Kit for molecular typing diagnosis of kidney cancer and application of kit

Abstract

The invention relates to the technical field of bioengineering, and discloses a kit for molecular typing diagnosis of kidney cancer and application of the kit. The biomarker comprises 75 genes including ABCB1, BRIP1, EGFR, FH, MITF and the like. The invention further provides a DNA probe set for detecting the biomarker, a detection reagent containing the DNA probe set and application of the detection reagent in preparation of a kit for detecting kidney cancer gene variation. The kit provided by the invention has the advantages of wide coverage of detectable biomarkers, high detection performance and low detection cost, and is helpful for screening potential therapeutic targets, exploring novel therapeutic drugs and optimizing clinical test design.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and more specifically, to a kit for molecular typing diagnosis of renal cancer and its application. Background Art

[0002] Renal cell carcinoma (RCC) is a malignant tumor originating from the renal tubular epithelium, accounting for 80% to 90% of renal malignant tumors. The most common histopathological type of renal cell carcinoma is clear cell carcinoma, followed by papillary renal cell carcinoma and chromophobe cell carcinoma, as well as rare types of renal cell carcinoma such as collecting duct carcinoma. Renal cell carcinoma can be divided into hereditary renal cell carcinoma and sporadic renal cell carcinoma based on its relationship with genetic syndromes. Hereditary renal cell carcinoma and sporadic renal cell carcinoma cannot be distinguished from pathological morphology alone.

[0003] The 2016 WHO fourth edition of the kidney tumor classification standard is currently used in clinical practice. It is a classification based on pathological histology, but this classification method cannot effectively distinguish certain types of renal cell carcinoma. For example, renal cell carcinoma can be divided into hereditary renal cell carcinoma and sporadic renal cell carcinoma based on its relationship with genetic syndromes, but hereditary renal cell carcinoma and sporadic renal cell carcinoma cannot be distinguished from each other based on pathological morphology alone. The 2025v1 NCCN Guidelines for Hereditary Renal Cell Carcinoma point out that hereditary renal cell carcinoma syndromes include VHL syndrome, HRPC syndrome, BHDS syndrome, TSC syndrome, HLRCC syndrome, BAP1 tumor susceptibility syndrome, and PGL / PCC syndrome. It is recommended to detect germline variations in genes such as VHL, MET, FLCN, TSC1 / 2, FH, BAP1, SDHA / B / C / D, and conduct genetic screening for patients and their relatives to detect potential risks as early as possible. The common hereditary renal cell carcinoma mutation sites recommended by the Guidelines for the Diagnosis and Treatment of Renal Cell Carcinoma (2022 Edition) include: VHL, MET, FLCN, FH, SDHB / C / D, PTEN, MITF, HRPT2, BAP1. The guidelines also recommend genetic screening for patients and their relatives. The fifth edition of the WHO renal tumor classification criteria in 2020 proposed for the first time a class of molecularly defined renal tumors, including TFE3 rearranged renal cell carcinoma, TFEB-altered renal cell carcinoma, ELOC (formerly TCEB1) mutation renal cell carcinoma, ALK rearranged renal cell carcinoma, SMARCB1-deficient medullary renal cell carcinoma, succinate dehydrogenase (SDH)-deficient renal cell carcinoma, hereditary leiomyomatosis and renal cell carcinoma syndrome (FH)-related renal cell carcinoma, fumarate hydratase-deficient renal cell carcinoma, and a total of 10 subtypes. This type of tumor is relatively rare, and each subtype has clear molecular changes. Its corresponding prognostic evaluation model and diagnosis and treatment plan are different from traditional pathological subtypes. The WHO classification recommends the detection of multiple variant forms of multiple genes, such as TFE3 rearrangement, TFEB rearrangement or amplification, TCEB1 deletion or mutation, FH gene germline or systemic variation, ALK rearrangement, SMARCB1 gene deletion or mutation, etc., to assist in the molecular typing of renal cell tumors.

