A gene combination for early diagnosis of kidney cancer, a kit and application thereof

Abstract

This invention discloses a gene composition, kit, and application for early diagnosis of renal cell carcinoma, relating to the field of molecular diagnostic technology. The gene composition consists of the PLIN2 gene, SLC25A25 gene, PITX1 gene, and SOX1 gene. The differential methylation site of the PLIN2 gene is cg13990947, the differential methylation site of the SLC25A25 gene is cg14294859, the differential methylation site of the PITX1 gene is cg02037307, and the differential methylation site of the SOX1 gene is cg1. 6705627; This invention, by screening four genes (PLIN2, SLC25A25, PITX1, and SOX1) and specific methylation site combinations, can accurately identify two major subtypes of renal cell carcinoma—clear cell and papillary renal cell carcinoma—compared to single-gene detection. The methylation difference between cancerous and normal tissues, Δβ-value, is greater than 0.2, solving the problem of heterogeneous renal cell carcinoma detection. The optimized urine free DNA lysis buffer and bisulfite conversion system improve the nucleic acid extraction and conversion rates. Combined with dedicated methylation-specific primers and probes, it enables non-invasive detection of urine samples.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically to a gene composition, kit, and application for the early diagnosis of renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCC) is the leading cause of death among malignant tumors of the urinary system, accounting for 2%-3% of adult malignant tumors. It comprises more than ten subtypes, with clear cell renal cell carcinoma and papillary renal cell carcinoma being the main pathological types, accounting for over 85% of cases and exhibiting poor prognosis, making them key areas for RCC diagnosis. Currently, the gold standard for pathological diagnosis of RCC in clinical practice is biopsy. While this method is accurate, it carries a high risk of complications and tumor metastasis, and the results are easily affected by the heterogeneity of RCC. Imaging diagnostic methods such as ultrasound, CT, and MRI also have their own drawbacks, including low specificity, radiation exposure, high cost, and limited detection capabilities. Non-invasive molecular testing based on body fluids offers a new direction for tumor diagnosis. Among these, DNA methylation markers have become a research hotspot due to their advantages of early diagnosis, high sensitivity, and high specificity, and are widely used in auxiliary tumor diagnosis. However, due to the significant molecular-level heterogeneity among RCC subtypes, existing technologies have not yet screened out RCC DNA methylation markers with excellent diagnostic performance, nor are there dedicated RCC gene methylation detection kits, failing to meet the clinical need for early, non-invasive, and accurate diagnosis of RCC.

[0003] Renal cell carcinoma (RCC) has multiple subtypes with significant molecular heterogeneity, posing a major challenge to its molecular detection. While current research has identified some gene methylation level changes as being associated with RCC development, there is a lack of high-performance DNA methylation biomarkers for diagnosis, and no dedicated gene methylation detection kits are available for auxiliary diagnosis of RCC. Clinically, there is a need for readily applicable RCC methylation biomarkers and highly accurate methylation detection kits to compensate for the shortcomings of existing diagnostic methods and improve the efficiency and accuracy of early RCC diagnosis. Therefore, developing a gene composition, kit, and its application for early RCC diagnosis is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a gene composition, kit, and application for the early diagnosis of renal cell carcinoma. It can accurately identify major subtypes of renal cell carcinoma by screening for combinations of four genes (PLIN2, SLC25A25, PITX1, and SOX1) and specific methylation sites, thus solving the molecular detection challenges caused by the heterogeneity of renal cell carcinoma. Through the use of dedicated methylation-specific primers and probes, and an optimized system for extracting cell-free DNA from urine and converting bisulfite, it achieves non-invasive detection based on urine samples. By establishing a logistic regression diagnostic model and combining it with a multiplex real-time quantitative PCR detection system, it provides high-sensitivity and high-specificity diagnostic assistance for the early diagnosis of renal cell carcinoma.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a gene composition for early diagnosis of renal cell carcinoma, the gene composition comprising the PLIN2 gene, the SLC25A25 gene, the PITX1 gene, and the SOX1 gene, wherein the differential methylation site of the PLIN2 gene is cg13990947, the differential methylation site of the SLC25A25 gene is cg14294859, the differential methylation site of the PITX1 gene is cg02037307, and the differential methylation site of the SOX1 gene is cg16705627.

[0006] Furthermore, the differential methylation sites of the PLIN2, SLC25A25, PITX1, and SOX1 genes each contain 200 bp of nucleotide sequences upstream and downstream of them. The gene composition uses the ACTB gene as an internal control gene to calibrate the detection results of gene methylation levels.

[0007] Furthermore, the gene composition is suitable for the early diagnosis of clear cell renal cell carcinoma and papillary renal cell carcinoma. Each differential methylation site is highly methylated in cancerous tissue, and the average methylation level β-value in adjacent or normal tissue is <0.2, while the differential methylation level Δβ-value is >0.2.

[0008] A kit for early diagnosis of renal cell carcinoma, comprising a methylation-specific primer-probe combination corresponding to the above-mentioned gene composition, and also comprising a urine cell-free DNA extraction reagent, a methylation detection sample pretreatment reagent, a PCR reaction solution, a primer-probe mixture, a positive control, and a negative control.

