Lung cancer methylation marker combination, detection product and application thereof

Abstract

The application belongs to the technical field of biology and relates to a set of biomarker combinations, detection products and application thereof. The biomarkers provided by the application comprise a combination of SHOX2 genes, PTGER4 genes, NXPH1 genes, HOXD9 genes, PCDH8 genes and TBR1 genes, and the marker combinations can be used for lung cancer diagnosis. The application also discloses detection primers and probes designed based on the marker combinations, wherein the sequences of the detection primers are shown as SEQ ID NO:1-12, and the sequences of the detection probes are shown as SEQ ID NO:13-18. The detection of the methylation level of related genes by using the biomarkers and detection products provided by the application is more accurate, has high sensitivity and specificity, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and relates to a lung cancer methylation marker combination, a detection product and application thereof. BACKGROUND

[0002] With the continuous development of biotechnology, the method of using gene detection to diagnose diseases has attracted widespread attention. DNA methylation is an important part of epigenetics. Changes in methylation status are an important factor causing cancer. This change includes low overall methylation degree of the genome and abnormal increase in local methylation degree of CpG island, thereby causing instability of the genome and non-expression of tumor suppressor genes.

[0003] In recent years, the progress of epigenetic research on lung cancer has far-reaching significance for the early diagnosis and treatment of lung cancer. There is evidence that high methylation of tumor suppressor gene promoter CpG island is an important mechanism of gene inactivation. Transcriptional inactivation of different genes will affect cell cycle, DNA repair, apoptosis, etc., and is closely related to the occurrence and development of cancer. Clinically, it is considered that detecting methylation gene markers in the tissues and cells of lung cancer patients and body fluids (such as sputum, serum and plasma, etc.) is an effective auxiliary detection method for lung cancer, and has the value of improving the detection rate of lung cancer. The methylation gene markers related to lung cancer reported so far still have the defects of low sensitivity and low specificity, which limits their application in clinical detection. The high sensitivity of methylation gene markers to a certain or certain cancer indicates a low misdiagnosis rate of the corresponding cancer; similarly, the high specificity of methylation gene markers indicates a low misdiagnosis rate of the corresponding cancer. The sensitivity and specificity of methylation gene marker detection are two important indicators for measuring the reference value.

[0004] Therefore, further in-depth research on lung cancer methylation gene markers is needed to develop high-sensitivity and high-specificity methylation genes and their combinations related to lung cancer for detection, so as to provide more effective means for the accuracy of lung cancer diagnosis, which is a problem to be solved in the current methylation gene marker detection technology. SUMMARY

[0005] In view of the defects of the prior art, the present application provides a lung cancer methylation marker combination, and specific detection primers and probes are designed for specific regions of the lung cancer methylation marker, and a corresponding detection system is established to realize the detection purposes of high accuracy, high sensitivity and high specificity of lung cancer samples based on the lung cancer methylation marker combination, thereby providing valuable reference information for lung cancer diagnosis.

[0006] To achieve the above object and other related objects, the present application provides a lung cancer methylation marker, a detection kit and application thereof.

[0007] An object of the present application is to provide a lung cancer diagnostic detection material comprising specific detection primers and / or specific detection probes of a methylation marker combination comprising a combination of SHOX2 gene, PTGER4 gene, NXPH1 gene, HOXD9 gene, PCDH8 gene and TBR1 gene.

[0008] Another object of the present application is to provide use of the detection material as described above in the preparation of a lung cancer diagnostic product.

[0009] Another object of the present application is to provide a lung cancer diagnostic product comprising the detection material as described above.

[0010] In a preferred embodiment, the lung cancer diagnostic product is a kit comprising the detection material as described above.

[0011] Another object of the present application is to provide a lung cancer methylation marker combination comprising a combination of SHOX2 gene, PTGER4 gene, NXPH1 gene, HOXD9 gene, PCDH8 gene and TBR1 gene.

[0012] Another object of the present application is to provide use of the gene methylation marker as described above or a substance for detecting the gene methylation marker as described above in the preparation of a lung cancer diagnostic product.

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

[0014] The lung cancer methylation marker combination and detection product provided by the present application have excellent performance, greatly improved detection performance compared to the prior art, high detection accuracy, sensitivity and specificity, are easy to carry out, convenient for clinical promotion, can be used for liquid biopsy, have low cost, can reduce the medical cost of liquid patients, and have other advantages. DETAILED DESCRIPTION

[0015] The embodiments of the present application are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure. The present application can also be implemented or applied in different specific embodiments, and various modifications or changes can be made to the details in the specification without departing from the spirit of the present application.

[0016] An object of the present application is to provide more efficient lung tumor-related methylation markers, improve the efficiency of early screening and diagnosis of lung tumors, and solve the problems of low early diagnosis rate of lung cancer and heavy clinical treatment burden. An effective lung tumor methylation marker combination is provided.

[0017] The present application utilizes fluorescent PCR detection technology to detect the methylation status of lung cancer tissue related markers in the white membrane layer (most of which are leukocytes). The potential of the marker for blood detection of lung cancer is preliminarily confirmed. The ideal lung cancer methylation detection marker should have the following characteristics:

[0018] (1) low DNA methylation level in the white membrane layer;

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

[0020] The methylation marker detection is performed using methylation specific PCR (MSP). The basic principle of MSP is that only the methylated sequence template produces an amplification signal in the detection, while the non-methylated sequence template does not produce an amplification signal. This method can be achieved by designing methylation specific sequences for primers or probes, or by designing methylation specific primer and probe pairs. The main steps include:

[0021] 1) According to the marker sequence, design detection primers and probes in the detection segment suitable for MSP (usually CpG rich region);

[0022] 2) Perform nucleic acid extraction of white membrane layer and lung cancer tissue samples;

[0023] 3) Perform bisulfite treatment on nucleic acid to convert unmethylated cytosine to uracil, while methylated cytosine maintains the sequence unchanged;

[0024] 4) Perform fluorescent PCR detection.

