WO2022127717A1 - Marqueur moléculaire de méthylation ou combinaison de ceux-ci pour détecter les nodules pulmonaires bénins et malins, et leur utilisation - Google Patents
Marqueur moléculaire de méthylation ou combinaison de ceux-ci pour détecter les nodules pulmonaires bénins et malins, et leur utilisation Download PDFInfo
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Definitions
- the present invention claims the priority of the Chinese patent application CN2020114961847 filed on December 17, 2020, entitled “Methylation Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Their Combinations and Applications", the entire specification and disclosure content of the invention Incorporated herein by reference; the present invention also claims the priority of PCT application PCT/CN2021/086902 filed on April 13, 2021, entitled “Methylation Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Their Combinations and Applications”
- PCT/CN2021/086902 filed on April 13, 2021, entitled “Methylation Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Their Combinations and Applications”
- the disclosure of this invention regarding the combination of methylated molecular markers M, N, O is incorporated herein by reference.
- the invention belongs to the field of biotechnology, and in particular relates to a methylation molecular marker for detecting benign and malignant pulmonary nodules, a combination and an application thereof, and the application includes a detection kit and a detection method.
- Pulmonary nodule solitary pulmonary nodule, refers to the image of a round shadow, a single, well-defined, diameter less than or equal to 3cm, surrounded by air-containing lung tissue surrounded by high and low density Solid or subsolid lesions without atelectasis, hilar enlargement, or pleural effusion. It often invades the lungs, bilateral hilar lymph nodes, eyes, skin and other organs, and its chest invasion rate is as high as 80% to 90%. A considerable number of pulmonary nodules cannot exclude the possibility of early malignancy.
- Pulmonary nodules are divided into two categories, benign and malignant, and often have no obvious symptoms. Benign nodules require treatment based on the cause, and malignant ones require early surgery. Benign causes are often associated with autoimmune diseases or various infections, and malignant causes are often associated with lung cancer.
- Lung cancer is one of the malignant tumors with the fastest growing morbidity and mortality, and the greatest threat to the health and life of the population.
- treating patients at an early stage of lung cancer can effectively improve their five-year survival rate.
- Statistical research data found that after receiving effective treatment, the five-year survival rate of patients with stage I lung cancer can reach more than 60% to 90%, while the five-year survival rate of patients with advanced lung cancer is less than 20%. Therefore, the diagnosis and treatment of early-stage cancer patients is particularly important.
- the clinical detection methods of lung cancer include: imaging detection, cytology detection and molecular marker detection.
- the detection samples for lung cancer molecular marker detection mainly come from tissue biopsy and liquid biopsy, both of which have their own characteristics. Tissue samples are mainly obtained directly from the tumor site through surgery or fiberoptic bronchoscopy, so the detection accuracy is higher.
- tissue biopsy due to the invasiveness of tissue biopsy, the existence of tumor heterogeneity and various reasons, it cannot be obtained. Tissue specimens or the amount of tissue specimens are insufficient to complete molecular detection, which makes the role of tissue biopsy in the early diagnosis, prediction of metastasis and prognosis of lung cancer to some extent limited.
- liquid biopsy has the advantages of simple operation, non-invasiveness, strong reproducibility, and dynamic monitoring of diseases.
- Lung cancer liquid biopsy uses the patient's blood, sputum and bronchoalveolar lavage fluid as specimens to detect and analyze the tumor cell DNA and its modification level, such as DNA methylation.
- the tumor cell DNA and its modification level such as DNA methylation.
- improving the sensitivity is a huge challenge for this detection method.
- DNA methylation is closely related to the occurrence of cancer, especially the promoter hypermethylation of CpG island regions may lead to the transcriptional silencing of tumor suppressor genes, thereby affecting the process of tumorigenesis, because DNA methylation is the most common in all cancers. There are findings, and they occur in precancerous or early stages of cancer, making them ideal markers for cancer diagnosis.
- the inventor of the present invention has been committed to finding DNA methylation molecular markers that can well detect benign and malignant pulmonary nodules.
- Malignant DNA methylation molecular markers can be used to detect benign and malignant pulmonary nodules with high sensitivity and specificity, thereby providing technical support for the diagnosis of lung cancer, especially early lung cancer.
- one of the objectives of the present invention is to provide a DNA methylation molecular marker for detecting benign and malignant pulmonary nodules, and the DNA methylation molecular marker is very good for the detection of benign and malignant pulmonary nodules
- the sensitivity and specificity can effectively improve the detection rate of malignant pulmonary nodules.
- a first aspect of the present invention provides a DNA methylation molecular marker or a combination thereof that can be used to detect benign and malignant pulmonary nodules, wherein the DNA methylation molecular marker is selected from the group consisting of SEQ ID NO. 1 to SEQ ID Any one or a combination of two or more of the sequences shown in NO.19; or any one or more of the fully complementary sequences selected from the sequences shown in SEQ ID NO.1 to SEQ ID NO.19 combination; or any one or a combination of two or more selected from the contiguous fragments of at least 55% of the full length of the sequence shown in SEQ ID NO.1 to SEQ ID NO.19; or selected from SEQ ID NO.1 Any one or a combination of two or more of the complete complementary sequences of at least 55% of the full length of the sequence shown in ⁇ SEQ ID NO.19.
- the second aspect of the present invention provides the application of the above DNA methylation molecular marker or a combination thereof or a reagent for detecting the methylation level thereof in the preparation of a kit for detecting benign and malignant pulmonary nodules.
