Ovarian cancer targeted medication related gene NGS detection kit and application

Abstract

The invention relates to an ovarian cancer targeted medication related gene NGS detection kit and application thereof, and the kit comprises a probe group used for detecting ovarian cancer targeted medication related gene variation conditions and corresponding reagents based on a next generation sequencing technology. By means of a hybrid capture method, variation site conditions of 12 genes (BRAF, NTRK1, NTRK2, NTRK3, RET, MLH1, MSH2, MSH6, PMS2, BRCA1, BRCA2 and KRAS) related to important targeted medication of ovarian cancer can be detected at a time. The probe used in the invention has the advantages of wide coverage, high sequencing depth and strong specificity to ovarian cancer patients, and compared with polygene solid tumor large panel detection, the resource waste caused by irrelevant gene detection is reduced, and the reduction of the detection cost and the improvement of the detection efficiency are realized.

Description

Technical Field

[0001] This invention relates to an NGS detection kit for genes related to targeted drug therapy for ovarian cancer and its application, belonging to the field of multi-gene detection technology. Background Technology

[0002] Ovarian cancer is currently one of the three major malignant tumors threatening women's reproductive health (the others being cervical cancer and endometrial cancer). Due to its insidious early symptoms and the difficulty in treating advanced stages, it is known as the "silent killer of women's health," and its disease burden has become a significant challenge in the prevention and treatment of female malignant tumors. In terms of incidence, ovarian cancer ranks sixth among female malignant tumors. In recent years, influenced by population aging, changes in reproductive patterns (late marriage and childbirth, fewer pregnancies and less breastfeeding), exposure to environmental endocrine disruptors (such as plastic products and certain ingredients in cosmetics), and family genetic factors (such as BRCA1 / 2 gene mutations), its incidence has been slowly increasing, placing continuous pressure on women's health protection systems.

[0003] In terms of mortality, ovarian cancer is far more malignant than breast cancer and cervical cancer. Due to the long treatment cycle and high cost of drugs in advanced stages (such as targeted drugs, the annual treatment cost can reach 100,000 to 300,000 yuan), it not only directly lowers the overall life expectancy of female residents, but also places a heavy economic burden on patients' families. At the same time, the loss of fertility caused by the disease, the decline in quality of life caused by ascites, and the psychological trauma caused by recurrence further create a significant emotional and social burden.

[0004] With advancements in medical technology, ovarian cancer treatment has evolved from the traditional single-mode of "surgery + platinum-based chemotherapy" to a comprehensive approach centered on "precision targeted therapy and multidisciplinary collaborative management." Targeted therapy precisely intervenes in ovarian cancer cells by targeting specific molecular markers (such as BRCA1 / 2 gene mutations), effectively inhibiting tumor growth while minimizing damage to normal ovarian tissue and the hematopoietic system. Compared to traditional chemotherapy, it offers significant advantages in "better efficacy and milder side effects"—for example, PARP inhibitors can reduce the incidence of nausea and vomiting in BRCA-mutated patients by more than 50% and decrease the risk of bone marrow suppression by 40%. The core prerequisite for achieving targeted therapy is to clearly identify the molecular characteristics of the patient's tumor through molecular testing for targeted drug administration. Only by accurately identifying targetable molecular abnormalities can doctors match patients with corresponding targeted drugs, avoiding ineffective treatment due to "blind drug use."

[0005] From a clinical perspective, molecular testing for targeted therapy is crucial throughout several key stages of ovarian cancer treatment. In the treatment planning phase, molecular testing results are a core basis for selecting patients suitable for targeted therapy. For example, approximately 20%-25% of ovarian cancer patients currently have BRCA1 / 2 gene mutations. These patients, when treated with PARP inhibitors after platinum-based chemotherapy, can extend progression-free survival by 12-18 months and reduce the risk of disease progression by more than 60%. Without molecular testing, these patients may miss out on targeted treatment options, leading to rapid disease progression.

[0006] Molecular testing for targeted therapy also plays an irreplaceable role in the treatment process. Some patients, after 1-2 years of PARP inhibitor treatment, may experience a decline in treatment efficacy or disease relapse due to drug resistance mutations in tumor cells (such as BRCA reversion mutations or PI3K / AKT pathway activating mutations). In such cases, timely detection of new drug resistance mutations through molecular testing (such as liquid biopsy to detect circulating tumor DNA) allows doctors to quickly adjust the treatment plan, such as switching to anti-angiogenic drugs (bevacizumab) combined with chemotherapy, or trying novel targeted drugs (such as ATR inhibitors), thereby slowing disease progression and prolonging patient survival.

