Gene composition for early diagnosis and pathological typing of kidney cancer, kit and application of gene composition

Abstract

The invention relates to the technical field of molecular diagnosis, and discloses a gene composition for early diagnosis and pathological typing of kidney cancer, a kit and application of the gene composition, provided markers can detect ccRCC and pRCC at the same time by jointly detecting the methylation level of main pathological typing related genes of the kidney cancer, the sensitivity and specificity of kidney cancer detection are remarkably improved, and the kit is suitable for clinical application. By setting an independent logistic regression interpretation formula, the marker and the kit provided by the invention also have the effect of assisting pathological typing of the kidney cancer, are convenient to use and are suitable for clinical popularization and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular diagnosis, and particularly relates to a gene combination for early diagnosis and pathological typing of renal cancer, a kit and application thereof. BACKGROUND

[0002] Renal cell carcinoma (RCC) accounts for 2-3% of adult malignant tumors, and is the malignant tumor with the highest mortality among the three major high-incidence cancers of the urinary system. According to the histopathological classification, RCC has more than ten subtypes, and the three subtypes with the highest incidence are clear cell RCC (ccRCC; 75-80%), papillary RCC (pRCC; 10-15%) and chromophobe RCC (chRCC; 5%), among which, ccRCC is the main pathological type with the highest proportion (75-80%) and the worst prognosis, pRCC is the second, and chRCC has the lowest proportion and is relatively inert. Therefore, ccRCC and pRCC are the main pathological types in the diagnosis of renal cancer. At present, puncture biopsy is the most reliable gold standard method for the diagnosis of renal cancer, determination of histopathological type and grading, but puncture has the disadvantages of high complication risk, increased risk of tumor metastasis, and the accuracy of puncture results is affected by the heterogeneity of renal cancer.

[0003] Non-invasive molecular detection based on body fluids has great application potential in tumor diagnosis and treatment. Liquid biopsy of different types of molecular markers has shown great potential in the application of auxiliary diagnosis, drug guidance, efficacy evaluation and recurrence monitoring of tumors. Especially, DNA methylation markers have the advantages of early diagnosis, high sensitivity and high specificity in tumor diagnosis. At present, methylation detection kits account for the majority of products used for auxiliary diagnosis of tumors.

[0004] Although existing studies have identified some changes in the methylation levels of genes associated with the occurrence of renal cancer through various techniques, renal cancer has multiple different subtypes, and the significant molecular level heterogeneity among subtypes makes it one of the most challenging tumors in molecular detection. There is a lack of DNA methylation markers with excellent diagnostic performance, and there is also no methylation detection kit for renal cancer-related genes.

[0005] The occurrence of different subtypes of renal cancer is regulated by multiple genes, therefore, for different subtypes of renal cancer, combined detection of the methylation levels of multiple genes can effectively improve the detection sensitivity of renal cancer. At present, there is still a need in the art to further develop DNA methylation markers of renal cancer-related genes that can be practically used for the diagnosis of renal cancer, and to develop detection kits with high sensitivity. SUMMARY

[0006] In view of the problem that the significant molecular level heterogeneity among different subtypes of renal cancer mentioned in the background causes difficulty in molecular detection, and the lack of DNA methylation markers and detection kits with excellent diagnostic performance, the present application provides a gene combination, a kit and its application for early diagnosis and pathological typing of renal cancer, aiming to develop a DNA methylation marker of renal cancer related genes and a high sensitivity detection kit for practical use in the diagnosis of renal cancer, so as to realize the combined detection of multiple gene methylation levels for different subtypes of renal cancer.

[0007] The technical scheme adopted by the present application is as follows: a gene combination for early diagnosis and pathological typing of renal cancer, comprising one or more of IRX4 gene, PLIN2 gene, ZNF154 gene and SOX1 gene.

