Marker for diagnosing lung cancer, nucleic acid product, and kit

Abstract

The application relates to a marker for diagnosing lung cancer, a nucleic acid product and a kit, the marker comprising one or more of SCARA5, ZNF323 and DLX1, and lung cancer is diagnosed by detecting the methylation level of the marker. The application finds that SCARA5, ZNF323 and DLX1 can be used as biomarkers for high-sensitivity and high-specificity screening and diagnosis of early lung cancer.

Description

Technical Field

[0001] This application relates to the field of molecular biotechnology, and in particular to a composition, kit, application, and method for screening lung cancer. Background Technology

[0002] Lung cancer is one of the most common and deadliest malignant tumors. In 2020, lung cancer accounted for 11.4% of all new cancer cases and 18% of all cancer deaths. Lung cancer is characterized by high incidence, high mortality, and low five-year survival rate. Therefore, early screening and diagnosis of lung cancer has become crucial for prolonging patient survival and saving lives.

[0003] Early lung cancer screening primarily relies on imaging techniques (such as chest X-rays and chest CT scans). While chest X-rays offer advantages like high penetration, ease of use, and low radiation dose, their low resolution makes it difficult to detect lesions smaller than 5mm-6mm in diameter, and they also have blind spots. Therefore, chest X-rays are not clinically recommended for lung cancer screening. Low-dose CT (LDCT) screening is currently the internationally recognized most effective method for detecting pulmonary nodules and diagnosing early lung cancer. However, due to its high sensitivity, it also easily detects many non-neoplastic pulmonary nodules. Therefore, the detection of a large number of false positive nodules is a significant problem that LDCT screening urgently needs to address. Summary of the Invention

[0004] This application study found that methylation of the SCARA5, ZNF323, and DLX1 genes is highly correlated with lung cancer. Based on this, this application provides a highly sensitive and specific biomarker, nucleic acid product, and kit for diagnosing lung cancer.

[0005] According to one aspect of this application, a biomarker for diagnosing lung cancer is provided, characterized in that the biomarker includes one or more of SCARA5, ZNF323 and DLX1, and lung cancer is diagnosed by detecting the methylation level of the biomarker.

[0006] In one embodiment, using GRCh38.p14 as a reference, lung cancer is diagnosed by detecting the methylation levels of one or more of the following regions:

[0007] Region 1: Chr8:27990673~27994673;

[0008] Region 2: Chr6:28354271~28358271;

[0009] Region 3: Chr2:172085507~172089674.

[0010] According to another aspect of this application, the use of a reagent for detecting the methylation level of the above-mentioned markers in the preparation of products for diagnosing lung cancer is provided.

[0011] According to another aspect of this application, a nucleic acid product for diagnosing lung cancer is provided, the nucleic acid product comprising primer pairs for detecting the methylation level of the aforementioned markers.

[0012] In one embodiment, the nucleic acid product further includes a detection probe corresponding to the primer pair.

[0013] In one embodiment, the primer pair comprises one or more of the following:

[0014] The nucleotide sequences of the first primer pair used to detect the methylation level of the SCARA5 gene are shown in SEQ ID No. 1 and SEQ ID No. 2;

[0015] The nucleotide sequences of the second primer pair, as shown in SEQ ID No. 4 and SEQ ID No. 5, are used to detect the methylation level of the ZNF323 gene; and

[0016] The nucleotide sequences of the third primer pair used to detect the methylation level of the DLX1 gene are shown in SEQ ID No. 7 and SEQ ID No. 8.

[0017] In one embodiment, the nucleotide sequence of the detection probe corresponding to the first primer pair is shown in SEQ ID No. 3; and / or,

[0018] The nucleotide sequence of the detection probe corresponding to the second primer pair is shown in SEQ ID No. 6; and / or

[0019] The nucleotide sequence of the detection probe corresponding to the third primer pair is shown in SEQ ID No. 9.

[0020] In one embodiment, it further includes an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair.

[0021] In one embodiment, the nucleotide sequences of the internal reference primer pair are shown in SEQ ID No. 10 and SEQ ID No. 11, and the nucleotide sequence of the internal reference probe is shown in SEQ ID No. 12.

[0022] According to another aspect of this application, a kit for diagnosing lung cancer is provided, comprising reagents for detecting the aforementioned lung cancer biomarkers.