[0009] Furthermore, the methylation-specific primer-probe combination includes methylation-specific forward primers, reverse primers, and fluorescent probes for the PLIN2 gene, SLC25A25 gene, PITX1 gene, SOX1 gene, and ACTB gene. The nucleotide sequence of the primers used to detect the PLIN2 gene is as follows: PLIN2-MF: 5'-TCGTTAAATAGTCGGGATTAGTTGG-3'; PLIN2-MR: 5'-CGATAACTCACGCCTAATCCTAA-3'; The nucleotide sequence of the probe for detecting the PLIN2 gene is as follows: PLIN2-MP: 5'-AAACCGAAACGAACGAATCACCAACCTAAC-3'; The nucleotide sequence of the primers used to detect the SLC25A25 gene is as follows: SLC25A25-MF: 5'-GGTAGTTGAGGAATTATAAGGCGT-3'; SLC25A25-MR: 5'-AACCCACTAACTAACTCGCGTCCT-3'; The nucleotide sequence of the probe for detecting the SLC25A25 gene is as follows: SLC25A25-MP: 5'-GTTTAAGAGAGAGGCGCGGTGGTGATTT-3'; The nucleotide sequence of the primers used to detect the PITX1 gene is as follows: PITX1-MF: 5'-TAAATAGTATTCGTCGTTTGGTTACG-3'; PITX1-MR: 5'-AACGATCAACTATTATACTAACACGC-3'; The nucleotide sequence of the probe for detecting the PITX1 gene is as follows: PITX1-MP: 5'-AAACCCGAAACCGAACCCTACAAACCG-3'; The nucleotide sequence of the primers used to detect the SOX1 gene is as follows: SOX1-MF: 5'-TTTATTTCGGTCGTTTATGTTTTAGGT-3'; SOX1-MR: 5'-GATCTCCATCATCATACTATACATCGA-3'; The nucleotide sequence of the probe for detecting the SOX1 gene is as follows: SOX1-MP: 5'-CTAACGAATTCACCGACACCGCGAA-3'; The nucleotide sequence of the primers used to detect the ACTB gene is as follows: ACTB-MF: 5'-TTTGGGTTTTATTTAGAGTGTAGATG-3'; ACTB-MR: 5'-CAACCCCAATAAAACATAACACC-3'; The nucleotide sequence of the probe for detecting the ACTB gene is as follows: ACTB-MP: 5'-CAAATAATCCCTTCCCACCTCCTCAAACAT-3'; The lysis buffer for the urine-free DNA extraction reagent is an aqueous solution at pH 5.6 consisting of 4M guanidine isothiocyanate, 0.1M Tris-HCl, 0.01M EDTA-Na2, 0.2M NaCl, 2% Tween 20 (v / v), and 0.5% Triton X-100 (v / v). The positive control is a mixture of methylated and unmethylated human genomic DNA, and the negative control is unmethylated human genomic DNA.

[0010] Furthermore, the methylation detection sample pretreatment reagent includes conversion solution, buffer A, buffer B, buffer C, binding solution, desulfonation solution, washing solution 1, washing solution 2, elution solution, nucleic acid adsorption column, and collection tube; The conversion solution is prepared by adding 1210 μL of buffer A to each tube of conversion solution, shaking at room temperature for 1 min until dissolved, then adding 110 μL of buffer B and 110 μL of buffer C, shaking to mix well and set aside. The PCR reaction solution contains Taq DNA polymerase, dNTPs, and Mg. 2+ The primer-probe mixture is the methylation-specific primer-probe mixture of claim 5, and during detection, it is divided into two groups of triple primer-probe mixtures: PLIN2 / SLC25A25 / ACTB and PITX1 / SOX1 / ACTB.

[0011] An application of a kit for early diagnosis of renal cell carcinoma is disclosed, applicable to the preparation of early diagnostic reagents for renal cell carcinoma. This application involves detecting the DNA methylation levels of the PLIN2, SLC25A25, PITX1, and SOX1 genes in urine samples to achieve early diagnosis of renal cell carcinoma. The detection process involves first extracting cell-free DNA from the urine sample, then converting the extracted cell-free DNA to bisulfite, followed by detecting the gene methylation level using multiplex real-time quantitative PCR, and finally calculating and interpreting the results using a logistic regression formula.

[0012] Furthermore, the pretreatment and preservation of the urine samples are as follows: freshly collected urine samples are immediately mixed with a urine free DNA preservation solution containing EDTA, and the samples are stored and / or transported at 2-8°C for no more than 7 days and frozen. The extraction steps for cell-free DNA in urine are as follows: the pretreated urine sample is centrifuged at 3000×g and 4℃ for 10 min, 4 mL of supernatant is taken and 3 mL of the lysis buffer described in claim 5 and 0.05 mL of 20 mg / mL proteinase K are added, and the mixture is incubated at 55℃ for 10 min. 2 mL of isopropanol and 30 μL of magnetic beads are added sequentially, mixed well, and the magnetic beads are adsorbed. After washing twice and drying at room temperature, the mixture is eluted with 50 μL of elution buffer preheated at 65℃ to obtain cell-free DNA solution in urine. The total volume of the bisulfite conversion reaction system was 150 μL, consisting of 130 μL of bisulfite solution, 10 ng - 2 μg of cell-free urinary DNA sample, and nuclease-free water to make up the volume. The bisulfite conversion adopted conversion program 2, which was as follows: keep the hot lid open at 105°C, denature at 98°C for 5 min, convert at 64°C for 40 min, denature at 95°C for 3 min, convert at 64°C for 20 min, repeat the 95°C denaturation for 3 min and 64°C conversion for 20 min steps twice, and finally keep warm at 4°C for a total denaturation time of 14 min and a total conversion time of 100 min. After conversion, the DNA solution after binding, washing, desulfonation, and elution steps was obtained.

