A combined marker for lung cancer detection and application thereof

Abstract

The application discloses a combined marker for lung cancer detection and application thereof. The application finds that the genes SHOX2, RASSF1, GRIK2, HOXA9 and PTGER4 are related to early lung cancer diagnosis, which are extracted from DNA and RNA of lung cancer tissues and normal tissues beside the cancer, and the original result is verified by screening another group of lung cancer tissue and normal tissue DNA by using whole gene methylation sequencing and expression analysis method, and GRIK2 is a newly found lung cancer methylation marker. The methylation state of each target gene is judged by using human methylation DNA standard product, then the methylation degree of the test sample is judged by using combined C5 2 mode, and whether the sample is from a lung cancer patient is further judged, the judgment method is stricter than the common method, and the false positive rate is reduced, but the detection rate and specificity are greater than 90%. The application can screen and assist in diagnosing early lung cancer, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a combination biomarker for lung cancer detection and its application. Background Technology

[0002] Lung cancer takes 5-10 years to develop from its initial lesion to metastasis. Early detection not only leads to better treatment outcomes but also lower medical costs. However, once this stage is exceeded, the disease progresses rapidly, treatment costs increase, and outcomes become poor. Therefore, early diagnosis and treatment are the best approach to managing lung cancer. Because early-stage lung cancer lacks specific clinical manifestations, although many early tumor signals appear, highly sensitive and specific early diagnostic techniques are lacking. Most patients are diagnosed at an advanced stage. For example, common ground-glass opacities in the lungs are primarily determined by chest CT scans, but differentiation is difficult, and final diagnosis often occurs in the middle or late stages.

[0003] Early diagnosis of tumors typically depends on two key factors: high sensitivity of the diagnostic markers and non-invasive, convenient testing methods. With rapid advancements in technology, tumor markers have emerged as a new field, presenting both challenges and hopes in tumor diagnosis and treatment. Tumor markers can be detected in body fluids or tissues and can reflect the presence, differentiation level, prognostic assessment, and treatment effectiveness of tumors.

[0004] With the continuous development of gene diagnostic technology, domestic and international studies have found that the level of gene methylated DNA in liquid biopsy ctDNA can be used for early diagnosis. Its sensitivity and specificity are superior to serum protein markers such as the CA199 series. Moreover, DNA methylation occurs in almost all tumors, appearing in the early stages of carcinogenesis and can be detected before the appearance of clinical symptoms. It is a potentially useful indicator for early tumor diagnosis, disease risk prediction, clinical course monitoring, and efficacy evaluation. As a novel molecular marker, DNA methylation has received increasing attention in tumor diagnosis due to its advantages: First, promoter hypermethylation occurs frequently during tumor formation, even more so than gene mutations, including the methylation of many oncogenes and tumor suppressor genes related to tumor formation; second, methylation is an important event in the early stages of tumor development; third, DNA methylation is stable and can be detected by PCR amplification; and fourth, it exhibits certain tissue specificity. Therefore, methylation detection has potential application value in early tumor diagnosis.

[0005] Currently, aberrantly methylated DNA of the same gene accounts for only a very small portion of total DNA in peripheral blood, approximately 0.1% to 1%. These unmethylated DNAs are only slightly different from methylated DNA, thus necessitating the detection of aberrantly methylated DNA from a highly complex "background." Furthermore, circulating blood DNA is typically degraded (usually tens to hundreds of base pairs), and plasma ctDNA has a half-life of only 2.5 hours; therefore, timely extraction and excellent extraction techniques are required to achieve high recovery rates. Since cancer development is multifactorial, multigeneic, and multistep, even clinically diagnosed lung cancer may be caused by smoking, family genetic factors, occupation, and lifestyle. The cancer mechanisms and causes in these patients are completely different. Therefore, a multigene detection kit is needed that can cover multiple signal transduction systems in the cancer mechanism, including various carcinogenic factors such as smoking, genetics, and environmental occupations. This kit should be able to simultaneously detect the methylation status of genes related to lung cancer caused by multiple factors. Summary of the Invention

[0006] The purpose of this invention is to diagnose lung cancer at an early stage.

[0007] This invention first protects the application of combined biomarkers in the preparation of lung cancer detection kits;

[0008] The combined biomarker may be composed of the SHOX2 gene, RASSF1 gene, GRIK2 gene, HOXA9 gene, and PTGER4 gene;

[0009] The GeneID of the SHOX2 gene is 6474;

[0010] The GeneID of the RASSF1 gene is 11186;

[0011] The GeneID of the GRIK2 gene is 2898;

[0012] The GeneID of the HOXA9 gene is 3205;

[0013] The GeneID of the PTGER4 gene is 5734.

[0014] The present invention also protects a lung cancer detection kit, which may include a detection reagent for the combined biomarkers.

[0015] The kit may specifically consist of detection reagents for the combined markers.

[0016] In any of the kits described above, the detection reagents for the combined biomarkers may include primer and probe sets for detecting the methylation level of the SHOX2 gene, primer and probe sets for detecting the methylation level of the RASSF1 gene, primer and probe sets for detecting the methylation level of the GRIK2 gene, primer and probe sets for detecting the methylation level of the HOXA9 gene, and primer and probe sets for detecting the methylation level of the PTGER4 gene.

[0017] In any of the kits described above, the detection reagent for the combined biomarker may specifically consist of a primer and probe set for detecting the methylation level of the SHOX2 gene, a primer and probe set for detecting the methylation level of the RASSF1 gene, a primer and probe set for detecting the methylation level of the GRIK2 gene, a primer and probe set for detecting the methylation level of the HOXA9 gene, and a primer and probe set for detecting the methylation level of the PTGER4 gene.

[0018] The primer and probe set described above for detecting the methylation level of the SHOX2 gene can be primer and probe set SHOX2-1, primer and probe set SHOX2-2, primer and probe set SHOX2-3 or primer and probe set SHOX2-4.

[0019] The primer-probe set SHOX2-1 described above consists of primer SHOX2-F1 shown in SEQ ID NO: 1, primer SHOX2-R1 shown in SEQ ID NO: 2, and probe SHOX2-P1 shown in SEQ ID NO: 3.

[0020] The primer-probe set SHOX2-2 described above consists of primer SHOX2-F2 shown in SEQ ID NO: 4, primer SHOX2-R2 shown in SEQ ID NO: 5, and probe SHOX2-P2 shown in SEQ ID NO: 6.