[0025] Through white membrane layer and tissue sample detection, the marker meeting the above characteristics proves the potential of detecting lung cancer through blood. Then, in the plasma sample, these markers are verified, and the detection samples include control group, lung cancer group, and lung nodule, lung inflammation interference population, etc. to analyze the reference level of these markers and the performance of marker combination.

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

[0027] 1) Nucleic acid extraction of plasma sample;

[0028] 2) Perform bisulfite treatment on nucleic acid to convert unmethylated cytosine to uracil, while methylated cytosine maintains the sequence unchanged;

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

[0030] 4) Perform fluorescent PCR detection.

[0031] The present inventors, through a large number of exploratory researches, provide a set of biomarkers for diagnosing lung cancer, which comprises a combination of SHOX2 gene, PTGER4 gene, NXPH1 gene, HOXD9 gene, PCDH8 gene and TBR1 gene. The biomarkers have excellent detection sensitivity and specificity, and can solve the problems of current lung cancer (including lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC)) diagnosis accuracy, sensitivity, specificity, etc., on the basis of which the present application is completed.

[0032] The present application provides a set of biomarkers for diagnosing lung cancer, which comprises a combination of SHOX2 gene, PTGER4 gene, NXPH1 gene, HOXD9 gene, PCDH8 gene and TBR1 gene. Specifically, the combination comprises a CpG island of the SHOX2 gene or a CpG island of the promoter of the SHOX2 gene, a CpG island of the PTGER4 gene or a CpG island of the promoter of the PTGER4 gene, a CpG island of the NXPH1 gene or a CpG island of the promoter of the NXPH1 gene, a CpG island of the HOXD9 gene or a CpG island of the promoter of the HOXD9 gene, a CpG island of the PCDH8 gene or a CpG island of the promoter of the PCDH8 gene, and a CpG island of the TBR1 gene or a CpG island of the promoter of the TBR1 gene. Through screening of the biomarkers, lung cancer can be diagnosed and / or whether an individual is susceptible to lung cancer can be assessed more accurately, at low cost and high depth.

[0033] In some preferred embodiments, the methylation 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;

[0034] The methylation region of the CpG island of the PTGER4 gene or the CpG island of the promoter of the PTGER4 gene is the sequence of chr5: 40681083-40681165, or an active fragment thereof;

[0035] The methylation region of the CpG island of the NXPH1 gene or the CpG island of the promoter of the NXPH1 gene is the sequence of chr7: 8482292-8482408, or an active fragment thereof;

[0036] The methylation region of the CpG island of the HOXD9 gene or the CpG island of the promoter of the HOXD9 gene is the sequence of chr2: 176987563-176987664, or an active fragment thereof;

[0037] The methylation region of the CpG island of the PCDH8 gene or the CpG island of the promoter of the PCDH8 gene is the sequence of chr13: 53421168-53421239, or an active fragment thereof.

[0038] The methylation region of the CpG island of the TBR1 gene or the CpG island of the promoter of the TBR1 gene is the sequence of chr2: 162280418-162280523, or an active fragment thereof.

[0039] In the present application, the active fragment generally refers to a substance that can be detected by the substance, and the corresponding gene related to the methylation level can be found, which can be, for example, DNA or its mRNA or homologs retaining its function, and the selection method of the active fragment should be known to those skilled in the art. The homolog of the gene can be a sequence with 80% (for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or more homology with the gene.

[0040] In other preferred embodiments, the methylation 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;

[0041] The methylation region of the CpG island of the PTGER4 gene or the CpG island of the promoter of the PTGER4 gene is the sequence of chr5: 40681083-40681165;

[0042] The methylation region of the CpG island of the NXPH1 gene or the CpG island of the promoter of the NXPH1 gene is the sequence of chr7: 8482292-8482408;

[0043] The methylation region of the CpG island of the HOXD9 gene or the CpG island of the promoter of the HOXD9 gene is the sequence of chr2: 176987563-176987664;

[0044] The methylation region of the CpG island of the PCDH8 gene or the CpG island of the promoter of the PCDH8 gene is the sequence of chr13: 53421168-53421239;

[0045] The methylation region of the CpG island of the TBR1 gene or the CpG island of the promoter of the TBR1 gene is the sequence of chr2: 162280418-162280523.

[0046] The present inventors have found that methylation of the region of SHOX2 gene chr3: 157821339-157821429, methylation of the region of PTGER4 gene chr5: 40681083-40681165, methylation of the region of NXPH1 gene chr7: 8482292-8482408, methylation of the region of HOXD9 gene chr2: 176987563-176987664, methylation of the region of PCDH8 gene chr13: 53421168-53421239, and methylation of the region of TBR1 gene chr2: 162280418-162280523 are closely related to the occurrence and development of lung cancer, and a patient with lung cancer will most probably have the above methylation at the same time.

[0047] The present application also provides the use of the gene methylation markers as described above in the preparation of a lung cancer diagnosis product. The diagnosis includes early differential diagnosis of lung cancer, evaluation and dynamic monitoring of micro-lesion residue, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation, etc. The lung cancer diagnosis product can be in any suitable product form, including but not limited to primers, probes, kits, chips, membrane strips, protein arrays, etc.

