Thyroid nodule benign and malignant detection library and kit

Abstract

The invention relates to a thyroid nodule benign and malignant detection library and a kit, and belongs to the technical field of library construction. The construction method of the thyroid nodule benign and malignant detection library comprises the following steps: extracting sample DNA and sample RNA from a sample to be detected, carrying out a round of PCR amplification by taking the extracted sample DNA and sample RNA as templates and taking amplification primers of gene mutation detection genes and gene fusion detection genes as primers to obtain a round of PCR amplification product, and purifying the round of PCR amplification product; performing Index primer amplification on the purified primary PCR amplification product to obtain a secondary PCR amplification product, and purifying the secondary PCR amplification product to obtain the target product. The establishment of the thyroid nodule benign and malignant detection library is completed by adopting two-step amplification and two-step purification. The gene mutation target detection specificity and uniformity (20%) can reach 95% or above, and the gene fusion target detection specificity reaches 100%.

Description

Technical Field

[0001] The present invention belongs to the technical field of library construction, and particularly relates to a library and a kit for detecting the benign and malignant of thyroid nodules. Background Art

[0002] The thyroid tissue is mainly composed of follicular epithelial cells and parafollicular cells. The follicular epithelial cells are responsible for synthesizing and secreting thyroid hormones, and the parafollicular cells secrete calcitonin, which participates in the regulation of calcium and phosphorus metabolism. A thyroid nodule refers to a scattered lesion formed by local abnormal proliferation of thyroid cells. Epidemiological data shows that the incidence rate of thyroid nodules in China is as high as 18.6%, and more than 200 million people carry thyroid nodules, among which about 7-15% are malignant nodules (thyroid cancer). Fine needle aspiration biopsy (FNA) is the gold standard for preoperatively identifying the benign and malignant of thyroid nodules, but still 25% of thyroid nodules cannot be clearly diagnosed by FNA due to insufficient cytological sample volume or atypical morphology, and molecular detection techniques need to be combined for risk stratification.

[0003] In recent years, important progress has been made in the research on the molecular mechanisms of thyroid nodules and thyroid cancer. Somatic mutations have been confirmed to be the key driving factors for the occurrence of thyroid cancer, and its molecular characteristics mainly focus on signal pathways such as MAPK and PI3K. Common gene variations include BRAF, HRAS / KRAS / NRAS, TP53 gene mutations, and RET / PTC and PAX8 / PPARG gene fusions, etc. The mutation rate of BRAF in papillary thyroid cancer can reach 80%, and rare mutations occur in benign nodules, which is a very important tumor marker for papillary thyroid cancer. The incidence rate of RET / PTC fusion in sporadic papillary cancer is 15-20%, and it is more common in ionizing radiation and childhood papillary cancer. The incidence rate of TERT promoter mutation in differentiated thyroid cancer is 10-15%, and it is 40-45% in more aggressive poorly differentiated cancer and undifferentiated cancer. The research on these gene mutations provides detailed basis for the molecular diagnosis of thyroid nodules. Thyroid cancer diagnosis and treatment guidelines and expert consensus at home and abroad both recommend incorporating molecular diagnosis into the differential diagnosis process of benign and malignant thyroid nodules.

[0004] There are usually two methods for targeted next-generation sequencing to enrich target regions: hybridization capture method and multiplex amplification method. Among them, the hybridization capture method requires more template amount, the whole detection experiment process takes a long time, and has higher requirements for the operation of experimental personnel. The multiplex amplification method requires less total amount of template nucleic acid, has a fast detection process and is easy to operate. The invention patent with the application publication number of CN116219016A discloses a method, a kit and an application for detecting the benign and malignant of thyroid nodules. The sensitivity of this kit for detecting the benign and malignant of thyroid nodules is 78.57%, and the accuracy is 80%, both the sensitivity and the accuracy are relatively low. Summary of the Invention

[0005] The first object of the present invention is to provide a library for detecting the benign and malignant of thyroid nodules to solve the technical problems of low accuracy and sensitivity in the detection of benign and malignant of thyroid.

[0006] The second object of the present invention is to provide a kit for detecting the benign and malignant of thyroid nodules.

[0007] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0008] A library for detecting the benign and malignant of thyroid nodules, and the construction method of the library includes the following steps:

[0009] S1: Extract sample DNA and sample RNA from the sample to be tested. Using the extracted sample DNA and sample RNA as templates, and using the amplification primers for the detection genes of gene mutations and the detection genes of gene fusions as primers, perform a round of PCR amplification to obtain a round of PCR amplification products, and purify the round of PCR amplification products;

[0010] S2: Perform Index primer amplification on the purified round of PCR amplification products to obtain a round of PCR amplification products. After purifying the round of PCR amplification products, it is obtained.

[0011] Further, the detection genes of gene mutations include BRAF gene, HRAS gene, KRAS gene, NRAS gene, RET gene, TERT gene, TP53 gene, PIK3CA gene, EIF1AX gene, CTNNB1 gene, IDH1 gene, ATK1 gene, EZH1 gene, SPOP gene and ZNF148 gene; the detection genes of gene fusions include NTRK1 gene, NTRK3 gene, BRAF gene, RET gene, ALK gene and PPARG gene.

[0012] Furthermore, the BRAF gene detection sites include: V600E, K601E; the HRAS gene detection sites include: Q61R / K, G12V, G13R; the KRAS gene detection sites include: Q61R, G12D / R / V, G13D; the NRAS gene detection sites include: Q61R / K / L, G12C; the RET gene detection sites include: M918T, C634R / W / Y; the TERT gene detection sites include: C228T, C250T; the TP53 gene detection sites include: G248W / Q, R175H, R273C / H, C135Y; the PIK3CA gene detection sites include: H1047R / L, E542K, E545K; the EIF1AX gene detection sites include: A113*; the CTNNB1 gene detection sites include: T40I, K49R; the IDH1 gene detection sites include: V178I, G70D; the AKT1 gene detection sites include: E17K; the EZH1 gene detection sites include: Q571R; the SPOP gene detection sites include: P94R; the ZNF148 gene detection sites include: K528fs; the NTRK1 gene fusion detection sites include: TPM3(7)-NTRK1(10); the NTRK3 gene fusion detection sites include: ETV6(4)-NTRK3(14), ETV6(5)-NTRK3(15); the BRAF gene fusion detection sites include: SND1(14)-BRAF(9), SND1(14)-BRAF(11); the RET gene fusion sites include: CCDC6(1)-RET(12), CCDC6(2)-RET(12), CCDC6(8)-RET(12), CCDC6(8)-RET(11), NCOA4(8)-RET(12), NCOA4(8)-RET(11); the ALK gene fusion detection sites include: STRN(3)-ALK(20), EML4(13)-ALK(20), EML4(6)-ALK(20); the PPARG gene fusion detection sites include: PAX8(8)-PPARG(2), PAX8(10)-PPARG(2), CREB3L2(2)-PPARG(2).