[0023] In one embodiment, the reagent detects the methylation level of the marker by one or more of the following methods:

[0024] Methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, methylation-specific quantitative PCR, and mass spectrometry.

[0025] In one embodiment, it includes one or more of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents, as well as the above-mentioned nucleic acid products.

[0026] This application collects and analyzes lung cancer-related methylation data from the TCGA and GEO databases, and after extensive research, identifies genes highly associated with lung cancer: SCARA5, ZNF323, and DLX1. These genes can serve as biomarkers for highly sensitive and specific screening and diagnosis of early-stage lung cancer.

[0027] By constructing a gene methylation detection system, nucleic acid products with excellent detection performance were screened. Experiments have shown that the nucleic acid product of this application has a sensitivity of >95% for screening lung cancer: 168 cases were detected in 176 lung cancer patient samples; and a specificity of >98%: 3 cases were detected in 192 healthy human samples. Attached Figure Description

[0028] Figure 1 The results show the sensitivity and specificity of the gene combinations detected in Example 4 of this application. Detailed Implementation

[0029] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0030] 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 to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0031] Some embodiments of this application provide a biomarker for diagnosing lung cancer, including one or more of SCARA5, ZNF323 and DLX1, and diagnose lung cancer by detecting the methylation level of the above biomarkers.

[0032] Methylation level refers to whether cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency / proportion / percentage of methylation. It represents both a qualitative and quantitative concept. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

[0033] DNA methylation is recognized as the most valuable biomarker for early cancer screening. Currently, there are already FDA or CFDA certifications for cancer screening and diagnosis products based on DNA methylation detection for colorectal cancer, gastric cancer, and other cancers.

[0034] Circulating tumor DNA (ctDNA) is cell-free DNA from tumor cells that exists in the bloodstream of a patient. After tumor cells die or undergo apoptosis, their DNA is released into the circulatory system and exists freely in the blood. By detecting the methylation status of related genes in ctDNA, information about tumor tissue in the body can be obtained, thus providing a basis for tumor diagnosis.

[0035] In one embodiment, using GRCh38.p14 as a reference, lung cancer is diagnosed by detecting the methylation levels of one or more of the following regions:

[0036] Region 1: Chr8:27990673~27994673;

[0037] Region 2: Chr6:28354271~28358271;

[0038] Region 3: Chr2:172085507~172089674.

[0039] One embodiment of this application also provides the application of a reagent for detecting the methylation level of the above-mentioned markers in the preparation of products for diagnosing lung cancer.

[0040] Furthermore, one embodiment of this application provides a nucleic acid product for diagnosing lung cancer, comprising primer pairs for detecting the methylation level of the aforementioned biomarkers.

[0041] In one embodiment, the above-mentioned nucleic acid product also includes a detection probe corresponding to the above-mentioned primer pair.

[0042] In one embodiment, the primer pairs comprise one or more of the following:

[0043] The first primer pair, with nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, is used to detect the methylation level of the SCARA5 gene; the second primer pair, with nucleotide sequences as shown in SEQ ID No. 4 and SEQ ID No. 5, is used to detect the methylation level of the ZNF323 gene; and the third primer pair, with nucleotide sequences as shown in SEQ ID No. 7 and SEQ ID No. 8, is used to detect the methylation level of the DLX1 gene. Specifically, the nucleotide sequence shown in SEQ ID No. 1 is 5'-TTAGATTTTATTGTAGAAGT-3'; the nucleotide sequence shown in SEQ ID No. 2 is 5'-TCTAAATACTACCTAAAATA-3'; the nucleotide sequence shown in SEQ ID No. 4 is 5'-AGTTTTAATAGAGTATAATAGT-3'; the nucleotide sequence shown in SEQ ID No. 5 is 5'-TTAAAACTATTTAAACACTTTC-3'; the nucleotide sequence shown in SEQ ID No. 7 is 5'-TATTGGAGGTGAGGAAGA-3'; and the nucleotide sequence shown in SEQ ID No. 8 is 5'-CAAACCCAAACCTATAAT-3'.

[0044] In one embodiment, the nucleotide sequence of the detection probe corresponding to the first primer pair is shown in SEQ ID No. 3; the nucleotide sequence of the detection probe corresponding to the second primer pair is shown in SEQ ID No. 6; and the nucleotide sequence of the detection probe corresponding to the third primer pair is shown in SEQ ID No. 9. Specifically, the nucleotide sequence shown in SEQ ID No. 3 is 5'-TGGCGGCGAGAGTAGTTGTAG-3'; the nucleotide sequence shown in SEQ ID No. 6 is 5'-TAATCGTAATAGAGTTTAACGGTTTTA-3'; and the nucleotide sequence shown in SEQ ID No. 9 is 5'-TGGTAACGAGAGACGTTTATATATAG-3'.

