Lung cancer methylation marker, detection kit and application thereof

Abstract

The present invention belongs to the field of biotechnology and relates to a group of biomarkers and related detection kits. The biomarkers provided by the present invention include a combination of FOXD3 gene, FOXI2 gene, RASSF1A gene, SHOX2 gene and SOX17 gene, and the marker combination can be used for lung cancer diagnosis. Detection primers and probes are designed based on the marker combination, and the kit further developed is used for diagnosing lung cancer and / or assessing lung cancer risk, etc. The use of the biomarkers described in the present invention can avoid whole genome sequencing, greatly save the amount of sequencing data, have excellent detection sensitivity and specificity, and are low in cost, high in depth, and more accurate in detecting the methylation levels of related genes. The present invention provides new ideas for lung cancer screening, postoperative and prognostic evaluation, and is of great significance for clarifying the molecular mechanisms related to lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and relates to a lung cancer methylation marker combination, a detection kit and applications thereof. Background Art

[0002] Currently, clinical lung cancer screening and diagnosis primarily rely on low-dose spiral CT (LDCT) and chest X-ray imaging. In addition, a variety of other adjunctive methods exist to support lung cancer diagnosis, such as fiberoptic bronchoscopy, sputum cytology, and tumor marker testing. However, long-term research has revealed that each of these methods has its pros and cons, including high cost, high investment, low sensitivity, high rates of missed diagnoses, high false-positive rates, and limitations in early diagnosis. Some are also difficult to perform and can cause pain and inconvenience to patients. Therefore, sensitive and specific biomarkers are urgently needed.

[0003] Recent advances in lung cancer epigenetic research have profound implications for the early diagnosis and treatment of lung cancer. Evidence suggests that hypermethylation of CpG islands in tumor suppressor gene promoters is a key mechanism of gene inactivation. Transcriptional inactivation of various genes can affect the cell cycle, DNA repair, apoptosis, and other processes, and is closely linked to the development and progression of cancer. Gene methylation refers to the process by which enzymes selectively add methyl groups to cytosine (C) within CpG dinucleotides on DNA molecules, forming 5'-methylcytosine. Methylation of CpG islands in gene promoter regions is a common phenomenon in lung cancer patients. Currently reported lung cancer-related methylation gene markers suffer from low sensitivity (35.5%) and low specificity (73%), limiting their clinical application. High sensitivity of a methylation gene marker for a specific cancer or specific groups indicates a low rate of missed diagnosis; similarly, high specificity indicates a low rate of misdiagnosis. Therefore, the sensitivity and specificity of a methylation gene marker test are two important indicators of its usefulness.

[0004] Therefore, further in-depth research on lung cancer methylation gene markers and the development of highly sensitive and specific detection reagents for lung cancer-related methylation genes and their combinations, thereby providing more effective means for the accuracy of lung cancer diagnosis, are issues that urgently need to be addressed in current methylation gene marker detection technology. Summary of the Invention

[0005] In response to the limitations of existing methods, the purpose of the present invention is to provide a lung cancer methylation marker, design primers and probes for specific regions of the screened lung cancer methylation marker, and establish a corresponding detection system to achieve the purpose of high accuracy, high sensitivity and high specificity in the detection of lung cancer samples based on the screened lung cancer methylation marker combination, providing valuable reference information for lung cancer diagnosis.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a lung cancer methylation marker, a detection kit and applications thereof.

[0007] One of the objects of the present invention is to provide a lung cancer methylation marker, characterized in that it comprises a combination of FOXD3 gene, FOXI2 gene, RASSF1A gene, SHOX2 gene and SOX17 gene.

[0008] Another object of the present invention is to provide use of a substance for detecting the gene methylation marker described above in the preparation of a lung cancer diagnosis product.

[0009] Another object of the present invention is to provide a detection material for diagnosing lung cancer, which comprises the specific detection primers and / or specific detection probes for the gene methylation markers described above.

[0010] Another object of the present invention is to provide the use of the above-mentioned detection substance in the preparation of lung cancer diagnosis products.

[0011] Another object of the present invention is to provide a lung cancer diagnosis product, which comprises the detection substance described above.

[0012] In a preferred embodiment, the lung cancer diagnostic product is a kit comprising the detection substance described above.

[0013] As described above, the lung cancer methylation marker, detection kit and application thereof of the present invention have the following beneficial effects:

[0014] The present invention provides a lung cancer methylation marker and detection kit with excellent performance. Compared with existing detection methods such as fluorescent PCR detection method, second-generation sequencing detection method, etc., the detection performance is greatly improved, and it has the characteristics of high detection accuracy, sensitivity and specificity. It is also easy to carry out and convenient for clinical promotion. It can be used for liquid biopsy, has low cost, and can reduce the medical costs of liquid patients. DETAILED DESCRIPTION

[0015] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0016] The purpose of this invention is to provide more effective lung tumor-related methylation markers, improve the efficiency of early lung tumor screening and diagnosis, and address the problems of low early lung cancer diagnosis rates and heavy clinical treatment burdens. The goal is to provide a set of effective lung tumor methylation markers and a highly effective marker combination.