[0021] The primer-probe set SHOX2-3 described above consists of primer SHOX2-F3 shown in SEQ ID NO: 7, primer SHOX2-R3 shown in SEQ ID NO: 8, and probe SHOX2-P3 shown in SEQ ID NO: 9.

[0022] The primer-probe set SHOX2-4 described above consists of primer SHOX2-F4 shown in SEQ ID NO: 10, primer SHOX2-R4 shown in SEQ ID NO: 11, and probe SHOX2-P4 shown in SEQ ID NO: 12.

[0023] The primer and probe set described above for detecting the methylation level of the RASSF1 gene can be primer and probe set RASSF1-1, primer and probe set RASSF1-2, or primer and probe set RASSF1-3.

[0024] The primer-probe set RASSF1-1 described above consists of primer RASSF1-F1 shown in SEQ ID NO: 13, primer RASSF1-R1 shown in SEQ ID NO: 14, and probe RASSF1-P1 shown in SEQ ID NO: 15.

[0025] The primer-probe set RASSF1-2 described above consists of primer RASSF1-F2 shown in SEQ ID NO: 16, primer RASSF1-R2 shown in SEQ ID NO: 17, and probe RASSF1-P2 shown in SEQ ID NO: 18.

[0026] The primer-probe set RASSF1-3 described above consists of primer RASSF1-F3 shown in SEQ ID NO: 19, primer RASSF1-R3 shown in SEQ ID NO: 20, and probe RASSF1-P3 shown in SEQ ID NO: 21.

[0027] The primer and probe sets described above for detecting the methylation level of the GRIK2 gene are primer and probe set GRIK2-1, primer and probe set GRIK2-2, or primer and probe set GRIK2-3.

[0028] The primer-probe set GRIK2-1 described above consists of primer GRIK2-F1 shown in SEQ ID NO: 22, primer GRIK2-R1 shown in SEQ ID NO: 23, and probe GRIK2-P1 shown in SEQ ID NO: 24.

[0029] The primer-probe set GRIK2-2 described above consists of primer GRIK2-F2 shown in SEQ ID NO: 25, primer GRIK2-R2 shown in SEQ ID NO: 26, and probe GRIK2-P2 shown in SEQ ID NO: 27.

[0030] The primer-probe set GRIK2-3 described above consists of primer GRIK2-F3 shown in SEQ ID NO: 28, primer GRIK2-R3 shown in SEQ ID NO: 29, and probe GRIK2-P3 shown in SEQ ID NO: 30.

[0031] The primer and probe sets described above for detecting the methylation level of the HOXA9 gene are primer and probe set HOXA9-1, primer and probe set HOXA9-2, primer and probe set HOXA9-3, or primer and probe set HOXA9-4.

[0032] The primer-probe set HOXA9-1 described above consists of primer HOXA9-F1 shown in SEQ ID NO: 31, primer HOXA9-R1 shown in SEQ ID NO: 32, and probe HOXA9-P1 shown in SEQ ID NO: 33.

[0033] The primer-probe set HOXA9-2 described above consists of primer HOXA9-F2 shown in SEQ ID NO: 34, primer HOXA9-R2 shown in SEQ ID NO: 35, and probe HOXA9-P2 shown in SEQ ID NO: 36.

[0034] The primer-probe set HOXA9-3 described above consists of primer HOXA9-F3 shown in SEQ ID NO: 37, primer HOXA9-R3 shown in SEQ ID NO: 38, and probe HOXA9-P3 shown in SEQ ID NO: 39.

[0035] The primer-probe set HOXA9-4 described above consists of primer HOXA9-F4 shown in SEQ ID NO: 40, primer HOXA9-R4 shown in SEQ ID NO: 41, and probe HOXA9-P4 shown in SEQ ID NO: 42.

[0036] The primer and probe sets described above for detecting the methylation level of the PTGER4 gene are primer and probe set PTGER4-1, primer and probe set PTGER4-2, or primer and probe set PTGER4-3.

[0037] The primer-probe set PTGER4-1 described above consists of primer PTGER4-F1 shown in SEQ ID NO: 43, primer PTGER4-R1 shown in SEQ ID NO: 44, and probe PTGER4-P1 shown in SEQ ID NO: 45.

[0038] The primer-probe set PTGER4-2 described above consists of primer PTGER4-F2 shown in SEQ ID NO: 46, primer PTGER4-R2 shown in SEQ ID NO: 47, and probe PTGER4-P2 shown in SEQ ID NO: 48.

[0039] The primer-probe set PTGER4-3 described above consists of primer PTGER4-F3 shown in SEQ ID NO: 49, primer PTGER4-R3 shown in SEQ ID NO: 50, and probe PTGER4-P3 shown in SEQ ID NO: 51.

[0040] Any of the above-described kits may further include an internal reference gene detection reagent. The internal reference gene may be the ACTB gene. The ACTB gene is available in GeneBank at index 60.

[0041] The kit described above may specifically consist of detection reagents for the combined biomarkers described above and detection reagents for the internal reference genes described above.

[0042] The internal reference gene detection reagent described above can be the primer and probe set ACTB-1 or the primer and probe set ACTB-2 used to detect the methylation level of the ACTB gene.

[0043] The primer-probe set ACTB-1 consists of primer ACTB-F1 shown in SEQ ID NO: 52, primer ACTB-R1 shown in SEQ ID NO: 53, and probe ACTB-P1 shown in SEQ ID NO: 54.

[0044] The primer-probe set ACTB-2 consists of primer ACTB-F2 shown in SEQ ID NO: 55, primer ACTB-R2 shown in SEQ ID NO: 56, and probe ACTB-P2 shown in SEQ ID NO: 57.

[0045] Each of the probes described above (such as probes for detecting internal reference genes or probes for detecting the methylation level of each gene in the combined biomarker) has a fluorescent label at one end and a fluorescent quenching label at the other end.

[0046] The specific target of detection for any of the above-mentioned kits can be plasma cfDNA, fecal cfDNA, or genomic DNA from colon tissue.

[0047] The kit described above may further include a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarker into the subject's dCT. X It is used to determine whether a person being tested has lung cancer.

[0048] The subject's dCT XThe calculation method is as follows: Genomic DNA from the plasma, fecal cfDNA, or lung tissue of the test subject is chemically modified. Then, using these as templates, fluorescent PCR amplification is performed using any of the primers and probes described above. Fluorescence signals are collected, and the CT values ​​of SHOX2, RASSF1, GRIK2, HOXA9, PTGER4, and ACTB are obtained, and denoted as CT values ​​respectively. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not S-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT value of each gene SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4. X =CT x -CT ACTB ;

[0049] The method for determination can be as follows: if at least two of the SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 genes of the test subject are methylated, then the test subject is a lung cancer patient; otherwise, the test subject is not a lung cancer patient; whether a gene is methylated is determined by comparing the dCT and dCT threshold values ​​of the genes in the test sample.

