CN111363814B - Lung cancer diagnosis reagent and reagent kit based on DMRTA2 and FOXD3 genes - Google Patents
Lung cancer diagnosis reagent and reagent kit based on DMRTA2 and FOXD3 genes Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention belongs to the field of biological medicine, and relates to a combined detection of DMTA 2 and FOXD3 genes, a lung cancer detection/diagnosis reagent and a kit, wherein the reagent or the kit comprises a detection reagent aiming at methylation of the DMTA 2 and FOXD3 genes, and the detection reagent is used for detecting sequences of the DMTA 2 and FOXD3 genes 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
Technical Field
The invention belongs to the field of gene diagnosis, and in particular relates to application of DMRT A2 and FOXD3 genes in lung cancer detection, a methylation detection/diagnosis reagent containing the DMRT A2 and FOXD3 genes, and a kit containing the reagent.
Background
Lung cancer is a malignant tumor of the lung that originates in the bronchial mucosa, glands or alveolar epithelium. 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 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. Currently, the sensitivity, specificity and application range of markers for lung cancer detection are still to be improved.
(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 biopsy, thoracoscopic biopsy, mediastinoscope biopsy, hydrothorax cytology, etc., can be used separately to aid diagnosis according to existing conditions in the presence of indications.
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.
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.
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 application of DMRTA2 and FOXD3 genes or nucleic acid fragments thereof in preparing tumor detection/diagnosis reagents or kits.
It is another object of the present invention to provide the use of a primer composition for the preparation of a tumor detection/diagnosis reagent or kit.
It is another object of the present invention to provide the use of a probe composition 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 DMRTA2 and FOXD3 genes.
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 aim of the invention is achieved by the following technical means:
in one aspect, the invention provides a DMRTA2 gene or a nucleic acid fragment thereof, and application of the FOXD3 gene or the nucleic acid fragment thereof in preparing a tumor detection/diagnosis reagent or a kit.
The DMRT-like family A2 gene, which belongs to the family of DMRT genes, encodes a protein that binds to DM-binding motif (DM-domain) and contains DM-domain (DNA sequence that binds to the proteins encoded by the Drosophila doublesex gene and the nematode mab-3 gene) that is common to both genes. DMRT genes are a type of transcription regulatory factor, and the products encoded by the genes are combined with specific DNA sequences through zinc finger structures to regulate gene expression.
FOXD3 gene (forkhead box D3), fork D3 gene, which belongs to the fork family of transcription factors.
At present, the combination of DMRTA2 and FOXD3 as tumor markers for lung cancer is not reported. The invention detects lung cancer based on DMRTA2 and FOXD3 genes, or nucleic acid fragments thereof for the first time. When the two are combined for detection, the detection rate can reach 100%, and the detection rate of lung cancer is greatly improved. In the prior art, related researches for detecting lung cancer by adopting polygene combination, such as patent CN201510203539, and combining human SHOX2 gene and RASSF1 gene are also available, in 20 cases of tumor samples, two cases are still undetected, the coverage rate of 100% cannot be achieved, and missed detection can occur. The combination of the DMRTA2 gene and the FOXD3 gene in the invention has the advantages that the arbitrary positive rate reaches 100%, the omission is not easy to occur, the early detection and early diagnosis are facilitated, and the survival rate of patients is improved.
As a preferred embodiment, further, the nucleic acid fragment of the DMRTA2 gene is selected from the group consisting of SEQ ID NO:22. SEQ ID NO: 24. SEQ ID NO:26 or SEQ ID NO:28; more preferably SEQ ID NO:22. the nucleic acid fragment of FOXD3 gene is selected from the group consisting of SEQ ID NO:30. SEQ ID NO: 32. or SEQ ID NO:34, more preferably SEQ ID NO:30.
in another aspect, the present invention also provides a primer combination, where the primer combination contains a primer pair a and a primer pair B;
the primer pair A is selected from any one of the following:
SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:53 and SEQ ID NO:54, seq ID NO:56 and SEQ ID NO:57, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:72, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:77 and SEQ ID NO:78;
the primer pair B is selected from any one of the following:
SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:111, seq ID NO:113 and SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:117, SEQ ID NO:119 and SEQ ID NO:120;
Preferably, the primer pair A is selected from any one of the following:
SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:71 and SEQ ID NO:72;
the primer pair B is selected from any one of the following:
SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93, SEQ ID NO:107 and SEQ ID NO:108, a step of;
more preferably, the primer pair a is selected from any one of the following:
SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:48;
the primer pair B is selected from any one of the following:
SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93;
most preferably, the primer pair a is selected from: SEQ ID NO:1 and SEQ ID NO:2, the primer pair B is selected from the following groups: SEQ ID NO:80 and SEQ ID NO:81.
Wherein, the primer pair A can be used for amplifying the nucleic acid fragment of the DMRT A2 gene, and the primer pair B can be used for efficiently amplifying the nucleic acid fragment of the FOXD3 gene.
In another aspect, the present invention also provides a nucleic acid probe set, the probe set comprising a probe C and a probe D;
the probe C is selected from any one of the following sequences:
SEQ ID NO：3，SEQ ID NO：40，SEQ ID NO：43，SEQ ID NO：46，SEQ ID NO：49，SEQ ID NO：52，SEQ ID NO：55，SEQ ID NO：58，SEQ ID NO：61，SEQ ID NO：64，SEQ ID NO：67，SEQ ID NO：70，SEQ ID NO：73，SEQ ID NO：76，SEQ ID NO：79；
the probe D is selected from any one of the following sequences:
SEQ ID NO：82，SEQ ID NO：85，SEQ ID NO：88，SEQ ID NO：91，SEQ ID NO：94，SEQ ID NO：97，SEQ ID NO：100，SEQ ID NO：103，SEQ ID NO：106，SEQ ID NO：109，SEQ ID NO：112，SEQ ID NO：115，SEQ ID NO：118，SEQ ID NO：121；
preferably, the probe C is selected from any one of the following sequences:
SEQ ID NO：3，SEQ ID NO：40，SEQ ID NO：43，SEQ ID NO：46，SEQ ID NO：49，SEQ ID NO：64，SEQ ID NO：73；
the probe D is selected from any one of the following sequences:
SEQ ID NO：82，SEQ ID NO：85，SEQ ID NO：88，SEQ ID NO：91，SEQ ID NO：94，SEQ ID NO：109；
more preferably, the probe C is selected from any one of the following sequences:
SEQ ID NO：3，SEQ ID NO：40，SEQ ID NO：43，SEQ ID NO：46，SEQ ID NO：49；
the probe D is selected from any one of the following sequences:
SEQ ID NO：82，SEQ ID NO：85，SEQ ID NO：88，SEQ ID NO：91，SEQ ID NO：94；
most preferably, the probe C is selected from the group consisting of SEQ ID NOs: 3, the probe D is selected from the group consisting of SEQ ID NOs: 82.
wherein, the probe C can be specifically combined with the nucleic acid fragment of the DMRTA2 gene, and the probe D can be specifically combined with the nucleic acid fragment of the FOXD3 gene.
On the other hand, the invention also provides application of the primer or the nucleic acid probe in preparing a tumor detection/diagnosis reagent or a kit. The methylation of the DMRTA2 and FOXD3 genes or their nucleic acid fragments can be detected based on qMSP by the primers and probes described above.