[0013] Furthermore, the reaction system for the multiplex real-time quantitative PCR consists of 8.25 μL of PCR reaction solution, 6.75 μL of primer-probe mixture, and 15 μL of DNA template converted with bisulfite per reaction, for a total reaction volume of 30 μL. The PCR reaction program is as follows: pre-denaturation at 95°C for 2 min, followed by denaturation at 95°C for 10 s and extension at 64°C for 30 s, with fluorescence acquisition constituting one cycle. A total of 45 cycles are performed. Using the ACTB gene as an internal reference, the Cp value difference between each target gene and the ACTB gene is calculated, where A = Cp. PLIN2 -Cp ACTB B=Cp SLC25A25 -Cp ACTB C=Cp PITX1 -Cp ACTB D=Cp SOX1 -Cp ACTB .

[0014] Furthermore, the logistic regression formula is Z. RCC =2.9804-0.368A-0.204B-0.286C-0.173D, interpret the result based on the formula, when Z RCC A Z-score ≥0.47 indicates a high risk of renal cell carcinoma. RCCA value <0.47 indicates a low risk of renal cell carcinoma. This application shows no cross-reactivity with urine samples from other urinary system tumors and has no effect on the detection results of levofloxacin hydrochloride, furosemide, uric acid, glucose, mitomycin C, Candida albicans, human serum albumin, ascorbic acid, unconjugated bilirubin, alcohol, and hemoglobin at their respective concentrations.

[0015] Compared with existing technologies, this gene composition, kit, and application for early diagnosis of renal cell carcinoma have the following beneficial effects: This invention, through screening four genes (PLIN2, SLC25A25, PITX1, and SOX1) and specific methylation site combinations, can accurately identify two major subtypes of renal cell carcinoma—clear cell and papillary renal cell carcinoma—compared to single-gene detection. The methylation difference between cancerous and normal tissues (Δβ-value > 0.2) solves the challenge of detecting renal cell carcinoma heterogeneity. An optimized urine-free DNA lysis buffer and bisulfite conversion system improve nucleic acid extraction and conversion rates. Combined with dedicated methylation-specific primers and probes, non-invasive detection of urine samples is achieved. A logistic regression model based on Cp value differences, combined with a multiplex real-time quantitative PCR system, achieves a renal cell carcinoma diagnostic sensitivity of 87.20% and a specificity of 83.94%, with no cross-reactivity to urinary system tumors and strong anti-interference properties. This fills the gap in renal cell carcinoma methylation detection kits and significantly improves the accuracy and practicality of early diagnosis.

[0016] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort. Figure 1 This refers to the average methylation level of the methylation sites in the PLIN2 gene in different tissue samples of ccRCC and pRCC in Example 1 of this invention; Figure 2 This refers to the average methylation level of the methylation sites in the SLC25A25 gene in different tissue samples of ccRCC and pRCC in Example 1 of this invention; Figure 3This refers to the average methylation level of methylation sites in the PITX1 gene in different tissue samples of ccRCC and pRCC in Example 1 of this invention; Figure 4 This refers to the average methylation level of the methylation sites in the SOX1 gene in different tissue samples of ccRCC and pRCC in Example 1 of this invention; Figure 5 This is a linear correlation diagram between the Cp values ​​obtained from the detection of ACTB, PLIN2, SLC25A25, PITX1, and SOX1 genes in Example 5 of this invention and the corresponding sample DNA concentrations. Figure 6 This is a precision diagram of the detection of ACTB, PLIN2, SLC25A25, PITX1, and SOX1 genes in Example 5 of this invention. Detailed Implementation

[0018] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0019] This invention relates to an early diagnostic method for renal cell carcinoma based on the methylation levels of specific genes (PLIN2, SLC25A25, PITX1, SOX1) in cell-free DNA in urine. The principle is to detect the hypermethylation status of these genes in the urine of renal cell carcinoma patients, perform quantitative analysis using real-time quantitative PCR technology, and combine this with a logistic regression model to determine the risk of cancer, thereby achieving a non-invasive, highly sensitive, and specific auxiliary diagnosis of renal cell carcinoma. Example 1: Selection of Renal Cell Carcinoma-Related Genes and Differential Methylation Sites

[0020] This invention collected whole-genome methylation data from 324 cases of ccRCC (clear cell renal cell carcinoma) and 160 cases of adjacent or normal tissues, and 226 cases of pRCC (papillary renal cell carcinoma) and 45 cases of adjacent or normal tissues from the TCGA (Cancer Genome Atlas) dataset. The samples were categorized by type, comparing methylation levels between cancerous and adjacent or normal tissues, and differential methylation analysis was performed on renal cancer-related genes. To ensure sufficient sensitivity and specificity in the samples, the selected genes and differentially methylated sites must meet the following requirements: (1) sufficient significant difference between the two renal cancer subtypes, ccRCC and pRCC (ccRCC p-value < 1E-20 and pRCC p-value < 1E-10); (2) differentially hypermethylated sites in cancerous tissues; (3) average methylation level β-value < 0.2 in adjacent or normal tissues; (4) differential methylation level Δβ-value > 0.2. See also Figure 1, Figure 2 , Figure 3 and Figure 4 .