- a third aspect of the present invention provides a kit for detecting benign and malignant pulmonary nodules, the kit comprising a reagent for detecting the methylation level of the above-mentioned DNA methylation molecular marker.
- the kit can be used for the following detection platforms: including the use of PCR amplification method, fluorescence quantitative PCR method, digital PCR method, liquid chip method, next-generation sequencing method, third-generation sequencing method, second-generation sequencing method, pyrosequencing method, repeat Reagents used in sulfite conversion sequencing, methylation chip, simplified bisulfite sequencing, or a combination thereof.
- the detection method is PCR amplification detection, fluorescence quantitative PCR detection, digital PCR detection, chip detection.
- the reagents in the kit for detecting the methylation level of the above DNA methylation molecular markers include primers and probes for fluorescence quantitative PCR detection of the DNA methylation molecular markers.
- a fourth aspect of the present invention provides a method for detecting the methylation level of the above-mentioned DNA methylation molecular marker or a combination thereof, comprising the following steps:
- step (3) The multiplex PCR product obtained in step (3) is detected by multiplex fluorescence quantitative PCR with the probe for the above-mentioned DNA methylation molecular marker.
- Detection can be performed using the primers and probes in the above kits.
- a fifth aspect of the present invention provides a method for detecting benign and malignant pulmonary nodules, comprising the following steps:
- the present invention has the following beneficial effects:
- the present invention finds DNA methylation-specific molecular markers that are highly related to lung cancer. By detecting the methylation levels of these DNA methylation molecular markers, benign and malignant lung nodules can be detected, with good sensitivity and specificity. sex.
- the molecular marker SEQ ID NO.9 alone has high specificity in lung tissue samples, which is similar to SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 , any combination of SEQ ID NO.13, SEQ ID NO.16 and SEQ ID NO.17 molecular markers has higher sensitivity and specificity, especially SEQ ID NO.9, and SEQ ID NO.17 correspond to Marker's
- the combination, or a further combination including other markers can well improve the sensitivity and specificity of the detection of benign and malignant pulmonary nodules, effectively improve the detection rate of early malignant pulmonary nodules, conduct early treatment and intervention, and improve patient outcomes. Survival rate; at the same time reduce the false positive rate of detection and avoid over-diagnosis of benign pulmonary nodules.
- the fluorescent quantitative PCR detection primers and probes for the DNA methylation molecular markers overcome the disadvantage that multiple primers and probes interfere with each other during multiple PCR amplification and detection.
- each DNA methylation molecular marker can be effectively amplified and enriched; when the multiplex PCR products are subsequently detected by multiplex fluorescence quantitative PCR, the corresponding DNA
- the kit is optimized so that the primers and probes for different DNA methylation molecular markers do not interfere with each other, and can successfully realize multiple PCR amplification and multiple fluorescence quantitative PCR detection, effectively improving the detection efficiency.
- the detection method for the DNA methylation molecular marker provided by the present invention can effectively enrich the target molecule by introducing multiple PCR amplification, overcome the limitation of low sample acquisition amount, and also amplify the detection signal while also amplifying the detection signal.
- the combined detection of multiple molecular markers can improve the detection sensitivity and detection efficiency, and can enhance the detection rate of lung cancer and the accuracy of detection of benign and malignant pulmonary nodules.
- Figure 1 is the amplification curve of one of the molecular markers (Marker17) in Example 3 detected by fluorescence quantitative PCR reaction.
- FIG. 2 is a ROC curve diagram of each molecular marker combination in Example 5.
- FIG. 2 is a ROC curve diagram of each molecular marker combination in Example 5.
- the "plurality” mentioned in the present invention means two or more.
- "And/or" which describes the association relationship of the associated objects means that there can be three kinds of relationships, for example, A and/or B, which can mean that A exists alone, A and B exist at the same time, and B exists alone.
- the character "/" generally indicates that the associated objects are an "or" relationship.
- the DNA methylation molecular markers for detecting benign and malignant pulmonary nodules include 19 formazan among 10 detection genes CDO1, DAPK, GHSR, HOXA11, HOXB4, LHX9, MIR196A1, PTGER4, SHOX2 and TBX15
- the methylation region in some embodiments of the present invention, relates to a DNA methylation molecular marker or a combination thereof that can be used to detect benign and malignant pulmonary nodules, wherein the DNA methylation molecular marker is selected from SEQ ID NO .1 to SEQ ID NO.19 or any one or a combination of two or more of the sequences shown in their complete complementary sequences; or selected from SEQ ID NO.1 to SEQ ID NO.19 or their complete complementary sequences. Any one or a combination of two or more contiguous fragments of at least 55% of the full length of the sequence.
- One embodiment of the present invention relates to a DNA methylation molecular marker suitable for the detection of benign and malignant pulmonary nodules in respiratory samples, including any one or a combination of two or more of 19 methylation detection regions.
- these DNA methylation molecular markers can be combined arbitrarily to realize the detection of benign and malignant pulmonary nodules on the test sample.
- the DNA methylation molecular marker or a combination thereof comprises the sequences shown in SEQ ID NO.9 and SEQ ID NO.17, or the complete sequence comprising SEQ ID NO.9 and SEQ ID NO.17 Complementary sequence, or a contiguous fragment comprising at least 55% of the full length of the sequence shown in SEQ ID NO.9 and SEQ ID NO.17, or comprising at least 55% of the full length of the sequence shown in SEQ ID NO.9 and SEQ ID NO.17 % of the complete complement of contiguous fragments.