[0045] In one embodiment, the above-mentioned nucleic acid product further includes an internal reference primer pair and a corresponding internal reference probe.

[0046] In one embodiment, the internal reference gene is selected from one or more of ACTB, GAPDH, ALDOA, and PGK1.

[0047] Furthermore, the internal reference gene was selected from ACTB.

[0048] In one embodiment, the nucleotide sequences of the internal reference primer pair are shown in SEQ ID No. 10 and SEQ ID No. 11, and the nucleotide sequence of the internal reference probe is shown in SEQ ID No. 12.

[0049] Specifically, the nucleotide sequence shown in SEQ ID No. 10 is 5'-TAGGTAAAGATGAGTTTATATAAGGA-3'; the nucleotide sequence shown in SEQ ID No. 11 is 5'-CAAACTATAACCTCTACAACCTTCAA-3'; and the nucleotide sequence shown in SEQ ID No. 12 is 5'-TGGAATTTGTGGTTAGGAAGGAGGTTG-3'.

[0050] The aforementioned nucleic acid products can be used clinically to screen for early-stage lung cancer with high sensitivity and specificity.

[0051] In addition, one embodiment of this application provides a kit for diagnosing lung cancer, including reagents for detecting the aforementioned biomarkers.

[0052] In one embodiment, the above reagents are used to detect the methylation level of the biomarker by one or more of the following methods: methylation-specific PCR, methylation chip detection, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, methylation-specific quantitative PCR, and mass spectrometry.

[0053] In one embodiment, the kit includes one or more of the following: nucleic acid extraction reagent, methylation conversion reagent, quality control reagent, PCR reaction reagent, and sequencing reagent, as well as the nucleic acid product for diagnosing lung cancer.

[0054] In one embodiment, the PCR reaction reagents include the above-mentioned nucleic acid product, PCR buffer, dNTP, MgCl2, and Taq DNA polymerase.

[0055] In one embodiment, the concentration of the nucleic acid product is 8 μM to 12 μM; the concentration of the PCR buffer can be 1×, 2×, 5× or 10×; the concentration of dNTP is 8 mM to 12 mM; the concentration of MgCl2 is 20 mM to 30 mM; and the concentration of Taq DNA polymerase is 4 U / μL to 6 U / μL.

[0056] In a specific example, the concentration of the nucleic acid product was 10 μM; the PCR buffer was 5×; the concentration of dNTPs was 10 mM; the MgCl2 concentration was 25 mM; and the Taq DNA polymerase concentration was 5 U / μL.

[0057] The above-mentioned kits for diagnosing lung cancer have at least the following advantages: rapid, simple, highly sensitive, and highly specific.

[0058] This application also provides a method for detecting the methylation level of the above-mentioned markers, including steps S10, S20, and S30. Specifically:

[0059] Step S10: Extract sample DNA.

[0060] In one embodiment, the above-mentioned sample is a biological sample selected from an individual, including but not limited to one or more of the following: cell lines, histological sections, tissue biopsy / paraffin-embedded tissue, body fluids, sputum, bronchoalveolar lavage fluid, urine, plasma, serum, whole blood, isolated blood cells, and cells isolated from blood.

[0061] Furthermore, the above sample is plasma. Cell-free DNA in plasma can be used to detect tumors, and it has the advantages of being minimally harmful to patients and having high specificity.

[0062] Step S20: The extracted DNA is methylated to obtain a transformed sample.

[0063] In one embodiment, methylation is converted to sulfite conversion or enzymatic conversion.

[0064] Step S30: Using the transformed sample obtained in step S20 as a template, the transformed template is tested using the above-mentioned nucleic acid product.

[0065] In one embodiment, the transformed template is detected by one or more of the following methods: methylation-specific PCR, methylation chip detection, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, methylation-specific quantitative PCR, and mass spectrometry.

[0066] The methods for detecting the methylation level of the above-mentioned biomarkers have at least the following advantages: they are simple and convenient, highly sensitive, highly specific, and can assist in the clinical diagnosis of early lung cancer. Specific Implementation

[0068] The present application will be further described below with reference to specific embodiments and comparative examples, but should not be construed as limiting the scope of protection of the present application.