[0004] In addition, renal cancer is a malignant tumor with high genetic heterogeneity. The biological behaviors of tumor cells with different gene mutation spectra and genetic phenotypes have different sensitivities to treatment. Therefore, the Chinese Expert Consensus on Genetic Testing for Renal Cancer (2021 Edition) points out that genetic testing of renal cancer is necessary, which is a new direction to improve the efficacy of renal cancer treatment and achieve precision treatment of renal cancer.

[0005] With the promotion and application of next-generation sequencing technology, cancer treatment has entered a new stage of development through the combined application of genetic diagnosis and customized drugs. Through genomic analysis of renal cancer cell lines and tumor tissues, researchers have discovered a variety of gene mutations and copy number variations associated with the occurrence and development of renal cancer. The results of genomic analysis showed that there were 19 genes with significant mutations in ccRCC, including VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR and TP53. Among them, mutation or inactivation of the VHL gene existed in approximately 90% of patients. In a phase II clinical trial, the objective response rate of beztifen for VHL syndrome-related renal cancer could reach 49%. The mutation rate of PBRM1 was approximately 41%, which was considered to be the second most common mutation gene in ccRCC after VHL. A study on the relationship between the gene mutation status and treatment effect of tumor patients found that patients with PBM1 mutations could obtain longer disease-free survival by using everolimus as the first line. In addition, approximately 10% of ccRCC patients carried BAP1 mutations; and approximately 13% of ccRCC patients had SETD2 mutations. Studies on the papillary subtype of nccRCC (PRCC) have shown that approximately 15% to 21% of patients have MET gene mutations, and the effects of approved treatments for clear cell renal cell carcinoma may be limited, while targeted therapy for MET mutations is considered to be one of the directions that can be explored. In summary, whether from the perspective of diagnosis or prognosis, key information based on molecular pathology is very important for doctors. Based on the accurately detected genetic information, doctors can more accurately select treatment methods, clarify the cause of the disease, improve the overall efficiency of treatment, and minimize possible adverse reactions during treatment.

[0006] Therefore, how to more accurately achieve molecular classification of renal cancer is one of the urgent problems to be solved in this field. Summary of the invention

[0007] The purpose of the present invention is to assist in the diagnosis and typing of renal cell carcinoma, help in prognosis judgment and molecular targeted therapy, and clarify the genetic genes of hereditary renal tumors. To this end, the present application provides a kit for molecular typing diagnosis of renal carcinoma and its application.

[0008] In a first aspect, the present application provides the use of biomarkers in the preparation of products for detecting renal cancer gene mutations, wherein the biomarkers include the following genes:

[0009] ABCB1, BRIP1, EGFR, FH, MITF, PARP1, SETD2, VHL, ALK, CDK12, ERCC2, FLCN, MLH1, PBRM1, SMARCA4, MET, ARID1A, CDK4, ERCC3, F GFR4, MRE11, PIK3CA, SMARCB1, SDHA, ARID1B, CDK6, ERCC4, KDM5C, MSH2, PMS1, TCEB1, SDHC, ATM, CDKN2A, ERCC5, KDM6A, MSH6, PMS2, TERT, SDHD, ATR, CHEK1, EWSR1, KEAP1, MTOR, POLE, TFE3, BAP1, CHEK2, FANCA, KMT2A, NBN, PTEN, TP53, BLM, CSMD2, FANCC , KMT2D, NF2, RAD51, TSC1, BRCA1, CSMD3, FAT1, KRAS, NFE2L2, RECQL4, TSC2, BRCA2, DNMT3A, FGFR3, MDM2, PALB2, SDHB, UGT1A1.

[0010] Furthermore, the genetic variations include single nucleotide variations (SNVs), insertions and deletions (Indels), copy number variations (CNVs), microsatellite instability (MSI) and tumor mutation burden (TMB).

[0011] Furthermore, the product for detecting renal cancer gene mutation includes a kit.

