Library and kit for detecting benign and malignant thyroid nodules

Abstract

The application relates to a thyroid nodule benignity and malignancy detection library and a kit, and belongs to the technical field of library construction. The construction method of the thyroid nodule benignity and malignancy detection library is as follows: sample DNA and sample RNA are extracted from a to-be-detected sample, the extracted sample DNA and sample RNA are used as templates, and a round of PCR amplification is carried out on the templates by using amplification primers of gene mutation detection genes and gene fusion detection genes as primers to obtain a round of PCR amplification product, and the round of PCR amplification product is purified; the purified round of PCR amplification product is subjected to Index primer amplification to obtain a second round of PCR amplification product, and the second round of PCR amplification product is purified, and the library is obtained. The establishment of the thyroid nodule benignity and malignancy detection library is completed by adopting two-step amplification and two-step purification. The specificity and uniformity (20%) of gene mutation target detection can reach more than 95%, and the specificity of gene fusion target detection can reach 100%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of library construction, and particularly relates to a thyroid nodule benign and malignant detection library and kit. BACKGROUND

[0002] Thyroid tissue is mainly composed of follicular epithelial cells and parafollicular cells. Follicular epithelial cells are responsible for the synthesis and secretion of thyroid hormones, and parafollicular cells secrete calcitonin and participate in the regulation of calcium and phosphorus metabolism. Thyroid nodule refers to a local abnormal proliferation of thyroid cells to form scattered lesions. Epidemiological data shows that the incidence of thyroid nodules in China is as high as 18.6%, and more than 200 million people carry thyroid nodules, of which about 7-15% are malignant nodules (thyroid cancer). Fine needle aspiration biopsy (FNA) is the gold standard for preoperative identification of benign and malignant thyroid nodules, but 25% of thyroid nodules cannot be diagnosed by FNA due to insufficient cytological sample size or atypical morphology, and need to be combined with molecular detection technology for risk stratification.

[0003] In recent years, important progress has been made in the molecular mechanism research of thyroid nodules and thyroid cancer. Somatic mutations have been confirmed as key drivers of the occurrence of thyroid cancer, and the molecular characteristics mainly focus on MAPK, PI3K and other signaling pathways. Common gene mutations include BRAF, HRAS / KRAS / NRAS, TP53 gene mutations, and RET / PTC and PAX8 / PPARG gene fusions. BRAF mutation rate in thyroid papillary carcinoma can reach 80%, and is rarely mutated in benign nodules, which is a very important tumor marker for thyroid papillary carcinoma. The incidence of RET / PTC fusion in sporadic papillary carcinoma is 15-20%, and is more common in ionizing radiation and pediatric papillary carcinoma. The incidence of TERT promoter mutation in differentiated thyroid carcinoma is 10-15%, and in more invasive poorly differentiated carcinoma and anaplastic carcinoma, it is 40-45%. The research on these gene mutations provides detailed basis for the molecular diagnosis of thyroid nodules. The domestic and foreign thyroid cancer diagnosis and treatment guidelines and expert consensus all recommend that molecular diagnosis be included in the differential diagnosis process of benign and malignant thyroid nodules.

[0004] Targeted next-generation sequencing usually has two methods to enrich the target region: hybrid capture method and multiplex amplification method. The hybrid capture method requires more template amount, the whole detection experiment process takes a long time, and the operation requirements of the experiment personnel are higher. The multiplex amplification method requires less total amount of template nucleic acid, the detection process is fast and the operation is simple. The invention patent with the application publication number CN116219016A discloses a thyroid nodule benign and malignant detection method, kit and application. The sensitivity of the kit for detecting the benign and malignant thyroid nodules is 78.57%, and the accuracy is 80%, both of which are low. SUMMARY

[0005] The first object of the present application is to provide a thyroid nodule benign and malignant detection library to solve the technical problem of low accuracy and sensitivity of thyroid benign and malignant detection.

[0006] The second object of the present application is to provide a thyroid nodule benign and malignant detection kit.

[0007] In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0008] The thyroid nodule benign and malignant detection library, the construction method of the library comprises the following steps:

[0009] S1: extracting 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 of the detection genes of gene mutation and gene fusion as primers to perform one round of PCR amplification to obtain one round of PCR amplification product, and purifying the one round of PCR amplification product;

[0010] S2: Index primer amplification is performed on the purified one round of PCR amplification product to obtain a two-round PCR amplification product, and the two-round PCR amplification product is purified to obtain the library.