[0017] This study uses fluorescent PCR to detect methylation of markers in the buffy coat (mostly white blood cells) and lung cancer tissue. This study has preliminarily confirmed the potential of this marker for blood-based lung cancer testing. An ideal lung cancer methylation marker should possess the following characteristics:

[0018] (1) Low DNA methylation level in the tunica buffy coat;

[0019] (2) The DNA methylation level in lung cancer tissue is high.

[0020] Methylation marker detection is performed using methylation-specific PCR (MSP). The basic principle of MSP is that only methylated sequence templates generate amplification signals during detection, while unmethylated sequence templates do not. This method can be achieved by designing methylation-specific sequences in primers or probes, or by designing methylation-specific primer and probe pairs simultaneously. The main steps include:

[0021] 1) Design primers and probes based on the marker sequence in a region suitable for MSP detection (usually a CpG-rich region);

[0022] 2) Extract nucleic acids from buffy coat and lung cancer tissue samples;

[0023] 3) Bisulfite treatment of nucleic acids converts unmethylated cytosine into uracil, while methylated cytosine maintains the sequence unchanged;

[0024] 4) Perform fluorescence PCR detection.

[0025] Markers meeting the aforementioned characteristics, as determined in buffy coat and tissue samples, demonstrated their potential for detecting lung cancer in blood. These markers were then validated in plasma samples, including controls, lung cancer patients, and individuals with lung nodules and inflammation, to analyze reference levels and the performance of these marker combinations.

[0026] Because the amount of free DNA in plasma from a single sample is limited, the target can be pre-amplified before performing fluorescent PCR detection to enable the detection of as many methylation sites as possible using the minimum amount of DNA. The main steps include:

[0027] 1) Nucleic acid extraction from plasma samples;

[0028] 2) Bisulfite treatment of nucleic acids converts unmethylated cytosine into uracil, while methylated cytosine maintains the sequence unchanged;

[0029] 3) Pre-amplification and dilution of the target;

[0030] 4) Perform fluorescence PCR detection.

[0031] Through extensive exploratory research, the present inventors have developed a panel of biomarkers for diagnosing lung cancer, comprising a combination of the FOXD3, FOXI2, RASSF1A, SHOX2, and SOX17 genes. These biomarkers exhibit excellent sensitivity and specificity, addressing the current challenges of low diagnostic accuracy, sensitivity, and specificity in lung cancer (including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)). This research led to the present invention.

[0032] The present invention provides a group of biomarkers for diagnosing lung cancer, which comprises a combination of the FOXD3 gene, the FOXI2 gene, the RASSF1A gene, the SHOX2 gene, and the SOX17 gene. Specifically, the group comprises the CpG island of the FOXD3 gene or the CpG island of the FOXD3 gene promoter, the CpG island of the FOXI2 gene or the CpG island of the FOXD3 gene promoter, the CpG island of the RASSF1A gene or the CpG island of the RASSF1A gene promoter, the CpG island of the SHOX2 gene or the CpG island of the SHOX2 gene promoter, and the CpG island of the SOX17 gene or the combination of the CpG island of the SOX17 gene promoter. By screening the biomarkers, lung cancer and / or the assessment of whether an individual is susceptible to lung cancer can be performed at low cost, with high depth, and more accurately.

[0033] In some preferred embodiments, the methylated region of the CpG island of the FOXD3 gene or the CpG island of the promoter of the FOXD3 gene is the sequence of chr1:63785908-63785999, or an active fragment thereof;

[0034] The methylated region of the CpG island of the FOXI2 gene or the CpG island of the promoter of the FOXI2 gene is the sequence of chr10:129534759-129534851, or an active fragment thereof;

[0035] The methylated region of the CpG island of the RASSF1A gene or the CpG island of the promoter of the RASSF1A gene is the sequence of chr3:50378061-50378154, or an active fragment thereof;

[0036] The methylated region of the CpG island of the SHOX2 gene or the CpG island of the promoter of the SHOX2 gene is the sequence of chr3:157821339-157821429, or an active fragment thereof;

[0037] The methylated region of the CpG island of the SOX17 gene or the CpG island of the promoter of the SOX17 gene is the sequence of chr8:55370987-55371098, or an active fragment thereof.

[0038] In the present invention, the active fragment generally refers to a substance that can be detected by detecting the relevant methylation level in the corresponding gene, and can specifically be, for example, DNA or its mRNA or a homolog that retains its function. The method for selecting active fragments should be known to those skilled in the art. The homolog of the gene can be a sequence having 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more homology with the gene.

[0039] In other preferred embodiments, the methylated region of the CpG island of the FOXD3 gene or the CpG island of the promoter of the FOXD3 gene is the sequence of chr1:63785908-63785999;

[0040] The methylated region of the CpG island of the FOXI2 gene or the CpG island of the promoter of the FOXI2 gene is the sequence of chr10:129534759-129534851;

[0041] The methylated region of the CpG island of the RASSF1A gene or the CpG island of the promoter of the RASSF1A gene is the sequence of chr3:50378061-50378154;

[0042] The methylated region of the CpG island of the SHOX2 gene or the CpG island of the promoter of the SHOX2 gene is the sequence of chr3:157821339-157821429;

[0043] The methylation region of the CpG island of the SOX17 gene or the CpG island of the promoter of the SOX17 gene is the sequence of chr8:55370987-55371098.