[0048] The present application also provides the use of the substance for detecting the gene methylation markers as described above in the preparation of a lung cancer diagnosis product. The diagnosis includes early differential diagnosis of lung cancer, evaluation and dynamic monitoring of micro-lesion residue, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation, etc. The lung cancer diagnosis product can be in any suitable product form, including but not limited to primers, probes, kits, chips, membrane strips, protein arrays, etc.

[0049] In the present application, 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 methylation of the region of chr3: 157821339-157821429 of the SHOX2 gene, the methylation of the region of chr5: 40681083-40681165 of the PTGER4 gene, the methylation of the region of chr7: 8482292-8482408 of the NXPH1 gene, the methylation of the region of chr2: 176987563-176987664 of the HOXD9 gene, the methylation of the region of chr13: 53421168-53421239 of the PCDH8 gene, and the methylation of the region of chr2: 162280418-162280523 of the TBR1 gene all exist in the genome of an individual, it is considered that the individual has lung cancer or is more likely to have lung cancer. For another example, when at least one of the methylation of the region of chr3: 157821339-157821429 of the SHOX2 gene, the methylation of the region of chr5: 40681083-40681165 of the PTGER4 gene, the methylation of the region of chr7: 8482292-8482408 of the NXPH1 gene, the methylation of the region of chr2: 176987563-176987664 of the HOXD9 gene, the methylation of the region of chr13: 53421168-53421239 of the PCDH8 gene, and the methylation of the region of chr2: 162280418-162280523 of the TBR1 gene does not exist in the genome of an individual, it is considered that the individual does not have lung cancer or is more likely to not have lung cancer. Since the early diagnosis of lung cancer is usually difficult, the introduction of the above screening can more accurately diagnose lung cancer in an individual.

[0050] In the present application, the micro-lesion residual evaluation and dynamic monitoring can be specifically for confirming whether there is a micro-lesion residual in the individual. For example, when the individual has the methylation of the chr3: 157821339-157821429 region of the SHOX2 gene, the methylation of the chr5: 40681083-40681165 region of the PTGER4 gene, the methylation of the chr7: 8482292-8482408 region of the NXPH1 gene, the methylation of the chr2: 176987563-176987664 region of the HOXD9 gene, the methylation of the chr13: 53421168-53421239 region of the PCDH8 gene, and the methylation of the chr2: 162280418-162280523 region of the TBR1 gene in the genome of the individual, it is considered that the individual has a greater possibility of residual micro-lesions. For another example, when at least one of the methylation of the chr3: 157821339-157821429 region of the SHOX2 gene, the methylation of the chr5: 40681083-40681165 region of the PTGER4 gene, the methylation of the chr7: 8482292-8482408 region of the NXPH1 gene, the methylation of the chr2: 176987563-176987664 region of the HOXD9 gene, the methylation of the chr13: 53421168-53421239 region of the PCDH8 gene, and the methylation of the chr2: 162280418-162280523 region of the TBR1 gene does not occur in the genome of the individual, it is considered that the individual has a smaller possibility of residual micro-lesions.

[0051] In the present application, the auxiliary judgment of lung cancer recurrence and prognosis refers to the prediction of the risk of lung cancer recurrence and the prognosis, and specifically can be used to confirm the possibility of lung cancer recurrence or the deterioration tendency of an individual, and can be used to guide clinical diagnosis and treatment. For example, when the methylation of the chr3: 157821339-157821429 region of the SHOX2 gene, the methylation of the chr5: 40681083-40681165 region of the PTGER4 gene, the methylation of the chr7: 8482292-8482408 region of the NXPH1 gene, the methylation of the chr2: 176987563-176987664 region of the HOXD9 gene, the methylation of the chr13: 53421168-53421239 region of the PCDH8 gene, and the methylation of the chr2: 162280418-162280523 region of the TBR1 gene exist in the genome of an individual at the same time, it is considered that the individual has a greater possibility of lung cancer recurrence or the lung cancer is more likely to deteriorate. For another example, when at least one of the methylation of the chr3: 157821339-157821429 region of the SHOX2 gene, the methylation of the chr5: 40681083-40681165 region of the PTGER4 gene, the methylation of the chr7: 8482292-8482408 region of the NXPH1 gene, the methylation of the chr2: 176987563-176987664 region of the HOXD9 gene, the methylation of the chr13: 53421168-53421239 region of the PCDH8 gene, and the methylation of the chr2: 162280418-162280523 region of the TBR1 gene does not exist in the genome of an individual, it is considered that the individual has a smaller possibility of recurrence or the lung cancer has a smaller possibility of deterioration.

[0052] In the present application, the drug efficacy evaluation can be used to confirm whether a certain drug or treatment method is effective for an individual. For example, when the individual's genome has the methylation of the region of chr3: 157821339-157821429 of the SHOX2 gene, the methylation of the region of chr5: 40681083-40681165 of the PTGER4 gene, the methylation of the region of chr7: 8482292-8482408 of the NXPH1 gene, the methylation of the region of chr2: 176987563-176987664 of the HOXD9 gene, the methylation of the region of chr13: 53421168-53421239 of the PCDH8 gene, and the methylation of the region of chr2: 162280418-162280523 of the TBR1 gene, it is considered that a certain drug or treatment method is not effective for the individual. For another example, when at least one of the methylation of the region of chr3: 157821339-157821429 of the SHOX2 gene, the methylation of the region of chr5: 40681083-40681165 of the PTGER4 gene, the methylation of the region of chr7: 8482292-8482408 of the NXPH1 gene, the methylation of the region of chr2: 176987563-176987664 of the HOXD9 gene, the methylation of the region of chr13: 53421168-53421239 of the PCDH8 gene, and the methylation of the region of chr2: 162280418-162280523 of the TBR1 gene does not exist in the individual's genome, it is considered that a certain drug or treatment method is effective for the individual.