[0011] Further, the detection genes of gene mutation 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; and the detection genes of gene fusion include NTRK1 gene, NTRK3 gene, BRAF gene, RET gene, ALK gene and PPARG gene.

[0012] Further, 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. Further, in the detection of the gene mutation, the BRAF gene detection site amplification primer pair includes a forward primer as shown in SEQ ID NO. 1 and a reverse primer as shown in SEQ ID NO. 2.

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

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

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

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

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

[0019] the TP53 first detection primer pair comprises a forward primer as shown in SEQ ID NO. 21 and a reverse primer as shown in SEQ ID NO. 22; the TP53 second detection primer pair comprises a forward primer as shown in SEQ ID NO. 23 and a reverse primer as shown in SEQ ID NO. 24; and the TP53 third detection primer pair comprises a forward primer as shown in SEQ ID NO. 25 and a reverse primer as shown in SEQ ID NO. 26;

[0020] the PIK3CA gene detection site amplification primer pair comprises a PIK3CA first detection primer pair and a PIK3CA second detection primer pair; the PIK3CA first detection primer pair comprises a forward primer as shown in SEQ ID NO. 27 and a reverse primer as shown in SEQ ID NO. 28; and the PIK3CA second detection primer pair comprises a forward primer as shown in SEQ ID NO. 29 and a reverse primer as shown in SEQ ID NO. 30;

[0021] the EIF1AX gene detection site amplification primer pair comprises an EIF1AX first detection primer pair and an EIF1AX second detection primer pair; the EIF1AX first detection primer pair comprises a forward primer as shown in SEQ ID NO. 31 and a reverse primer as shown in SEQ ID NO. 32; and the EIF1AX second detection primer pair comprises a forward primer as shown in SEQ ID NO. 33 and a reverse primer as shown in SEQ ID NO. 34;

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

[0023] the IDH1 gene detection site amplification primer pair comprises an IDH1 first detection primer pair and an IDH1 second detection primer pair; the IDH1 first detection primer pair comprises a forward primer as shown in SEQ ID NO. 37 and a reverse primer as shown in SEQ ID NO. 38; and the IDH1 second detection primer pair comprises a forward primer as shown in SEQ ID NO. 39 and a reverse primer as shown in SEQ ID NO. 40;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0045] Further, the final concentration ratio of the amplification primer of the detection gene of the gene mutation to the amplification primer of the detection gene of the gene fusion is 4:1.

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

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

[0048] Further, the quality ratio of the sample DNA and the sample RNA is 1:1.

[0049] Further, a housekeeping gene for judging whether the amplification primers of the detection genes of the gene fusion work is further included; the housekeeping gene includes a POLR2A gene amplification primer pair and a 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] The kit for detecting the benign and malignant thyroid nodules includes the above-mentioned library for detecting the benign and malignant thyroid nodules.

[0051] The beneficial effects of the present application are as follows:

[0052] The application can complete DNA and RNA co-detection by a tube method, is simple and convenient to operate, and is high in accuracy. The application adopts 2-step amplification and 2-step purification to complete establishment of a thyroid nodule benign and malignant detection library. The specificity and uniformity (20%) of gene mutation target detection can reach more than 95%, the specificity of gene fusion target detection reaches 100%, the sensitivity of the kit of the application in the detection of thyroid nodule benign and malignant is 92.8%, the specificity is 90.9%, and the accuracy is 92%.

[0053] The kit of the application includes 30 gene mutation sites of 15 DNA genes and 17 gene fusion sites of 6 RNA genes, is low in material cost, is low in target point number, and is high in sensitivity, accuracy and specificity.

[0054] The application adopts a non-equal proportion mixing mode for primers of different mutation sites and fusion sites, and creatively designs the matching amount of different primers, compared with an equal proportion mixing mode, the application has achieved good effects in improving library concentration and primer uniformity. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 A library concentration comparison chart of the thyroid nodule benign and malignant detection library established by mixing of the primer mixing scheme 1 and the primer mixing scheme 2 in example 3;

[0056] Figure 2 A primer uniformity chart of the thyroid nodule benign and malignant detection library established by mixing of the primer mixing scheme 1 and the primer mixing scheme 2 in example 3. DETAILED DESCRIPTION

[0057] The application will be further described below by combining with the examples of the application and the drawings.