[0008] A kit for early diagnosis and pathological typing of renal cancer, comprising reagents for detecting the differential methylation sites of the gene combination of IRX4 gene, PLIN2 gene, ZNF154 gene and SOX1 gene.

[0009] Further, the differential methylation sites of the gene combination of IRX4 gene, PLIN2 gene, ZNF154 gene and SOX1 gene are respectively: cg05658487, cg13990947, cg01268824, cg16705627 methylation sites and their ±200bp sequences.

[0010] Further, the reagents further comprise probes and primers for detecting IRX4 gene, probes and primers for detecting PLIN2 gene, probes and primers for detecting ZNF154 gene and probes and primers for detecting SOX1 gene.

[0011] Further, the nucleotide sequences of the primers for detecting IRX4 gene are shown as IRX4-MF and IRX4-MR, and the nucleotide sequence of the probe for detecting IRX4 gene is shown as IRX4-MP; the nucleotide sequences of the primers for detecting PLIN2 gene are shown as PLIN2-MF and PLIN2-MR, and the nucleotide sequence of the probe for detecting PLIN2 gene is shown as PLIN2-MP; the nucleotide sequences of the primers for detecting ZNF154 gene are shown as ZNF154-MF and ZNF154-MR, and the nucleotide sequence of the probe for detecting ZNF154 gene is shown as ZNF154-MP; the nucleotide sequences of the primers for detecting SOX1 gene are shown as SOX1-MF and SOX1-MR, and the nucleotide sequence of the probe for detecting SOX1 gene is shown as SOX1-MP.

[0012] Further, the detection kit further comprises a lysis solution, an extraction reagent, a conversion reagent, a PCR reaction solution, a primer probe mixture, a positive control and a negative control.

[0013] Further, the positive control is a sample containing methylated human genomic DNA and non-methylated human genomic DNA; and the negative control is a sample containing non-methylated human genomic DNA.

[0014] The application of a kit for early diagnosis and pathological typing of renal cancer, characterized by the following steps: 1) mixing a freshly collected sample with an EDTA-containing free DNA storage solution to obtain a pretreated sample, and storing the pretreated sample at 2-8°C; 2) lysing the pretreated sample with a lysis solution of a urine free DNA extraction reagent, and then extracting free DNA using an extraction reagent to obtain an extracted free DNA solution; 3) converting the extracted free DNA solution using a conversion reagent, and collecting a converted free DNA solution; 4) performing qPCR detection on the converted free DNA solution, and interpreting the results.

[0015] Preferably, the formula of the lysis solution of the urine free DNA extraction reagent is as follows: guanidine isothiocyanate concentration of 1.5-4.5 M; Tris-HCl concentration of 0.08-1.2 M; EDTA-Na2 concentration of 0.008-0.012 M; NaCl concentration of 0.2-1.2 M; Tween 20 concentration of 1-2.2% by volume; Triton X-100 concentration of 0.5-1.2% by volume; and pH 5.6-8.2.

[0016] Preferably, the formula of the lysis solution of the urine free DNA extraction reagent is as follows: guanidine isothiocyanate concentration of 1.5-4.0 M; Tris-HCl concentration of 0.08-1.0 M; EDTA-Na2 concentration of 0.008-0.010 M; NaCl concentration of 0.2-1.0 M; Tween 20 concentration of 1-2.0% by volume; Triton X-100 concentration of 0.5-1.0% by volume; and pH 5.6-8.0.

[0017] Preferably, the formula of the lysis solution of the urine free DNA extraction reagent is as follows: guanidine isothiocyanate concentration of 1.5-4.0 M; Tris-HCl concentration of 0.08-1.0 M; EDTA-Na2 concentration of 0.008-0.010 M; NaCl concentration of 0.2-1.0 M; Tween 20 concentration of 1-2.0% by volume; Triton X-100 concentration of 0.5-1.0% by volume; and pH 5.6-8.0.

