Capture probe combination, kit and application for detecting thyroid cancer-related genes

Abstract

The present invention discloses a capture probe combination for detecting thyroid cancer-related genes, including at least one of the sequences shown in SEQ ID NOs 1 to 101. A kit comprising the capture probe combination is disclosed to detect thyroid cancer-related genes using the kit. The present invention can detect 8 thyroid cancer-related genes RET, TERT, BRAF, TP53, NRAS, HRAS, KRAS, and PIK3CA at one time using a hybrid capture method. The capture probe and kit provided by the present invention have a wide coverage, a high sequencing depth, a starting amount of samples required for library construction as low as 20 ng, a short experimental process, and the advantages of high efficiency and high precision, and have great application potential and market value.

Description

Technical Field

[0001] The present invention relates to the technical field of multi-gene detection, and in particular to a capture probe combination, a kit and applications for detecting eight genes highly related to the diagnosis and treatment of thyroid cancer. Background Art

[0002] The incidence of thyroid nodules in China is about 20%-35%. Nodules are divided into benign and malignant. Benign nodules are mostly caused by thyroid infectious inflammation, autoimmune diseases and other thyroid diseases; malignant thyroid nodules are thyroid cancer, accounting for about 7% to 15% of the population with thyroid nodules. According to the origin and differentiation of the tumor, thyroid cancer is divided into: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC) and anaplastic thyroid cancer (ATC). PTC is the most common, accounting for about 85%-90% of all thyroid cancers. PTC and FTC are collectively called differentiated thyroid carcinoma (DTC). DTC has mild biological behavior and a good prognosis. ATC is extremely malignant, with a median survival time of only 7 to 10 months. The prognosis of MTC is between the two.

[0003] Different pathological types of thyroid cancer are significantly different in pathogenesis, biological behavior, histological morphology, clinical manifestations, treatment methods, and prognosis. 20%-25% of fine needle aspirations (FNA) cannot be judged as benign or malignant by cytological means, and molecular diagnosis is required to clarify the classification. According to the TBSRTC classification of malignant risks and management recommendations, molecular testing is required to help clarify the benign and malignant nature of grade III, IV, and V thyroid cancers. According to the fifth edition of the WHO Diagnosis + Prognosis of Thyroid Tumors and relevant domestic and foreign literature, BRAF, RAS, RET, TP53, PIK3CA, and TERT are auxiliary diagnostic typing to evaluate the genes that must be tested for subsequent surgical plans.

[0004] Taking BRAF and TERT mutations as an example, BRAF belongs to the RAF kinase family downstream of KRAS, and BRAF V600E accounts for more than 90% of BRAF mutations. BRAF gene mutation is currently the most common type of gene mutation in thyroid cancer. The BRAF gene encodes an intracellular serine-threonine kinase, which activates downstream targets in the MAPK / ERK signaling pathway, such as MEK, after phosphorylation. Current studies believe that BRAF gene mutations are positively correlated with the high invasiveness, lymph node metastasis rate, and recurrence rate of thyroid cancer. According to a meta-analysis study by Lee et al., 12 independent studies were included, with a total of 1,168 patients with papillary thyroid cancer. The results showed that BRAF V600E mutations were associated with the histological subtypes, extrathyroidal invasion, and advanced clinical stages of papillary thyroid cancer, but were not related to race, age, gender, and tumor size. Other studies have found that thyroid cancer with BRAF mutations often has low iodine uptake rates and poor radioactive iodine treatment effects. The false positive rate of BRAF mutations is very low, so FNA cell specimens with BRAF mutations can be highly suspected of malignant nodules. The TERT gene is located on chromosome 5 and encodes telomerase reverse transcriptase. This enzyme plays an important role in the telomere maintenance mechanism of cells. TERT promoter mutations can increase intracellular telomerase activity and prevent telomere shortening, playing an important role in cell immortality and tumorigenesis. TERT promoter mutations are more common in highly invasive FTC and PTC, and are closely associated with high-risk clinical pathological features, often indicating a poor prognosis. In papillary thyroid carcinoma, TERT promoter mutations can occur in combination with other oncogene mutations such as BRAF mutations and RAS mutations, and the invasiveness is higher and the prognosis is worse. According to the 5th edition of the WHO Classification of Endocrine and Neuroendocrine Tumors, patients with co-mutations of BRAF V600E and TERT promoter in papillary thyroid carcinoma (PTC) have a poor prognosis; patients with co-mutations of BRAF V600E and TERT promoter in anaplastic thyroid carcinoma (ATC) have a poor prognosis. According to the Guangdong expert consensus on thyroid cancer gene detection and clinical application, patients with BRAF V600E and TERT promoter co-mutations are closely related to iodine resistance in recurrent thyroid cancer, which may lead to failure of iodine treatment. Patients with differentiated thyroid cancer with BRAF and TERT can undergo total thyroidectomy and ipsilateral central lymph node dissection.

