Composition and kit for detecting methylation of SOX15 gene and EME1 gene of human plasma for lung cancer

Abstract

The invention discloses a composition for detecting methylation of a human plasma SOX15 gene and an EME1 gene for lung cancer, which can be used for realizing early screening and early diagnosis of the lung cancer through detection of plasma DNA (Deoxyribonucleic Acid). Meanwhile, the invention further provides a kit for methylation of the SOX15 gene and the EME1 gene, the sensitivity of detection of lung cancer patients reaches 83.49%, and the specificity is 92.10%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a composition for detecting methylation of the SOX15 and EME1 genes in human plasma for lung cancer. Background Technology

[0002] Lung cancer is a leading cause of cancer-related deaths worldwide, accounting for nearly 30% of cancer deaths in China. Despite recent advancements in novel treatments such as molecularly targeted drugs and immune checkpoint inhibitors, improving the 5-year survival rate for patients with advanced lung cancer remains challenging. Therefore, early diagnosis of lung cancer plays a crucial role in improving cancer survival rates. In early lung cancer screening, low-dose computed tomography (LDCT) is currently the most effective method for improving cancer survival and reducing mortality. However, it also has drawbacks such as insufficient sensitivity and a high false-positive rate, requiring further invasive screening. Therefore, combining a low-invasive, highly sensitive, and highly specific detection method with LDCT screening can effectively improve the early diagnosis rate of lung cancer. Summary of the Invention

[0003] The purpose of this invention is to provide a composition and kit for detecting methylation of the SOX15 and EME1 genes in human plasma for early screening and diagnosis of lung cancer. The detection targets CpG sites in the promoter regions of the SOX15 and EME1 genes, offering high accuracy, convenient sampling, and minimal invasiveness.

[0004] To achieve the above objectives, one technical solution adopted by the present invention is: a composition for detecting methylation of the SOX15 gene and EME1 gene in human plasma for lung cancer, wherein the composition comprises the SOX15 gene and the EME1 gene.

[0005] Furthermore, the primer sequence for the SOX15 gene is SEQ ID No: 1; and the primer sequence for the EME1 gene is SEQ ID No: 2.

[0006] Furthermore, the probe sequence of the SOX15 gene is SEQ ID No: 4; and the probe sequence of the EME1 gene is SEQ ID No: 5.

[0007] To elaborate further, the internal reference for quantitative real-time PCR detection is the ACTB gene, the primer sequence for the ACTB gene is SEQ ID No: 3, and the probe primer sequence is SEQ ID No: 6.

[0008] A kit for detecting early-stage liver cancer includes SOX15 gene primers and probes, EME1 gene primers and probes, ACTB internal reference gene primers and probes, and PCR amplification enzyme reaction solution.

[0009] The beneficial effects of this invention are:

[0010] This invention provides a combined detection kit for SOX15 and EME1 gene dimethylation, which can be used for early screening and diagnosis of lung cancer through plasma DNA detection; specifically:

[0011] First, we designed primers and probes to establish a methylation amplification system targeting CG-rich regions of the SOX15 and EME1 genes. We then tested the sensitivity and specificity of the PCR system for lung cancer using FFPE samples.

[0012] Then, the performance of the kit was validated through extensive testing of clinical plasma samples, including comprehensive testing of benign lung lesion samples and other tumor samples.

[0013] This invention develops a novel dual-gene methylation detection kit, which achieves a sensitivity of 83.49% and a specificity of 92.10% for lung cancer detection by detecting SOX15 and EME1 genes. The method is simple and non-invasive, enabling early screening and diagnosis of lung cancer. Attached Figure Description

[0014] Figure 1 This is the ROC curve of the PCR system for detecting the SOX15 gene and EME1 gene in the sample according to the present invention. Detailed Implementation

[0015] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0016] Example 1

[0017] The specific preparation method of the detection kit provided by this invention is as follows:

[0018] We established amplification systems for the target regions. For the SOX15 gene (Gene ID: 6665) and the EME1 gene (Gene ID: 146956), we screened two CpG-rich regions from each of the two targets. For each region, we designed a pair of primers and screened the optimal amplification system using whole-genome methylation quality control materials.

[0019] The amplification reaction system is shown in Table 1-1 below:

[0020] Table 1-1

[0021] Components Specification volume PCR Mix 2× 10μL SOX15 upstream amplification primers 20μM 0.4μl SOX15 downstream amplification primers 20μM 0.4μl SOX15 fluorescent probe 20μM 0.2μl EME1 upstream amplification primers 20μM 0.4μl EME1 downstream amplification primers 20μM 0.4μl EME1 fluorescent probe 20μM 0.2μl upstream amplification primers for internal control 20μM 0.4μl Downstream amplification primers for internal control 20μM 0.4μl Internal control fluorescent probe 20μM 0.2μl Nuclease-free water 2μl DNA 5μl Overall system 20μl

[0022] The upstream and downstream primer sequences for the SOX15 gene, EME1 gene, and internal reference gene are shown in Table 1-2 below:

[0023] Table 1-2

[0024]

[0025] The amplification reaction procedure is shown in Table 1-3 below:

[0026] Table 1-3

[0027]

[0028] Example 2

[0029] To validate the target's detection performance for lung cancer, we collected tissue sections (including cancerous and adjacent tissues) from 60 lung cancer patients and 32 samples from other cancers (including colorectal cancer, esophageal cancer, liver cancer, and lung cancer).