[0013] 1. Furthermore, among the genes for detecting gene mutations: the amplification primer pair for the BRAF gene detection site includes the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2;

[0014] The amplification primer pair for the HRAS gene detection site includes a first HRAS detection primer pair and a second HRAS detection primer pair. The first HRAS detection primer pair includes a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4; the second HRAS detection primer pair includes a forward primer as shown in SEQ ID NO.5 and a reverse primer as shown in SEQ ID NO.6;

[0015] The amplification primer pair for the KRAS gene detection site includes a first KRAS detection primer pair and a second KRAS detection primer pair; the first KRAS detection primer pair includes a forward primer as shown in SEQ ID NO.7 and a reverse primer as shown in SEQ ID NO.8; the second KRAS detection primer pair includes a forward primer as shown in SEQ ID NO.9 and a reverse primer as shown in SEQ ID NO.10;

[0016] The amplification primer pair for the NRAS gene detection site includes a first NRAS detection primer pair and a second NRAS detection primer pair; the first NRAS detection primer pair includes a forward primer as shown in SEQ ID NO.11 and a reverse primer as shown in SEQ ID NO.12; the second NRAS detection primer pair includes a forward primer as shown in SEQ ID NO.13 and a reverse primer as shown in SEQ ID NO.14;

[0017] The amplification primer pair for the RET gene detection site includes a first RET detection primer pair and a second RET detection primer pair; the first RET detection primer pair includes a forward primer as shown in SEQ ID NO.15 and a reverse primer as shown in SEQ ID NO.16; the second RET detection primer pair includes a forward primer as shown in SEQ ID NO.17 and a reverse primer as shown in SEQ ID NO.18;

[0018] The amplification primer pair for the TERT gene detection site includes a forward primer as shown in SEQ ID NO.19 and a reverse primer as shown in SEQ ID NO.20;

[0019] The amplification primer pairs for the TP53 gene detection sites include a first TP53 detection primer pair, a second TP53 detection primer pair, and a third TP53 detection primer pair; the first TP53 detection primer pair includes a forward primer as shown in SEQ ID NO.21 and a reverse primer as shown in SEQ ID NO.22; the second TP53 detection primer pair includes a forward primer as shown in SEQ ID NO.23 and a reverse primer as shown in SEQ ID NO.24; the third TP53 detection primer pair includes a forward primer as shown in SEQ ID NO.25 and a reverse primer as shown in SEQ ID NO.26;

[0020] The amplification primer pairs for the PIK3CA gene detection sites include a first PIK3CA detection primer pair and a second PIK3CA detection primer pair; the first PIK3CA detection primer pair includes a forward primer as shown in SEQ ID NO.27 and a reverse primer as shown in SEQ ID NO.28; the second PIK3CA detection primer pair includes a forward primer as shown in SEQ ID NO.29 and a reverse primer as shown in SEQ ID NO.30;

[0021] The amplification primer pairs for the EIF1AX gene detection sites include a first EIF1AX detection primer pair and a second EIF1AX detection primer pair; the first EIF1AX detection primer pair includes a forward primer as shown in SEQ ID NO.31 and a reverse primer as shown in SEQ ID NO.32; the second EIF1AX detection primer pair includes a forward primer as shown in SEQ ID NO.33 and a reverse primer as shown in SEQ ID NO.34;

[0022] The amplification primer pair for the CTNNB1 gene detection site includes a forward primer as shown in SEQ ID NO.35 and a reverse primer as shown in SEQ ID NO.36;

[0023] The amplification primer pairs for the IDH1 gene detection sites include a first IDH1 detection primer pair and a second IDH1 detection primer pair; the first IDH1 detection primer pair includes a forward primer as shown in SEQ ID NO.37 and a reverse primer as shown in SEQ ID NO.38; the second IDH1 detection primer pair includes a forward primer as shown in SEQ ID NO.39 and a reverse primer as shown in SEQ ID NO.40;

[0024] The amplification primer pair for the AKT1 gene detection site includes a forward primer as shown in SEQ ID NO.41 and a reverse primer as shown in SEQ ID NO.42;

[0025] The amplification primer pair for the EZH1 gene detection site includes a forward primer as shown in SEQ ID NO.43 and a reverse primer as shown in SEQ ID NO.44;

[0026] The amplification primer pair for the SPOP gene detection site includes a forward primer as shown in SEQ ID NO.45 and a reverse primer as shown in SEQ ID NO.46;

[0027] The amplification primer pair for the ZNF148 gene detection site includes a forward primer as shown in SEQ ID NO.47 and a reverse primer as shown in SEQ ID NO.48;

[0028] The amplification primer pair for the TPM3(7)-NTRK1(10) detection site includes a forward primer as shown in SEQ ID NO.49 and a reverse primer as shown in SEQ ID NO.50;

[0029] The amplification primer pair for the ETV6(4)-NTRK3(14) detection site includes a forward primer as shown in SEQ ID NO.51 and a reverse primer as shown in SEQ ID NO.52;

[0030] The amplification primer pair for the ETV6(5)-NTRK3(15) detection site includes a forward primer as shown in SEQ ID NO.53 and a reverse primer as shown in SEQ ID NO.54;

[0031] The amplification primer pair for the SND1(14)-BRAF(9) detection site includes a forward primer as shown in SEQ ID NO.55 and a reverse primer as shown in SEQ ID NO.56;

[0032] The amplification primer pair for the SND1(14)-BRAF(11) detection site includes a forward primer as shown in SEQ ID NO.57 and a reverse primer as shown in SEQ ID NO.58;

[0033] The amplification primer pair for the CCDC6(1)-RET(12) detection site includes a forward primer as shown in SEQ ID NO.59 and a reverse primer as shown in SEQ ID NO.60;

[0034] The amplification primer pair for the CCDC6(2)-RET(12) detection site includes a forward primer as shown in SEQ ID NO.61 and a reverse primer as shown in SEQ ID NO.62;

[0035] The amplification primer pair for the CCDC6(8)-RET(12) detection site includes the forward primer shown in SEQ ID NO.63 and the reverse primer shown in SEQ ID NO.64;

[0036] The amplification primer pair for the CCDC6(8)-RET(11) detection site includes the forward primer shown in SEQ ID NO.65 and the reverse primer shown in SEQ ID NO.66;

[0037] The amplification primer pair for the NCOA4(8)-RET(12) detection site includes the forward primer shown in SEQ ID NO.67 and the reverse primer shown in SEQ ID NO.68;

[0038] The amplification primer pair for the NCOA4(8)-RET(11) detection site includes the forward primer shown in SEQ ID NO.69 and the reverse primer shown in SEQ ID NO.70;

[0039] The amplification primer pair for the STRN(3)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.71 and the reverse primer shown in SEQ ID NO.72;

[0040] The amplification primer pair for the EML4(13)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.73 and the reverse primer shown in SEQ ID NO.74;

[0041] The amplification primer pair for the EML4(6)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.75 and the reverse primer shown in SEQ ID NO.76;

[0042] The amplification primer pair for the PAX8(8)-PPARG(2) detection site includes the forward primer shown in SEQ ID NO.77 and the reverse primer shown in SEQ ID NO.78;

[0043] The amplification primer pair for the PAX8(10)-PPARG(2) detection site includes the forward primer shown in SEQ ID NO.79 and the reverse primer shown in SEQ ID NO.80;

[0044] The amplification primer pair for the CREB3L2(2)-PPARG(2) detection site includes the forward primer shown in SEQ ID NO.81 and the reverse primer shown in SEQ ID NO.82.

