A gene combination for early diagnosis of papillary renal cell carcinoma, kit and application thereof

Abstract

This invention relates to the field of molecular diagnostics, disclosing a gene composition, kit, and its application for the early diagnosis of papillary renal cell carcinoma (pRCC). It provides a non-invasive and highly accurate diagnostic biomarker for pRCC, achieving a sensitivity, specificity, and accuracy of 86.05%, 90.51%, and 88.79%, respectively, demonstrating excellent clinical diagnostic performance and effectively addressing the problem of low accuracy in pRCC diagnosis. This invention also provides a kit for the combined detection of DNA methylation levels of the ZNF154, OXR1, TMEM178A, and LINE1 genes, which is simple to operate and has stable performance.

Description

Technical Field

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

[0002] Papillary renal cell carcinoma (pRCC) accounts for 10%–15% of renal cell carcinomas and is the second most common subtype. Its prognosis is highly correlated with the timing of diagnosis. Early-stage patients with grade I or II disease and no metastasis have a significantly higher 5-year survival rate after standardized treatment, while the prognosis of late-stage or metastatic patients is significantly worse. However, this cancer often has no specific symptoms in its early stages, and most patients are discovered incidentally through imaging examinations such as ultrasound and CT during physical examinations. Current clinical early diagnostic methods have significant limitations—enhanced CT and MRI are not sensitive enough to identify small or atypical early lesions and are prone to missed diagnoses. Although ultrasound-guided renal biopsy is a pathological confirmation method, it is an invasive procedure and is not suitable for early screening of high-risk groups such as those with a family history of hereditary papillary renal cell carcinoma, carriers of MET gene mutations, and those who smoke, are obese, or have hypertension. Although gene testing can screen for related mutations, its use alone cannot balance convenience and accurate identification of early lesions, and cannot meet the clinical need for early, non-invasive, and efficient detection of this cancer.

[0003] Although existing research has identified several gene methylation level changes associated with renal cell carcinoma using various techniques, renal cell carcinoma has multiple subtypes. The significant molecular heterogeneity among these subtypes makes it one of the most challenging tumors for molecular detection. There is a lack of high-performance DNA methylation biomarkers, especially those related to pRCC (p-RCC), with significantly fewer reported biomarkers than ccRCC (cc-RCC). Furthermore, there are currently no methylation detection kits for renal cell carcinoma-related genes. Further research in this field is needed to develop DNA methylation biomarkers that can be effectively used for the diagnosis of papillary renal cell carcinoma, as well as detection kits with high sensitivity and specificity. Summary of the Invention

[0004] Addressing the issue mentioned in the background art that there is still a need for further development of DNA methylation biomarkers that can be effectively used for the diagnosis of papillary renal cell carcinoma (pRCC), as well as detection kits with high sensitivity and specificity, this invention provides a gene composition, kit, and application for the early diagnosis of pRCC. It offers a non-invasive and highly accurate diagnostic biomarker for pRCC, achieving a sensitivity, specificity, and accuracy of 86.05%, 90.51%, and 88.79%, respectively, demonstrating excellent clinical diagnostic performance and effectively solving the problem of low accuracy in pRCC diagnosis. This invention also provides a kit for the combined detection of DNA methylation levels of the ZNF154, OXR1, TMEM178A, and LINE1 genes, which is simple to operate and has stable performance.

[0005] The technical solution adopted in this invention is as follows: A gene combination for the early diagnosis of papillary renal cell carcinoma, comprising one or more of the ZNF154 gene, OXR1 gene, TMEM178A gene, and LINE1 gene.

[0006] A kit for the early diagnosis of papillary renal cell carcinoma, the kit comprising reagents for detecting differentially methylated sites in combinations of the ZNF154, OXR1, TMEM178A, and LINE1 genes.

[0007] The kit used in this invention exhibits extremely high detection sensitivity and specificity for papillary renal cell carcinoma by jointly detecting multiple DNA methylation markers.

[0008] Furthermore, the differentially methylated sites of the ZNF154 gene, OXR1 gene, TMEM178A gene and LINE1 gene combination are cg01268824, cg17176732, cg10708761, cg11056055 methylation sites and their ±200bp sequences, respectively.

[0009] Furthermore, the kit includes probes and primers for detecting the ZNF154 gene, the OXR1 gene, the TMEM178A gene, and the LINE1 gene.

