CN111363811B

CN111363811B - Lung cancer diagnostic agent and kit based on FOXD3 gene

Info

Publication number: CN111363811B
Application number: CN201811591944.5A
Authority: CN
Inventors: 李仕良; 牛智通; 赵荣淞; 黄龙武; 吴幽治; 邱浩纯; 邹鸿志
Original assignee: Guangzhou Kangliming Biotechnology Co ltd
Current assignee: Guangzhou Kangliming Biotechnology Co ltd
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2024-02-02
Anticipated expiration: 2038-12-25
Also published as: CN111363811A

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 methylation of a FOXD3 gene and is used for detecting a sequence of the FOXD3 gene modified by bisulfite or hydrazine salt. The reagent of the invention is proved by experiments to be capable of detecting and diagnosing lung cancer with high sensitivity and high specificity, and has extremely high clinical application value.

Description

Lung cancer diagnostic agent and kit based on FOXD3 gene

Technical Field

The invention belongs to the field of gene diagnosis, and in particular relates to a detection/diagnosis reagent for methylation of human FOXD3 gene 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. According to the pathological type, it can be classified into: 1) Small cell lung cancer (small cell lung cancer, SCLC): lung cancer of a special pathological type has obvious distant metastasis tendency and 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 is a certain difference in biological behavior and clinical course of disease. The generation positions can be further divided into: 1) Central lung cancer (central lung cancer): lung cancer growing in and above the bronchus opening of the lung segment; 2) Surrounding lung cancer (peripheral lung cancer): lung cancer growing far beyond the bronchus opening in the lung segment.

In recent years, the incidence and mortality of Chinese lung cancer are increased year by year due to the influence of factors such as aging population, atmospheric pollution, smoking and the like, and about 7 people in China are diagnosed with cancer according to 2017 Chinese tumor registration annual report issued by the national cancer center, wherein the incidence and mortality of the lung cancer are the first. China has become the country with the largest number of lung cancer in the world, and experts predict that the number of lung cancer in the 2025 China will reach 100 ten thousand. And from epidemiological studies, it has been shown that: smoking is an important factor in causing lung cancer. About 80% -90% of lung cancer worldwide can be attributed to smoking. The relative risk of lung cancer in 1-19 and over 20 persons who smoke daily is 4.27 and 8.61 compared to non-smokers, and the relative risk of death from lung cancer in 1-19 and over 20 persons who smoke daily over a long period is 6.14 and 10.73 compared to non-smokers. Although the technology for treating lung cancer is very different day by day, the survival rate of 5 years is only increased from 4% to about 12%, the existing antitumor drugs still only play a role in relieving the illness state, the progression-free survival time of patients is only prolonged by 3 months to 5 months on average, but for patients with stage I lung cancer, the survival rate of 5 years after operation is as high as about 60% -70%. Therefore, early diagnosis and early operation of lung cancer are one of the most effective methods for improving the 5-year survival rate and reducing the death rate of lung cancer.

The existing clinical auxiliary diagnosis of lung cancer mainly comprises the following steps, but all the lung cancer cannot be completely found and diagnosed early:

(1) Biochemical examination of blood: for primary lung cancer, there is no specific biochemical blood examination at present. Elevated blood alkaline phosphatase or blood calcium in lung cancer patients considers the potential for bone metastasis, elevated blood alkaline phosphatase, glutamic-oxaloacetic transaminase, lactate dehydrogenase or bilirubin considers the potential for liver metastasis.

(2) Tumor marker examination: 1) CEA:30% -70% of lung cancer patients have abnormally high levels of CEA in serum, but are mainly seen in more advanced lung cancer patients. At present, CEA in serum is mainly used for estimating lung cancer prognosis and monitoring treatment process. 2) NSE: is the first-choice marker of the small cell lung cancer, is used for diagnosing and monitoring treatment response of the small cell lung cancer, and has different reference values according to different detection methods and different using reagents. 3) CYFRA21-1: is the first choice marker of small cell lung cancer, has the sensitivity to lung squamous cancer diagnosis of 60 percent and has different reference values according to different detection methods and using reagents.

(3) Imaging examination: 1) Chest X-ray examination: chest orthoses and lateral panels should be included. In primary hospitals, chest positive side tablets are still the most basic and preferred image diagnosis method in the initial diagnosis of lung cancer. Once lung cancer is diagnosed or suspected, a chest CT examination is performed. 2) CT examination: chest CT is the most commonly used and most important examination method for lung cancer diagnosis and differential diagnosis, stage and treatment follow-up diagnosis. Lung puncture biopsy under CT guidance is an important diagnostic technique for lung cancer, and conditional hospitals can be used for diagnosis of difficult-to-qualify lung lesions, and cases where clinical diagnosis of lung cancer requires cytology and histological confirmation and other methods are difficult to obtain. In recent years, multi-layer helical CT and Low Dose CT (LDCT) are effective screening tools for the discovery of early lung cancer and reduction of mortality, and full-american national lung cancer screening studies (NLST) have shown that LDCT can reduce mortality of 20% lung cancer compared to chest X-ray screening. Low-dose helical CT is recommended as an important means for early lung cancer screening, but has many human factors and a very high false positive rate. 3) Ultrasonic inspection: the method is mainly used for finding out important organs of the abdomen, whether metastasis exists in the peritoneal cavity and the retroperitoneal lymph nodes, and also used for checking cervical lymph nodes. For the lung internal lesions or chest wall lesions which are close to the chest wall, the cyst solidity of the lung internal lesions or chest wall lesions can be identified, and puncture biopsy under ultrasonic guidance can be performed; ultrasound is also commonly used for chest water extraction positioning. 4) Bone scanning: has higher sensitivity to the detection of the bone metastasis of the lung cancer, but has certain false positive rate. Can be used for the following cases: preoperative examination of lung cancer; patients with localized symptoms.

