Primer set, kit and method for detecting fusion gene mutation

Abstract

The present invention discloses a primer set, a kit, and a method for detecting fusion gene mutations. A specific amplification primer set is designed based on 140 pairs of hotspot exons that are prone to breakage in fusion genes commonly found in 15 solid tumors. The specific amplification primer set can cover as many fusion gene partners as possible. Using the specific amplification primers, a single-tube multiplex nested PCR amplification method is used to detect fusion genes, which can reduce primer redundancy, improve primer amplification efficiency, and reduce nonspecific amplification, thereby improving detection accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology technology, and more specifically, the present invention relates to a primer set, a kit and a method for detecting fusion gene mutations. Background Art

[0002] Gene fusion mutation is a common type of genetic abnormality, accounting for approximately 5% to 9%. Gene fusion detection is an important molecular marker for auxiliary diagnosis of cancer patients. For example, the ALK fusion gene mutation has more than 50 fusion partners and is commonly seen in lung cancer (77.8%), colorectal cancer (5.5%), liver cancer (4.2%), gallbladder cancer, melanoma, sarcoma, and other tumors such as inflammatory fibroma (2.8%). The ALK fusion gene is one of the important therapeutic targets for non-small cell lung cancer (NSCLC). To date, a total of 6 ALK-TKIs have been approved for clinical use in China, namely crizotinib, alectinib, ceritinib, brigatinib, lorlatinib, and ensartinib.

[0003] FISH was initially used as the gold standard for fusion gene detection, but FISH is subject to subjective factors, and its results carry a high risk of misjudgment. Furthermore, FISH is based on the detection of gene fusion variants at the DNA level, and can only detect one target at a time. This results in high detection costs, low efficiency, and low sensitivity. NGS technology has improved sensitivity compared to FISH, but NGS technology is also based on the DNA level. Fusion breakpoints typically occur in longer intronic regions, and the fusion breakpoints may vary from patient to patient. Detecting fusion genes at the DNA level inevitably presents the following limitations and challenges: 1) Fusion gene detection must cover very long intronic regions containing a large number of repetitive sequences in order to accurately locate the fusion breakpoints; 2) High GC content prevents probes from effectively capturing target region fragments; and 3) Introns of different genes contain very similar repetitive sequences, a feature that hinders accurate sequence comparison and affects detection accuracy.

[0004] Therefore, it is very necessary to provide a method for detecting fusion gene mutations with higher accuracy. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a primer set, a kit and a method for detecting fusion gene mutations. Using the primer set, accurate detection of 140 pairs of fusion genes and 349 fusion subtype mutations can be achieved.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] The first aspect of the present invention provides a primer set for detecting fusion gene mutations, the primer set comprising:

[0008] A primer pair for detecting the fusion gene ACTB-GLI1, the primer pair comprising a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO.2, a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.4, a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.2, and a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.5;

[0009] A primer pair for detecting the fusion gene AGK-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.6 and a reverse primer shown in SEQ ID NO.7;

[0010] A primer pair for detecting the fusion gene AKAP9-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.8 and a reverse primer shown in SEQ ID NO.9;

[0011] A primer pair for detecting the fusion gene ASPSCR1-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.10 and a reverse primer shown in SEQ ID NO.11, and a forward primer shown in SEQ ID NO.10 and a reverse primer shown in SEQ ID NO.12;

[0012] A primer pair for detecting the fusion gene ATIC-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 13 and a reverse primer shown in SEQ ID NO. 14;

[0013] A primer pair for detecting the BCOR-CCNB3 fusion gene, the primer pair comprising a forward primer shown in SEQ ID NO. 15 and a reverse primer shown in SEQ ID NO. 16;

[0014] A primer pair for detecting the fusion gene BRD3-C15orf55, the primer pair comprising a forward primer shown in SEQ ID NO. 17 and a reverse primer shown in SEQ ID NO. 18;

[0015] A primer pair for detecting the fusion gene BRD4-C15orf55, the primer pair comprising a forward primer represented by SEQ ID NO. 19 and a reverse primer represented by SEQ ID NO. 20, and a forward primer represented by SEQ ID NO. 19 and a reverse primer represented by SEQ ID NO. 18;

[0016] A primer pair for detecting the fusion gene CARS-ALK, the primer pair comprising a forward primer shown in SEQ ID NO.21 and a reverse primer shown in SEQ ID NO.14;

[0017] A primer pair for detecting the fusion gene CCDC6-RET, the primer pair comprising a forward primer shown in SEQ ID NO.22 and a reverse primer shown in SEQ ID NO.23, and a forward primer shown in SEQ ID NO.24 and a reverse primer shown in SEQ ID NO.23;

[0018] A primer pair for detecting the fusion gene CD74-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 25 and a reverse primer shown in SEQ ID NO. 26;

[0019] A primer pair for detecting the fusion gene CDC27-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 27 and a reverse primer shown in SEQ ID NO. 9;

[0020] A primer pair for detecting the fusion gene CDH11-USP6, the primer pair comprising a forward primer shown in SEQ ID NO.28 and a reverse primer shown in SEQ ID NO.29, a forward primer shown in SEQ ID NO.28 and a reverse primer shown in SEQ ID NO.30, a forward primer shown in SEQ ID NO.31 and a reverse primer shown in SEQ ID NO.29, and a forward primer shown in SEQ ID NO.31 and a reverse primer shown in SEQ ID NO.30;

[0021] A primer pair for detecting the fusion gene CHCHD7-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO. 32 and a reverse primer shown in SEQ ID NO. 33, and a forward primer shown in SEQ ID NO. 32 and a reverse primer shown in SEQ ID NO. 34;

[0022] A primer pair for detecting the fusion gene CIC-DUX4, the primer pair comprising a forward primer shown in SEQ ID NO. 35 and a reverse primer shown in SEQ ID NO. 36;

[0023] A primer pair for detecting the fusion gene CIC-FOXO4, the primer pair comprising a forward primer shown in SEQ ID NO.35 and a reverse primer shown in SEQ ID NO.36, and a forward primer shown in SEQ ID NO.37 and a reverse primer shown in SEQ ID NO.38;

[0024] A primer pair for detecting the fusion gene CIC-FOXO4, the primer pair comprising a forward primer shown in SEQ ID NO. 39 and a reverse primer shown in SEQ ID NO. 38;

[0025] A primer pair for detecting the fusion gene CLCN6-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.40 and a reverse primer shown in SEQ ID NO.41;

[0026] A primer pair for detecting the fusion gene CLTC-ALK, the primer pair comprising a forward primer shown in SEQ ID NO.42 and a reverse primer shown in SEQ ID NO.43;

[0027] A primer pair for detecting the fusion gene CLTC-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.44 and a reverse primer shown in SEQ ID NO.12;

[0028] A primer pair for detecting the fusion gene CNBP-USP6, the primer pair comprising a forward primer shown in SEQ ID NO.45 and a reverse primer shown in SEQ ID NO.29, and a forward primer shown in SEQ ID NO.45 and a reverse primer shown in SEQ ID NO.30;

[0029] A primer pair for detecting the fusion gene COL1A1-PDGFB, the primer pair comprising a forward primer shown in SEQ ID NO.46 and a reverse primer shown in SEQ ID NO.47, and a forward primer shown in SEQ ID NOs.48 to 73 and a reverse primer shown in SEQ ID NO.47;

[0030] A primer pair for detecting the fusion gene COL1A1-USP6, comprising a forward primer shown in SEQ ID NO. 74 and reverse primers shown in SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 75;

[0031] A primer pair for detecting the fusion gene COL1A2-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO. 76 and a reverse primer represented by SEQ ID NO. 33, and a forward primer represented by SEQ ID NO. 76 and a reverse primer represented by SEQ ID NO. 34;

[0032] A primer pair for detecting the fusion gene CRTC1-MAML2, the primer pair comprising a forward primer of SEQ ID NO. 77 and a reverse primer of SEQ ID NO. 78, and a forward primer of SEQ ID NO. 77 and a reverse primer of SEQ ID NO. 79;

[0033] A primer pair for detecting the fusion gene CRTC3-MAML2, the primer pair comprising a forward primer shown in SEQ ID NO.80 and a reverse primer shown in SEQ ID NO.79;

[0034] A primer pair for detecting the fusion gene CTNNB1-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO.81 and a reverse primer shown in SEQ ID NO.33, and a forward primer shown in SEQ ID NO.81 and a reverse primer shown in SEQ ID NO.34;

[0035] A primer pair for detecting the fusion gene DNAJB1-PRKACA, the primer pair comprising a forward primer shown in SEQ ID NO.82 and a reverse primer shown in SEQ ID NO.83, and a forward primer shown in SEQ ID NO.84 and a reverse primer shown in SEQ ID NO.85;

[0036] A primer pair for detecting the fusion gene EPC1-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.86 and a reverse primer shown in SEQ ID NO.87;

[0037] A primer pair for detecting the fusion gene ERC1-RET, the primer pair comprising forward primers shown in SEQ ID NOs. 88 to 92 and a reverse primer shown in SEQ ID NO. 23;