[0045] Further, the final concentration ratio of the amplification primers for the genes with gene mutations to the amplification primers for the genes with gene fusions is 4:1.

[0046] Further, the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the HRAS gene detection site amplification primer for HRAS is 1.5:8; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the HRAS gene detection site amplification primer for HRAS is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the KRAS gene detection site amplification primer for KRAS is 1.5:1.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the KRAS gene detection site amplification primer for KRAS is 1.5:0.6; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the NRAS gene detection site amplification primer for NRAS is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the NRAS gene detection site amplification primer for NRAS is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair and the second detection primer pair of the RET gene detection site amplification primer for RET is 1.5:1.5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the TERT gene detection site is 1.5:15; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair, the second detection primer pair, and the third detection primer pair of the TP53 gene detection site amplification primer for TP53 is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the PIK3CA gene detection site amplification primer for PIK3CA is 1.5:0.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the PIK3CA gene detection site amplification primer for PIKC A is 1.5:0.8; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the EIF1AX gene detection site amplification primer for EIF1AX is 1.5:1.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the EIF1AX gene detection site amplification primer for EIF1AX is 1.5:2; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first detection primer pair of the IDH1 gene detection site amplification primer for IDH1 is 1.5:1.2, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second detection primer pair of the IDH1 gene detection site amplification primer for IDH1 is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the AKT1 gene detection site is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the EZH1 gene detection site is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the SPOP gene detection site is 1.5:0.6; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the ZNF148 gene detection site is 1.5:0.8.

[0047] Further, in the amplification primers for the detection genes of gene fusion, the amplification primer pairs for the TPM3(7)-NTRK1(10) detection site, ETV6(4)-NTRK3(14) detection site, ETV6(5)-NTRK3(15) detection site, SND1(14)-BRAF(9) detection site, SND1(14)-BRAF(11) detection site, CCDC6(1)-RET(12) detection site, CCDC6(2)-RET(12) detection site, CCDC6(8)-RET(12) detection site, CCDC6(8)-RET(11) detection site, NCOA4(8)-RET(12) detection site, NCOA4(8)-RET(11) detection site, STRN(3)-ALK(20) detection site, EML4(13)-ALK(20) detection site, EML4(6)-ALK(20) detection site, PAX8(8)-PPARG(2) detection site, PAX8(10)-PPARG(2) detection site, CREB3L2(2)-PPARG(2) detection site, etc. are mixed in equal mass.

[0048] Further, the mass ratio of the sample DNA to the sample RNA is 1:1.

[0049] Further, it also includes a housekeeping gene for judging whether the amplification primers for the detection genes of gene fusion work; the housekeeping genes include the POLR2A gene amplification primer pair and the DDX5 amplification primer pair. The POLR2A gene amplification primer pair includes a forward primer as shown in SEQ ID NO.83 and a reverse primer as shown in SEQ ID NO.84; the DDX5 amplification primer pair includes a forward primer as shown in SEQ ID NO.85 and a reverse primer as shown in SEQ ID NO.86.

[0050] A kit for detecting the benign and malignant of thyroid nodules, comprising the above-mentioned library for detecting the benign and malignant of thyroid nodules.

[0051] The beneficial effects of the present invention:

[0052] The present invention can complete the co-detection of DNA and RNA through a one-tube method, with simple operation and high accuracy. The present invention uses two-step amplification and two-step purification to establish a library for detecting the benign and malignant of thyroid nodules. The specificity and uniformity (20%) of the gene mutation target detection can both reach over 95%, and the specificity of the gene fusion target detection reaches 100%. The sensitivity of the kit of the present invention in detecting the benign and malignant of thyroid nodules is 92.8%, the specificity is 90.9%, and the accuracy is 92%.

[0053] The kit of the present invention includes 30 gene mutation sites of 15 DNA genes and 17 gene fusion sites of 6 RNA genes, with low material cost, few target points, and high sensitivity, accuracy and specificity.

[0054] The primers for different mutation sites and fusion sites of the present invention are mixed in a non-equal proportion manner, and the ratio and dosage of different primers are creatively designed. Compared with the equal-proportion mixing method, the present invention has achieved good results in improving the library concentration and primer uniformity. Description of the Drawings

[0055] Figure 1 It is a comparison chart of the library concentrations of the libraries for detecting the benign and malignant of thyroid nodules established by mixing Primer Mixing Scheme 1 and Primer Mixing Scheme 2 in Example 3;

[0056] Figure 2 It is a primer uniformity chart of the libraries for detecting the benign and malignant of thyroid nodules established by mixing Primer Mixing Scheme 1 and Primer Mixing Scheme 2 in Example 3. Detailed Embodiments

[0057] The present invention will be further described below in conjunction with the embodiments of the present invention and the drawings.

[0058] Example 1

[0059] The steps for establishing the library for detecting the benign and malignant of thyroid nodules in Example 1 are as follows:

[0060] The detection of benign and malignant thyroid nodules includes the detection of gene mutations and gene fusions. The genes for detecting gene mutations include BRAF gene, HRAS gene, KRAS gene, NRAS gene, RET gene, TERT gene, TP53 gene, PIK3CA gene, EIF1AX gene, CTNNB1 gene, IDH1 gene, ATK1 gene, EZH1 gene, SPOP gene, ZNF148 gene. The detection sites for BRAF gene mutations include: V600E, K601E; the detection sites for HRAS gene include: Q61R / K, G12V, G13R; the detection sites for KRAS gene mutations include: Q61R, G12D / R / V, G13D; the detection sites for NRAS gene include: Q61R / K / L, G12C; the detection sites for RET gene mutations include: M918T, C634R / W / Y; the detection sites for TERT gene include: C228T, C250T; the detection sites for TP53 gene mutations include: G248W / Q, R175H, R273C / H, C135Y; the detection sites for PIK3CA gene include: H1047R / L, E542K, E545K; the detection sites for EIF1AX gene mutations include: A113*; the detection sites for CTNNB1 gene include: T40I, K49R; the detection sites for IDH1 gene include: V178I, G70D; the detection sites for AKT1 gene include: E17K; the detection sites for EZH1 gene include: Q571R; the detection sites for SPOP gene mutations include: P94R; the detection sites for ZNF148 gene include: K528fs.