(4) Other examinations: 1) Sputum cytology examination: the existing noninvasive diagnosis method for lung cancer is simple and convenient, the positive rate can be improved by about 60% by continuous smear examination, and the noninvasive diagnosis method is a conventional diagnosis method for suspected lung cancer cases. 2) Fiberoptic bronchoscopy: one of the most important means in lung cancer diagnosis plays an important role in qualitative positioning diagnosis of lung cancer and selection of surgical schemes. Routine examination items necessary for patients undergoing surgical treatment. While transbronchoscopic needle biopsy (TBNA) is beneficial for pre-treatment staging, due to the greater technical difficulty and risk, the patient should go to a higher-level hospital for further examination. 3) Other: such as percutaneous lung puncture biopsy, thoracoscopic biopsy, mediastinal biopsy, hydrothorax cytology, etc., can be used separately to assist diagnosis according to existing conditions in the presence of indications.

In clinical practice work, the success and failure of any lung cancer screening project is determined by the identification of high risk groups, and a risk prediction model integrating multiple high risk factors is known as one of methods for identifying the high risk groups of lung cancer. Along with the rapid development of technology, tumor marker detection becomes a new field of tumor diagnosis and treatment after imaging diagnosis and pathological diagnosis, and can have great influence on tumor diagnosis, detection and treatment. The tumor markers can be detected in body fluid or tissues, and can reflect the existence, differentiation degree, prognosis estimation, personalized medicine application, treatment effect and the like of tumors. Early lung cancer patients have no obvious symptoms, are difficult to be perceived by doctors and patients, and have no obvious specific markers on blood or biochemical projects, so that early detection and early diagnosis are difficult to carry out by a conventional diagnosis method, and the early diagnosis of lung cancer, especially the screening of large-scale application groups, is difficult.

More and more studies have shown that two broad classes of mechanisms are involved in the tumor formation process. One is the formation of mutations by DNA nucleotide sequence changes, i.e., genetic mechanisms. Tumors have been demonstrated in the field of molecular biology as a genetic disease. The other is the epigenetic (epigenetics) mechanism, i.e., independent of DNA sequence changes leading to changes in gene expression levels, which play an increasing role in the tumor formation process. The two mechanisms of genetics and epigenetic are crossed mutually, and the formation of tumors is promoted together. Abnormal methylation of genes can occur early in tumorigenesis and the degree of abnormal methylation of genes increases during the progressive progression of tumors. The genome of 98 common human primary tumors was analyzed and found to have at least 600 abnormally methylated CpG islands per tumor.

Many studies have shown that promoter aberrant methylation is a frequent early event in the development of many tumors, and thus the methylation state of tumor-associated genes is an early sensitive indicator of tumorigenesis and is considered to be a promising tumor molecular biomarker (biomarker). More importantly, cancerous cells can release DNA into the peripheral blood. Nanogram-scale free DNA is present in normal human peripheral blood. It was found that abnormal methylation of the promoter of tumor-associated genes present in tumor tissue can be detected also in peripheral blood plasma/serum, tumor-associated body fluids (e.g., saliva, sputum, etc.). These biological samples are relatively easy to obtain, and DNA in the biological samples can be sensitively detected after a large amount of DNA is amplified by PCR technology, so that the methylation state of the promoter region of some tumor related genes can be detected, and the biological samples can provide valuable information for early diagnosis of tumors. Detection of abnormal methylation of promoters has further advantages over other types of tumor molecular markers. The abnormal methylation areas of the promoter of a certain gene are the same in different types of tumors, so that the detection is convenient; in addition, abnormal methylation is a positive signal compared to markers such as allele deletions, and is readily distinguishable from negative background in normal tissue. Esteler et al detected abnormal methylation status of promoter regions of p16, DAPK, GSTP1, MGM T and other genes in tumor tissues and serum of 22 cases of non-small cell lung cancer (NSCLC), and found that 68% (15/22) of tumor tissues had promoter methylation of at least one gene; in 15 tissue positive cases, 11 cases were detected with the presence of abnormal methylation of the promoter in the serum. In addition, many researchers have detected promoter methylation of certain tumor-associated genes from tumor tissues and serum of patients with liver cancer, head and neck cancer, esophageal cancer and colon cancer, respectively. Palmesano et al examined abnormal methylation of p16 and MGMT promoters in tumor tissues and sputum of 21 lung squamous carcinoma patients, and found that abnormal methylation of promoter regions of one or two genes was present in all sputum samples. Wherein 10 cases of sputum samples are collected after tumor diagnosis; another 11 sputum samples were from high risk groups with a history of smoking or other exposure, and these 11 subjects were diagnosed with lung cancer at 5-35 months later. While these 21 sputum samples were positive by sputum cell morphology only in 4 cases. Thus, detection of abnormal methylation in the promoter region of a gene is a very sensitive indicator. The results of these studies indicate that: detection of DNA methylation can be used as a means of early signs and risk assessment of cancer.