[0038] A primer pair for detecting the fusion gene ETV6-NTRK3, the primer pair comprising a forward primer shown in SEQ ID NO.93 and a reverse primer shown in SEQ ID NO.94, and a forward primer shown in SEQ ID NO.95 and a reverse primer shown in SEQ ID NO.94;

[0039] A primer pair for detecting the fusion gene EWSR1-ATF1, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.97, a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.98, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.98, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.100, and a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.97;

[0040] A primer pair for detecting the fusion gene EWSR1-CREB1, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.102;

[0041] A primer pair for detecting the fusion gene EWSR1-DDIT3, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.105, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.103;

[0042] A primer pair for detecting the fusion gene EWSR1-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.106, a forward primer shown in SEQ ID NO.107 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.106, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.109;

[0043] A primer pair for detecting the fusion gene EWSR1-ETV1, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.110;

[0044] A primer pair for detecting the fusion gene EWSR1-ETV4, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.111;

[0045] A primer pair for detecting the fusion gene EWSR1-FEV, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.112, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.112;

[0046] A primer pair for detecting the fusion gene EWSR1-FLI1, the primer pair comprising a forward primer represented by SEQ ID NO.96 and a reverse primer represented by SEQ ID NOs.113 to 116, a forward primer represented by SEQ ID NO.99 and a reverse primer represented by SEQ ID NOs.113 to 117, a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.113, a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.115, a forward primer represented by SEQ ID NO.96 and a reverse primer represented by SEQ ID NO.118, a forward primer represented by SEQ ID NO.96 and a reverse primer represented by SEQ ID NO.115, and a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.113;

[0047] A primer pair for detecting the fusion gene EWSR1-NFATC2, the primer pair comprising a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.119;

[0048] A primer pair for detecting the fusion gene EWSR1-NR4A3, the primer pair comprising a forward primer shown in SEQ ID NO.120 and a reverse primer shown in SEQ ID NO.121, a forward primer shown in SEQ ID NO.122 and a reverse primer shown in SEQ ID NO.121, a forward primer shown in SEQ ID NO.122 and a reverse primer shown in SEQ ID NO.123, a forward primer shown in SEQ ID NO.124 and a reverse primer shown in SEQ ID NO.123, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.123, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.121;

[0049] A primer pair for detecting the fusion gene EWSR1-PATZ1, the primer pair comprising a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.125;

[0050] A primer pair for detecting the fusion gene EWSR1-PBX1, the primer pair comprising a forward primer represented by SEQ ID NO.99 and a reverse primer represented by SEQ ID NO.126, and a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.126;

[0051] A primer pair for detecting the fusion gene EWSR1-POU5F1, the primer pair comprising a forward primer set forth in SEQ ID NO.107 and a reverse primer set forth in SEQ ID NO.127, a forward primer set forth in SEQ ID NO.99 and a reverse primer set forth in SEQ ID NO.127, and a forward primer set forth in SEQ ID NO.99 and a reverse primer set forth in SEQ ID NO.128;

[0052] A primer pair for detecting the fusion gene EWSR1-SP3, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.129, and a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.130;

[0053] A primer pair for detecting the fusion gene EWSR1-WT1, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.131, a forward primer shown in SEQ ID NO.132 and a reverse primer shown in SEQ ID NO.133, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.131, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.134, a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.133, a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.131, and a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.131;

[0054] A primer pair for detecting the fusion gene EWSR1-ZNF444, the primer pair comprising a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.135, and a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.136;

[0055] A primer pair for detecting the fusion gene FAM131B-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.137 and a reverse primer shown in SEQ ID NO.9, and a forward primer shown in SEQ ID NO.138 and a reverse primer shown in SEQ ID NO.9;

[0056] A primer pair for detecting the fusion gene FCHSD1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.139 and a reverse primer shown in SEQ ID NO.9;

[0057] A primer pair for detecting the fusion gene FGFR1-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO.140 and a reverse primer represented by SEQ ID NO.33, and a forward primer represented by SEQ ID NO.141 and a reverse primer represented by SEQ ID NO.33;

[0058] A primer pair for detecting the fusion gene FCHSD1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.139 and a reverse primer shown in SEQ ID NO.9;

[0059] A primer pair for detecting the fusion gene FUS-ATF1, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.98;

[0060] A primer pair for detecting the fusion gene FUS-CREB3L2, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.143, a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.144, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.146, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.143, a forward primer shown in SEQ ID NO.147 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.148 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.149 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.150 and a reverse primer shown in SEQ ID NO. The reverse primer shown in SEQ ID NO.103, the forward primer shown in SEQ ID NO.145, and the reverse primer shown in SEQ ID NO.103;

[0061] A primer pair for detecting the fusion gene FUS-DDIT3, the primer pair comprising a forward primer shown in SEQ ID NO.151 and a reverse primer shown in SEQ ID NO.105;

[0062] A primer pair for detecting the fusion gene FUS-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.106, a forward primer shown in SEQ ID NO.152 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.109, and a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.106;

[0063] A primer pair for detecting the GNAI1-BRAF fusion gene, the primer pair comprising a forward primer represented by SEQ ID NO. 153 and a reverse primer represented by SEQ ID NO. 9;

[0064] A primer pair for detecting the fusion gene GOLGA5_PTC5-RET, the primer pair comprising a forward primer shown in SEQ ID NO.154 and a reverse primer shown in SEQ ID NO.23;

[0065] A primer pair for detecting the fusion gene HAS2-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO.155 and a reverse primer shown in SEQ ID NO.33;

[0066] A primer pair for detecting the fusion gene HERPUD1-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.156 and a reverse primer shown in SEQ ID NO.157;

[0067] A primer pair for detecting the fusion gene HEY1-NCOA2, the primer pair comprising a forward primer shown in SEQ ID NO.158 and a reverse primer shown in SEQ ID NO.159;

[0068] A primer pair for detecting the fusion gene HMGA2-ALDH2, the primer pair comprising a forward primer shown in SEQ ID NO. 160 and a reverse primer shown in SEQ ID NO. 161;

[0069] A primer pair for detecting the fusion gene HMGA2-CCNB1IP1, the primer pair comprising a forward primer shown in SEQ ID NO.162 and a reverse primer shown in SEQ ID NO.163;

[0070] A primer pair for detecting the fusion gene HMGA2-COX6C, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.164;

[0071] A primer pair for detecting the fusion gene HMGA2-EBF1, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.165;

[0072] A primer pair for detecting the fusion gene HMGA2-FHIT, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.166;

[0073] A primer pair for detecting the fusion gene HMGA2-LHFP, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.167;

[0074] A primer pair for detecting the fusion gene HMGA2-LPP, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.168, and a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.169;

[0075] A primer pair for detecting the fusion gene HMGA2-NFIB, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.170;

[0076] A primer pair for detecting the fusion gene HMGA2-RAD51B, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.171;

[0077] A primer pair for detecting the fusion gene HMGA2-WIF1, the primer pair comprising a forward primer shown in SEQ ID NO. 160 and reverse primers shown in SEQ ID NOs. 172 to 175, and a forward primer shown in SEQ ID NO. 176 and a reverse primer shown in SEQ ID NO. 173;

[0078] A primer pair for detecting the fusion gene JAZF1-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.177 and a reverse primer shown in SEQ ID NO.87;

[0079] A primer pair for detecting the fusion gene JAZF1-SUZ12, the primer pair comprising a forward primer shown in SEQ ID NO. 177 and a reverse primer shown in SEQ ID NO. 178;

[0080] A primer pair for detecting the fusion gene KCTD7-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 179 and a reverse primer represented by SEQ ID NO. 7;

[0081] A primer pair for detecting the fusion gene KIAA1549-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.180 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.181 and a reverse primer shown in SEQ ID NO.41, a forward primer shown in SEQ ID NO.181 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.183, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.41, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.184 and a reverse primer shown in SEQ ID NO.9, and a forward primer shown in SEQ ID NO.185 and a reverse primer shown in SEQ ID NO.9;

[0082] A primer pair for detecting the fusion gene KTN1-RET, the primer pair comprising a forward primer shown in SEQ ID NO.186 and a reverse primer shown in SEQ ID NO.23;

[0083] A primer pair for detecting the fusion gene LIFR-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO. 187 and a reverse primer represented by SEQ ID NO. 33, and a forward primer represented by SEQ ID NO. 187 and a reverse primer represented by SEQ ID NO. 34;

[0084] A primer pair for detecting the fusion gene LMNA-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.188 and a reverse primer shown in SEQ ID NO.189;

[0085] A primer pair for detecting the fusion gene MACF1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO.190 and a reverse primer represented by SEQ ID NO.9, and a forward primer represented by SEQ ID NO.191 and a reverse primer represented by SEQ ID NO.9;

[0086] A primer pair for detecting the fusion gene MEAF6-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.192 and a reverse primer shown in SEQ ID NO.87;

[0087] A primer pair for detecting the fusion gene MIR548F1-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 193 and a reverse primer shown in SEQ ID NO. 189;

[0088] A primer pair for detecting the fusion gene MKRN1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 194 and a reverse primer shown in SEQ ID NO. 41;