[0007] Currently, authoritative domestic and international guidelines for the diagnosis and treatment of ovarian cancer (such as the NCCN Ovarian Cancer Guidelines and the Chinese Society of Clinical Oncology (CSCO) Ovarian Cancer Guidelines) have clearly recommended that all newly diagnosed advanced (stage III / IV) ovarian cancer patients routinely undergo BRCA1 / 2 gene testing and HRD status assessment before treatment; for patients with recurrent ovarian cancer, it is recommended to perform molecular testing again to identify the cause of drug resistance and guide adjustments to subsequent treatment plans. This recommendation, from an industry standard perspective, further establishes the core position of molecular testing for targeted drug therapy in the precision treatment of ovarian cancer. Summary of the Invention

[0008] On the one hand, the purpose of this invention is to provide a probe set for detecting gene mutations related to targeted therapy for ovarian cancer.

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] A probe set for detecting gene mutations related to targeted therapy for ovarian cancer, comprising nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.234, wherein the ovarian cancer targeted therapy-related genes include BRAF, NTRK1, NTRK2, NTRK3, RET, MLH1, MSH2, MSH6, PMS2, BRCA1, BRCA2, and KRAS.

[0011] The multi-gene panel for detecting genes related to targeted therapy for ovarian cancer provided by this invention includes genes from multiple databases, relevant guidelines, and expert consensus, covering genes related to targeted therapy for ovarian cancer.

[0012] On the other hand, another objective of this invention is to provide an NGS detection kit and method for using genes related to targeted therapy for ovarian cancer. The provided probes cover the full coding region sequences of all involved genes; fusion genes additionally cover intron sequences: BRAF covers introns 8-10, RET covers intron 11, and NTRK1 covers introns 8-14. This invention improves the detection rate of fusion genes by detecting their introns, avoiding missed diagnoses due to insufficient detection range.

[0013] A NGS detection kit for genes related to targeted therapy in ovarian cancer includes a capture probe set, the capture probe set having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.234, wherein the 5' or 3' end of the capture probe is labeled with biotin. Preferably, the detection kit as described above further includes a genomic DNA extraction reagent, a DNA library construction reagent, and a library capture reagent.

[0014] Furthermore, the genomic DNA extraction reagent can be the Novizan FFPE sample DNA extraction kit, the DNA library construction reagent can be the Twist Library Preparation EF Kit 1, 2.0 from Twist Company, and the library capture reagent can be the Twist Liquid Hybridization Capture Kit from Twist Company.

[0015] The test kits described above also include the instruments or reagents required for sequencing (such as the Illumina high-throughput sequencing platform and human genome sequences).

[0016] A method for detecting gene mutations related to targeted therapy for ovarian cancer, wherein the method is not for diagnostic / therapeutic purposes, and includes the following steps:

[0017] S1. Genomic DNA was extracted from the ex vivo tumor tissue of the ovarian cancer subject. The genomic DNA was fragmented, end-repaired, dA-tailed, and ligated with adapters. The ligated products were then amplified to obtain a DNA library.

[0018] S2. The DNA library was hybridized with probes whose nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.234. All probes were labeled with biotin. After hybridization, streptavidin magnetic beads were used for capture. After purification and mixing, the sequencing library of genes related to ovarian cancer targeted drugs was obtained.

[0019] S3. Perform next-generation sequencing and data analysis on the sequencing library to obtain the variation information of genes related to targeted drug therapy for ovarian cancer.

[0020] In the detection method described above, preferably, in S1, the product with added dA tail is ligated to the Twist universal adapter; after purification, the purified ligation adapter product is amplified by PCR using UDI primers, and the PCR reaction product is purified again to obtain a DNA library.

[0021] In S2, the purified DNA library is concentrated and hybridized with the probe set shown in SEQ ID NO.1-SEQ ID NO.234. The DNA is captured using streptavidin magnetic beads, and the captured product is amplified using primers corresponding to the universal Twist sequence. After amplification, the DNA is purified again to obtain the sequencing library.

[0022] In the detection method described above, preferably, in steps S1 and S2, the amplification program is as follows: pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; extension: 72℃ for 1min, followed by holding at 4℃.