[0044] The inventors discovered that methylation of the chr1:63785908-63785999 region of the FOXD3 gene, methylation of the chr10:129534759-129534851 region of the FOXI2 gene, methylation of the chr3:50378061-50378154 region of the RASSF1A gene, methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and methylation of the chr8:55370987-55371098 region of the SOX17 gene are closely related to the occurrence and development of lung cancer. Patients with lung cancer are highly likely to have the above-mentioned methylations at the same time.

[0045] The present invention also provides the use of the gene methylation markers described above in the preparation of lung cancer diagnostic products. Such diagnostics include early differential diagnosis of lung cancer, assessment and dynamic monitoring of residual microlesions, auxiliary assessment of lung cancer recurrence and prognosis, and evaluation of drug efficacy. These lung cancer diagnostic products can be in any suitable form, including but not limited to primers, probes, kits, chips, membrane strips, protein arrays, and the like.

[0046] The present invention also provides the use of a substance for detecting the aforementioned gene methylation marker in the preparation of a lung cancer diagnostic product. Such diagnostics include early differential diagnosis of lung cancer, assessment and dynamic monitoring of residual microlesions, auxiliary assessment of lung cancer recurrence and prognosis, and evaluation of drug efficacy. Such lung cancer diagnostic products can take any suitable form, including but not limited to primers, probes, kits, chips, membrane strips, protein arrays, and the like.

[0047] In the present invention, the early differential diagnosis of lung cancer can specifically be used to confirm whether an individual has lung cancer or is more likely to have lung cancer. For example, when the individual's genome simultaneously contains methylation of the chr1:63785908-63785999 region of the FOXD3 gene, methylation of the chr10:129534759-129534851 region of the FOXI2 gene, methylation of the chr3:50378061-50378154 region of the RASSF1A gene, methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and methylation of the chr8:55370987-55371098 region of the SOX17 gene, the individual is considered to have lung cancer or is more likely to have lung cancer. For example, if at least one of the following is absent in an individual's genome: methylation of the chr1:63785908-63785999 region of the FOXD3 gene, methylation of the chr10:129534759-129534851 region of the FOXI2 gene, methylation of the chr3:50378061-50378154 region of the RASSF1A gene, methylation of the chr3:157821339-157821429 region of the SHOX2 gene, or methylation of the chr8:55370987-55371098 region of the SOX17 gene, the individual is considered to be free of lung cancer or more likely to be free of lung cancer. Since early diagnosis of lung cancer is often difficult, the introduction of such screening can more accurately diagnose lung cancer in individuals.

[0048] In the present invention, the assessment and dynamic monitoring of residual microlesions can be specifically used to confirm whether an individual has residual microlesions. For example, when the individual's genome simultaneously contains methylation of the chr1:63785908-63785999 region of the FOXD3 gene, methylation of the chr10:129534759-129534851 region of the FOXI2 gene, methylation of the chr3:50378061-50378154 region of the RASSF1A gene, methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and methylation of the chr8:55370987-55371098 region of the SOX17 gene, then the individual is considered to have a high probability of residual microlesions. For another example, when at least one of the methylation regions of the FOXD3 gene's chr1:63785908-63785999, the FOXI2 gene's chr10:129534759-129534851, the RASSF1A gene's chr3:50378061-50378154, the SHOX2 gene's chr3:157821339-157821429, and the SOX17 gene's chr8:55370987-55371098 is not present in an individual's genome, it is considered unlikely that the individual has residual microlesions.

[0049] In the present invention, the auxiliary judgment of lung cancer recurrence and prognosis refers to the prediction of the risk and prognosis of lung cancer recurrence, which can be specifically used to confirm the possibility of recurrence or worsening tendency of individual lung cancer, and can be used to guide clinical diagnosis and treatment. For example, when the individual's genome is simultaneously methylated in the chr1:63785908-63785999 region of the FOXD3 gene, the chr10:129534759-129534851 region of the FOXI2 gene, the chr3:50378061-50378154 region of the RASSF1A gene, the chr3:157821339-157821429 region of the SHOX2 gene, and the chr8:55370987-55371098 region of the SOX17 gene, it is considered that the individual's lung cancer is more likely to recur or the lung cancer is more likely to worsen. For another example, when at least one of the methylation patterns in the chr1:63785908-63785999 region of the FOXD3 gene, the methylation pattern in the chr10:129534759-129534851 region of the FOXI2 gene, the methylation pattern in the chr3:50378061-50378154 region of the RASSF1A gene, the methylation pattern in the chr3:157821339-157821429 region of the SHOX2 gene, and the methylation pattern in the chr8:55370987-55371098 region of the SOX17 gene in an individual's genome is not present, it is considered that the individual is unlikely to relapse or the possibility of lung cancer worsening is low.

[0050] In the present invention, the drug efficacy evaluation can be specifically used to determine whether a certain drug or treatment is effective for an individual. For example, if the individual's genome simultaneously contains methylation of the chr1:63785908-63785999 region of the FOXD3 gene, methylation of the chr10:129534759-129534851 region of the FOXI2 gene, methylation of the chr3:50378061-50378154 region of the RASSF1A gene, methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and methylation of the chr8:55370987-55371098 region of the SOX17 gene, then the drug or treatment is considered to be ineffective for the individual. For another example, when at least one of the methylation regions of the FOXD3 gene's chr1:63785908-63785999, the FOXI2 gene's chr10:129534759-129534851, the RASSF1A gene's chr3:50378061-50378154, the SHOX2 gene's chr3:157821339-157821429, and the SOX17 gene's chr8:55370987-55371098 is not present in an individual's genome, a certain drug or treatment is considered to be effective for the individual.