[0005] The detection and typing of genes related to thyroid cancer has practical significance for clinical and medical research, so it is necessary to develop a panel that can comprehensively and deeply cover thyroid cancer-related gene mutations.

[0006] Probe capture sequencing is one of the commonly used target gene multi-site detection schemes. This scheme designs and synthesizes effective specific probes, hybridizes with genomic DNA, captures and enriches the target region sequence, and then uses mainstream sequencing platforms for high-throughput sequencing. The probe hybridization capture efficiency and the coverage depth of the genomic region are affected by many factors. The development and verification of disease-related target gene panels and key sites rely on the study of target regions of a large number of samples. Only probes with high sequence accuracy can specifically bind to the target region sequence, greatly improving the capture efficiency of the probe and achieving the purpose of enriching the target fragment. Summary of the invention

[0007] In view of the above-mentioned problems, the purpose of the present invention is to provide a multi-gene panel for detecting thyroid cancer-related genes and a set of capture probes designed based on this gene panel, as well as a kit including the capture probes, for accurately, quickly and effectively capturing thyroid cancer-related gene mutations.

[0008] The technical solution adopted by the present invention is:

[0009] A capture probe combination for detecting thyroid cancer-related genes, the capture probe combination comprising at least one of the sequences shown in SEQ ID NOs: 1 to 101.

[0010] Preferably, the capture probe combination includes all of the sequences shown in SEQ ID NOs: 1 to 101.

[0011] The thyroid cancer-related genes detected by the capture probe combination of SEQ ID NOs: 1 to 101 include at least one of the following eight genes: RET, TERT, BRAF, TP53, NRAS, HRAS, KRAS, and PIK3CA.

[0012] Among them, BRAF, RAS, RET, TP53, PIK3CA, and TERT are auxiliary diagnostic typing to evaluate the necessary genes for subsequent surgical plans.

[0013] The designed capture probe combination covers the entire coding region sequence of the gene involved.

[0014] Another object of the present invention is to provide a kit for detecting thyroid cancer-related genes, wherein the kit comprises the capture probe combination.

[0015] The kit may also include genomic DNA extraction reagents, DNA library construction reagents, capture DNA library construction reagents, etc., and corresponding commercially available kits may be used. For example, the genomic DNA extraction reagent may use the Tiangen Blood Genome Extraction Kit, the DNA library construction reagent may use the Twist Library Preparation Enzymatic Fragmentation Kit 2.0 of Twist, and the capture DNA library construction reagent may use the liquid phase hybridization capture kit of Twist. It may also include instruments or reagents required for sequencing (such as Illumina high-throughput sequencing platform and human genome sequence).

[0016] The present invention also provides the use of a kit for detecting thyroid cancer-related genes in detecting thyroid cancer-related genes. Further, the method of the application includes detecting eight genes related to thyroid cancer and their mutations.

[0017] The present invention also provides a method for detecting thyroid cancer-related genes using the kit, the method comprising the following steps:

[0018] (1) extracting DNA from the sample to be tested;

[0019] (2) constructing a DNA library;

[0020] The construction of DNA library generally includes DNA fragmentation, end repair, A-tailing, universal adapter connection, PCR amplification, and finally purification to obtain DNA library;

[0021] (3) using a capture probe combination for detecting thyroid cancer-related genes to perform hybridization capture and PCR amplification to obtain a hybridization capture library;

[0022] (4) The library after hybridization capture is sequenced, and the sequencing data is analyzed and tested by bioinformatics to obtain the mutation status of thyroid cancer-related genes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention utilizes hybridization capture method to detect 8 genes related to thyroid cancer and their mutations. Only a trace amount of genomic DNA is needed to construct a DNA library, and the template starting amount can be as low as 20 ng. The rapid hybridization system of the kit of the present invention can achieve the efficiency of general overnight hybridization in a short hybridization time of 15 minutes, and the final library fragment size is about 300 to 450 bp, which can be compatible with various Illumina sequencing platforms for sequencing.