[0018] Further, the result interpretation formula of the ccRCC is ZccRCC = 1.3702 - 0.151A - 0.103B - 0.094D, and the result interpretation formula of the pRCC is ZpRCC = 1.1085 - 0.139B - 0.187C - 0.076D; wherein A is the Cp value difference between the IRX4 and ACTB genes, B is the Cp value difference between the PLIN2 and ACTB genes, C is the Cp value difference between the ZNF154 and ACTB genes, and D is the Cp value difference between the SOX1 and ACTB genes.

[0019] Further, when ZccRCC is greater than or equal to 0.16, the subject has a higher risk of ccRCC, and vice versa; when ZpRCC is greater than or equal to 0.44, the subject has a higher risk of pRCC, and vice versa; when ZccRCC is greater than or equal to 0.16 and / or ZpRCC is greater than or equal to 0.44, the subject has a higher risk of kidney cancer; and when ZccRCC is less than 0.16 and ZpRCC is less than 0.44, the subject has a lower risk of kidney cancer.

[0020] Further, the sample source of the cell-free DNA is one of blood and urine.

[0021] In summary, due to the adoption of the above technical solutions, the present application has the following beneficial effects: the markers provided by the present application can detect ccRCC and pRCC at the same time by jointly detecting the methylation levels of the genes related to the main pathological types of kidney cancer, thereby significantly improving the sensitivity and specificity of kidney cancer detection; by setting independent logistic regression interpretation formulas, the markers and kits provided by the present application also have the function of assisting in the pathological typing of kidney cancer, are easy to use, and are suitable for clinical popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0022] The present application will be described by way of example and with reference to the accompanying drawings, in which: Figure 1 is the average methylation level of the methylation site in the IRX4 gene in the ccRCC and pRCC different tissue samples in Example 1 of the present application.

[0023] Figure 2 is the average methylation level of the methylation site in the PLIN2 gene in the ccRCC and pRCC different tissue samples in Example 1 of the present application.

[0024] Figure 3 is the average methylation level of the methylation site in the ZNF154 gene in the ccRCC and pRCC different tissue samples in Example 1 of the present application.

[0025] Figure 4is the average methylation level of the methylation site in the SOX1 gene in ccRCC and pRCC different tissue samples in Example 1 of the present application.

[0026] Figure 5 is the linear correlation diagram between the Cp value of the gene detected by detection system 1 (A-E), detection system 2 (F-J) and detection system 3 (K-O) and the corresponding sample DNA concentration in Example 5 of the present application.

[0027] Figure 6 is the precision diagram of detection system 1 (A), detection system (2) and detection system (3) in Example 5 of the present application. DETAILED DESCRIPTION

[0028] All features disclosed in this specification, and / or the steps of any method or process specified in this specification, can be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive.

[0029] The following is described in conjunction with the examples.

[0030] Example 1 Selection of kidney cancer related genes and differential methylation sites The present application collected 324 cases of ccRCC and 160 cases of cancer adjacent or normal tissues, and 226 cases of pRCC and 45 cases of cancer adjacent or normal tissues from the TCGA data set (https: / / www.cancer.gov / tcga) whole genome methylation data. Classify by sample type, that is, compare the methylation levels between cancer tissues and cancer adjacent or normal tissues, and perform differential methylation analysis on kidney cancer related genes.

[0031] To ensure that the detection in the sample can have sufficient sensitivity and specificity, the selected genes and differential methylation sites need to meet the following requirements: (1) It has sufficient differential significance in at least one subtype of ccRCC or pRCC (ccRCC p-value < 1E-50, pRCC p-value < 1E-10); (2) It is a site with differential hypermethylation in cancer tissues; (3) The average methylation level in cancer adjacent or normal tissues is less than 0.2; (4) The gene and differential methylation site with higher methylation difference significance in two subtypes of kidney cancer and lower average methylation level in cancer adjacent or normal tissues can be used as a marker for kidney cancer diagnosis; the gene and differential methylation site with significant methylation difference in one subtype and insignificant methylation difference in another subtype can be used as a marker for classification of two subtypes of kidney cancer.