[0061] Genes for detecting gene fusions include the NTRK1 gene, NTRK3 gene, BRAF gene, RET gene, ALK gene, and PPARG gene. Detection sites for NTRK1 gene fusions include: TPM3(7)-NTRK1(10); Detection sites for NTRK3 gene fusions include: ETV6(4)-NTRK3(14), ETV6(5)-NTRK3(15); Detection sites for BRAF gene fusions include: SND1(14)-BRAF(9), SND1(14)-BRAF(11); RET gene fusion sites include: CCDC6(1)-RET(12), CCDC6(2)-RET(12), CCDC6(8)-RET(12), CCDC6(8)-RET(11), NCOA4(8)-RET(12), NCOA4(8)-RET(11); Detection sites for ALK gene fusions include: STRN(3)-ALK(20), EML4(13)-ALK(20), EML4(6)-ALK(20); Detection sites for PPARG gene fusions include: PAX8(8)-PPARG(2), PAX8(10)-PPARG(2), CREB3L2(2)-PPARG(2). According to the level of human housekeeping gene expression, POLR2A and DDX5 were respectively designed to have a pair of internal reference primers for evaluating whether the primers at the RNA level were amplified normally. The primer sequences for gene mutations, gene fusions, and housekeeping genes are shown in Table 1.

[0062] Table 1 Primer sequences for gene mutations, gene fusions, and housekeeping genes