- the DNA methylation molecular marker or a combination thereof comprises the sequences shown in SEQ ID NO.9 and SEQ ID NO.17, or the complete sequence comprising SEQ ID NO.9 and SEQ ID NO.17 Complementary sequence, or a contiguous fragment comprising at least 55% of the full length of the sequence shown in SEQ ID NO.9 and SEQ ID NO.17, or comprising at least 55% of the full length of the sequence shown in SEQ ID NO.9 and SEQ ID NO.17 % of the complete complement of the contiguous fragment; and selected from
- the DNA methylation molecular marker or a combination thereof comprises the sequences shown in SEQ ID NO. 9, SEQ ID NO. 13 and SEQ ID NO. 17, or comprises SEQ ID NO. 9, SEQ ID NO.
- the DNA methylation molecular marker or a combination thereof comprises the sequences shown in SEQ ID NO. 9, SEQ ID NO. 13 and SEQ ID NO. 17, or comprises SEQ ID NO. 9, SEQ ID NO.
- SEQ ID NO.1 ⁇ SEQ ID NO.8 SEQ ID NO.10 ⁇ SEQ ID NO.12, SEQ ID NO.14 ⁇ SEQ ID NO.16, and SEQ ID NO.18-SEQ ID NO.19 at least one of the sequence or its complementary sequence, or SEQ ID NO.1 to SEQ ID NO.8, SEQ ID NO.10 to SEQ ID NO.12, SEQ ID NO.14 to SEQ ID NO.16, and SEQ ID NO.14 to SEQ ID NO.16 At least one of the continuous fragments of at least 55% of the full length of the sequence shown in ID NO.18-SEQ ID NO.19, or SEQ ID NO.1 ⁇ SEQ ID NO.8, SEQ ID NO.10 ⁇ SEQ ID NO. 12. At least one of the complete complementary sequences of SEQ ID NO.14 to SEQ ID NO.16, and at least 55% of the full-length contiguous fragments of the sequences shown in SEQ ID NO.18 to SEQ ID NO.19.
- the combination of DNA methylation molecular markers further includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.13; or further includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.13.
- DNA methylation molecular markers further includes the sequences shown in SEQ ID NO.9 and SEQ ID NO.17; or further includes SEQ ID NO.9 and SEQ ID NO.17 The complete complement of the sequence shown; or a continuous fragment comprising at least 55% of the full length of the sequence shown in SEQ ID NO. 9 and SEQ ID NO. A contiguous fragment of at least 55% of the fully complementary sequence of the indicated sequence.
- the combination of DNA methylation molecular markers includes the sequences shown in SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17, or includes SEQ ID NO.17 ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and the full complement of SEQ ID NO.17, or including SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17 A contiguous fragment of at least 55% of the full length of the sequence shown in ID NO.17, or at least a contiguous fragment comprising the full length of the sequence shown in SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17 55% of the complete complement of contiguous fragments.
- the combination of DNA methylation molecular markers comprises SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17 SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17, or the complete complement of SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO.
- SEQ ID NO.9, SEQ ID NO.13 a continuous fragment of at least 55% of the full length of the sequences shown in SEQ ID NO.17, or including SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.17
- SEQ ID NO.17 The complete complement of a contiguous fragment of at least 55% of the full length of the sequence shown in ID NO. 9, SEQ ID NO. 13, and SEQ ID NO. 17.
- the combination of DNA methylation molecular markers includes SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 10, SEQ ID NO. Sequences shown in ID NO. 11, SEQ ID NO. 16, and SEQ ID NO. 17, or including SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO.
- the combination of DNA methylation molecular markers further comprises the sequences shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, and SEQ ID NO.16; or Including SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, and the complete complement of the sequence shown in SEQ ID NO.16; or also including SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, and a contiguous fragment of at least 55% of the full length of the sequence shown in SEQ ID NO.16, or further comprising a sequence selected from the group consisting of SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, and SEQ ID NO. . A contiguous fragment of at least 55% of the complete complement of the sequence shown in 16.
- the combination of DNA methylation molecular markers further comprises the sequences shown in SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.11, and SEQ ID NO.13; or Including the complete complement of the sequences shown in SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.11, and SEQ ID NO.13; or including SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.
- the combination of DNA methylation molecular markers includes SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 10, SEQ ID NO. SEQ ID NO. SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.16 and SEQ ID NO.17, or including SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO. 9.
- the combination of DNA methylation molecular markers comprises SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.11, and SEQ ID NO.17 The sequence shown, or the complete complement of SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.11, and SEQ ID NO.17, or SEQ ID NO.3, SEQ ID NO.
- SEQ ID NO.6 SEQ ID NO.9, SEQ ID NO.11, and a contiguous fragment of at least 55% of the full length of the sequences shown in SEQ ID NO.17, or including SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.17
- SEQ ID NO.9 SEQ ID NO.9, SEQ ID NO.11
- SEQ ID NO.17 The complete complement of a contiguous fragment of at least 55% of the full length of the sequences shown in ID NO. 9, SEQ ID NO. 11, and SEQ ID NO. 17.