[0050] If the dCT of the SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4 genes in the subject is less than or equal to the dCT threshold, then the subject is methylated based on that gene.

[0051] The dCT threshold value for each gene is the average statistical value obtained by comparing the dCT values ​​of lung cancer tissue and adjacent normal tissue. It is a threshold value that can best distinguish between tumors and non-tumors (for example, the median value of the dCt values ​​of each target gene among all validated positive and negative lung cancer samples is the dCt threshold value; a Ct value of methylation region detection of the gene ≤ the threshold value is positive; a Ct value of methylation region detection of the gene ≥ the threshold value is negative).

[0052] Experiments have shown that five genes—SHOX2, RASSF1, GRIK2, and HOXA9—in lung cancer tissue can be used to diagnose early-stage lung cancer. The procedure is simple, time-efficient, and exhibits high sensitivity and specificity, while effectively improving the detection rate and reducing false positives. This invention has significant application value. Attached Figure Description

[0053] Figure 1 The results are the ROC analysis results from Example 4. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0056] In the following embodiments, "sample to be tested" refers to the nucleic acid sample to be tested; specifically, the sample to be tested can be isolated blood cells, one or more of the cells isolated from blood, cell lines, bronchoalveolar lavage fluid, tissue sections, surgical tissue, biopsy tissue, paraffin-embedded tissue, body fluids, feces, urine, plasma, serum, whole blood, etc., including gDNA, cfDNA, and ctDNA.

[0057] In the following examples, "lung cancer" includes adenocarcinoma and squamous cell carcinoma, which are common malignant tumors of the respiratory tract, especially non-small cell lung cancer. Early symptoms are not obvious, and it takes about 5 to 7 years from the onset of cancer to the clinical discovery of a mass; the 5-year survival rate for early-stage lung cancer is over 90%, while that for late-stage lung cancer is only 10%.

[0058] In the following examples, "target nucleic acid" or "target gene" refers to nucleic acid fragments of five lung cancer-related genes, namely, methylated DNA-specific fragments of the human GRIK2, HOXA9, PTGER4, RASSF1, and SHOX2 genes. Highly specific primers were designed to amplify different target fragments, and highly specific probes were used to identify them. The designed primers and probes are complementary to the target sites.

[0059] In the following embodiments, a "probe" refers to a single-stranded nucleic acid with a known nucleotide sequence whose nucleotide sequence structure is substantially complementary to that of the target nucleic acid, allowing it to form a double strand with the target nucleic acid. The 5' end of the probe may carry a fluorescent group and / or the 3' end may carry a quenching group marker. The binding of the primer and probe to the methylation-specific sequence in the sample DNA enables the molecular marker to detect the disease—lung cancer.

[0060] In the method of this invention, the extracted DNA needs to be chemically modified to obtain a converted DNA fragment as the test sample. Bisulfite, bisulfite, or hydrazine modification can all be applied to this chemical modification to convert cytosine in the DNA sample to uracil, while leaving 5'-methylcytosine unchanged, thus distinguishing between methylated and unmethylated gene fragments, enabling the designed primers and probes to recognize and perform PCR amplification.

[0061] Those skilled in the art can use quantitative measurements to determine the methylation level at a specific CpG site, where the methylation level exceeds a certain threshold, wherein the threshold may be a value representing the average or median methylation level of a given population, or it is preferably an optimal threshold.

[0062] The method of this invention involves performing a 6-channel quantitative fluorescent probe PCR amplification reaction in a single reaction tube. The Ct value of each gene is obtained through fluorescence signal. The dCt (ΔCt) value of the target gene in the lung cancer positive clinical sample is obtained by subtracting the Ct value of the internal reference gene ACTB from the Ct value of each gene. This dCt value is then compared with the dCT threshold (critical value) for methylation of the gene obtained from a large number of known lung cancer tissue and control tissue DNA samples. If the dCt value is less than the threshold, it indicates that the target gene is methylated, thus determining the methylation status of the target gene.

[0063] The combined application of dCT values ​​of each target gene to the C5 combination 2 The mathematical model determines the methylation status of the test sample. A sample is considered methylated if two or more of the five target genes show methylation, indicating a methylation-positive sample. This can preliminarily indicate that the sample comes from a high-risk lung cancer patient or a lung cancer patient. Single-gene positivity results are for follow-up. The interpretation of these results is more complex than the usual "any gene positive" method (C5). 1 The method is strictly controlled (a positive result for any one of the five genes is considered a tumor), reducing false positives by one-fold. This means the method needs higher sensitivity and specificity to achieve the usual results (e.g., C5). 1 Judgment criteria.

[0064] Considering the needs of clinical testing, liquid biopsy can effectively reduce harm to patients. When using real-time fluorescent PCR for detection, the probe is attached with a fluorescent group suitable for identifying methylated DNA fragments of different genes. One end of the probe is labeled with a fluorescent group, and the other end with a quencher group; the quencher group quenches the fluorescence emitted by the fluorescent group. During the PCR amplification reaction, the positive exonuclease activity of the polymerase cleaves the bases carrying the fluorescent group. The freed fluorescent group is no longer affected by the quencher group and emits a fluorescence signal of a certain wavelength under excitation light. As the PCR product accumulates, the fluorescence signal continuously strengthens, thereby detecting the presence of specifically methylated DNA. In a preferred embodiment of this invention, during detection, six specific probes labeled with six different fluorescent groups are added to the same reaction tube, corresponding to human SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4, as well as the internal reference gene ACTB, respectively, simultaneously indicating the presence of methylated DNA fragments of five target genes within the same reaction tube. In a preferred embodiment of the present invention, the fluorescent group labeled on the detection probe can be VIC, ROX, FAM, Cy5, Cy5.5, HEX, TET, JOE, NED, or TAMRA, etc.; while the quenching group can be BHQ, MGB, or Dabcy1. This invention is applicable to the multichannel PCR detection technology commonly used in clinical testing, enabling multichannel fluorescence detection in a single reaction tube.

[0065] In the examples described below, all patients provided informed consent and the experiments were ethically approved.