[0012] In a second aspect, the present application provides a DNA probe set for detecting the biomarkers described in the first aspect, wherein the DNA probe set comprises DNA probes with sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 272.

[0013] In a third aspect, the present application provides a detection reagent comprising the DNA probe set described in the second aspect.

[0014] Furthermore, the detection reagent also includes a hybridization reaction solution.

[0015] In a fourth aspect, the present application provides a use of the detection reagent described in the third aspect in the preparation of a kit for detecting renal cancer gene mutations.

[0016] In a fifth aspect, the present application provides a kit for molecular typing and diagnosis of renal cancer, comprising the detection reagent described in the third aspect.

[0017] Furthermore, the kit also includes a DNA reagent for extracting samples.

[0018] Furthermore, the sample is renal cancer tumor tissue.

[0019] Compared with the prior art, this application has the following beneficial effects:

[0020] Compared with traditional imaging, histopathological examinations and laboratory tests, the evolving genomics provides more possibilities for the diagnosis and treatment of renal cancer. Among them, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) and next-generation sequencing (NGS) are currently widely used in molecular biology and clinical diagnosis. However, FISH is usually used to detect specific chromosomal structural abnormalities or the physical location of genes. The operation is complicated and not suitable for multi-target detection. PCR is usually only for single-gene detection and cannot detect copy number variation and structural variation. In addition, the molecular information provided by the two is not as comprehensive as that of next-generation sequencing. They are not as good as NGS in supporting personalized medicine and precision treatment, and are not suitable for continuous detection of disease progression.

[0021] NGS has many significant advantages in genomic analysis, such as higher resolution and sensitivity, more sensitive detection of copy number variation and structural variation, and higher sensitivity in detecting low-frequency variation; it has high throughput and high efficiency, and can analyze thousands or even millions of gene sequences simultaneously in a single run. Its high throughput may reduce the sequencing cost of multiple samples; it provides support for personalized medicine and treatment, and can serve as a companion diagnostic tool to assist targeted therapy and immunotherapy.

[0022] In summary:

[0023] a) The kit provided by the present invention can detect a wide range of biomarkers and has high detection performance;

[0024] The kit provided by the present invention comprises specific probes for detecting biomarkers, which include various forms of variation in 562 genes, including single nucleotide variations (SNVs), insertions and deletions (Indels), copy number variations (CNVs), microsatellite instability (MSI) and tumor mutation burden (TMB).

[0025] b) Low testing cost and wider application scope;

[0026] The kidney cancer biomarker detection system provided by the present invention can detect various forms of variation in the DNA of tumor tissue samples of kidney cancer patients, including single nucleotide variations (SNVs), insertions and deletions (Indels), copy number variations (CNVs) and microsatellite instability (MSI), by only detecting a small amount of DNA samples once. One detection and analysis process can detect multiple types, with lower costs, wider application range, and is more conducive to promotion.

[0027] c) Wide range of sample types to be tested;

[0028] The kit provided by the present invention can be used to detect different sample types, including FFPE and blood samples, detect patient germline and system mutations, and provide more comprehensive genetic information.

[0029] d) Discover new therapeutic targets and develop targeted drugs;

[0030] The molecular typing test provided by the present invention is helpful to screen potential therapeutic targets and discover new therapeutic drugs, thereby optimizing clinical trial design. DETAILED DESCRIPTION

[0031] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0032] Example 1: Screening of biomarkers

[0033] First, according to the current molecular classification of renal cancer, 75 directly related genes were included in 10 major categories (Homologous Recombination Repair (HRR), Mismatch Repair (MMR), SWISNF, TP53signal, Pathological classification, Renal cancer related gene, Oncogenicpathway gene, Renal cancer related gene, Cell Cycle, Oncogene). Then, according to the literature reports and the relevant information of the current clinical trials, after screening, a combination of biomarkers for renal cancer detection was obtained, as shown in Table 1 below.

