagggag 180
aaagccggcg gccggttcct gagccgagag cggccgcgga aaaatcctct gcctccgctg 240
gaaatcgata ttaggccggc gcgggcgcgg gacgtcgggg ccgcagccag taggttgtgc 300
acgtctcatc atttagctaa tcgagtcgaa aagtttctgt aagggccg 348
<210>20
<211>348
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
agttcgatat ttagtcggtt ttcggtagga ttttgtatcg cgtcgggatc ggattttttc 60
gttggggcgg ttttttgcgt taaggttagt aggaattttt ttgtcgtttt tttcggtcgt 120
cgtttcgttt ttttttcgtt ttcggaggtt gtgtaggcgt tatggttcgt ttggagggag 180
aaagtcggcg gtcggttttt gagtcgagag cggtcgcgga aaaatttttt gttttcgttg 240
gaaatcgata ttaggtcggc gcgggcgcgg gacgtcgggg tcgtagttag taggttgtgt 300
acgttttatt atttagttaa tcgagtcgaa aagtttttgt aagggtcg 348
<210>21
<211>476
<212>DNA
<213>Homo sapiens
<400>21
ctgagctccg tggcagcccc cgaacaccct catcgcccgc tgccccctcc ccgccgccgc 60
taccaacccc gaggagggat gaccctctcc ggcggcggca gcgccagcga catgtccggc 120
cagacggtgc tgacggccga ggacgtggac atcgatgtgg tgggcgaggg cgacgacggg 180
ctggaagaga aggacagcga cgcaggttgc gatagccccg cggggccgcc ggagctgcgc 240
ctggacgagg cggacgaggt gcccccggcg gcaccccatc acggacagcc tcagccgccc 300
caccagcagc ccctgacatt gcccaaggag gcggccggag ccggggccgg accggggggc 360
gacgtgggcg cgccggaggc ggacggctgc aagggcggtg ttggcggcga ggagggcggc 420
gcgagcggcg gcgggcctgg cgcgggcagc ggttcggcgg gaggcctggc cccgag 476
<210>22
<211>476
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
ttgagtttcg tggtagtttt cgaatatttt tatcgttcgt tgtttttttt tcgtcgtcgt 60
tattaatttc gaggagggat gatttttttc ggcggcggta gcgttagcga tatgttcggt 120
tagacggtgt tgacggtcga ggacgtggat atcgatgtgg tgggcgaggg cgacgacggg 180
ttggaagaga aggatagcga cgtaggttgc gatagtttcg cggggtcgtc ggagttgcgt 240
ttggacgagg cggacgaggt gttttcggcg gtattttatt acggatagtt ttagtcgttt 300
tattagtagt ttttgatatt gtttaaggag gcggtcggag tcggggtcgg atcggggggc 360
gacgtgggcg cgtcggaggc ggacggttgt aagggcggtg ttggcggcga ggagggcggc 420
gcgagcggcg gcgggtttgg cgcgggtagc ggttcggcgg gaggtttggt ttcgag 476
<210>23
<211>780
<212>DNA
<213>Homo sapiens
<400>23
cggaagggag agggggcggg gaaggcaggg cagcgacagt cgcacagtcc cgcggacgct 60
cccaggccca cgccctgact cgctcacacc cacccacact cacacccacc cgctccctgg 120
gccccagggc ccggatccag cctgggtggg ggggtctccg ggcgggccgc agcgccctcc 180
gtgccccggg gatgctggcg cacagtgcgg agcggagttg cgcgtctctc gtccctttgt 240
tgacaattcc ctgaaccaac ttgagtttgg ccggctcggc cgcggccctg acgtcacgca 300
cggtcacgtg gccccgcctc ccgctggatc tttaagtaga aagtaatcta tcaggccagt 360
ccttaaaacg ggactttcga ctaccggggc ttcggcgtcc ctgacaccca gccccctgcc 420
cccccgctac tgtccctgcc cgcgccctcc cgagctgctc ggcgcccggc gtcccgcgcc 480
cgcctggacc gctcctgcgc cccacgccag ggccagaggc cgaggaaggc gggctaagtg 540
agggggcgcg gcgtggagaa ccgccggggc cgggagcggt agcgagcgcc tagtaccgag 600
cgccagggac ggcaggagtt cgcggagcgc ggccgctggg ggcggacggc agagcccgcg 660
ccacgcgatg cggggccgcc gagtgtgagc tgagcccagc gggccccaag ccacctgcgg 720
ccccctcccc tctccctgcc ccccatcttt cgggggcact caaaccctct tcccctgagc 780
<210>24
<211>780
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
cggaagggag agggggcggg gaaggtaggg tagcgatagt cgtatagttt cgcggacgtt 60
tttaggttta cgttttgatt cgtttatatt tatttatatt tatatttatt cgttttttgg 120
gttttagggt tcggatttag tttgggtggg ggggttttcg ggcgggtcgt agcgtttttc 180
gtgtttcggg gatgttggcg tatagtgcgg agcggagttg cgcgtttttc gtttttttgt 240
tgataatttt ttgaattaat ttgagtttgg tcggttcggt cgcggttttg acgttacgta 300
cggttacgtg gtttcgtttt tcgttggatt tttaagtaga aagtaattta ttaggttagt 360
ttttaaaacg ggattttcga ttatcggggt ttcggcgttt ttgatattta gttttttgtt 420
ttttcgttat tgtttttgtt cgcgtttttt cgagttgttc ggcgttcggc gtttcgcgtt 480
cgtttggatc gtttttgcgt tttacgttag ggttagaggt cgaggaaggc gggttaagtg 540
agggggcgcg gcgtggagaa tcgtcggggt cgggagcggt agcgagcgtt tagtatcgag 600
cgttagggac ggtaggagtt cgcggagcgc ggtcgttggg ggcggacggt agagttcgcg 660
ttacgcgatg cggggtcgtc gagtgtgagt tgagtttagc gggttttaag ttatttgcgg 720
tttttttttt tttttttgtt ttttattttt cgggggtatt taaatttttt ttttttgagt 780
<210>25
<211>972
<212>DNA
<213>Homo sapiens
<400>25
agcccggggc ggggtggggc tggagctcct gtctcttggc cagctgaatg gaggcccagt 60
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>26