[0066] In the following examples, the human whole-genome methylated DNA standard (EpiTect PCR Control DNA Set, Qiagen CatNo. / ID:59695, where methylated DNA is represented by +ve ctr) served as the positive control DNA. The human whole-genome unmethylated DNA standard (EpiTect PCR Control DNA Set, Qiagen CatNo. / ID:59695, where unmethylated DNA is represented by -ve ctr) or water served as the negative control DNA.

[0067] Example 1: Obtaining biomarkers for lung cancer detection

[0068] The inventors of this invention extracted DNA and RNA from lung cancer tissue (i.e., surgical tissue from lung cancer patients (pathologically confirmed, C), as a tumor-positive sample) and adjacent normal tissue (adjacent normal tissue from the same case during surgery (pathologically confirmed, A), as a tumor-negative sample). Using whole-genome methylation sequencing and expression analysis, combined with multiple databases and comprehensive clinical information, large-scale screening was conducted to design probes to enrich the methylation sites of relevant genes. By comparing differences in methylation levels, biomarkers for lung cancer detection were obtained. The biomarkers for lung cancer detection consist of six genes: SHOX2 (GeneID: 6474), RASSF1 (GeneID: 11186), GRIK2 (GeneID: 2898), HOXA9 (GeneID: 3205), PTGER4 (GeneID: 5734), and ACTB (GeneID: 60).

[0069] Based on the nucleotide sequences of the above genes, primers and probes used in Table 1 were designed and synthesized by Nanjing Genscript Biotech Co., Ltd.

[0070] Table 1

[0071]

[0072]

[0073]

[0074] Note: Primer names containing "F" indicate upstream primers, "R" indicate downstream primers, and "P" indicate probes; CY5.5 indicates CY5.5 labeling; VIC indicates VIC labeling; ROX indicates ROX labeling; FAM indicates FAM labeling; TAMRA indicates TAMRA labeling; CY5 indicates CY5 labeling; MGB indicates fluorescence quenching labeling; ACTB (GeneID: 60) is the internal reference gene.

[0075] Example 2: Detection of gene methylation markers in lung cancer (A) and adjacent normal tissue (C)

[0076] 1. Dilute the upstream primers, downstream primers, and probes of SHOX2, RASSF1, GRIK2, HOXA9, PTGER4, and ACTB in Table 1 with water.

[0077] 2. The samples to be tested (14 cases of clinical lung cancer (LUAD) paired samples (lung cancer tissue (represented by C) and adjacent normal tissue (represented by A)), 10 cases of benign lung nodules paired samples (benign lung nodule tissue (also represented by C) and adjacent normal tissue (also represented by A)), and 3 human lung cancer cell lines (A549 cells, H1299 cells, and HCC827, respectively) were homogenized. Then, genomic DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (Beijing Tiangen Biotech (Beijing) Co., Ltd., Cat.#DP304-03) to obtain the genomic DNA of the samples to be tested.

[0078] 3. Obtain genomic DNA from the sample to be tested using EZ DNAMethylation-Direct. TM The DNA of the test subject was modified with bisulfite using KIT (ZYMORESEARCH, D5001 / D5002) to obtain the transformed DNA.

[0079] 4. Prepare the reaction system shown in Table 2 (total 25 μL), where the template is the DNA transformed from the test subject, positive control DNA, negative control DNA, or blank control; then perform fluorescent PCR amplification according to the reaction program. The 6-channel quantitative PCR instruments used were QuantStudio 5 (Applied Biosystems, Thermo Fisher Scientific, USA) and QuantGene 9600 (Hangzhou, China, Borui Technology Co., Ltd.). The reaction program was as follows: Stage 1: 95℃ for 5 min, 1 cycle; Stage 2: 95℃ for 15 sec; 60℃ for 30 sec; 45 cycles; Stage 3: Collect fluorescence signals at 58℃, and obtain the CT values ​​of SHOX2, RASSF1, GRIK2, HOXA9, PTGER4, and ACTB, respectively, and record them as CT. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45.

[0080] Table 2. Reaction System

[0081]

[0082]

[0083] Note: The DNA enzyme is Accurate Tag HS DNA polymerase (CM0008, 5u / ul, AGL Biosciences, China).

[0084] The DNA sequences of the amplified regions of primer and probe sets SHOX2-1, RASSF1-1, GRIK2-1, HOXA9-1, PTGER4-1 and ACTB-1 after sulfite conversion are shown in Table 3.

[0085] Table 3

[0086]

[0087]

[0088]

[0089]

[0090] The CT values ​​of the detection results are shown in Table 4. Further calculation of the dCT for each gene is denoted as dCT. X (dCT X =CT X -CT ACTB ).

[0091] Table 4-1. Detection results of gene methylation markers in tumor tissue (A) and adjacent normal tissue (C)

[0092]

[0093]

[0094] Note: TE is 10mM Tris HCl-EDTA (10mM / 1mM) buffer, used as a blank control, and dCT Th is the dCT cutoff value.

[0095] Table 4-2. Detection results of gene methylation markers in tumor tissue (A) and adjacent normal tissue (C)

[0096]

[0097]

[0098] Note: TE is 10mM Tris HCl-EDTA (10mM / 1mM) buffer, used as a blank control, and dCT Th is the dCT cutoff value.

[0099] 5. Results of gene methylation marker detection in lung cancer tissue DNA

[0100] (1) According to Table 4, lung cancer tissue (C), human lung cancer cell lines A549, HCC827, and H1299 cells are gene methylation-positive DNA. Adjacent normal tissue (A) is gene methylation-negative DNA. BS-treated DNA was amplified by five-gene fluorescent PCR. The CT value obtained, minus the CT value of the internal control ACTB, is the dCT value of the gene. The dCT values ​​of the positive sample group, together with pathologically confirmed lung cancer tissue and positive control DNA, constitute the positive sample group; the dCT values ​​of the negative sample group, together with adjacent normal tissue and negative control DNA, constitute the negative sample group.

[0101] (2) Whether gene methylation has occurred is determined by comparing the dCT values ​​(denoted as dCT) of SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 in the test samples. X To achieve: dCT X =CT x -CT ACTB The dCT value is the CT value obtained by subtracting the CT value of ACTB from the detected CT value. If the dCT value of the SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4 genes in the test subject is less than or equal to the dCT threshold value, then the test subject has methylation based on that gene. The dCT threshold value for each gene is a statistically averaged dCT value obtained by comparing the dCT values ​​of lung cancer tissue and adjacent normal tissue confirmed by a large number of cases. It is the critical value (threshold) that can best distinguish between tumors and non-tumor tissues.

[0102] The results showed that, under the given PCR reaction conditions, the dCT critical values ​​for SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 were 5, 5, 5, 5, and 5, respectively.