[0034] Table 1 List of genes for DNA mutation detection

[0035]

[0036]

[0037] Probes were designed for the above biomarkers, and the probe sequence information is shown in SEQ ID NO: 1 to SEQ ID NO: 272.

[0038] Example 2: Application of biomarkers

[0039] 1. Sample DNA extraction

[0040] FFPE and blood samples were extracted and purified using a magnetic bead paraffin total nucleic acid extraction kit and a genomic DNA extraction kit according to the manufacturer's instructions. The extracted DNA was accurately quantified (Qubit fluorescence quantification instrument), and the extracted DNA was stored at -20°C.

[0041] 2. Prelibrary preparation

[0042] A. DNA disruption

[0043] 1) According to the sample concentration, take 100 ng of gDNA and 35 μL of total volume for fragmentation. If the sample volume is insufficient, use 10 mM Tris-HCl, PH 8.0 solution to make up to 35 μL.

[0044] 2) Prepare the fragmentation reaction solution according to the following table:

[0045] Component name Volume (μL) Shearing enzyme buffer (10×) 5 Shear enzyme 10 total 15

[0046] 3) Take 15 μL of the mixed fragmentation reaction solution and add it to the sample to be tested, mix well, centrifuge briefly to collect the reaction solution to the bottom of the tube, and immediately put the PCR tube into the PCR instrument, set the program as follows and run: (heat cover temperature: 50°C)

[0047]

[0048] 4) After the reaction is completed, take out the PCR tube, centrifuge briefly, and place it on an ice box for later use.

[0049] B. End repair + A

[0050] 1) Prepare the end repair reaction mix according to the following table:

[0051]

[0052]

[0053] 2) Take 10 μL of the end repair reaction mix and add it to the sample tube, mix well, centrifuge briefly to collect the reaction solution to the bottom of the tube, and immediately put the PCR tube into the PCR instrument, set the program and run it as follows:

[0054] temperature time 65℃ ∞ 65℃ 30min 4℃ ∞

[0055] 3) After the reaction is completed, take out the PCR tube, centrifuge briefly, and place it on an ice box for later use.

[0056] C. Connector connection

[0057] 1) Prepare the ligation reaction mix according to the table below, oscillate and mix thoroughly, then centrifuge briefly to collect the mixture at the bottom of the tube for later use

[0058] Component name Volume (μL) Ligase buffer 30 DNA ligase 10 Nuclease-free water 5 total 45

[0059] 2) Add 5 μL of universal adapter to each sample tube, vortex to mix, and centrifuge briefly to collect to the bottom of the tube.

[0060] 3) Add 45 μL of the mixed ligation reaction mix to the sample tube, shake and mix, centrifuge briefly to collect the reaction solution to the bottom of the tube, and immediately put the PCR tube into the PCR instrument, set the program as follows and run: (heat cover temperature: 50°C)

[0061] temperature time 20℃ ∞ 20℃ 15min 4℃ ∞

[0062] 4) After the run is completed, take out the PCR tube, centrifuge briefly, and place on an ice box for later use.

[0063] D. Purification of ligation products

[0064] 1) It is recommended to use KAPA HyperPure Beads (Roche). Take 88 μL of purified magnetic beads and add them to the PCR tube of the connected product. Perform magnetic bead purification according to the instructions of the purification kit.

[0065] 2) The purified product was eluted with 22 μL of 10 mM Tris-HCl, pH 8.0 solution that had been equilibrated to room temperature. 20 μL of the supernatant was transferred to a new PCR tube without touching the magnetic beads. The tube was placed on ice for later use.

[0066] E. Library amplification (PCR1)

[0067] 1) Add 25 μL of PCR amplification reaction solution 1 and 5 μL of PCR amplification primer (including sample tag sequence) to each sample, mix well, centrifuge briefly to collect the reaction solution to the bottom of the tube, and immediately put the PCR tube into the PCR instrument, set the program as follows and run: (heat cover temperature: 105°C)

[0068]

[0069] 2) After the run is complete, remove the PCR tube, centrifuge briefly, and place on ice for later use.