[0023] As described above, preferably, in S2, the hybridization system includes: a purified DNA library, a linker blocking agent, a blocking solution, and a probe set. The hybridization reaction program is 95°C for 5 min, 60°C for 2 h to 16 h, and then incubated at 60°C.

[0024] The sequencing library obtained using the detection method described above was sequenced using Illumina's Nextseq2000. After quality control of the sequenced data, bioinformatics analysis was performed using software such as BWA / GATK to identify gene mutation types such as point mutations, insertions, deletions, and fusions, thereby providing molecular evidence for matching corresponding targeted drugs in clinical practice.

[0025] The reference genome version used in this invention is grch37 / hg19.

[0026] (III) Beneficial Effects

[0027] The beneficial effects of this invention are:

[0028] The probe set provided by this invention for detecting gene mutations related to targeted therapy for ovarian cancer is based on next-generation sequencing and uses a hybridization capture method. It can simultaneously detect mutations in all exon regions of 12 genes in the sample, providing broad coverage and detecting mutations in all exon regions. Probes are designed in intron regions where fusion breakpoints are prone to occur, such as BRAF (introns 8-10), RET (intron 11), and NTRK1 (introns 8-14) in fusion genes. This effectively detects gene fusion mutations, achieving comprehensive coverage of unknown and fusion mutations and avoiding missed detections.

[0029] The ovarian cancer targeted drug-related gene NGS detection kit provided by this invention can detect the mutation sites of 12 genes (BRAF, NTRK1 / 2 / 3, RET, MLH1, MSH2, MSH6, PMS2, BRCA1 / 2, KRAS) with clear drug targets in ovarian cancer in one go. It has high specificity for ovarian cancer patients. Compared with large panel detection of multi-gene solid tumors, it reduces the waste of resources caused by irrelevant gene detection, realizes the reduction of detection costs and the improvement of detection efficiency, and achieves cost reduction and efficiency improvement.

[0030] The NGS detection kit for ovarian cancer targeted drug-related genes provided by this invention has a 100% concordance rate in detecting clinical samples and verifying the results of mutation sites by ARMS-PCR. It can accurately identify pathogenic mutations in genes such as BRCA2 and match corresponding targeted drugs (such as olaparib, niraparib, etc.), providing a core basis for precise targeted treatment of ovarian cancer patients. Attached Figure Description

[0031] Figure 1 To construct a good document library for fragment analysis results. Detailed Implementation

[0032] This invention provides a set of detection panels, kits, and applications for ovarian cancer-related genes. The kit contains relevant probes and corresponding reagents. Based on next-generation sequencing technology, it utilizes hybridization capture to simultaneously detect important ovarian cancer-related genes for targeted drug therapy, including BRAF, NTRK1, NTRK2, NTRK3, RET, MLH1, MSH2, MSH6, PMS2, BRCA1, BRCA2, and KRAS. The probes designed in this invention have broad coverage and high sequencing depth, enabling the detection of all variants in the designed gene probe capture regions. Finally, through sequencing and bioinformatics analysis, all variants of ovarian cancer-related genes for targeted drug therapy can be obtained, and the corresponding target drug information for each variant can be provided.

[0033] According to the 2017 Chinese Expert Consensus on Next-Generation Sequencing in Clinical Molecular Pathology Laboratories and the 2017 AMP / ASCO / CAP Standards and Guidelines for the Interpretation and Reporting of Tumor Cell Variations, the interpretation and annotation of the clinical significance of tumor somatic cell variations are divided into the following four levels of evidence:

[0034] Category A: Targets approved for drug use by the U.S. Food and Drug Administration (FDA) or the National Medical Products Administration (NMPA); variants with clear diagnostic / therapeutic / prognostic significance as included in domestic and international treatment guidelines.

[0035] Category B: Variants that have not yet been included in treatment guidelines but have been incorporated into expert consensus in the field.

[0036] Category C: Genetic variants approved by the FDA or NMPA for predictable treatment outcomes in other cancers, or variant sites in ongoing clinical trials.

[0037] Grade D: Preclinical studies or a small number of case reports, without expert consensus.

[0038] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1: Design and Synthesis of Gene Probes Related to Targeted Drug Therapy for Ovarian Cancer

[0040] I. Screening of genes related to targeted therapy for ovarian cancer

[0041] By manually reading the National Comprehensive Cancer Network (NCCN) guidelines, the CSCO guidelines organized, screened, and summarized the information on genes related to targeted therapy in the guidelines, as detailed in Table 1.