[0051] In the present invention, the substance is specifically a substance used to detect the methylation level of the gene or its active fragment in samples such as peripheral or intratumoral blood, plasma, serum, urine, feces, sputum, fresh tissue, extracts of fresh tissue or fecal matter, paraffin sections, and coarse needle aspiration samples. Those skilled in the art can use appropriate methods to detect the methylation present in the above samples. For example, the specific methods that can be used can be Sanger sequencing, whole genome sequencing, whole exome sequencing, targeted sequencing, and the like. The substance used to detect methylation in a gene or its active fragment is generally corresponding to the detection method.

[0052] For example, the substance for detecting methylation in the chr1:63785908-63785999 region of the FOXD3 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr1:63785908-63785999 region of the FOXD3 gene or its active fragment. For another example, the substance for detecting methylation in the chr10:129534759-129534851 region of the FOXI2 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr10:129534759-129534851 region of the FOXI2 gene or its active fragment. For another example, the substance for detecting methylation in the chr3:50378061-50378154 region of the RASSF1A gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr3:50378061-503781 For example, the substance for detecting methylation in the chr3:157821339-157821429 region of the SHOX2 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr3:157821339-157821429 region of the SHOX2 gene or its active fragment; for example, the substance for detecting methylation in the chr8:55370987-55371098 region of the SOX17 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr8:55370987-55371098 region of the SOX17 gene or its active fragment; for example, the specific probe preferably includes an isolated polynucleotide that is at least partially complementary to the target sequence, so that it can hybridize with the target sequence to detect whether there is specific methylation on the target. The substance for detecting the methylation level in a gene or its active fragment may also include various other related detection reagents.

[0053] In some preferred embodiments, the substance for detecting the gene methylation markers as described above includes a substance for detecting methylation of the chr1:63785908-63785999 region of the FOXD3 gene, a substance for detecting methylation of the chr10:129534759-129534851 region of the FOXI2 gene, a substance for detecting methylation of the chr3:50378061-50378154 region of the RASSF1A gene, a substance for detecting methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and a combination of substances for detecting methylation of the chr8:55370987-55371098 region of the SOX17 gene.

[0054] In some preferred embodiments, the substance for detecting the gene methylation markers described above includes specific detection primers and / or specific detection probes for detecting the gene methylation markers described above.

[0055] The specific detection primers include at least any one of the following primer pairs A to E:

[0056] Primer pair A targeting the methylated region of the FOXD3 gene, the sequences of which are shown in SEQ ID NOs: 1-2;

[0057] Primer pair B targeting the methylated region of the FOXI2 gene, the sequences of which are shown in SEQ ID NOs: 3-4;

[0058] Primer pair C targeting the methylated region of the RASSF1A gene, the sequences of which are shown in SEQ ID NOs: 5-6;

[0059] Primer pair D targeting the methylated region of the SHOX2 gene, the sequences of which are shown in SEQ ID NOs: 7-8;

[0060] Primer pair E targeting the methylated region of the SOX17 gene, the sequences of which are shown in SEQ ID NOs: 9-10;

[0061] The specific detection probe includes at least any one of the following probes A to E:

[0062] Probe A targeting the methylated region of the FOXD3 gene, the sequence of which is shown in SEQ ID NO: 11;

[0063] Probe B targeting the methylated region of the FOXI2 gene, the sequence of which is shown in SEQ ID NO: 12;

[0064] Probe C targeting the methylated region of the RASSF1A gene, the sequence of which is shown in SEQ ID NO: 13;

[0065] Probe D targeting the methylated region of the SHOX2 gene, the sequence of which is shown in SEQ ID NO: 14;

[0066] The sequence of probe E targeting the methylated region of the SOX17 gene is shown in SEQ ID NO: 15.

[0067] The present invention also provides a detection object for lung cancer detection, which comprises the specific detection primers and / or specific detection probes of the gene methylation markers described above.

[0068] In some preferred embodiments, the specific detection primers include at least any one of the following primer pairs A to E:

[0069] Primer pair A targeting the methylated region of the FOXD3 gene, the sequences of which are shown in SEQ ID NOs: 1-2;

[0070] Primer pair B targeting the methylated region of the FOXI2 gene, the sequences of which are shown in SEQ ID NOs: 3-4;

[0071] Primer pair C targeting the methylated region of the RASSF1A gene, the sequences of which are shown in SEQ ID NOs: 5-6;

[0072] Primer pair D targeting the methylated region of the SHOX2 gene, the sequences of which are shown in SEQ ID NOs: 7-8;

[0073] Primer pair E targeting the methylated region of the SOX17 gene, the sequences of which are shown in SEQ ID NOs: 9-10;

[0074] The specific detection probe includes at least any one of the following probes A to E:

[0075] Probe A targeting the methylated region of the FOXD3 gene, the sequence of which is shown in SEQ ID NO: 11;

[0076] Probe B targeting the methylated region of the FOXI2 gene, the sequence of which is shown in SEQ ID NO: 12;

[0077] Probe C targeting the methylated region of the RASSF1A gene, the sequence of which is shown in SEQ ID NO: 13;

[0078] Probe D targeting the methylated region of the SHOX2 gene, the sequence of which is shown in SEQ ID NO: 14;

[0079] The sequence of probe E targeting the methylated region of the SOX17 gene is shown in SEQ ID NO: 15.