<211>972
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
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>27
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
ggcggtcggt ttttgagtc 19
<210>28
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
ctactaacta cgaccccgac g 21
<210>29
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
cgttggaaat cgatattagg tcggc 25
<210>30
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
cggttttttg cgttaaggtt ag 22
<210>31
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
acgcctacac aacctccg 18
<210>32
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
ttttcggtcg tcgtttcgtt t 21
<210>33
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
gttgtgtagg cgttatggtt cg 22
<210>34
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
cgacctaata tcgatttcca acg 23
<210>35
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
cggtttttga gtcgagagcg gtcg 24
<210>36
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
ttcgatattt agtcggtttt cg 22
<210>37
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
aaaattccta ctaaccttaa cg 22
<210>38
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
cgcgtcggga tcggattttt 20
<210>39
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
aggatagcga cgtaggttgc 20
<210>40
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
atccgtaata aaataccgcc g 21
<210>41
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
tcgtcggagt tgcgtttgga c 21
<210>42
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
tcgaggacgt ggatatcg 18
<210>43
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
gaaactatcg caacctacg 19
<210>44
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
cgggttggaa gagaaggata gcga 24
<210>45
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>45
gttggaagag aaggatagcg 20
<210>46
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
gactaaaact atccgtaata aaataccg 28
<210>47
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
cgatagtttc gcggggtcgt cg 22
<210>48
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
ttaaggaggc ggtcggagtc 20
<210>49
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
ctcgaaacca aacctccc 18
<210>50
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
gcggtgttgg cggcgaggag 20
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gagtttcgtg gtagttttcg 20
<210>52
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
atcgatatcc acgtcctc 18
<210>53
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
cgtcaacacc gtctaaccga a 21
<210>54
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
agcgagcgtt tagtatcg 18
<210>55
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>55
gctaaactca actcacactc g 21
<210>56
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>56
cggacggtag agttcgcgtt acg 23
<210>57
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>57
ttcgattatc ggggtttcg 19
<210>58
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>58
aaaacgatcc aaacgaacg 19
<210>59
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>59
cgagttgttc ggcgttcggc 20
<210>60
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>60
cgcggttttg acgttacg 18
<210>61
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>61
ccgaaacccc gataatcg 18
<210>62
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>62
cggttacgtg gtttcgtttt tcgt 24
<210>63
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>63
cgagttgttc ggcgttcg 18
<210>64
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>64
ctcgacctct aaccctaacg 20
<210>65
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>65
cgcgttcgtt tggatcgttt ttgc 24
<210>66
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>66
ttttggattt aaggggaaga taaa 24
<210>67
<211>27
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>67
tttttccttc tctacatctt tctacct 27
<210>68
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>68
aagggaaatt gagaaatgagagaaggga 28
<210>69
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>69
tttaaagggt tcgtcgttta agtc 24
<210>70
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>70