[0058] Example 1

[0059] The establishment steps of the thyroid nodule benign and malignant detection library of example 1 are as follows:

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

[0061] The detection genes of the gene fusion include NTRK1 gene, NTRK3 gene, BRAF gene, RET gene, ALK gene and PPARG gene. The NTRK1 gene fusion detection site includes: TPM3(7)-NTRK1(10); the NTRK3 gene fusion detection site includes: ETV6(4)-NTRK3(14), ETV6(5)-NTRK3(15); the BRAF gene fusion detection site includes: SND1(14)-BRAF(9), SND1(14)-BRAF(11); the RET gene fusion site includes: 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 site includes: STRN(3)-ALK(20), EML4(13)-ALK(20), EML4(6)-ALK(20); the PPARG gene fusion detection site includes: PAX8(8)-PPARG(2), PAX8(10)-PPARG(2), CREB3L2(2)-PPARG(2). According to the level of expression of the human housekeeping gene, one pair of internal reference primers of POLR2A and DDX5 are selected respectively to evaluate whether the primers on the RNA level are normally amplified. The primer sequences of the gene mutation, gene fusion and housekeeping gene are shown in Table 1.

[0062] Table 1 Primer sequences of gene mutation, gene fusion and housekeeping gene

[0063] Primer Name Nucleotide Sequence Sequence Number Primer Name Primer Name Sequence Number BRAF-F ACACGACGCTCTTCCGATCTCACAAAATGGATCCAGACAACTGT SEQ IDNO.1 BRAF-R GACGTGTGCTCTTCCGATCTTGCTTGCTCTGATAGGAAAATGAG SEQ IDNO.2 HRAS-F1 ACACGACGCTCTTCCGATCTTGTCCTCAAAAGACTTGGTGTTG SEQ IDNO.3 HRAS-R1 GACGTGTGCTCTTCCGATCTCGGAAGCAGGTGGTCATTGA SEQ IDNO.4 HRAS-F2 ACACGACGCTCTTCCGATCTGGCTCACCTCTATAGTGGGGT SEQ IDNO.5 HRAS-R2 GACGTGTGCTCTTCCGATCTGCGATGACGGAATATAAGCTGG SEQ IDNO.6 KRAS-F1 ACACGACGCTCTTCCGATCTCAGTCCTCATGTACTGGTCCC SEQ IDNO.7 KRAS-R1 GACGTGTGCTCTTCCGATCTAGGTGCACTGTAATAATCCAGACT SEQ IDNO.8 KRAS-F2 ACACGACGCTCTTCCGATCTGGTCCTGCACCAGTAATATGCA SEQ IDNO.9 KRAS-R2 GACGTGTGCTCTTCCGATCTAGGCCTGCTGAAAATGACTGA SEQ IDNO.10 NRAS-F1 ACACGACGCTCTTCCGATCTTTGATGGCAAATACACAGAGGA SEQ IDNO.11 NRAS-R1 GACGTGTGCTCTTCCGATCTACCCCCAGGATTCTTACAGAA SEQ IDNO.12 NRAS-F2 ACACGACGCTCTTCCGATCTCCACTGGGCCTCACCTCTAT SEQ IDNO.13 NRAS-R2 GACGTGTGCTCTTCCGATCTTCCAACAGGTTCTTGCTGGT SEQ IDNO.14 RET-F1 ACACGACGCTCTTCCGATCTCCCCTCCTTCCTAGAGAGTTAGA SEQ IDNO.15 RET-R1 GACGTGTGCTCTTCCGATCTCTCCACCCCAAGAGAGCAAC SEQ IDNO.16 RET-F2 ACACGACGCTCTTCCGATCTTGGAGAGCCATGAGGCAGA SEQ ID NO. 17 RET-R2 GACGTGTGCTCTTCCGATCTTGGAGAGCCATGAGGCAGA 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) -F ACACGACGCTCTTCCGATCTTTCACCATTCTTCCACCCTGG 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 ACACGACGCTCTTCCGATCTTGCAGAAGGAGAAAGAAACCCT ACACGACGCTCTTCCGATCTTCACTGACTAGAGCTGGAATGTC SEQ ID NO. 65 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)-F ACACGACGCTCTTCCGATCTCACCCTGACCCCTTCCAAC 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 Figure 1 Figure 2 Figure 1 Figure 2 Figure 1 Figure 2 ​ ​ ​

[0064] Example 2

[0065] The sample DNA and sample RNA of the clinical sample were extracted by using the FFPE DNA & RNA extraction kit, respectively. After Qubit detection, 10 ng of sample DNA and sample RNA were taken to carry out the co-library construction process. The specific library construction process is as follows:

[0066] 1. RNA denaturation

[0067] The RNA denaturation reaction system in the PCR tube was prepared according to Table 2.