[0021] After screening, the renal cell carcinoma-related genes and their differentially methylated sites, as well as the methylation levels and significant differences in different renal cell carcinoma subtypes and tissues, are shown in the table below: Example 2: Design and Screening of Primer and Probe Combinations Based on the differentially methylated sites and their ±200bp sequences of the renal cell carcinoma-related genes PLIN2 (cg13990947), SLC25A25 (cg14294859), PITX1 (cg02037307), and SOX1 (cg16705627) screened in Example 1, and the gDNA sequence of the internal reference gene ACTB (β-actin gene), multiple methylation-specific forward and reverse primers were designed and synthesized. Using methylated and unmethylated human genomic DNA after bisulfite conversion as templates, qPCR (real-time quantitative polymerase chain reaction) amplification was performed using the SYBR Green dye method.

[0022] Based on the difference in Cp values ​​between each target gene and the internal reference gene in methylated and unmethylated templates (ΔCp=Cp... 靶基因 -Cp 内参基因 The peak temperature and number of peaks of the melting curves were used to evaluate the amplification efficiency and specificity of each primer combination, and primer combinations with high amplification efficiency and good specificity were screened out.

[0023] Based on the selected primer combinations, methylation-specific fluorescent probes were designed and synthesized between the forward and reverse primers. Using methylated and unmethylated human genomic DNA converted to bisulfite as templates, qPCR amplification was performed using the probe method. The amplification curves of each primer-probe combination in methylated and unmethylated templates, as well as the Cp value difference (ΔCp) between each target gene and the internal reference gene, were analyzed. 靶基因 =Cp 靶基因 -Cp 内参基因 The amplification efficiency of each primer-probe combination is evaluated based on the difference in ΔCp values ​​between the two templates for each primer-probe combination. 靶基因 =△Cp 非甲基化 -△Cp 甲基化 The study aimed to evaluate the specificity of each primer-probe combination for methylated templates and its ability to distinguish between methylated and unmethylated templates.

[0024] After screening primer and probe combinations in methylated and unmethylated templates, one set of forward primers, reverse primers, and fluorescent probes with high amplification efficiency and specificity was selected for each of the four target genes. The primer and probe sequences are as follows: PLIN2-MF: 5'-TCGTTAAATAGTCGGGATTAGTTGG-3' PLIN2-MR: 5'-CGATAACTCACGCCTAATCCTAA-3' PLIN2-MP: 5'-AAACCGAAACGAACGAATCACCAACCTAAC-3' SLC25A25-MF:5'-GGTAGTTGAGGAATTATAAGGCGT-3' SLC25A25-MR: 5'-AACCCACTAACTAACTCGCGTCCT-3' SLC25A25-MP: 5'-GTTTAAGAGAGAGGCGCGGTGGTGATTT-3' PITX1-MF: 5'-TAAATAGTATTCGTCGTTTGGTTACG-3' PITX1-MR:5'-AACGATCAACTATTATACTAACACGC-3' PITX1-MP: 5'-AAACCCGAAACCGAACCCTACAAACCG-3' SOX1-MF: 5'-TTTATTTCGGTCGTTTATGTTTTAGGT-3' SOX1-MR: 5'-GATCTCCATCATCATACTATACATCGA-3' SOX1-MP: 5'-CTAACGAATTCACCGACACCGCGAA-3' ACTB-MF: 5'-TTTGGGTTTTATTTAGAGTGTAGATG-3' ACTB-MR: 5'-CAACCCCAATAAAACATAACACC-3' ACTB-MP: 5'-CAAATAATCCCTTCCCACCTCCTCAAACAT-3' Example 3: A lysis buffer and extraction method for a urine-derived cell-free DNA extraction reagent.

[0025] The operation steps are as follows: 1. Processing and preservation of urine samples: Freshly collected urine samples should be immediately mixed with a urine cell-free DNA preservation solution containing EDTA (ethylenediaminetetraacetic acid) to inhibit the activity of DNase in the urine sample, maintain the stability of urine cell-free DNA (ucfDNA), preserve the integrity of exfoliated cells in the urine, and prevent the release of intracellular nucleic acids.

[0026] Before further processing, samples should be stored at 2-8℃ and / or transported for no more than 7 days, avoiding freezing.