aaacgattac tttcgcccg 19
<210>71
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>71
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 or SEQ ID NO: 23; 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: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ id no: 36 and SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO: 64; preferably, the primer is selected from SEQ id no: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, seq id NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 51 and SEQ ID NO: 52; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37; 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: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 41. SEQ ID NO: 44. SEQ ID NO: 47. SEQ ID NO: 50. SEQ ID NO: 53. SEQ ID NO: 56. SEQ ID NO: 59. SEQ ID NO: 62. SEQ ID NO: 65; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 53; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. any one of SEQ ID NOs: 38; 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 is characterized in that the reagent contains a detection reagent for FOXD3 gene methylation.
6. The detection/diagnostic reagent according to claim 5, wherein the detection reagent for methylation of FOXD3 gene detects the sequence of FOXD3 gene modified with bisulfite or hydrazine;
preferably, it is bisulphite modified.
7. The detection/diagnostic reagent according to claim 5, wherein the detection region of the reagent against FOXD3 gene is as shown in SEQ ID NO: 19. SEQ ID NO: 21 or SEQ ID NO: 23 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: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ id no: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO: 64; preferably, the primer is selected from SEQ ID NO: 1 and SEQ ID NO: 2, SEQ id no: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, seq id NO: 36 and SEQ ID NO: 37, SEQ ID NO: 51 and SEQ ID NO: 52; more preferably, the primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 36 and SEQ ID NO: 37; 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 as shown in SEQ ID NO: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 41. SEQ ID NO: 44. SEQ ID NO: 47. SEQ ID NO: 50. SEQ ID NO: 53. SEQ ID NO: 56. SEQ ID NO: 59. SEQ ID NO: 62. SEQ ID NO: 65; preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38. SEQ ID NO: 53; more preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NOs: 3. SEQ ID NO: 29. SEQ ID NO: 32. SEQ ID NO: 35. SEQ ID NO: 38; 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 reference gene is β -actin, COL2A 1;
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: 66. SEQ ID NO: 67 and the primer set shown in SEQ ID NO: 68.
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, large cell carcinoma.
16. A method for detecting DNA methylation of FOXD3 gene, which comprises the following steps:
(1) processing a sample to be detected by bisulfite or hydrazine to obtain a modified sample to be detected;
(2) carrying out the FOXD3 gene methylation detection on the modified sample to be detected in the step (1) by using the reagent or the kit as described in 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 means for detecting DNA methylation of the FOXD3 gene, and,
b. a result judgment system;
preferably, the DNA methylation detection means of FOXD3 gene comprises the reagent or the kit according to any one of claims 5 to 14;
preferably, the result judging means is used for outputting the risk of lung cancer and/or the type of lung cancer according to the DNA methylation result of the FOXD3 gene detected by the detection system;
more preferably, the disease risk is that the methylation results of the to-be-detected sample and the normal sample are compared according to the result, and when the methylation of the to-be-detected sample and the methylation of the normal sample have a significant difference or a very significant difference, the result judges that the disease risk of the to-be-detected sample is high.
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