[0069] Example 1: Screening of methylated genes and design of primer pairs and probes

[0070] This application constructs a large dataset of lung cancer-related methylation by mining data from the TCGA database and performing high-throughput methylation sequencing on clinical samples. It then uses bioinformatics methods to build analytical models for different methylation sites, screens out plasma cell-free DNA methylation biomarkers with application value, and further screens out methylation biomarkers with high specificity and high sensitivity through validation on clinical samples.

[0071] Specifically, this application screens candidate targets suitable for plasma free DNA methylation through the following steps: Based on differential analysis of methylation data from lung cancer patients and healthy individuals in the TCGA database and high-throughput methylation sequencing data of clinical samples, differential methylation sites showing high methylation in lung cancer patients and low methylation in healthy individuals are screened; further, differential methylation sites obtained above are analyzed for methylation differences in 18 other cancers in the TCGA database, and differential methylation sites showing low methylation in other cancers are extracted; further, the screened differential methylation sites are clinically validated, and finally, SCARA5, ZNF323, and DLX1 gene methylation are determined as the optimal detection targets, and a nucleic acid product for early screening and diagnosis of lung cancer is designed.

[0072] Primer pairs and probes were designed using Primer Express 3.0 and Methyl Primer Express v1.0 software, based on the human whole genome sequence GRCh38.p14 published by the National Center for Biotechnology Information (NCBI). The primer pairs and probes include those for detecting methylation of the SCARA5 gene (referencing GRCh38.p14, region Chr8:27990673–27994673), ZNF323 (region Chr6:28354271–28358271), and DLX1 (region Chr2:172085507–172089674), as well as a primer pair and probe for detecting the internal reference gene ACTB. The nucleotide sequences of the primer pairs and probes are shown in Table 1.

[0073] Table 1. Nucleotide sequence of primers and probes

[0074]

[0075]

[0076] Among them, the probe SEQ ID No. 3 for detecting SCARA5 has a reporter fluorescent group FAM at its 5' end and a quencher fluorescent group BHQ at its 3' end; the probe SEQ ID No. 6 for detecting ZNF323 has a reporter fluorescent group FAM at its 5' end and a quencher fluorescent group BHQ at its 3' end; the probe SEQ ID No. 9 for detecting DLX1 has a reporter fluorescent group FAM at its 5' end and a quencher fluorescent group BHQ at its 3' end; and the probe SEQ ID No. 12 for detecting ACTB has a reporter fluorescent group JOE at its 5' end and a quencher fluorescent group BHQ at its 3' end.

[0077] Example 2: A kit for lung cancer screening and diagnosis

[0078] This kit includes primer pairs and probes as shown in Table 1 for lung cancer screening and diagnosis. The kit also includes nucleic acid extraction reagents, methylation conversion reagents, PCR reaction solution, positive control, and negative control. The PCR reaction solution includes primers and probes as shown in SEQ ID No. 1–SEQ ID No. 3, SEQ ID No. 4–SEQ ID No. 6, SEQ ID No. 7–SEQ ID No. 9, and SEQ ID No. 10–SEQ ID No. 12, PCR buffer, dNTPs, MgCl2, and Taq DNA polymerase. The specific correspondence between primers and probes is shown in Table 1. The concentration of primers and probes is 10 μM; the PCR buffer is 5×; the concentration of dNTPs is 10 mM; the concentration of MgCl2 is 25 mM; and the concentration of Taq DNA polymerase is 5 U / μL. The positive control uses human methylated genomic DNA, and the negative control uses human unmethylated genomic DNA.

[0079] Example 3: Application of the reagent kit in screening and diagnosing lung cancer

[0080] I. Materials, Reagents, and Instruments

[0081] Nucleic acid extraction reagents and methylation conversion kits were all developed in-house; PCR buffer, dNTPs, and Taq DNA polymerase were purchased from Takara; MgCl2 was purchased from Sigma; primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd.; and the real-time PCR instrument was an ABI 7500.

[0082] II. Sample Preparation

[0083] The positive reference is human methylated genomic DNA, the negative reference is human unmethylated genomic DNA, and the sensitivity reference is 0.1% human methylated genomic DNA in 10 ng / mL human unmethylated genomic DNA; the samples to be processed are plasma samples from one confirmed lung cancer patient and one normal healthy person.