[0080] The present invention also provides a lung cancer diagnosis product, which includes the detection substance described above.

[0081] In the present invention, the lung cancer diagnostic product can be in any suitable product form, including but not limited to a kit, a chip, a membrane strip, a protein array, etc.

[0082] When the lung cancer diagnostic product is a kit, it comprises the detection substances as described above, which are used to amplify the methylated regions, namely, substances for detecting methylation of the chr1:63785908-63785999 region of the FOXD3 gene, substances for detecting methylation of the chr10:129534759-129534851 region of the FOXI2 gene, substances for detecting methylation of the chr3:50378061-50378154 region of the RASSF1A gene, substances for detecting methylation of the chr3:157821339-157821429 region of the SHOX2 gene, and substances for detecting methylation of the chr8:55370987-55371098 region of the SOX17 gene; reagents for processing the methylated regions; and primers for sequencing the amplified products.

[0083] In the detection kit provided by the present invention, the substance for detecting methylation in a gene or its active fragment generally corresponds to the detection method. For example, the substance for detecting methylation in the chr1:63785908-63785999 region of the FOXD3 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr1:63785908-63785999 region of the FOXD3 gene or its active fragment. For another example, the substance for detecting methylation in the chr10:129534759-129534851 region of the FOXI2 gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr10:129534759-129534851 region of the FOXI2 gene or its active fragment. For another example, the substance for detecting methylation in the chr3:50378061-50378154 region of the RASSF1A gene or its active fragment includes a specific primer and / or probe for detecting methylation in the chr10:129534759-129534851 region of the FOXI2 gene or its active fragment. A specific primer and / or probe for methylation in the chr3:50378061-50378154 region of the gene A or its active fragment; for another example, the substance for detecting methylation in the chr3:157821339-157821429 region of the gene SHOX2 or its active fragment includes a specific primer and / or probe for detecting methylation in the chr3:157821339-157821429 region of the gene SHOX2 or its active fragment; for another example, the substance for detecting methylation in the chr8:55370987-55371098 region of the gene SOX17 or its active fragment includes a specific primer and / or probe for detecting methylation in the chr8:55370987-55371098 region of the gene SOX17 or its active fragment; for another example, the specific probe preferably includes an isolated polynucleotide that is at least partially complementary to the target sequence, so that it can hybridize with the target sequence to detect whether specific methylation exists on the target.

[0084] Optionally, the kit may also include various other related detection reagents, including but not limited to one or more of nucleic acid extraction reagents, reagents for amplifying targets, bisulfite conversion reagents, reagents for evaluating the methylation status of targets, internal reference genes, negative controls and positive controls.

[0085] Wherein, the reagent for amplifying the target comprises an enzyme, for example, an enzyme for a polynucleotide amplification reaction, wherein the polynucleotide amplification reaction is selected from the following group: polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), primer extension, rolling circle amplification (RCA), self-sustaining sequence replication (3SR) and loop-mediated isothermal amplification (LAMP).

[0086] Among them, the reagent for evaluating the methylation status of the target is a reagent to be used in a polynucleotide methylation detection method, and the detection method is selected from the following group: mass spectrometry, bisulfite sequencing, methylation-specific PCR (MSP), methylated DNA immunoprecipitation (MeDIP or mDIP), pyrophosphate sequencing, HpaII small fragment enrichment by ligation-mediated PCR (HELP assay), landmark genome scanning (RLGS), molecular break-light assay of DNA adenine methyltransferase activity, methyl-sensitive Southern blotting and high-resolution dissolution (HRM) analysis, etc.

[0087] In one embodiment, the reagent used to assess the methylation status of a target is a chemical reagent, such as bisulfite or sodium bisulfite.

[0088] In one embodiment, the reagent used to assess the methylation status of a target is a biological reagent, such as a polypeptide or an enzyme. In another embodiment, the enzyme is a polynucleotide polymerase; the polynucleotide polymerase is configured for PCR; the polynucleotide polymerase can be a DNA polymerase, such as a DNA polymerase that does not have 3' to 5' exonuclease activity.

[0089] In one embodiment, the internal reference gene is ACTB.

[0090] Kits provided herein may also comprise separate containers (eg, vials) for one or more components and / or instructions for using the kit or system.

[0091] The present invention also provides uses of the kit as described above, for early differential diagnosis (early screening and early diagnosis) of lung cancer, assessment of minimal residual lesions (MRD) and dynamic monitoring, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation and drug resistance monitoring, etc.

[0092] The primers, probes, kits, detection systems, systems, or articles of manufacture described herein can be configured for any appropriate use or purpose. For example, the primers, probes, kits, detection systems, systems, or articles of manufacture described herein can be configured for use in assessing the presence of lung cancer in a subject, or for use in analyzing or profiling lung cancer in a subject.