[0053] In the present application, the substance is specifically a substance for detecting the methylation level of the gene or the active fragment thereof in a sample such as peripheral or intratumoral blood, plasma, serum, urine, feces, sputum, extract of fresh tissue, fresh tissue or fecal material, paraffin section, and coarse needle puncture sample. Those skilled in the art can detect the methylation existing in the above-mentioned sample by using a suitable method, for example, the method that can be specifically used can be Sanger sequencing, whole genome sequencing, whole exon sequencing, targeted sequencing, etc. The substance for detecting the methylation of the gene or the active fragment thereof is usually corresponding to the detection method,

[0054] For example, the substance for detecting methylation in the region of SHOX2 gene chr3: 157821339-157821429 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of SHOX2 gene chr3: 157821339-157821429 or an active fragment thereof, for another example, the substance for detecting methylation in the region of PTGER4 gene chr5: 40681083-40681165 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of PTGER4 gene chr5: 40681083-40681165 or an active fragment thereof, for another example, the substance for detecting methylation in the region of NXPH1 gene chr7: 8482292-8482408 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of NXPH1 gene chr7: 8482292-8482408 or an active fragment thereof, for another example, the substance for detecting methylation in the region of HOXD9 gene chr2: 176987563-176987664 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of HOXD9 gene chr2: 176987563-176987664 or an active fragment thereof, for another example, the substance for detecting methylation in the region of PCDH8 gene chr13: 53421168-53421239 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of PCDH8 gene chr13: 53421168-53421239 or an active fragment thereof, for another example, the substance for detecting methylation in the region of TBR1 gene 162280418-162280523 or an active fragment thereof includes specific primers and / or probes for detecting methylation in the region of TBR1 gene 162280418-162280523 or an active fragment thereof, for another example, the specific probe preferably includes an isolated polynucleotide which is at least partially complementary to the target sequence, so as to be hybridized with the target sequence, so as to realize detection of whether the specific methylation exists on the target. The substance for detecting methylation level in the gene or an active fragment thereof can also include other various related detection reagents.

[0055] In some preferred embodiments, the substance for detecting the gene methylation markers as described above comprises a combination of a substance for detecting methylation of the region of chr3: 157821339-157821429 of the SHOX2 gene, a substance for detecting methylation of the region of chr5: 40681083-40681165 of the PTGER4 gene, a substance for detecting methylation of the region of chr7: 8482292-8482408 of the NXPH1 gene, a substance for detecting methylation of the region of chr2: 176987563-176987664 of the HOXD9 gene, a substance for detecting methylation of the region of chr13: 53421168-53421239 of the PCDH8 gene, and a substance for detecting methylation of the region of chr2: 162280418-162280523 of the TBR1 gene.

[0056] In some preferred embodiments, the substance for detecting the gene methylation markers as described above comprises specific detection primers and / or specific detection probes for detecting the gene methylation markers as described above.

[0057] The specific detection primers comprise at least any one of the following primer pairs A-F:

[0058] Primer pair A for the methylation region of the SHOX2 gene, the sequences of which are shown in SEQ ID NOs: 1-2;

[0059] Primer pair B for the methylation region of the PTGER4 gene, the sequences of which are shown in SEQ ID NOs: 3-4;

[0060] Primer pair C for the methylation region of the NXPH1 gene, the sequences of which are shown in SEQ ID NOs: 5-6;

[0061] Primer pair D for the methylation region of the HOXD9 gene, the sequences of which are shown in SEQ ID NOs: 7-8;

[0062] Primer pair E for the methylation region of the PCDH8 gene, the sequences of which are shown in SEQ ID NOs: 9-10;

[0063] Primer pair F for the methylation region of the TBR1 gene, the sequences of which are shown in SEQ ID NOs: 11-12;

[0064] The specific detection probes comprise at least any one of the following probes A-F:

[0065] Probe A for the methylation region of the SHOX2 gene, the sequence of which is shown in SEQ ID NO: 13;

[0066] Probe B for a methylation region of PTGER4 gene, the sequence of which is shown as SEQ ID NO: 14;

[0067] Probe C for a methylation region of NXPH1 gene, the sequence of which is shown as SEQ ID NO: 15;

[0068] Probe D for a methylation region of HOXD9 gene, the sequence of which is shown as SEQ ID NO: 16;

[0069] Probe E for a methylation region of PCDH8 gene, the sequence of which is shown as SEQ ID NO: 17;

[0070] Probe F for a methylation region of TBR1 gene, the sequence of which is shown as SEQ ID NO: 18.

[0071] The present application also provides a detection substance for lung cancer detection, which comprises specific detection primers and / or specific detection probes of the gene methylation markers as described above.