[0032] After screening, the kidney cancer related genes and the differentially methylated sites thereof, and the methylation levels and the difference significance in different kidney cancer subtypes and tissues are shown in Table 1.

[0033] Table 1. Kidney cancer related genes and the differentially methylated sites thereof, and the methylation levels and the difference significance in different kidney cancer subtypes and tissues Example 2. Design and screening of primer and probe combination According to the differentially methylated sites of the kidney cancer related genes IRX4 (cg05658487), PLIN2 (cg13990947), ZNF154 (cg01268824), SOX1 (cg16705627) and the sequences of ±200 bp thereof screened in Example 1, and the gDNA sequence of the internal reference gene ACTB, a plurality of methylation specific forward primers and reverse primers were designed and synthesized. Using the bisulfite converted methylated and non-methylated human genomic DNA as templates, SYBR Green dye method was used for qPCR amplification, and the detection results are shown in Table 2.

[0034] Table 2. qPCR amplification detection results of primers According to the C p value difference (△C p =C p靶基因 –C p内参基因 ) of each target gene and the internal reference gene, and the peak temperature and the number of peaks of the melting curve in the methylated and non-methylated templates, the amplification efficiency and specificity of each primer combination were evaluated, and the primer combination with high amplification efficiency and good amplification specificity was screened.

[0035] According to the methylation specific fluorescent probe designed and synthesized between the forward primer and the reverse primer of the screened primer combination, the bisulfite converted methylated and non-methylated human genomic DNA was used as the template, and the probe method was used for qPCR amplification. According to the amplification curve linearity of each primer probe combination in the methylated and non-methylated templates, and the C p value difference (△C p靶基因 =C p靶基因 –C p内参基因 ) of each target gene and the internal reference gene, the amplification efficiency of each primer probe combination was evaluated; according to the △C p value difference (△△C p靶基因 =△C p非甲基化 –△C p甲基化 ) of each primer probe combination between the two templates, the specificity of each primer probe combination to the methylated template, and the detection discrimination ability between the non-methylated template were evaluated, and the detection results are shown in Table 3.

[0036] Table 3 Primer screening After the test screening of the primer and probe combination in the methylated and non-methylated templates, 1 set of forward primer, reverse primer and fluorescent probe combination with high amplification efficiency and good amplification specificity was screened for each of the 4 target genes, and the primer and probe sequences are as follows: IRX4-MF: 5'-CGACGATAAGGGTGGTTATTTTCG-3'; IRX4-MR: 5'-TTAACCAAACGCTAAACTCGCTAC-3'; IRX4-MP: 5'-CCCTCGACCCACGAAACCCCTCTC-3'; PLIN2-MF: 5'-TCGTTAAATAGTCGGGATTAGTTGG-3'; PLIN2-MR: 5'-CGATAACTCACGCCTATAATCCTAA-3'; PLIN2-MP: 5'-AAACCGAAACGAACGAATCACCAACCTAAC-3'; ZNF154-MF: 5'-AAACTTAAATAAACCATTTCTCATCGC-3'; ZNF154-MR: 5'-TTTATAGATTTCGAGGTGGGTGTC-3'; ZNF154-MP: 5'-ACGCCCTAAAACTTCGCTTTTACTCCAAAA-3'; SOX1-MF: 5'-TTTATTTCGGTCGTTTATGTTTTAGGT-3'; SOX1-MR: 5'-GATCTCCATCATCATACTATACATCGA-3'; SOX1-MP: 5'-CTAACGAATTCACCGACACCGCGAA-3'; ACTB-MF: 5'-TTTGGGTTTATTTAGAGTGTAGATG-3'; ACTB-MR: 5'-CAACCCCAATAAAACATAACACC-3'; ACTB-MP: 5'-CAAATAATCCCTTCCCACCTCCTCAAACAT-3'.