[0068] Table 2 RNA denaturation reaction system

[0069]

[0070] Mix the RNA denaturation reaction system well with a pipette or vortex mixer, centrifuge to collect the reaction solution to the bottom of the PCR tube, and place the PCR tube in a PCR instrument: 70°C, 5 min, hot cover 80°C, immediately after the reaction is over, place on ice for 3 min, and 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 well with a pipette or vortex mixer, centrifuge to collect the reaction solution to the bottom of the PCR tube for PCR amplification, and obtain the reverse transcription reaction product.

[0076] 3. One round of PCR amplification

[0077] Prepare the one round amplification reaction system in the PCR tube according to Table 4, and the DNA amplification primer in Table 4 is the amplification primer of the detection gene of gene mutation, and the RNA amplification primer is the amplification primer of the detection gene of gene fusion. The DNA amplification primer and the RNA amplification primer in Table 4 are mixed to prepare according to the method of scheme 2.

[0078] Table 4 One round amplification reaction system and amplification program

[0079]

[0080] 4. One round of magnetic bead purification

[0081] Use the magnetic beads that have been equilibrated at room temperature for 30 min to purify the one round PCR amplification product in step 3:

[0082] A. Add 39 μL of magnetic beads equilibrated at room temperature to 30 μL of one round PCR amplification product in step 3, and mix gently with a pipette for 20 times;

[0083] B. After incubation at room temperature for 5 min, place the PCR tube on the magnetic stand for 3 min;

[0084] C. Remove the supernatant, continue to place the PCR tube on the magnetic stand, and add 200 μL of 80% ethanol solution to the PCR tube, stand for 30 s;

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

[0086] E. Stand at room temperature for 3 min to completely evaporate the residual ethanol;

[0087] F. Add 15 μL of nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and gently pipette the resuspended magnetic beads with a pipette to avoid generating bubbles, and stand at room temperature for 2 min;

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

[0089] H. Use a pipette to aspirate 13 μL of 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 reaction system for the second round of amplification according to Table 5 in the PCR tube.

[0092] Table 5. Second round of amplification reaction system and reaction procedure

[0093]

[0094] 6. Second round of magnetic bead purification

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

[0096] A. Add 30 μL of room temperature equilibrated magnetic beads to 30 μL of second round PCR amplification product, and gently pipette the mixture 20 times with a pipette;

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

[0098] C. Remove the supernatant, continue to place the PCR tube on the magnetic stand, add 200 μL of 80% ethanol solution to the tube, stand for 30 s;

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

[0100] E. Stand at room temperature for 3 min to completely evaporate the residual ethanol;

[0101] F. Add 22.5 μL nuclease-free water to the PCR tube, remove the PCR tube from the magnetic stand, and gently pipette the resuspended magnetic beads with the pipette to avoid bubbles, and stand at room temperature for 2 min;

[0102] G. Put the PCR tube back on the magnetic stand and stand for 3 min;

[0103] H. Use the pipette to suck 20 μL supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared thyroid nodule benign and malignant detection library.

[0104] 7. Library quality control

[0105] Use Qubit 4.0 to determine the concentration. Use Agilent 2100 Bioanalyzer to detect the peak type of the library.

[0106] Example 3

[0107] The initial concentration of each amplification primer of the detection gene of gene mutation and the detection gene of gene fusion is 100 μM, which is mixed according to the method of scheme 1 and scheme 2.

[0108] Scheme 1: Equal mass mixing of amplification primers of detection gene of gene mutation, and equal mass mixing of amplification primers of detection gene of gene fusion;

[0109] Scheme 2: Non-equal mass mixing of amplification primers of detection gene of gene mutation, and equal mass mixing of amplification primers of detection gene of gene fusion;

[0110] The amplification primers of the detection gene of gene mutation in scheme 2 are mixed according to the mode of table 6.

[0111] Table 6 Non-equal mass mixing of DNA amplification primers in scheme 2

[0112]

[0113] Comparing the two primer strategies of scheme 1 and scheme 2, the library concentration and primer amplification uniformity are shown in ​ and ​ It can be seen from ​ and ​ that the library concentrations of scheme 1 and scheme 2 are not much different, but the primer amplification uniformity (20 %) of scheme 2 is significantly improved. ​ and ​ Samples 1-10 in and

[0114] are clinical samples 1-10.

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

[0116] Table 7 Detection results of 10 cancer tissue samples by the reagent kit of Example 2

[0117]

[0118] Example 5

[0119] The reagent kit of Example 2 was used to conduct preoperative, postoperative and comparative detection of the reagent kit of Example 2 for 25 clinical samples. The results are shown in Table 8.