[0025] The capture probe designed by the present invention covers the entire coding region sequence of the gene involved, the coverage of the target region can reach 100%, and the average sequencing depth is as high as 100x. Based on the second-generation sequencing method, the present invention can complete the detection of thyroid cancer-related genes in one step, detect the mutation of the target gene, have high specificity, comprehensive coverage, high efficiency, high precision, and has great application potential and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram showing the fragment analysis results of the hybridization library in Example 1.

[0027] Figure 2 This is a diagram showing the fragment analysis results of the hybridization library of Example 2. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0029] Example 1

[0030] Gene screening of thyroid cancer-related genes was conducted by studying the NCCN and WHO guidelines, as well as the consensus of domestic and foreign experts, and reading relevant literature, including 8 genes such as RET, TERT, BRAF, TP53, NRAS, HRAS, KRAS, and PIK3CA. The pathogenic / potentially pathogenic variants of related genes were screened through the ClinVar database, and these variants must be included in the probe design.

[0031] (1) Using thyroid puncture fluid (FNA) samples from clinical thyroid cancer patients, positive and negative samples with known results by fluorescent PCR were screened out, and genomic DNA was extracted (kit: Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit).

[0032] (2) Library construction: Twist Library Preparation Enzymatic Fragmentation Kit 2.0 (lot number 34000002908) from Twist was used.

[0033] 1. DNA fragmentation (enzymatic digestion), end repair and dA tailing.

[0034] The concentration of gDNA was determined using the Qubit quantitative analysis kit. The reaction solution was prepared (on ice) and kept on ice for later use. The reaction solution was configured as follows:

[0035] Reagents Single reaction volume Frag / ATBuffer 4uL Frag / ATEnzyme 6uL Total volume 10uL

[0036] Set the following program on the PCR instrument, with the heated cover temperature at 70°C:

[0037] temperature time 37℃ 20min 65℃ 30min 4℃ hold

[0038] Add 40 μL of DNA sample to the above reaction solution, with a total amount of 50-200 ng. Seal the reaction tube or cover it with a lid, gently shake and mix, centrifuge for a moment, and run it on a PCR instrument with a preset program.

[0039] 2. Connect the universal connector

[0040] Universal adapter (Twist Bioscience) and DNA Ligation Mix (Twist Bioscience) were thawed on ice; the PCR program was set to 20°C hold with the heated lid closed.

[0041] In each sample plate well or single tube containing DNA fragments with dA tails, add 5μl of UniversalAdapter, mix gently by pipetting, and then place on ice. Add 20μl of Ligation Mix, mix gently by pipetting, seal the reaction tube or cover it with a lid, centrifuge for a moment, and run on a pre-programmed PCR instrument.

[0042] 3. First step magnetic bead purification

[0043] After connecting the universal adapter, add 60 μL of mixed DNA purification magnetic beads (0.8x) to each sample, vortex to mix, incubate at room temperature for 5 minutes, and then stand on the magnetic rack for 5 minutes after centrifugation.

[0044] Aspirate the supernatant, add 200 μl of freshly prepared 80% ethanol, and incubate for one minute. Repeat once.

[0045] Aspirate the supernatant and carefully remove the remaining ethanol using a 10μl pipette, avoiding touching the magnetic beads. Dry the magnetic beads on a magnetic rack until they are non-reflective and cracked. Add 18μl of ddH2O to each sample, pipette to mix, and incubate at room temperature for 25min; after a quick spin, place on a magnetic rack and let stand for 3min. Transfer 15μl of supernatant to a PCR tube and place on ice for later use.

[0046] 4. UDI Primer PCR Amplification

[0047] Thaw Equinox LibraryAmp Mix (2x) and UDI primers (TwistBioscience) on ice; set the following program on the PCR instrument with a heated lid temperature of 105°C:

[0048]

[0049] Add 10 μl of UDI primers and 25 μl of Equinox Library Amp Mix (2x) after inversion to each of the libraries purified in the first step, and gently pipette to mix. After centrifugation, transfer to a PCR instrument and run on the PCR instrument according to the preset program.

[0050] 5. Second step magnetic bead purification

[0051] Vortex the pre-equilibrated DNA purification beads until mixed. Add 50μl (1x) of pre-mixed DNA purification beads to each ligated sample after connecting the UDI connector. Vortex to mix and incubate at room temperature for 5 minutes. After centrifugation, let it stand on the magnetic rack for 5 minutes, aspirate the supernatant, add 200μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once. Use a 10μl pipette to aspirate the remaining ethanol. Dry the beads on the magnetic rack until the beads are non-reflective and dry. Add 24μl of ddH2O to each sample, pipette to mix, incubate at room temperature for 5 minutes, centrifuge, place on the magnetic rack, and let it stand for 2 minutes.