In another aspect, the present invention also provides a tumor detection/diagnosis reagent, wherein the reagent contains a reagent for detecting methylation of DMRTA2 gene and FOXD3 gene.
Further, the reagent detects a sequence modified with bisulfite or hydrazine salt.
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).
DNA methylation in eukaryotes occurs predominantly at C in CpG dinucleotides and exists predominantly in the form of 5-methylcytosine (m 5C). The distribution of CpG dinucleotides in the human genome is very heterogeneous, with CpG remaining at or above normal probability in a partial region of the genome. CpG islands are mainly located in the promoter and the first exon of the gene, are regions rich in CpG dinucleotides, and have the length of 300-3000bp. 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.
As a preferred embodiment, the reagent targets the detection region of the DMRTA2 gene as set forth in SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO:26 or SEQ ID NO: 28; more preferably, the sequence set forth in SEQ ID NO: shown at 22; the detection region of the reagent aiming at the FOXD3 gene is shown as SEQ ID NO: 30. SEQ ID NO: 32. or SEQ ID NO: shown at 34; more preferably, the sequence set forth in SEQ ID NO: shown at 30.
The inventor finds that the selection of the detection regions of the DMRTA2 and FOXD3 genes can influence the detection efficiency of tumors.
The reagent of the present invention contains primers for amplifying DMRTA2 and FOXD3 genes.
As a preferred embodiment, the primer for detecting DMRTA2 gene is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:53 and SEQ ID NO:54, seq ID NO:56 and SEQ ID NO:57, SEQ ID NO:59 and SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:72, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:77 and SEQ ID NO:78, any pair of which; more preferably, the sequence selected from SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:63, SEQ ID NO:71 and SEQ ID NO:72, a primer pair shown in FIG. 72; further preferred is a sequence selected from the group consisting of SEQ ID NOs: 1 and SEQ ID NO:2, seq ID NO:38 and SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:45, SEQ ID NO:47 and SEQ ID NO: 48; most preferably, the sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and a primer pair shown in the following.
The primer for detecting the FOXD3 gene is selected from SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:96, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:102, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO:107 and SEQ ID NO:108, SEQ ID NO:110 and SEQ ID NO:111, seq ID NO:113 and SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:117, SEQ ID NO:119 and SEQ ID NO:120, any pair of the two; more preferably, the sequence selected from SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93, SEQ ID NO:107 and SEQ ID NO:108, a primer pair shown in seq id no; further preferred is a sequence selected from the group consisting of SEQ ID NOs: 80 and SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:84, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:90, seq ID NO:92 and SEQ ID NO:93, any pair of the two; most preferably, the sequence selected from SEQ ID NO:80 and SEQ ID NO:81.
The primer is used for amplifying specific regions of the DMRTA2 and FOXD3 genes. 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.
In a preferred embodiment, the reagent of the present invention further comprises a probe that specifically binds to DMRTA2 and FOXD3 genes.
Further, the probe for detecting DMRTA2 gene is selected from SEQ ID NO:3, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, and a sequence of any one of seq id no; more preferably, the sequence selected from SEQ ID NO:3, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:73, and a sequence of any one of seq id no; further preferred is a sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, and a sequence of any one of seq id no; most preferably, the sequence selected from SEQ ID NO:3.
the probe for detecting the FOXD3 gene is selected from the group consisting of SEQ ID NO:82, seq ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, seq ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, seq ID NO:115, seq ID NO:118, seq ID NO: 121; more preferably, the sequence selected from SEQ ID NO:82, seq ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:109, and a sequence of any one of seq id no; further preferred is a sequence selected from the group consisting of SEQ ID NOs: 82, seq ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, or a sequence of any one of seq id no; most preferably, the sequence selected from SEQ ID NO:82.
As a preferred embodiment, the reagent further comprises a detection reagent for an internal reference gene, wherein the internal reference gene is beta-actin or COL2A1.
Further, the detection reagent of the reference gene is a primer and a probe aiming at the reference gene. In a preferred embodiment, the detection reagent of the reference gene beta-actin is SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO: 21.
The detection reagent of the reference gene COL2A1 is SEQ ID NO:122 and SEQ ID NO:123, a primer pair set forth in SEQ ID NO: 124.
In a preferred embodiment, the reagent further comprises bisulphite, bisulfite or hydrazine salt to modify the DMRTA2 and FOXD3 genes, although this may not be the case.
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 DMRTA2 and FOXD3 genes.
In another aspect, the present invention also provides a method for detecting DNA methylation of DMRTA2 gene and 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) Detecting the methylation conditions of the DMRTA2 gene and the FOXD3 of the sample to be detected modified in the step (1) by using the reagent or the kit;
in the step (2), real-time fluorescence quantitative methylation specific polymerase chain reaction is adopted for detection.
In another aspect, the present invention also provides a lung cancer detection/diagnosis system, which is characterized in that the system comprises:
DNA methylation detection means of DMRTA2 gene and FOXD3 gene, and,
b. a result judging system;
the DNA methylation detection component of the DMRTA2 gene and the FOXD3 gene contains the reagent or the kit;
the result judging component is used for outputting the disease risk and/or the lung cancer type of the lung cancer according to the DNA methylation results of the DMRTA2 gene and the FOXD3 gene detected by the detection system;
more preferably, the risk of illness is that methylation results of the sample to be detected and the normal sample are compared according to judgment of the passing results, and when methylation of the sample to be detected and the normal sample has significant difference or extremely significant difference, the result judges that the risk of illness of the sample to be detected is high.
In the present invention, the test sample or sample to be tested may be selected from alveolar lavage fluid, 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 tumor is selected from lung cancer; further, the lung cancer is selected from small cell lung cancer and non-small cell lung cancer; the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma or large cell carcinoma.
The invention has the beneficial effects that:
although, in the prior art, methylation of DMRTA2 gene and FOXD3 gene, respectively, has been reported to be methylated in 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. Among the methylation genes possibly related to lung cancer, the DMRTA2 gene and the FOXD3 gene are combined for the first time and serve as tumor markers of lung cancer, so that the lung cancer detection rate is greatly improved, and in the embodiment of the invention, the combined detection rate is 100%. The invention also provides an optimized detection reagent aiming at the DMRTA2 gene and the FOXD3 gene, which has high sensitivity and specificity and is hopefully applied to clinical diagnosis of lung cancer.
When the detection sample is tissue, the specificity of the single detection of the DMTA 2 gene and the FOXD3 gene on all lung cancers reaches 100%, however, even the optimized detection reagent, the sensitivity of the DMTA 2 gene on the lung cancers is 89.3%, and the sensitivity of the FOXD3 is only 67.9%. By combining the DMRTA2 gene and the FOXD3 detection, the sensitivity to all lung cancers is improved to 92.9%, and the sensitivity is very rare in the existing lung cancer markers.
At present, lung cancer detection kits based on SHOX2 genes are available on the market. In one embodiment of the present invention, the combined detection effect of DMRTA2 and 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 is 44.4% for the combined detection rate of DMRTA2 and FOXD3, and 0% for the detection rate of SHOX2 gene. In another embodiment of the invention, the lung cancer is compared and analyzed as a whole by detecting the alveolar lavage fluid, and the combined detection of the DMRT A2 and the FOXD3 can obviously improve the detection rate of the lung cancer, and the combined detection rate is as high as 85.7%. According to the comparison analysis of the subtype of lung cancer, the sensitivity of the detection effect on the adenocarcinoma is up to 81.8%, which is far higher than 36.4% of that of SHOX 2.