[0037] A lysis solution and extraction method of a urine cell-free DNA extraction reagent 1) Processing and preservation of urine samples: Freshly collected urine samples should be immediately mixed with 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), maintain the integrity of exfoliated cells in urine, and avoid the release of intracellular nucleic acids.

[0038] The sample should be stored and / or transported at 2-8°C for no more than 7 days before further processing to avoid cryopreservation.

[0039] 2) The lysis solution formula of the urine cell-free DNA extraction reagent includes the following four: 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 extraction steps of urine cell-free DNA are as follows: (1) The urine sample is centrifuged at 3000xg for 10min at 4°C, and 4mL supernatant is transferred to a new 10mL centrifuge tube; (2) Add 3mL lysis solution and 0.05mL 20mg / mL proteinase K, vortex to mix, and incubate at 55°C for 10min; (3) Add 2mL isopropanol and 30μL magnetic beads in order, vortex to mix, and mix on a inversion mixer for 3min at room temperature; (4) Insert the centrifuge tube into the magnetic stand, stand for 2 min to attract the magnetic beads, and then use a pipette to remove the supernatant after the magnetic beads are completely adsorbed; (5) Take the centrifuge tube off the magnetic stand, add 600 μL of washing solution, fully resuspend the magnetic beads after shaking, and then transfer to a 1.5 mL centrifuge tube, stand for 1 min; (6) Insert the centrifuge tube into the magnetic stand, stand for 1 min to attract the magnetic beads, and then use a pipette to remove the supernatant after the magnetic beads are completely adsorbed; (7) Take the centrifuge tube off the magnetic stand, add 600 μL of washing solution, fully resuspend the magnetic beads after shaking, and then stand for 1 min; (8) Insert the centrifuge tube into the magnetic stand, stand for 1 min to attract the magnetic beads, and then use a pipette to remove the supernatant after the magnetic beads are completely adsorbed; (9) Open the cover and air dry for 10 min at room temperature, avoid too long air drying time, and excessive drying will seriously reduce the nucleic acid elution efficiency; (10) Add 50 μL of elution solution preheated at 65°C, fully shake and disperse the magnetic beads, incubate at 65°C for 5 min, and vortex shake for 5 s every 2 min during the incubation; (11) After instantaneous centrifugation for 5 s on a palm centrifuge, insert the centrifuge tube into the magnetic stand, stand for 1 min to attract the magnetic beads, and then use a pipette to transfer the supernatant to a new centrifuge tube, which is the extracted ucfDNA solution, ready for use or storage at -80°C.

[0040] Qubit and qPCR detection methods were used to compare the concentration and extraction efficiency of the ucfDNA solution extracted by the above four kinds of lysis solution formulations. The qPCR detection method uses the conventional internal reference gene ACTB primer probe, and the Cp value of the internal reference gene is used to compare the ucfDNA extraction efficiency of the four kinds of lysis solution formulations. The smaller the Cp value, the higher the detection efficiency. The lysis solution formulation experiment results of the urine free DNA extraction reagent are shown in Table 4.

[0041] The conventional internal reference gene ACTB primer and probe sequences are as follows: ACTB-F: 5'-TGACTTAGTTGCGTTACACCCTT-3' ACTB-R: 5'-GACTGCTGTCACCTTCACCG-3' ACTB-P: 5'-CCTAACTTGCGCAGAAAACAAGATG-3'.

[0042] Table 4 Lysis solution formulation experiment results of urine free DNA extraction reagent From the results of Table 4, it can be concluded that the ucfDNA extraction rate of Formula 2 is the highest, and Formula 2 is used as the lysing solution for subsequent experiments.