[0052] Take 2 μl and dilute 10 times, then use Qubit to measure the library concentration and use fragment analyzer to measure the fragment size. The average fragment length should be between 350 and 425 bp.

[0053] (3) Hybridization capture:

[0054] 1. Library hybridization

[0055] The amount of library pool input is calculated according to the library DNA concentration, and the library pool is obtained by mixing. The total amount of library pool DNA is 1.5 to 4 ug.

[0056] Add pre-hybridization reagent to the mixed sample according to the table below, gently shake to mix, and centrifuge briefly:

[0057]

[0058] The sequence of the thyroid cancer panel capture probe is as follows:

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] Open the cap of the reaction tube and place it in a vacuum concentrator. After balancing, dry it at 45°C until the solution in the tube is completely dried. The drying process takes about 30 to 60 minutes.

[0068] Incubate Fast Hybridization Mix (Twist Bioscience) at 65°C for 10 min until the precipitate is completely dissolved; set the PCR instrument program according to the table below and heat the lid at 85°C.

[0069] temperature time 95℃ 5min 60℃ 2h

[0070] After drying, remove the centrifuge tube from the vacuum concentrator, tear off the sealing film, quickly vortex the preheated Fast Hybridization Mix (Twist Bioscience) and take 20 μl of the resuspended sample, flick to mix, and let stand at room temperature for 5 minutes. Use a pipette to blow up and down 10 times. After the DNA is completely dissolved, transfer all the solution in the tube to a new PCR tube as much as possible, add 30 μl of paraffin oil to the tube, and spin to remove bubbles. Transfer the PCR tube to the preheated PCR instrument and run it on the preset PCR instrument.

[0071] 2. Library capture and cleaning

[0072] Prepare DNA-binding magnetic beads Streptavidin Binding Beads (TwistBioscience) while running the hybridization reaction program, shake well to mix, and equilibrate at room temperature for at least 30 min; preheat 450 μl Fast Wash Buffer 1 at 66°C on a thermostatic metal bath; preheat 700 μl Wash Buffer 2 at 48°C; dispense the enrichment reagent into centrifuge tubes according to the table below.

[0073] Reagents Single reaction volume FastBindingBuffer(TwistBioscience) 900ul FastWashBuffer1(TwistBioscience) 450ul WashBuffer2(TwistBioscience) 700ul

[0074] Vortex the pre-equilibrated Binding Beads (Twist Bioscience) until completely mixed, and take 100μl of magnetic beads to a 1.5mL centrifuge tube. Add 200μl Fast Binding Buffer to the centrifuge tube and mix with a pipette tip; place the centrifuge tube on the magnetic rack for 1 minute or until the solution is clear, discard the supernatant, and remove the centrifuge tube; repeat the above washing steps 2 times, a total of 3 times. After 3 washes, add 200μl Fast Binding Buffer and vortex to resuspend and mix thoroughly.

[0075] After the hybridization reaction is completed, open the PCR instrument cover and quickly transfer all the hybridization solution to the balanced magnetic bead solution, and immediately blow and mix. Close the centrifuge tube and seal the tube mouth with sealing film, then place it on the Biocate oscillator, 1200rpm, and mix thoroughly for 30 minutes at room temperature.

[0076] Centrifuge the tube quickly, place it on the magnetic rack for 1 minute, and remove the supernatant. Remove the tube from the magnetic rack, add 200 μl of preheated FW1, mix gently, and then quickly incubate at 66°C for 5 minutes. Repeat once.

[0077] Transfer all the liquid in the tube to a new 1.5mL centrifuge tube, place it on the magnetic rack for 1 minute, and remove the supernatant. Remove the centrifuge tube from the magnetic rack, add 200μl preheated W2, mix gently and quickly place it at 48℃ for 5 minutes, place it on the magnetic rack for 1 minute, remove the supernatant and repeat the washing with W2 twice, a total of three times. After a quick centrifugation, place it on the magnetic rack and use a 10μl pipette tip to absorb all the residual liquid; add 45μl ddH2O, mix it by pipetting, and incubate it on ice.