- the combination of the DNA methylation molecular markers is the sequences shown in SEQ ID NO.1 to SEQ ID NO.11, and SEQ ID NO.13 to -SEQ ID NO.19; or SEQ ID NO.1 to SEQ ID NO.11, and the complete complement of the sequence shown in the sequence shown in SEQ ID NO.13 to-SEQ ID NO.19; or SEQ ID NO.1 to SEQ ID NO.11, and SEQ ID NO. 13 to -SEQ ID NO. 19 is a continuous fragment of at least 55% of the full length of the sequence shown, or SEQ ID NO. 1 to SEQ ID NO. 11, and SEQ ID NO. 13 to - a contiguous fragment of at least 55% of the complete complementarity of the sequence shown in the sequence shown in SEQ ID NO. 19.
- the combination of DNA methylation molecular markers is the sequence shown in SEQ ID NO.1 to SEQ ID NO.19; or the sequence shown in SEQ ID NO.1 to SEQ ID NO.19 or the DNA methylation molecular marker is at least 55% of the full length of the sequence shown in SEQ ID NO.1 ⁇ SEQ ID NO.19 continuous fragment; or SEQ ID NO.1 ⁇ The complete complement of a contiguous fragment of at least 55% of the full length of the sequence shown in ID NO. 19.
- a contiguous fragment of at least 55% of the full length of the sequence shown above may be at least 55% of the full length of the sequence, or at least 58%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc. contiguous fragments.
- the DNA methylation molecular markers are at least 55% of the full-length contiguous fragments of the sequences shown in SEQ ID NO.1 to SEQ ID NO.19, respectively:
- Fragments amplified with primers for any set of SEQ ID NO.20 and SEQ ID NO.21, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.26 and SEQ ID NO.27 are in SEQ ID NO.
- the DNA methylation molecular markers are at least 55% of the full-length contiguous fragments of the sequences shown in SEQ ID NO.1 to SEQ ID NO.19, respectively:
- the DNA methylation molecular marker is a molecular marker for respiratory samples.
- the respiratory tract sample is a lung tissue sample or a respiratory fluid sample.
- kits for detecting benign and malignant pulmonary nodules comprising a reagent for detecting the methylation level of the above-mentioned DNA methylation molecular marker.
- the kit can be applied to detection platforms such as PCR amplification, fluorescence quantitative PCR diagnosis, digital PCR (digital PCR) or detection chips, preferably a platform that can realize high-throughput detection.
- the kit can be used for the following detection platforms: including PCR amplification, fluorescence quantitative PCR, digital PCR, liquid chip method, next-generation sequencing, third-generation sequencing, and second-generation sequencing , Pyrosequencing, Bisulfite Conversion Sequencing, Methylation Chips, Simplified Bisulfite Sequencing, or a combination thereof.
- the detection method is PCR amplification detection, fluorescence quantitative PCR detection, digital PCR detection, chip detection.
- primers and probes are designed for the DNA methylation molecular markers in the above-mentioned specific methylation regions.
- the genomic DNA (gDNA) treated with bisulfite is subjected to multiple PCR amplification; the methylation signal of the detection region is detected by fluorescence quantitative PCR using the specific probe of the DNA methylation molecular marker, and then The naive Bayesian algorithm was used to establish a benign and malignant prediction model, and finally the benign and malignant pulmonary nodules were diagnosed by the established model.
- the reagents in the kit for detecting the methylation level of the above-mentioned DNA methylation molecular markers include primers and probes for fluorescence quantitative PCR detection of the DNA methylation molecular markers, and the Primers and probes are:
- the primers and probes for SEQ ID NO.1 are selected from at least one group of: primers shown in SEQ ID NO.20 and SEQ ID NO.21, and probes shown in SEQ ID NO.22; SEQ ID NO.23 and Primer shown in SEQ ID NO.24, and probe shown in SEQ ID NO.25; primer shown in SEQ ID NO.26 and SEQ ID NO.27, and probe shown in SEQ ID NO.28;
- the primers and probes for SEQ ID NO.2 are selected from at least one group of the following: primers shown in SEQ ID NO.29 and SEQ ID NO.30, and probes shown in SEQ ID NO.31; SEQ ID NO.31; The primers shown in NO.32 and SEQ ID NO.33, and the probe shown in SEQ ID NO.34; the primers shown in SEQ ID NO.35 and SEQ ID NO.36, and the probe shown in SEQ ID NO.37;
- the primers and probes for SEQ ID NO.3 are selected from at least one group of the following: primers shown in SEQ ID NO.38 and SEQ ID NO.39, and probes shown in SEQ ID NO.40; SEQ ID NO.40; The primers shown in NO.41 and SEQ ID NO.42, and the probe shown in SEQ ID NO.43; the primers shown in SEQ ID NO.44 and SEQ ID NO.45, and the probe shown in SEQ ID NO.46;
- the primers and probes for SEQ ID NO.4 are selected from at least one group of the following: primers shown in SEQ ID NO.47 and SEQ ID NO.48, and probes shown in SEQ ID NO.49; SEQ ID NO.49 The primers shown in NO.50 and SEQ ID NO.51, and the probe shown in SEQ ID NO.52; the primers shown in SEQ ID NO.53 and SEQ ID NO.54, and the probe shown in SEQ ID NO.55;