[0093] The inventors have conducted extensive experimental studies and provided a set of biomarkers including FOXD3, FOXI2, RASSF1A, SHOX2, and SOX17 genes through a high-throughput screening method. The biomarkers provided by the present invention can avoid whole-genome sequencing, greatly saving the amount of sequencing data required. Detection primers and probes are designed for the methylation regions of the screened biomarkers. By detecting the methylation levels of the main pathogenic genes related to lung cancer at one time, the method has excellent detection sensitivity and specificity, is simple and rapid to operate, and has low cost, high depth, and more accurate detection of related methylation. Based on the biomarkers, corresponding detection kits can be further developed for clinical molecular diagnosis of lung cancer. They can be potentially applied to early differential diagnosis (early screening and early diagnosis) of lung cancer, assessment of minimal residual disease (MRD) and dynamic monitoring, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation, and drug resistance monitoring. Bioinformatics and other technical means can also be used to further explore the functions and internal mechanisms of lung cancer-related genes, providing a theoretical basis for early detection, early prevention, early diagnosis, early treatment, and future related targeted therapies of lung cancer. In addition, when analyzing target combinations, mathematical models of machine learning can also be introduced, such as linear regression, support vector regression, ridge regression, random forest, etc.

[0094] The present invention designs specific primers and probes targeting specific lung cancer methylation marker regions identified through screening, constructs a detection system, and then tests the performance of the identified lung cancer markers in collected lung cancer and normal samples. Testing has shown that the biomarkers of the present invention can detect lung cancer with high accuracy and specificity, suggesting that the biomarker combination has the potential to serve as a noninvasive diagnostic and prognostic marker for lung cancer.

[0095] The present invention also provides a detection system, which includes: 10 μL of converted DNA obtained after treatment with a methylation reagent, 2.5 μL of a primer and probe premix containing a detection region; and 12.5 μL of a PCR reagent; wherein in the primer and probe premix, the primer sequences are as shown in SEQ ID NOs: 1 to 10, and the final concentration of each primer is 500 nM; the probe sequences are as shown in SEQ ID NOs: 11 to 15, and the final concentration of each probe is 200 nM.

[0096] The present invention also provides a detection system, which comprises: 10 μL of converted DNA obtained after bisulfite treatment, 2.5 μL of primer and probe premix containing the detection region; 12.5 μL of PCR reagent ( Universal Probe qPCR Master Mix (NEB); wherein the primer and probe premix comprises primer sequences as shown in SEQ ID NOs: 1 to 10, with a final concentration of each primer of 500 nM, and probe sequences as shown in SEQ ID NOs: 11 to 15, with a final concentration of each probe of 200 nM.

[0097] The present invention also provides an in vitro detection method, which includes collecting a sample to be tested; extracting and purifying DNA in the sample; converting the purified DNA sample with bisulfite; amplifying the sample using a primer and probe combination; analyzing the amplification results to determine the methylation level of the sample; and judging the individual's disease status based on the methylation level of the sample.

[0098] In one embodiment, the individual may be a subject suspected of having lung cancer.

[0099] In one embodiment, the primers used in the PCR amplification are shown in SEQ ID NOs: 1 to 10.

[0100] In one embodiment, the probes include probes as shown in SEQ ID NOs: 11 to 15 that target lung cancer-specific regions.

[0101] In the present invention, each marker gene is detected by PCR amplification reaction;

[0102] Set the Ct positive judgment interval for each gene. If any gene is positive, the sample to be tested will be judged as positive; if all genes are negative, the sample to be tested will be judged as negative.

[0103] Among them, the positive judgment intervals of each gene are: FOXD3 gene: Ct≤26.02, FOXI2 gene: Ct≤23.26, RASSF1A gene: Ct≤23.43, SHOX2 gene: Ct≤28.18, and SOX17 gene: Ct≤28.60.

[0104] As used in this article, DNA methylation refers to the methylation process that occurs at the 5th carbon atom of cytosine in CpG dinucleotides. As a relatively stable modification state, it can be inherited to new offspring DNA during DNA replication under the action of DNA methyltransferase. It is an important epigenetic mechanism. During DNA methylation, methylation of the gene promoter region can lead to transcriptional silencing of tumor suppressor genes, and therefore it is closely related to the occurrence of lung cancer.

[0105] In some embodiments, the term "methylation state" or "methylation status" refers to the presence or absence of 5-methylcytosine ("5-mC" or "5-mCyt") at one or more CpG dinucleotides within a DNA sequence. The methylation status at one or more specific CpG methylation sites (each with two CpG dinucleotide sequences) within a DNA sequence includes "unmethylated," "fully methylated," and "hemimethylated."

[0106] As used herein, the terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. A "subject" can be an organism, or a portion or component of an organism, to which the provided compositions, methods, kits, devices, and systems are administered or applied. For example, the subject can be a mammal, or a cell, tissue, organ, or portion thereof.

[0107] As used herein, the term "sample" refers to any substance that may contain a target molecule to be analyzed, including biological samples.

[0108] As used herein, a "primer" can be a natural or synthetic oligonucleotide that, after forming a duplex with a polynucleotide template, can serve as a starting point for nucleic acid synthesis and extend from its 3' end along the template to form an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are typically extended by a polymerase such as a DNA polymerase.

[0109] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0110] Furthermore, it should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified. It should also be understood that the combination connection relationship between one or more devices / apparatuses mentioned in the present invention does not exclude the presence of other devices / apparatuses before or after the combination device / apparatus, or the insertion of other devices / apparatuses between two explicitly mentioned devices / apparatuses, unless otherwise specified. Furthermore, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be considered within the scope of the present invention.