Early lung cancer patients often have no obvious symptoms and signs, are easily ignored by patients, and are rarely treated by symptoms. The clinical routine chest X-ray and sputum shedding cytology examination can not meet the requirement of screening early lung cancer far away, and the clinical routine chest X-ray and sputum shedding cytology examination can not be proved to reduce the death rate. The screening omission rate of chest X-ray can reach 54% -90%, and sputum shedding cytology is low in cost and does not need expensive equipment, but the omission rate is high, human factor interference of result judgment is more, and multiple times of detection are needed. Low-dose helical CT is recommended as an important means for early lung cancer screening, but has many human factors and a very high false positive rate.

Currently, there are many studies to detect the methylation state of cells or DNA in blood, sputum, alveolar lavage fluid in an effort to find markers for early diagnosis of lung cancer. Although some genes have been found in the prior art to have DNA methylation associated with lung cancer, there is still a need in the art to further study related genes that can be practically used for lung cancer diagnosis and to develop detection reagents with higher detection accuracy.

Disclosure of Invention

The invention aims to provide an application of a nucleic acid fragment of FOXD3 gene in preparing a tumor detection/diagnosis reagent or kit.

Another object of the invention is to provide an application of the primer pair in preparing a tumor detection/diagnosis reagent or kit.

It is another object of the present invention to provide the use of a probe for the preparation of a tumor detection/diagnostic reagent or kit.

It is a further object of the present invention to provide a reagent, kit and method for diagnosing methylation of the human FOXD3 gene.

The invention further aims to provide a lung cancer detection/diagnosis reagent and a kit with strong specificity and high sensitivity.

It is a further object of the present invention to provide a lung cancer detection/diagnostic reagent and kit that have a wide range of application to lung cancer.

It is a further object of the present invention to provide a convenient to use lung cancer detection/diagnostic reagent and kit.

The above object of the present invention is achieved by the following technical means:

in one aspect, the invention provides the use of a nucleic acid fragment in the preparation of a tumor detection/diagnostic reagent or kit. Wherein the nucleic acid fragment is derived from the FOXD3 gene, and specifically from the fact that the FOXD3 gene is located on chromosome 1: 63788730-63790797 (with GRCh37 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 invention, the nucleic acid fragment is selected from the group consisting of SEQ ID NO:19.

In another aspect, the invention also provides a primer pair 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, any one shown in fig. 64; 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, SEQ ID NO:51 and SEQ ID NO:52, a primer pair shown in seq id no; 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. As a preferred embodiment of the invention, the primer is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2.

In another aspect, the 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, any one of which is shown in phantom; 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 invention, the 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 a kit.

In another aspect, the present invention provides a tumor detection/diagnosis reagent comprising a methylation detection reagent of the FOXD3 gene.

FOXD3 gene (forkhead box D3), fork D3 gene, which belongs to the fork family of transcription factors. It has been reported that FOXD3 is expressed mainly in embryonic stem cells or pluripotent stem cells, has a close relationship with embryonic development and multipotency of stem cells, and is a marker member of tumor stem cells. In addition, FOXD3 has been reported to have an effect of inhibiting cancer.

Methylation occurs when cytosine is treated with bisulfite or hydrazine salt to form uracil, which is recognized as thymine when PCR amplification is performed because uracil is similar to thymine, and methylation is not performed when cytosine is changed to thymine (C to T) in the PCR amplification sequence, and methylation is not performed when cytosine is changed to cytosine (C). The technique for PCR detection of methylated genes is typically methylation specific PCR (Methylmion Specific PCR, MSP), and PCR amplification is performed by designing primers for the treated methylated fragments (i.e., unchanged C in the fragments), if amplified, this indicates methylation, and if not amplified, this indicates methylation.

Further, the methylation detection reagent of the FOXD3 gene detects the sequence of the FOXD3 gene modified with bisulfite or hydrazine salt.

As an exemplary embodiment, the sequence of the FOXD3 gene modified with bisulfite was detected.

In a preferred embodiment, the detection region of the FOXD3 gene to which the reagent is directed is specifically derived from the FOXD3 gene located on chromosome 1: 63788730-63790797 (with GRCh37 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: indicated at 23; more preferably as set forth in SEQ ID NO:19 as detection areas.

The inventor finds that the selection of the detection region of the FOXD3 gene can have an influence on the detection efficiency of tumors. As to the FOXD3 gene of the present invention, the inventors performed RRBS methylation sequencing to obtain the FOXD3 gene itself and the methylation of the base within 5000bp upstream of the gene. Through preliminary analysis, methylation conditions of different areas of the gene can be obviously found to be different in lung cancer and non-lung cancer control groups, so that the selection of different areas to design primers and probes has important influence on diagnosis of lung cancer and non-lung cancer. As shown in FIG. 1, the left square region is designed with primers and probes that are better for detection of both adenocarcinoma and squamous carcinoma, while the right square region is better for detection of adenocarcinoma than squamous carcinoma. Although all were in the vicinity of the FOXD3 gene, it was apparent that the effect of designing the primer probe was better by selecting the left region.

The reagent of the present invention contains an amplification primer.

As a preferred embodiment, the primer is shown 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, any one shown in fig. 64; 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, SEQ ID NO:51 and SEQ ID NO:52, a primer pair shown in seq id no; 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, a primer pair shown in seq id no; most preferably, the primer is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, and a primer pair shown in the following.