[0103] Following the steps above, replace "primers and probes of primer and probe set SHOX2-1" with "primers and probes of primer and probe set SHOX2-2", "primers and probes of primer and probe set SHOX2-3", or "primers and probes of primer and probe set SHOX2-4"; replace "primers and probes of primer and probe set RASSF1-1" with "primers and probes of primer and probe set RASSF1-2" or "primers and probes of primer and probe set RASSF1-3"; and replace "primers and probes of primer and probe set GRIK2-1" with "primers and probes of primer and probe set GRIK2-2" or "primers and probes of primer and probe set GRIK2-1". Replace "primers and probes for primers and probes of HOXA9-1" with "primers and probes for HOXA9-2", "primers and probes for HOXA9-3", or "primers and probes for HOXA9-4"; replace "primers and probes for PTGER4-1" with "primers and probes for PTGER4-2" or "primers and probes for PTGER4-3"; and replace "primers and probes for ACTB-1" with "primers and probes for ACTB-2". All other steps remain unchanged. The results show that under the established PCR reaction conditions, the dCT critical values ​​for SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 are 5, 5, 5, 5, and 5, respectively.

[0104] The DNA sequences of the amplified regions of primer probe sets SHOX2-2, SHOX2-3, SHOX2-4, RASSF1-2, RASSF1-3, GRIK2-2, GRIK2-3, HOXA9-2, HOXA9-3, HOXA9-4, PTGER4-2, PTGER4-3, and ACTB-2 after sulfite conversion are shown in Table 3.

[0105] (3) Determining whether a sample comes from a lung cancer patient is done by comparing the dCT and critical values ​​of each gene in the sample using the group summation method C5. 2 The determination is as follows: if the dCt values ​​of at least two of the five genes SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 in the sample are less than or equal to the threshold, it indicates that the sample is methylated, and the sample is positive, meaning it comes from a lung cancer patient; otherwise (i.e., the dCt value is greater than the dCt threshold value of the gene), the sample is not methylated, and the sample is negative, meaning it does not come from a lung cancer patient.

[0106] Using a combined model to determine if a sample originates from a tumor: If any two or more of the five target genes in the tested sample show methylation, then the sample is determined to be from a tumor patient, i.e., using C5... 2 The combination method results in a positive result (red), while a result with only a single gene or no gene involved in methylation is negative (green).

[0107] In 14 paired cancer tissue samples, methylation was detected in 13 cases, with a sensitivity or detection rate of 92%. In 10 benign nodule samples, only 1 case showed a false positive due to gene methylation, with a specificity of 90%, indicating that the 5-gene methylation method can differentiate between benign and malignant nodules. Therefore, the synergistic detection of GRIK2, HOXA9, PTGER4, RASSF1, and SHOX2 genes helps improve tumor detection and differentiation rates.

[0108] The 5-gene methylation method improves tumor detection rates, while combined methods reduce false positives, making test results from high-risk or lung cancer patients more reliable. The interpretation of results from the 5-gene methylation method is more robust, and individuals with a single positive gene are followed up. This method's sensitivity is twice that of the usual "any one gene positive" method, meaning that this method requires higher sensitivity and specificity to obtain the results of the usual (e.g., C5) methods. 1 Judgment criteria.

[0109] Therefore, it can be seen that the synergistic detection of GRIK2, HOXA9, PTGER4, RASSF1 and SHOX2 genes in lung cancer tissue can improve the detection rate of lung cancer.

[0110] Example 3, Sensitivity Experiment

[0111] The samples to be tested were 100% MetBisDNA (100% + VE ctr DNA), 10% MetBisDNA (10% + VE ctr DNA with 90% - VE ctr DNA), 1% MetBisDNA (1% + VE ctr DNA with 99% - VE ctr DNA), 0% MetBisDNA (100% - VE ctr DNA), and TE (no DNA, only TE buffer).

[0112] Each sample to be tested underwent the following experiment:

[0113] 1. Dilute the primers and probes of “SHOX2-1”, “RASSF1-1”, “GRIK2-1”, “HOXA9-1”, “PTGER4-1” and “ACTB-1” in Table 1 with water respectively.

[0114] 2. Take the sample to be tested and use EZ DNAMethylation-Direct TM KIT was used for bisulfite modification to obtain the DNA transformed from the sample to be tested.

[0115] 3. Prepare the reaction system shown in Table 2 (total 25 μL), where the template is the DNA transformed from the sample to be tested, positive control DNA, or negative control DNA; then perform PCR amplification according to the reaction program. The reaction program is as follows: Stage 1: 95℃ for 5 min, 1 cycle; Stage 2: 95℃ for 15 sec; 60℃ for 30 sec; 45 cycles; Stage 3: Collect fluorescence signals at 58℃, and obtain the CT values ​​of GRIK2, HOXA9, PTGER4, RASSF1, SHOX2, and ACTB, respectively, and record them as CT. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not S-shaped or the CT value is blank, the CT value is recorded as 45. Further calculate the dCT value (denoted as dCT) for each gene (GRIK2, HOXA9, PTGER4, RASSF1, or SHOX2). X ), dCT X =CT x -CT ACTB .

[0116] The test results for the five dilution samples are shown in Table 5.

[0117] Table 5

[0118]

[0119] Following step 5 of Example 2, determine whether the five test samples are positive or negative. The results of the five test samples are shown in Table 5. The results indicate that the method provided by this invention can detect whether methylation has occurred, and the limit of detection is 10% MetBisDNA, equivalent to 0.5 ng cfDNA.

[0120] Following the steps above, replace "primers and probes of primer and probe set SHOX2-1" with "primers and probes of primer and probe set SHOX2-2", "primers and probes of primer and probe set SHOX2-3", or "primers and probes of primer and probe set SHOX2-4"; replace "primers and probes of primer and probe set RASSF1-1" with "primers and probes of primer and probe set RASSF1-2" or "primers and probes of primer and probe set RASSF1-3"; and replace "primers and probes of primer and probe set GRIK2-1" with "primers and probes of primer and probe set GRIK2-2" or "primers and probes of primer and probe set GRIK2-1". Replace “primers and probes for primers ...

[0121] The results showed that any primer-probe combination consisting of any one of the four primer-probe sets of SHOX2, any one of the three primer-probe sets of RASSF1, any one of the three primer-probe sets of GRIK2, any one of the four primer-probe sets of HOXA9, any one of the three primer-probe sets of PTGER4, and any one of the two primer-probe sets of ACTB could detect whether methylation had occurred, with a minimum detection limit of 10% MetBisDNA, equivalent to 0.5 ng cfDNA.