[0010] Furthermore, the nucleotide sequences of the primers for detecting the ZNF154 gene are shown as ZNF154-MF and ZNF154-MR, and the nucleotide sequence of the probe for detecting the ZNF154 gene is shown as ZNF154-MP; the nucleotide sequences of the primers for detecting the OXR1 gene are shown as OXR1-MF and OXR1-MR, and the nucleotide sequence of the probe for detecting the OXR1 gene is shown as OXR1-MP; the nucleotide sequences of the primers for detecting the TMEM178A gene are shown as TMEM178A-MF and TMEM178A-MR, and the nucleotide sequence of the probe for detecting the TMEM178A gene is shown as TMEM178A-MP; the nucleotide sequences of the primers for detecting the LINE1 gene are shown as LINE1-MF and LINE1-MR, and the nucleotide sequence of the probe for detecting the LINE1 gene is shown as LINE1-MP.

[0011] Furthermore, the test kit also includes lysis buffer, extraction reagent, transformation reagent, PCR reaction solution, primer-probe mixture, positive control and negative control.

[0012] Furthermore, the positive control is a sample containing methylated human genomic DNA and unmethylated human genomic DNA; the negative control is a sample containing unmethylated human genomic DNA.

[0013] The application of a kit for the early diagnosis of papillary renal cell carcinoma, the method of use includes the following steps: 1) Mix freshly collected samples with EDTA-containing cell-free DNA preservation solution to obtain pretreated samples, and store them at 2~8℃; 2) The pretreated sample was lysed with the lysis buffer of the cell-free DNA extraction reagent, and then the cell-free DNA was extracted using the extraction reagent to obtain the extracted cell-free DNA solution; 3) The extracted free DNA solution was transformed with a transformation reagent, and the transformed free DNA solution was collected; 4) Perform qPCR detection on the transformed cell-free DNA solution and interpret the results according to the interpretation formula.

[0014] Furthermore, the interpretation formula is: Z pRCC =6.8937-1.317A–1.047B-0.814C+1.426D; where A is the Cp value difference between ZNF154 and ACTB genes, B is the Cp value difference between OXR1 and ACTB genes, C is the Cp value difference between TMEM178A and ACTB genes, and D is the Cp value difference between LINE1 and ACTB genes.

[0015] Furthermore, when ZpRCC ≥ 2.87, the result is interpreted as positive, indicating that the subject has a high risk of pRCC; conversely, when ZpRCC < 2.87, the result is interpreted as negative, indicating that the subject has a low risk of pRCC.

[0016] The kit of this invention uses multiplex qPCR detection technology to simultaneously detect the methylation levels of ZNF154, OXR1, TMEM178A and LINE1 genes in a single urine sample, while using the ACTB gene as an internal control to establish a logistic regression model as an early diagnostic biomarker for papillary renal cell carcinoma.

[0017] Furthermore, the sample source for cell-free DNA is blood or urine.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1) This invention provides a non-invasive and highly accurate diagnostic marker for papillary renal cell carcinoma (pRCC). The diagnostic sensitivity, specificity, and accuracy for pRCC reach 86.05%, 90.51%, and 88.79%, respectively, demonstrating excellent clinical diagnostic performance and effectively solving the problem of low accuracy in papillary renal cell carcinoma.

[0019] 2) This invention provides a kit for the combined detection of DNA methylation levels of ZNF154, OXR1, TMEM178A and LINE1 genes, which is simple to operate and has stable performance. Attached Figure Description

[0020] The present invention will be described by way of example and with reference to the accompanying drawings, wherein: Figure 1 This represents the average methylation level of the methylation sites in the ZNF154 gene in different pRCC tissue samples in Example 1 of this invention.

[0021] Figure 2 This represents the average methylation level of the methylation sites in the OXR1 gene in different pRCC tissue samples in Example 1 of this invention.

[0022] Figure 3 This refers to the average methylation level of the methylation site in the TMEM178A gene in different pRCC tissue samples in Example 1 of this invention.

[0023] Figure 4 This represents the average methylation level of the LINE1 gene methylation site in different pRCC tissue samples in Example 1 of this invention.

[0024] Figure 5This is a linear correlation diagram between the gene Cp values ​​obtained from detection systems 1 (AE), 2 (FJ), and 3 (KO) in Example 5 of this invention and the corresponding sample DNA concentrations; Figure 6 This is a precision diagram of detection system 1 (A), detection system (2) and detection system (3) in Example 5 of this invention. Detailed Implementation

[0025] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0026] Example 1: Selection of Papillary Renal Cell Carcinoma-Related Genes and Differential Methylation Sites This invention collected whole-genome methylation data from 226 pRCC cases and 45 adjacent or normal tissues from the TCGA dataset (https: / / www.cancer.gov / tcga). The samples were categorized by type, comparing methylation levels between cancerous and adjacent or normal tissues, and differential methylation analysis was performed on pRCC-related genes. To ensure sufficient sensitivity and specificity in the samples, the selected genes and differential methylation sites met the following requirements: (1) sufficient significance in pRCC (p-value < 1E-10); (2) differential methylation level |Δβ-value| > 0.3; (3) methylation level β-value < 0.2 or > 0.8 in adjacent or normal tissues.