[0084] III. DNA Extraction

[0085] 1. Add 2 mL of plasma sample, 5 mL of nucleic acid lysis adsorption buffer and 100 μL of magnetic beads to a 15 mL centrifuge tube, vortex to mix, and incubate the centrifuge tube at 56 °C for 10 minutes.

[0086] 2. Place the centrifuge tube in a magnetic rack for 2 minutes to magnetically aspirate, remove all waste liquid, add 1 mL of washing solution A, mix well to ensure complete resuspension of the magnetic beads;

[0087] 3. Place the centrifuge tubes on a magnetic rack for 2 minutes to magnetically attract them, remove all the waste liquid, and use a 10μL to 100μL pipette tip to remove as much residual liquid as possible. Transfer the centrifuge tubes to a non-magnetic test tube rack, open the tube caps, and let them dry at room temperature for 5 minutes.

[0088] 4. Add 40 μL of elution buffer, tighten the cap, vortex to mix, resuspend the magnetic beads, and incubate the centrifuge tube at 56°C for 5 min.

[0089] 5. Place the centrifuge tube on a magnetic rack for 2 minutes to magnetically absorb the elution solution, then transfer all the elution solution to a new 0.2 mL PCR tube.

[0090] IV. Sulfite Conversion

[0091] 1. Add 110 μL of sulfite solution to a 0.2 mL PCR tube containing 40 μL of DNA. After sealing the centrifuge tube, vortex to mix and then centrifuge briefly. Place the centrifuge tube in a regular PCR instrument for reaction. Set the reaction conditions to 95℃ for 5 min, 60℃ for 10 min, 95℃ for 5 min, and 60℃ for 10 min.

[0092] 2. After the reaction is complete, transfer the DNA solution to a new 1.5 mL centrifuge tube, add 600 μL of binding buffer and 10 μL of magnetic beads, vortex to mix, and let stand at room temperature for 5 min.

[0093] 3. Place the centrifuge tube in a magnetic rack for 2 minutes to magnetically absorb all the waste liquid, add 500 μL of washing solution A, and vortex to mix to ensure that the magnetic beads are completely resuspended;

[0094] 4. Place the centrifuge tubes on a magnetic rack for 2 minutes to magnetically suspend them. Remove all the waste liquid, add 200 μL of desulfonation solution, vortex to mix and ensure that the magnetic beads are completely resuspended, and let stand at room temperature for 15 minutes.

[0095] 5. Place the centrifuge tube in a magnetic rack for 2 minutes to magnetically absorb the liquid, remove all the waste liquid, add 500 μL of washing solution B, and vortex to mix to ensure that the magnetic beads are completely resuspended.

[0096] 6. Place the centrifuge tube in a magnetic rack for 2 minutes to magnetically absorb the liquid, remove all waste liquid, add 500 μL of washing solution C, and vortex to mix to ensure that the magnetic beads are completely resuspended.

[0097] 7. Place the centrifuge tube in a magnetic rack for 2 minutes to magnetically absorb the liquid, remove all the waste liquid, add 500 μL of washing solution D, and vortex to mix to ensure that the magnetic beads are completely resuspended.

[0098] 8. Place the centrifuge tubes on a magnetic rack for 2 minutes to remove all the waste liquid. Use a 10μL to 100μL pipette tip to remove as much residual liquid as possible. Transfer the centrifuge tubes to a non-magnetic test tube rack, open the caps, and let them dry at room temperature for 5 minutes.

[0099] 9. Add 50 μL of elution buffer, tighten the cap, vortex to mix, resuspend the magnetic beads, and incubate the centrifuge tube at 56 °C for 5 min.

[0100] 10. Place the centrifuge tubes on a magnetic rack and allow them to magnetically adhere for 2 minutes. Transfer all the eluent to a new centrifuge tube for later use.