[0089] A primer pair for detecting the fusion gene MPRIP-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.195 and a reverse primer shown in SEQ ID NO.26, a forward primer shown in SEQ ID NO.196 and a reverse primer shown in SEQ ID NO.26, a forward primer shown in SEQ ID NO.197 and a reverse primer shown in SEQ ID NO.26, and a forward primer shown in SEQ ID NO.197 and a reverse primer shown in SEQ ID NO.198;

[0090] A primer pair for detecting the fusion gene MYB-NFIB, the primer pair comprising a forward primer shown in SEQ ID NO.199 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.199 and a reverse primer shown in SEQ ID NO.170, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.202, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.170, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.203, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.204, a forward primer shown in SEQ ID NO.205 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.205 and a reverse primer shown in SEQ ID NO.170, and a reverse primer shown in SEQ ID NO. The forward primer shown in SEQ ID NO.205 and the reverse primer shown in SEQ ID NO.204;

[0091] A primer pair for detecting the fusion gene MYH9-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 206 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 206 and a reverse primer represented by SEQ ID NO. 30;

[0092] A primer pair for detecting the fusion gene NAB2-STAT6, the primer pair comprising a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.208, a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.209, a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.210, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.212, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.208, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.213, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.214, a forward primer shown in SEQ ID NO.215 and a reverse primer shown in SEQ ID NO.216, a forward primer shown in SEQ ID NO.217 and a reverse primer shown in SEQ ID NO.218, a forward primer shown in SEQ ID NO.219 and a reverse primer shown in SEQ ID NO.220, The forward primer shown in SEQ ID NO. 217 and the reverse primer shown in SEQ ID NO. 216, the forward primer shown in SEQ ID NO. 217 and the reverse primer shown in SEQ ID NO. 218, and the forward primer shown in SEQ ID NO. 219 and the reverse primer shown in SEQ ID NO. 208;

[0093] A primer pair for detecting the fusion gene NACC2-NTRK2, the primer pair comprising a forward primer shown in SEQ ID NO.220 and a reverse primer shown in SEQ ID NO.221;

[0094] A primer pair for detecting the fusion gene NCOA4-RET, the primer pair comprising a forward primer shown in SEQ ID NO.222 and a reverse primer shown in SEQ ID NO.23, a forward primer shown in SEQ ID NO.223 and a reverse primer shown in SEQ ID NO.224, a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.23, a forward primer shown in SEQ ID NO.226 and a reverse primer shown in SEQ ID NO.227, a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.228, and a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.23;

[0095] A primer pair for detecting the fusion gene NDRG1-ERG, the primer pair comprising a forward primer represented by SEQ ID NO.229 and a reverse primer represented by SEQ ID NO.157;

[0096] A primer pair for detecting the fusion gene NFASC-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 230 and a reverse primer represented by SEQ ID NO. 189;

[0097] A primer pair for detecting the fusion gene NONO-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.231 and a reverse primer shown in SEQ ID NO.12;

[0098] A primer pair for detecting the fusion gene NUDCD3-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 232 and a reverse primer shown in SEQ ID NO. 9;

[0099] A primer pair for detecting the fusion gene OMD-USP6, the primer pair comprising a forward primer shown in SEQ ID NO. 233 and a reverse primer shown in SEQ ID NO. 29;

[0100] A primer pair for detecting the fusion gene PAPSS1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 234 and a reverse primer represented by SEQ ID NO. 9;

[0101] A primer pair for detecting the fusion gene PAX3-AFX, the primer pair comprising a forward primer shown in SEQ ID NO. 235 and a reverse primer shown in SEQ ID NO. 38;

[0102] A primer pair for detecting the fusion gene PAX3-FOXO1, the primer pair comprising a forward primer shown in SEQ ID NO. 235 and a reverse primer shown in SEQ ID NO. 236;

[0103] A primer pair for detecting the fusion gene PAX3-NCOA1, the primer pair comprising a forward primer of SEQ ID NO. 237 and a reverse primer of SEQ ID NO. 238, a forward primer of SEQ ID NO. 235 and a reverse primer of SEQ ID NO. 239, and a forward primer of SEQ ID NO. 235 and a reverse primer of SEQ ID NO. 240;

[0104] A primer pair for detecting the fusion gene PAX7-FOXO1, the primer pair comprising a forward primer shown in SEQ ID NO. 241 and a reverse primer shown in SEQ ID NO. 236;

[0105] A primer pair for detecting the fusion gene PAX8-PPARG, the primer pair comprising a forward primer represented by SEQ ID NO. 242 and a reverse primer represented by SEQ ID NO. 243, a forward primer represented by SEQ ID NO. 242 and a reverse primer represented by SEQ ID NO. 244, and forward primers represented by SEQ ID NOs. 245 to 247 and a reverse primer represented by SEQ ID NO. 244;

[0106] A primer pair for detecting the fusion gene PLIN3-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 248 and a reverse primer shown in SEQ ID NO. 9;

[0107] A primer pair for detecting the fusion gene POR-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.249 and a reverse primer shown in SEQ ID NO.41;

[0108] A primer pair for detecting the fusion gene PRCC-TFE3, the primer pair comprising a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.251, a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.11, a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.12, and a forward primer represented by SEQ ID NO.252 and a reverse primer represented by SEQ ID NO.251;

[0109] A primer pair for detecting the fusion gene PRKAR1A-RET, the primer pair comprising forward primers shown in SEQ ID NOs. 253-254 and a reverse primer shown in SEQ ID NO. 23;

[0110] A primer pair for detecting the fusion gene QKI-NTRK2, the primer pair comprising a forward primer shown in SEQ ID NO.255 ​​and a reverse primer shown in SEQ ID NO.256;

[0111] A primer pair for detecting the fusion gene RANBP2-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 257 and a reverse primer represented by SEQ ID NO. 14;

[0112] A primer pair for detecting the fusion gene RNF130-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 258 and a reverse primer shown in SEQ ID NO. 9;

[0113] A primer pair for detecting the fusion gene SEC31A-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 259 and a reverse primer represented by SEQ ID NO. 43;

[0114] A primer pair for detecting the fusion gene SERPINE1-FOSB, the primer pair comprising a forward primer shown in SEQ ID NO. 260 and reverse primers shown in SEQ ID NOs. 261 to 262;

[0115] A primer pair for detecting the fusion gene SFPQ-TFE3, the primer pair comprising a forward primer represented by SEQ ID NO.263 and a reverse primer represented by SEQ ID NO.11, a forward primer represented by SEQ ID NO.264 and a reverse primer represented by SEQ ID NO.11, and a forward primer represented by SEQ ID NO.264 and a reverse primer represented by SEQ ID NO.12;

[0116] A primer pair for detecting the fusion gene SLC45A3-BRAF, the primer pair comprising a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 7, a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 9, and a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 157;

[0117] A primer pair for detecting the fusion gene SND1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 266 and a reverse primer represented by SEQ ID NO. 9;

[0118] A primer pair for detecting the SOX6-BRAF fusion gene, the primer pair comprising a forward primer shown in SEQ ID NO. 267 and a reverse primer shown in SEQ ID NO. 9;

[0119] A primer pair for detecting the fusion gene SQSTM1-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 268 and a reverse primer represented by SEQ ID NO. 189;

[0120] A primer pair for detecting the fusion gene SS18-SSX1, the primer pair comprising a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.270, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.271, a forward primer shown in SEQ ID NO.272 and a reverse primer shown in SEQ ID NO.273, a forward primer shown in SEQ ID NO.272 and a reverse primer shown in SEQ ID NO.270, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.274, a forward primer shown in SEQ ID NO.275 and a reverse primer shown in SEQ ID NO.274, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.276, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.277, and a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO. Reverse primer shown in No. 278;

[0121] A primer pair for detecting the fusion gene SSBP2-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 279 and a reverse primer represented by SEQ ID NO. 26;

[0122] A primer pair for detecting the fusion gene STRN-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 280 and a reverse primer shown in SEQ ID NO. 14;

[0123] A primer pair for detecting the fusion gene TAF15-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO.281 and a reverse primer represented by SEQ ID NO.123;

[0124] A primer pair for detecting the fusion gene TAX1BP1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 282 and a reverse primer represented by SEQ ID NO. 41;

[0125] A primer pair for detecting the TCEA1-PLAG1 fusion gene, the primer pair comprising a forward primer represented by SEQ ID NO. 283 and a reverse primer represented by SEQ ID NO. 34;

[0126] A primer pair for detecting the fusion gene TCF12-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO. 284 and a reverse primer represented by SEQ ID NO. 123;

[0127] A primer pair for detecting the fusion gene TFG-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO.285 and a reverse primer represented by SEQ ID NO.123;

[0128] A primer pair for detecting the fusion gene TFG-NTRK1, the primer pair comprising a forward primer set forth in SEQ ID NO. 286 and a reverse primer set forth in SEQ ID NO. 189, a forward primer set forth in SEQ ID NO. 286 and a reverse primer set forth in SEQ ID NO. 287, and a forward primer set forth in SEQ ID NO. 288 and a reverse primer set forth in SEQ ID NO. 189;

[0129] A primer pair for detecting the fusion gene THRAP3-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 75;

[0130] A primer pair for detecting the fusion gene THRAP3-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 75;