- primers and probes for SEQ ID NO.5 are selected from at least one group of the following: primers shown in SEQ ID NO.56 and SEQ ID NO.57, and probes shown in SEQ ID NO.58; EQ ID The primers shown in NO.59 and SEQ ID NO.60, and the probe shown in SEQ ID NO.61; the primers shown in SEQ ID NO.62 and SEQ ID NO.63, and the probe shown in SEQ ID NO.64;
- primers and probes for SEQ ID NO.6 are selected from at least one group of: primers shown in SEQ ID NO.65 and SEQ ID NO.66, and probes shown in SEQ ID NO.67; SEQ ID NO.67 The primers shown in NO.68 and SEQ ID NO.69, and the probe shown in SEQ ID NO.70; the primers shown in SEQ ID NO.71 and SEQ ID NO.72, and the probe shown in SEQ ID NO.73;
- the primers and probes for SEQ ID NO.7 are selected from at least one group of the following: primers shown in SEQ ID NO.74 and SEQ ID NO.75, and probes shown in SEQ ID NO.76; SEQ ID NO.76 The primers shown in NO.77 and SEQ ID NO.78, and the probe shown in SEQ ID NO.79; the primers shown in SEQ ID NO.80 and SEQ ID NO.81, and the probe shown in SEQ ID NO.82;
- primers and probes for SEQ ID NO.8 are selected from at least one group of: primers shown in SEQ ID NO.83 and SEQ ID NO.84, and probes shown in SEQ ID NO.85; SEQ ID NO.85; The primers shown in NO.86 and SEQ ID NO.87, and the probe shown in SEQ ID NO.88; the primers shown in SEQ ID NO.89 and SEQ ID NO.90, and the probe shown in SEQ ID NO.91;
- the primers and probes for SEQ ID NO.9 are selected from at least one group of the following: primers shown in SEQ ID NO.92 and SEQ ID NO.93, and probes shown in SEQ ID NO.94; SEQ ID NO.94 The primers shown in NO.95 and SEQ ID NO.96, and the probe shown in SEQ ID NO.97; the primers shown in SEQ ID NO.98 and SEQ ID NO.99, and the probe shown in SEQ ID NO.100;
- primers and probes for SEQ ID NO.10 are selected from at least one group of: primers shown in SEQ ID NO.101 and SEQ ID NO.102, and probes shown in SEQ ID NO.103; SEQ ID NO.103 The primers shown in NO.104 and SEQ ID NO.105, and the probe shown in SEQ ID NO.106; the primers shown in SEQ ID NO.107 and SEQ ID NO.108, and the probe shown in SEQ ID NO.109;
- primers and probes for SEQ ID NO.11 are selected from at least one group of: primers shown in SEQ ID NO.110 and SEQ ID NO.111, and probes shown in SEQ ID NO.112; SEQ ID NO.112 The primers shown in NO.113 and SEQ ID NO.114, and the probe shown in SEQ ID NO.115; the primers shown in SEQ ID NO.116 and SEQ ID NO.117, and the probe shown in SEQ ID NO.118;
- primers and probes for SEQ ID NO.12 are selected from at least one group of: primers shown in SEQ ID NO.119 and SEQ ID NO.120, and probes shown in SEQ ID NO.121; SEQ ID NO.121 The primers shown in NO.122 and SEQ ID NO.123, and the probe shown in SEQ ID NO.124; the primers shown in SEQ ID NO.125 and SEQ ID NO.126, and the probe shown in SEQ ID NO.127;
- primers and probes for SEQ ID NO.13 are selected from at least one group of: primers shown in SEQ ID NO.128 and SEQ ID NO.129, and probes shown in SEQ ID NO.130; SEQ ID NO.130; The primers shown in NO.131 and SEQ ID NO.132, and the probe shown in SEQ ID NO.133; the primers shown in SEQ ID NO.134 and SEQ ID NO.135, and the probe shown in SEQ ID NO.136;
- primers and probes for SEQ ID NO.14 are selected from at least one group of: primers shown in SEQ ID NO.137 and SEQ ID NO.138, and probes shown in SEQ ID NO.139; SEQ ID NO.139 The primers shown in NO.140 and SEQ ID NO.141, and the probe shown in SEQ ID NO.142; the primers shown in SEQ ID NO.143 and SEQ ID NO.144, and the probe shown in SEQ ID NO.145;
- the primers and probes for SEQ ID NO.15 are selected from at least one group of: primers shown in SEQ ID NO.146 and SEQ ID NO.147, and probes shown in SEQ ID NO.148; SEQ ID NO.148; 149 and SEQ ID NO.150, and the probe shown in SEQ ID NO.151; the primers shown in SEQ ID NO.152 and SEQ ID NO.153, and the probe shown in SEQ ID NO.154;
- primers and probes for SEQ ID NO.16 are selected from at least one group of: primers shown in SEQ ID NO.155 and SEQ ID NO.156, and probes shown in SEQ ID NO.157; SEQ ID NO.157 The primers shown in NO.158 and SEQ ID NO.159, and the probe shown in SEQ ID NO.160; the primers shown in SEQ ID NO.161 and SEQ ID NO.162, and the probe shown in SEQ ID NO.163;
- primers and probes for SEQ ID NO.17 are selected from at least one group of: primers shown in SEQ ID NO.164 and SEQ ID NO.165, and probes shown in SEQ ID NO.166; SEQ ID NO.166 The primers shown in NO.167 and SEQ ID NO.168, and the probe shown in SEQ ID NO.169; the primers shown in SEQ ID NO.170 and SEQ ID NO.171, and the probe shown in SEQ ID NO.172;
- primers and probes for SEQ ID NO.18 are selected from at least one group of: primers shown in SEQ ID NO.173 and SEQ ID NO.174, and probes shown in SEQ ID NO.175; SEQ ID NO.175 The primers shown in NO.176 and SEQ ID NO.177, and the probe shown in SEQ ID NO.178; the primers shown in SEQ ID NO.179 and SEQ ID NO.180, and the probe shown in SEQ ID NO.181;
- primers and probes for SEQ ID NO.19 are selected from at least one group of: primers shown in SEQ ID NO.182 and SEQ ID NO.183, and probes shown in SEQ ID NO.184; SEQ ID NO.184; The primers shown in NO.185 and SEQ ID NO.186, and the probe shown in SEQ ID NO.187; the primers shown in SEQ ID NO.188 and SEQ ID NO.189, and the probe shown in SEQ ID NO.190;
- primers and probes that have at least 70%, 80%, 90%, 95% or 99% sequence identity with multiple contiguous nucleotides of the above sequences.