[0101] V. PCR Procedure

[0102] 1. The PCR reaction solution system is shown in Tables 2-8;

[0103] Table 2. SCARA5 gene methylation reaction solution

[0104]

[0105]

[0106] Table 3. ZNF323 gene methylation reaction solution

[0107] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.4 (10μM) 0.25μL SEQ ID No.5 (10μM) 0.25μL SEQ ID No. 6 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 2.5μL Total 15μL

[0108] Table 4. DLX1 gene methylation reaction solution

[0109] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.7 (10μM) 0.25μL SEQ ID No. 8 (10μM) 0.25μL SEQ ID No.9 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 2.5μL Total 15μL

[0110] Table 5. SCARA5 / ZNF323 gene methylation reaction solution

[0111] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.1 (10μM) 0.25μL SEQ ID No.2 (10μM) 0.25μL SEQ ID No.3 (10μM) 0.25μL SEQ ID No.4 (10μM) 0.25μL SEQ ID No.5 (10μM) 0.25μL SEQ ID No. 6 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 1.75μL Total 15μL

[0112] Table 6. SCARA5 / DLX1 gene methylation reaction solution

[0113] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.1 (10μM) 0.25μL SEQ ID No.2 (10μM) 0.25μL SEQ ID No.3 (10μM) 0.25μL SEQ ID No.7 (10μM) 0.25μL SEQ ID No. 8 (10μM) 0.25μL SEQ ID No.9 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 1.75μL Total 15μL

[0114] Table 7. Methylation reaction solution for ZNF323 / DLX1 gene

[0115] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.4 (10μM) 0.25μL SEQ ID No.5 (10μM) 0.25μL SEQ ID No. 6 (10μM) 0.25μL SEQ ID No.7 (10μM) 0.25μL SEQ ID No. 8 (10μM) 0.25μL SEQ ID No.9 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 1.75μL Total 15μL

[0116] Table 8. Methylation reaction solution for SCARA5 / ZNF323 / DLX1 genes

[0117] Components of a single PCR reaction Amount of a single PCR reaction SEQ ID No.1 (10μM) 0.25μL SEQ ID No.2 (10μM) 0.25μL SEQ ID No.3 (10μM) 0.25μL SEQ ID No.4 (10μM) 0.25μL SEQ ID No.5 (10μM) 0.25μL SEQ ID No. 6 (10μM) 0.25μL SEQ ID No.7 (10μM) 0.25μL SEQ ID No. 8 (10μM) 0.25μL SEQ ID No.9 (10μM) 0.25μL SEQ ID No. 10 (10μM) 0.25μL SEQ ID No. 11 (10μM) 0.25μL SEQ ID No. 12 (10μM) 0.25μL Taq DNA polymerase (5 U / μL) 0.5μL dNTPs (10mM) 0.5μL <![CDATA[MgCl2(25mM)]]> 5μL PCR buffer (5×) 5μL <![CDATA[dd H2O]]> 1μL Total 15μL

[0118] 2. Sample addition

[0119] Add 15 μL of PCR reaction solution and 10 μL of sample DNA to the prepared PCR reaction tubes, tighten the caps, and centrifuge briefly at low speed. The negative and positive controls are loaded in the same manner as the test samples.

[0120] 3. Quantitative real-time PCR detection

[0121] (1) Fluorescence channel selection: Two channels, FAM and JOE, were selected for each sample. The reference fluorescence (PassiveReference) was set to none;

[0122] (2) The reaction conditions are set as shown in Table 9 (the reaction system is 25 μL);

[0123] Table 9. Fluorescent PCR Reaction Conditions

[0124]

[0125] VI. Results Analysis

[0126] 1. PCR Result Analysis

[0127] After the reaction is complete, the results are automatically saved. The results are then automatically analyzed using the instrument's software. If any channel in the PCR amplification has an amplification curve, its Ct value (Cycle threshold) is calculated. The magnitude of the Ct value reflects the relative content of the detected gene.

[0128] 2. Determination of test results

[0129] A result with a Ct value of ≤32 for the ACTB gene is considered valid, indicating sufficient DNA content in the sample; otherwise, it is considered invalid. A positive result with a Ct value of ≤43 for the FAM channel indicates a high risk of lung cancer, while a negative result with a Ct value of >43 or ND indicates a low risk of lung cancer. ND is an abbreviation for "Not Detected".