[0042] Table 1 lists 12 genes and related targeted drugs associated with ovarian cancer targeted therapy.

[0043]

[0044]

[0045]

[0046] II. Probe Design and Synthesis

[0047] Hybridization capture probe sequences were designed based on the relevant genes in Table 1, see SEQ ID NO.1-SEQ ID NO.234. The probes cover the full coding region sequences of all the above genes and additionally cover the intron sequences of the fusion genes: BRAF covers introns 8-10, RET covers intron 11, and NTRK1 covers introns 8-14.

[0048] The probe sequences targeting the NTRK1 gene are shown in SEQ ID NO. 1-36, the probe sequences targeting the MSH2 gene are shown in SEQ ID NO. 37-45, the probe sequences targeting the MSH6 gene are shown in SEQ ID NO. 46-58, the probe sequences targeting the MLH1 gene are shown in SEQ ID NO. 59-68, the probe sequences targeting the PMS2 gene are shown in SEQ ID NO. 69-78, the probe sequences targeting the BRAF gene are shown in SEQ ID NO. 79-117, the probe sequences targeting the NTRK2 gene are shown in SEQ ID NO. 118-128, the probe sequences targeting the RET gene are shown in SEQ ID NO. 129-161, the probe sequences targeting the KRAS gene are shown in SEQ ID NO. 162-164, the probe sequences targeting the BRCA2 gene are shown in SEQ ID NO. 165-199, the probe sequences targeting the NTRK3 gene are shown in SEQ ID NO. 200-211, and the probe sequences targeting the BRCA1 gene are shown in SEQ ID NO. 212-234.

[0049] All probes can be synthesized by the biotechnology company, and during synthesis, each probe is labeled with biotin at its 5' end.

[0050] Example 2

[0051] This embodiment is a workflow for NGS detection of ovarian cancer targeted drug-related genes based on the probe set provided in Example 1. It includes: first, establishing a DNA library; the library construction process includes extraction of genomic DNA (gDNA), DNA fragmentation, end repair and dA tailing, adapter ligation, PCR enrichment (using universal sequencing primers adapted to the sequencer), and obtaining a DNA library; then, hybridizing the DNA library with the probe for library capture; after the probe captures the target fragment, specific amplification is performed on the target fragment using amplification primers corresponding to the adapter; after purification, the obtained file is sequenced and subjected to bioinformatics analysis. The specific operations are as follows:

[0052] 1. Genomic DNA extraction

[0053] Ovarian cancer tumor tissue was taken from the subjects, prepared into paraffin blocks, sectioned, and genomic DNA was extracted using the Novizan FFPE Sample DNA Extraction Kit.

[0054] 2. Constructing a DNA library

[0055] The genomic DNA obtained in step 1 above was used to construct a DNA library using Twist's Twist Library Preparation EF Kit 1, 2.0 (lot number 34000002908). The specific library construction process is as follows:

[0056] 1) DNA fragmentation, end repair, and dA tailing

[0057] The reaction system consisted of: X μL (200 ng) genomic DNA, 4 μL Frag / AT Buffer, 6 μL Frag / AT Enzymes, 40-X μL nucleic acid-free water, and a total of 50 μL. After preparation, the mixture was thoroughly mixed by pipetting and incubated in a PCR instrument for DNA fragmentation, end repair, and dA tailing. The specific program was: 37℃ for 10 min, 65℃ for 30 min, and 4℃ Hold to obtain the DNA fragmentation, end repair, and dA tailing products.

[0058] 2) Connect the Twist universal connector

[0059] The dA tailing product obtained above was prepared into a reaction system with a total volume of 75 μL: 50 μL of DNA fragmentation, end repair, and dA tailing product, 5 μL of sequencing adapters (Twist universal Adapters), and 20 μL of Ligation Master Mix, for a total volume of 75 μL. After preparing the system, it was mixed thoroughly by pipetting and then incubated in a PCR instrument. The adapter ligation incubation program was: 20 °C for 15 min, followed by hold at 4 °C.