[0070] F. Purification of library amplification products

[0071] 1) It is recommended to use KAPA HyperPure Beads (Roche). Take 45 μL of purified magnetic beads and add them to a PCR tube. Perform magnetic bead purification according to the instructions of the purification kit.

[0072] 2) The purified product was eluted with 32 μL 10 mM Tris-HCl, pH 8.0 solution, and 30 μL of supernatant was transferred to a new PCR tube without touching the magnetic beads. The tube was placed on ice for later use.

[0073] G. Library quality control

[0074] 1) Library concentration quantification. Use nucleic acid quantification kit The dsDNA HS Assay Kit and its supporting instruments determine the concentration of the sample DNA library, and the calculated total library concentration must be ≥500ng.

[0075] 2) Take the sample DNA library and use the capillary electrophoresis reagent High Sensitivity D5000 Reagents and supporting instruments to perform fragment quality control. The average fragment length of the library is between 200-600bp, and there are no obvious small and large fragment peaks.

[0076] 3. Library capture

[0077] A. Hybridization

[0078] 1) Prepare the amplified DNA library. Use a 1.5mL centrifuge tube to mix 2-8 libraries with different PCR amplification primers (including sample tag sequences). The hybridization input volume for each library is 500ng. If the volume of the pool is greater than or equal to 45μL, proceed with the experiment directly; if the volume of the pool is less than 45μL, use 10mM Tris-HCl, PH8.0 solution to make up to 45μL for the experiment.

[0079] 2) Add 20 μL of DNA blocking solution that has been equilibrated to room temperature and mixed and centrifuged into the pooled library, vortex to mix, and centrifuge briefly.

[0080] 3) Add 2 times the volume of magnetic beads (recommended to use KAPA HyperPure Beads (Roche)) that have been equilibrated to room temperature and mixed (pooling volume + 20 μL DNA blocking solution), mix well, centrifuge briefly, and incubate at room temperature for 10 minutes.

[0081] 4) Centrifuge briefly and place the sample tube on a magnetic rack. After the solution is clear, carefully aspirate and discard the supernatant to avoid aspirating the magnetic beads.

[0082] 5) Keep the sample tube on the magnetic rack, add 1000 μL of freshly prepared 80% ethanol to the 1.5 mL tube, let it stand at room temperature for 30 seconds, and carefully aspirate and discard the supernatant to avoid aspirating the magnetic beads.

[0083] 6) Centrifuge briefly, place the sample tube back on the magnetic rack, and let it stand for 30 seconds.

[0084] 7) Use a 20 μL pipette tip to aspirate and discard the remaining ethanol solution.

[0085] 8) Place a 1.5 mL centrifuge tube on a magnetic rack, open the lid and dry the magnetic beads at room temperature until no ethanol remains (make sure the magnetic beads do not dry out, otherwise it will affect the elution efficiency).

[0086] 9) Add 13.4 μL of blocking sequence that has been equilibrated to room temperature, remove the sample tube from the magnetic stand, vortex to fully resuspend the magnetic beads, and centrifuge briefly.

[0087] 10) Mix the thawed reagents by vortexing, centrifuge briefly to collect the mixture to the bottom of the tube, prepare the hybridization mixture according to the number of hybridization reactions according to the table below, vortex to mix, and centrifuge briefly to collect the mixture to the bottom of the tube.

[0088] Component name Volume (μL) Hybridization Buffer 1 28 Hybridization Buffer 2 12 Nuclease-free water 3 total 43

[0089] 11) Add 43 μL of hybridization mixture to the resuspended sample tube, vortex to mix, centrifuge briefly, and incubate at room temperature for 2 minutes.

[0090] 12) Place the sample tube on a magnetic rack to adsorb the magnetic beads. After the solution is clarified, transfer 56.4 μL of the supernatant to a PCR tube containing 4 μL of capture probe. Vortex thoroughly to mix. After a brief centrifugation, place the PCR tube in a thermal cycler and set the program as follows and run: (heat cover temperature: 105°C).