In addition, the detection marker has very high specificity and sensitivity for different types of lung cancer, including squamous carcinoma, large cell carcinoma and adenocarcinoma 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 DMRT A2 gene and the FOXD3 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 ROC curve of combination genes of DMRTA2, FOXD3, SIX3, PCDHGA12, HOXD8, GATA3, DMRTA2 and FOXD3 for detecting lung cancer
FIG. 2 ROC curve of clinical tissue sample lung cancer combined detection by DMRTA2, FOXD3, DMRTA2 and FOXD3 genes
FIG. 3 ROC curve of combination of DMRTA2 and FOXD3 genes, detection of DMRTA2, FOXD3 and SHOX2 genes in sputum samples
FIG. 4 ROC curve of combination of DMRTA2 and FOXD3 genes, detection of DMRTA2, FOXD3 and SHOX2 genes in lavage fluid sample
Detailed Description
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 DMRTA2, FOXD3, SIX3, PCDHGA12, HOXD8 and GATA3 as candidate detection genes, takes beta-actin genes as internal reference genes, researches the distribution situation of methylation sites of all genes, and designs detection primer probes for detection. The detection primer probes of each gene are as follows:
the detection primers and probes of the DMRTA2 are as follows:
SEQ ID NO:1 DMRTA2 primer MF1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO:2 DMRTA2 primer MR1: TACGACCTAACCGCGCTCTCA
SEQ ID NO:3 dmrt 2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
The detection primers and probes of FOXD3 are:
SEQ ID NO:80 FOXD3 primer F: CGTCGGGATCGGATTTTTTC
SEQ ID NO:81 FOXD3 primer R: TCTCGACTCAAAAACCGACCG
SEQ ID NO:82 FOXD3 probe: FAM-CGGTTTTTTGCGTTAAGGTTAG-BQ1
The detection primers and probes of the SIX3 are as follows:
SEQ ID NO:7 SIX3 primer F: CGTTTTATATTTTTGGCGAGTAGC
SEQ ID NO:8 SIX3 primer R: ACTCCGCCAACACCG
SEQ ID NO:9 SIX3 probe: FAM-CGGCGGCGGCGCGGGAGGCGG-BQ1
The detection primers and probes of PCDHGA12 are:
SEQ ID NO:10 PCDHGA12 primer F: TTGGTTTTTACGGTTTTCGAC
SEQ ID NO:11 PCDHGA12 primer R: AAATTCTCCGAAACGCTCG
SEQ ID NO:12 PCDHGA12 probe:
FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1
the detection primers and probes for the HOXD8 were:
SEQ ID NO:13 HOXD8 primer F: TTAGTTTCGGCGCGTAGC
SEQ ID NO:14 HOXD8 primer R: CCTAAAACCGACGCGATCTA
SEQ ID NO:15 HOXD8 probe:
FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
the detection primers and probes for GATA3 are:
SEQ ID NO:16 GATA3 primer F: TTTCGGTAGCGGGTATTGC
SEQ ID NO:17 GATA3 primer R: AAAATAACGACGAACCAACCG
SEQ ID NO:18 GATA3 probe:
FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1
the detection primers and probes of the beta-actin are as follows:
SEQ ID NO: 19. beta-actin primer F: TTTTGGATTGTGAATTTGTG
SEQ ID NO: 20. beta-actin primer R: AAAACCTACTCCTCCCTTAAA
SEQ ID NO: 21. beta-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
Sample information: a total of 36 lung tissue samples, of which 11 lung tissue samples as controls, including 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 (3) calculating methylation copy numbers of genes in the samples respectively by using a standard curve, judging the methylation degree of tissues by adopting the ratio=target gene copy number/ACTB copy number of 100, and finally selecting a threshold value as a standard for judging a cancer group and a control group, wherein any one of the converted ratios exceeds or is equal to a judgment value, the judgment can be positive, and both markers are smaller than the judgment value and can 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
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Note that: "+" is positive for methylated DNA detection and "-" is negative for methylated DNA detection; when the specimens are cooperatively detected, the specimens are judged as negative when the specimens are negative, and the specimens are judged as positive when the specimens are negative and the specimens are positive when the specimens are positive.
TABLE 4 statistical results
In 11 lung tissue control samples, when the specificity is 100%, in 25 lung cancer samples, the positive rates of the DMRTA2 and FOXD3 methylation are 96% (24/25) and 84% (21/25), respectively, the detection rate is far higher than that of other genes, the detection rate can be up to 100% (25/25) through the combined detection of the DMRTA2 and the FOXD3, and the lung cancer detection rate is obviously improved through the combined analysis of the two genes. In squamous cell carcinoma, the methylation positive rate of DMRT A2 and FOXD3 is 100% and 75%, respectively, while in adenocarcinoma, the methylation positive rate of DMRT A2 gene is 95.2%, the methylation positive rate of FOXD3 is 85.7%, and the combined detection can have complementary effect, so that the positive rate is increased to 100%.
Therefore, DMRTA2 and FOXD3 gene methylation were selected for synergistic detection. The inventors have also optimised their detection conditions, see in particular example 2.
The ROC curve of the combination genes of DMRTA2, FOXD3, SIX3, PCDHGA12, HOXD8, GATA3, DMRTA2 and FOXD3 for detecting lung cancer is shown in FIG. 1, and the area under the ROC curve of the combination genes of DMRTA2 and FOXD3 is 1.
Example 2: detection of DMRTA2 Gene and FOXD3 in clinical tissue specimens
a. The detection primer probe is as follows:
the detection primers and probes of the DMRTA2 are as follows:
SEQ ID NO:1 DMRTA2 primer MF1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO:2 DMRTA2 primer MR1: TACGACCTAACCGCGCTCTCA
SEQ ID NO:3 dmrt 2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
The detection primers and probes of FOXD3 are:
SEQ ID NO:80 FOXD3 primer F: CGTCGGGATCGGATTTTTTC
SEQ ID NO:81 FOXD3 primer R: TCTCGACTCAAAAACCGACCG
SEQ ID NO:82 FOXD3 probe: FAM-CGGTTTTTTGCGTTAAGGTTAG-BQ1
The detection primers and probes of the beta-actin are as follows:
SEQ ID NO: 19. beta-actin primer F: TTTTGGATTGTGAATTTGTG
SEQ ID NO: 20. beta-actin primer R: AAAACCTACTCCTCCCTTAAA
SEQ ID NO: 21. beta-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
b. Sample information: the lung cancer tissue sample and the corresponding paracancerous tissue are taken as non-lung cancer control, and 56 cases of lung paraffin tissue samples are combined. Among them, 6 squamous cell carcinomas and 22 adenocarcinomas were included. The test process comprises the following steps:
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 and 6:
TABLE 5 liquid formulation
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f. Calculating methylation copy number of the DMRT A2 gene in a sample by using a standard curve, judging methylation degrees of two groups of tissues by adopting a ratio of = copy number/ACTB copy number of 100, and finally judging that the methylation rate of DMRT A2 exceeds '4.3' or the methylation rate of FOXD3 exceeds '21.5' after conversion by taking a numerical value of '4.3' and a numerical value of '21.5' of FOXD3 gene as standards for judging cancer groups and control groups, wherein the methylation rate of DMRT A2 is less than or equal to '4.3' and the methylation rate of FOXD3 is less than or equal to '21.5' and the methylation rate of the DMRT A2 is less than or equal to '21.5' and the methylation rate of the FOXD3 is negative. According to this standard, the detection results of 56 tissue specimens are shown in Table 7:
TABLE 7 detection results
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Note that: "+" is positive for methylated DNA detection and "-" is negative for methylated DNA detection; when the specimens are cooperatively detected, the specimens are judged as negative when the specimens are negative, and the specimens are judged as positive when the specimens are negative and the specimens are positive when the specimens are positive.