[0072] In some preferred embodiments,

[0073] The specific detection primers comprise at least any one of the following primer pairs A-F:

[0074] Primer pair A for a methylation region of SHOX2 gene, the sequences of which are shown as SEQ ID NO: 1-2;

[0075] Primer pair B for a methylation region of PTGER4 gene, the sequences of which are shown as SEQ ID NO: 3-4;

[0076] Primer pair C for a methylation region of NXPH1 gene, the sequences of which are shown as SEQ ID NO: 5-6;

[0077] Primer pair D for a methylation region of HOXD9 gene, the sequences of which are shown as SEQ ID NO: 7-8;

[0078] Primer pair E for a methylation region of PCDH8 gene, the sequences of which are shown as SEQ ID NO: 9-10;

[0079] Primer pair F for a methylation region of TBR1 gene, the sequences of which are shown as SEQ ID NO: 11-12;

[0080] The specific detection primers comprise at least any one of the following probes A-F:

[0081] Probe A for a methylation region of SHOX2 gene, the sequence of which is shown as SEQ ID NO: 13;

[0082] Probe B for the methylation region of PTGER4 gene, the sequence of which is shown as SEQ ID NO: 14;

[0083] Probe C for the methylation region of NXPH1 gene, the sequence of which is shown as SEQ ID NO: 15;

[0084] Probe D for the methylation region of HOXD9 gene, the sequence of which is shown as SEQ ID NO: 16;

[0085] Probe E for the methylation region of PCDH8 gene, the sequence of which is shown as SEQ ID NO: 17;

[0086] Probe F for the methylation region of TBR1 gene, the sequence of which is shown as SEQ ID NO: 18.

[0087] The present application also provides a lung cancer diagnostic product, which comprises the detection substance as described above.

[0088] In the present application, 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.

[0089] When the lung cancer diagnostic product is a kit, it comprises the detection substance as described above, which is used for amplifying the methylation region, i.e. comprises substances for detecting the methylation of the region chr3: 157821339-157821429 of SHOX2 gene, substances for detecting the methylation of the region chr5: 40681083-40681165 of PTGER4 gene, substances for detecting the methylation of the region chr7: 8482292-8482408 of NXPH1 gene, substances for detecting the methylation of the region chr2: 176987563-176987664 of HOXD9 gene, substances for detecting the methylation of the region chr13: 53421168-53421239 of PCDH8 gene, and substances for detecting the methylation of the region chr2: 162280418-162280523 of TBR1 gene; reagents for processing the methylation region; and primers for sequencing the amplification product.

[0090] The substance for detecting methylation in the gene or its active fragment in the detection kit provided by the application is usually corresponding to the detection method, for example, the substance for detecting methylation in the region of chr3: 157821339-157821429 of SHOX2 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of chr3: 157821339-157821429 of SHOX2 gene or its active fragment, for another example, the substance for detecting methylation in the region of chr5: 40681083-40681165 of PTGER4 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of chr5: 40681083-40681165 of PTGER4 gene or its active fragment, for another example, the substance for detecting methylation in the region of chr7: 8482292-8482408 of NXPH1 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of chr7: 8482292-8482408 of NXPH1 gene or its active fragment, for another example, the substance for detecting methylation in the region of chr2: 176987563-176987664 of HOXD9 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of chr2: 176987563-176987664 of HOXD9 gene or its active fragment, for another example, the substance for detecting methylation in the region of chr13: 53421168-53421239 of PCDH8 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of chr13: 53421168-53421239 of PCDH8 gene or its active fragment, for another example, the substance for detecting methylation in the region of 162280418-162280523 of TBR1 gene or its active fragment includes specific primers and / or probes for detecting methylation in the region of 162280418-162280523 of TBR1 gene or its active fragment, for another example, the specific probe preferably includes an isolated polynucleotide which is at least partially complementary to the target sequence, so as to be hybridized with the target sequence, and the specific methylation on the target sequence can be detected.

[0091] Optionally, the kit can further include other various related detection reagents, including but not limited to one or more of nucleic acid extraction reagents, reagents for amplifying the target, bisulfite conversion reagents, reagents for evaluating the methylation state of the target, internal reference genes, negative controls and positive controls, etc.

[0092] wherein the reagent for amplifying the target comprises an enzyme, for example, an enzyme for a polynucleotide amplification reaction selected from the group consisting of 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-sustained sequence replication (3SR), and loop-mediated isothermal amplification (LAMP).

[0093] wherein the reagent for assessing the methylation status of the target is a reagent for a polynucleotide methylation detection method selected from the group consisting of mass spectrometry, bisulfite sequencing, methylation-specific PCR (MSP), methylation DNA immunoprecipitation (MeDIP or mDIP), pyrosequencing, HpaII tiny fragment-enriched PCR (HELP assay), restriction landmark genomic scanning (RLGS), molecular break light assay of DNA adenine methyltransferase activity, methylation-sensitive Southern blotting, and high-resolution melting (HRM) analysis, and the like.

[0094] In one embodiment, the reagent for assessing the methylation status of the target is a chemical reagent, for example, bisulfite or sodium bisulfite.

[0095] In one embodiment, the reagent for assessing the methylation status of the target is a biological reagent, for example, a polypeptide or an enzyme. In another embodiment, wherein the enzyme is a polynucleotide polymerase; the polynucleotide polymerase is configured for PCR; the polynucleotide polymerase can be a DNA polymerase, for example, a DNA polymerase without 3' to 5' exonuclease activity.

[0096] In one embodiment, the internal reference gene is ACTB.

[0097] The kit provided by the present application further comprises a separate container (e.g., a vial) for one or more components and / or instructions for using the kit or system.

[0098] The present application also provides the use of the kit as described above for early differential diagnosis (early screening and early diagnosis), micro-lesion residual evaluation (MRD) and dynamic monitoring, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation and drug resistance monitoring, and the like.

[0099] The primers, probes, kits, detection systems, systems, or articles of manufacture described herein can be configured for any suitable use or purpose. For example, the primers, probes, kits, detection systems, systems, or articles of manufacture described herein can be configured for assessing the presence of lung cancer in a subject, or for lung cancer analysis or profiling of a subject.