[0027] 2. The lysis buffer formulations for urine-free DNA extraction reagents include the following four types: Formula 1: Guanidine isothiocyanate concentration is 4M; Tris-HCl concentration is 0.1M; EDTA-Na2 concentration is 0.01M; NaCl concentration is 1M; Tween 20 concentration is 1% by volume; Triton X-100 concentration is 1% by volume; pH 8.0; Formula 2: Guanidine isothiocyanate concentration is 4M; Tris-HCl concentration is 0.1M; EDTA-Na2 concentration is 0.01M; NaCl concentration is 0.2M; Tween 20 concentration is 2% by volume; Triton X-100 concentration is 0.5% by volume; pH 5.6; Formula 3: Guanidine isothiocyanate concentration is 2.5M; Tris-HCl concentration is 0.1M; EDTA-Na2 concentration is 0.01M; NaCl concentration is 1M; Tween 20 concentration is 2% by volume; Triton X-100 concentration is 1% by volume; pH 8.0; Formula 4: Guanidine isothiocyanate concentration is 1.5M; guanidine hydrochloride concentration is 2.5M; Tris-HCl concentration is 0.1M; EDTA-Na2 concentration is 0.01M; NaCl concentration is 0.2M; Tween 20 concentration is 1% by volume; Triton X-100 concentration is 0.5% by volume; pH 5.6; 3. The steps for extracting cell-free DNA from urine are as follows: 1) Centrifuge the urine sample at 3000×g at 4℃ for 10 min, and transfer 4 mL of the supernatant to a new 10 mL centrifuge tube; 2) Add 3 mL of lysis buffer and 0.05 mL of 20 mg / mL proteinase K, vortex to mix, and incubate at 55 °C for 10 min for lysis; 3) Add 2 mL of isopropanol and 30 μL of magnetic beads in sequence, vortex to mix, and mix at room temperature for 3 min on an inverted mixer. 4) Insert the centrifuge tube into the magnetic rack and let it stand for 2 minutes to attract the magnetic beads. Once the magnetic beads are completely attracted, use a pipette to remove the supernatant. 5) Remove the centrifuge tube from the magnetic rack, add 600 μL of cleaning solution, shake well to resuspend the magnetic beads, transfer to a 1.5 mL centrifuge tube, and let stand for 1 min; 6) Insert the centrifuge tube into the magnetic rack and let it stand for 1 minute to attract the magnetic beads. Once the magnetic beads are completely attracted, use a pipette to remove the supernatant. 7) Remove the centrifuge tube from the magnetic rack, add 600 μL of cleaning solution, shake well to resuspend the magnetic beads, and let stand for 1 min; 8) Insert the centrifuge tube into the magnetic rack and let it stand for 1 minute to attract the magnetic beads. Once the magnetic beads are completely attracted, use a pipette to remove the supernatant. 9) Let it air dry at room temperature for 10 minutes. Avoid drying for too long, as excessive drying will severely reduce the efficiency of nucleic acid elution. 10) Add 50 μL of preheated elution buffer at 65℃, shake thoroughly to disperse the magnetic beads, and incubate at 65℃ for 5 min, vortexing for 5 s every 2 min during this period; 11) After centrifuging briefly for 5 seconds on a hand centrifuge, insert a magnetic rack and let it stand for 1 minute to attract the magnetic beads. Once the magnetic beads are completely attracted, use a pipette to transfer the supernatant to a new centrifuge tube. This is the extracted ucfDNA solution, which can be used or stored at -80℃.

[0028] Two detection methods, Qubit and qPCR, were used to compare the concentration and extraction efficiency of the ucfDNA solutions extracted from the four lysis buffer formulations. The qPCR method used standard ACTB primers and probes as the internal reference gene, and the extraction efficiency of the ucfDNA from the four lysis buffer formulations was compared based on the Cp value of the internal reference gene; a lower Cp value indicated higher extraction efficiency.

[0029] The conventional internal reference gene ACTB primer and probe sequences used are as follows: ACTB-F: 5'-TGACTTAGTTGCGTTACACCCTT-3' ACTB-R: 5'-GACTGCTGTCACCTTCACCG-3' ACTB-P: 5'-CCTAACTTGCGCAGAAAACAAGATG-3' The test results are shown in the table below: The results showed that Formula 2 had the highest ucfDNA extraction rate, and Formula 2 was used as the lysis buffer for subsequent experiments. Example 4: A sample pretreatment reagent for methylation detection and a method for bisulfite conversion.

[0030] A sample pretreatment reagent for methylation detection includes conversion buffer, buffer A, buffer B, buffer C, binding buffer, desulfonation buffer, washing buffer 1, washing buffer 2, elution buffer, nucleic acid adsorption column, and collection tube.

[0031] The specific steps for the conversion of bisulfite are as follows: 1) Prepare the bisulfite solution according to your experimental requirements. When preparing the solution, add 1210 μL of buffer A to each tube of conversion solution, shake at room temperature for about 1 minute until dissolved, then add 110 μL of buffer B and 110 μL of buffer C, shake to mix well and set aside. 2) Prepare the bisulfite conversion reaction system in 200 μL sterile centrifuge tubes. The specific preparation system is as follows: 3) Mix the above solution by pipetting, briefly centrifuge, and then place it in a PCR instrument to perform the following bisulfite conversion reaction procedure: 4) Add 600 μL of binding solution and approximately 150 μL of the converted reaction product to the adsorption column (placed in the collection tube). Gently invert and mix 6-8 times, then let stand at room temperature for 2 minutes. 5) Centrifuge at 12000×g for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; 6) Add 500 μL of washing solution 1 to the adsorption column, centrifuge at 12000×g for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; 7) Add 500 μL of desulfurization solution to the adsorption column, let it stand at room temperature (15~25℃) for 15 min, centrifuge at 12000×g for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube. 8) Add 500 μL of washing solution 2 to the adsorption column, centrifuge at 12000×g for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; 9) Repeat step 8; 10) Centrifuge an empty column at 12000×g for 2 min; 11) Transfer the adsorption column to a new 1.5 mL centrifuge tube, open the cap for 10 min, add 20-40 μL of elution buffer to the center of the adsorption column membrane, let stand at room temperature for 2 min, centrifuge at 12000×g for 2 min, and collect the DNA solution. This is the ucfDNA solution obtained by bisulfite conversion and recovery, which can be used or stored at -80℃. Methylated and unmethylated human genomic DNA were used as bisulfite transformation samples, and transformed nucleic acid samples were obtained through the above-described steps. The transformation and recovery efficiencies of the six bisulfite transformation procedures were compared using qPCR detection with the methylation-specific primers and probes (ACTB-MF / ACTB-MR / ACTB-MP) for the ACTB internal reference gene shown in Example 2 and the conventional primers and probes (ACTB-F / ACTB-R / ACTB-P) for the ACTB internal reference gene shown in Example 3. The difference in Cp values ​​obtained using the conventional primers and methylation-specific primers for the ACTB internal reference gene (ΔCp = Cp) was used as the basis for the comparison. ACTB -Cp ACTB-M According to the formula: Conversion rate = 1 - 2 -△Cp Calculate the conversion rate; compare the nucleic acid recovery rates of the above 6 bisulfite conversion reaction procedures based on the Cp values ​​obtained from the methylation-specific primer probes of the ACTB internal reference gene. The smaller the Cp value, the higher the recovery rate.