Table 8 results statistics
The ROC curves of the combination detection of the genes DMRTA2, FOXD3 and the genes DMRTA2 and FOXD3 for lung cancer are shown in figure 2, and the lower parts of the ROC curves of the combination detection of the genes DMRTA2, FOXD3 and the genes are respectively 0.953, 0.878 and 0.959.
The result shows that in 28 cases of lung cancer side control, under the condition that the negative detection rate is 100%, the positive rates of the methylation of the DMRTA2 and the FOXD3 are 89.3% and 67.9%, respectively, the joint detection sensitivity of the two genes is as high as 92.9%, and only 2 cases of adenocarcinoma is missed, so that the joint detection rate of the lung cancer can be obviously improved by the DMRTA2 and the FOXD 3.
By combining the examples, it can be fully demonstrated that the combination of DMRTA2 and FOXD3 has a better detection effect on lung cancer detection and diagnosis.
Example 3: detection of DMRTA2 and FOXD3 genes in sputum specimens
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. In order to verify the detection effect of the combined detection of the DMRTA2 and FOXD3 genes, the present inventors detected the detection efficiency of the DMRTA2, 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 the DMRTA2 are as follows:
SEQ ID NO:1 DMRTA2 primer MF1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO:2 DMRTA2 primer MR1: TACGACCTAACCGCGCTCTCA
SEQ ID NO:3 dmrt 2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
The detection primers and probes of FOXD3 are:
SEQ ID NO:4 FOXD3 primer F: CGTCGGGATCGGATTTTTTC
SEQ ID NO:5 FOXD3 primer R: TCTCGACTCAAAAACCGACCG
SEQ ID NO:6 FOXD3 probe: 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 a ratio of = copy number/ACTB copy number of 100, and finally selecting a threshold value of 25.9 of DMRTA2, a threshold value of 8.9 of FOXD3 and a threshold value of 5.1 of SHOX2 as standards for judging cancer groups and control groups, 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
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Note that: "+" indicates that the detection result is a positive sample; "-" indicates that the detection result is a negative sample. When the specimens are detected in combination, the specimens are judged as negative when the specimens are negative, and the specimens are judged as positive when the specimens are negative and the specimens are positive.
f. Analysis of results
TABLE 12 statistical results
The ROC curves for the detection of lung cancer in sputum samples for DMRTA2, FOXD3, SHOX2 and combinations of DMRTA2 and FOXD3 genes are shown in fig. 3, and the lower part of the ROC curves for the combined detection of DMRTA2, FOXD3, SHOX2 and DMRTA2 and FOXD3 genes are 0.868, 0.894, 0.847, 0.897, respectively.
From the above results, it can be seen that the detection effect of DMRTA2 and FOXD3 is superior to that of the SHOX2 gene, both when comparing and analyzing lung cancer as a whole and when comparing and analyzing lung cancer according to the subtype of lung cancer. Meanwhile, the combination detection of the DMRTA2 and the FOXD3 can effectively improve the detection rate of squamous cell carcinoma and adenocarcinoma, particularly the detection rate of adenocarcinoma is 44.4 percent, the detection rate of SHOX2 gene is 0 percent, the adenocarcinoma is generally peripheral, and due to the tree-shaped physiological structure of bronchi, the exfoliated cells in the deep part of the lung are more difficult to expectorate through sputum, so that the detection of the part is more difficult and significant.
Example 4: detection of DMRTA2 and FOXD3 genes in lavage fluid samples
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).
b. Bisulphite modification of DNA was performed using the ZYMO reserve organism company DNA transformation kit (EZ DNA Methylation Kit, D5002).
c. The liquid preparation system is shown in Table 9.
d. The amplification detection system is shown 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 a ratio of = copy number/ACTB copy number of 100, and finally selecting a threshold value of 2.1 of DMRTA2, a threshold value of 2.6 of FOXD3 and a threshold value of 0.6 of SHOX2 as standards for judging cancer groups and control groups, 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 test results of 79 lavage samples are shown in Table 13:
TABLE 13 lavage fluid sample detection results
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Note that: "+" indicates that the detection result is a positive sample; "-" indicates that the detection result is a negative sample. When the specimens are detected in combination, the specimens are judged as negative when the specimens are negative, and the specimens are judged as positive when the specimens are negative and the specimens are positive.
TABLE 14 statistical results
The ROC curves for the detection of lung cancer in alveolar lavage fluid samples for DMRTA2, FOXD3, SHOX2 and combinations of DMRTA2 and FOXD3 genes are shown in fig. 4, and the ROC curves for the combined detection of DMRTA2, FOXD3, SHOX2 and DMRTA2 and FOXD3 genes are 0.870, 0.791, 0.784, 0.931, respectively, below.
From the above results, it can be seen that the detection effect of DMRTA2 and FOXD3 is better than that of the SHOX2 gene, both by comparing and analyzing lung cancer as a whole and by comparing and analyzing the lung cancer subtype. Meanwhile, the combination detection of the DMRTA2 and the FOXD3 can obviously improve the detection rate of lung cancer, and the combined detection rate is as high as 85.7%. Especially for the detection of adenocarcinoma, the sensitivity reaches 81.8%, which is far higher than 36.4% 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.
The comprehensive examples 1-4 can fully demonstrate that the combination of DMRTA2 and FOXD3 has 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 DMRTA2 and FOXD3 Gene regions
Various research data show that the methylation state 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, the diagnosis and detection effects of the same tumor are different for the same sample, and even the selected regions are unsuitable sometimes, so that the diagnosis effect on the tumor is completely absent. After repeated researches and comparisons, the sequence of the promoter region of the selection gene is as follows:
the inventors selected different regions of promoters of DMRTA2 and FOXD3 genes for detection, see in particular table 15 below.
TABLE 15 selection of detection regions of DMRTA2 and FOXD3 genes
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Different methylation primers and probes were designed based on regions 1, 2, 3, 4 of the DMRTA2 sequence, and FOXD3 sequences, region 1, 2, and 3, with each primer probe information shown in table 16.
For DMRTA2 sequences, wherein group 1D, group 2D, group 3D, group 4D, group 5D are methylation primers and probes designed according to region 1; set 6D, set 7D, set 8D, set 9D are methylation primers and probes designed according to region 2; set 10D, set 11D, set 12D are methylation primers and probes designed according to region 3; set 13D, set 14D, set 15D are methylation primers and probes designed according to region 4.