[0100] The inventors provide a set of biomarkers including SHOX2 gene, PTGER4 gene, NXPH1 gene, HOXD9 gene, PCDH8 gene and TBR1 gene through a large number of experimental researches and a high-throughput screening method. The biomarkers provided in the application can avoid whole genome sequencing and greatly save the amount of required sequencing data. The detection primers and probes are designed according to the screened methylation regions of the biomarkers, the methylation levels of the main pathogenic genes related to lung cancer are detected at one time, and the detection has excellent sensitivity and specificity, is simple and fast to operate, and is low in cost, high in depth and accurate in detection of related methylation. Based on the biomarkers, a corresponding detection kit can be further developed for clinical molecular diagnosis of lung cancer, and can be potentially applied to early differential diagnosis (early screening and early diagnosis), micro-lesion residual evaluation (MRD) and dynamic monitoring of lung cancer, auxiliary judgment of recurrence and prognosis of lung cancer, drug efficacy evaluation and drug resistance monitoring, and the like. In addition, the functions and internal mechanisms of the lung cancer related genes can be further explored by means of bioinformatics and other technical means, so as to provide a theoretical basis for early discovery, early prevention, early diagnosis, early treatment and future targeted treatment of lung cancer. In addition, in the target point combination analysis, a mathematical model of machine learning can also be introduced, such as linear regression, support vector regression, ridge regression, random forest and the like.

[0101] The application designs specific primers and probes for the specific lung cancer methylation marker regions obtained through screening, constructs a detection system, and detects the performance of the lung cancer markers obtained through screening according to the collected lung cancer samples and normal samples. The detection finds that the biomarkers of the application can detect lung cancer with high accuracy and high specificity, which indicates that the biomarker combination has the potential to serve as a non-invasive lung cancer diagnosis and prognosis marker.

[0102] The application also provides a detection system, which comprises: 10 μL of transformed DNA obtained after methylation reagent treatment, 2.5 μL of primer and probe premix liquid containing a detection region; and 12.5 μL of PCR reagent.

[0103] The application also provides a detection system, which comprises: 10 μL of transformed DNA obtained after methylation reagent treatment, 2.5 μL of primer and probe premix liquid containing a detection region; and 12.5 μL of PCR reagent. Probe qPCR Master Mix (NEB); wherein the primer sequences are shown in SEQ ID NO: 1-12, and the final concentration of each primer is 500nM, and the probe sequences are shown in SEQ ID NO: 13-18, and the final concentration of each probe is 200nM.

[0104] The present application also provides an in vitro detection method, which comprises collecting a sample to be detected; extracting and purifying DNA in the sample; transforming the purified DNA sample with bisulfite; amplifying the sample by using a primer and probe combination; analyzing the amplification result to determine the methylation level of the sample; and determining the disease condition of the individual based on the methylation level of the sample.

[0105] In an embodiment, the individual can be a subject suspected of having lung cancer.

[0106] In an embodiment, the primer used in the PCR amplification is shown in SEQ ID NO: 1-12.

[0107] In an embodiment, the probe comprises a probe targeting a lung cancer specific region, shown in SEQ ID NO: 13-18.

[0108] In the present application, each marker gene is detected by PCR amplification reaction;

[0109] The Ct positive interpretation interval of each gene is set, and when any gene is positive, the sample to be detected is determined to be positive; when all genes are negative, the sample to be detected is determined to be negative.

[0110] The positive interpretation interval of each gene is as follows: SHOX2 gene: Ct≤28.12, PTGER4 gene: Ct≤25.58, NXPH1 gene: Ct≤23.94, HOXD9 gene: Ct≤24.59, PCDH8 gene: Ct≤24.08, and TBR1 gene: Ct≤23.42.

[0111] As used herein, DNA methylation refers to the methylation process occurring on the 5th carbon atom of cytosine in CpG dinucleotide, which is a relatively stable modification state, can be inherited to the newly born daughter DNA during the DNA replication process under the action of DNA methyltransferase, is an important epigenetic mechanism, and when DNA methylation occurs, the methylation of the gene promoter region can lead to the transcriptional silence of tumor suppressor genes, and therefore it is closely related to the occurrence of lung cancer.

[0112] 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 state at one or more particular CpG methylation sites (each having two CpG dinucleotide sequences) within a DNA sequence includes "unmethylated," "fully methylated," and "hemimethylated."

[0113] 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 part or component of the 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 part of the mammal.

[0114] As used herein, the term "sample" refers to any substance that can contain a target molecule that needs to be analyzed, including a biological sample.

[0115] As used herein, a "primer" can be a natural or synthetic oligonucleotide that, upon annealing to a polynucleotide template, is capable of acting as a point of initiation for nucleic acid synthesis and extending along the template from its 3' end, thereby forming an extended duplex. The sequence of the nucleotides added during extension is determined by the sequence of the template polynucleotide. Primers are typically extended by a polymerase, such as a DNA polymerase.

[0116] Before further description of the specific embodiments of the application, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; and that the description and drawings are to be regarded as illustrative in nature.

[0117] When numerical ranges are given, it is understood that every numerical range encompassing the lower value and the upper value of the range is contemplated, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any method, device, material, or the like that is similar in principle to methods, devices, materials, etc. described in the embodiments of the present application can be used in place of those described in the embodiments of the present application, according to the knowledge of the skilled in the art and the description of the present application, in addition to the specific methods, devices, materials, etc. used in the embodiments of the present application.

[0118] Example 1 Methylation biomarker combination, specific primer and probe design

[0119] The gene annotation section and the sequence of 5kb upstream and downstream thereof are derived from a gene annotation database (NCBI, Ensemble, UCSC, etc.), and a CpG-rich region is selected for primer and probe design. The CpG-rich region sequence (or its complementary sequence) is manually or software-assisted converted into a sequence after simulated bisulfite treatment, according to the design principles of methylation-specific PCR primers and probes, wherein the C of CpG is considered to be methylated, and the C of non-CpG is considered to be non-methylated. After obtaining the simulated bisulfite-treated sequence, conventional primer and probe design methods can be used for design and synthesis.