[0131] A primer pair for detecting the fusion gene TMPRSS2-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.291, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.292, a forward primer shown in SEQ ID NO.293 and a reverse primer shown in SEQ ID NO.291, a forward primer shown in SEQ ID NO.293 and a reverse primer shown in SEQ ID NO.294, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.295 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.296 and a reverse primer shown in SEQ ID NO.157, and a forward primer shown in SEQ ID NO.297 and a reverse primer shown in SEQ ID NO. Reverse primer shown in NO.157;

[0132] A primer pair for detecting the fusion gene TP53-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 298 and a reverse primer represented by SEQ ID NO. 287;

[0133] A primer pair for detecting the fusion gene TPM3-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 299 and a reverse primer represented by SEQ ID NO. 14;

[0134] A primer pair for detecting the fusion gene TPM3-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 299 and a reverse primer shown in SEQ ID NO. 190;

[0135] A primer pair for detecting the fusion gene TPM4-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 300 and a reverse primer shown in SEQ ID NO. 14;

[0136] A primer pair for detecting the fusion gene TPR-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.301 and a reverse primer shown in SEQ ID NO.190;

[0137] A primer pair for detecting the fusion gene TRIM24-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.302 and a reverse primer shown in SEQ ID NO.7, a forward primer shown in SEQ ID NO.303 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.304 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.305 and a reverse primer shown in SEQ ID NO.23, and a forward primer shown in SEQ ID NO.304 and a reverse primer shown in SEQ ID NO.23;

[0138] A primer pair for detecting the fusion gene WWTR1-CAMTA1, the primer pair comprising a forward primer represented by SEQ ID NO. 306 and a reverse primer represented by SEQ ID NO. 307, and a forward primer represented by SEQ ID NO. 306 and a reverse primer represented by SEQ ID NO. 308;

[0139] A primer pair for detecting the fusion gene YAP1-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.309 and a reverse primer shown in SEQ ID NO.251;

[0140] A primer pair for detecting the fusion gene YWHAE-FAM22A, the primer pair comprising a forward primer shown in SEQ ID NO.310 and a reverse primer shown in SEQ ID NO.311;

[0141] A primer pair for detecting the fusion gene ZC3H7B-BCOR, the primer pair comprising a forward primer shown in SEQ ID NO.312 and a reverse primer shown in SEQ ID NO.313;

[0142] A primer pair for detecting the fusion gene ZKSCAN5-BRAF, wherein the primer pair includes a forward primer shown in SEQ ID NO.314 and a reverse primer shown in SEQ ID NO.9.

[0143] The second aspect of the present invention is to provide a kit for detecting fusion gene mutations.

[0144] The kit for detecting fusion gene variation includes the above-mentioned primer set for detecting fusion gene variation.

[0145] The third aspect of the present invention is to provide a method for detecting fusion gene mutations.

[0146] The method for detecting fusion gene variation comprises the following steps: using the cDNA of the sample to be tested as a template and using the above-mentioned kit for detecting fusion gene variation to perform nested PCR amplification.

[0147] In the present invention, a specific amplification primer set was designed based on the hotspot exons that are prone to breakage in 140 pairs of fusion genes (including core genes and gene partners fused to core genes) commonly found in 15 solid tumors. The specific amplification primer set can cover as many fusion gene partners as possible; using the specific amplification primers, a single-tube multiplex nested PCR amplification method was adopted to detect the fusion genes, which can reduce primer redundancy, improve primer amplification efficiency, and make the detection more accurate. DETAILED DESCRIPTION

[0148] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0150] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0151] In some embodiments of the present invention, a primer set for detecting fusion gene mutation is disclosed. The primer set sequence is shown in Table 2, and the fusion gene targeted is shown in Table 1.

[0152] In some embodiments, a universal primer as shown in SEQ ID NO. 335 is added to the 5' end of the forward primer of the primer pair, and a universal primer as shown in SEQ ID NO. 336 is added to the 5' end of the reverse primer of the primer pair.

[0153] In other embodiments of the present invention, a kit for detecting fusion gene variation is provided, wherein the kit comprises the above-mentioned primer set for detecting fusion gene variation.

[0154] In some embodiments, the kit further comprises a primer set for detecting internal reference genes, wherein the internal reference genes include ITGB7, MRPL13, HPRT1, TBP, JUN, MYC, IPO8, LRP1, HMBS, and YWHAZ.

[0155] In some embodiments, the primer set for detecting the internal reference gene includes: a primer pair for detecting ITGB7, the primer pair including the forward primer shown in SEQ ID NO.315 and the reverse primer shown in SEQ ID NO.316; a primer pair for detecting MRPL13, the primer pair including the forward primer shown in SEQ ID NO.317 and the reverse primer shown in SEQ ID NO.318; a primer pair for detecting HPRT1, the primer pair including the forward primer shown in SEQ ID NO.319 and the reverse primer shown in SEQ ID NO.320; a primer pair for detecting TBP, the primer pair including the forward primer shown in SEQ ID NO.321 and the reverse primer shown in SEQ ID NO.322; a primer pair for detecting JUN, the primer pair including the forward primer shown in SEQ ID NO.323 and the reverse primer shown in SEQ ID NO.324; a primer pair for detecting MYC, the primer pair including the forward primer shown in SEQ ID NO.325 and the reverse primer shown in SEQ ID NO. NO.326; a primer pair for detecting IPO8, the primer pair comprising the forward primer shown in SEQ ID NO.327 and the reverse primer shown in SEQ ID NO.328; a primer pair for detecting LRP1, the primer pair comprising the forward primer shown in SEQ ID NO.329 and the reverse primer shown in SEQ ID NO.330; a primer pair for detecting HMBS, the primer pair comprising the forward primer shown in SEQ ID NO.331 and the reverse primer shown in SEQ ID NO.332; and a primer pair for detecting YWHAZ, the primer pair comprising the forward primer shown in SEQ ID NO.333 and the reverse primer shown in SEQID NO.334.

[0156] In other embodiments of the present invention, a method for detecting fusion gene variation is also provided, comprising the following steps: using the cDNA of the sample to be tested as a template and using the above-mentioned kit for detecting fusion gene variation to perform multiple nested PCR amplification.

[0157] In some embodiments, the reaction system of the multiplex nested PCR includes: 5X HiFi Mix 3-5 μL, primer premix 3-5 μL, and cDNA 11-13 μL; the primer premix includes a primer set for detecting the fusion gene and a primer set for detecting the internal reference gene in a volume ratio of 0.8-1.2::0.8-1.2.

[0158] In some embodiments, the reaction procedure of the multiplex nested PCR includes: 99° C., 2 min; 99° C., 15 s, 60° C., 4 min, 30 cycles; 10° C., insulation.

[0159] The present invention is described in detail below with reference to specific embodiments.

[0160] Example 1 Primer pairs for detecting fusion gene mutations

[0161] The following steps are involved:

[0162] 1. Screening of fusion genes and fusion gene partners

[0163] Using data from the COSMIC Fusion Curation database, the TCGA database, and research literature, we screened 140 common fusion gene pairs across 15 solid tumors, including soft tissue sarcoma (Sarcoma), perivascular epithelioid cell tumor (PEComa), extraskeletal myxoid chondrosarcoma, bone cancer, and renal cell carcinoma. These fusion genes included 61 core fusion genes, including ALK, BRAF, and RET. Also included were 98 previously reported fusion gene partners, i.e., another gene fused to the core gene, such as the TPM3 / TPM4 gene fusion with ALK. The specific fusion genes and fusion gene partners are shown in Table 1.

[0164] Table 1

[0165]

[0166]

[0167]

[0168] 2. Design specific primer pairs for fusion genes

[0169] Based on the transcript sequence of the fusion gene, specific amplification primers for the breakpoint region of the fusion gene are designed and synthesized, wherein the forward primer and reverse primer match the fusion partner on both sides of the fusion gene breakpoint and its core fusion gene, respectively.

[0170] ①. Screen the hotspot exons where the fusion gene breaks according to the database, and obtain the sequence of the hotspot exons as a template for the design of specific primers.

[0171] ② Design and synthesize specific amplification primers for the breakpoint region of the fusion gene. The primer sequences are shown in Table 2. In Table 2, fusion gene subtypes are named as partner gene (located exon) - core gene (located exon).

[0172] Table 2

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] ③. Add universal primer A (CTTGACTCCGCCCCCTTATC, SEQ ID NO. 335) and universal primer B (CTAGTGACGCTGTGTCCTCC, SEQ ID NO. 336) to the 5' end of the forward primer and reverse primer shown in Table 2, respectively.

[0190] 3. Design specific primers for internal reference genes

[0191] Ten housekeeping genes were selected as internal reference genes, and specific primers were designed using their transcripts as templates. The reference genes were: ITGB7, MRPL13, HPRT1, TBP, JUN, MYC, IPO8, LRP1, HMBS, and YWHAZ. The specific primer sequences are shown in Table 3.