For FOXD3, where group 1F, group 2F, group 3F, group 4F, group 5F are methylation primers and probes designed according to region 1; set 6F, set 7F, set 8F, set 9F, set 10F are methylation primers and probes designed according to region 2; set 11F, set 12F, set 13F, set 14F are methylation primers and probes designed according to region 3.
Table 16 primers and probes
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The above 29 groups of primer probe combinations are detected in 36 lung tissue samples, wherein 11 normal tissue samples, 25 cancer tissue samples and 21 adenocarcinoma are obtained from 25 cancer group samples. The results of the measurements are shown in tables 17-18 below.
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 DMRTA2 in tissues
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TABLE 18 detection results of FOXD3 in tissues
| The area where is located | Group of | Primer probe combination | Specificity (specificity) | Sensitivity of |
| Zone 1 | Group 1F | FOXD3-F1,FOXD3-R1,FOXD3-P1 | 100％ | 84％ |
| Zone 1 | Group 2F | FOXD3-F2,FOXD3-R2,FOXD3-P2 | 100％ | 68％ |
| Zone 1 | Group 3F | FOXD3-F3,FOXD3-R3,FOXD3-P3 | 100％ | 80％ |
| Zone 1 | Group 4F | FOXD3-F4,FOXD3-R4,FOXD3-P4 | 100％ | 72％ |
| Zone 1 | Group 5F | FOXD3-F5,FOXD3-R5,FOXD3-P5 | 100％ | 84％ |
| Zone 2 | Group 6F | FOXD3-F6,FOXD3-R6,FOXD3-P6 | 100％ | 40％ |
| Zone 2 | Group 7F | FOXD3-F7,FOXD3-R7,FOXD3-P7 | 100％ | 40％ |
| Zone 2 | Group 8F | FOXD3-F8,FOXD3-R8,FOXD3-P8 | 100％ | 52％ |
| Zone 2 | Group 9F | FOXD3-F9,FOXD3-R9,FOXD3-P9 | 100％ | 48％ |
| Zone 2 | Group 10F | FOXD3-F10,FOXD3-R10,FOXD3-P10 | 100％ | 68％ |
| Zone 3 | Group 11F | FOXD3-F11,FOXD3-R11,FOXD3-P11 | 100％ | 64％ |
| Zone 3 | Group 12F | FOXD3-F12,FOXD3-R12,FOXD3-P12 | 100％ | 52％ |
| Zone 3 | Group 13F | FOXD3-F13,FOXD3-R13,FOXD3-P13 | 100％ | 36％ |
| Zone 3 | Group 14F | FOXD3-F14,FOXD3-R14,FOXD3-P14 | 100％ | 40％ |
The results show that for DMRTA2, group 1D, group 2D, group 3D, group 4D, group 5D, group 10D, and group 13D all have higher detection rates. Regardless of the primers and probes designed by the invention, the detection sensitivity of the region 1 can be as low as 72%, and the detection sensitivity of the region 1 can be as high as 96%, wherein the detection rate is far higher than that of a plurality of pairs of primers designed for the region 2, and the detection sensitivity of most of the primers of the region 1 is higher than that of the primers of the region 3 and the region 4, so that the detection rate of the region 1 of the DMRTA2 is obviously higher than that of other regions (see Table 17).
For FOXD3, the detection rates of group 1F, group 2F, group 3F, group 4F, group 5F and group 10F are all higher, the detection sensitivity of the region 1 can reach 68% at the lowest and 84% at the highest no matter which primer and probe are designed by the invention, and therefore, the detection rate of the region 1 of the FOXD3 is obviously higher than that of other regions (see Table 18).
Example 6 selection of primer and Probe combinations
To further verify the detection rate 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 carcinomas in 15 lung cancers, 7 adenocarcinomas, and 1 large cell carcinoma, with the detection results shown in tables 19-20 below.
TABLE 19 detection results of DMRTA2 in sputum
| Group of | Primer probe combination | Specificity (specificity) | Sensitivity of |
| Group 1D | DM2-F1,DM2-R1,DM2-P1 | 100％ | 80％ |
| Group 2D | DM2-F2,DM2-R2,DM2-P2 | 100％ | 60％ |
| Group 3D | DM2-F3,DM2-R3,DM2-P3 | 100％ | 66.7％ |
| Group 4D | DM2-F4,DM2-R4,DM2-P4 | 100％ | 46.7％ |
| Group 5D | DM2-F5,DM2-R5,DM2-P5 | 100％ | 66.7％ |
| Group 10D | DM2-F10,DM2-R10,DM2-P10 | 100％ | 60％ |
| Group 13D | DM2-F13,DM2-R13,DM2-P13 | 100％ | 46.7％ |
TABLE 20 detection results of FOXD3 in sputum
| Group of | Primer probe combination | Specificity (specificity) | Sensitivity of |
| Group 1F | FOXD3-F1,FOXD3-R1,FOXD3-P1 | 100％ | 73.3％ |
| Group 2F | FOXD3-F2,FOXD3-R2,FOXD3-P2 | 100％ | 40％ |
| Group 3F | FOXD3-F3,FOXD3-R3,FOXD3-P3 | 100％ | 53.3％ |
| Group 4F | FOXD3-F4,FOXD3-R4,FOXD3-P4 | 100％ | 46.7％ |
| Group 5F | FOXD3-F5,FOXD3-R5,FOXD3-P5 | 100％ | 53.3％ |
| Group 10F | FOXD3-F10,FOXD3-R10,FOXD3-P10 | 100％ | 46.7％ |
From the detection results of 22 sputum specimens, group 1D of DMRTA2 showed: the detection rate of DM2-F1, DM2-R1 and DM2-P1 is highest and reaches 80%. Although the sensitivity of group 1D reached 96% and the sensitivity of group 2D reached 88% in the tissue samples, the sensitivity of group 2D was greatly reduced to 60% for the sputum test samples.
FOXD3 group 1F: FOXD3-F1, FOXD3-R1 and FOXD3-P1 were detected at the highest rate, reaching 73.3%. Although the sensitivity of both group 1F and group 5F reached 84% in the tissue samples, the sensitivity of group 5F was greatly reduced to 53.3% for the sputum test samples.