[0091] step temperature time transsexual 95℃ 5min Hybridization 55℃ 16-20h

[0092] B. Capture

[0093] 1) Prepare washing buffer: Prepare the amount of washing buffer required for each pool according to the table below.

[0094]

[0095]

[0096] 2) Washing of capture magnetic beads

[0097] i. Vortex the capture beads that have been equilibrated to room temperature to mix thoroughly.

[0098] ii. Pipette n*50μL capture magnetic beads into a new 1.5mL centrifuge tube, where n represents the number of hybridization capture reactions. Each 1.5mL centrifuge tube can perform up to 6 reactions for washing capture magnetic beads.

[0099] iii. Place the centrifuge tube on a magnetic rack and carefully discard the supernatant after the solution becomes clear, avoiding the magnetic beads.

[0100] iv. Keep 1.5 centrifuge tube on the magnetic rack, add n*100μL 1X magnetic bead wash buffer, vortex to mix and centrifuge briefly, place on the magnetic rack until the solution is clear, and discard the supernatant with a pipette.

[0101] v. Repeat the previous step and rinse twice in total using 1X Magnetic Bead Wash Buffer.

[0102] vi. Add n*50μL 1X magnetic bead wash buffer, remove the tube, vortex to mix, and dispense all the contents into new 0.2mL PCR tubes at 50μL / rxn, and place on the magnetic rack until the solution is clear.

[0103] vii. Carefully discard the supernatant to avoid aspirating the magnetic beads. The washed capture beads can be used to bind the DNA library.

[0104] 3) Magnetic bead binding

[0105] i. Keep the PCR machine running according to the following settings (heat cover temperature: 105°C):

[0106] step temperature time capture 55℃ ∞

[0107] ii. Transfer 60.4 μL of hybridization product to the freshly cleaned magnetic bead tube, vortex to mix, resuspend the captured magnetic beads, centrifuge briefly, and incubate at 55°C for 15 min.

[0108] iii. After the incubation, take out the centrifuge tube, centrifuge briefly, and keep the PCR tube on the PCR instrument.

[0109] iv. Add 100 μL of preheated 1X wash buffer 2 to the magnetic bead capture system, vortex to mix, centrifuge briefly, place on a magnetic stand, wait for the solution to become clear, and carefully discard the supernatant.

[0110] v. Keep the PCR tube on the PCR instrument, add 200 μL of preheated 1X Wash Buffer 1, vortex to mix, centrifuge briefly, and incubate at 55°C for 5 min.

[0111] vi. After the incubation, remove the PCR tube from the PCR instrument, centrifuge briefly and place it on a magnetic rack to adsorb the magnetic beads. After the solution is clear, carefully aspirate and discard the supernatant to avoid aspirating the magnetic beads.

[0112] vii. Repeat steps v. and vi. once.

[0113] viii. At room temperature, add 200 μL of 1X Wash Buffer 2 to the PCR tube, vortex to mix, centrifuge briefly, incubate at room temperature for 1 min, and place on a magnetic rack until the solution is clear. Carefully aspirate and discard the supernatant to avoid aspirating the magnetic beads.

[0114] ix. At room temperature, add 200 μL of room temperature 1X Wash Buffer 3 to the PCR tube, vortex to mix, centrifuge briefly, transfer the capture system to a new PCR tube, incubate at room temperature for 1 min, place on a magnetic rack until the solution is clear, and carefully aspirate the supernatant to avoid aspirating the magnetic beads.

[0115] x. At room temperature, add 200 μL of 1X Wash Buffer 4 to the PCR tube, vortex to mix, incubate at room temperature for 1 min, and place on a magnetic rack until the solution is clear. Carefully aspirate and discard the supernatant to avoid aspirating the magnetic beads.