[0027] After screening, the papillary renal cell carcinoma-related genes and their differentially methylated sites, as well as the methylation levels and significant differences between pRCC and normal tissues, are shown in Table 1 below.

[0028] Table 1. Screening of papillary renal cell carcinoma-related genes and their differentially methylated sites. The average methylation level of methylation sites in the ZNF154 gene in different pRCC tissue samples is as follows: Figure 1 As shown, the average methylation level of methylation sites in the OXR1 gene in different pRCC tissue samples is as follows: Figure 2 As shown; the average methylation level of methylation sites in the TMEM178A gene in different pRCC tissue samples is as follows. Figure 3 As shown; the average methylation level of methylation sites in the LINE1 gene in different tissue samples of pRCC is as follows. Figure 4 As shown.

[0029] Example 2: Design and Screening of Primer and Probe Combinations Based on the differentially methylated sites and their ±200bp sequences of the papillary renal cell carcinoma-related genes ZNF154 (cg01268824), OXR1 (cg17176732), TMEM178A (cg10708761), and LINE1 (cg11056055) screened in Example 1, and the gDNA sequence of the internal reference gene ACTB, multiple methylation-specific forward and reverse primers were designed and synthesized. Using methylated and unmethylated human genomic DNA after bisulfite conversion as templates, qPCR amplification was performed using the SYBR Green dye method.

[0030] 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.

[0031] 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.

[0032] 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: ZNF154-MF: 5'-AAACTTAAATAAACCATTTCTCATCGC-3' ZNF154-MR:5'-TTTATAGATTTCGAGGTGGGTGTC-3' ZNF154-MP:5'-ACGCCCTAAAACTTCGCTTTTACTCCAAAA-3' OXR1-MF: 5'-TTTTGTAGTTTTTGAAAGTTCGGTTCG-3' OXR1-MR: 5'-ACAAACTAAACGAATCCTCTCAACG-3' OXR1-MP: 5'-CCCACCCGCCTAACGCCAATAAAACTAA-3' TMEM178A-MF: 5'-TCGAACTCCAAATCAAAATTCCAAA-3' TMEM178A-MR: 5'-TGATTAGTGTTTCGTTTTTGTATTTAGGA-3' TMEM178A-MP: 5'-TAAACGCTACCACGCAACCCGACCTA-3' LINE1-MF:5'-TGTCGTTTTGCGGTTTGATTTTAG-3' LINE1-MR:5'-GAAATTATATCCCTCACCTAACTCGAA-3' LINE1-MP: 5'-TCCTACGCCCACGAAATCCCGCTAA-3' ACTB-MF: 5'-TTTGGGTTTTATTTAGAGTGTAGATG-3' ACTB-MR: 5'-CAACCCCAATAAAACATAACACC-3' ACTB-MP: 5'-CAAATAATCCCTTCCCACCTCCTCAAACAT-3'.

[0033] Example 3: A lysis buffer and extraction method for a free DNA extraction reagent 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 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.

[0034] Before further processing, samples should be stored at 2-8℃ and / or transported for no more than 7 days, avoiding freezing.

[0035] 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; EDTANa2 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.

[0036] 3) Extraction of cell-free DNA from urine (1) 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. After 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. After 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. After the magnetic beads are completely attracted, use a pipette to remove the supernatant. (9) Open the lid and air dry at room temperature for 10 minutes. Avoid drying for too long, as excessive drying will seriously reduce the efficiency of nucleic acid elution. (10) Add 50 μL of elution buffer preheated at 65℃, shake 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 for 5 seconds on a hand centrifuge, insert a magnetic rack and let it stand for 1 minute to attract the magnetic beads. After 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℃.

[0037] The concentration and extraction efficiency of the ucfDNA solutions extracted from the four lysis buffer formulations were compared using both qPCR and qubit methods. The results are shown in Table 2. For the qPCR method, the conventional internal reference gene ACTB primers and probes were used. 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 a higher extraction efficiency.

[0038] 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'.