[0032] The test results are shown in the table below: The results showed that program 1 had a lower conversion rate, while the other programs had higher conversion rates with little difference. Program 2 had the highest nucleic acid recovery rate, but the difference between it and the other programs was small. Considering the conversion rate, recovery rate, and time of the conversion reaction program, program 2 was used for subsequent experiments. Example 5: A real-time quantitative PCR detection system for detecting methylation of PLIN2, SLC25A25, PITX1, and SOX1 genes. Using the methylation-specific primers and probes for the PLIN2, SLC25A25, PITX1, SOX1 genes and the ACTB internal reference gene shown in Example 2, qPCR was performed in two PCR wells according to the following three detection systems, in triple combinations of PLIN2 / SLC25A25 / ACTB and PITX1 / SOX1 / ACTB, to detect the methylation level of the target gene in the test sample after bisulfite conversion.

[0033] Yin-Yang property control products: Note: In each test, both positive and negative control samples must be converted to bisulfite and detected by qPCR simultaneously with the sample to be tested for quality control of the test results. 1) Testing System 1: The qPCR reaction system is shown in the table below: The qPCR reaction procedure is shown in the table below: 2) Detection System 2: The qPCR reaction system is shown in the table below: The qPCR reaction procedure is shown in the table below: 3) Detection System 3: The qPCR reaction system is shown in the table below: The qPCR reaction procedure is shown in the table below: Evaluation of the testing system: 1) The accuracy of the above three detection systems was evaluated using the following methods: The three detection systems described above were used to detect human genomic DNA solutions with methylation ratios of 10% and concentrations of 0.25, 2, 16, 128, and 1024 ng / μL. Linear fitting was performed with the logarithm of DNA concentration as the X-axis and the detection Cp value as the Y-axis. The detection results are shown below. Figure 5 As shown: The results showed that there were some differences among the five genes in detection system 3. Overall, the detection accuracy of detection system 3 was slightly better than that of detection systems 1 and 2.

[0034] 2) The precision of the above three detection systems was evaluated using the following methods: The three detection systems described above were used to detect human genomic DNA solutions with a methylation rate of 10% and a concentration of 16 ng / μL, with 20 repeated tests. The coefficient of variation (CV) of the Cp values ​​of each target gene in the three detection systems was calculated. The detection results are as follows: Figure 6 As shown.

[0035] In summary, detection system 3 has better accuracy and precision than detection systems 1 and 2, and this detection system was selected for subsequent experiments. Example 6: Clinical validation of the early diagnostic performance of a combination of PLIN2, SLC25A25, PITX1, and SOX1 genes in renal cell carcinoma. A total of 441 urine samples were collected from subjects, including 289 subjects with RCC confirmed by histopathology, 137 subjects with non-RCC, and 15 subjects with other urinary system tumors. All samples were obtained from the First Affiliated Hospital and the Second Affiliated Hospital of Zhejiang University School of Medicine. Methylation detection was performed on the above clinical samples according to the sample processing and detection system described in Examples 2-5.

[0036] 1) Performance analysis model for auxiliary diagnosis of renal cell carcinoma: Substitute the Cp values ​​of each gene detected by qPCR into the following logistic regression formula and interpret the results: Z RCC = 2.9804 - 0.368A - 0.204B - 0.286C - 0.173D, where A is the Cp difference between the PLIN2 and ACTB genes, B is the Cp difference between the SLC25A25 and ACTB genes, C is the Cp difference between the PITX1 and ACTB genes, and D is the Cp difference between the SOX1 and ACTB genes. That is: A = Cp PLIN2 -Cp ACTB B=Cp SLC25A25 -Cp ACTB C=Cp PITX1 -Cp ACTB D=Cp SOX1 -Cp ACTB When Z RCC A result ≥0.47 is interpreted as positive, indicating a higher risk of RCC in the subject; conversely, when Z... RCC When the value is <0.47, the result is interpreted as negative, indicating that the subject has a low risk of RCC.