[0063] Primer Name Nucleotide Sequence Sequence Number Primer Name Primer Name Sequence Number BRAF-F ACACGACGCTCTTCCGATCTCACAAAATGGATCCAGACAACTGT SEQ ID NO.1 BRAF-R GACGTGTGCTCTTCCGATCTTGCTTGCTCTGATAGGAAAATGAG SEQ ID NO.2 HRAS-F1 ACACGACGCTCTTCCGATCTTGTCCTCAAAAGACTTGGTGTTG SEQ ID NO.3 HRAS-R1 GACGTGTGCTCTTCCGATCTCGGAAGCAGGTGGTCATTGA SEQ ID NO.4 HRAS-F2 ACACGACGCTCTTCCGATCTGGCTCACCTCTATAGTGGGGT SEQ ID NO.5 HRAS-R2 GACGTGTGCTCTTCCGATCTGCGATGACGGAATATAAGCTGG SEQ ID NO.6 KRAS-F1 ACACGACGCTCTTCCGATCTCAGTCCTCATGTACTGGTCCC SEQ ID NO.7 KRAS-R1 GACGTGTGCTCTTCCGATCTAGGTGCACTGTAATAATCCAGACT SEQ ID NO.8 KRAS-F2 ACACGACGCTCTTCCGATCTGGTCCTGCACCAGTAATATGCA SEQ ID NO.9 KRAS-R2 GACGTGTGCTCTTCCGATCTAGGCCTGCTGAAAATGACTGA SEQ ID NO.10 NRAS-F1 ACACGACGCTCTTCCGATCTTTGATGGCAAATACACAGAGGA SEQ ID NO.11 NRAS-R1 GACGTGTGCTCTTCCGATCTACCCCCAGGATTCTTACAGAA SEQ ID NO.12 NRAS-F2 ACACGACGCTCTTCCGATCTCCACTGGGCCTCACCTCTAT SEQ ID NO.13 NRAS-R2 GACGTGTGCTCTTCCGATCTTCCAACAGGTTCTTGCTGGT SEQ ID NO.14 RET-F1 ACACGACGCTCTTCCGATCTCCCCTCCTTCCTAGAGAGTTAGA SEQ ID NO.15 RET-R1 GACGTGTGCTCTTCCGATCTCTCCACCCCAAGAGAGCAAC SEQ ID NO.16 RET-F2 ACACGACGCTCTTCCGATCTTGGAGAGCCATGAGGCAGA SEQ ID NO.17 RET-R2 GACGTGTGCTCTTCCGATCTATGCAGAAGGCAGACAGCAG SEQ ID NO.18 TERT-F ACACGACGCTCTTCCGATCTAGCGCTGCCTGAAACTCG SEQ ID NO.19 TERT-R GACGTGTGCTCTTCCGATCTCGTCCTGCCCCTTCACCTT SEQ ID NO.20 TP53-F1 ACACGACGCTCTTCCGATCTGCTCACCATCGCTATCTGAG SEQ ID NO.21 TP53-R1 GACGTGTGCTCTTCCGATCTCCCTGCCCTCAACAAGATG SEQ ID NO.22 TP53-F2 ACACGACGCTCTTCCGATCTAAGTGGCTCCTGACCTGGA SEQ ID NO.23 TP53-R2 GACGTGTGCTCTTCCGATCTTCTTGGGCCTGTGTTATCTCC SEQ ID NO.24 TP53-F3 ACACGACGCTCTTCCGATCTCTCCACCGCTTCTTGTCCTG SEQ ID NO.25 TP53-R3 GACGTGTGCTCTTCCGATCTCTTTTCCTATCCTGAGTAGTGGT SEQ ID NO.26 PIK3CA-F1 ACACGACGCTCTTCCGATCTACAGAGTAACAGACTAGCTAGA SEQ ID NO.27 PIK3CA-R1 GACGTGTGCTCTTCCGATCTAGCACTTACCTGTGACTCCAT SEQ ID NO.28 PIK3CA-F2 ACACGACGCTCTTCCGATCTACATTCGAAAGACCCTAGCCT SEQ ID NO.29 PIK3CA-R2 GACGTGTGCTCTTCCGATCTCCAGAGTGAGCTTTCATTTTCTCAG SEQ ID NO.30 EIF1AX-F1 ACACGACGCTCTTCCGATCTACCTAACCAGCAACACATAACCT SEQ ID NO.31 EIF1AX-R1 GACGTGTGCTCTTCCGATCTGTCTCTATTTAAGGATAACAAAGCTGA SEQ ID NO.32 EIF1AX-F2 ACACGACGCTCTTCCGATCTACGTCATCAATATCTTCATCATCATCT SEQ ID NO.33 EIF1AX-R2 GACGTGTGCTCTTCCGATCTAGGAATGCTCTTATGATGAAACTTTGA SEQ ID NO.34 CTNNB1-F1 ACACGACGCTCTTCCGATCTACCAGACAGAAAAGCGGCTG SEQ ID NO.35 CTNNB1-R1 GACGTGTGCTCTTCCGATCTAGGACTGAGAAAATCCCTGTTCC SEQ ID NO.36 IDH1-F1 ACACGACGCTCTTCCGATCTCCGTGCCACCCAGAATATTTC SEQ ID NO.37 IDH1-R1 GACGTGTGCTCTTCCGATCTGAATCGTGATGCCACCAACG SEQ ID NO.38 IDH1-F2 ACACGACGCTCTTCCGATCTAGACAGAGCCATTTGGAAGGA SEQ ID NO.39 IDH1-R2 GACGTGTGCTCTTCCGATCTTCATTTGGTTGTGGTGGGTGA SEQ ID NO.40 AKT1-F ACACGACGCTCTTCCGATCTCTTGAGGAGGAAGTAGCGTGG SEQ ID NO.41 AKT1-R GACGTGTGCTCTTCCGATCTGCATCCCAGGCACATCTGT SEQ ID NO.42 EZH1-F ACACGACGCTCTTCCGATCTCGCTGGATGCTGCAGTTTTT SEQ ID NO.43 EZH1-R GACGTGTGCTCTTCCGATCTCCCTTCCCAGGTCAGAATCG SEQ ID NO.44 SPOP-F ACACGACGCTCTTCCGATCTAGAGGAGAACATTTACCCATAGCTT SEQ ID NO.45 SPOP-R GACGTGTGCTCTTCCGATCTTTTGCGAGTAAACCCCAAAGG SEQ ID NO.46 ZNF148-F ACACGACGCTCTTCCGATCTACTGAAGGATATCTCATGCTGTCC SEQ ID NO.47 ZNF148-R GACGTGTGCTCTTCCGATCTATGAAAGTACCACGGCATCCA SEQ ID NO.48 TPM3(7)-NTRK1(10)-F ACACGACGCTCTTCCGATCTAAACTCAAGGAGGCAGAGACC SEQ ID NO.49 TPM3(7)-NTRK1(10)-R GACGTGTGCTCTTCCGATCTCCGAGACCCCAAAAGGTGTT SEQ ID NO.50 ETV6(4)-NTRK3(14)-F ACACGACGCTCTTCCGATCTTTCACCATTCTTCCACCCTGG SEQ ID NO.51 ETV6(4)-NTRK3(14)-R GACGTGTGCTCTTCCGATCTTGGTGATGCCGTGGTTGATG SEQ ID NO.52 ETV6(5)-NTRK3(15)-F ACACGACGCTCTTCCGATCTATCGGGAAGACCTGGCTTAC SEQ ID NO.53 ETV6(5)-NTRK3(15)-R GACGTGTGCTCTTCCGATCTTTTCCAAAGGCTCCCTCACC SEQ ID NO.54 SND1(14)-BRAF(9)-F ACACGACGCTCTTCCGATCTCCAGAGCTATTAAGAATGGCAAAGG SEQ ID NO.55 SND1(14)-BRAF(9)-R GACGTGTGCTCTTCCGATCTGGGGGTAGCAGACAAACCTG SEQ ID NO.56 SND1(14)-BRAF(11)-F ACACGACGCTCTTCCGATCTCCAGAGCTATTAAGAATGGCAAAGG SEQ ID NO.57 SND1(14)-BRAF(11) GACGTGTGCTCTTCCGATCTGATCCAATTCTTTGTCCCACTGT SEQ ID NO.58 CCDC6(1)-RET(12)-F ACACGACGCTCTTCCGATCTACAAACTGAAGTGCAAGGCA SEQ ID NO.59 CCDC6(1)-RET(12)-R GACGTGTGCTCTTCCGATCTTGCCTTGACCACTTTTCCAAA SEQ ID NO.60 CCDC6(2)-RET(12)-F ACACGACGCTCTTCCGATCTTGCAGAAGGAGAAAGAAACCCT SEQ ID NO.61 CCDC6(2)-RET(12)-R GACGTGTGCTCTTCCGATCTTGCCTTGACCACTTTTCCAAA SEQ ID NO.62 CCDC6(8)-RET(12)-F ACACGACGCTCTTCCGATCTCAACTTCACTGACTAGAGCTGGA SEQ ID NO.63 CCDC6(8)-RET(12)-R GACGTGTGCTCTTCCGATCTTCCAAATTCGCCTTCTCCTAGAG SEQ ID NO.64 CCDC6(8)-RET(11)-F ACACGACGCTCTTCCGATCTTCACTGACTAGAGCTGGAATGTC SEQ ID NO.65 CCDC6(8)-RET(11)-R GACGTGTGCTCTTCCGATCTCAGCACCGAGACGATGAAGG SEQ ID NO.66 NCOA4(8)-RET(12)-F ACACGACGCTCTTCCGATCTCCTGCCAGTGGTTATCAAGC SEQ ID NO.67 NCOA4(8)-RET(12)-R GACGTGTGCTCTTCCGATCTTGCCTTGACCACTTTTCCAAA SEQ ID NO.68 NCOA4(8)-RET(11)-F ACACGACGCTCTTCCGATCTCCTGCCAGTGGTTATCAAGCT SEQ ID NO.69 NCOA4(8)-RET(11)-R GACGTGTGCTCTTCCGATCTGCACCGAGACGATGAAGGAG SEQ ID NO.70 STRN(3)-ALK(20)-F ACACGACGCTCTTCCGATCTACAGGAAAGAGCCAAATACCACA SEQ ID NO.71 STRN(3)-ALK(20)-R GACGTGTGCTCTTCCGATCTGCTTGCTCAGCTTGTACTCAG SEQ ID NO.72 EML4(13)-ALK(20)-F ACACGACGCTCTTCCGATCTTCTTACTGGAGACTCAGGTGGA SEQ ID NO.73 EML4(13)-ALK(20)-R GACGTGTGCTCTTCCGATCTGCTTGCTCAGCTTGTACTCAG SEQ ID NO.74 EML4(6)-ALK(20)-F ACACGACGCTCTTCCGATCTTGTCGAAAATACCTTCAACACCC SEQ ID NO.75 EML4(6)-ALK(20)-R GACGTGTGCTCTTCCGATCTGCTTGCTCAGCTTGTACTCAG SEQ ID NO.76 PAX8(8)-PPARG(2)-F ACACGACGCTCTTCCGATCTCGGATACCCACCCCACATC SEQ ID NO.77 PAX8(8)-PPARG(2)-R GACGTGTGCTCTTCCGATCTCCATTACGGAGAGATCCACGG SEQ ID NO.78 PAX8(10)-PPARG(2)-F ACACGACGCTCTTCCGATCTCACCCTGACCCCTTCCAAC SEQ ID NO.79 PAX8(10)-PPARG(2)-R GACGTGTGCTCTTCCGATCTCCATTACGGAGAGATCCACGG SEQ ID NO.80 CREB3L2(2)-PPARG(2)-F ACACGACGCTCTTCCGATCTCTGAGCACAGCTACTCCCTG SEQ ID NO.81 CREB3L2(2)-PPARG(2)-R GACGTGTGCTCTTCCGATCTCCATTACGGAGAGATCCACGG SEQ ID NO.82 POLR2A-F ACACGACGCTCTTCCGATCTTAAGAAGGCCAAGCAGGACG SEQ ID NO.83 POLR2A-R GACGTGTGCTCTTCCGATCTGAGCAGAGGAGCCAGTCTTG SEQ ID NO.84 DDX5-F ACACGACGCTCTTCCGATCTCAAGAGCGTGACTGGGTTCT SEQ ID NO.85 DDX5-R GACGTGTGCTCTTCCGATCTACTGCGAGCAGTTCTTCCAA SEQ ID NO.86

[0064] Example 2

[0065] Use the FFPE DNA&RNA extraction kit to complete the extraction of sample DNA and sample RNA from clinical samples respectively. After Qubit detection, 10 ng of each of the sample DNA and sample RNA was taken to carry out the co-library construction process. The specific library construction process is as follows:

[0066] 1. RNA denaturation

[0067] Prepare the RNA denaturation reaction system in a PCR tube according to Table 2.