[0122] Example 4: Lung Cancer Detection Using Blood as a Sample

[0123] The samples to be tested included whole blood samples from 44 patients with ethically approved and pathologically confirmed lung cancer, 11 patients with benign lung nodules, and 140 healthy volunteers.

[0124] 1. Dilute the primers and probes of “SHOX2-1”, “RASSF1-1”, “GRIK2-1”, “HOXA9-1”, “PTGER4-1” and “ACTB-1” in Table 1 with water respectively.

[0125] 2. Take 8 ml of the sample to be tested into an EDTA anticoagulant vacuum blood collection tube and centrifuge twice within 2 hours (first centrifuge at 1600g for 15 min, second centrifuge at 15000g for 15 min) to obtain cell-free plasma.

[0126] 3. Take the cell-free plasma separately and extract free DNA using a plasma free DNA centrifugation kit (D3182-03S, Meiji Company, Guangzhou) to obtain cfDNA from the plasma of the test subject.

[0127] 4. Collect plasma cfDNA from the subjects to be tested and apply EZ DNA Methylation-Direct. TM KIT (Cat.no.D5002, Zymo Research, USA) was modified with bisulfite to obtain the transformed cfDNA from the test subject.

[0128] 5. Prepare the reaction system shown in Table 2 (total 25 μL), where the template is the transformed cfDNA from the test subject, positive control DNA, or negative control DNA; then perform PCR amplification according to the reaction program. The reaction program is as follows: Stage 1: 95℃ for 5 min, 1 cycle; Stage 2: 95℃ for 15 sec; 60℃ for 30 sec; 45 cycles; Stage 3: Collect fluorescence signals at 58℃, and obtain the CT values ​​of GRIK2, HOXA9, PTGER4, RASSF1, SHOX2, and ACTB, respectively, and record them as CT. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not S-shaped or the CT value is blank, the CT value is recorded as 45. Further calculate the dCT value (denoted as dCT) for each gene (GRIK2, HOXA9, PTGER4, RASSF1, or SHOX2). X ), dCT X =CT x -CT ACTB That is, the CT value obtained by subtracting the CT value of the internal control ACTB from the detected CT value.

[0129] 6. Following step 5 in Example 2, determine whether the sample to be tested is positive or negative.

[0130] The results are shown in Table 6. The results indicate that, comparing the CT value (dCT / dCT cutoff value) of individual gene methylation in the plasma of lung cancer patients, the detection rate was relatively high: 84% for the HOXA9 gene and 80% for the PTGER4 gene. In contrast, the methylation detection rates of these genes in the plasma of healthy volunteers were very low, indicating high specificity (above 93%). SHOX2 showed a false positive rate of 0 in the plasma of healthy volunteers (100% specificity), but its detection rate in the plasma of lung cancer patients was also low (36%). Therefore, the five target genes can detect lung cancer at an early stage in plasma cfDNA samples. The low methylation of individual genes in the blood of patients with benign pulmonary nodules indicates that these five target gene markers can differentiate between malignant and benign pulmonary nodules; that is, the methylation of the five target genes in plasma can distinguish between lung cancer patients, patients with benign pulmonary nodules, and healthy volunteers. When using a combined method to determine the methylation results of samples, the detection rate in the plasma of lung cancer patients was 93%, and the specificity was also 93%.

[0131] Table 6

[0132]

[0133] The results were analyzed using the ROC method; the detection results are shown below. Figure 1 ROC curves and area under the curve (AUC) showed that all five gene methylation markers in lung cancer tissue had high value for diagnosing early-stage lung cancer. The AUC value of synergistic detection of GRIK2, HOXA9, PTGER4, RASSF1, and SHOX2 reached 0.912. Therefore, synergistic detection of GRIK2, HOXA9, PTGER4, RASSF1, and SHOX2 genes in peripheral blood can improve the detection rate of lung cancer, and all five gene methylation markers in plasma have high value for diagnosing early-stage lung cancer.

[0134] The results above indicate that the combined use of the five markers can cover multiple carcinogenic mechanisms and various signal transduction systems in carcinogenesis, and can improve the methylation positivity rate or detection rate of samples. The combination of methods can reduce false positives and improve the accuracy of the test.

[0135] Therefore, it is evident that plasma cfDNA testing can identify whether a person is a lung cancer patient or not.