[0120] In this embodiment, the biomarker combination is shown in Table 1, including the gene name, gene annotation position, and PCR detection section. The primer and probe sequences designed for the biomarker combination are shown in Table 2.

[0121] Table 1. Gene name, gene annotation position, and PCR detection section involved in the present application

[0122] Gene Gene Hg19 Pos. PCR region SHOX2 chr3:157814948-157824292 chr3:157821339-157821429 PTGER4 chr5:40679600-40693837 chr5:40681083-40681165 NXPH1 chr7:8473585-8792593 chr7:8482292-8482408 HOXD9 chr2:176987088-176989853 chr2:176987563-176987664 PCDH8 chr13:53418109-53422775 chr13:53421168-53421239 TBR1 chr2:162272605-162282381 chr2:162280418-162280523

[0123] Table 2. Gene name, primer and probe sequence involved in the present application

[0124]

[0125] Example 2 Performance evaluation of lung cancer marker detection for plasma samples

[0126] Ninety plasma samples from healthy controls with no lung abnormalities and ninety-five plasma samples from lung cancer patients before surgery were selected.

[0127] Commercial Qiagen QIAamp Circulating Nucleic Acid Kit was used to extract extracellular free DNA in the above-mentioned plasma samples. Commercial bisulfite conversion reagent MethylCodeTM Bisulfite Conversion Kit was used to perform bisulfite conversion treatment on the extracted extracellular free DNA to obtain converted DNA.

[0128] Alternatively, the above-mentioned converted DNA was used for pre-amplification, and a premix liquid (primer pool) containing the target primer and internal reference (ACTB) primer pairs shown in Table 2 was used for PCR amplification with the converted DNA as the template, wherein the final concentration of each primer was 100 nM.

[0129] PCR reaction system contains: 10 μL of the converted DNA after bisulfite treatment, 2.5 μL of the premix containing the primers described above; 12.5 μL of PCR reagent (Takara) Probe qPCR Master Mix (NEB).

[0130] PCR reaction conditions are as follows: 95℃ for 5 minutes; 95℃ for 30 seconds, 56℃ for 60 seconds, for 15 cycles.

[0131] The obtained pre-amplification product is diluted 10 times for fluorescence PCR detection. The primer and probe sequences shown in Table 2 are used, and the internal reference gene ACTB is detected at the same time (as a control).

[0132] The final concentration of primers in the primer and probe premix is 500 nM, and the final concentration of probes is 200 nM.

[0133] PCR reaction system contains: 10 μL of the converted DNA after bisulfite treatment, 2.5 μL of the premix containing the primers described above; 12.5 μL of PCR reagent (Takara) Probe qPCR Master Mix (NEB).

[0134] PCR reaction conditions are as follows: 95℃ for 5 minutes; 95℃ for 15 seconds, 56℃ for 40 seconds (fluorescence collection), 50 cycles. Different probes for different genes are designed with different fluorescence modifications. The fluorescence channel corresponding to the probe modification for different genes is selected. The Ct value of the target point for which no amplification signal is detected is set to 50. Ct value refers to the cycle number when the fluorescence signal reaches the set threshold in the PCR reaction.

[0135] When the specificity of each target is about 90%, the detection sensitivity and specificity of each target are as shown in Table 5.

[0136] Table 3. Detection performance statistics of each target at about 90% specificity

[0137] Gene Sensitivity Specificity Positive call interval SHOX2 71.58％ 90.00％ Ct < 28.12 PTGER4 52.63％ 88.89％ Ct < 25.58 NXPH1 68.42％ 91.11％ Ct < 23.94 HOXD9 65.26％ 90.00％ Ct < 24.59 PCDH8 62.11％ 88.89％ Ct < 24.08 TBR1 66.32％ 90.00％ Ct < 23.42

[0138] In the combined analysis of the targets, the data analysis can set a positive judgment threshold for a single target. When the targets are combined, any positive target sample is judged as positive; when all the detected targets are negative, the sample is judged as negative. In order to balance the sensitivity and specificity, the positive judgment interval of each target is adjusted, and the specific judgment is as follows:

[0139] Table 4. Positive interval of each target in comprehensive judgment

[0140] Gene Positive call interval SHOX2 Ct < 25.0 PTGER4 Ct < 24.1 NXPH1 Ct < 23.1 HOXD9 Ct < 23.4 PCDH8 Ct < 23.2 TBR1 Ct < 22.1

[0141] The sensitivity of lung cancer detection can reach 81.05% and the specificity of healthy control can reach 88.89% when all the detection targets are combined.

[0142] In conclusion, the methylation marker combination for lung cancer diagnosis provided by the application can be used for early screening, prognosis and the like of lung cancer, has higher accuracy, sensitivity and specificity, and can realize real-time monitoring of lung cancer and effectively prolong the survival period of patients.