[0068] Table 2 RNA denaturation reaction system

[0069]

[0070] Mix the RNA denaturation reaction system thoroughly with a pipette or vortex mixer. Centrifuge to collect the reaction solution at the bottom of the PCR tube. Place the PCR tube in a PCR instrument: 70 °C for 5 min, with the heated lid at 80 °C. Immediately place it on ice for 3 min after the reaction to obtain the denatured product.

[0071] 2. cDNA Synthesis

[0072] Prepare the reverse transcription reaction system in the PCR tube according to Table 3.

[0073] Table 3 Reverse Transcription Reaction System and Amplification Program

[0074]

[0075] Mix the reverse transcription reaction system thoroughly with a pipette or vortex mixer. Centrifuge to collect the reaction solution at the bottom of the PCR tube for PCR amplification to obtain the reverse transcription reaction product.

[0076] 3. First-round PCR Amplification

[0077] Prepare the first-round amplification reaction system in the PCR tube according to Table 4. The DNA amplification primers in Table 4 are the amplification primers for the genes with gene mutations to be detected, and the RNA amplification primers are the amplification primers for the genes with gene fusions to be detected. The DNA amplification primers and RNA amplification primers in Table 4 are prepared by mixing in the manner of Scheme 2.

[0078] Table 4 First-round Amplification Reaction System and Amplification Program

[0079]

[0080] 4. First-round Magnetic Bead Purification

[0081] Purify the first-round PCR amplification product in step 3 using magnetic beads that have been equilibrated at room temperature for 30 min:

[0082] A. Add 39 μL of the magnetic beads equilibrated at room temperature to the 30 μL of the first-round PCR amplification product in step 3. Gently pipette and mix 20 times with a pipette.

[0083] B. Incubate at room temperature for 5 min, then place the PCR tube on a magnetic stand and let it stand for 3 min.

[0084] C. Remove the supernatant. Keep the PCR tube on the magnetic stand. Add 200 μL of an ethanol solution with a volume fraction of 80% to the PCR tube and let it stand for 30 s.

[0085] D. Remove the supernatant. Keep the PCR tube on the magnetic stand and add 200 μL of 80% ethanol solution to the tube. After standing for 30 s, completely remove the supernatant. Use a 10-μL pipette to remove the residual ethanol solution at the bottom of the PCR tube;

[0086] E. Let it stand at room temperature for 3 min to allow the residual ethanol to completely evaporate;

[0087] F. Add 15 μL of nuclease-free water to the PCR tube. Remove the PCR tube from the magnetic stand and gently pipette to resuspend the magnetic beads, avoiding the generation of bubbles. Let it stand at room temperature for 2 min;

[0088] G. Place the PCR tube back on the magnetic stand and let it stand for 3 min;

[0089] H. Use a pipette to aspirate 13 μL of the supernatant and transfer it to a new 200-μL PCR tube. The supernatant in the tube is the multiplex PCR product.

[0090] 5. Index Primer Amplification

[0091] Prepare the second-round amplification reaction system in the PCR tube according to Table 5.

[0092] Table 5 Second-round Amplification Reaction System and Reaction Program

[0093]

[0094] 6. Second-round Magnetic Bead Purification

[0095] Use the magnetic beads that have been equilibrated at room temperature for 30 min to purify the second-round PCR amplification product in Step 5:

[0096] A. Add 30 μL of the equilibrated magnetic beads at room temperature to 30 μL of the second-round PCR amplification product and gently pipette to mix evenly 20 times;

[0097] B. After incubating at room temperature for 5 min, place the PCR tube on the magnetic stand and let it stand for 3 min;

[0098] C. Remove the supernatant. Keep the PCR tube on the magnetic stand and add 200 μL of 80% ethanol solution to the tube. Let it stand for 30 s;

[0099] D. Remove the supernatant. Keep the PCR tube on the magnetic stand and add 200 μL of 80% ethanol solution to the tube. After standing for 30 s, completely remove the supernatant. Use a 10-μL pipette to remove the residual ethanol solution at the bottom of the PCR tube;

[0100] E. Let it stand at room temperature for 3 min to allow the residual ethanol to completely evaporate;

[0101] F. Add 22.5 μL of nuclease-free water to the PCR tube. Remove the PCR tube from the magnetic stand, and gently pipette to resuspend the magnetic beads, avoiding the generation of bubbles. Let it stand at room temperature for 2 min.

[0102] G. Place the PCR tube back on the magnetic stand and let it stand for 3 min.

[0103] H. Use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared library for detecting the benign and malignant thyroid nodules.

[0104] 7. Library Quality Control

[0105] Use Qubit 4.0 for concentration measurement. Use an Agilent 2100 Bioanalyzer for library peak detection.

[0106] Example 3

[0107] The initial concentration of each amplification primer for the genes detecting gene mutations and the genes detecting gene fusions is 100 μM, and they are mixed according to the methods of Protocol 1 and Protocol 2.

[0108] Protocol 1: The amplification primers for the genes detecting gene mutations are mixed with equal mass, and the amplification primers for the genes detecting gene fusions are mixed with equal mass.

[0109] Protocol 2: The amplification primers for the genes detecting gene mutations are mixed with unequal mass, and the amplification primers for the genes detecting gene fusions are mixed with equal mass.

[0110] In Protocol 2, the amplification primers for the genes detecting gene mutations are mixed according to the manner in Table 6.

[0111] Table 6 Unequal mass mixing of DNA amplification primers in Protocol 2

[0112]

[0113] Comparing the two primer strategies of Protocol 1 and Protocol 2, after Qubit measurement, the library concentration and primer amplification uniformity are as shown in Figure 1 and Figure 2 It can be seen from Figure 1 and Figure 2 that the difference in library concentration between Protocol 1 and Protocol 2 is not significant, but the primer amplification uniformity of Protocol 2 (20%) is significantly improved. Figure 1 and Figure 2 Samples 1 - 10 in

[0114] Example 4

[0115] The kit prepared in Example 2 was used to retrospectively detect cancer tissue samples surgically removed from 10 patients with thyroid cancer. As can be seen from Table 7, the consistency between the detection results of the kit in Example 2 and the pathological detection results was 100%.

[0116] Table 7 Detection results of retrospectively detecting 10 cancer tissue samples with the kit in Example 2

[0117]

[0118] Example 5

[0119] The kit in Example 2 was used to conduct comparative detections before and after surgery on 25 clinical samples. The results are shown in Table 8.

[0120] Table 8 Detection results of 25 clinical samples

[0121] Based on the comparison of the detection results, the statistics are shown in Table 9.