[0136] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of the combined biomarker in the preparation of a lung cancer detection kit; the combined biomarker is composed of the SHOX2 gene, RASSF1 gene, GRIK2 gene, HOXA9 gene and PTGER4 gene; The GeneID of the SHOX2 gene is 6474; The GeneID of the RASSF1 gene is 11186; The GeneID of the GRIK2 gene is 2898; The GeneID of the HOXA9 gene is 3205; The GeneID of the PTGER4 gene is 5734; The lung cancer detection kit includes detection reagents for the combined biomarkers, which include primer and probe sets for detecting the methylation level of the SHOX2 gene, the methylation level of the RASSF1 gene, the methylation level of the GRIK2 gene, the methylation level of the HOXA9 gene, and the methylation level of the PTGER4 gene. The primer and probe set used to detect the methylation level of the SHOX2 gene is primer and probe set SHOX2-1, primer and probe set SHOX2-2, primer and probe set SHOX2-3 or primer and probe set SHOX2-4; The primer and probe set used to detect the methylation level of the RASSF1 gene is primer and probe set RASSF1-1, primer and probe set RASSF1-2, or primer and probe set RASSF1-3; The primer and probe set used to detect the methylation level of the GRIK2 gene is primer and probe set GRIK2-1, primer and probe set GRIK2-2, or primer and probe set GRIK2-3. The primer and probe set used to detect the methylation level of the HOXA9 gene is primer and probe set HOXA9-1, primer and probe set HOXA9-2, primer and probe set HOXA9-3 or primer and probe set HOXA9-4. The primer and probe set used to detect the methylation level of the PTGER4 gene is primer and probe set PTGER4-1, primer and probe set PTGER4-2, or primer and probe set PTGER4-3. The primer-probe set SHOX2-1 consists of primer SHOX2-F1 shown in SEQ ID NO: 1, primer SHOX2-R1 shown in SEQ ID NO: 2, and probe SHOX2-P1 shown in SEQ ID NO: 3; The primer-probe set SHOX2-2 consists of primer SHOX2-F2 shown in SEQ ID NO: 4, primer SHOX2-R2 shown in SEQ ID NO: 5, and probe SHOX2-P2 shown in SEQ ID NO: 6; The primer-probe set SHOX2-3 consists of primer SHOX2-F3 shown in SEQ ID NO: 7, primer SHOX2-R3 shown in SEQ ID NO: 8, and probe SHOX2-P3 shown in SEQ ID NO: 9; The primer-probe set SHOX2-4 consists of primer SHOX2-F4 shown in SEQ ID NO: 10, primer SHOX2-R4 shown in SEQ ID NO: 11, and probe SHOX2-P4 shown in SEQ ID NO: 12; The primer-probe set PTGER4-1 consists of primer PTGER4-F1 shown in SEQ ID NO: 43, primer PTGER4-R1 shown in SEQ ID NO: 44, and probe PTGER4-P1 shown in SEQ ID NO: 45; The primer-probe set PTGER4-2 consists of primer PTGER4-F2 shown in SEQ ID NO: 46, primer PTGER4-R2 shown in SEQ ID NO: 47, and probe PTGER4-P2 shown in SEQ ID NO: 48; The primer-probe set PTGER4-3 consists of primer PTGER4-F3 shown in SEQ ID NO: 49, primer PTGER4-R3 shown in SEQ ID NO: 50, and probe PTGER4-P3 shown in SEQ ID NO: 51; The primer-probe set RASSF1-1 consists of primer RASSF1-F1 shown in SEQ ID NO: 13, primer RASSF1-R1 shown in SEQ ID NO: 14, and probe RASSF1-P1 shown in SEQ ID NO: 15; The primer-probe set RASSF1-2 consists of primer RASSF1-F2 shown in SEQ ID NO: 16, primer RASSF1-R2 shown in SEQ ID NO: 17, and probe RASSF1-P2 shown in SEQ ID NO: 18; The primer-probe set RASSF1-3 consists of primer RASSF1-F3 shown in SEQ ID NO: 19, primer RASSF1-R3 shown in SEQ ID NO: 20, and probe RASSF1-P3 shown in SEQ ID NO: 21; The primer-probe set GRIK2-1 consists of primer GRIK2-F1 shown in SEQ ID NO: 22, primer GRIK2-R1 shown in SEQ ID NO: 23, and probe GRIK2-P1 shown in SEQ ID NO: 24; The primer-probe set GRIK2-2 consists of primer GRIK2-F2 shown in SEQ ID NO: 25, primer GRIK2-R2 shown in SEQ ID NO: 26, and probe GRIK2-P2 shown in SEQ ID NO:

27. The primer-probe set GRIK2-3 consists of primer GRIK2-F3 shown in SEQ ID NO: 28, primer GRIK2-R3 shown in SEQ ID NO: 29, and probe GRIK2-P3 shown in SEQ ID NO: 30; The primer-probe set HOXA9-1 consists of primer HOXA9-F1 shown in SEQ ID NO: 31, primer HOXA9-R1 shown in SEQ ID NO: 32, and probe HOXA9-P1 shown in SEQ ID NO: 33; The primer-probe set HOXA9-2 consists of primer HOXA9-F2 shown in SEQ ID NO: 34, primer HOXA9-R2 shown in SEQ ID NO: 35, and probe HOXA9-P2 shown in SEQ ID NO:

36. The primer-probe set HOXA9-3 consists of primer HOXA9-F3 shown in SEQ ID NO: 37, primer HOXA9-R3 shown in SEQ ID NO: 38, and probe HOXA9-P3 shown in SEQ ID NO:

39. The primer-probe set HOXA9-4 consists of primer HOXA9-F4 shown in SEQ ID NO: 40, primer HOXA9-R4 shown in SEQ ID NO: 41, and probe HOXA9-P4 shown in SEQ ID NO:

42. The kit also includes an internal reference gene detection reagent; the internal reference gene is the ACTB gene. The kit also includes a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarkers into the subject's dCT. X It is used to determine whether a person being tested has lung cancer. The subject's dCT X The calculation method is as follows: Genomic DNA from the plasma, fecal cfDNA, or lung tissue of the test subject is chemically modified. Then, using these as templates, fluorescent PCR amplification is performed using the aforementioned primers and probes. Fluorescent signals are collected, and the CT values ​​of SHOX2, RASSF1, GRIK2, HOXA9, PTGER4, and ACTB are obtained, and denoted as CT values ​​respectively. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT value of each gene SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4. X =CT X -CT ACTB ; The method for determination is as follows: if at least two of the SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 genes in the test subject are methylated, then the test subject is a lung cancer patient; otherwise, the test subject is not a lung cancer patient. Whether a gene has undergone methylation is determined by comparing the dCT and dCT threshold of the gene in the test sample; If the dCT of the SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4 genes in the subject is less than or equal to the dCT threshold, then the subject is methylated based on that gene. The dCT threshold value for each gene is the average statistical value obtained by comparing the dCT values ​​of lung cancer tissue and adjacent normal tissue. It is a threshold value that can best distinguish between tumors and non-tumors.