[0120] Table 8 Detection results of 25 clinical samples

[0121]

[0122] Based on the comparison of the detection results, the statistics are shown in Table 9.

[0123] Table 9 Comparison of detection results

[0124]

[0125] As shown in Table 8 and Table 9, the detection sensitivity of the reagent kit of 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 benign and malignant thyroid nodules, characterized in that, The method for constructing the library includes 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 of the gene mutation detection gene and the gene fusion detection gene as primers, perform one round of PCR amplification to obtain one round of PCR amplification products. Purify the one round of PCR amplification products. S2: The purified first-round PCR amplification product is amplified with index primers to obtain the second-round PCR amplification product. After purification, the second-round PCR amplification product is obtained. The genes for detecting gene mutations include BRAF, HRAS, KRAS, NRAS, RET, TERT, TP53, PIK3CA, EIF1AX, CTNNB1, IDH1, ATK1, EZH1, SPOP, and ZNF148; the genes for detecting gene fusions include NTRK1, NTRK3, BRAF, RET, ALK, and PPARG. 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; and the TP53 gene detection sites include: G248W / Q, R175 H, R273C / H, C135Y; PIK3CA gene detection sites include: H1047R / L, E542K, E545K; EIF1AX gene detection sites include: A113*; CTNNB1 gene detection sites include: T40I, K49R; IDH1 gene detection sites include: V178I, G70D; AKT1 gene detection sites include: E17K; EZH1 gene detection sites include: Q571R; SPOP gene detection sites include: P94R; ZNF148 gene detection sites include: K52 8fs; 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), NCOOA4(8)-RET(12), NCOOA4(8)-RET(11); ALK gene fusion detection sites include: STRN(3)-ALK(20), EML4(13)-ALK(20), EML4(6)-ALK(20); PPARG gene fusion detection sites include: PAX8(8)-PPARG(2), PAX8(10)-PPARG(2), CREB3L2(2)-PPARG(2); The amplification primers for the gene mutation detection gene are shown in sequences SEQ ID NO.1-SEQ ID NO.48, and the amplification primers for the gene fusion detection gene are shown in sequences SEQ ID NO.49-SEQ ID NO.

82.

2. The thyroid nodule benign / malignant detection library according to claim 1, characterized in that, The final concentration ratio of the amplification primers for detecting gene mutations to the amplification primers for detecting gene fusions is 4:

1.

3. The thyroid nodule benign / malignant detection library according to claim 1, characterized in that, The primers used to amplify the gene fusion detection genes include the following primer pairs: TPM3(7)-NTRK1(10) detection site amplification primer pair, ETV6(4)-NTRK3(14) detection site amplification primer pair, ETV6(5)-NTRK3(15) detection site amplification primer pair, SND1(14)-BRAF(9) detection site amplification primer pair, SND1(14)-BRAF(11) detection site amplification primer pair, CCDC6(1)-RET(12) detection site amplification primer pair, CCDC6(2)-RET(12) detection site amplification primer pair, CCDC6(8)-RET(12) detection site amplification primer pair, and CCDC6 The following primer pairs were mixed of equal quality: (8)-RET(11) detection site amplification primer pairs, NCOA4(8)-RET(12) detection site amplification primer pairs, NCOA4(8)-RET(11) detection site amplification primer pairs, STRN(3)-ALK(20) detection site amplification primer pairs, EML4(13)-ALK(20) detection site amplification primer pairs, EML4(6)-ALK(20) detection site amplification primer pairs, PAX8(8)-PPARG(2) detection site amplification primer pairs, PAX8(10)-PPARG(2) detection site amplification primer pairs, and CREB3L2(2)-PPARG(2) detection site amplification primer pairs.

4. The thyroid nodule benign / malignant detection library according to claim 1, characterized in that, The mass ratio of the sample DNA to the sample RNA was 1:

1.

5. The thyroid nodule benign / malignant detection library according to claim 1, characterized in that, It also includes housekeeping genes for determining whether the amplification primers of the detection genes are working; the housekeeping genes include the POLR2A gene and DDX5, wherein the amplification primer pair of the POLR2A gene includes a forward primer as shown in SEQ ID NO.83 and a reverse primer as shown in SEQ ID NO.84; and the amplification primer pair of the DDX5 includes a forward primer as shown in SEQ ID NO.85 and a reverse primer as shown in SEQ ID NO.86.

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