[0030] We first obtained DNA from tissue sections using a commercially available kit: the Blood / Cell / Tissue Gene DNA Extraction Kit (Centrifuge Column Type) (Catalog No. YDP304) manufactured by Tiangen Biotech (Beijing) Co., Ltd. After extraction, the extracted DNA underwent methylation treatment using the commercially available EZ DNA Methylation Kit (Catalog No. D5005) manufactured by ZYMO Research. The specific kit transformation procedure was performed according to the kit instructions. The transformed DNA was then detected using methylation PCR.

[0031] After treatment with the methylation kit, the methylated SOX15 or EME1 genes will produce fluorescent signals during the quantitative real-time PCR detection process, while the unmethylated genes will not produce fluorescent signals.

[0032] The results of SOX15 or EME1 gene methylation detection in 92 tissue section samples are shown in Table 2-1 below:

[0033] Table 2-1

[0034]

[0035]

[0036]

[0037]

[0038] The results above show that SOX15 and EME1 genes were methylated positive in all 40 lung cancer tissue samples, while they were methylated negative in adjacent tissues and other cancer samples. This indicates that the two target genes can effectively detect lung cancer. The following experiment will use plasma samples to test the sensitivity and specificity of the detection.

[0039] Example 3

[0040] To verify the clinical performance of the dual-gene methylation assay kit, we collected plasma samples from several patients clinically diagnosed with lung cancer, healthy individuals, and patients with benign lung lesions, and performed the assay using the SOX15 and EME1 dual-gene methylation system.

[0041] A total of 75 plasma samples from lung adenocarcinoma, 28 plasma samples from lung squamous cell carcinoma, 6 plasma samples from pulmonary tuberculosis, 20 plasma samples from pneumonia, and 50 plasma samples from healthy individuals were tested.

[0042] First, DNA was extracted from the plasma sample. The plasma DNA extraction used a commercially available kit: the Magnetic Bead Serum / Plasma Cell-Free DNA Extraction Kit (catalog number DP709) manufactured by Tiangen Biotech (Beijing) Co., Ltd. After extraction, the extracted DNA underwent methylation treatment. The DNA methylation treatment used a commercially available kit: the EZ DNA Methylation Kit (catalog number D5005) manufactured by ZYMO Research. The specific kit transformation procedure was performed according to the kit instructions. The transformed DNA was then detected by methylation PCR.

[0043] After treatment with the methylation kit, the methylated SOX15 or EME1 genes produced fluorescent signals during quantitative real-time PCR detection, while unmethylated genes did not produce fluorescent signals. The methylation detection results of SOX15 or EME1 genes in all plasma samples are shown in Table 3-1 below:

[0044] Table 3-1

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] The optimal positive cutoff value of the kit was determined based on the above data using dual-target combined detection of plasma samples, as follows:

[0052] 1. An EME1 gene CT value ≤ 38 indicates EME1 methylation positivity; a CT value > 38 or Unde indicates EME1 methylation negativity.

[0053] 2. A CT value ≤ 38 for the SOX15 gene indicates positive SOX15 methylation; a CT value > 38 or Unde indicates negative SOX15 gene methylation.

[0054] 3. The method for determining the sample test results is shown in Table 3-2 below:

[0055] Table 3-2

[0056]

[0057] Using the above-mentioned positive / negative determination method, 86 out of 103 lung cancer plasma samples were positive for methylation, with a sensitivity of 83.49%; and 6 out of 76 non-lung cancer samples were positive for methylation, with a specificity of 92.10%.

[0058] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A composition for detecting methylation of the SOX15 and EME1 genes in human plasma for lung cancer, characterized in that: The composition consists of the SOX15 gene and the EME1 gene.

2. The composition for detecting SOX15 and EME1 gene methylation in human plasma according to claim 1, characterized in that: The primer sequence for the SOX15 gene is SEQ ID No: 1; the primer sequence for the EME1 gene is SEQ ID No:

2.

3. The composition for detecting SOX15 and EME1 gene methylation in human plasma according to claim 2, characterized in that: The probe sequence for the SOX15 gene is SEQ ID No: 4; the probe sequence for the EME1 gene is SEQ ID No:

5.

4. The composition for detecting SOX15 and EME1 gene methylation in human plasma according to claim 3, characterized in that: The internal control for quantitative real-time PCR detection is the ACTB gene. The primer sequence for the ACTB gene is SEQ ID No: 3, and the probe primer sequence is SEQ ID No:

6.

5. A kit for detecting early-stage lung cancer, characterized in that: This includes primers and probes for the SOX15 gene, primers and probes for the EME1 gene, primers and probes for the ACTB internal reference gene, and PCR amplification enzyme reaction solution.

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