[0192] Table 3

[0193]

[0194]

[0195] Example 2 Method for Detecting Fusion Gene Mutations

[0196] Detection of fusion gene mutation using the primers in Table 2 and Table 3 of Example 1 specifically includes the following steps:

[0197] (1) MAGMAX FFPE paraffin embedding extraction reagent (Thermo Fisher Scientific) was used to extract RNA from the samples to be tested (paraffin block tissue, fresh tissue, peripheral blood, bone marrow blood, and other biological samples);

[0198] (2) RNA was reverse transcribed into cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0199] Table 4

[0200]

[0201] Table 5

[0202] Reaction procedure [25℃, 10min]; [42℃, 30min]; [85℃, 5min]; [10℃, keep warm]

[0203] (3) Mix equal volumes of fusion gene breakpoint-specific primers (add universal primer A to the 5' end of the forward primer and universal primer B to the 5' end of the reverse primer) and internal reference gene primers to obtain a primer premix (5 pmol / μL).

[0204] (4) Construction of fusion gene library

[0205] Using IonAmpliSeq TM Library construction was performed using the PCR-based Library Kit 2.0 (Thermo Fisher Scientific) reagents. Fusion-gene-specific primers were used to enrich the target fusion gene. When the target gene undergoes fusion and transcription to produce transcripts, it can be amplified by the specific primers; if fusion does not occur, there is no amplification. Simultaneously, universal primers A and B will amplify the fusion gene amplification product using it as a template, effectively amplifying the abundance of the fusion gene amplification product and thus increasing the sensitivity of fusion gene detection. The PCR reaction system and reaction procedure are shown in Tables 6 and 7.

[0206] Table 6

[0207] Components System (μL) <![CDATA[5X Ion AmpliSeq TM HiFi Mix]]> 4.0 Primer premix 4.0 cDNA 12

[0208] Table 7

[0209] program [99℃, 2min]; [99℃, 15s, 60℃, 4min; 30 cycles]; [10℃, keep warm]

[0210] (5) Primer digestion

[0211] Add 2uL Fupa (Ion AmpliSeq TMLibrary Kit 2.0, Thermo Fisher Scientific); operating program: [50°C, 10 min]; [55°C, 10 min]; [60°C, 20 min]; [10°C, keep warm].

[0212] (6) Sequencing

[0213] The reagents used were Ion AmpliSeq TM Library Kit 2.0, Thermo Fisher Scientific.

[0214] a. Connect sequencing adapters and sample tags at both ends of the amplified product

[0215] The ligation reagents are shown in Table 8, and the ligation reaction is shown in Table 9.

[0216] Table 8

[0217] Components System (μL) Switch Solution 4 DNA Ligase 2 connector 2 Primer digestion products 22

[0218] Table 9

[0219] Reaction procedure [22℃, 30min]; [68℃, 5min]; [72℃, 5min]; [10℃, keep warm]

[0220] b. After the program is completed, perform 1.8x magnetic bead purification using 50ul PCR SuperMix HighFidelity elution.

[0221] c. Amplify the purified product. The amplification system is shown in Table 10, and the amplification program is shown in Table 11.

[0222] Table 10

[0223] Components System (μL) Library Amplification Primer Mix 2 Previous step eluent 48

[0224] Table 11

[0225] Reaction procedure [98℃, 2min]; [98℃, 15sec; 60℃, 1min; 6 cycles]; [10℃, keep warm]

[0226] d. Add 25 μL Mix the magnetic beads and take the supernatant; add 50 μL Magnetic bead purification; 25ul low TE elution.

[0227] e. Using Ion PGM TM Hi-Q TM The library was sequenced using the View OT2 Kit. The sequencing data was cleaned and aligned with the fusion gene reference sequence for identification, identifying the fusion gene, its fusion partner, the exon where the breakpoint was located, and the fusion gene subtype.

[0228] Example 3 Accuracy of the method of the present invention

[0229] Using the method of Example 2, 17 samples with known fusion genes from the College of American Pathologists' external quality control review and 14 clinical patient samples with known fusion genes detected by FISH were analyzed. The results for 30 of these samples were consistent with expectations. One clinical sample, which was negative for the fusion gene by FISH, tested positive for the EWSR1-WT1.COSF226 fusion gene using the method of the present invention. This sample was confirmed to be positive for the EWSR1-WT1.COSF226 fusion gene using Sanger sequencing. Specific results are shown in Table 13.

[0230] Table 13

[0231]

[0232]

[0233] As shown in Table 13, the method of the present invention has extremely high accuracy and better sensitivity than traditional methods.

[0234] Example 4 Sensitivity of the method of the present invention

[0235] Using the method of Example 2, one negative clinical patient sample (NT21R0005) and two positive clinical patient samples (NT21R0037, carrying COL1A1-PDGFB; NT21R0036, carrying BCOR-CCNB3) were mixed and diluted to create three gradient reference samples with tumor contents of 1% (PanFusion_L1), 5% (PanFusion_L5), and 15% (PanFusion_L15), respectively. Repeat testing was then performed, with five replicate wells set for each reference sample. The specific results are shown in Table 14.

[0236] Table 14

[0237]

[0238]

[0239] From the results in Table 14, it can be seen that the sensitivity of the method of the present invention is 1% of the tumor content ratio. This method has extremely high sensitivity and is better than the traditional method (FISH: 10% to 20%).

[0240] Amplification efficiency of the experiment example with and without universal primers A / B added to both ends of the specific primers

[0241] Using an internal reference gene as the evaluation target, five samples were analyzed using the reaction system and procedure of Example 2. The amplification efficiency was evaluated with and without universal primers A / B added to both ends of the fusion gene-specific primers. A higher amplification product concentration indicates a higher amplification efficiency. The results are shown in Table 12.

[0242] Table 12

[0243]

[0244] As can be seen from Table 12, when universal primers A / B are added, the library concentration is higher, indicating that the amplification efficiency of the primers can be improved by nested amplification, thereby achieving the purpose of improving detection sensitivity.