[0111] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0112] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0113] Example 1 Methylation biomarker combination, specific primers and probe design

[0114] Gene annotation regions and their 5 kb upstream and downstream sequences are exported from gene annotation databases (NCBI, Ensemble, UCSC, etc.). CpG-rich regions are selected for primer and probe design. The CpG-rich region sequences (or their complementary sequences) are converted manually or with software assistance to simulate bisulfite-treated sequences. Based on methylation-specific PCR primer and probe design principles, CpG C residues are assumed to be methylated, while non-CpG C residues are assumed to be unmethylated. Once the simulated bisulfite-treated sequences are obtained, conventional primer and probe design methods can be used for design and synthesis.

[0115] In this example, the biomarker combination, including gene name, gene annotation position, and PCR detection segment, is shown in Table 1. The primer and probe sequences designed for the biomarker combination are shown in Table 2.

[0116] Table 1. Gene names, gene annotation positions, and PCR detection segments involved in the present invention

[0117] Gene Gene Hg19 Pos. PCR region FOXD3 chr1:63778730-63790797 chr1:63785908-63785999 FOXI2 chr10:129525499-129539450 chr10:129534759-129534851 RASSF1A chr3:50367219-50378411 chr3:50378061-50378154 SHOX2 chr3:157814948-157824292 chr3:157821339-157821429 SOX17 chr8:55370495-55373448 chr8:55370987-55371098

[0118] Table 2. Gene names, primers and probe sequences involved in the present invention

[0119]

[0120] Example 2 Evaluation of the detection performance of lung cancer markers in plasma samples

[0121] 120 plasma samples from healthy controls with no abnormalities in the lungs and 120 plasma samples from lung cancer patients before surgery were selected.

[0122] Extracellular DNA was extracted from the plasma samples using the Qiagen QIAamp Circulating Nucleic Acid Kit and then converted using the MethylCode™ Bisulfite Conversion Kit.

[0123] Optionally, the converted DNA is used for pre-amplification, and PCR amplification is performed using a premix (primer pool) containing the target primers and internal reference (ACTB) primer pairs shown in Table 2, with the converted DNA as a template, wherein the final concentration of each primer is 100 nM.

[0124] The PCR reaction system includes: 10 μL of converted DNA obtained after bisulfite treatment, 2.5 μL of premix containing the above primers; 12.5 μL of PCR reagent ( Universal Probe qPCR Master Mix (NEB).

[0125] The PCR reaction conditions were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 60 seconds, for 15 cycles.

[0126] The obtained pre-amplification product was diluted 10-fold and then used for fluorescence PCR detection. The primer and probe sequences shown in Table 2 were used, and the internal reference gene ACTB was detected at the same time (as a control).

[0127] In the primer and probe premix, the final concentration of the primer is 500 nM and the final concentration of the probe is 200 nM.

[0128] The PCR reaction system includes: 10 μL pre-amplification diluted product, 2.5 μL of primer and probe premix containing the detection site; 12.5 μL PCR reagent ( Universal Probe qPCR Master Mix (NEB).

[0129] PCR reaction conditions were as follows: 95°C for 5 minutes; 95°C for 15 seconds, 56°C for 40 seconds (for fluorescence collection), for 50 cycles. Different gene probes were designed with different fluorescent modifications, and corresponding fluorescence detection channels were selected for each modified gene probe. The Ct value for targets where no amplification signal was detected was set to 50. The Ct value refers to the cycle number at which the fluorescence signal in the PCR reaction reached the set threshold.

[0130] When the specificity of each target is uniformly required to be approximately 90%, the statistical data of the detection sensitivity and specificity of each target are shown in Table 5 below.

[0131] Table 3. Detection performance statistics for each target at approximately 90% specificity

[0132] Gene Sensitivity Specificity Positive interpretation interval FOXD3 60.83％％ 90.00％％ Ct≤26.02 FOXI2 59.17％ 90.00％ Ct≤23.26 RASSF1A 56.67％ 90.00％ Ct≤23.43 SHOX2 67.50％％ 89.17％％ Ct≤28.18 SOX17 60.00％％ 90.00％％ Ct≤28.60

[0133] When performing combined analysis of targets, data analysis can use a positive judgment threshold set for a single target. When combining targets, if any one target is positive, the sample is judged as positive; if all detected targets are negative, the sample is judged as negative. In order to balance sensitivity and specificity, the positive judgment interval of each target has been adjusted, and the specific judgment is:

[0134] Table 4. Positive intervals of each target in comprehensive interpretation

[0135] Gene Positive interpretation interval FOXD3 Ct≤25.2 FOXI2 Ct≤22.6 RASSF1A Ct≤23.4 SHOX2 Ct≤24.8 SOX17 Ct≤24.2

[0136] Combining all of the above genes in this way was found to result in a lung cancer detection sensitivity of 80.0% and a specificity of 89.2% for healthy controls.

[0137] In summary, addressing the technical limitations of current lung cancer detection products, the present invention provides a marker combination for lung cancer diagnosis. This combination offers enhanced accuracy, sensitivity, and specificity for early lung cancer screening, enabling real-time monitoring of lung cancer and effectively prolonging patient survival. This invention effectively overcomes the shortcomings of existing technologies and possesses high industrial value.