[0116] xi. Centrifuge briefly, place the sample tube back on the magnetic stand, let it stand for 30 seconds, and discard the remaining solution with a 20μL pipette tip.

[0117] xii. Remove the PCR tube from the magnetic rack, add 20 μL of nuclease-free water, vortex to mix, resuspend the magnetic beads, centrifuge briefly, and incubate at room temperature for 2 minutes before proceeding with subsequent experiments.

[0118] C. Capture Library Amplification

[0119] 1) Add 5 μL of PCR primer 2 and 25 μL of PCR reaction solution to each sample, vortex, mix thoroughly and centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0120] 2) Place the PCR tube into the PCR instrument, set the program according to the table below and run:

[0121]

[0122] Note: 2-5 DNA libraries form a pool, and 12 cycles are recommended; 6-8 DNA libraries form a pool, and 11 cycles are recommended.

[0123] D. Library Purification

[0124] 1) Place the amplified product on a magnetic rack until the solution becomes clear.

[0125] 2) Carefully transfer 50 μL of supernatant containing the enriched library after PCR amplification to a new PCR tube and discard the capture beads.

[0126] 3) Vortex the purified magnetic beads (KAPA HyperPure Beads (Roche) is recommended), mix thoroughly, add 70 μL of purified magnetic beads to the PCR product, and purify the magnetic beads according to the purification instructions of the kit.

[0127] 4) Elute the purified product with 22 μL of 10 mM Tris-HCl, pH 8.0, which has been equilibrated to room temperature. Pipette 20 μL into a new PCR tube, making sure not to touch the magnetic beads. Place on ice for later use.

[0128] E. Capture Library Quality Control

[0129] 1) Use nucleic acid quantification kit The dsDNAHS Assay Kit and its supporting instruments measure the concentration of sample capture library.

[0130] 2) Take the captured library and use capillary electrophoresis reagent High Sensitivity D5000 Reagents and supporting instruments to perform library fragment quality control. The average fragment length of the library is between 200-600bp, and there are no obvious small and large fragment peaks.

[0131] F. Sequencing

[0132] The sequencing library was sequenced using the gene sequencer NextSeq 550Dx or Novasek 6000 and the matching sequencing reagents according to the manufacturer's instructions to generate sequencing data.

[0133] G. Data Analysis

[0134] Use analysis software to analyze the original offline data. The bioinformatics analysis process can be divided into the following steps:

[0135] 1) Data preprocessing: Use software to analyze the data sequencing quality parameter Q30 base ratio. If the Q30 ratio is ≥60%, the quality control passes; otherwise, the quality control fails. Then use Illumina's software to convert the BCL file generated by NextSeq550Dx / Novaseq 6000 sequencing into a Fastq file. Then use Trimmomatic-0.36 software to remove the adapter sequences and low-quality base fragments introduced during library construction.

[0136] 2) Data alignment: Remove low-quality bases and adapter sequences from the original offline data, perform base quality statistics on the filtered data, read the sequencing fragments back to the reference genome and count the read number, alignment rate, sequencing depth and coverage, and detect variants.

[0137] 3) Data quality control: The sample sequencing quality is determined based on the sample Q30 base ratio, the ratio of sequence alignment to the reference genome, the average sequencing depth of the target region and other parameters. If Q30 ≥ 75%, the ratio of sequence alignment to the reference genome ≥ 90%, and the average sequencing depth ≥ 300X, the sample sequencing data quality control passes; otherwise, it fails, the test is judged to have failed, and resequencing is required.

[0138] 4) Mutation analysis: SNV / Indels mutation detection was performed using the detection software Vardict V1.8.2-2 and MuTect2V4.0.12.0; MSI detection was performed using the software MSIsensor2; and CNV detection was performed using the software cnvkit V0.9.6. The analysis parameter settings required sequence alignment quality ≥ 20, base quality ≥ 30, and the rest were default settings.