[0060] 3) Purification

[0061] a. Add 60 μL of DNA Purification Beads (0.8 times the sample volume) equilibrated to room temperature and thoroughly mixed to the sample from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min;

[0062] b. Instantaneous detachment, place on the magnetic rack for 5 minutes, discard the supernatant;

[0063] c. Wash with 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0064] d. Repeat step c;

[0065] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 3-5 minutes until the alcohol has completely evaporated;

[0066] f. Add 18 μL of nuclease-free water, mix well by pipetting, and incubate at room temperature for 5 min to obtain adapter-ligated DNA;

[0067] g. Instantly separate the tube and place it on a magnetic rack for 2 minutes until the liquid is clear. Take 15 μL of the supernatant and transfer it to a new PCR tube for PCR amplification. The PCR amplification reaction system is as follows: 15 μL of Adapter Ligated DNA, 25 μL of Equinox Library Amp Mix (2X), and 10 μL of UDI primers (Twist Bioscience), for a total volume of 50 μL.

[0068] After mixing the above-mentioned system by pipetting, incubate it in a PCR instrument. The PCR program is as follows: pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s, 8 cycles; extension: 72℃ for 1min, then incubate at 4℃.

[0069] 4) Purify again

[0070] a. Add 50 μL of DNA Purification Beads equilibrated to room temperature and thoroughly mixed to the PCR product from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min;

[0071] b. Instantly detach the sample, place it on a magnetic rack for 5 minutes, and discard the supernatant;

[0072] c. Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0073] d. Repeat step c;

[0074] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 3-5 minutes until the alcohol has completely evaporated;

[0075] f. Add 24 μL of nuclease-free water, mix well by pipetting, and let stand at room temperature for 5 min;

[0076] g. Instantly centrifuge, place on a magnetic rack for 2 minutes until the liquid is clear, take 22 μL of supernatant into a new centrifuge tube to obtain the DNA library, and vortex to mix;

[0077] Take 2 μL of the above DNA library, dilute it 10-fold, and quantify it using the Qubit dsDNA HS Assay Kit. Record the library concentration. Take 1 μL of the DNA library diluted 10-fold in the previous step and determine the library fragment length using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length is approximately between 220-320 bp.

[0078] 3. Library pooling, hybridization capture, PCR amplification, and purification

[0079] The library capture reagent used was the liquid phase hybridization capture kit from Twist, and the specific operation was as follows:

[0080] Library Pooling (1-8 samples)

[0081] a. The total amount of each library pool is 1.5-4.0 μg, and the pooling volume of each library is calculated based on the concentration of each library;

[0082] b. Mix the libraries in a 1.5 mL centrifuge tube, gently shake, and centrifuge.

[0083] 1) Library hybridization and capture

[0084] a. Library concentration, the system is as follows: pooled library X (the volume of 1-8 samples added together), 8 μL of universal blockers, 5 μL of blocking solution, and 4 μL of probes (nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.234).

[0085] The reaction tube was dried completely at 45°C in a vacuum dryer.

[0086] 2) Library hybridization

[0087] a. After drying, add 20 μL of the Fast Hybridization Mix (do not allow it to return to room temperature) to a centrifuge tube, gently tap to mix, let stand at room temperature for 5 min, and then transfer to a new PCR tube;

[0088] b. Add 30 μL of Hyb Enhancer to the surface of the liquid in the PCR tube, briefly remove air bubbles, and then place it in the PCR instrument. The amplification program for the hybridization reaction system is: 95℃ for 5 min, 60℃ for 2 h, and 60℃ Hold.

[0089] 3) Document capture

[0090] a. Take 100 μL of streptavidin binding beads into a 1.5 mL tube (after being placed at room temperature for 30 min and shaken to mix).

[0091] b. Add 200 μL of Fast Binding Buffer, mix well, place on a magnetic rack for 1 min, discard the supernatant, and repeat this washing step 2 times for a total of 3 times; then add 200 μL of Fast Binding Buffer, vortex to resuspend and mix thoroughly.

[0092] c. After library hybridization is complete, open the PCR tube cap on the PCR instrument, quickly transfer all the hybridization solution to the magnetic bead solution, and immediately mix by pipetting.

[0093] d. Incubate the centrifuge tubes on a centrifuge at room temperature and 1200 rpm for 30 min. Centrifuge the centrifuge tubes rapidly on a magnetic rack for 1 min, then discard the supernatant.

[0094] e. Add 200 μL of Fast Wash Buffer 1 preheated at 66°C (do not remove from the metal bath), mix gently, and then quickly incubate at 66°C for 5 min.