The primer is used for amplifying a specific region of the FOXD3 gene. It is well known in the art that the successful design of primers is critical to PCR. Compared with the general PCR, in the methylation detection of genes, the design influence of the primer is more critical, because the methylation reaction promotes the conversion of C in a DNA chain into U, so that the GC content is reduced, long continuous T appears in the sequence after the PCR reaction, the breakage of the DNA chain is easy to cause, and the primer with proper Tm value and stability is difficult to select; on the other hand, in order to distinguish between sulfured and non-sulfured and incompletely treated DNA, a sufficient number of "C" primers are required, which all increase the difficulty in selecting stable primers. Thus, in DNA methylation detection, the choice of amplified fragment for which the primer is directed, such as the length and position of the amplified fragment, and the choice of primer, among others, all affect the sensitivity and specificity of the detection. The inventor also finds that different amplification target fragments and primer pairs have different detection effects through experiments. Many times, it has been found that certain genes or nucleic acid fragments have expression differences between tumors and non-tumors, however, their distance translates into markers of tumors, which remain a long distance in clinic. The most important reasons are that the detection sensitivity and the specificity of the potential tumor markers are difficult to meet detection requirements due to the limitation of detection reagents, or the detection method is complex in operation and high in cost and is difficult to be applied to large-scale clinic.

As an alternative embodiment, the kit of the present invention further comprises a probe. As a preferred embodiment, 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, any one of which is shown in phantom; 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 of the SEQ ID NOs: 38, a step of carrying out the process; most preferably, the nucleic acid probe is selected from the group consisting of SEQ ID NO:3. as a preferred embodiment, the kit of the invention contains both primers and probes, preferably, the primers are as shown in SEQ ID NO:1 and SEQ ID NO:2, and simultaneously, the probe is shown as SEQ ID NO:3.

As an alternative embodiment, the reagent contains a detection reagent comprising a reference gene. As a preferred embodiment, the internal reference is beta-actin or COL2A1. As a preferred embodiment, the detection reagent for the reference gene is a primer or a probe for the reference gene. In a more preferred embodiment, the detection reagent of the reference gene is SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO: 18.

As a preferred embodiment, the reagent further comprises bisulphite, bisulfite or hydrazine salt to modify the FOXD3 gene, although this may not be included.

As a preferred embodiment, the reagent contains a DNA polymerase, dNTPs, mg ²⁺ One or more of ion and buffer solution, preferably including DNA polymerase, dNTPs, mg ²⁺ The PCR reaction system of the ion and the buffer solution is used for amplifying the modified FOXD3 gene.

The sample to be tested by the test/diagnostic reagent of the present invention may be selected from alveolar lavage, tissue, hydrothorax, sputum, blood, serum, plasma, urine, prostatic fluid or stool. As a preferred embodiment, the sample is selected from 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 against a tumor selected from lung cancer tissue and paracancerous normal tissue (or benign lung disease tissue).

In another aspect, the invention also provides a kit comprising the above detection/diagnostic reagent.

In another aspect, the present invention also provides a method for detecting DNA methylation of FOXD3 gene, comprising the steps of:

(1) Carrying out bisulphite or hydrazine salt treatment on the sample to be detected to obtain a modified sample to be detected;

(2) And (3) detecting the methylation of the FOXD3 gene of the modified sample to be tested in the step (1) by using the reagent or the kit.

In a preferred embodiment, in step (2), the detection is performed using real-time fluorescence quantitative methylation-specific polymerase chain reaction.

On the other hand, the invention also provides a lung cancer detection/diagnosis system. The system comprises:

(1) DNA methylation detecting means of FOXD3 gene, and,

(2) A result judging system;

preferably, the means for detecting DNA methylation of the FOXD3 gene comprises the reagent or 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 risk of illness is that the methylation results of the sample to be tested and the normal sample are compared by the result judging component, and when the methylation of the sample to be tested and the methylation of the normal sample have a significant difference or a very significant difference, the result judging component outputs that the risk of illness of the sample to be tested is high.

As a preferred embodiment, if the FOXD3 gene DNA methylation is positive, the provider of the sample to be tested is indicated to be a high risk or lung cancer patient. As a preferred embodiment, the positive means that the obtained detection result is compared with the detection result of the normal sample, and when the amplification result of the sample to be tested is significantly or extremely significantly different from that of the normal sample, the donor of the sample to be tested is positive.

The invention has the beneficial effects that:

although, in the prior art, FOXD3 gene methylation has been reported as one of tumor markers for lung cancer. However, the reports of tumor markers of lung cancer are numerous, and the tumor markers can be truly used in clinic, but are few as markers for detecting lung cancer. The detection reagent for the FOXD3 gene has high sensitivity and specificity for lung cancer, and is hopeful to be used as a tumor marker for clinical diagnosis of lung cancer.

The FOXD3 gene has very high sensitivity and specificity to lung cancer, and the detection area and the detection reagent based on the methylation of the FOXD3 gene optimized by the invention can enable the marker to reach the lung cancer detection rate with the specificity of 95% and the sensitivity of 77% in a tissue sample. Wherein the detection rate of squamous carcinoma reaches 91.3%; among the most difficult adenocarcinomas to detect, the detection rate of adenocarcinomas was 73.1% and all large cell carcinomas were detected.