[0139] 5) Mutation annotation: The software ANNOVAR V20160425snpEff V5.0C was used to annotate SNV / Indels and gene fusions in HGVS format. The COSMIC database (v91), ClinVar database (v20191112), dbSNP database (v145), 1000Genomes database (v201508), and ExAC database (v0.3) were used for annotation.

[0140] 6) After the above processing, if the mutation meets the quality requirements, if the mutation abundance of SNV / Indels is ≥0.5%, it is judged as positive; otherwise, it is negative; if the MSI score is ≥10%, it is judged as MSI-H; otherwise, it is MSS (microsatellite stable); if the CNV CN is ≥3.25, it is judged as positive, otherwise, it is negative.

[0141] Example 3: Performance Verification

[0142] 1. Accuracy: 28 samples were tested, and the types of variation included single nucleotide variation (SNV), insertion and deletion (Indel), copy number variation (CNV) and microsatellite instability (MSI). SNV, Indel, and CNV were verified using digital PCR or next-generation sequencing (NGS), and MSI was verified using multiplex fluorescence PCR-capillary electrophoresis or NGS. The results showed that the consistency of the test results with the reference method was 98.6%. The test results are as follows:

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] 2. Repeatability: 3 repeatability references were tested, and the types of variation included SNV, Indel, CNV and MSI. The intra-batch repetitions were repeated 3 times, the inter-batch repetitions were repeated within 2 days, and the inter-operator repetitions were performed by 2 operators. The results showed that all target variations were detected, and the variation CV was ≤15%. The test results are as follows:

[0150]

[0151]

[0152]

[0153]

[0154] 3. Minimum detection limit: Three samples were tested, and the variant types included SNV, Indel, CNV and MSI. The test was repeated 10 times. The results showed that the detection rate of each variant type was 100%.

[0155]

[0156] The above embodiments are only preferred embodiments of the present invention and are not any formal or substantial limitations of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a biomarker in the preparation of a product for detecting renal cancer gene mutation, characterized in that: The biomarkers include the following genes: ABCB1, BRIP1, EGFR, FH, MITF, PARP1, SETD2, VHL, ALK, CDK12, ERCC2, FLCN, MLH1, PBRM1, SMARCA4, MET, ARID1A, CDK4, ERCC3, F GFR4, MRE11, PIK3CA, SMARCB1, SDHA, ARID1B, CDK6, ERCC4, KDM5C, MSH2, PMS1, TCEB1, SDHC, ATM, CDKN2A, ERCC5, KDM6A, MSH6, PMS2, TERT, SDHD, ATR, CHEK1, EWSR1, KEAP1, MTOR, POLE, TFE3, BAP1, CHEK2, FANCA, KMT2A, NBN, PTEN, TP53, BLM, CSMD2, FANCC , KMT2D, NF2, RAD51, TSC1, BRCA1, CSMD3, FAT1, KRAS, NFE2L2, RECQL4, TSC2, BRCA2, DNMT3A, FGFR3, MDM2, PALB2, SDHB, UGT1A1.

2. The use according to claim 1, characterized in that: The genetic variations include single nucleotide variations (SNVs), insertions and deletions (Indels), copy number variations (CNVs), microsatellite instability (MSI) and tumor mutation burden (TMB).

3. The use according to claim 1 or 2, characterized in that: The product for detecting renal cancer gene mutation includes a kit.

4. A DNA probe set for detecting the biomarker according to claim 1, characterized in that: The DNA probe set includes DNA probes with sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

272.

5. A detection reagent comprising the DNA probe set according to claim 4.

6. The detection reagent according to claim 5, characterized in that Also included is a hybridization reaction solution.

7. Use of the detection reagent according to claim 5 or 6 in the preparation of a kit for detecting renal cancer gene mutation.

8. A kit for molecular typing diagnosis of renal cancer, characterized in that: Comprising the detection reagent according to claim 5 or 6.

9. The kit according to claim 8, characterized in that Also included are DNA reagents used to extract samples.

10. The kit according to claim 9, characterized in that The sample is renal cancer tumor tissue.

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