[0138] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention. Sequence Listing <110> West China Hospital of Sichuan University Jiangsu Xunyuan Biotechnology Co., Ltd. Chengdu West China Precision Medicine Industry Technology Research Institute Co., Ltd. Jiangsu Xunyuan Biotechnology Co., Ltd. <120> Lung cancer methylation marker, detection kit and application thereof <160> 15 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 agggagttta gagagttag 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 ctacaaaact cccctcac 18 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 ttgttttgaa ttgttgggg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 aaaacaacga actaacatct 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gtgttaacgc gttgcgtatc 20 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6 aaccccgcga actaaaaacg a 21 <210> 7 <211> twenty four <212> DNA <213> Artificial Sequence <400> 7 gttttttgga tagttaggta attt 24 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <400> 8 ccctttaaac aaccaacata acgtaa 26 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 gagttgagta agatgttgg 19 <210> 10 <211> twenty two <212> DNA <213> Artificial Sequence <400> 10 tcaaactcgc aaaaaacaat tt 22 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 ttgcggacgg cggaatcga 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 tttcgtgggg tacgcgcga 19 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 accaactacc gtataaaatt acacgcgata 30 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <400> 14 ctcgtacgac cccgatcg 18 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 tcgagtcgta gatttaggcg gtcgg 25

Claims

1. Use of a substance for detecting methylation markers in the preparation of a lung cancer diagnostic product; the methylation markers comprise a combination of the FOXD3 gene, the FOXI2 gene, the RASSF1A gene, the SHOX2 gene, and the SOX17 gene.

2. The method according to claim 1, wherein: The methylation marker includes the CpG island of the FOXD3 gene or the CpG island of the promoter of the FOXD3 gene, the CpG island of the FOXI2 gene or the CpG island of the promoter of the FOXI2 gene, the CpG island of the RASSF1A gene or the CpG island of the promoter of the RASSF1A gene, the CpG island of the SHOX2 gene or the CpG island of the promoter of the SHOX2 gene, and the CpG island of the SOX17 gene or a combination of CpG islands of the promoter of the SOX17 gene.

3. The method according to claim 2, wherein: The methylated region of the CpG island of the FOXD3 gene or the CpG island of the promoter of the FOXD3 gene is the sequence of chr1:63785908-63785999; The methylated region of the CpG island of the FOXI2 gene or the CpG island of the promoter of the FOXI2 gene is the sequence of chr10:129534759-129534851; The methylated region of the CpG island of the RASSF1A gene or the CpG island of the promoter of the RASSF1A gene is the sequence of chr3:50378061-50378154; The methylated region of the CpG island of the SHOX2 gene or the CpG island of the promoter of the SHOX2 gene is the sequence of chr3:157821339-157821429; The methylation region of the CpG island of the SOX17 gene or the CpG island of the promoter of the SOX17 gene is the sequence of chr8:55370987-55371098.

4. The use according to claim 1, wherein Each marker gene was detected by PCR amplification reaction; Set the Ct positive judgment interval for each gene. If any gene is positive, the sample to be tested will be judged as positive; if all genes are negative, the sample to be tested will be judged as negative. Among them, the positive judgment intervals of each gene are: FOXD3 gene: Ct≤25.2, FOXI2 gene: Ct≤22.6, RASSF1A gene: Ct≤23.4, SHOX2 gene: Ct≤24.8, and SOX17 gene: Ct≤24.

2.

5. Use of the test substance in the preparation of a lung cancer diagnostic product, characterized in that: The detection object comprises a specific detection primer and / or a specific detection probe for the methylation marker for the use according to any one of claims 1 to 3.

6. The use according to claim 5, characterized in that The specific detection primers include at least any one of the following primer pairs A to E: Primer pair A targeting the methylated region of the FOXD3 gene, the sequences of which are shown in SEQ ID NOs: 1-2; Primer pair B targeting the methylated region of the FOXI2 gene, the sequences of which are shown in SEQ ID NOs: 3-4; Primer pair C targeting the methylated region of the RASSF1A gene, the sequences of which are shown in SEQ ID NOs: 5-6; Primer pair D targeting the methylation region of the SHOX2 gene, the sequence of which is shown in SEQ ID NOs: 7-8; primer pair E targeting the methylation region of the SOX17 gene, the sequence of which is shown in SEQ ID NOs: 9-10; The specific detection probe includes at least any one of the following probes A to E: Probe A targeting the methylated region of the FOXD3 gene, the sequence of which is shown in SEQ ID NO: 11; Probe B targeting the methylated region of the FOXI2 gene, the sequence of which is shown in SEQ ID NO: 12; Probe C targeting the methylated region of the RASSF1A gene, the sequence of which is shown in SEQ ID NO: 13; Probe D targeting the methylated region of the SHOX2 gene, the sequence of which is shown in SEQ ID NO: 14; The sequence of probe E targeting the methylated region of the SOX17 gene is shown in SEQ ID NO:

15.

7. The use according to claim 5, characterized in that Samples used for lung cancer diagnosis come from peripheral or intratumoral blood, plasma, serum, urine, feces, sputum, fresh tissue, extracts of fresh tissue or fecal material, paraffin sections, and puncture specimens.

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