[0130] 3. Test Results

[0131] (1) SCARA5 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0132] Table 10. Results of SCARA5 gene methylation detection

[0133]

[0134] (2) ZNF323 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0135] Table 11. Results of ZNF323 gene methylation detection

[0136]

[0137] (3) DLX1 gene methylation was not detected in negative reference materials and normal samples, resulting in a negative result. It was detected in positive reference materials, sensitivity reference materials, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0138] Table 12. Results of DLX1 gene methylation detection

[0139]

[0140] (4) SCARA5 / ZNF323 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0141] Table 13. SCARA5 / ZNF323 gene methylation detection results

[0142]

[0143] (5) SCARA5 / DLX1 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0144] Table 14. Results of SCARA5 / DLX1 gene methylation detection

[0145]

[0146] (6) ZNF323 / DLX1 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0147] Table 15. Results of ZNF323 / DLX1 gene methylation detection

[0148]

[0149]

[0150] (7) SCARA5 / ZNF323 / DLX1 gene methylation was not detected in negative reference and normal samples, resulting in a negative result; however, it was detected in positive reference, sensitivity reference, and lung cancer samples, resulting in a positive result. The results are shown in the table below:

[0151] Table 16. Results of SCARA5 / ZNF323 / DLX1 gene methylation detection

[0152]

[0153] Example 4: Primers and probes used to detect clinical samples

[0154] Several clinical plasma samples were collected, including 176 newly diagnosed lung cancer patients and 192 plasma samples from healthy individuals. The clinical samples were tested using the kit shown in Example 3. The specific steps were the same as in Example 3, and the test results are shown in the table below:

[0155] Table 17. Results of SCARA5 gene methylation detection in clinical samples

[0156]

[0157] Table 18. Results of clinical sample detection of ZNF323 gene methylation

[0158]

[0159] Table 19. Results of DLX1 gene methylation detection in clinical samples

[0160]

[0161] Table 20. SCARA5 / ZNF323 gene methylation detection results

[0162]

[0163] Table 21. Results of SCARA5 / DLX1 gene methylation detection

[0164]

[0165] Table 22. Results of ZNF323 / DLX1 gene methylation detection

[0166]

[0167] Table 23. Results of SCARA5 / ZNF323 / DLX1 gene methylation detection

[0168]

[0169] like Figure 1 As shown, the primers and probes of this application exhibit a sensitivity of over 84% and a specificity of over 98% for detecting the methylation status of any one of the SCARA5, ZNF323, and DLX1 genes. The sensitivity for detecting the methylation status of any two of these genes is over 89%, and the specificity is over 98%. The combined detection of the methylation of all three genes showed a sensitivity >95% (detecting 168 out of 176 lung cancer patients) and a specificity >98% (detecting 3 out of 192 healthy individuals). These results demonstrate that SCARA5, ZNF323, and DLX1, as biomarkers, can detect early-stage lung cancer with high specificity and sensitivity.

[0170] As can be seen from the above embodiments, the primers, probes, and kits of this application can detect the methylation levels of SCARA5, ZNF323, and DLX1 genes in samples with high specificity and high sensitivity, providing a reference for the screening and diagnosis of early lung cancer.

[0171] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A nucleic acid product for diagnosing lung cancer, characterized in that, The nucleic acid product includes primer pairs for detecting the methylation level of a biomarker and detection probes corresponding to the primer pairs; the primer pairs include: The nucleotide sequences of the first primer pair used to detect the methylation level of the SCARA5 gene are shown in SEQ ID No. 1 and SEQ ID No. 2; The nucleotide sequences of the second primer pair, as shown in SEQ ID No. 4 and SEQ ID No. 5, are used to detect the methylation level of the ZNF323 gene; The nucleotide sequences of the third primer pair used to detect the methylation level of the DLX1 gene are shown in SEQ ID No. 7 and SEQ ID No. 8; The nucleotide sequence of the detection probe corresponding to the first primer pair is shown in SEQ ID No. 3; The nucleotide sequence of the detection probe corresponding to the second primer pair is shown in SEQ ID No. 6; The nucleotide sequence of the detection probe corresponding to the third primer pair is shown in SEQ ID No.

9.

2. The nucleic acid product according to claim 1, characterized in that, The nucleic acid product also includes an internal reference primer pair and an internal reference probe corresponding to the internal reference primer pair.

3. The nucleic acid product according to claim 2, characterized in that, The nucleotide sequences of the internal reference primer pair are shown in SEQ ID No. 10 and SEQ ID No. 11, and the nucleotide sequence of the internal reference probe is shown in SEQ ID No.

12.

4. The use of the nucleic acid product for diagnosing lung cancer as described in any one of claims 1 to 3 in the preparation of products for diagnosing lung cancer.

5. A reagent kit for diagnosing lung cancer, characterized in that, It includes one or more of nucleic acid extraction reagents, methylation conversion reagents, quality control reagents, PCR reaction reagents and sequencing reagents, as well as the nucleic acid product according to any one of claims 1 to 3.

Images