2. A lung cancer detection kit, characterized in that, The detection reagent includes a combination of biomarkers; the detection reagent includes a primer and probe set for detecting the methylation level of the SHOX2 gene, a primer and probe set for detecting the methylation level of the RASSF1 gene, a primer and probe set for detecting the methylation level of the GRIK2 gene, a primer and probe set for detecting the methylation level of the HOXA9 gene, and a primer and probe set for detecting the methylation level of the PTGER4 gene. The primer and probe set used to detect the methylation level of the SHOX2 gene is primer and probe set SHOX2-1, primer and probe set SHOX2-2, primer and probe set SHOX2-3 or primer and probe set SHOX2-4; The primer and probe set used to detect the methylation level of the RASSF1 gene is primer and probe set RASSF1-1, primer and probe set RASSF1-2, or primer and probe set RASSF1-3; The primer and probe set used to detect the methylation level of the GRIK2 gene is primer and probe set GRIK2-1, primer and probe set GRIK2-2, or primer and probe set GRIK2-3. The primer and probe set used to detect the methylation level of the HOXA9 gene is primer and probe set HOXA9-1, primer and probe set HOXA9-2, primer and probe set HOXA9-3 or primer and probe set HOXA9-4. The primer and probe set used to detect the methylation level of the PTGER4 gene is primer and probe set PTGER4-1, primer and probe set PTGER4-2, or primer and probe set PTGER4-3. The primer-probe set SHOX2-1 consists of primer SHOX2-F1 shown in SEQ ID NO: 1, primer SHOX2-R1 shown in SEQ ID NO: 2, and probe SHOX2-P1 shown in SEQ ID NO: 3; The primer-probe set SHOX2-2 consists of primer SHOX2-F2 shown in SEQ ID NO: 4, primer SHOX2-R2 shown in SEQ ID NO: 5, and probe SHOX2-P2 shown in SEQ ID NO: 6; The primer-probe set SHOX2-3 consists of primer SHOX2-F3 shown in SEQ ID NO: 7, primer SHOX2-R3 shown in SEQ ID NO: 8, and probe SHOX2-P3 shown in SEQ ID NO: 9; The primer-probe set SHOX2-4 consists of primer SHOX2-F4 shown in SEQ ID NO: 10, primer SHOX2-R4 shown in SEQ ID NO: 11, and probe SHOX2-P4 shown in SEQ ID NO: 12; The primer-probe set PTGER4-1 consists of primer PTGER4-F1 shown in SEQ ID NO: 43, primer PTGER4-R1 shown in SEQ ID NO: 44, and probe PTGER4-P1 shown in SEQ ID NO: 45; The primer-probe set PTGER4-2 consists of primer PTGER4-F2 shown in SEQ ID NO: 46, primer PTGER4-R2 shown in SEQ ID NO: 47, and probe PTGER4-P2 shown in SEQ ID NO: 48; The primer-probe set PTGER4-3 consists of primer PTGER4-F3 shown in SEQ ID NO: 49, primer PTGER4-R3 shown in SEQ ID NO: 50, and probe PTGER4-P3 shown in SEQ ID NO: 51; The primer-probe set RASSF1-1 consists of primer RASSF1-F1 shown in SEQ ID NO: 13, primer RASSF1-R1 shown in SEQ ID NO: 14, and probe RASSF1-P1 shown in SEQ ID NO: 15; The primer-probe set RASSF1-2 consists of primer RASSF1-F2 shown in SEQ ID NO: 16, primer RASSF1-R2 shown in SEQ ID NO: 17, and probe RASSF1-P2 shown in SEQ ID NO: 18; The primer-probe set RASSF1-3 consists of primer RASSF1-F3 shown in SEQ ID NO: 19, primer RASSF1-R3 shown in SEQ ID NO: 20, and probe RASSF1-P3 shown in SEQ ID NO: 21; The primer-probe set GRIK2-1 consists of primer GRIK2-F1 shown in SEQ ID NO: 22, primer GRIK2-R1 shown in SEQ ID NO: 23, and probe GRIK2-P1 shown in SEQ ID NO: 24; The primer-probe set GRIK2-2 consists of primer GRIK2-F2 shown in SEQ ID NO: 25, primer GRIK2-R2 shown in SEQ ID NO: 26, and probe GRIK2-P2 shown in SEQ ID NO:

27. The primer-probe set GRIK2-3 consists of primer GRIK2-F3 shown in SEQ ID NO: 28, primer GRIK2-R3 shown in SEQ ID NO: 29, and probe GRIK2-P3 shown in SEQ ID NO: 30; The primer-probe set HOXA9-1 consists of primer HOXA9-F1 shown in SEQ ID NO: 31, primer HOXA9-R1 shown in SEQ ID NO: 32, and probe HOXA9-P1 shown in SEQ ID NO: 33; The primer-probe set HOXA9-2 consists of primer HOXA9-F2 shown in SEQ ID NO: 34, primer HOXA9-R2 shown in SEQ ID NO: 35, and probe HOXA9-P2 shown in SEQ ID NO:

36. The primer-probe set HOXA9-3 consists of primer HOXA9-F3 shown in SEQ ID NO: 37, primer HOXA9-R3 shown in SEQ ID NO: 38, and probe HOXA9-P3 shown in SEQ ID NO:

39. The primer-probe set HOXA9-4 consists of primer HOXA9-F4 shown in SEQ ID NO: 40, primer HOXA9-R4 shown in SEQ ID NO: 41, and probe HOXA9-P4 shown in SEQ ID NO:

42. The kit also includes an internal reference gene detection reagent; the internal reference gene is the ACTB gene. The kit also includes a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarkers into the subject's dCT. X It is used to determine whether a person being tested has lung cancer. The subject's dCT X The calculation method is as follows: Genomic DNA from the plasma, fecal cfDNA, or lung tissue of the test subject is chemically modified. Then, using these as templates, fluorescent PCR amplification is performed using the aforementioned primers and probes. Fluorescent signals are collected, and the CT values ​​of SHOX2, RASSF1, GRIK2, HOXA9, PTGER4, and ACTB are obtained, and denoted as CT values ​​respectively. SHOX2 CT scan RASSF1 CT scan GRIK2 CT scan HOXA9 CT scan PTGER4 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT value of each gene SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4. X =CT X -CT ACTB ; The method for determination is as follows: if at least two of the SHOX2, RASSF1, GRIK2, HOXA9, and PTGER4 genes in the test subject are methylated, then the test subject is a lung cancer patient; otherwise, the test subject is not a lung cancer patient. Whether a gene has undergone methylation is determined by comparing the dCT and dCT threshold of the gene in the test sample; If the dCT of the SHOX2, RASSF1, GRIK2, HOXA9, or PTGER4 genes in the subject is less than or equal to the dCT threshold, then the subject is methylated based on that gene. The dCT threshold value for each gene is the average statistical value obtained by comparing the dCT values ​​of lung cancer tissue and adjacent normal tissue. It is a threshold value that can best distinguish between tumors and non-tumors.

3. The reagent kit according to claim 2, characterized in that: The GeneBank accession number for the ACTB gene is 60.

4. The reagent kit according to claim 3, characterized in that: The internal reference gene detection reagent is the primer and probe set ACTB-1 or the primer and probe set ACTB-2 used to detect the methylation level of the ACTB gene. The primer-probe set ACTB-1 consists of primer ACTB-F1 shown in SEQ ID NO: 52, primer ACTB-R1 shown in SEQ ID NO: 53, and probe ACTB-P1 shown in SEQ ID NO: 54; The primer-probe set ACTB-2 consists of primer ACTB-F2 shown in SEQ ID NO: 55, primer ACTB-R2 shown in SEQ ID NO: 56, and probe ACTB-P2 shown in SEQ ID NO:

57.

5. The kit according to claim 2 or 4, characterized in that: Each probe has a fluorescent label at one end and a fluorescent quenching label at the other end.

6. The reagent kit according to claim 2, characterized in that: The kit detects plasma cfDNA, fecal cfDNA, or genomic DNA from lung tissue.

Images