[0043] Example 4: A methylation detection sample pretreatment reagent and bisulfite conversion method A methylation detection sample pretreatment reagent includes a conversion solution, buffer A, buffer B, buffer C, a binding solution, a desulfonation solution, a washing solution 1, a washing solution 2, an elution solution, a nucleic acid adsorption column, and a collection tube.

[0044] The specific operation steps of bisulfite conversion are as follows: 1) When preparing, 1210 μL of buffer A needs to be added to each tube of conversion solution, shaken at room temperature for about 1 min to dissolve, then 110 μL of buffer B, 110 μL of buffer C are added, and shaken to mix evenly for standby; 2) The bisulfite conversion reaction system is prepared in a 200 μL sterile centrifuge tube, including: 130 μL of bisulfite solution, DNA sample X (10 ng-2 μg), and 20-X of nuclease-free water (the maximum volume of DNA and nuclease-free water is 20 μL).

[0045] 3) The above solution is blown and mixed evenly, centrifuged briefly, and then placed in a PCR instrument. The bisulfite conversion reaction program is shown in Table 5.

[0046] Table 5: Bisulfite conversion reaction program 4) Add 600 μL of the binding solution and the converted reaction product (about 150 μL) to the adsorption column (the adsorption column is placed in the collection tube). Gently mix 6-8 times, and stand at room temperature for 2 min; 5) Centrifuge at 12000xg 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 12000xg for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; 7) Add 500 μL of desulfonation solution to the adsorption column, stand at room temperature (15-25°C) for 15 min, centrifuge at 12000xg 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 12000xg for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; 9) Repeat step 8; 10) Centrifuge the empty column at 12000xg for 2 min; 11) Move the adsorption column to a new 1.5 mL centrifuge tube, open the cap for 10 min, add 20-40 μL elution solution to the center of the adsorption column membrane, stand at room temperature for 2 min, centrifuge at 12000 x g for 2 min, collect the DNA solution, which is the ucfDNA solution obtained by bisulfite conversion and recovery, for use or storage at -80 °C.

[0047] The above-mentioned operation steps were used to obtain the nucleic acid sample after conversion by taking the methylated and non-methylated human genomic DNA as the bisulfite conversion sample. The conversion and recovery efficiency of the above-mentioned six bisulfite conversion reaction procedures were compared by using the methylation-specific primer probe of the internal reference gene ACTB (ACTB-MF / ACTB-MR / ACTB-MP) shown in Example 2 and the conventional primer probe of the internal reference gene ACTB (ACTB-F / ACTB-R / ACTB-P) shown in Example 3 by using the qPCR detection method. According to the difference (△Cp = CpACTB - CpACTB-M) between the Cp values of the conventional primer probe and the methylation-specific primer probe of the ACTB internal reference gene, the conversion rate was calculated according to the formula: conversion rate = 1 - 2-△Cp; and the nucleic acid recovery rate of the above-mentioned six bisulfite conversion reaction procedures was compared according to the Cp value of the methylation-specific primer probe of the ACTB internal reference gene, and the smaller the Cp value, the higher the recovery rate.

[0048] Table 6 Experimental detection results of bisulfite conversion As shown from the results in Table 6, the results show that the conversion rate of procedure 1 is low, the conversion rates of the remaining procedures are high and the difference is small, and the nucleic acid recovery rate of procedure 2 is the highest, but the difference with other procedures is small. Considering the conversion rate, recovery rate and time consumption of the conversion reaction procedure, procedure 2 is used for subsequent experiments.

[0049] Example 5 A real-time fluorescent quantitative PCR detection system for detecting the methylation of IRX4, PLIN2, ZNF154 and SOX1 genes The methylation-specific primer probes of the IRX4, PLIN2, ZNF154, SOX1 genes and the ACTB internal reference gene shown in Example 2 were used, combined in IRX4 / PLIN2 / ACTB and ZNF154 / SOX1 / ACTB triplets, respectively in two PCR holes, and the qPCR detection of the methylation level of the target genes in the bisulfite conversion sample was carried out according to the following three detection systems.