At present, lung cancer detection kits based on SHOX2 genes are available on the market. In one embodiment of the present invention, the detection effect of FOXD3 is superior to that of the SHOX2 gene, regardless of whether the lung cancer is compared as a whole or the subtype of lung cancer is compared. In particular, the detection effect on adenocarcinoma was 22.2% for FOXD3 and 0% for SHOX 2. In another embodiment of the invention, lung cancer as a whole is compared and analyzed, 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 SHOX2, according to the comparison and analysis of the subtype of lung cancer. In particular to the detection effect of the adenocarcinoma, the sensitivity of the kit reaches 54.5 percent, which is far higher than 36.4 percent of SHOX 2.

In addition, the detection marker has very high specificity and sensitivity for different types of lung cancer, including squamous carcinoma, adenocarcinoma and large cell carcinoma in small cell lung cancer and non-small cell lung cancer, and has wide application range and can be basically used as tumor markers of all lung cancers. However, the existing clinical lung cancer markers are generally only suitable for detecting one type of lung cancer, such as NSE for diagnosing small cell lung cancer and monitoring treatment response, and CYFRA21-1 is a first-choice marker of non-small cell lung cancer.

The detection reagent and the method containing the FOXD3 gene can conveniently and accurately judge lung cancer and lung benign disease patients, and the detection method of the gene is hopefully converted into a gene detection kit and is used for screening, clinical detection and prognosis monitoring of lung cancer.

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 for detecting lung cancer in clinical tissue specimen

FIG. 5 ROC curve of FOXD3 and SHOX2 genes detected in sputum samples

FIG. 6 ROC curve of FOXD3 and SHOX2 genes detected in lavage fluid samples

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

Example 1: detection of selection of target genes

Methylated DNA has obvious advantages as a detection target, and compared with protein markers, DNA can be amplified and is easy to detect; compared with mutation markers, the methylation sites of DNA are all located at specific sites of the gene, generally in the promoter region, so that detection becomes easier and more convenient. In order to complete the invention, the inventor screens hundreds of genes, selects better FOXD3, SIX3, PCDHGA12, HOXD8 and GATA3 as candidate detection genes, takes beta-actin gene as internal reference genes, researches the distribution situation of methylation sites of each gene, and designs detection primer probes for detection. The detection primer probes of each gene are as follows:

The detection primers and probes of FOXD3 are:

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

The detection primers and probes of the SIX3 are as follows:

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 PCDHGA12 are:

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 the 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 for 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

The detection primers and probes of the beta-actin are as follows:

SEQ ID NO: 16. beta-actin primer F: TTTTGGATTGTGAATTTGTG

SEQ ID NO: 17. beta-actin primer R: AAAACCTACTCCTCCCTTAAA

SEQ ID NO: 18. beta-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1

Sample information: the lung paraffin tissue samples are 36 in total, wherein 11 lung tissue samples serving as a control comprise 4 paracancerous normal tissues and 7 benign lung disease tissues; the cancer tissue samples were 25 cases, including 4 cases of squamous cell carcinoma and 21 cases of adenocarcinoma.

The test process comprises the following steps:

a. collecting the operation cutting specimens of lung cancer or benign lung, embedding with paraffin, staining pathological tissue section, and identifying the tissue type and purity. Tissue sections were used to extract DNA using the Magen DNA extraction kit (HiPure FFPE DNA Kit, D3126-03).

b. Bisulphite modification of DNA was performed using the ZYMO reserve organism company DNA transformation kit (EZ DNA Methylation Kit, D5002).

c. Amplification detection systems and detection systems are shown in tables 1-2:

TABLE 1 liquid formulation

TABLE 2 PCR reaction procedure

d. And calculating the methylation copy number of the genes in the sample by using a standard curve, judging the methylation degree of two groups of tissues by adopting the ratio=copy number/ACTB copy number of 100, and finally selecting a threshold value as a standard for judging the cancer group and the control group, wherein the converted ratio exceeds a set threshold value to be positive, and the ratio is equal to or smaller than the set threshold value to be negative. According to this standard, the detection results of 36 tissue specimens are shown in tables 3 to 4:

TABLE 3 detection results in tissue

/>

Note that: "+" indicates that the detection result is a positive sample; "-" indicates that the detection result is a negative sample

TABLE 4 statistical results

All specimens of the control group were judged to be FOXD3 methylation negative, whereas of the 25 lung cancer specimens, 21 were judged to be positive, and only 3 lung adenocarcinomas and 1 lung squamous carcinoma were judged to be negative. The detection sensitivity of the FOXD3 gene was 84%, the specificity was 100%, and the positive detection rate was far higher than that of the other 4 genes. The FOXD3 gene is indicated to have important significance in clinical detection and diagnosis of lung cancer.

The ROC curves of five candidate genes FOXD3, SIX3, PCDHGA12, HOXD8 and GATA3 for detecting lung cancer are shown in FIG. 2, and the area under the ROC curve for detecting FOXD3 gene is 0.916.

Therefore, the inventors selected FOXD3 as a candidate gene, optimized the detection conditions, widened the detection range, and collected 122 lung cancer tissue samples and their corresponding paracancerous tissues as non-lung cancer controls, together with 244 lung paraffin tissue samples. Among them, 23 squamous cell carcinomas, 93 adenocarcinomas, 3 large cell carcinomas, 1 mixed carcinoma, 2 have not been clearly diagnosed with lung cancer types. See in particular example 2.