[0039] Table 2. Concentration and extraction efficiency of ucfDNA solution The data in Table 2 show that Formula 2 has the highest UCF DNA extraction yield, and Formula 2 was used as the lysis buffer for subsequent experiments. Example 4: A sample pretreatment reagent for methylation detection and a bisulfite conversion method. 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. 1) When preparing, add 1210 μL of buffer A to each tube of conversion buffer, shake at room temperature for about 1 min 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 a 200 μL sterile centrifuge tube, including: 130 μL of bisulfite solution, DNA sample X (10 ng ~ 2 μg), and 20-X nuclease-free water (the maximum volume of DNA and nuclease-free water is 20 μL). 3) Mix the above solution by pipetting, briefly centrifuge, and then place it in a PCR instrument to perform the bisulfite conversion reaction program (as shown in Table 3). Table 3 Bisulfite Conversion Reaction Program 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℃.

[0040] 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 reaction procedures were compared using qPCR detection methods 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 results are shown in Table 4. The difference in Cp values ​​obtained from 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.

[0041] Table 4. Results of conversion and recovery efficiency tests for the bisulfite conversion reaction process. The data in Table 4 show that Procedure 1 had a lower conversion rate, while the conversion rates of the other procedures were all relatively high with little difference. Procedure 2 had the highest nucleic acid recovery rate, but the difference between it and the other procedures was small. Considering the conversion rate, recovery rate, and time consumed in the transformation reaction, Procedure 2 was selected for subsequent experiments.

[0042] Example 5: A real-time quantitative PCR detection system for detecting methylation of ZNF154, OXR1, TMEM178A, and LINE1 genes. Using the methylation-specific primers and probes for the ZNF154, OXR1, TMEM178A, LINE1 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 the ZNF154 / OXR1 / ACTB and TMEM178A / LINE1 / ACTB triple combinations, to detect the methylation level of the target gene in the test sample after bisulfite conversion.

[0043] Table 5 Components of negative and positive quality control samples 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.

[0044] 1) Detection System 1 The qPCR reaction system for detection system 1 is shown in Table 6, and the qPCR reaction procedure for detection system 1 is shown in Table 7.

[0045] Table 6. qPCR reaction system for detection system 1 Table 7. qPCR reaction procedure for detection system 1 2) Detection System 2 The qPCR reaction system for detection system 2 is shown in Table 8, and the qPCR reaction procedure for detection system 2 is shown in Table 9.

[0046] Table 8. qPCR reaction system for detection system 2 Table 9. qPCR reaction procedure for detection system 2 2) Detection system 3 The qPCR reaction system for detection system 2 is shown in Table 10, and the qPCR reaction procedure for detection system 2 is shown in Table 11.

[0047] Table 10 qPCR reaction system for detection system 2 Component Name Dosage μL / reaction Main ingredients PCR reaction solution 8.25 Taq DNA polymerase, dNTPs, Mg2+ Primer-probe mixture 6.75 Primers and probes template 15 DNA after bisulfite conversion Total volume 30 Table 11 qPCR reaction procedure for detection system 2 Evaluation of the testing system: a. Accuracy evaluation of the testing system 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, detection system 3 had the highest R2 value among the five genes, indicating that the detection accuracy of detection system 3 was better than that of detection systems 1 and 2.

[0048] b. Precision evaluation of the detection system 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, detection system 3 had the lowest CV values ​​among the five genes, indicating that its precision was superior to that of detection systems 1 and 2.

[0049] 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.

[0050] Example 6: Clinical validation of the early diagnostic performance of a combination of ZNF154, OXR1, TMEM178A, and LINE1 genes in renal cell carcinoma. A total of 238 urine samples were collected from the subjects, including 86 cases of pRCC confirmed by histopathology, 137 cases of non-RCC, and 15 cases of 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.

[0051] 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 pRCC =6.8937-1.317A–1.047B-0.814C+1.426D Where A represents the Cp value difference between the ZNF154 and ACTB genes, B represents the Cp value difference between the OXR1 and ACTB genes, C represents the Cp value difference between the TMEM178A and ACTB genes, and D represents the Cp value difference between the LINE1 and ACTB genes. That is: A=Cp ZNF154 –Cp ACTB B=Cp OXR1 –Cp ACTB C=Cp TMEM178A –Cp ACTB D=Cp LINE1 –Cp ACTB When Z pRCC A result ≥2.87 is interpreted as positive, indicating a higher risk of pRCC in the subject; conversely, when Z... pRCC When the value is <2.87, the result is interpreted as negative, indicating that the subject has a low risk of pRCC.

[0052] 2) Clinical analysis performance The diagnostic value of combined detection of methylation levels of ZNF154, OXR1, TMEM178A and LINE1 genes in urine samples for papillary renal cell carcinoma is shown in Table 12, and the clinical analysis results are shown in Table 13.