[0050] Table 7 Components of negative and positive quality control 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.

[0051] 1) Testing System 1 The qPCR reaction system for detection system 1 is shown in Table 8, and the qPCR reaction procedure for detection system 1 is shown in Table 9.

[0052] Table 8. qPCR reaction system for detection system 1 Table 9. qPCR reaction procedure for detection system 1 2) Detection System 2 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.

[0053] Table 10 qPCR reaction system for detection system 2 Table 11 qPCR reaction procedure for detection system 2 2) Testing System 3 The qPCR reaction system for detection system 2 is shown in Table 12, and the qPCR reaction procedure for detection system 2 is shown in Table 13.

[0054] Table 12 qPCR reaction system for detection system 2 Table 13 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.

[0055] b. Precision evaluation of the detection system The three detection systems described above were used to detect a human genomic DNA solution with a methylation ratio of 10% and a concentration of 16 ng / μL, and the detection was repeated 20 times. The coefficients of variation (CV) of the Cp values of each target gene of the three detection systems were calculated, and the detection results are shown in Table 2. Figure 6 As shown in Table 2, the CV values of the five genes of detection system 3 were the smallest, indicating that the precision of detection system 3 was better than that of detection systems 1 and 2.

[0056] In summary, the accuracy and precision of detection system 3 are better than those of detection systems 1 and 2, and this detection system is selected for subsequent experiments.

[0057] Example 6 Clinical verification of the early diagnosis performance and pathological typing role of a combination of IRX4, PLIN2, ZNF154, and SOX1 genes for kidney cancer A total of 441 urine samples of subjects were collected, including 203 ccRCC samples, 86 pRCC samples, 137 non-RCC samples, 15 other subtype RCC samples, and urine samples of subjects with urinary system tumors confirmed by histopathology. All samples were from the First Affiliated Hospital of Zhejiang University School of Medicine and the Second Affiliated Hospital of Zhejiang University School of Medicine. The above clinical samples were subjected to methylation detection according to the sample processing and detection system described in Examples 2-5.

[0058] 1) Kidney cancer auxiliary pathological typing and diagnosis performance analysis model The Cp value results of each gene detected by qPCR were substituted into the following logistic regression formula and the results were interpreted: Z ccRCC = 1.3702 - 0.151A - 0.103B - 0.094D Z pRCC = 1.1085 - 0.139B - 0.187C - 0.076D Wherein A is the Cp value difference between IRX4 and ACTB genes, B is the Cp value difference between PLIN2 and ACTB genes, C is the Cp value difference between ZNF154 and ACTB genes, and D is the Cp value difference between SOX1 and ACTB genes. That is: A = Cp IRX4 – Cp ACTB B = Cp PLIN2 – Cp ACTB C = Cp ZNF154 – Cp ACTB D = Cp SOX1 – Cp ACTB When Z ccRCC≥0.16, the subject has a higher risk of ccRCC, otherwise, the subject has a lower risk of ccRCC; when Z pRCC ≥0.44, the subject has a higher risk of pRCC, otherwise, the subject has a lower risk of pRCC; When Z ccRCC ≥0.16 and / or Z pRCC ≥0.44, the subject has a higher risk of kidney cancer; when Z ccRCC <0.16 and Z pRCC <0.44, the subject has a lower risk of kidney cancer.

[0059] 2) Clinical analysis performance The diagnostic value of the combined detection of the methylation levels of the IRX4, PLIN2, ZNF154 and SOX1 genes in the urine sample for kidney cancer was detected, and the detection results are shown in Table 14, and the clinical analysis performance results are shown in Table 15.