[0037] 2) Clinical analysis performance: The diagnostic value of combined detection of methylation levels of PLIN2, SLC25A25, PITX1, and SOX1 genes in urine samples for renal cell carcinoma is shown in the table below: Clinical sample test results: Clinical analytical performance: The results showed that the combined detection reagent for methylation of PLIN2, SLC25A25, PITX1 and SOX1 genes had a diagnostic sensitivity, specificity and accuracy of 87.20%, 83.94% and 86.15% for RCC, respectively, demonstrating excellent clinical diagnostic performance.

[0038] 3) Analytical specificity: Urine samples from 15 other subjects with urinary system tumors were tested, and all results were negative with no cross-reactivity. The detection of endogenous and exogenous interfering substances indicated the presence of levofloxacin hydrochloride (1.08 mg / mL), furosemide (0.5 mg / mL), uric acid (1.4 mmol / L), glucose (55 mmol / L), mitomycin C (0.1 mg / mL), and Candida albicans (2.5 × 10⁻⁶). 10Eleven interfering substances, including CFU / mL, human serum albumin (10 mg / mL), ascorbic acid (50 mg / mL), unconjugated bilirubin (2 mg / mL), alcohol (1% (V / V)), and hemoglobin (100 mg / mL), had no effect on the detection results at their respective concentrations. The results indicate that the combined methylation detection reagent for the PLIN2, SLC25A25, PITX1, and SOX1 genes exhibits excellent analytical specificity.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A gene composition for early diagnosis of renal cell carcinoma, characterized in that, The gene composition consists of the PLIN2 gene, SLC25A25 gene, PITX1 gene, and SOX1 gene. The differential methylation site of the PLIN2 gene is cg13990947, the differential methylation site of the SLC25A25 gene is cg14294859, the differential methylation site of the PITX1 gene is cg02037307, and the differential methylation site of the SOX1 gene is cg16705627.

2. The gene composition for early diagnosis of renal cell carcinoma according to claim 1, characterized in that, The differential methylation sites of the PLIN2, SLC25A25, PITX1, and SOX1 genes each contain 200 bp of nucleotide sequences upstream and downstream of them. The gene composition uses the ACTB gene as an internal control gene to calibrate the detection results of gene methylation levels.

3. The gene composition for early diagnosis of renal cell carcinoma according to claim 1, characterized in that, The gene composition is suitable for early diagnosis of clear cell renal cell carcinoma and papillary renal cell carcinoma. Each differential methylation site is highly methylated in cancerous tissue, and the average methylation level β-value in adjacent or normal tissue is <0.2, while the differential methylation level Δβ-value is >0.

2.

4. A reagent kit for the early diagnosis of renal cell carcinoma, characterized in that, The kit contains a methylation-specific primer-probe combination corresponding to the gene composition described in any one of claims 1 to 3, and also contains a urine cell-free DNA extraction reagent, a methylation detection sample pretreatment reagent, a PCR reaction solution, a primer-probe mixture, a positive control, and a negative control.

5. A reagent kit for early diagnosis of renal cell carcinoma according to claim 4, characterized in that, The methylation-specific primer-probe combination includes methylation-specific forward primers, reverse primers, and fluorescent probes for the PLIN2 gene, SLC25A25 gene, PITX1 gene, SOX1 gene, and ACTB gene. The nucleotide sequence of the primers used to detect the PLIN2 gene is as follows: PLIN2-MF: 5'-TCGTTAAATAGTCGGGATTAGTTGG-3'; PLIN2-MR: 5'-CGATAACTCACGCCTAATCCTAA-3'; The nucleotide sequence of the probe for detecting the PLIN2 gene is as follows: PLIN2-MP: 5'-AAACCGAAACGAACGAATCACCAACCTAAC-3'; The nucleotide sequence of the primers used to detect the SLC25A25 gene is as follows: SLC25A25-MF: 5'-GGTAGTTGAGGAATTATAAGGCGT-3'; SLC25A25-MR: 5'-AACCCACTAACTAACTCGCGTCCT-3'; The nucleotide sequence of the probe for detecting the SLC25A25 gene is as follows: SLC25A25-MP: 5'-GTTTAAGAGAGAGGCGCGGTGGTGATTT-3'; The nucleotide sequence of the primers used to detect the PITX1 gene is as follows: PITX1-MF: 5'-TAAATAGTATTCGTCGTTTGGTTACG-3'; PITX1-MR: 5'-AACGATCAACTATTATACTAACACGC-3'; The nucleotide sequence of the probe for detecting the PITX1 gene is as follows: PITX1-MP: 5'-AAACCCGAAACCGAACCCTACAAACCG-3'; The nucleotide sequence of the primers used to detect the SOX1 gene is as follows: SOX1-MF: 5'-TTTATTTCGGTCGTTTATGTTTTAGGT-3'; SOX1-MR: 5'-GATCTCCATCATCATACTATACATCGA-3'; The nucleotide sequence of the probe for detecting the SOX1 gene is as follows: SOX1-MP: 5'-CTAACGAATTCACCGACACCGCGAA-3'; The nucleotide sequence of the primers used to detect the ACTB gene is as follows: ACTB-MF: 5'-TTTGGGTTTTATTTAGAGTGTAGATG-3'; ACTB-MR: 5'-CAACCCCAATAAAACATAACACC-3'; The nucleotide sequence of the probe for detecting the ACTB gene is as follows: ACTB-MP: 5'-CAAATAATCCCTTCCCACCTCCTCAAACAT-3'; The lysis buffer for the urine-free DNA extraction reagent is an aqueous solution at pH 5.6 consisting of 4M guanidine isothiocyanate, 0.1M Tris-HCl, 0.01M EDTA-Na2, 0.2M NaCl, 2% Tween 20 (v / v), and 0.5% Triton X-100 (v / v). The positive control is a mixture of methylated and unmethylated human genomic DNA, and the negative control is unmethylated human genomic DNA.