The final preferred detection primer probes are as follows:
the detection primers and probes of the DMRTA2 are as follows:
SEQ ID NO:1 DMRTA2 primer F1: TTCGGTTTAGTGTGCGTCGTC
SEQ ID NO:2 DMRTA2 primer R1: TACGACCTAACCGCGCTCTCA
SEQ ID NO:3 dmrt 2 probe P1: FAM-ACTACTCCGCCTCCGATCCC-BQ1
The detection primers and probes of FOXD3 are:
SEQ ID NO:80 FOXD3 primer F1: CGTCGGGATCGGATTTTTTC
SEQ ID NO:81 FOXD3 primer R1: TCTCGACTCAAAAACCGACCG
SEQ ID NO:82 FOXD3 probe P1: FAM-CGGTTTTTTGCGTTAAGGTTAG-BQ1
The detection primers and probes of the beta-actin are as follows:
SEQ ID NO: 19. beta-actin primer F: TTTTGGATTGTGAATTTGTG
SEQ ID NO: 20. beta-actin primer R: AAAACCTACTCCTCCCTTAAA
SEQ ID NO: 21. beta-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
Sequence listing
<110> Guangzhou city Kang Liming Biotechnology Co of Limited liability
<120> diagnostic reagent for lung cancer based on DMRTA2 and FOXD3 genes and kit
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<400> 24
cgtcggattt gggagcgcgc cgaaacatag caggaagaag gcaggaaaaa tcgtcctggc 60
atttatgcct tgaacaccct cccttcccca cccccgcacc cttgtcccgc ccctaccccg 120
accgcagtaa ttgaattgtc acctgccccc catcccgcct cagggctcca gagccacttg 180
ggaagctcgg ctttgccaag cgccccaggc cagtaagagc ggtgcagccg agccggcgcc 240
ccagaagccg agaggttaca caagtctacg cagagaggcg caccggacac ggtaattagg 300
agcaattcac acgctctcgg gcgcacggaa actacttgtc aggaaatttt caacgctcct 360
cctcgcccac ttttttccac tcctttcctg gcgcgccccc tcccctccca gaggcacgca 420
gtggagcttt tggagtttcg aatctttcgg acactgcaga atgcggggct ccccgggcgc 480
gggcccggcc aatcagagcg ccgtctgcct cccctggcca atggcaggcg ccacattgtt 540
gtccaattct gtctaatgga gccctaagga ccaacaatag agcgggcgag cggctcattg 600
agagtctggc agcgccgggc aactgggccc cggctgcgcg cagcgtccgg cgcgcaccgc 660
gaggggaccc ggcacggcga gagtcaggtc gcgggttcca accacgcggc ccgcagacga 720
gcctccgacc cggatttctg aagggagggt tggactgcgc tgcgttctga ggggtccgga 780
agttcggaca tggtgactga aggagggtga cgtggtcaga taaggggccg ggggcaaagg 840
gaagcggcca agagctccag cgtcgccagt ggagccggga ggcgcgtgca gcgccagcgg 900
aggaggagga tcccggagcc caag 924
<210> 25
<211> 924
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cgtcggattt gggagcgcgt cgaaatatag taggaagaag gtaggaaaaa tcgttttggt 60
atttatgttt tgaatatttt ttttttttta ttttcgtatt tttgtttcgt ttttatttcg 120
atcgtagtaa ttgaattgtt atttgttttt tatttcgttt tagggtttta gagttatttg 180
ggaagttcgg ttttgttaag cgttttaggt tagtaagagc ggtgtagtcg agtcggcgtt 240
ttagaagtcg agaggttata taagtttacg tagagaggcg tatcggatac ggtaattagg 300
agtaatttat acgttttcgg gcgtacggaa attatttgtt aggaaatttt taacgttttt 360
tttcgtttat ttttttttat tttttttttg gcgcgttttt ttttttttta gaggtacgta 420
gtggagtttt tggagtttcg aatttttcgg atattgtaga atgcggggtt tttcgggcgc 480
gggttcggtt aattagagcg tcgtttgttt tttttggtta atggtaggcg ttatattgtt 540
gtttaatttt gtttaatgga gttttaagga ttaataatag agcgggcgag cggtttattg 600
agagtttggt agcgtcgggt aattgggttt cggttgcgcg tagcgttcgg cgcgtatcgc 660
gaggggattc ggtacggcga gagttaggtc gcgggtttta attacgcggt tcgtagacga 720
gttttcgatt cggatttttg aagggagggt tggattgcgt tgcgttttga ggggttcgga 780
agttcggata tggtgattga aggagggtga cgtggttaga taaggggtcg ggggtaaagg 840
gaagcggtta agagttttag cgtcgttagt ggagtcggga ggcgcgtgta gcgttagcgg 900
aggaggagga tttcggagtt taag 924
<210> 26
<211> 1001
<212> DNA
<213> Homo sapiens
<400> 26
caaagggccg aggtgctgtg actgctgtct tctagctaac cgctcctcac gacacgtttt 60
ctcccctccc agaggccaag ttgcagaagt ttgacctgtt tcctaagacg ctgctgcagg 120
caggccgccc gggcagcccg ctgccgccgc cggtgaagcc cttatcaccc gacggcgcag 180
actcggggcc cgggacgtcg tccccagagg tgcggcccgg ctcaggctcg gagaacggcg 240
atggcgagtc cttttctggt tcgcccctag ctcgggcctc caaagaggca ggtggcagct 300
gcccaggcag cgctggccct ggcggcggcg gcgaggagga cagcccgggc tccgctagcc 360
ctctgggctc tgaatccggt tcagaggctg acaaagaaga gggtgaggcc gcgccggcgc 420
cagggctggg cggaggctcg ggtccacggc agcggacgcc gctggatatc ttgacacgcg 480
tgttcccagg ccaccggcga ggcgtcctgg agctggtgtt gcagggctgc ggcggcgacg 540
tggtgcaggc catcgagcag gtgctgaacc accaccgtgg gggcctggcg gccggcctgg 600
gccctgcggc gcccccagat aaggccgccg tgggtgctgc agcagctgca gacgacgcgt 660
ggcccagccg cgtcgacgcc gccgccgccg ccgccgccgc cgccgggggg cctgggctgc 720
ctgcgccgct gcaggcgggg cccgccgcac ctccgcacca cagacccttg ctggccggcg 780
ccatggcgcc tggggcgctg ggctcgctga gcagccgctc ggccttctcg ccgctgcagc 840
ccaacgccag tcacttcggt gccgacgcgg gcgcctaccc gctgggcgcg ccgctcggcc 900
tcagccccct gcgcctggcc tactccgcgg cggcggcgca cagccgcggt ctggccttca 960
tggcgcccta ctccactgcc ggcttggtgc ccacgctcgg c 1001
<210> 27
<211> 1001
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
taaagggtcg aggtgttgtg attgttgttt tttagttaat cgttttttac gatacgtttt 60
tttttttttt agaggttaag ttgtagaagt ttgatttgtt ttttaagacg ttgttgtagg 120
taggtcgttc gggtagttcg ttgtcgtcgt cggtgaagtt tttattattc gacggcgtag 180
attcggggtt cgggacgtcg tttttagagg tgcggttcgg tttaggttcg gagaacggcg 240
atggcgagtt tttttttggt tcgtttttag ttcgggtttt taaagaggta ggtggtagtt 300
gtttaggtag cgttggtttt ggcggcggcg gcgaggagga tagttcgggt ttcgttagtt 360
ttttgggttt tgaattcggt ttagaggttg ataaagaaga gggtgaggtc gcgtcggcgt 420
tagggttggg cggaggttcg ggtttacggt agcggacgtc gttggatatt ttgatacgcg 480
tgtttttagg ttatcggcga ggcgttttgg agttggtgtt gtagggttgc ggcggcgacg 540
tggtgtaggt tatcgagtag gtgttgaatt attatcgtgg gggtttggcg gtcggtttgg 600
gttttgcggc gtttttagat aaggtcgtcg tgggtgttgt agtagttgta gacgacgcgt 660
ggtttagtcg cgtcgacgtc gtcgtcgtcg tcgtcgtcgt cgtcgggggg tttgggttgt 720
ttgcgtcgtt gtaggcgggg ttcgtcgtat tttcgtatta tagatttttg ttggtcggcg 780
ttatggcgtt tggggcgttg ggttcgttga gtagtcgttc ggttttttcg tcgttgtagt 840
ttaacgttag ttatttcggt gtcgacgcgg gcgtttattc gttgggcgcg tcgttcggtt 900
ttagtttttt gcgtttggtt tatttcgcgg cggcggcgta tagtcgcggt ttggttttta 960
tggcgtttta ttttattgtc ggtttggtgt ttacgttcgg t 1001
<210> 28
<211> 1001
<212> DNA
<213> Homo sapiens
<400> 28
aggaacagga gagtctgtgg ttgtgcggga aaacgcgtgt agggcaaccc gtggagattc 60
cttctcccaa aggcactttc cccctttcct ctgcgcccca ttcccgggcg gagagtatca 120
gccaaactaa cggaatccgg cgcggccagg gaaggggtgg gtgcgggagg ggccattccg 180
tcccggagcc accggtttgg ggataattac ttttaatgtc agaaagattt agtttaatat 240
catctctatt aggctgccgg gagggtaatt aaacgggacg cgtcgcagcc ggcaaacaga 300
tggcgtctgc ttgctccgag gccgcgggca aacagcgcaa aagtcaatgc ctcccgggtc 360
ccccatcggc gcccctggct cttttactca gagctacact actcaccctc ggcccccttc 420
cccacgccgc gtctccagcc ggctctgcgc cctaggccct caaccccggc ctcaccccaa 480
cgcagtccct ttggacgcca ccagttcctg ggcgtgggcg tgggggatgg ggccgggggc 540
gaatagggat ggggtggatt ttcttggctg ggccctctca cccgggcccc cagcccctgg 600
cacagcagag gcgtcagcaa atgattctga gctaagagta cgcgtgtgga tgtgtgagga 660
agaccctctg ctctcagccg cctcatttgt cccggagggt tttggctttt tctgcccggg 720
gcccaactgt agaagtaaga ctggagctcc gcgcataaac cagggcatac ctgcgtaaga 780
atgtgctcct gtcggcgtgt gctatcaccg aggtggaaat ctgtgggagt ctgggaatgt 840
cacacaccaa gtggatggcc caggatgggg acatgcttat aagaacactg tgaggtcaaa 900
ctcactgaag gggtcccctt gagccggagg gtagcatcag agttgagcct gggcccggtg 960
gtgctctctg aatgctggcg tgagagtgtg tcccaaactt t 1001
<210> 29
<211> 1001
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
aggaatagga gagtttgtgg ttgtgcggga aaacgcgtgt agggtaattc gtggagattt 60
ttttttttaa aggtattttt tttttttttt ttgcgtttta ttttcgggcg gagagtatta 120
gttaaattaa cggaattcgg cgcggttagg gaaggggtgg gtgcgggagg ggttatttcg 180