[0245] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0246] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A primer set for detecting fusion gene mutation, characterized in that: The primer set includes: A primer pair for detecting the fusion gene ACTB-GLI1, the primer pair comprising a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO.2, a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.4, a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.2, and a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO.5; A primer pair for detecting the fusion gene AGK-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.6 and a reverse primer shown in SEQ ID NO.7; A primer pair for detecting the fusion gene AKAP9-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.8 and a reverse primer shown in SEQ ID NO.9; A primer pair for detecting the fusion gene ASPSCR1-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.10 and a reverse primer shown in SEQ ID NO.11, and a forward primer shown in SEQ ID NO.10 and a reverse primer shown in SEQ ID NO.12; A primer pair for detecting the fusion gene ATIC-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 13 and a reverse primer shown in SEQ ID NO. 14; A primer pair for detecting the BCOR-CCNB3 fusion gene, the primer pair comprising a forward primer shown in SEQ ID NO. 15 and a reverse primer shown in SEQ ID NO. 16; A primer pair for detecting the fusion gene BRD3-C15orf55, the primer pair comprising a forward primer shown in SEQ ID NO. 17 and a reverse primer shown in SEQ ID NO. 18; A primer pair for detecting the fusion gene BRD4-C15orf55, the primer pair comprising a forward primer represented by SEQ ID NO. 19 and a reverse primer represented by SEQ ID NO. 20, and a forward primer represented by SEQ ID NO. 19 and a reverse primer represented by SEQ ID NO. 18; A primer pair for detecting the fusion gene CARS-ALK, the primer pair comprising a forward primer shown in SEQ ID NO.21 and a reverse primer shown in SEQ ID NO.14; A primer pair for detecting the fusion gene CCDC6-RET, the primer pair comprising a forward primer shown in SEQ ID NO.22 and a reverse primer shown in SEQ ID NO.23, and a forward primer shown in SEQ ID NO.24 and a reverse primer shown in SEQ ID NO.23; A primer pair for detecting the fusion gene CD74-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 25 and a reverse primer shown in SEQ ID NO. 26; A primer pair for detecting the fusion gene CDC27-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 27 and a reverse primer shown in SEQ ID NO. 9; A primer pair for detecting the fusion gene CDH11-USP6, the primer pair comprising a forward primer shown in SEQ ID NO.28 and a reverse primer shown in SEQ ID NO.29, a forward primer shown in SEQ ID NO.28 and a reverse primer shown in SEQ ID NO.30, a forward primer shown in SEQ ID NO.31 and a reverse primer shown in SEQ ID NO.29, and a forward primer shown in SEQ ID NO.31 and a reverse primer shown in SEQ ID NO.30; A primer pair for detecting the fusion gene CHCHD7-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO. 32 and a reverse primer shown in SEQ ID NO. 33, and a forward primer shown in SEQ ID NO. 32 and a reverse primer shown in SEQ ID NO. 34; A primer pair for detecting the fusion gene CIC-DUX4, the primer pair comprising a forward primer shown in SEQ ID NO. 35 and a reverse primer shown in SEQ ID NO. 36; A primer pair for detecting the fusion gene CIC-FOXO4, the primer pair comprising a forward primer shown in SEQ ID NO.35 and a reverse primer shown in SEQ ID NO.36, and a forward primer shown in SEQ ID NO.37 and a reverse primer shown in SEQ ID NO.38; A primer pair for detecting the fusion gene CIC-FOXO4, the primer pair comprising a forward primer shown in SEQ ID NO. 39 and a reverse primer shown in SEQ ID NO. 38; A primer pair for detecting the fusion gene CLCN6-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.40 and a reverse primer shown in SEQ ID NO.41; A primer pair for detecting the fusion gene CLTC-ALK, the primer pair comprising a forward primer shown in SEQ ID NO.42 and a reverse primer shown in SEQ ID NO.43; A primer pair for detecting the fusion gene CLTC-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.44 and a reverse primer shown in SEQ ID NO.12; A primer pair for detecting the fusion gene CNBP-USP6, the primer pair comprising a forward primer shown in SEQ ID NO.45 and a reverse primer shown in SEQ ID NO.29, and a forward primer shown in SEQ ID NO.45 and a reverse primer shown in SEQ ID NO.30; A primer pair for detecting the fusion gene COL1A1-PDGFB, the primer pair comprising a forward primer represented by SEQ ID NO.46 and a reverse primer represented by SEQ ID NO.47, and a forward primer represented by SEQ ID NOs.48 to 73 and a reverse primer represented by SEQ ID NO.47; A primer pair for detecting the fusion gene COL1A1-USP6, comprising a forward primer shown in SEQ ID NO. 74 and reverse primers shown in SEQ ID NO. 29, SEQ ID NO. 30, and SEQ ID NO. 75; A primer pair for detecting the fusion gene COL1A2-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO. 76 and a reverse primer represented by SEQ ID NO. 33, and a forward primer represented by SEQ ID NO. 76 and a reverse primer represented by SEQ ID NO. 34; A primer pair for detecting the fusion gene CRTC1-MAML2, the primer pair comprising a forward primer of SEQ ID NO. 77 and a reverse primer of SEQ ID NO. 78, and a forward primer of SEQ ID NO. 77 and a reverse primer of SEQ ID NO. 79; A primer pair for detecting the fusion gene CRTC3-MAML2, the primer pair comprising a forward primer shown in SEQ ID NO. 80 and a reverse primer shown in SEQ ID NO. 79; A primer pair for detecting the fusion gene CTNNB1-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO.81 and a reverse primer shown in SEQ ID NO.33, and a forward primer shown in SEQ ID NO.81 and a reverse primer shown in SEQ ID NO.34; A primer pair for detecting the fusion gene DNAJB1-PRKACA, the primer pair comprising a forward primer shown in SEQ ID NO.82 and a reverse primer shown in SEQ ID NO.83, and a forward primer shown in SEQ ID NO.84 and a reverse primer shown in SEQ ID NO.85; A primer pair for detecting the fusion gene EPC1-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.86 and a reverse primer shown in SEQ ID NO.87; A primer pair for detecting the fusion gene ERC1-RET, the primer pair comprising forward primers shown in SEQ ID NOs. 88 to 92 and a reverse primer shown in SEQ ID NO. 23; A primer pair for detecting the fusion gene ETV6-NTRK3, the primer pair comprising a forward primer shown in SEQ ID NO.93 and a reverse primer shown in SEQ ID NO.94, and a forward primer shown in SEQ ID NO.95 and a reverse primer shown in SEQ ID NO.94; A primer pair for detecting the fusion gene EWSR1-ATF1, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.97, a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.98, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.98, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.100, and a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.97; A primer pair for detecting the fusion gene EWSR1-CREB1, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.102; A primer pair for detecting the fusion gene EWSR1-DDIT3, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.105, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.103; A primer pair for detecting the fusion gene EWSR1-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.106, a forward primer shown in SEQ ID NO.107 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.106, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.109; A primer pair for detecting the fusion gene EWSR1-ETV1, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.110; A primer pair for detecting the fusion gene EWSR1-ETV4, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.111; A primer pair for detecting the fusion gene EWSR1-FEV, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.112, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.112; A primer pair for detecting the fusion gene EWSR1-FLI1, the primer pair comprising a forward primer set forth in SEQ ID NO.96 and a reverse primer set forth in SEQ ID NOs.113 to 116, a forward primer set forth in SEQ ID NO.99 and a reverse primer set forth in SEQ ID NOs.113 to 117, a forward primer set forth in SEQ ID NO.101 and a reverse primer set forth in SEQ ID NO.113, a forward primer set forth in SEQ ID NO.101 and a reverse primer set forth in SEQ ID NO.115, a forward primer set forth in SEQ ID NO.96 and a reverse primer set forth in SEQ ID NO.118, a forward primer set forth in SEQ ID NO.96 and a reverse primer set forth in SEQ ID NO.115, and a forward primer set forth in SEQ ID NO.101 and a reverse primer set forth in SEQ ID NO.113; A primer pair for detecting the fusion gene EWSR1-NFATC2, the primer pair comprising a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.119; A primer pair for detecting the fusion gene EWSR1-NR4A3, the primer pair comprising a forward primer shown in SEQ ID NO.120 and a reverse primer shown in SEQ ID NO.121, a forward primer shown in SEQ ID NO.122 and a reverse primer shown in SEQ ID NO.121, a forward primer shown in SEQ ID NO.122 and a reverse primer shown in SEQ ID NO.123, a forward primer shown in SEQ ID NO.124 and a reverse primer shown in SEQ ID NO.123, a forward primer shown in SEQ ID NO.104 and a reverse primer shown in SEQ ID NO.123, and a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.121; A primer pair for detecting the fusion gene EWSR1-PATZ1, the primer pair comprising a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.125; A primer pair for detecting the fusion gene EWSR1-PBX1, the primer pair comprising a forward primer represented by SEQ ID NO.99 and a reverse primer represented by SEQ ID NO.126, and a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.126; A primer pair for detecting the fusion gene EWSR1-POU5F1, the primer pair comprising a forward primer set forth in SEQ ID NO.107 and a reverse primer set forth in SEQ ID NO.127, a forward primer set forth in SEQ ID NO.99 and a reverse primer set forth in SEQ ID NO.127, and a forward primer set forth in SEQ ID NO.99 and a reverse primer set forth in SEQ ID NO.128; A primer pair for detecting the fusion gene EWSR1-SP3, the primer pair comprising a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.129, and a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.130; A primer pair for detecting the fusion gene EWSR1-WT1, the primer pair comprising a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.131, a forward primer shown in SEQ ID NO.132 and a reverse primer shown in SEQ ID NO.133, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.131, a forward primer shown in SEQ ID NO.99 and a reverse primer shown in SEQ ID NO.134, a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.133, a forward primer shown in SEQ ID NO.101 and a reverse primer shown in SEQ ID NO.131, and a forward primer shown in SEQ ID NO.96 and a reverse primer shown in SEQ ID NO.131; A primer pair for detecting the fusion gene EWSR1-ZNF444, the primer pair comprising a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.135, and a forward primer represented by SEQ ID NO.101 and a reverse primer represented by SEQ ID NO.136; A primer pair for detecting the fusion gene FAM131B-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.137 and a reverse primer shown in SEQ ID NO.9, and a forward primer shown in SEQ ID NO.138 and a reverse primer shown in SEQ ID NO.9; A primer pair for detecting the fusion gene FCHSD1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.139 and a reverse primer shown in SEQ ID NO.9; A primer pair for detecting the fusion gene FGFR1-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO.140 and a reverse primer represented by SEQ ID NO.33, and a forward primer represented by SEQ ID NO.141 and a reverse primer represented by SEQ ID NO.33; A primer pair for detecting the fusion gene FCHSD1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.139 and a reverse primer shown in SEQ ID NO.9; A primer pair for detecting the fusion gene FUS-ATF1, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.98; A primer pair for detecting the fusion gene FUS-CREB3L2, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.143, a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.144, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.146, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.143, a forward primer shown in SEQ ID NO.147 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.148 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.149 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.103, a forward primer shown in SEQ ID NO.150 and a reverse primer shown in SEQ ID NO. The reverse primer shown in SEQ ID NO. 103, the forward primer shown in SEQ ID NO. 145, and the reverse primer shown in SEQ ID NO. 103; A primer pair for detecting the fusion gene FUS-DDIT3, the primer pair comprising a forward primer shown in SEQ ID NO.151 and a reverse primer shown in SEQ ID NO.105; A primer pair for detecting the fusion gene FUS-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.142 and a reverse primer shown in SEQ ID NO.106, a forward primer shown in SEQ ID NO.152 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.108, a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.109, and a forward primer shown in SEQ ID NO.145 and a reverse primer shown in SEQ ID NO.106; A primer pair for detecting the GNAI1-BRAF fusion gene, the primer pair comprising a forward primer represented by SEQ ID NO. 153 and a reverse primer represented by SEQ ID NO. 9; A primer pair for detecting the fusion gene GOLGA5_PTC5-RET, the primer pair comprising a forward primer shown in SEQ ID NO.154 and a reverse primer shown in SEQ ID NO.23; A primer pair for detecting the fusion gene HAS2-PLAG1, the primer pair comprising a forward primer shown in SEQ ID NO.155 and a reverse primer shown in SEQ ID NO.33; A primer pair for detecting the fusion gene HERPUD1-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.156 and a reverse primer shown in SEQ ID NO.157; A primer pair for detecting the fusion gene HEY1-NCOA2, the primer pair comprising a forward primer shown in SEQ ID NO.158 and a reverse primer shown in SEQ ID NO.159; A primer pair for detecting the fusion gene HMGA2-ALDH2, the primer pair comprising a forward primer shown in SEQ ID NO. 160 and a reverse primer shown in SEQ ID NO. 161; A primer pair for detecting the fusion gene HMGA2-CCNB1IP1, the primer pair comprising a forward primer shown in SEQ ID NO.162 and a reverse primer shown in SEQ ID NO.163; A primer pair for detecting the fusion gene HMGA2-COX6C, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.164; A primer pair for detecting the fusion gene HMGA2-EBF1, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.165; A primer pair for detecting the fusion gene HMGA2-FHIT, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.166; A primer pair for detecting the fusion gene HMGA2-LHFP, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.167; A primer pair for detecting the fusion gene HMGA2-LPP, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.168, and a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.169; A primer pair for detecting the fusion gene HMGA2-NFIB, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.170; A primer pair for detecting the fusion gene HMGA2-RAD51B, the primer pair comprising a forward primer shown in SEQ ID NO.160 and a reverse primer shown in SEQ ID NO.171; A primer pair for detecting the fusion gene HMGA2-WIF1, the primer pair comprising a forward primer shown in SEQ ID NO. 160 and reverse primers shown in SEQ ID NOs. 172 to 175, and a forward primer shown in SEQ ID NO. 176 and a reverse primer shown in SEQ ID NO. 173; A primer pair for detecting the fusion gene JAZF1-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.177 and a reverse primer shown in SEQ ID NO.87; A primer pair for detecting the fusion gene