[0053] Table 12 Clinical Sample Detection Results Table 13 Clinical analysis performance results Detection target Sensitivity Specificity Accuracy ZpRCC 86.05% 90.51% 88.79% The data in Table 13 show that the combined methylation detection reagent for the ZNF154, OXR1, TMEM178A and LINE1 gene combinations has a diagnostic sensitivity, specificity and accuracy of 86.05%, 90.51% and 88.79% for pRCC, respectively, demonstrating excellent clinical diagnostic performance.

[0054] 3) Specificity analysis Urine samples from 15 subjects with other urinary system tumors were tested, and all results were negative with no cross-reactivity. The detection of endogenous and exogenous interfering substances showed that 11 interfering substances—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), Candida albicans (2.5 × 10¹⁰ 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 ZNF154, OXR1, TMEM178A, and LINE1 genes has excellent analytical specificity.

[0055] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A gene combination for the early diagnosis of papillary renal cell carcinoma, characterized in that, Including one or more of the ZNF154 gene, OXR1 gene, TMEM178A gene and LINE1 gene.

2. A kit for early diagnosis of papillary renal cell carcinoma according to claim 1, characterized in that, The kit includes reagents for detecting differentially methylated sites in combinations of the ZNF154, OXR1, TMEM178A, and LINE1 genes.

3. The kit for early diagnosis of papillary renal cell carcinoma according to claim 2, characterized in that, The test kit also includes lysis buffer, extraction reagent, transformation reagent, PCR reaction solution, primer-probe mixture, positive control and negative control.

4. A kit for early diagnosis of papillary renal cell carcinoma according to claim 2, characterized in that, The differentially methylated sites of the ZNF154, OXR1, TMEM178A, and LINE1 gene combinations are cg01268824, cg17176732, cg10708761, and cg11056055 methylation sites and their ±200bp sequences, respectively.

5. A kit for early diagnosis of papillary renal cell carcinoma according to claim 4, characterized in that, The kit includes probes and primers for detecting the ZNF154 gene, the OXR1 gene, the TMEM178A gene, and the LINE1 gene.

6. A kit for early diagnosis of papillary renal cell carcinoma according to claim 5, characterized in that, The nucleotide sequences of the primers for detecting the ZNF154 gene are shown in ZNF154-MF and ZNF154-MR, and the nucleotide sequence of the probe for detecting the ZNF154 gene is shown in ZNF154-MP; the nucleotide sequences of the primers for detecting the OXR1 gene are shown in OXR1-MF and OXR1-MR, and the nucleotide sequence of the probe for detecting the OXR1 gene is shown in OXR1-MP; the nucleotide sequences of the primers for detecting the TMEM178A gene are shown in TMEM178A-MF and TMEM178A-MR, and the nucleotide sequence of the probe for detecting the TMEM178A gene is shown in TMEM178A-MP; the nucleotide sequences of the primers for detecting the LINE1 gene are shown in LINE1-MF and LINE1-MR, and the nucleotide sequence of the probe for detecting the LINE1 gene is shown in LINE1-MP.

7. The application of a kit for early diagnosis of papillary renal cell carcinoma according to any one of claims 2 to 6, characterized in that, The usage method includes the following steps: 1) Mix freshly collected samples with EDTA-containing cell-free DNA preservation solution to obtain pretreated samples, and store them at 2~8℃; 2) The pretreated sample was lysed with the lysis buffer of the cell-free DNA extraction reagent, and then the cell-free DNA was extracted using the extraction reagent to obtain the extracted cell-free DNA solution; 3) The extracted free DNA solution was transformed with a transformation reagent, and the transformed free DNA solution was collected; 4) Perform qPCR detection on the transformed cell-free DNA solution and interpret the results according to the interpretation formula.

8. The application of the kit for early diagnosis of papillary renal cells according to claim 7, characterized in that, Cell-free DNA samples are derived from blood and urine.

9. The application of the kit for early diagnosis of papillary renal cell carcinoma according to claim 8, characterized in that, The interpretation formula is: Z pRCC =6.8937-1.317A–1.047B-0.814C+1.426D; where A is the Cp value difference between ZNF154 and ACTB genes, B is the Cp value difference between OXR1 and ACTB genes, C is the Cp value difference between TMEM178A and ACTB genes, and D is the Cp value difference between LINE1 and ACTB genes.

10. The application of the kit for early diagnosis of papillary renal cells according to claim 8, characterized in that, When Z pRCC A result ≥2.87 is interpreted as positive, indicating a higher risk of pRCC in the subject; conversely, when Z... pRCC When the value is <2.87, the result is interpreted as negative, indicating that the subject has a low risk of pRCC.

Images