Example 2: detection of FOXD3 gene in clinical specimen

a. The detection primer probe is as follows:

the detection primers and probes of FOXD3 are:

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

The detection primers and probes of the beta-actin are as follows:

SEQ ID NO: 16. beta-actin primer F: TTTTGGATTGTGAATTTGTG

SEQ ID NO: 17. beta-actin primer R: AAAACCTACTCCTCCCTTAAA

SEQ ID NO: 18. beta-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1

b. Sample information: 122 as a non-lung cancer control, 244 cases of lung paraffin tissue specimens were pooled for lung cancer tissue samples and corresponding paracancerous tissues. Among them, 23 squamous cell carcinomas, 93 adenocarcinomas, 3 large cell carcinomas, 1 mixed carcinoma, 2 have not been clearly diagnosed with lung cancer types.

c. Collecting the operation excision specimen of the lung cancer, separating the cancer tissue and the adjacent cancer tissue, embedding the cancer tissue and the adjacent cancer tissue with paraffin, staining pathological tissue sections, and identifying the tissue type and the purity of the pathological tissue sections. Tissue sections were used to extract DNA using the Magen DNA extraction kit (HiPure FFPE DNA Kit, D3126-03).

d. Bisulphite modification of DNA was performed using the ZYMO reserve organism company DNA transformation kit (EZ DNA Methylation Kit, D5002).

e. Amplification detection systems and detection systems are shown in tables 5-6:

TABLE 5 liquid formulation

TABLE 6 PCR reaction procedure

f. Detection result

Calculating methylation copy number of FOXD3 gene in a sample by using a standard curve, judging methylation degree of two groups of tissues by adopting the ratio of FOXD3 copy number/ACTB copy number of 100, and finally selecting a value of '13.8' as a standard for judging cancer groups and a control group, wherein the converted ratio exceeds '13.8', can be judged positive, and is equal to or smaller than '13.8', and can be judged negative. According to this standard, 244 tissue specimens were tested as follows:

TABLE 7 detection results

/>

TABLE 8 statistical results

As a result, 122 cases were lung cancer specimens, and 94 cases of methylated DNA positive were detected with a specificity of 95.0% and a sensitivity of 77.0% among 122 cases. Wherein the detection rate of squamous carcinoma reaches 91.3%, and 2 cases of squamous carcinoma are missed; the detection rate of adenocarcinoma is 73.1%, and all large cell cancers can be detected. From the box-plot and scatter plot (FIG. 3), the FOXD3 gene detection DNA methylation was more clearly distinguished in the lung cancer group and the non-lung cancer group. The FOXD3 gene is proved to be a molecular marker for clinical diagnosis of lung cancer.

Especially for adenocarcinoma, the specificity reaches 95.0%, the sensitivity reaches 73.1%, and the method has extremely high clinical application value. Lung adenocarcinoma is easier to occur in females and non-smokers, no obvious clinical symptoms are usually found in the early stage, and the detection of the lung adenocarcinoma is more difficult and meaningful due to higher omission rate of the surrounding adenocarcinoma due to the tree-shaped physiological structure of bronchi. And FOXD3 was assayed in tissue specimens with a ROC curve as shown in FIG. 4 and an AUC of 0.937.

Example 3: detection of FOXD3 gene in sputum specimen

A large number of documents show that SHOX2 can be used as a marker for detecting lung cancer, and the SHOX2 has higher detection rate in samples such as alveolar lavage fluid, lesion tissue, hydrothorax, sputum and the like. To verify the detection effect of FOXD3, the present inventors examined the detection efficiency of FOXD3 and SHOX2 genes in sputum at the same time.

The detection primer probes of each gene are as follows:

the detection primers and probes of FOXD3 are:

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 are:

SHOX2_T_MF3 primer F: TTTAAAGGGTTCGTCGTTTAAGTC

SHOX2_T_MR3 primer R: AAACGATTACTTTCGCCCG

SHOX2_Taq_P3_Probe: FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1

Sample information: the total of the test sputum samples is 60, wherein 31 samples of a normal control group, 29 samples of a cancer group and 4 samples of lung cancer which are not clearly classified are 9 cancer cells, 6 cancer cells, 9 cancer cells and 1 cancer cell.

The test process comprises the following steps:

a. sputum samples of patients diagnosed with lung cancer and non-lung cancer were collected, and after thickening with DTT, the pellet was centrifuged to isolate cells, washed 2 times with PBS, and DNA was extracted using the DNA extraction kit of Magen (HiPure FFPE DNA Kit, D3126-03).

b. The bisulfite modification of the DNA was performed using the DNA transformation kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Bio Inc.

c. The liquid preparation system is shown in Table 9:

TABLE 9 liquid distribution System

/>

d. The amplification system is as in table 10:

TABLE 10 PCR reaction procedure

e. The detection results are as follows:

and calculating methylation copy numbers of the genes in the samples by using a standard curve, judging methylation degrees of two groups of tissues by adopting the ratio=copy number/ACTB copy number of 100, and finally selecting the threshold value of FOXD3 as 8.9 and the threshold value of SHOX2 as 5.1, wherein the converted ratio exceeds a set threshold value and can be judged as positive, and the converted ratio is equal to or smaller than the set threshold value and can be judged as negative. According to this standard, the detection results of 60 sputum specimens are shown in Table 11:

TABLE 11 sputum specimen test results

/>

Note that: "+" indicates that the detection result is a positive sample; "-" indicates that the detection result is a negative sample.

f. Analysis of results

TABLE 12 statistical results

As can be seen from the above results, the detection effect of FOXD3 was superior to that of SHOX2 gene, regardless of whether lung cancer as a whole was subjected to comparative analysis or a subtype of lung cancer was subjected to comparative analysis. In particular, the detection effect on adenocarcinoma is 22.2% for FOXD3 and 0% for SHOX2, and adenocarcinoma is generally peripheral, and due to the dendritic physiological structure of bronchi, it is more difficult and meaningful to detect the deep lung exfoliated cells by expectoration of sputum. ROC curves for FOXD3 and SHOX2 detected in sputum specimens are shown in fig. 5, with FOXD3 AUC value of 0.894 and SHOX2 AUC value of 0.847.