JAZF1-SUZ12, the primer pair comprising a forward primer shown in SEQ ID NO. 177 and a reverse primer shown in SEQ ID NO. 178; A primer pair for detecting the fusion gene KCTD7-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 179 and a reverse primer represented by SEQ ID NO. 7; A primer pair for detecting the fusion gene KIAA1549-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.180 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.181 and a reverse primer shown in SEQ ID NO.41, a forward primer shown in SEQ ID NO.181 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.183, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.41, a forward primer shown in SEQ ID NO.182 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.184 and a reverse primer shown in SEQ ID NO.9, and a forward primer shown in SEQ ID NO.185 and a reverse primer shown in SEQ ID NO.9; A primer pair for detecting the fusion gene KTN1-RET, the primer pair comprising a forward primer shown in SEQ ID NO.186 and a reverse primer shown in SEQ ID NO.23; A primer pair for detecting the fusion gene LIFR-PLAG1, the primer pair comprising a forward primer represented by SEQ ID NO. 187 and a reverse primer represented by SEQ ID NO. 33, and a forward primer represented by SEQ ID NO. 187 and a reverse primer represented by SEQ ID NO. 34; A primer pair for detecting the fusion gene LMNA-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.188 and a reverse primer shown in SEQ ID NO.189; A primer pair for detecting the fusion gene MACF1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO.190 and a reverse primer represented by SEQ ID NO.9, and a forward primer represented by SEQ ID NO.191 and a reverse primer represented by SEQ ID NO.9; A primer pair for detecting the fusion gene MEAF6-PHF1, the primer pair comprising a forward primer shown in SEQ ID NO.192 and a reverse primer shown in SEQ ID NO.87; A primer pair for detecting the fusion gene MIR548F1-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 193 and a reverse primer shown in SEQ ID NO. 189; A primer pair for detecting the fusion gene MKRN1-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 194 and a reverse primer shown in SEQ ID NO. 41; A primer pair for detecting the fusion gene MPRIP-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.195 and a reverse primer shown in SEQ ID NO.26, a forward primer shown in SEQ ID NO.196 and a reverse primer shown in SEQ ID NO.26, a forward primer shown in SEQ ID NO.197 and a reverse primer shown in SEQ ID NO.26, and a forward primer shown in SEQ ID NO.197 and a reverse primer shown in SEQ ID NO.198; A primer pair for detecting the fusion gene MYB-NFIB, the primer pair comprising a forward primer shown in SEQ ID NO.199 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.199 and a reverse primer shown in SEQ ID NO.170, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.202, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.170, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.203, a forward primer shown in SEQ ID NO.201 and a reverse primer shown in SEQ ID NO.204, a forward primer shown in SEQ ID NO.205 and a reverse primer shown in SEQ ID NO.200, a forward primer shown in SEQ ID NO.205 and a reverse primer shown in SEQ ID NO.170, and a reverse primer shown in SEQ ID NO. The forward primer shown in SEQ ID NO.205 and the reverse primer shown in SEQ ID NO.204; A primer pair for detecting the fusion gene MYH9-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 206 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 206 and a reverse primer represented by SEQ ID NO. 30; A primer pair for detecting the fusion gene NAB2-STAT6, the primer pair comprising a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.208, a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.209, a forward primer shown in SEQ ID NO.207 and a reverse primer shown in SEQ ID NO.210, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.212, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.208, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.213, a forward primer shown in SEQ ID NO.211 and a reverse primer shown in SEQ ID NO.214, a forward primer shown in SEQ ID NO.215 and a reverse primer shown in SEQ ID NO.216, a forward primer shown in SEQ ID NO.217 and a reverse primer shown in SEQ ID NO.218, a forward primer shown in SEQ ID NO.219 and a reverse primer shown in SEQ ID NO.220, The forward primer shown in SEQ ID NO. 217 and the reverse primer shown in SEQ ID NO. 216, the forward primer shown in SEQ ID NO. 217 and the reverse primer shown in SEQ ID NO. 218, and the forward primer shown in SEQ ID NO. 219 and the reverse primer shown in SEQ ID NO. 208; A primer pair for detecting the fusion gene NACC2-NTRK2, the primer pair comprising a forward primer shown in SEQ ID NO.220 and a reverse primer shown in SEQ ID NO.221; A primer pair for detecting the fusion gene NCOA4-RET, the primer pair comprising a forward primer shown in SEQ ID NO.222 and a reverse primer shown in SEQ ID NO.23, a forward primer shown in SEQ ID NO.223 and a reverse primer shown in SEQ ID NO.224, a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.23, a forward primer shown in SEQ ID NO.226 and a reverse primer shown in SEQ ID NO.227, a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.228, and a forward primer shown in SEQ ID NO.225 and a reverse primer shown in SEQ ID NO.23; A primer pair for detecting the fusion gene NDRG1-ERG, the primer pair comprising a forward primer represented by SEQ ID NO.229 and a reverse primer represented by SEQ ID NO.157; A primer pair for detecting the fusion gene NFASC-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 230 and a reverse primer represented by SEQ ID NO. 189; A primer pair for detecting the fusion gene NONO-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.231 and a reverse primer shown in SEQ ID NO.12; A primer pair for detecting the fusion gene NUDCD3-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 232 and a reverse primer shown in SEQ ID NO. 9; A primer pair for detecting the fusion gene OMD-USP6, the primer pair comprising a forward primer shown in SEQ ID NO. 233 and a reverse primer shown in SEQ ID NO. 29; A primer pair for detecting the fusion gene PAPSS1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 234 and a reverse primer represented by SEQ ID NO. 9; A primer pair for detecting the fusion gene PAX3-AFX, the primer pair comprising a forward primer shown in SEQ ID NO. 235 and a reverse primer shown in SEQ ID NO. 38; A primer pair for detecting the fusion gene PAX3-FOXO1, the primer pair comprising a forward primer shown in SEQ ID NO. 235 and a reverse primer shown in SEQ ID NO. 236; A primer pair for detecting the fusion gene PAX3-NCOA1, the primer pair comprising a forward primer of SEQ ID NO. 237 and a reverse primer of SEQ ID NO. 238, a forward primer of SEQ ID NO. 235 and a reverse primer of SEQ ID NO. 239, and a forward primer of SEQ ID NO. 235 and a reverse primer of SEQ ID NO. 240; A primer pair for detecting the fusion gene PAX7-FOXO1, the primer pair comprising a forward primer shown in SEQ ID NO. 241 and a reverse primer shown in SEQ ID NO. 236; A primer pair for detecting the fusion gene PAX8-PPARG, the primer pair comprising a forward primer represented by SEQ ID NO. 242 and a reverse primer represented by SEQ ID NO. 243, a forward primer represented by SEQ ID NO. 242 and a reverse primer represented by SEQ ID NO. 244, and forward primers represented by SEQ ID NOs. 245 to 247 and a reverse primer represented by SEQ ID NO. 244; A primer pair for detecting the fusion gene PLIN3-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 248 and a reverse primer shown in SEQ ID NO. 9; A primer pair for detecting the fusion gene POR-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.249 and a reverse primer shown in SEQ ID NO.41; A primer pair for detecting the fusion gene PRCC-TFE3, the primer pair comprising a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.251, a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.11, a forward primer represented by SEQ ID NO.250 and a reverse primer represented by SEQ ID NO.12, and a forward primer represented by SEQ ID NO.252 and a reverse primer represented by SEQ ID NO.251; A primer pair for detecting the fusion gene PRKAR1A-RET, comprising SEQ ID Forward primers shown in SEQ ID NO. 253-254 and reverse primer shown in SEQ ID NO. 23; A primer pair for detecting the fusion gene QKI-NTRK2, the primer pair comprising a forward primer shown in SEQ ID NO.255 ​​and a reverse primer shown in SEQ ID NO.256; A primer pair for detecting the fusion gene RANBP2-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 257 and a reverse primer represented by SEQ ID NO. 14; A primer pair for detecting the fusion gene RNF130-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO. 258 and a reverse primer shown in SEQ ID NO. 9; A primer pair for detecting the fusion gene SEC31A-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 259 and a reverse primer represented by SEQ ID NO. 43; A primer pair for detecting the fusion gene SERPINE1-FOSB, the primer pair comprising a forward primer shown in SEQ ID NO. 260 and reverse primers shown in SEQ ID NOs. 261 to 262; A primer pair for detecting the fusion gene SFPQ-TFE3, the primer pair comprising a forward primer represented by SEQ ID NO.263 and a reverse primer represented by SEQ ID NO.11, a forward primer represented by SEQ ID NO.264 and a reverse primer represented by SEQ ID NO.11, and a forward primer represented by SEQ ID NO.264 and a reverse primer represented by SEQ ID NO.12; A primer pair for detecting the fusion gene SLC45A3-BRAF, the primer pair comprising a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 7, a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 9, and a forward primer set forth in SEQ ID NO. 265 and a reverse primer set forth in SEQ ID NO. 157; A primer pair for detecting the fusion gene SND1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 266 and a reverse primer represented by SEQ ID NO. 9; A primer pair for detecting the SOX6-BRAF fusion gene, the primer pair comprising a forward primer shown in SEQ ID NO. 267 and a reverse primer shown in SEQ ID NO. 9; A primer pair for detecting the fusion gene SQSTM1-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 268 and a reverse primer represented by SEQ ID NO. 189; A primer pair for detecting the fusion gene SS18-SSX1, the primer pair comprising a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.270, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.271, a forward primer shown in SEQ ID NO.272 and a reverse primer shown in SEQ ID NO.273, a forward primer shown in SEQ ID NO.272 and a reverse primer shown in SEQ ID NO.270, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.274, a forward primer shown in SEQ ID NO.275 and a reverse primer shown in SEQ ID NO.274, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.276, a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO.277, and a forward primer shown in SEQ ID NO.269 and a reverse primer shown in SEQ ID NO. Reverse primer shown in No. 278; A primer pair for detecting the fusion gene SSBP2-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 279 and a reverse primer represented by SEQ ID NO. 26; A primer pair for detecting the fusion gene STRN-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 280 and a reverse primer shown in SEQ ID NO. 14; A primer pair for detecting the fusion gene TAF15-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO.281 and a reverse primer represented by SEQ ID NO.123; A primer pair for detecting the fusion gene TAX1BP1-BRAF, the primer pair comprising a forward primer represented by SEQ ID NO. 282 and a reverse primer represented by SEQ ID NO. 41; A primer pair for detecting the TCEA1-PLAG1 fusion gene, the primer pair comprising a forward primer represented by SEQ ID NO. 283 and a reverse primer represented by SEQ ID NO. 34; A primer pair for detecting the fusion gene TCF12-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO. 284 and a reverse primer represented by SEQ ID NO. 123; A primer pair for detecting the fusion gene TFG-NR4A3, the primer pair comprising a forward primer represented by SEQ ID NO.285 and a reverse primer represented by SEQ ID NO.123; A primer pair for detecting the fusion gene TFG-NTRK1, the primer pair comprising a forward primer set forth in SEQ ID NO. 286 and a reverse primer set forth in SEQ ID NO. 189, a forward primer set forth in SEQ ID NO. 286 and a reverse primer set forth in SEQ ID NO. 287, and a forward primer set forth in SEQ ID NO. 288 and a reverse primer set forth in SEQ ID NO. 189; A primer pair for detecting the fusion gene THRAP3-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 75; A primer pair for detecting the fusion gene THRAP3-USP6, the primer pair comprising a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 29, and a forward primer represented by SEQ ID NO. 289 and a reverse primer represented by SEQ ID NO. 75; A primer pair for detecting the fusion gene TMPRSS2-ERG, the primer pair comprising a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.291, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.292, a forward primer shown in SEQ ID NO.293 and a reverse primer shown in SEQ ID NO.291, a forward primer shown in SEQ ID NO.293 and a reverse primer shown in SEQ ID NO.294, a forward primer shown in SEQ ID NO.290 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.295 and a reverse primer shown in SEQ ID NO.157, a forward primer shown in SEQ ID NO.296 and a reverse primer shown in SEQ ID NO.157, and a forward primer shown in SEQ ID NO.297 and a reverse primer shown in SEQ ID NO. Reverse primer shown in NO.157; A primer pair for detecting the fusion gene TP53-NTRK1, the primer pair comprising a forward primer represented by SEQ ID NO. 298 and a reverse primer represented by SEQ ID NO. 287; A primer pair for detecting the fusion gene TPM3-ALK, the primer pair comprising a forward primer represented by SEQ ID NO. 299 and a reverse primer represented by SEQ ID NO. 14; A primer pair for detecting the fusion gene TPM3-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO. 299 and a reverse primer shown in SEQ ID NO. 190; A primer pair for detecting the fusion gene TPM4-ALK, the primer pair comprising a forward primer shown in SEQ ID NO. 300 and a reverse primer shown in SEQ ID NO. 14; A primer pair for detecting the fusion gene TPR-NTRK1, the primer pair comprising a forward primer shown in SEQ ID NO.301 and a reverse primer shown in SEQ ID NO.190; A primer pair for detecting the fusion gene TRIM24-BRAF, the primer pair comprising a forward primer shown in SEQ ID NO.302 and a reverse primer shown in SEQ ID NO.7, a forward primer shown in SEQ ID NO.303 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.304 and a reverse primer shown in SEQ ID NO.9, a forward primer shown in SEQ ID NO.305 and a reverse primer shown in SEQ ID NO.23, and a forward primer shown in SEQ ID NO.304 and a reverse primer shown in SEQ ID NO.23; A primer pair for detecting the fusion gene WWTR1-CAMTA1, the primer pair comprising a forward primer represented by SEQ ID NO. 306 and a reverse primer represented by SEQ ID NO. 307, and a forward primer represented by SEQ ID NO. 306 and a reverse primer represented by SEQ ID NO. 308; A primer pair for detecting the fusion gene YAP1-TFE3, the primer pair comprising a forward primer shown in SEQ ID NO.309 and a reverse primer shown in SEQ ID NO.251; A primer pair for detecting the fusion gene YWHAE-FAM22A, the primer pair comprising a forward primer shown in SEQ ID NO.310 and a reverse primer shown in SEQ ID NO.311; A primer pair for detecting the fusion gene ZC3H7B-BCOR, the primer pair comprising a forward primer shown in SEQ ID NO.312 and a reverse primer shown in SEQ ID NO.313; A primer pair for detecting the fusion gene ZKSCAN5-BRAF, comprising a forward primer shown in SEQ ID NO.314 and a reverse primer shown in SEQ ID NO.