- the primers and probes are:
- the primers and probes for SEQ ID NO.1 are: primers shown in SEQ ID NO.23 and SEQ ID NO.24, and probes shown in SEQ ID NO.25;
- primers and probes for SEQ ID NO.2 are: primers shown in SEQ ID NO.35 and SEQ ID NO.36, and probes shown in SEQ ID NO.37;
- primers and probes for SEQ ID NO.3 are: primers shown in SEQ ID NO.38 and SEQ ID NO.39, and probes shown in SEQ ID NO.40;
- primers and probes for SEQ ID NO.4 are: primers shown in SEQ ID NO.47 and SEQ ID NO.48, and probes shown in SEQ ID NO.49;
- primers and probes for SEQ ID NO.5 are: primers shown in SEQ ID NO.59 and SEQ ID NO.60, and probes shown in SEQ ID NO.61;
- primers and probes for SEQ ID NO.6 are: primers shown in SEQ ID NO.71 and SEQ ID NO.72, and probes shown in SEQ ID NO.73;
- primers and probes for SEQ ID NO.7 are: primers shown in SEQ ID NO.80 and SEQ ID NO.81, and probes shown in SEQ ID NO.82;
- primers and probes for SEQ ID NO.8 are: primers shown in SEQ ID NO.89 and SEQ ID NO.90, and probes shown in SEQ ID NO.91;
- primers and probes for SEQ ID NO.9 are: primers shown in SEQ ID NO.92 and SEQ ID NO.93, and probes shown in SEQ ID NO.94;
- primers and probes for SEQ ID NO.10 are: primers shown in SEQ ID NO.104 and SEQ ID NO.105, and probes shown in SEQ ID NO.106;
- primers and probes for SEQ ID NO.11 are: primers shown in SEQ ID NO.110 and SEQ ID NO.111, and probes shown in SEQ ID NO.112;
- primers and probes for SEQ ID NO.12 are: primers shown in SEQ ID NO.125 and SEQ ID NO.126, and probes shown in SEQ ID NO.127;
- primers and probes for SEQ ID NO.13 are: primers shown in SEQ ID NO.128 and SEQ ID NO.129, and probes shown in SEQ ID NO.130;
- primers and probes for SEQ ID NO.14 are: primers shown in SEQ ID NO.143 and SEQ ID NO.144, and probes shown in SEQ ID NO.145;
- primers and probes for SEQ ID NO.15 are: primers shown in SEQ ID NO.152 and SEQ ID NO.153, and probes shown in SEQ ID NO.154;
- primers and probes for SEQ ID NO.16 are: primers shown in SEQ ID NO.158 and SEQ ID NO.159, and probes shown in SEQ ID NO.160;
- primers and probes for SEQ ID NO.17 are: primers shown in SEQ ID NO.167 and SEQ ID NO.168, and probes shown in SEQ ID NO.169;
- primers and probes for SEQ ID NO.18 are: primers shown in SEQ ID NO.179 and SEQ ID NO.180, and probes shown in SEQ ID NO.181;
- primers and probes for SEQ ID NO.19 are: primers shown in SEQ ID NO.188 and SEQ ID NO.189, and probes shown in SEQ ID NO.190;
- primers and probes that have at least 70%, 80%, 90%, 95% or 99% sequence identity with multiple contiguous nucleotides of the above sequences.
- the kit further comprises primers and probes for real-time PCR detection of the internal reference gene ACTB, preferably, the primers and probes are: SEQ ID NO.191 and SEQ ID NO.192 The primers shown, and the probe shown in SEQ ID NO. 193.
- the test sample of the kit is a respiratory sample.
- the respiratory tract sample is a lung tissue sample or a respiratory fluid sample.
- a method for detecting the methylation level of the above-mentioned DNA methylation molecular marker or a combination thereof comprising the following steps:
- step (3) The multiplex PCR product obtained in step (3) is detected by multiplex fluorescence quantitative PCR with the probe for the above-mentioned DNA methylation molecular marker.
- the multiplex PCR reaction conditions are as follows: 98°C for 30s; 15-35 cycles: 98°C for 15s, 58°C-66°C for 15-30s; 72°C for 15-30s; 72°C for 5min; and/ Or, the fluorescent quantitative PCR reaction conditions are as follows: 95°C for 30s; 35 to 50 cycles: 95°C for 10s; 60°C to 64°C for 30s.
- the primers and probes in the above kits are used for detection.