[0095] f. Place on a magnetic rack for 1 min, remove the supernatant, add 200 μL of preheated Fast Wash Buffer 1, mix lightly, and then quickly incubate at 66°C for 5 min.

[0096] g. Transfer all liquid in the tube to a new 1.5 mL tube, place on a magnetic rack for 1 min, and discard the supernatant.

[0097] h. Add 200 μL of preheated Wash Buffer 2 (do not remove from the metal bath), mix gently, and then quickly incubate at 48°C for 5 min, on a magnetic rack for 1 min. Remove the supernatant and wash twice more with Wash Buffer 2, for a total of three times.

[0098] I. After detaching, place the liquid on a magnetic rack and use a 10μL pipette tip to aspirate any remaining liquid.

[0099] J. Add 45 μL H2O, mix well by pipetting, and incubate on ice (Note: If PCR is not performed immediately, store the magnetic bead mixture at -20°C).

[0100] 4) PCR amplification

[0101] The PCR amplification reaction system consisted of 22.5 μL of magnetic bead mixture, 25 μL of high-fidelity hot-start PCR premix (KAPA HIFIHotStart ReadyMix), 2.5 μL of amplification primers, and a total volume of 50 μL.

[0102] After preparing the reaction system, mix it thoroughly by pipetting and place it on a PCR instrument for reaction. The PCR reaction program is as follows: pre-denaturation: 98℃ for 45s; denaturation-annealing-extension: 98℃ for 15s, 60℃ for 30s, 72℃ for 30s, with 12 cycles; final extension: 72℃ for 1min; hold at 4℃.

[0103] 5) Purification

[0104] a. Add 90 μL of DNA Purification Beads equilibrated to room temperature and thoroughly mixed to the PCR product from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min.

[0105] b. Instantaneous detachment, place on the magnetic rack for 5 minutes, discard the supernatant;

[0106] c. Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0107] d. Repeat step c;

[0108] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 3-5 minutes until the alcohol has completely evaporated;

[0109] f. Add 34 μL of nuclease-free water, mix well by pipetting, and let stand at room temperature for 5 min;

[0110] g. Immediately separate the sample and place it on a magnetic rack for 2 minutes until the liquid becomes clear. Take 32 μL of the supernatant into a new centrifuge tube to obtain the hybridized and captured library. Shake to mix.

[0111] Take 2 μL of the amplified DNA capture library and quantify it using the Qubit dsDNAHS Assay Kit. Record the library concentration, which is approximately 1-15 ng / μL. Take 1 μL of the amplified DNA capture library and determine the library fragment length using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length is approximately 300-450 bp.

[0112] 4. Sequencing

[0113] The amplified captured DNA library obtained in step 3 above was sequenced using Illumina's Nextseq 2000 with sequencing parameters PE: 2×150. After quality control of the data, bioinformatics analysis was performed using software such as BWA / GATK, with the reference genome version being grch37 / hg19. Relevant databases such as gnomAD / Clinvar / OncoKB were annotated. Based on the 2017 Chinese Expert Consensus on Next-Generation Sequencing Detection in Clinical Molecular Pathology Laboratories and the 2017 AMP / ASCO / CAP Standards and Guidelines for the Interpretation and Reporting of Tumor Cell Variations, the interpretation and annotation of the clinical significance of tumor somatic cell variations were divided into four levels of evidence:

[0114] Category A: Targets approved for drug use by the U.S. Food and Drug Administration (FDA) or the National Medical Products Administration (NMPA); variants with clear diagnostic / therapeutic / prognostic significance as included in domestic and international treatment guidelines.

[0115] Category B: Variants that have not yet been included in treatment guidelines but have been incorporated into expert consensus in the field.

[0116] Category C: Genetic variants approved by the FDA or NMPA for predictable treatment outcomes in other cancers, or variant sites in ongoing clinical trials.

[0117] Grade D: Preclinical studies or a small number of case reports, without expert consensus.