tttcggagtt atcggtttgg ggataattat ttttaatgtt agaaagattt agtttaatat 240
tatttttatt aggttgtcgg gagggtaatt aaacgggacg cgtcgtagtc ggtaaataga 300
tggcgtttgt ttgtttcgag gtcgcgggta aatagcgtaa aagttaatgt ttttcgggtt 360
ttttatcggc gtttttggtt tttttattta gagttatatt atttattttc ggtttttttt 420
tttacgtcgc gtttttagtc ggttttgcgt tttaggtttt taatttcggt tttattttaa 480
cgtagttttt ttggacgtta ttagtttttg ggcgtgggcg tgggggatgg ggtcgggggc 540
gaatagggat ggggtggatt tttttggttg ggttttttta ttcgggtttt tagtttttgg 600
tatagtagag gcgttagtaa atgattttga gttaagagta cgcgtgtgga tgtgtgagga 660
agattttttg tttttagtcg ttttatttgt ttcggagggt tttggttttt tttgttcggg 720
gtttaattgt agaagtaaga ttggagtttc gcgtataaat tagggtatat ttgcgtaaga 780
atgtgttttt gtcggcgtgt gttattatcg aggtggaaat ttgtgggagt ttgggaatgt 840
tatatattaa gtggatggtt taggatgggg atatgtttat aagaatattg tgaggttaaa 900
tttattgaag gggttttttt gagtcggagg gtagtattag agttgagttt gggttcggtg 960
gtgttttttg aatgttggcg tgagagtgtg ttttaaattt t 1001
<210> 30
<211> 348
<212> DNA
<213> Homo sapiens
<400> 30
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> 31
<211> 348
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
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> 32
<211> 476
<212> DNA
<213> Homo sapiens
<400> 32
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> 33
<211> 476
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
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> 34
<211> 780
<212> DNA
<213> Homo sapiens
<400> 34
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> 35
<211> 780
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
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> 36
<211> 972
<212> DNA
<213> Homo sapiens
<400> 36
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> 37
<211> 972
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
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> 38
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
ttaagtgtac gtttatcgcg gag 23
<210> 39
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
cgcacactaa accgaaaacc tc 22
<210> 40
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
cgttaacgac gaccaacgcc aa 22
<210> 41
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gtcgttgtat cgttatcggt gag 23
<210> 42
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gacaccacgc cataattacg aca 23
<210> 43
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ccgcaacaaa ccgcctacca c 21
<210> 44
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cgttgtatcg ttatcggtga gc 22
<210> 45
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gcacaaacaa tccttccaac gac 23
<210> 46
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
acttctcgac tacccgcaac aaca 24
<210> 47
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
cggtcgttgt atcgttatcg 20
<210> 48
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
ctacccgcaa caacaataac g 21
<210> 49
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
cgtggtaggc ggtttgttgc g 21
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
cgggttttaa ttacgcggtt c 21
<210> 51
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
cgcctcccga ctccactaac 20
<210> 52
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
ccccgacccc ttatctaacc ac 22
<210> 53
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
taagagcggt gtagtcgagt c 21
<210> 54
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
aatttccgta cgcccgaaa 19
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
cgttttagaa gtcgagaggt tat 23
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
ggttaattag agcgtcgttt 20
<210> 57
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gcaaccgaaa cccaattacc c 21
<210> 58
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
gacgctacca aactctcaat aaaccg 26
<210> 59
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ttttcggata ttgtagaatg c 21
<210> 60
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
gaaacccaat tacccgac 18
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
acgctctaat taaccgaacc 20
<210> 62
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
ggggttcgtc gtattttcg 19
<210> 63
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
aaccgaacga ctactcaacg 20
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
cgttatggcg tttggggcgt tg 22
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
tattcgacgg cgtagattcg 20
<210> 66
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
gttctccgaa cctaaaccg 19
<210> 67
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
cgtcgttttt agaggtgcgg tt 22
<210> 68
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
cgtcgttgga tattttgata cg 22
<210> 69
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
ctacaacacc aactccaaaa cg 22
<210> 70
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
cgtgttttta ggttatcggc gagg 24
<210> 71
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
gtcgggaggg taattaaacg 20
<210> 72
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
cgacctcgaa acaaacaaac g 21
<210> 73
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
cgcgtcgtag tcggtaaata gatgg 25
<210> 74
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
tggttgtgcg ggaaaacg 18
<210> 75
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
ctccgcccga aaataaaacg 20
<210> 76
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
cgtgtagggt aattcgtgga gattt 25
<210> 77
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
gtaaatgatt ttgagttaag agtacg 26
<210> 78
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
ccctccgaaa caaataaaac g 21
<210> 79
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
cgtgtggatg tgtgaggaag attt 24
<210> 80
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
cgtcgggatc ggattttttc 20
<210> 81
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
tctcgactca aaaaccgacc g 21
<210> 82
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
cggttttttg cgttaaggtt ag 22
<210> 83
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
ggcggtcggt ttttgagtc 19
<210> 84
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
ctactaacta cgaccccgac g 21
<210> 85
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
cgttggaaat cgatattagg tcggc 25
<210> 86
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
cggttttttg cgttaaggtt ag 22
<210> 87
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
acgcctacac aacctccg 18
<210> 88
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
ttttcggtcg tcgtttcgtt t 21
<210> 89
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
gttgtgtagg cgttatggtt cg 22
<210> 90
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
cgacctaata tcgatttcca acg 23
<210> 91
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
cggtttttga gtcgagagcg gtcg 24
<210> 92
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
ttcgatattt agtcggtttt cg 22
<210> 93
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
aaaattccta ctaaccttaa cg 22
<210> 94
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
cgcgtcggga tcggattttt 20
<210> 95
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
aggatagcga cgtaggttgc 20
<210> 96
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
atccgtaata aaataccgcc g 21