- a method for detecting benign and malignant pulmonary nodules comprising the following steps:
- the sample in the step (2), if the CT value of the internal reference gene is between 10-25, the sample is judged to be a valid sample; otherwise, it is an invalid sample; then the CT value of the internal reference gene is used. Correct the CT value of each DNA methylation molecular marker in the valid samples; if the CT value of the target DNA methylation molecular marker is less than 40, it is judged that the DNA methylation molecular marker has been detected, and the obtained DNA methylation molecular marker is obtained.
- a logistic regression (Logistic Regression) algorithm is used to establish a benign and malignant prediction model of pulmonary nodules.
- the cross-validation method is used to randomly divide the data set into 3 equal parts, and any 2 of them are combined as the training set, and the remaining 1 is used as the test set.
- 3 different training-test set combinations can be obtained, and then the logistic regression algorithm is used to establish a benign and malignant prediction model for the combinations containing different DNA methylation molecular markers in the training set, and when the combination contains a specific A test set of combinations of DNA methylation molecular markers evaluated the classification ability of the model.
- 100 random and independent experiments were performed, and the final classification ability of the model containing specific DNA methylation molecular markers was determined by the average classification ability of the 100 models.
- DNA methylation molecular markers described in SEQ ID NO. 1 to SEQ ID NO. 19 are the corresponding markers described, and the corresponding markers amplified by the corresponding primers.
- a kit for the detection of benign and malignant pulmonary nodules in respiratory samples containing DNA for 19 methylated regions of 10 detection genes CDO1, DAPK, GHSR, HOXA11, HOXB4, LHX9, MIR196A1, PTGER4, SHOX2 and TBX15
- the detection primers and probes of the methylation molecular markers Marker1 to Marker19, the detection region sequence and sequence number of each DNA methylation molecular marker are specifically shown in Table 1 (wherein the underlined part of each region is the following examples of the present invention
- the marker of the corresponding sequence of the fragment amplified by the primers used is preferred:
- the kit designed three pairs of primers and three probes for each of the specific methylation sites in the 19 molecular markers Marker1 to Marker19 used for the detection of benign and malignant pulmonary nodules in respiratory samples (the probes can be fluorescently labeled with Fluorophore such as FAM, VIC and NED), and labeled as combination 1, 2, 3, respectively.
- the combination of primers and probes selected in each molecular marker can be arbitrarily selected and combined with the combinations 1, 2, and 3 of primers and probes in other molecular markers and detected on the same platform.
- the specific primer and probe sequences corresponding to each molecular marker are shown in Table 2:
- primers and probes are as follows:
- Marker1 primer and probe combination 2 Marker2 primer and probe combination 3, Marker3 primer and probe combination 1, Marker4 primer and probe combination 1, Marker5 primer and probe combination 2, Marker6 primer and probe combination Needle combination 3, Marker7 primer and probe combination 3, Marker8 primer and probe combination 3, Marker9 primer and probe combination 1, Marker10 primer and probe combination 2, Marker11 primer and probe combination 1, Marker12 Primer and probe set 3 of Marker13, Primer and probe set 1 of Marker13, Primer and probe set 3 of Marker14, Primer and probe set 3 of Marker15, Primer and probe set 2 of Marker16, Primer and probe set of Marker17 Combination 2, primer and probe combination 3 of Marker18, primer and probe combination 3 of Marker19.
- the corresponding primers and probes will be selected according to different combinations of methylated molecular markers.
- the kit also includes primers and probes of the internal reference gene ACTB, and its sequence is specifically shown in Table 3:
- the kit described in Example 1 is used to detect the methylation levels of Marker1 to Marker19 in respiratory samples.
- a method for detecting methylation levels of DNA methylation molecular markers comprising the following steps:
- the extracted gDNA is converted into bisulfite, and 50-100 ng gDNA is put into, preferably 75 ng in this embodiment, and is operated according to the Zymo DNA Methylation-Direct MagPrep instruction manual, so that the deamination of cytosine that does not occur methylation in the DNA is converted into. uracil, while methylated cytosine remains unchanged. All bisulfite-converted DNA products were used for multiplex PCR amplification.
- the transformed DNA products are all amplified by multiplex PCR, and the reaction components are: a specific combination of molecular markers and a primer mixture of an internal reference gene, wherein the concentration of each primer is 200nM-300nM, and this embodiment is preferably 300nM ; the concentration of magnesium ions is 1-3mM, preferably 1.5mM in this embodiment; the concentration of dNTP mixture is 200-600uM, preferably 400uM in this embodiment; the reaction enzyme is Hot Start High-Fidelity DNA Polymerase (NEB, Cat#M0515), the unit number of one reaction is 1-3U, and 2U is preferred in this embodiment.
- the preparation of the multiplex PCR reaction system is shown in Table 4:
- the specific reaction conditions are: pre-denaturation, 98°C, 30s; 5-10 cycles of reaction 1, preferably 5 cycles in this embodiment: denaturation, 98°C, 15s; annealing, 58-66°C, 15-30s, this embodiment It is preferably 58°C, 15s; the extension is 68°C, 15-30s, and this embodiment is preferably 15s. 10-15 cycles of reaction 2, preferably 13 cycles in this embodiment: denaturation, 98°C, 15s; annealing, 58-66°C, 15-30s, this embodiment is preferably 62°C, 15s; extension at 68°C, 15-30s , 15s is preferred in this embodiment.
- the multiplex PCR products are diluted 1-5 times, preferably 5 times in this embodiment.
- the fluorescent quantitative PCR reaction components are: primer-probe mixture, wherein the primer concentration is 200-900nM, preferably 400nM in this embodiment; the probe concentration is 100-200nM, this embodiment is preferably 200nM.