[0122] Table 9 Comparison of the detection results

[0123]

[0124] As can be seen from Table 8 and Table 9, the detection sensitivity of the kit in Example 2 was 92.8% (13 / 14), the specificity was 90.9% (10 / 11), and the accuracy was 92% (23 / 25).

Claims

1. A library for detecting the benign and malignant nature of thyroid nodules, characterized in that, The method for constructing the library comprises the following steps: S1: Extract sample DNA and sample RNA from the sample to be tested. Using the extracted sample DNA and sample RNA as templates, and using the amplification primers for the genes for detecting gene mutations and the genes for detecting gene fusions as primers, perform a round of PCR amplification to obtain a round of PCR amplification products, and purify the round of PCR amplification products; S2: Perform Index primer amplification on the purified round of PCR amplification products to obtain a second round of PCR amplification products. After purifying the second round of PCR amplification products, it is obtained; The genes for detecting gene mutations include BRAF gene, HRAS gene, KRAS gene, NRAS gene, RET gene, TERT gene, TP53 gene, PIK3CA gene, EIF1AX gene, CTNNB1 gene, IDH1 gene, ATK1 gene, EZH1 gene, SPOP gene and ZNF148 gene; The genes for detecting gene fusions include NTRK1 gene, NTRK3 gene, BRAF gene, RET gene, ALK gene and PPARG gene; The BRAF gene detection sites include: V600E, K601E; the HRAS gene detection sites include: Q61R / K, G12V, G13R; the KRAS gene detection sites include: Q61R, G12D / R / V, G13D; the NRAS gene detection sites include: Q61R / K / L, G12C; the RET gene detection sites include: M918T, C634R / W / Y; the TERT gene detection sites include: C228T, C250T; the TP53 gene detection sites include: G248W / Q, R175H, R273C / H, C135Y; the PIK3CA gene detection sites include: H1047R / L, E542K, E545K; the EIF1AX gene detection sites include: A113*; the CTNNB1 gene detection sites include: T40I, K49R; the IDH1 gene detection sites include: V178I, G70D; the AKT1 gene detection sites include: E17K; the EZH1 gene detection sites include: Q571R; the SPOP gene detection sites include: P94R; the ZNF148 gene detection sites include: K528fs; the NTRK1 gene fusion detection sites include: TPM3(7)-NTRK1(10); the NTRK3 gene fusion detection sites include: ETV6(4)-NTRK3(14), ETV6(5)-NTRK3(15); the BRAF gene fusion detection sites include: SND1(14)-BRAF(9), SND1(14)-BRAF(11); the RET gene fusion sites include: CCDC6(1)-RET(12), CCDC6(2)-RET(12), CCDC6(8)-RET(12), CCDC6(8)-RET(11), NCOA4(8)-RET(12), NCOA4(8)-RET(11); the ALK gene fusion detection sites include: STRN(3)-ALK(20), EML4(13)-ALK(20), EML4(6)-ALK(20); the PPARG gene fusion detection sites include: PAX8(8)-PPARG(2), PAX8(10)-PPARG(2), CREB3L2(2)-PPARG(2).

2. The library for detecting the benign and malignant thyroid nodules according to claim 1, wherein Among the genes for detecting gene mutations: the amplification primer pair for the BRAF gene detection site includes a forward primer as shown in SEQ ID NO.1 and a reverse primer as shown in SEQ ID NO.2; The amplification primer pair for the HRAS gene detection site includes a first HRAS detection primer pair and a second HRAS detection primer pair. The first HRAS detection primer pair includes a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.4; the second HRAS detection primer pair includes a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO.6; The amplification primer pair for the KRAS gene detection site includes a first KRAS detection primer pair and a second KRAS detection primer pair; the first KRAS detection primer pair includes a forward primer shown in SEQ ID NO.7 and a reverse primer shown in SEQ ID NO.8; the second KRAS detection primer pair includes a forward primer shown in SEQ ID NO.9 and a reverse primer shown in SEQ ID NO.10; The amplification primer pair for the NRAS gene detection site includes a first NRAS detection primer pair and a second NRAS detection primer pair; the first NRAS detection primer pair includes a forward primer shown in SEQ ID NO.11 and a reverse primer shown in SEQ ID NO.12; the second NRAS detection primer pair includes a forward primer shown in SEQ ID NO.13 and a reverse primer shown in SEQ ID NO.14; The amplification primer pair for the RET gene detection site includes a first RET detection primer pair and a second RET detection primer pair; the first RET detection primer pair includes a forward primer shown in SEQ ID NO.15 and a reverse primer shown in SEQ ID NO.16; the second RET detection primer pair includes a forward primer shown in SEQ ID NO.17 and a reverse primer shown in SEQ ID NO.18; The amplification primer pair for the TERT gene detection site includes a forward primer shown in SEQ ID NO.19 and a reverse primer shown in SEQ ID NO.20; The amplification primer pair for the TP53 gene detection site includes a first TP53 detection primer pair, a second TP53 detection primer pair, and a third TP53 detection primer pair; the first TP53 detection primer pair includes a forward primer shown in SEQ ID NO.21 and a reverse primer shown in SEQ ID NO.22; The second TP53 detection primer pair includes a forward primer shown in SEQ ID NO.23 and a reverse primer shown in SEQ ID NO.24; the third TP53 detection primer pair includes a forward primer shown in SEQ ID NO.25 and a reverse primer shown in SEQ ID NO.26; The amplification primer pair for the PIK3CA gene detection site includes a PIK3CA first detection primer pair and a PIK3CA second detection primer pair; the PIK3CA first detection primer pair includes a forward primer shown in SEQ ID NO.27 and a reverse primer shown in SEQ ID NO.28; the PIK3CA second detection primer pair includes a forward primer shown in SEQ ID NO.29 and a reverse primer shown in SEQ ID NO.30; The amplification primer pair for the EIF1AX gene detection site includes an EIF1AX first detection primer pair and an EIF1AX second detection primer pair; the EIF1AX first detection primer pair includes a forward primer shown in SEQ ID NO.31 and a reverse primer shown in SEQ ID NO.32; the EIF1AX second detection primer pair includes a forward primer shown in SEQ ID NO.33 and a reverse primer shown in SEQ ID NO.34; The amplification primer pair for the CTNNB1 gene detection site includes a forward primer shown in SEQ ID NO.35 and a reverse primer shown in SEQ ID NO.36; The amplification primer pair for the IDH1 gene detection site includes an IDH1 first detection primer pair and an IDH1 second detection primer pair; the IDH1 first detection primer pair includes a forward primer shown in SEQ ID NO.37 and a reverse primer shown in SEQ ID NO.38; the IDH1 second detection primer pair includes a forward primer shown in SEQ ID NO.39 and a reverse primer shown in SEQ ID NO.40; The amplification primer pair for the AKT1 gene detection site includes a forward primer shown in SEQ ID NO.41 and a reverse primer shown in SEQ ID NO.42; The amplification primer pair for the EZH1 gene detection site includes a forward primer shown in SEQ ID NO.43 and a reverse primer shown in SEQ ID NO.44; The amplification primer pair for the SPOP gene detection site includes a forward primer shown in SEQ ID NO.45 and a reverse primer shown in SEQ ID NO.46; The amplification primer pair for the ZNF148 gene detection site includes a forward primer shown in SEQ ID NO.47 and a reverse primer shown in SEQ ID NO.48; The amplification primer pair for the TPM3(7)-NTRK1(10) detection site includes a forward primer shown in SEQ ID NO.49 and a reverse primer shown in SEQ ID NO.50; The amplification primer pair for the ETV6(4)-NTRK3(14) detection site includes a forward primer shown in SEQ ID NO.51 and a reverse primer shown in SEQ ID NO.52; The amplification primer pair for the ETV6(5)-NTRK3(15) detection site includes the forward primer shown in SEQ ID NO.53 and the reverse primer shown in SEQ ID NO.54; The amplification primer pair for the SND1(14)-BRAF(9) detection site includes the forward primer shown in SEQ ID NO.55 and the reverse primer shown in SEQ ID NO.56; The amplification primer pair for the SND1(14)-BRAF(11) detection site includes the forward primer shown in SEQ ID NO.57 and the reverse primer shown in SEQ ID NO.58; The amplification primer pair for the CCDC6(1)-RET(12) detection site includes the forward primer shown in SEQ ID NO.59 and the reverse primer shown in SEQ ID NO.60; The amplification primer pair for the CCDC6(2)-RET(12) detection site includes the forward primer shown in SEQ ID NO.61 and the reverse primer shown in SEQ ID NO.62; The amplification primer pair for the CCDC6(8)-RET(12) detection site includes the forward primer shown in SEQ ID NO.63 and the reverse primer shown in SEQ ID NO.64; The amplification primer pair for the CCDC6(8)-RET(11) detection site includes the forward primer shown in SEQ ID NO.65 and the reverse primer shown in SEQ ID NO.66; The amplification primer pair for the NCOA4(8)-RET(12) detection site includes the forward primer shown in SEQ ID NO.67 and the reverse primer shown in SEQ ID NO.68; The amplification primer pair for the NCOA4(8)-RET(11) detection site includes the forward primer shown in SEQ ID NO.69 and the reverse primer shown in SEQ ID NO.70; The amplification primer pair for the STRN(3)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.71 and the reverse primer shown in SEQ ID NO.72; The amplification primer pair for the EML4(13)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.73 and the reverse primer shown in SEQ ID NO.74; The amplification primer pair for the EML4(6)-ALK(20) detection site includes the forward primer shown in SEQ ID NO.75 and the reverse primer shown in SEQ ID NO.76; The amplification primer pair for the PAX8(8)-PPARG(2) detection site includes the forward primer shown in SEQ ID NO.77 and the reverse primer shown in SEQ ID NO.78; The amplification primer pair for the PAX8(10)-PPARG(2) detection site includes the forward primer shown in SEQ ID NO.79 and the reverse primer shown in SEQ ID NO.80; The amplification primer pair for the CREB3L2(2)-PPARG(2) detection site includes a forward primer shown in SEQ ID NO.81 and a reverse primer shown in SEQ ID NO.