<210> 97
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
tcgtcggagt tgcgtttgga c 21
<210> 98
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
tcgaggacgt ggatatcg 18
<210> 99
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
gaaactatcg caacctacg 19
<210> 100
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
cgggttggaa gagaaggata gcga 24
<210> 101
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
gttggaagag aaggatagcg 20
<210> 102
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
gactaaaact atccgtaata aaataccg 28
<210> 103
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
cgatagtttc gcggggtcgt cg 22
<210> 104
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
ttaaggaggc ggtcggagtc 20
<210> 105
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
ctcgaaacca aacctccc 18
<210> 106
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
gcggtgttgg cggcgaggag 20
<210> 107
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
gagtttcgtg gtagttttcg 20
<210> 108
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
atcgatatcc acgtcctc 18
<210> 109
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
cgtcaacacc gtctaaccga a 21
<210> 110
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
agcgagcgtt tagtatcg 18
<210> 111
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
gctaaactca actcacactc g 21
<210> 112
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
cggacggtag agttcgcgtt acg 23
<210> 113
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
ttcgattatc ggggtttcg 19
<210> 114
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
aaaacgatcc aaacgaacg 19
<210> 115
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 115
cgagttgttc ggcgttcggc 20
<210> 116
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 116
cgcggttttg acgttacg 18
<210> 117
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 117
ccgaaacccc gataatcg 18
<210> 118
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 118
cggttacgtg gtttcgtttt tcgt 24
<210> 119
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 119
cgagttgttc ggcgttcg 18
<210> 120
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 120
ctcgacctct aaccctaacg 20
<210> 121
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 121
cgcgttcgtt tggatcgttt ttgc 24
<210> 122
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 122
ttttggattt aaggggaaga taaa 24
<210> 123
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 123
tttttccttc tctacatctt tctacct 27
<210> 124
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 124
aagggaaatt gagaaatgag agaaggga 28
<210> 125
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 125
tttaaagggt tcgtcgttta agtc 24
<210> 126
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 126
aaacgattac tttcgcccg 19
<210> 127
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 127
ttagaaggta ggaggcggaa aattag 26
Claims (17)
1. A reagent for detecting methylation of a DMRTA2 gene and a FOXD3 gene, wherein the reagent comprises a primer combination and a probe, and the primer combination comprises a primer pair A and a primer pair B;
the primer pair A is shown as SEQ ID NO:1 and SEQ ID NO:2, the primer pair B is shown as SEQ ID NO:80 and SEQ ID NO: shown at 81;
the probe is shown as SEQ ID NO:3 and SEQ ID NO: 82;
the detection reagent for the methylation of the DMTA 2 gene and the FOXD3 gene is used for respectively detecting the sequences of the DMTA 2 gene and the FOXD3 gene after being subjected to bisulfite modification.
2. The test reagent of claim 1, wherein the reagent further comprises a test reagent for a reference gene.
3. The test reagent of claim 2, wherein the reference gene is β -actin or COL2A1.
4. The detection reagent according to claim 2, wherein the detection reagent for the reference gene is a primer or a probe for the reference gene.
5. The detection reagent according to claim 3, wherein the detection reagent of the reference gene beta-actin is as shown in SEQ ID NO:19 and SEQ ID NO:20, and the primer pair is shown as SEQ ID NO: 21.
6. The detection reagent according to claim 3, wherein the detection reagent for the internal reference gene COL2A1 is as shown in SEQ ID NO:122 and SEQ ID NO:123, as set forth in SEQ ID NO: 124.
7. 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.
8. The test reagent of claim 1, wherein the sample is selected from alveolar lavage fluid, tissue, sputum.
9. Use of a detection reagent according to any one of claims 1-8 in the preparation of a lung cancer diagnostic reagent or kit.
10. The use according to claim 9, wherein the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.
11. The use of claim 10, wherein the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma, or large cell carcinoma.
12. A lung cancer diagnostic reagent comprising the detection reagent according to any one of claims 1 to 8.
13. The lung cancer diagnostic reagent of claim 12, wherein the lung cancer is selected from the group consisting of small cell lung cancer and non-small cell lung cancer.
14. The lung cancer diagnostic reagent of claim 13, wherein the non-small cell lung cancer is selected from squamous cell carcinoma, adenocarcinoma, or large cell carcinoma.
15. A system for diagnosing lung cancer, said system comprising:
DNA methylation detection means of DMRTA2 gene and FOXD3 gene, and,
b. a result judging system;
the DNA methylation detection component of the DMRTA2 gene and the FOXD3 gene contains the detection reagent according to any one of claims 1 to 8.
16. The system according to claim 15, wherein the result judging means is for outputting the risk of lung cancer based on the DNA methylation results of the DMRTA2 gene and FOXD3 gene detected by the detecting system.
17. The system of claim 16, wherein the risk of illness is a result of comparing the methylation of the test sample with the methylation of the normal sample based on the pass result, and the result judging means outputs that the risk of illness of the test sample is high when the methylation of the test sample has a significant difference or a very significant difference from the methylation of the normal sample.
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| CN201811593899.7A CN111363814B (en) | 2018-12-25 | 2018-12-25 | Lung cancer diagnosis reagent and reagent kit based on DMRTA2 and FOXD3 genes |
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| US8399193B2 (en) * | 2007-08-30 | 2013-03-19 | City Of Hope | DNA methylation biomarkers for lung cancer |
| US20140271455A1 (en) * | 2013-03-14 | 2014-09-18 | City Of Hope | Dna methylation biomarkers for small cell lung cancer |
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| AU2000260630A1 (en) * | 2000-06-30 | 2002-01-14 | The Procter & Gamble Company | Detergent compositions comprising a cyclodextrin glucanotransferase enzyme |
| CN105886657A (en) * | 2010-09-13 | 2016-08-24 | 临床基因组学股份有限公司 | Epigenetic Markers Of Colorectal Cancers And Diagnostic Methods Using The Same |
| CN106232833A (en) * | 2014-01-30 | 2016-12-14 | 加利福尼亚大学董事会 | The haplotyping that methylates (MONOD) for non-invasive diagnostic |
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