9.

2. The primer set for detecting fusion gene variation according to claim 1, characterized in that: A universal primer as shown in SEQ ID NO. 335 is added to the 5' end of the forward primer of the primer pair, and a universal primer as shown in SEQ ID NO. 336 is added to the 5' end of the reverse primer of the primer pair.

3. Use of the primer set according to claim 1 or 2 in preparing a kit for detecting fusion gene mutations.

4. A kit for detecting fusion gene mutation, characterized in that: The kit comprises the primer set for detecting fusion gene variation according to claim 1 or 2.

5. The kit for detecting fusion gene variation according to claim 4, characterized in that: The kit also includes a primer set for detecting internal reference genes, and the internal reference genes include ITGB7, MRPL13, HPRT1, TBP, JUN, MYC, IPO8, LRP1, HMBS, and YWHAZ.

6. The kit for detecting fusion gene variation according to claim 5, characterized in that: The primer set for detecting the internal reference gene includes: a primer pair for detecting ITGB7, the primer pair including the forward primer shown in SEQ ID NO.315 and the reverse primer shown in SEQ ID NO.316; a primer pair for detecting MRPL13, the primer pair including the forward primer shown in SEQ ID NO.317 and the reverse primer shown in SEQ ID NO.318; a primer pair for detecting HPRT1, the primer pair including the forward primer shown in SEQ ID NO.319 and the reverse primer shown in SEQ ID NO.320; a primer pair for detecting TBP, the primer pair including the forward primer shown in SEQ ID NO.321 and the reverse primer shown in SEQ ID NO.322; a primer pair for detecting JUN, the primer pair including the forward primer shown in SEQ ID NO.323 and the reverse primer shown in SEQ ID NO.324; a primer pair for detecting MYC, the primer pair including the forward primer shown in SEQ ID NO.325 and the reverse primer shown in SEQ ID NO.

326. NO.326; a primer pair for detecting IPO8, the primer pair comprising the forward primer shown in SEQ ID NO.327 and the reverse primer shown in SEQ ID NO.328; a primer pair for detecting LRP1, the primer pair comprising the forward primer shown in SEQ ID NO.329 and the reverse primer shown in SEQ ID NO.330; a primer pair for detecting HMBS, the primer pair comprising the forward primer shown in SEQ ID NO.331 and the reverse primer shown in SEQ ID NO.332; and a primer pair for detecting YWHAZ, the primer pair comprising the forward primer shown in SEQ ID NO.333 and the reverse primer shown in SEQ ID NO.334.

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