[0078] 3. PCR Amplification

[0079] Prepare DNA purification magnetic beads, shake and mix thoroughly, and equilibrate at room temperature for at least 30 minutes; prepare 80% ethanol; thaw Equinox Library Amp Mix (2x) and Amplification Primers (TwistBioscience) on ice; set the following program on the PCR instrument, with the hot cover temperature at 105°C.

[0080]

[0081] Prepare PCR tubes and add 25μl of Equinox LibraryAmp Mix (2x) and 2.5μl of Amplification Primers to each tube. Take 22.5μl of the magnetic bead suspension prepared in step 2 and add it to the PCR tube. Gently shake and mix, and centrifuge it momentarily. Transfer the PCR tube to the preheated PCR instrument and run the above program.

[0082] 4. Magnetic Bead Purification

[0083] Vortex the pre-equilibrated DNA purification magnetic beads until mixed. Add 90μl (1.8x) of mixed DNA purification magnetic beads to each enriched sample prepared in 2.3. Vortex to mix and incubate at room temperature for 5 minutes. Centrifuge and place on the magnetic rack for 5 minutes, then discard the supernatant. Add 200μl of freshly prepared 80% ethanol, incubate for one minute, and discard the supernatant. Repeat once. Use a 10μl pipette to absorb the remaining ethanol. Dry the magnetic beads on the magnetic rack until the beads are non-reflective and dry. Add 32μl of ddH2O to each sample, mix by pipetting, incubate at room temperature for 5 minutes, centrifuge and place on the magnetic rack, then let it stand for 2 minutes.

[0084] Transfer 30 μl of supernatant to a clean 1.5 ml centrifuge tube and use 2 μl The library concentration was determined by dsDNA HS Assay Kit (Vazyme), and the library concentration should be greater than 0.5 ng / μl. The library fragment length distribution was detected by 2100 fragment analyzer, and the average fragment length should be around 300-400 bp.

[0085] The fragment analysis results of the hybridization library of the sample in this example are as follows Figure 1 .

[0086] Sequencing on the machine:

[0087] The NextSeq 2000 instrument of the Illumina platform was used for sequencing, and the sequencing parameters were PE 2x150. After quality control of the data, bioinformatics analysis of the data was performed using related software such as BWA / GATK, and relevant databases such as gnomAD / Clinvar / OMIM were annotated.

[0088] The sequencing results are shown below:

[0089]

[0090] The NGS sequencing results were compared with the QPCR results, and the results were consistent.

[0091] Example 2

[0092] Tissue samples from patients with thyroid cancer were used for testing. Tissue samples were extracted using the Novozymes FFPE extraction kit. dDNA concentration was determined using dsDNA HS Assay Kit (Vazyme). Hybridization library construction was performed according to Example 1. The fragment analysis results of the hybridization library are shown in Figure 2 .

[0093] Data analysis was performed according to Example 1, and the test results obtained are shown in the following table.

[0094]

[0095] It can be seen that the probe panel and kit of the present invention can accurately detect thyroid cancer-related genes and mutations.

Claims

1. A capture probe combination for detecting thyroid cancer-related genes, the capture probe combination comprising at least one of the sequences shown in SEQ ID NOs: 1 to 101 2. The capture probe combination according to claim 1, characterized in that The capture probe combination includes all the sequences shown in SEQ ID NOs 1 to 101.

3. The capture probe combination according to claim 1, characterized in that The thyroid cancer-related genes detected by the capture probe combination of SEQ ID NOs 1 to 101 include at least one of the following eight genes: RET, TERT, BRAF, TP53, NRAS, HRAS, KRAS, and PIK3CA.

4. A kit for detecting thyroid cancer-related genes, the kit comprising the capture probe combination as described in claim 1 or 2.

5. Use of the kit for detecting thyroid cancer-related genes as described in claim 4 in detecting thyroid cancer-related genes.

6. The use according to claim 5, characterized in that The application method includes detecting 8 genes related to thyroid cancer and their mutations.

7. A method for detecting thyroid cancer-related genes, characterized in that Utilizing the kit for detecting thyroid cancer-related genes as claimed in claim 4, the method comprises the following steps: (1) extracting DNA from the sample to be tested; (2) constructing a DNA library; (3) using a capture probe combination for detecting thyroid cancer-related genes to perform hybridization capture and PCR amplification to obtain a hybridization capture library; (4) The library after hybridization capture is sequenced, and the sequencing data is analyzed and tested by bioinformatics to obtain the mutation status of thyroid cancer-related genes.

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