Example 4: detection of FOXD3 gene in lavage fluid specimen

Sample information: the total of 79 samples of the tested alveolar lavage fluid is 58 samples of a normal control group, 21 samples of a cancer group, 6 samples of squamous cell carcinoma, 4 samples of small cell carcinoma and 11 samples of adenocarcinoma in 21 samples of the cancer group.

The test process comprises the following steps:

a. alveolar lavage fluid samples, which were confirmed as lung cancer patients and non-lung cancer patients, were collected, cells were centrifuged, and DNA was extracted using the DNA extraction kit of Magen (HiPure FFPE DNA Kit, D3126-03).

c. The liquid preparation system is the same as in Table 9.

d. The amplification detection system is as in Table 10.

e. The detection results are as follows:

and calculating methylation copy numbers of the genes in the samples by using a standard curve, judging methylation degrees of two groups of tissues by adopting the ratio=copy number/ACTB copy number of 100, and finally selecting the threshold value of FOXD3 as 2.6 and the threshold value of SHOX2 as 0.6, wherein the converted ratio exceeds a set threshold value and can be judged positive, and the converted ratio is equal to or smaller than the set threshold value and can be judged negative. According to this standard, the test results of 79 lavage samples are shown in Table 13:

TABLE 13 lavage fluid sample detection results

/>

TABLE 14 statistical results

/>

As can be seen from the above results, the detection of FOXD3 and SHOX2 was performed simultaneously, and the lung cancer as a whole was analyzed by comparison, the detection rate of FOXD3 was 61.9%, which was far higher than that of SHOX2 by 47.6%, and the detection result of squamous cell carcinoma group FOXD3 was 16.7% higher than that of SHOX2 by comparison analysis according to the subtype of lung cancer. In particular to the detection effect of the adenocarcinoma, the sensitivity of the kit reaches 54.5 percent, which is far higher than 36.4 percent of SHOX 2. Because adenocarcinomas are generally peripheral, alveolar lavage fluid is not readily accessible to deep lung alveoli or cancerous tissue due to the dendritic physiological structure of the bronchi, and detection of this portion is therefore more difficult and meaningful. ROC curves for FOXD3 and SHOX2 detected in alveolar lavage fluid samples are shown in fig. 6, with FOXD3 AUC of 0.791 and SHOX2 AUC of 0.784.

By combining examples 1 to 4, it can be fully demonstrated that FOXD3 has a better detection effect on lung cancer detection and diagnosis, especially on biological samples such as sputum, alveolar lavage fluid and the like. Can be more easily applied to large-scale crowd screening. Has more excellent socioeconomic value.

Example 5: selection of region of FOXD3 gene, detection primer and probe

Various research data show that methylation status and distribution of the same gene are not uniform, so that methylation primers and probe detection systems designed by different regions are selected for the same gene, diagnostic detection efficacy of the same tumor is different for the same sample, even if the selected region is unsuitable, so that the diagnostic effect on the tumor is not at all generated, and table 15 lists different regions of FOXD3 genes selected in the experimental process of the invention.

TABLE 15 sequences of different regions of FOXD3 gene

/>

Designing different methylation primers and probes according to the region 1, the region 2 and the region 3, wherein the probe information of each primer is shown in a table 16, and the methylation primers and probes designed according to the region 1 are shown in the group 1, the group 2, the group 3, the group 4 and the group 5; set 6, set 7, set 8, set 9, set 10 are methylation primers and probes designed according to region 2; set 11, set 12, set 13, set 14 are methylation primers and probes designed according to region 3. All primers and probes were synthesized by the Uygur crassier (Shanghai) trade company, inc.

TABLE 16 primers and probes designed according to different regions of FOXD3 gene

/>

The 14 sets of primer probe combinations in the 36 lung tissue sample detection table 16, wherein the 11 normal tissue samples, the 25 cancer tissue samples and the 21 cancer samples are respectively provided with 4 squamous cell carcinomas and 21 adenocarcinomas. The test results are shown in Table 17.

Sample processing, detection result judgment and statistics are the same as in example 1; the PCR liquid preparation system and the reaction process are routine operation in the field.