[0060] Table 14 Detection results of clinical samples Table 15 Clinical analysis performance results As shown in the results of Table 15, the accuracy of the combined methylation detection reagent of the IRX4, PLIN2, ZNF154 and SOX1 genes for ccRCC and pRCC reached 90.00% and 88.79%, respectively, both with high sensitivity and specificity; the comprehensive detection sensitivity for kidney cancer reached 84.78%, and the specificity reached 78.83%, both with high diagnostic sensitivity and specificity, and the typing accuracy for ccRCC and pRCC reached 91.44% and 78.38%, respectively.

[0061] 3) Specificity analysis The urine samples of 15 subjects with other subtypes of RCC and urinary system tumors were detected, and the detection results were all negative, without cross-reaction; endogenous and exogenous interfering substances were detected, indicating that 11 interfering substances including 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 x 1010 CFU / mL), human serum albumin (10 mg / mL), ascorbic acid (50 mg / mL), non-conjugated bilirubin (2 mg / mL), alcohol (1% (V / V)), and hemoglobin (100 mg / mL) had no effect on the detection results at the corresponding concentrations. The results showed that the combined methylation detection reagent of the IRX4, PLIN2, ZNF154 and SOX1 genes had good analysis specificity.

[0062] The above-described embodiments are merely illustrative of the present application, which is described in more detail and specifically, but should not be interpreted as a limitation on the scope of protection of the present application. It should be noted that for those skilled in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A gene combination for early diagnosis and pathological classification of renal cell carcinoma, characterized in that, This includes one or more of the IRX4, PLIN2, ZNF154, and SOX1 genes.

2. A reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 1, characterized in that, The kit includes reagents for detecting differentially methylated sites in combinations of the IRX4, PLIN2, ZNF154, and SOX1 genes.

3. The reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 2, characterized in that, The differentially methylated sites of the gene combinations of IRX4, PLIN2, ZNF154 and SOX1 are cg05658487, cg13990947, cg01268824, and cg16705627, and their ±200bp sequences, respectively.

4. The kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 3, characterized in that, The kit includes probes and primers for detecting the IRX4 gene, the PLIN2 gene, the ZNF154 gene, and the SOX1 gene.

5. A reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 4, characterized in that, The nucleotide sequences of the primers for detecting the IRX4 gene are shown as IRX4-MF and IRX4-MR, and the nucleotide sequence of the probe for detecting the IRX4 gene is shown as IRX4-MP; the nucleotide sequences of the primers for detecting the PLIN2 gene are shown as PLIN2-MF and PLIN2-MR, and the nucleotide sequence of the probe for detecting the PLIN2 gene is shown as PLIN2-MP; 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 SOX1 gene are shown as SOX1-MF and SOX1-MR, and the nucleotide sequence of the probe for detecting the SOX1 gene is shown as SOX1-MP.

6. A reagent kit for early diagnosis and pathological classification of 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.

7. A reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 6, characterized in that, 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.

8. The application of a reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to any one of claims 2 to 7, 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 urine 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.

9. The application of the reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 8, characterized in that, The interpretation formula is: The result interpretation formula for ccRCC is: Z ccRCC = 1.3702 - 0.151A - 0.103B -0.094D; The formula for interpreting the pRCC result is: Z pRCC = 1.1085 - 0.139B - 0.187C - 0.076D; where A is the Cp value difference between the IRX4 and ACTB genes, B is the Cp value difference between the PLIN2 and ACTB genes, C is the Cp value difference between the ZNF154 and ACTB genes, and D is the Cp value difference between the SOX1 and ACTB genes.

10. The application of the reagent kit for early diagnosis and pathological classification of renal cell carcinoma according to claim 8, characterized in that, When Z ccRCC When the value is ≥0.16, the subject has a higher risk of ccRCC, and vice versa. When Z pRCC When the value is ≥0.44, the subject has a higher risk of developing pRCC, and vice versa. When Z ccRCC ≥0.16 and / or Z pRCC When Z is ≥0.44, the subject has a higher risk of developing kidney cancer; when Z ccRCC <0.16 and Z pRCC When the value is <0.44, the risk of kidney cancer in the subjects is low.

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