[0143] The above is only a preferred embodiment of the application, and is not intended to limit the application in any form or substance. SEQUENCE LISTING <110> West China Hospital of Sichuan University Shanghai Yuyuan Biotech Co., Ltd. Chengdu West China Precision Medicine Industry Technology Research Institute Co., Ltd. Jiangsu Yuyuan Biotech Co., Ltd. <120> Lung cancer methylation marker combination, detection product and application thereof <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <400> 1 gttttttgga tagttaggta attt 24 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <400> 2 ccctttaaac aaccaacata acgtaa 26 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 gttagttatt attatgttta tttt 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 aaataatcac taaactattc aa 22 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <400> 5 tgtatttgtg tttttataaa tttgt 25 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 caaaccccga aatttccc 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 gattcgttta taggttatga g 21 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <400> 8 cgaaccatca accacac 17 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <400> 9 ggtagttatt tggtggtga 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 caccaacgtc aaattatact 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 ggcgttaatt tttatttggg 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <400> 12 aatcgaacaa atccttaaac tt 22 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <400> 13 ctcgtacgac cccgatcg 18 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <400> 14 cgtttttttg agtttcgatc g 21 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 cgagatcgtt cgaggttatt tgcga 25 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <400> 16 cgacgaggtg ttcgcgg 17 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 tcgcggcgtc gttggatc 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <400> 18 tggtcgtcga gcgttcgt 18

Claims

1. Use of a test substance for lung cancer diagnosis in the production of a lung cancer diagnosis product, characterized in that, The detection reagent is a specific detection primer and / or a specific detection probe for a lung cancer methylation marker combination; the methylation marker combination is a combination comprising a SHOX2 gene, a PTGER4 gene, a NXPH1 gene, a HOXD9 gene, a PCDH8 gene, and a TBR1 gene; The SHOX2 gene is a CpG island of a SHOX2 gene or a CpG island of a promoter of a SHOX2 gene; a methylation region of the CpG island of the SHOX2 gene or the CpG island of the promoter of the SHOX2 gene is a sequence of chr3: 157821339-157821429; The PTGER4 gene is a CpG island of a PTGER4 gene or a CpG island of a promoter of a PTGER4 gene; a methylation region of the CpG island of the PTGER4 gene or the CpG island of the promoter of the PTGER4 gene is a sequence of chr5: 40681083-40681165; The NXPH1 gene is a CpG island of a NXPH1 gene or a CpG island of a promoter of a NXPH1 gene; a methylation region of the CpG island of the NXPH1 gene or the CpG island of the promoter of the NXPH1 gene is a sequence of chr7: 8482292-8482408; The HOXD9 gene is a CpG island of a HOXD9 gene or a CpG island of a promoter of a HOXD9 gene; a methylation region of the CpG island of the HOXD9 gene or the CpG island of the promoter of the HOXD9 gene is a sequence of chr2: 176987563-176987664; The PCDH8 gene is a CpG island of a PCDH8 gene or a CpG island of a promoter of a PCDH8 gene; a methylation region of the CpG island of the PCDH8 gene or the CpG island of the promoter of the PCDH8 gene is a sequence of chr13: 53421168-53421239; The TBR1 gene is a CpG island of a TBR1 gene or a CpG island of a promoter of a TBR1 gene; a methylation region of the CpG island of the TBR1 gene or the CpG island of the promoter of the TBR1 gene is a sequence of chr2: 162280418-162280523.

2. Use as claimed in claim 1, characterised in that, The specific detection primer comprises at least any one of the following primer pairs A-F: The primer pair A for the methylation region of the SHOX2 gene has sequences as shown in SEQ ID NOs: 1-2; The primer pair B for the methylation region of the PTGER4 gene has sequences as shown in SEQ ID NOs: 3-4; The primer pair C for the methylation region of the NXPH1 gene has sequences as shown in SEQ ID NOs: 5-6; The primer pair D for the methylation region of the HOXD9 gene has sequences as shown in SEQ ID NOs: 7-8; The primer pair E for the methylation region of the PCDH8 gene has sequences as shown in SEQ ID NOs: 9-10; and The primer pair F for the methylation region of the TBR1 gene has sequences as shown in SEQ ID NOs: 11-12. a primer pair F for a methylation region of the TBR1 gene, the sequence of which is shown as SEQ ID NO: 11-12; the specific detection probe comprises at least any one of the following probes A-F: a probe A for a methylation region of the SHOX2 gene, the sequence of which is shown as SEQ ID NO: 13; a probe B for a methylation region of the PTGER4 gene, the sequence of which is shown as SEQ ID NO: 14; a probe C for a methylation region of the NXPH1 gene, the sequence of which is shown as SEQ ID NO: 15; a probe D for a methylation region of the HOXD9 gene, the sequence of which is shown as SEQ ID NO: 16; a probe E for a methylation region of the PCDH8 gene, the sequence of which is shown as SEQ ID NO: 17; a probe F for a methylation region of the TBR1 gene, the sequence of which is shown as SEQ ID NO:

18.

3. The use according to claim 1, wherein The diagnosis comprises early differential diagnosis of lung cancer, auxiliary judgment of recurrence and prognosis of lung cancer.

4. The use according to claim 1, wherein The sample for lung cancer diagnosis is derived from peripheral or intratumoral blood, plasma, serum, urine, feces, sputum, fresh tissue, extract of fresh tissue or fecal material, paraffin section, or puncture sample.

5. The use according to claim 1, wherein The lung cancer diagnosis product is a kit comprising the detection object of claim 1 or 2 for amplifying the methylation region, reagents for treating the methylation region, and primers for sequencing the amplification product.

6. Use according to any one of claims 1 to 5, wherein Each marker gene is detected by PCR amplification reaction; Ct positive reading interval of each gene is set, and when any gene is positive, the sample to be tested is judged as positive; when all genes are negative, the sample to be tested is judged as negative. The positive reading interval of each gene is as follows: SHOX2 gene: Ct≤28.12, PTGER4 gene: Ct≤25.58, NXPH1 gene: Ct≤23.94, HOXD9 gene: Ct≤24.59, PCDH8 gene: Ct≤24.08, and TBR1 gene: Ct≤23.42.

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