TABLE 17 detection results of different primer probe combinations in tissue

The area where is located	Group of	Primer probe combination	Specificity (specificity)	Sensitivity of
					Zone 1	Group 1	FOXD3-F1,FOXD3-R1,FOXD3-P1	100％	84％
Zone 1	Group 2	FOXD3-F2,FOXD3-R2,FOXD3-P2	100％	68％
					Zone 1	Group 3	FOXD3-F3,FOXD3-R3,FOXD3-P3	100％	80％
Zone 1	Group 4	FOXD3-F4,FOXD3-R4,FOXD3-P4	100％	72％
					Zone 1	Group 5	FOXD3-F5,FOXD3-R5,FOXD3-P5	100％	84％
Zone 2	Group 6	FOXD3-F6,FOXD3-R6,FOXD3-P6	100％	40％
					Zone 2	Group 7	FOXD3-F7,FOXD3-R7,FOXD3-P7	100％	40％
Zone 2	Group 8	FOXD3-F8,FOXD3-R8,FOXD3-P8	100％	52％
					Zone 2	Group 9	FOXD3-F9,FOXD3-R9,FOXD3-P9	100％	48％
Zone 2	Group 10	FOXD3-F10,FOXD3-R10,FOXD3-P10	100％	68％
					Zone 3	Group 11	FOXD3-F11,FOXD3-R11,FOXD3-P11	100％	64％
Zone 3	Group 12	FOXD3-F12,FOXD3-R12,FOXD3-P12	100％	52％
					Zone 3	Group 13	FOXD3-F13,FOXD3-R13,FOXD3-P13	100％	36％
Zone 3	Group 14	FOXD3-F14,FOXD3-R14,FOXD3-P14	100％	40％

The results show that the detection rates of group 1, group 2, group 3, group 4, group 5 and group 10 are higher. However, no matter what kind of primer and probe is designed by the invention, the detection sensitivity of the region 1 can be 68% at the lowest and 84% at the highest, and the detection rate is higher than that of a plurality of pairs of primers designed for the region 2 and the region 3, so that the detection rate of the region 1 is obviously higher than that of other regions (see Table 17).

Example 6 selection of primer and Probe combinations

To further verify the detection rate of different primer and probe combinations in sputum, the inventors selected 22 sputum specimens, and used the primers and probes in table 16 for verification, including 7 normal controls, 15 lung cancer controls, 7 squamous cell carcinoma in 15 lung cancers, 7 adenocarcinoma, 1 large cell carcinoma, and the detection results are shown in table 18.

Sample processing, detection result judgment and statistics are the same as in example 3; the PCR liquid preparation system and the reaction process are routine operation in the field.

TABLE 18 detection results in sputum

From the detection results of 22 sputum specimens, group 1: FOXD3-F1, FOXD3-R1 and FOXD3-P1 were detected at the highest rate, reaching 73.3%.

Although the sensitivity of both group 1 and group 5 reached 84% in the tissue samples, the sensitivity of group 5 was greatly reduced to 53.3% for the sputum test samples, which also proved that it was particularly difficult to design a test reagent with high sensitivity for the sputum samples from one aspect.

Finally, according to the detection result of each primer probe, the most preferred primer probe sequence is the combination of the group 1: FOXD3-F1, FOXD3-R1, FOXD3-P1.

Sequence listing

<110> Guangzhou city Kang Liming Biotechnology Co of Limited liability

<120> FOXD3 gene-based lung cancer diagnostic agent and kit

<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 gagaaatgag agaaggga 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

1. A reagent for detecting methylation of a FOXD3 gene, wherein the reagent for detecting methylation of the FOXD3 gene detects a sequence of the FOXD3 gene modified by bisulfite, and the reagent comprises a primer pair and a probe combination, and the primer is shown as SEQ ID NO:1 and SEQ ID NO:2 is shown in the figure; the probe is shown as SEQ ID NO: 3.

2. The test reagent of claim 1, wherein the reagent is directed against the detection region of the FOXD3 gene as set forth in SEQ ID NO: 19.

3. The test reagent of claim 1, wherein the reagent further comprises a reference gene test reagent.

4. The test reagent according to claim 3, wherein the reference gene is β -actin.

5. The detection reagent according to claim 3, wherein the detection reagent for the reference gene is a primer or a probe for the reference gene.

6. The detection reagent according to claim 3, wherein the detection reagent for the reference gene is a nucleotide sequence represented by SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO: 18.

7. The test reagent of claim 1, wherein the reagent further comprises bisulfite, or a hydrazine salt.

8. The detection reagent according to claim 1, wherein the reagent further comprises DNA polymerase, dNTPs, mg ²⁺ One or more of ions and buffer solution.

9. The test reagent of claim 1, wherein the test sample of the reagent is selected from alveolar lavage fluid, tissue, sputum.

10. Use of a detection reagent according to any one of claims 1-9 in the preparation of a lung cancer diagnostic reagent or kit.

11. The use according to claim 10, wherein the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.

12. The use of claim 11, wherein the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma, large cell carcinoma.

13. A lung cancer diagnostic reagent comprising the detection reagent of any one of claims 1-9.

14. The lung cancer diagnostic reagent of claim 13, wherein the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.

15. The lung cancer diagnostic reagent of claim 14, wherein the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma, large cell carcinoma.

16. A system for diagnosing lung cancer, said system comprising:

DNA methylation detecting means of FOXD3 gene, and,

b. a result judging system;

the DNA methylation detecting means of FOXD3 gene comprises the detecting reagent according to any one of claims 1 to 9.

17. The system according to claim 16, wherein the result judging means is for outputting the risk of lung cancer based on the result of DNA methylation of FOXD3 gene detected by the detecting system.

18. The system of claim 17, wherein the risk of disease is determined to be high based on comparing the methylation results of the test sample with the methylation results of the normal sample when the methylation of the test sample is significantly different or very significantly different from the methylation of the normal sample.