[0118] Example 3

[0119] This embodiment uses the probe set provided in Example 1 and follows the NGS detection method in Example 2 to detect ovarian cancer targeted therapy-related genes in clinical samples. The detection method is as follows:

[0120] Two FFPE samples pathologically diagnosed as ovarian cancer were collected, one from each sample. The mutation information of the targeted drug-producing genes in these two samples was detected. Genomic DNA was first extracted from the two FFPE samples, and then libraries were constructed according to the method described in Example 1. Libraries were successfully constructed (see...). Figure 1The library was sequenced on an Illumina high-throughput sequencer with sequencing parameters PE: 2×150. After quality control of the data, bioinformatics analysis was performed using software such as BWA / GATK, with the reference genome version being grch37 / hg19. Relevant databases such as gnomAD / Clinvar / OncoKB were annotated. Based on the 2017 Chinese Expert Consensus on Next-Generation Sequencing Detection in Clinical Molecular Pathology Laboratories and the 2017 AMP / ASCO / CAP Standards and Guidelines for the Interpretation and Reporting of Tumor Cell Variations, the interpretation and annotation of the clinical significance of tumor somatic cell variations were divided into four levels of evidence. Finally, ARMS-PCR was used to validate each locus, with a 100% concordance rate. Sequencing quality is shown in Table 2. The detection results for Sample 1 are shown in Table 3, and the detection results for the other sample are shown in Table 4.

[0121] Table 2: Sequencing Quality

[0122]

[0123] Table 3: Detection results of Sample 1

[0124]

[0125] Table 4: Detection results of Sample 2

[0126]

[0127] The test results show that the probe coverage of the detection kit provided by this invention can reach 100%, and the average depth of the target region is above 500x, with quality control meeting the requirements. The test results on routine samples indicate that the probes, methods, and kits described in this invention can accurately detect mutations in genes related to targeted therapy for ovarian cancer, providing indications for related diseases.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A set of probes for detecting gene mutations related to targeted therapy for ovarian cancer, characterized in that, It includes nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.234; the ovarian cancer targeted drug-related genes include BRAF, NTRK1, NTRK2, NTRK3, RET, MLH1, MSH2, MSH6, PMS2, BRCA1, BRCA2 and KRAS.

2. A NGS detection kit for genes related to targeted therapy for ovarian cancer, characterized in that, It includes a set of capture probes, which are probes with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.234, and each probe is labeled with biotin at its 5' or 3' end.

3. The detection kit as described in claim 2, characterized in that, It also includes genomic DNA extraction reagents, DNA library construction reagents, and library capture reagents.

4. The detection kit as described in claim 3, characterized in that, Genomic DNA extraction was performed using the Novizan FFPE Sample DNA Extraction Kit; DNA library construction was performed using the Twist Library Preparation EF Kit 1, 2.0 from Twist; and library capture was performed using the Twist Liquid Hybridization Capture Kit from Twist.

5. A method for detecting gene mutations related to targeted drug therapy for ovarian cancer for non-diagnostic / therapeutic purposes, characterized in that, Includes the following steps: S1. Genomic DNA was extracted from the ex vivo tumor tissue of the ovarian cancer subject. The genomic DNA was fragmented, end-repaired, and dA tailed, and then ligated with adapters. The ligated products were amplified to obtain a DNA library. S2. The DNA library is hybridized with the probe set described in claim 1, wherein each probe in the probe set is labeled with biotin, and after hybridization, streptavidin magnetic beads are used for capture, and after purification and mixing, a sequencing library of genes related to ovarian cancer targeted drug therapy is obtained. S3. Perform next-generation sequencing and data analysis on the sequencing library to obtain the variation information of genes related to targeted drug therapy for ovarian cancer.

6. The detection method as described in claim 5, characterized in that, In S1, the product with added dA tailing is ligated to the Twist universal adapter; after purification, the purified ligation adapter product is amplified by PCR using UDI primers, and the PCR reaction product is purified again to obtain a DNA library. In S2, the purified DNA library was concentrated and hybridized with the probe set shown in SEQ ID NO.1-SEQ ID NO.

234. The DNA was captured using streptavidin magnetic beads. The captured product was amplified using primers corresponding to the universal sequence of the Twist universal adapter. After amplification, the DNA was purified again to obtain the sequencing library.

7. The detection method as described in claim 6, characterized in that, In S1 and S2, the amplification procedure is as follows: Pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; Extension: 72℃ for 1min, followed by 4℃ heat preservation.

8. The detection method as described in claim 6, characterized in that, In S2, the hybridization system includes: purified DNA library, adapter blocking agent, blocking solution and probe set. The hybridization reaction program is 95℃ for 5 min, 60℃ for 2 h to 16 h, and 60℃ for incubation.

Images