- the reaction enzyme mixture used is 1 times Universal qPCR Master Mix (NEB, Cat#M3003), one reaction is 10ul system.
- the specific reaction conditions are: pre-denaturation, 95°C, 5 min; 40-50 cycles, preferably 40 cycles in this embodiment: denaturation, 95°C for 15s; annealing, 60-64°C, preferably 62°C, 30s in this embodiment, signal collect.
- the configuration of the qPCR fluorescence quantitative reaction system is shown in Table 6:
- test sample is a valid sample according to the CT value of the internal reference gene in the sample determined by the fluorescence quantitative PCR reaction. If the CT value of the internal reference gene in the test sample is between 10-25, the sample is judged as a valid sample. ; If the CT value of the internal reference gene in the test sample is less than 10, the initial input amount of the sample is excessive; if the CT value of the internal reference gene in the test sample is >25, the initial sample input amount is insufficient. Samples with excess or insufficient initial input are judged as invalid samples and will not be included in testing and analysis.
- This embodiment provides a method for detecting molecular markers in standard products, and the detection steps are as follows:
- NA12878 DNA was treated with Single Cell Kit (Qiagen, Cat#150343) and Mung Bean Nuclease (NEB, Cat#M0250L) to prepare 0% methylation standard;
- the prepared 0% methylation standard was treated with CpG Methyltransferase (M.SssI) to give a 100% methylation standard.
- the multiplex PCR primer mixture is primers for 21 molecular markers and an internal reference gene.
- test sample is a valid sample according to the CT value of the internal reference gene ACTB in the sample determined by the fluorescence quantitative PCR reaction. If the CT value of the internal reference gene in the test sample is between 10-25, the sample is judged to be valid. sample;
- the primer-probe combination of each molecular marker is the preferred combination in Example 1.
- a negative control is set for each test.
- the negative control uses water as a template to perform multiplex PCR, and the obtained negative control multiplex PCR products are then subjected to fluorescence quantitative PCR for each specific molecular marker. If there is no detection signal in the negative control, it is determined that there is no exogenous contamination in the entire experimental operation.
- DNA extraction was performed on paraffin section samples of lung tissue, as described in Example 2.
- Example 5 Performance of different molecular marker combinations in the detection of benign and malignant pulmonary nodules in lung tissue samples
- 19 molecular markers including Marker1 to Marker19 are used to detect and analyze the 57 pulmonary nodule tissue samples in Example 4 through the experimental method of Example 2.
- the specific detection kits, test methods and data judgment Treatment was as described in Example 2, and primer and probe combinations were as preferred in Example 1.
- 3 different training-test set combinations can be obtained, and then the logistic regression algorithm is used to establish a benign and malignant prediction model for the combinations containing different DNA methylation molecular markers in the training set, and when the combination contains a specific A test set of combinations of DNA methylation molecular markers evaluated the classification ability of the model.
- 100 random and independent experiments were performed, and the final classification ability of the model containing specific DNA methylation molecular markers was determined by the average classification ability of the 100 models.
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Abstract
L'invention concerne un marqueur moléculaire de méthylation d'ADN pour détecter des nodules pulmonaires bénins et malins. Le marqueur moléculaire de méthylation de l'ADN est choisi parmi les séquences présentées dans SEQ ID NO : 1 à SEQ ID NO : 19, ou l'une quelconque ou une combinaison d'au moins deux fragments continus comprenant au moins 55 % de la longueur totale de la séquence. En variante, le marqueur moléculaire de méthylation de l'ADN est choisi parmi les séquences complémentaires complètes des séquences présentées dans SEQ ID NO : 1 à SEQ ID NO : 19, ou l'une quelconque ou une combinaison d'au moins deux fragments continus comprenant au moins 55 % de la longueur totale de la séquence. La présente invention concerne également un kit de détection du marqueur moléculaire de méthylation de l'ADN susmentionné et un procédé de détection. Le marqueur moléculaire de méthylation de l'ADN est fortement corrélé au cancer du poumon. Particulièrement lorsqu'il est utilisé en combinaison, la sensibilité et la spécificité de la détection des nodules pulmonaires bénins et malins peuvent être encore améliorées, le taux de détection des nodules pulmonaires malins est amélioré et le taux de faux positifs est réduit. Les amorces et les sondes du kit permettent de surmonter l'inconvénient des amorces et des sondes multiples interférant entre elles lors de l'amplification et de la détection par PCR multiplex, et les performances quantitatives sont équivalentes à celles d'une région unique.
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US18/337,000 US20240084393A1 (en) | 2020-12-17 | 2023-06-17 | Dna methelation molecular markers for identifying benignity or malignancy of lung nodule and applications of the same |
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CNPCT/CN2021/086902 | 2021-04-13 |
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US18/337,000 Continuation-In-Part US20240084393A1 (en) | 2020-12-17 | 2023-06-17 | Dna methelation molecular markers for identifying benignity or malignancy of lung nodule and applications of the same |
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CN110229908B (zh) * | 2019-07-03 | 2023-01-31 | 四川沃文特生物技术有限公司 | 用于早期检测肺癌基因甲基化的引物、探针及试剂盒 |
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CN113637745B (zh) * | 2020-04-27 | 2022-12-06 | 广州市基准医疗有限责任公司 | 用于检测肺结节良恶性的甲基化分子标记物或其组合和应用 |
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