82.

3. The library for detecting benign and malignant thyroid nodules according to claim 1, wherein, The final concentration ratio of the amplification primer for the detected gene with gene mutation to the amplification primer for the detected gene with gene fusion is 4:

1.

4. The thyroid nodule benign and malignant detection library according to claim 2, characterized in that The final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first HRAS detection primer pair of the amplification primer for the HRAS gene detection site is 1.5:8; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second HRAS detection primer pair of the amplification primer for the HRAS gene detection site is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first KRAS detection primer pair of the amplification primer for the KRAS gene detection site is 1.5:1.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second KRAS detection primer pair of the amplification primer for the KRAS gene detection site is 1.5:0.6; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first NRAS detection primer pair of the amplification primer for the NRAS gene detection site is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second NRAS detection primer pair of the amplification primer for the NRAS gene detection site is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first RET detection primer pair and the second RET detection primer pair of the amplification primer for the RET gene detection site is 1.5:1.5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the TERT gene detection site is 1.5:15; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first TP53 detection primer pair, the second TP53 detection primer pair, and the third TP53 detection primer pair of the amplification primer for the TP53 gene detection site is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first PIK3CA detection primer pair of the amplification primer for the PIK3CA gene detection site is 1.5:0.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second PIK3CA detection primer pair of the amplification primer for the PIK3CA gene detection site is 1.5:0.8; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first EIF1AX detection primer pair of the amplification primer for the EIF1AX gene detection site is 1.5:1.5, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second EIF1AX detection primer pair of the amplification primer for the EIF1AX gene detection site is 1.5:2; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the first IDH1 detection primer pair of the amplification primer for the IDH1 gene detection site is 1.5:1.2, and the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the second IDH1 detection primer pair of the amplification primer for the IDH1 gene detection site is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the AKT1 gene detection site is 1.5:5; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the EZH1 gene detection site is 1.5:1; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the SPOP gene detection site is 1.5:0.6; the final concentration ratio of the amplification primer pair for the BRAF gene detection site to the amplification primer pair for the ZNF148 gene detection site is 1.5:0.

8.

5. The thyroid nodule benign and malignant detection library according to claim 2, wherein The amplification primers for the detected genes with gene fusion, including the amplification primer pairs for the TPM3(7)-NTRK1(10) detection site, ETV6(4)-NTRK3(14) detection site, ETV6(5)-NTRK3(15) detection site, SND1(14)-BRAF(9) detection site, SND1(14)-BRAF(11) detection site, CCDC6(1)-RET(12) detection site, CCDC6(2)-RET(12) detection site, CCDC6(8)-RET(12) detection site, CCDC6(8)-RET(11) detection site, NCOA4(8)-RET(12) detection site, NCOA4(8)-RET(11) detection site, STRN(3)-ALK(20) detection site, EML4(13)-ALK(20) detection site, EML4(6)-ALK(20) detection site, PAX8(8)-PPARG(2) detection site, PAX8(10)-PPARG(2) detection site, CREB3L2(2)-PPARG(2) detection site, are mixed in equal mass.

6. The library for detecting the benign and malignant thyroid nodules according to claim 1, wherein The mass ratio of the sample DNA to the sample RNA is 1:

1.

7. The library for detecting benign and malignant thyroid nodules according to claim 1, characterized in that, It also includes a housekeeping gene for determining whether the amplification primers for the detected genes with gene fusion work; the housekeeping gene includes the POLR2A gene amplification primer pair and the DDX5 amplification primer pair. The POLR2A gene amplification primer pair includes a forward primer shown in SEQ ID NO.83 and a reverse primer shown in SEQ ID NO.84; the DDX5 amplification primer pair includes a forward primer shown in SEQ ID NO.85 and a reverse primer shown in SEQ ID NO.

86.

8. A kit for detecting the benign and malignant nature of thyroid nodules, characterized in that, It includes the library for detecting the benign and malignant thyroid nodules described in claim 1.

Images