6. A reagent kit for early diagnosis of renal cell carcinoma according to claim 5, characterized in that, The sample pretreatment reagents for methylation detection include conversion solution, buffer A, buffer B, buffer C, binding solution, desulfonation solution, washing solution 1, washing solution 2, elution solution, nucleic acid adsorption column, and collection tube; The conversion solution is prepared by adding 1210 μL of buffer A to each tube of conversion solution, shaking at room temperature for 1 min until dissolved, then adding 110 μL of buffer B and 110 μL of buffer C, shaking to mix well and set aside. The PCR reaction solution contains Taq DNA polymerase, dNTPs, and Mg. 2+ The primer-probe mixture is the methylation-specific primer-probe mixture of claim 5, and during detection, it is divided into two groups of triple primer-probe mixtures: PLIN2 / SLC25A25 / ACTB and PITX1 / SOX1 / ACTB.

7. The application of a reagent kit for early diagnosis of renal cell carcinoma, applicable to the use of the reagent kit according to any one of claims 4 to 6 in the preparation of a diagnostic reagent for early renal cell carcinoma, characterized in that, This application assists in the early diagnosis of renal cell carcinoma by detecting the DNA methylation levels of the PLIN2, SLC25A25, PITX1, and SOX1 genes in urine samples. The detection process involves first extracting cell-free DNA from the urine sample, then converting the extracted cell-free DNA to bisulfite, followed by using multiplex real-time quantitative PCR to detect the gene methylation level, and finally calculating and interpreting the results using a logistic regression formula.

8. The application of the reagent kit for early diagnosis of renal cell carcinoma according to claim 7, characterized in that, The pretreatment and preservation of the urine samples are as follows: freshly collected urine samples are immediately mixed with urine free DNA preservation solution containing EDTA, and the samples are stored and / or transported at 2-8°C for no more than 7 days and frozen. The extraction steps for cell-free DNA in urine are as follows: the pretreated urine sample is centrifuged at 3000×g and 4℃ for 10 min, 4 mL of supernatant is taken and 3 mL of the lysis buffer described in claim 5 and 0.05 mL of 20 mg / mL proteinase K are added, and the mixture is incubated at 55℃ for 10 min. 2 mL of isopropanol and 30 μL of magnetic beads are added sequentially, mixed well, and the magnetic beads are adsorbed. After washing twice and drying at room temperature, the mixture is eluted with 50 μL of elution buffer preheated at 65℃ to obtain cell-free DNA solution in urine. The total volume of the bisulfite conversion reaction system was 150 μL, consisting of 130 μL of bisulfite solution, 10 ng - 2 μg of cell-free urinary DNA sample, and nuclease-free water to make up the volume. The bisulfite conversion adopted conversion program 2, which was as follows: keep the hot lid open at 105°C, denature at 98°C for 5 min, convert at 64°C for 40 min, denature at 95°C for 3 min, convert at 64°C for 20 min, repeat the 95°C denaturation for 3 min and 64°C conversion for 20 min steps twice, and finally keep warm at 4°C for a total denaturation time of 14 min and a total conversion time of 100 min. After conversion, the DNA solution after binding, washing, desulfonation, and elution steps was obtained.

9. The application of the reagent kit for early diagnosis of renal cell carcinoma according to claim 8, characterized in that, The reaction system for the multiplex real-time quantitative PCR consisted of 8.25 μL of PCR reaction solution, 6.75 μL of primer-probe mixture, and 15 μL of DNA template converted to bisulfite per reaction, with a total reaction volume of 30 μL. The PCR reaction program was as follows: 95℃ pre-denaturation for 2 min, followed by 95℃ denaturation for 10 s and 64℃ extension for 30 s, with fluorescence acquisition constituting one cycle. A total of 45 cycles were performed. Using the ACTB gene as an internal reference, the Cp value difference between each target gene and the ACTB gene was calculated, where A = Cp. PLIN2 -Cp ACTB B=Cp SLC25A25 -Cp ACTB C=Cp PITX1 -Cp ACTB D=Cp SOX1 -Cp ACTB .

10. The application of the reagent kit for early diagnosis of renal cell carcinoma according to claim 9, characterized in that, The logistic regression formula is Z. RCC =2.9804-0.368A-0.204B-0.286C-0.173D, interpret the result based on the formula, when Z RCC A Z-score ≥0.47 indicates a high risk of renal cell carcinoma. RCC A value <0.47 indicates a low risk of renal cell carcinoma. This application shows no cross-reactivity with urine samples from other urinary system tumors and has no effect on the detection results of levofloxacin hydrochloride, furosemide, uric acid, glucose, mitomycin C, Candida albicans, human serum albumin, ascorbic acid, unconjugated bilirubin, alcohol, and hemoglobin at their respective concentrations.

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